Synergistic Antibiotic‒Antibiotic and Antibiotic‒Phage Combinations Against Biofilm-Associated Staphylococcus aureus in Dual-Species Biofilms

Synergistic Antibiotic‒Antibiotic and Antibiotic‒Phage Combinations Against Biofilm-Associated Staphylococcus aureus in Dual-Species Biofilms | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Synergistic Antibiotic‒Antibiotic and Antibiotic‒Phage Combinations Against Biofilm-Associated Staphylococcus aureus in Dual-Species Biofilms Paria Mojarrad, Hanna Tiainen, Päivi Tammela, Timo Laaksonen, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8986145/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 10 You are reading this latest preprint version Abstract Treating polymicrobial biofilms often requires the combination of antibiotics and the participating species play a major role in the response of these bacteria to antimicrobial agents. S. aureus and P. aeruginosa are among the most common species found in polymicrobial biofilms. We used in-house dual-species biofilms of S. aureus and P. aeruginosa , to evaluate their response to antibiotics, combinations of antibiotics and selected antibiotics‒phage combinations. The interaction of antibiotic combinations on the inhibition and eradication of biofilms were assessed by calculating the fractional inhibitory concentration index (FICI). Coculture of P. aeruginosa and S. aureus altered their antibiotic sensitivity and S. aureus became highly tolerant to antibiotics. Most antibiotic combinations have synergistic effects on biofilm inhibition. The combinations of vancomycin‒ofloxacin and vancomycin‒ciprofloxacin had FICI values of 0.034 and 0.055 against S. aureus respectively. Combinations of glycopeptide and fluoroquinolone antibiotics also showed synergistic interactions in biofilm eradication. The addition of phage Stab 21 restored the efficacy of ciprofloxacin and vancomycin against S. aureus in dual-species biofilms, reducing their biofilm inhibitory concentration to 0.5 µg/mL from 33 µg/mL for ciprofloxacin and > 2048 µg/mL for vancomycin. In summary, we describe the susceptibility profile of S. aureus to combination treatments within a dual-species biofilm and report effective antibiotic–antibiotic and phage–antibiotic combinations against antibiotic-tolerant S. aureus . Antimicrobial combination dual-species biofilm Staphylococcus aureus Pseudomonas aeruginosa antimicrobial resistance (AMR) bacterial interaction bacteriophage therapy Figures Figure 1 Figure 2 1. Introduction Polymicrobial biofilms are major cause of treatment failure in chronic infections by reducing bacterial susceptibility to both antimicrobial agents and host immune systems [ 1 ]. Staphylococcus aureus and Pseudomonas aeruginosa are the two most frequently isolated species from chronic infections including chronic wound infections, respiratory infections in cystic fibrosis (CF), endocarditis, and implantable device-associated infections [ 2 , 3 ]. Clinical observations show a complex and dynamic interspecies interaction between these two species. P. aeruginosa outcompete S. aureus and inhibits its growth in early cloning by secreting several staphylolytic compounds such as siderophores and 4-hydroxy-2-heptylquinoline-N-oxide (HQNO) [ 4 , 5 ]. In later stage of colonization S. aureus defense mechanism drive it to develop metabolic variants such as small-colony variant (SCV), with fermentative growth state and profoundly increased antibiotic tolerance [ 4 – 6 ]. This complex interspecies interaction has inspired several researchers to develop in vitro biofilm models of these two bacteria. However, in vitro co-culture of the two species is challenging as P. aeruginosa strongly inhibit the growth of S. aureus , which does not reflect what observed in vivo. The modification of growth media is needed to support co-culture of both species. Monotherapy often fails to eradicate polymicrobial biofilms because they exhibit higher resilience and drug tolerance compared to mono-species biofilms [ 7 ]. Combination therapy involving the simultaneous use of two or three antibiotics or nonantibiotic agents is the leading clinical approach against polymicrobial infections [ 8 ]. Combination of antibiotic regimens enhances efficacy against biofilms through synergistic interaction, reduced resistance incidence, and improved penetration into the biofilm matrix [ 8 – 10 ]. To target polymicrobial biofilms of S. aureus and P. aeruginosa , antibiotic combinations should include either species-specific agents or broad-spectrum antibiotics effective against both organisms. Biological antibacterial agents are rapidly gaining interest in fighting against chronic infections. Phage therapy, in particular, has emerged as a promising addition to antibiotic treatment for chronic biofilm owing to its ability to replicate within bacterial populations and penetrate biofilm structures [ 11 ]. The phage Stab 21 belongs to the genus Kayvirus in the subfamily Twortvirinae of the family Herelleviridae. It has a broad host range primarily among S. aureus strains and thus poses great potential for therapeutic use against S. aureus infections [ 12 , 13 ]. Combination therapy is ideal if the interaction of combined agents is synergistic or additive. There are previous reports on synergistic interaction of phage–antibiotic combinations against biofilm [ 31 , 32 ]. Several studies have established in vitro dual-species biofilm of S. aureus and P. aeruginosa to investigate their interspecies interaction and antibacterial susceptibility profile [ 14 , 15 ]. However, employing such models to investigate their response to combination strategies remains limited. We previously reported a dual-species biofilm model of P. aeruginosa and S. aureus , developed on fibroblast-derived cell matrix (CDM) [ 16 ]. CDMs are decellularized extracellular matrices that help to mimic the microenvironments of in vivo biofilms. We used this model to investigate the response of P. aeruginosa and S. aureus to single and combined antibiotics. As S. aureus became highly antibiotic-tolerant in the biofilm, it was our primary target in this study. The antibiotics were chosen from glycopeptides, β-lactams, aminoglycosides, fluoroquinolones (FQs), rifampicin, and polymyxins which are effective against either S. aureus or P. aeruginosa or both [ 7 , 17 , 18 ]. Colistin is a clinically relevant antipseudomonal antibiotic [ 19 ]. Vancomycin, teicoplanin, penicillin, and ampicillin are effective against S. aureus , while gentamicin, tobramycin, ciprofloxacin, levofloxacin, ofloxacin, and rifampicin are active against both species. Vancomycin is among the most included antibiotic in combination therapy against staphylococcal biofilms [ 20 ] and has well-documented synergy against both P. aeruginosa and S. aureus [ 21 , 22 ]. We further assessed the effect of adding the phage Stab 21 to the best antibiotic‒antibiotic combination against S. aureus . Finally, we evaluated selected antibiotic combinations on the biofilm developed on CDM. 2. Materials and Methods 2.1. Strains and culture conditions P. aeruginosa (PAO1 [ATCC 47085]), ATCC 33352) and S. aureus (ATCC 25923) were stored at − 80°C and revived on tryptic soy agar (TSA; Catalog No. CM0131B; Thermo Fisher Scientific) before use. Overnight cultures were prepared by inoculating a single colony from each bacterial species into 10 mL of tryptic soy broth (TSB)[ 16 , 23 , 24 ] and incubating at 37°C with shaking (200 rpm) for 20 h. Biofilm formation was performed in TSB (mono-species biofilm) and TSB+ fetal bovine serum (FBS) (dual-species biofilm). TSB is used as a standard, nutrient-rich medium that promotes S. aureus biofilm and we showed in our previous study that TSB enriched with NaCl and glucose in combination with FBS is the optimum media for culture of S. aureus in presence of P. aeruginosa [ 25 ]. Four-hour subcultures were prepared via a 1:1000 dilution of the overnight cultures in fresh TSB and incubated for an additional 4 h under the same conditions. Freshly prepared overnight and 4-hour cultures were used for all the experiments. All the experiments in this study were performed with two biological replicates, each of which was measured in technical duplicate unless otherwise stated. Most of the experiments were also repeated independently at least two times to ensure their reproducibility. 2.1. Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) MIC assays were performed by the broth dilution method according to Hacek et al . with minor modifications [ 26 ]. Serial two-fold dilution of antibiotics was done in TSB in 96-well microtiter polystyrene plates (catalog No. 167008; Thermo Fisher Scientific, Nunclon). Wells were inoculated with a 4-h culture of either S. aureus or P. aeruginosa to a final concentration of ~ 10 6 CFU/ml. Positive (inoculated TSB without antibiotic) and negative (TSB) controls were included. The plates were incubated at 37°C for 20 h. Bacterial growth was monitored by measuring optical density (OD) at 600 nm using Multiskan GO Microplate Reader (Thermo Fisher Scientific, Waltham, MA, USA). The MIC was defined as the lowest antibiotic concentration resulting in no visible increase in turbidity compared to the negative control (TSB). To determine MBC, wells without visible growth were spotted (3*5 µL) on TSA plates and incubated at 37°C for 20 h to determine the MBC values. MBC was defined as the lowest concentration of antibiotic resulting in no growth on the agar plates [ 27 ]. 2.2. Dual-species biofilm formation Dual-species biofilms were developed according to our previous work with minor modifications [ 16 ]. Briefly, TSB supplemented with 1% (w/v) glucose and 2.5% (w/v) sodium chloride was mixed 1:1 with fetal bovine serum (FBS; Catalog No. A5256701, Thermo Fisher Scientific) to support co-growth of S. aureus with P. aeruginosa . In each well, 180 µL of the 1:1 TSB-FBS mixture was inoculated with 10 µL of a 4-hour S. aureus mixture (~ 10 7 CFU/mL) and 1 µL of a 4-hour P. aeruginosa mixture (~ 10 6 CFU/mL), resulting in a final volume of 191 µL per well. The growth media without inoculation was used as negative controls. The plates were subsequently incubated at 37°C for 20 h without shaking to allow biofilm development. The pH of the biofilm was measured after biofilm formation. 2.3. Colony counting Dual-species biofilm formation was confirmed by colony counting. Overnight and 4-hour cultures (100 µL) were serially tenfold diluted in phosphate-buffered saline (PBS) and plated (3×10 µl) on TSA. For dual-species biofilms, 100 µl aliquots of planktonic cells from the supernatant and biofilm (redispersed in PBS), serially ten-fold diluted and cultured on MacConkey agar (MCA; Catalog No. CM0007, Thermo Fisher Scientific) and mannitol salt agar (MSA; Catalog No. CM0085B, Thermo Fisher Scientific). The biofilm samples were mechanically disrupted by pipetting and vertexing to minimize cell aggregation and ensure single-cell suspension. MCA and MSA were used as selective growth media for Gram-negative bacteria including P . aeruginosa and S. aureus , respectively. The plates were incubated at 37°C for 20 h (TSA, MCA), or 48–72 h (MSA) to account for the slower growth rate of S. aureus . The colony counts are expressed as log 10 CFU/mL [ 16 ]. 2.4. Mono-species biofilm formation Mono-species biofilms were established in polystyrene 96-well plates in 180 µL TSB inoculated with 4-hour culture of S. aureus or P. aeruginosa (~ 10 7 CFU/mL). Wells with TSB without inoculation were used as negative controls. The plates were incubated at 37°C for 20 h. Bacterial growth was verified by OD 600 measurement. 2.5. Phages, host bacteria, and phage propagation The phage Stab 21 was selected to target S. aureus , and the phage PA8P1 was selected to target P. aeruginosa [ 12 , 13 , 28 , 29 ]. The host bacterial strains S. xylosus DD-34 [ 13 ] for Stab 21 and P. aeruginosa PA8 [ 29 ] for PA8P1, S. aureus and P. aeruginosa (ATCC 33352), were stored at ‑70°C and retrieved on Lysogeny agar (LA) plates, i.e., Lysogeny Broth (LB) solidified with 1.5% (w/v) agar [ 28 – 30 ]. For phage propagation, one colony of the host strain was inoculated into 5 mL LB and incubated overnight at 37°C in a tube rotator (Fisherbrand Mini Tube Rotator, Thermo Fisher Scientific, USA) at 15 rpm. The phage lysates were prepared by adding 400–500 µL of overnight bacterial culture and 40 µL of phage stock (in DMSO or glycerol) stored at ‑70°C into 10 mL LB and incubating at 37°C in a tube rotator at 15 rpm. Once the lysis had occurred (within 3–5 h), the tubes were centrifuged for 5 min at 5000 rpm using a Thermo Fisher Scientific SL16R centrifuge (rotor TX-400) to separate the remaining bacterial cells and cellular debris. The supernatant was passed through an LLG-syringe filter (SPHEROS, LLG Labware) with a pore size of 0.22 µm to remove any remaining bacterial cells. To enhance phage stability, 40% sucrose was added to a final concentration of 8% (v/v) and the resulting phage lysate was stored at 4°C throughout the study. 2.6. Phage titration by the double-layer method A well-established double-layer agar plating method (Sambrook, 2001) with slight modifications was used to determine the titer of the phage lysate and the quantity of plaque-forming units (PFU) per well [ 30 ]. In brief, single colonies of each host bacterial species from agar plates were grown in 1.3 mL LB in 2 mL Eppendorf tubes for 2–3 h at 37˚C in a tube rotator (Fisherbrand Mini Tube Rotator, Thermo Fischer Scientific, USA) at 15 rpm. The absorbance at 600 nm (A 600 ) was measured via a DSM Cell Density Meter (Laxco, USA), and the volume of bacterial culture for one titration plate was calculated as µL = 45 / A 600 . A tenfold dilution series of up to 10 − 8 bacteriophage lysates in LB was prepared. Appropriate amounts of host bacteria and 3 mL of melted and tempered (at 55°C) soft agar with 50 µL of the appropriate dilution of phage lysate were mixed gently and poured onto LA plates evenly. The plates were left at room temperature (RT) for 30 min to solidify before being incubated overnight at 37°C. The average density of infectious bacteriophage particles in a phage lysate was calculated as PFU/mL. 2.7. Phage susceptibility testing by a drop test and a liquid culture assay Preliminary phage susceptibility testing was performed by a drop test on a solid surface. Briefly, host bacterial strains were grown in 1.3 mL of LB at 37°C until the exponential growth phase (A 600 = 0.2-1) was reached. Absorbance at 600 nm (A 600 ) was measured with a DSM Cell Density Meter (Laxco, USA), and the volume of bacterial culture needed for one plate was calculated as µL = 45 / A 600 . Appropriate amounts of host bacteria and 3 mL of melted and tempered (at 55°C) soft agar were mixed gently and poured onto LA plates evenly. The plates were left at RT for 30 min to solidify before pipetting 5 µL of phage lysate in dilutions of 10 − 2 to 10 − 7 PFU/mL. Phage infections against their original bacterial host strain were used as positive control. The plates were left to settle for an additional 30 min at RT, followed by overnight incubation at 37˚C. A liquid-culture phage screening assay was used as a complementary assay to verify the efficiency of infection by measuring bacterial growth kinetic curves with a microscopy plate reader, oCelloScope (BioSense Solutions Aps, Denmark). It captures time-lapse digital microscopy images of microbial cultures and quantifies changes in cell density over time using automated image-analysis. Previously optimized species-specific growth conditions from Patpatia et al . (2022) were used, and the screening time was 18 h at 37˚C without shaking [ 31 ]. The efficiency of infection was determined by comparing the growth of uninfected bacteria with that of phage-treated bacteria at a given time point. 2.8. Minimum biofilm inhibitory concentration (MBIC) Serial dilution of antibiotics was prepared in TSB for mono-species biofilms or enriched TSB + FBS (50:50) for dual-species biofilms. Enriched TSB + FBS media did not change the MBIC values compared to TSB for mono-species biofilms, therefore unnecessary usage of FBS was avoided. For phage–antibiotic combinations, phage solution (10 8 PFU/mL) was added to achieve 10 7 PFU per well, and the volume of antibiotic solution was adjusted accordingly. Mono- or dual-species biofilms were formed as described in sections 2.4 and 2.2, with positive controls, inoculated media without antibiotic, and negative controls, non-inoculated media, included. After incubation, wells were homogenized by pipetting and spotted (3×5 µL per well) onto TSA (mono-species biofilms) or MSA and MCA (dual-species biofilms) [ 32 ]. In dual-species biofilms, S. aureus biofilm was spotted after refilling the wells and redispersing the biofilm in 50 µL of fresh media. We assumed the biofilm formation starts at 0 h time of bacterial culture and biofilm and planktonic growth are highly codependent [ 33 ]. Therefore, the MBIC was defined as the minimum antibiotic concentration that prevented planktonic regrowth on the corresponding agar plates. 2.9. Minimum biofilm eradication concentration (MBEC) Mono- or dual-species biofilms were formed in polystyrene 96-well plate as described in sections 2.4 and 2.2. After 20 h of biofilm growth, the supernatant from the preformed biofilm plate was completely removed, and the plate was washed with PBS to remove loose bacterial cells. The antibiotic–antibiotic or phage–antibiotic solutions were added to the wells in the biofilm plate and incubated at 37°C for 20 h. After incubation, 3×5 µL from each well was spotted onto TSA for mono-species biofilms, or onto MSA and MCA for dual-species biofilms [ 32 ]. In dual-species biofilms, S. aureus biofilm was cultured after refilling the wells with 50 µL of fresh media to minimize the interference of P. aeruginosa . The MBEC was defined as having the lowest antibiotic concentration resulting in no planktonic re-growth on the corresponding agar from the treated pre-formed biofilm. 2.10. Checkerboard assay to evaluate antibiotic interactions Checkerboard assay was conducted to evaluate the interaction between the two antibiotics against the dual-species biofilm. In 96-well plates 90 µL of antibiotic A (8× maximum concentration) solution in enriched TSB was added to column 1 of the plate. Serial twofold dilutions were performed across columns 1–10. For antibiotic B, 90 µL of a 4× maximum concentration of antibiotic in enriched TSB was added to row A, columns 1–11. Twofold serial dilutions were carried out vertically down to row G. Enriched TSB were added to column 12 for the negative and positive controls. For inoculation, 10 µL of 4-hour S. aureus culture and 1 µL of 4-hour P. aeruginosa culture were added to each well, excluding the negative control wells. Fresh non-inoculated media and inoculated media without antibiotics were used as negative and positive controls, respectively. Finally, 90 µL of FBS was added to all the wells including the control wells. The plate was incubated at 37°C for 20 h. Following incubation, 3×5µL from each well was spotted onto selective agar plates MCA for P. aeruginosa , and for S. aureus , the wells were first emptied and refilled with 50 µL of fresh media before spotting on MSA to minimize P. aeruginosa interference. The plates were incubated for 20 h (48–72 h for S. aureus ). The fractional inhibitory concentration index (FICI) was calculated via the following Eq. (1) to assess the interaction between the antibiotics [ 34 ]. $$FICI=\frac{{MBIC}_{AB1incombinationwithAB2}}{{MBIC}_{AB1alone}}+\frac{{MBIC}_{AB2incombinationwithAB1}}{{MBIC}_{AB2alone}}\left(1\right)$$ The FICI values were interpreted as follows: synergistic when FICI ≤ 0.5; sub-synergistic when 0.5 < FICI ≤ 1.0; indifferent when 1.0 4.0, indicating antibiotic interactions against the dual-species biofilm. 2.11. Development of the CDM CDMs were prepared following a method adapted from Kaukonen et al. with minor modifications [ 35 ]. Glass coverslips were placed in 24-well plates, sterilized with 70% ethanol, treated with 0.2% (w/v) gelatin in PBS (800 µL) pre-warmed to 37°C, and incubated for 1 h at 37°C. After gelatin was aspirated, the coverslips were washed gently with PBS. Gelatin was crosslinked by adding 1% (v/v) glutaraldehyde solution (500 µL) for 30 min at room temperature (RT), followed by glutaraldehyde aspiration and two PBS washes. The residual glutaraldehyde was quenched with 1 M glycine (500 µL) in PBS for 20 min at RT. Following glycine removal, the coverslips were washed twice with PBS and incubated with pre-warmed Dulbecco's Modified Eagle Medium (DMEM, high glucose) (800 µL, 1 h, 37°C). Human dermal fibroblasts (ATTC PCS-201-010) were then seeded at 5 × 10⁴ cells per well on coverslips and cultured at 37°C until monolayer confluence. The growth medium was then replaced with DMEM supplemented with 50 µg/mL freshly prepared ascorbic acid (0.45 µm-filtered). Cultures were maintained for 14 to 21 days in an incubator (37°C) with ascorbic acid renewed every two days to stimulate extracellular matrix (ECM) production. For decellularization, the wells were rinsed with PBS and incubated with pre-warmed (37°C) standard extraction solution (1 mL of NH₄OH, 250 µL of Triton X-100, and 48.75 mL of PBS: 500 µL per well) [ 35 ]. After ~ 2 min, once the cells were fully detached, the extraction solution was discarded, and the coverslips were washed twice with PBS. The residual cellular DNA was removed by incubation with DNase I was added to each well (10 µg/mL in deionized water; 500 µL per well) and incubated at 37°C for 30 min. After incubation, the CDMs were washed twice with PBS. CDMs were stored at 4°C in PBS containing 1% (v/v) penicillin–streptomycin until further use, for up to 4 weeks [ 35 ]. 2.12. Forming dual-species biofilms on CDMs We developed the dual-species biofilms on CDMs using the method described in our previous work [ 17 ]. The enriched TSB supplemented with 1% glucose and 2.5% NaCl was mixed 1:1 with FBS was used. CDMs were placed in 24-well plates and washed with PBS [ 17 ]. A total of 800 µL of inoculated media containing S. aureus (10⁷ CFU/mL in each well) and P. aeruginosa (10⁶ CFU/mL in each well) was added to the wells. Wells filled with either inoculated or non-inoculated media were used as positive and negative controls, respectively. The plates were incubated at 37°C for 20 h, without shaking, to allow biofilm formation. After incubation, the planktonic bacteria were removed, and the biofilm was gently washed three times with PBS to remove any loosely adherent cells. The pH of the biofilm formed on CDM was measured. 2.13. Combined antibiotics on the dual-species biofilm formed on CDM Antibiotic concentrations for the CDM-biofilm assays were selected based on the MBIC and MBEC data obtained from 2.8 and 2.9 Combinations of antibiotics A and B were tested at their respective MBIC/MBEC values, as well as twofold higher and up to eightfold lower concentrations. Biofilms were established in 24-well plates on predeveloped CDM following the abovementioned biofilm formation protocols (section 2.2). The antibiotic combinations were either added to the media before developing biofilms on CDM for biofilm inhibition assays or to biofilms formed on CDM for biofilm eradication assays. The plates were incubated at 37°C for 20 h without shaking. Following incubation, biofilm viability was assessed by spotting 3×5 µL from each well on MSA and MCA plates. S. aureus biofilm was cultured after refilling the wells with 50 µL of fresh media to minimize the interference of P. aeruginosa . 2.14. Scanning electron microscopy (SEM) Three different samples were prepared for SEM imaging: (i) CDM alone, (ii) a dual-species biofilm formed overnight on CDM, and (iii) a dual-species biofilm formed overnight on CDM and was subsequently treated with a combination of ciprofloxacin and vancomycin at their effective inhibitory concentrations. Each condition was prepared in separate wells of a 24-well plate. The samples were initially gently washed five times with PBS to remove any loosely attached cells or debris. Then they were fixed by adding 500 µL of a fixation solution containing 4% paraformaldehyde and 1% glutaraldehyde for 2 h at room temperature [ 36 ]. Following fixation, the samples were washed three times with 500 µL of PBS and finally rinsed with deionized water [ 37 ]. Dehydration was carried out by sequential incubation in 50%, 75%, 90%, and 100% ethanol, each for 10 min at room temperature [ 38 ]. After dehydration, the samples were left to air dry overnight at room temperature. The dried samples were mounted onto SEM stubs and sputter-coated with a thin layer of carbon to increase conductivity. SEM imaging was performed using Quanta™ 250 FEG SEM (Thermo Fisher Scientific, Hillsboro, OR, USA) instrument equipped with an Everhardt Thornley SED (secondary electron detector) to assess the surface morphology of the CDMs and the structure of the dual-species biofilms with and without antibiotic treatment [ 16 ]. SEM images were obtained at an accelerating voltage of 5 kV, with a dwell time of 5 µs, a spot size of 3.5, and a working distance of 10.6 mm. Images were captured at magnifications of 1,000× and 10,000×. 3. Results 3.1. Antibiotic susceptibility profiles of S. aureus and P. aeruginosa The MICs and MBCs of ten antibiotics were determined for both P. aeruginosa and S. aureus to confirm their susceptibility to these antibiotics. P. aeruginosa was highly sensitive to most of the tested antibiotics (MIC and MBC < 1 µg/mL); however, it presented relatively high MIC and MBC values to rifampicin, consistent with typical susceptibility of P. aeruginosa PAO1 strains to rifampicin (Table 1 ). Table 1 MIC and MBC values for selected antibiotics against P. aeruginosa. P. aeruginosa GEN TOB OFX CIP LVX CST RIF MIC ( µg/mL ) ≤ 1 ≤ 1 ≤ 1 ≤ 1 ≤ 1 ≤ 1 32 MBC ( µg/mL ) 2 ≤ 1 ≤ 1 ≤ 1 ≤ 1 ≤ 1 256 *Abbreviations: GEN, gentamicin; TOB, tobramycin; OFX, ofloxacin; CIP, ciprofloxacin; LVX, levofloxacin; CST, colistin; and RIF, rifampicin. S. aureus showed sensitivity to vancomycin, ampicillin, gentamicin, tobramycin, ofloxacin, ciprofloxacin, levofloxacin, rifampicin, and penicillin, with MIC and MBC values of < 1 µg/mL (Table 2). A lower susceptibility was observed for teicoplanin (MIC of 4 µg/mL; MBC of 6 µg/mL), which can be attributed to the growth media, bacterial strains and other test-dependent factors. TSB strongly supports the growth of S. aureus , often leading to increased biomass and doubling time [ 39 ]. Nevertheless, MIC assays confirmed the susceptibility of bacteria to the tested antibiotics. Table 2. MIC and MBC values of selected antibiotics against S. aureus . S. aureus VAN TEC AMP PEN GEN TOB OFX CIP LVX RIF MIC (µg/mL) 2 4 ≤ 1 ≤ 1 ≤ 1 ≤ 1 ≤ 1 ≤ 1 ≤ 1 ≤ 1 MBC (µg/mL) 2 6 ≤ 1 ≤ 1 2 ≤ 1 ≤ 1 2 ≤ 1 ≤ 1 *Abbreviations: VAN, vancomycin; TEC, teicoplanin; AMP, ampicillin; PEN, penicillin; GEN, gentamicin; TOB, tobramycin; OFX, ofloxacin; CIP, ciprofloxacin; LVX, levofloxacin; and RIF, rifampicin. Coculture of S. aureus and P. aeruginosa were confirmed and quantified via colony counting after an overnight (20 h), 4 h, and after dual-species biofilm formation (20 h) (Fig. 1 ). Both species reached ~ 10 10 CFU/mL overnight and ~ 10 9 CFU/mL after 4 h culture. Following dual-species biofilm formation, the planktonic cell counts were just below 10 11 CFU/mL, while the biofilm-associated cell counts were 2×10 9 CFU/mL for S. aureus and ~ 8×10 9 CFU/mL for P. aeruginosa . The pH of the biofilm was measured 5.8. 3.3. Coculture with S. aureus altered the response of P. aeruginosa to antibiotics MBIC and MBEC assays were performed to compare the response of mono-species biofilms and dual-species biofilms to antibiotics. The MBIC and MBEC values of antibiotics against P. aeruginosa mono-species and dual-species biofilm are presented in Table 3 . Notable increases were observed for rifampicin, gentamicin, and colistin, with MBICs of > 2048, 56, and 20 µg/mL, respectively. Tobramycin, ofloxacin, ciprofloxacin, and levofloxacin presented MBICs of 8, 4, 3, and 3 µg/mL, respectively, slightly exceeding their MICs. The presence of S. aureus in dual-species biofilms slightly altered the P. aeruginosa biofilm response to antibiotics. Compared to those in mono-species biofilms, The MBIC values of most antibiotics for P. aeruginosa increased 2- or 3-fold. The MBIC value of the broad-spectrum antibiotic gentamicin decreased almost 2-fold in dual-species biofilms compared with that in mono-species biofilms, which is consistent with previous reports [ 40 ]. The MBEC values of antibiotics in mono-species biofilms increased relative to their MBICs, with those of gentamicin, colistin, and tobramycin increasing 5-, 5-, and 9-fold, respectively. In contrast, the MBEC values for ofloxacin, ciprofloxacin, and levofloxacin did not change significantly compared with their MBICs. In dual-species biofilms, MBEC values of FQ antibiotics increased to 7-fold compared to those of mono-species biofilms. In dual species biofilms, P. aeruginosa showed high tolerance to colistin with an MBEC value of > 2048. Table 3 Antibiotic susceptibility of P. aeruginosa in mono- and dual-species biofilms (MBIC and MBEC, µg/mL) P. aeruginosa GEN TOB OFX CIP LVX CST RIF Mono-species MBIC 56 8 4 3 3 20 > 2048 MBEC 256 76 5 3 3 192 > 2048 Dual-species MBIC 32 19 8 7 5 64 > 2048 MBEC 128 48 38 9 18 > 2048 > 2048 *Abbreviations: GEN, gentamicin; TOB, tobramycin; OFX, ofloxacin; CIP, ciprofloxacin; LVX, levofloxacin; CST, colistin; and RIF, rifampicin. 3.4. Coculture with P. aeruginosa reduced S. aureus biofilm susceptibility to most of the tested antibiotics The MBIC and MBEC values of antibiotics against mono-species and dual-species biofilms of S. aureus are presented in Table 4 . The MBIC values of ampicillin, teicoplanin, tobramycin, gentamicin, ofloxacin, and vancomycin were > 2048, 96, 64, 56, 52, and 48 µg/mL, respectively for the mono-species biofilms of S. aureus . The MBEC values were even higher than MBICs, with rifampicin and vancomycin showing values of 64 and 192 µg/mL, respectively. Compared with the MBIC values, the FQs presented up to a 4-fold increase in their MBEC values in mono-species biofilms. Table 4 Antibiotic susceptibility of S. aureus in mono- and dual-species biofilms (MBIC and MBEC, µg/mL) S. aureus VAN TEC AMP PEN GEN TOB OFX CIP LVX RIF Mono-species MBIC 48 96 > 2048 4 56 64 52 33 8 1 MBEC 192 > 2048 > 2048 4 64 40 100 60 30 64 Dual-species MBIC > 2048 > 2048 > 2048 > 2048 347 512 320 192 40 48 MBEC > 2048 > 2024 > 2048 > 2048 > 2048 > 2048 > 2048 > 2048 > 2048 > 2048 *Abbreviations: VAN, vancomycin; TEC, teicoplanin; AMP, ampicillin; PEN, penicillin; GEN, gentamicin; TOB, tobramycin; OFX, ofloxacin; CIP, ciprofloxacin; LVX, levofloxacin; and RIF, rifampicin. The tolerance profile of S. aureus significantly increased in dual-species biofilms with P. aeruginosa (Table 4 ). The MBIC values significantly increased for the dual-species biofilms, indicating high tolerance to vancomycin, teicoplanin, ampicillin, and penicillin (≥ 2048 µg/mL). The MBIC for gentamicin, tobramycin, ofloxacin, and ciprofloxacin increased to 347 µg/mL, 512 µg/mL, 320 µg/mL, and 192 µg/mL, respectively. The MBEC values indicated the resistance of S. aureus in the dual-species biofilms to the tested concentrations of antibiotics, with no eradication achieved even at the highest tested concentration of 2048 µg/mL. These results suggest that the protective effect afforded by the dual-species biofilm matrix is particularly due to the presence of P. aeruginosa . 3.5. Phage activity against S. aureus was different on the solid surface compared to liquid culture Preliminary phage susceptibility testing of Stab 21 against S. aureus showed varying results. Stab 21 showed lytic activity against S. aureus on solid media based on drop testing; however, in liquid culture, Stab21 (in 10 5 PFU/well) could not infect S. aureus during 18 h of incubation, while its original host strain, Staphylococcus xylosus was infected (supplementary information, Figure S1 ). Higher phage concentrations (10 6 and 10 7 PFU/well) contributed to higher lytic activity, although at 10 7 PFU/well, the emergence of phage-resistance bacteria was observed after 10 hours (Supplementary information, Figure S1 ). Phage susceptibility testing against P. aeruginosa using phage PA8P1 demonstrated strong lytic activity on both solid media and in liquid culture (supplementary information, Figure S2). 3.6. S. aureus showed high susceptibility to phage–antibiotic combination treatment in the MBIC assay, but significantly lower susceptibility in the MBEC assay The effects of phage treatment alone and in combination with ciprofloxacin or vancomycin were investigated against S. aureus and P. aeruginosa in mono and dual-species biofilms. Under single-species biofilm growth conditions, S. aureus and P. aeruginosa were treated with their respective phages at the four highest concentrations tested (10⁴ to 10⁷ PFU/well for PA8P1 and 10⁵ to 10⁸ PFU/well for Stab 21), and the biofilm formation was assessed following an 18-hour incubation period. Based on visual assessment, none of the phage treatments were effective in preventing biofilm development. Both phages were chosen to be used at a concentration of 10 7 PFU/well throughout the following experiments. Phage Stab21 was subsequently tested in combination with sub-biofilm-inhibitory concentrations of vancomycin and ciprofloxacin against S. aureus biofilms, and phage PA8P1 was tested in combination with ciprofloxacin and colistin against P. aeruginosa biofilms. The addition of Stab 21 significantly reduced the MBIC of ciprofloxacin from 33 µg/mL (mono-treatment) to 0.5 µg/mL. Similarly, with vancomycin, the MBIC decreased from 48 µg/mL in monotreatment to 10 µg/mL in the presence of Stab21 (Table 5 ). As for phage PA8P1, phage‑antibiotic synergy was not confirmed with either of the tested antibiotics against P. aeruginosa biofilms. Compared to antibiotic treatment alone, the addition of PA8P1 increased the MBIC of ciprofloxacin, whereas the MBIC of colistin remained unaffected by the presence of phages (supplementary information, Table S1 ). Thus, PA8P1 was excluded from further evaluation in dual-species biofilm assays. Table 5 MBIC of S. aureus in biofilms treated with Stab 21 plus ciprofloxacin or vancomycin S. aureus Ciprofloxacin Vancomycin MBIC (µg/mL) Mono-species biofilm AB control 33 48 AB + Stab 21 0.5 10 Dual-species biofilm AB control 192 > 2048 AB + Stab 21 < 0.25 < 0.5 In the dual-species biofilm model, the addition of Stab 21 further decreased the MBIC values of both antibiotics. In the dual-species biofilm, S. aureus exhibited increased resistance to ciprofloxacin, with the MBIC increasing from 33 µg/mL (mono-species) to 192 µg/mL. However, the addition of Stab 21 significantly restored its susceptibility to ciprofloxacin, reducing the MBIC to 2048 µg/mL. Upon the introduction of Stab 21, the susceptibility of vancomycin was regained, lowering the MBIC to < 0.5 µg/mL. Notably, we did not test lower concentrations of antibiotics. In summary, based on the MBIC of ciprofloxacin and vancomycin against S. aureus growth in both mono- and dual-species biofilm models, the addition of Stab 21 significantly reduced the dose of both ciprofloxacin and vancomycin needed to inhibit the biofilm growth (Table 5 ). The effectiveness of Stab 21 combined with ciprofloxacin in eradicating S. aureus from dual-species biofilms was subsequently evaluated. The addition of Stab 21 reduced the MBEC of ciprofloxacin from 2048 µg/mL to 1024 µg/mL. As expected, the addition of Stab 21 had no effect on P. aeruginosa , and only the inhibitory activity of the antibiotics was observed (Table 7 ). 3.7. Most combinations had synergistic interactions on the inhibition of the dual-species biofilm The efficacy of antibiotic combinations against dual-species biofilm formation was assessed by calculating the FICI (Fig. 2 ). Multiple antibiotic pairs showed synergistic interactions (FICI < 0.5) on the inhibition of S. aureus within dual-species biofilms (Table 6 ). Combinations such as vancomycin–ciprofloxacin, rifampicin–colistin, vancomycin–colistin, and penicillin-ciprofloxacin demonstrated synergistic effects against both P. aeruginosa and S. aureus . The vancomycin–ciprofloxacin combination had strong synergistic effects against S. aureus (FICI = 0.055), which was further enhanced by the addition of the phage Stab 21 (FICI = 0.002). However, it should be noted that the same MBIC was reached by combining either ciprofloxacin or vancomycin alone with phages, indicating that the combination of antibiotics with phages would not be necessary. Therefore, the most significant synergy against S. aureus was observed with vancomycin-ciprofloxacin-stab 21 (FICI = 0.002), followed by combinations of vancomycin-ofloxacin (FICI = 0.034), ciprofloxacin-levofloxacin (FICI = 0.047), vancomycin-ciprofloxacin (FICI = 0.055), vancomycin-levofloxacin (FICI = 0.083), rifampin-colistin (FICI = 0.093), and vancomycin-colistin (FICI = 0.094). The MBIC data for both species are presented in Supplementary Information Table S2. Table 6 Interaction of the chosen combinations on the inhibition of the dual-species biofilms. Antibiotics P. aeruginosa S. aureus FIC a FIC b ∑FICI Interaction FIC a FIC b ∑FICI Interaction Vancomycin-colistin 0.002 0.093 0.095 Synergistic 0.023 0.071 0.094 Synergistic Vancomycin-gentamicin 0.002 1.000 1.002 Indifference 0.750 0.375 1.125 Indifference Vancomycin-ciprofloxacin 0.043 0.270 0.313 Synergistic 0.050 0.005 0.055 Synergistic Vancomycin-levofloxacin 0.002 0.750 0.752 Sub-synergistic 0.021 0.062 0.083 Synergistic Vancomycin-ofloxacin 0.002 0.500 0.502 Sub-synergistic 0.019 0.015 0.034 Synergistic Vancomycin-tobramycin 0.002 1.000 1.002 Indifference 0.019 0.125 0.144 Synergistic Teicoplanin-levofloxacin 0.251 0.750 1.001 Indifference 0.035 0.150 0.185 Synergistic Penicillin-colistin 0.129 0.625 0.754 Sub-synergistic 0.012 0.375 0.387 Synergistic Penicillin-ciprofloxacin 0.156 0.250 0.406 Synergistic 0.187 0.024 0.211 Synergistic Penicillin-levofloxacin 0.004 0.512 0.516 Sub-synergistic 0.002 0.500 0.502 Sub-synergistic Ampicillin-colistin 0.375 0.187 0.562 Sub-synergistic 0.004 0.250 0.254 Synergistic Ampicillin-ciprofloxacin 0.008 0.250 0.258 Synergistic 0.002 0.250 0.252 Synergistic Ciprofloxacin-levofloxacin 0.500 0.250 0.750 Sub-synergistic 0.020 0.027 0.047 Synergistic Rifampin-colistin 0.004 0.281 0.285 Synergistic 0.062 0.031 0.093 Synergistic Vancomycin-ciprofloxacin-Stab21 0.107 0.500 0.607 Sub-synergistic 0.001 0.0002 0.002 Synergistic 3.8. Only a few of the tested combinations showed synergy on the eradication of the dual-species biofilm The efficacy of antimicrobial combinations used to eradicate pre-formed dual-species biofilms of S. aureus and P. aeruginosa was not as successful as their ability to inhibit them. As shown in Table 7 , no combination had synergistic interaction against both species, and only five combinations were synergistic against S. aureus within the biofilm. This highlights the challenge resulting from the reduced susceptibility of S. aureus to antibiotics following biofilm formation. Among the tested pairs, vancomycin-ciprofloxacin showed the strongest synergistic effects (FICI = 0.156), suggesting that the vancomycin-ciprofloxacin combination has enhanced efficacy in targeting S. aureus within the complex biofilm structure. However, the addition of the phage Stab 21 to the ciprofloxacin-vancomycin combination did not improve their synergy (FICI = 0.250). Interestingly, the highest concentrations of both antibiotics did not result in synergistic effects with the phage, suggesting that phage efficacy depends on optimal, rather than maximal, antibiotic concentrations for the most favorable outcome (Supplementary Information, Figure S3). Similarly, vancomycin-ofloxacin (FICI = 0.199) and vancomycin-levofloxacin (FICI = 0.250) also had synergistic effects. Additionally, the rifampicin-colistin combination demonstrated only a sub-synergistic interaction (FICI = 0.750). The MBEC values for both species are provided in Supplementary Information (Table S3). Table 7 Interaction of the chosen combinations on the eradication of dual-species biofilms. Antibiotics P. aeruginosa S. aureus FIC a FIC b ∑FICI Interaction FIC a FIC b ∑FICI Interaction Vancomycin-colistin 0.008 1.000 1.008 Indifference 0.125 1.000 1.125 Indifference Vancomycin-gentamicin 0.002 1.000 1.002 Indifference 0.125 1.000 1.125 Indifference Vancomycin-ciprofloxacin 0.066 0.500 0.566 Sub-synergistic 0.125 0.031 0.156 Synergistic Vancomycin-levofloxacin 0.187 0.750 0.937 Sub-synergistic 0.156 0.094 0.250 Synergistic Vancomycin-ofloxacin 0.002 1.000 1.002 Indifference 0.187 0.012 0.199 Synergistic Vancomycin-tobramycin 0.002 1.000 1.002 Indifference 0.094 1.000 1.094 Indifference Teicoplanin-levofloxacin 0.156 0.375 0.531 Sub-synergistic 0.008 0.039 0.047 Synergistic Penicillin-colistin 0.979 1.000 1.979 Indifference 0.979 1.000 1.979 Indifference Penicillin-ciprofloxacin 0.126 0.250 0.376 Synergistic 0.125 1.000 1.125 Indifference Penicillin-levofloxacin 0.500 1.000 1.500 Indifference 0.979 1.000 1.979 Indifference Ampicillin-colistin 0.250 1.000 1.250 Indifference 0.500 1.000 1.500 Indifference Ampicillin-ciprofloxacin 0.063 0.375 0.438 Synergistic 0.979 1.000 1.979 Indifference Ciprofloxacin-levofloxacin 1.250 0.078 1.328 Indifference 0.375 1.000 1.375 Indifference Rifampin-colistin 0.979 1.000 1.979 Indifference 0.250 0.500 0.750 Sub-synergistic Vancomycin-ciprofloxacin-Stab21 0.500 1.000 1.500 Indifference 0.001 0.250 0.250 Synergistic 3.9. The presence of CDM in the biofilm model altered the susceptibility of the biofilm to antibiotic combinations in biofilm inhibition and eradication assays Table 8 presents the response of S. aureus within dual-species biofilms grown on CDM and treated with the most effective antibiotic combinations at varying concentrations relative to their MBICs (Fig. 3 ). The vancomycin–colistin combination exhibited reduced efficacy, failing to inhibit S. aureus in biofilms grown on CDM even when either antibiotic was used at twice its MBIC. In contrast, the MBIC values were lower for other combinations against biofilms on CDMs than on plate, indicating increased antibiotic susceptibility on CDM. The combination of ciprofloxacin- or levofloxacin- with vancomycin, and penicillin with levofloxacin, showed slightly greater efficacy, whereas vancomycin-ofloxacin and teicoplanin-levofloxacin inhibited S. aureus growth at substantially lower concentrations. None of the single antibiotics tested at their MBICs inhibited S. aureus growth in the dual-species biofilm in CDM. As shown in Fig. 3 , treatment of the biofilms formed on the CDM with vancomycin–ciprofloxacin at their MBIC effectively reduced P. aeruginosa , but S. aureus bacteria remained attached to the CDM surface (Fig. 3 . F). The pH of the biofilm was measured 8.1 after formation on CDM. Table 8 S. aureus response to the selected antibiotic combinations in inhibition of dual-species biofilm on CDM Antibiotic combinations MBIC AB1/AB2 (µg/mL) Coefficient by which the MBIC of AB1 (Top Row) and AB2 (Bottom Row) is multiplied 2 1 1 1 0.5 0.5 0.5 0.25 0.25 0.25 0.125 0.125 0.125 0.063 1 2 1 0.5 1 0.5 0.25 0.5 0.25 0.125 0.25 0.125 0.063 0.125 Vancomycin-colistin 4/16 + + + + + + + + + + + + + + Vancomycin-ciprofloxacin 4/1 − − − − − + + + + + + + + + Vancomycin-levofloxacin 43/2.5 − − − − − + + + + + + + + + Vancomycin-ofloxacin 64/51 − − − − − − − − − − + + + + Penicillin-levofloxacin 4/64 − − − − − + + + + + + + + + Teicoplanin-levofloxacin 72/6 − − − − − − − − − − − − + + Column “ MBIC AB1/AB2 ” indicates MBIC values of antibiotic 1 (AB1) and antibiotic 2 (AB2) when used in combination against S. aureus in dual-species biofilm without CDM (MBICs values can be found in SI table S2). The coefficients in columns 3–16 indicate the number by which MBIC values of each antibiotic are multiplied (upper row AB1 and bottom row AB2). For example, in the case of vancomycin-colistin, coefficients 2 (top) and 1 (bottom) indicate that vancomycin (AB1) is used at twofold of its MBIC (2×4 = 8 µg/mL), and colistin (AB2) at onefold of its MBIC (1×16 = 16 µg/mL). "+" indicates bacterial growth on MSA; "−" indicates no growth. Biofilm eradication assays for biofilms on CDMs also indicated altered susceptibility of S. aureus to antibiotic combinations (Table 9 ). The vancomycin–colistin pair exhibited reduced efficacy, requiring higher antibiotic concentrations for biofilm eradication. In contrast, vancomycin-ciprofloxacin and teicoplanin-levofloxacin were slightly more effective at eradicating S. aureus when the biofilm was formed on CDM. The combination of vancomycin with levofloxacin or ofloxacin achieved significantly greater eradication of S. aureus in CDM-grown biofilms. These results suggest that the structural properties of CDM may increase antibiotic penetration or activity in biofilms. Single antibiotics failed to eradicate S. aureus in the dual-species biofilms. The corresponding data for P. aeruginosa are provided in Supplementary Information (Table S4 and Table S5). Table 9 S. aureus response to the selected antibiotic combination in eradication of dual-species biofilm on CDM Antibiotic combinations MBEC AB1/AB2 (µg/mL) Coefficient by which MBEC of AB1 (Top Row) and AB2 (Bottom Row) is multiplied 2 1 1 1 0.5 0.5 0.5 0.25 0.25 0.25 0.125 1 2 1 0.5 1 0.5 0.25 0.5 0.25 0.125 0.25 Vancomycin-colistin 256/64 + + + + + + + + + + + Vancomycin-ciprofloxacin 256/2 − − − − + + + + + + + Vancomycin-levofloxacin 320/6 − − − − − − − − − + + Vancomycin-ofloxacin 384/24 − − − − − − − − − + + Teicoplanin-levofloxacin 16/2.5 − − − − − + + + + + + Column “ MBEC AB1/AB2 ” indicates MBEC values of antibiotic 1 (AB1) and antibiotic 2 (AB2) when used in combination against S. aureus in dual-species biofilm without CDM (MBECs can be found in SI table S3). The coefficient in columns 3–13 indicates the number by which MBEC values of each antibiotic are multiplied (upper row AB1 and bottom row AB2). For example, in the case of vancomycin-colistin, coefficients 2 (top) and 1 (bottom) indicate that vancomycin (AB1) is used at twofold of its MBEC (2×256 µg/mL), and colistin (AB2) at onefold of its MBEC (1×64 µg/mL). "+" indicates bacterial growth on MSA; "−" indicates no growth. 4. Discussion The MIC and MBC results confirmed the sensitivity of both P. aeruginosa and S. aureus to almost all the tested antibiotics, which is consistent with previous reports on the same strains [ 41 – 43 ]. In biofilms, P. aeruginosa PAO1 is less susceptible to rifampicin, colistin, and gentamicin [ 44 , 45 ]. S. aureus , however, showed increased tolerance to all the tested antibiotics. Increased tolerance was expected as biofilm formation reduces bacterial sensitivity to antibiotics by enhancing the mutagenesis, adaptation, and resistance of bacterial species. The observed additional antibiotic tolerance of S. aureus in dual-species biofilms was attributed to the presence of P. aeruginosa . Several previous studies reported altered susceptibility of S. aureus to antibiotics in co-culture with P. aeruginosa [ 46 ]. According to previous reports, polysaccharides produced by P. aeruginosa play a critical role in shielding both species from antibiotics [ 46 ]. P. aeruginosa produces three key exopolysaccharides—alginate, Pel, and Psl—that form its extracellular matrix, providing both structural support and protection for bacterial cells [ 47 – 49 ]. The high tolerance of S. aureus to antibiotics can also result from the protective effect of other exoproducts of P. aeruginosa , such as HQNO [ 47 ]. HQNO suppresses S. aureus respiration, reducing its susceptibility to aminoglycosides such as tobramycin and gentamycin, and it also protects S. aureus from vancomycin [ 47 ]. Consistent with our findings, DeLeon et al . reported increased tolerance of S. aureus to gentamicin in cocultures with P. aeruginosa [ 49 ]. However, Trizna et al. reported the high efficiency of aminoglycosides, including gentamicin, in the eradication of both species in mixed biofilms [ 50 ]. We observed high tolerance of S. aureus to penicillin in dual-species biofilms, which can be explained by the effect of the β-lactamase enzyme produced by P. aeruginosa [ 49 ]. Orazi et al reported that cell-free cultures of P. aeruginosa can increase the sensitivity of S. aureus biofilms to FQs while increasing their tolerance to other antibiotic classes, such as glycopeptides and β-lactams [ 51 ]. However, our results and those of Trizna et al., showed higher tolerance of S. aureus to FQs in multi-species biofilms than in mono-species biofilms [ 50 ]. S. aureus showed the lowest tolerance to levofloxacin and rifampicin in our dual-species biofilms, although neither antibiotic achieved complete biofilm eradication. Hiltunen et al . (2019) reported a higher efficiency of levofloxacin than vancomycin against S. aureus biofilms in prosthetic joint infections [ 52 ]. The variation in the response of S. aureus to antibiotics reported in different studies may be due to strain-specific differences and biofilm growth conditions. Among the combined antibiotics, FQs including ciprofloxacin, levofloxacin, and ofloxacin, combined with glycopeptides, vancomycin and teicoplanin, demonstrated significant efficacy against S. aureus in dual-species biofilms. Glycopeptides, such as vancomycin and teicoplanin, inhibit cell wall peptidoglycan synthesis in S. aureus [ 53 ]. FQs, on the other hand, target bacterial DNA synthesis by inhibiting DNA gyrase and topoisomerase IV [ 54 ]. Moreover, FQs are particularly effective against P. aeruginosa because of their ability to penetrate EPS and target sessile cells, even in mature biofilms [ 54 ]. Thus, the synergistic interaction of FQs and glycopeptides against dual-species biofilms is not surprising. Kamble et al . reported synergistic interactions between ciprofloxacin and vancomycin against clinical strains of S. aureus biofilms and their persister cells [ 8 ]. The synergy between FQs and glycopeptides in inhibiting and eradicating S. aureus within the dual-species biofilm may also be attributed to early elimination of P. aeruginosa by FQs and thereby loss of its protective effect on S. aureus [ 55 ]. Reports on the efficacy of combinations (FQs) and glycopeptides are variable among different studies. For example, in the study by Weber et al. , the combination of ofloxacin and vancomycin was neither synergistic nor antagonistic and did not result in improved outcomes compared with ofloxacin alone against S. aureus [ 56 ]. Consistent with our findings, many other in vitro studies using resistant clinical S. aureus isolates have shown strong synergy for drug pairs such as vancomycin-gentamicin, ciprofloxacin-vancomycin, and vancomycin-tobramycin [ 8 , 57 ]. Moreover, in vitro data is not always supported by preclinical and clinical studies. Rifampicin-based combinations are the combination therapy most approved for preclinical models of implant-associated infections [ 58 – 60 ]. Compared with monotherapy, rifampin with glycopeptides such as vancomycin has synergistic interactions and reduced resistance against prosthetic joint infections and osteomyelitis [ 58 – 60 ]. Although many in vitro studies have indicated the potential of FQ–glycopeptide combinations against both sensitive and tolerant S. aureus , supporting in vivo studies is limited. Phage–antibiotic combination treatment, supported by several successful clinical cases, was also evaluated in this study [ 61 – 63 ]. The phage Stab 21 restored the susceptibility of S. aureus to ciprofloxacin and vancomycin, enhancing the inhibition of S. aureus biofilm formation within the dual-species biofilm. However, the effectiveness of phage–antibiotic combinations against S. aureus in preformed biofilms remains low. Combining Stab 21 with both ciprofloxacin and vancomycin demonstrated that the phage requires an optimal antibiotic concentration for optimal eradication, as higher antibiotic concentrations did not yield superior biofilm eradication (SI, Fig. 2 ). Several rationales for phage–antibiotic synergy have been proposed, including bacterial invasion by targeting several bacterial receptors and associated genetic trade-offs, cell morphological changes, phage-mediated enhancement of antibiotic penetration via depolymerase production, and stress-induced phage production [ 64 , 65 ]. However, antibiotics targeting, e.g., protein synthesis machinery, may suppress phage replication, as phages rely on bacterial ribosomes and other protein synthesis machinery. Nevertheless, in this study, ciprofloxacin was effective when combined with Stab 21, with the critical factor being the identification of a sublethal concentration allowing phage propagation. Our findings also highlight the strong influence of the environmental context on phage infectivity. The inability of Stab 21 to infect S. aureus in liquid culture despite successful infection on solid media during preliminary screening may reflect altered expression of surface receptors or enzymatic activity across planktonic and surface-attached states [ 66 ]. Thus, phage lytic activity should be considered not only as a phage characteristic but a result of interplay between phage, bacterial host, and environmental factors, including biofilms. To our surprise, the presence of CDM, in most of the tested combinations, reduced the tolerance of biofilms to antibiotic combinations. Previous reports have shown exacerbated antibiotic tolerance of S. aureus and P. aeruginosa by EPS and the surrounding human-derived matrix (HDM) [ 49 ]. Antibiotic penetration into biofilms is significantly influenced by the physicochemical properties (e.g., pH and surface charge) of both antibiotics and the EPS of biofilms [ 49 ]. The biofilm pH was ~ 5.8, which increased to ~ 8.1 when the biofilm formed on CDM. The alkaline shift could partially explain the increased susceptibility of S. aureus to some antibiotics under these conditions. Colistin and vancomycin have isoelectric points (PIs) of approximately 10 and 8.3, respectively, which makes them slightly positive at pH values of 6–8 [ 67 , 68 ]. The PI of teicoplanin is approximately 5.5 and can be assumed to have a neutral charge at pH 6 and a small negative charge at pH 8 [ 69 ]. The rest of the tested antibiotics tested against biofilms on CDM have a PI of approximately 7, which might make them slightly positive at pH 6 and negative at pH 8 [ 70 , 71 ]. Bacterial cell surfaces and biofilms typically possess a net negative charge due to the abundance of carboxyl, amino, and phosphate groups [ 72 , 73 ]. During biofilm maturation, particularly in strong biofilm formers such as S. aureus , this negative surface potential increases, creating a substantial barrier to the diffusion of positively charged molecules [ 73 ]. Thus, it can be assumed that more negatively charged combinations, such as teicoplanin-levofloxacin, resulted in greater matrix penetration. Another study reported the reduced efficacy of positively charged gentamicin in coagulated wound-like matrices (WLMs) [ 49 ]. The effect of CDM on response of biofilms to antibiotics need to be further investigated in our future studies. Overall, our study demonstrated that interspecies interaction of bacteria alters their response to antibiotics. Our results further highlighted the complex response of bacterial species to combined antibiotics in inhibition and eradication of multi-species biofilm, as the inhibitory combinations were not necessarily efficient in eradication of biofilm. The combination of glycopeptides and FQs may be a promising candidate for treatment of multi-species biofilm but remains to be confirmed by in vivo and clinical studies. Compared with phage or antibiotic treatment alone, the addition of phage Stab 21 together with ciprofloxacin, vancomycin, or both, was significantly more effective at preventing the formation of S. aureus biofilms in both the mono and dual-species models, suggesting the high potential of phage therapy in treatment of chronic infections. Nevertheless, further in vitro , in vivo , and clinical studies are needed to establish viable treatment recommendations against multi-species biofilms. Abbreviations AMR Antimicrobial resistance ATCC American Type Culture Collection CDM Cell-derived matrix CF Cystic fibrosis CFU Colony-forming units DMEM Dulbecco's modified eagle medium ECM Extracellular matrix EPS Extracellular polymeric substances FICI Fractional inhibitory concentration index LA Lysogeny agar LB Lysogeny broth MBC Minimum bactericidal concentration MBEC Minimum biofilm eradication concentration MBIC Minimum biofilm inhibitory concentration MCA MacConkey agar MIC Minimum inhibitory concentration MRSA Methicillin-resistant Staphylococcus aureus MSA Mannitol salt agar OD Optical density PBS Phosphate-buffered saline PFU Plaque-forming units SEM Scanning electron microscopy TSA Tryptic soy agar TSB Tryptic soy broth. Declarations Declarations Ethics approval Not applicable. No human or animal subjects were used in this study. Consent for publication Not applicable. Availability of data and materials Most data generated in this study are included in this article. Other raw data will be available upon reasonable requests from the corresponding author. Declaration of competing interests The authors declare that they have no competing financial interests and personal relationships with other people or organizations that could inappropriately influence or bias the work reported in this paper. Funding This work was supported by the Research Council of Finland [grant number 361647; and grant number 362584]; and PROFI7 funding to the University of Helsinki. Moreover, the research was supported by the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation program (ERC CoG), [grant number 101001016] and the Otto A. Malm Foundation. Authors’ contributions Paria Mojarrad: Writing – original draft, Methodology, Investigation, Visualization, Software, Investigation, Formal analysis, Data curation. Hanna Tiainen: Writing – original draft, Writing, Methodology, Visualization, Software, Investigation, Formal analysis. Päivi Tammela: Methodology, Validation, Funding acquisition, Writing – review & editing. Timo Laaksonen: Methodology, Validation, Supervision, Formal analysis, Funding acquisition, Writing – review & editing, Resources, Project administration. Saija J. Kiljunen: Methodology, Conceptualization, Validation, Writing – review & editing, Funding acquisition. Zahra Gounani: Methodology, Conceptualization, Validation, Supervision, Formal analysis, Funding acquisition, Writing – review & editing, Resources, Project administration. Acknowledgments The authors thank Kai Puhakainen, Alexandra Correia, and Jéssica Ferreira Amorim for their technical support. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8986145","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":633561590,"identity":"e27ef836-fc1d-4ae6-a74f-0353acce0949","order_by":0,"name":"Paria Mojarrad","email":"","orcid":"","institution":"University of Helsinki","correspondingAuthor":false,"prefix":"","firstName":"Paria","middleName":"","lastName":"Mojarrad","suffix":""},{"id":633561591,"identity":"9197c510-729d-4948-8921-962c82066452","order_by":1,"name":"Hanna Tiainen","email":"","orcid":"","institution":"University of Helsinki","correspondingAuthor":false,"prefix":"","firstName":"Hanna","middleName":"","lastName":"Tiainen","suffix":""},{"id":633561592,"identity":"14d2cee2-2c68-4397-9534-a7c9de42b01b","order_by":2,"name":"Päivi Tammela","email":"","orcid":"","institution":"University of Helsinki","correspondingAuthor":false,"prefix":"","firstName":"Päivi","middleName":"","lastName":"Tammela","suffix":""},{"id":633561593,"identity":"3cd76293-1a50-41f5-ba40-6e7c8bb6d35e","order_by":3,"name":"Timo Laaksonen","email":"","orcid":"","institution":"University of Helsinki","correspondingAuthor":false,"prefix":"","firstName":"Timo","middleName":"","lastName":"Laaksonen","suffix":""},{"id":633561594,"identity":"65bf7960-6c8b-482d-bf62-f6b83e184e17","order_by":4,"name":"Saija Kiljunen","email":"","orcid":"","institution":"University of Helsinki","correspondingAuthor":false,"prefix":"","firstName":"Saija","middleName":"","lastName":"Kiljunen","suffix":""},{"id":633561595,"identity":"dd628c58-f76e-448c-a2a2-1ba0c4ba73e4","order_by":5,"name":"Zahra Gounani","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABEElEQVRIie3PMUvEMBTA8RcKmUqzvoC0XyFHQBc/TIPDLUWc5IazBoTcUnA9QfwMN3WuBHqL7gVvuFs6dXCSG26wVxwEyXmjYP5LMrwfLwHw+f5k4bd7OqkgAqKJFsDoceSlAvpFuDmKELMn/akBuHaAZPZav2/hJhZvd+1687SKKbNmM78CjByEFJcXDwUspVjVZ0KVraSoZqOFAHT9JYBM9m+r1aJJT1GVVhkkhq8F5C5CWSfJbiDjD1SP9taw54E4t4SYySCEaU+yfou2KQVl+KGHIXYyOBGV5E12jWltRwZ7MhfoJMl9Jkk3yeOoGZd8O7UJY8uWF7tzTLTDDAn7c/2h+X35bwM+n8/3n/sEKFFU7yQufuMAAAAASUVORK5CYII=","orcid":"","institution":"University of Helsinki","correspondingAuthor":true,"prefix":"","firstName":"Zahra","middleName":"","lastName":"Gounani","suffix":""}],"badges":[],"createdAt":"2026-02-27 09:38:18","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8986145/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8986145/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":108492581,"identity":"f1161669-cfff-449b-b8d3-150f2942ff0f","added_by":"auto","created_at":"2026-05-05 09:58:06","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":449146,"visible":true,"origin":"","legend":"\u003cp\u003eColony counts of \u003cem\u003eP. aeruginosa\u003c/em\u003e and \u003cem\u003eS. aureus\u003c/em\u003e in planktonic cultures and dual-species biofilms. Colony counting results for bacteria \u003cem\u003eP. aeruginosa\u003c/em\u003e and \u003cem\u003eS. aureus \u003c/em\u003eafter an overnight culture, 4-hour culture, and after formation of the dual-species biofilm in planktonic phase and biofilm phase. To selectively differentiate the two species, samples were plated on MCA for \u003cem\u003eP. aeruginosa\u003c/em\u003eand MSA for \u003cem\u003eS. aureus\u003c/em\u003e.\u003c/p\u003e","description":"","filename":"1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8986145/v1/5ffb1a5bf0d5fad17fb1aae4.jpg"},{"id":108492767,"identity":"2c706f75-3a1c-4531-a1e9-3c9291064650","added_by":"auto","created_at":"2026-05-05 09:58:35","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":151440,"visible":true,"origin":"","legend":"\u003cp\u003eSynergistic inhibition of dual-species biofilm formation by the ampicillin–colistin combination. (A) Schematic representation of the 96-well plate layout used for the FICI assay, illustrating the concentration gradient of ampicillin and colistin. (B) Experimental results demonstrate synergistic inhibition of biofilm formation. The red line indicates the wells where \u003cem\u003eS. aureus\u003c/em\u003egrowth was observed using the spotting technique.\u003c/p\u003e","description":"","filename":"2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8986145/v1/11544da47028eb3d77ce5a8f.jpg"},{"id":108494645,"identity":"292d18f6-8c14-423f-bc48-f300b639020c","added_by":"auto","created_at":"2026-05-05 10:06:16","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1545320,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8986145/v1/bf1e68b8-d1f6-47bb-b9c9-864db2c8eab7.pdf"},{"id":108420654,"identity":"2792b142-e88a-4a78-adb6-09f62436f0ba","added_by":"auto","created_at":"2026-05-04 12:37:38","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":4048536,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryMaterialMBC.docx","url":"https://assets-eu.researchsquare.com/files/rs-8986145/v1/a503a1e56777524c182977a2.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Synergistic Antibiotic‒Antibiotic and Antibiotic‒Phage Combinations Against Biofilm-Associated Staphylococcus aureus in Dual-Species Biofilms","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003ePolymicrobial biofilms are major cause of treatment failure in chronic infections by reducing bacterial susceptibility to both antimicrobial agents and host immune systems [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. \u003cem\u003eStaphylococcus aureus\u003c/em\u003e and \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e are the two most frequently isolated species from chronic infections including chronic wound infections, respiratory infections in cystic fibrosis (CF), endocarditis, and implantable device-associated infections [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Clinical observations show a complex and dynamic interspecies interaction between these two species. \u003cem\u003eP. aeruginosa\u003c/em\u003e outcompete \u003cem\u003eS. aureus\u003c/em\u003e and inhibits its growth in early cloning by secreting several staphylolytic compounds such as siderophores and 4-hydroxy-2-heptylquinoline-N-oxide (HQNO) [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. In later stage of colonization \u003cem\u003eS. aureus\u003c/em\u003e defense mechanism drive it to develop metabolic variants such as small-colony variant (SCV), with fermentative growth state and profoundly increased antibiotic tolerance [\u003cspan additionalcitationids=\"CR5\" citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. This complex interspecies interaction has inspired several researchers to develop in vitro biofilm models of these two bacteria. However, in vitro co-culture of the two species is challenging as \u003cem\u003eP. aeruginosa\u003c/em\u003e strongly inhibit the growth of \u003cem\u003eS. aureus\u003c/em\u003e, which does not reflect what observed in vivo. The modification of growth media is needed to support co-culture of both species.\u003c/p\u003e \u003cp\u003eMonotherapy often fails to eradicate polymicrobial biofilms because they exhibit higher resilience and drug tolerance compared to mono-species biofilms [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Combination therapy involving the simultaneous use of two or three antibiotics or nonantibiotic agents is the leading clinical approach against polymicrobial infections [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Combination of antibiotic regimens enhances efficacy against biofilms through synergistic interaction, reduced resistance incidence, and improved penetration into the biofilm matrix [\u003cspan additionalcitationids=\"CR9\" citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eTo target polymicrobial biofilms of \u003cem\u003eS. aureus\u003c/em\u003e and \u003cem\u003eP. aeruginosa\u003c/em\u003e, antibiotic combinations should include either species-specific agents or broad-spectrum antibiotics effective against both organisms. Biological antibacterial agents are rapidly gaining interest in fighting against chronic infections. Phage therapy, in particular, has emerged as a promising addition to antibiotic treatment for chronic biofilm owing to its ability to replicate within bacterial populations and penetrate biofilm structures [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. The phage Stab 21 belongs to the genus \u003cem\u003eKayvirus\u003c/em\u003e in the subfamily \u003cem\u003eTwortvirinae\u003c/em\u003e of the family \u003cem\u003eHerelleviridae.\u003c/em\u003e It has a broad host range primarily among \u003cem\u003eS. aureus\u003c/em\u003e strains and thus poses great potential for therapeutic use against \u003cem\u003eS. aureus\u003c/em\u003e infections [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Combination therapy is ideal if the interaction of combined agents is synergistic or additive. There are previous reports on synergistic interaction of phage\u0026ndash;antibiotic combinations against biofilm [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eSeveral studies have established in vitro dual-species biofilm of \u003cem\u003eS. aureus\u003c/em\u003e and \u003cem\u003eP. aeruginosa\u003c/em\u003e to investigate their interspecies interaction and antibacterial susceptibility profile [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. However, employing such models to investigate their response to combination strategies remains limited. We previously reported a dual-species biofilm model of \u003cem\u003eP. aeruginosa\u003c/em\u003e and \u003cem\u003eS. aureus\u003c/em\u003e, developed on fibroblast-derived cell matrix (CDM) [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. CDMs are decellularized extracellular matrices that help to mimic the microenvironments of in vivo biofilms. We used this model to investigate the response of \u003cem\u003eP. aeruginosa\u003c/em\u003e and \u003cem\u003eS. aureus\u003c/em\u003e to single and combined antibiotics. As \u003cem\u003eS. aureus\u003c/em\u003e became highly antibiotic-tolerant in the biofilm, it was our primary target in this study. The antibiotics were chosen from glycopeptides, β-lactams, aminoglycosides, fluoroquinolones (FQs), rifampicin, and polymyxins which are effective against either \u003cem\u003eS. aureus\u003c/em\u003e or \u003cem\u003eP. aeruginosa\u003c/em\u003e or both [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Colistin is a clinically relevant antipseudomonal antibiotic [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Vancomycin, teicoplanin, penicillin, and ampicillin are effective against \u003cem\u003eS. aureus\u003c/em\u003e, while gentamicin, tobramycin, ciprofloxacin, levofloxacin, ofloxacin, and rifampicin are active against both species. Vancomycin is among the most included antibiotic in combination therapy against \u003cem\u003estaphylococcal\u003c/em\u003e biofilms [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e] and has well-documented synergy against both \u003cem\u003eP. aeruginosa\u003c/em\u003e and \u003cem\u003eS. aureus\u003c/em\u003e [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. We further assessed the effect of adding the phage Stab 21 to the best antibiotic‒antibiotic combination against \u003cem\u003eS. aureus\u003c/em\u003e. Finally, we evaluated selected antibiotic combinations on the biofilm developed on CDM.\u003c/p\u003e"},{"header":"2. Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1. Strains and culture conditions\u003c/h2\u003e \u003cp\u003e \u003cem\u003eP. aeruginosa\u003c/em\u003e (PAO1 [ATCC 47085]), ATCC 33352) and \u003cem\u003eS. aureus\u003c/em\u003e (ATCC 25923) were stored at \u0026minus;\u0026thinsp;80\u0026deg;C and revived on tryptic soy agar (TSA; Catalog No. CM0131B; Thermo Fisher Scientific) before use. Overnight cultures were prepared by inoculating a single colony from each bacterial species into 10 mL of tryptic soy broth (TSB)[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e] and incubating at 37\u0026deg;C with shaking (200 rpm) for 20 h. Biofilm formation was performed in TSB (mono-species biofilm) and TSB+ fetal bovine serum (FBS) (dual-species biofilm). TSB is used as a standard, nutrient-rich medium that promotes \u003cem\u003eS. aureus\u003c/em\u003e biofilm and we showed in our previous study that TSB enriched with NaCl and glucose in combination with FBS is the optimum media for culture of \u003cem\u003eS. aureus\u003c/em\u003e in presence of \u003cem\u003eP. aeruginosa\u003c/em\u003e [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Four-hour subcultures were prepared via a 1:1000 dilution of the overnight cultures in fresh TSB and incubated for an additional 4 h under the same conditions. Freshly prepared overnight and 4-hour cultures were used for all the experiments. All the experiments in this study were performed with two biological replicates, each of which was measured in technical duplicate unless otherwise stated. Most of the experiments were also repeated independently at least two times to ensure their reproducibility.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.1. Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC)\u003c/h2\u003e \u003cp\u003eMIC assays were performed by the broth dilution method according to Hacek \u003cem\u003eet al\u003c/em\u003e. with minor modifications [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Serial two-fold dilution of antibiotics was done in TSB in 96-well microtiter polystyrene plates (catalog No. 167008; Thermo Fisher Scientific, Nunclon). Wells were inoculated with a 4-h culture of either \u003cem\u003eS. aureus\u003c/em\u003e or \u003cem\u003eP. aeruginosa\u003c/em\u003e to a final concentration of ~\u0026thinsp;10\u003csup\u003e6\u003c/sup\u003e CFU/ml. Positive (inoculated TSB without antibiotic) and negative (TSB) controls were included. The plates were incubated at 37\u0026deg;C for 20 h. Bacterial growth was monitored by measuring optical density (OD) at 600 nm using Multiskan GO Microplate Reader (Thermo Fisher Scientific, Waltham, MA, USA). The MIC was defined as the lowest antibiotic concentration resulting in no visible increase in turbidity compared to the negative control (TSB). To determine MBC, wells without visible growth were spotted (3*5 \u0026micro;L) on TSA plates and incubated at 37\u0026deg;C for 20 h to determine the MBC values. MBC was defined as the lowest concentration of antibiotic resulting in no growth on the agar plates [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.2. Dual-species biofilm formation\u003c/h2\u003e \u003cp\u003eDual-species biofilms were developed according to our previous work with minor modifications [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Briefly, TSB supplemented with 1% (w/v) glucose and 2.5% (w/v) sodium chloride was mixed 1:1 with fetal bovine serum (FBS; Catalog No. A5256701, Thermo Fisher Scientific) to support co-growth of \u003cem\u003eS. aureus\u003c/em\u003e with \u003cem\u003eP. aeruginosa\u003c/em\u003e. In each well, 180 \u0026micro;L of the 1:1 TSB-FBS mixture was inoculated with 10 \u0026micro;L of a 4-hour \u003cem\u003eS. aureus\u003c/em\u003e mixture (~\u0026thinsp;10\u003csup\u003e7\u003c/sup\u003e CFU/mL) and 1 \u0026micro;L of a 4-hour \u003cem\u003eP. aeruginosa\u003c/em\u003e mixture (~\u0026thinsp;10\u003csup\u003e6\u003c/sup\u003e CFU/mL), resulting in a final volume of 191 \u0026micro;L per well. The growth media without inoculation was used as negative controls. The plates were subsequently incubated at 37\u0026deg;C for 20 h without shaking to allow biofilm development. The pH of the biofilm was measured after biofilm formation.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.3. Colony counting\u003c/h2\u003e \u003cp\u003eDual-species biofilm formation was confirmed by colony counting. Overnight and 4-hour cultures (100 \u0026micro;L) were serially tenfold diluted in phosphate-buffered saline (PBS) and plated (3\u0026times;10 \u0026micro;l) on TSA. For dual-species biofilms, 100 \u0026micro;l aliquots of planktonic cells from the supernatant and biofilm (redispersed in PBS), serially ten-fold diluted and cultured on MacConkey agar (MCA; Catalog No. CM0007, Thermo Fisher Scientific) and mannitol salt agar (MSA; Catalog No. CM0085B, Thermo Fisher Scientific). The biofilm samples were mechanically disrupted by pipetting and vertexing to minimize cell aggregation and ensure single-cell suspension. MCA and MSA were used as selective growth media for Gram-negative bacteria including \u003cem\u003eP\u003c/em\u003e. \u003cem\u003eaeruginosa\u003c/em\u003e and \u003cem\u003eS. aureus\u003c/em\u003e, respectively. The plates were incubated at 37\u0026deg;C for 20 h (TSA, MCA), or 48\u0026ndash;72 h (MSA) to account for the slower growth rate of \u003cem\u003eS. aureus\u003c/em\u003e. The colony counts are expressed as log\u003csub\u003e10\u003c/sub\u003e CFU/mL [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.4. Mono-species biofilm formation\u003c/h2\u003e \u003cp\u003eMono-species biofilms were established in polystyrene 96-well plates in 180 \u0026micro;L TSB inoculated with 4-hour culture of \u003cem\u003eS. aureus\u003c/em\u003e or \u003cem\u003eP. aeruginosa\u003c/em\u003e (~\u0026thinsp;10\u003csup\u003e7\u003c/sup\u003e CFU/mL). Wells with TSB without inoculation were used as negative controls. The plates were incubated at 37\u0026deg;C for 20 h. Bacterial growth was verified by OD\u003csub\u003e600\u003c/sub\u003e measurement.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.5. Phages, host bacteria, and phage propagation\u003c/h2\u003e \u003cp\u003eThe phage Stab 21 was selected to target \u003cem\u003eS. aureus\u003c/em\u003e, and the phage PA8P1 was selected to target \u003cem\u003eP. aeruginosa\u003c/em\u003e [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. The host bacterial strains \u003cem\u003eS. xylosus\u003c/em\u003e DD-34 [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e] for Stab 21 and \u003cem\u003eP. aeruginosa\u003c/em\u003e PA8 [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e] for PA8P1, \u003cem\u003eS. aureus and P. aeruginosa\u003c/em\u003e (ATCC 33352), were stored at ‑70\u0026deg;C and retrieved on Lysogeny agar (LA) plates, i.e., Lysogeny Broth (LB) solidified with 1.5% (w/v) agar [\u003cspan additionalcitationids=\"CR29\" citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. For phage propagation, one colony of the host strain was inoculated into 5 mL LB and incubated overnight at 37\u0026deg;C in a tube rotator (Fisherbrand Mini Tube Rotator, Thermo Fisher Scientific, USA) at 15 rpm. The phage lysates were prepared by adding 400\u0026ndash;500 \u0026micro;L of overnight bacterial culture and 40 \u0026micro;L of phage stock (in DMSO or glycerol) stored at ‑70\u0026deg;C into 10 mL LB and incubating at 37\u0026deg;C in a tube rotator at 15 rpm. Once the lysis had occurred (within 3\u0026ndash;5 h), the tubes were centrifuged for 5 min at 5000 rpm using a Thermo Fisher Scientific SL16R centrifuge (rotor TX-400) to separate the remaining bacterial cells and cellular debris. The supernatant was passed through an LLG-syringe filter (SPHEROS, LLG Labware) with a pore size of 0.22 \u0026micro;m to remove any remaining bacterial cells. To enhance phage stability, 40% sucrose was added to a final concentration of 8% (v/v) and the resulting phage lysate was stored at 4\u0026deg;C throughout the study.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.6. Phage titration by the double-layer method\u003c/h2\u003e \u003cp\u003eA well-established double-layer agar plating method (Sambrook, 2001) with slight modifications was used to determine the titer of the phage lysate and the quantity of plaque-forming units (PFU) per well [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. In brief, single colonies of each host bacterial species from agar plates were grown in 1.3 mL LB in 2 mL Eppendorf tubes for 2\u0026ndash;3 h at 37˚C in a tube rotator (Fisherbrand Mini Tube Rotator, Thermo Fischer Scientific, USA) at 15 rpm. The absorbance at 600 nm (A\u003csub\u003e600\u003c/sub\u003e) was measured via a DSM Cell Density Meter (Laxco, USA), and the volume of bacterial culture for one titration plate was calculated as \u0026micro;L\u0026thinsp;=\u0026thinsp;45 / A\u003csub\u003e600\u003c/sub\u003e. A tenfold dilution series of up to 10\u003csup\u003e\u0026minus;\u0026thinsp;8\u003c/sup\u003e bacteriophage lysates in LB was prepared. Appropriate amounts of host bacteria and 3 mL of melted and tempered (at 55\u0026deg;C) soft agar with 50 \u0026micro;L of the appropriate dilution of phage lysate were mixed gently and poured onto LA plates evenly. The plates were left at room temperature (RT) for 30 min to solidify before being incubated overnight at 37\u0026deg;C. The average density of infectious bacteriophage particles in a phage lysate was calculated as PFU/mL.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e2.7. Phage susceptibility testing by a drop test and a liquid culture assay\u003c/h2\u003e \u003cp\u003ePreliminary phage susceptibility testing was performed by a drop test on a solid surface. Briefly, host bacterial strains were grown in 1.3 mL of LB at 37\u0026deg;C until the exponential growth phase (A\u003csub\u003e600\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;0.2-1) was reached. Absorbance at 600 nm (A\u003csub\u003e600\u003c/sub\u003e) was measured with a DSM Cell Density Meter (Laxco, USA), and the volume of bacterial culture needed for one plate was calculated as \u0026micro;L\u0026thinsp;=\u0026thinsp;45 / A\u003csub\u003e600\u003c/sub\u003e. Appropriate amounts of host bacteria and 3 mL of melted and tempered (at 55\u0026deg;C) soft agar were mixed gently and poured onto LA plates evenly. The plates were left at RT for 30 min to solidify before pipetting 5 \u0026micro;L of phage lysate in dilutions of 10\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e to 10\u003csup\u003e\u0026minus;\u0026thinsp;7\u003c/sup\u003e PFU/mL. Phage infections against their original bacterial host strain were used as positive control. The plates were left to settle for an additional 30 min at RT, followed by overnight incubation at 37˚C. A liquid-culture phage screening assay was used as a complementary assay to verify the efficiency of infection by measuring bacterial growth kinetic curves with a microscopy plate reader, oCelloScope (BioSense Solutions Aps, Denmark). It captures time-lapse digital microscopy images of microbial cultures and quantifies changes in cell density over time using automated image-analysis. Previously optimized species-specific growth conditions from Patpatia \u003cem\u003eet al\u003c/em\u003e. (2022) were used, and the screening time was 18 h at 37˚C without shaking [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. The efficiency of infection was determined by comparing the growth of uninfected bacteria with that of phage-treated bacteria at a given time point.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e2.8. Minimum biofilm inhibitory concentration (MBIC)\u003c/h2\u003e \u003cp\u003eSerial dilution of antibiotics was prepared in TSB for mono-species biofilms or enriched TSB\u0026thinsp;+\u0026thinsp;FBS (50:50) for dual-species biofilms. Enriched TSB\u0026thinsp;+\u0026thinsp;FBS media did not change the MBIC values compared to TSB for mono-species biofilms, therefore unnecessary usage of FBS was avoided. For phage\u0026ndash;antibiotic combinations, phage solution (10\u003csup\u003e8\u003c/sup\u003e PFU/mL) was added to achieve 10\u003csup\u003e7\u003c/sup\u003e PFU per well, and the volume of antibiotic solution was adjusted accordingly. Mono- or dual-species biofilms were formed as described in sections 2.4 and 2.2, with positive controls, inoculated media without antibiotic, and negative controls, non-inoculated media, included. After incubation, wells were homogenized by pipetting and spotted (3\u0026times;5 \u0026micro;L per well) onto TSA (mono-species biofilms) or MSA and MCA (dual-species biofilms) [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. In dual-species biofilms, \u003cem\u003eS. aureus\u003c/em\u003e biofilm was spotted after refilling the wells and redispersing the biofilm in 50 \u0026micro;L of fresh media. We assumed the biofilm formation starts at 0 h time of bacterial culture and biofilm and planktonic growth are highly codependent [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. Therefore, the MBIC was defined as the minimum antibiotic concentration that prevented planktonic regrowth on the corresponding agar plates.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e2.9. Minimum biofilm eradication concentration (MBEC)\u003c/h2\u003e \u003cp\u003eMono- or dual-species biofilms were formed in polystyrene 96-well plate as described in sections 2.4 and 2.2. After 20 h of biofilm growth, the supernatant from the preformed biofilm plate was completely removed, and the plate was washed with PBS to remove loose bacterial cells. The antibiotic\u0026ndash;antibiotic or phage\u0026ndash;antibiotic solutions were added to the wells in the biofilm plate and incubated at 37\u0026deg;C for 20 h. After incubation, 3\u0026times;5 \u0026micro;L from each well was spotted onto TSA for mono-species biofilms, or onto MSA and MCA for dual-species biofilms [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. In dual-species biofilms, \u003cem\u003eS. aureus\u003c/em\u003e biofilm was cultured after refilling the wells with 50 \u0026micro;L of fresh media to minimize the interference of \u003cem\u003eP. aeruginosa\u003c/em\u003e. The MBEC was defined as having the lowest antibiotic concentration resulting in no planktonic re-growth on the corresponding agar from the treated pre-formed biofilm.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e2.10. Checkerboard assay to evaluate antibiotic interactions\u003c/h2\u003e \u003cp\u003eCheckerboard assay was conducted to evaluate the interaction between the two antibiotics against the dual-species biofilm. In 96-well plates 90 \u0026micro;L of antibiotic A (8\u0026times; maximum concentration) solution in enriched TSB was added to column 1 of the plate. Serial twofold dilutions were performed across columns 1\u0026ndash;10. For antibiotic B, 90 \u0026micro;L of a 4\u0026times; maximum concentration of antibiotic in enriched TSB was added to row A, columns 1\u0026ndash;11. Twofold serial dilutions were carried out vertically down to row G. Enriched TSB were added to column 12 for the negative and positive controls. For inoculation, 10 \u0026micro;L of 4-hour \u003cem\u003eS. aureus\u003c/em\u003e culture and 1 \u0026micro;L of 4-hour \u003cem\u003eP. aeruginosa\u003c/em\u003e culture were added to each well, excluding the negative control wells. Fresh non-inoculated media and inoculated media without antibiotics were used as negative and positive controls, respectively. Finally, 90 \u0026micro;L of FBS was added to all the wells including the control wells. The plate was incubated at 37\u0026deg;C for 20 h. Following incubation, 3\u0026times;5\u0026micro;L from each well was spotted onto selective agar plates MCA for \u003cem\u003eP. aeruginosa\u003c/em\u003e, and for \u003cem\u003eS. aureus\u003c/em\u003e, the wells were first emptied and refilled with 50 \u0026micro;L of fresh media before spotting on MSA to minimize \u003cem\u003eP. aeruginosa\u003c/em\u003e interference. The plates were incubated for 20 h (48\u0026ndash;72 h for \u003cem\u003eS. aureus\u003c/em\u003e). The fractional inhibitory concentration index (FICI) was calculated via the following Eq.\u0026nbsp;(1) to assess the interaction between the antibiotics [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e].\u003cdiv id=\"Equa\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equa\" name=\"EquationSource\"\u003e\n$$FICI=\\frac{{MBIC}_{AB1incombinationwithAB2}}{{MBIC}_{AB1alone}}+\\frac{{MBIC}_{AB2incombinationwithAB1}}{{MBIC}_{AB2alone}}\\left(1\\right)$$\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e \u003cp\u003eThe FICI values were interpreted as follows: synergistic when FICI\u0026thinsp;\u0026le;\u0026thinsp;0.5; sub-synergistic when 0.5\u0026thinsp;\u0026lt;\u0026thinsp;FICI\u0026thinsp;\u0026le;\u0026thinsp;1.0; indifferent when 1.0\u0026thinsp;\u0026lt;\u0026thinsp;FICI\u0026thinsp;\u0026le;\u0026thinsp;4.0; and antagonistic when FICI\u0026thinsp;\u0026gt;\u0026thinsp;4.0, indicating antibiotic interactions against the dual-species biofilm.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e2.11. Development of the CDM\u003c/h2\u003e \u003cp\u003eCDMs were prepared following a method adapted from Kaukonen \u003cem\u003eet al.\u003c/em\u003e with minor modifications [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. Glass coverslips were placed in 24-well plates, sterilized with 70% ethanol, treated with 0.2% (w/v) gelatin in PBS (800 \u0026micro;L) pre-warmed to 37\u0026deg;C, and incubated for 1 h at 37\u0026deg;C. After gelatin was aspirated, the coverslips were washed gently with PBS. Gelatin was crosslinked by adding 1% (v/v) glutaraldehyde solution (500 \u0026micro;L) for 30 min at room temperature (RT), followed by glutaraldehyde aspiration and two PBS washes. The residual glutaraldehyde was quenched with 1 M glycine (500 \u0026micro;L) in PBS for 20 min at RT. Following glycine removal, the coverslips were washed twice with PBS and incubated with pre-warmed Dulbecco's Modified Eagle Medium (DMEM, high glucose) (800 \u0026micro;L, 1 h, 37\u0026deg;C). Human dermal fibroblasts (ATTC PCS-201-010) were then seeded at 5 \u0026times; 10⁴ cells per well on coverslips and cultured at 37\u0026deg;C until monolayer confluence. The growth medium was then replaced with DMEM supplemented with 50 \u0026micro;g/mL freshly prepared ascorbic acid (0.45 \u0026micro;m-filtered). Cultures were maintained for 14 to 21 days in an incubator (37\u0026deg;C) with ascorbic acid renewed every two days to stimulate extracellular matrix (ECM) production. For decellularization, the wells were rinsed with PBS and incubated with pre-warmed (37\u0026deg;C) standard extraction solution (1 mL of NH₄OH, 250 \u0026micro;L of Triton X-100, and 48.75 mL of PBS: 500 \u0026micro;L per well) [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. After ~\u0026thinsp;2 min, once the cells were fully detached, the extraction solution was discarded, and the coverslips were washed twice with PBS. The residual cellular DNA was removed by incubation with DNase I was added to each well (10 \u0026micro;g/mL in deionized water; 500 \u0026micro;L per well) and incubated at 37\u0026deg;C for 30 min. After incubation, the CDMs were washed twice with PBS. CDMs were stored at 4\u0026deg;C in PBS containing 1% (v/v) penicillin\u0026ndash;streptomycin until further use, for up to 4 weeks [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e2.12. Forming dual-species biofilms on CDMs\u003c/h2\u003e \u003cp\u003eWe developed the dual-species biofilms on CDMs using the method described in our previous work [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. The enriched TSB supplemented with 1% glucose and 2.5% NaCl was mixed 1:1 with FBS was used. CDMs were placed in 24-well plates and washed with PBS [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. A total of 800 \u0026micro;L of inoculated media containing \u003cem\u003eS. aureus\u003c/em\u003e (10⁷ CFU/mL in each well) and \u003cem\u003eP. aeruginosa\u003c/em\u003e (10⁶ CFU/mL in each well) was added to the wells. Wells filled with either inoculated or non-inoculated media were used as positive and negative controls, respectively. The plates were incubated at 37\u0026deg;C for 20 h, without shaking, to allow biofilm formation. After incubation, the planktonic bacteria were removed, and the biofilm was gently washed three times with PBS to remove any loosely adherent cells. The pH of the biofilm formed on CDM was measured.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e2.13. Combined antibiotics on the dual-species biofilm formed on CDM\u003c/h2\u003e \u003cp\u003eAntibiotic concentrations for the CDM-biofilm assays were selected based on the MBIC and MBEC data obtained from 2.8 and 2.9 Combinations of antibiotics A and B were tested at their respective MBIC/MBEC values, as well as twofold higher and up to eightfold lower concentrations. Biofilms were established in 24-well plates on predeveloped CDM following the abovementioned biofilm formation protocols (section 2.2). The antibiotic combinations were either added to the media before developing biofilms on CDM for biofilm inhibition assays or to biofilms formed on CDM for biofilm eradication assays. The plates were incubated at 37\u0026deg;C for 20 h without shaking. Following incubation, biofilm viability was assessed by spotting 3\u0026times;5 \u0026micro;L from each well on MSA and MCA plates. \u003cem\u003eS. aureus\u003c/em\u003e biofilm was cultured after refilling the wells with 50 \u0026micro;L of fresh media to minimize the interference of \u003cem\u003eP. aeruginosa\u003c/em\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003e2.14. Scanning electron microscopy (SEM)\u003c/h2\u003e \u003cp\u003eThree different samples were prepared for SEM imaging: (i) CDM alone, (ii) a dual-species biofilm formed overnight on CDM, and (iii) a dual-species biofilm formed overnight on CDM and was subsequently treated with a combination of ciprofloxacin and vancomycin at their effective inhibitory concentrations. Each condition was prepared in separate wells of a 24-well plate.\u003c/p\u003e \u003cp\u003eThe samples were initially gently washed five times with PBS to remove any loosely attached cells or debris. Then they were fixed by adding 500 \u0026micro;L of a fixation solution containing 4% paraformaldehyde and 1% glutaraldehyde for 2 h at room temperature [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. Following fixation, the samples were washed three times with 500 \u0026micro;L of PBS and finally rinsed with deionized water [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. Dehydration was carried out by sequential incubation in 50%, 75%, 90%, and 100% ethanol, each for 10 min at room temperature [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. After dehydration, the samples were left to air dry overnight at room temperature.\u003c/p\u003e \u003cp\u003eThe dried samples were mounted onto SEM stubs and sputter-coated with a thin layer of carbon to increase conductivity. SEM imaging was performed using Quanta\u0026trade; 250 FEG SEM (Thermo Fisher Scientific, Hillsboro, OR, USA) instrument equipped with an Everhardt Thornley SED (secondary electron detector) to assess the surface morphology of the CDMs and the structure of the dual-species biofilms with and without antibiotic treatment [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. SEM images were obtained at an accelerating voltage of 5 kV, with a dwell time of 5 \u0026micro;s, a spot size of 3.5, and a working distance of 10.6 mm. Images were captured at magnifications of 1,000\u0026times; and 10,000\u0026times;.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e\n\u003ch2\u003e3.1. Antibiotic susceptibility profiles of \u003cem\u003eS. aureus\u003c/em\u003e and \u003cem\u003eP. aeruginosa\u003c/em\u003e\u003c/h2\u003e\n\u003cp\u003eThe MICs and MBCs of ten antibiotics were determined for both \u003cem\u003eP. aeruginosa\u003c/em\u003e and \u003cem\u003eS. aureus\u003c/em\u003e to confirm their susceptibility to these antibiotics. \u003cem\u003eP. aeruginosa\u003c/em\u003e was highly sensitive to most of the tested antibiotics (MIC and MBC\u0026thinsp;\u0026lt;\u0026thinsp;1 \u0026micro;g/mL); however, it presented relatively high MIC and MBC values to rifampicin, consistent with typical susceptibility of \u003cem\u003eP. aeruginosa\u003c/em\u003e PAO1 strains to rifampicin (Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e\n\u003cdiv class=\"gridtable\"\u003e\n\u003cdiv class=\"colspec\" align=\"left\"\u003e\u0026nbsp;\u003c/div\u003e\n\u003ctable id=\"Tab1\" border=\"1\"\u003e\u003ccaption\u003e\n\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\n\u003cdiv class=\"CaptionContent\"\u003e\n\u003cp\u003eMIC and MBC values for selected antibiotics against P. aeruginosa.\u003c/p\u003e\n\u003c/div\u003e\n\u003c/caption\u003e\n\u003cthead\u003e\n\u003ctr\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003e\u003cem\u003eP. aeruginosa\u003c/em\u003e\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eGEN\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eTOB\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eOFX\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eCIP\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eLVX\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eCST\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eRIF\u003c/p\u003e\n\u003c/th\u003e\n\u003c/tr\u003e\n\u003c/thead\u003e\n\u003ctbody\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003eMIC (\u003c/strong\u003e\u0026micro;g/mL\u003cstrong\u003e)\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u0026le;\u0026thinsp;1\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u0026le;\u0026thinsp;1\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u0026le;\u0026thinsp;1\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u0026le;\u0026thinsp;1\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u0026le;\u0026thinsp;1\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u0026le;\u0026thinsp;1\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e32\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003eMBC (\u003c/strong\u003e\u0026micro;g/mL\u003cstrong\u003e)\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e2\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u0026le;\u0026thinsp;1\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u0026le;\u0026thinsp;1\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u0026le;\u0026thinsp;1\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u0026le;\u0026thinsp;1\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u0026le;\u0026thinsp;1\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e256\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003c/tbody\u003e\n\u003ctfoot\u003e\n\u003ctr\u003e\n\u003ctd colspan=\"8\"\u003e*Abbreviations: GEN, gentamicin; TOB, tobramycin; OFX, ofloxacin; CIP, ciprofloxacin; LVX, levofloxacin; CST, colistin; and RIF, rifampicin.\u003c/td\u003e\n\u003c/tr\u003e\n\u003c/tfoot\u003e\n\u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003e\u003cem\u003eS. aureus\u003c/em\u003e showed sensitivity to vancomycin, ampicillin, gentamicin, tobramycin, ofloxacin, ciprofloxacin, levofloxacin, rifampicin, and penicillin, with MIC and MBC values of \u0026lt;\u0026thinsp;1 \u0026micro;g/mL (Table\u0026nbsp;2). A lower susceptibility was observed for teicoplanin (MIC of 4 \u0026micro;g/mL; MBC of 6 \u0026micro;g/mL), which can be attributed to the growth media, bacterial strains and other test-dependent factors. TSB strongly supports the growth of \u003cem\u003eS. aureus\u003c/em\u003e, often leading to increased biomass and doubling time [\u003cspan class=\"CitationRef\"\u003e39\u003c/span\u003e]. Nevertheless, MIC assays confirmed the susceptibility of bacteria to the tested antibiotics.\u003c/p\u003e\n\u003cdiv class=\"gridtable\"\u003e\n\u003cdiv class=\"colspec\" align=\"left\"\u003eTable\u0026nbsp;2. MIC and MBC values of selected antibiotics against \u003cem\u003eS. aureus\u003c/em\u003e.\u003c/div\u003e\n\u003cdiv class=\"colspec\" align=\"left\"\u003e\u0026nbsp;\u003c/div\u003e\n\u003ctable id=\"Taba\" border=\"1\"\u003e\n\u003ctbody\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003eS. aureus\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003eVAN\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003eTEC\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003eAMP\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003ePEN\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003eGEN\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003eTOB\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003eOFX\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003eCIP\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003eLVX\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003eRIF\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003eMIC\u003c/strong\u003e (\u0026micro;g/mL)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e2\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e4\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u0026le;\u0026thinsp;1\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u0026le;\u0026thinsp;1\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u0026le;\u0026thinsp;1\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u0026le;\u0026thinsp;1\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u0026le;\u0026thinsp;1\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u0026le;\u0026thinsp;1\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u0026le;\u0026thinsp;1\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" align=\"left\"\u003e\n\u003cp\u003e\u0026le;\u0026thinsp;1\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003eMBC\u003c/strong\u003e (\u0026micro;g/mL)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e2\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e6\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u0026le;\u0026thinsp;1\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u0026le;\u0026thinsp;1\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e2\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u0026le;\u0026thinsp;1\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u0026le;\u0026thinsp;1\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e2\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u0026le;\u0026thinsp;1\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" align=\"left\"\u003e\n\u003cp\u003e\u0026le;\u0026thinsp;1\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003c/tbody\u003e\n\u003ctfoot\u003e\n\u003ctr\u003e\n\u003ctd colspan=\"12\"\u003e*Abbreviations: VAN, vancomycin; TEC, teicoplanin; AMP, ampicillin; PEN, penicillin; GEN, gentamicin; TOB, tobramycin; OFX, ofloxacin; CIP, ciprofloxacin; LVX, levofloxacin; and RIF, rifampicin.\u003c/td\u003e\n\u003c/tr\u003e\n\u003c/tfoot\u003e\n\u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003eCoculture of \u003cem\u003eS. aureus\u003c/em\u003e and \u003cem\u003eP. aeruginosa\u003c/em\u003e were confirmed and quantified via colony counting after an overnight (20 h), 4 h, and after dual-species biofilm formation (20 h) (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e). Both species reached\u0026thinsp;~\u0026thinsp;10\u003csup\u003e10\u003c/sup\u003e CFU/mL overnight and ~\u0026thinsp;10\u003csup\u003e9\u003c/sup\u003e CFU/mL after 4 h culture. Following dual-species biofilm formation, the planktonic cell counts were just below 10\u003csup\u003e11\u003c/sup\u003e CFU/mL, while the biofilm-associated cell counts were 2\u0026times;10\u003csup\u003e9\u003c/sup\u003e CFU/mL for \u003cem\u003eS. aureus\u003c/em\u003e and ~\u0026thinsp;8\u0026times;10\u003csup\u003e9\u003c/sup\u003e CFU/mL for \u003cem\u003eP. aeruginosa\u003c/em\u003e. The pH of the biofilm was measured 5.8.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec20\" class=\"Section2\"\u003e\n\u003ch2\u003e3.3. Coculture with \u003cem\u003eS. aureus\u003c/em\u003e altered the response of \u003cem\u003eP. aeruginosa\u003c/em\u003e to antibiotics\u003c/h2\u003e\n\u003cp\u003eMBIC and MBEC assays were performed to compare the response of mono-species biofilms and dual-species biofilms to antibiotics. The MBIC and MBEC values of antibiotics against \u003cem\u003eP. aeruginosa\u003c/em\u003e mono-species and dual-species biofilm are presented in Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e. Notable increases were observed for rifampicin, gentamicin, and colistin, with MBICs of \u0026gt;\u0026thinsp;2048, 56, and 20 \u0026micro;g/mL, respectively. Tobramycin, ofloxacin, ciprofloxacin, and levofloxacin presented MBICs of 8, 4, 3, and 3 \u0026micro;g/mL, respectively, slightly exceeding their MICs. The presence of \u003cem\u003eS. aureus\u003c/em\u003e in dual-species biofilms slightly altered the \u003cem\u003eP. aeruginosa\u003c/em\u003e biofilm response to antibiotics. Compared to those in mono-species biofilms, The MBIC values of most antibiotics for \u003cem\u003eP. aeruginosa\u003c/em\u003e increased 2- or 3-fold. The MBIC value of the broad-spectrum antibiotic gentamicin decreased almost 2-fold in dual-species biofilms compared with that in mono-species biofilms, which is consistent with previous reports [\u003cspan class=\"CitationRef\"\u003e40\u003c/span\u003e].\u003c/p\u003e\n\u003cp\u003eThe MBEC values of antibiotics in mono-species biofilms increased relative to their MBICs, with those of gentamicin, colistin, and tobramycin increasing 5-, 5-, and 9-fold, respectively. In contrast, the MBEC values for ofloxacin, ciprofloxacin, and levofloxacin did not change significantly compared with their MBICs. In dual-species biofilms, MBEC values of FQ antibiotics increased to 7-fold compared to those of mono-species biofilms. In dual species biofilms, \u003cem\u003eP. aeruginosa\u003c/em\u003e showed high tolerance to colistin with an MBEC value of \u0026gt;\u0026thinsp;2048.\u003c/p\u003e\n\u003cdiv class=\"gridtable\"\u003e\n\u003cdiv class=\"colspec\" align=\"left\"\u003e\u0026nbsp;\u003c/div\u003e\n\u003cdiv class=\"colspec\" align=\"left\"\u003e\u0026nbsp;\u003c/div\u003e\n\u003ctable id=\"Tab2\" border=\"1\"\u003e\u003ccaption\u003e\n\u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e\n\u003cdiv class=\"CaptionContent\"\u003e\n\u003cp\u003eAntibiotic susceptibility of P. aeruginosa in mono- and dual-species biofilms (MBIC and MBEC, \u0026micro;g/mL)\u003c/p\u003e\n\u003c/div\u003e\n\u003c/caption\u003e\n\u003cthead\u003e\n\u003ctr\u003e\n\u003cth colspan=\"2\" align=\"left\"\u003e\n\u003cp\u003e\u003cem\u003eP. aeruginosa\u003c/em\u003e\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eGEN\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eTOB\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eOFX\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eCIP\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eLVX\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eCST\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eRIF\u003c/p\u003e\n\u003c/th\u003e\n\u003c/tr\u003e\n\u003c/thead\u003e\n\u003ctbody\u003e\n\u003ctr\u003e\n\u003ctd rowspan=\"2\" align=\"left\"\u003e\n\u003cp\u003eMono-species\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003eMBIC\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e56\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e8\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e4\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e3\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e3\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e20\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u0026gt;\u0026thinsp;2048\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003eMBEC\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e256\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e76\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e5\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e3\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e3\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e192\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u0026gt;\u0026thinsp;2048\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd rowspan=\"2\" align=\"left\"\u003e\n\u003cp\u003eDual-species\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003eMBIC\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e32\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e19\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e8\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e7\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e5\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e64\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u0026gt;\u0026thinsp;2048\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003eMBEC\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e128\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e48\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e38\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e9\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e18\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u0026gt;\u0026thinsp;2048\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u0026gt;\u0026thinsp;2048\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003c/tbody\u003e\n\u003ctfoot\u003e\n\u003ctr\u003e\n\u003ctd colspan=\"9\"\u003e*Abbreviations: GEN, gentamicin; TOB, tobramycin; OFX, ofloxacin; CIP, ciprofloxacin; LVX, levofloxacin; CST, colistin; and RIF, rifampicin.\u003c/td\u003e\n\u003c/tr\u003e\n\u003c/tfoot\u003e\n\u003c/table\u003e\n\u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec21\" class=\"Section2\"\u003e\n\u003ch2\u003e3.4. Coculture with \u003cem\u003eP. aeruginosa\u003c/em\u003e reduced \u003cem\u003eS. aureus\u003c/em\u003e biofilm susceptibility to most of the tested antibiotics\u003c/h2\u003e\n\u003cp\u003eThe MBIC and MBEC values of antibiotics against mono-species and dual-species biofilms of \u003cem\u003eS. aureus\u003c/em\u003e are presented in Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e. The MBIC values of ampicillin, teicoplanin, tobramycin, gentamicin, ofloxacin, and vancomycin were \u0026gt;\u0026thinsp;2048, 96, 64, 56, 52, and 48 \u0026micro;g/mL, respectively for the mono-species biofilms of \u003cem\u003eS. aureus\u003c/em\u003e. The MBEC values were even higher than MBICs, with rifampicin and vancomycin showing values of 64 and 192 \u0026micro;g/mL, respectively. Compared with the MBIC values, the FQs presented up to a 4-fold increase in their MBEC values in mono-species biofilms.\u003c/p\u003e\n\u003cdiv class=\"gridtable\"\u003e\n\u003cdiv class=\"colspec\" align=\"left\"\u003e\u0026nbsp;\u003c/div\u003e\n\u003cdiv class=\"colspec\" align=\"left\"\u003e\u0026nbsp;\u003c/div\u003e\n\u003ctable id=\"Tab3\" border=\"1\"\u003e\u003ccaption\u003e\n\u003cdiv class=\"CaptionNumber\"\u003eTable 4\u003c/div\u003e\n\u003cdiv class=\"CaptionContent\"\u003e\n\u003cp\u003eAntibiotic susceptibility of S. aureus in mono- and dual-species biofilms (MBIC and MBEC, \u0026micro;g/mL)\u003c/p\u003e\n\u003c/div\u003e\n\u003c/caption\u003e\n\u003cthead\u003e\n\u003ctr\u003e\n\u003cth colspan=\"2\" align=\"left\"\u003e\n\u003cp\u003e\u003cem\u003eS. aureus\u003c/em\u003e\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eVAN\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eTEC\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eAMP\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003ePEN\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eGEN\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eTOB\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eOFX\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eCIP\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eLVX\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eRIF\u003c/p\u003e\n\u003c/th\u003e\n\u003c/tr\u003e\n\u003c/thead\u003e\n\u003ctbody\u003e\n\u003ctr\u003e\n\u003ctd rowspan=\"2\" align=\"left\"\u003e\n\u003cp\u003eMono-species\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003eMBIC\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e48\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e96\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u0026gt;\u0026thinsp;2048\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e4\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e56\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e64\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e52\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e33\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e8\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e1\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003eMBEC\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e192\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u0026gt;\u0026thinsp;2048\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u0026gt;\u0026thinsp;2048\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e4\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e64\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e40\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e100\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e60\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e30\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e64\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd rowspan=\"2\" align=\"left\"\u003e\n\u003cp\u003eDual-species\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003eMBIC\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u0026gt;\u0026thinsp;2048\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u0026gt;\u0026thinsp;2048\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u0026gt;\u0026thinsp;2048\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u0026gt;\u0026thinsp;2048\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e347\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e512\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e320\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e192\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e40\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e48\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003eMBEC\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u0026gt;\u0026thinsp;2048\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u0026gt;\u0026thinsp;2024\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u0026gt;\u0026thinsp;2048\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u0026gt;\u0026thinsp;2048\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u0026gt;\u0026thinsp;2048\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u0026gt;\u0026thinsp;2048\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u0026gt;\u0026thinsp;2048\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u0026gt;\u0026thinsp;2048\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u0026gt;\u0026thinsp;2048\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u0026gt;\u0026thinsp;2048\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003c/tbody\u003e\n\u003ctfoot\u003e\n\u003ctr\u003e\n\u003ctd colspan=\"12\"\u003e*Abbreviations: VAN, vancomycin; TEC, teicoplanin; AMP, ampicillin; PEN, penicillin; GEN, gentamicin; TOB, tobramycin; OFX, ofloxacin; CIP, ciprofloxacin; LVX, levofloxacin; and RIF, rifampicin.\u003c/td\u003e\n\u003c/tr\u003e\n\u003c/tfoot\u003e\n\u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003eThe tolerance profile of \u003cem\u003eS. aureus\u003c/em\u003e significantly increased in dual-species biofilms with \u003cem\u003eP. aeruginosa\u003c/em\u003e (Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e). The MBIC values significantly increased for the dual-species biofilms, indicating high tolerance to vancomycin, teicoplanin, ampicillin, and penicillin (\u0026ge;\u0026thinsp;2048 \u0026micro;g/mL). The MBIC for gentamicin, tobramycin, ofloxacin, and ciprofloxacin increased to 347 \u0026micro;g/mL, 512 \u0026micro;g/mL, 320 \u0026micro;g/mL, and 192 \u0026micro;g/mL, respectively. The MBEC values indicated the resistance of \u003cem\u003eS. aureus\u003c/em\u003e in the dual-species biofilms to the tested concentrations of antibiotics, with no eradication achieved even at the highest tested concentration of 2048 \u0026micro;g/mL. These results suggest that the protective effect afforded by the dual-species biofilm matrix is particularly due to the presence of \u003cem\u003eP. aeruginosa\u003c/em\u003e.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec22\" class=\"Section2\"\u003e\n\u003ch2\u003e3.5. Phage activity against \u003cem\u003eS. aureus\u003c/em\u003e was different on the solid surface compared to liquid culture\u003c/h2\u003e\n\u003cp\u003ePreliminary phage susceptibility testing of Stab 21 against \u003cem\u003eS. aureus\u003c/em\u003e showed varying results. Stab 21 showed lytic activity against \u003cem\u003eS. aureus\u003c/em\u003e on solid media based on drop testing; however, in liquid culture, Stab21 (in 10\u003csup\u003e5\u003c/sup\u003e PFU/well) could not infect \u003cem\u003eS. aureus\u003c/em\u003e during 18 h of incubation, while its original host strain, \u003cem\u003eStaphylococcus xylosus\u003c/em\u003e was infected (supplementary information, Figure \u003cspan class=\"InternalRef\"\u003eS1\u003c/span\u003e). Higher phage concentrations (10\u003csup\u003e6\u003c/sup\u003e and 10\u003csup\u003e7\u003c/sup\u003e PFU/well) contributed to higher lytic activity, although at 10\u003csup\u003e7\u003c/sup\u003e PFU/well, the emergence of phage-resistance bacteria was observed after 10 hours (Supplementary information, Figure \u003cspan class=\"InternalRef\"\u003eS1\u003c/span\u003e). Phage susceptibility testing against \u003cem\u003eP. aeruginosa\u003c/em\u003e using phage PA8P1 demonstrated strong lytic activity on both solid media and in liquid culture (supplementary information, Figure S2).\u003c/p\u003e\n\u003ch3\u003e3.6. S. aureus showed high susceptibility to phage\u0026ndash;antibiotic combination treatment in the MBIC assay, but significantly lower susceptibility in the MBEC assay\u003c/h3\u003e\n\u003cp\u003eThe effects of phage treatment alone and in combination with ciprofloxacin or vancomycin were investigated against \u003cem\u003eS. aureus\u003c/em\u003e and \u003cem\u003eP. aeruginosa\u003c/em\u003e in mono and dual-species biofilms. Under single-species biofilm growth conditions, \u003cem\u003eS. aureus\u003c/em\u003e and \u003cem\u003eP. aeruginosa\u003c/em\u003e were treated with their respective phages at the four highest concentrations tested (10⁴ to 10⁷ PFU/well for PA8P1 and 10⁵ to 10⁸ PFU/well for Stab 21), and the biofilm formation was assessed following an 18-hour incubation period. Based on visual assessment, none of the phage treatments were effective in preventing biofilm development. Both phages were chosen to be used at a concentration of 10\u003csup\u003e7\u003c/sup\u003e PFU/well throughout the following experiments.\u003c/p\u003e\n\u003cp\u003ePhage Stab21 was subsequently tested in combination with sub-biofilm-inhibitory concentrations of vancomycin and ciprofloxacin against \u003cem\u003eS. aureus\u003c/em\u003e biofilms, and phage PA8P1 was tested in combination with ciprofloxacin and colistin against \u003cem\u003eP. aeruginosa\u003c/em\u003e biofilms. The addition of Stab 21 significantly reduced the MBIC of ciprofloxacin from 33 \u0026micro;g/mL (mono-treatment) to 0.5 \u0026micro;g/mL. Similarly, with vancomycin, the MBIC decreased from 48 \u0026micro;g/mL in monotreatment to 10 \u0026micro;g/mL in the presence of Stab21 (Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003e). As for phage PA8P1, phage‑antibiotic synergy was not confirmed with either of the tested antibiotics against \u003cem\u003eP. aeruginosa\u003c/em\u003e biofilms. Compared to antibiotic treatment alone, the addition of PA8P1 increased the MBIC of ciprofloxacin, whereas the MBIC of colistin remained unaffected by the presence of phages (supplementary information, Table \u003cspan class=\"InternalRef\"\u003eS1\u003c/span\u003e). Thus, PA8P1 was excluded from further evaluation in dual-species biofilm assays.\u003c/p\u003e\n\u003cdiv class=\"gridtable\"\u003e\n\u003cdiv class=\"colspec\" align=\"left\"\u003e\u0026nbsp;\u003c/div\u003e\n\u003cdiv class=\"colspec\" align=\"left\"\u003e\u0026nbsp;\u003c/div\u003e\n\u003ctable id=\"Tab4\" border=\"1\"\u003e\u003ccaption\u003e\n\u003cdiv class=\"CaptionNumber\"\u003eTable 5\u003c/div\u003e\n\u003cdiv class=\"CaptionContent\"\u003e\n\u003cp\u003eMBIC of S. aureus in biofilms treated with Stab 21 plus ciprofloxacin or vancomycin\u003c/p\u003e\n\u003c/div\u003e\n\u003c/caption\u003e\n\u003cthead\u003e\n\u003ctr\u003e\n\u003cth colspan=\"2\" align=\"left\"\u003e\n\u003cp\u003e\u003cem\u003eS. aureus\u003c/em\u003e\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\u0026nbsp;\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eCiprofloxacin\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eVancomycin\u003c/p\u003e\n\u003c/th\u003e\n\u003c/tr\u003e\n\u003c/thead\u003e\n\u003ctbody\u003e\n\u003ctr\u003e\n\u003ctd rowspan=\"4\" align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003eMBIC\u003c/strong\u003e (\u0026micro;g/mL)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd rowspan=\"2\" align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003eMono-species biofilm\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003eAB control\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e33\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e48\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003eAB\u0026thinsp;+\u0026thinsp;Stab 21\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.5\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e10\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd rowspan=\"2\" align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003eDual-species biofilm\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003eAB control\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e192\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u0026gt;\u0026thinsp;2048\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003eAB\u0026thinsp;+\u0026thinsp;Stab 21\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u0026lt;\u0026thinsp;0.25\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u0026lt;\u0026thinsp;0.5\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003c/tbody\u003e\n\u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003eIn the dual-species biofilm model, the addition of Stab 21 further decreased the MBIC values of both antibiotics. In the dual-species biofilm, \u003cem\u003eS. aureus\u003c/em\u003e exhibited increased resistance to ciprofloxacin, with the MBIC increasing from 33 \u0026micro;g/mL (mono-species) to 192 \u0026micro;g/mL. However, the addition of Stab 21 significantly restored its susceptibility to ciprofloxacin, reducing the MBIC to \u0026lt;\u0026thinsp;0.25 \u0026micro;g/mL. A similar trend was observed with vancomycin, where \u003cem\u003eS. aureus\u003c/em\u003e displayed elevated resistance in the mixed biofilm, with an increase in the MBIC from 48 \u0026micro;g/mL (mono-species) to \u0026gt;\u0026thinsp;2048 \u0026micro;g/mL. Upon the introduction of Stab 21, the susceptibility of vancomycin was regained, lowering the MBIC to \u0026lt;\u0026thinsp;0.5 \u0026micro;g/mL. Notably, we did not test lower concentrations of antibiotics. In summary, based on the MBIC of ciprofloxacin and vancomycin against \u003cem\u003eS. aureus\u003c/em\u003e growth in both mono- and dual-species biofilm models, the addition of Stab 21 significantly reduced the dose of both ciprofloxacin and vancomycin needed to inhibit the biofilm growth (Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003e). The effectiveness of Stab 21 combined with ciprofloxacin in eradicating \u003cem\u003eS. aureus\u003c/em\u003e from dual-species biofilms was subsequently evaluated. The addition of Stab 21 reduced the MBEC of ciprofloxacin from 2048 \u0026micro;g/mL to 1024 \u0026micro;g/mL. As expected, the addition of Stab 21 had no effect on \u003cem\u003eP. aeruginosa\u003c/em\u003e, and only the inhibitory activity of the antibiotics was observed (Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003e).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec23\" class=\"Section2\"\u003e\n\u003ch2\u003e3.7. Most combinations had synergistic interactions on the inhibition of the dual-species biofilm\u003c/h2\u003e\n\u003cp\u003eThe efficacy of antibiotic combinations against dual-species biofilm formation was assessed by calculating the FICI (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e). Multiple antibiotic pairs showed synergistic interactions (FICI\u0026thinsp;\u0026lt;\u0026thinsp;0.5) on the inhibition of \u003cem\u003eS. aureus\u003c/em\u003e within dual-species biofilms (Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003e). Combinations such as vancomycin\u0026ndash;ciprofloxacin, rifampicin\u0026ndash;colistin, vancomycin\u0026ndash;colistin, and penicillin-ciprofloxacin demonstrated synergistic effects against both \u003cem\u003eP. aeruginosa\u003c/em\u003e and \u003cem\u003eS. aureus\u003c/em\u003e. The vancomycin\u0026ndash;ciprofloxacin combination had strong synergistic effects against \u003cem\u003eS. aureus\u003c/em\u003e (FICI\u0026thinsp;=\u0026thinsp;0.055), which was further enhanced by the addition of the phage Stab 21 (FICI\u0026thinsp;=\u0026thinsp;0.002). However, it should be noted that the same MBIC was reached by combining either ciprofloxacin or vancomycin alone with phages, indicating that the combination of antibiotics with phages would not be necessary. Therefore, the most significant synergy against \u003cem\u003eS. aureus\u003c/em\u003e was observed with vancomycin-ciprofloxacin-stab 21 (FICI\u0026thinsp;=\u0026thinsp;0.002), followed by combinations of vancomycin-ofloxacin (FICI\u0026thinsp;=\u0026thinsp;0.034), ciprofloxacin-levofloxacin (FICI\u0026thinsp;=\u0026thinsp;0.047), vancomycin-ciprofloxacin (FICI\u0026thinsp;=\u0026thinsp;0.055), vancomycin-levofloxacin (FICI\u0026thinsp;=\u0026thinsp;0.083), rifampin-colistin (FICI\u0026thinsp;=\u0026thinsp;0.093), and vancomycin-colistin (FICI\u0026thinsp;=\u0026thinsp;0.094). The MBIC data for both species are presented in Supplementary Information Table S2.\u003c/p\u003e\n\u003cdiv class=\"gridtable\"\u003e\n\u003cdiv class=\"colspec\" align=\"left\"\u003e\u0026nbsp;\u003c/div\u003e\n\u003cdiv class=\"colspec\" align=\"left\"\u003e\u0026nbsp;\u003c/div\u003e\n\u003ctable id=\"Tab5\" border=\"1\"\u003e\u003ccaption\u003e\n\u003cdiv class=\"CaptionNumber\"\u003eTable 6\u003c/div\u003e\n\u003cdiv class=\"CaptionContent\"\u003e\n\u003cp\u003eInteraction of the chosen combinations on the inhibition of the dual-species\u0026nbsp;biofilms.\u003c/p\u003e\n\u003c/div\u003e\n\u003c/caption\u003e\n\u003cthead\u003e\n\u003ctr style=\"height: 35px;\"\u003e\n\u003cth style=\"height: 73px;\" rowspan=\"2\" align=\"left\"\u003e\n\u003cp\u003eAntibiotics\u003c/p\u003e\n\u003c/th\u003e\n\u003cth style=\"height: 35px;\" colspan=\"4\" align=\"left\"\u003e\n\u003cp\u003e\u003cem\u003eP. aeruginosa\u003c/em\u003e\u003c/p\u003e\n\u003c/th\u003e\n\u003cth style=\"height: 35px;\" colspan=\"5\" align=\"left\"\u003e\n\u003cp\u003e\u003cem\u003eS. aureus\u003c/em\u003e\u003c/p\u003e\n\u003c/th\u003e\n\u003c/tr\u003e\n\u003ctr style=\"height: 38px;\"\u003e\n\u003cth style=\"height: 38px;\" align=\"left\"\u003e\n\u003cp\u003eFIC \u003csub\u003ea\u003c/sub\u003e\u003c/p\u003e\n\u003c/th\u003e\n\u003cth style=\"height: 38px;\" align=\"left\"\u003e\n\u003cp\u003eFIC \u003csub\u003eb\u003c/sub\u003e\u003c/p\u003e\n\u003c/th\u003e\n\u003cth style=\"height: 38px;\" align=\"left\"\u003e\n\u003cp\u003e\u0026sum;FICI\u003c/p\u003e\n\u003c/th\u003e\n\u003cth style=\"height: 38px;\" colspan=\"2\" align=\"left\"\u003e\n\u003cp\u003eInteraction\u003c/p\u003e\n\u003c/th\u003e\n\u003cth style=\"height: 38px;\" align=\"left\"\u003e\n\u003cp\u003eFIC \u003csub\u003ea\u003c/sub\u003e\u003c/p\u003e\n\u003c/th\u003e\n\u003cth style=\"height: 38px;\" align=\"left\"\u003e\n\u003cp\u003eFIC \u003csub\u003eb\u003c/sub\u003e\u003c/p\u003e\n\u003c/th\u003e\n\u003cth style=\"height: 38px;\" align=\"left\"\u003e\n\u003cp\u003e\u0026sum;FICI\u003c/p\u003e\n\u003c/th\u003e\n\u003cth style=\"height: 38px;\" align=\"left\"\u003e\n\u003cp\u003eInteraction\u003c/p\u003e\n\u003c/th\u003e\n\u003c/tr\u003e\n\u003c/thead\u003e\n\u003ctbody\u003e\n\u003ctr style=\"height: 35px;\"\u003e\n\u003ctd style=\"height: 35px;\" align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003eVancomycin-colistin\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" align=\"left\"\u003e\n\u003cp\u003e0.002\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" align=\"left\"\u003e\n\u003cp\u003e0.093\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" align=\"left\"\u003e\n\u003cp\u003e0.095\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" colspan=\"2\" align=\"left\"\u003e\n\u003cp\u003eSynergistic\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" align=\"left\"\u003e\n\u003cp\u003e0.023\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" align=\"left\"\u003e\n\u003cp\u003e0.071\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" align=\"left\"\u003e\n\u003cp\u003e0.094\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" align=\"left\"\u003e\n\u003cp\u003eSynergistic\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr style=\"height: 35px;\"\u003e\n\u003ctd style=\"height: 35px;\" align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003eVancomycin-gentamicin\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" align=\"left\"\u003e\n\u003cp\u003e0.002\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" align=\"left\"\u003e\n\u003cp\u003e1.000\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" align=\"left\"\u003e\n\u003cp\u003e1.002\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" colspan=\"2\" align=\"left\"\u003e\n\u003cp\u003eIndifference\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" align=\"left\"\u003e\n\u003cp\u003e0.750\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" align=\"left\"\u003e\n\u003cp\u003e0.375\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" align=\"left\"\u003e\n\u003cp\u003e1.125\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" align=\"left\"\u003e\n\u003cp\u003eIndifference\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr style=\"height: 35px;\"\u003e\n\u003ctd style=\"height: 35px;\" align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003eVancomycin-ciprofloxacin\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" align=\"left\"\u003e\n\u003cp\u003e0.043\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" align=\"left\"\u003e\n\u003cp\u003e0.270\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" align=\"left\"\u003e\n\u003cp\u003e0.313\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" colspan=\"2\" align=\"left\"\u003e\n\u003cp\u003eSynergistic\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" align=\"left\"\u003e\n\u003cp\u003e0.050\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" align=\"left\"\u003e\n\u003cp\u003e0.005\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" align=\"left\"\u003e\n\u003cp\u003e0.055\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" align=\"left\"\u003e\n\u003cp\u003eSynergistic\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr style=\"height: 35px;\"\u003e\n\u003ctd style=\"height: 35px;\" align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003eVancomycin-levofloxacin\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" align=\"left\"\u003e\n\u003cp\u003e0.002\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" align=\"left\"\u003e\n\u003cp\u003e0.750\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" align=\"left\"\u003e\n\u003cp\u003e0.752\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" colspan=\"2\" align=\"left\"\u003e\n\u003cp\u003eSub-synergistic\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" align=\"left\"\u003e\n\u003cp\u003e0.021\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" align=\"left\"\u003e\n\u003cp\u003e0.062\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" align=\"left\"\u003e\n\u003cp\u003e0.083\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" align=\"left\"\u003e\n\u003cp\u003eSynergistic\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr style=\"height: 35px;\"\u003e\n\u003ctd style=\"height: 35px;\" align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003eVancomycin-ofloxacin\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" align=\"left\"\u003e\n\u003cp\u003e0.002\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" align=\"left\"\u003e\n\u003cp\u003e0.500\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" align=\"left\"\u003e\n\u003cp\u003e0.502\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" colspan=\"2\" align=\"left\"\u003e\n\u003cp\u003eSub-synergistic\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" align=\"left\"\u003e\n\u003cp\u003e0.019\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" align=\"left\"\u003e\n\u003cp\u003e0.015\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" align=\"left\"\u003e\n\u003cp\u003e0.034\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" align=\"left\"\u003e\n\u003cp\u003eSynergistic\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr style=\"height: 35px;\"\u003e\n\u003ctd style=\"height: 35px;\" align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003eVancomycin-tobramycin\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" align=\"left\"\u003e\n\u003cp\u003e0.002\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" align=\"left\"\u003e\n\u003cp\u003e1.000\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" align=\"left\"\u003e\n\u003cp\u003e1.002\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" colspan=\"2\" align=\"left\"\u003e\n\u003cp\u003eIndifference\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" align=\"left\"\u003e\n\u003cp\u003e0.019\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" align=\"left\"\u003e\n\u003cp\u003e0.125\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" align=\"left\"\u003e\n\u003cp\u003e0.144\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" align=\"left\"\u003e\n\u003cp\u003eSynergistic\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr style=\"height: 35px;\"\u003e\n\u003ctd style=\"height: 35px;\" align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003eTeicoplanin-levofloxacin\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" align=\"left\"\u003e\n\u003cp\u003e0.251\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" align=\"left\"\u003e\n\u003cp\u003e0.750\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" align=\"left\"\u003e\n\u003cp\u003e1.001\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" colspan=\"2\" align=\"left\"\u003e\n\u003cp\u003eIndifference\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" align=\"left\"\u003e\n\u003cp\u003e0.035\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" align=\"left\"\u003e\n\u003cp\u003e0.150\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" align=\"left\"\u003e\n\u003cp\u003e0.185\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" align=\"left\"\u003e\n\u003cp\u003eSynergistic\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr style=\"height: 35px;\"\u003e\n\u003ctd style=\"height: 35px;\" align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003ePenicillin-colistin\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" align=\"left\"\u003e\n\u003cp\u003e0.129\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" align=\"left\"\u003e\n\u003cp\u003e0.625\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" align=\"left\"\u003e\n\u003cp\u003e0.754\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" colspan=\"2\" align=\"left\"\u003e\n\u003cp\u003eSub-synergistic\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" align=\"left\"\u003e\n\u003cp\u003e0.012\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" align=\"left\"\u003e\n\u003cp\u003e0.375\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" align=\"left\"\u003e\n\u003cp\u003e0.387\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" align=\"left\"\u003e\n\u003cp\u003eSynergistic\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr style=\"height: 35px;\"\u003e\n\u003ctd style=\"height: 35px;\" align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003ePenicillin-ciprofloxacin\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" align=\"left\"\u003e\n\u003cp\u003e0.156\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" align=\"left\"\u003e\n\u003cp\u003e0.250\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" align=\"left\"\u003e\n\u003cp\u003e0.406\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" colspan=\"2\" align=\"left\"\u003e\n\u003cp\u003eSynergistic\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" align=\"left\"\u003e\n\u003cp\u003e0.187\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" align=\"left\"\u003e\n\u003cp\u003e0.024\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" align=\"left\"\u003e\n\u003cp\u003e0.211\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" align=\"left\"\u003e\n\u003cp\u003eSynergistic\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr style=\"height: 35px;\"\u003e\n\u003ctd style=\"height: 35px;\" align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003ePenicillin-levofloxacin\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" align=\"left\"\u003e\n\u003cp\u003e0.004\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" align=\"left\"\u003e\n\u003cp\u003e0.512\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" align=\"left\"\u003e\n\u003cp\u003e0.516\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" colspan=\"2\" align=\"left\"\u003e\n\u003cp\u003eSub-synergistic\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" align=\"left\"\u003e\n\u003cp\u003e0.002\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" align=\"left\"\u003e\n\u003cp\u003e0.500\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" align=\"left\"\u003e\n\u003cp\u003e0.502\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" align=\"left\"\u003e\n\u003cp\u003eSub-synergistic\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr style=\"height: 35px;\"\u003e\n\u003ctd style=\"height: 35px;\" align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003eAmpicillin-colistin\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" align=\"left\"\u003e\n\u003cp\u003e0.375\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" align=\"left\"\u003e\n\u003cp\u003e0.187\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" align=\"left\"\u003e\n\u003cp\u003e0.562\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" colspan=\"2\" align=\"left\"\u003e\n\u003cp\u003eSub-synergistic\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" align=\"left\"\u003e\n\u003cp\u003e0.004\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" align=\"left\"\u003e\n\u003cp\u003e0.250\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" align=\"left\"\u003e\n\u003cp\u003e0.254\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" align=\"left\"\u003e\n\u003cp\u003eSynergistic\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr style=\"height: 35px;\"\u003e\n\u003ctd style=\"height: 35px;\" align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003eAmpicillin-ciprofloxacin\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" align=\"left\"\u003e\n\u003cp\u003e0.008\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" align=\"left\"\u003e\n\u003cp\u003e0.250\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" align=\"left\"\u003e\n\u003cp\u003e0.258\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" colspan=\"2\" align=\"left\"\u003e\n\u003cp\u003eSynergistic\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" align=\"left\"\u003e\n\u003cp\u003e0.002\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" align=\"left\"\u003e\n\u003cp\u003e0.250\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" align=\"left\"\u003e\n\u003cp\u003e0.252\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" align=\"left\"\u003e\n\u003cp\u003eSynergistic\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr style=\"height: 35.7248px;\"\u003e\n\u003ctd style=\"height: 35.7248px;\" align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003eCiprofloxacin-levofloxacin\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35.7248px;\" align=\"left\"\u003e\n\u003cp\u003e0.500\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35.7248px;\" align=\"left\"\u003e\n\u003cp\u003e0.250\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35.7248px;\" align=\"left\"\u003e\n\u003cp\u003e0.750\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35.7248px;\" colspan=\"2\" align=\"left\"\u003e\n\u003cp\u003eSub-synergistic\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35.7248px;\" align=\"left\"\u003e\n\u003cp\u003e0.020\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35.7248px;\" align=\"left\"\u003e\n\u003cp\u003e0.027\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35.7248px;\" align=\"left\"\u003e\n\u003cp\u003e0.047\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35.7248px;\" align=\"left\"\u003e\n\u003cp\u003eSynergistic\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr style=\"height: 35px;\"\u003e\n\u003ctd style=\"height: 35px;\" align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003eRifampin-colistin\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" align=\"left\"\u003e\n\u003cp\u003e0.004\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" align=\"left\"\u003e\n\u003cp\u003e0.281\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" align=\"left\"\u003e\n\u003cp\u003e0.285\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" colspan=\"2\" align=\"left\"\u003e\n\u003cp\u003eSynergistic\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" align=\"left\"\u003e\n\u003cp\u003e0.062\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" align=\"left\"\u003e\n\u003cp\u003e0.031\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" align=\"left\"\u003e\n\u003cp\u003e0.093\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" align=\"left\"\u003e\n\u003cp\u003eSynergistic\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr style=\"height: 35px;\"\u003e\n\u003ctd style=\"height: 35px;\" align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003eVancomycin-ciprofloxacin-Stab21\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" align=\"left\"\u003e\n\u003cp\u003e0.107\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" align=\"left\"\u003e\n\u003cp\u003e0.500\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" align=\"left\"\u003e\n\u003cp\u003e0.607\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" colspan=\"2\" align=\"left\"\u003e\n\u003cp\u003eSub-synergistic\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" align=\"left\"\u003e\n\u003cp\u003e0.001\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" align=\"left\"\u003e\n\u003cp\u003e0.0002\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" align=\"left\"\u003e\n\u003cp\u003e0.002\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" align=\"left\"\u003e\n\u003cp\u003eSynergistic\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003c/tbody\u003e\n\u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cstrong\u003e3.8. Only a few of the tested combinations showed synergy on the eradication of the dual-species biofilm\u003c/strong\u003e\u003cbr /\u003e\n\u003cp\u003eThe efficacy of antimicrobial combinations used to eradicate pre-formed dual-species biofilms of \u003cem\u003eS. aureus\u003c/em\u003e and \u003cem\u003eP. aeruginosa\u003c/em\u003e was not as successful as their ability to inhibit them. As shown in Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003e, no combination had synergistic interaction against both species, and only five combinations were synergistic against \u003cem\u003eS. aureus\u003c/em\u003e within the biofilm. This highlights the challenge resulting from the reduced susceptibility of \u003cem\u003eS. aureus\u003c/em\u003e to antibiotics following biofilm formation. Among the tested pairs, vancomycin-ciprofloxacin showed the strongest synergistic effects (FICI\u0026thinsp;=\u0026thinsp;0.156), suggesting that the vancomycin-ciprofloxacin combination has enhanced efficacy in targeting \u003cem\u003eS. aureus\u003c/em\u003e within the complex biofilm structure. However, the addition of the phage Stab 21 to the ciprofloxacin-vancomycin combination did not improve their synergy (FICI\u0026thinsp;=\u0026thinsp;0.250). Interestingly, the highest concentrations of both antibiotics did not result in synergistic effects with the phage, suggesting that phage efficacy depends on optimal, rather than maximal, antibiotic concentrations for the most favorable outcome (Supplementary Information, Figure S3). Similarly, vancomycin-ofloxacin (FICI\u0026thinsp;=\u0026thinsp;0.199) and vancomycin-levofloxacin (FICI\u0026thinsp;=\u0026thinsp;0.250) also had synergistic effects. Additionally, the rifampicin-colistin combination demonstrated only a sub-synergistic interaction (FICI\u0026thinsp;=\u0026thinsp;0.750). The MBEC values for both species are provided in Supplementary Information (Table S3).\u0026nbsp;\u003c/p\u003e\n\u003cdiv class=\"gridtable\"\u003e\n\u003cdiv class=\"colspec\" align=\"left\"\u003e\u0026nbsp;\u003c/div\u003e\n\u003ctable id=\"Tab6\" border=\"1\"\u003e\u003ccaption\u003e\n\u003cdiv class=\"CaptionNumber\"\u003eTable 7\u003c/div\u003e\n\u003cdiv class=\"CaptionContent\"\u003e\n\u003cp\u003eInteraction of the chosen combinations on the eradication of dual-species\u0026nbsp;biofilms.\u003c/p\u003e\n\u003c/div\u003e\n\u003c/caption\u003e\n\u003cthead\u003e\n\u003ctr\u003e\n\u003cth rowspan=\"2\" align=\"left\"\u003e\n\u003cp\u003eAntibiotics\u003c/p\u003e\n\u003c/th\u003e\n\u003cth colspan=\"4\" align=\"left\"\u003e\n\u003cp\u003e\u003cem\u003eP. aeruginosa\u003c/em\u003e\u003c/p\u003e\n\u003c/th\u003e\n\u003cth colspan=\"9\" align=\"left\"\u003e\n\u003cp\u003e\u003cem\u003eS. aureus\u003c/em\u003e\u003c/p\u003e\n\u003c/th\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eFIC \u003csub\u003ea\u003c/sub\u003e\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eFIC \u003csub\u003eb\u003c/sub\u003e\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003e\u0026sum;FICI\u003c/p\u003e\n\u003c/th\u003e\n\u003cth colspan=\"2\" align=\"left\"\u003e\n\u003cp\u003eInteraction\u003c/p\u003e\n\u003c/th\u003e\n\u003cth colspan=\"2\" align=\"left\"\u003e\n\u003cp\u003eFIC \u003csub\u003ea\u003c/sub\u003e\u003c/p\u003e\n\u003c/th\u003e\n\u003cth colspan=\"2\" align=\"left\"\u003e\n\u003cp\u003eFIC \u003csub\u003eb\u003c/sub\u003e\u003c/p\u003e\n\u003c/th\u003e\n\u003cth colspan=\"2\" align=\"left\"\u003e\n\u003cp\u003e\u0026sum;FICI\u003c/p\u003e\n\u003c/th\u003e\n\u003cth colspan=\"2\" align=\"left\"\u003e\n\u003cp\u003eInteraction\u003c/p\u003e\n\u003c/th\u003e\n\u003c/tr\u003e\n\u003c/thead\u003e\n\u003ctbody\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003eVancomycin-colistin\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.008\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e1.000\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e1.008\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" align=\"left\"\u003e\n\u003cp\u003eIndifference\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" align=\"left\"\u003e\n\u003cp\u003e0.125\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" align=\"left\"\u003e\n\u003cp\u003e1.000\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" align=\"left\"\u003e\n\u003cp\u003e1.125\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" align=\"left\"\u003e\n\u003cp\u003eIndifference\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003eVancomycin-gentamicin\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.002\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e1.000\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e1.002\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" align=\"left\"\u003e\n\u003cp\u003eIndifference\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" align=\"left\"\u003e\n\u003cp\u003e0.125\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" align=\"left\"\u003e\n\u003cp\u003e1.000\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" align=\"left\"\u003e\n\u003cp\u003e1.125\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" align=\"left\"\u003e\n\u003cp\u003eIndifference\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003eVancomycin-ciprofloxacin\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.066\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.500\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.566\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" align=\"left\"\u003e\n\u003cp\u003eSub-synergistic\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" align=\"left\"\u003e\n\u003cp\u003e0.125\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" align=\"left\"\u003e\n\u003cp\u003e0.031\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" align=\"left\"\u003e\n\u003cp\u003e0.156\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" align=\"left\"\u003e\n\u003cp\u003eSynergistic\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003eVancomycin-levofloxacin\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.187\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.750\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.937\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" align=\"left\"\u003e\n\u003cp\u003eSub-synergistic\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" align=\"left\"\u003e\n\u003cp\u003e0.156\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" align=\"left\"\u003e\n\u003cp\u003e0.094\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" align=\"left\"\u003e\n\u003cp\u003e0.250\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" align=\"left\"\u003e\n\u003cp\u003eSynergistic\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003eVancomycin-ofloxacin\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.002\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e1.000\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e1.002\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" align=\"left\"\u003e\n\u003cp\u003eIndifference\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" align=\"left\"\u003e\n\u003cp\u003e0.187\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" align=\"left\"\u003e\n\u003cp\u003e0.012\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" align=\"left\"\u003e\n\u003cp\u003e0.199\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" align=\"left\"\u003e\n\u003cp\u003eSynergistic\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003eVancomycin-tobramycin\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.002\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e1.000\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e1.002\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" align=\"left\"\u003e\n\u003cp\u003eIndifference\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" align=\"left\"\u003e\n\u003cp\u003e0.094\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" align=\"left\"\u003e\n\u003cp\u003e1.000\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" align=\"left\"\u003e\n\u003cp\u003e1.094\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" align=\"left\"\u003e\n\u003cp\u003eIndifference\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003eTeicoplanin-levofloxacin\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.156\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.375\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.531\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" align=\"left\"\u003e\n\u003cp\u003eSub-synergistic\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.008\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" align=\"left\"\u003e\n\u003cp\u003e0.039\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" align=\"left\"\u003e\n\u003cp\u003e0.047\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" align=\"left\"\u003e\n\u003cp\u003eSynergistic\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"1\" align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003ePenicillin-colistin\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.979\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e1.000\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e1.979\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" align=\"left\"\u003e\n\u003cp\u003eIndifference\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" align=\"left\"\u003e\n\u003cp\u003e0.979\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" align=\"left\"\u003e\n\u003cp\u003e1.000\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" align=\"left\"\u003e\n\u003cp\u003e1.979\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" align=\"left\"\u003e\n\u003cp\u003eIndifference\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003ePenicillin-ciprofloxacin\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.126\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.250\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.376\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" align=\"left\"\u003e\n\u003cp\u003eSynergistic\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" align=\"left\"\u003e\n\u003cp\u003e0.125\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" align=\"left\"\u003e\n\u003cp\u003e1.000\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" align=\"left\"\u003e\n\u003cp\u003e1.125\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" align=\"left\"\u003e\n\u003cp\u003eIndifference\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003ePenicillin-levofloxacin\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.500\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e1.000\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e1.500\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" align=\"left\"\u003e\n\u003cp\u003eIndifference\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" align=\"left\"\u003e\n\u003cp\u003e0.979\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" align=\"left\"\u003e\n\u003cp\u003e1.000\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" align=\"left\"\u003e\n\u003cp\u003e1.979\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" align=\"left\"\u003e\n\u003cp\u003eIndifference\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003eAmpicillin-colistin\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.250\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e1.000\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e1.250\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" align=\"left\"\u003e\n\u003cp\u003eIndifference\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" align=\"left\"\u003e\n\u003cp\u003e0.500\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" align=\"left\"\u003e\n\u003cp\u003e1.000\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" align=\"left\"\u003e\n\u003cp\u003e1.500\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" align=\"left\"\u003e\n\u003cp\u003eIndifference\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003eAmpicillin-ciprofloxacin\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.063\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.375\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.438\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" align=\"left\"\u003e\n\u003cp\u003eSynergistic\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" align=\"left\"\u003e\n\u003cp\u003e0.979\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" align=\"left\"\u003e\n\u003cp\u003e1.000\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" align=\"left\"\u003e\n\u003cp\u003e1.979\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" align=\"left\"\u003e\n\u003cp\u003eIndifference\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003eCiprofloxacin-levofloxacin\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e1.250\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.078\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e1.328\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" align=\"left\"\u003e\n\u003cp\u003eIndifference\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" align=\"left\"\u003e\n\u003cp\u003e0.375\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" align=\"left\"\u003e\n\u003cp\u003e1.000\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" align=\"left\"\u003e\n\u003cp\u003e1.375\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" align=\"left\"\u003e\n\u003cp\u003eIndifference\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003eRifampin-colistin\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.979\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e1.000\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e1.979\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" align=\"left\"\u003e\n\u003cp\u003eIndifference\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" align=\"left\"\u003e\n\u003cp\u003e0.250\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" align=\"left\"\u003e\n\u003cp\u003e0.500\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" align=\"left\"\u003e\n\u003cp\u003e0.750\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" align=\"left\"\u003e\n\u003cp\u003eSub-synergistic\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003eVancomycin-ciprofloxacin-Stab21\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.500\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e1.000\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e1.500\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" align=\"left\"\u003e\n\u003cp\u003eIndifference\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" align=\"left\"\u003e\n\u003cp\u003e0.001\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" align=\"left\"\u003e\n\u003cp\u003e0.250\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" align=\"left\"\u003e\n\u003cp\u003e0.250\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" align=\"left\"\u003e\n\u003cp\u003eSynergistic\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003c/tbody\u003e\n\u003c/table\u003e\n\u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv class=\"Section2\"\u003e\u0026nbsp;\u003c/div\u003e\n\u003cdiv class=\"Section2\"\u003e\u003cstrong\u003e3.9. The presence of CDM in the biofilm model altered the susceptibility of the biofilm to antibiotic combinations in biofilm inhibition and eradication assays\u003c/strong\u003e\u003cbr /\u003e\n\u003cp\u003eTable\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e8\u003c/span\u003e presents the response of \u003cem\u003eS. aureus\u003c/em\u003e within dual-species biofilms grown on CDM and treated with the most effective antibiotic combinations at varying concentrations relative to their MBICs (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e). The vancomycin\u0026ndash;colistin combination exhibited reduced efficacy, failing to inhibit \u003cem\u003eS. aureus\u003c/em\u003e in biofilms grown on CDM even when either antibiotic was used at twice its MBIC. In contrast, the MBIC values were lower for other combinations against biofilms on CDMs than on plate, indicating increased antibiotic susceptibility on CDM. The combination of ciprofloxacin- or levofloxacin- with vancomycin, and penicillin with levofloxacin, showed slightly greater efficacy, whereas vancomycin-ofloxacin and teicoplanin-levofloxacin inhibited \u003cem\u003eS. aureus\u003c/em\u003e growth at substantially lower concentrations. None of the single antibiotics tested at their MBICs inhibited \u003cem\u003eS. aureus\u003c/em\u003e growth in the dual-species biofilm in CDM. As shown in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e, treatment of the biofilms formed on the CDM with vancomycin\u0026ndash;ciprofloxacin at their MBIC effectively reduced \u003cem\u003eP. aeruginosa\u003c/em\u003e, but \u003cem\u003eS. aureus\u003c/em\u003e bacteria remained attached to the CDM surface (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e. F). The pH of the biofilm was measured 8.1 after formation on CDM.\u003c/p\u003e\n\u003cdiv class=\"gridtable\"\u003e\n\u003cdiv class=\"colspec\" align=\"left\"\u003e\u0026nbsp;\u003c/div\u003e\n\u003ctable id=\"Tab7\" border=\"1\"\u003e\u003ccaption\u003e\n\u003cdiv class=\"CaptionNumber\"\u003eTable 8\u003c/div\u003e\n\u003cdiv class=\"CaptionContent\"\u003e\n\u003cp\u003eS. aureus response to the selected antibiotic combinations in inhibition of dual-species biofilm on CDM\u003c/p\u003e\n\u003c/div\u003e\n\u003c/caption\u003e\n\u003ctbody\u003e\n\u003ctr\u003e\n\u003ctd rowspan=\"3\" align=\"left\"\u003e\n\u003cp\u003eAntibiotic combinations\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd rowspan=\"3\" align=\"left\"\u003e\n\u003cp\u003eMBIC AB1/AB2\u003c/p\u003e\n\u003cp\u003e(\u0026micro;g/mL)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"14\" align=\"left\"\u003e\n\u003cp\u003eCoefficient by which the MBIC of AB1 (Top Row) and AB2 (Bottom Row) is multiplied\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e2\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e1\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e1\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e1\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.5\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.5\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.5\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.25\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.25\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.25\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.125\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.125\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.125\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.063\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e1\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e2\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e1\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.5\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e1\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.5\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.25\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.5\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.25\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.125\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.25\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.125\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.063\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.125\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003eVancomycin-colistin\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cem\u003e4/16\u003c/em\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e+\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e+\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e+\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e+\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e+\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e+\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e+\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e+\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e+\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e+\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e+\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e+\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e+\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e+\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003eVancomycin-ciprofloxacin\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cem\u003e4/1\u003c/em\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u0026minus;\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u0026minus;\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u0026minus;\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u0026minus;\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u0026minus;\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e+\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e+\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e+\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e+\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e+\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e+\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e+\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e+\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e+\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003eVancomycin-levofloxacin\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cem\u003e43/2.5\u003c/em\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u0026minus;\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u0026minus;\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u0026minus;\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u0026minus;\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u0026minus;\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e+\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e+\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e+\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e+\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e+\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e+\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e+\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e+\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e+\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003eVancomycin-ofloxacin\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cem\u003e64/51\u003c/em\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u0026minus;\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u0026minus;\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u0026minus;\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u0026minus;\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u0026minus;\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u0026minus;\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u0026minus;\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u0026minus;\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u0026minus;\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u0026minus;\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e+\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e+\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e+\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e+\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003ePenicillin-levofloxacin\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cem\u003e4/64\u003c/em\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u0026minus;\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u0026minus;\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u0026minus;\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u0026minus;\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u0026minus;\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e+\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e+\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e+\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e+\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e+\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e+\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e+\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e+\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e+\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003eTeicoplanin-levofloxacin\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cem\u003e72/6\u003c/em\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u0026minus;\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u0026minus;\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u0026minus;\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u0026minus;\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u0026minus;\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u0026minus;\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u0026minus;\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u0026minus;\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u0026minus;\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u0026minus;\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u0026minus;\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u0026minus;\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e+\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e+\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd colspan=\"16\"\u003eColumn \u0026ldquo;\u003cem\u003eMBIC AB1/AB2\u003c/em\u003e\u0026rdquo; indicates MBIC values of antibiotic 1 (AB1) and antibiotic 2 (AB2) when used in combination against \u003cem\u003eS. aureus\u003c/em\u003e in dual-species biofilm without CDM (MBICs values can be found in SI table S2). The coefficients in columns 3\u0026ndash;16 indicate the number by which MBIC values of each antibiotic are multiplied (upper row AB1 and bottom row AB2). For example, in the case of vancomycin-colistin, coefficients 2 (top) and 1 (bottom) indicate that vancomycin (AB1) is used at twofold of its MBIC (2\u0026times;4\u0026thinsp;=\u0026thinsp;8 \u0026micro;g/mL), and colistin (AB2) at onefold of its MBIC (1\u0026times;16\u0026thinsp;=\u0026thinsp;16 \u0026micro;g/mL). \"+\" indicates bacterial growth on MSA; \"\u0026minus;\" indicates no growth.\u003c/td\u003e\n\u003c/tr\u003e\n\u003c/tbody\u003e\n\u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eBiofilm eradication assays for biofilms on CDMs also indicated altered susceptibility of \u003cem\u003eS. aureus\u003c/em\u003e to antibiotic combinations (Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e9\u003c/span\u003e). The vancomycin\u0026ndash;colistin pair exhibited reduced efficacy, requiring higher antibiotic concentrations for biofilm eradication. In contrast, vancomycin-ciprofloxacin and teicoplanin-levofloxacin were slightly more effective at eradicating \u003cem\u003eS. aureus\u003c/em\u003e when the biofilm was formed on CDM. The combination of vancomycin with levofloxacin or ofloxacin achieved significantly greater eradication of \u003cem\u003eS. aureus\u003c/em\u003e in CDM-grown biofilms. These results suggest that the structural properties of CDM may increase antibiotic penetration or activity in biofilms. Single antibiotics failed to eradicate \u003cem\u003eS. aureus\u003c/em\u003e in the dual-species biofilms. The corresponding data for \u003cem\u003eP. aeruginosa\u003c/em\u003e are provided in Supplementary Information (Table S4 and Table S5).\u003c/p\u003e\n\u003cdiv class=\"gridtable\"\u003e\n\u003cdiv class=\"colspec\" align=\"left\"\u003e\u0026nbsp;\u003c/div\u003e\n\u003ctable id=\"Tab8\" border=\"1\"\u003e\u003ccaption\u003e\n\u003cdiv class=\"CaptionNumber\"\u003eTable 9\u003c/div\u003e\n\u003cdiv class=\"CaptionContent\"\u003e\n\u003cp\u003eS. aureus response to the selected antibiotic combination in eradication of dual-species biofilm on CDM\u003c/p\u003e\n\u003c/div\u003e\n\u003c/caption\u003e\n\u003ctbody\u003e\n\u003ctr\u003e\n\u003cth rowspan=\"3\" align=\"left\"\u003e\n\u003cp\u003eAntibiotic combinations\u003c/p\u003e\n\u003c/th\u003e\n\u003cth rowspan=\"3\" align=\"left\"\u003e\n\u003cp\u003eMBEC AB1/AB2\u003c/p\u003e\n\u003cp\u003e(\u0026micro;g/mL)\u003c/p\u003e\n\u003c/th\u003e\n\u003cth colspan=\"11\" align=\"left\"\u003e\n\u003cp\u003eCoefficient by which MBEC of AB1 (Top Row) and AB2 (Bottom Row) is multiplied\u003c/p\u003e\n\u003c/th\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003e2\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003e1\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003e1\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003e1\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003e0.5\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003e0.5\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003e0.5\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003e0.25\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003e0.25\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003e0.25\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003e0.125\u003c/p\u003e\n\u003c/th\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003e1\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003e2\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003e1\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003e0.5\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003e1\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003e0.5\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003e0.25\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003e0.5\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003e0.25\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003e0.125\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003e0.25\u003c/p\u003e\n\u003c/th\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003eVancomycin-colistin\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cem\u003e256/64\u003c/em\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e+\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e+\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e+\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e+\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e+\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e+\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e+\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e+\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e+\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e+\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e+\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003eVancomycin-ciprofloxacin\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cem\u003e256/2\u003c/em\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u0026minus;\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u0026minus;\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u0026minus;\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u0026minus;\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e+\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e+\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e+\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e+\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e+\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e+\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e+\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003eVancomycin-levofloxacin\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cem\u003e320/6\u003c/em\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u0026minus;\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u0026minus;\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u0026minus;\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u0026minus;\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u0026minus;\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u0026minus;\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u0026minus;\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u0026minus;\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u0026minus;\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e+\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e+\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003eVancomycin-ofloxacin\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cem\u003e384/24\u003c/em\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u0026minus;\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u0026minus;\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u0026minus;\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u0026minus;\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u0026minus;\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u0026minus;\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u0026minus;\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u0026minus;\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u0026minus;\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e+\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e+\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003eTeicoplanin-levofloxacin\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cem\u003e16/2.5\u003c/em\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u0026minus;\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u0026minus;\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u0026minus;\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u0026minus;\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u0026minus;\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e+\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e+\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e+\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e+\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e+\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e+\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003c/tbody\u003e\n\u003ctfoot\u003e\n\u003ctr\u003e\n\u003ctd colspan=\"13\"\u003eColumn \u0026ldquo;\u003cem\u003eMBEC AB1/AB2\u003c/em\u003e\u0026rdquo; indicates MBEC values of antibiotic 1 (AB1) and antibiotic 2 (AB2) when used in combination against \u003cem\u003eS. aureus\u003c/em\u003e in dual-species biofilm without CDM (MBECs can be found in SI table S3). The coefficient in columns 3\u0026ndash;13 indicates the number by which MBEC values of each antibiotic are multiplied (upper row AB1 and bottom row AB2). For example, in the case of vancomycin-colistin, coefficients 2 (top) and 1 (bottom) indicate that vancomycin (AB1) is used at twofold of its MBEC (2\u0026times;256 \u0026micro;g/mL), and colistin (AB2) at onefold of its MBEC (1\u0026times;64 \u0026micro;g/mL). \"+\" indicates bacterial growth on MSA; \"\u0026minus;\" indicates no growth.\u003c/td\u003e\n\u003c/tr\u003e\n\u003c/tfoot\u003e\n\u003c/table\u003e\n\u003c/div\u003e\n\u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eThe MIC and MBC results confirmed the sensitivity of both \u003cem\u003eP. aeruginosa\u003c/em\u003e and \u003cem\u003eS. aureus\u003c/em\u003e to almost all the tested antibiotics, which is consistent with previous reports on the same strains [\u003cspan additionalcitationids=\"CR42\" citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. In biofilms, \u003cem\u003eP. aeruginosa\u003c/em\u003e PAO1 is less susceptible to rifampicin, colistin, and gentamicin [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e, \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. \u003cem\u003eS. aureus\u003c/em\u003e, however, showed increased tolerance to all the tested antibiotics. Increased tolerance was expected as biofilm formation reduces bacterial sensitivity to antibiotics by enhancing the mutagenesis, adaptation, and resistance of bacterial species. The observed additional antibiotic tolerance of \u003cem\u003eS. aureus\u003c/em\u003e in dual-species biofilms was attributed to the presence of \u003cem\u003eP. aeruginosa\u003c/em\u003e. Several previous studies reported altered susceptibility of \u003cem\u003eS. aureus\u003c/em\u003e to antibiotics in co-culture with \u003cem\u003eP. aeruginosa\u003c/em\u003e [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. According to previous reports, polysaccharides produced by \u003cem\u003eP. aeruginosa\u003c/em\u003e play a critical role in shielding both species from antibiotics [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. \u003cem\u003eP. aeruginosa\u003c/em\u003e produces three key exopolysaccharides\u0026mdash;alginate, Pel, and Psl\u0026mdash;that form its extracellular matrix, providing both structural support and protection for bacterial cells [\u003cspan additionalcitationids=\"CR48\" citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]. The high tolerance of \u003cem\u003eS. aureus\u003c/em\u003e to antibiotics can also result from the protective effect of other exoproducts of \u003cem\u003eP. aeruginosa\u003c/em\u003e, such as HQNO [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. HQNO suppresses \u003cem\u003eS. aureus\u003c/em\u003e respiration, reducing its susceptibility to aminoglycosides such as tobramycin and gentamycin, and it also protects \u003cem\u003eS. aureus\u003c/em\u003e from vancomycin [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. Consistent with our findings, DeLeon \u003cem\u003eet al\u003c/em\u003e. reported increased tolerance of \u003cem\u003eS. aureus\u003c/em\u003e to gentamicin in cocultures with \u003cem\u003eP. aeruginosa\u003c/em\u003e [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]. However, Trizna \u003cem\u003eet al.\u003c/em\u003e reported the high efficiency of aminoglycosides, including gentamicin, in the eradication of both species in mixed biofilms [\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e]. We observed high tolerance of \u003cem\u003eS. aureus\u003c/em\u003e to penicillin in dual-species biofilms, which can be explained by the effect of the β-lactamase enzyme produced by \u003cem\u003eP. aeruginosa\u003c/em\u003e [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]. Orazi \u003cem\u003eet al\u003c/em\u003e reported that cell-free cultures of \u003cem\u003eP. aeruginosa\u003c/em\u003e can increase the sensitivity of \u003cem\u003eS. aureus\u003c/em\u003e biofilms to FQs while increasing their tolerance to other antibiotic classes, such as glycopeptides and β-lactams [\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e]. However, our results and those of Trizna et al., showed higher tolerance of \u003cem\u003eS. aureus\u003c/em\u003e to FQs in multi-species biofilms than in mono-species biofilms [\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e]. \u003cem\u003eS. aureus\u003c/em\u003e showed the lowest tolerance to levofloxacin and rifampicin in our dual-species biofilms, although neither antibiotic achieved complete biofilm eradication. Hiltunen \u003cem\u003eet al\u003c/em\u003e. (2019) reported a higher efficiency of levofloxacin than vancomycin against \u003cem\u003eS. aureus\u003c/em\u003e biofilms in prosthetic joint infections [\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e]. The variation in the response of \u003cem\u003eS. aureus\u003c/em\u003e to antibiotics reported in different studies may be due to strain-specific differences and biofilm growth conditions.\u003c/p\u003e \u003cp\u003eAmong the combined antibiotics, FQs including ciprofloxacin, levofloxacin, and ofloxacin, combined with glycopeptides, vancomycin and teicoplanin, demonstrated significant efficacy against \u003cem\u003eS. aureus\u003c/em\u003e in dual-species biofilms. Glycopeptides, such as vancomycin and teicoplanin, inhibit cell wall peptidoglycan synthesis in \u003cem\u003eS. aureus\u003c/em\u003e [\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e]. FQs, on the other hand, target bacterial DNA synthesis by inhibiting DNA gyrase and topoisomerase IV [\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e]. Moreover, FQs are particularly effective against \u003cem\u003eP. aeruginosa\u003c/em\u003e because of their ability to penetrate EPS and target sessile cells, even in mature biofilms [\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e]. Thus, the synergistic interaction of FQs and glycopeptides against dual-species biofilms is not surprising. Kamble \u003cem\u003eet al\u003c/em\u003e. reported synergistic interactions between ciprofloxacin and vancomycin against clinical strains of \u003cem\u003eS. aureus\u003c/em\u003e biofilms and their persister cells [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. The synergy between FQs and glycopeptides in inhibiting and eradicating \u003cem\u003eS. aureus\u003c/em\u003e within the dual-species biofilm may also be attributed to early elimination of \u003cem\u003eP. aeruginosa\u003c/em\u003e by FQs and thereby loss of its protective effect on \u003cem\u003eS. aureus\u003c/em\u003e [\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eReports on the efficacy of combinations (FQs) and glycopeptides are variable among different studies. For example, in the study by Weber \u003cem\u003eet al.\u003c/em\u003e, the combination of ofloxacin and vancomycin was neither synergistic nor antagonistic and did not result in improved outcomes compared with ofloxacin alone against \u003cem\u003eS. aureus\u003c/em\u003e [\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e]. Consistent with our findings, many other \u003cem\u003ein vitro\u003c/em\u003e studies using resistant clinical \u003cem\u003eS. aureus\u003c/em\u003e isolates have shown strong synergy for drug pairs such as vancomycin-gentamicin, ciprofloxacin-vancomycin, and vancomycin-tobramycin [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e]. Moreover, \u003cem\u003ein vitro\u003c/em\u003e data is not always supported by preclinical and clinical studies. Rifampicin-based combinations are the combination therapy most approved for preclinical models of implant-associated infections [\u003cspan additionalcitationids=\"CR59\" citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e]. Compared with monotherapy, rifampin with glycopeptides such as vancomycin has synergistic interactions and reduced resistance against prosthetic joint infections and osteomyelitis [\u003cspan additionalcitationids=\"CR59\" citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e]. Although many \u003cem\u003ein vitro\u003c/em\u003e studies have indicated the potential of FQ\u0026ndash;glycopeptide combinations against both sensitive and tolerant \u003cem\u003eS. aureus\u003c/em\u003e, supporting \u003cem\u003ein vivo\u003c/em\u003e studies is limited.\u003c/p\u003e \u003cp\u003ePhage\u0026ndash;antibiotic combination treatment, supported by several successful clinical cases, was also evaluated in this study [\u003cspan additionalcitationids=\"CR62\" citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e]. The phage Stab 21 restored the susceptibility of \u003cem\u003eS. aureus\u003c/em\u003e to ciprofloxacin and vancomycin, enhancing the inhibition of \u003cem\u003eS. aureus\u003c/em\u003e biofilm formation within the dual-species biofilm. However, the effectiveness of phage\u0026ndash;antibiotic combinations against \u003cem\u003eS. aureus\u003c/em\u003e in preformed biofilms remains low. Combining Stab 21 with both ciprofloxacin and vancomycin demonstrated that the phage requires an optimal antibiotic concentration for optimal eradication, as higher antibiotic concentrations did not yield superior biofilm eradication (SI, Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Several rationales for phage\u0026ndash;antibiotic synergy have been proposed, including bacterial invasion by targeting several bacterial receptors and associated genetic trade-offs, cell morphological changes, phage-mediated enhancement of antibiotic penetration via depolymerase production, and stress-induced phage production [\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e, \u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e]. However, antibiotics targeting, e.g., protein synthesis machinery, may suppress phage replication, as phages rely on bacterial ribosomes and other protein synthesis machinery. Nevertheless, in this study, ciprofloxacin was effective when combined with Stab 21, with the critical factor being the identification of a sublethal concentration allowing phage propagation. Our findings also highlight the strong influence of the environmental context on phage infectivity. The inability of Stab 21 to infect \u003cem\u003eS. aureus\u003c/em\u003e in liquid culture despite successful infection on solid media during preliminary screening may reflect altered expression of surface receptors or enzymatic activity across planktonic and surface-attached states [\u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e]. Thus, phage lytic activity should be considered not only as a phage characteristic but a result of interplay between phage, bacterial host, and environmental factors, including biofilms.\u003c/p\u003e \u003cp\u003eTo our surprise, the presence of CDM, in most of the tested combinations, reduced the tolerance of biofilms to antibiotic combinations. Previous reports have shown exacerbated antibiotic tolerance of \u003cem\u003eS. aureus\u003c/em\u003e and \u003cem\u003eP. aeruginosa\u003c/em\u003e by EPS and the surrounding human-derived matrix (HDM) [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]. Antibiotic penetration into biofilms is significantly influenced by the physicochemical properties (e.g., pH and surface charge) of both antibiotics and the EPS of biofilms [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]. The biofilm pH was ~\u0026thinsp;5.8, which increased to ~\u0026thinsp;8.1 when the biofilm formed on CDM. The alkaline shift could partially explain the increased susceptibility of \u003cem\u003eS. aureus\u003c/em\u003e to some antibiotics under these conditions.\u003c/p\u003e \u003cp\u003eColistin and vancomycin have isoelectric points (PIs) of approximately 10 and 8.3, respectively, which makes them slightly positive at pH values of 6\u0026ndash;8 [\u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e, \u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e68\u003c/span\u003e]. The PI of teicoplanin is approximately 5.5 and can be assumed to have a neutral charge at pH 6 and a small negative charge at pH 8 [\u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e69\u003c/span\u003e]. The rest of the tested antibiotics tested against biofilms on CDM have a PI of approximately 7, which might make them slightly positive at pH 6 and negative at pH 8 [\u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e70\u003c/span\u003e, \u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e71\u003c/span\u003e]. Bacterial cell surfaces and biofilms typically possess a net negative charge due to the abundance of carboxyl, amino, and phosphate groups [\u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e72\u003c/span\u003e, \u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e73\u003c/span\u003e]. During biofilm maturation, particularly in strong biofilm formers such as \u003cem\u003eS. aureus\u003c/em\u003e, this negative surface potential increases, creating a substantial barrier to the diffusion of positively charged molecules [\u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e73\u003c/span\u003e]. Thus, it can be assumed that more negatively charged combinations, such as teicoplanin-levofloxacin, resulted in greater matrix penetration. Another study reported the reduced efficacy of positively charged gentamicin in coagulated wound-like matrices (WLMs) [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]. The effect of CDM on response of biofilms to antibiotics need to be further investigated in our future studies.\u003c/p\u003e \u003cp\u003eOverall, our study demonstrated that interspecies interaction of bacteria alters their response to antibiotics. Our results further highlighted the complex response of bacterial species to combined antibiotics in inhibition and eradication of multi-species biofilm, as the inhibitory combinations were not necessarily efficient in eradication of biofilm. The combination of glycopeptides and FQs may be a promising candidate for treatment of multi-species biofilm but remains to be confirmed by in vivo and clinical studies. Compared with phage or antibiotic treatment alone, the addition of phage Stab 21 together with ciprofloxacin, vancomycin, or both, was significantly more effective at preventing the formation of \u003cem\u003eS. aureus\u003c/em\u003e biofilms in both the mono and dual-species models, suggesting the high potential of phage therapy in treatment of chronic infections. Nevertheless, further \u003cem\u003ein vitro\u003c/em\u003e, \u003cem\u003ein vivo\u003c/em\u003e, and clinical studies are needed to establish viable treatment recommendations against multi-species biofilms.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cdiv class=\"DefinitionList\"\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eAMR\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eAntimicrobial resistance\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eATCC\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eAmerican Type Culture Collection\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eCDM\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eCell-derived matrix\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eCF\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eCystic fibrosis\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eCFU\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eColony-forming units\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eDMEM\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eDulbecco's modified eagle medium\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eECM\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eExtracellular matrix\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eEPS\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eExtracellular polymeric substances\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eFICI\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eFractional inhibitory concentration index\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eLA\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eLysogeny agar\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eLB\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eLysogeny broth\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eMBC\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eMinimum bactericidal concentration\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eMBEC\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eMinimum biofilm eradication concentration\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eMBIC\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eMinimum biofilm inhibitory concentration\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eMCA\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eMacConkey agar\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eMIC\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eMinimum inhibitory concentration\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eMRSA\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eMethicillin-resistant Staphylococcus aureus\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eMSA\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eMannitol salt agar\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eOD\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eOptical density\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003ePBS\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ePhosphate-buffered saline\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003ePFU\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ePlaque-forming units\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eSEM\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eScanning electron microscopy\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eTSA\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eTryptic soy agar\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eTSB\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eTryptic soy broth.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eDeclarations Ethics approval\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable. No human or animal subjects were used in this study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMost data generated in this study are included in this article. Other raw data will be available upon reasonable requests from the corresponding author.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDeclaration of competing interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing financial interests and personal relationships with other people or organizations that could inappropriately influence or bias the work reported in this paper.\u0026nbsp;\u003c/p\u003e\n\u003ch1\u003eFunding\u003c/h1\u003e\n\u003cp\u003eThis work was supported by the Research Council of Finland [grant number 361647; and grant number 362584]; and PROFI7 funding to the University of Helsinki. Moreover, the research was supported by the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation program (ERC CoG), [grant number 101001016] and the Otto A. Malm Foundation.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors’ contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eParia Mojarrad:\u0026nbsp;Writing – original draft, Methodology,\u0026nbsp;Investigation, Visualization, Software, Investigation, Formal analysis, Data curation. Hanna Tiainen:\u0026nbsp;Writing – original draft, Writing, Methodology, Visualization, Software, Investigation, Formal analysis. Päivi Tammela:\u0026nbsp;Methodology, Validation, Funding acquisition, Writing – review \u0026amp; editing. Timo Laaksonen: Methodology, Validation, Supervision, Formal analysis, Funding acquisition, Writing – review \u0026amp; editing, Resources, Project administration. Saija J. Kiljunen:\u0026nbsp;Methodology, Conceptualization, Validation, Writing – review \u0026amp; editing, Funding acquisition. Zahra Gounani:\u0026nbsp;Methodology, Conceptualization, Validation, Supervision, Formal analysis, Funding acquisition, Writing – review \u0026amp; editing, Resources, Project administration.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors thank Kai Puhakainen, Alexandra Correia, and Jéssica Ferreira Amorim for their technical support. Authors sincerely acknowledge the Helsinki Bioimaging (HBI) the research infrastructure platforms of the Helsinki Institute of Life Science (HiLIFE) of the University of Helsinki. Moreover, Precision Phage Ltd (Jyväskylä, Finland) is acknowledged for providing phage PA8P1.\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eLiu HY, Prentice EL, Webber MA. 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Front Bioeng Biotechnol. 2021;9. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3389/fbioe.2021.643722\u003c/span\u003e\u003cspan address=\"10.3389/fbioe.2021.643722\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLavoie T, Daffinee KE, Vicent ML, LaPlante KL. \u003cem\u003eStaphylococcus\u003c/em\u003e biofilm dynamics and antibiotic resistance: insights into biofilm stages, zeta potential dynamics, and antibiotic susceptibility. Microbiol Spectr. 2025. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1128/spectrum.02915-24\u003c/span\u003e\u003cspan address=\"10.1128/spectrum.02915-24\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bmc-microbiology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"mcro","sideBox":"Learn more about [BMC Microbiology](http://bmcmicrobiol.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/mcro","title":"BMC Microbiology","twitterHandle":"#bmcmicrobiology","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Antimicrobial combination, dual-species biofilm, Staphylococcus aureus, Pseudomonas aeruginosa, antimicrobial resistance (AMR), bacterial interaction, bacteriophage therapy","lastPublishedDoi":"10.21203/rs.3.rs-8986145/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8986145/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eTreating polymicrobial biofilms often requires the combination of antibiotics and the participating species play a major role in the response of these bacteria to antimicrobial agents. \u003cem\u003eS. aureus\u003c/em\u003e and \u003cem\u003eP. aeruginosa\u003c/em\u003e are among the most common species found in polymicrobial biofilms. We used in-house dual-species biofilms of \u003cem\u003eS. aureus\u003c/em\u003e and \u003cem\u003eP. aeruginosa\u003c/em\u003e, to evaluate their response to antibiotics, combinations of antibiotics and selected antibiotics‒phage combinations. The interaction of antibiotic combinations on the inhibition and eradication of biofilms were assessed by calculating the fractional inhibitory concentration index (FICI). Coculture of \u003cem\u003eP. aeruginosa\u003c/em\u003e and \u003cem\u003eS. aureus\u003c/em\u003e altered their antibiotic sensitivity and \u003cem\u003eS. aureus\u003c/em\u003e became highly tolerant to antibiotics. Most antibiotic combinations have synergistic effects on biofilm inhibition. The combinations of vancomycin‒ofloxacin and vancomycin‒ciprofloxacin had FICI values of 0.034 and 0.055 against \u003cem\u003eS. aureus\u003c/em\u003e respectively. Combinations of glycopeptide and fluoroquinolone antibiotics also showed synergistic interactions in biofilm eradication. The addition of phage Stab 21 restored the efficacy of ciprofloxacin and vancomycin against \u003cem\u003eS. aureus\u003c/em\u003e in dual-species biofilms, reducing their biofilm inhibitory concentration to 0.5 \u0026micro;g/mL from 33 \u0026micro;g/mL for ciprofloxacin and \u0026gt;\u0026thinsp;2048 \u0026micro;g/mL for vancomycin. In summary, we describe the susceptibility profile of \u003cem\u003eS. aureus\u003c/em\u003e to combination treatments within a dual-species biofilm and report effective antibiotic\u0026ndash;antibiotic and phage\u0026ndash;antibiotic combinations against antibiotic-tolerant \u003cem\u003eS. aureus\u003c/em\u003e.\u003c/p\u003e","manuscriptTitle":"Synergistic Antibiotic‒Antibiotic and Antibiotic‒Phage Combinations Against Biofilm-Associated Staphylococcus aureus in Dual-Species Biofilms","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-05-04 12:37:29","doi":"10.21203/rs.3.rs-8986145/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"editorInvitedReview","content":"","date":"2026-05-13T13:25:45+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"258683366259962792921379608387415478372","date":"2026-05-11T07:59:54+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"164744364493162270727767586281540207814","date":"2026-05-06T14:48:26+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-04-28T23:32:25+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"263158972822463477466966007282864251277","date":"2026-04-25T15:01:48+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-04-23T08:45:02+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2026-04-14T10:57:16+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-03-02T14:16:56+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-03-02T14:06:38+00:00","index":"","fulltext":""},{"type":"submitted","content":"BMC Microbiology","date":"2026-02-27T09:27:36+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-microbiology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"mcro","sideBox":"Learn more about [BMC Microbiology](http://bmcmicrobiol.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/mcro","title":"BMC Microbiology","twitterHandle":"#bmcmicrobiology","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"84678b5a-3564-48dd-9808-88d18b58713f","owner":[],"postedDate":"May 4th, 2026","published":true,"recentEditorialEvents":[{"type":"editorInvitedReview","content":"","date":"2026-05-13T13:25:45+00:00","index":75,"fulltext":""},{"type":"reviewerAgreed","content":"258683366259962792921379608387415478372","date":"2026-05-11T07:59:54+00:00","index":74,"fulltext":""},{"type":"reviewerAgreed","content":"164744364493162270727767586281540207814","date":"2026-05-06T14:48:26+00:00","index":73,"fulltext":""}],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-05-04T12:37:29+00:00","versionOfRecord":[],"versionCreatedAt":"2026-05-04 12:37:29","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8986145","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8986145","identity":"rs-8986145","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}