Triclosan alters biofilm structures and confers antibiotic tolerance in Staphylococcus aureus using multiple regulatory pathways

Triclosan alters biofilm structures and confers antibiotic tolerance in Staphylococcus aureus using multiple regulatory pathways | 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 Article Triclosan alters biofilm structures and confers antibiotic tolerance in Staphylococcus aureus using multiple regulatory pathways Kim Hardie, Dean Walsh, Andrea Salzer, Parvati Iyer, Christiane Wolz, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3954016/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted You are reading this latest preprint version Abstract The biocide triclosan is used extensively in both household and hospital settings. The chronic exposure to the biocide occurring in individuals that use triclosan-containing products results in low levels of triclosan present in the human body that has been linked to induction of antibiotic tolerance and altered biofilm formation. Here we aimed to unravel the molecular mechanisms involved in triclosan-induced antibiotic tolerance and biofilm formation in Staphylococcus aureus . Triclosan treatment prior to planktonic exposure to bactericidal antibiotics resulted in 1,000 fold higher viable cell counts compared to non-pretreated cultures. Triclosan pretreatment also protected S. aureus biofilms against otherwise lethal doses of antibiotics as shown by live/dead cell staining and viable cell counting. Triclosan mediated antibiotic tolerance in S. aureus biofilms required an active stringent response because biofilms of a pppGpp 0 strain were not protected from antibiotic killing. Incubation of S. aureus with triclosan also altered biofilm structure due to SarA-mediated overproduction of the polysaccharide intercellular adhesin (PIA) in the biofilm matrix. Thus, physiologically relevant concentrations of triclosan can trigger (p)ppGpp dependent antibiotic tolerance as well as SarA dependent biofilm formation. Biofilms Antibiotic resistance Antibiotic tolerance Biocides Triclosan Staphylococcus aureus stringent response SarA polysaccharide intercellular adhesin Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Importance The prevalent bacterium Staphylococcus aureus infects skin lesions and indwelling devices, and this can cause sepsis with 33% mortality. Intrinsic to this is the formation of co-ordinated communities (biofilms) protected by a polysaccharide coat. S. aureus is increasingly difficult to eradicate due to its antibiotic resistance. Protection against Methicillin Resistant S. aureus (MRSA) includes pre-hospital admission washing with products containing biocides. The biocide triclosan is the predominant antibacterial compound in sewage in Ontario due to its use in household and hospital settings. Levels of triclosan accumulate with exposure in humans. The significance of our research is in identifying the mechanisms triggered by exposure of S. aureus to physiological levels of triclosan that go on to raise the tolerance of S. aureus to antibiotics and promote the formation of biofilms. This understanding will inform future criteria used to determine effective antimicrobial treatments. Introduction Biofilms are surface attached communities of bacteria enclosed in an exopolymeric matrix. For the pathogen Staphylococcus aureus , this matrix is composed of surface proteins ( 1 ), polysaccharide intercellular adhesin (PIA) ( 2 ), and extracellular DNA (eDNA) ( 3 , 4 ). Biofilms can act as reservoirs of antibiotic tolerance and use numerous mechanisms to withstand antimicrobial treatment, including reduced penetration of antimicrobials into the biofilm matrix ( 5 ), and a generally slow growth rate ( 6 ). This reduced rate of growth serves to protect bacteria by diminishing the efficacy of the majority of antimicrobials. The enduring resilience of biofilms has made them a severe clinical concern, particular with regards to chronic infections caused by biofilm forming bacteria such as S. aureus ( 7 ). Antibiotic tolerance is the process by which an entire bacterial population can survive transient exposure to antibiotics that would otherwise be lethal ( 8 , 9 ). Antibiotic tolerance distinguishes itself from antibiotic resistance in numerous ways. For example, resistant bacteria may need higher concentrations of an antimicrobial to achieve bacterial killing, tolerant bacteria require a longer exposure time to an antimicrobial to provide the same level of killing achieved against susceptible bacteria ( 8 ). Additionally, resistant bacteria are typically protected against a single antibiotic or a small group of closely related antibiotics, whilst tolerant bacteria can typically better withstand a broad array of antimicrobials ( 8 , 9 ). The presence of antibiotic tolerant bacteria leads to flawed antimicrobial treatments, risking recurrent infection in patients ( 10 , 11 ). Antibiotic tolerance in S. aureus is becoming increasingly relevant clinically, as exemplified by 6%-43% of S. aureus clinical strains being vancomycin tolerant ( 12 ). Drug tolerant S. aureus can cause prolonged fevers and extended bacteraemia duration, increased treatment failures and even increased mortality rates ( 13 , 14 ). There is a broad range of molecular strategies employed by S. aureus to facilitate antibiotic tolerance ( 15 ). The stringent response is one such strategy and is conserved among most bacterial species as a means to combat nutritional deficiencies ( 16 , 17 ). During the stringent response the alarmones ppGpp and pppGpp (collectively known as (p)ppGpp) are synthesised from ATP and GTP (pppGpp) or GDP (ppGpp) ( 18 ). In S. aureus , alarmone synthesis is driven by three alarmone synthetases: two small alarmone synthetases; RelP and RelQ, and a larger protein Rel. Rel is bifunctional and equipped with a synthetase domain for alarmone synthesis and a hydrolase domain for the purpose of alarmone degradation. Hydrolysis of these alarmones in S. aureus is essential for survival, as accumulation of (p)ppGpp results in cell death ( 19 , 20 ). Rel, RelP, and RelQ are triggered by different inducing stresses, including various antibiotics. Numerous studies have used the antibiotic mupirocin, an antibiotic that induces amino acid starvation, to induce Rel activity ( 19 , 21 , 22 ). RelP and RelQ expression has been triggered by exposure to the cell wall targeting antibiotics ampicillin, oxacillin, and vancomycin ( 21 , 23 ). Once the stringent response has been triggered, (p)ppGpp orchestrates global changes in gene expression, with steep downregulation of genes associated with proliferative functions, such as protein synthesis and DNA replication ( 24 , 25 ). As bactericidal antibiotics disrupt the function of active targets, the metabolic shutdown and quiescence induced by the stringent response renders these drugs largely ineffectual ( 26 ). The synthetic biocide triclosan inhibits type II fatty acid synthesis (FASII) at the enoylacyl carrier protein reductase (FabI) step( 27 , 28 ). At high concentrations, triclosan provokes membrane permeability and non-specific membrane damage (29). In hospitals, triclosan is used as an MRSA decolonisation therapy (30), antimicrobial hand wash (30), antiseptic ointment (31), and is impregnated into surgical sutures (32) and urinary catheters (33). Triclosan is also widely used in domestic settings, being found in many household products including soaps, toothpastes (34), cosmetics and laundry detergents (35). This widespread use of triclosan means that both people and environments are chronically exposed to the biocide. Absorption of triclosan from triclosan containing toothpastes (TCT) is high, with triclosan blood concentration increasing from 0.81 ng/mL to 296 ng/mL after 14 days of TCT use (36). The average urine concentration of triclosan in patients exposed to the biocide during their hospital stay was 245 ng/mL, with concentrations reaching as high as 505 ng/mL (34). Previous studies have suggested that pretreatment with low levels of triclosan can induce antibiotic tolerance in both Escherichia coli ( 37 , 38 ) and S. aureus ( 37 ) to multiple different antibiotics. Moreover, insufficiently dosed treatments can alter biofilm formation ( 39 ). S. aureus has been shown to grow thicker biofilms in the presence of sub-MIC mupirocin ( 40 ), clindamycin ( 41 ), and β-lactams ( 42 ) due to increased eDNA release, whilst vancomycin exposure caused increased biofilm formation due to elevated levels of both PIA and eDNA in the biofilm matrix ( 43 ). Salicylic acid, the active component of aspirin, has been shown to increase the production of PIA in S. aureus biofilms ( 44 ). Although triclosan has not yet been observed to alter the biofilm formation of S. aureus , the biocide does stimulate cellulose production in established Salmonella typhimirium biofilms ( 45 ). As biocides are globally accessible and both biofilm formation and antibiotic tolerance have been associated with antibiotic treatment failure, augmentation or induction of these processes by biocide exposure could be a serious clinical concern. This study aimed to investigate how triclosan exposure affects not just planktonic S. aureus , but also S. aureus biofilms. We show that low levels of triclosan can induce antibiotic tolerance via the stringent response in S. aureus biofilms, thereby increasing their resilience against antibiotic killing. In addition, triclosan exposure throughout biofilm formation led to a significant increase in biofilm polysaccharide production in a SarA-dependent manner. Overall, low levels of triclosan, consistent with those found accumulating within the human body, can trigger multiple molecular mechanisms to drastically alter the phenotype of S. aureus biofilms, protecting them against antibiotics and possibly additional threats. Materials and Methods Bacterial strains and culture conditions S. aureus strains (see Table 1 ) were grown at 37°C on brain heart infusion (BHI) agar plates for 16–20 hours. For growth in liquid media S. aureus was grown at 37°C in BHI broth with shaking at 200 rpm unless stated otherwise. Table 1 S. aureus strains used in this study Strain Description Reference HG001 Derivative of NCTC8325; rsbU repaired, tcaR defective, carries prophages Φ11, Φ12, and Φ13. ( 46 ) HG001 (p)ppGpp 0 HG001 rel/relP/relQ triple mutant (Δ rel syn , Δ relP syn , Δ relQ syn ) ( 21 ) HG001 sarA HG001 sarA :: Bursa aurealis ( erm ) ( 24 ) Antibiotic susceptibility testing Antibiotics tested - Antimicrobials used for these experiments were obtained from Sigma-Aldrich: triclosan (Igrasan), ciprofloxacin, vancomycin, rifampicin. Minimum inhibitory concentration (MIC) determination − 96 well microtitre plates were inoculated with S. aureus strains at an optical density of 600 nm (OD 600 ) of 0.05 in Mueller-Hinton broth. Minimum inhibitory concentrations (MICs) were determined by the broth microdilution method in accordance with EUCAST guidelines ( 47 ). Minimum biofilm eradication concentration (MBEC) determination - MBEC experiments were conducted by inoculating 96 well microtitre plates with S. aureus at an OD 600 of 0.05 in Mueller-Hinton broth and incubating for 24 hours at 37°C to allow biofilm formation. Following this, a 2-fold dilution series of antibiotics was added to wells and biofilms incubated in Mueller-Hinton broth with antibiotics for a further 24 hours at 37°C. After incubation, biofilms were removed from wells by vigorous scraping with pipette tips and sonicating at 30 kHz for 1 minute in sonication bath. Disrupted biofilms were then spotted out onto Mueller-Hinton agar plates and the MBEC defined as the minimum antibiotic concentration at which viable colonies are not detected. Time-kill assays S. aureus strains at OD 600 0.01 were inoculated into either untreated BHI broth or BHI broth supplemented with triclosan (500 ng/mL; 0.25x MIC). For conditions requiring fatty acid supplementation, 500 µM oleic acid and 0.1% v/v Brij 58 for solubilisation of oleic acid was also added. Cultures were then incubated at 37°C in shaking conditions (200 rpm) for a period of 30 minutes to allow for acclimatisation to biocide and/or fatty acid supplementation. Conditions that did not include triclosan or oleic acid were also incubated for 30 minutes. Following triclosan pretreatment, the first samples are taken from all conditions to determine initial OD 600 and colony forming units (CFUs). Then, inhibitory concentrations of antibiotics were added to relevant conditions. Throughout this study the antibiotics ciprofloxacin (1 µg/mL), vancomycin (2 µg/mL), rifampicin (40 ng/mL) were used. This resulted in four conditions per strain being present in each experiment, consisting of untreated S. aureus , biocide exposed, antibiotic treated, biocide exposed + antibiotic treated. Cultures were then returned to the incubator and samples were taken every hour for 6–8 hours depending on the experiment. Each hour, CFUs were determined and the OD 600 recorded to determine viability and growth, respectively. Cell viability was determined by normalising the CFU at each time point against the CFU of the initial time point (following pretreatment, but just before antibiotic addition). This corrected for the increased growth of untreated conditions relative to triclosan exposed conditions that occurred during the 30 minute pretreatment step. Biofilm imaging Biofilm preparation - Overnight cultures of S. aureus were diluted in BHI to OD 600 0.05 and loaded into µ-slide 8-well glass bottomed chambers (ibidi, glass bottom). 500 ng/mL triclosan, was added to relevant wells, allowing biofilms to form in the presence of triclosan. For conditions requiring fatty acid supplementation, 500 µM oleic acid and 0.1% Brij 58 was also added. Chambers were then placed in a 37°C incubator for 48 hours in static conditions. Following this incubation period, growth medium was removed from the biofilms and replaced with fresh BHI growth medium. Live/dead staining - To investigate antibiotic tolerance either 4096 µg/mL ciprofloxacin, 2048 µg/mL vancomycin or 2048 µg/mL rifampicin were added to wells for 3 hours. Next, live/dead staining was carried out using 6 µM syto 9 and 30 µM propidium iodide (BacLight, Molecular Probes). Biofilms were imaged using a Zeiss LSM 700 compact confocal laser scanning microscope using the 40x objective and appropriate fluorescence settings (syto 9 = 488 nm laser, propidium iodide = 555 nm laser). Staining of biofilm components - To examine biofilm structure, the nucleic acid stain 4',6-diamidino-2-phenylindole (DAPI) was added at a final concentration of 10 µg/mL and the polysaccharide stain fluorescein-conjugated wheat germ agglutinin (WGA) (Invitrogen™) was added at a final concentration of 10 µg/mL. Biofilms were imaged using a Zeiss LSM 700 compact confocal laser scanning microscope using the 40x objective and appropriate fluorescence settings (DAPI = 405 nm laser, fluorescein = 488 nm laser). Quantification using Comstat2 software - Biomass of live cells, dead cells, and stained matrix polysaccharide was analysed using Comstat2 software ( 48 ). Biofilm characterisation Biofilm preparation − 0.5 mL of BHI was added to 24 well plates and inoculated with OD 600 0.05 S. aureus . Wells were treated with either 500 ng/mL triclosan, 500 µM oleic acid solubilised in 0.1% Brij 58, or a combination of both triclosan and solubilised oleic acid. Biofilms were grown statically at 37°C for 48 hours. For biofilm experiments including antibiotics, 4096 µg/mL ciprofloxacin, 2048 µg/mL vancomycin or 2048 µg/mL rifampicin were added to wells for the last 12 hours of biofilm incubation. Following incubation, growth medium was removed and biofilms washed with PBS. Calcofluor white staining to quantify polysaccharide – Calcofluor white staining was carried out as previously described ( 49 ) with minor alterations. 200 µL of calcofluor white (1 mg/mL in dH 2 O) was added to wells and incubated in the dark for 1 hour. Calcofluor white was then removed and biofilms washed with PBS to remove unbound calcofluor white. 200 µL of 96% ethanol was then added to solubilise biofilm-bound calcofluor white. Fluorescence intensity was then measured using the 360 nm excitation filter and 460 nm emission filter. CFU determination – PBS was added to wells and biofilms were sonicated at 30 kHz for 1 minute, diluted, and CFUs determined. For experiments involving antibiotics, the % viability of the biofilm was defined as the CFU of the antibiotic treated sample (for instance WT + cip or WT T + cip) divided by the CFU of the corresponding control (WT untreated or WT T). This corrected for inherent differences in cell number when comparing an untreated S. aureus biofilm to a triclosan exposed S. aureus biofilm. Results Triclosan induces antibiotic tolerance towards ciprofloxacin and vancomycin, but not rifampicin in planktonically grown S. aureus . Triclosan was added to planktonic S. aureus cultures at a concentration of 500 ng/mL (5× MIC HG001; Table S1 ), corresponding to physiologically relevant concentrations of triclosan found in the urine of users of triclosan-containing products ( 50 ). Triclosan pretreatment provided protection from inhibitory concentrations of ciprofloxacin (4× MIC) (Fig. 1 B) and vancomycin (Fig. 1 D). Cell viability in triclosan-exposed (T + C, T + V) conditions remained relatively constant throughout the time-kill assays, whilst conditions treated with ciprofloxacin (C) or vancomycin (V) alone displayed a sharp decrease in cell viability over time. Of note, antibiotic treatment did not result in a concomitant decrease in OD, indicating that bacteria are not lysed under these conditions. Triclosan induced protection resulted in a 100-fold increase in cell survival in the presence of ciprofloxacin, a DNA gyrase inhibitor, by 3 hours of antibiotic exposure, and a 1000-fold difference by 5 hours (T + C, Fig. 1 B). Likewise, triclosan exposure also provided 1000-fold higher cell survival in the presence of the cell wall synthesis targeting antibiotic vancomycin (1× MIC) by 6 hours (T + V, Fig. 1 D). Notably, triclosan was unable to protect against killing by rifampicin (4× MIC) (Fig S1 B), despite the combination of triclosan and rifampicin displaying the same pattern of growth inhibition seen in previous experiments with ciprofloxacin or vancomycin (Fig S1 A). This may suggest that triclosan-induced tolerance only protects against bactericidal antibiotics such as ciprofloxacin and vancomycin, but not bacteriostatic antibiotics such as rifampicin. Antibiotic tolerance is often associated with nutritional starvation ( 51 ). As triclosan is a fatty acid synthesis inhibitor, further investigation addressed whether oleic acid supplementation could counter fatty acid starvation caused by triclosan, thereby preventing triclosan-induced antibiotic tolerance. A physiologically relevant concentration of oleic acid (500 µM), consistent with the levels of fatty acids found in human serum ( 52 ), was added alongside triclosan pretreatment. Oleic acid supplementation completly reverted the protective effect of triclosan. Triclosan pretreated cultures with oleic acid were as sensitive to ciprofloxacin (OA + T + C) and vancomycin (OA + T + V) as cultures that received no triclosan pretreatment (OA + C, OA + V). Oleic acid had no effect on the growth or viability of untreated or antibiotic alone conditions (OA, OA + C, OA + V, OA + R) (Fig. 1 A, Fig. 1 C). Thus, the protective effect of triclosan is clearly linked to fatty acid availability. Triclosan can protect S. aureus biofilms from eradication by high concentrations of ciprofloxacin, vancomycin, and rifampicin. Following the findings that triclosan exposure could induce antibiotic tolerance in planktonic S. aureus , it was investigated whether triclosan exposure could induce antibiotic tolerance in S. aureus biofilms also. Biofilms grown in the presence of triclosan and treated with ciprofloxacin (1× MBEC) (T + C, Fig. 2 A), vancomycin (1× MBEC) (T + V, Fig S2B) and rifampicin (1× MBEC) (T + R, Fig S2C) displayed significantly less cell death compared to biofilms that were not pretreated with triclosan (C, V, R) (Fig. 2 B). Just as in planktonic experiments, oleic acid supplementation alongside triclosan pretreatment prevented triclosan-induced antibiotic tolerance, with OA + T + C, OA + T + V, and OA + T + R biofilms having live cell percentages comparable to C, V, and R (Fig. 2 B). Live dead microscopy shows that triclosan alone (T; Fig. 2 A), oleic acid alone (OA; Fig S2A) and the fatty acid with triclosan (OA + T; Fig S2A) had little to no effect on cell viability compared to untreated biofilms (U) (Fig. 2 B). Exposure to triclosan throughout biofilm formation results in increased polysaccharide production. Fluorescent staining was used to characterise the composition of triclosan exposed S. aureus biofilms (Fig. 3 A). Alongside DAPI to visualise cells, fluorescein-conjugated WGA was used to stain the PNAG residues of the PIA polysaccharide that is prevalent in the S. aureus biofilm matrix. CLSM revealed that triclosan exposure significantly alters the production of polysaccharide in the S. aureus biofilm matrix. Echoing the findings of previous antibiotic tolerance experiments, oleic acid supplementation was able to largely negate the triclosan-induced changes, with enhanced polysaccharide production not being observed in OA + T biofilms. Comstat2 quantification showed that triclosan biofilms are composed of significantly more WGA stained PNAG compared to untreated (U), oleic acid only (OA) or oleic acid and triclosan (OA + T) pretreatments (Fig. 3 B), indicative of more PIA polysaccharide. SarA coordinates triclosan-induced polysaccharide synthesis. Following the findings that physiologically relevant levels of triclosan could induce antibiotic tolerance and alter biofilm formation, the molecular mechanism behind these changes was investigated. The effect of the staphylococcal accessory regulator (Sar) was first explored, as Sar controls the production of PIA in the biofilm matrix of S. aureus . Furthermore, PIA can impede the penetration and killing of numerous antibiotics – including vancomycin, ciprofloxacin, and rifampicin ( 53 ). Imaging sarA biofilms using confocal microscopy revealed that triclosan exposure did not result in an increase in the quantity of polysaccharide within the biofilm matrix (Fig. 4 A), unlike in the WT, in which triclosan exposure resulted in significantly higher proportions of PIA in the biofilm matrix (Fig. 4 B). Calcofluor white staining of biofilms, in which the binding of the stain to β(1→4) linked ᴅ-glucose or derivatives was used to quantify polysaccharide concentrations, confirmed the microscopy results (Fig. 4 C). The lack of increased matrix polysaccharide of the sarA mutant biofilms in the presence of triclosan, suggests triclosan-induced stimulation of SarA results in increased polysaccharide in triclosan exposed biofilms. Using the sarA mutant strain in planktonic kill-curve experiments, sar was ruled out from orchestrating triclosan-induced antibiotic tolerance since the triclosan exposed sarA mutant, like the WT, exhibited tolerance to vancomycin and ciprofloxacin (Fig S3). The stringent response is essential for triclosan-induced antibiotic tolerance in S. aureus biofilms. After experiments investigating the action of SarA in response to triclosan exposure, the mechanism behind triclosan-induced antibiotic tolerance remained elusive. The stringent response has been associated with triclosan-induced antibiotic tolerance in planktonic E. coli ( 37 ). This study sought to determine whether this was also the case in S. aureus through the use of an S. aureus stringent response (p)ppGpp 0 mutant incapable of producing (p)ppGpp. In planktonic kill-curve experiments, the stringent response was found to play no role in coordinating triclosan-induced antibiotic tolerance in planktonic culture (Fig S4). Although the (p)ppGpp 0 strain was less viable compared to the WT when treated with triclosan and antibiotic combinations, this was because of the increased sensitivity of the (p)ppGpp 0 mutant to triclosan, rather than the stringent response negating triclosan-induced antibiotic tolerance. Conversely, the stringent response was essential for coordinating triclosan-induced antibiotic tolerance in S. aureus biofilms. Live/dead staining shows the (p)ppGpp 0 strain failing to withstand ciprofloxacin (1× MBEC), vancomycin (1× MBEC), and rifampicin (1× MBEC) treatment despite triclosan exposure throughout biofilm maturation ((p)ppGpp 0 T + C, Fig S5B; (p)ppGpp 0 T + V, Fig. 5 A; (p)ppGpp 0 T + R, Fig S5C). Comstat2 quantification of live cell biomass shows triclosan significantly protects HG001 WT against ciprofloxacin, vancomycin, and rifampicin (Fig. 5 B), whilst the (p)ppGpp 0 T + C, (p)ppGpp 0 T + V, and (p)ppGpp 0 T + R biofilms demonstrate levels of viability comparative to WT C, WT V, and WT R respectively. Moreover, the inability of the (p)ppGpp 0 biofilm to handle stress is highlighted by the reduced viability of the untreated ((p)ppGpp 0 ) and triclosan exposed ((p)ppGpp 0 T), relative the same conditions in the WT (WT, WT T; Fig S5A, 5B). Endpoint CFUs of S. aureus biofilms also verified that for the HG001 WT, triclosan exposed biofilms were protected against killing by high concentrations of ciprofloxacin (Fig. 5 C), vancomycin (Fig. 5 D), and rifampicin (Fig. 5 E). Additionally, neither oleic acid supplementation nor the use of the (p)ppGpp 0 mutant resulted in triclosan-induced antibiotic tolerance, further validating the live/dead imaging. In HG001 WT, triclosan was able to induce a 1000-fold increase in tolerance to ciprofloxacin (Fig. 5 C) and rifampicin (Fig. 5 E), and a 100-fold increase in vancomycin tolerance (Fig. 5 D). These findings are even more striking when considering that triclosan pretreated biofilms had been exposed to otherwise lethal concentrations of antibiotics for 12 hours, as opposed to the 3 hours of antibiotic treatment seen in the live/dead biofilm experiments. This could suggest that triclosan exposed S. aureus can withstand treatment with extensive levels of antibiotics for prolonged periods of time. Despite these significant changes to antibiotic susceptibility in (p)ppGpp 0 biofilms, triclosan exposure still resulted in excess polysaccharide being produced in these biofilms (Fig S6A, S6B, S6C), further underlining that triclosan-induced antibiotic tolerance and triclosan-induced polysaccharide production are distinct mechanisms. Discussion This study aimed to characterise the effects of physiologically relevant levels of triclosan on S. aureus . Here, we show that 500 ng/mL triclosan, well within the limits of triclosan previously detected in human urine (2.4–3,790 ng/mL) ( 50 ), can trigger antibiotic tolerance in S. aureus biofilms, and alter biofilm formation. Whereas triclosan-induced biofilm formation was dependent on the global regulator SarA, triclosan-induced antibiotic tolerance in biofilms was (p)ppGpp dependent. Triclosan-induced biofilm formation as well as antibiotic tolerance were remediated by oleic acid, demonstrating that interruption of fatty acid biosynthesis is the main mode of triclosan action. Antimicrobial induced biofilm formation has been previously described ( 39 , 42 , 54 , 55 ). Here, we shed light on underlying mechanisms by demonstrating that triclosan increases the proportions of polysaccharide present in the biofilm matrix of S. aureus . Typically, biofilm formation is stimulated by sub-MIC levels of an antibiotic ( 54 , 55 ). However, in this study, 500 ng/mL of triclosan, whilst a physiologically relevant concentration of the biocide, is not sub-MIC. This is evidenced by triclosan halting the growth of planktonic cultures and decreasing biofilm cell density. A decrease in cell mass and concurrent stimulation of biofilm matrix production is contrary to many examples of antibiotic induced biofilm formation, but is not a complete anomaly. Skogman and colleagues (2012) found similar results when treating S. aureus biofilms with penicillin. They hypothesised that some antimicrobials decrease cell viability whilst increasing production of biofilm matrix components. However, these changes can only be confirmed through parallel measurement of biofilm viability, biomass, and quantifying matrix components, as in this study. Therefore, it may be that more antimicrobials previously associated with stimulating biofilm formation fall into this category, but this is yet to be fully characterised ( 56 ). Live/dead staining of triclosan exposed biofilms did not detect any notable increase in the proportions of dead cells. Instead, it appears that triclosan exposed biofilms consist of a reduced population that produces and exports far more PIA, relative to unexposed biofilms. SarA is a known regulator of icaADBC expression, thereby altering the production of PIA synthesis enzymes ( 57 – 62 ). SarA played a key role in triclosan-induced PIA overproduction since a sarA mutant was unable to overproduce PIA following triclosan exposure. PIA overproduction could be beneficial for numerous reasons ( 53 ), including increased tolerance to mechanical forces and resistance to immunological stresses, such as killing by host antimicrobial peptides and polymorphonuclear leukocytes ( 63 , 64 ), opsonisation by antibodies and complement ( 65 – 67 ), and phagocytosis by macrophages ( 63 ). PIA producing Staphylococci have previously been shown to be less susceptible to killing by some antibiotics, including vancomycin and ciprofloxacin ( 53 , 68 , 69 ). Accordingly, this study hypothesised that triclosan-induced polysaccharide production protected S. aureus from killing by antibiotics, and was therefore the cause of triclosan-induced antibiotic tolerance. However, since the sarA mutant displayed antibiotic tolerance despite no longer producing excess polysaccharide, there does not appear to be a direct link between polysaccharide production and antibiotic tolerance following triclosan exposure. In biofilms, triclosan-induced antibiotic tolerance was orchestrated by the stringent response. A (p)ppGpp 0 strain still overproduces PIA upon triclosan treatment, but no longer benefits from triclosan-induced antibiotic tolerance. Westfall et al. (2019) found that triclosan pretreatment protected planktonic E. coli against ampicillin, kanamycin, streptomycin and ciprofloxacin ( 37 ), our lab has also previously demonstrated that triclosan can protect E. coli from both ciprofloxacin mediated killing and changes to cell morphology ( 38 ). Triclosan-induced antibiotic tolerance in planktonic E. coli was mediated by the stringent response ( 37 ). In S. aureus , triclosan exposure increased the tolerance of S. aureus biofilms against high doses of ciprofloxacin, vancomycin, and rifampicin. When S. aureus is in its planktonic lifestyle the loss of its stringent response was unable to abolish triclosan-induced antibiotic tolerance but almost entirely negated triclosan-induced antibiotic tolerance in S. aureus biofilms. This suggests that triclosan-induced antibiotic tolerance is initially coordinated by a currently unidentified stress response in planktonic S. aureus , whilst the stringent response is important in maintaining triclosan-induced antibiotic tolerance in S. aureus biofilms. Fatty acid starvation might activate (p)ppGpp synthetase in S. aureus ( 70 ) and B. subtilis ( 71 ). However, mechanisms and conditions related to how fatty acid starvation could interfere with Rel activity remain unclear. Our data indicate that such putative conditions differ between planktonic and biofilm grown bacteria. The triclosan-induced changes to S. aureus physiology appear to originate from fatty acid starvation. When growth medium is supplemented with concentrations of oleic consistent with those found in human serum ( 52 ), the antibiotic susceptibility of triclosan pretreated S. aureus HG001 is restored and biofilm formation unchanged relative to untreated controls. The restoration of antibiotic susceptibility when fatty acid starvation is negated is logical, as fatty acid starvation is one of the numerous nutritional deficiencies capable of instigating the stringent response. However, the link between fatty acid starvation and SarA is less clear, and may suggest the effects of fatty acid starvation are broader than previously thought. The observation that oleic acid supplementation was able to override triclosan-induced affects at all is striking, as the notion that exogenous fatty acids can overcome the effects of fatty acid synthesis inhibitors has been viewed as controversial ( 71 – 75 ). Since the concentration of triclosan used in this experiment was low in comparison to triclosan concentrations in healthcare and household products, it cannot be concluded whether fatty acid supplementation is sufficient to save S. aureus from higher concentrations of triclosan. However, the data does suggest serum concentrations of oleic acid ( 52 ) would be sufficient to overcome tolerance induced by concentrations of triclosan that have accumulated in the human body ( 34 , 36 ). Triclosan pretreatment was able to reduce the efficacy of ciprofloxacin against E. coli by 100-fold in an in-vivo murine model, illustrating that triclosan-induced tolerance can endure outside of in-vitro settings ( 37 ). However, as E. coli and S. aureus are known to utilise exogenous fatty acids by different mechanisms ( 74 ), in-vivo work using triclosan treated S. aureus would be needed to determine this. This present study shows that exposure to physiologically relevant levels of triclosan can drive S. aureus to trigger multiple, divergent stress responses that alter numerous facets of S. aureus physiology. These physiological changes are rooted in the stress caused by triclosan-induced fatty acid starvation, before branching off. These diverging responses provide protection against antibiotics, facilitated by the stringent response, and potentially protect from other threats mediated by SarA controlled polysaccharide synthesis. Skogman and colleagues (2012) advised that the criteria for determining an effective antimicrobial treatment should be based on bacterial viability, biofilm biomass, plus matrix composition. We suggest going further to incorporate potential antibiotic tolerance. If the concentration of triclosan used in this study was to be evaluated based only on viability, triclosan would be deemed an effective therapy. However, when factoring in the increased matrix production and pleiotropic antibiotic tolerance induced by the biocide, triclosan appears far less alluring. This is further compounded by the finding that triclosan-induced affects occurred at an inhibitory concentration, rather than at sub-MIC levels. Thereby emphasising that accumulated or residual antimicrobials in the human body may cause large scale physiological change to pathogens. This reemphasises the need for stricter control on biocide use globally. Declarations Acknowledgements DW was funded via the Biotechnology and Biological Sciences Research Council (BBSRC), UK Doctoral Training Programme studentship (BB/M008770/1: www.bbsrc.ac.uk) held jointly by the School of Life Sciences and School of Pharmacy, University of Nottingham. KH and JA are partly funded by the National Biofilms Innovation Centre (NBIC) which is an Innovation and Knowledge Centre funded by the BBSRC and InnovateUK (Award Number BB/R012415/1). We thank the School of Life Sciences Imaging Facility (SLIM) for input on image analysis (particularly Robert Markus, Seema Bagia and Tim Self). CW was funded by Deutsche Forschungsgemeinschaft, Schwerpunktprogramm Spp1879 to CW (Project 423246275) and by infrastructural funding from the Deutsche Forschungsgemeinschaft (DFG), Cluster of Excellence EXC 2124 “Controlling Microbes to Fight Infections” (Project 390838134). The project was also supported by the University of Nottingham and the University of Tübingen’s funding as part of the Excellence Strategy of the German Federal and State Governments, in close collaboration with the University of Nottingham. Author Contributions KH and DW conceptualized and drove the study. KH, JA and CW supervised the generation and curation of the data by DW, PI and AS. DW created the original manuscript draft, and all authors contributed to the reviewing and editing of the manuscript. References Speziale P, Pietrocola G, Foster TJ, Geoghegan JA. 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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-3954016","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":275440543,"identity":"0e2fc262-1323-4a69-ba9b-2bf746da5b7b","order_by":0,"name":"Kim Hardie","email":"data:image/png;base64,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","orcid":"https://orcid.org/0000-0002-4892-2739","institution":"University of Nottingham","correspondingAuthor":true,"prefix":"","firstName":"Kim","middleName":"","lastName":"Hardie","suffix":""},{"id":275440544,"identity":"8255faa3-20c8-436a-a3be-50fc2ccb3a32","order_by":1,"name":"Dean Walsh","email":"","orcid":"","institution":"University of Warwick","correspondingAuthor":false,"prefix":"","firstName":"Dean","middleName":"","lastName":"Walsh","suffix":""},{"id":275440545,"identity":"d4678ac9-f584-47b6-83a0-af61b3fbfa75","order_by":2,"name":"Andrea Salzer","email":"","orcid":"","institution":"University of Tübingen","correspondingAuthor":false,"prefix":"","firstName":"Andrea","middleName":"","lastName":"Salzer","suffix":""},{"id":275440546,"identity":"a686335b-0d5e-4a0d-a7bb-c6514be39790","order_by":3,"name":"Parvati Iyer","email":"","orcid":"","institution":"University of Nottingham","correspondingAuthor":false,"prefix":"","firstName":"Parvati","middleName":"","lastName":"Iyer","suffix":""},{"id":275440547,"identity":"c2e64791-fdb7-4895-916c-c65e5eef12ed","order_by":4,"name":"Christiane Wolz","email":"","orcid":"https://orcid.org/0000-0003-3909-5281","institution":"Interfaculty Institute of Microbiology and Infection Medicine, University of Tübingen","correspondingAuthor":false,"prefix":"","firstName":"Christiane","middleName":"","lastName":"Wolz","suffix":""},{"id":275440548,"identity":"a2ccf50b-947b-4c8d-b379-e4e2a14d971e","order_by":5,"name":"Jonathan Aylott","email":"","orcid":"","institution":"University of Nottingham","correspondingAuthor":false,"prefix":"","firstName":"Jonathan","middleName":"","lastName":"Aylott","suffix":""}],"badges":[],"createdAt":"2024-02-13 17:10:26","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3954016/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3954016/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":53416669,"identity":"aeb7d45d-eb12-4675-ac07-4fe81207ea7d","added_by":"auto","created_at":"2024-03-25 17:58:20","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":260175,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePretreatment with triclosan protects S. aureus planktonic cultures from antibiotic treatment, this protection is negated by oleic acid supplementation. \u003c/strong\u003eFor planktonic experiments, \u003cem\u003eS. aureus\u003c/em\u003e strain HG001 was incubated in BHI in the presence or absence of triclosan, with or without 500 μM oleic acid solubilised in 0.1% Brij 58, for 30 mins at 37°C. Inhibitory concentrations of antibiotics were added and incubation continued. Untreated (U, OA), 500 ng/mL triclosan pretreated (T, OA+T), 1 μg/mL ciprofloxacin treated (C, OA+C), 500 ng/mL triclosan treated and 1 μg/mL ciprofloxacin treated (T+C, OA+T+C), 2 µg/mL vancomycin treated (V, OA+V), 500 ng/mL triclosan treated and 2 µg/mL vancomycin treated (T+V, OA+T+V) conditions are shown.\u0026nbsp; Growth curves with ciprofloxacin \u003cstrong\u003e(A)\u003c/strong\u003e and vancomycin \u003cstrong\u003e(C)\u003c/strong\u003e showing optical densities (OD\u003csub\u003e600nm\u003c/sub\u003e) plotted against time. Error bars display ±SD, n=3. \u003cstrong\u003eB)\u003c/strong\u003e Ciprofloxacin time-kill assays, \u003cstrong\u003eD)\u003c/strong\u003e vancomycin time-kill assays with cell viability (%) plotted against time. Cell viability for antibiotic treated conditions was determined by normalising CFU/mL at each time point against the CFU/mL of the initial time point for each condition (after pretreatment step, but before addition of antibiotics). The line labelled LOD denotes the limit of detection. Error bars display ±SD, n=3.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-3954016/v1/0d8134907e8e61b08f1fe4a9.png"},{"id":53416671,"identity":"678a7c84-3b99-40fd-8012-3c20967691c3","added_by":"auto","created_at":"2024-03-25 17:58:20","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":2999601,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTriclosan exposure protects S. aureus biofilms from antibiotic treatment, this protection is negated by oleic acid supplementation. \u003c/strong\u003eCLSM was used to assess the effect of triclosan on the antibiotic tolerance of \u003cem\u003eS. aureus \u003c/em\u003eHG001 WT. Syto 9 (green) was used to visualise live cells, whilst propidium iodide (red) was used to visualise cell death. \u003cstrong\u003eA)\u003c/strong\u003e Untreated biofilms (U), 500 ng/mL triclosan exposed biofilms (T), biofilms grown in the presence or absence of 500 μM oleic acid and treated with 4096 μg/mL ciprofloxacin (C, OA+C) and 500 ng/mL triclosan exposed biofilms treated with 4096 μg/mL ciprofloxacin (T+C, OA+T+C) are shown. For each condition a 2D image of a selected z-plane is shown for live, dead, and overlay images. A 3D image of each condition is also shown. Images are representative of multiple experiments and were taken using the 40x objective (n = 3). \u003cstrong\u003eB) \u003c/strong\u003eQuantification of the percentage of live cells in biofilms was carried out using Comstat2 image analysis software. Statistical significance was calculated using a two-way ANOVA followed by Tukey’s multiple comparisons test. Error bars represent SD, n=3. *** denotes P≤0.001, ** denotes P≤0.01, * denotes P≤0.05.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-3954016/v1/089c708fa9aa20d29ae9486a.png"},{"id":53416675,"identity":"d48df8d2-4748-48b3-88b0-c62c80da84d0","added_by":"auto","created_at":"2024-03-25 17:58:21","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":2577884,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eBiofilms grown in the presence of physiologically relevant levels of triclosan produce more matrix polysaccharide, though oleic acid supplementation prevents this. A) \u003c/strong\u003eCLSM was used to assess the effect of triclosan and oleic acid supplementation on the biofilm formation of \u003cem\u003eS. aureus \u003c/em\u003eHG001 WT biofilms. Conditions include untreated biofilms (U), biofilms supplemented with 500 μΜ oleic acid (OA), exposed to 500 ng/mL triclosan (T), supplemented with 500 μΜ oleic acid and exposed to 500 ng/mL triclosan (OA+T). DAPI (blue) was used to visualise cells, fluorescein-conjugated WGA (green) was used to visualise PNAG residues of polysaccharide. For each condition a 2D image of a selected z-plane is shown for DAPI, WGA, and overlay images. A 3D image of each condition is also shown. Images are representative of multiple experiments and were taken using the 40x objective (n=3). \u003cstrong\u003eB) \u003c/strong\u003eQuantification of polysaccharide biomass was carried out using Comstat2 image analysis software. Statistical significance was calculated using a one-way ANOVA followed by Tukey’s multiple comparisons test. Error bars represent SD, n=3 * denotes P≤0.05, ** denotes P≤0.01.\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-3954016/v1/40251c507acda7d464a1e1ef.png"},{"id":53416670,"identity":"8e93de3e-76f7-4368-b01b-3a90abd0f637","added_by":"auto","created_at":"2024-03-25 17:58:20","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1099498,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSarA is responsible for triclosan-induced polysaccharide production in S. aureus biofilms. \u003c/strong\u003eCLSM was used to assess the effect of triclosan on the biofilm formation of \u003cem\u003eS. aureus \u003c/em\u003eHG001 \u003cem\u003esarA\u003c/em\u003e biofilms \u003cstrong\u003eA)\u003c/strong\u003e. Conditions include untreated biofilms (HG001 \u003cem\u003esarA\u003c/em\u003e) and biofilms exposed to 500 ng/mL triclosan (HG001 \u003cem\u003esarA \u003c/em\u003eT). DAPI (blue) was used to visualise cells, fluorescein-conjugated WGA (green) was used to visualise PNAG residues of polysaccharide. For each condition a 2D image of a selected z-plane is shown for DAPI, WGA, and overlay images. A 3D image of each condition is also shown. Images are representative of multiple experiments and were taken using the 40x objective (n=3). \u003cstrong\u003eB) \u003c/strong\u003eQuantification of polysaccharide biomass in HG001 WT and HG001 \u003cem\u003esarA \u003c/em\u003ebiofilms was carried out using Comstat2 image analysis software. \u003cstrong\u003eC) \u003c/strong\u003eQuantification of the fluorescence intensity of matrix polysaccharide stained with calcofluor white. Statistical significance was calculated using a two-way ANOVA followed by Sidak’s multiple comparisons test.\u003cstrong\u003e \u003c/strong\u003eError bars represent SD, n=3. **** denotes P≤0.0001, ** denotes P≤0.01, * denotes P≤0.05.\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-3954016/v1/71eeef0ccf77d2c480607990.png"},{"id":53416678,"identity":"5c75a725-57c6-4674-9efe-4813d1612925","added_by":"auto","created_at":"2024-03-25 17:58:22","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1736483,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe stringent response plays a role in triclosan-induced antibiotic tolerance against multiple antibiotics in S. aureus biofilms.\u003c/strong\u003e\u003cem\u003e \u003c/em\u003eCLSM was used to assess the effect of triclosan on the antibiotic tolerance of \u003cem\u003eS. aureus \u003c/em\u003eHG001 WT and HG001 (p)ppGpp\u003csup\u003e0\u003c/sup\u003e. Syto 9 (green) was used to visualise live cells, whilst\u0026nbsp;propidium iodide (red) was used to visualise cell death. \u003cstrong\u003eA)\u003c/strong\u003e Biofilms treated with 2048 μg/mL vancomycin (WT V, (p)ppGpp\u003csup\u003e0 \u003c/sup\u003eV) and 500 ng/mL triclosan exposed biofilms treated with 2048 μg/mL vancomycin (WT T+V, (p)ppGpp\u003csup\u003e0 \u003c/sup\u003eT+V) are shown. For each condition a 2D image of a selected z-plane is shown for live, dead, and overlay images. A 3D image of each condition is also shown. Images are representative of multiple experiments and were taken using the 40x objective (n = 3). \u003cstrong\u003eB) \u003c/strong\u003eQuantification of the percentage of live cells in biofilms was carried out using Comstat2 image analysis software. Endpoint CFUs of S. aureus HG001 (WT), HG001 + 500 μM oleic acid (WT+OA), HG001 (p)ppGpp\u003csup\u003e0\u003c/sup\u003e ((p)ppGpp\u003csup\u003e0\u003c/sup\u003e) biofilms. Each strain/condition is either untreated or exposed to 500 ng/mL triclosan (T). Statistical significance was calculated using a two-way ANOVA followed by Tukey’s multiple comparisons test. \u003cstrong\u003eC)\u003c/strong\u003e Viability of \u003cem\u003eS. aureus \u003c/em\u003ebiofilms following 12 hours of treatment with 4096 μg/mL ciprofloxacin (cip). \u003cstrong\u003eD)\u003c/strong\u003e Viability of \u003cem\u003eS. aureus \u003c/em\u003ebiofilms following 12 hours of treatment with 2048 μg/mL vancomycin (van). \u003cstrong\u003eE)\u003c/strong\u003e Viability of \u003cem\u003eS. aureus \u003c/em\u003ebiofilms following 12 hours of treatment with 2048 μg/mL rifampicin (rif). Statistical significance was calculated using a two-way ANOVA followed by Sidak’s multiple comparisons test.\u003cstrong\u003e \u003c/strong\u003eError bars represent SD, n=3. **** denotes P≤0.0001, *** denotes P≤0.001, ** denotes P≤0.01, * denotes P≤0.05.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-3954016/v1/49106049eba417234ec1a089.png"},{"id":53418196,"identity":"35358a00-55c2-4d13-827e-fbed4f1cf427","added_by":"auto","created_at":"2024-03-25 18:06:23","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":4003219,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3954016/v1/9e4788f1-8f45-4bc2-9e26-99633a4a3eb9.pdf"},{"id":53416679,"identity":"437510eb-b7dc-433c-86e3-e8adafeb54f4","added_by":"auto","created_at":"2024-03-25 17:58:22","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":5540932,"visible":true,"origin":"","legend":"","description":"","filename":"Triclosanpaperdraft2024finalsupplementary.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3954016/v1/dcac48ae962c505d201de66e.pdf"}],"financialInterests":"(Not answered)","formattedTitle":"Triclosan alters biofilm structures and confers antibiotic tolerance in Staphylococcus aureus using multiple regulatory pathways","fulltext":[{"header":"Importance ","content":"\u003cp\u003eThe prevalent bacterium \u003cem\u003eStaphylococcus aureus\u003c/em\u003e infects skin lesions and indwelling devices, and this can cause sepsis with 33% mortality. Intrinsic to this is the formation of co-ordinated communities (biofilms) protected by a polysaccharide coat. \u003cem\u003eS. aureus\u003c/em\u003e is increasingly difficult to eradicate due to its antibiotic resistance. Protection against Methicillin Resistant \u003cem\u003eS. aureus\u003c/em\u003e (MRSA) includes pre-hospital admission washing with products containing biocides. The biocide triclosan is the predominant antibacterial compound in sewage in Ontario due to its use in household and hospital settings. Levels of triclosan accumulate with exposure in humans. The significance of our research is in identifying the mechanisms triggered by exposure of \u003cem\u003eS. aureus\u003c/em\u003e to physiological levels of triclosan that go on to raise the tolerance of \u003cem\u003eS. aureus\u003c/em\u003e to antibiotics and promote the formation of biofilms. This understanding will inform future criteria used to determine effective antimicrobial treatments.\u0026nbsp;\u003c/p\u003e"},{"header":"Introduction","content":"\u003cp\u003eBiofilms are surface attached communities of bacteria enclosed in an exopolymeric matrix. For the pathogen \u003cem\u003eStaphylococcus aureus\u003c/em\u003e, this matrix is composed of surface proteins (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e), polysaccharide intercellular adhesin (PIA) (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e), and extracellular DNA (eDNA) (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e). Biofilms can act as reservoirs of antibiotic tolerance and use numerous mechanisms to withstand antimicrobial treatment, including reduced penetration of antimicrobials into the biofilm matrix (\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e), and a generally slow growth rate (\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e). This reduced rate of growth serves to protect bacteria by diminishing the efficacy of the majority of antimicrobials. The enduring resilience of biofilms has made them a severe clinical concern, particular with regards to chronic infections caused by biofilm forming bacteria such as \u003cem\u003eS. aureus\u003c/em\u003e (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eAntibiotic tolerance is the process by which an entire bacterial population can survive transient exposure to antibiotics that would otherwise be lethal (\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e). Antibiotic tolerance distinguishes itself from antibiotic resistance in numerous ways. For example, resistant bacteria may need higher concentrations of an antimicrobial to achieve bacterial killing, tolerant bacteria require a longer exposure time to an antimicrobial to provide the same level of killing achieved against susceptible bacteria (\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e). Additionally, resistant bacteria are typically protected against a single antibiotic or a small group of closely related antibiotics, whilst tolerant bacteria can typically better withstand a broad array of antimicrobials (\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e). The presence of antibiotic tolerant bacteria leads to flawed antimicrobial treatments, risking recurrent infection in patients (\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e). Antibiotic tolerance in \u003cem\u003eS. aureus\u003c/em\u003e is becoming increasingly relevant clinically, as exemplified by 6%-43% of \u003cem\u003eS. aureus\u003c/em\u003e clinical strains being vancomycin tolerant (\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e). Drug tolerant \u003cem\u003eS. aureus\u003c/em\u003e can cause prolonged fevers and extended bacteraemia duration, increased treatment failures and even increased mortality rates (\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThere is a broad range of molecular strategies employed by \u003cem\u003eS. aureus\u003c/em\u003e to facilitate antibiotic tolerance (\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e). The stringent response is one such strategy and is conserved among most bacterial species as a means to combat nutritional deficiencies (\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e). During the stringent response the alarmones ppGpp and pppGpp (collectively known as (p)ppGpp) are synthesised from ATP and GTP (pppGpp) or GDP (ppGpp) (\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e). In \u003cem\u003eS. aureus\u003c/em\u003e, alarmone synthesis is driven by three alarmone synthetases: two small alarmone synthetases; RelP and RelQ, and a larger protein Rel. Rel is bifunctional and equipped with a synthetase domain for alarmone synthesis and a hydrolase domain for the purpose of alarmone degradation. Hydrolysis of these alarmones in \u003cem\u003eS. aureus\u003c/em\u003e is essential for survival, as accumulation of (p)ppGpp results in cell death (\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e). Rel, RelP, and RelQ are triggered by different inducing stresses, including various antibiotics. Numerous studies have used the antibiotic mupirocin, an antibiotic that induces amino acid starvation, to induce Rel activity (\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e). RelP and RelQ expression has been triggered by exposure to the cell wall targeting antibiotics ampicillin, oxacillin, and vancomycin (\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e). Once the stringent response has been triggered, (p)ppGpp orchestrates global changes in gene expression, with steep downregulation of genes associated with proliferative functions, such as protein synthesis and DNA replication (\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e). As bactericidal antibiotics disrupt the function of active targets, the metabolic shutdown and quiescence induced by the stringent response renders these drugs largely ineffectual (\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe synthetic biocide triclosan inhibits type II fatty acid synthesis (FASII) at the enoylacyl carrier protein reductase (FabI) step(\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e). At high concentrations, triclosan provokes membrane permeability and non-specific membrane damage (29). In hospitals, triclosan is used as an MRSA decolonisation therapy (30), antimicrobial hand wash (30), antiseptic ointment (31), and is impregnated into surgical sutures (32) and urinary catheters (33). Triclosan is also widely used in domestic settings, being found in many household products including soaps, toothpastes (34), cosmetics and laundry detergents (35). This widespread use of triclosan means that both people and environments are chronically exposed to the biocide. Absorption of triclosan from triclosan containing toothpastes (TCT) is high, with triclosan blood concentration increasing from 0.81 ng/mL to 296 ng/mL after 14 days of TCT use (36). The average urine concentration of triclosan in patients exposed to the biocide during their hospital stay was 245 ng/mL, with concentrations reaching as high as 505 ng/mL (34).\u003c/p\u003e \u003cp\u003ePrevious studies have suggested that pretreatment with low levels of triclosan can induce antibiotic tolerance in both \u003cem\u003eEscherichia coli\u003c/em\u003e (\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e) and \u003cem\u003eS. aureus\u003c/em\u003e (\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e) to multiple different antibiotics. Moreover, insufficiently dosed treatments can alter biofilm formation (\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e). \u003cem\u003eS. aureus\u003c/em\u003e has been shown to grow thicker biofilms in the presence of sub-MIC mupirocin (\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e), clindamycin (\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e), and β-lactams (\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e) due to increased eDNA release, whilst vancomycin exposure caused increased biofilm formation due to elevated levels of both PIA and eDNA in the biofilm matrix (\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e). Salicylic acid, the active component of aspirin, has been shown to increase the production of PIA in \u003cem\u003eS. aureus\u003c/em\u003e biofilms (\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e). Although triclosan has not yet been observed to alter the biofilm formation of \u003cem\u003eS. aureus\u003c/em\u003e, the biocide does stimulate cellulose production in established \u003cem\u003eSalmonella typhimirium\u003c/em\u003e biofilms (\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e). As biocides are globally accessible and both biofilm formation and antibiotic tolerance have been associated with antibiotic treatment failure, augmentation or induction of these processes by biocide exposure could be a serious clinical concern.\u003c/p\u003e \u003cp\u003eThis study aimed to investigate how triclosan exposure affects not just planktonic \u003cem\u003eS. aureus\u003c/em\u003e, but also \u003cem\u003eS. aureus\u003c/em\u003e biofilms. We show that low levels of triclosan can induce antibiotic tolerance via the stringent response in \u003cem\u003eS. aureus\u003c/em\u003e biofilms, thereby increasing their resilience against antibiotic killing. In addition, triclosan exposure throughout biofilm formation led to a significant increase in biofilm polysaccharide production in a SarA-dependent manner. Overall, low levels of triclosan, consistent with those found accumulating within the human body, can trigger multiple molecular mechanisms to drastically alter the phenotype of \u003cem\u003eS. aureus\u003c/em\u003e biofilms, protecting them against antibiotics and possibly additional threats.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eBacterial strains and culture conditions\u003c/h2\u003e \u003cp\u003e \u003cem\u003eS. aureus\u003c/em\u003e strains (see Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e) were grown at 37\u0026deg;C on brain heart infusion (BHI) agar plates for 16\u0026ndash;20 hours. For growth in liquid media \u003cem\u003eS. aureus\u003c/em\u003e was grown at 37\u0026deg;C in BHI broth with shaking at 200 rpm unless stated otherwise.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003e\u003cem\u003eS. aureus\u003c/em\u003e strains used in this study\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eStrain\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eDescription\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eReference\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHG001\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eDerivative of NCTC8325; \u003cem\u003ersbU\u003c/em\u003e repaired, \u003cem\u003etcaR\u003c/em\u003e defective, carries prophages Φ11, Φ12, and Φ13.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e(\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHG001 (p)ppGpp\u003csup\u003e0\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eHG001 rel/relP/relQ triple mutant (Δ\u003cem\u003erel\u003c/em\u003e\u003csub\u003e\u003cem\u003esyn\u003c/em\u003e\u003c/sub\u003e, Δ\u003cem\u003erelP\u003c/em\u003e\u003csub\u003e\u003cem\u003esyn\u003c/em\u003e\u003c/sub\u003e, Δ\u003cem\u003erelQ\u003c/em\u003e\u003csub\u003e\u003cem\u003esyn\u003c/em\u003e\u003c/sub\u003e)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e(\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHG001 \u003cem\u003esarA\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eHG001 \u003cem\u003esarA\u003c/em\u003e::\u003c/p\u003e \u003cp\u003e\u003cem\u003eBursa aurealis\u003c/em\u003e\u003c/p\u003e \u003cp\u003e(\u003cem\u003eerm\u003c/em\u003e)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e(\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eAntibiotic susceptibility testing\u003c/h2\u003e \u003cp\u003e \u003cb\u003eAntibiotics tested -\u003c/b\u003e Antimicrobials used for these experiments were obtained from Sigma-Aldrich: triclosan (Igrasan), ciprofloxacin, vancomycin, rifampicin.\u003c/p\u003e \u003cp\u003e \u003cb\u003eMinimum inhibitory concentration (MIC) determination \u0026minus;\u003c/b\u003e\u0026thinsp;96 well microtitre plates were inoculated with \u003cem\u003eS. aureus\u003c/em\u003e strains at an optical density of 600 nm (OD\u003csub\u003e600\u003c/sub\u003e) of 0.05 in Mueller-Hinton broth. Minimum inhibitory concentrations (MICs) were determined by the broth microdilution method in accordance with EUCAST guidelines (\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cb\u003eMinimum biofilm eradication concentration (MBEC) determination -\u003c/b\u003e MBEC experiments were conducted by inoculating 96 well microtitre plates with \u003cem\u003eS. aureus\u003c/em\u003e at an OD\u003csub\u003e600\u003c/sub\u003e of 0.05 in Mueller-Hinton broth and incubating for 24 hours at 37\u0026deg;C to allow biofilm formation. Following this, a 2-fold dilution series of antibiotics was added to wells and biofilms incubated in Mueller-Hinton broth with antibiotics for a further 24 hours at 37\u0026deg;C. After incubation, biofilms were removed from wells by vigorous scraping with pipette tips and sonicating at 30 kHz for 1 minute in sonication bath. Disrupted biofilms were then spotted out onto Mueller-Hinton agar plates and the MBEC defined as the minimum antibiotic concentration at which viable colonies are not detected.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eTime-kill assays\u003c/h2\u003e \u003cp\u003e \u003cem\u003eS. aureus\u003c/em\u003e strains at OD\u003csub\u003e600\u003c/sub\u003e 0.01 were inoculated into either untreated BHI broth or BHI broth supplemented with triclosan (500 ng/mL; 0.25x MIC). For conditions requiring fatty acid supplementation, 500 \u0026micro;M oleic acid and 0.1% v/v Brij 58 for solubilisation of oleic acid was also added. Cultures were then incubated at 37\u0026deg;C in shaking conditions (200 rpm) for a period of 30 minutes to allow for acclimatisation to biocide and/or fatty acid supplementation. Conditions that did not include triclosan or oleic acid were also incubated for 30 minutes.\u003c/p\u003e \u003cp\u003eFollowing triclosan pretreatment, the first samples are taken from all conditions to determine initial OD\u003csub\u003e600\u003c/sub\u003e and colony forming units (CFUs). Then, inhibitory concentrations of antibiotics were added to relevant conditions. Throughout this study the antibiotics ciprofloxacin (1 \u0026micro;g/mL), vancomycin (2 \u0026micro;g/mL), rifampicin (40 ng/mL) were used. This resulted in four conditions per strain being present in each experiment, consisting of untreated \u003cem\u003eS. aureus\u003c/em\u003e, biocide exposed, antibiotic treated, biocide exposed\u0026thinsp;+\u0026thinsp;antibiotic treated. Cultures were then returned to the incubator and samples were taken every hour for 6\u0026ndash;8 hours depending on the experiment. Each hour, CFUs were determined and the OD\u003csub\u003e600\u003c/sub\u003e recorded to determine viability and growth, respectively. Cell viability was determined by normalising the CFU at each time point against the CFU of the initial time point (following pretreatment, but just before antibiotic addition). This corrected for the increased growth of untreated conditions relative to triclosan exposed conditions that occurred during the 30 minute pretreatment step.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eBiofilm imaging\u003c/h2\u003e \u003cp\u003e \u003cb\u003eBiofilm preparation -\u003c/b\u003e Overnight cultures of \u003cem\u003eS. aureus\u003c/em\u003e were diluted in BHI to OD\u003csub\u003e600\u003c/sub\u003e 0.05 and loaded into \u0026micro;-slide 8-well glass bottomed chambers (ibidi, glass bottom). 500 ng/mL triclosan, was added to relevant wells, allowing biofilms to form in the presence of triclosan. For conditions requiring fatty acid supplementation, 500 \u0026micro;M oleic acid and 0.1% Brij 58 was also added. Chambers were then placed in a 37\u0026deg;C incubator for 48 hours in static conditions. Following this incubation period, growth medium was removed from the biofilms and replaced with fresh BHI growth medium.\u003c/p\u003e \u003cp\u003e \u003cb\u003eLive/dead staining -\u003c/b\u003e To investigate antibiotic tolerance either 4096 \u0026micro;g/mL ciprofloxacin, 2048 \u0026micro;g/mL vancomycin or 2048 \u0026micro;g/mL rifampicin were added to wells for 3 hours. Next, live/dead staining was carried out using 6 \u0026micro;M syto 9 and 30 \u0026micro;M propidium iodide (BacLight, Molecular Probes). Biofilms were imaged using a Zeiss LSM 700 compact confocal laser scanning microscope using the 40x objective and appropriate fluorescence settings (syto 9\u0026thinsp;=\u0026thinsp;488 nm laser, propidium iodide\u0026thinsp;=\u0026thinsp;555 nm laser).\u003c/p\u003e \u003cp\u003e \u003cb\u003eStaining of biofilm components -\u003c/b\u003e To examine biofilm structure, the nucleic acid stain 4',6-diamidino-2-phenylindole (DAPI) was added at a final concentration of 10 \u0026micro;g/mL and the polysaccharide stain fluorescein-conjugated wheat germ agglutinin (WGA) (Invitrogen\u0026trade;) was added at a final concentration of 10 \u0026micro;g/mL. Biofilms were imaged using a Zeiss LSM 700 compact confocal laser scanning microscope using the 40x objective and appropriate fluorescence settings (DAPI\u0026thinsp;=\u0026thinsp;405 nm laser, fluorescein\u0026thinsp;=\u0026thinsp;488 nm laser).\u003c/p\u003e \u003cp\u003e \u003cb\u003eQuantification using Comstat2 software -\u003c/b\u003e Biomass of live cells, dead cells, and stained matrix polysaccharide was analysed using Comstat2 software (\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eBiofilm characterisation\u003c/h2\u003e \u003cp\u003e \u003cb\u003eBiofilm preparation \u0026minus;\u003c/b\u003e\u0026thinsp;0.5 mL of BHI was added to 24 well plates and inoculated with OD\u003csub\u003e600\u003c/sub\u003e 0.05 \u003cem\u003eS. aureus\u003c/em\u003e. Wells were treated with either 500 ng/mL triclosan, 500 \u0026micro;M oleic acid solubilised in 0.1% Brij 58, or a combination of both triclosan and solubilised oleic acid. Biofilms were grown statically at 37\u0026deg;C for 48 hours. For biofilm experiments including antibiotics, 4096 \u0026micro;g/mL ciprofloxacin, 2048 \u0026micro;g/mL vancomycin or 2048 \u0026micro;g/mL rifampicin were added to wells for the last 12 hours of biofilm incubation. Following incubation, growth medium was removed and biofilms washed with PBS.\u003c/p\u003e \u003cp\u003e \u003cb\u003eCalcofluor white staining to quantify polysaccharide \u0026ndash;\u003c/b\u003e Calcofluor white staining was carried out as previously described (\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e) with minor alterations. 200 \u0026micro;L of calcofluor white (1 mg/mL in dH\u003csub\u003e2\u003c/sub\u003eO) was added to wells and incubated in the dark for 1 hour. Calcofluor white was then removed and biofilms washed with PBS to remove unbound calcofluor white. 200 \u0026micro;L of 96% ethanol was then added to solubilise biofilm-bound calcofluor white. Fluorescence intensity was then measured using the 360 nm excitation filter and 460 nm emission filter.\u003c/p\u003e \u003cp\u003e \u003cb\u003eCFU determination \u0026ndash;\u003c/b\u003e PBS was added to wells and biofilms were sonicated at 30 kHz for 1 minute, diluted, and CFUs determined. For experiments involving antibiotics, the % viability of the biofilm was defined as the CFU of the antibiotic treated sample (for instance WT\u0026thinsp;+\u0026thinsp;cip or WT T\u0026thinsp;+\u0026thinsp;cip) divided by the CFU of the corresponding control (WT untreated or WT T). This corrected for inherent differences in cell number when comparing an untreated \u003cem\u003eS. aureus\u003c/em\u003e biofilm to a triclosan exposed \u003cem\u003eS. aureus\u003c/em\u003e biofilm.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003e \u003cb\u003eTriclosan induces antibiotic tolerance towards ciprofloxacin and vancomycin, but not rifampicin in planktonically grown\u003c/b\u003e \u003cb\u003eS. aureus\u003c/b\u003e.\u003c/p\u003e \u003cp\u003eTriclosan was added to planktonic \u003cem\u003eS. aureus\u003c/em\u003e cultures at a concentration of 500 ng/mL (5\u0026times; MIC HG001; Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e), corresponding to physiologically relevant concentrations of triclosan found in the urine of users of triclosan-containing products (\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e). Triclosan pretreatment provided protection from inhibitory concentrations of ciprofloxacin (4\u0026times; MIC) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB) and vancomycin (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD). Cell viability in triclosan-exposed (T\u0026thinsp;+\u0026thinsp;C, T\u0026thinsp;+\u0026thinsp;V) conditions remained relatively constant throughout the time-kill assays, whilst conditions treated with ciprofloxacin (C) or vancomycin (V) alone displayed a sharp decrease in cell viability over time. Of note, antibiotic treatment did not result in a concomitant decrease in OD, indicating that bacteria are not lysed under these conditions. Triclosan induced protection resulted in a 100-fold increase in cell survival in the presence of ciprofloxacin, a DNA gyrase inhibitor, by 3 hours of antibiotic exposure, and a 1000-fold difference by 5 hours (T\u0026thinsp;+\u0026thinsp;C, Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). Likewise, triclosan exposure also provided 1000-fold higher cell survival in the presence of the cell wall synthesis targeting antibiotic vancomycin (1\u0026times; MIC) by 6 hours (T\u0026thinsp;+\u0026thinsp;V, Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD). Notably, triclosan was unable to protect against killing by rifampicin (4\u0026times; MIC) (Fig \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eB), despite the combination of triclosan and rifampicin displaying the same pattern of growth inhibition seen in previous experiments with ciprofloxacin or vancomycin (Fig \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eA). This may suggest that triclosan-induced tolerance only protects against bactericidal antibiotics such as ciprofloxacin and vancomycin, but not bacteriostatic antibiotics such as rifampicin.\u003c/p\u003e \u003cp\u003eAntibiotic tolerance is often associated with nutritional starvation (\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e). As triclosan is a fatty acid synthesis inhibitor, further investigation addressed whether oleic acid supplementation could counter fatty acid starvation caused by triclosan, thereby preventing triclosan-induced antibiotic tolerance. A physiologically relevant concentration of oleic acid (500 \u0026micro;M), consistent with the levels of fatty acids found in human serum (\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e), was added alongside triclosan pretreatment. Oleic acid supplementation completly reverted the protective effect of triclosan. Triclosan pretreated cultures with oleic acid were as sensitive to ciprofloxacin (OA\u0026thinsp;+\u0026thinsp;T\u0026thinsp;+\u0026thinsp;C) and vancomycin (OA\u0026thinsp;+\u0026thinsp;T\u0026thinsp;+\u0026thinsp;V) as cultures that received no triclosan pretreatment (OA\u0026thinsp;+\u0026thinsp;C, OA\u0026thinsp;+\u0026thinsp;V). Oleic acid had no effect on the growth or viability of untreated or antibiotic alone conditions (OA, OA\u0026thinsp;+\u0026thinsp;C, OA\u0026thinsp;+\u0026thinsp;V, OA\u0026thinsp;+\u0026thinsp;R) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA, Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). Thus, the protective effect of triclosan is clearly linked to fatty acid availability.\u003c/p\u003e \u003cp\u003e \u003cb\u003eTriclosan can protect\u003c/b\u003e \u003cb\u003eS. aureus\u003c/b\u003e \u003cb\u003ebiofilms from eradication by high concentrations of ciprofloxacin, vancomycin, and rifampicin.\u003c/b\u003e\u003c/p\u003e \u003cp\u003eFollowing the findings that triclosan exposure could induce antibiotic tolerance in planktonic \u003cem\u003eS. aureus\u003c/em\u003e, it was investigated whether triclosan exposure could induce antibiotic tolerance in \u003cem\u003eS. aureus\u003c/em\u003e biofilms also. Biofilms grown in the presence of triclosan and treated with ciprofloxacin (1\u0026times; MBEC) (T\u0026thinsp;+\u0026thinsp;C, Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA), vancomycin (1\u0026times; MBEC) (T\u0026thinsp;+\u0026thinsp;V, Fig S2B) and rifampicin (1\u0026times; MBEC) (T\u0026thinsp;+\u0026thinsp;R, Fig S2C) displayed significantly less cell death compared to biofilms that were not pretreated with triclosan (C, V, R) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). Just as in planktonic experiments, oleic acid supplementation alongside triclosan pretreatment prevented triclosan-induced antibiotic tolerance, with OA\u0026thinsp;+\u0026thinsp;T\u0026thinsp;+\u0026thinsp;C, OA\u0026thinsp;+\u0026thinsp;T\u0026thinsp;+\u0026thinsp;V, and OA\u0026thinsp;+\u0026thinsp;T\u0026thinsp;+\u0026thinsp;R biofilms having live cell percentages comparable to C, V, and R (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). Live dead microscopy shows that triclosan alone (T; Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA), oleic acid alone (OA; Fig S2A) and the fatty acid with triclosan (OA\u0026thinsp;+\u0026thinsp;T; Fig S2A) had little to no effect on cell viability compared to untreated biofilms (U) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003e \u003cb\u003eExposure to triclosan throughout biofilm formation results in increased polysaccharide production.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eFluorescent staining was used to characterise the composition of triclosan exposed \u003cem\u003eS. aureus\u003c/em\u003e biofilms (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). Alongside DAPI to visualise cells, fluorescein-conjugated WGA was used to stain the PNAG residues of the PIA polysaccharide that is prevalent in the \u003cem\u003eS. aureus\u003c/em\u003e biofilm matrix. CLSM revealed that triclosan exposure significantly alters the production of polysaccharide in the \u003cem\u003eS. aureus\u003c/em\u003e biofilm matrix. Echoing the findings of previous antibiotic tolerance experiments, oleic acid supplementation was able to largely negate the triclosan-induced changes, with enhanced polysaccharide production not being observed in OA\u0026thinsp;+\u0026thinsp;T biofilms. Comstat2 quantification showed that triclosan biofilms are composed of significantly more WGA stained PNAG compared to untreated (U), oleic acid only (OA) or oleic acid and triclosan (OA\u0026thinsp;+\u0026thinsp;T) pretreatments (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB), indicative of more PIA polysaccharide.\u003c/p\u003e \u003cp\u003e \u003cb\u003eSarA coordinates triclosan-induced polysaccharide synthesis.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eFollowing the findings that physiologically relevant levels of triclosan could induce antibiotic tolerance and alter biofilm formation, the molecular mechanism behind these changes was investigated. The effect of the staphylococcal accessory regulator (Sar) was first explored, as Sar controls the production of PIA in the biofilm matrix of \u003cem\u003eS. aureus\u003c/em\u003e. Furthermore, PIA can impede the penetration and killing of numerous antibiotics \u0026ndash; including vancomycin, ciprofloxacin, and rifampicin (\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e). Imaging \u003cem\u003esarA\u003c/em\u003e biofilms using confocal microscopy revealed that triclosan exposure did not result in an increase in the quantity of polysaccharide within the biofilm matrix (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA), unlike in the WT, in which triclosan exposure resulted in significantly higher proportions of PIA in the biofilm matrix (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). Calcofluor white staining of biofilms, in which the binding of the stain to β(1\u0026rarr;4) linked ᴅ-glucose or derivatives was used to quantify polysaccharide concentrations, confirmed the microscopy results (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). The lack of increased matrix polysaccharide of the \u003cem\u003esarA\u003c/em\u003e mutant biofilms in the presence of triclosan, suggests triclosan-induced stimulation of SarA results in increased polysaccharide in triclosan exposed biofilms. Using the \u003cem\u003esarA\u003c/em\u003e mutant strain in planktonic kill-curve experiments, \u003cem\u003esar\u003c/em\u003e was ruled out from orchestrating triclosan-induced antibiotic tolerance since the triclosan exposed \u003cem\u003esarA\u003c/em\u003e mutant, like the WT, exhibited tolerance to vancomycin and ciprofloxacin (Fig S3).\u003c/p\u003e \u003cp\u003e \u003cb\u003eThe stringent response is essential for triclosan-induced antibiotic tolerance in\u003c/b\u003e \u003cb\u003eS. aureus\u003c/b\u003e \u003cb\u003ebiofilms.\u003c/b\u003e\u003c/p\u003e \u003cp\u003eAfter experiments investigating the action of SarA in response to triclosan exposure, the mechanism behind triclosan-induced antibiotic tolerance remained elusive. The stringent response has been associated with triclosan-induced antibiotic tolerance in planktonic \u003cem\u003eE. coli\u003c/em\u003e (\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e). This study sought to determine whether this was also the case in \u003cem\u003eS. aureus\u003c/em\u003e through the use of an \u003cem\u003eS. aureus\u003c/em\u003e stringent response (p)ppGpp\u003csup\u003e0\u003c/sup\u003e mutant incapable of producing (p)ppGpp. In planktonic kill-curve experiments, the stringent response was found to play no role in coordinating triclosan-induced antibiotic tolerance in planktonic culture (Fig S4). Although the (p)ppGpp\u003csup\u003e0\u003c/sup\u003e strain was less viable compared to the WT when treated with triclosan and antibiotic combinations, this was because of the increased sensitivity of the (p)ppGpp\u003csup\u003e0\u003c/sup\u003e mutant to triclosan, rather than the stringent response negating triclosan-induced antibiotic tolerance.\u003c/p\u003e \u003cp\u003eConversely, the stringent response was essential for coordinating triclosan-induced antibiotic tolerance in \u003cem\u003eS. aureus\u003c/em\u003e biofilms. Live/dead staining shows the (p)ppGpp\u003csup\u003e0\u003c/sup\u003e strain failing to withstand ciprofloxacin (1\u0026times; MBEC), vancomycin (1\u0026times; MBEC), and rifampicin (1\u0026times; MBEC) treatment despite triclosan exposure throughout biofilm maturation ((p)ppGpp\u003csup\u003e0\u003c/sup\u003e T\u0026thinsp;+\u0026thinsp;C, Fig S5B; (p)ppGpp\u003csup\u003e0\u003c/sup\u003e T\u0026thinsp;+\u0026thinsp;V, Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA; (p)ppGpp\u003csup\u003e0\u003c/sup\u003e T\u0026thinsp;+\u0026thinsp;R, Fig S5C). Comstat2 quantification of live cell biomass shows triclosan significantly protects HG001 WT against ciprofloxacin, vancomycin, and rifampicin (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB), whilst the (p)ppGpp\u003csup\u003e0\u003c/sup\u003e T\u0026thinsp;+\u0026thinsp;C, (p)ppGpp\u003csup\u003e0\u003c/sup\u003e T\u0026thinsp;+\u0026thinsp;V, and (p)ppGpp\u003csup\u003e0\u003c/sup\u003e T\u0026thinsp;+\u0026thinsp;R biofilms demonstrate levels of viability comparative to WT C, WT V, and WT R respectively. Moreover, the inability of the (p)ppGpp\u003csup\u003e0\u003c/sup\u003e biofilm to handle stress is highlighted by the reduced viability of the untreated ((p)ppGpp\u003csup\u003e0\u003c/sup\u003e) and triclosan exposed ((p)ppGpp\u003csup\u003e0\u003c/sup\u003e T), relative the same conditions in the WT (WT, WT T; Fig S5A, 5B).\u003c/p\u003e \u003cp\u003eEndpoint CFUs of \u003cem\u003eS. aureus\u003c/em\u003e biofilms also verified that for the HG001 WT, triclosan exposed biofilms were protected against killing by high concentrations of ciprofloxacin (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC), vancomycin (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD), and rifampicin (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE). Additionally, neither oleic acid supplementation nor the use of the (p)ppGpp\u003csup\u003e0\u003c/sup\u003e mutant resulted in triclosan-induced antibiotic tolerance, further validating the live/dead imaging. In HG001 WT, triclosan was able to induce a 1000-fold increase in tolerance to ciprofloxacin (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC) and rifampicin (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE), and a 100-fold increase in vancomycin tolerance (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD). These findings are even more striking when considering that triclosan pretreated biofilms had been exposed to otherwise lethal concentrations of antibiotics for 12 hours, as opposed to the 3 hours of antibiotic treatment seen in the live/dead biofilm experiments. This could suggest that triclosan exposed \u003cem\u003eS. aureus\u003c/em\u003e can withstand treatment with extensive levels of antibiotics for prolonged periods of time. Despite these significant changes to antibiotic susceptibility in (p)ppGpp\u003csup\u003e0\u003c/sup\u003e biofilms, triclosan exposure still resulted in excess polysaccharide being produced in these biofilms (Fig S6A, S6B, S6C), further underlining that triclosan-induced antibiotic tolerance and triclosan-induced polysaccharide production are distinct mechanisms.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThis study aimed to characterise the effects of physiologically relevant levels of triclosan on \u003cem\u003eS. aureus\u003c/em\u003e. Here, we show that 500 ng/mL triclosan, well within the limits of triclosan previously detected in human urine (2.4\u0026ndash;3,790 ng/mL) (\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e), can trigger antibiotic tolerance in \u003cem\u003eS. aureus\u003c/em\u003e biofilms, and alter biofilm formation. Whereas triclosan-induced biofilm formation was dependent on the global regulator SarA, triclosan-induced antibiotic tolerance in biofilms was (p)ppGpp dependent. Triclosan-induced biofilm formation as well as antibiotic tolerance were remediated by oleic acid, demonstrating that interruption of fatty acid biosynthesis is the main mode of triclosan action.\u003c/p\u003e \u003cp\u003eAntimicrobial induced biofilm formation has been previously described (\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e, \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e, \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e). Here, we shed light on underlying mechanisms by demonstrating that triclosan increases the proportions of polysaccharide present in the biofilm matrix of \u003cem\u003eS. aureus\u003c/em\u003e. Typically, biofilm formation is stimulated by sub-MIC levels of an antibiotic (\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e, \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e). However, in this study, 500 ng/mL of triclosan, whilst a physiologically relevant concentration of the biocide, is not sub-MIC. This is evidenced by triclosan halting the growth of planktonic cultures and decreasing biofilm cell density. A decrease in cell mass and concurrent stimulation of biofilm matrix production is contrary to many examples of antibiotic induced biofilm formation, but is not a complete anomaly. Skogman and colleagues (2012) found similar results when treating \u003cem\u003eS. aureus\u003c/em\u003e biofilms with penicillin. They hypothesised that some antimicrobials decrease cell viability whilst increasing production of biofilm matrix components. However, these changes can only be confirmed through parallel measurement of biofilm viability, biomass, and quantifying matrix components, as in this study. Therefore, it may be that more antimicrobials previously associated with stimulating biofilm formation fall into this category, but this is yet to be fully characterised (\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e). Live/dead staining of triclosan exposed biofilms did not detect any notable increase in the proportions of dead cells. Instead, it appears that triclosan exposed biofilms consist of a reduced population that produces and exports far more PIA, relative to unexposed biofilms.\u003c/p\u003e \u003cp\u003eSarA is a known regulator of \u003cem\u003eicaADBC\u003c/em\u003e expression, thereby altering the production of PIA synthesis enzymes (\u003cspan additionalcitationids=\"CR58 CR59 CR60 CR61\" citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e). SarA played a key role in triclosan-induced PIA overproduction since a \u003cem\u003esarA\u003c/em\u003e mutant was unable to overproduce PIA following triclosan exposure. PIA overproduction could be beneficial for numerous reasons (\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e), including increased tolerance to mechanical forces and resistance to immunological stresses, such as killing by host antimicrobial peptides and polymorphonuclear leukocytes (\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e, \u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e), opsonisation by antibodies and complement (\u003cspan additionalcitationids=\"CR66\" citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e), and phagocytosis by macrophages (\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e). PIA producing Staphylococci have previously been shown to be less susceptible to killing by some antibiotics, including vancomycin and ciprofloxacin (\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e, \u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e68\u003c/span\u003e, \u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e69\u003c/span\u003e). Accordingly, this study hypothesised that triclosan-induced polysaccharide production protected \u003cem\u003eS. aureus\u003c/em\u003e from killing by antibiotics, and was therefore the cause of triclosan-induced antibiotic tolerance. However, since the \u003cem\u003esarA\u003c/em\u003e mutant displayed antibiotic tolerance despite no longer producing excess polysaccharide, there does not appear to be a direct link between polysaccharide production and antibiotic tolerance following triclosan exposure.\u003c/p\u003e \u003cp\u003eIn biofilms, triclosan-induced antibiotic tolerance was orchestrated by the stringent response. A (p)ppGpp\u003csup\u003e0\u003c/sup\u003e strain still overproduces PIA upon triclosan treatment, but no longer benefits from triclosan-induced antibiotic tolerance. Westfall et al. (2019) found that triclosan pretreatment protected planktonic \u003cem\u003eE. coli\u003c/em\u003e against ampicillin, kanamycin, streptomycin and ciprofloxacin (\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e), our lab has also previously demonstrated that triclosan can protect \u003cem\u003eE. coli\u003c/em\u003e from both ciprofloxacin mediated killing and changes to cell morphology (\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e). Triclosan-induced antibiotic tolerance in planktonic \u003cem\u003eE. coli\u003c/em\u003e was mediated by the stringent response (\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e). In \u003cem\u003eS. aureus\u003c/em\u003e, triclosan exposure increased the tolerance of \u003cem\u003eS. aureus\u003c/em\u003e biofilms against high doses of ciprofloxacin, vancomycin, and rifampicin. When \u003cem\u003eS. aureus\u003c/em\u003e is in its planktonic lifestyle the loss of its stringent response was unable to abolish triclosan-induced antibiotic tolerance but almost entirely negated triclosan-induced antibiotic tolerance in \u003cem\u003eS. aureus\u003c/em\u003e biofilms. This suggests that triclosan-induced antibiotic tolerance is initially coordinated by a currently unidentified stress response in planktonic \u003cem\u003eS. aureus\u003c/em\u003e, whilst the stringent response is important in maintaining triclosan-induced antibiotic tolerance in \u003cem\u003eS. aureus\u003c/em\u003e biofilms. Fatty acid starvation might activate (p)ppGpp synthetase in \u003cem\u003eS. aureus\u003c/em\u003e (\u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e70\u003c/span\u003e) and \u003cem\u003eB. subtilis\u003c/em\u003e (\u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e71\u003c/span\u003e). However, mechanisms and conditions related to how fatty acid starvation could interfere with Rel activity remain unclear. Our data indicate that such putative conditions differ between planktonic and biofilm grown bacteria.\u003c/p\u003e \u003cp\u003eThe triclosan-induced changes to \u003cem\u003eS. aureus\u003c/em\u003e physiology appear to originate from fatty acid starvation. When growth medium is supplemented with concentrations of oleic consistent with those found in human serum (\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e), the antibiotic susceptibility of triclosan pretreated \u003cem\u003eS. aureus\u003c/em\u003e HG001 is restored and biofilm formation unchanged relative to untreated controls. The restoration of antibiotic susceptibility when fatty acid starvation is negated is logical, as fatty acid starvation is one of the numerous nutritional deficiencies capable of instigating the stringent response. However, the link between fatty acid starvation and SarA is less clear, and may suggest the effects of fatty acid starvation are broader than previously thought. The observation that oleic acid supplementation was able to override triclosan-induced affects at all is striking, as the notion that exogenous fatty acids can overcome the effects of fatty acid synthesis inhibitors has been viewed as controversial (\u003cspan additionalcitationids=\"CR72 CR73 CR74\" citationid=\"CR71\" class=\"CitationRef\"\u003e71\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR75\" class=\"CitationRef\"\u003e75\u003c/span\u003e). Since the concentration of triclosan used in this experiment was low in comparison to triclosan concentrations in healthcare and household products, it cannot be concluded whether fatty acid supplementation is sufficient to save \u003cem\u003eS. aureus\u003c/em\u003e from higher concentrations of triclosan. However, the data does suggest serum concentrations of oleic acid (\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e) would be sufficient to overcome tolerance induced by concentrations of triclosan that have accumulated in the human body (\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e). Triclosan pretreatment was able to reduce the efficacy of ciprofloxacin against \u003cem\u003eE. coli\u003c/em\u003e by 100-fold in an \u003cem\u003ein-vivo\u003c/em\u003e murine model, illustrating that triclosan-induced tolerance can endure outside of \u003cem\u003ein-vitro\u003c/em\u003e settings (\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e). However, as \u003cem\u003eE. coli\u003c/em\u003e and \u003cem\u003eS. aureus\u003c/em\u003e are known to utilise exogenous fatty acids by different mechanisms (\u003cspan citationid=\"CR74\" class=\"CitationRef\"\u003e74\u003c/span\u003e), \u003cem\u003ein-vivo\u003c/em\u003e work using triclosan treated \u003cem\u003eS. aureus\u003c/em\u003e would be needed to determine this.\u003c/p\u003e \u003cp\u003eThis present study shows that exposure to physiologically relevant levels of triclosan can drive \u003cem\u003eS. aureus\u003c/em\u003e to trigger multiple, divergent stress responses that alter numerous facets of \u003cem\u003eS. aureus\u003c/em\u003e physiology. These physiological changes are rooted in the stress caused by triclosan-induced fatty acid starvation, before branching off. These diverging responses provide protection against antibiotics, facilitated by the stringent response, and potentially protect from other threats mediated by SarA controlled polysaccharide synthesis.\u003c/p\u003e \u003cp\u003eSkogman and colleagues (2012) advised that the criteria for determining an effective antimicrobial treatment should be based on bacterial viability, biofilm biomass, plus matrix composition. We suggest going further to incorporate potential antibiotic tolerance. If the concentration of triclosan used in this study was to be evaluated based only on viability, triclosan would be deemed an effective therapy. However, when factoring in the increased matrix production and pleiotropic antibiotic tolerance induced by the biocide, triclosan appears far less alluring. This is further compounded by the finding that triclosan-induced affects occurred at an inhibitory concentration, rather than at sub-MIC levels. Thereby emphasising that accumulated or residual antimicrobials in the human body may cause large scale physiological change to pathogens. This reemphasises the need for stricter control on biocide use globally.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDW was funded via the Biotechnology and Biological Sciences Research Council (BBSRC), UK Doctoral Training Programme studentship (BB/M008770/1: www.bbsrc.ac.uk) held jointly by the School of Life Sciences and School of Pharmacy, University of Nottingham. KH and JA are partly funded by the National Biofilms Innovation Centre (NBIC) which is an Innovation and Knowledge Centre funded by the BBSRC and InnovateUK (Award Number BB/R012415/1). We thank the School of Life Sciences Imaging Facility (SLIM) for input on image analysis (particularly Robert Markus, Seema Bagia and Tim Self). CW was funded by Deutsche Forschungsgemeinschaft, Schwerpunktprogramm Spp1879 to CW (Project 423246275) and by infrastructural funding from the Deutsche Forschungsgemeinschaft (DFG), Cluster of Excellence EXC 2124 \u0026ldquo;Controlling Microbes to Fight Infections\u0026rdquo; (Project 390838134). The project was also supported by the University of Nottingham and the University of T\u0026uuml;bingen\u0026rsquo;s funding as part of the Excellence Strategy of the German Federal and State Governments, in close collaboration with the University of Nottingham.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eKH and DW conceptualized and drove the study. KH, JA and CW supervised the generation and curation of the data by DW, PI and AS. DW created the original manuscript draft, and all authors contributed to the reviewing and editing of the manuscript.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eSpeziale P, Pietrocola G, Foster TJ, Geoghegan JA. Protein-based biofilm matrices in Staphylococci. Front Cell Infect Microbiol. 2014;4:171.\u003c/li\u003e\n\u003cli\u003eO\u0026apos;Gara JP. ica and beyond: biofilm mechanisms and regulation in Staphylococcus epidermidis and Staphylococcus aureus. FEMS Microbiol Lett. 2007;270(2):179-88.\u003c/li\u003e\n\u003cli\u003eMoormeier DE, Bose JL, Horswill AR, Bayles KW. Temporal and stochastic control of Staphylococcus aureus biofilm development. mBio. 2014;5(5):e01341-14.\u003c/li\u003e\n\u003cli\u003eMoormeier DE, Bayles KW. Staphylococcus aureus biofilm: a complex developmental organism. Mol Microbiol. 2017;104(3):365-76.\u003c/li\u003e\n\u003cli\u003eLimoli DH, Jones CJ, Wozniak DJ. 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Biochimie. 2017;141:30-9.\u003c/li\u003e\n\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":"npj-antimicrobials-and-resistance","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"npjamar","sideBox":"Learn more about [npj Antimicrobials and Resistance](http://www.nature.com/npjamar/)","snPcode":"44259","submissionUrl":"https://submission.springernature.com/new-submission/44259/3","title":"npj Antimicrobials and Resistance","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"NPJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Biofilms, Antibiotic resistance, Antibiotic tolerance, Biocides, Triclosan, Staphylococcus aureus, stringent response, SarA, polysaccharide intercellular adhesin","lastPublishedDoi":"10.21203/rs.3.rs-3954016/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3954016/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe biocide triclosan is used extensively in both household and hospital settings. The chronic exposure to the biocide occurring in individuals that use triclosan-containing products results in low levels of triclosan present in the human body that has been linked to induction of antibiotic tolerance and altered biofilm formation. Here we aimed to unravel the molecular mechanisms involved in triclosan-induced antibiotic tolerance and biofilm formation in \u003cem\u003eStaphylococcus aureus\u003c/em\u003e. Triclosan treatment prior to planktonic exposure to bactericidal antibiotics resulted in 1,000 fold higher viable cell counts compared to non-pretreated cultures. Triclosan pretreatment also protected \u003cem\u003eS. aureus\u003c/em\u003e biofilms against otherwise lethal doses of antibiotics as shown by live/dead cell staining and viable cell counting. Triclosan mediated antibiotic tolerance in \u003cem\u003eS. aureus\u003c/em\u003e biofilms required an active stringent response because biofilms of a pppGpp\u003csup\u003e0\u003c/sup\u003e strain were not protected from antibiotic killing. Incubation of \u003cem\u003eS. aureus\u003c/em\u003e with triclosan also altered biofilm structure due to SarA-mediated overproduction of the polysaccharide intercellular adhesin (PIA) in the biofilm matrix. Thus, physiologically relevant concentrations of triclosan can trigger (p)ppGpp dependent antibiotic tolerance as well as SarA dependent biofilm formation.\u003c/p\u003e","manuscriptTitle":"Triclosan alters biofilm structures and confers antibiotic tolerance in Staphylococcus aureus using multiple regulatory pathways","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-03-25 17:58:15","doi":"10.21203/rs.3.rs-3954016/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"npj-antimicrobials-and-resistance","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"npjamar","sideBox":"Learn more about [npj Antimicrobials and Resistance](http://www.nature.com/npjamar/)","snPcode":"44259","submissionUrl":"https://submission.springernature.com/new-submission/44259/3","title":"npj Antimicrobials and Resistance","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"NPJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"c31470cb-224f-4976-b60e-7bf51bbe8fa5","owner":[],"postedDate":"March 25th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2025-04-19T22:45:08+00:00","versionOfRecord":[],"versionCreatedAt":"2024-03-25 17:58:15","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-3954016","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3954016","identity":"rs-3954016","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}