The modulating effect on Staphylococcus species and Pseudomonas aeruginosa biofilm development of salivary pellicle conditioning titanium surfaces

The modulating effect on Staphylococcus species and Pseudomonas aeruginosa biofilm development of salivary pellicle conditioning titanium surfaces | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article The modulating effect on Staphylococcus species and Pseudomonas aeruginosa biofilm development of salivary pellicle conditioning titanium surfaces Miryam Martínez-Hernández, Polet Reyes-Mendoza, Mariana Chávez-Esparza, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6638066/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Objectives To evaluate the modulating effect of salivary pellicle (SP) on titanium (Ti) surfaces on the development of Staphylococcus species and Pseudomonas aeruginosa biofilm. Materials and Methods Ti substrates were incubated for 2 hours with whole saliva samples to induce the salivary pellicle formation. After conditioning with SP, Ti substrates were incubated for 12 hours with P. aeruginosa , S. aureus , S. epidermidis strains individually, in addition to co-culture of the three bacteria. The biofilm development on the Ti substrates was visualized by scanning electron microscopy (SEM). To measure the metabolic activity and vitality of cells within the biofilm the XTT assay was used, while the proportion of the species tested in the biofilms was determined throughout DNA-DNA hybridizations (checkerboard). Results Salivary pellicle modulated the biofilm development on the Ti surfaces, favouring the formation of P. aeruginosa biofilms, while inhibiting the growth of S. aureus and S. epidermidis . In the case of the coculture biofilms, a predominance of P. aeruginosa cells over the Staphylococcus strains was observed. Conclusions When the bacterial strains were tested individually, the SP importantly reduced the development of biofilms of Staphylococcus species, whereas it favored the development of P. aeruginosa biofilms on Ti surfaces. Similarly, when mixed biofilms developed, SP favored the selective expansion of P. aeruginosa and S. aureus growth over S. epidermidis growth. Clinical relevance: The results of this study provide valuable information on the modulatory effect of the SP on the development of opportunistic bacteria biofilms on Ti surfaces used for dental and oral implantology. Salivary pellicle titanium surfaces dental implants Staphylococcus aureus Staphylococcus epidermidis Pseudomonas aeruginosa Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction Biofilm formation on biomedical surfaces is always a concern when they are in operation in the oral cavity, mainly because, the oral cavity, has one of the largest and most diverse microbiotas of the body, harbouring a diverse set of over 774 different bacterial species [ 1 , 2 ]. The vast majority of the oral cavity bacteria exists in the form of biofilms. In general, dental biofilm maintain a harmonious relationship with the host, providing benefits to overall health [ 3 , 4 ], nevertheless, alterations in biofilm composition caused by certain stress factors, such as tobacco use or lack of plaque removal, can alter the biofilm homeostasis leading to the development of oral diseases, such as periodontitis or peri-implantitis [ 5 ]. Peri-implantitis is the main biological complication that can negatively affect long term outcomes of the osseointegrated dental implants [ 6 ]. Even though the long-term survival rates of dental implants are high (≥ 10 years) [ 7 ], it is estimated that approximately 1/3 of all patients and 1/5 of all implants placed will experience peri-implantitis [ 8 ]. The peri-implant infections have often been regarded as pathologies analogous to gingivitis and periodontitis, mainly because those pathologies share similarities in clinical characteristics and aetiology [ 9 ]. However, there is increasing evidence supporting the notion of periimplantitis is not periodontitis [ 10 ], such as critical histopathological differences between peri-implantitis and periodontitis have been found [ 11 ], in addition to current microbial identification methods have revealed an unexpected difference between the microbiome surrounding teeth and that surrounding implants [ 12 ]. Regarding the characterization of the microbiota associated with the presence of peri-implant damage, through the use of DNA probes and culture analysis, it has been possible to isolate common periodontopathogenic bacteria in diseased peri-implant sites, including Porphyromonas gingivalis and Tannerella forsythia [ 13 ], however, it has recently been reported that the development of peri-implant lesions could be associated with bacterial species other than canonical bacterial associated with periodontitis, namely P. gingivalis , Treponema denticola and T. forsythia [ 14 ]. In this regard, it has been pointed out that the presence, in high proportions, of pathogenic-opportunistic and antibiotic-resistant bacteria such as Pseudomonas aeruginosa , Staphylococcus aureus and S. epidermidis could play an important role in the etiopathogenesis of peri-implantitis [ 15 – 18 ]. These reports indicate that the presence of such bacterial strains on dental implant surfaces is due to a certain “affinity for Titanium (Ti)” [ 15 , 19 , 20 ]. However, the underlying mechanisms of this affinity have not been explored in depth. Ti surfaces of dental implants vs. cementum or natural tooth enamel represent chemically different substrates; differences in surface energy, topography, wettability, and electrochemical charges of these substrates will modulate bacterial adhesion, ultimately resulting in the formation of distinct biofilms on the different substrates [ 21 ]. An essential prerequisite for bacterial adhesion and subsequent biofilm development in the oral cavity is salivary pellicle (SP) formation. SP is a thin film coating all oral surfaces, and mainly consists of amino and fatty acids, proteins and carbohydrates derived mainly from saliva, but also from microorganisms [ 22 , 23 ]. The formation of SP involves a specific and non-random complex process that will result in the selective adsorption of biomolecules [ 24 ]. The SP formed on Ti surfaces have aroused great interest due to their importance in peri-implant health [ 25 , 26 ]. SP formed on dental enamel and the one formed on titanium surfaces have showed important molecular differences [ 27 ], which could explain the presence of S. epidermidis , S. aureus [ 15 , 17 , 28 ], and P. aeruginosa [ 29 – 31 ] in peri-implantitis sites. In a study by El-Telbany, et al .[ 31 ] it was possible to isolate P. aeruginosa in 10% of cases of peri-implantitis evaluated, indicating that the presence of these pathogens would be associated to a higher prevalence of disease [ 32 ]. Since the modulating effect of SP formed on titanium surfaces in the formation of oral biofilms is now recognized [ 33 ], a deeper understanding of how this protein film affects the adhesion of non-canonical periodontal pathogens, such as the opportunistic pathogens Staphylococcus species and Pseudomonas aeruginosa , to which the aetiology of peri-implantitis has even been attributed, is now needed. Materials and methods Ti surfaces tested Ti disks with diameter of 15 mm were prepared from 1-mm-thick sheets of grade 2 unalloyed Ti (ASTM F67). The methods used to produce the pretreatment (PT) surfaces have been previously reported [ 27 ]. The Ti surfaces are relatively smooth with an average roughness of (Ra) < 0.19 µm and a water contact angle of 81.9° ± 0.4°. Ti disks were manufactured by Institut Straumann AG (Basel, Switzerland) and delivered to us ready to use. For each experiment, the number of Ti disks was adjusted to normalize the total surface area evaluated to 8 cm 2 . Salivary pellicle formation assays Whole human saliva samples used for salivary pellicle formation assays on titanium substrates were obtained from a healthy, middle-aged volunteer with no active caries lesions or history of periodontal disease. The healthy saliva donor was currently nonsmoker, with more than 20 natural teeth (excluding third molars). Clinical measurements were taken from the healthy volunteer at six sites per tooth (mesiobuccal, buccal, distobuccal, distolingual, lingual, and mesiolingual) at all teeth excluding third molars (approximately 168 sites evaluated) as previously described [ 34 ]. Before saliva samples were collected, the volunteer was asked to rinse their mouth with clean water for 30 seconds to remove desquamated epithelial cells, microorganisms and food debris. Sterile 50 ml tubes were used to collect passive drooling saliva for 3 minutes. Saliva samples were centrifuged at 15,000 g, 4°C, for 15 minutes; the resulting clean supernatants, supplemented with the complete EDTA-free protease inhibitor mixture (RocheDiagnostics, Penzberg, Germany), were used for salivary pellicle formation assays on Ti substrates. The saliva donor gave informed consent acknowledging his willingness to participate. This study was performed under the guidelines of the Declaration of Helsinki and approved by the Ethics Committee for Human Studies of the Division of Postgraduate Studies and Research, School of Dentistry, National Autonomous University of Mexico (CIE/0108/09/2023). To induced salivary pellicle formation, Ti surfaces were placed individually in 24-well plates with 500 µL of whole saliva and incubated for two hours at 37°C in a chamber with 10% humidity. After incubation, the surfaces were washed twice with 1 mL of H 2 O dd to remove non-adsorbed proteins. Another set of surfaces incubated under the same conditions without salivary film formation, was used as a control. Subsequently, the surfaces were transferred to new sterile 24-well cell culture plates and were immediately used in biofilm development assays. Biofilm development assays of Staphylococcus and Pseudomonas aeruginosa species Three reference strains (Table 1 ) were used for in vitro biofilm development assays on the tested surfaces. All strains were obtained as lyophilized cultures from the American Type Culture Collection (ATCC, Rockville, MD, USA). Table 1 Reference strains used for the biofilm development assays Specie Strain a Gram Staphylococcus aureus 25923 Positive Staphylococcus epidermidis 14990 Pseudomonas aeruginosa 43636 Negative a American Type Culture Collection, Rockville, MD, USA. Pure cultures of each of the three bacterial strains were obtained by seeding individually on plates with Trypticase Soy Agar (TSA; BBL, Becton-Dickinson) enriched with 0.3 µg/mL menadione (Sigma-Aldrich) and 5 µg/mL hemin (Sigma-Aldrich) and incubated for 24 h at 37°C under aerobic conditions. After incubation period, the different bacterial cultures were harvested and individually suspended in tubes contained enriched TSB broth (TSB added with 0.3 µg/mL menadione and 5 µg/mL hemin). The optical density (OD) in each tube was adjusted to 1 at λ = 600 nm in a spectrophotometer (FilterMax F5 Multi-Mode Microplate Reader, Thermo Fisher) to obtain a bacterial suspension with 1 x10 6 cells/mL. To test biofilm development, Ti surfaces conditioned with SP (experimental surfaces) or uncoated with SP (control surfaces) were independently incubated with 1 mL of bacterial suspension of S. aureus , S. epidermidis or P. aeruginosa or co-culture of those strains. The inoculated surfaces were incubated for 12 h at 37°C under aerobic conditions in an orbital shaker (≈ 160 rpm), under aerobic atmosphere. After aerobic incubation, each surface was washed twice with 1 mL of enriched broth to detach bacterial cells not adhered to the surfaces. All experiments were run in triplicate and repeated at least two different times. Scanning Electron Microscopy (SEM) observations The microscopic appearance of the biofilm of S. aureus , S. epidermidis , and P. aeruginosa or bacterial coculture developed on Ti surfaces covered and uncovered (control surfaces) by the salivary pellicle was observed by SEM. In brief, Samples were fixed in 2.0% glutaraldehyde 24 h at room temperature. Then washed three times with phosphate buffer solution (pH 7.4), followed by dehydration through a series of graded ethanol solutions of 20, 40, 60, 80, and 100%. Then, the specimens were stored in a desiccator for 24 hours, and sputter-coated with a gold layer (SDC 050; Bal-Tec AG, Balzers, Germany). Thereafter, the specimens were fixed with carbon tape to stubs, placed into the vacuum chamber of a scanning electron microscope (SEM (JEOL JSM5600-LV, Japan), and the central areas of the specimens were photographed at different magnification. Images were obtained in high vacuum mode with secondary electrons at 20–25 kV. Viability analysis The viability of the biofilm cells of S. aureus , S. epidermidis , and P. aeruginosa , or the co-culture of these strains, on salivary pellicle-covered or uncovered Ti surfaces was estimated by was analysed by using the XTT (2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide) assay (Sigma, MO, USA). Briefly, after aerobic incubation, the inoculated surfaces were rinsed twice with PBS to remove non-adhered bacteria and transferred to sterile well plates. Then, the surfaces were incubated for 3 h in 1 mL of enriched TSB broth added with 20 µL of the XTT solution. Finally, the O.D of 100 µL aliquots of the supernatants were read at λ = 450 nm in a FilterMaxF5 multi-mode microplate reader (Molecular Devices, USA), and the number of viable bacteria was calculated according to standard curves previously performed. Checkerboard analysis After aerobic bacterial incubation, each Ti specimen was washed twice with TSB broth. After washing, 1 ml of enriched broth was added and samples were sonicated for 5 periods of 10 s each to detach the adherent bacteria to each Ti surface, coated and not covered by salivary pellicle. To determine the proportion of each bacterial strain of the biofilm developed on the Ti surfaces, 100 µl of the bacterial suspension obtained after sonication of each sample were placed in individual Eppendorf tubes with 100 µl of 0.5 M NaOH. Bacterial species form the biofilm developed were identified and quantified using the checkerboard DNA–DNA hybridization technique previously described [ 34 , 35 ]. Briefly, DNA probes were prepared using the growth from 3- to 7-day cultures of the three reference strains used for the biofilm development assays ( Table I ). Bacterial growth was harvested and placed in tubes containing 1 mL of TE buffer. Cells were washed twice and lysed at 37 ºC for 1 h with either 10% sodium dodecyl sulfate (Sigma-Aldrich) (SDS) plus proteinase K (Sigma-Aldrich) (20 mg/ml) for Gram-negative strains or lysozyme (Sigma-Aldrich) (15 mg/mL) plus achromopeptidase (Sigma-Aldrich) (5 mg/ml) for Gram-positive strains. DNA was isolated and purified. Whole-genomic DNA probes were prepared for each species by labeling 1 mg DNA with digoxigenin (Roche Diag- nostics, Mannheim, Germany) using the random primer technique [ 36 ]. The sensitivity of the assay was set to allow the detection of approximately 10 4 cells of a given species by adjusting the concentration of each individual DNA probe. For the DNA–DNA hybridization, each sample was thawed at room temperature, boiled for 10 min, and neutralized with 800 µl 5 M ammonium acetate (Sigma-Aldrich). The released DNA from each sample was then placed into individual lanes (Min- islot-30, Immunetics, Cambridge, MA), concentrated onto a 15×15 cm positively charged nylon membrane (Roche Diagnosis), and fixed to the membrane by cross-linking under ultraviolet light. Two lanes on each membrane contained standards consisting of a mixture at 10 5 and 10 6 cells of each bacterial species tested. The membranes were prehybridized at 42 ºC for 2 h and placed in a second device (Miniblotter-45, Immunetics) with the sample lanes rotated 90º to the channels of the apparatus. Probes were hybridized in two sets of three consecutive channels, leaving empty channels (hybridization solution only) to allow noise and background correction of signals. The membranes were washed twice at high stringency for 20 min each time at 68 ºC in phosphate buffer (0.1X SSC and 0.1% SDS). Membranes were blocked by 1-h incubation in blocking buffer containing 1% casein in maleate buffer [100 m M maleic acid (Sigma-Aldrich), and 150 m M NaCl, pH 7.5]. Hybrids were detected by exposing the membranes to a 1:50,000 dilution of antidigoxigenin antibody conjugated to alkaline phosphatase (Roche Diagnostics) for 30 min. Signals were detected by chemiluminescence using a chemiluminescent agent (CDP-Star, Roche Diagnostics) for 30 min on the membranes at room temperature and exposed to films in autoradiographic cassettes for 30 min. Signals were detected with specialized software (Quantity One, BioRad Laboratories), adjusted by subtracting the average plus two standard deviations of the noise and background detected in the empty lanes, and converted to absolute counts by comparison with the standards on the membrane. Failure to detect a signal was recorded as zero. Statistical analysis Data for XTT values and total bacterial counts are presented as the Mean ± Standard Error of the Mean (SEM). All data were analysed by Student´s t -test followed by Bonferroni correction for multiple comparisons, for paired comparisons and differences between strains with ANOVA test by IBM-SPSS 21 software. Significant differences ( p > 0.05) were determined. Results In the present work, the modulating effect of the salivary film formed on titanium surfaces on the development of biofilms of Staphylococcus species and Pseudomonas aeruginosa was investigated. Saliva donor For the present investigation, a donor subject was selected to reduce the variability in the protein profile that could result from the inclusion of different volunteers, as has been previously reported [ 37 ]. The volunteer underwent a full periodontal evaluation to confirm the diagnosis of periodontal health, which he defined as the absence of sites with probing depths ≤ 3mm and bleeding values at full-mouth probing < 10% [ 5 ]. The results of the full periodontal evaluation are presented in a supplementary Table . Biofilm morphology evaluated by SEM The following are images obtained under Scanning Electron Microscopy using secondary electrons of biofilms developed by S. aureus , S. epidermidis , P. aeruginosa , and their coculture, formed on Ti surfaces conditioned with SP (experimental) and unconditioned by SP (control). (Fig. 1 – 4 ). As can be seen in the previous image, the biofilms developed by S. aureus were slightly more complex on the control Ti substrates, i.e., they were not conditioned with the SP, compared to the S. aureus biofilms developed on the Ti substrates conditioned with the SP. In micrograph 2A , corresponding to the Ti surfaces conditioned with the salivary film, it is possible to observe S. aureus cells forming grape-like clusters, periodically distributed on the conditioned substrate, however, on these surfaces it was not possible to observe the formation of a highly complex biofilms, this finding can be seen in greater detail mainly in image 2B . Regarding to the control Ti substrates (not conditioned with the SP), images 2C and 2D show that greater bacterial adhesion occurred on these surfaces, also observing a more homogeneous distribution on the surface of cocci clusters. Like what was observed in the formation of S. aureus biofilms on Ti substrates, the development of S. epidermidis biofilms was higher on the control substrates (Fig. 2 ), i.e., those that were not conditioned with the SP. On the Ti surfaces that were conditioned with the SP (images 3A and 3B ), it is possible to appreciate few bacterial grape-like clusters adhered on the conditioned surface, compared to the Ti surfaces that were not conditioned with the SP; on these surfaces it was possible to observe a remarkable increase in bacterial adhesion and exopolysaccharides formation, in addition to the formation of complex biofilms covering a large area of the Ti surfaces (images 3C and 3D ). In Fig. 3 it is possible to observe the biofilm developed by Pseudomonas aeruginosa on the Ti surfaces conditioned with the salivary pellicle compared to the biofilm of P. aeruginosa developed on Ti surfaces that were not conditioned with the PS (control). As can be seen in images 1A and 1B a greater number of bacterial cells adhered to the surface can be seen, in addition to the development of a complex biofilm on these surfaces, in comparison with the surfaces that were not conditioned with the PS (images 1C and 1D ), where it is evident that there was minor biofilm development. Finally, Fig. 4 shows the representative images of biofilm formation developed by the co-culture of P. aeruginosa , S. aureus and S. epidermidis , in which it is possible to observe the differences in biofilm formation on the Ti surfaces conditioned with the salivary film compared to the biofilms formed on the Titanium surfaces not conditioned with the SP. As can be seen, in terms of the number of cells adhering to the surfaces, it is not possible to identify differences in bacterial adhesion on the substrates conditioned with the SP (images 4A and 4B ) compared to the substrates that were not conditioned with the saliva pellicle (control). However, regarding the bacterial species predominantly colonizing the surfaces, there was an important difference, on the surfaces conditioned with the salivary film (image 4A, B ) a predominance in the adhesion of P. aeruginosa (bacilli), while on the surfaces that were not conditioned by the SP, it was possible to observe the presence of a greater number of bacterial cells in the form of cocci, which would correspond to a greater adhesion of S. aureus and/or S. epidermidis to these samples. Biofilm metabolic activity evaluated by XTT assay Optical density values representing bacterial viability in the XTT assay are shown in Fig. 5 . The figure shows the results of the biofilm developed by each of the bacteria separately, in addition to the three bacterial species evaluated in coculture on the Ti surfaces conditioned with the salivary pellicle. A similar trend was observed in the metabolic activity of the biofilms composed of Staphylococcus species on Ti surfaces. That is, the presence of the salivary pellicle on the substrates reduced the bacterial viability of both strains. The presence of SP reduced the viability of S. aureus cells attached to Ti surfaces (3.7 × 10 5 ± 1.4), this compared to that observed on substrates that were not conditioned. (3.1 × 10 5 ± 0.1) ( NS ). In general, S. epidermidis biofilm development was found to be higher than that of S. aureus on the surfaces tested. Furthermore, the presence of a salivary pellicle on Ti substrates had been demonstrated to reduce the viability of S. epidermidis bacteria, as opposed to that observed for unconditioned substrates, 3.0 × 10 5 ± 0.1 vs 7.4 × 10 5 ± 0.2, respectively ( p < 0.001). The study revealed a noteworthy finding, among the three species assessed individually, the species that exhibited the highest degree of substrate affinity, regardless of whether they had been conditioned or not, was P. aeruginosa. Moreover, in contrast with the findings observed for Staphylococcus species, the presence of the salivary pellicle on the Ti surfaces tested actually enhanced the P. aeruginosa viability (7.8 × 10 5 ± 1.6), when compared to the findings observed on the Ti surfaces that were not conditioned with the SP (5.5 ×10 5 ± 0.8) ( NS ). Finally, when biofilm formation of P. aeruginosa , S. aureus and S. epidermidis was evaluated in co-culture, it was found that there was less bacterial adhesion on surfaces that were coated with the salivary pellicle (4.3 × 10 5 ± 0.2) compared to the adhesion observed on surfaces that were not coated with the PS (4.8 × 10 5 ± 0.4), however, such difference was not statistically significant. Biofilm composition evaluated by DNA-DNA hybridizations To determine the proportion of each bacterial strain adhered to the Ti surfaces, DNA-DNA hybridizations were performed. Different patterns of biofilm formation were observed on the Ti disk conditioned or unconditioned by the SP pellicle (Fig. 6 ). As can be seen in the figure above, the development of biofilms on titanium discs showed different trends, depending on the presence of the conditioning salivary film and the bacterial species evaluated. Regarding S. aureus , unlike what was observed with the XTT assay, which evaluates cellular activity, the results derived from DNA-DNA hybridizations indicated that there was a slight increase in the number of cells colonizing the conditioned Ti surfaces compared to the numbers of cells colonizing the unconditioned surfaces (3.0 × 10 5 ± 0.9 vs 0.8 × 10 5 ± 0.3) ( NS ). For S. epidermis , total DNA probe counts correlated with the level of cellular metabolic activity (as indicated by the TXX assay result). Lower counts were observed on Ti surfaces conditioned with the salivary film than on unconditioned Ti discs, confirming that the protein film exerted an inhibitory effect on this bacterial strain (5.2 × 10 5 ± 1.1 vs 14.9 × 10 5 ± 3.3) ( p < 0.001). In contrast to what was observed for the S. epidermidis biofilm, the total DNA probe counts of the P. aeruginosa are slightly higher in the biofilm developed on the substrates conditioned with the salivary film, in comparison with what was observed in the non-conditioned Ti disks (difference not statistically significant). The above finding is further corroborated by the higher metabolic activity of P. aeruginosa observed on the conditioned Ti substrates (see TXX assay results above). This would indicate that the protein pellicle would have a stimulatory effect on the development of P. aeruginosa biofilms on Ti substrates. Mean proportions of bacterial species assessed when cocultured on Ti surfaces The mean proportions (% of the total DNA probe count) of the bacterial strains evaluated in coculture in the biofilm developed on the Ti conditioned or unconditioned by the salivary film, it was observed that the growth of the S. epidermidis was not detected, which would not necessarily indicate that it was entirely suppressed when it was co-cultured with S. aureus and P. aeruginosa , but that its numbers were likely to be lower than detection level of the DNA-DNA hybridizations technique of 1 × 10 4 . Besides, it was confirmed that for both salivary film conditioned Ti surfaces and the unconditioned ones, the biofilm developed was dominated by P. aeruginosa species: 80.4% and 68.8%, respectively, this in comparison to the counts of S. aureus cells detected on those substrates: 19.1% and 31.3%, respectively. These differences were significant ( p < 0.05) with lower proportions of S. aureus and higher proportions of P. aeruginosa , on both, experimental and control surfaces. Discussion Titanium remains the metal of choice in dental implantology due to its physicochemical and biocompatibility properties. Once dental implants are placed in the oral cavity and exposed to biofluids such as blood and saliva, their surface is subject to the adsorption of biomolecules, with the consequent formation of salivary pellicle (SP) on them, modifying their biological behaviors [ 38 ]. In general terms, dental implants exhibit high success rates. However, bacterial interactions and the subsequent biofilm development on the surfaces of these biomedical devices play a key role in the emergence of peri-implant infections, compromising the longevity of the functional dental implants in the oral cavity. There is no clear understanding of the exact mechanism of microbial interaction during peri-implantitis [ 10 , 39 ]. While some studies conclude that the microbial profile is similar to periodontitis [ 40 , 41 ], contradicted studies have reported that opportunistic bacteria, such as Staphylococcus aureus , which are not part of the most common species in the oral cavity [ 42 ], play a role in the progression of the disease [ 15 , 17 ]. In recent decades, it has been identified that the development of peri-implantitis lesions is associated with the presence, in high proportions, of pathogen-opportunistic and antibiotic-resistant bacteria such as Pseudomonas aeruginosa , Staphylococcus aureus , and Staphylococcus epidermidis [ 16 , 43 ]. Although different reports indicate that they show some affinity for Ti, the underlying mechanisms of this so call “affinity” have not been explored in depth [ 19 , 20 ]. Understanding the ecological triggers underlying the microbial pathogenesis of peri-implantitis is essential for the development of better preventive, diagnostic and therapeutic strategies, since peri-implant tissues are more susceptible to endogenous oral infections. Due to the above, the main objective of the present investigation was to evaluate the modulating effect of the salivary film formed on Ti surfaces on the development of P. aeruginosa , S. aureus and S. epidermidis biofilms. According to the overall results obtained through the tests performed in this research: SEM, XTT assay, and DNA-DNA hybridizations, when the biofilm development of bacterial strains was evaluated individually, it was observed that the highest affinity for the substrates, regardless of whether they were conditioned or unconditioned, was for the P. aeruginos a strain, followed by S. epidermidis , while S. aureus showed the lowest biofilm development. The enhanced adhesion and subsequent biofilm development by P. aeruginosa on the surfaces tested can be attributed to the ability of the bacterial cell membrane to stretch over the surfaces, together with a concomitant increase in the level of extracellular polymeric substance (EPS) produced by this microorganism, as previously reported [ 19 ]. P. aeruginosa is known to be a strong producer of the glycocalyx, which provides a local environment that favours adhesion, community cohesion, and communication through highly specific interactions between and within cells of both bacterial and host origin [ 44 ]. In clinical settings, P. aeruginosa has been isolated from the palate and dorsum of the tongue in a slightly elevated proportion in implant-bearing patients [ 45 ]. Given the pathogenicity associated with this bacterial strain, the fact that the SP formed on titanium surfaces does not reduce the formation of P. aeruginosa biofilms, but rather promotes it, is alarming. Regarding Streptococcus species biofilm development on the Ti substrates, a marked difference was found between the higher affinity of S. epidermidis for the substrates compared to the affinity shown by the S. aureus strain. However, this affinity changed as the presence of S. epidermidis was masked by S. aureus and especially P. aeruginosa in the biofilms developed by the strains studied in coculture. It has previously been reported that S. epidermidis , along with S. aureus , are isolated in high proportions from Ti dental implant surfaces [ 46 , 47 ], Indeed, both S. aureus and S. epidermidis have been associated with subgingival flora around failed implants [ 16 , 48 ]. The adhesion of S. aureus to Ti dental implant surfaces is regarded as favoured by the chemical characteristics of the substrate [ 49 ]. S. aureus is not only isolated more frequently in sites with peri-implantitis [ 50 ], associated with implant loss [ 32 ] but has also been associated with cases of treatment-resistant periodontitis [ 51 ]. Regarding the effect of the salivary film on the biofilm development of the strains studied individually, it was interesting to note that the conditioning of the Ti substrates with the PS only had a strong modulating effect by reducing the growth of S. epidermidis , whereas it had no significant effect on the development of S. aureus or P. aeruginosa biofilms. This seems to contrast with earlier reports [ 33 ], where the salivary film formed on Ti surfaces significantly reduced the adhesion of specific periodontal bacteria such as Fusobacterium nucleatum and Porphyromonas gingivalis . This would therefore be an indication of a selective antimicrobial effect of the salivary pellicle conditioning of Ti surfaces. Evaluating the development of biofilms generated by co-culturing P. aeruginosa , S. aureus and S. epidermidis on Ti surfaces, it was found that saliva film allowed greater P. aeruginosa strain development compared to S. aureus and S. epidermidis growth. Previously, it was found that pre-conditioning Ti surfaces with saliva increased bacterial adhesion and biofilm production of P. aeruginosa [ 20 ]. The above would confirm that certain salivary proteins adsorbed on Ti surfaces could provide binding receptors for adhesion of this bacterial specie, as previously hypothesised [ 52 ]. Our results highlight the role of the salivary pellicle in P. aeruginosa adhesion and suggest that differences in biofilm formation in the oral environment are specific to each bacterial species. The precedent that extracellular products of P. aeruginosa can inhibit the growth of S. epidermidis and S. aureus in cocultures [ 53 ] explains what was observed in the images obtained by Scanning Electron Microscopy in the present investigation, where it was possible to see the predominance of adherent bacilli on the experimental and control Ti surfaces. This finding was confirmed by DNA-DNA hybridization analysis, the bacterial strain colonising the experimental substrates was mainly P. aeruginosa , followed by S. aureus , while the presence of S. epidermis could not be confirmed. While the literature reports that S. aureus and S. epidermidis can proliferate abundantly on Ti and its alloys when found together [ 54 ], there are few reports evaluating biofilm formation of these bacteria together with P. aeruginosa , individually or in co-culture, on titanium surfaces, and even fewer on titanium surfaces conditioned with salivary pellicle. When the adhesion of the same bacterial strains tested here was evaluated on conventional Ti [ 55 ] with a contact angle of ∼70.6, close to the contact angle values of the experimental surfaces in this study: ∼81.9° ± 0.4°, a fairly close number of cells of each of the tested bacteria, S. aureus , S. epidermidis and P. aeruginosa , was observed, the latter being the strain that showed the lowest affinity for Ti substrates. However, given that in the study cited above [ 55 ], the Ti surfaces were not conditioned with the SP, we can highlight the effect of salivary conditioning of the Ti surfaces under the experimental conditions described in the present study. Finally, it is important to note that as there are few reports evaluating the modular effect of the salivary pellicle on the development of biofilms on Ti surfaces used in implant dentistry, further research is needed on the role of the salivary pellicle constituents in the microbial patterns currently associated with peri-implantitis in clinical practice, not only in periodontally healthy subjects but also in subjects with periodontitis. Conclusions From the results of the present investigation, it can be concluded that the presence of salivary pellicle on commercially pure Ti substrates selectively modulates the development of P. aeruginosa , S. aureus and S. epidermidis biofilms on Ti surfaces when cultured individually. The presence of the salivary pellicle positively favored P. aeruginosa biofilm development on Ti substrates, while inhibiting S. epidermidis biofilm formation. In the case of the biofilm developed on Ti substrates by the co-culture of P. aeruginosa , S. aureus and S. epidermidis , it was found that the salivary pellicle favored the growth mainly of P. aeruginosa over the growth of S. aureus or even S. epidermidis , this under a probable common ecological scenario. Declarations Funding This work was supported by UNAM-PAPIIT IN209324, IA20064 and IN229223. Data Availability The data that support the findings of this study are available from the corresponding author upon reasonable request. Ethics approval and consent to participate. The collection of the saliva sample was carried out under the acceptance and approval of the corresponding Ethics Committee. The authors declare that the procedures followed conform to the standards of the Ethics Committee on Human Studies of the Division of Postgraduate Studies and Research of the Faculty of Dentistry of the National Autonomous University of Mexico (CIE/0108/09/2023), which was conducted in accordance with the Declaration of Helsinki. Conflict of Interest The authors declare no competing interests. Acknowledgments The authors thank to collaboration of the laboratory technician Leticia Cruz Fonseca of the Universidad Nacional Autónoma de México (UNAM). ORCID Martínez-Hernández, Miryam https://orcid.org/0000-0002-1589-8605 Reyes-Mendoza, Paulina-Polet Chávez-Esparza, Mariana Rodríguez-Hernández, Adriana-Patricia https://orcid.org/0000-0002-9473-1749 García-Pérez, Víctor I. https://orcid.org/0000-0003-2999-6385 Author Contribution Author Contributions StatementConceptualization, validation, formal analysis, data curation, supervision, project administration, resources and funding acquisition: Martínez-Hernández, Miryam, Rodríguez-Hernández, Adriana-Patricia, and García-Pérez, V. I.; methodology and writing, and editing—original draft: Rodríguez-Hernández, Adriana-Patricia, Martínez-Hernández, Miryam, García-Pérez, V. I, Chávez-Esparza, M and Reyes-Mendoza, P; review, and editing—original draft: Rodríguez-Hernández, Adriana-Patricia, Martínez-Hernández, Miryam, García-Pérez, V. I, Chávez-Esparza, M and Reyes-Mendoza, P. All authors have read and agreed to the published version of the manuscript. 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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-6638066","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":455370607,"identity":"4e025791-9b2f-45ec-b297-3df5f0c1e4a0","order_by":0,"name":"Miryam Martínez-Hernández","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABBElEQVRIiWNgGAWjYDACZihtwN4ApnkYDjAYEKmF5wCxWmDAQCIByiKkRbed95nEzxyGaHPJxw8f3Wy7I8N3/PAGZt4ddtEMYmew6jU7zG4m2buNIXfn7DRj49y2ZzySZ9IKmHnPJOc2SKclYNfCxibBC9Sy4XYOm3Ru22EegwM5Bsy8bcxALckHcGmR/AvScvMMVMv5NyAt9UAtiQ24tEiDbbnBA9VyA2zLYXy2MFvLgrScAfol59xhHskbzwoOzm07ntuGyy/njzHefAvScvzww8c5ZYft+c4nb3zwtq06t186B1dos0gwMPxHFQI7iA2HeiBg/oBbbhSMglEwCkYBEAAA3mpiA7+wAJEAAAAASUVORK5CYII=","orcid":"","institution":"National Autonomous University of Mexico","correspondingAuthor":true,"prefix":"","firstName":"Miryam","middleName":"","lastName":"Martínez-Hernández","suffix":""},{"id":455370610,"identity":"d698a4da-1564-4d3b-a618-bef54d683eca","order_by":1,"name":"Polet Reyes-Mendoza","email":"","orcid":"","institution":"National Autonomous University of Mexico","correspondingAuthor":false,"prefix":"","firstName":"Polet","middleName":"","lastName":"Reyes-Mendoza","suffix":""},{"id":455370611,"identity":"2953000a-8613-4a93-987c-34af3fd2c1d9","order_by":2,"name":"Mariana Chávez-Esparza","email":"","orcid":"","institution":"National Autonomous University of Mexico","correspondingAuthor":false,"prefix":"","firstName":"Mariana","middleName":"","lastName":"Chávez-Esparza","suffix":""},{"id":455370612,"identity":"b6505ff0-fad3-4271-9a05-745b4e839f6b","order_by":3,"name":"Adriana-Patricia Rodríguez-Hernández","email":"","orcid":"","institution":"National Autonomous University of Mexico","correspondingAuthor":false,"prefix":"","firstName":"Adriana-Patricia","middleName":"","lastName":"Rodríguez-Hernández","suffix":""},{"id":455370617,"identity":"49056cc5-33be-4853-8f38-63aaad6c0c4d","order_by":4,"name":"Víctor I. 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Biofilm developed on Ti surfaces conditioned with salivary pellicle (magnification x1000; 5K), and \u003cstrong\u003eB\u003c/strong\u003e. a higher magnification (x2500; 5Kv). \u003cstrong\u003eC\u003c/strong\u003e. Biofilm developed on unconditioned Ti surfaces with the salivary pellicle (magnification x1000; 5K), and \u003cstrong\u003eD\u003c/strong\u003e. a higher magnification (x2500; 5Kv).\u003c/p\u003e","description":"","filename":"floatimage134.png","url":"https://assets-eu.researchsquare.com/files/rs-6638066/v1/0fe4889aaa506b5da53d716d.png"},{"id":82609446,"identity":"bf4b4889-99d4-429f-ab51-dffe84b52ae0","added_by":"auto","created_at":"2025-05-13 10:28:33","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1070846,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eStaphylococcus epidermidis\u003c/em\u003ebiofilm developed on Ti surfaces. \u003cstrong\u003eA\u003c/strong\u003e. Biofilm developed on Ti surfaces conditioned with salivary pellicle (magnification x1000; 5K), and \u003cstrong\u003eB\u003c/strong\u003e. a higher magnification (x2500; 5Kv). \u003cstrong\u003eC\u003c/strong\u003e. Biofilm developed on unconditioned Ti surfaces with the salivary pellicle (magnification x1000; 5K), a \u003cstrong\u003eD\u003c/strong\u003e. a higher magnification (x2500; 5Kv).\u003c/p\u003e","description":"","filename":"floatimage220.png","url":"https://assets-eu.researchsquare.com/files/rs-6638066/v1/7d191ee030ad34dae3b96da5.png"},{"id":82609449,"identity":"a44607bc-be46-49fd-8201-c22fceade930","added_by":"auto","created_at":"2025-05-13 10:28:33","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1170739,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e biofilm developed on Ti surfaces. \u003cstrong\u003eA\u003c/strong\u003e. Biofilm developed on Ti surfaces conditioned with salivary pellicle (magnification x1000; 5K), and \u003cstrong\u003eB\u003c/strong\u003e. a higher magnification (x2500; 5Kv). \u003cstrong\u003eC\u003c/strong\u003e. Biofilm developed on unconditioned Ti surfaces with the salivary pellicle (magnification x1000; 5K); and \u003cstrong\u003eD\u003c/strong\u003e. a higher magnification (x2500; 5Kv).\u003c/p\u003e","description":"","filename":"floatimage319.png","url":"https://assets-eu.researchsquare.com/files/rs-6638066/v1/1cb47476176759b253ce0c9b.png"},{"id":82610792,"identity":"abcb50b3-7404-4943-93cb-8ac75f92dc31","added_by":"auto","created_at":"2025-05-13 10:44:33","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1147209,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eS. aureus\u003c/em\u003e, \u003cem\u003eS. epidermidis\u003c/em\u003e and \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e coculture biofilm developed on Ti surfaces. \u003cstrong\u003eA\u003c/strong\u003e. Biofilm developed on Ti surfaces conditioned with salivary pellicle (magnification x1000; 5K), and \u003cstrong\u003eB\u003c/strong\u003e. a higher magnification (x2500; 5Kv). \u003cstrong\u003eC\u003c/strong\u003e. Biofilm developed on unconditioned Ti surfaces with the salivary pellicle (magnification x1000; 5K), and \u003cstrong\u003eD\u003c/strong\u003e. a higher magnification (x2500; 5Kv).\u003c/p\u003e","description":"","filename":"floatimage412.png","url":"https://assets-eu.researchsquare.com/files/rs-6638066/v1/3d8d1b326981f06b24233ead.png"},{"id":82609443,"identity":"71169aac-89f7-43b2-8ac8-1ae061d2749b","added_by":"auto","created_at":"2025-05-13 10:28:33","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":142104,"visible":true,"origin":"","legend":"\u003cp\u003eBacterial cells \u003cstrong\u003e× \u003c/strong\u003e10\u003csup\u003e5\u003c/sup\u003e SEM (Standard error of the mean) of the biofilms, of the three individual species or the coculture of them, developed on the titanium surfaces conditioned with salivary pellicle or without pellicle conditioning analysed by XTT assay. Student´s \u003cem\u003et\u003c/em\u003e-test using Bonferroni´s modification for paired comparisons.\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-6638066/v1/2dc114dd1c64415dffbaa57a.png"},{"id":82610010,"identity":"3a1684b4-f524-4b47-aa15-119394e1c8e3","added_by":"auto","created_at":"2025-05-13 10:36:33","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":159305,"visible":true,"origin":"","legend":"\u003cp\u003eMean individual levels (total DNA probe count × 105 SEM: Standard error of the mean) of the biofilms, of the three individual species and total levels in co-culture, developed on the titanium surfaces conditioned with salivary pellicle (SP) or without protein conditioning analysed by DNA-DNA hybridization Checkerboard technique. Student´s \u003cem\u003et\u003c/em\u003e-test using Bonferroni´s modification for paired comparisons and differences between strains with ANOVA of SP or without protein assays.\u003c/p\u003e","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-6638066/v1/7ed7866498f02fc5e14c927a.png"},{"id":82609454,"identity":"2f2f2531-154b-464d-98b9-de50c294fc95","added_by":"auto","created_at":"2025-05-13 10:28:33","extension":"jpeg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":453664,"visible":true,"origin":"","legend":"\u003cp\u003ePie charts of the mean proportions (% of total DNA probe count) of individual species biofilm developed on the titanium surfaces conditioned with salivary pellicle or without protein pellicle conditioning. Student´s \u003cem\u003et\u003c/em\u003e-test using Bonferroni´s modification for paired comparisons * \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05 of \u003cem\u003eS. aureus\u003c/em\u003e and \u003cem\u003eP. aeruginosa\u003c/em\u003e.\u003c/p\u003e","description":"","filename":"floatimage7.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6638066/v1/b7496281731f2ebd8d14d733.jpeg"},{"id":83479599,"identity":"730ebea4-ca22-4241-952a-1d499c917ebb","added_by":"auto","created_at":"2025-05-27 06:16:59","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":6527718,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6638066/v1/40932062-6a47-43d5-95e4-c1a3dfabe380.pdf"},{"id":82609442,"identity":"439a2098-49ca-4e04-8410-b2ba2a34f913","added_by":"auto","created_at":"2025-05-13 10:28:33","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":14862,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryTable.docx","url":"https://assets-eu.researchsquare.com/files/rs-6638066/v1/c085359f719bd548bba6afc4.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"The modulating effect on Staphylococcus species and Pseudomonas aeruginosa biofilm development of salivary pellicle conditioning titanium surfaces","fulltext":[{"header":"Introduction","content":"\u003cp\u003eBiofilm formation on biomedical surfaces is always a concern when they are in operation in the oral cavity, mainly because, the oral cavity, has one of the largest and most diverse microbiotas of the body, harbouring a diverse set of over 774 different bacterial species [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. The vast majority of the oral cavity bacteria exists in the form of biofilms.\u003c/p\u003e \u003cp\u003eIn general, dental biofilm maintain a harmonious relationship with the host, providing benefits to overall health [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e], nevertheless, alterations in biofilm composition caused by certain stress factors, such as tobacco use or lack of plaque removal, can alter the biofilm homeostasis leading to the development of oral diseases, such as periodontitis or peri-implantitis [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e].\u003c/p\u003e \u003cp\u003ePeri-implantitis is the main biological complication that can negatively affect long term outcomes of the osseointegrated dental implants [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Even though the long-term survival rates of dental implants are high (\u0026ge;\u0026thinsp;10 years) [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e], it is estimated that approximately 1/3 of all patients and 1/5 of all implants placed will experience peri-implantitis [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe peri-implant infections have often been regarded as pathologies analogous to gingivitis and periodontitis, mainly because those pathologies share similarities in clinical characteristics and aetiology [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. However, there is increasing evidence supporting the notion of periimplantitis is not periodontitis [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e], such as critical histopathological differences between peri-implantitis and periodontitis have been found [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e], in addition to current microbial identification methods have revealed an unexpected difference between the microbiome surrounding teeth and that surrounding implants [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eRegarding the characterization of the microbiota associated with the presence of peri-implant damage, through the use of DNA probes and culture analysis, it has been possible to isolate common periodontopathogenic bacteria in diseased peri-implant sites, including \u003cem\u003ePorphyromonas gingivalis\u003c/em\u003e and \u003cem\u003eTannerella forsythia\u003c/em\u003e [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e], however, it has recently been reported that the development of peri-implant lesions could be associated with bacterial species other than canonical bacterial associated with periodontitis, namely \u003cem\u003eP. gingivalis\u003c/em\u003e, \u003cem\u003eTreponema denticola\u003c/em\u003e and \u003cem\u003eT. forsythia\u003c/em\u003e [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. In this regard, it has been pointed out that the presence, in high proportions, of pathogenic-opportunistic and antibiotic-resistant bacteria such as \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e, \u003cem\u003eStaphylococcus aureus\u003c/em\u003e and \u003cem\u003eS. epidermidis\u003c/em\u003e could play an important role in the etiopathogenesis of peri-implantitis [\u003cspan additionalcitationids=\"CR16 CR17\" citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. These reports indicate that the presence of such bacterial strains on dental implant surfaces is due to a certain \u0026ldquo;affinity for Titanium (Ti)\u0026rdquo; [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. However, the underlying mechanisms of this affinity have not been explored in depth.\u003c/p\u003e \u003cp\u003eTi surfaces of dental implants \u003cem\u003evs.\u003c/em\u003e cementum or natural tooth enamel represent chemically different substrates; differences in surface energy, topography, wettability, and electrochemical charges of these substrates will modulate bacterial adhesion, ultimately resulting in the formation of distinct biofilms on the different substrates [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. An essential prerequisite for bacterial adhesion and subsequent biofilm development in the oral cavity is salivary pellicle (SP) formation. SP is a thin film coating all oral surfaces, and mainly consists of amino and fatty acids, proteins and carbohydrates derived mainly from saliva, but also from microorganisms [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. The formation of SP involves a specific and non-random complex process that will result in the selective adsorption of biomolecules [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. The SP formed on Ti surfaces have aroused great interest due to their importance in peri-implant health [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eSP formed on dental enamel and the one formed on titanium surfaces have showed important molecular differences [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e], which could explain the presence of \u003cem\u003eS. epidermidis\u003c/em\u003e, \u003cem\u003eS. aureus\u003c/em\u003e [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e], and \u003cem\u003eP. aeruginosa\u003c/em\u003e [\u003cspan additionalcitationids=\"CR30\" citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e] in peri-implantitis sites. In a study by El-Telbany, \u003cem\u003eet al\u003c/em\u003e.[\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e] it was possible to isolate \u003cem\u003eP. aeruginosa\u003c/em\u003e in 10% of cases of peri-implantitis evaluated, indicating that the presence of these pathogens would be associated to a higher prevalence of disease [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eSince the modulating effect of SP formed on titanium surfaces in the formation of oral biofilms is now recognized [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e], a deeper understanding of how this protein film affects the adhesion of non-canonical periodontal pathogens, such as the opportunistic pathogens \u003cem\u003eStaphylococcus\u003c/em\u003e species and \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e, to which the aetiology of peri-implantitis has even been attributed, is now needed.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eTi surfaces tested\u003c/h2\u003e \u003cp\u003eTi disks with diameter of 15 mm were prepared from 1-mm-thick sheets of grade 2 unalloyed Ti (ASTM F67). The methods used to produce the pretreatment (PT) surfaces have been previously reported [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. The Ti surfaces are relatively smooth with an average roughness of (Ra)\u0026thinsp;\u0026lt;\u0026thinsp;0.19 \u0026micro;m and a water contact angle of 81.9\u0026deg; \u0026plusmn; 0.4\u0026deg;. Ti disks were manufactured by Institut Straumann AG (Basel, Switzerland) and delivered to us ready to use. For each experiment, the number of Ti disks was adjusted to normalize the total surface area evaluated to 8 cm\u003csup\u003e2\u003c/sup\u003e.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eSalivary pellicle formation assays\u003c/h3\u003e\n\u003cp\u003e Whole human saliva samples used for salivary pellicle formation assays on titanium substrates were obtained from a healthy, middle-aged volunteer with no active caries lesions or history of periodontal disease. The healthy saliva donor was currently nonsmoker, with more than 20 natural teeth (excluding third molars). Clinical measurements were taken from the healthy volunteer at six sites per tooth (mesiobuccal, buccal, distobuccal, distolingual, lingual, and mesiolingual) at all teeth excluding third molars (approximately 168 sites evaluated) as previously described [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eBefore saliva samples were collected, the volunteer was asked to rinse their mouth with clean water for 30 seconds to remove desquamated epithelial cells, microorganisms and food debris. Sterile 50 ml tubes were used to collect passive drooling saliva for 3 minutes. Saliva samples were centrifuged at 15,000 g, 4\u0026deg;C, for 15 minutes; the resulting clean supernatants, supplemented with the complete EDTA-free protease inhibitor mixture (RocheDiagnostics, Penzberg, Germany), were used for salivary pellicle formation assays on Ti substrates. The saliva donor gave informed consent acknowledging his willingness to participate. This study was performed under the guidelines of the Declaration of Helsinki and approved by the Ethics Committee for Human Studies of the Division of Postgraduate Studies and Research, School of Dentistry, National Autonomous University of Mexico (CIE/0108/09/2023).\u003c/p\u003e \u003cp\u003eTo induced salivary pellicle formation, Ti surfaces were placed individually in 24-well plates with 500 \u0026micro;L of whole saliva and incubated for two hours at 37\u0026deg;C in a chamber with 10% humidity. After incubation, the surfaces were washed twice with 1 mL of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003edd\u003c/sub\u003e to remove non-adsorbed proteins. Another set of surfaces incubated under the same conditions without salivary film formation, was used as a control. Subsequently, the surfaces were transferred to new sterile 24-well cell culture plates and were immediately used in biofilm development assays.\u003c/p\u003e \u003cp\u003e \u003cb\u003eBiofilm development assays of\u003c/b\u003e \u003cb\u003eStaphylococcus\u003c/b\u003e \u003cb\u003eand\u003c/b\u003e \u003cb\u003ePseudomonas aeruginosa\u003c/b\u003e \u003cb\u003especies\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThree reference strains (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e) were used for \u003cem\u003ein vitro\u003c/em\u003e biofilm development assays on the tested surfaces. All strains were obtained as lyophilized cultures from the American Type Culture Collection (ATCC, Rockville, MD, USA).\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\u003eReference strains used for the biofilm development assays\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=\"char\" char=\".\" 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\u003eSpecie\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eStrain\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eGram\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eStaphylococcus aureus\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e25923\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003ePositive\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eStaphylococcus epidermidis\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e14990\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e43636\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eNegative\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003e \u003csup\u003ea\u003c/sup\u003e American Type Culture Collection, Rockville, MD, USA.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cp\u003ePure cultures of each of the three bacterial strains were obtained by seeding individually on plates with Trypticase Soy Agar (TSA; BBL, Becton-Dickinson) enriched with 0.3 \u0026micro;g/mL menadione (Sigma-Aldrich) and 5 \u0026micro;g/mL hemin (Sigma-Aldrich) and incubated for 24 h at 37\u0026deg;C under aerobic conditions.\u003c/p\u003e \u003cp\u003eAfter incubation period, the different bacterial cultures were harvested and individually suspended in tubes contained enriched TSB broth (TSB added with 0.3 \u0026micro;g/mL menadione and 5 \u0026micro;g/mL hemin). The optical density (OD) in each tube was adjusted to 1 at λ\u0026thinsp;=\u0026thinsp;600 nm in a spectrophotometer (FilterMax F5 Multi-Mode Microplate Reader, Thermo Fisher) to obtain a bacterial suspension with 1 x10\u003csup\u003e6\u003c/sup\u003e cells/mL. To test biofilm development, Ti surfaces conditioned with SP (experimental surfaces) or uncoated with SP (control surfaces) were independently incubated with 1 mL of bacterial suspension of \u003cem\u003eS. aureus\u003c/em\u003e, \u003cem\u003eS. epidermidis\u003c/em\u003e or \u003cem\u003eP. aeruginosa\u003c/em\u003e or co-culture of those strains. The inoculated surfaces were incubated for 12 h at 37\u0026deg;C under aerobic conditions in an orbital shaker (\u0026asymp;\u0026thinsp;160 rpm), under aerobic atmosphere. After aerobic incubation, each surface was washed twice with 1 mL of enriched broth to detach bacterial cells not adhered to the surfaces. All experiments were run in triplicate and repeated at least two different times.\u003c/p\u003e\n\u003ch3\u003eScanning Electron Microscopy (SEM) observations\u003c/h3\u003e\n\u003cp\u003eThe microscopic appearance of the biofilm of \u003cem\u003eS. aureus\u003c/em\u003e, \u003cem\u003eS. epidermidis\u003c/em\u003e, and \u003cem\u003eP. aeruginosa\u003c/em\u003e or bacterial coculture developed on Ti surfaces covered and uncovered (control surfaces) by the salivary pellicle was observed by SEM. In brief, Samples were fixed in 2.0% glutaraldehyde 24 h at room temperature. Then washed three times with phosphate buffer solution (pH 7.4), followed by dehydration through a series of graded ethanol solutions of 20, 40, 60, 80, and 100%. Then, the specimens were stored in a desiccator for 24 hours, and sputter-coated with a gold layer (SDC 050; Bal-Tec AG, Balzers, Germany). Thereafter, the specimens were fixed with carbon tape to stubs, placed into the vacuum chamber of a scanning electron microscope (SEM (JEOL JSM5600-LV, Japan), and the central areas of the specimens were photographed at different magnification. Images were obtained in high vacuum mode with secondary electrons at 20\u0026ndash;25 kV.\u003c/p\u003e\n\u003ch3\u003eViability analysis\u003c/h3\u003e\n\u003cp\u003eThe viability of the biofilm cells of \u003cem\u003eS. aureus\u003c/em\u003e, \u003cem\u003eS. epidermidis\u003c/em\u003e, and \u003cem\u003eP. aeruginosa\u003c/em\u003e, or the co-culture of these strains, on salivary pellicle-covered or uncovered Ti surfaces was estimated by was analysed by using the XTT (2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide) assay (Sigma, MO, USA). Briefly, after aerobic incubation, the inoculated surfaces were rinsed twice with PBS to remove non-adhered bacteria and transferred to sterile well plates. Then, the surfaces were incubated for 3 h in 1 mL of enriched TSB broth added with 20 \u0026micro;L of the XTT solution. Finally, the O.D of 100 \u0026micro;L aliquots of the supernatants were read at λ\u0026thinsp;=\u0026thinsp;450 nm in a FilterMaxF5 multi-mode microplate reader (Molecular Devices, USA), and the number of viable bacteria was calculated according to standard curves previously performed.\u003c/p\u003e\n\u003ch3\u003eCheckerboard analysis\u003c/h3\u003e\n\u003cp\u003eAfter aerobic bacterial incubation, each Ti specimen was washed twice with TSB broth. After washing, 1 ml of enriched broth was added and samples were sonicated for 5 periods of 10 s each to detach the adherent bacteria to each Ti surface, coated and not covered by salivary pellicle.\u003c/p\u003e \u003cp\u003eTo determine the proportion of each bacterial strain of the biofilm developed on the Ti surfaces, 100 \u0026micro;l of the bacterial suspension obtained after sonication of each sample were placed in individual Eppendorf tubes with 100 \u0026micro;l of 0.5 \u003cem\u003eM\u003c/em\u003e NaOH. Bacterial species form the biofilm developed were identified and quantified using the checkerboard DNA\u0026ndash;DNA hybridization technique previously described [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. Briefly, DNA probes were prepared using the growth from 3- to 7-day cultures of the three reference strains used for the biofilm development assays (\u003cb\u003eTable I\u003c/b\u003e). Bacterial growth was harvested and placed in tubes containing 1 mL of TE buffer. Cells were washed twice and lysed at 37 \u0026ordm;C for 1 h with either 10% sodium dodecyl sulfate (Sigma-Aldrich) (SDS) plus proteinase K (Sigma-Aldrich) (20 mg/ml) for Gram-negative strains or lysozyme (Sigma-Aldrich) (15 mg/mL) plus achromopeptidase (Sigma-Aldrich) (5 mg/ml) for Gram-positive strains. DNA was isolated and purified. Whole-genomic DNA probes were prepared for each species by labeling 1 mg DNA with digoxigenin (Roche Diag- nostics, Mannheim, Germany) using the random primer technique [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. The sensitivity of the assay was set to allow the detection of approximately 10\u003csup\u003e4\u003c/sup\u003e cells of a given species by adjusting the concentration of each individual DNA probe. For the DNA\u0026ndash;DNA hybridization, each sample was thawed at room temperature, boiled for 10 min, and neutralized with 800 \u0026micro;l 5 \u003cem\u003eM\u003c/em\u003e ammonium acetate (Sigma-Aldrich). The released DNA from each sample was then placed into individual lanes (Min- islot-30, Immunetics, Cambridge, MA), concentrated onto a 15\u0026times;15 cm positively charged nylon membrane (Roche Diagnosis), and fixed to the membrane by cross-linking under ultraviolet light. Two lanes on each membrane contained standards consisting of a mixture at 10\u003csup\u003e5\u003c/sup\u003e and 10\u003csup\u003e6\u003c/sup\u003e cells of each bacterial species tested. The membranes were prehybridized at 42 \u0026ordm;C for 2 h and placed in a second device (Miniblotter-45, Immunetics) with the sample lanes rotated 90\u0026ordm; to the channels of the apparatus. Probes were hybridized in two sets of three consecutive channels, leaving empty channels (hybridization solution only) to allow noise and background correction of signals. The membranes were washed twice at high stringency for 20 min each time at 68 \u0026ordm;C in phosphate buffer (0.1X SSC and 0.1% SDS). Membranes were blocked by 1-h incubation in blocking buffer containing 1% casein in maleate buffer [100 m\u003cem\u003eM\u003c/em\u003e maleic acid (Sigma-Aldrich), and 150 m\u003cem\u003eM\u003c/em\u003e NaCl, pH 7.5]. Hybrids were detected by exposing the membranes to a 1:50,000 dilution of antidigoxigenin antibody conjugated to alkaline phosphatase (Roche Diagnostics) for 30 min. Signals were detected by chemiluminescence using a chemiluminescent agent (CDP-Star, Roche Diagnostics) for 30 min on the membranes at room temperature and exposed to films in autoradiographic cassettes for 30 min. Signals were detected with specialized software (Quantity One, BioRad Laboratories), adjusted by subtracting the average plus two standard deviations of the noise and background detected in the empty lanes, and converted to absolute counts by comparison with the standards on the membrane. Failure to detect a signal was recorded as zero.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eData for XTT values and total bacterial counts are presented as the Mean\u0026thinsp;\u0026plusmn;\u0026thinsp;Standard Error of the Mean (SEM). All data were analysed by Student\u0026acute;s \u003cem\u003et\u003c/em\u003e-test followed by Bonferroni correction for multiple comparisons, for paired comparisons and differences between strains with ANOVA test by IBM-SPSS 21 software. Significant differences (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05) were determined.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003eIn the present work, the modulating effect of the salivary film formed on titanium surfaces on the development of biofilms of \u003cem\u003eStaphylococcus\u003c/em\u003e species and \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e was investigated.\u003c/p\u003e\n\u003ch3\u003eSaliva donor\u003c/h3\u003e\n\u003cp\u003eFor the present investigation, a donor subject was selected to reduce the variability in the protein profile that could result from the inclusion of different volunteers, as has been previously reported [\u003cspan class=\"CitationRef\"\u003e37\u003c/span\u003e]. The volunteer underwent a full periodontal evaluation to confirm the diagnosis of periodontal health, which he defined as the absence of sites with probing depths\u0026thinsp;\u0026le;\u0026thinsp;3mm and bleeding values at full-mouth probing\u0026thinsp;\u0026lt;\u0026thinsp;10% [\u003cspan class=\"CitationRef\"\u003e5\u003c/span\u003e]. The results of the full periodontal evaluation are presented in a \u003cstrong\u003esupplementary\u003c/strong\u003e \u003cstrong\u003eTable\u003c/strong\u003e.\u003c/p\u003e\n\u003ch2\u003eBiofilm morphology evaluated by SEM\u003c/h2\u003e\n\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\n \u003cp\u003eThe following are images obtained under Scanning Electron Microscopy using secondary electrons of biofilms developed by \u003cem\u003eS. aureus\u003c/em\u003e, \u003cem\u003eS. epidermidis\u003c/em\u003e, \u003cem\u003eP. aeruginosa\u003c/em\u003e, and their coculture, formed on Ti surfaces conditioned with SP (experimental) and unconditioned by SP (control). (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e\n \u003cp\u003eAs can be seen in the previous image, the biofilms developed by \u003cem\u003eS. aureus\u003c/em\u003e were slightly more complex on the control Ti substrates, i.e., they were not conditioned with the SP, compared to the \u003cem\u003eS. aureus\u003c/em\u003e biofilms developed on the Ti substrates conditioned with the SP. In micrograph \u003cstrong\u003e2A\u003c/strong\u003e, corresponding to the Ti surfaces conditioned with the salivary film, it is possible to observe \u003cem\u003eS. aureus\u003c/em\u003e cells forming grape-like clusters, periodically distributed on the conditioned substrate, however, on these surfaces it was not possible to observe the formation of a highly complex biofilms, this finding can be seen in greater detail mainly in image \u003cstrong\u003e2B\u003c/strong\u003e. Regarding to the control Ti substrates (not conditioned with the SP), images \u003cstrong\u003e2C\u003c/strong\u003e and \u003cstrong\u003e2D\u003c/strong\u003e show that greater bacterial adhesion occurred on these surfaces, also observing a more homogeneous distribution on the surface of cocci clusters.\u003c/p\u003e\n \u003cp\u003eLike what was observed in the formation of \u003cem\u003eS. aureus\u003c/em\u003e biofilms on Ti substrates, the development of \u003cem\u003eS. epidermidis\u003c/em\u003e biofilms was higher on the control substrates (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e), i.e., those that were not conditioned with the SP. On the Ti surfaces that were conditioned with the SP (images \u003cstrong\u003e3A\u003c/strong\u003e and \u003cstrong\u003e3B\u003c/strong\u003e), it is possible to appreciate few bacterial grape-like clusters adhered on the conditioned surface, compared to the Ti surfaces that were not conditioned with the SP; on these surfaces it was possible to observe a remarkable increase in bacterial adhesion and exopolysaccharides formation, in addition to the formation of complex biofilms covering a large area of the Ti surfaces (images \u003cstrong\u003e3C\u003c/strong\u003e and \u003cstrong\u003e3D\u003c/strong\u003e).\u003c/p\u003e\n \u003cp\u003eIn Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e it is possible to observe the biofilm developed by \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e on the Ti surfaces conditioned with the salivary pellicle compared to the biofilm of \u003cem\u003eP. aeruginosa\u003c/em\u003e developed on Ti surfaces that were not conditioned with the PS (control). As can be seen in images \u003cstrong\u003e1A\u003c/strong\u003e and \u003cstrong\u003e1B\u003c/strong\u003e a greater number of bacterial cells adhered to the surface can be seen, in addition to the development of a complex biofilm on these surfaces, in comparison with the surfaces that were not conditioned with the PS (images \u003cstrong\u003e1C\u003c/strong\u003e and \u003cstrong\u003e1D\u003c/strong\u003e), where it is evident that there was minor biofilm development.\u003c/p\u003e\n \u003cp\u003eFinally, Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e shows the representative images of biofilm formation developed by the co-culture of \u003cem\u003eP. aeruginosa\u003c/em\u003e, \u003cem\u003eS. aureus\u003c/em\u003e and \u003cem\u003eS. epidermidis\u003c/em\u003e, in which it is possible to observe the differences in biofilm formation on the Ti surfaces conditioned with the salivary film compared to the biofilms formed on the Titanium surfaces not conditioned with the SP. As can be seen, in terms of the number of cells adhering to the surfaces, it is not possible to identify differences in bacterial adhesion on the substrates conditioned with the SP (images \u003cstrong\u003e4A\u003c/strong\u003e and \u003cstrong\u003e4B\u003c/strong\u003e) compared to the substrates that were not conditioned with the saliva pellicle (control). However, regarding the bacterial species predominantly colonizing the surfaces, there was an important difference, on the surfaces conditioned with the salivary film (image \u003cstrong\u003e4A, B\u003c/strong\u003e) a predominance in the adhesion of \u003cem\u003eP. aeruginosa\u003c/em\u003e (bacilli), while on the surfaces that were not conditioned by the SP, it was possible to observe the presence of a greater number of bacterial cells in the form of cocci, which would correspond to a greater adhesion of \u003cem\u003eS. aureus\u003c/em\u003e and/or \u003cem\u003eS. epidermidis\u003c/em\u003e to these samples.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\n \u003ch2\u003eBiofilm metabolic activity evaluated by XTT assay\u003c/h2\u003e\n \u003cp\u003eOptical density values representing bacterial viability in the XTT assay are shown in Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003e. The figure shows the results of the biofilm developed by each of the bacteria separately, in addition to the three bacterial species evaluated in coculture on the Ti surfaces conditioned with the salivary pellicle.\u003c/p\u003e\n \u003cp\u003eA similar trend was observed in the metabolic activity of the biofilms composed of \u003cem\u003eStaphylococcus\u003c/em\u003e species on Ti surfaces. That is, the presence of the salivary pellicle on the substrates reduced the bacterial viability of both strains. The presence of SP reduced the viability of S. aureus cells attached to Ti surfaces (3.7 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e \u0026plusmn; 1.4), this compared to that observed on substrates that were not conditioned. (3.1 \u003cstrong\u003e\u0026times;\u003c/strong\u003e 10\u003csup\u003e5\u003c/sup\u003e \u0026plusmn; 0.1) (\u003cem\u003eNS\u003c/em\u003e). In general, \u003cem\u003eS. epidermidis\u003c/em\u003e biofilm development was found to be higher than that of \u003cem\u003eS. aureus\u003c/em\u003e on the surfaces tested. Furthermore, the presence of a salivary pellicle on Ti substrates had been demonstrated to reduce the viability of \u003cem\u003eS. epidermidis\u003c/em\u003e bacteria, as opposed to that observed for unconditioned substrates, 3.0 \u003cstrong\u003e\u0026times;\u003c/strong\u003e 10\u003csup\u003e5\u003c/sup\u003e \u0026plusmn; 0.1 \u003cem\u003evs\u003c/em\u003e 7.4 \u003cstrong\u003e\u0026times;\u003c/strong\u003e 10\u003csup\u003e5\u003c/sup\u003e \u0026plusmn; 0.2, respectively (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001).\u003c/p\u003e\n \u003cp\u003eThe study revealed a noteworthy finding, among the three species assessed individually, the species that exhibited the highest degree of substrate affinity, regardless of whether they had been conditioned or not, was \u003cem\u003eP. aeruginosa.\u003c/em\u003e Moreover, in contrast with the findings observed for \u003cem\u003eStaphylococcus\u003c/em\u003e species, the presence of the salivary pellicle on the Ti surfaces tested actually enhanced the \u003cem\u003eP. aeruginosa\u003c/em\u003e viability (7.8 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e \u0026plusmn; 1.6), when compared to the findings observed on the Ti surfaces that were not conditioned with the SP (5.5 \u0026times;10\u003csup\u003e5\u003c/sup\u003e \u0026plusmn; 0.8) (\u003cem\u003eNS\u003c/em\u003e).\u003c/p\u003e\n \u003cp\u003eFinally, when biofilm formation of \u003cem\u003eP. aeruginosa\u003c/em\u003e, \u003cem\u003eS. aureus\u003c/em\u003e and \u003cem\u003eS. epidermidis\u003c/em\u003e was evaluated in co-culture, it was found that there was less bacterial adhesion on surfaces that were coated with the salivary pellicle (4.3 \u003cstrong\u003e\u0026times;\u003c/strong\u003e 10\u003csup\u003e5\u003c/sup\u003e \u0026plusmn; 0.2) compared to the adhesion observed on surfaces that were not coated with the PS (4.8 \u003cstrong\u003e\u0026times;\u003c/strong\u003e 10\u003csup\u003e5\u003c/sup\u003e \u0026plusmn; 0.4), however, such difference was not statistically significant.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\n \u003ch2\u003eBiofilm composition evaluated by DNA-DNA hybridizations\u003c/h2\u003e\n \u003cp\u003eTo determine the proportion of each bacterial strain adhered to the Ti surfaces, DNA-DNA hybridizations were performed. Different patterns of biofilm formation were observed on the Ti disk conditioned or unconditioned by the SP pellicle (Fig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003e).\u003c/p\u003e\n \u003cp\u003eAs can be seen in the figure above, the development of biofilms on titanium discs showed different trends, depending on the presence of the conditioning salivary film and the bacterial species evaluated. Regarding \u003cem\u003eS. aureus\u003c/em\u003e, unlike what was observed with the XTT assay, which evaluates cellular activity, the results derived from DNA-DNA hybridizations indicated that there was a slight increase in the number of cells colonizing the conditioned Ti surfaces compared to the numbers of cells colonizing the unconditioned surfaces (3.0 \u003cstrong\u003e\u0026times;\u003c/strong\u003e 10\u003csup\u003e5\u003c/sup\u003e \u0026plusmn; 0.9 \u003cem\u003evs\u003c/em\u003e 0.8 \u003cstrong\u003e\u0026times;\u003c/strong\u003e 10\u003csup\u003e5\u003c/sup\u003e \u0026plusmn; 0.3) (\u003cem\u003eNS\u003c/em\u003e). For \u003cem\u003eS. epidermis\u003c/em\u003e, total DNA probe counts correlated with the level of cellular metabolic activity (as indicated by the TXX assay result). Lower counts were observed on Ti surfaces conditioned with the salivary film than on unconditioned Ti discs, confirming that the protein film exerted an inhibitory effect on this bacterial strain (5.2 \u003cstrong\u003e\u0026times;\u003c/strong\u003e 10\u003csup\u003e5\u003c/sup\u003e \u0026plusmn; 1.1 \u003cem\u003evs\u003c/em\u003e 14.9 \u003cstrong\u003e\u0026times;\u003c/strong\u003e 10\u003csup\u003e5\u003c/sup\u003e \u0026plusmn; 3.3) (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001).\u003c/p\u003e\n \u003cp\u003eIn contrast to what was observed for the \u003cem\u003eS. epidermidis\u003c/em\u003e biofilm, the total DNA probe counts of the \u003cem\u003eP. aeruginosa\u003c/em\u003e are slightly higher in the biofilm developed on the substrates conditioned with the salivary film, in comparison with what was observed in the non-conditioned Ti disks (difference not statistically significant). The above finding is further corroborated by the higher metabolic activity of P. aeruginosa observed on the conditioned Ti substrates (see TXX assay results above). This would indicate that the protein pellicle would have a stimulatory effect on the development of \u003cem\u003eP. aeruginosa\u003c/em\u003e biofilms on Ti substrates.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\n \u003ch2\u003eMean proportions of bacterial species assessed when cocultured on Ti surfaces\u003c/h2\u003e\n \u003cp\u003eThe mean proportions (% of the total DNA probe count) of the bacterial strains evaluated in coculture in the biofilm developed on the Ti conditioned or unconditioned by the salivary film, it was observed that the growth of the \u003cem\u003eS. epidermidis\u003c/em\u003e was not detected, which would not necessarily indicate that it was entirely suppressed when it was co-cultured with \u003cem\u003eS. aureus\u003c/em\u003e and \u003cem\u003eP. aeruginosa\u003c/em\u003e, but that its numbers were likely to be lower than detection level of the DNA-DNA hybridizations technique of 1 \u003cstrong\u003e\u0026times;\u003c/strong\u003e 10\u003csup\u003e4\u003c/sup\u003e. Besides, it was confirmed that for both salivary film conditioned Ti surfaces and the unconditioned ones, the biofilm developed was dominated by \u003cem\u003eP. aeruginosa\u003c/em\u003e species: 80.4% and 68.8%, respectively, this in comparison to the counts of \u003cem\u003eS. aureus\u003c/em\u003e cells detected on those substrates: 19.1% and 31.3%, respectively. These differences were significant (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) with lower proportions of \u003cem\u003eS. aureus\u003c/em\u003e and higher proportions of \u003cem\u003eP. aeruginosa\u003c/em\u003e, on both, experimental and control surfaces.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eTitanium remains the metal of choice in dental implantology due to its physicochemical and biocompatibility properties. Once dental implants are placed in the oral cavity and exposed to biofluids such as blood and saliva, their surface is subject to the adsorption of biomolecules, with the consequent formation of salivary pellicle (SP) on them, modifying their biological behaviors [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn general terms, dental implants exhibit high success rates. However, bacterial interactions and the subsequent biofilm development on the surfaces of these biomedical devices play a key role in the emergence of peri-implant infections, compromising the longevity of the functional dental implants in the oral cavity. There is no clear understanding of the exact mechanism of microbial interaction during peri-implantitis [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. While some studies conclude that the microbial profile is similar to periodontitis [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e], contradicted studies have reported that opportunistic bacteria, such as \u003cem\u003eStaphylococcus aureus\u003c/em\u003e, which are not part of the most common species in the oral cavity [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e], play a role in the progression of the disease [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. In recent decades, it has been identified that the development of peri-implantitis lesions is associated with the presence, in high proportions, of pathogen-opportunistic and antibiotic-resistant bacteria such as \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e, \u003cem\u003eStaphylococcus aureus\u003c/em\u003e, and \u003cem\u003eStaphylococcus epidermidis\u003c/em\u003e [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. Although different reports indicate that they show some affinity for Ti, the underlying mechanisms of this so call \u0026ldquo;affinity\u0026rdquo; have not been explored in depth [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eUnderstanding the ecological triggers underlying the microbial pathogenesis of peri-implantitis is essential for the development of better preventive, diagnostic and therapeutic strategies, since peri-implant tissues are more susceptible to endogenous oral infections. Due to the above, the main objective of the present investigation was to evaluate the modulating effect of the salivary film formed on Ti surfaces on the development of \u003cem\u003eP. aeruginosa\u003c/em\u003e, \u003cem\u003eS. aureus\u003c/em\u003e and \u003cem\u003eS. epidermidis\u003c/em\u003e biofilms.\u003c/p\u003e \u003cp\u003eAccording to the overall results obtained through the tests performed in this research: SEM, XTT assay, and DNA-DNA hybridizations, when the biofilm development of bacterial strains was evaluated individually, it was observed that the highest affinity for the substrates, regardless of whether they were conditioned or unconditioned, was for the \u003cem\u003eP. aeruginos\u003c/em\u003ea strain, followed by \u003cem\u003eS. epidermidis\u003c/em\u003e, while \u003cem\u003eS. aureus\u003c/em\u003e showed the lowest biofilm development. The enhanced adhesion and subsequent biofilm development by \u003cem\u003eP. aeruginosa\u003c/em\u003e on the surfaces tested can be attributed to the ability of the bacterial cell membrane to stretch over the surfaces, together with a concomitant increase in the level of extracellular polymeric substance (EPS) produced by this microorganism, as previously reported [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. \u003cem\u003eP. aeruginosa\u003c/em\u003e is known to be a strong producer of the glycocalyx, which provides a local environment that favours adhesion, community cohesion, and communication through highly specific interactions between and within cells of both bacterial and host origin [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. In clinical settings, \u003cem\u003eP. aeruginosa\u003c/em\u003e has been isolated from the palate and dorsum of the tongue in a slightly elevated proportion in implant-bearing patients [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. Given the pathogenicity associated with this bacterial strain, the fact that the SP formed on titanium surfaces does not reduce the formation of \u003cem\u003eP. aeruginosa\u003c/em\u003e biofilms, but rather promotes it, is alarming.\u003c/p\u003e \u003cp\u003eRegarding \u003cem\u003eStreptococcus\u003c/em\u003e species biofilm development on the Ti substrates, a marked difference was found between the higher affinity of \u003cem\u003eS. epidermidis\u003c/em\u003e for the substrates compared to the affinity shown by the \u003cem\u003eS. aureus\u003c/em\u003e strain. However, this affinity changed as the presence of \u003cem\u003eS. epidermidis\u003c/em\u003e was masked by \u003cem\u003eS. aureus\u003c/em\u003e and especially \u003cem\u003eP. aeruginosa\u003c/em\u003e in the biofilms developed by the strains studied in coculture.\u003c/p\u003e \u003cp\u003eIt has previously been reported that \u003cem\u003eS. epidermidis\u003c/em\u003e, along with \u003cem\u003eS. aureus\u003c/em\u003e, are isolated in high proportions from Ti dental implant surfaces [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e, \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e], Indeed, both \u003cem\u003eS. aureus\u003c/em\u003e and \u003cem\u003eS. epidermidis\u003c/em\u003e have been associated with subgingival flora around failed implants [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe adhesion of \u003cem\u003eS. aureus\u003c/em\u003e to Ti dental implant surfaces is regarded as favoured by the chemical characteristics of the substrate [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]. \u003cem\u003eS. aureus\u003c/em\u003e is not only isolated more frequently in sites with peri-implantitis [\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e], associated with implant loss [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e] but has also been associated with cases of treatment-resistant periodontitis [\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eRegarding the effect of the salivary film on the biofilm development of the strains studied individually, it was interesting to note that the conditioning of the Ti substrates with the PS only had a strong modulating effect by reducing the growth of \u003cem\u003eS. epidermidis\u003c/em\u003e, whereas it had no significant effect on the development of \u003cem\u003eS. aureus\u003c/em\u003e or \u003cem\u003eP. aeruginosa\u003c/em\u003e biofilms. This seems to contrast with earlier reports [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e], where the salivary film formed on Ti surfaces significantly reduced the adhesion of specific periodontal bacteria such as \u003cem\u003eFusobacterium nucleatum\u003c/em\u003e and \u003cem\u003ePorphyromonas gingivalis\u003c/em\u003e. This would therefore be an indication of a selective antimicrobial effect of the salivary pellicle conditioning of Ti surfaces.\u003c/p\u003e \u003cp\u003eEvaluating the development of biofilms generated by co-culturing \u003cem\u003eP. aeruginosa\u003c/em\u003e, \u003cem\u003eS. aureus\u003c/em\u003e and \u003cem\u003eS. epidermidis\u003c/em\u003e on Ti surfaces, it was found that saliva film allowed greater \u003cem\u003eP. aeruginosa\u003c/em\u003e strain development compared to \u003cem\u003eS. aureus\u003c/em\u003e and \u003cem\u003eS. epidermidis\u003c/em\u003e growth. Previously, it was found that pre-conditioning Ti surfaces with saliva increased bacterial adhesion and biofilm production of \u003cem\u003eP. aeruginosa\u003c/em\u003e [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. The above would confirm that certain salivary proteins adsorbed on Ti surfaces could provide binding receptors for adhesion of this bacterial specie, as previously hypothesised [\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e]. Our results highlight the role of the salivary pellicle in \u003cem\u003eP. aeruginosa\u003c/em\u003e adhesion and suggest that differences in biofilm formation in the oral environment are specific to each bacterial species.\u003c/p\u003e \u003cp\u003eThe precedent that extracellular products of \u003cem\u003eP. aeruginosa\u003c/em\u003e can inhibit the growth of \u003cem\u003eS. epidermidis\u003c/em\u003e and \u003cem\u003eS. aureus\u003c/em\u003e in cocultures [\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e] explains what was observed in the images obtained by Scanning Electron Microscopy in the present investigation, where it was possible to see the predominance of adherent bacilli on the experimental and control Ti surfaces. This finding was confirmed by DNA-DNA hybridization analysis, the bacterial strain colonising the experimental substrates was mainly \u003cem\u003eP. aeruginosa\u003c/em\u003e, followed by \u003cem\u003eS. aureus\u003c/em\u003e, while the presence of \u003cem\u003eS. epidermis\u003c/em\u003e could not be confirmed.\u003c/p\u003e \u003cp\u003eWhile the literature reports that \u003cem\u003eS. aureus\u003c/em\u003e and \u003cem\u003eS. epidermidis\u003c/em\u003e can proliferate abundantly on Ti and its alloys when found together [\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e], there are few reports evaluating biofilm formation of these bacteria together with \u003cem\u003eP. aeruginosa\u003c/em\u003e, individually or in co-culture, on titanium surfaces, and even fewer on titanium surfaces conditioned with salivary pellicle. When the adhesion of the same bacterial strains tested here was evaluated on conventional Ti [\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e] with a contact angle of \u0026sim;70.6, close to the contact angle values of the experimental surfaces in this study: \u0026sim;81.9\u0026deg; \u0026plusmn; 0.4\u0026deg;, a fairly close number of cells of each of the tested bacteria, \u003cem\u003eS. aureus\u003c/em\u003e, \u003cem\u003eS. epidermidis\u003c/em\u003e and \u003cem\u003eP. aeruginosa\u003c/em\u003e, was observed, the latter being the strain that showed the lowest affinity for Ti substrates. However, given that in the study cited above [\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e], the Ti surfaces were not conditioned with the SP, we can highlight the effect of salivary conditioning of the Ti surfaces under the experimental conditions described in the present study.\u003c/p\u003e \u003cp\u003eFinally, it is important to note that as there are few reports evaluating the modular effect of the salivary pellicle on the development of biofilms on Ti surfaces used in implant dentistry, further research is needed on the role of the salivary pellicle constituents in the microbial patterns currently associated with peri-implantitis in clinical practice, not only in periodontally healthy subjects but also in subjects with periodontitis.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eFrom the results of the present investigation, it can be concluded that the presence of salivary pellicle on commercially pure Ti substrates selectively modulates the development of \u003cem\u003eP. aeruginosa\u003c/em\u003e, \u003cem\u003eS. aureus\u003c/em\u003e and \u003cem\u003eS. epidermidis\u003c/em\u003e biofilms on Ti surfaces when cultured individually. The presence of the salivary pellicle positively favored \u003cem\u003eP. aeruginosa\u003c/em\u003e biofilm development on Ti substrates, while inhibiting \u003cem\u003eS. epidermidis\u003c/em\u003e biofilm formation.\u003c/p\u003e \u003cp\u003eIn the case of the biofilm developed on Ti substrates by the co-culture of \u003cem\u003eP. aeruginosa\u003c/em\u003e, \u003cem\u003eS. aureus\u003c/em\u003e and \u003cem\u003eS. epidermidis\u003c/em\u003e, it was found that the salivary pellicle favored the growth mainly of \u003cem\u003eP. aeruginosa\u003c/em\u003e over the growth of \u003cem\u003eS. aureus\u003c/em\u003e or even \u003cem\u003eS. epidermidis\u003c/em\u003e, this under a probable common ecological scenario.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e This work was supported by UNAM-PAPIIT IN209324, IA20064 and IN229223.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability\u0026nbsp;\u003c/strong\u003eThe data that support the findings of this study are available from the corresponding author upon reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate.\u0026nbsp;\u003c/strong\u003eThe collection of the saliva sample was carried out under the acceptance and approval of the corresponding Ethics Committee. The authors declare that the procedures followed conform to the standards of the Ethics Committee on Human Studies of the Division of Postgraduate Studies and Research of the Faculty of Dentistry of the National Autonomous University of Mexico (CIE/0108/09/2023), which was conducted in accordance with the Declaration of Helsinki.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of Interest\u0026nbsp;\u003c/strong\u003eThe authors declare no competing interests.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors thank to collaboration of the laboratory technician Leticia Cruz Fonseca of the Universidad Nacional Aut\u0026oacute;noma de M\u0026eacute;xico (UNAM).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eORCID\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMart\u0026iacute;nez-Hern\u0026aacute;ndez, Miryam https://orcid.org/0000-0002-1589-8605\u003c/p\u003e\n\u003cp\u003eReyes-Mendoza, Paulina-Polet\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eCh\u0026aacute;vez-Esparza, Mariana\u003csup\u003e\u0026nbsp;\u003c/sup\u003e\u003c/p\u003e\n\u003cp\u003eRodr\u0026iacute;guez-Hern\u0026aacute;ndez, Adriana-Patricia https://orcid.org/0000-0002-9473-1749\u003c/p\u003e\n\u003cp\u003eGarc\u0026iacute;a-P\u0026eacute;rez, V\u0026iacute;ctor I. https://orcid.org/0000-0003-2999-6385\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eAuthor Contributions StatementConceptualization, validation, formal analysis, data curation, supervision, project administration, resources and funding acquisition: Mart\u0026iacute;nez-Hern\u0026aacute;ndez, Miryam, Rodr\u0026iacute;guez-Hern\u0026aacute;ndez, Adriana-Patricia, and Garc\u0026iacute;a-P\u0026eacute;rez, V. I.; methodology and writing, and editing\u0026mdash;original draft: Rodr\u0026iacute;guez-Hern\u0026aacute;ndez, Adriana-Patricia, Mart\u0026iacute;nez-Hern\u0026aacute;ndez, Miryam, Garc\u0026iacute;a-P\u0026eacute;rez, V. I, Ch\u0026aacute;vez-Esparza, M and Reyes-Mendoza, P; review, and editing\u0026mdash;original draft: Rodr\u0026iacute;guez-Hern\u0026aacute;ndez, Adriana-Patricia, Mart\u0026iacute;nez-Hern\u0026aacute;ndez, Miryam, Garc\u0026iacute;a-P\u0026eacute;rez, V. I, Ch\u0026aacute;vez-Esparza, M and Reyes-Mendoza, P. 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Biomaterials 31:706\u0026ndash;713. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.biomaterials.2009.09.081\u003c/span\u003e\u003cspan address=\"10.1016/j.biomaterials.2009.09.081\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"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":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Salivary pellicle, titanium surfaces, dental implants, Staphylococcus aureus, Staphylococcus epidermidis, Pseudomonas aeruginosa","lastPublishedDoi":"10.21203/rs.3.rs-6638066/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6638066/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eObjectives\u003c/h2\u003e \u003cp\u003eTo evaluate the modulating effect of salivary pellicle (SP) on titanium (Ti) surfaces on the development of \u003cem\u003eStaphylococcus\u003c/em\u003e species and \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e biofilm.\u003c/p\u003e\u003ch2\u003eMaterials and Methods\u003c/h2\u003e \u003cp\u003eTi substrates were incubated for 2 hours with whole saliva samples to induce the salivary pellicle formation. After conditioning with SP, Ti substrates were incubated for 12 hours with \u003cem\u003eP. aeruginosa\u003c/em\u003e, \u003cem\u003eS. aureus\u003c/em\u003e, \u003cem\u003eS. epidermidis\u003c/em\u003e strains individually, in addition to co-culture of the three bacteria. The biofilm development on the Ti substrates was visualized by scanning electron microscopy (SEM). To measure the metabolic activity and vitality of cells within the biofilm the XTT assay was used, while the proportion of the species tested in the biofilms was determined throughout DNA-DNA hybridizations (checkerboard).\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eSalivary pellicle modulated the biofilm development on the Ti surfaces, favouring the formation of \u003cem\u003eP. aeruginosa\u003c/em\u003e biofilms, while inhibiting the growth of \u003cem\u003eS. aureus\u003c/em\u003e and \u003cem\u003eS. epidermidis\u003c/em\u003e. In the case of the coculture biofilms, a predominance of \u003cem\u003eP. aeruginosa\u003c/em\u003e cells over the \u003cem\u003eStaphylococcus\u003c/em\u003e strains was observed.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003eWhen the bacterial strains were tested individually, the SP importantly reduced the development of biofilms of \u003cem\u003eStaphylococcus\u003c/em\u003e species, whereas it favored the development of \u003cem\u003eP. aeruginosa\u003c/em\u003e biofilms on Ti surfaces. Similarly, when mixed biofilms developed, SP favored the selective expansion of \u003cem\u003eP. aeruginosa\u003c/em\u003e and \u003cem\u003eS. aureus\u003c/em\u003e growth over \u003cem\u003eS. epidermidis\u003c/em\u003e growth.\u003c/p\u003e\u003ch2\u003eClinical relevance:\u003c/h2\u003e \u003cp\u003eThe results of this study provide valuable information on the modulatory effect of the SP on the development of opportunistic bacteria biofilms on Ti surfaces used for dental and oral implantology.\u003c/p\u003e","manuscriptTitle":"The modulating effect on Staphylococcus species and Pseudomonas aeruginosa biofilm development of salivary pellicle conditioning titanium surfaces","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-05-13 10:28:28","doi":"10.21203/rs.3.rs-6638066/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"12f86826-ba63-4fac-a66e-676639967102","owner":[],"postedDate":"May 13th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-05-27T06:08:41+00:00","versionOfRecord":[],"versionCreatedAt":"2025-05-13 10:28:28","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6638066","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6638066","identity":"rs-6638066","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}