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High levels of antibiotic tolerance are a hallmark of bacterial biofilms. In contrast to well-characterized inherited antibiotic resistance, molecular mechanisms leading to reversible and transient antibiotic tolerance displayed by biofilm bacteria are still poorly understood. The physiological heterogeneity of biofilms influences the formation of transient specialized subpopulations that may be more tolerant to antibiotics. In this study, we used random transposon mutagenesis to identify biofilm-specific tolerant mutants normally exhibited by subpopulations located in specialized niches of heterogeneous biofilms. Using Escherichia coli as a model organism, we demonstrated, through identification of amino acid auxotroph mutants, that starved biofilms exhibited significantly greater tolerance towards fluoroquinolone ofloxacin than their planktonic counterparts. We demonstrated that the biofilm-associated tolerance to ofloxacin was fully dependent on a functional SOS response upon starvation to both amino acids and carbon source and partially dependent on the stringent response upon leucine starvation. However, the biofilm-specific ofloxacin increased tolerance did not involve any of the SOS-induced toxin-antitoxin systems previously associated with formation of highly tolerant persisters. We further demonstrated that ofloxacin tolerance was induced as a function of biofilm age, which was dependent on the SOS response. Our results therefore show that the SOS stress response induced in heterogeneous and nutrient-deprived biofilm microenvironments is a molecular mechanism leading to biofilm-specific high tolerance to the fluoroquinolone ofloxacin.

This study aimed to investigate the effect of erythritol air-polishing on implant surface topography and bacterial colonization, and to determine the antimicrobial activity of erythritol powder. Titanium implants, with machined/acid-etched hybrid design, were divided into three groups: erythritol air-polishing for 1 min (E1), 5 min (E5), and untreated control. Surface analysis was performed using a stylus profilometer and scanning electron microscope (SEM). To test the ability to prevent biofilm formation, four bacteria strains (Staphylococcus aureus, Klebsiella pneumoniae, Streptococcus mutans, Streptococcus sanguinis) were separately cultured on five implants per group and colony counting was performed. The intrinsic erythritol antibacterial activity was investigated by means of minimum inhibitory concentration against the same strains. At SEM analysis implant surfaces appeared unaltered by air-polishing and presented increasing amount of residues depending on the treatment duration. Machined surfaces exhibited no significant differences in roughness parameters between the groups. On acid-etched surfaces, E5 presented significantly lower Ra (vs. E1 and control) and Rz (vs. control). The count of colonies was significantly lower for all bacterial strains on treated implants as compared to control, with E1 and E5 being equally capable to reduce by 1.5 log bacteria growth. Erythritol antimicrobial activity against all tested bacterial strains was confirmed. The proposed erythritol air-polishing protocols did not alter implant surfaces and the antimicrobial properties of erythritol are conserved by the titanium implant surfaces. Erythritol air-polishing could be repeatedly used in supportive peri-implant care programmes.

The effect of two dietary sugars, glucose and galactose, on biofilm formation of the oral fungal pathogen Candida on denture acrylic strips coated with saliva and serum pellicles was examined in vitro using Candida albicans (3 isolates), C. glabrata (2 isolates) and C. tropicalis (2 isolates). The degree of biofilm activity was affected by both the dietary sugar and the nature of the pellicle (ANOVA, p < 0.01). With most isolates the glucose grown yeasts demonstrated significantly more bioflim activity than the galactose grown fungi, in the presence of pellicles (ANOVA, p < 0.01 or P < 0.01). In contrast, one isolate of galactose-grown yeast elicited significantly higher biofilm activity than glucose-grown yeasts on the control strips (ANOVA, p < 0.01). Taken together, these results imply that a saliva or a serum pellicle, and the carbon source in the environment, act a complex manner modulating Candida bioflim formation.