Tolerant Small-colony Variants Form Prior to Resistance Within a Staphylococcus aureus Biofilm Based on Antibiotic Selective Pressure

BACKGROUND: The treatment of periprosthetic joint infection (PJI) is focused on the surgical or chemical removal of biofilm. Antibiotics in isolation are typically ineffective against PJI. Bacteria survive after antibiotic administration because of antibiotic tolerance, resistance, and persistence that arise in the resident bacteria of a biofilm. Small-colony variants are typically slow-growing bacterial subpopulations that arise after antibiotic exposure and are associated with persistent and chronic infections such as PJI. The role of biofilm-mediated antibiotic tolerance in the emergence of antibiotic resistance remains poorly defined experimentally. QUESTIONS/PURPOSES: We asked: (1) Does prior antibiotic exposure affect how Staphylococcus aureus survives within a developing biofilm when exposed to an antibiotic that penetrates biofilm, like rifampicin? (2) Does exposure to an antibiotic with poor biofilm penetration, such as vancomycin, affect how S. aureus survives within a developing biofilm? (3) Do small-colony variants emerge from antibiotic-tolerant or -resistant bacteria in a S. aureus biofilm? METHODS: We used a porous membrane as an in vitro implant model to grow luminescent S. aureus biofilms and simultaneously track microcolony expansion. We evaluated the impact of tolerance on the development of resistance by comparing rifampicin (an antibiotic that penetrates S. aureus biofilm) with vancomycin (an antibiotic that penetrates biofilm poorly). We performed viability counting after membrane dissociation to discriminate among tolerant, resistant, and persistent bacteria. Biofilm quantification and small-colony morphologies were confirmed using scanning electron microscopy. Because of experimental variability induced by the starting bacterial inoculum, relative changes were compared since absolute values may not have been statistically comparable. RESULTS: Antibiotic-naïve S. aureus placed under the selective pressure of rifampicin initially survived within an emerging biofilm by using tolerance given that biofilm resident cell viability revealed 1.0 x 108 CFU, of which 7.5 x 106 CFU were attributed to the emergence of resistance and 9.3 x 107 CFU of which were attributed to the development of tolerance. Previous exposure of S. aureus to rifampicin obviated tolerance-mediate survival when rifampicin resistance was present, since the number of viable biofilm resident cells (9.5 x 109 CFU) nearly equaled the number of rifampicin-resistant bacteria (1.1 x 1010 CFU). Bacteria exposed to an antibiotic with poor biofilm penetration, like vancomycin, survive within an emerging biofilm by using tolerance as well because the biofilm resident cell viability for vancomycin-naïve (1.6 x 1010 CFU) and vancomycin-resistant (1.0 x 1010 CFU) S. aureus could not be accounted for by emergence of resistance. Adding rifampicin to vancomycin resulted in a nearly 500-fold reduction in vancomycin-tolerant bacteria from 1.5 x 1010 CFU to 3.3 x 107 CFU. Small-colony variant S. aureus emerged within the tolerant bacterial population within 24 hours of biofilm-penetrating antibiotic administration. Scanning electron microscopy before membrane dissociation confirmed the presence of small, uniform cells with biofilm-related microstructures when unexposed to rifampicin as well as large, misshapen, lysed cells with a small-colony variant morphology [29, 41, 42, 63] and a lack of biofilm-related microstructures when exposed to rifampicin. This visually confirmed the rapid emergence of small-colony variants within the sessile niche of a developing biofilm when exposed to an antibiotic that exerted selective pressure. CONCLUSION: Tolerance explains why surgical and nonsurgical modalities that rely on antibiotics to "treat" residual microscopic biofilm may fail over time. The differential emergence of resistance based on biofilm penetration may explain why some suppressive antibiotic therapies that do not penetrate biofilm well may rely on bacterial control while limiting the emergence of resistance. However, this strategy fails to address the tolerant bacterial niche that harbors persistent bacteria with a small-colony variant morphology. CLINICAL RELEVANCE: Our work establishes biofilm-mediated antibiotic tolerance as a neglected feature of bacterial communities that prevents the effective treatment of PJI.