Biofilms: A developmental niche for vancomycin-intermediate Staphylococcus aureus.

Staphylococcus aureus are gram-positive bacteria responsible for a wide array of diseases, ranging from skin and soft tissue infections to more chronic illnesses such as toxic shock syndrome, osteomyelitis, and endocarditis. Vancomycin is currently one of the most effective antibiotics available in treating patients infected with methicillin-resistant S. aureus (MRSA), however the emergence of vancomycin-resistant S. aureus (VRSA), and more commonly vancomycin-intermediate S. aureus (VISA), threaten the future efficacy of vancomycin. Intermediate resistance to vancomycin occurs due to mutations within the loci of Staphylococcal genes involved in cell wall formation such as rpoB, graS, and yycG. We hypothesized the VISA phenotype may also arise as a result of the natural stress occurring within S. aureus biofilms, and that this phenomenon is mediated by the RecA/SOS response. Wildtype and recA null mutant/lexAG94E strains of S. aureus biofilms were established in biofilm microtiter assays or planktonic cultures with or without the addition of sub-inhibitory concentrations of vancomycin (0.063 mg/l - 0.25 mg/L ciprofloxacin, 0.5 mg/l vancomycin). Efficiency of plating techniques were used to quantify the subpopulation of biofilm-derived S. aureus cells that developed vancomycin-intermediate resistance. The results indicated that a greater subpopulation of cells from wildtype biofilms (4.16 × 102 CFUs) emerged from intermediate-resistant concentrations of vancomycin (4 µg/ml) compared with the planktonic counterpart (1.53 × 101 CFUs). Wildtype biofilms (4.16 × 102 CFUs) also exhibited greater resistance to intermediate-resistant concentrations of vancomycin compared with strains deficient in the recA null mutant (8.15 × 101 CFUs) and lexA genes (8.00 × 101 CFUs). While the VISA phenotype would be an unintended consequence of genetic diversity and potentially gene transfer in the biofilm setting, it demonstrates that mutations occurring within biofilms allow for S. aureus to adapt to new environments, including the presence of widely used antibiotics.

Electrically polarized nanoscale surfaces generate reactive oxygenated and chlorinated species for deactivation of microorganisms.

Because of the decreasing supply of new antibiotics, recent outbreaks of infectious diseases, and the emergence of antibiotic-resistant microorganisms, it is imperative to develop new effective strategies for deactivating a broad spectrum of microorganisms and viruses. We have implemented electrically polarized nanoscale metallic (ENM) coatings that deactivate a wide range of microorganisms including Gram-negative and Gram-positive bacteria with greater than 6-log reduction in less than 10 minutes of treatment. The electrically polarized devices were also effective in deactivating lentivirus and Candida albicans. The key to the high deactivation effectiveness of ENM devices is electrochemical production of micromolar cuprous ions, which mediated reduction of oxygen to hydrogen peroxide. Formation of highly damaging species, hydroxyl radicals and hypochlorous acid, from hydrogen peroxide contributed to antimicrobial properties of the ENM devices. The electric polarization of nanoscale coatings represents an unconventional tool for deactivating a broad spectrum of microorganisms through in situ production of reactive oxygenated and chlorinated species.