RESUMO
Surface disinfectants are regularly used in prophylactic and infection control measures. Concern has been raised whether residues of sub-inhibitory disinfectant concentrations may constitute a selective pressure and could contribute to the development of strains which are tolerant and/or resistant to biocides including antibiotics. The current study investigated whether Staphylococcus (S.) aureus ATCC® 29213™ and ATCC® 6538™ would change their growth characteristics and antimicrobial susceptibility profiles after prolonged treatment with sub-inhibitory concentrations of sodium hypochlorite (NaOCl). NaOCl is a fast-acting disinfectant with a broad-spectrum activity, inexpensive and widely used in healthcare and the food production industry. Minimum inhibitory concentration (MIC) for NaOCl was determined by broth macrodilution according to the guidelines for disinfectant efficacy testing provided by the German Veterinary Medical Society. Serial passages after 24 h and 72 h, respectively, in defined sub-inhibitory concentrations of NaOCl resulted in a number of phenotypic variants. Two of these variants, derived from S. aureus ATCC® 29213™, showed elevated MICs of oxacillin and were considered as in vitro-generated borderline oxacillin-resistant S. aureus (BORSA). Transmission electron microscopy revealed a significantly thickened cell wall in these isolates, a phenomenon that has also been described for Listeria monocytogenes after low-level exposure to NaOCl. Whole genome sequencing revealed an early stop codon in the gene coding for the GdpP protein and thereby abolishing the function of this gene. GdpP represents a phosphodiesterase that regulates gene expression, and loss of function of the GdpP protein has been described in association with borderline oxacillin resistance. Our findings suggest that a mutation in the GdpP protein gene and morphological changes of the cell wall were induced by repeated exposure to sub-lethal NaOCl concentrations, and most likely accounted for a BORSA phenotype in two variants derived from S. aureus ATCC® 29213™.
RESUMO
In comparison to biocide efficacy testing, biocide susceptibility testing of bacteria so far lacks standardized methods for routine use. The aims of the present study were to develop a broth macrodilution method to test bacterial pathogens for their biocide susceptibility and to evaluate this method in an interlaboratory trial. Staphylococcus aureus ATCC®6538 was tested for its susceptibility to benzalkonium chloride, chlorhexidine and isopropanol comparing test strain suspension preparations, test volumes and incubation times. The use of 2â¯mL volumes for the testing and an incubation time of 24â¯h were proposed. Ten German laboratories participated in the interlaboratory trial. Four reference strains (S. aureus ATCC®6538, Enterococcus hirae ATCC®10541, Escherichia coli ATCC®10536 and Pseudomonas aeruginosa ATCC®15442) commonly used for biocide activity testing, were included. Strains were tested three times at independent occasions for their susceptibility to benzalkonium chloride, glutardialdehyde and isopropanol. In total, 360 data points were obtained (30 per strain/biocide combination). The modal minimal inhibitory concentration ± one dilution step was defined as acceptable range. For the four reference strains and the three biocides 80-100% of the values were considered as acceptable. The deviations within the laboratories for a strain/biocide combination were rather consistent. In general, the testing was performed without difficulties by the laboratories. Although inoculum plate counts of four laboratories were outside the acceptable range, this did not have a large impact on the results. The proposed method was stable and easy to perform. It may contribute to a harmonization and standardization of biocide susceptibility testing.
