Development and evaluation of a broth macrodilution method to determine the biocide susceptibility of bacteria

Abstract

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 2 mL volumes for the testing and an incubation time of 24 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.

Keywords: Benzalkonium chloride; Chlorhexidine; Enterococcus hirae; Escherichia coli; Glutardialdehyde; Isopropanol; Pseudomonas aeruginosa; Staphylococcus aureus.