Effect of low doses of biocides on the antimicrobial resistance and the biofilms of Cronobacter sakazakii and Yersinia enterocolitica

The susceptibility of Cronobacter sakazakii ATCC 29544 (CS) and Yersinia enterocolitica ATCC 9610 (YE) to sodium hypochlorite (10% of active chlorine; SHY), peracetic acid (39% solution of peracetic acid in acetic acid; PAA) and benzalkonium chloride (BZK) was tested. Minimum inhibitory concentration (MIC) values (planktonic cells; microdilution broth method) of 3,800 ppm (SHY), 1,200 ppm (PAA) and 15 ppm (BZK) for CS, and 2,500 ppm (SHY), 1,275 ppm (PAA) and 20 ppm (BZK) for YE, were found. In some instances, an increase in growth rate was observed in presence of sub-MICs (0.25MIC, 0.50MIC or 0.75MIC) of biocides relative to the samples without biocides. The cultures exhibited an acquired tolerance to biocides and an increase in antibiotic resistance after exposure to sub-MICs of such disinfectants. Strains were able to form strong biofilms on polystyrene after 48 hours (confocal laser scanning microscopy), with average biovolumes in the observation field (14,161 µm(2)) of 242,201.0 ± 86,570.9 µm(3) (CS) and 190,184.5 ± 40,860.3 µm(3) (YE). Treatment of biofilms for 10 minutes with disinfectants at 1MIC or 2MIC reduced the biovolume of live cells. PAA (YE) and BZK (CS and YE) at 1MIC did not alter the percentage of dead cells relative to non-exposed biofilms, and their effect of countering biofilm was due principally to the detachment of cells. These results suggest that doses of PAA and BZK close to MICs might lead to the dissemination of live bacteria from biofilms with consequent hazards for public health.