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Effect of multiple passages of S. typhimurium in the presence of disinfectants on susceptibility to antimicrobials, on persistence in the one-day-old chick model and efflux systems

Abstract number: 1733_382

Bagnall M., Randall L., Coldham N., Karatzas A., Humphrey T., Piddock L., Woodward M.

Objectives: Reduced susceptibility to certain disinfectants has been linked with reduced susceptibility to some antibiotics. If persistence and accumulation of such mutants persist within the food chain this would be of concern. In this study, mutants derived from Salmonella typhimurium following multiple exposures to two different farm disinfectants (either a combination formaldehyde, glutaraldehyde and quaternary ammonium compound aldehyde-based disinfectant (F/G/QAC) or an oxidising compound disinfectant (OXC) disinfectant) were analysed for their susceptibility to antimicrobials and fitness in the day old chick model. The proteomes were also compared and molecular flux investigated using the fluorescent dye bisbenzimide (H33342).

Methods: Minimum Inhibitory Concentrations (MIC) of various disinfectants and antibiotics were determined by the BSAC agar dilution method. In competitive index experiments one-day-old chicks were infected by oral gavage (c.104 cfu per bird, c.1:1 mix of mutant and parent strain). Infection was monitored by cloacal swabbing at 3, 10, 17, 24, 31 and 35 days post-infection. Infection was calculated as cfu/g of faecal matter. In conjunction with the cloacal swabs 5 birds/group were sacrificed at 7, 14, 21 and 28 days post-infection and the caecal contents enumerated as previously described. Proteomes were determined by 2D-LC-MS and uptake of bisbenzimide (2.5 mM) using a fluorescent plate reader in a 96 well format at 350 and 460 nm respectively.

Results: The mutant strains were significantly less able to persist in the one-day-old chick model than their parent strains. Both mutants showed reduced susceptibility (circa x 4) to some clinical antibiotics (cip, chlor, tet and amp) and were also cyclohexane resistant. This suggests that the mechanism of resistance is due to up-regulated efflux and/or reduced porins. The proteomics confirmed increased expression of the AcrAB/TolC efflux pump. Reduced uptake of the dye bisbenzimide was consistent with these changes in protein expression.

Conclusions: Repeated exposure of Salmonella Typhimurium to disinfectants gave rise to mutants with reduced susceptibility to disinfectants and/or antibiotics. This was coupled with increased efflux, as measured by bisbenzimide uptake assays, and increased differences in key efflux proteins.

Session Details

Date: 31/03/2007
Time: 00:00-00:00
Session name: European Society of Clinical Microbiology and Infectious Diseases
Subject:
Location: ICC, Munich, Germany
Presentation type:
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