Peter GILBERT, B.Sc., Ph.D University of Manchester, UK Laboratory Studies: Resistance / Cross-resistance Development in Microcosm Communities

Bacteria Grow as Biofilms Ubiquitous Polymicrobial (skin, gut, mouth,envirnoment) Functional consortia of microbial cells forming at surfaces Show both phenotypic and clonal heterogeneity COMMUNITIES

Drain Fermentation Rig Feeding regime: Biofilms fed 4 x daily, 20 min. perfusion, 0.5 ml / min., synthetic dish water (feast) Fermenter pans were continuously wetted with untreated tap water (1 ml / min) (famine) Biocide Studies Parallel Fermenters run to steady state, Dosing for 6months Triclosan-containing detergent QAC-based detergent Four Year Study Stable Polymicrobial Community Regular Analysis by DGGE Culturables archived, 16S Identities Susceptibility testing (MIC, MBC) to large panel of biocides and antibiotics

TRICLOSAN DEGRADATION Baseline communities Log 10 CFU/g Mainly: Pseudomonads Enteric species Bacilli Also, sulphate reducers Clostridia Prevotella sp. Triclosan exposure (Palmolive washing- up liquid) (3 months at 4  g / ml, 3 at 8  g / ml) Expansion of: Tolerant and putatively degradative organisms Loss of: Hyper- sensitive species Clonal expansion of: “Unidentified eubacterium” (Homology to Alcaligenes xylosoxidans D88005) Reduction in susceptible strains - expansion of innately insensitive strains Lab trained TCS tolerant E.coli fails to establish

Microcosm Isolates Strains archived over 4 years Retrospective analysis of susceptibility Culturable isolates unchanged with respect to susceptiblity towards a range of antibiotics

40 Selected isolates subjected to Triclosan “training”. Exposure of E. coli markedly decreased triclosan susceptibility, significant but small changes in existing insusceptibility of Klebsiella oxytoca, Aranicola proteolyticus and Stenotrophomonas maltophilia. Susceptibility of the remaining 35 strains to triclosan and antibiotics remained unchanged. Conclusions: Selection for insensitivity to triclosan exposure is not widespread but is confined to certain enteric bacteria. Change in TCS susceptibility did not affect susceptibility towards chemically unrelated antimicrobials. Effects of chronic triclosan exposure upon the antimicrobial susceptibility of 40 ex-situ environmental and human isolates

Conclusions Natural communities are fiercely competitive Chronic exposure to antibacterials (triclosan, QAC) leads to clonal expansion of pre-existing insensitive strains No evidence of emergence of de-novo resistance traits Lab-selected mutants incur fitness cost and cannot survive

Efflux Induction E coli SPC105 MarO 11 -LacZ X-gal top-agar More than half induced Mar CONTROL 53 Household products purchased and tested as inducers Multi-Drug Efflux Pumps the microbial vomit response

Microcosm Studies MCBAIN, A.J., S.A. BARTOLO, R., CATRENICH, C., CHARBONNEAU, D., LEDDER, R. & P. GILBERT (2003) Growth and molecular characterisation of dental plaque microcosms, Journal of Applied Microbiology, 94, MCBAIN, A.J., S.A. BARTOLO, R., CATRENICH, C., CHARBONNEAU, D., LEDDER, R. & P. GILBERT (2003) Effects of triclosan-containing rinse upon the dynamics and antimicrobial susceptibility of in-vitro plaque ecosystems. Antimicrobial Agents and Chemotherapy, 47, McBAIN, A.J., BARTOLO R.G., CATRENICH, C.E., CHARBONNEAU, D., LEDDER, R.L., PRICE, B.B. AND P. GILBERT (2003) Long-term exposure of sink drain microcosms to triclosan: effects upon bacterial vitality and antimicrobial susceptibility. Applied and Environmental Microbiology, 69, McBAIN, A.J., BARTOLO R.G., CATRENICH, C.E., CHARBONNEAU, D., LEDDER, R.L., AND P. GILBERT (2003) Effects of a chlorhexidine gluconate-containing mouthwash upon the vitality and antimicrobial susceptibility of in-vitro plaque ecosystems. Applied and Environmental Microbiology, 69, McBain, A.J., R.G Ledder, P. Sreenivasan & P. GILBERT (2004) Selection for Resistance by Chronic Triclosan Exposure is not Universal. Journal of Antimicrobial Chemotherapy 53, Ledder, Gilbert, Willis & McBain (2005) Effects of chronic triclosan exposure upon the antimicrobial susceptibility of 40 ex-situ environmental and human isolates J. Appl. Microbiol In Press Adaptation by Ex-vivo Isolates Rickard, A.H., Lindsay, S., Lockwood, G.B. & P. GILBERT (2004) Induction of multiple antibiotic resistance operon by miscellaneous groceries Journal of Applied Microbiology, 97, Inducers of Efflux BIBLIOGRAPHY