# Presentation: 049350

## Peter GILBERT, B.Sc., Ph.D
University of Manchester, UK

*University of Manchester, UK*

- Laboratory Studies: Resistance / Cross-resistance Development in Microcosm Communities

## Bacteria Grow as Biofilms

**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)***** **** **

## TRICLOSAN DEGRADATION

**TRICLOSAN DEGRADATION**

**Baseline communities**
**10.6 - 11.4 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)**

## 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. 

- 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**

## Multi-Drug Efflux Pumpsthe microbial vomit response

**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,** 655-664
- 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,** 3531-3538
- 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,** 5433-5442.
- 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,** 4770-4776.

- 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, 772-777

- 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,** 1063-1068

**Inducers of Efflux**

**BIBLIOGRAPHY**