Peter GILBERT, B.Sc., Ph.D

1
Laboratory Studies Resistance / Cross-resistance
Development in Microcosm Communities

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

2
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
3
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)
4
TRICLOSAN DEGRADATION
Baseline communities 10.6 - 11.4 Log10
CFU/g Mainly Pseudomonads Enteric
species Bacilli Also, sulphate reducers
Clostridia Prevotella sp.
Triclosan exposure (Palmolive washing-up
liquid) (3 months at 4 mg / ml, 3 at 8 mg /
ml) Expansion of Tolerant and putatively
degradative organisms Loss of Hyper- sensitive
species
Clonal expansion of Unidentified
eubacterium (Homology to Alcaligenes
xylosoxidans D88005)
5
Microcosm Isolates

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

6
Effects of chronic triclosan exposure upon the
antimicrobial susceptibility of 40 ex-situ
environmental and human isolates

• 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.

7
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

8
Multi-Drug Efflux Pumpsthe microbial vomit
response
Efflux Induction
53 Household products purchased and tested as
inducers
CONTROL
More than half induced Mar



E coli SPC105
MarO11-LacZ
X-gal top-agar
9
Microcosm Studies
BIBLIOGRAPHY

• 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.

Adaptation by Ex-vivo Isolates
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
Inducers of Efflux
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