Transcript Document 7578756

Laboratory Studies:
Resistance / Cross-resistance
Development in Microcosm
Communities
Peter GILBERT, B.Sc., Ph.D
University of Manchester, UK
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
Four Year Study
Stable
Polymicrobial
Community
Feeding
regime:
Regular Analysis by DGGE
Biofilms fedarchived,
4 x daily,16S
20 Identities
Culturables
min. perfusion,
0.5 ml(MIC,
/
Susceptibility
testing
MBC)
min., synthetic dish
to
large panel of biocides and antibiotics
water (feast)
Biocide Studies
Fermenter
pans were
Parallel Fermenters
continuously
wettedrun to
steady
state, tap water
with
untreated
Dosing
for 6months
(1
ml / min)
(famine)
Triclosan-containing detergent
QAC-based detergent
TRICLOSAN DEGRADATION
A. caviae (X74674)
Aeromonas caviae
Aeromonas jandei
Aeromonas hydrophila
Aeromonas hydrophila
Aeromonas hydrophila (X60404)
Serratia grimesii (AJ233430)
Serratia sp.
Serratia proteamaculans (AJ233435)
Serratia plymuthica (AF286872)
Serratia plymuthica (AJ233433)
K. pneumoniae (X87276)
Pseudomonas aeruginosa (AB037547)
Baseline communities
10.6 - 11.4 Log10 CFU/g
80 Achromobacter xylosoxidans (AF411021)
42 Alcaligenes xylosoxidans (D88005)
22 Blackwater bioreactor bacterium (AF394171)
13 unidentified eubacterium
Mainly:
Pseudomonads
Enteric species
Bacilli
100 unidentified eubacterium
unidentified eubacterium
95
28
48
46
42
86
unidentified eubacterium
unidentified eubacterium
unidentified eubacterium
unidentified eubacterium
100 Delftia acidovorans
Delftia acidovorans (AF149849)
Ralstonia oxalatica (AF155567)
100
Ralstonia basilensis (AF312022)
50
61 Ralstonia sp.
Ralstonia sp. (AF301897)
Pseudomonas aeruginosa (AB037547)
100 Pseudomonas nitroreducens (D84021)
86
59 Pseudomonas sp.
Also, sulphate reducers
Clostridia
Prevotella sp.
42
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Pseudomonas nitroreducens (D84021)
Pseudomonas nitroreducens
Delftia acidovorans (AF149849)
Delftia acidovorans
Comamonas sp. AJ002803
Ralstonia oxalatica (AF155567)
Ralstonia sp. (AF301897)
Ralstonia sp.
Achromobacter xylosoxidans
Achromobacter xylosoxidans (AF411021)
Alcaligenes xylosoxidans (D88005)
Blackwater bioreactor bacterium (AF394171)
Uncultured eubacterium clone (AY038619)
Unidentified eubacterium
Stenotrophomonas maltophilia (AJ2956730
Xanthomonas sp. Oral clone (AJ13904)
Stenotrophomonas sp.
Stenotrophomonas sp. (AJ300772)
Xanthomonas campestris (AF123092)
Stenotrophomonas matophilia
Stenotrophomonas maltophilia
Stenotrophomonas maltophilia (X95923)
Stenotrophomonas maltophilia
Stenotrophomonas maltophilia
Microbacterium phyllosphaerae (AJ277840)
Microbacterium phyllosphaerae
Microbacterium imperialis (X77442)
Staphylococcus warneri (L37603)
Bacillus licheniformis (AF391127)
Bacillus sphaericus AF435435
Bacillus sp.
Bacillus fusiformis (AF169537)
Bacillus sp.
Bacillus sp.
Bacillus cereus
Bacillus cereus (AF155952)
Bacillus anthracis (AF176321)
Bacillus sp.
Bosea thiooxidans (AJ250798)
unidentified alpha proteobacterium
Afipia sp. (AJ29922)
Afipia sp.
Chelatococcus asaccharovorans (AJ294349)
Flavobacterium thalpophilum (D14020)
Sphingobacterium multivorum (AB020205)
Chryseobacterium scophthalmum (AJ271009)
Chryseobacterium joostei
87
94 unidentified gamma proteobacterium
Uncultured gamma proteobacterium
100 Bacillus sphaericus AF435435
96 Bacillus cereus
100
Bacillus fusiformis (AF169537)
Bacillus licheniformis (AF391127)
Bacillus anthracis (AF176321)
100 Bacillus cereus (AJ310098)
Bacillus cereus
Afipia sp. (AJ29922)
87
97 Bosea thiooxidans (AJ250798)
Afipia sp.
100 90 Afipia sp. (AJ29922)
Chelatococcus asaccharovorans (AJ294349)
93 Sphingobacterium multivorum (AB020205)
Uncultured Sphingobacterium (AB076874)
100
Flavobacterium thalpophilum (D14020)
100
Chryseobacterium scophthalmum (AJ271009)
45
Chryseobacterium indologenes AY050493
100
Chryseobacterium scophthalmum (AJ271009)
47
Triclosan exposure (Palmolive washingup liquid)
100
24
(3 months at 4 mg / ml, 3 at 8 mg / ml)
63
93
Expansion of:
Clonal expansion of:
99 Chryseobacterium joostei(AJ271010)
Chryseobacterium joostei
Thermus thermophilus (outgroup)
Tolerant and putatively degradative “Unidentified eubacterium”
organisms
(Homology to Alcaligenes
expansion
of D88005)
innately
xylosoxidans
• Reduction in
strains Hyper- sensitive species
insensitive strains
• Lab trained TCS tolerant E.coli fails to establish
Chryseobacterium sp.
Chryseobacterium sp.
Chryseobacterium sp.
Thermus thermophilus (outgroup)
Loss of:
susceptible
Microcosm Isolates
• Strains archived over 4 years
• Retrospective analysis of susceptibility
• Culturable isolates unchanged with
respect to susceptiblity towards a range of
antibiotics
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.
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
53 Household
products
purchased and
tested as inducers
More than
half induced
Mar
E coli SPC105
MarO -LacZ
11
X-gal top-agar
CONTROL
BIBLIOGRAPHY
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.
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