Peter GILBERT, B.Sc., Ph.DUniversity 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 StudiesParallel Fermenters run to steady state, Dosing for 6monthsTriclosan-containing detergent QAC-based detergent

Four Year Study Stable Polymicrobial CommunityRegular Analysis by DGGECulturables archived, 16S IdentitiesSusceptibility testing (MIC, MBC)to large panel of biocides and antibiotics

A erom onas hydroph ila

A erom onas cav iae (X 74674) A . caviae

A erom onas hydroph ila

A erom onas jandei

P seudom onas aerug inosa (A B 037547)

D elftia ac idovorans ( A F149849)

A erom onas hydroph ila ( X 60404) Serra tia grim es ii (A J233430)

(A J233435) Serra tia proteam acu lansSerra tia p lym uthica ( A F286872)

Serra tia p lym uthica (A J233433)

K . pneum oniae ( X 87276)

P seudom onas n itroreducens (D 84021) P seudom onas n itroreducens

D elftia ac idovorans C om am onas sp . A J002803

R alstonia oxala tica ( A F155567) sp . (A F301897)R alstonia sp . R alstonia

A chrom obac ter xylosoxidans ( A F411021) A chrom obac ter xylosoxidans

A lcaligenes xy losoxidans ( D 88005) B lackw a te r b io reactor bac te rium (A F394171)U ncu ltu red eubacterium clone (AY 038619) U n iden tified eubacte rium

S teno trophom onas m a ltoph ilia (A J2956730 X anthom onas sp. O ra l clone (A J13904) S teno trophom onas sp .S teno trophom onas sp . (A J300772)

X anthom onas cam pestr is (A F123092) S teno trophom onas m a toph ilia

S teno trophom onas m a ltoph ilia S teno trophom onas m a ltoph ilia ( X 95923)

S teno trophom onas m a ltoph iliaS teno trophom onas m a ltoph ilia

M icrobac te rium phyllosphaerae (A J277840)M icrobac te rium phyllosphaerae M icrobac te rium im peria lis (X 77442)

S taphylococcus w arneri (L 37603) B acillu s lichen iform is ( A F391127)

A F435435 B ac illus sphaer icus sp .B ac illus

B acillu s fu sifo rm is (A F169537) sp .B ac illus

sp .B ac illus B ac illus cereus

(A F155952) B ac illus cereus A F 176321)B ac illus an thrac is (

sp .B ac illus (A J250798) B osea th ioox idans un iden tified a lpha p ro teobac te rium

A J29922) A fip ia sp. ( A fip ia sp.

C hela tococcus asaccharovorans ( A J294349) (D 14020)F lavobacterium tha lpoph ilum

Sph ingobac terium m ultivorum (A B 020205)C hryseobacter ium scoph tha lm um ( A J271009)

C hryseobacter ium joostei

sp .C hryseobacter ium sp .C hryseobacter ium sp .C hryseobacter ium

sp . Serra tia

T herm us therm oph ilus (ou tgroup)

S p h in g o b a c te r iu m m u ltiv o ru m (A B 0 2 0 2 0 5 )U n c u ltu re d S p h in g o b a c te r iu m (A B 0 7 6 8 7 4 )

A fip ia sp . (A J250798) B osea th iooxidans

embl_TTRN1embl_SWRGDembl_AF39149a reversepseBacil_sp._embl_CSC27embl_AH16Sembl_CAS29embl_CJO27embl_AF411embl_AB037embl_SGR23Ahydroph4 Afipia_sp.hgall embl_AXD00embl_SSP30Steno13A 18a a.jandei_2embl_ASP298a embl_CSPAJembl_AF155embl_AF394embl_AF155Steno29A embl_AF169embl_SMRR9embl_SPR23reversekleembl_MI16S27a embl_AC16S50A embl_AF286embl_SPL23embl_AF463embl_KP16Sembl_PSPIPP.seud16A embl_PNIAMp.nitro embl_AF149deftia embl_AF301Ralstoniasembl_AY038Eubacteriuembl_SMA13embl_AF385embl_AF123Steno5A Steno48A Steno31A embl_MPH27M.phyl embl_AF435Bacil_sp._Bacil_sp.3B.cereus_5embl_AF17632b embl_BTH25Alphaprot_embl_STD02embl_AB02014a 29b 100279274844999839210053100587110010010010045789995996994829998100388210096919392781001005846979972649110049691009646501008369385772996199100

1 0 0

4 2

8 6

2 4

4 6

6 3

4 8

2 8

9 5

1 0 0

1 0 0

1 0 0

4 2

1 0 0

1 0 0

1 3

9 7

1 0 0

1 0 0

4 7

2 2

9 3

8 6

1 0 0

1 0 0

9 6

5 0

8 7

4 28 0

9 4

1 0 0

6 1

5 9

9 7

1 0 0

8 7

9 0

9 3

4 5

9 9

A chrom obacte r xylosoxidans (A F411021) A lcaligenes xy losox idans (D 88005) B lackw a te r b ioreac to r bacte rium (A F394171)un identified eubacterium

un identified gam m a p ro teobacterium

un identified eubacteriumun identified eubacterium

un identified eubacteriumun identified eubacteriumun identified eubacterium

D elftia ac idovorans

un identified eubacterium

D elftia ac idovorans (A F149849) R alstonia oxala tica (A F155567) R alstonia basilensis (A F 312022)

R alstonia .sp R alstonia sp. (A F301897)

P se u d o m o n a s a e ru g in o sa (A B 0 3 7 5 4 7 ) P se u d o m o n a s n i tro re d u c e n s (D 8 4 0 2 1 )

P se u d o m o n a s sp .

U n c u ltu re d g a m m a p ro te o b a c te r iu m

A F435435 B ac illus sphaer icus B acillu s cereus B acillu s fu sifo rm is (A F169537)

B ac illus lichen ifo rm is (A F391127) A F 176321)B ac illus an thrac is ( B acillu s cereus ( A J310098)

B acillu s cereus A J29922) A fipia sp. (

C hela tococcus asaccharovorans ( A J294349)

(D 1 4 0 2 0 )F la v o b a c te r iu m th a lp o p h i lu mC h ry se o b a c te r iu m sc o p h th a lm u m ( A J2 7 1 0 0 9 )

C h ry se o b a c te r iu m in d o lo g e n e s AY 0 5 0 4 9 3C h ry se o b a c te r iu m sc o p h th a lm u m ( A J2 7 1 0 0 9 )

C hryseobacterium jooste i

A J29922) A fipia sp. (

C hry se o b a c te riu m jo o s te i(A J2 7 1 0 1 0 )

T herm us therm ophilu s (ou tgroup)

TRICLOSAN DEGRADATION

Baseline communities10.6 - 11.4 Log10 CFU/g

Mainly:PseudomonadsEnteric speciesBacilli

Also, sulphate reducers ClostridiaPrevotella 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

MarO11-LacZ

X-gal top-agar

More than half induced

Mar

CONTROL

53 Household products purchased and tested as inducers

Multi-Drug Efflux Pumpsthe 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