Transcript John Brazier Presentation

A sentinel study of antibiotic
resistance in Gram-Positive Anaerobic
Cocci (GPAC)
J.S. Brazier, V. Hall, T.E. Morris, M. Gal
and B.I. Duerden
Anaerobe Reference Laboratory
NPHS Microbiology Cardiff,
University Hospital of Wales, Cardiff
Gram-positive Anaerobic Cocci
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GPAC are a heterogeneous group that are common
members of human normal flora in various sites.
They are opportunist pathogens and can be found in a
wide variety of infections including deep-seated soft
tissue abscesses, ulcers, prosthetic, joint, bone,
bloodstream and pelvic infections.
They are often not identified beyond descriptive terms
such as “Anaerobic streptococcus” (sic)
GPAC Genera
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Peptostreptococcus
Micromonas
Anaerococcus
Peptoniphilus
Finegoldia
Slackia
Peptococcus
Aim of Study
• To obtain fresh clinical isolates of GPAC and
to perform identification and susceptibility
tests to establish the level of antibiotic
resistance in this group of anaerobes and to
note resistance in particular species.
Components of the Study
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Recruitment of sentinels
Collection and referral of isolates
Verification and identification of GPAC
Susceptibility testing
Collate data
Report back to sentinels
Publish results
Sentinel Recruitment
• A letter explaining the study was issued to 48
PHLS or PHL collaborating laboratories in
England and Wales.
• 18 replied in the affermative; Cambridge,
Carlisle, Coventry, Gloucester, Hereford,
Ipswich, Leeds, Lincoln, Manchester,
Nottingham, Peterborough, Plymouth,
Preston, Rhyl, Salisbury, Southampton, and
UCH and St. George’s in London.
Collection and referral
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Labs were asked to collect up to 10 isolates of GPAC
from clinical materials irrespective of their potential
clinical significance. Transport swabs provided.
Selection criteria: Gram positive coccus that does not
grow in air or CO2 enriched atmosphere. Asked to
ignore susceptibility/resistance to a 5ug metronidazole
disc on primary plates.
Sampling was done simultaneously over a one-month
period (point-prevalence study) during Feb. 2002.
Submit isolates to ARL with records of their source.
Verification and identification of
GPAC
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All isolates were checked for growth in air/CO2, cellular morphology.
Identified according to criteria in VPI Anaerobe Lab. manual and
Murdoch (1998) using API 32A kit, analysis of VFA metabolites,
odour, UV fluorescence, indole and colonial charactertistics.
In total, 113 isolates were verified as GPAC and included in the study.
GPAC originated from a wide range of sources including; leg
ulcers, sebaceous cysts, ears, B/C’s, HVS, cervix, penis,
placenta, prostate, toe, heel and foot wounds, knee and leg
wounds, laparotomy wounds, pilonidal sinus, perineum and
psoas wounds, etc.
GPAC received from each lab:
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St. George’s = 9
Preston = 9
Southampton = 10
Carlisle = 9
Ipswich = 4
Rhyl = 7
Hereford = 8
Lincoln = 10
Leeds = 10
Cambridge = 8
Nottingham = 6
Coventry = 10
Manchester = 10
UCH = 9
Plymouth = 6
Gloucester = 5
Salisbury = 2
Peterborough = 1
Identifications:
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Finegoldia magna = 43
Ps. anaerobius = 25
An. vaginalis = 11
Mic. micros = 5
Pep. harei = 4
Pep. assacharolyticus = 3
Pep ivorii = 3
An. prevotii = 2
Pep lacrimalis = 1
Sl. heliotrinrdeucens = 1
Ps. sp. (butyrate group) = 15
MIC testing
• MIC’s against 10 drugs were determined
using the E test method – ARL modification.
• McFarland 5.0 suspensions made in saline
and swabbed on half plate of FAA blood agar.
• Control organism F. magna (NCTC 11804)
similarly prepared and swabbed on other half.
E test strip placed diametrically between the
test and control organisms.
ARL “Stokes” E test
Test organism
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F. magna (NCTC 11804)
Agents tested:
• Penicillin, tetracycline, erythromycin,
cefoxitin, clindamycin, chloramphenicol,
imipenem, co-amoxyclav, piperacillintazobactam and metronidazole.
• Plates were incubated anaerobically and read
after 48h. in batches of 10 (= 100 plates).
Results
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MIC50 and MIC90 values were calculated for each
drug/species combination that had 10 or more
examples.
Resistance to an agent was defined as an MIC above
the breakpoint as listed in the Wadsworth Anaerobe
Lab. Manual.
MIC’s of the control (F. magna NCTC 11804) did not
vary by more than 1-2 dilutions for each drug between
batches
Breakpoint MIC’s as listed in
Wadsworth Anaerobe Manual
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Penicillin = 2mg/L
Chloramphenicol = 32mg/L
Tetracycline = 16mg/L
Co-amoyxclav = 16mg/L
Erythromycin = 8mg/L
Imipenem = 16mg/l
Cefoxitin = 64mg/L
Pip/tazobactam = 128mg/L
Clindamycin = 8mg/L
Metronidazole = 32mg/L
Summary of overall GPAC resistance
levels (n=113):
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Penicillin = 7.1%
Tetracycline = 41.6%
Erythromycin = 27.4%
Cefoxitin = 0%
Clindamycin = 7.1%
Chloramphenicol = 0%
Imipenem = 0%
Co-amoxclav = 3.5%
Pip.tazobactam = 0%
Metronidazole = 0%
F. magna (n=43)
• Penicillin = 0% resistant
• Tetracycline = 37.2% resistant
• Erythromycin = 30.2% resistant
• Clindamycin = 6.9% resistant
Ps. anaerobius (n=25)
• Penicillin = 28% resistant
• Tetracycline = 60% resistant
• Co-amoxyclav = 16% resistant
• Butyrate Group of GPAC (n= 15):
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Tetracycline = 53% resistant
Penicillin = 27% resistant
Cefoxitin = 13% resistant
Macrolide-lincosamide linked resistance
in GPAC
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Previously reported by Reig et al (1992) who found
17.7% of GPAC with this phenotype.
We found 7 isolates (6.2%) belonging to four different
species with MIC’s >256mg/L to both erythromycin and
clindamycin. These were; A.prevotii, F. magna, P.harei,
and a Ps. sp. (butyrate group).
Reig et al reported that 80% of macrolide resistance in
Peptostreptococcus sp. was due to the ermTR gene
and that these organisms might be an important
reservoir of macrolide resistance for transfer to
pathogens such as Strep. pyogenes.
Conclusions
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This is one of the largest susceptibility studies
specifically on GPAC ever performed.
Significant levels of GPAC tetracycline and macrolide
resistance was found in the most commonly isolated
species. No Mz resistance found.
Comparisons to other studies included: Wren (1996) in
London found 16% resistance of F. magna to penicillin
compared to our nil resistance, and 9% resistance to
clindamycin compared to our 7.1% resistance.
Sanchez’s (1992) study in USA >10% resistance of F.
magna to clindamycin.
Acknowledgments
• Sentinel laboratories, ARL staff: Val, Mic, Tref,
Carol and Brian.
• Publication: “ Antibiotic susceptibilities of
Gram-positive anaerobic cocci: results of a
sentinel study in England and Wales” Journal
of Antimicrobial Chemotherapy 2003;52:224228. Brazier, Hall, Morris, Gal and Duerden.