A sentinel study of antibiotic resistance in Gram-Positive Anaerobic Cocci (GPAC)

1
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

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Gram-positive Anaerobic Cocci

• 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)

3
GPAC Genera

• Peptostreptococcus
• Micromonas
• Anaerococcus
• Peptoniphilus
• Finegoldia
• Slackia
• Peptococcus

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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.

5
Components of the Study

• Recruitment of sentinels
• Collection and referral of isolates
• Verification and identification of GPAC
• Susceptibility testing
• Collate data
• Report back to sentinels
• Publish results

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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. Georges in London.

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Collection and referral

• 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.

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Verification and identification of GPAC

• 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/Cs, HVS, cervix, penis, placenta, prostate,
  toe, heel and foot wounds, knee and leg wounds,
  laparotomy wounds, pilonidal sinus, perineum and
  psoas wounds, etc.

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GPAC received from each lab

• St. Georges 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

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Identifications

• 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

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MIC testing

• MICs 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.

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ARL Stokes E test
Test organism

• F. magna (NCTC 11804)

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Agents tested

• Penicillin, tetracycline, erythromycin,
  cefoxitin, clindamycin, chloramphenicol,
  imipenem, co-amoxyclav, piperacillin-tazobactam
  and metronidazole.
• Plates were incubated anaerobically and read
  after 48h. in batches of 10 ( 100 plates).

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Results

• 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.
• MICs of the control (F. magna NCTC 11804) did
  not vary by more than 1-2 dilutions for each drug
  between batches

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Breakpoint MICs as listed in Wadsworth Anaerobe
Manual

• 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

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Summary of overall GPAC resistance levels (n113)

• 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

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F. magna (n43)

• Penicillin 0 resistant
• Tetracycline 37.2 resistant
• Erythromycin 30.2 resistant
• Clindamycin 6.9 resistant

18
Ps. anaerobius (n25)

• Penicillin 28 resistant
• Tetracycline 60 resistant
• Co-amoxyclav 16 resistant
• Butyrate Group of GPAC (n 15)
• Tetracycline 53 resistant
• Penicillin 27 resistant
• Cefoxitin 13 resistant

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Macrolide-lincosamide linked resistance in GPAC

• 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 MICs gt256mg/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.

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Conclusions

• 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. Sanchezs (1992) study in USA gt10
  resistance of F. magna to clindamycin.

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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 200352224-228.
  Brazier, Hall, Morris, Gal and Duerden.