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

• 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

• 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

• 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

• 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

• 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:

• 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:• 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

• F. magna (NCTC 11804)

Test organism

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).

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.

• 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

• 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):

• 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):

• Tetracycline = 53% resistant

• Penicillin = 27% resistant

• Cefoxitin = 13% resistant

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 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

• 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;52:224-

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