Anaerobe Reference Laboratory, National Public Health Service Wales, Microbiology Cardiff, University Hospital of Wales, Cardiff CF14 4XW, UK
Received 19 February 2003; returned 20 April 2003; revised 2 May 2003; accepted 3 May 2003
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Abstract |
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Methods: A total of 113 GPAC isolates consisting predominantly of current or former members of the genus Peptostreptococcus was obtained from 17 sentinel laboratories in England and one in Wales. Minimum inhibitory concentrations (MICs) of 10 antimicrobial agents were determined by the Etest method. The agents tested were: penicillin, tetracycline, erythromycin, cefoxitin, clindamycin, chloramphenicol, imipenem, co-amoxiclav, piperacillin/tazobactam and metronidazole. MIC50 and MIC90 values for each drug-species combination were calculated whenever suitable numbers of each species were obtained.
Results: Excellent spectra of activity (0% resistance) against GPAC were seen for metronidazole, piperacillin/tazobactam, cefoxitin, imipenem and chloramphenicol. Low degrees of resistance to co-amoxiclav (3.5%), clindamycin (7.1%), penicillin (7.1%) and significant degrees of resistance to tetracycline (41.6%) and erythromycin (27.4%) were detected. Some examples of putative macrolide-lincosamide linked resistance were noted in seven (6.2%) isolates of GPAC.
Conclusion: This study is one of the largest susceptibility studies specifically on GPAC carried out to date and the resulting data may be of value to those involved in the empirical treatment of infections involving Gram-positive anaerobic cocci.
Keywords: Peptostreptococcus spp., Etests, anaerobes, MICs
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Introduction |
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Materials and methods |
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Eighteen sentinel clinical diagnostic microbiology laboratories were recruited for the study. They consisted of 17 former Public Health Laboratories (PHL) or PHL collaborating laboratories in England and one in Wales. The participating laboratories were in: Cambridge, Carlisle, Coventry, Gloucester, Hereford, Ipswich, Leeds, Lincoln, Manchester, Nottingham, Peterborough, Plymouth, Preston, Rhyl, Salisbury and Southampton. The former PHL collaborating laboratories were St Georges Hospital and University College Hospital, London.
Selection of GPAC isolates
Participants were asked to collect up to 10 Gram-positive anaerobic cocci (GPAC) isolated from clinical material over a 1 month period during February 2002, irrespective of their potential clinical significance. Selection criteria for GPAC was based on Gram stain reaction, cellular morphology and the inability to grow in air or 5% carbon dioxide in air at 37°C. Participants were requested not to base selection on the presence of a zone of susceptibility to a metronidazole 5 µg disc (which is commonly placed directly on primary isolation plates as an indicator of the presence of anaerobes) as this would prejudice against bona fide metronidazole-resistant GPAC. Isolates of putative GPAC were sent to the Anaerobe Reference Laboratory (ARL) in Cardiff for identification and determination of minimum inhibitory concentrations (MICs) of 10 antimicrobial agents.
Identification of GPAC
Isolates were identified according to the criteria of Holdeman et al.7 including analysis of volatile metabolic end-products by gasliquid chromatography supplemented with the criteria of Murdoch1 using the API ATB 32A Rapid ID anaerobe identification kit (bioMérieux Laboratories, Marcy lÉtoile, France). In total, 113 isolates were confirmed as GPAC and included in the study.
Isolates were identified as: F. magna, n = 43; P. anaerobius, n = 25; A. vaginalis, n = 11; M. micros, n = 5; P. harei, n = 4; P. asaccharolyticus, n = 3; P. ivorii, n= 3; A. prevotii, n = 2; P. lacrimalis, n = 1; Slackia heliotrinireducens (formerly P. heliotrinireducens), n = 1; and 15 unidentified butyrate-producing species of GPAC. MIC determination of penicillin, tetracycline, erythromycin, cefoxitin, clindamycin, chloramphenicol, imipenem, co-amoxiclav, piperacillin/tazobactam and metronidazole was by the Etest method (AB Biodisk, Solna, Sweden) which is the preferred method used in the ARL. Organisms for testing, usually in batches of 10, were harvested from a 48 h anaerobic culture on Fastidious Anaerobe agar (FAA, Lab M Ltd, Bury, UK) supplemented with 6% defibrinated horse blood, and a suspension was made in sterile distilled water to a turbidity equivalent to a McFarland 5.0 Standard. A sterile cotton swab was used to apply this suspension to one-half of a fresh FAA blood agar plate. A similar suspension of a control organism, F. magna (NCTC 11804), was applied to the other half of the plate and the Etest strip of the drug under test was placed along the diametric interface of the two inocula. This enabled each batch to be checked for reproducibility of results. Plates were incubated in an anaerobic chamber at 37°C for 48 h before reading of the MIC at the intersection of inhibition of growth and the Etest strip as is standard practice.
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Results |
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Discussion |
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This sentinel antimicrobial susceptibility study on 113 fresh clinical isolates is one of the largest carried out specifically on GPAC. Previous susceptibility data on GPAC were recorded mainly from susceptibility studies on a range of anaerobes and most of these were carried out outside the UK as summarized in the review article by Murdoch.1 This study is also one of the largest in terms of the number of antimicrobial agents tested against GPAC, equalling Bowker et al.s9 study of nine agents plus trovafloxacin against 75 GPAC isolates. Other studies include that of Panchini et al.10 who tested seven agents against 32 GPAC isolates and the report of Greenwood & Palfreyman11 on the susceptibility of 50 GPAC isolates to penicillin and vancomycin in 1987.
The most recent comparable UK susceptibility study on an unspecified number of Peptostreptococcus spp. isolated in a London hospital was published by Wren.12 He reported 16% resistance amongst isolates of P. magnus (F. magna) to penicillin. This compares with no resistance to penicillin in this species found in this study. Wren also noted 12% resistance of P. asaccharolyticus to clindamycin and 8% resistance of P. micros to penicillin, which is in contrast to the absence of resistance to both agents found in this study. Regarding potential international differences in clindamycin resistance in GPAC, results in both a French multicentre study13 that reported 28% clindamycin resistance amongst Peptostreptococcus spp., and an American study by Sanchez et al.3 who noted >10% resistance of P. magnus to clindamycin, were higher than the 7.1% overall GPAC resistance and 7% resistance of F. magna found in this study in the UK. However, Wren12 recorded 9% of P. magnus (F. magna) resistant to clindamycin in London, a level similar to our results suggesting that clindamycin resistance is lower in the UK than in France or the USA. However, it should be considered that methodological differences may have influenced these results.
Amongst the individual species or groups of GPAC, there were significant levels of resistance to certain antibiotics. For example, 15/25 (60%) of P. anaerobius were resistant to tetracycline, as were 16/43 (37.2%) of F. magna and 3/5 (60%) of M. micros. Thirteen of 43 (30.2%) F. magna were also resistant to erythromycin. Although the overall activity of erythromycin in GPAC was quite poor with 27.4% resistance, activity against P. anaerobius individually was better with only 1/25 (4%) resistant. Examples of macrolide-lincosamide linked resistance in GPAC has previously been reported by Reig et al.14 and Sanchez et al.3 Reig et al. reported 17.7% of this phenotype whereas our study identified seven strains (6.2%) belonging to four different species or groups with putative macrolide-lincosamide linked resistance of >256 mg/L to both erythromycin and clindamycin. These were A. prevotii (2), F. magna (2), butyrate-producing GPAC (2) and one strain of P. harei. There were 12 examples (10.6%) representing dissociated resistance (clindamycin MIC < 1.0 mg/L; erythromycin MIC > 8 mg/L) compared with 5.1% examples of dissociated resistance in the study of Reig et al.14 Reig et al.15 reported that an incidence of 80% macrolide resistance in 21 Peptostreptococcus strains was due to the erm(TR) gene and suggested these anaerobic members of the normal oropharyngeal flora may be an important reservoir of this gene for transfer to macrolide resistant Streptococcus pyogenes. Chopra & Roberts16 also highlighted the potential mobility of the genetic determinants associated with resistance to tetracycline and this study suggests that GPAC may act as reservoirs of tetracycline resistance for transfer to other endogenous bacteria. Although the numbers are small, there is a suggestion that some GPAC species are commonly resistant to certain agents as both A. prevotii isolates were resistant to erythromycin, clindamycin and tetracycline; similarly 75% (three of four) of P. harei isolates were resistant to tetracycline. This compares with no resistance to any agents in P. lacrimalis, S. heliotrinireducens and P. ivorii. Further investigations are warranted on greater numbers of these species.
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Acknowledgements |
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Footnotes |
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References |
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2
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9 . Bowker, K. E., Wootton, M., Holt, H. A. et al. (1996). The in-vitro activity of trovafloxacin and nine other antimicrobials against 413 anaerobic bacteria. Journal of Antimicrobial Chemotherapy 38, 27181.[Abstract]
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Reig, M., Galan, J., Baquero, F. et al. (2001). Macrolide resistance in Peptostreptococcus spp. mediated by ermTR: possible source of macrolide-lincosamide-streptogramin B resistance in Streptococcus pyogenes. Antimicrobial Agents and Chemotherapy 45, 6302.
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Chopra, I. & Roberts, M. (2001). Tetracycline antibiotics: mode of action, applications, molecular biology, and epidemiology of bacterial resistance. Microbiology and Molecular Biology Reviews 65, 23260.
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