Assessment of the Etest method for detection of penicillin resistance in acute suppurative oral infection

M. A. O. Lewisa,*, C. L. Pankhurstb, C. W. I. Douglasc, M. V. Martind, E. G. Absia, P. B. Bishopa and S. A. Jonese

a Department of Oral Surgery, Medicine & Pathology, Dental School, University of Wales College of Medicine, Cardiff; b Combined Microbiology Research Unit, GKT, King's College Dental School Campus, University of London, London; c Department of Oral Pathology, School of Clinical Dentistry, University of Sheffield, Sheffield; d Department of Dentistry, Dental School, University of Liverpool, Liverpool; e Medical Department, SmithKline Beecham Pharmaceuticals (UK Division), Welwyn Garden City, Hertfordshire, UK

Sir,

The microflora of acute suppurative dental infections usually involves four or five bacterial species, with a predominance of strict anaerobes.1 In recent years, concern has been expressed regarding an increase in the incidence of penicillin-resistant organisms, in particular Prevotella spp., within the flora of acute dentoalveolar abscess.2 The availability of a rapid and reliable assessment of MIC would be helpful in monitoring resistance patterns. The development of the Etest (AB Biodisk, Solna, Sweden) has provided a simple method of providing MIC data. Although the Etest has been employed in the assessment of a variety of human infections, little information is available on the application of this method to acute suppurative dental infections.

Aspirates of pus, obtained from dental abscesses at four centres (Dental School, University of Wales College of Medicine, Cardiff; King's College Dental School, University of London; School of Clinical Dentistry, University of Sheffield and Dental School, University of Liverpool) were studied. Samples in each centre were inoculated on to plates of blood agar containing 5% sheep blood and fastidious anaerobe agar (FAA; Lab M, Bury, UK), which were incubated at 37°C in air plus 10% CO2 and in an anaerobic chamber, respectively. Aerobic growth was examined after 48 h and anaerobic growth daily for 10 days. Susceptibility to penicillin, ampicillin and co-amoxiclav was determined for each isolate using the Etest method on FAA, supplemented with 5% sheep blood, and incubated anaerobically. MIC was read after 48 h.

The MIC for strains demonstrating resistance to one or more of the antibiotics (penicillin, MIC > 1 mg/L; ampicillin, MIC > 2 mg/L and co-amoxiclav, MIC > 2 mg/L) and a selection of strains classified as either intermediate or sensitive (Table) was subsequently determined at the Oral Microbiology Laboratory in Cardiff, using a reference agar dilution method on Wilkins-Chalgren agar (Difco, East Molesey, UK).3,4 Control organisms employed were Bacteroides fragilis ATCC 25285 and Clostridium perfringens NCTC 11229. Susceptibility was interpreted as described previously.2

Samples from 78 infections yielded a total of 331 isolates, predominantly Streptococcus and Prevotella spp. The Etest method indicated that 64 (19%) strains were resistant to penicillin, 31 (9%) strains were resistant to ampicillin and 13 (4%) strains were resistant to co-amoxiclav. It was possible to perform a comparison of the MIC obtained by Etest method and that of the agar dilution method for 65 strains with penicillin, 70 strains with ampicillin and 72 strains with co-amoxiclav. The MIC obtained by each method was the same or within ±2 doubling (log2) dilutions of each other for 56 (86%) strains to penicillin, for 59 (84%) strains to ampicillin and for 60 (83%) strains to co-amoxiclav. A categorical discrepancy (resistant/sensitive or vice versa) occurred for one strain (1%) to penicillin, five strains (6%) to ampicillin and four strains (5%) to co-amoxiclav.

The Etest plates were easy to read and had clear, sharp endpoints for the majority of the bacteria–antibiotic combinations, including strains that were found to be capable of producing ß-lactamases. The haziness and pinpoint growth, previously reported for ß-lactamase-producing strains of the B. fragilis group, was rarely encountered for any of the bacterial species investigated here. In the majority of cases the MIC value obtained by Etest was in agreement with that obtained by agar dilution. Overall, there was a trend for the MIC values obtained by Etest to be lower than those recorded using the agar dilution method. However, when the MIC results were converted into categories of susceptibility there was good correlation between the methods. The category of penicillin susceptibility by Etest was the same as that obtained by the agar dilution method for the majority (82%) of isolates.

The spectrum of microbial species involved in the present study is representative of that characteristically isolated from acute dentoalveolar abscess.1 The use of Etest on FAA provided MIC values for a range of bacterial species, including slow-growing strict anaerobes, that were similar to those obtained by the more laborious agar dilution method. The Etest reliably detected penicillin resistance, which indicates that it can provide a simple and rapid method of assessing quantitative susceptibility for those bacterial isolates from acute suppurative oral infection. It is suggested that if penicillin-resistant strains are detected by routine disc-diffusion test in pus samples from dental infections then the Etest should be used to provide an MIC value for penicillin for such isolates. In addition, it is recommended that such isolates should be assessed with an Etest to co-amoxiclav since such resistance is likely to be due to ß-lactamase production.2


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Table. Identity of the 77 bacterial strains that demonstrated resistance to one or more of the antibiotics by the Etest and subsequently had MIC determined by agar dilution
 
Acknowledgments

This study was supported by SmithKline Beecham Pharmaceuticals (UK).

Notes

J Antimicrob Chemother 2000; 46: 328–330

* Corresponding author. Tel: +44-29-20742541; Fax: +44-29-20742442; E-mail: oralsurgery{at}cardiff.ac.uk Back

References

1 . Lewis, M. A. O., MacFarlane, T. W. & McGowan, D. A. (1986). Quantitative bacteriology of acute dento-alveolar abscesses. Journal of Medical Microbiology 21, 101–4.[Abstract]

2 . Lewis, M. A. O., Pankhurst, C. L., Douglas, C. W. I., Martin, M. V., Absi, E. G., Bishop, P. A. et al. (1995). Prevalence of penicillinresistant bacteria in acute suppurative oral infection. Journal of Antimicrobial Chemotherapy 35, 785–91.[Abstract]

3 . Washington, J. A. (1985). Susceptibility tests: agar dilution. In Manual for Clinical Microbiology, 4th edn, (Lenette, E. H., Balows, A., Hauser, W. J. & Shadomy, H. J., Eds), pp. 967–71. American Society for Microbiology, Washington, DC.

4 . National Committee for Clinical Laboratory Standards. (1983). Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria that Grow Aerobically—Tentative Standard M7-T. NCCLS, Villanova, PA.





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