Susceptibility testing Pasteurella multocida by BSAC standardized methodology

J. M. Andrews*, G. Jevons, N. Brenwald and A. Fraise for the BSAC Working Party on Sensitivity Testing

The BSAC Standardized Method Development Centre, Microbiology, City Hospital, Dudley Road, Birmingham B18 7QH, UK

Keywords: P. multocida , disc testing

Sir,

Pasteurella multocida are associated with wound infections following dog or cat bites, scratches or licking of skin lesions and more serious invasive infections.1 Currently there are no recommendations for susceptibility testing this organism by BSAC methodology. In this study, we describe a disc susceptibility testing method based on the BSAC standardized method.2 A total of 83 clinical isolates provided by 16 centres around the UK (see the Acknowledgements) and two control strains, P. multocida NCTC 8489 (following cat bite) and P. multocida NCTC 8281 (human, dog bite) were studied. Identification of the clinical isolates was confirmed by API 20NE (bioMérieux, Basingstoke, UK) and the presence of ß-lactamase was detected by the nitrocefin test (Oxoid, Basingstoke, UK). For MIC determinations and disc susceptibility testing, Iso-Sensitest agar (Oxoid) supplemented with 5% defibrinated horse blood (TCS) and 20 mg/L NAD (Mast Laboratories, Merseyside, UK) was used. An inoculum of 104 cfu/spot and an inoculum equivalent to semi-confluence (an organism suspension equivalent to a 0.5 McFarland standard diluted 1:100 in distilled water before inoculation) were used for MICs and disc susceptibility testing, respectively. All tests were incubated at 35–37°C for 18–20 h in an atmosphere enriched with 4–6% CO2. The antibiotics studied were penicillin, ampicillin, cefotaxime, ciprofloxacin and tetracycline. Acceptable limits for control strain P. multocida NCTC 8281 were not determined because zone diameters were ≥40 mm for ampicillin and cefotaxime; this would mean that routinely two plates would be necessary for testing to avoid zone merging. However, acceptable limits for control strain P. multocida NCTC 8489 were determined by disc testing 50 times on pre-poured plates from Oxoid and bioMérieux and media poured in-house to a depth of 3.5, 4 and 4.5 mm. Zone diameters from the five media types were combined and 95 percentiles calculated.3 Applying the BSAC MIC breakpoints, susceptibility was determined.4

All of the organisms studied were ß-lactamase negative. Mode MICs for each of the antibiotics tested were: penicillin, 0.06 mg/L (range 0.015–0.12 mg/L); ampicillin, 0.12 mg/L (range 0.03–0.25 mg/L); cefotaxime, 0.004 mg/L (range 0.001–0.004 mg/L); ciprofloxacin, 0.008 mg/L (range 0.004–0.12 mg/L); and tetracycline, 0.25 mg/L (range 0.06–0.5 mg/L). All of the isolates were susceptible to each of the antibiotics tested based on comparison with the BSAC MIC breakpoints, except for one isolate (Z1469) that had a ciprofloxacin MIC of 0.12 mg/L which was 16-fold higher than the mode MIC of 0.008 mg/L. Currently the BSAC recommends the use of a 30 µg nalidixic acid disc for the detection of low-level fluoroquinolone resistance in Gram-negatives.2 For Z1469 with reduced fluoroquinolone susceptibility, a zone of 14 mm was observed (a zone of 26 mm with a ciprofloxacin 1 µg disc). For the control NCTC 8489 strain and for susceptible isolates, zones of ≥28 mm were seen. It is therefore possible to use a 30 µg nalidixic acid disc to detect reduced susceptibility to fluoroquinolones. The presumptive quinolone resistance-determining regions (QRDR) of gyrA and parC for Z1469 and NCTC 8489 were amplified by PCR; direct sequencing of purified PCR products was carried out by automated fluorescent sequencing (MWG Biotech UK Ltd, Milton Keynes, UK). No QRDR nucleotide sequence differences were seen between Z1469 and NCTC 8489. Similar strains of Haemophilus influenzae showing reduced quinolone susceptibility, but lacking gyrA or parC mutations have been described.5 Recommendations for interpreting susceptibility and acceptable limits for the control strain are given in Table 1.


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Table 1. MIC and zone diameter breakpoints for interpreting the susceptibility of Pasteurella multocida by BSAC methodology and expected MIC and zone diameter ranges for control strain P. multocida NCTC 8489

 

Acknowledgements

Centres providing clinical isolates: Derriford Hospital, Plymouth; William Harvey Hospital, Ashford; Crawley Hospital, Crawley; Cheltenham Hospital, Cheltenham; Gloucester Royal Hospital, Gloucester; Derbyshire Royal Infirmary, Derby; Macclesfield District General Hospital, Macclesfield; University Hospital of Wales, Cardiff; Queen Elizabeth 2 Hospital, Welwyn Garden City; Leicester Royal Infirmary, Leicester; Blackpool Victoria Hospital, Blackpool; Colchester General Hospital, Colchester; Bassetlaw District General Hospital, Worksop; Truro Hospital, Truro; Royal Preston Hospital, Preston; City Hospital, Birmingham.

Footnotes

* Corresponding author. Tel: +44-121-507-5693; Fax: +44-121-551-7763; Email: jenny.andrews{at}swbh.nhs.uk

References

1 . Murray, P. R. (Ed.). (1995). Manual of Clinical Microbiology, 6th edn, p. 612. American Society for Microbiology, Washington, DC, USA.

2 . Andrews, J. M. (2004). BSAC standardized disc susceptibility testing method (version 3). Journal of Antimicrobial Chemotherapy 53, 713–28.[Free Full Text]

3 . Andrews, J. M. (2001). The development of the BSAC standardized method of disc diffusion testing. Journal of Antimicrobial Chemotherapy 48, Suppl. S1, 29–42.[Abstract/Free Full Text]

4 . MacGowan, A. P. & Wise, R. (2001). Establishing MIC breakpoints and the interpretation of in vitro susceptibility tests. Journal of Antimicrobial Chemotherapy 48, Suppl. S1, 17–28.[Abstract/Free Full Text]

5 . Perez-Vazquez, M., Roman, F., Aracil, B. et al. (2004). Laboratory detection of Haemophilus influenzae with decreased susceptibility to nalidixic acid, ciprofloxacin, levofloxacin, and moxifloxacin due to gyrA and parC mutations. Journal of Clinical Microbiology 42, 1185–91.[Abstract/Free Full Text]





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