Department of Microbiology, City Hospital, Dudley Road, Birmingham B18 7QH, UK
Keywords: Haemophilus influenzae, ciprofloxacin, susceptibility testing
Sir,
Although rare, ciprofloxacin resistance has been described in Haemophilus influenzae. Resistance can occur as a result of mutations in the quinolone resistance-determining regions (QRDRs) of gyrA and parC.1 Isolates harbouring a mutation in the QRDR of gyrA alone show reduced susceptibility to ciprofloxacin; mutations in the QRDRs of both gyrA and parC are required to give MICs above the resistance breakpoint.
It has been recommended that the most reliable way to determine the quinolone susceptibility of Neisseria gonorrhoeae is to use nalidixic acid; isolates with reduced quinolone susceptibility give no zone of inhibition with a 30 µg nalidixic acid disc.2 Nalidixic acid has also been used to test the quinolone susceptibility of H. influenzae. Corkill et al.3 found that in H. influenzae nalidixic acid was a better indicator of reduced ciprofloxacin susceptibility than ciprofloxacin because of the enhanced reduction in zone size to nalidixic acid compared with ciprofloxacin.
With this study, we intended to confirm and highlight the usefulness of nalidixic acid in detecting reduced quinolone resistance in H. influenzae using the BSAC recommended disc diffusion methodology.2 We examined two clinical isolates of H. influenzae (A1405 and A1012), which had previously been found to show reduced ciprofloxacin susceptibility by the BSAC standardized agar dilution method.4 A ciprofloxacin-susceptible strain NCTC 11931 was used for comparison. The MICs of ciprofloxacin, levofloxacin and moxifloxacin were determined by the BSAC standardized agar dilution method.4 The BSAC disc diffusion method2 was used to determine the susceptibility of the isolates to ciprofloxacin (1 µg disc) and nalidixic acid (30 µg disc). The QRDRs of gyrA and parC of the isolates were amplified by PCR using primers and conditions as previously described.5,6 Nucleotide sequencing of the amplimers was carried out by automated sequencing (Lark Technologies Ltd, Saffron Walden, UK). The nucleotide sequences of the QRDRs were compared with those of a quinolone-susceptible strain (NCTC 11931) and published nucleotide sequences.
The H. influenzae isolates A1405 and A1012 both had significantly higher MICs of ciprofloxacin than the susceptible H. influenzae strain NCTC 11931 (Table ). Only A1012 would be considered resistant; it had an MIC of ciprofloxacin equal to the BSAC resistance breakpoint of
2.0 mg/L2 and gave a zone diameter of 13 mm for ciprofloxacin that was considerably smaller than the BSAC recommended breakpoint zone diameter of 28 mm.2 Isolate A1405 had a zone diameter of 29 mm that was larger than the breakpoint and would be considered susceptible. In contrast, both A1012 and A1405 showed no zone with nalidixic acid, whilst the susceptible NCTC 11931 strain had a 31 mm zone.
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We have shown that the nalidixic acid disc diffusion test was capable of detecting reduced ciprofloxacin susceptibility in an isolate with a single mutation in the QRDR of gyrA. This resistance would not have been detected using ciprofloxacin as both the MIC and zone diameter would classify the isolate as susceptible on the basis of current BSAC breakpoints. Detection of low-level quinolone resistance is important as it highlights those isolates for which a quinolone may not be the most appropriate therapy. Using a quinolone to treat such isolates increases the likelihood of selecting mutants with high-level quinolone resistance; only one further mutation is required to produce strains that are resistant to ciprofloxacin.
Nalidixic acid appears to be a very useful indicator compound for testing quinolone susceptibility of H. influenzae as it will detect both low- and high-level quinolone resistance. The large differences in zone sizes for susceptible isolates compared with isolates showing reduced susceptibility makes interpretation easy, which is useful in a diagnostic setting. It also alleviates the need to test several different quinolones.
Acknowledgements
We would like to thank Dr I. Gould for supplying isolate A1012.
Footnotes
* Corresponding author. Tel: +44-121-507-4228; Fax: +44-121-551-7763; E-mail: n.p.brenwald{at}bham.ac.uk
References
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