JAC Advance Access originally published online on January 23, 2006
Journal of Antimicrobial Chemotherapy 2006 57(3):577-578; doi:10.1093/jac/dki488
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Correspondence |
Characterization of a clinical isolate of Haemophilus influenzae with a high level of fluoroquinolone resistance
1 School of Medicine, University of Seville, Seville, Spain; 2 Hospital Virgen Macarena, Seville, Spain
* Corresponding author. Tel: +34-954552863; Fax: +34-954177413; E-mail: jmrodriguez{at}us.es
Keywords: antibiotic resistance , susceptibility , ciprofloxacin
Sir,
Haemophilus influenzae is one of the bacterial pathogens most likely to be involved in community-acquired respiratory infections in patients with chronic obstructive pulmonary disease. In this species, resistance to fluoroquinolones occurs at very low frequency although in such cases treatment failure has been reported.1 In 2003, a fluoroquinolone-resistant H. influenzae isolate was recovered from the sputum of an 80-year-old male patient with a history of bronchiectasis and residual lesions from old tuberculosis but with no history of treatment with fluoroquinolones. Identification of this H. influenzae (strain number 79105) was carried out by Gram stain, tests for catalase, oxidase, X and V factor requirements (MAST), Api NH (bioMerieux, Madrid, Spain) and antisera to serotypes a-f (Difco, Sparks, MD, USA).
Susceptibility tests were performed by disc diffusion and Etest on HTM agar and Chocolate agar at 35°C and 5% CO2 and by microdilution in HTM broth according to CLSI (formerly NCCLS) guidelines.2 H. influenzae ATCC 49247 was used as a reference strain. Isolate 79105 was resistant to nalidixic acid, fluoroquinolones and co-trimoxazole and showed MICs of >256, 32, 32, 8 and 2 mg/L for nalidixic acid, ciprofloxacin, norfloxacin, levofloxacin and moxifloxacin, respectively, and >4/76 mg/L for co-trimoxazole. Moreover, this strain was non-typeable and a non-ß-lactamase producer like most of the previously described fluoroquinolone-resistant isolates.
Interestingly, this clinical strain showed dependence with nalidixic acid, ciprofloxacin and co-trimoxazole on HTM agar, growing only round the disc or Etest strip containing these antibiotics. In contrast this effect was not seen with organisms grown on Chocolate agar. Surprisingly, the dependence disappeared after several subcultures.
In our hospital the proportion of isolates of H. influenzae resistant to new quinolones is <1% and even lower rates have been reported in other studies.3,4 At the present time only a few isolates of H. influenzae with a high level of fluoroquinolone resistance (MIC of ciprofloxacin >16 mg/L) have been described.5 Since mutations in the DNA gyrase and topoisomerase IV genes are the most common mechanisms of fluoroquinolone resistance, the corresponding regions of the gyrA and parC genes of H. influenzae ATCC 49247 (as control) and of H. influenzae 79105 strains were sequenced (the primers used have been described previously).1 The sequence of the quinolone-resistant isolate showed two substitutions in GyrA (S84Y and D88A) and two in ParC (S84N and E88A). This combination of mutations has not been described previously and only one H. influenzae isolate has previously shown four substitutions in these genes.5 Additionally, the QRDR regions of the gyrB and parE genes were analysed with the set of previously described primers.1 The sequence of the quinolone-resistant isolate showed the E559G substitution in GyrB and the L445F substitution in ParE. In summary, this strain contained six mutations in four different genes associated with quinolone resistance. In all cases, the sequence of the ATCC 49247 H. influenzae strain was identical to those obtained from the database.
To explain observed differences in susceptibility between ciprofloxacin and moxifloxacin, we tried to identify the presence of a possible efflux pump with a different affinity for those antimicrobials. Efflux of fluoroquinolones was determined indirectly by measuring the intracellular accumulation of norfloxacin by fluorometric assay.6 A standardized cell suspension (OD520 = 2.0) was preincubated with 10 mg/L norfloxacin, following which half of the cell suspension was exposed to 0.2 mM carbonyl cyanide chlorophenylhydrazone (CCCP). Cell-fluorescence was measured with a fluorescence spectrophotometer (Hitachi 2000; at 279/445 nm). The addition of CCCP to the cells did not increase the level of intracellular norfloxacin. Thus, there was no evidence that efflux pumps inhibited by CCCP contributed to the observed differences in susceptibility of the strain to ciprofloxacin, norfloxacin and moxifloxacin.
In conclusion, an H. influenzae isolate resistant to fluoroquinolones by means of a new combination of mutations in topoisomerase genes has been described. It is unknown whether other different efflux pumps or additional mechanisms are implicated in quinolone resistance and explain the different susceptibility to ciprofloxacin and moxifloxacin in this strain.
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None to declare.
Acknowledgements
This study was developed under the auspices of REIPI (Spanish Network for Research in Infectious Diseases C013/14), Instituto de Salud Carlos III, Ministerio de Salud y Consumo, Spain.
References
1. Bastida T, Perez-Vazquez M, Campos J et al. Levofloxacin treatment failure in Haemophilus influenzae pneumonia. Emerg Infect Dis 2003; 9: 1475–8.[Medline]
2. National Committee for Clinical Laboratory Standards. Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria that Gow Aerobically—Sixth Edition: Approved Standard M7-A6. NCCLS, Wayne, PA, USA, 2003.
3. Biedenbach DJ, Jones RN. Fluoroquinolone-resistant Haemophilus influenzae: frequency of occurrence and analysis of confirmed strains in the SENTRY antimicrobial surveillance program (North and Latin America). Diagn Microbiol Infect Dis 2000; 36: 255–9.[CrossRef][Web of Science][Medline]
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Elliott E, Oosthuizen D, Johnson MM et al. Fluoroquinolone resistance in Haemophilus influenzae. J Antimicrob Chemother 2003; 52: 734–5.
5. Biedenbach DJ, Jones RN. Five-year analysis of Haemophilus influenzae isolates with reduced susceptibility to fluoroquinolones: prevalence results from the SENTRY antimicrobial surveillance program. Diagn Microbiol Infect Dis 2003; 46: 55–61.[CrossRef][Web of Science][Medline]
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Martínez-Martínez L, Pascual A, Conejo Mdel C et al. Energy-dependent accumulation of norfloxacin and porin expression in clinical isolates of Klebsiella pneumoniae and relationship to extended-spectrum ß-lactamase production. Antimicrob Agents Chemother 2002; 46: 3926–3.
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