1 Centro Nacional de Microbiología, Instituto de Salud Carlos III, Carretera Majadahonda a Pozuelo, Km. 2, 28220 Majadahonda, Madrid; 2 Servicio de Microbiología, Hospital Ramón y Cajal, Madrid, Spain
Received 21 May 2002; returned 8 August 2002; revised 19 September 2002; accepted 19 October 2002
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Abstract |
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Keywords: Haemophilus influenzae, antibiotic resistance, quinolones, susceptibility testing methods
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Introduction |
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Methods used in routine clinical laboratories to test the activity of antimicrobials against pathogens comprise broth microdilution, agar dilution, Etest and disc diffusion.
The aims of this study were to provide comparative data about the activities of new and old quinolones against H. influenzae populations with different ciprofloxacin susceptibility, to compare Etest and agar dilution susceptibility testing methods with the NCCLS standard broth microdilution method for H. influenzae, and to assess cross-susceptibility and/or cross-resistance among evaluated quinolones.
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Materials and methods |
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Sixty-two clinical H. influenzae isolates and two reference strains from the American Type Culture Collection were included: ATCC 49247, recommended by the NCCLS for susceptibility testing, and ATCC 51907, whose genome has been sequenced.2 Two American clinical strains3 and 30 Spanish clinical isolates from the collection of our Haemophilus Reference Laboratory were selected according to their reduced susceptibility to ciprofloxacin (MIC 0.12 mg/L) and/or nalidixic acid (MIC
4 mg/L). The last group was obtained from respiratory specimens from patients with cystic fibrosis or chronic respiratory infections, as a result of antibiotic resistance surveillance in Spanish clinical isolates, including the resistant isolates already studied by our group.4,5 As a fully susceptible group, we included 30 strains with ciprofloxacin MICs of
0.03 mg/L and nalidixic acid MICs of
2 mg/L. Both ATCC strains were included for quality control and comparative purposes.
Susceptibility testing
All H. influenzae strains were studied by the three methods. The reference broth microdilution method was carried out according to the NCCLS guidelines,6,7 except that the final test volume in each well was 200 µL and the inoculated plates were incubated for 2024 h at 35°C with 5% CO2 before reading results because many strains did not grow well without these modifications.
Haemophilus test medium (HTM) was prepared with MuellerHinton broth (Oxoid Ltd, Basingstoke, UK) supplemented with HTM Supplement (Oxoid) and yeast extract (0.5%) (Difco, Detroit, MI, USA). Microtitre plates were inoculated to produce a final inoculum density of 5 x 105 cfu/mL. The agar dilution technique was a modification of the standard method recommended for other organisms by the NCCLS.7 HTM Base (Oxoid) supplemented with HTM Supplement (Oxoid) was used. Plates were inoculated to obtain a final inoculum on the agar of
104 cfu per spot. The inoculated plates were incubated for 2024 h at 35°C with 5% CO2. The MIC was defined as the lowest concentration of quinolone that inhibited growth. Quinolones were supplied as powders of known potencies by the following manufacturers: nalidixic acid and norfloxacin (Sigma S.A., Madrid, Spain); ciprofloxacin and moxifloxacin (Bayer Hispania S.A., Barcelona, Spain); sparfloxacin and levofloxacin (Aventis, Madrid, Spain); grepafloxacin (GlaxoSmithKline, Spain); gatifloxacin (Andromaco, Barcelona, Spain); clinafloxacin (Parke-Davis, MI, USA).
The Etest susceptibility method was carried out according to the manufacturers instructions (AB Biodisk, Solna, Sweden). Two 150 mm HTM (Oxoid) agar plates were inoculated with each bacterial suspension adjusted to a McFarland standard of 0.5 and allowed to air dry. Six Etest strips were placed on to each plate. Plates were incubated for 1824 h at 35°C with 5% CO2. Etest MICs were read from the intersection of the inhibition ellipse with the test strip, and then were transformed into the standard double dilution MIC values to permit appropriate comparison with the other methods used.
The quinolones evaluated by each method are indicated in Table 1. In all methods final colony counts were carried out to check bacterial inocula.
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In the comparative study of MIC values obtained by the standard microdilution, HTM-agar dilution and Etest methods, essential agreement was considered when MICs were identical or within one dilution of the reference microdilution method. Management of data and statistical calculations were carried out using the Whonet (WHO/CSR/DRS/99.1, World Health Organization) and GraphPad Prism (GraphPad Software, Inc.) computer programs. Pearson correlation coefficients (r values) and regression slope equations were generated for each susceptibility test method.
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Results |
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Table 2 shows the comparison of Etest and agar dilution methods for MIC calculations with the standard NCCLS microdilution method. Essential agreement (±1 log2 dilution) with the reference technique was 86.4% for Etest (range 30.6100%) and 82.1% for HTM-agar dilution (range 58.090%). The corresponding correlation coefficient values were 0.96 in both cases. Etest and agar dilution techniques gave lower MIC values than microdilution for nalidixic acid (Etest) and norfloxacin (agar dilution). Discrepancies with Etest determinations were essentially due to nalidixic acid; excluding the nalidixic acid results, the essential agreement increases to 95.1% (Table 2).
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When H. influenzae isolates with lower ciprofloxacin MICs including the ATCC strains (n = 34, MIC 0.06 mg/L, a concentration without amino acid changes in the quinolone resistance-determining regions (QRDRs) of GyrA and ParC58) were compared with strains with high ciprofloxacin MICs (n = 19, MICs
2 mg/L, a concentration with amino acid changes both in GyrA and ParC58), all quinolones increased their MIC90 in the latter group by a magnitude of 28 to 210 (2561000 times), suggesting that cross-resistance to all quinolones was the rule.
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Discussion |
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Our data confirm the excellent activities of all quinolones against susceptible populations of H. influenzae; with the exception of nalidixic acid, all had MICs 0.12 mg/L. The most active quinolones against susceptible strains were clinafloxacin, moxifloxacin, gatifloxacin, ciprofloxacin, trovafloxacin and levofloxacin, all with MICs
0.06 mg/L. However, with less ciprofloxacin-susceptible strains all assayed quinolones lost activity, and only clinafloxacin and gatifloxacin retained good in vitro activity (MIC90 0.25 mg/L), and to a lesser extent, moxifloxacin and levofloxacin (MIC90 1.0 mg/L). With the most resistant strains (range 232 mg/L), none of the quinolones assayed in this study presented good activity.
We found excellent agreement among the three susceptibility testing methods that are commonly used in clinical microbiology laboratories. Etest was a reliable method for the determination of quinolone MICs for H. influenzae, and it correlated very well with the NCCLS reference method. In addition, the Etest is less labour-intensive than other susceptibility testing methods, so we consider the utility of this method as a simple way to detect lost of susceptibility to quinolones in H. influenzae. The rapid detection of this reduced susceptibility to quinolones in H. influenzae is very important in antibiotic resistance surveillance programmes.
In summary, decreased susceptibility to ciprofloxacin in H. influenzae strongly correlated with decreased susceptibility to a large number of new and old quinolones; moreover, we have also shown that all quinolones undergo a similar magnitude of activity loss. Therefore, we believe that, for screening purposes, the determination of ciprofloxacin MIC should be sufficient to detect the decreased susceptibility to the whole group of quinolones. Nevertheless, nalidixic acid MIC determination may also be included to detect strains that are nalidixic acid resistant and ciprofloxacin susceptible (three of 32 resistant strains in this study) as they would be undetected with only ciprofloxacin MIC determination.
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Acknowledgements |
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Footnotes |
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References |
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