Activities of 13 quinolones by three susceptibility testing methods against a collection of Haemophilus influenzae isolates with different levels of susceptibility to ciprofloxacin: evidence for cross-resistance

María Pérez-Vázquez1, Federico Román1, M. Carmen Varela2, Rafael Cantón2 and José Campos1,*

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


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The activities of nalidixic acid, ciprofloxacin, norfloxacin, ofloxacin, pefloxacin, flerofloxacin, sparfloxacin, grepafloxacin, gatifloxacin, moxifloxacin, trovafloxacin, levofloxacin and clinafloxacin against a panel of Haemophilus influenzae strains were assessed by three susceptibility testing methods: Etest, agar dilution and the reference broth microdilution method using Haemophilus test medium (HTM) in all cases. The panel included 62 clinical and two reference H. influenzae strains; 32 had decreased susceptibility to ciprofloxacin (MIC >= 0.12 mg/L) and 30 were susceptible to this antibiotic (MIC <= 0.06 mg/L). Both Etest and HTM agar dilution results (r = 0.96; 86.61% and 82.1% of MICs within + one log2, respectively) correlated well with the reference microdilution method. The MIC90 of ciprofloxacin was 4.0 mg/L (range 0.007–32.0 mg/L). Trovafloxacin activity was similar to that of ciprofloxacin but sparfloxacin, grepafloxacin, ofloxacin, pefloxacin and flerofloxacin activities were higher (with MIC values one log2 dilution lower than ciprofloxacin). The least active were norfloxacin (MIC90 16 mg/L) and nalidixic acid (MIC90 128 mg/L). Levofloxacin and moxifloxacin were more active than ciprofloxacin (MIC90 2 mg/L); clinafloxacin and gatifloxacin were the most active with an MIC90 of 0.25 mg/L. Cross-susceptibility among all quinolones was observed (r > 0.9). Resistance to ciprofloxacin was associated with a similar magnitude of activity loss to other new and old quinolones. Ciprofloxacin MIC determination should be sufficient to detect the decreased susceptibility to the whole group of quinolones.

Keywords: Haemophilus influenzae, antibiotic resistance, quinolones, susceptibility testing methods


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Fluoroquinolones remain among the most powerful in vitro antimicrobial agents against Haemophilus influenzae, and are also highly effective as oral treatments of respiratory tract infections.1 However, the emergence of H. influenzae isolates with reduced susceptibility to ciprofloxacin and other quinolones is of clinical and public health concern, particularly in countries like Spain with high levels of antibiotic resistance to respiratory tract pathogens.

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.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Test isolates

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 20–24 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 Mueller–Hinton 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 20–24 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 manufacturer’s 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 18–24 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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Table 1.  Activities of 13 quinolones against a panel of 64 strains of H. influenzae
 
Analysis of the results

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.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The in vitro activities of the assayed quinolones against a panel of 64 strains of H. influenzae are shown in Table 1. If we only consider microdilution as the reference susceptibility testing method, the most active agent was clinafloxacin (MIC90 0.25 mg/L), followed by (in order of decreasing activity) levofloxacin and moxifloxacin (MIC90 2 mg/L), ciprofloxacin (MIC90 4 mg/L), grepafloxacin and sparfloxacin (MIC90 8 mg/L), norfloxacin (MIC90 16 mg/L) and nalidixic acid (MIC90 128 mg/L).

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 30.6–100%) and 82.1% for HTM-agar dilution (range 58.0–90%). 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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Table 2.  Comparison of Etest and agar dilution susceptibility testing methods using microdilution as reference method
 
To determine whether or not the cross-susceptibility to the whole group of assayed quinolones could be inferred from the susceptibility data of one of them, different correlation coefficients of paired results were calculated. The correlation coefficient between ciprofloxacin MICs obtained by the Etest method and each one of the remaining quinolone MICs also obtained by Etest was 90.2–95% (average 93%) (P < 0.001), except for nalidixic acid, which was 82.0%. This could be due to three strains that had nalidixic acid MICs of 8 and 16 mg/L with ciprofloxacin MICs of 0.06 mg/L.

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 (256–1000 times), suggesting that cross-resistance to all quinolones was the rule.


    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Fluoroquinolone resistance is an emerging problem in respiratory tract pathogens, including H. influenzae.9,10 Natural populations of H. influenzae are very susceptible to quinolones; in most general surveillance studies resistance is <1%.19 However, this resistance rate could be higher in isolates recovered from patients with chronic respiratory infections, as the clinical management of these patients often includes repetitive antibiotic treatments with oral quinolones3 and quinolone resistance could be induced as a consequence of longer antibiotic pressure.

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 2–32 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.


    Acknowledgements
 
We thank Maite Camacho and Enrique Moguel for their collaboration. We thank Janet Hindler for donating two quinolone-resistant H. influenzae isolates. This work was supported by a grant from the FIS (99/0304), Ministerio de Sanidad (Spain). M. Pérez-Vázquez is a recipient of a grant from the ISCIII (00/0069) and another one from the ‘Sociedad Española de Quimioterapia’.


    Footnotes
 
* Corresponding author. Tel: +34-91-509-7901, ext. 3650, 3643; Fax: +34-91-509-7966; E-mail: jcampos{at}isciii.es Back


    References
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
1 . García-Rodriguez, J. A., Baquero, F., García de Lomas, J. & Aguilar, L. (1999). Antimicrobial susceptibility of 1422 H. influenzae isolates from respiratory tract infections in Spain results of 1-year multicenter surveillance study. Infection 27, 265–7.[CrossRef][ISI][Medline]

2 . Fleischmann, R. D., Adams, M. D., White, O., Clayton, R. A., Kirkness, E. F., Kerlavage, A. R. et al. (1995). Whole-genome random sequencing and assembly of H. influenzae Rd. Science 269, 449–604.

3 . Barriere, S. L. & Hindler, J. (1993). Ciprofloxacin resistant Haemophilus influenzae in a patient with chronic lung disease. Annals of Pharmacotherapy 27, 309–10.[Abstract]

4 . Campos, J., Román, F., Georgiou, M., García, C., Gómez-Lus, R., Cantón, R. et al. (1996). Long-term persistence of ciprofloxacin-resistant Haemophilus influenzae in patients with cystic fibrosis. Journal of Infectious Diseases 174, 1345–7.[ISI][Medline]

5 . Georgiou, M., Muñoz, R., Román, F., Cantón, R., Gómez-Lus, R., Campos, J. et al. (1996). Ciprofloxacin-resistant Haemophilus influenzae possess mutations in analogous positions of GyrA and ParC. Antimicrobial Agents and Chemotherapy 40, 1741–4.[Abstract]

6 . National Committee for Clinical Laboratory Standards. (2000). Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria that Grow Aerobically—Sixth Edition: Approved Standard M7-A5. NCCLS, Wayne, PA, USA.

7 . National Committee for Clinical Laboratory Standards. (2002). Performance Standards for Antimicrobial Susceptibility Testing: Eleventh Informational Supplement M100-S12. NCCLS, Wayne, PA, USA.

8 . Pérez-Vázquez, M., Camacho, T., Roman, F., Campos, J. & Canton, R. (2001). Mutations in quinolone-resistance determining regions (QRDR) of gyrA, gyrB, parC and parE genes in Haemophilus influenzae clinical isolates. In Program and Abstracts of the Forty-first Interscience Conference on Antimicrobial Agents and Chemotherapy, Chicago, IL, USA, 2001. Abstract 145, p. 79. American Society for Microbiology, Washington, DC, USA.

9 . Biedenbach, D. J. &. Jones, R. N. (2000). Fluoroquinolone-resistant Haemophilus influenzae: frequency of occurrence and analysis of confirmed strains in the SENTRY antimicrobial surveillance program (North and Latin America). Diagnostic Microbiology and Infectious Disease 36, 255–6.[CrossRef][ISI][Medline]

10 . Bootsma, H. J., Troelstra, A., van Veen-Rutgers, A., Mooi, F. R., de Neeling, A. J. & Overbeek, B. P. (1997). Isolation and characterization of a ciprofloxacin-resistant isolate of Haemophilus influenzae from The Netherlands. Journal of Antimicrobial Chemotherapy 39, 292–3.[Free Full Text]