JAC Advance Access originally published online on February 22, 2005
Journal of Antimicrobial Chemotherapy 2005 55(4):596-597; doi:10.1093/jac/dki048
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Correspondence |
Emergence of Neisseria meningitidis with decreased susceptibility to ciprofloxacin in Argentina
1 Servicio Antimicrobianos, Dpto Bacteriología, Instituto Nacional de Enfermedades Infecciosas (INEI)–ANLIS ‘Dr Carlos G. Malbrán’, Av. Velez Sarsfield 563 (1281), Buenos Aires; 2 Hospital Area Cipolleti, Rio Negro; 3 Hospital Sor María Ludovica, La Plata, Buenos Aires, Argentina; 4 Reference Laboratory for Meningococci. Servicio de Bacteriología. Instituto de Salud Carlos III, Majadahonda, Spain
* Corresponding author. Tel/Fax: +54-11-4303-2812; Email: acorso{at}anlis.gov.ar
Keywords: N. meningitidis , fluoroquinolones , efflux , QRDRs
Sir,
To date, only three Neisseria meningitidis clinical isolates showing decreased susceptibility to ciprofloxacin (DSC) have been reported in France (1999), Australia (2000) and Spain (2003).1
The mechanisms of resistance were mutations in the quinolone resistance determining region (QRDR) of the gyrA gene, resulting in amino acid substitutions Asp-95
Gly, Asp-95Asn and Thr-91Ile, respectively.1
During 1997–2003, 873 meningococcal strains from invasive disease were submitted to the National Reference Laboratory (INEI) as part of the National Surveillance Programme for serogroup and antimicrobial resistance in N. meningitidis. In 2002, we detected one strain (M5191) with DSC (MIC 0.12 mg/L), isolated from the CSF of a 63-year-old woman at Hospital Area Cipolleti, Rio Negro Province. The woman suffered from diabetes and chronic urinary tract infections, and she had been previously treated with antibiotics, including fluoroquinolones. During 2003 a second strain (M5507) with DSC (MIC 0.06 mg/L) was isolated from both CSF and blood, in a 1-year-old child from Hospital Sor María Ludovica, La Plata, Buenos Aires Province. In this case, the patient had not been previously exposed to any antibiotic. Treatment with ceftriaxone resulted in a good clinical outcome in both cases. All other meningococci examined were susceptible to ciprofloxacin (MICs 
0.015 mg/L).
Serogroup and serotype/serosubtype were determined by slide agglutination and ELISA, respectively. N. meningitidis M5191 was typed as Y:non-typeable:P1.5 and M5507 as B:1:P1.non-subtypeable. Determination of MICs by agar dilution and disc diffusion testing were both performed using Mueller–Hinton agar supplemented with 5% sheep blood, with incubation for 24 h at 35°C in air containing 5% CO2. N. meningitidis EMGM-2, EMGM-10 and EMGM-13 were used as control strains.2 The MICs for M5191 and M5507 were (mg/L): penicillin, 0.03/0.12; ampicillin, 0.06/0.25; ceftriaxone, 0.001/0.002; rifampicin, 0.008/0.008; chloramphenicol, 0.5/0.5; and tetracycline, 0.12/0.12, respectively. Both meningococci showed resistance to nalidixic acid (MICs 64 mg/L).
Sequencing of QRDRs in gyrA and parC,3
and gyrB and parE,4
was performed by standard methods. As described for the Australian isolate, N. meningitidis M5507 contained a mutation in the gyrA gene that resulted in the amino acid substitution Asp-95Asn. No mutations were detected in the QRDR of the parC gene in M5507. Unexpectedly, no mutations were detected in N. meningitidis M5191 when the four QRDRs were analysed.
The major mechanisms of fluoroquinolone resistance identified in bacterial strains are chromosomal mutations in DNA gyrase and topoisomerase IV, and overexpression of endogenous efflux pumps.5 Therefore, we phenotypically assessed the possibility of increased efflux of quinolones in this strain, by determining ciprofloxacin MICs with and without 6.25 mg/L of reserpine (an inhibitor of multidrug efflux pumps). In N. meningitidis M5191, the addition of reserpine significantly reduced the ciprofloxacin and nalidixic acid MICs 30-fold and 256-fold, respectively, reaching the same levels as those for the susceptible control strains (Table 1). Reserpine alone had no effect on growth or colony morphology. Although accumulation and efflux have not been specifically investigated here, the absence of mutations in the QRDRs of gyrA, parC, gyrB and parE genes analysed and the reduction in quinolone MICs in the presence of reserpine, strongly suggest that an efflux mechanism is responsible for DSC in N. meningitidis M5191. In fact, the sequence of the mtrRCDE gene complex from this strain showed a deletion affecting most of the mtrR gene (data not shown). Although some authors have reported that this pump appears less active in meningococci than in gonococci,6 the deletion detected in M5191 might be involved in the expression of the efflux system. Alternatively, we cannot discard a combination of mechanisms conferring DSC.
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Nalidixic acid has been used to detect DSC in Neisseria gonorrhoeae.7 Therefore, we evaluated nalidixic acid (30 µg) and ciprofloxacin (5 µg) by the disc diffusion method, to detect DSC in meningococci. N. meningitidis with DSC showed zones of inhibition of 6–7 mm with nalidixic acid and of 31–35 mm with ciprofloxacin (Table 2). In contrast, susceptible strains displayed bigger zones: 33–34 mm for nalidixic acid and 38–42 mm for ciprofloxacin. Thus, the nalidixic acid disc seems to be a useful screen for DSC, independent of the resistance mechanism involved. The lack of clear guidelines for susceptibility breakpoints in this species would impair the detection of emerging resistance mechanisms. Therefore, we propose the inclusion of a 30 µg disc in the antibiogram, in order to detect the emergence of DSC in N. meningitidis isolates. When reduced zones of inhibition are noted, the MIC of ciprofloxacin should be determined in order to confirm the DSC and monitor trends in susceptibility to this antimicrobial agent in meningococcal strains.
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Rifampicin is commonly used as chemoprophylaxis to eradicate meningococci carriage. However, there is a tendency to replace rifampicin by ciprofloxacin in adults, because it can be used in single doses, whereas rifampicin requires twice-daily administration for 2 days. To date, we cannot attribute the use of ciprofloxacin in meningococci chemoprophylaxis as a cause of the emergence of DSC, but it seems reasonable to speculate that fluoroquinolone consumption in the community, for a range of infections such as urinary tract infections or community-acquired respiratory infections, may be, in part, responsible for the emergence of fluoroquinolones resistance.
Acknowledgements
The work was performed at Servicio Antimicrobianos, Instituto Nacional de Enfermedades Infecciosas—ANLIS ‘Dr Carlos G. Malbrán’, Av. Velez Sarsfield 563 (1281), Buenos Aires, Argentina. This study was presented in part at the Forty-fourth Interscience Conference on Antimicrobial Agents and Chemotherapy, Washington, DC, USA, 2004.
References
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