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Emerging Infectious Diseases

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Article Contents

The Study
Conclusions
Acknowledgments
References
Figure 1
Figure 2
Table
Appendix Table
Suggested Citation

Volume 13, Number 10–October 2007

Dispatch

Ciprofloxacin-Resistant Neisseria meningitidis, Delhi, India

Smita Singhal,* Kedar P. Purnapatre,* Vandana Kalia,* Smita Dube,* Deepti Nair,† Monorama Deb,† Pushpa Aggarwal,‡ Sunil Gupta,§ Dilip J. Upadhyay,* Ashok Rattan,¶ and V. Samuel Raj*
*Ranbaxy Research Laboratories, Gurgaon, India; †Vardhman Mahaveer Medical College and Safdarjung Hospital, New Delhi, India; ‡Ministry of Health and Family Welfare, New Delhi, India; §National Institute of Communicable Diseases, Delhi, India; and ¶Caribbean Epidemiology Centre, Port of Spain, Trinidad and Tobago

The Study

A total of 444 meningococcal cases and 62 deaths due to meningococcal meningitis serogroup A were reported in Delhi during April–July 2005. The reappearance of meningococcal cases was reported in Delhi during January–March 2006 (177 meningococcal cases and 17 deaths). The meningococcal cases were reported from the major hospitals and were characterized by sudden onset of fever with petechial rash, neck stiffness, and altered sensory functions. All age groups were affected, but the highest proportion was in those 15–30 years of age; male patients accounted for 71% of cases and female patients 29%.

Fourteen N. meningitidis clinical isolates were collected from the major hospitals in Delhi. All these isolates were from patients with meningococcal meningitis from the outbreak. The strains were isolated mainly from cerebrospinal fluid and a few from skin swabs; the primary culture, isolation, and serogrouping (Latex agglutination kit, Wellcogen, Wellcome, Dartford, UK) were performed in the respective hospitals. N. meningitidis strains from Ranbaxy Research Laboratories culture collection (RRL-1 and RRL-2) were used as reference serogroup A strains. In addition, 11 clinical isolates collected during the January–March 2006 recurrent outbreak were also included for selective antimicrobial drug susceptibility testing in this study.

The MICs of antimicrobial agents against these isolates were determined by the agar dilution method on Mueller-Hinton agar plates with 5% sheep blood as recommended by Clinical and Laboratory Standard Institute (CLSI) guidelines (7). The pulsed-field gel electrophoresis (PFGE) method used in this study was based on the procedures described by Popovic et al. (8). Two isolates (Ap-II 420 and Ir-I 442) from the outbreak were used for genotyping as described by Maiden et al. (9) and analyzed by using the multilocus sequence typing (MLST) database (10).

Because the N. meningitidis isolates showed resistance to ciprofloxacin, the DNA gyrase A quinolone-resistance–determining regions (QRDRs) of 2 ciprofloxacin-sensitive and 7 ciprofloxacin-resistant meningococcal strains from this outbreak and 2 reference strains were sequenced. The following primers were used for QRDR study. The forward primer was 5´-CGTACTGTACGCGATGCACGA-3´; the reverse primer was 5´-TTTCGCCATGCGGATTTCGGT-3´. The genomic DNA was isolated from the strains and PCR was performed as described by Shultz et al. (11).

The MIC pattern of the clinical isolates of N. meningitidis from the outbreak showed that they were susceptible to β-lactam antibiotics penicillin, ampicillin, and a third-generation cephalosporin, ceftriaxone. The MIC against ceftriaxone for all the clinical N. meningitidis isolates was <0.001 μg/mL. Of 14 strains, 12 showed decreased susceptibility to all the fluoroquinolones, especially to ciprofloxacin (MIC 0.25 μg/mL) (Appendix Table. In addition, all these 12 strains were resistant to nalidixic acid (MIC >16 μg/ml). A notable shift in antimicrobial susceptibility was not observed in these isolates against protein synthesis inhibitors (Appendix Table). Eleven meningococcal isolates from the recurrent outbreak showed resistance to ciprofloxacin as well as nalidixic acid (Table). The break points are based on the CLSI guidelines (7).

Figure 1

Figure 1. Pulsed-field gel electrophoresis (PFGE) pattern of the Neisseria meningitidis isolates from the outbreak. The chromosomal DNA digested with SpeI enzyme...

Figure 2

Figure 2. Comparison of GyrA quinolone-resistance–determining region (QRDR) for the alteration of amino acids in Neisseria meningitidis isolates...

The PFGE patterns of the strains from the outbreak (9 strains) were indistinguishable, and they appeared to be from the same origin (Figure 1). The antimicrobial drug resistance patterns were not distinguishable from the PFGE types. The PFGE pattern of the strains from the outbreak was different from that of the reference strains RRL-1 and RRL-2. The housekeeping genes of the ciprofloxacin-resistant strain (Ir-I 442) showed no alteration or mutation, and they were identical to the ciprofloxacin-sensitive strain (Ap-II 420). Neisseria MLST analysis with the MLST database showed that these isolates were similar to the outbreak strains from Dhaka, Bangladesh (2002), and Nigeria (2003) (10). In addition to the endemicity in Delhi, the migration of persons or visitors from other countries may have contributed to the spread of the serogroup A outbreak.

The sequencing of the gyrA QRDRs of ciprofloxacin-resistant strains of N. meningitidis showed 4 amino acid differences. One of these encoded a conservative threonine substitution at position 91 (Thr-91 → Ile); the other 3 changes were synonymous: Asn-103 → Asp, Ile-111 → Val, and Val-120 → Ile (Figure 2). Gyrase A changes have been reported in clinical isolates of resistant meningococci in positions 91 (Thr-91 → Ile) and 95 (Asp-95 → Asn and Asp-95 → Gly) (4–6). The N. meningitidis gyrA gene shares 95% identity with the N. gonorrhoeae gyrA gene. The GyrA substitution in meningococci at position 91 (Thr-91 → Ile) was equivalent to Ser-91 → Ile, reported in fluoroquinolone-resistant N. gonorrhoeae from Japan (12). In meningococci as well as in gonococci, the mutation at position 91 causes fluoroquinolone resistance. For example, 1 N. meningitidis strain from 2005 outbreak, IR-II 440, showed the mutation only at position 91 (Thr-91 → Ile), and ciprofloxacin resistance was observed.

Conclusions

The antimicrobial drug susceptibility of the clinical isolates and further sequencing of the QRDRs of gyrase A of the strains confirms the emergence of ciprofloxacin resistance in the outbreak in Delhi. In addition to chemoprophylaxis, fluoroquinolone consumption in the community, for a range of infections may be, in part, responsible for the emergence of ciprofloxacin resistance in N. meningitidis isolates. Lack of N. gonorrhoeae isolate response to ciprofloxacin has been reported in Delhi (13). Drift in susceptibility of N. gonorrhoeae to ciprofloxacin caused therapeutic failure. Ciprofloxacin treatment failure in cases of typhoid fever has also been reported (14). To date, to our knowledge, no failure of ciprofloxacin as chemoprophylaxis for N. meningitidis has been reported. To predict clinical outcome, further detailed pharmacokinetic/pharmacodynamic (PK/PD) analysis is needed to determine the impact of the PK/PD parameters on resistance selectivity (15).

Acknowledgments

We thank Pradip Kumar Bhatnagar and Kulvinder Singh Saini for critical review of this article and R. Madhubala for providing the laboratory facility for PFGE experiments. We appreciate the useful discussions regarding the epidemiology of meningococcal outbreaks with Shiv Lal, Sashi Khare, and R. Gaind.

Dr Singhal is a senior research scientist at Ranbaxy Research Laboratories, Gurgaon, India, and coordinator of the microbial in vitro susceptibility testing program. Her major research interest is the emergence of bacterial resistance to existing antimicrobial agents.

References

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Figures

Figure 1. Pulsed-field gel electrophoresis (PFGE) pattern of the Neisseria meningitidis isolates from the outbreak. The chromosomal DNA digested with SpeI enzyme...
Figure 2. Comparison of GyrA quinolone-resistance–determining region (QRDR) for the alteration of amino acids in Neisseria meningitidis isolates...

Tables

Table. Antimicrobial susceptibility pattern of Neisseria meningitidis isolates from the recurrent outbreak during January–March 2006, Delhi, India
Appendix Table. Antimicrobial susceptibility pattern of Neisseria meningitidis isolates from the recurrent outbreak during January–March 2006, Delhi, India