Emergence of fluoroquinolone resistance in Salmonella enterica serovar Typhi in Andaman and Nicobar Islands, India

Sir,

Enteric fever, due to infection with Salmonella enterica serotype Typhi and Paratyphi A, is estimated to cause more than 27 million infections each year worldwide with 216,000 deaths12. Almost 80 per cent of cases and deaths occur in Asia. Annual incidence as high as 1,100 cases per 100,000 population has been documented in developing countries34.

It is a major public health problem in India, accounting for more than 300,000 cases per year5. Decreased susceptibility of Salmonella enterica serovar Typhi isolates to fluoroquinolones and treatment failure with these drugs have been reported frequently in the past several years6–10. In India, resistance of S. Typhi to chloramphenicol was first reported from Kerala in 197211. According to a recent report, re-emergence of chloramphenicol susceptible Salmonella enterica serovar Typhi and Paratyphi A was observed in Chennai12.

Enteric fever cases occur regularly at Port Blair and at a few other endemic pockets in the rural areas in the archipelago of Andaman and Nicobar Islands, a Union Territory of India. Though treatment failure with fluoroquinolones is frequently reported by clinicians, the drug sensitivity status of Salmonella Typhi isolates is not known. A study was, therefore, initiated to assess this, and this communication presents the initial results of this study.

The study was conducted during May 2009 - October 2010. Blood and stool samples were collected from suspected patients (both adult and children) of typhoid fever attending the outpatient departments or admitted to two referral hospitals viz., G.B. Pant Hospital and INHS Dhanwantari, in Port Blair, prior to the administration of antimicrobial drugs. Written consent was obtained from the patient/guardian prior to collection of samples. The study protocol was approved by the ethical committee of RMRC (ICMR), Port Blair.

Blood samples were inoculated into brain heart infusion broth and incubated at 37°C. Subcultures were made onto MacConkey agar and Hektoen Enteric Agar plates (Hi-Media, Mumbai, India), first after overnight incubation of the broth and thereafter daily for seven days. Stool samples were collected in sterile containers (Hi-media, Mumbai), inoculated into selenite-F broth and subcultured onto MacConkey agar and Hektoen Enteric Agar plates after 18 h incubation13. The isolates obtained were identified by conventional biochemical tests14 and confirmed by agglutination with Salmonella antisera (Denka Seiken Co, Ltd., Japan). The antibiotic susceptibility tests were done by Kirby-Bauer disk diffusion method15 according to Clinical and Laboratory Standards Institute (CLSI) guidelines16 using commercially available disks (Hi-media Laboratories, Mumbai) of ampicillin (10 μg, AMP), carbenicillin (100 μg, CAR), imipenem (10 μg, IMP), amikacin (30 μg, AMK), chloramphenicol (30 μg, CHL), co-trimoxazole (25 μg, CoT), nalidixic acid (30 μg, NAL), norfloxacin (10 μg, NOR), ciprofloxacin (5 μg, CIP), ofloxacin (5 μg, OFX), gatifloxacin (5 μg, GAT), gentamycin (10 μg, GEN), nitrofurantoin (300 μg, NIT), and tetracycline (30 μg, TET), azithromycin (30 μg, AZT), cephalothin (30 μg, CEF), cefuroxime (5 μg, CXM), cefixime (30 μg, CFM), ceftriaxone (30 μg, CRO). Escherichia coli ATCC 25922 and Staphylococcus aureus ATCC 25923 strains were used in the assay as quality control strains. Minimum inhibitory concentration (MIC) for fluoroquinolone of the resistant isolates was assessed by E-Test (AB BIODISK, Sweden). Carbenicillin, imipenem and nitrofurantoin were included though these are not recommended for treatment of typhoid fever, because resistance to these drugs could be used as a phenotypic characteristic to study the clonal relatedness of the isolates.

DNA was extracted from the isolates following standard protocol and the quinolone resistance determining region (QRDR; gyrA, gyrB, parC and parE) was amplified using specific primers17. The amplified products were then cycle sequenced (PCR machine: ABI, 9700). The products were sequenced (Genetic Analyzer, ABI 3130, USA) and analyzed using SeqScape software (Applied Biosystem, USA). MEGA5 software18 was used to determine mutations in these DNA gyrase and topoisomerase IV genes. All isolates were screened for the presence of plasmid mediated quinolone resistant determinants (PMQR), qnrA, qnrB, qnrC, qnrS, qepA and aac (6’)-Ib-cr genes by polymerase chain reaction19.

Blood and stool samples from 68 patients were cultured during the study period. Six (8.82%, 95% CI: 3.31, 18.22) isolates of S. Typhi were obtained, four from stool cultures and two from blood cultures. These isolates showed five different drug resistance patterns. All the isolates were (100%, 95% CI: 54.07, 100.0) resistant to co-trimoxazole, ampicillin, carbenicillin, azithromycin and nalidixic acid (Table).

Table Antibiotic resistance pattern and minimum inhibitory concentration (MIC) for quinolones of six isolates of Salmonella enterica serovar Typhi obtained from Andaman and Nicobar Islands, India

One of the six isolates (95% CI: 0.42, 64.12) was resistant to all the four fluoroquinolones tested viz. norfloxacin, ciprofloxacin, ofloxacin and gatifloxacin. The MICs of NAL, NOR and CIP for this isolate were >256 μg/ml. The remaining five isolates showed intermediate resistance to norfloxacin and ciprofloxacin. The MICs of ciprofloxacin and norfloxacin for all these isolates were 0.25 and 1 μg/ml, respectively. While these five isolates were sensitive to gatifloxacin, only one was sensitive to ofloxacin, the remaining four showing intermediate level resistance. All Salmonella enterica serovar Typhi isolates were sensitive to third generation cephalosporins, chloramphenicol and imipenem.

All six isolates had double mutations in gyrA gene, one at position 83 that resulted in the substitution of serine with lysine and the other one at position 87 that led to the substitution of aspartic acid with asparagine (Genebank Accession Number- HQ318777). All isolates also had a functional mutation at position 80 of parC gene resulting in the substitution of serine with isoleucine (Genebank Accession Number- HQ318779). No mutations were detected in gyrB and parE genes of the six isolates. None of the isolates were found to harbour the PMQR determinants.

Fluoroquinolones, particularly ciprofloxacin, ofloxacin and gatifloxacin are used as the first line drugs for the treatment of enteric fever in Andaman and Nicobar Islands. All the isolates obtained so far were either fully resistant or showed intermediate level resistance to the most common drug used to treat enteric fever viz. ciprofloxacin with one showing an MIC above the break-point for resistance and the remaining above the level (0.125 μg/ml) that is considered to confer intermediate level resistance. This corroborates with the frequent treatment failures observed in the islands.

Mutations in gyrA are seen almost invariably in isolates with MIC >0.125 μg/ml20, which is also seen in the present study. All these isolates had multiple mutations in topoisomerase gene, which is considered as a pre-requisite for conferring high level of resistance to quinolones21. The present preliminary data show that multiple mutations in topoisomerase genes, including double mutations in gyrA, need not always confer high level of resistance to ciprofloxacin as reported earlier22; rather it may result only in reduced susceptibility to the drug. In a study conducted in Hong Kong, it was reported that 19 isolates of S. enterica that had the same double mutations in gyrA as seen in our isolates, along with a mutation in parC had ciprofloxacin MIC in the range of 0.12 - 0.5 μg/ml, though the mutation in parC was different from the one observed here23. Vibrio cholerae with reduced susceptibility towards ciprofloxacin (0.25 to 0.5 mg/l) harboured one mutation each in gyrA and parC, respectively24.

Enteric fever is a common occurrence in Andaman and Nicobar Islands with an estimated 1,100 cases occurring every year among the 350,000 population giving an incidence of > 450 cases/100,000 (unpublished data). Strains with genetic characteristics that confer resistance to fluoroquinolones appear to be the dominant clone of Salmonella enterica serovar Typhi causing enteric fever in the Islands. Resistance to third generation cephalosporins does not appear to have emerged in the Islands yet. However, there is every possibility that the bacteria may acquire such properties in near future, leaving little options for the physician for specific therapy for enteric fever. Routine monitoring of drug resistance among Salmonella enterica serovar Typhi is essential from public health point of view.