Lekshmi, Narendrakumar and Sabu, Thomas (2018) Vibrio cholerae O1 gaining reduced susceptibility to doxycycline, India. Journal of global antimicrobial resistance, 12. pp. 141-142. ISSN 2213-7173

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Abstract

Sir, In Vibrio cholerae, the causative agent of cholera (acute watery diarrhoea), the antibacterial drug susceptibility pattern, resistance determinants and cholera toxin genotype are continuously changing. Commonly used antibiotics for the treatment of cholera are doxycycline, ciprofloxacin and azithromycin. However, overuse of antibiotics has resulted in the emergence of multidrug-resistant V. cholerae strains owing to the selective pressure of therapeutic treatment [1]. Local evolution of V. cholerae strains, producing novel variants such as O139 and altered El Tor with different pathogenicity and antibiotic resistance patterns, is well documented in India [2]. In India, the homeland for cholera, a single dose of doxycycline or azithromycin is currently used for treating the disease, whereas neighbouring cholera-endemic countries such as Bangladesh and Nepal use ciprofloxacin/azithromycin and cotrimoxazole/tetracycline for cholera prophylaxis. Here we report the growing reduced susceptibility of V. cholerae O1 to doxycycline, the most effective and commonly used antibiotic for the disease in India. Sporadic cases of cholera are reported every year from south India owing to the lush monsoon rains received over a span of 3–4 months. Among the total V. cholerae isolates collected during the period 2000–2016, 36 isolates were randomly selected representing each year. The isolates were subjected to bacteriological analysis and their toxigenicity was confirmed by amplifying the major virulence genes such as ctxA, ctxB, tcpA, hylA and toxR. Genotyping was achieved by ctxB gene sequencing. Antibiogram profiling was performed by the disk diffusion assay according to Clinical and Laboratory Standards Institute (CLSI) guidelines for V. cholerae[3]. Minimum inhibitory concentrations (MICs) of ciprofloxacin, azithromycin and doxycycline were determined by Etest (AB bioMérieux, Solna, Sweden) according to the manufacturer’s instructions. Furthermore, the MIC was validated by microtitre plate-based antibacterial assay incorporating resazurin dye. All of the isolates were identified as V. cholerae O1 El Tor Ogawa by phenotypic, genotypic and serological tests. Sequencing of the ctxB gene of these isolates revealed that 28 isolates, collected from 2007 onwards, possessed an additional mutation at amino acid position 20 similar to that of the Haitian outbreak strains. Whilst all of the isolates revealed complete resistance to cotrimoxazole, nalidixic acid, trimethoprim and streptomycin, several showed multidrug resistance to up to six antibiotics including ampicillin and erythromycin. All of the isolates were found to harbour the SXT element, strB and sul2 on PCR amplification. Whole-genome sequencing of one of the isolates (W4-13) used in the study detected the aminoglycoside resistance gene strB and a sulfonamide resistance gene sul2, with 100% identity to that of the prototype resistance genes from the BLAST database, and an aminoglycoside resistance gene (strA), phenicol resistance genes (floR and catB9) and a trimethoprim resistance gene (dfrA1), with 99% identity, as in our previous study [4]. In the present study, the MIC of doxycycline ranged between 0.125–3 μg/mL. The Haitian variants (isolated from 2007 to 2016) consistently showed reduced susceptibility to doxycycline with MICs of 0.75 μg/mL, whereas the altered El Tor strains isolated between 2009 and 2011 (n = 4) showed a maximum MIC of 3 μg/mL. Similarly, reduced susceptibility to azithromycin and ciprofloxacin was also determined in these isolates, with MICs in the range of 0.19–1 μg/mL and 0.19–0.50 μg/mL respectively. The maximum doxycycline breakpoint used in the study was 4 μg/mL. Resistance to tetracycline and related antibiotics such as doxycycline is identified to be due to acquisition of genes coding for active efflux pumps or proteins that protect the bacterial ribosome (Tet and Otr) [5]. In this study, we report reduced susceptibility to doxycycline (3 μg/mL) in circulating strains of altered El Tor V. cholerae that is at the borderline of susceptibility breakpoint as well as Haitian variant strains gaining reduced susceptibility at 0.75 μg/mL (Fig. 1). Compared with the prototype strains, the test isolates analysed showed an increasing MIC of doxycycline; the reason for this increase is under further investigation. Although doxycycline resistance in environmental non-O1/non-O139 V. cholerae has been documented [6], reports on V. cholerae O1 resistant to doxycycline is sparse [7]. Although tetracycline resistance is considered a proxy indicator for doxycycline resistance, in the present study all of the isolates were sensitive to tetracycline. However, Etest revealed a progressing pattern of reduced susceptibility of V. cholerae O1 to doxycycline. To the best of our knowledge, this is the first report of increasing reduced susceptibility of V. cholerae O1 to doxycycline in India. Reduced susceptibility to ciprofloxacin, tetracycline and azithromycin among the circulating strains of India and Africa has been reported lately, causing concern both to the medical and scientific communities [8]. Global dissemination of such strains having reduced susceptibility to doxycycline, azithromycin and ciprofloxacin to cholera-endemic countries may create pandemonium in treatment efficiency. Henceforth, this study advocates furnishing strict restrictions on the indiscriminate use of antibiotics for treating cholera and promotes global surveillance of antibiotic resistance progression in V. cholerae.

Item Type:	Article
Subjects:	Cholera And Biofilm
Depositing User:	Central Library RGCB
Date Deposited:	23 Jul 2018 06:01
Last Modified:	23 Jul 2018 06:01
URI:	http://rgcb.sciencecentral.in/id/eprint/661

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