Mutations in gyrA & parC genes of Shigella flexneri 2a determining the fluoroquinolone resistance

Shigellosis or bacillary dysentery is caused by a group of facultative anaerobic Gram-negative rods of the genus Shigella. Worldwide, about 165 million cases of shigellosis were reported annually (99% occurring in the developing world) with one million associated deaths 1. Approximately 60 per cent of deaths involve children younger than five years 2. Four Shigella species, S. dysenteriae, S. flexneri, S. boydii, S. sonnei cause shigellosis in humans. Of these, S. flexneri is the most frequently isolated species in developing countries, which has six serotypes and two variants (X, Y) including subserotypes 3-6. Antibiotic therapy lessens the risk of serious complications and death, shortens the duration of symptoms and hastens the elimination of Shigella from the stool. Multidrug resistance is widespread in Shigella and the current treatment of shigellosis is with ciprofloxacin and with three second-line antibiotics; pivmecillinam, azithromycin and ceftriaxone 7. Since 2002, there has been an alarming increase in S. flexneri resistant to fluoroquinolones in India, thereby limiting the treatment options 8-11. We report here two isolates of S. flexneri type 2a isolated from a hospital in Kerala in 2010 which were found to be resistant to fluoroquinolones and possessed mutations in gyrA and parC genes. Two isolates of Shigella (SF1 and SF2) were isolated from a 62 yr old female and a 52 yr old male dysentery patients who presented with severe bleeding per rectum, fever and inflammatory bowel disease, admitted to Medical College Hospital, Thiruvananthapuram, Kerala, India in 2010. There was no history of travelling and they were taking protein powder as food supplement. Both patients responded well with injection of cefotaxime. The isolates were confirmed as Shigella species by biochemical tests and serotyped using commercially available antisera (Denka Seiken, Tokyo, Japan). These were stored in Luria–Bertani broth (BD, Difco, MD, USA) containing 50 per cent glycerol at-80°C. Antibiotic susceptibility testing was performed using the Kirby–Bauer disc susceptibility method 12 according to Clinical Laboratory Standards Institute guidelines 13. The antibiotic discs (µg) (Hi-Media Laboratories, Mumbai, India) used were ampicillin Escherichia coli ATCC 25922 was used as control. The minimum inhibitory concentrations (MICs) of nalidixic acid, norfloxacin and ciprofloxacin were determined using E test (AB Biodisk, Solna, Sweden). The bacterial cell lysate was used as a template for PCR analysis. The bacteria grown overnight at 37 °C in Luria-Bertani broth were boiled, snap-cooled and stored at-20°C until use. Quinolone resistance determining regions (QRDR) of gyrA, gyrB, parC …