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

Sir,

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 deaths1. Approximately 60 per cent of deaths involve children younger than five years2. 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 subserotypes3456. 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 ceftriaxone7. Since 2002, there has been an alarming increase in S. flexneri resistant to fluoroquinolones in India, thereby limiting the treatment options891011. 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 method12 according to Clinical Laboratory Standards Institute guidelines13. The antibiotic discs (µg) (Hi-Media Laboratories, Mumbai, India) used were ampicillin (10), ceftriaxone (30), nalidixic acid (30), ciprofloxacin (5), ofloxacin (5), norfloxacin (10), trimethoprim (5), tetracycline (30), chloramphenicol (30), streptomycin (10), gentamicin (10) and co-trimoxazole (1.25 + 23.75). 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 and parE were amplified as described previously1415. The amplified products were separated on a 1 per cent agarose gel, stained with ethidium bromide and visualized using a BIORAD Gel Doc EZ Imager (Bio Rad, USA). PCR products were purified using ExosapIT (USB) and sequencing reactions were carried out using the Big Dye Terminator Cycle Sequencing kit (Applied Biosystems, USA). Nucleotide sequencing was performed in both directions with the same PCR primers used for the amplification of the target genes in an automatic sequencer (ABI Prism 3200; Applied Biosystems). Sequences were edited with BIOEDIT v7.1.3 (http://www.mbio.ncsu.edu/bioedit/bioedit.html) and compared in BLAST of the NCBI database (www.ncbi.nlm.nih.gov/BLAST).

The two Shigella isolates (SF1 and SF2) were identified as S. flexneri 2a by serotyping with specific antisera. Antibiogram revealed that both the isolates were resistant to ampicillin, co-trimoxazole, nalidixic acid, ciprofloxacin, ofloxacin, norfloxacin, trimethoprim, chloramphenicol, streptomycin and gentamicin. The isolates did not show resistance to ceftriaxone. Antimicrobial resistance has been increasing among Shigella and the choice of antibiotics becoming limited with the emergence of multidrug resistant strains. In the present investigation, the test isolates were resistant to two or more classes of antibiotics, including ampicillin and co-trimoxazole. Similar findings have been reported from different parts of the country8910. Both isolates showed high level resistance to ciprofloxacin (MICs of 24 and 12 µg/ml) and for norfloxacin (MICs of 32 µg/ml for both). Both isolates possessed mutations in gyrA at position 87 with the replacement of D-aspartic acid with N-asparagine and at position 80 of parC with the replacement of serine by isoleucine. None of the isolates had any mutation in gyrB or parE genes. The mutation reported in parC gene is in accordance with the previous reports915. In gyrA gene, mutation initially happens at 83^rd position (S83L) followed by D87N16. But in the present study, the isolates did not show any change at 83^rd position of gyrA gene. The resistance to fluoroquinolones may be due to the acquisition of mutations in the QRDR of DNA gyrase (gyrA and gyrB) and topoisomerase IV (parC and parE), plasmid-mediated quinolone resistance (PMQR) determinants, such as (qnr), aminoglycoside aac(6′)-Ib-cr and active efflux of quinolones17. Since only the QRDR genes were sequenced, the possibility of other mechanisms in the observed resistance to fluoroquinolones was not ruled out.

Shigellosis is a major public health problem in India and several reports have identified S. flexneri to be the predominant circulating serotype in different parts of the country810111819. A study conducted in 1978 identified 16 Shigellae from Calicut and Trivandrum, of which eight were S. flexneri. Three of these showed resistance to ampicillin, chloramphenicol, streptomycin, sulphadiazine and tetracycline20. The emergence of fluoroquinolone resistant S. flexneri type 2a is a therapeutic challenge in the treatment of shigellosis. Periodic monitoring and reporting of Shigella serotypes circulating in the country and their antibiotic susceptibility will help the clinicians in the proper selection of drugs and their judicious use for shigellosis.