|Year : 2015 | Volume
| Issue : 6 | Page : 836-838
Mutations in gyrA & parC genes of Shigella flexneri 2a determining the fluoroquinolone resistance
MP Divya1, P Deepa Mathew1, R Jyothi2, Ramani Bai2, Sabu Thomas1
1 Cholera & Environmental Microbiology Laboratory, Rajiv Gandhi Centre for Biotechnology, Thiruvananthapuram 695 014, India
2 Department of Microbiology, Government Medical College Hospital, Thiruvananthapuram 695 011, Kerala, India
|Date of Web Publication||14-Jul-2015|
Cholera & Environmental Microbiology Laboratory, Rajiv Gandhi Centre for Biotechnology, Thiruvananthapuram 695 014
Source of Support: None, Conflict of Interest: None
|How to cite this article:|
Divya M P, Mathew P D, Jyothi R, Bai R, Thomas S. Mutations in gyrA & parC genes of Shigella flexneri 2a determining the fluoroquinolone resistance. Indian J Med Res 2015;141:836-8
|How to cite this URL:|
Divya M P, Mathew P D, Jyothi R, Bai R, Thomas S. Mutations in gyrA & parC genes of Shigella flexneri 2a determining the fluoroquinolone resistance. Indian J Med Res [serial online] 2015 [cited 2020 Jul 16];141:836-8. Available from: http://www.ijmr.org.in/text.asp?2015/141/6/836/160722
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  . Approximately 60 per cent of deaths involve children younger than five years  . 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 ,,, . 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  . Since 2002, there has been an alarming increase in S. flexneri resistant to fluoroquinolones in India, thereby limiting the treatment options ,,, . 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  according to Clinical Laboratory Standards Institute guidelines  . 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 previously , . 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 country ,, . 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 reports , . In gyrA gene, mutation initially happens at 83 [rd] position (S83L) followed by D87N  . 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 quinolones  . 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 country ,,,, . 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 tetracycline  . 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.
| Acknowledgment|| |
The authors acknowledge the National Institute of Cholera and Enteric Diseases (NICED), Kolkata, for serotyping the isolates. The authors are thankful to Prof. M. Radhakrishna Pillai, Director, Rajiv Gandhi Centre for Biotechnology, Thiruvananthapuram for the support and facilities provided. The first author (DMP) acknowledges the financial assistance provided by the Indian Council of Medical Research (ICMR), New Delhi in form of Junior Research Fellowship.
| References|| |
Kweon MN. Shigellosis: the current status of vaccine development. Curr Opin Infect Dis
Bardhan P, Faruque ASG, Naheed A, Sack DA. Decrease in shigellosis-related death without Shigella
spp.-specific interventions, Asia. Emerg Infect Dis
Sack DA, Lyke C, McLaughlin C, Suwanvanichkij V. Antimicrobial resistance in shigellosis, cholera and campylobacteriosis. Available from http://www.who.int/drugresistance/Antimicrobial_resistance_in_shigellosis_cholera_and_cmpdf
, accessed on January 7, 2013.
Van Der Ploeg CA, Viñas MR, Terragno R, Bruno SB, Binsztein N. Laboratory protocol: Serotyping of Shigella
spp. WHO Global Foodborne Infections Network 2010. Available from: http://www.antimicrobialresistance.dk/data/images/protocols/gfn_shigellaserotypification-final-29-06-10.pdf
, accessed on January 18, 2013.
Zhang W, Luo Y, Li J, Lin L, Ma Y, Hu C, et al
. Wide dissemination of multidrug resistant Shigella
isolates in China. J Antimicrob Chemother
Zhu JY, Duan GC, Yang HY, Fan QT, Xi YL. Atypical class 1 integron coexists with Class 1 and Class 2 integrons in multidrug resistant Shigella flexneri
isolates from China. Curr Microbiol
World Health Organization. Guidelines for the Control of Shigellosis, including epidemics due to Shigella dysenteriae
Geneva: WHO; 2005. Available from: http://whqlibdoc.who.int/publications/2005/9241592330.pdf
, accessed on January 7, 2013.
Mandal J, GaneshV, Emelda J, Mahadevan S, Parija SC. The recent trends in shigellosis: A JIPMER perspective. J Clin Diagn
Pazhani GP, Niyogi SK, Singh AK, Sen B, Taneja N, Kundu M, et al
. Molecular characterization of multidrug-resistant Shigella
species isolated from epidemic and endemic cases of shigellosis in India. J Med Microbiol
Srinivasa H, Baijayanti M, RakshaY. Magnitude of drug resistant shigellosis: A report from Bangalore. Indian J Med Microbiol
Taneja N. Changing epidemiology of shigellosis and emergence of ciprofloxacin-resistant shigellae in India. J Clin Microbiol
Bauer AW, Kirby WM, Sherris JC, Truc M. Antibiotic susceptibility testing by a standardized single disk method. Am J Clin Pathol
Clinical l0 aboratory Standards Institute (CLSI). Performance standards for antimicrobial susceptibility testing
; 21 [st]
Informational Supplement, M100- S21. Wayne, PA, USA: CLSI; 2011.
Baranwal S, Dey K, Ramamurthy T, Nair GB, Kundu M. Role of active efflux in association with target gene mutations in fluoroquinolone resistance in clinical isolates of Vibrio cholerae. Antimicrob Agents Chemother
Dutta S, Kawamura Y, Ezaki T, Nair GB, Yoshida S. Alteration in the GyrA
subunit of DNA gyrase and the parC
subunit of topoisomerase IV in quinolone-resistant Shigella dysenteriae
serotype 1 clinical isolates from Kolkata, India. Antimicrob Agents Chemother
Jacoby GA. Mechanisms of resistance to quinolones. Clin Infect Dis
(Suppl 2): S120-6.
Strahilevitz J, Jacoby GA, Hooper DC, Robicsek A. Plasmid-mediated quinolone resistance: a multifaceted threat. Clin Microbiol Rev
Dhodapkar R, Acharya NS, Harish BN, Parija SC. Shigellosis in Puducherry. Indian J Med Res
Nandy S, Mitra U , Rajendran K, Dutta P, Dutta S. Subtype prevalence, plasmid profiles and growing fluoroquinolone resistance in Shigella
from Kolkata, India (2001-2007): a hospital-based study. Trop Med Int Health
Paniker CK, Vimala KN, Bhat P, Stephen S. Drug resistant shigellosis in south India. Indian J Med Res