Original Research Article DOI: 10.18231/2455-6807.2017.0040 International Journal of Medical Microbiology and Tropical Diseases, October-December, 2017;3(4):167-170 167 Detection of gyrA ser83 mutation and parC gene amplification in E.coli and Klebsiella pneumoniae from tertiary care hospital, Puducherry G. Muthu 1,* , T. Mangaiyarkarasi 2 , Vishnuprabu 3 , E.Sathishkumar 4 , S. Srivani 5 1, 2 Scientist-C, 2 Associate Professor, 3 Scientist, 1 Model Rural Health Research Unit (NIE-ICMR/DHR), Linked to Tirunelveli Medical College, Primary Health Centre, Kallur, Tirunelveli, 2 Sri Manakula Vinayagar Medical College and Hospital, Puducherry, 3 Centre for Biotechnology, Anna University, Chennai, 4,5 Department of Microbiology, University of Madras, Chennai. *Corresponding Author: Email: gopalmuthukrishnan@gmail.com Abstract Introduction: Fluoroquinolones are the broad spectrum antibiotics and recently the clinical isolates of Enterobacteriaceae species had slowly extend the high resistance towards them. The point mutation in gyrA gene had showed decreased susceptibility to ciprofloxacin and resistant to nalidixic acid. The aim of the present study was to identify gyrA and parC genes and gyrA ser83 mutation among clinical isolates of Escherichia coli and Klebsiella pneumoniae by RFLP. Methods: A total of 100 clinical strains of both Escherichia coli and Klebsiella pneumoniae were collected from tertiary care hospital from Pondicherry. All the strains were subjected to antimicrobial susceptibility pattern testing by disc diffusion method as per the Clinical Laboratory Standard Institute (CLSI2012). PCR based screening of gyrA and parC was carried out and additional analysis of gyrA ser83-phe mutation in E.coli and K.pneumoniae by agarose gel electrophoresis. Results: Variation in both ciprofloxacin and nalidixic acid resistance were showed in the (100) clinical isolates of E.coli and K. pneumoniae were reported earlier. GyrA and parC genes were detected in both susceptible and resistant isolates of E.coli and K.pneumoniae. Among them, E.coli showed 100% (50/50) and K.pneumoniae showed 84% (42/50) positivity for gyrA while 78% (39/50) and 64% (32/50) positivity for parC. PCR-RFLP results indicated gyrA ser83 mutation in 84% (42/50) of E.coli and 66% (33/50). The results also shows that E.coli is less susceptible to fluoroquinolones compared to K.pneumoniae. Conclusion: The overall resistance were increased for the ciprofloxacin and nalidixic acid among clinical isolates of E.coli and K. pneumoniae due to gyrA ser83-phe mutation. The antimicrobial resistance to fluoroquinolone drugs indicate the rapid genetic modifications undertaken by Enterobacteriaceae species and need for routine screening for resistance to develop next generation drugs. Keywords: E.coli, Klebsiella species, Ciprofloxacin, gyrA, parC and PCR-RFLP. Introduction Among the Enterobacteriaceae species, the prevalence of resistance to fluoroquinolone is becoming a major problem in India. Fluoroquinolones and third and fourth generation cephalosporins are the most frequently prescribed antimicrobial agents for both gram negative and positive bacterial infections in all over the world. (1) However, antimicrobial resistance to drugs at present is increasing at an alarming rate, and is a global health concern, posing distinctive challenges to clinical microbiologists, clinicians, infection control professionals. Fluoroquinolones resistance has increased in number in almost all gram negative bacteria, particularly in E.coli, Klebsiella species, Salmonella species etc. (2,3) In recent years ESBL producing E.coli and Klebsiella pneumoniae have been increasing owing to infection and providing treatment to become a serious issue and also due to multi drug resistance (MDR). Eventhough fluoroquinolones are the effective drug for extended spectrum betalactamases producing E.coli and Klebsiella pneumoniae and also to treat the nosocomial, urinary tract infection, enteric fever and other bacterial infections. Moreover, many studies reported that the ESBL-EK strains became susceptible to the fluoroquinolones drugs, also aspect of treatment these agents are very effective to them. (4) Although a current studies have established that 40–45% of such isolates are resistant to fluoroquinolones. (5,6) An exposure and risk factors for fluoroquinolone resistant among ESBL-EK infection is most important and looks at the reason for the emergence of resistance. Fluoroquinolones are the most important drug which can have main role in treating ESBL-EK infection. In addition, consequently assist to reduce the dependence on other drugs like carbapenem and it can be limit the emergency of resistance in carbapenem drugs. ESBL-producing strains have been shown to be significantly more frequent among ciprofloxacin- resistant E. coli than among ciprofloxacin-susceptible E. coli strains. (7) Moreover, prior use of fluoroquinolones, an indwelling urinary catheter, and an invasive procedure within 72 hr prior to bacteraemia have been identified as independent risk factors for ciprofloxacin resistance in bloodstream infections due to ESBL E. coli and Klebsiella spp. (8-10) Fluroquinolones exert an antibacterial effect through inhibiting DNA synthesis by interacting with