Detection of blaTEM and blaCTX-M genes by multiplex polymerase chain reaction amongst uropathogenic Escherichia coli strains isolated from hospitalized patients in Kolkata, India
Paper Details
Detection of blaTEM and blaCTX-M genes by multiplex polymerase chain reaction amongst uropathogenic Escherichia coli strains isolated from hospitalized patients in Kolkata, India
Abstract
Production of extended-spectrum β-lactamase (ESBL) is one of the most important resistance mechanisms that hamper the antimicrobial treatment of infections caused by uropathogenic Escherichia coli (UPEC). The objective of the study was to determine the blaTEM and blaCTX-M producers simultaneously in a single tube reaction by multiplex PCR technique using gene specific primer sets. A total of 100 E. coli strains, isolated from hospitalized patients were phenotypically screened for ESBL production using drug-inhibitor combination disks, namely, ceftazidime-clavulanate (30+10μg) and cefotaxime-clavulanate (30+10μg) following CLSI guidelines. Out of the phenotypically screened 85 ESBL producers, multiplex PCR carried out on plasmid DNA alone indicated 50.5 % and 55.3% positivity for blaCTX-M and blaTEM gene respectively, whereas PCR on both plasmid and genomic DNA showed 81.1 % positivity for blaCTX-M and 83.5% positivity for blaTEM genes. A high percentage (52.9%) of the isolates was found to harbor both genes simultaneously. Moreover amplification of both plasmid and genomic DNA from the non-ESBL producers indicated that 6 of the isolates harbored blaTEM gene. Amplification of whole genomic DNA increased the positivity of detection, compared to amplification of plasmid DNA alone, suggesting β-lactamase expression was controlled by both chromosomal and plasmid DNA. This study for the first time reports the prevalence of the two most common ESBL responsive genes using multiplex PCR in hospitalized patients from Kolkata, India. This technique provided an efficient and rapid differentiation of ESBLs and could be used as a rapid tool for epidemiological studies.
Bush, K. 2002. The impact of beta-lactamases on the development of novel antimicrobial agents. Current Opinion Investigative Drugs 3,1284–1290.
Clinical Laboratory Standards Institute 2006. Performance standards for antimicrobial disc susceptibility tests, XVI international Supplement (M100-S16). Wayne Pa, USA: National Committee for Clinical Laboratory Standards.
Harada S, Ishii Y, Yamaguchi K. 2008. Extended-spectrum β- Lactamases: Implications for the Clinical Laboratory and Therapy. Korean Journal of Laboratory Medine 28, 401-412.
Hosoglu S, Gundes S, Kolayh F, Karadenizli A, Demirdag K, Gunaydm M, Altmdis M, Caylan R, Uemak H. 2007. Extended-spectrum β-Lactamases in ceftazidime resistant Escherichia coli and Klebsiella pneumoniae isolates in Turkish hospitals. Indian Journal of Medical Microbiology 25, 346-350.
Jemima SA, Varghese S. 2008. Multiplex PCR for blaCTX-M & blaSHV in the extended spectrum beta lactamase (ESBL) producing Gram-negative isolates Indian Journal of Medical Research 128, 313-317.
Kahan NR, Chinitz DP, Waitman DA, Dushnitzky D, Kahan E, Shapiro M. 2006. Empiric treatment of uncomplicated urinary tract infection with fluoroquinolones in older women in Israel: another lost treatment option? Annals of Pharmacotherapy 40, 2223-2227.
Kumar MS, Lakshmi V, Rajagopalan R. 2006. Occurrence of extended spectrum beta-lactamases among Enterobacteriaceae spp. isolated at a tertiary care institute. Indian Journal of Medical Microbiology 24, 208-211.
Manges AR, Natarajan P, Solberg OD, Dietrich PS, Riley LW. 2006. The changing prevalence of drug-resistant Escherichia coli clonal groups in a community: evidence for community outbreaks of urinary tract infections. Epidemiology Infection 134, 425-431.
Moura A, Nicolau A, Hooton T, Azeredo J. 2009. Antibiotherapy and pathogenesis of uncomplicated difficult relationships UTI : Journal of Applied Microbiology 106, 1779–1791.
Myer and Koshi. 2001. Methods in biochemical identification of bacteria. Myer’s and Koshi’s manual of diagnostic procedures in medical microbiology and immunology/ serology, 2nd ed. Vellore: Department of Clinical Microbiology, Christian Medical College 195-202.
Navon-Vanenzia S, Hammer-Munz O, Schwartz D, Turner D, Kuzmenko B, Carmeli Y. 2003. Occurrence and phenotypic characteristics of extended-spectrum β-lactamases among members of family Enterobacteriaceae at the Tel-Aviv Medical Center (Israel) and evaluation of diagnostic tests. Journal of Clinical Microbiology 41, 155-158.
Paterson DL, Yu VL. 1999. Extended spectrum beta lactamases: a call for improved detection and control. Clinical Infectious Diseases 29, 1419-22.
Perez F, Endimiani A, Hujer KM, Bonomo RA. 2007. The continuing challenge of ESBLs. Current Opinion in Pharmacology 7, 459–469.
Sambrook J, Fritsch EF, Maniatis T. 2001. Molecular cloning: A laboratory manual, 2nd ed. Cold Spring Harbor, New York, USA.
Sharma J, Sharma M, Ray P. 2010. Detection of TEM & SHV genes in Escherichia coli & Klebsiella pneumoniae isolates in a tertiary care hospital from India. Indian Journal of Medical Research 132, 332-336.
Mandira Mukherjee, Shreya Basu, Monalisa Majumdar, 2011. Detection of blaTEM and blaCTX-M genes by multiplex polymerase chain reaction amongst uropathogenic Escherichia coli strains isolated from hospitalized patients in Kolkata, India. Int. J. Biosci., 1(6), 64-69.
Copyright © 2011 by the Authors. This article is an open access article and distributed under the terms and conditions of the Creative Commons Attribution 4.0 (CC BY 4.0) license.