https://imcjms.com/public Ibrahim Medical College Journal of Medical Science https://imcjms.com/public/registration/journal_full_text/253 Wed, 12 Jul 2017 08:42:13 +0000 Extended spectrum beta lactamases (ESBLs) produced by Gram negative bacteria are mainly mediated by three important genes, namely TEM, SHV and CTX-M. In this study, we used a multiplex PCR to determine the prevalence of CTX-M and its subgroups CTX-M-3, CTX-M-14, among the members of Enterobacteriaceae family and in Pseudomonas spp that were isolated from different clinical samples in a tertiary care hospital in Bangladesh. Out of 300 isolates tested, 71.3% were positive for ESBL production by DDDT. The rate of positivity for TEM, SHV and CTX-M genes in 107 randomely selected isolates was 83.2%. Among these, 56.2% (50/89) was positive for CTX-M. Among the CTX-M positive isolates, CTX-M-3 and CTX-M-14 were 78.0% (39/50) and 80.0% (40/50) respectively. Our study demonstrated that CTX-M variants were common in Enterobacteriaceae and Pseudomonas spp prevalent in the hospital of Bangladesh. Ibrahim Med. Coll. J. 2015; 9(1): 26-30     Extended spectrum beta-lactamases (ESBLs) are enzymes that mediate resistance to third generation cephalosporins as well as monobactams and are inhibited in vitro by b-lactamase inhibitors such as clavulanic acid and tazobactam.1 Most ESBLs are mutants of TEM and SHV enzymes, but CTX-M enzymes are also increasingly becoming important. These CTX-M enzymes predominantly hydrolyze cefotaxime.2 In clinical strains, CTX-M encoding genes have commonly been located on plasmids that vary in size from 7 to 160 kb.3 ESBLs have been reported worldwide in many different genera of Enterobactericeae and Pseudomonas spp.4 ESBL producing organisms have been reported from different parts of the world and ESBLs production rates are now very high in Asia compared to Europe.5 Epidemiological reports demonstrate that some enzymes are more frequently reported than others. Predominant enzyme type varies with country and that diverse CTX-M types often exist within a single country.6 CTX-M-3, a variant of CTX-M-5 has also been reported from India.3 There was no systematic study about ESBL in Bangladesh until 2004. In 2004, it was first reported that 43.2% and 39.5% Esch. coli and K. pneumoniae isolated from clinical samples were positive for ESBL respectively7 and in 2010 it increased to 57.89%.8   Total 300 clinical isolates were collected from both the outpatients and inpatients departments of Mymensingh Medical College Hospital (MMCH) over a period of 6 months from January 2011 to June 2011. Urine and pus from skin wound were used as specimen. Specimens were collected aseptically. All samples were routinely cultured on MacConkey and blood agar plates at 370C aerobically for 18 hours. Gram negative isolates were identified by standard biochemical tests.10 The susceptibility to antibiotics was determined by Kirby Bauer method on Muller Hinton agar according to CLSI 2010 protocols for Gram negative panels.11 ESBL production was determined by double disc diffusion test (DDDT).12     Electrophoresis of the amplified product was done in 1.0% agarose gel and visualized by staining with ethidium bromide (0.5 mg/ml). A 100 bp molecular weight ladder (Roche, USA) was used to measure the molecular weights of the amplified products. The images of ethidium bromide stained DNA bands were digitized using a gel documentation system (AlphaimagerTM 3400, USA). All th laboratory works were carried out in the department of Microbiology at Mymensingh Medical College. Results     Table-3: Distribution of CTX-M gene among 89 genotypic positive ESBL organisms   Table-4: Pattern of CTX-M-3, CTX-M-14 distribution in CTX-M positive isolates  Discussion In Europe, CTX-M is also predominant and among them CTX-M-3 and CTX-M-14 are most frequently detected.21 In 2002, CTX-M-1, CTX-M-3 and CTX-M 14 were isolated from Enterobacteriaceae in France and China.20,22 Similar predominance of CTX-M-3 and CTX-M-14 has been reported from other countries of Asia and America.15,23-26 In our isolates the CTX-M-3 and CTX-M-14 were found to co-exist in both Esch. coli, Klebsiella and other Enterobacteriaceae. The co-existence of two or more kinds of ESBLs in a single isolates also common in study done by Lin et al.27 Other variants of CTX-M genes may exist elsewhere in Bangladesh. Regular screening and national surveillance characterizing the CTX-M genes needs to be instituted at different geographical locations and healthcare settings to monitor the transmission and spread of ESBL mediated resistance. References 2.    Woodford N, Ward ME, Kaufmann ME, Turton J, Fagan EJ, James D, et al. Community and hospital spread of Escherichia coli producing CTX-M extended-spectrum beta-lactamases in the UK. J Antimicrob Chemother 2004; 54: 735-43. 4.    Pitout JDD, Laupland KB. Extended Spectrum b Lactamase producing Enterobacteriaceae: an emerging public health concern. Lancet Infectious Disease 2008; 8: 159-66. 6.    Livermore DM, Woodford N. The b-lactamase threat in Enterobacteriaceae, Pseudomonas and Acinetobacter. Trends Microbiol 2006; 14: 413–20. 8.    Haque R, Salam MA. Detection of ESBL producing nosocomial gram negative bacteria from a tertiary care hospital in Bangladesh. Pakistan J Med Sci 2010; 26(4): 887-891. 10.  Cheesbrough M. District laboratory practice in tropical countries. Vol 2,  Cambridge University Press, UK 2006. 12.  Clinical Laboratory Standards Institute. Performance standards for antimicrobial susceptibility testing. 16th Informational supplement 2006; M100-S15. 14.  Pagani L, Amico ED, Migliavacca R, D’Andrea MM, Giacobone E, Amicosante G, Romero E, and Rossolini GM. Multiple CTX-M-Type Extended-Spectrum b-Lactamases in Nosocomial Isolates of Enterobacteriaceae from a Hospital in Northern Italy. Journal of Clinical Microbiology 2003; 41(9): 4264-4269. 16.  Lal P, Kapil A, Das BK and Sood S. Occurence of TEM and SHV gene in extended spectrum beta lactamases (ESBLs) producing Klebsiella spp.isolated from a tertiary care hospital. Indian Journal Medcal Research 2007; 125: 173-178. 18.  Livermore DM, Woodford N. The beta-lactamase threat in Enterobacteriaceae, Pseudomonas and Acinetobacter. Trends Microbial 2006; 14(9): 413-420. 20.  Chanawong A, Lulitanond A, Kaewkes W, Lulitanond V, Srigulbutr S, Homchampa P. CTX-M Extended spectrum b lactamases among clinical isolates of Enterobacteriaceae in a thai university hospital. Southeast Asian J Trop Med Public Health 2007; 38(3): 493-500. 22.  Dutour C, Bonnet R, Marchandin H, Boyer M, Chanal C, Sirot D and Sirot J. CTX-M-1, CTX-M-3, and CTX-M-14 b-Lactamases from Enterobacteriaceae Isolated in France. Antimicrobial Agents and Chemotherapy 2002; 46(2): 534-537. 24.  Jabeen K, Zafar A, Hasan R, et al. Frequency and sensitivity pattern of Extended spectrum beta lactamase producing isolates in a tertiary care hospital laboratory of Pakistan. Journal Pakistan Medical Association 2005; 55(10): 436-9. 26.  Pitout JDD, Gregson DB, Church DL, Elsayed S, and Laupland KB. Community wide outbreaks of Clonally Related CTX-M-14 b-lactamase-producing Escherichia coli Strains in the Calgery health region. Journal Clinical Microbiology 2005; 43(6): 2844-2849. 2026 Ibrahim Medical College. All rights reserved.