Molecular Detection of blaTEM, blaCTX-M and blaSHV Genes in Extended Spectrum β-Lactamase (ESBL) Escherichia coli from Clinical Samples

Introduction: Extended spectrum β-lactamases are the group of β-lactamase enzymes which confer resistance to the oxyimino-cephalosporins and monobactams. study Methodology: Clinical samples were processed in microbiology laboratory and culture isolates were characterized by standard microbiological techniques following standard procedures . Antibiotic susceptibility testing was performed by modified Kirby-Bauer disc diffusion method as recommended by Clinical and Laboratory Standard Institute. Extended spectrum βlactamases were phenotypically confirmed by combined disc method. ESBL producing genes i.e. blaTEM, blaCTX-M and blaSHV were confirmed by PCR. Results: Of the 1449 total Escherichia coli isolates, 323/1449(22.29%) isolates were multidrug resistance. Among total MDR E. coli isolates, 215/323(66.56%) isolates were ESBL producers. The maximum number of ESBL E. coli was isolated from urine 194(90.23%), followed by sputum Original Research Article 12(5.58%), swab 5 (2.32%), pus 2 (0.93%) and blood 2 (0.93%). Antibiotic susceptibility pattern of ESBL E. coli producers showed highest sensitivity towards tigecycline (100%) followed by polymyxin B, colistin and meropenem. Out of 215 phenotypically confirmed ESBL E. coli , only 186(86.51%) isolates were found to positive by PCR. The last 29(13.49%) were negative for any of the resistant genes. Among the ESBL genotypes, most common was blaTEM 118(63.4%) followed by blaCTX-M 68(36.6%). Conclusion: The emergence of ESBL producing E. coli isolates with high antibiotic resistant rates to commonly used antibiotics and increased predominance of major gene types bla TEM is a serious concern to the clinicians as well as microbiologist. This study forwarded a real massage to all the clinicians for the emergence of XDR and PDR resistant bacteria and preservation of antibiotics for their proper use in near future, if past experience with MDR and ESBLs is any indicator. Aims: This study was focused to find out the ESBL producing Escherichia coli and detection of TEM, SHV and CTXM genes by Polymerase Chain Reaction (PCR).

The first ESBL was detected from Germany in 1983 and in 1985 from France and at the end of 1980s,and the beginning of the 1990s from United States [3]. The emergence of New TEM and SHV enzymes are still existing in Europe, and found to have distinct epidemic clones, for example SHV-12 detected in Escherichia coli and Klebsiella pneumoniae isolates in Italy [4]. CTX-M-9 group are commonly found in isolates in Spain and CTX-M-3 enzymes have been identified the major strains in Eastern Europe, although variants of CTX-M group 1 (including the CTX-M-15 type) are the most widespread throughout Europe [3,4].
The rapid increase in extended spectrum βlactamases with the existence of multidrug resistant organisms is a global problem. The prevalence of ESBL producing organisms is more than 20% in Asia and South Africa. The detection of major genes such as blaTEM, blaCTX-M and blaSHV in ESBL producing E. coli by molecular methods and their antibiotic resistance pattern can provide valuable information about their epidemiology and help in formulation of rational antimicrobial therapy [5].
In Developing country like Nepal also due to the increasing incidence of ESBL producing Escherichia coli, the cost associated with the consequences also rises, so considers as an economic burden on the patients both in community and in hospital. Therefore, this study was conducted with the objectives of studying the spectrum of MDR and ESBL Escherichia coli producing strains and detection of TEM,SHV and CTX-M genes by Polymerase Chain Reaction. Characterization of ESBL Escherichia coli at molecular level may be beneficial to analyze the root cause of ESBL pattern which may help to make a positive contribution to current understanding and knowledge of the situation caused by ESBL Escherichia coli producing strains and for the development of better treatment strategy and prevention of the disease.

Screening of ESBL
The screening was done by disc diffusion technique using 3 rd generation cephalosporins (ceftazidime, cefotaxime and ceftriaxone). Isolates resistant to more than one of these agents were identified as possible ESBL producers [7].

Confirmation of ESBL
For confirmation, combined disc test was performed using Ceftazidime (30µg) alone and ceftazidime with clavulanic acid (30µg/10µg) and cefotaxime (30µg) and cefotaxime with clavulanic acid (30µg/10µg). A difference in zone of inhibition by ≥5mm of either of ceftazidime clavulanic acid with ceftazidime alone and cefotaxime clavulanic acid with cefotaxime alone was interpreted as confirmed ESBL [7].

Gene Identification
From confirmed ESBL E. coli, plasmid DNA was extracted using alkaline hydrolysis method.
These plasmid DNA served as a template for PCR amplification using blaTEM, blaCTX-M and blaSHV specific primers (Marcogen, Korea). For PCR amplification, 1.5µl plasmid DNA was added to 25 µl mixture containing 13 µl master mixture (Solis Biodyne, Estonia),10.5µl nuclease free water and 0.5µl each of reverse and forward primers. PCR was performed in 5 Prime/02 thermal cycler using optimized condition. Bibby Scientific, U.K.using optimized condition. For blaTEM gene identification,initial denaturation at .The amplified product was subjected to gel electrophoresis (2% gel stained with ethidium bromide) at 70v for 45 minutes.DNA ladder (100bp) was used to estimate the molecular weight of amplified products. After electrophoresis, gel doc system was used for photo documentation.

Control
For ESBL test, Escherichai coli (ATCC 25922), Klebsiella pneumoniae (ATCC 700603) were taken as negative control and positive control respectively. Confirmed Escherichia coli strains harbouring blaTEM, blaSHV, blaCTX-M were taken as positive control and nuclease free water as negative control.

Statistical Analysis
Data were entered and percentage were calculated and analyzed by using Statistical Package for Social Science (SPSS) version 21.

CONCLUSION
In conclusion, the present study highlights the emergence of MDR and ESBL producing E. coli isolates with high antibiotic resistant rates to commonly used antibiotics and increased predominance of major gene types blaTEM is a serious concern to the clinicians as well as microbiologist. Since the spread of MDR and ESBL producing E. coli has been increasing rapidly worldwide including developing country like Nepal, treatment options for resistant bacteria have been increasingly sorted. In the present study, no resistance was documented to tigecycline, polymyxin B, and colistin suggesting the suitable drug of choice for treating ESBL producing E. coli causing life threatening infections. Therefore, molecular detection and identification of ESBL producing bacterial isolates should be essential at routine laboratory level. Of particular concern, our findings emphasizes the need for implementation of strict antibiotic policy, clinical care management and antibiotic stewardship program absolutely required in each and every health sectors to minimize the increasing trends of MDR and ESBL isolates by all concern authorities which will help in reduction of mortality of patients. This study forwards a real massage to all clinicians for the emergence of XDR and PDR resistant bacteria and preservation of antibiotics for their proper use in near future, if past experience with MDR and ESBLs is any indicator.

CONSENT
It is not applicable.