Mechanism Of Antibiotic Resistance Among Clinical Isolates Of Gram Negative Bacteria By Phenotypic And Genotypic Methods

Aim of the Study:To study the mechanism of antibiotic resistance among clinical isolates of gram negative bacteria by phenotypic and genotypic methods.Background:Nosocomial infections caused by multi-drug resistant pathogens are creating major problem to the hospitalized patients. Especially in Gram Negative Bacteria the emergence of extended-spectrum β-lactamases (ESBLs) and metallo β-lactamases (MBLs) are at an increasing rate, while the development of new antimicrobial agents has not made any improvements. A body of experience exists with the detection and treatment of extended-spectrum B-lactamase producing organisms (Klebsiella pneumoniae and Escherichia coli), suggesting that knowledge of their existence and dissemination might have an effect on therapeutic choices and patient outcomes via targeted empirical antimicrobial selection and infection control practices. Among gram negative bacteria, dissemination of extended spectrum β-lactamases and metallo-B-lactamses affects the choice of antimicrobial therapy. The challenges given by these resistance determinants to clinicians should not be neglected. Gram negative bacteria possessing ESBLs and MBLs challenge the most powerful antimicrobials (oxyiminocephalosporins and carbapenems).B-lactam antibiotics are the most commonly used antibiotics worldwide because of their efficacy, broad spectra and low toxicity. The selective pressures which are generated by the indiscriminate use of the B-lactam antibiotics have led to the selection of a variety of mutated forms of beta-lactamases such as the ESBLs, Amp C B-lactamases and metallo-B-lactamases. These β-lactamases have emerged as the most problematic mechanism which poses a therapeutic challenge to the health care settings. Therefore, resistance to this very important class of agent poses an increasingly complex problem for physicians. Among the variety of mechanisms that can provide resistance to β-lactam antibiotics in gram-negative bacilli, the production of B-lactamase is by far one of the most important factor. With the introduction of newer B-lactams, observed changes in β-lactamases include the increased prevalence of older enzymes, the appearance of new enzymes and alteration in the level of expression of the enzymes. These changes have been responsible for resistance to newer cephalosporins, monobactams, carbapenems, and β-lactamase inhibitor/β-lactam drug combinations. Resistance to β-lactam antibiotics has also emerged through alterations in the targets of the drugs, the penicillin-binding proteins, and through alterations in outer membrane permeability of the organisms to the drugs. With some β-lactam agents, multiple mechanisms must be acquired before clinically relevant levels of resistance are obtained. This is especially true for carbapenems and fourth generation cephalosporins. Gradually, resistance to β-lactam antibiotics is on the rise among clinical isolates of gram-negative bacilli, and only through more careful use of these agents, can their usefulness for treatment and prevention of infections be preserved?Objectives:1. To find the prevalence and various risk factors of multidrug resistant gram negative bacteria.2. To find mechanisms of antibiotic resistance by phenotypic methods which include ESBL and MBL production.3. To detect the gene responsible for ESBL and MBL production among different gram negative isolates and characterized them by direct sequencing of PCR products.Method:A total of136gram negative isolates obtained from various clinical samples that includes sputum, urine, pus, catheter tips and other body fluids.The study was started from October2011to February2013at China Japan Union Hospital (Third Clinical Hospital), Jilin University from various wards like respiratory medicine, ICU, pediatrics, orthopedics, nephrology, neurosurgery, hepato-biliary and pancreatic department, cardiovascular department, urology, medical department etc. All the isolates were cultured and identified as gram negative bacteria.All the isolates were screened for resistance to third generation cephalosporins (oxyimino-cephalosporins) and carbapenems by Modified Kirby Bauer Disc diffusion method. The ESBL status was confirmed by double disc synergy test (DDST) comprising CAZ/CAZ+CLA and CTX/CTX/CLA while MBL producing strains were confirmed by Imipenem-EDTA combined disc method. Molecular characterization of the ESBL producing strains were performed by PCR methods specific for TEM,SHV,CTX-M genes as well as IMP-1and VIM-2specific for the MBL genes.Results:Among136gram negative bacteria, Pseudomonas aeruginosa71(52.2%), Escherichia coli23(16.9%), Klebsiella pneumoniae14(10.3%), Acinetobacter baumanii12(8.8%), Enterobacter aerogenes4(2.9%), Serratia marcescens3(2.2%),Stenotrophomonas maltophilia4(2.9%), Morganella morganii2(1.5%) froteus mirabilis3(2.2%), were isolated. The present study revealed a higher occurrence of multidrug resistant ESBL in Pseudomonas aeruginosa24(41.4%) followed by Escherichia coli12(20.9%), Klebsiella pneumoniae9(15.5%), Acinetobacter baumanii7(12.3%), Stenotrophomonas maltophilia3(5.2%), Morganella morganii1(1.7%), Proteus mirabilis2(3.4%) while among20MBL positive strains, Acinetobacter baumanii was the predominant MBL producer9(45%), followed by Pseudomonas aeruginosa and Stenotrophomonas maltophilia4(20%) each, Klebsiella pneumoniae2(10%) and Escherichia coli1(5%). The respiratory tract infections73.5%were the most common infections followed by urinary tract infections (14.7%), catheter tips (8.1%), pus and other body fluids (2.9%) and stool (0.73%) in the hospital wards. Ampicillin was the most resistant antibiotic while imipenem and meropenem were the least resistant one. Among118third generation cephalosporin resistant clinical isolates49.1%(58/118) were ESBL positive isolates while57.1%(20/35) were MBL positive in carbapenem resistant strains by phenotypic methods. The PCR results in all the isolates show expression of BlaTEM-1positive34.6%(47/136),BlaSHV-11positive2.9%(4/136),Bla CTX-M-1positive8.1%(11/136) and Bla CTX-M-9positive8.8%(12/136).Among20MBL producing strains, there was occurrence of1case of VIM-2and IMP-1. There was co-existence of ESBL and MBL in9gram negative strains by phenotypic methods. However, PER-1gene was not detected in any strains.Conclusion:Therefore, it can be concluded that there was a high prevalence of multi-drug resistance gram negative bacterial infection in nosocomial infection. ESBL and MBL are the main cause for the resistance of third generation cephalosporins. TEM and SHV type are the major resistant genotypecarrying broad spectrum beta-lactamase. CTX-M are extended spectrum betalactamase type while VIM-2and IMP-1belongs to MBL type.