Multidrug Resistance Among Various Clinical Bacterial Isolates and Production of Different Types of ß-Lactamases with Subsequent Transfer Mechanism by Plasmid DNA Analysis

Abstract

In the present study 710 urine, 551 blood, 91 pus, 28 fluid, 59 sputum, 13 CSF, 2 tissue, 16 stool, 30throat swab and 3 perianal swab samples were collected from symptomatic patientsvisiting KathmanduModel Hospital fromMay 2007 toAugust 2007. The samples were processed for routine culture. Theisolated bacteria were subjected for antimicrobial susceptibility testing by modified Kirby-Bauermethod. The production of extended-spectrumβ-lactamases (ESBLs), AmpC β-lactamases (ABLs) andmetallo-β-lactamases (MBLs) in the multidrug resistant isolates likely toproduce these β–lactamaseswere determined by Double Disk Synergy test (DDST), AmpC Disk test (ADT) and EDTA-DiskSynergy test (EDST) methods respectively. Plasmid DNA anlysis of the multidrug resiatant ESBL, ABL and MBL producing isolates of the bacteriawere performed. The plasmid encoded β–lactamases and multidrug resistance transfer mechanism/swere also studied by conjugation and transformation method. The work was carried out at the laboratoryof Central Department of Microbiology, Tribhuvan University, Kirtipur, Kathmandu, Nepal. All together,1503 samples were received in the laboratory, of which predominant were urine 710(47.24%) samples. Out of 1503 samples, 336(22.35%) showedbacterial growth(urine -30.84 %,blood-9.25 %, pus-61.53 %, fluid-7.14 %, sputum-1.69 %, CSF-7.69 %, tissue-100.0 %, stool-0.0 %,throat swab-3.34 % and perianal swab-100.0 %). The most predominant bacterial pathogen among allthese samples wasE.coliwith 193/336 (57.44 %) isolates. Imipenem was the drug of choice with a susceptibility of 98.43%for allGram negative isolates, whereasamikacin and vancomycin were the drugs of choice with susceptibility 100.0% each for Gram positiveisolates.Out of 336 isolates, 41.07 % (138/336) MDR isolates were found. E.coli was found mostpredominant MDR isolates with 46.12% (89/193) MDR strains among all samples. E. coli was foundmost predominant ESBL producers with 100.0% (27/27) ESBL producing MDR strains, ABL producerswith 81.03 % (47/58) ABL producing MDR strains and MBL producers with 75.75% (25/33) MBLproducing MDR strains. All MDR β-lactamase producing isolates ofE.coli (30) andC. freundii (3) selected for plasmid DNAanalysis harbored megaplasmid with other small size plasmids. Most of them were found responsible forthe transfer of plasmid mediated β-lactamase production with MDR traits frequently via conjugation.Themost common plasmid type responsible for different β-lactamase production and mutidrug resistancewas 32.5 kb. Key Words:Double Disk Synergy test,AmpC Disk test, EDTA Disk Synergy test, Plasmid,Conjugation