Genotypic Characterization of Antibiotic Resistant Klebsiella Species

Abstract

The rapidly growing drug resistance among bacterial strains has become a challenge for scientific community for treating life-threatening infections. In the past few years, the extensive and indiscriminate use of broad spectrum drugs has led to the emergence and spread of multiple drug resistance among bacterial flora. β-lactam drugs are considered as the most widely prescribed antibiotics for treating infections caused by gram-negative bacterial strains of Enterobacteriaceae family. The emerging resistance in the strains of Enterobacteriaceae family has been observed due to the production of various resistance enzymes like beta lactamases, extended spectrum beta lactamases and carbapenemases. In this study, our primary focus was to study drug resistance amongst strains of the genus Klebsiella. Klebsiella species have gained significant clinical importance due to high rates of mortality and morbidity among the nosocomial and community acquired infections caused by them. Resistance enzymes like beta lactamases, extended spectrum beta lactamases and carbapenemases producing strains of Klebsiella species are capable of hydrolyzing Beta Lactam, Extended Spectrum Cephalosporin and Carbapenem drugs respectively. Studies have revealed that these enzymes produced by Klebsiella species are found to be plasmid mediated and can confer resistance from one bacterial strain to another via horizontal gene transfer. They can also mediate resistance against antibiotics other than beta lactams including aminoglycosides, quinolones, fluoroquinolones, etc. In our study, we performed a comprehensive resistance profiling of 125 Klebsiella strains and detected the prevalence of resistance enzyme producers by phenotypic and genotypic methods. Drug susceptibility testing was performed by Kirby Bauer Disc Diffusion method and minimum inhibitory concentrations were also determined by Macrobroth Dilution method. Phenotypic detection of Extended Spectrum Beta Lactamase (ESBL) was performed by the techniques of Double Disc Synergy and Combination Disc Test, whereas production of Carbapenemase enzyme was detected by Modified Hodge Test. Genotypic characterization of drug resistant Klebsiella strains was performed by identifying the prevalence of resistance genes including blaTEM, blaSHV, blaCTX-M blaNDM, blaKPC, blaOXA-23, blaOXA-51, and AmpC. Polymerase chain reaction (PCR) was performed for the detection of resistance genes and DNA sequencing was performed for selected gene to verify the variant of the gene. In our study, we identified multiple drug resistant strains of Klebsiella, with high resistance potential against Amoxicillin (100%), Tetracycline (86%), Nalidixic Acid (86%), Cephradine (82%), Gentamicin (82%) and Co-amoxyclav (80%) respectively. Antimicrobial drugs that showed good inhibitory results against Klebsiella strains were found as Amikacin (96%), Meropenem (94%) and Piperacillin-tazobactam (91%). Minimum inhibitory concentrations (MICs) were determined to validate the susceptibility results obtained through disc diffusion method. Resistance percentages with Macrobroth dilution method were detected as 81.6%, 81.6%, 63.2%, 36%, 28.8% and 25.6% against gentamicin, cephradine, co-amoxyclav, cefuroxime, ciprofloxacin, cefotaxime and cefoperazone respectively. Carbapenems were found as the most effective drugs against Klebsiella isolates. The drug resistant Klebsiella strains were further analyzed phenotypically to detect the production of extended spectrum beta lactamase and Carbapenemase enzymes. It was found that 64.8% and 5% of Klebsiella isolates were extended spectrum beta lactamase and carbapenemase producers, respectively. Furthermore, we performed molecular detection of drug resistance genes to confirm phenotypic findings of our study. The polymerase chain reaction method revealed the presence of resistance genes predominantly blaSHV, followed by blaTEM, blaKPC, AmpC, blaOXA-51, blaCTX-M, blaNDM-1 and blaOXA-23 in 48%, 43.2%, 38%, 23%, 3.2%, 2.4%, 2.4% and 1.6% in Klebsiella isolates, respectively. We randomly selected a drug resistant ESBL producing Klebsiella strain to identify the variant of the gene through DNA sequencing. Complete sequence of the blaTEM positive PCR product revealed 94% similarity with TEM-198 of Klebsiella pneumoniae KUN5033. In this study, we determined high prevalence of drug resistance among Klebsiella strains due to the production of plasmid mediated enzymes predominantly extended spectrum beta lactamase. It was found that these resistant strains cannot be easily detected through phenotypic screening methods. Therefore, identification and confirmation of these strains through molecular methods are preferred. This study provides the phenotypic and genotypic statistical data of drug resistant Klebsiella strains circulating among the population of Karachi-Pakistan, which will facilitate the clinicians to design a correct empirical therapy and to implement infection control policy.