Antimicrobial Resistance and Genotyping of Extended Spectrum Beta-Lactamase Producing Clinical Isolates

Abstract

The development of resistance mechanism in Gram-Negative bacteria is growing all over the world affecting developing countries the most. Extended Spectrum Beta-lactamase (ESBLs) production is one of the widespread mechanisms of resistance associated with irrational or excessive use of extended spectrum cephalosporins. The ground objective of this study was to investigate the prescribing pattern of broad spectrum antibiotics in hospital setup and the prevalence of ESBL production in Gram-Negative clinical isolates collected from an outpatient source by both phenotypic and genotypic detection. The study also focused on the treatment options available to treat resistant clinical isolates of Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa and Proteus mirabilis. Total 685 in-patient data were collected over a period of three months for the prescribing patterns of extended spectrum antibiotics used alone or in combination. The data were analyzed for the utilization of extended spectrum antibiotics in various wards either empiric or prophylactic use of broad spectrum cephalosporins. For the prevalence of ESBL production, 1005 clinical isolates of E. coli, K. pneumoniae, P. aeruginosa and P. mirabilis were collected from an out-patient source. Antimicrobial susceptibility and resistance patterns were determined by Kirby-Bauer disc diffusion method under the recommended guidelines of Clinical Laboratory Standard Institute (CLSI). Phenotypic detection of ESBL was performed on 352 clinical isolates using the double disc synergy test between amoxicillin/clavulanic acid (10μg) and ceftazidime or cefotaxime (30μg) disc. Multidrug resistance among ESBL positive isolates was also determined. Pearson’s or Fisher’s exact Chi-square test was used to analyze statistical association between ESBLs and Non ESBLs at 0.05 level of significance. Furthermore, polymerase chain reaction was used for the identification of TEM, SHV and CTX-M gene in ESBL positive clinical isolates. The prescribing pattern in hospital setup revealed that 75% of the overall antibiotics were prescribed for prophylactic treatments, whereas, among all prescribed antibiotics the frequency of broad spectrum cephalosporins alone or in combination was found to be the highest. Out of 1005 gram-negative clinical isolates, prevalence was found to be E. coli 680 (67.66 %), K. pneumoniae 248 (24.67%), P. mirabilis 3 (0.3 %) and P. aeruginosa XIII 74 (7.36%). Out of 352 clinical isolates, 96 (27.27%) were ESBL positive, female preponderance was found in 62 (67%), whereas 32 (33%) were males. E. coli was the most prevalent ESBL producers 79 (82%), followed by K. pneumoniae 16 (17%) and P. mirabilis 1 (1%). The urinary tract infections were the most commonly recovered infections i.e., 72 (75%), and among them 63 and 9 were E. coli and K. pneumoniae respectively. Age groups of 16-30 and 46-60 years were most affected with ESBL producing uropathogenic E. coli with a high prevalence in females. A high rate of resistance was observed against broad spectrum cephalosporins, fluoroquinolones, amoxicillin/clavulanic acid, sulfamethoxazole/trimethoprim and ampicillin. Whereas, amikacin, imipenem, tazobactam/piperacillin and fosfomycin were found effective antibiotic choices. The rate of multidrug-resistance was found to be 95%, 62.5% and 100% in ESBL producing E. coli, K. pneumoniae and P. mirabilis respectively. Statistical evaluation between ESBLs and Non ESBLs revealed significant association in E. coli (p 0.0004) and K. pneumoniae (p 0.032). Genotype identification revealed the presence of TEM, SHV and CTX-M genes in 82.14% (69/84) of the isolates. CTX-M was the most prevalent gene found in 59.5% (50/84) of the isolates, followed by TEM 41.6% (35/84) and SHV 13% (11/84). CTX-M was dominant among uropathogenic E. coli related to community acquired urinary tract infections. The study concluded an increased prevalence rate of ESBLs in E. coli, most of them recovered from urinary tract infections. The study also found an increased rate of multidrug-resistance among Gram-Negative clinical isolates. These incidences can be reduced by the restricted use of broad spectrum cephalosporins. The spread of ESBL producing uropathogenic E. coli can be controlled by implementing ESBL screening along with limiting the use of empiric therapy. Nevertheless, hygienic practices must be employed to reduce the incidence of community onset urinary tract infections.