Molecular characterization of plasmid-encoded blaTEM, blaSHV and blaCTX-M among extended spectrum β-lactamases [ESBLs] producing Acinetobacter baumannii

Acinetobacter baumannii associated with nosocomial infections are among the top six drug resistant microbes. Extensive use of the β-lactam group of antibiotics has resulted in the emergence of drug resistance and has raised a major clinical crisis. Among the newer β-lactamases, extended spectrum β-lactamases (ESBLs) have emerged as a major cause of resistance against cephalosporins. A variety of molecular mechanisms such as mutations of penicillin-binding proteins (PBP’s), alterations of membrane permeability and chromosomal or plasmid borne cephalosporinases induced by β-lactamases (bla-genes) viz., blaTEM, blaSHV and blaCTX-M in inducing resistance among A.baumannii have been reported [1]. Plasmid-encoded ESBLs are commonly reported to mediate resistance to penicillin, cephalosporins and monobactams, and have been found worldwide among Enterobacteriaceae, Pseudomonas aeruginosa and A.baumannii species [2]. Detection and molecular characterization of these genes are routinely performed by PCR, specific multiplex PCR assays, and multi-locus sequence typing (MLST), which optimize and facilitate the monitoring of the spread and emergence of ESBL producers of A.baumannii. Since these ESBL genes are transmissible and ESBL-related cephalosporin resistance being reported in many parts of the world [3], there is now a developing need to detect the ESBL producers among potentially important and under-researched uropathogens such as A.baumannii that are especially encountered in patients with long term hospital stay [4]. Our study is designed to contribute to mapping the ESBL molecular signature of A.baumannii by screening for ESBL producers and its genetic relatedness of resistance associated with plasmid-encoded blaTEM, blaSHV and blaCTX-M genes. From 1000 urine samples of patients with severe urinary tract infections (UTI), we isolated 73 consecutive, non-repetitive A.baumannii strains. Estimation of the sample size was done based on the formula n = (Z1-α)2 (P (1 − P)/D2) [5]. The prevalence was set approximately at 50%, with a 95% confidence interval, 5% precision and power set as 80%. Inclusion criteria were the selection of the UTI patients who manifested with specific urinary tract symptoms viz., frequency and urgency of urination with suprapubic discomfort, dysuria, flank pain and without prior administration of antibiotics. All the other strains were excluded from the study. Collection of specimens included random sampling technique regardless of the demographic data. Ethical committee approval and informed consent from all participants were obtained. All the A.baumannii strains were phenotypically and genotypically confirmed by conventional microbiological analytical tests and by PCR, respectively. Confirmed strains were further subjected for specific analysis for ESBL producers among A. baumannii. Ceftazidime [30 μg], cefotaxime [30 μg], ceftriaxone [30μg] and aztreonam [30 μg] were used for the initial screening of ESBL producers, and to improve the sensitivity of the ESBL detection, more than one antibiotic agent in a Kirby–Bauer disc diffusion assay was used [6]. Briefly, 0.5 McFarland standards of A.baumannii isolates were made as lawn culture on a sterile Mueller Hinton agar plate. The discs were placed on the surface of the plate and the zone diameter was interpreted in comparison with the CLSI criteria, after incubation at 37 °C/24 hrs [6]. Double disc synergy testing (DDST) was performed to detect the ESBL production among the A.baumannii isolates by standard Kirby–Bauer disc diffusion method. Briefly, broth suspension of A.baumannii isolates was adjusted to 0.5 McFarland standards. Lawn cultures of the broth were made onto sterile Mueller Hinton agar plates. Cephalosporin discs viz., ceftazidime (30 μg), cefotaxime (30 μg) and ceftriaxone (30 μg) [HiMedia Paper discs, Mumbai, India] were placed around the amoxicillin-clavulanate [20/10 μg] discs at a centre-to-