Genetic Diversity and Molecular Basis of Multidrug Resistance in Acinetobacter Baumannii Clinical Isolates

Abstract

Acinetobacter baumannii is an increasingly important hospital-acquired Gramnegative bacterium that can thrive in the environment of healthcare facilities, and possess a significant public health concern. These features accompanied by its inherent capacity of resistance to antimicrobial agents, acquisition of diverse resistance mechanisms and the aptitude for epidemic spread greatly contribute to the success of A. baumannii as the most important nosocomial pathogen. The main aim of the present study was to investigate the molecular mechanisms underlying the multidrug-resistant phenotypes and the molecular epidemiology of this ignored pathogen of high clinical importance from Pakistan. A total of 319 A. baumannii isolates obtained from various clinical specimens were identified by routine microbiological procedures and further confirmed by multiplex PCR for the amplification of recA gene and internal transcribed spacer (ITS) region along with the amplification of blaOXA-51-like gene. The antimicrobial susceptibility pattern was determined through disc diffusion method and MIC was measured using agar dilution, broth microdilution and E-test® methods. The presence of genes encoding resistance to beta-lactams, 16S rRNA methylases, aminoglycoside modifying enzymes, fluoroquinolones, tetracyclines and sulfonamides were evaluated by PCR followed by sequencing. Repetitive extragenic palindromic PCR (REP-PCR) and multilocus sequence typing (MLST) was performed to investigate the genetic diversity. According to the results, the 96.6% isolates were multidrug-resistant (MDR) and 84.3% were extreme drug-resistant (XDR); 298 (93.4%) were resistant to carbapenems. The blaOXA-51was identified in all A. baumannii strains confirmed by multiplex PCR whereas the acquired blaOXA-23 gene was identified in 284 (89%) isolates. Higher rates of resistance were observed to the extended-spectrum cephalosporins including ceftazidime, cefotaxime, ceftriaxone and cefepime with MIC50 ≥ 128 μg/ml. The blaOXA-23 gene with an upstream ISAba1 was the foremost mechanism of carbapenem resistance that was found in 279 (87.5%) isolates and the blaNDM was found in only 7 strains belonging to a single MLST type. The genes encoding plasmid-mediated quinolone resistant were not detected in any isolate and the mutations in the gyrA and parC genes were the main underlying mechanism responsible for fluoroquinolone resistance. The 209 (65.5%) isolates were resistant to tetracyclines and 94.3% of these isolates were found positive for tetB gene. Among the sulfonamide resistance determinants, sul2 (85.2%) was the most common gene followed by sul1 (32.8%) whereas the combination of sul1 and sul2 genes was detected in 24.6% isolates. All the XIX isolates were found susceptible to polymyxins (polymyxin B and colistin) with MIC50 as 0.5 μg/ml as well as tigecycline with MIC50 (1 μg/ml). On the basis of REP-PCR the indigenous isolates were separated into 8 distant clones whereas MLST demonstrated the presence of seven already reported STs (ST642, ST589, ST2, ST600, ST338, ST103, and ST615) from different parts of world and eight new sequence type that were single or double locus variants to each other. The predominant STs i.e. ST642 and ST589 belonged to clonal complex I according to the Pasteur scheme and were found to harbor blaOXA-23 gene. The overall multidrug resistance was almost common among the isolates of already established STs whereas the isolates belonging to ST338 and the new STs were mainly susceptible. This dichotomy specifies the major selective advantage exerted by the antimicrobial resistance that drives the enduring clonal expansion of multidrug-resistant pathogens. The study revealed the alarming trends of multidrug resistance and substantial genetic diversity among A. baumannii clinical isolates from Pakistan. The differences in the distribution of various antimicrobial resistance mechanisms among various clones demonstrate the capacity of A. baumannii to acquire and express a wider range of resistance determinants. The deeper understanding of the genetic and biochemical basis of antibiotic resistance is of principal importance to design the policies for the effective control of emergence, spread, and development of innovative approaches for the therapeutic management of these multidrug-resistant pathogens.