Molecular Epidemiology and Genetic Characterization of Multi drug-Resistant Acinetobacter baumanni

Abstract

A. baumannii infections are an emerging serious health issue, owing to the increasing development of antibiotic resistance across the globe including Pakistan. Particularly A. baumannii that produce extended spectrum beta-lactamases (ESBLs) and resist carbapenems are recognized threat to public health. These multi drug-resistant (MDR) A. baumannii result in serious life-threatening infections, increased treatment cost due to prolonged hospital stay, and increased mortality rate. However, a comprehensive national level data regarding A. baumannii is lacking. Keeping this in view, the present study encompass epidemiology, resistance traits, virulence markers and clonal diversity, to understand the current trend of antibiotic resistance and clonal relatedness which is necessary for planning therapeutic strategies. In this study, a total of 375 isolates of Acinetobacter genus were obtained from the Microbiology laboratory of Pakistan Institute of Medical Sciences (PIMS), a tertiary care hospital in Islamabad, Pakistan. Several species of bacteria are included in this genus. Among them, A. baumannii is considered as the primary pathogen in clinical settings. We identified A. baumannii to be the most prevalent species (n=240, 64%) in our sample using various phenotypic and genotypic tests [colony morphology, Gram staining, biochemical (API 20 NE kit) and genetic (16S rRNA and blaOXA-51-like genes) tests]. The basic demographic data of patients such as gender, age, specimen sites and hospital wards were also recorded. Antibiotic susceptibility assay was done for all isolates by disk diffusion method against a panel of antibiotics. Phenotypic assays for ESBLs, metallo beta-lactamases (MBLs), carbapenemases and AmpC were performed. PCR was carried out to detect ESBLs encoding genes, blaTEM, blaCTX-M and blaSHV, blaM-PER, blaM-GES, blaM-VEB; MBLs carbapenemase encoding genes, blaVIM, blaSIM, blaNDM, blaKPC, blaAIM, blaSPM, blaBIC, blaIMP, blaDIM, and blaGIM ; ADC (Class C) β-lactamase; Oxacillinases included blaOXA-51, blaOXA-23, blaOXA-24, blaOXA-48 and blaOXA-58. PCR was also done to detect resistance genes against aminoglycosides (aadB, aphA6, aadA1, and aacC1) and tetracycline (tetA and tetB genes) along with a set of virulence genes including csgA, iutA, cnf1 and cvaC. PCR amplifications were performed for fluoroquinolone resistance genes (qnrA, qnrB and qnrS), insertion sequence ISAba1 and outer membrane protein-encoding gene CarO. Phenotypic viii efflux pump activity was analyzed by ethidium bromide cartwheel assay and PCR was done to detect efflux pump encoding genes. ERIC-PCR was performed for all A. baumannii isolates and dendrograms were made by bionumeric software (version 7.6) to investigate clonal relatedness among MDR isolates. Selected isolates were screened by PFGE analysis for clonal diversity. Multi-locus sequence typing (MLST) was carried out on subgroup of isolates. MLST sequences were uploaded to A. baumannii database (http://pubmlst.org/abaumannii/) for determination of the allele and subsequent sequence type (ST). Phylogenetic tree was generated by PHYLOVIZ software 2.0 to compare STs with clonal complexes. Demographic data revealed that most of the patients were adult males (59%) mostly admitted in intensive care unit (ICU) with high number of isolates extracted from tracheal secretions i.e. 48%. The phenotypic antibiotic susceptibility data exhibited that isolates were resistant to all classes (Penicllins, Cephalosporins, Fluoroquinolones, Aminoglycoside, Carbapenems and Tetracyclines) with tigecycline being the most effective with 50% of the isolates being susceptible. Among all isolates, 54% were multi drug-resistant (MDR), 33% were pan drug-resistant (PDR) and 13% were categorized as extreme drug-resistant (XDR). None of the isolates showed ESBL production using phenotypic DDST (Double disk synergy test). However, 59% of the isolates produced AmpC by AmpC disk test (n=141), 65% were carbapenemase producers using Modified hodge test (MHT) (n=157) and 42% were MBLs producers using MEM EDTA test (n=101). Furthermore 31% of the isolates showed efflux pump activity (Ethidium bromide cartwheel method). The phenotypic assays were further confirmed with genotypic analysis, which showed varying results. The results regarding overall antibiotic resistance genes suggest that blaOXA-51-like was present in all of the isolates followed by AmpC-ADC (71%), ISAba1 (31%), blaOXA-23-like (29%), blaIMP (26%), adeS (21%), aacC1 (20%), qnrB (17%), tet B (12%), blaNDM (9%), blaOXA-48-like (8%), qnrS (7%) and blaVIM (6%). Among the various virulence genes (csgA, iutA, cnf1 and cvaC), only a single isolate carried csgA. Using ERIC-PCR, 240 isolates were grouped into eighteen clusters. A large number of isolates appeared as singleton and doubles suggesting high diversity among the A. baumannii isolates. In case of PFGE analysis, subset of isolates was divided into 10 pulsotypes belonging to MDR group. Among them, 3 major pulsotypes, B (n=23), C (n=16), and pulsotype H (n=15) were found in MDR A. baumannii. The MLST analysis showed the presence of four ix different sequence types (STs) i.e. ST409, ST164, ST101 and ST241; all of which belong to Clonal Complex I (CC1), as per Pasteur scheme. It is need of the hour to evaluate the presence of MDR A. baumannii in healthcare settings and understand the molecular epidemiology of these MDR A. baumannii to form strategies to decrease the overall burden of infections caused by these opportunistic bacteria. Hence, the present study provides comprehensive report on demography, resistotypes (phenotypic and genotypic) and clonal diversity of A. baumannii isolates present at the site of study. This information can be used for devising effective management strategies to counter the spread of A. baumannii infections.