Genomic Polymorphism And Resistome Of Multidrug-Resistant Acinetobacter Baumannii From Clinical Isolates

Background:Multidrug-resistant (MDR) Acinetobacter baumannii has emerged as an important nosocomial pathogen worldwide and its increasing resistance to carbapenems may significantly reduce the choice of effective antibiotics. Given the tremendous challenge posed by MDR A. baumannii, prevention and control of infections due to this microorganism is becoming a serious clinical concern. In the present study, we conducted a detailed investigation of the molecular epidemiology and genetic basis of MDR A. baumannii clinical isolates identified at our laboratory over a 3-year period. The whole genome of epidemic MDR A. baumannii AB07104, AB0868, and AB0715, representative of three prevalent clones, respectively, have been entirely sequenced, and the collection and analysis of all antibiotic resistance genes in these three MDR A. baumannii isolates be known as resistome have also been performed.Methods:We developed a multiplex polymerase chain reaction (PCR) assay to rapidly detect and distinguish MDR A. baumannii clinical isolates. Molecular typing results of all 194 MDR A. baumannii isolates were obtained by the DiversiLab system of automated repetitive sequenced-based PCR, multiplex PCRs for identification of sequence type group, and multilocus sequence typing (MLST), respectively. We constructed a rapid molecular genotyping system based on these three assays. Coding DNA sequences (CDSs) identification and genome annotation of three MDR A. baumannii isolates (AB07104, AB0868, and AB0715) were performed through a publicly available web server for bacterial genome annotation called BASys (Bacterial Annotation System). Insertion sequences (ISs) were identified by submission of the whole genomes to the IS Finder website (http://www-is.biotoul.fr/). Resistance-associated genes in three MDR A. baumannii strains were scanned through whole genome sequences. The study of resistome has been designed to investigate the various resistance mechanisms involved in MDR A. baumannii clinical isolates.Results:The present study showed that all strains were positive for blaOXA-51-like genes. One hundred forty-eight (76.29%) of these strains had blaOXA-23-like genes, three (1.55%) had the gene blaOXA-24-like, three (1.55%) had the gene blaOXA-58-like, and one hundred seventy-three (89.18%) had the gene intI1. Nine types of resistance-associated genes combinations were found, and the most prevalent type was positive for genes blaOXA-51-like,blaOXA-23-like, and intI1. The MDR A. baumannii strains representing two prevalent clones (A and H clones identified by PFGE) in 6 military hospitals of China were grouped by Institute Pasteur MLST scheme into the sequence 2 (ST2), and the allelic profile was cpn60-2,fusA-2, gltA-2, pyrG-2, recA-2, rplB-2 and rpoB-2. This allelic profile corresponds to European cloneⅡrecommended to designated by ST2 or CC2 (where CC stands for clonal complex) for uniform nomenclature, and is widely disseminated in the world. According to Bartual et al. MLST scheme, isolate AB07104 (representing PFGE clone A) is ST218, which corresponds to UK OXA-23 clone 1. A novel clone (PFGE clone F) ST23 (Institute Pasteur MLST scheme)/ST91 (Bartual et al. MLST scheme) carrying the plasmid-borne blaOXA-58-like gene was found. The whole-genome sequenced strains (AB07104, AB0868, and AB0715) contain a high number of ISAbal copies. ISAbal was found upstream of blaOXA-23-like, blaampC, and sulⅡgenes in the chromosomes of three strains. Transposon Tn2008 was identified as genetic structure harboring the gene blaOXA-23-like, which is similar to Tn2006 but lacks the second copy of ISAbal. Resistome of three whole-genome sequenced strains contain a broad range of different types and combinations of resistance related determinants distributed throughout the genome. These genes were enriched forβ-lactamases, multidrug efflux pumps, multidrug resistance proteins, outer membrane proteins, heavy metals resistance proteins, integrons, and mobile genetic elements that are multiple independent genetic mechanisms leading to MDR in A. baumannii. The mutations of resistance-associated Ser83Leu in gyrA and Ser80Ile in parC were found in three MDR A.baumannii strains. Another unexpected finding was the presence of a similar AbaR-type structure in the genomes of three MDR A. baumannii strains, identically inserted in the homologous ATPase-like ORF. This genomic island was found in an "empty" state, exhibiting mobility-associated genes but no resistance markers.Conclusion:The combination of markers detected in the multiplex described provides powerful information not only on identification as A. baumannii but also on probable multidrug resistant potential and allows, to some extent, prediction of likely genotype. In this study we constructed a combinational genotyping system, which can be easily used in routine clinical microbiology laboratories for rapidly identifying and tracked the source of the most prevalent clones in nosocomial outbreaks of MDR A. baumannii. We have identified the prevalence of MDR A. baumannii ST2 (CC2), belonging to European cloneⅡ, with serious multidrug resistance in 6 military hospitals of China. The AbaR-type resistance island was not present in the genomes of strains AB07104, AB0868 and AB0715, suggesting that it is not an important contributor to the MDR phenotype for the prevalent clones in China. In the present work, from the resistome perspective, a high-throughput analysis of the elements involved in the multidrug resistance of A. baumannii is made. The obtained data are important for further predicting and fighting resistance and for understanding the combinational resistance mechanisms in A. baumannii clinical isolates.