Investigation of resistance and virulence mechanisms of multidrug resistant Acinetobacter baumannii strain by whole genome sequencing

Acinetobacter baumannii is one of the multi-drug resistant (MDR) opportunistic nosocomial pathogens, ıt is the main cause of nosocomial infections and is of worldwide concern due to its resistance to a wide variety of antibacterial agents. The aim of this study was to identify antimicrobial resistance and virulence factor genes exhibited by multidrug resistant Acinetobacter baumannii, to analyze biofilm formation and to investigate clonal subtypes of isolate. In this study, A. baumannii strain was isolated from Trabzon Fatih State Hospital and antibacterial susceptibility of the isolate was determined with VITEK 2 Compact system. The presence of β-lactamase encoding genes and class 1 integrons in the isolate was investigated by PCR method. The sample with positive PCR result (blaOXA-23) was cloned into pGEM-T easy vector and sent for base sequence analysis and the results were analyzed using bioinformatics programs. Whole genome sequencing was performed with the Illumina NovaSeq 6000 platform and MLST analysis was evaluated using the Oxford and Pasteur typing schemes. All virulence factor genes in A. baumannii A73 strain were identified using the VFDB database program. Imipenem and levofloxacin MIC values were determined by liquid microdilution method. Biofilm formation and induction of biofilm formation were performed at imipenem and levofloxacin (1/2, 1/4, 1/8*MIC) concentrations in 96-well plates in triplicate. The ability of the isolate to move was performed in petri dishes in triplicate and visualized with 0.2% crystal violet. According to the antibiotic susceptibility test of the strain; It was defined as multidrug resistant (MDR) with resistance to carbapenems, penicillins, aminoglycosides, and fluoroquinolones. According to the results of whole genome sequencing, it was determined that the isolate carried blaOXA-23, blaOXA-51, blaampC and blaTEM beta-lactamase genes. The sequence of the blaOXA-51-like gene was evaluated with bioinformatics programs, and it was determined to be blaOXA-66. According to the Pasteur MLST scheme, the strain showed the ST2 allelic profile. A nucleotide change was detected in gdhB, one of the genes used in the Oxford MLST scheme (G189A) and named gdhB-227. When MLST analysis was performed using the newly formed variant and other MLST Oxford scheme genes, the new ST clone was determined. The resulting new clone was named ST2121. An amino acid change (Met638Leu) was also detected in the gyrA gene. It was determined that multi-drug resistant isolate carried bap, basABCDFGHIJ, csuA/BABCDE, bauABCDEF, plcD, pgaABCD, entE, barAB, ompA, abaIR, piT2EAFTE/AUBl, fimADT, cvaC, bfmR, bfmS virulence factor genes. In our study imipenem induced the highest biofilm formation at a concentration of 32 µg/mL and levofloxacin at a concentration of 16 µg/mL. In conclusion, the cause of multidrug resistance in A. baumannii was investigated. A new clone of STOxf, ST2121 has been detected. In this study, the ST2Pas/ST2121 strain harboring the blaOXA-66 gene was reported for the first time in A. baumannii in Turkey.