Molecular characterization of multidrug resistant Acinetobacter species in Universiti Kebangsaan Malaysia Medical Centre

Abstract

This study was carried out to investigate the molecular characteristic of carbapenems, cephalosporins and quinolones resistance and genetic diversity of Acinetobacter spp. clinical isolates in UKMMC. A total 178 Acinetobacter spp. were isolated from clinical specimens. Sequencing of the 16S rRNA and rpoB genes were carried out to determine the species of Acinetobacter. The minimum inhibitory concentration (MIC) for imipenem, meropenem, ceftazidime, cefepime and ciprofloxacin was determined by E-test method. The presence of carbapenemase and cephalosporinase genes was performed by PCR. Cloning and sequencing were carried out for detection of mutation in quinolone resistance determining region (QRDR). Fingerprinting was performed by REP-PCR and analyzed using Fingerprinting II software. In the present study, Acinetobacter clinical isolates were assigned into eight genospecies with the majority of isolates (93.8%) were A. baumannii. Other species were A. pitti, A. nosocomialis, A. haemolyticus, A. schindleri, A. ursingii, A. junnii and A. bereziniae. Overall, more than 73% of the A. baumannii isolates showed resistance to all five antibiotics. MIC50 and MIC90 of the antibiotics showed that A. baumannii were highly resistance to quinolones (> 32 mg/L), carbapenems (> 32 mg/L) and cephalosporins (> 256 mg/L). More than 80% of the non-A. baumannii isolates were susceptible to all five antibiotics. Resistance genes for carbapenems were detected in 97.7%, 95.4% and 3.8% of the non-susceptible carbapenem A. baumannii for blaOXA-51-like, blaOXA-23-like and blaIMP, respectively. The resistance genes for blaOXA-58-like, blaOXA-24-like and blaVIM were not detected. The blaOXA-51-like gene was identified in both non-susceptible and susceptible A. baumannii. Among the isolates harboring blaOXA-51-like, ISAba1 was detected in 46.3% (75 of 162) of the isolates. Seventy-three of the isolates harboring ISAba1/blaOXA-51 were non-susceptible to carbapenems. From 137 A. baumannii isolates carrying blaOXA-23–like, 128 of them were positive for ISAba1/blaOXA-23-like. The blaADC genes were present in 93.7% of cephalosporin non-susceptible A. baumannii isolates. ISAba1/blaADC was detected in 85.8% of cephalosporin non-susceptible A. baumannii isolates. Point mutation in gyrA (Ser-83 to Leu-83) and parC (Ser-80 to Leu-80) genes were identified in all non-susceptible A. baumannii isolates. REP-PCR analysis from 167 A. baumannii isolates gave 129 REP-types generates 18 clusters namely as cluster A to R. Cluster A was the largest comprising of 64 isolates followed by cluster E with 28 isolates. Isolates in both clusters A and E showed high rates of antibiotic resistance and majority of the isolates were from patients hospitalized in the ICU. REP-PCR analysis of 11 non-A. baumannii isolates showed 11 REP-types with high susceptibility rates and the dendrogram generates two clusters (I and II). In conclusion, A. baumannii is the commonest species isolated from clinical specimens with high resistance rates towards carbapenemes, cephalosporins and quinolone which is due to the presence of ISAba1/blaOXA-23, ISAba1/blaADC and mutation in gyrA and parC genes, respectively. REP-PCR fingerprinting can be used as a tool for classification of Acinetobacter spp. into different clusters. The results show that A. baumannii isolates in the hospital were genetically diverse and widespread in different wards with high rates of antimicrobial resistance.