Whole-genome sequencing of Acinetobacter baumannii clinical isolates from a tertiary hospital in Terengganu, Malaysia (2011-2020), revealed the predominance of the Global Clone 2 lineage

Abstract

Carbapenem-resistant Acinetobacter baumannii is recognized by the World Health Organization (WHO) as one of the top priority pathogens. Despite its public health importance, genomic data of clinical isolates from Malaysia remain scarce. In this study, whole-genome sequencing was performed on 126 A. baumannii isolates collected from the main tertiary hospital in the state of Terengganu, Malaysia, over a 10-year period (2011-2020). Antimicrobial susceptibilities determined for 20 antibiotics belonging to 8 classes showed that 77.0% (n=97/126) of the isolates were categorized as multidrug resistant (MDR), with all MDR isolates being carbapenem resistant. Multilocus sequence typing analysis categorized the Terengganu A. baumannii clinical isolates into 34 Pasteur and 44 Oxford sequence types (STs), with ST2_Pasteur of the Global Clone 2 lineage identified as the dominant ST (n=76/126; 60.3%). The ST2_Pasteur isolates could be subdivided into six Oxford STs with the majority being ST195_Oxford (n=35) and ST208_Oxford (n=17). Various antimicrobial resistance genes were identified with the bla _OXA-23-encoded carbapenemase being the predominant acquired carbapenemase gene (n=90/126; 71.4%). Plasmid-encoded rep genes were identified in nearly all (n=122/126; 96.8%) of the isolates with the majority being Rep_3 family (n=121). Various virulence factors were identified, highlighting the pathogenic nature of this bacterium. Only 14/126 (11.1%) of the isolates were positive for the carriage of CRISPR-Cas arrays with none of the prevalent ST2_Pasteur isolates harbouring them. This study provided a genomic snapshot of the A. baumannii isolates obtained from a single tertiary healthcare centre in Malaysia over a 10-year period and showed the predominance of a single closely related ST2_Pasteur lineage, indicating the entrenchment of this clone in the hospital.

Keywords: Acinetobacter baumannii; GC2 lineage; Malaysia; genome sequencing; multidrug resistant.

Fig. 1.. Antibiotic resistance profiles of the 126 A. baumannii clinical isolates from HSNZ, Terengganu, from 2011 to 2020.

Fig. 2.. The distribution of Pasteur MLST by year of isolation for the 126 A. baumannii clinical isolates from HSNZ, Terengganu. The predominance of ST2_Pasteur (depicted in orange) in each year was evident.

Fig. 3.. Midpoint-rooted ML core-genome phylogenetic tree of the Terengganu A. baumannii isolates together with other Malaysian A. baumannii genomes (as reported by Rao et al. [85] and Muzahid et al. [84] and indicated in the outermost ring of the tree as the towns and states within Malaysia) and several A. baumannii reference genomes representing known major GC types GC1 to GC8 as listed in Data S1.

Fig. 4.. The phenotypic AMR profiles and corresponding carriage of AMR genes of the 126 Terengganu A. baumannii isolates plotted along with the ML core-genome phylogenetic tree of the isolates. Each isolate was also annotated with their corresponding Pasteur and Oxford MLST. The antibiotics are labelled as follows: ampicillin/sulbactam (SAM), piperacillin/tazobactam (TZP), ceftazidime (CAZ), cefepime (FEP), cefotaxime (CTX), ceftriaxone (CRO), doripenem (DOR), imipenem (IPM), meropenem (MEM), gentamicin (CN), tobramycin (TOB), amikacin (AK), doxycycline (DO), minocycline (MH), tetracycline (TE), ciprofloxacin (CIP), levofloxacin (LEV) and trimethoprim/sulfamethoxazole (SXT), polymyxin B (PB) and colistin (CT). The assigned AMR phenotypes are colour coded as light red for resistance, green for intermediate resistance and yellow for susceptible. The genotypic AMRs are sorted by antibiotic classes to which they confer resistance, and the colourless squares represent the absence of AMR genes. UNK indicates unknown.

Fig. 5.. Carriage of potential plasmids in the 126 sequenced Terengganu A. baumannii isolates. The genome sequences were screened for the presence of the Acinetobacter plasmid-encoded replicase genes, which were classified either as the Rep_1 (blue squares), Rep_3 (red squares) or PriCT_1 (purple squares) families and further subtyped as detailed in [28] and [46]. Each isolate was also annotated with their corresponding Pasteur and Oxford STs, and whether they are categorized as MDR isolates.

Fig. 6.. The ML core-genome phylogenetic tree of 126 Terengganu A. baumannii isolates was constructed using FastTree with 1000 bootstraps under the generalized time-reversible model and was then visualized using iTOL (https://itol.embl.de/). The tree was annotated with resistance profile, Oxford MLST, Pasteur MLST, the CPS chromosomal KL and OCL biosynthesis types and the virulence factor genes. The virulence genes are sorted by their virulence factors, and the colourless squares represent the absence of virulence genes. UNK indicates unknown.