Whole-Genome-Sequence-Based Characterization of Extensively Drug-Resistant Acinetobacter baumannii Hospital Outbreak

Abstract

Carbapenem-resistant Acinetobacter baumannii (CRAB) is an important opportunistic pathogen linked to a variety of nosocomial infections and hospital outbreaks worldwide. This study aimed at investigating and characterizing a CRAB outbreak at a large tertiary hospital in Lebanon. A total of 41 isolates were collected and analyzed using pulsed-field gel electrophoresis (PFGE). Whole-genome sequencing (WGS) was performed on all the isolates, and long-read PacBio sequencing was used to generate reference genomes. The multilocus sequence types (MLST), repertoire of resistance genes, and virulence factors were determined from the sequencing data. The plasmid content was analyzed both in silico and using the A. baumannii PCR-based replicon typing (AB-PBRT) method. Genome analysis initially revealed two clones, one carrying bla_OXA-23 on Tn2006 (ST-1305, ST-195, and ST-218) and another carrying bla_OXA-72 on pMAL-1 (ST-502 and ST-2059, a new ST), with the latter having two subclones, as revealed using the Bayesian transmission network. All isolates were extensively drug resistant (XDR). WGS analysis revealed the transmission pathways and demonstrated the diversity of CRAB isolates and mobile genetic elements in this health care setting. Outbreak detection using WGS and immediate implementation of infection control measures contribute to restraining the spread and decreasing mortality.IMPORTANCE Carbapenem-resistant Acinetobacter baumannii (CRAB) has been implicated in hospital outbreaks worldwide. Here, we present a whole-genome-based investigation of an extensively drug-resistant CRAB outbreak rapidly spreading and causing high incidences of mortality at numerous wards of a large tertiary hospital in Lebanon. This is the first study of its kind in the region. Two circulating clones were identified using a combination of molecular typing approaches, short- and long-read sequencing and Bayesian transmission network analysis. One clone carried bla_OXA-23 on Tn2006 (ST-1305, ST-195, and ST-218), and another carried bla_OXA-72 on a pMAL-1 plasmid (ST-502 and ST-2059, a new ST). A pMAL-2 plasmid was circulating between the two clones. The approaches implemented in this study and the obtained findings facilitate the tracking of outbreak scenarios in Lebanon and the region at large.

Keywords: A. baumannii; CRAB; Tn2006; hospital outbreak; pMAL-1.

FIG 1

PFGE dendrogram, isolates’ STs, and patients’ information. PT, pulsotype; M, male; F, female; DTA, deep tracheal aspiration; P. fluid, peritoneal fluid; BW, bronchial wash; abdm, abdomen; umbl, umbilical; +, died; ICU, intensive care unit; ERICU, emergency room intensive care unit; CCU, coronary care unit; PED, pediatrics; NICU, neonatal intensive care unit; ONCO, oncology; PED(OR), pediatrics operation room; MED1, medical floor 1; MED2, medical floor 2.

FIG 2

Maximum-likelihood phylogeny based on SNPs across A. baumannii isolates. (A) Branch lengths are proportional to the number of nucleotide substitutions per site. Bootstrap values are indicated by numbers below branches as well as by branch coloring (green, high confidence levels; red, low confidence levels). Dashed lines connect labels with particular leaf nodes in order to avoid labels to overlap the branches. Classes of antibiotics are given the following markings: A, aminoglycosides; B, β-lactams; Q, quinolones; T, tetracyclines; GM, gentamicin; AK, amikacin; TZP, piperacillin-tazobactam; CAZ, ceftazidime; FEP, cefepime; IMP, imipenem; MEM, meropenem; CIP, ciprofloxacin; LEV, levofloxacin. Dark blue indicates resistant, light blue indicates intermediate susceptibility, and blank indicates sensitive. (B) Unrooted phylograms inferred from SNP data from core genomes of strains belonging to the Tn2006-mediated clone and cluster pMAL-1-mediated clone, respectively. Red circle in the pMAL-1-mediated clone highlights ST-2059 isolates. Bootstrap values are indicated by numbers below branches as well as by branch coloring (green, high confidence levels; red, low confidence levels). Dashed lines connect labels with particular leaf nodes in order to avoid labels to overlap the branches.

FIG 3

Comparative schematic representation of pMAL-1 and its closely related plasmids. pMAL-1 was aligned and compared with the three most closely related plasmids, pA105-2 (GenBank accession no. KR535993.1), p2ABST25 (GenBank accession no. AEPA01000396.1), and pAB0057 (GenBank accession no. NC_011585.1). Genes in pMAL-1 are annotated and colored with the following scheme: repB, blue; repA, light blue; XerC/XerD recombination sites, black; bla_OXA-72, red; ISAba31, yellow; apkA-apkB, purple; tonB, green; sep, dark blue; hypothetical proteins, gray.

FIG 4

Bayesian transmission network analysis of the pMAL-1-mediated clone. (A) Histogram showing the distribution of pairwise genetic distances of the isolates and the relative frequencies of the genetic distances of the isolates of the pMAL-1-mediated clone. (B) Box-and-whisker plots of the estimated dates of infection. The cases are arranged based on how ancestral their core genomes are. (C) Isolates of the pMAL-1-mediated clone and their relative SNP distances from the other isolates of the cluster. At a hamming distance of 20, the isolates were arranged into two apparent subclones based on SNPs shared by some isolates in their core genomes introduced during the progression of the outbreak. (D) Graph showing the density of the SNPs relative to their positions in the genomes of the sequenced isolates: A (bp 784121), BapA prefix-like domain-containing protein; B (bp 1521949), DUF2750 domain-containing protein FDN01_10955; C (bp 2162660), stress-induced protein; D (bp 2971361), putative pilus assembly protein FilE.