Genomic surveillance, characterization and intervention of a polymicrobial multidrug-resistant outbreak in critical care

Abstract

Background. Infections caused by carbapenem-resistant Acinetobacter baumannii (CR-Ab) have become increasingly prevalent in clinical settings and often result in significant morbidity and mortality due to their multidrug resistance (MDR). Here we present an integrated whole-genome sequencing (WGS) response to a persistent CR-Ab outbreak in a Brisbane hospital between 2016-2018.Methods. A. baumannii, Klebsiella pneumoniae, Serratia marcescens and Pseudomonas aeruginosa isolates were sequenced using the Illumina platform primarily to establish isolate relationships based on core-genome SNPs, MLST and antimicrobial resistance gene profiles. Representative isolates were selected for PacBio sequencing. Environmental metagenomic sequencing with Illumina was used to detect persistence of the outbreak strain in the hospital.Results. In response to a suspected polymicrobial outbreak between May to August of 2016, 28 CR-Ab (and 21 other MDR Gram-negative bacilli) were collected from Intensive Care Unit and Burns Unit patients and sent for WGS with a 7 day turn-around time in clinical reporting. All CR-Ab were sequence type (ST)1050 (Pasteur ST2) and within 10 SNPs apart, indicative of an ongoing outbreak, and distinct from historical CR-Ab isolates from the same hospital. Possible transmission routes between patients were identified on the basis of CR-Ab and K. pneumoniae SNP profiles. Continued WGS surveillance between 2016 to 2018 enabled suspected outbreak cases to be refuted, but a resurgence of the outbreak CR-Ab mid-2018 in the Burns Unit prompted additional screening. Environmental metagenomic sequencing identified the hospital plumbing as a potential source. Replacement of the plumbing and routine drain maintenance resulted in rapid resolution of the secondary outbreak and significant risk reduction with no discernable transmission in the Burns Unit since.Conclusion. We implemented a comprehensive WGS and metagenomics investigation that resolved a persistent CR-Ab outbreak in a critical care setting.

Keywords: Acinetobacter baumannii; CR-Ab; WGS; burns ward; carbapenem resistance; genomics; intensive care unit; metagenomics; surveillance; whole-genome sequencing.

Fig. 1.

Patient relationship matrix describing 2016 outbreak of CR-Ab: (a) Each circle represents a patient, where the size of the circle correlates to the number of isolates from that patient. Colours correspond to bacterial species. Straight lines connecting circles represent patients with identical isolates (with the colour of the line indicating the specific species) at the core-genome level (and as such directionality of transmission cannot be inferred). Lines with arrows (also coloured by species) represent predicted direction of transmission based on the accumulation of SNPs between patients’ isolates. Circular arrows represent changes in individual patient’s isolates, (b) timeline of patient samples, as well as location and surgery dates.

Fig. 2.

Large ~40 kb tandem duplication found in MS14413: Duplication of part of the capsule (k) region in the MS14413 complete genome (top line), resulting in three chromosomal copies of Tn2006 (third copy at alternate locus). This duplication appears to have arisen in some of the index patient isolates, but not other isolates involved in the outbreak (e.g. MS14407 concatenated draft genome, central line; vertical double black lines represent contig break in the draft assembly, presumed to be caused by the same IS as in MS14413). The wzy gene in the capsule region was found to be interrupted by an ISAba125 element, however a secondary wzy gene was identified at the start of the capsule region. Neither the ISAba125 insertion or secondary wzy gene is found in the KL12 capsule locus of A. baumannii strain D36 (bottom line).

Fig. 3.

Ongoing CR-Ab surveillance from 2016 to 2018: (a) timeline of CR-Ab cases and dates of environmental swabbing between 2016–2018. (b) Relationship matrix of all CR-Ab isolates related to the initial outbreak. Col-R=predicted colistin resistance via mutation in pmrB. Isolates within the same circle are identical at the core genome. Branches represent 1 SNP difference (except where specified). Isolates from the original 2016 outbreak are in yellow. Purple isolates were collected in late 2016–2017. Isolates in blue were collected in 2018. Isolate M88825 was isolated from an Antechamber environment in 2018 and found to be identical at the core SNP level to M7120, isolated in August 2017.

Fig. 4.

Burns bath 3 floor trap and metagenomic read abundance profiles: (a) an example of the biomass uncovered under the floor trap in a Burns Unit bathroom. Areas of high biomass (such as this one) were targeted for environmental screening. (b) Each column shows the relative abundance of paired-end reads for each environmental sample that were classified at a bacterial genus level by comparing against a database of bacterial genomes from RefSeq. Only bacterial genera with a relative abundance >0.5% are shown as distinct. Genera with an abundance of <0.5% are grouped together as ‘Other’ (grey). Boxes outlined in black represent abundance of ‘ Acinetobacter ’.

Fig. 5.

Clustering of MAGs with outbreak strains: Mid-point rooted core genome SNP phylogenetic tree contextualizing the metagenome assembled genomes (MAGs) with de novo assemblies of the outbreak strains and publicly available complete A. baumannii genomes (yellow) showing clustering of the MAGs (blue) within the outbreak clade (pink).