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

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.

Quality control and de novo assembly of Illumina WGS data and comparative genome 25 analysis were carried out as described in the main document. Raw reads were analysed 26 using Nullarbor (https://github.com/tseemann/nullarbor) to determine MLST,27 antibiotic resistance gene profile, and core SNP phylogeny using species-specific 28 reference sequences. Closest publicly available complete genomes were chosen as 29 reference sequences where available (Acinetobacter baumannii Global Clone (GC) 2 30 now reassigned as E. hormaechei), an ST90 strain isolated from a burns patient at the 35 RBWH ICU in 2015. Antibiotic resistance gene content and MLST was further checked 36 using srst2[1] against the ARG-ANNOT [2] database and the Oxford MLST scheme [3], 37 respectively. Plasmid Inc Typing was done based on the relaxase gene as described by 38 Compain et al. [4]. 39 40 SNP differences between strains were determined using Nesoni 41 (https://github.com/Victorian-Bioinformatics-Consortium/nesoni) and evolutionary 42 relationships were determined shown as phylograms or Eburst-style matrices in which 43 nodes of identical isolates were separated by branches representing one or more core 44 SNP differences radiating from a founder (index) node. This format provided 45 consistency across reports, enabling a progressive expansion of the display figure from 46 a common anchor as each WGS batch was reported, in contrast to phylograms where 47 topology and isolate order could change substantially as the data was updated. For 48 in August 2016 and forms the anchor to Figure 3B which shows all ST1050 CR-Ab in 50 the study. 51 Methodologies used for subsequent reports (Nov 4 2016, Mar 9 2017, Jun 20 2017, Oct 52 10 2017) were essentially the same except that the concatenated draft genome of 53 ST1050 CR-Ab MS8436 or MS14413 was used as reference sequences, Abricate (v0. 6 PacBio genomes were assembled using Canu [5] v1.3 and manually closed using 77 Artemis [6]. A large duplicated region of ~40 kb was identified in the CR-Ab isolate 78 and resolved using read-mapping and PCR at unique borders of the duplication (see 79 below). The SMRT Analysis suite (v7.0.1.66975) was used to generate methylated 80 motif summaries and polished assemblies using the PacBio reads. Indels were further 81 corrected with Pilon [7] v1.22 using the trimmed Illumina reads mapped to the assembly 82 using BWA [8] v0.7.16a-r1181. Complete PacBio genomes were annotated using 83 Prokka [9] v1.12-beta. Insertion sequences were identified using ISSaga [10].  Table 2). The final P. aeruginosa isolate from the index patient 131 (MS14412) was found to be more resistant to carbapenems, likely due to a nonsense 132 mutation in the outer membrane porin oprD, as well as a non-conservative amino acid 133 change in the response regulator parR. Initial S. marcescens isolates appeared to only 134 carry aac(6')-Ic, blaSRT-2, oqxB, and qnrE. However, later acquisition of an IncHI2 135 plasmid in two S. marcescens isolates was associated with large number of additional 136 resistance genes, including the ESBL blaCTX-M-15 (Supplementary Table 3      This report is for research use only -methods described here are not NATA accredited.

Preliminary report: Genome sequencing of Carbapenem-resistant Acinetobacter baumannii (CRAB) and companion isolates from RBWH (version 1)
Summary: Genome sequencing of 18 clinical isolates (11 carbapenem-resistant Acinetobacter baumannii, 6 K. pneumoniae and 1 carbapenem-resistant Pseudomonas aeruginosa) was carried out at The University of Queensland (UQ). These were cultured from patients at RBWH, 6 involved in the current suspected CRAB outbreak and 3 identified earlier in 2016 as CRAB-colonised or infected. Two historical CRAB strains from 2006 were also sequenced for comparison.

Two separate introductions of CRAB into the ICU / Burns Unit have occurred in 2016.
The first in March-April involved 3 confirmed cases infected or colonized with closely related ST930 strains carrying an OXA-134 carbapenemase. The more recent introduction in May-June involved a divergent ST1050 strain belonging to the International Clone 2 (IC2), and carried OXA-23 carbapenemase. This has been confirmed by genomics in 5 patients from the current outbreak during May-June 2016, although a further 3 cases remain to be analysed. These current strains tightly cluster by single nucleotide polymorphism (SNP) profiling, confirming transmission between patients. To date, no CRAB strains have yet been identified from the environmental sampling. All 2016 clusters were different from the 2006 outbreak strains, which were ST208 with OXA-23.
Simultaneously there has been evidence of transmission between 4 patients in the current outbreak of an ESBL (CTX-M-15)-producing Klebsiella pneumoniae which are also closely related (all are ST515). There was no evidence of inter-species transmission of carbapenemase genes. The single carbapenem-resistant Pseudomonas aeruginosa isolated in blood from one patient did not carry any acquired carbapenemase genes of significance.
This preliminary report will be updated with further analysis and results following additional sequencing of strains delivered to UQ after preparation of the first batch of samples.
This report is for research use only -methods described here are not NATA accredited. This report is for research use only -methods described here are not NATA accredited. The strains all carry between 13-14 acquired resistance genes (see Table 2). The May-June cluster strains and the 2006 strains carried the carbapenemase OXA-23 (a carbapenemase gene commonly seen in CR-A. baumannii 2,3 ), whereas these were absent in the March-April cluster. The March-April strains carried the OXA-134 carbapenemase, which is usually acquired from Acinetobacter lwoffii, 4 as well as an IMP-26 carbapenemase -this is of significance, as the IMP gene appears closely related to the IMP-4-like carbapenemase commonly seen in Queensland Enterobacter strains. 5 This will undergo additional genomic investigation. All A. baumannii possess various OXA-51-like beta-lactamases, which may confer carbapenem resistance if highly expressed. 2 All A.
baumanii carry an ampC beta-lactamase, ADC-25, which may be expressed at high level by the addition of an insertion sequence (ISAba1) in proximity to the gene, conferring resistance to cephalosporins (and will be sought during ongoing analysis of the data). 6 It is also notable that recent strains carried the armA methylase gene, which confers broad aminoglycoside resistance (including amikacin), which conforms to the high MICs observed for aminoglycosides in many of these strains.
All strains also carried additional resistance genes for antibiotic classes such as sulphonamides, chloramphenicol and macrolides/lincosamides. The March-April 2016 strains also possessed resistance genes for folate inhibitors (such as trimethoprim) and rifampicin. In contrast to the 2006, recent 2016 isolates have no resistance genes to tetracyclines (also reflected in the MIC data).
Fluoroquinolone resistance mutations are not readily detected by most automated resistance gene annotation pipelines, and will require some additional data analysis to be confirmed.
This report is for research use only -methods described here are not NATA accredited.

K. pneumoniae (ESBL-producer)
All the K. pneumoniae strains identified as companion isolates from CRAB infected or colonised patients were also found to be closely related, being all of the same sequence type (ST515) and were similar by SNP profiling (details to follow). All carried 15 identical resistance genes, including the common CTX-M-15 type ESBL, as well as OXA-1, TEM-1 and SHV-1 (as always seen in K. pneumoniae) narrow spectrum beta-lactamases. However, if these latter genes are highly expressed, they may further contribute to broad-spectrum beta-lactam resistance.
This confirms that transmission of this significant pathogen between patients was likely to have occurred in parallel to the CRAB outbreak.