Genomic Investigation Reveals Contaminated Detergent as the Source of an Extended-Spectrum-β-Lactamase-Producing Klebsiella michiganensis Outbreak in a Neonatal Unit

Abstract

Klebsiella species are problematic pathogens in neonatal units and may cause outbreaks, for which the sources of transmission may be challenging to elucidate. We describe the use of whole-genome sequencing (WGS) to investigate environmental sources of transmission during an outbreak of extended-spectrum-β-lactamase (ESBL)-producing Klebsiella michiganensis colonizing neonates. Ceftriaxone-resistant Klebsiella spp. isolated from neonates (or their mothers) and the hospital environment were included. Short-read sequencing (Illumina) and long-read sequencing (MinION; Oxford Nanopore Technologies) were used to confirm species taxonomy, to identify antimicrobial resistance genes, and to determine phylogenetic relationships using single-nucleotide polymorphism profiling. A total of 21 organisms (10 patient-derived isolates and 11 environmental isolates) were sequenced. Standard laboratory methods identified the outbreak strain as an ESBL-producing Klebsiella oxytoca, but taxonomic assignment from WGS data suggested closer identity to Klebsiella michiganensis Strains isolated from multiple detergent-dispensing bottles were either identical or closely related by single-nucleotide polymorphism comparison. Detergent bottles contaminated by K. michiganensis had been used for washing milk expression equipment. No new cases were identified once the detergent bottles were removed. Environmental reservoirs may be an important source in outbreaks of multidrug-resistant organisms. WGS, in conjunction with traditional epidemiological investigation, can be instrumental in revealing routes of transmission and guiding infection control responses.

Keywords: Klebsiella michiganensis; Klebsiella oxytoca; extended-spectrum β-lactamase; outbreak; whole-genome sequencing.

FIG 1

Evolutionary relationship of K. michiganensis isolates. The maximum likelihood phylogenetic tree was built using 38 core genome SNPs, relative to K. michiganensis strain M82255, and was rooted using K. michiganensis strain CAV1374 (not shown). The month of isolation, source (neonatal or environmental), and specific site of isolation are indicated. Branches (black lines) represent the genetic distances between isolates in terms of SNPs. The scale is indicated. The outbreak timeline is presented adjacent to the tree. The timescale is represented at the bottom, with alternate months colored gray and yellow. The black triangle indicates the date on which the outbreak was declared and screening was switched from weekly to every 48 h. Each box in the timeline represents a period of 1 day, starting on 15 September 2017. Black bars represent the admission periods for colonized patients, and red dots represent dates on which samples were isolated (including environmental isolates). Blue shading represents periods in which there was no overlap in patient stays, and green shading represents periods in which multiple colonized patients were present in the SCN at the same time.

FIG 2

Evolutionary relationship of K. michiganensis strains. The minimal spanning tree shows relationships between environmental (red) and patient (blue) samples. Numbers attached to branches represent the numbers of SNPs between genomes. Isolate codes are represented within each node; if >1 code is listed, then the strains were identical at the core genome level.