Rapid identification and subsequent contextualization of an outbreak of methicillin-resistant Staphylococcus aureus in a neonatal intensive care unit using nanopore sequencing

Abstract

Outbreaks of methicillin-resistant Staphylococcus aureus (MRSA) are well described in the neonatal intensive care unit (NICU) setting. Genomics has revolutionized the investigation of such outbreaks; however, to date, this has largely been completed retrospectively and has typically relied on short-read platforms. In 2022, our laboratory established a prospective genomic surveillance system using Oxford Nanopore Technologies sequencing for rapid outbreak detection. Herein, using this system, we describe the detection and control of an outbreak of sequence-type (ST)97 MRSA in our NICU. The outbreak was identified 13 days after the first MRSA-positive culture and at a point where there were only two known cases. Ward screening rapidly defined the extent of the outbreak, with six other infants found to be colonized. There was minimal transmission once the outbreak had been detected and appropriate infection control measures had been instituted; only two further ST97 cases were detected, along with three unrelated non-ST97 MRSA cases. To contextualize the outbreak, core-genome single-nucleotide variants were identified for phylogenetic analysis after de novo assembly of nanopore data. Comparisons with global (n=45) and national surveillance (n=35) ST97 genomes revealed the stepwise evolution of methicillin resistance within this ST97 subset. A distinct cluster comprising nine of the ten ST97-IVa genomes from the NICU was identified, with strains from 2020 to 2022 national surveillance serving as outgroups to this cluster. One ST97-IVa genome presumed to be part of the outbreak formed an outgroup and was retrospectively excluded. A second phylogeny was created using Illumina sequencing, which considerably reduced the branch lengths of the NICU isolates on the phylogenetic tree. However, the overall tree topology and conclusions were unchanged, with the exception of the NICU outbreak cluster, where differences in branch lengths were observed. This analysis demonstrated the ability of a nanopore-only prospective genomic surveillance system to rapidly identify and contextualize an outbreak of MRSA in a NICU.

Keywords: antibiotic resistance; genomic surveillance; infection control; outbreak detection; phylogenetic analysis.

Fig. 1.. Line chart of neonatal intensive care unit MRSA ST97 outbreak. Days are displayed on the x-axis, with the day the first isolate was collected denoted as day 0 (actual dates have been omitted for patient privacy). Each horizontal shaded bar represents the days spent by the numbered infant in the room as indicated on the y-axis. Infants with non-ST97 MRSA are shown in lighter shaded bars and denoted with letters instead of numbers. Infant 7 was ST97 but was excluded from the outbreak based on the phylogenetic analysis, so is also shown in light grey.

Fig. 2.. Maximum-likelihood phylogeny of S. aureus ST97. The phylogeny was inferred from 28 634 core-genome single-nucleotide variants (SNVs) from 679 assembled genomes. SNVs were derived from a core-genome alignment of 2 087 573 bp and are called against the chromosome of sample 23MR1425. The phylogenetic tree is rooted at the midpoint, which corresponds to the actual root by S. aureus ST834 strain 70017 (SRA accession: DRR291698), which has been omitted for visualization.

Fig. 3.. Maximum-parsimony phylogeny of a subset of Clade 1.1 S. aureus ST97 isolates. (a) The phylogeny is based on nanopore data for the neonatal intensive care unit (NICU) isolates (denoted by ONT in taxon labels). The phylogeny was inferred from 4797 core-genome single-nucleotide variants (SNVs) from 103 genomes. SNVs were derived from a core-genome alignment of ~2 560 000 bp and were called against the chromosome of sample 23MR1425. The consistency index for the tree was 0.96. (b) The phylogeny is based on Illumina data for the NICU isolates. The phylogeny was inferred from 4651 core-genome SNVs from 103 genomes. SNVs were derived from a core-genome alignment of ~2 602 000 bp and are called against the chromosome of sample 23MR1425. The genome for sample sa220609barcode87 (*) represents nanopore-only sequence data. The consistency index for the tree is 0.99. SNV density filtering in SPANDx (excluded regions with three or more SNVs in a 10 bp window). Both phylogenetic trees were rooted according to the ERR4911723 outgroup. Bootstrap values >80 % (1000 replicates) are shown.

Fig. 4.. Evolutionary reconstruction of a subset of Clade 1.1 S. aureus ST97 isolates. A time-calibrated maximum clade credibility tree was inferred from 4189 core-genome single-nucleotide variants (SNVs) from 97 ST97 genomes. SNVs were derived from a core-genome alignment of ~2 605 600 bp and were called against the chromosome of sample 23MR1425 (bold). SNV density filtering in SPANDx (excluded regions with three or more SNVs in a 10 bp window). The x-axis represents the emergence time estimates.