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.


Sampling and extraction of DNA
This dataset comprises two distinct sets of Staphylococcus aureus samples.The first set consists of two methicillin-sensitive ST97 S. aureus cases collected in May 2022 and February 2023.These initial cases, represented by samples sa220609barcode87 and sa230215barcode55, were obtained from eye swabs using the standard bacterial swab technique in Amies transport medium.Subsequently, these swabs were cultured on 5% sheep blood agar at 37°C for 48 hours.For methicillin-resistant S. aureus (MRSA) screening, swabs were collected from various sites, including the nose, axilla, umbilicus, groin, and any open wounds.These swabs underwent culture for 18-24 hours on the CHROMagar™ MRSA (St-Denis, France) and enrichment in 7% salt broth for 18 hours, with subculture onto CHROMagar™ MRSA for a further 18-24 hours.CHROMagar MRSA is a selective and differential agar, which inhibits the growth of MSSA and most other bacteria.Mauvecoloured colonies were followed up as possible MRSA.Susceptibility to multiple other agents was determined for isolates confirmed as MRSA, using the Vitek II instrument (bioMerieux, Marcy-l'Etoile, France) and the AST-P656 card.In both groups of samples, the primary samples were promptly processed upon arrival at Awanui Laboratories Wellington.
Gram-positive DNA extraction involves suspending a 10µL loop of bacteria in 300uL of phosphate-buffered saline (PBS) and freezing at −20°C for at least 24 hours.The thawed solution is vortexed for one minute with a small quantity of 0.1mm zirconia/silica beads (dnature, Gisborne, NZ; SKU 11079101Z) and then centrifuged at ~12,000×g for 30 seconds at room temperature, with the supernatant used for sequencing without further clean-up.

Quality control for the nanopore sequence data
To perform taxonomic profiling and detect S. aureus in the raw nanopore sequence data, we used Kraken v2.1.3 [1] with default parameters and an NCBI Reference Sequence (RefSeq) database [2], Standard (https://benlangmead.github.io/aws-indexes/k2,accessed on 15 September 2023).The database contained references for archaea, bacteria, human, viruses, plasmids, and the 'UniVec core' subset of the UniVec database (a database of vector, adaptor, linker, and primer sequences).NanoStat v1.6.0 from Nanopack v1.6.0 [3] was used to perform an initial quality assessment on the raw nanopore reads.Additionally, NanoQC v0.9.4 from NanoPack was used to assess the overall quality of the sequencing data.NanoFilt v2.8.0, also from NanoPack, was used for read trimming.Initially, 50 nucleotides were trimmed from the start and end of each read to remove low-quality regions from the reads.Subsequently, NanoFilt was used again to filter out reads with a quality score below either Q10, Q12, or Q15.Any remaining reads that were less than 100 base pairs in length were also removed.
In the absence of an available ST97 reference genome representing New Zealand S. aureus strains, we selected 23MR1425, a clinical isolate collected from a neonatal eye swab, as our reference genome (index case on the WRH NICU identified in June 2023).The assembly for 23MR1425 underwent five rounds of additional polishing by mapping the corresponding Illumina reads to each contig using the Burrows-Wheeler Aligner (BWA) v0.7.17 [11], and then correcting SNVs and insertions and deletions (INDELs) with Pilon v1.24 [12].

Dataset curation of publicly available data
In addition to the S. aureus ST97 genomes sequenced in this study (Supplementary Materials, Table S1), six complete ST97 genome assemblies were downloaded from the NCBI Assembly database.Another 468 publicly available draft S. aureus genome assemblies were downloaded using the PathogenWatch platform (https://pathogen.watch/,accessed on 25 August 2023).Furthermore, this study included an additional 18 publicly available bovine-derived genomes [17] to this dataset, including 16 ST97 strains and one each of ST6164 and ST6162 (both clonal complex 97).

Illumina library construction and next-generation sequencing
As part of the Institute of Environmental Science and Research (ESR) national staphylococcal surveillance surveys, Staphylococcus cultures are plated on blood agar and incubated at 35°C with 5% carbon dioxide (CO2) for 18 hours.Following incubation, the cultures are examined for viability and purity.A subculture of a single colony pick is then transferred to another blood agar plate and incubated at the same conditions for 18 hours.
The heat-killed cell suspensions from an overnight culture of a single colony pick were extracted using the chemagic™ 360 instrument (PerkinElmer Inc., Waltham, Massachusetts, United States).The DNA library was created using the PlexWell Library Preparation kit (seqWell™, Boston, Massachusetts, United States), and sequenced as 2×151 bp paired-end reads on the NextSeq 550 platform using V2.5 chemistry (Illumina Inc., San Diego, California, United States) at ESR (Kenepuru, Porirua, New Zealand).
For the Livestock Improvement Corporation (LIC) (Newstead, New Zealand), S. aureus isolates were cultured from bulk tank milk samples collected as part of a nationwide bulk tank trial for LIC Milkomics®, bulk tank milk samples undergoing animal health testing and from individual quarter foremilk samples.Ethics approval for the bulk tank milk sampling was not required, however ethics approval for the individual quarter foremilk samples was obtained from the Ruakura Animal Ethics Committee, Hamilton/Kirikiriroa, North Island/Te Ikaa-Māui, (Approval Numbers: 14240 and 15575).The bulk tank milk samples for the Milkomics® trial were collected in a 35mL pottle containing 0.1mL of bronopol preservative.Pottles were delivered to the LIC Animal Health Laboratory located in Hamilton/Kirikiriroa, North Island/Te Ika-a-Māui by LIC herd testing.
The bulk tank milk samples for animal health testing were collected from the animal health laboratory and a subsample was used for bacteriology.100µl of bulk tank milk was initially spread plated onto Esculin Sheep Blood Agar plates using a Copan spreader.These plates were then incubated at 37°C for 48 hours.The individual quarter foremilk samples were collected from selected cows based on elevated somatic cell counts.
The milk samples were collected using standard aseptic technique by LIC technicians (North Island/Te Ika-a-Māui) and were taken back to the LIC Animal Health Laboratory.For South Island/Te Waka-o-Māui farms accredited veterinarians collected samples and the pottles were couriered chilled at 4°C to the LIC Animal Health Laboratory.10µl of foremilk was spread on Esculin Sheep Blood Agar plate using a Copan spreader.
The plates were incubated for 48 hours at 37°C.Presumptive S. aureus isolates for bulk tank/foremilk quarter samples were identified based on the presence of incomplete or complete haemolysis zones, positive catalase reactions, and positive rabbit-plasma coagulase reactions.To ensure the purity of the positive S. aureus isolates, strains were sub-cultured on Esculin Sheep Blood Agar plates twice.After confirming their purity, two to three colonies from each isolate were transferred into Laboratoire de Santé Publique du Québec (LSPQ) preservation medium and stored at −20°C for future use.
Thirteen human S. aureus isolates were obtained from the Pathlab on nutrient agar slopes.The Pathlab identified these isolates as methicillin-susceptible S. aureus, isolated from the Waikato, Lakes, and Bay of Plenty/Te Moana-a-Toi regions and no identifying information was obtained, therefore no ethics approval was required.Human isolates were prepared in the same manner as the bovine isolates with storage in LSPQ media.
For genomic DNA extraction, samples were cultivated from LSPQ stocks.Genomic DNA extraction was performed using a custom BioSprint® 96 DNA kit (Qiagen), and a Kingfisher machine (ThermoFisher, New Zealand).Samples were eluted into 100µl of elution buffer.
Following extraction, DNA concentrations were assessed for all samples, including both positive and negative controls, using a Victor Nivo in a 96-well plate format using the manufacturer's protocol for the 1dsDNA Broad Range Assay Kit (ThermoFisher, New Zealand).All isolates exhibited DNA concentrations exceeding 100ng/µL, while negative controls returned negative results.Subsequently, DNA concentrations were normalised to approximately 15ng/µL before proceeding with Illumina sequencing.The DNA was then stored at −20°C.The S. aureus genomes were sequenced in-house at LIC on an Illumina Novaseq 6000 using an S1 flow cell and utilising XP loading.For all 140 isolates, 150 bp paired-end Illumina sequencing reads were generated.The DNA library was created using the Illumina DNA preparation kit with Integrated DNA Technologies (IDT) for Illumina UD Indexes (Plate A/Set 1, Plate B/Set 2, Plate C/Set 3, Plate D/Set 4), and sequenced as 2×151 bp paired-end reads on the Novaseq 6000 on an S1 flow cell utilising XP loading (Illumina Inc., San Diego, California, United States) at LIC (Newstead, New Zealand).[23], BWA v0.7.17 [11]; and Pilon v1.24 [12].Shovill was used with parameters set to: (i) estimate the genome size to 2.8 Mb; (ii) remove contiguous sequences (contigs) with a sequence coverage below 20-fold; and (iii) enable single-cell mode.Assembly metrics were assessed using QUAST v5.0.2 [24].The quality metrics were assessed and are outlined in the Supplementary Materials (Table S8).