Infection Control & Hospital Epidemiology
(WGS) can be valuable for investigation of MRSA outbreaks in NICUs, providing better resolution than conventional typing techniques.7–13 In this study, we describe an apparent outbreak of MRSA colonization and/or infection in a NICU over a 3-year period. We compare the results of WGS and PFGE for investigating strain relatedness; we also describe how these data can be used to guide infection prevention and control (IPAC) interventions.
Methods Setting
The NICU at Saint Mary’s Campus, Mayo Clinic Hospital in Rochester, Minnesota, is a 26-bed level IV neonatal unit caring for ~370 infants per year. The unit is divided into 4 rooms, with 4–6 bed spaces in each room plus 4 additional adjoining single-room bed spaces used for patients requiring isolation precautions. The average daily census is 18–20 patients.
Screening
Prior to April 2016, targeted screening for S. aureus colonization with reflexive testing for MRSA if positive was performed on infants who were hospitalized in the NICU for 2 weeks or longer and had a central line in place. A single swab sampling the anterior nares, axilla, and groin, was obtained. Clinical cultures were collected as indicated. If MRSA colonization or infection was identified by either a screening or clinical culture, infants occu- pying adjacent bed spaces were also screened (ie, ring screening). From April 2016 to November 2017, universal weekly screening of all infants was performed. HCWs who had had contact with MRSA-positive infants were identified by clinical documentation and were screened once in the summer or fall of 2016 with swabs of their anterior nares.
Culture collection and processing
Infant swabs were plated on sheep blood agar and Columbia nalidixic acid (CNA) with sheep blood agar plates, incubated at 35°C with CO2,and examined forgrowthat 16–24 hours and, if negative, at 48 hours. Colonies were identified by Gram stain and coagulase testing, or by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). Staphylococcus aureus isolates were identified as MRSA by oxacillin susceptibility testing by agar dilution. HCW swabs were plated on BBL CHROMagar MRSA II (Becton Dickinson, Sparks, MD) and incubated at 35°C in ambient air. Results were reported as positive if characteristic mauve colonies were present after 20–26 hours of incubation. Supplemental testing, including Gram stain and/or coagulase testing, was performed if necessary. Results of screening swabs were reported in the medical record for infants, and the IPAC team was notified of positive results for both infants and HCWs. Environmental sampling was performed by collecting swabs of high-touch surfaces. They were processed as described above for HCW samples. All isolates studied were archived at −80°C in 0.9% sterile saline (Baxter, Deerfield, IL). Only the first MRSA iso- late from each infant and HCW was studied. Agar dilution was used for susceptibility testing to all antimicrobial agents studied.
1413
Contact precautions and decolonization for MRSA-positive infants and HCWs
Infants identified as colonized or infected with MRSA were cared for using contact precautions in single bed space rooms or were cohorted in a single room with multiple bed spaces. Colonized or infected infants were treated with nasal mupirocin twice daily for 5 days and remained in contact precautions for the remaining duration of their hospitalization; they did not undergo further screening. Colonized HCWs were treated with nasal mupirocin twice daily and chlorhexidine baths once daily for 5 days and were rescreened after decolonization to demon- strate MRSA eradication.
Pulsed-field gel electrophoresis
Pulsed-field gel electrophoresis (PFGE) was performed by nor- malizing a broth culture in brain-heart infusion broth to a tur- bidity of 0.58–0.63 using a turbidometer (Dade Behring, Deerfield, IL). The normalized suspension was concentrated and resuspended in 500 μL of EET buffer (100mM EDTA, 10mM EGTA, 10mM TRIS, pH 8.0). A 500 μL volume of agarose was added to each sample to prepare a plug. Sample plugs were subjected to cell lysis, restriction endonuclease digestion (using SmaI), and electrophoresis. Determination of clonal groups was performed as previously described.14 Further details are available in the supplemental content.
Whole-genome sequencing
Archived isolates of MRSA were recultured on trypic soy agar with 5% sheep blood and incubated at 35°C in room air. Iso- lates were passaged once prior to WGS. DNA was extracted from cultured isolates using the Zymo Research Quick-DNA Fungal/Bacterial Miniprep kit (Zymo Research, Irvine, CA) with a modified final elution volume of 200 µL. DNA was quantified with a Quantus fluorometer (Promega, Madison, WI) using the QunatiFluor ONE dsDNA System (Promega). DNA was normalized to 0.5 µM and paired-end (PE), dual- indexed DNA libraries were prepared with a Nextera XT PE library preparation kit (Illumina, San Diego, CA). Sequencing was performed on an Illumina MiSeq with V2 2 × 250-bp chemistry and a maximum of 12 sample libraries multiplexed per flow cell. Raw sequencing reads were processed for adapter and index cleaning using the MiSeq reporter software in real time.
Genome assembly and core-genome multilocus sequence typing
Following delivery of the processed data, read files were imported into SeqSphere+, version 4.0 software (Ridom, Munster, DE). The vendor derived de novo assembly, and the genome assembly and core-genome multilocus sequence typing (cgMLST) pipeline option was exercised in the software suite following default set- tings and an optional cgMLST target threshold of 90%. Minimum spanning trees were generated from the typing data table for visualization. Thresholds of ≤8 (related), 9–29 (possibly related), and ≥30 (unrelated) allelic differences were applied.15 Groupings obtained using WGS typing were qualitatively compared to PFGE groupings.
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