Setting and routine microbiology methods regarding MRSA
The Elisabeth-TweeSteden hospital, Tilburg, the Netherlands is a teaching hospital with 796 beds. Around 85 new cases of MRSA carriage or infection are identified each year. Upon hospital admission, all patients are screened for risk factors for MRSA carriage using a questionnaire. Such risk factors are recent hospital admission abroad, professional contact with livestock, intensive contact with a MRSA carrier or a stay in a refugee center in the last two months . In case of a high or intermediate risk, swabs are taken to test for MRSA carriage . This screening is part of the ‘search and destroy’ policy in the Netherlands and is followed by strict isolation and treatment of MRSA carriers [11, 12].
Routine microbiology methods regarding MRSA
For MRSA carriage screening swabs of the anterior nares, throat, perineum and, if present, catheters, drains and cutaneous lesions were collected using eSwab medium (Copan, Murrieta, USA) . The swabs were inoculated on a chromogenic MRSA2 Brilliance agar (Oxoid Ltd., Basingstoke, UK), on which MRSA isolates appear as blue colonies after overnight incubation at 35 ± 1 °C, and on a blood agar plate as growth control. The remaining eSwab medium was added to Mueller Hinton Broth (BD Diagnostics, Sparks, USA) supplemented with 6.5% sodium chloride. After overnight incubation at 35 ± 1 °C, the broth was inoculated on a chromogenic MRSA2 Brilliance agar. Species determination of presumptive MRSA colonies was performed by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (Bruker Daltonics, Leipzig Germany). Antibiotic susceptibility testing was performed of S. aureus isolates using either BD Phoenix 100 system (BD Diagnostics, Sparks, USA) or disc diffusion (BD Diagnostics, Sparks, USA) according to EUCAST . An in-house real-time PCR was performed on isolates with a cefoxitin MIC values > 4 mg/L or cefoxitin (30 μg) disc diffusion diameter < 22 mm to confirm the MRSA identification, detecting the Sa442 DNA fragment , S. aureus nuclease (nuc) , Panton-Valentine leukocidine (PVL) , and methicillin resistance genes MecA and MecC [17, 18]. Additionally, in selected samples (e.g., in case of limited patient isolation capacity) direct molecular screening for MRSA presence can be performed using the Xpert® MRSA NxG detection kit (Cepheid, Sunnyvale, USA). For each patient where MRSA was cultured, the isolate was sent to the National Institute for Public Health and the Environment (RIVM) for further genotyping by MLVA as described by Schouls et al. .
Source and extent of laboratory contamination
Source of laboratory contamination
Laboratory staff members were screened for MRSA carriage by sampling of the anterior nares, throat and perineum. These samples were cultured as described above.
Extend of laboratory contamination
The laboratory data system was searched for all MRSA isolates cultured in the Elisabeth-TweeSteden hospital from January 2008 until May 2019 with the same MLVA-type as the index MRSA isolate. For each of the detected MRSA isolates with an identical MLVA-type, the likelihood of (laboratory) contamination (likely or unlikely) was determined. Contamination with a MRSA isolate was deemed likely if the MRSA isolate was only cultured once and not in any other sample of the same patient.
Whole genome sequencing (WGS) and wgMLST
The MRSA index isolate, the MRSA isolates from the laboratory staff members, the MRSA isolates detected in the laboratory data system and the control strain ATCC43300 were selected for WGS. WGS was performed using Nextera XT chemistry on a Miseq sequencer (Illumina, San Diego, CA, USA). After error-correction and de novo genome assembly on CLC genomics workbench 20.0.4 (Qiagen, Germantown, MD, USA), the number of allelic differences between the MRSA isolates was determined using the wgMLST tools of Ridom SeqSphere + version 7.7.5 (Ridom GmbH, Munich, Germany). A total of 2574 alleles were included in the pairwise comparison, in which missing values were ignored. For data visualization, a neighbor-joining tree was created. A maximum allelic difference of 24 alleles was used to identify clusters .