Setting and background
As a result of continuous recording of newly detected multidrug-resistant bacteria doubled in comparison so previous years of CA-MRSA cases was observed in an 80 bed tertiary care children's hospital in the Eastern area of Switzerland during first semester of 2018. Furthermore, susceptibility test of most of the CA-MRSA cases revealed an efflux-mediated, non-inducible macrolide -streptogramin B antibiotic resistance pattern which further substantiate our observation. Epidemiological outbreak investigations were initiated which prompted the hypothesis that the outbreak was associated with a primary and secondary school in St. Gallen.
Microbial culture and MRSA detection
Copan eSwabs™ (Copan, Brescia, Italy) were used for swabbing and sent to the laboratory immediately after sampling. Enrichment broth (Brain heart infusion (BHI) with 6% NaCl, manufactured in house) was inoculated with 10 ml of the liquid medium and incubated overnight. Of the enriched broth 10 ml was inoculated onto a chromID chromogenic MRSA plate (bioMérieux, Marcy l'Etoile, France) with a WASP instrument (Copan, Brescia, Italy). After incubating the plates for 24 hours in the smart incubators of a WASPLab™ high-resolution images of media plates were inspected using the WASPLab™ WebApp software. Colonies, indicative for MRSA were identified by a software algorithm (“segregation”) and sent to reading and picking for further workup .
Identification was done with a MALDI-ToF instrument using the BDAL 9.0 database (MALDI Biotyper Smart System, Bruker Daltonics, Bremen, Germany) with the colony transfer method (“direct smear”). Colonies, identified as S. aureus were further tested with a PBP2a (penicillin-binding protein 2a, which confers methicillin-resistance) lateral flow immunochromatography assay (Alere Clearview PBP2a Culture Colony Test, Alere Inc. Waltham MA, USA), which confirmed MRSA. A BD™ Phoenix instrument (Becton Dickinson, Sparks, MD, USA) with PMIC-88 cartridge (tailored for the susceptibility testing of staphylococci and other Gram-positive bacteria) was used for susceptibility testing, except vancomycin, which was tested with epsilometer (E-) test (bioMérieux, Marcy l'Etoile, France). Antimicrobial susceptibility testing (AST) data were interpreted according to respective EUCAST guidelines (version 8.0 or 9.0 in 2018 and 2019, AST data not shown). This procedure applies to all isolates processed by the Centre for Laboratory Medicine in St. Gallen. Five isolates were tested elsewhere (i.e. available as laboratory report, only) and the data could not be verified.
Whole Genome Sequencing
DNA was quantified using the Qubit dsDNA BR HS Assay Kit and Qubit fluorometers (Invitrogen, https://www.thermofisher.com). WGS was performed using Illumina MiSeq with the Nextera XTlibrary preparation kit (Illumina Inc., USA), according to the manufacturer’s procedure. Trimming and assembly of raw reads was performed using the Velvet assembler of the SeqSphere software (Ridom, https://www.ridom.de, version 7.7.5 using S.aureus cgMLST v1.3 and S.aureus v1.1). The analysis included MLST, cgMLST (1`861 targets) and detecting resistance/virulence genes (mecA, macrolide resistance genes (ermA, ermC, mphC, msrA) and PVL (LukS-PV and LukF-PV)) by using SeqSphere. Coverage was at least 50-fold. We defined cgMLST clusters as groups of isolates with ≤5 different SNPs between neighboring isolates. To generate phylogenetic SNP trees, we used SeqSphere (Ridom; Münster, Germany) in the pairwise ignore missing values mode and an unweighted pair group method. The WGS data has been submitted to NCBI under the following submission number PRJNA738326. The genome assemblies were compared to a collection of 1861 MRSa isolates mostly originating from Switzerland collected at the University hospital Basel using the SeqSphere software.
The following case definition was used to categorize MRSA cases. First, all culture positive MRSA cases were “suspected cases”. Second, presence of the specific efflux-mediated macrolide resistance (phenotype) was assessed. Cases with an alternative antimicrobial susceptibility testing result were categorized as “non-cluster cases”. "Suspected cases" having efflux-mediated macrolide resistance were "probable cases". In "probable cases" where strains were available, WGS was performed. Finally, if strains were ST5 using cgMLST, cases were "confirmed cases" and considered either as “core cluster” (≤5 single nucleotide polymorphisms (SNPs)) or "cluster" (>5 but <15 SNPs). All cases not belonging to ST5 or ST5 with >15 SNPs were again classified as “non-cluster cases” (see Figure 1).
Identification of risk factors
Risk factors for CA-MRSA were assessed using a questionnaire sent by email and/or mail to the families of confirmed and probable cases. This first questionnaire was sent in spring to summer 2019. Questions covered schooling (nursery, primary and secondary school), contact with the health-care systems (e.g., inpatient care), current, previous, or recurrent infections with MRSA or skin infections of the case and household contacts, profession, workplace, pets, leisure activities including sports, events, school or holiday camps or trips and attendance at the local children’s festival held in June 2018.
Given persistent transmission throughout 18 months (2018 - 2019), a cross-sectional study was initiated. Results of the epidemiological investigation and the first questionnaire supported the hypothesis that the outbreak was linked to a particular primary and secondary school. Accordingly, all students and all staff at the affected school with proven direct contact to the students (i.e. teachers, pre-schooling teachers) were included in the study in November 2019. Clinical examination for skin lesions was performed in all students and staff. Swabs for MRSA (from axilla/inguinal region, nose/throat, wounds) were taken from all individuals with SSTI on clinical examination. These individuals also had to answer a second questionnaire, which was adapted from the previous one in summer 2018. Questions focused on school activities and leisure activities. Furthermore, every fifth student and staff without symptoms of CA-MRSA infection were randomly assigned to the cross-sectional study to receive swabbing and answer the questionnaire.
Continuous variables were assessed by t tests or Mann-Whitney U tests as appropriate. For categorical values, comparison was done by Fisher's exact test. Missing data were not imputed. A two-sided p value < 0.05 was considered as statistically significant. Given the small number of each outcome, only the univariate but not the multivariable analyses are reported. Data analysis was performed with SAS (version 9.3, SAS Institute Inc., Cary, NC, USA).