Computational protocol: Environmental surveillance identifies multiple introductions of MRSA CC398 in an Equine Veterinary Hospital in the UK, 2011–2016

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Protocol publication

[…] Presumptive MRSA colonies grown overnight on SBA were used for biochemical identification using gram-positive identification plates (GPID) (TREK Diagnostic Systems Ltd., Cleveland, OH, USA) and to prepare cell lysates for DNA extraction by heating a suspension of cells at 100 °C for 10 minutes. A multiplex PCR assay targeting the femA , nucA and mecA genes was used for confirmation of MRSA status.Molecular typing was performed on a selection of isolates (n = 6) by MLST as previously described. Confirmation of sequence type (ST) 398 by MLST in the isolates, prompted retrospective screening of all the MRSA isolates obtained from the environment (n = 62), SSI (n = 13) and hand plates (n = 6) in the past five years with a CC398-specific PCR. Non-duplicate isolates identified as CC398 were further characterised by staphylococcal chromosomal cassette mec (SCCmec) and spa gene typing. SCCmec typing (type I to type V) was performed according to Zhang et al.. S. aureus protein A (spa) typing was performed as previously described. Amplification was followed by Sanger sequencing to identify sequence variation of the polymorphic region X of the spa gene and spa types were determined using the spaTyper software (http://spatyper.fortinbras.us).Based Upon Repeat Pattern (BURP) was used to determine clonal relatedness from spa repeat regions and cluster spa types (spa CCs) of isolates by using Ridom StaphType (version 2.2.1) software (Ridom GmbH, Würzburg, Germany). The default parameters for BURP analysis were applied as previously described. Interpretation of BURP clusters was performed according to Mellman et al. where a group founder in clusters of at least three different spa types, is described as the spa type with the highest founder score.All PCR-confirmed CC398 MRSA isolates were screened for the presence of virulence genes lukS-PV, lukF-PV which encode the Panton-Valentine leukocidin, for genes associated with biofilm production (bap, icaA, icaD) and antimicrobial resistance genes (ermA, ermB, ermC, tetK, tetM, aacA-aphD), . Positive and negative controls for PCR reactions were included in each assay. […]

Pipeline specifications

Software tools spaTyper, StaphType
Application WGS analysis