High frequency of occupied attB regions in Norwegian Staphylococcus aureus isolates supports a two-step MRSA screening algorithm

Abstract

Rapid nucleic acid amplification tests for methicillin-resistant Staphylococcus aureus (MRSA) diagnostics commonly target the mec resistance gene, genes specific for S. aureus, and the integration site for the SCCmec resistance cassette, orfX. Due to poor specificity when these target genes are used individually, additional culture is required to verify positive results. The combination of these targets is useful, but the optimal algorithm may depend on the presence of the genetic markers in S. aureus isolates, as well as the prevalence of MRSA in a population. The aim of the present study was to identify a rapid, low-cost, and functional screening algorithm in order to reduce the response time for MRSA diagnostics. An in-house orfX-SCCmec polymerase chain reaction (PCR) assay was established and evaluated. The results were compared with an existing mec/nuc PCR assay and traditional culture. Methicillin-sensitive S. aureus (MSSA) that tested false-positive in the orfX-SCCmec PCR assay were further investigated with full genome sequencing using the Ion PGM™ System to verify results and causality. Based on these data, a two-step screening algorithm with initial mec/nuc PCR followed by orfX-SCCmec PCR on positive samples was suggested and tested on 1443 patient samples. 22.5 % of MSSA isolates tested false-positive with the orfX-SCCmec PCR. Full genome sequencing of these isolates identified genetic variation in the attB region of S. aureus, including empty cassette variants and non-mec SCC. The suggested two-step MRSA screening algorithm allowed us to report MRSA results for 95.6 % of all samples and 99 % of MRSA-negative samples after one day.

Updating Molecular Diagnostics for Detecting Methicillin-Susceptible and Methicillin-Resistant Staphylococcus aureus Isolates in Blood Culture Bottles

“…Although molecular diagnostic tests can provide rapid answers to guide therapeutic decisions for positive blood cultures that contain Gram-positive cocci in clusters, S. aureus strains containing a variety of genetic variations, such as insertions, deletions, and mutations within target sequences, can affect the accuracy of results (19, 20, 22–25). In this study, we noted a diverse set of genetic insertions leading to an MRSA-negative, S.…”

“…aureus-positive result with Xpert MRSA/S BC 2013 before the new algorithms were introduced. The first three cases were isolates of MRSA in which the orfX -SCC mec junction sequence was altered by insertions of additional genetic elements using the same attachment site as SCC mec to integrate in orfX , as previously described (16, 23). This prevented the formation of PCR products.…”

Challenges in the identification of methicillin‐resistant Staphylococcus argenteus by routine diagnostics

“…Whole genome sequencing (WGS) of the three isolates was performed using the Ion PGM™ System (Thermo Fisher Scientific, Waltham, MA, USA) as previously described . The sequencing data were quality checked, trimmed, and aligned to the S. argenteus reference strain NZ_CP015758.1 as previously described .…”

“…Whole genome sequencing (WGS) of the three isolates was performed using the Ion PGM™ System (Thermo Fisher Scientific, Waltham, MA, USA) as previously described . The sequencing data were quality checked, trimmed, and aligned to the S. argenteus reference strain NZ_CP015758.1 as previously described . Fasta and fastq files were submitted to the Centre for Genomic Epidemiology (CGE) (http://www.genomicepidemiology.org/) for species detection, multilocus sequence type identification (MLST), and the detection of plasmids, resistance, and virulence genes (Table ).…”

Introduction of a hydrolysis probe PCR assay for high-throughput screening of methicillin-resistant Staphylococcus aureus with the ability to include or exclude detection of Staphylococcus argenteus

“…aureus lacking the antibiotic resistance gene mecA [ 7 , 8 ]. Commercial PCR systems utilizing the detection of SCC mec are available yet expensive, while in-house assays detecting SCC mec require frequent oligonucleotide updates by the user due to the polymorphism within the cassette [ 9 ].…”