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

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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.

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Acknowledgments

We thank Johanne Bettina Garstad and Silje Eltvik Thommasen who performed the PCR tests as a part of their bachelor project, Nina M Clausen and Yngve Ness for testing the MRSA algorithm, and Tone Møller Tannæs for the good discussions.

Special thanks go to Anneke van der Zee and Lieuwe Roorda, Maasstad Hospital, Rotterdam, the Netherlands, for designing and sharing the primer sequences for the orfX-SCCmec PCR. The MRSA reference strains COL, BK2464, and ANS46 were kindly provided by J. Etienne, Lyon, France. Strain WIS was kindly provided by T. Ito, Juntendo, Japan.

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Correspondence to H. S. Tunsjø.

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The study was funded by the Department of Microbiology and Infection Control, Akershus University Hospital.

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The authors declare no conflict of interest.

The study was approved by the regional data protection officer at Akershus University Hospital.

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Tunsjø, H.S., Kalyanasundaram, S., Worren, M.M. et al. High frequency of occupied attB regions in Norwegian Staphylococcus aureus isolates supports a two-step MRSA screening algorithm. Eur J Clin Microbiol Infect Dis 36, 65–74 (2017). https://doi.org/10.1007/s10096-016-2771-0

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  • DOI: https://doi.org/10.1007/s10096-016-2771-0

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