Unusual Resistance Patterns Related to Alterations in the Erm(A) Attenuator In

Supplementary Information

Methods: Preparation of MRSA and non-MRSA mixtures and analyses on GeneOhm and Xpert

Twenty-onemixtures of MRSA/non-MRSA,and 8 mixtures with only non-MRSA strains were prepared followingCLSI guidelines for quality-control exercises (1). The two MRSA strains used in mixtures were a hospital-acquired strain with SCCmec type III (strain 8, Supplementary Table 1) and a community-acquired strain with SCCmecIV (strain 17). Non-MRSAutilized were MRCoNS (n = 2, S. epidermidis and S. sciuri), enterococci (n = 2, E. faecalis and E. faecium), and one strain each of MSSA, E. coli, K. pneumoniae, Acinetobacter spp., and P. aeruginosa.MRSA-containing mixtures were prepared by mixing either one of the 2 MRSA strain suspensions in brain heart infusion broth at 106 cfu/ml with 2 or 3 non-MRSA strain suspensions at 107 or 106 cfu/ml concentrations. Each of these MRSA-containing ‘parent’ mixtures were further diluted serially (101 to 106-fold) to obtain 105 to 100 cfu/ml concentrations of MRSA.Similar to the MRSA pure strain samples, MRSA-containing mixtures were also tested on the two assays from 100 to 105 cfu/ml MRSA concentrations until a positive result was obtained. The 8 mixtures that did not contain MRSA were tested at a single high concentration and were prepared by mixing one each of MRCoNS (105 cfu/ml), MSSA (106 cfu/ml), and a non-staphylococcalstrain(106 cfu/ml) suspension. Non-MRSA mixtures showing false-positive results on either molecular assay were further tested on 7 serial dilutions.

Supplementary Table 1: Characteristics of MRSA and non-MRSA strains tested in the study. *, Clone designation is according to refs(2,3).NA, non applicable; NT, non-typable; ND, not determined.

Supplementary Table 2: A comparison of CTvalues obtained on GeneOhm and Xpertfor select MRSA and for falsely detected non-MRSAstrains.Pure MRSA strains were tested in serial dilutions on these assays from 1 through 105 cfu/mluntil a positive result was obtained.Pure MRCoNS strains were testedat all dilutions. ̶ , not detected by assay; UR, unresolved; NA, data not available from manufacturer; strain concentrations that were not tested are colored grey.

Reference List

1. Quality assurance, quality control, laboratory records, and water quality. 2007. InH.D. Isenberg (ed.) Clinical Microbiology Procedures Handbook, 2nd ed. ASM Press.

2. Deurenberg, R. H. and E. E. Stobberingh. 2009. The molecular evolution of hospital- and community-associated methicillin-resistant Staphylococcus aureus. Curr.Mol.Med. 9:100-115.

3. Luczak-Kadlubowska, A., A. Sulikowska, J. Empel, A. Piasecka, M. Orczykowska, A. Kozinska, and W. Hryniewicz. 2008. Countrywide molecular survey of methicillin-resistant Staphylococcus aureus strains in Poland. J.Clin.Microbiol. 46:2930-2937.