DNA microarray profiling of a diverse collection of nosocomial methicillin-resistant staphylococcus aureus isolates assigns the majority to the correct sequence type and staphylococcal cassette chromosome mec (SCCmec) type and results in the subsequent identification and characterization of novel SCCmec-SCCM1 composite islands

Abstract

One hundred seventy-five isolates representative of methicillin-resistant Staphylococcus aureus (MRSA) clones that predominated in Irish hospitals between 1971 and 2004 and that previously underwent multilocus sequence typing (MLST) and staphylococcal cassette chromosome mec (SCCmec) typing were characterized by spa typing (175 isolates) and DNA microarray profiling (107 isolates). The isolates belonged to 26 sequence type (ST)-SCCmec types and subtypes and 35 spa types. The array assigned all isolates to the correct MLST clonal complex (CC), and 94% (100/107) were assigned an ST, with 98% (98/100) correlating with MLST. The array assigned all isolates to the correct SCCmec type, but subtyping of only some SCCmec elements was possible. Additional SCCmec/SCC genes or DNA sequence variation not detected by SCCmec typing was detected by array profiling, including the SCC-fusidic acid resistance determinant Q6GD50/fusC. Novel SCCmec/SCC composite islands (CIs) were detected among CC8 isolates and comprised SCCmec IIA-IIE, IVE, IVF, or IVg and a ccrAB4-SCC element with 99% DNA sequence identity to SCC(M1) from ST8/t024-MRSA, SCCmec VIII, and SCC-CI in Staphylococcus epidermidis. The array showed that the majority of isolates harbored one or more superantigen (94%; 100/107) and immune evasion cluster (91%; 97/107) genes. Apart from fusidic acid and trimethoprim resistance, the correlation between isolate antimicrobial resistance phenotype and the presence of specific resistance genes was ≥97%. Array profiling allowed high-throughput, accurate assignment of MRSA to CCs/STs and SCCmec types and provided further evidence of the diversity of SCCmec/SCC. In most cases, array profiling can accurately predict the resistance phenotype of an isolate.

Fig 1

Based Upon Repeat Pattern (BURP) analysis of the spa types identified among 175 MRSA isolates representative of the different antibiogram-resistogram, sequence type, and SCCmec type combinations recovered from patients in Irish hospitals between 1971 and 2004. Twenty-six of the 35 spa types were grouped into four cluster groups using the BURP algorithm. spa types were clustered together if they contained five or more spa repeats and if they had a cost value of ≤4, where cost accounts for the number of steps of evolution between spa types. A low cost value indicates close evolutionary relatedness between two spa types. The following six spa types were defined as singletons by BURP (t045, t160, t902, t3504, t3506, and t4253), as they could not be clustered with any other spa type, i.e., cost value of ≥5. The spa types t727, t1802, and t2196 were excluded, as they consisted of four repeat units only. Cost values are shown in parentheses. Group founders and cofounders (spa types with the second-highest group founder score) are shown in blue and are determined based on the spa type that shares the highest sequence identity with the greatest number of spa types within that cluster as determined by the cost values. No founder was assigned to cluster 4.

Fig 2

Schematic diagram showing the genomic organization of the novel SCCmec-SCC composite island (CI) SCCmec IIE-SCC_M1 identified in the ST8-MRSA spa type t190 isolate AR13.1/3330.2 (GenBank accession number HE858191) (a), the SCC_M1-SCC_arc-SCCmec element previously reported in ST8-MRSA spa type t024 isolate M1 (HM030720) (b), the ACME- and SCC-CIs previously reported in S. epidermidis ATCC 12228 (NC004461) (c), and SCCmec VIII previously described in ST8-MRSA spa type t008 isolate C10682 (FJ390057) (d). The structure of the novel SCCmec IIE-SCC_M1 element was determined by high-throughput whole-genome sequencing of AR13.1/3330.2 and was confirmed using primers spanning the SCCmec/SCC region. The areas surrounded by the red square indicate the regions of high similarity between SCC_M1 identified in isolate AR13.1/3330.2 and the other SCCmec/SCC elements.