Characterization of a new SCCmec element in Staphylococcus cohnii

Abstract

Background: Many SCCmec elements of coagulase-negative staphylococci (CoNS) could not be typed using multiplex PCR. Such a 'non-typable' SCCmec was encountered in a Staphylococcus cohnii isolate.

Methodology/principal findings: The SCCmec type of methicillin-resistant S. cohnii clinical isolate WC28 could not be assigned using multiplex PCR. Newly-designed primers were used to amplify ccrA and ccrB genes. The whole SCCmec was obtained by three overlapping long-range PCR, targeting regions from left-hand inverted repeat (IRL) to ccrA/B, from ccrA/B to mecA and from mecA to orfX. The region abutting IRL was identified using inverse PCR with self-ligated enzyme-restricted WC28 fragments as the template. WC28 SCCmec had a class A mec gene complex (mecI-mecR1-mecA). The ccrA and ccrB genes were closest (89.7% identity) to ccrA(SHP) of Staphylococcus haemolyticus strain H9 and to ccrB3 (90% identity) of Staphylococcus pseudintermedius strain KM241, respectively. Two new genes potentially encoding AAA-type ATPase were found in J1 region and a ψTn554 transposon was present in J2 region, while J3 region was the same as many SCCmec of Staphylococcus aureus. WC28 SCCmec abutted an incomplete SCC element with a novel allotype of ccrC, which was closest (82% identity) to ccrC1 allele 9 in Staphylococcus saprophyticus strain ATCC 15305. Only two direct target repeat sequences, one close to the 3'-end of orfX and the other abutting the left end of WC28 SCCmec, could be detected.

Conclusions/significance: A new 35-kb SCCmec was characterized in a S. cohnii isolate, carrying a class A mec gene complex, new variants of ccrA5 and ccrB3 and two novel genes in the J1 region. This element is flanked by 8-bp perfect inverted repeats and is similar to type III SCCmec in S. aureus and a SCCmec in S. pseudintermedius but with different J1 and J3 regions. WC28 SCCmec was arranged in tandem with an additional SCC element with ccrC, SCC(WC28), but the two elements might have integrated independently rather than constituted a composite. This study adds new evidence of the diversity of SCCmec in CoNS and highlights the need for characterizing the 'non-typable' SCCmec to reveal the gene pool associated with mecA.

Figure 1. Structure of and PCR mapping for WC28 SCCmec and adjacent regions.

Numbers and alphabets represent gene names in SCCmec (listed in Table S1) and SCC_WC28 (listed in Table 3), respectively. ψTn554 contains tnpB, tnpC, cadC and cadB. The 15 bp sequences abutting the IR are shown with nucleotides that differ in lower case. The region similar to type III SCCmec (85/2082) and the SCCmec of S. pseudintermedius KM241 is highlighted with a grey background. PCR primers and amplicon sizes are indicated. Several self-ligated restricted fragments were used as templates for inverse PCR with the names and restriction locations of the enzymes being shown.

Figure 2. A proposed model for double crossover-mediated exchange between two SCCmec.

When two different SCCmec (not to scale) contain two sequences of homology, exemplified by ccrB3 and IS431 here, two homologous recombination events (the upper panel) occurring between the two sequences can result in exchange of the intervening components (lines of different thicknesses) between the two SCCmec (the lower panel).