Multilocus sequence typing for characterization of methicillin-resistant and methicillin-susceptible clones of Staphylococcus aureus

Abstract

A multilocus sequence typing (MLST) scheme has been developed for Staphylococcus aureus. The sequences of internal fragments of seven housekeeping genes were obtained for 155 S. aureus isolates from patients with community-acquired and hospital-acquired invasive disease in the Oxford, United Kingdom, area. Fifty-three different allelic profiles were identified, and 17 of these were represented by at least two isolates. The MLST scheme was highly discriminatory and was validated by showing that pairs of isolates with the same allelic profile produced very similar SmaI restriction fragment patterns by pulsed-field gel electrophoresis. All 22 isolates with the most prevalent allelic profile were methicillin-resistant S. aureus (MRSA) isolates and had allelic profiles identical to that of a reference strain of the epidemic MRSA clone 16 (EMRSA-16). Four MRSA isolates that were identical in allelic profile to the other major epidemic MRSA clone prevalent in British hospitals (clone EMRSA-15) were also identified. The majority of isolates (81%) were methicillin-susceptible S. aureus (MSSA) isolates, and seven MSSA clones included five or more isolates. Three of the MSSA clones included at least five isolates from patients with community-acquired invasive disease and may represent virulent clones with an increased ability to cause disease in otherwise healthy individuals. The most prevalent MSSA clone (17 isolates) was very closely related to EMRSA-16, and the success of the latter clone at causing disease in hospitals may be due to its emergence from a virulent MSSA clone that was already a major cause of invasive disease in both the community and hospital settings. MLST provides an unambiguous method for assigning MRSA and MSSA isolates to known clones or assigning them as novel clones via the Internet.

FIG. 1

Polymorphic sites in four of the gene fragments. The nucleotides present at each variable site among the 155 S. aureus isolates are shown for allele 1. For the other alleles, only those sites that differ are shown; sites that are the same as those in allele 1 are shown by periods. Nucleotide sites that are the same in all alleles are not shown. The position of each polymorphic site within the sequenced fragment is shown above, in vertical format. The polymorphisms that are synonymous (S) and nonsynonymous (N) are shown below. Allele 5 of glpF is not shown as this allele was found in S. aureus isolates that are not described in this paper. The glpF and gmk fragments were the most uniform of the seven housekeeping gene fragments used in the MLST scheme, and arcC and aroE were the most variable.

FIG. 2

PFGE of pairs of isolates with identical allelic profiles. Chromosomal DNA from pairs of isolates of STs 25 (lanes b and c), 30 (lanes d and e), 34 (lanes f and g), 36 (lanes h and i), 39 (lanes j and k), 45 (lanes l and m), 47 (lanes n and o), and 49 (lanes p and q) were digested with SmaI and were separated by PFGE. Concatenated bacteriophage lambda molecular size markers were run in lanes a and r. Numbers on the right are in kilobase pairs.

FIG. 3

Dendrogram showing the genetic relatedness of the 155 S. aureus isolates. The STs that are represented by multiple isolates are shown by the open rectangles and are labeled on the right, with the number of isolates in the ST and, in parentheses, the number of MRSA isolates in the ST (M), followed by a colon and the number of isolates from patients with community-acquired infections (C), followed by a colon and the number of isolates from patients with hospital-acquired infections (H). For example, ST1 - 7 (0M:5C:2H) contains seven isolates, none of which are methicillin resistant, five isolates from patients with community-acquired infections, and two isolates from patients with hospital-acquired infections. The asterisks show the nodes that define the major clonal complexes, which include a group of isolates with the same allelic profile plus those closely related isolates that differ at only one of the seven loci. The node that defines the large cluster of closely related isolates of STs 29 to 38 is shown by a hash sign.

FIG. 4

Relatedness among the major cluster of isolates. The STs within the large clonal complex defined by the node indicated with a hash sign in Fig. 3 (STs 29 to 38) are shown, with the number of isolates in each ST indicated by the subscript and the susceptibility (S) or resistance (R) to methicillin indicated as a superscript. The circles indicate the two major STs in the cluster, and the STs that differ from these two major STs at only a single locus are shown by arrows. ST36 (clone EMRSA-16) differs from ST30 at only a single locus but includes exclusively MRSA isolates. ST36 is hypothesized to have arisen as a minor variant of ST30 that acquired the mec determinant by horizontal gene transfer and that has subsequently diversified slightly to give rise to the single-locus variants ST35 and ST38, which are also MRSA. All of the isolates that are not linked by arrows differ at more than one locus. ST37 differs from both ST30 and ST36 at a single locus (all three STs have different pta alleles) but, on parsimony grounds, is considered to be derived from ST30 since it is susceptible to methicillin. ST40 is included since it differs from ST30 at a single locus, although this is not apparent in Fig. 3 as it clustered anomalously when UPGMA was used.