Genetic diversity in CC398 methicillin-resistant Staphylococcus aureus isolates of different geographical origin

Authors


Corresponding author and reprint requests: M. Stegger, National Centre for Antimicrobials and Infection Control, Statens Serum Institut, 5 Artillerivej, DK-2300 Copenhagen S, Denmark
E-mail: mtg@ssi.dk

Abstract

Clin Microbiol Infect 2010; 16: 1017–1019

Abstract

Staphylococcus aureus of sequence type 398 has emerged in Europe, North America and Asia, and has typically been associated with livestock and their human contacts. We analysed two Panton–Valentine leukocidin (PVL)-negative t034-ST398 isolates from humans in contact with pigs and two t034-ST398 PVL-positive isolates from two unrelated, adopted Chinese children, using multistrain microarrays to determine genomic variability between the two sets of isolates. The ST398 isolates clearly belong to the same lineage when compared to other clonal lineages. However, the four isolates cluster into two distinct groups corresponding to differences in epidemiology based on mobile genetic elements and resistance patterns, suggesting that the two groups are epidemiologically distinct.

Staphylococcus aureus has generally been classified as an opportunistic human pathogen causing nosocomial and, in recent years, increasing numbers of community-acquired infections worldwide [1,2]. However, methicillin-resistant S. aureus (MRSA) has recently been found also in both pet animals and a variety of livestock animals, such as poultry, cattle and pigs [3,4]. A new clone discovered in 2004, of sequence type (ST) 398, has been found since in several European countries, the USA, Canada, Singapore and China [5,6]. The carriage prevalence of this clone is very high in livestock farmers and veterinary personnel [7]. Molecular analysis has shown isolates belonging to clonal complex (CC) 398, including ST398, to be generally non-typeable by standard SmaI pulsed-field gel electrophoresis analysis, due to DNA methylation, and to encode spa type t011, spa type t034 or close variants thereof, the majority of which carry SCCmec V [6,8,9]. This clone has shown a rapid increase in prevalence in The Netherlands [7], as well as in Denmark (manuscript in preparation), and has caused a nosocomial outbreak in The Netherlands [10]. A significant number of successful community-acquired lineages encode Panton–Valentine leukocidin (PVL), which has been associated with severe skin infections and necrotizing pneumonia [11,12]. So far, CC398 isolates from pigs and people in contact with pigs have been identified as PVL-negative. However, PVL-positive CC398 MRSA isolates have recently been described sporadically in China [5] and in Europe [13,19]. As part of a national surveillance of MRSA-positive persons, we identified two PVL-positive MRSA CC398 isolates in two unrelated children adopted from China with skin and soft tissue infections. These two isolates were compared with two isolates of the predominant type found in people with contact with pigs in Denmark by multistrain microarray analysis covering 3623 open reading frames, to estimate the genetic diversity of this epidemic clone.

The S aureus isolates were spa-typed, and the presence of mecA and lukF-PV was confirmed as previously described [14,15]. SCCmec types were annotated using multiplex PCR one and two, as described by Kondo et al. [16]. Testing of susceptibility to penicillin, cefoxitin, streptomycin, gentamicin, kanamycin, erythromycin, clindamycin, tetracycline, fusidic acid, rifampicin and norfloxacin was performed using Neo-Sensitabs (Rosco, Taastrup, Denmark) on Danish Blood Agar (SSI Diagnostica, Hillerod, Denmark). DNA was extracted using the Invitrogen PureLink kit (Carlsbad, CA, USA) and hybridized to the SAv1 microarray, as previously described [17,18]. The microarray contains PCR products for each predicted open reading frame from seven sequenced S. aureus genomes representing four CCs (CC1, CC5, CC8, and CC30). DNA from the four isolates was labelled with Cy3, mixed with Cy5-labelled DNA from MRSA252, hybridized, washed, and scanned. GeneSpring 7.3.1 software was used for data analysis. Microarray data are deposited in BμG@SBase and ArrayExpress. The array design is available at BμG@Sbase (accession number A-BUGS-17; http://bugs.sgul.ac.uk/A-BUGS-17) and ArrayExpress (accession number A-BUGS-17). Fully annotated microarray data have been deposited at BμG@Sbase (accession number E-BUGS-84; http://bugs.sgul.ac.uk/E-BUGS-84) and ArrayExpress (accession number E-BUGS-84).

The CC398 isolates contain the S. aureus core genome when compared to c. 475 different S. aureus isolates (data not shown). Spearman clustering analysis of the four CC398 isolates using core variable genes showed that they all cluster closely together in one lineage. CC398 is a typical S. aureus lineage on the basis of the array data, but constitutes a lineage distinct from other human lineages, similar to what has been found by van Belkum et al. [19]. Interestingly, CC398 isolates were also distinct from other S. aureus animal lineages [20], although we cannot discount the presence of lineage-specific genes that are not present on the multistrain microarray. Common traits of this lineage, based on the four isolates, include a capsule type 5, agr type I/IV, carriage of a novel fnbA gene, and the presence of cna, sasA and a GIβ region with a hysA variant. All isolates were negative for egc, sarT, sea, seb, sec, sed, see, sek, sel and tst [17].

There was significant variation in mobile genetic element carriage between the two identical Chinese isolates and the two identical Danish isolates (Table 1). The Danish isolates have a phage related to the ϕSa2 phage variant that are PV-luk negative, but with an integrase that originates from the ϕSa4 or ϕSa6 family. They both carry a pT181-like plasmid including the replication genes, which corresponds to a tetracycline resistant phenotype. In contrast, the two Chinese PVL-positive isolates encode three phages: a phage related to ϕSa1 from Mu50, a phage of the ϕSa2 family that is closely related to MW2, probably encompassing the PVL toxin gene, and a ϕSa3 phage related to MRSA252. These two isolates also contain a plasmid encoding a penicillinase-like enzyme and cadmium resistance. None of the tested isolates showed any evidence of pathogenicity islands. All isolates were SCCmec V as determined by PCR. The comparison of the Danish and Chinese isolates showed a highly conserved lineage background but evidence of at least six mobile genetic element movement events. This suggests a common progenitor but independent evolution of each geographical type.

Table 1.   Summary of differences in mobile genetic elements between isolates as detected by multistrain microarray and phenotypic antibiotic resistance testing
OriginDenmarkChina
  1. bla, β-lactamase resistance; chp, chemotaxis inhibitory protein; cadDX, cadmium resistance; PV-luk, Panton–Valentine leukocidin; scn, staphylococcal complement inhibitor; sak, staphylokinase; tet, tetracycline resistance; SaPI, staphylococcal pathogenicity island; pen, penicillin; strep, streptomycin; ery, erythromycin; clind, clindamycin; tet, tetracycline.

  2. Mobile genetic elements have been clustered into families according to Lindsay and Holden [20].

  3. aThe Danish isolates carry a lukF/S-PV-negative variant of ϕSa2.

  4. bThe closest match is MRSA252, sea-negative.

  5. cBased on the identification of the replication genes.

  6. d bla genes were also found in Danish isolates but without evidence that they were Tn552-positive.

spa typet034t034
ϕSa1+
ϕSa2a+ PV-luk
ϕSa3b chp, scn, sak
SaPI
SCCmecVV
Plasmid class Ic tet
Plasmid class II cadDX
Tn552d bla
Resistancepen, ery, clind, tet, streppen, ery, clind

PVL gene carriage in CC398 MRSA has been reported in both China and Europe, where these isolates all caused human disease. It is possible that the toxin contributes to human pathogenicity, as these strains caused skin and soft tissue infection in the two children living in Denmark. Therefore, the spread of such strains is of public health concern, especially if the spread is amplified through livestock populations. The majority of CC398 isolates from Denmark and other countries are tetracycline-resistant, most likely because of the use of tetracycline in livestock. Tetracycline resistance in the Danish isolates appeared to be mediated by a small, highly mobile, rolling circle pT181-like plasmid containing tetK [21]. The Chinese isolates were tetracycline-sensitive, and this may prevent their spread to livestock. However, tetracycline-resistant, PVL-positive MRSA CC398 isolates from hospitalized patients have been reported in China [5]. Both scenarios could lead to the rapid spread, to and among animals, of MRSA strains that are capable of causing severe skin and soft tissue infection in healthy human populations.

Acknowledgements

The Wellcome Trust supports the Bacterial Microarray Group at St George’s (http://www.bugs.sgul.ac.uk). We thank D. Waldron, A. Witney, J. Hinds and P. Butcher for additional assistance with microarrays.

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None of the authors has any associations that could pose a possible conflict of interest.

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