SummaryBackgroundAnimals can act as a reservoir and source for the emergence of novel meticillin-resistant Staphylococcus aureus (MRSA) clones in human beings. Here, we report the discovery of a strain of S aureus (LGA251) isolated from bulk milk that was phenotypically resistant to meticillin but tested negative for the mecA gene and a preliminary investigation of the extent to which such strains are present in bovine and human populations.MethodsIsolates of bovine MRSA were obtained from the Veterinary Laboratories Agency in the UK, and isolates of human MRSA were obtained from diagnostic or reference laboratories (two in the UK and one in Denmark). From these collections, we searched for mecA PCR-negative bovine and human S aureus isolates showing phenotypic meticillin resistance. We used whole-genome sequencing to establish the genetic basis for the observed antibiotic resistance.FindingsA divergent mecA homologue (mecALGA251) was discovered in the LGA251 genome located in a novel staphylococcal cassette chromosome mec element, designated type-XI SCCmec. The mecALGA251 was 70% identical to S aureus mecA homologues and was initially detected in 15 S aureus isolates from dairy cattle in England. These isolates were from three different multilocus sequence type lineages (CC130, CC705, and ST425); spa type t843 (associated with CC130) was identified in 60% of bovine isolates. When human mecA-negative MRSA isolates were tested, the mecALGA251 homologue was identified in 12 of 16 isolates from Scotland, 15 of 26 from England, and 24 of 32 from Denmark. As in cows, t843 was the most common spa type detected in human beings.InterpretationAlthough routine culture and antimicrobial susceptibility testing will identify S aureus isolates with this novel mecA homologue as meticillin resistant, present confirmatory methods will not identify them as MRSA. New diagnostic guidelines for the detection of MRSA should consider the inclusion of tests for mecALGA251.FundingDepartment for Environment, Food and Rural Affairs, Higher Education Funding Council for England, Isaac Newton Trust (University of Cambridge), and the Wellcome Trust.

A defined allelic-replacement mutant of the sly gene, encoding a thiol-activated cytolysin, from a European isolate of Streptococcus suis serotype 2 was generated and characterized. Unlike the parental strain, it is nonhemolytic, noncytotoxic for cultured macrophage-like cells, avirulent in a mouse infection model, yet only slightly attenuated in a porcine model of systemic infection. Streptococcus suis has been described as the etiological agent for a number of infectious disease syndromes in pigs, including arthritis, septicemia, meningitis, and pneumonia. S. suis produces a secreted hemolysin (suilysin) which has been suggested as playing a role in virulence (2,(4)(5)(6)(7)10). In order to investigate the role played by suilysin in the pathogenesis of a European serotype 2 isolate of S. suis, we generated a defined allelic-replacement mutant of the sly gene and compared the wild-type organism with the sly mutant in a number of assays.Bacterial strains and media. S. suis type 2 strain P1/7 was grown on Columbia agar (Oxoid) containing 10% defibrinated horse blood or in liquid cultures of Todd-Hewitt broth (Oxoid) supplemented with 7% fetal calf serum (FCS) (Gibco). Allelicreplacement mutants of S. suis were maintained on 1 g of erythromycin (Sigma) per ml.Mutagenesis of the suilysin gene from S. suis type 2. An erythromycin resistance gene cassette was introduced into an EcoRV site within a 1,278-bp fragment of the sly gene, contained in the vector pT7-Blue (Stratagene), amplified by PCR using primers suis1 (5Ј-AGCTTGACTTACGAGCCACAAG AG-3Ј) and suis2 (5Ј-CCACCATTCCCAAGCTAATCCTGT-3Ј) with chromosomal DNA from P1/7 as a template. The resulting plasmid, pSUI-erm, contains the erm gene in the same orientation as the sly gene. Plasmid pSUI-erm was introduced into P1/7 by electroporation (22.5 V/cm, 25 F, and 1,000 ⍀). A transformant which had undergone a doublecrossover event, confirmed by Southern hybridization and PCR, with concomitant insertion mutation of the sly gene, was isolated and named S7c.Phenotypic analysis of S7c. Overnight growth of suilysin mutant S7c on Columbia blood agar plates revealed no ␤-hemolysis. Secreted proteins from anaerobically grown overnight cultures of P1/7 and S7c were concentrated 100-fold by ammonium sulfate (50%, wt/vol) precipitation; then 10 l each of these preparations was spotted onto a Columbia horse blood agar plate and incubated at 37°C for 30 min. The proteins from P1/7 show clear zones of hemolysis, whereas there is a complete absence of hemolytic activity in the proteins obtained from S7c (Fig. 1a). The hemolytic activity from the wild-type parental bacteria was enhanced by the addition of ␤-mercaptoethanol, as previously reported (7) (Fig. 1a).The lack of expression of suilysin was confirmed by Western blotting. Supernatants from aerobically grown cultures of both P1/7 and S7C (sterilized using a 0.22-m-pore-size filter and concentrated 25-fold using Amicon filters) were probed with a monoclonal antibody (INT-STS-28-02; A. C. Jacobs, Intervet) raised against purified s...

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Mouse DNA methyltransferase is able to catalyse the transfer of a methyl group to certain CG-containing single-stranded oligonucleotides. The presence of a methylcytosine is required for efficient transfer. This methylcytosine may or may not be on the same oligonucleotide as that containing the accepting CG dinucleotide. When the accepting CG dinucleotide forms part of an unmethylated CG dinucleotide pair, its accepting activity is dramatically reduced. This provides the potential for methylation to spread along the DNA when it is rendered single-stranded at replication. It could also help to maintain fully methylated CG islands and asymmetrically methylated sites.

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