Antimicrobial resistance of Staphylococcus aureus: genetic basis.

The strong restriction barrier present in Staphylococcus aureus and Staphylococcus epidermidis has limited functional genomic analysis to a small subset of strains that are amenable to genetic manipulation. Recently, a conserved type IV restriction system termed SauUSI (which specifically recognizes cytosine methylated DNA) was identified as the major barrier to transformation with foreign DNA. Here we have independently corroborated these findings in a widely used laboratory strain of S. aureus. Additionally, we have constructed a DNA cytosine methyltransferase mutant in the high-efficiency Escherichia coli cloning strain DH10B (called DC10B). Plasmids isolated from DC10B can be directly transformed into clinical isolates of S. aureus and S. epidermidis. We also show that the loss of restriction (both type I and IV) in an S. aureus USA300 strain does not have an impact on virulence. Circumventing the SauUSI restriction barrier, combined with an improved deletion and transformation protocol, has allowed the genetic manipulation of previously untransformable strains of these important opportunistic pathogens. IMPORTANCE Staphylococcal infections place a huge burden on the health care sector due both to their severity and also to the economic impact of treating the infections because of prolonged hospitalization. To improve the understanding of Staphylococcus aureus and Staphylococcus epidermidis infections, we have developed a series of improved techniques that allow the genetic manipulation of strains that were previously refractory to transformation. These developments will speed up the process of mutant construction and increase our understanding of these species as a whole, rather than just a small subset of strains that could previously be manipulated.

Transforming the Untransformable: Application of Direct Transformation To Manipulate Genetically Staphylococcus aureus and Staphylococcus epidermidis

Products of the intercellular adhesion (ica) operon in Staphylococcus aureus and Staphylococcus epidermidis synthesize a linear β-1,6-linked glucosaminylglycan. This extracellular polysaccharide mediates bacterial cell-cell adhesion and is required for biofilm formation, which is thought to increase the virulence of both pathogens in association with prosthetic biomedical implants. The environmental signal(s) that triggers ica gene product and polysaccharide expression is unknown. Here we demonstrate that anaerobic in vitro growth conditions lead to increased polysaccharide expression in both S. aureus and S. epidermidis, although the regulation is less stringent in S. epidermidis. Anaerobiosis also dramatically stimulates ica-specific mRNA expression in ica- and polysaccharide-positive strains of both S. aureus and S. epidermidis. These data suggest a mechanism whereby ica gene expression and polysaccharide production may act as a virulence factor in an anaerobic environment in vivo.

Anaerobic Conditions Induce Expression of Polysaccharide Intercellular Adhesin in Staphylococcus aureus and Staphylococcus epidermidis

Restriction and modification enzymes and their recognition sequences

Phage K is a polyvalent phage of the Myoviridae family which is active against a wide range of staphylococci. Phage genome sequencing revealed a linear DNA genome of 127,395 bp, which carries 118 putative open reading frames. The genome is organized in a modular form, encoding modules for lysis, structural proteins, DNA replication, and transcription. Interestingly, the structural module shows high homology to the structural module from Listeria phage A511, suggesting intergenus horizontal transfer. In addition, phage K exhibits the potential to encode proteins necessary for its own replisome, including DNA ligase, primase, helicase, polymerase, RNase H, and DNA binding proteins. Phage K has a complete absence of GATC sites, making it insensitive to restriction enzymes which cleave this sequence. Three introns (lys-I1, pol-I2, and pol-I3) encoding putative endonucleases were located in the genome. Two of these (pol-I2 and pol-I3) were found to interrupt the DNA polymerase gene, while the other (lys-I1) interrupts the lysin gene. Two of the introns encode putative proteins with homology to HNH endonucleases, whereas the other encodes a 270-amino-acid protein which contains two zinc fingers (CX2CX22CX2C and CX2CX23CX2C). The availability of the genome of this highly virulent phage, which is active against infective staphylococci, should provide new insights into the biology and evolution of large broad-spectrum polyvalent phages.

Genome of Staphylococcal Phage K: a New Lineage of Myoviridae Infecting Gram-Positive Bacteria with a Low G+C Content

The occurrence of class II restriction endonucleases (enzymes that both recognize and cleave a specific nucleotide sequence in deoxyribonucleic acid [DNA] in Staphylococcus aureus has been investigated by analysis of crude extracts obtained from different propagating strains of the International Phage Typing System. Of the four main groups of strains in the International System, only extracts of group II strains were found to contain class II restriction endonucleases. The identical cleavage patterns obtained by incubation of different DNAs with cell extracts of group II strains suggest that these enzymes all recognize and cleave the same nucleotide sequence. This recognition site has been determined to be 5'-G-A-T-C-3'-3'-C-T-A-G-5' for the prototype of these enzymes, Sau3AI (J. S. Sussenbach et al., Nucleic Acids Res. 3:3192-3202, 1976). Evidence is presented that the classification of group II strains is based on restriction modification and is correlated with the presence of a class II restriction enoduclease that recognizes and cleaves the above sequence.

Occurrence of a class II restriction endonuclease in Staphylococcus aureus.

A specific endonuclease, Sau 3AI, has been partially purified from Staphylococcus aureus strain 3A by DEAE-cellulose chromatography. The enzyme cleaves adenovirus type 5 DNA many times, SV40 DNA eight times but does not cleave double-stranded phi X174 DNA. It recognizes the sequence (see article) and cleaves as indicated by the arrows. Evidence is presented that this enzyme plays a role in the biological restriction-modification system of Staphylococcus aureus strain 3A.

A restriction endonuclease from Staphylococcus aureus

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Occurrence of a class II restriction endonuclease in Staphylococcus aureus.

A restriction endonuclease from Staphylococcus aureus