Comparative genomics of Staphylococcus aureus musculoskeletal isolates

Much of the research aimed at defining the pathogenesis of Staphylococcus aureus has been done with a limited number of strains, most notably the 8325-4 derivative RN6390. Several lines of evidence indicate that this strain is unique by comparison to clinical isolates of S. aureus. Based on this, we have focused our efforts on two clinical isolates (UAMS-1 and UAMS-601), both of which are hypervirulent in our animal models of musculoskeletal infection. In this study, we used comparative genomic hybridization to assess the genome content of these two isolates relative to RN6390 and each of seven sequenced S. aureus isolates. Our comparisons were done by using an amplicon-based microarray from the Pathogen Functional Genomics Resource Center and an Affymetrix GeneChip that collectively represent the genomes of all seven sequenced strains. Our results confirmed that UAMS-1 and UAMS-601 share specific attributes that distinguish them from RN6390. Potentially important differences included the presence of cna and the absence of isaB, sarT, sarU, and sasG in the UAMS isolates. Among the sequenced strains, the UAMS isolates were most closely related to the dominant European clone EMRSA-16. In contrast, RN6390, NCTC 8325, and COL formed a distinct cluster that, by comparison to the other four sequenced strains (Mu50, N315, MW2, and SANGER-476), was the most distantly related to the UAMS isolates and EMRSA-16.     

Influences of sigmaB and agr on expression of staphylococcal enterotoxin B (seb) in Staphylococcus aureus

In Staphylococcus aureus, enterotoxin B (SEB) is a superantigen that activates host interleukins and induces adverse responses, ranging from food poisoning to toxic shock. The alternate sigma factor, sigmaB (SigmaB), and agr are two known regulators of S. aureus. Northern blots of strain COL, a sigB-positive strain, showed an inverse correlation between sigmaB expression and seb message. seb expression was also measured as a function of a seb promoter linked to green fluorescent protein (GFP) expression in RN6390, COL, and Newman. In sigB mutants of RN6390, SH1000, COL, and Newman, seb promoter activities, as measured by GFP expression, increased relative to the respective parental types but at differing levels, suggesting alternate strain-specific regulation. In agr mutants of RN6390 and Newman, seb promoter activities were intermediate between the high level seen for the sigB mutant and the low level in the sigB active strains. A sigB agr double mutant of RN6390 displayed lower GFP expression than the agr mutant. These results suggest that while sigmaB and agr regulate seb expression in a divergent manner, other activator(s) of seb that depend on sigB expression may be present in S. aureus.     

Staphylococcus aureus SH1000 and 8325‐4: comparative genome sequences of key laboratory strains in staphylococcal research

Aims: To provide comparative genome sequence data for two related model strains of Staphylococcus aureus (SH1000 and 8325‐4) that are used extensively in laboratory research. Methods and Results: Comparative genome sequencing was used to identify genetic differences between Staph. aureus SH1000 and the fully genome‐sequenced ancestral strain, Staph. aureus NCTC 8325. PCR amplification and DNA sequencing were employed to determine which of the genetic polymorphisms identified were also present in Staph. aureus 8325‐4, a direct derivative of 8325 and the parent strain of SH1000. Aside from known genetic differences between these strains, Staph. aureus SH1000 harboured 15 single‐nucleotide polymorphisms compared with 8325 (of which 12 were also found in 8325‐4), and a 63‐bp deletion upstream of the spa gene not present in either 8325 or 8325‐4. Conclusions: Staphylococcus aureus SH1000 and 8325‐4 contain a number of genetic polymorphisms relative to the progenitor strain of the lineage (8325) and to each other. Significance and Impact of the Study: The comparative genome sequences of SH1000 and 8325‐4 presented here define the genotypes of two key strains in staphylococcal laboratory research and reveal genetic polymorphisms that may impact their phenotypic properties.     

Transfer of resistance plasmids from Staphylococcus epidermidis to Staphylococcus aureus: evidence for conjugative exchange of resistance.

The ability of Staphylococcus epidermidis to transfer antimicrobial resistance to Staphylococcus aureus was tested by mixed culture on filter membranes. Two of six clinical isolates examined were able to transfer resistance to S. aureus strains 879R4RF, RN450RF, and UM1385RF. Subsequent S.aureus transconjugants resulting from matings with S. epidermidis donors were able to serve as donors to other S. aureus strains at similar frequencies. Cell-free and mitomycin C-induced filtrates of donors and transconjugants showed no plaque-forming ability. Addition of DNase I, citrate, EDTA, calcium chloride, and human sera to mating mixes and agar showed no effect on transfer. Nonviable donor cells were unable to transfer resistance and transfer did not occur at 4 degrees C. Cell-to-cell contact was required since transfer did not occur in broth or when filters of donor and recipient, respectively, were placed back-to-back so cells were not in direct contact. Analysis of DNA from S. epidermidis isolate UM899, its subsequent S. aureus transconjugants, and cured derivatives demonstrated that all resistance markers which transferred resided on plasmids. Mating experiments suggested a central role for the gentamicin plasmid pAM899-1 in the transfer process. It is concluded that our results are consistent with a conjugative transfer of resistance from S. epidermidis to S. aureus analogous to plasmid transfer demonstrated in streptococcal species for plasmids such as pAM beta 1. This represents a novel mechanism for gene exchange among staphylococci.     

Direct and synergistic hemolysis caused by Staphylococcus phenol-soluble modulins: implications for diagnosis and pathogenesis

Phenol-soluble modulins are secreted staphylococcal peptides with an amphipathic α-helical structure. Some PSMs are strongly cytolytic toward human neutrophils and represent major virulence determinants during Staphylococcus aureus skin and blood infection. However, capacities of PSMs to lyse human erythrocytes have not been investigated. Here, we demonstrate that many S. aureus and Staphylococcus epidermidis PSMs lyse human erythrocytes. Furthermore, synergism with S. aureus β-toxin considerably increased the hemolytic capacities of several PSMs. This synergism may be of key importance in PSM and β-toxin-producing S. aureus or in mixed-strain or -species infections with PSM and β-toxin producers. Of specific interest, several PSMs, in particular PSMα peptides, contributed to a considerable extent to synergistic hemolysis with β-toxin or when using the β-toxin-producing strain RN4220 in CAMP assays. Thus, CAMP-type assays should not be used to detect or quantify S. aureus δ-toxin production, but may be used for an overall assessment of Agr functionality. Our study suggests an additional role of PSMs in staphylococcal pathogenesis and demonstrates that the repertoire of staphylococcal hemolysins is not limited to S. aureus and is much larger and diverse than previously thought.     

Invasion of bone cells by Staphylococcus epidermidis

Bone implants infected with Staphylococcus epidermidis often require surgical intervention because of the failure of antibiotic treatment. The reasons why such infections are resistant to therapy are poorly understood. We have previously reported that another bacterium, Staphylococcus aureus, can invade bone cells and thereby evade antimicrobial therapy. In this study we have investigated the hypothesis that S. epidermidis can also invade bone cells and may therefore explain the difficulties of treating infections with this organism. We found that S. epidermidis was capable of invading bone cells but that there were significant strain dependent differences in this capacity. A recombinant protein encompassing the D1-D4 repeat region of S. aureus fibronectin-binding protein B completely inhibited internalization of S. aureus but failed to block internalization of S. epidermidis. Similarly a blocking antibody to alpha5beta1 integrin inhibited internalization of S. aureus by bone cells but had no effect on the uptake of S. epidermidis. Therefore unlike S. aureus, S. epidermidis does not gain entrance into bone cells through a fibronectin bridge between the alpha5beta1 integrin and a bacterial adhesin.     

A novel peptide screened by phage display can mimic TRAP antigen epitope against Staphylococcus aureus infections

Staphylococcus aureus is a major human pathogen. Pathogenic effects are largely due to production of bacterial toxins, whose synthesis is controlled by an mRNA molecule termed RNAIII. The S. aureus protein called RAP (RNAIII-activating protein) is secreted and activates RNAIII production by inducing the phosphorylation of its target protein TRAP (target of RAP). Antibodies to TRAP have been shown to suppress exotoxin production by S. aureus in vitro, suggesting that TRAP may be a useful vaccine target site. Here we showed that a peptide TA21 was identified by screening a phage display library using anti-TRAP antibodies. Mice vaccinated with Escherichia coli engineered to express TA21 on their surface (FTA21) were protected from S. aureus infections, using sepsis and cellulitis mice models. By sequence analysis, it was found that the TA21 is highly homologous to the C-terminal sequence of TRAP which is conserved among S. aureus and Staphylococcus epidermidis, suggesting that peptide TA21 may be a useful broad vaccine to protect from infection caused by various staphylococcal strains.     

The production of extracellular proteins is regulated by ribonuclease III via two different pathways in Staphylococcus aureus

Staphylococcus aureus ribonuclease III belongs to the enzyme family known to degrade double-stranded RNAs. It has previously been reported that RNase III cannot influence cell growth but regulates virulence gene expression in S. aureus. Here we constructed an RNase III inactivation mutant (Δrnc) from S. aureus 8325-4. It was found that the extracellular proteins of Δrnc were decreased. Furthermore, we explored how RNase III regulated the production of the extracellular proteins in S. aureus. We found during the lag phase of the bacterial growth cycle RNase III could influence the extracellular protein secretion via regulating the expression of secY2, one component of accessory secretory (sec) pathway. After S. aureus cells grew to exponential phase, RNase III can regulate the expression of extracellular proteins by affecting the level of RNAIII. Further investigation showed that the mRNA stability of secY2 and RNAIII was affected by RNase III. Our results suggest that RNase III could regulate the pathogenicity of S. aureus by influencing the level of extracellular proteins via two different ways respectively at different growth phases.     

Some Properties of the Beta-Lactamase Genes in Staphylococcus epidermidis

Eighty-three per cent of 200 different freshly isolated cultures of Staphylococcus epidermidis produced beta lactamase. Growth in the presence of acridine orange or ethidium bromide or growth at 44 C resulted in a high frequency of loss of the beta lactamase genes in some strains of S. epidermidis. The relationship between beta-lactamase production and resistance to mercuric, cadmium and arsenate ions differed from that observed in Staphylococcus aureus. It is postulated that the genes for beta lactamase in certain strains of S. epidermidis are on a plasmid.     

Cloning of an agr homologue of Staphylococcus saprophyticus

An agr homologue of Staphylococcus saprophyticus was identified, cloned and sequenced. The gene locus shows homologies to other staphylococcal agr systems, especially to those of S. epidermidis and S. lugdunensis. A putative RNAIII was identified and found to be differentially expressed during the growth phases. In contrast to the RNAIII molecules of S. epidermidis and S. aureus it does not contain an open reading frame that codes for a protein with homologies to the delta-toxin. Using PCR, the agr was found to be present in clinical isolates of S. saprophyticus.