Targeting the R domain of coagulase by active vaccination protects mice against lethal Staphylococcus aureus infection

Coagulase (Coa) secreted by Staphylococcus aureus is associated with the establishment of staphylococcal disease, which activates host prothrombin and generates fibrin shields. The R domain of Coa, consisting of several conserved repeats, is important in immune evasion during S. aureus infection. However, previous research showed that the Coa R domain induced very weak specific antibody responses. In this study, we constructed a new R domain, CoaR6, consisting of 6 repeats that occur most frequently in clinical isolates. By fusing CoaR6 with Hc, the C-terminal fragment of the heavy chain of tetanus neurotoxin, we successfully increased anti-CoaR6 IgG levels in immunized mice which were hardly detected in mice immunized with CoaR6 plus alum. To further improve anti-CoaR6 responses, the combination adjuvants alum plus CpG were formulated with the antigen and exhibited a significantly higher specific antibody response. Moreover, active Th1/Th17 immune responses were observed in Hc-CoaR6 immunized group rather than CoaR6. Active immunization of Hc-CoaR6 with alum plus CpG showed protective effects in a peritonitis model induced by two S. aureus strains with different coagulase types. Our results provided strategies to improve the immunogenicity of R domain and supporting evidences for R domain to be an S. aureus vaccine candidate.     

Potentiation of methicillin activity against methicillin-resistant Staphylococcus aureus by diterpenes

Totarol is a diterpene compound extracted from the totara tree. Totarol and eight other diterpenes were found to potentiate methicillin, one reducing the minimum inhibitory concentration of methicillin against resistant Staphylococcus aureus 256-fold. Totarol did not inhibit the synthesis of DNA or peptidoglycan in S. aureus, but reduced the respiration rate by 70%. Under potentiation conditions, diterpenes had only a slight effect on the respiration rate, but had a significant effect on expression of PBP 2a. We conclude that the primary staphylococcal target for totarol is the respiratory chain, but that potentiation of methicillin by diterpenes is by interference with PBP 2a expression.     

Relatedness between the coagulase gene 3'-end region and coagulase serotypes among Staphylococcus aureus strains

The 3'-end region of the coagulase gene from 22 strains of Staphylococcus aureus including 10 standard serotype strains was sequenced, and five subgroups with 4-8 tandem repeating units were distinguished among the tested strains. Phylogenetic analysis of the 3'-end region of the coagulase gene indicated that strains belonging to the same serotype were clustered in the same branch. A phylogenetic tree of the deduced amino acid sequences revealed that the C-terminal region might not be responsible for the epitope of the coagulase protein.     

Improved methods for detection and serotyping of coagulase from Staphylococcus aureus

Improved methods for detection and serotyping of staphylocoagulase were concomitantly devised. We devised an improved method for detection of coagulase activity on agarose film in the same manner as single radial immunodiffusion. The amounts of reagents required for detection of coagulase on agarose film were successfully diminished by adding polyethylene glycol (PEG) to the original formula described by Boothby et al. Using microplates in another improved method for coagulase serotyping, the amount of reaction fluid required was considerably less compared with the conventional tube method. PEG was found to be also effective to increase the efficacy of coagulase serotyping. In the presence or absence of anti-coagulase antisera, culture supernatants of staphylococcal strain grown in brain heart infusion broth were incubated with the reaction fluid containing bovine fibrinogen, rabbit plasma, 6-amino caproic acid, polyethylene glycol 6,000. Coagulase activity was visualized as a turbid mass formed in the wells. Turbid mass formation due to coagulase activity was type-specifically inhibited in the presence of type-specific antisera. Detailed procedures of the methods are precisely described with some preliminary results obtained by the methods.     

Simple and direct detection of Staphylococcus aureus in milk by a tube coagulase test

A tube coagulase test (TCT) is described as a simple and non-expensive system for detection of Staphylococcus aureus directly in milk. The procedure is characterized by mixing milk samples with rabbit citrate plasma followed by incubation at 37 °C for clot formation. The tube coagulase test demonstrated 91·5% accuracy, 88·5% sensitivity and 100% specificity for the direct recognition of Staph. aureus in milk samples from quarters with subclinical mastitis, when compared with plating of milk on blood agar. The TCT has the potential to detect other coagulase positive staphylococci in milk. It is concluded that TCT may be of use to veterinary practitioners with limited laboratory facilities, or to dairy farmers as a simple diagnostic test on site.     

Mobilization of recombinant plasmids from Staphylococcus aureus into coagulase negative Staphylococcus species.

pC221, a small nonconjugative staphylococcal plasmid, can be mobilized between staphylococci by pG01, a larger conjugative plasmid. pC221 carries the two transacting genes, mobA and mobB, which are needed for its mobilization. The products of these genes create a site-specific single-stranded nick (mobA) and then facilitate DNA transfer (mobB). Several useful Escherichia coli-staphylococcal shuttle plasmids containing the cloned single-stranded nick site were created and successfully mobilized into Staphylococcus aureus and two coagulase-negative staphylococci, S. epidermidis and S. saprophyticus, by providing mob genes (pC221) and conjugative transfer genes (pG01) in trans in the donor. These vectors may offer a genetic system for the introduction of recombinant plasmids into coagulase negative staphylococci.     

Wild-type variants of exopenicillinase from Staphylococcus aureus

1. Three variants of staphylococcal exopenicillinase (types A, B and C) can be distinguished on chemical, enzymological and immunological grounds. 2. Enzyme type A has a higher specific activity than that of type B, but has a similar combination affinity with anti-(exopenicillinase type A) serum. 3. Enzyme types A and C have a similar specific activity, but enzyme type C has a lower combination affinity for anti-(exopenicillinase type A) serum than has enzyme type A. 4. The sedimentation coefficients and amino acid analyses of the three enzyme types are similar. 5. All three enzyme types have small but significant differences in kinetics of action when hydrolysing benzylpenicillin, methicillin, cloxacillin and cephalosporin C. 6. Peptide maps, obtained from enzyme types A and C after digestion with trypsin, show that these two variants probably differ in the nature of only a very few amino acid residues. 7. Enzyme type B seems to be confined to staphylococci that are members of staphylococcal phage group II. Enzyme types A and C are produced by staphylococci that are members either of phage group I or III, but never group II. 8. The low specific enzyme activity and affinity of enzyme type B towards all penicillins tested suggest that this enzyme type has a lower `efficiency' in hydrolysing penicillin and therefore in protecting bacteria from the action of penicillin. This could account for the low incidence among `hospital staphylococci' of penicillin-resistant staphylococci that are members of phage group II.     

Enhanced thermal destruction of Listeria monocytogenes and Staphylococcus aureus by the lactoperoxidase system

The lactoperoxidase system (LPS) enhanced thermal destruction of Listeria monocytogenes and Staphylococcus aureus. After LPS activation, biphasic survival curves were observed for L. monocytogenes at 57.8 degrees C and for S. aureus at 55.2 degrees C. The data were consistent with a model that assumed two bacterial populations differing in heat sensitivity. The more heat-sensitive fractions (93% of the L. monocytogenes, 92% of the S. aureus) were killed almost instantly. For these biphasic survival curves, D values were based on the much smaller, less-heat-sensitive fractions. For L. monocytogenes, the D52.2 degrees C values were 30.2 min (untreated milk) and 10.7 min (LPS activated); corresponding D55.2 degrees C values were 8.2 and 1.6 min; corresponding D57.8 degrees C values were 2.3 and 0.5 min. For S. aureus, the D52.2 degrees C values were 33.3 min (untreated milk) and 2.2 min (LPS activated), and the corresponding D55.2 degrees C values were 7.6 and 1.1 min, respectively. The most rapid killing of L. monocytogenes occurred when samples were heated soon after activation of the LPS. Activation of the LPS followed by heating can increase the margin of safety with respect to milkborne pathogens.     

Circadian rhythms of antibacterial resistance, coagulase and antilysozyme activity of Staphylococcus aureus

AIM: To determine daily dynamics of antibacterial resistance as well as antilysozyme and coagulase activity of S. aureus strains. MATERIALS AND METHODS: On an example of clinical strains of S.aureus isolated from patients with surgical infections daily dynamics of biological characteristics of staphylococci was studied. After 12 hours of incubation strains were tested for coagulase activity by standard method (test tube method), antilysozyme activity by photometric method, and antibacterial resistance by method of serial dilutions in agar. Tests were repeated each 3-hours during a day. RESULTS: Variation of levels of studied biological characteristics of staphylococci during a day was revealed. Structures of coagulase and antilysozyme circadian rhythms had some differences in different S. aureus strains. Alongside with it, similarity in temporal expression of such biological characteristics of staphylococci as antibacterial resistance and antilysozyme activity was noted. CONCLUSION: Obtained data open prospect to use biorhythmological approach in study of biological characteristics of microorganisms during evaluation of their mechanisms of adaptation to changing environmental conditions. Chronobiological approach allows to reveal periods of maximal expression of S. aureus characteristics that could be used for increasing of effectiveness of antibacterial treatment by the choice of optimal time for administration of antibiotic.     

Quercetin protects rats from catheter‐related Staphylococcus aureus infections by inhibiting coagulase activity

Coagulase (Coa) activity is essential for the virulence of Staphylococcus aureus (S aureus), one of the most important pathogenic bacteria leading to catheter‐related bloodstream infections (CRBSI). We have demonstrated that the mutation of coagulase improved outcomes in disease models of S aureus CRBSI, suggesting that targeting Coa may represent a novel antiinfective strategy for CRBSI. Here, we found that quercetin, a natural compound that does not affect S aureus viability, could inhibit Coa activity. Chemical biological analysis revealed that the direct engagement of quercetin with the active site (residues Tyr187, Leu221 and His228) of Coa inhibited its activity. Furthermore, treatment with quercetin reduced the retention of bacteria on catheter surfaces, decreased the bacterial load in the kidneys and alleviated kidney abscesses in vivo. These data suggest that antiinfective therapy targeting Coa with quercetin may represent a novel strategy and provide a new leading compound with which to combat bacterial infections.     

Tandem coagulase/thermonuclease agar method for the detection of Staphylococcus aureus.

In optimizing previously reported coagulase agar media to obtain a rapid, reliable, and inexpensive coagulase test agar, variations in plasmas, pH, buffer system, fibrinogen, and fibrinolytic inhibitor were investigated. The agar with the following composition was determined best for the demonstration of coagulase production by Staphylococcus aureus: 25 ml of 15% bovine fibrinogen (fraction I, type I, citrated, Sigma Chemical Co.), 25 ml of rehydrated rabbit plasma (coagulase plasma ethylenediaminetetraacetic acid, Difco), 10.0 mg of soybean trypsin inhibitor (Schwarz/Mann), and 450 ml of brain heart infusion agar (Difco). In additional studies involving 7 different temperatures and 11 heating times, the thermal destruction of microbial nucleases on plate count agar and coagulase test agar was investigated. Heating the plates for 2.5 h at 65 degrees C destroyed all heatlabile nucleases, but not thermonucleases of S. aureus. A tandem agar plate method for the identification of S. aureus was developed. Coagulase and thermonuclease activity of 50 colonies can be detected on a single agar plate. Suspect S. aureus colonies isolated on various selective media are transferred to coagulase test agar, the plates are incubated at 37 degrees C for 18 h, and the coagulase reaction is recorded. The plates are then heated at 65 degrees C for 2.5 h, overlaid with toluidine blue-metachromatic diffusion agar, and reincubated at 37 degrees C for 3 h, and the thermonuclease reaction is recorded. Studies based on 88 enterotoxigenic S. aureus strains and 133 and 48 suspect S. aureus strains isolated from fresh salami mixtures on mannitol salt and tellurite-polymyxin-egg yolk agars, respectively, demonstrated 100% agreement between the tandem agar plate method and standard coagulase and thermonuclease tests. Overall, the tandem agar plate method is a rapid and convenient approach contributing to the identification of S. aureus from foods.     

Cold-sensitive variants of Staphylococcus aureus and their stimulation by penicillins and cephalosporins.

Cold-sensitive (Cs) variants were obtained from ageing broth cultures of the Oxford strain of Staphylococcus aureus (NCTC 6571) and other penicillin-sensitive strains of this species. Growth of all strains was severely retarded on certain media at 30 degrees C but was stimulated by the addition of penicillins and cephalosporins. Revertants were derived which behaved as the parent cells did with respect to growth temperature requirements and response to these antibiotics.