Role of Staphylococcus aureus coagulase and clumping factor in pathogenesis of experimental endocarditis.

The pathogenic role of staphylococcal coagulase and clumping factor was investigated in the rat model of endocarditis. The coagulase-producing and clumping factor-producing parent strain Staphylococcus aureus Newman and a series of mutants defective in either coagulase, clumping factor, or both were tested for their ability (i) to attach in vitro to either rat fibrinogen or platelet-fibrin clots and (ii) to produce endocarditis in rats with catheter-induced aortic vegetations. In vitro, the clumping factor-defective mutants were up to 100 times less able than the wild type strain to attach to fibrinogen and also significantly less adherent than the parents to platelet-fibrin clots. Coagulase-defective mutants, in contrast, were not altered in their in vitro adherence phenotype. The rate of in vivo infection was inoculum dependent. Clumping factor-defective mutants produced ca. 50% less endocarditis than the parent organisms when injected at inoculum sizes infecting, respectively, 40 and 80% (ID40 and ID80, respectively) of rats with the wild-type strain. This was a trend at the ID40 but was statistically significant at the ID80 (P < 0.05). Coagulase-defective bacteria were not affected in their infectivity. Complementation of a clumping factor-defective mutant with a copy of the wild-type clumping factor gene restored both its in vitro adherence and its in vivo infectivity. These results show that clumping factor plays a specific role in the pathogenesis of S. aureus endocarditis. Nevertheless, the rate of endocarditis with clumping factor-defective mutants increased with larger inocula, indicating the contribution of additional pathogenic determinants in the infective process.     

Isolation of Staphylococcus aureus clumping factor.

Immunochemically identical components were isolated from water-soluble phases of five Staphylococcus aureus strains by affinity chromatography on fibrinogen-linked Sepharose 4B. The elution was performed with 1 M MgCl2. The component could be isolated from sonicated preparations of whole cells, cell walls, and extracellular products of S. aureus but not from sonicated preparations of staphylococcal L-forms or from Staphylococcus epidermidis. Investigations of the eluted component by immunoelectrophoresis and Western blot analysis by use of different polyspecific antibodies to S. aureus raised in rabbits revealed only one immunoprecipitate or one band. By means of gel filtration on Sepharose CL 6B and sodium dodecyl sulfate-polyacrylamide gel electrophoresis a molecular mass of 420,000 and 360,000 was found, respectively. Chemical analysis showed a carbohydrate content of about 20% by weight. By crossed immunoelectrophoresis the isolated component was demonstrated to bind to human fibrinogen. The finding that this purified component inhibited the fibrinogen-induced clumping of staphylococci strongly suggests that the component is the S. aureus clumping factor.     

Fibrinogen-binding protein/clumping factor from Staphylococcus aureus.

The binding of staphylococcal components to fibrinogen was studied. Fibrinogen-binding material from lysed staphylococcal cells or culture supernatants was affinity purified on fibrinogen-Sepharose and analyzed on Western (immuno-) blots by the use of fibrinogen and antifibrinogen antibodies. Two main bands of 87 and 19 kilodaltons (kDa) and a weaker band of 35 kDa bound specifically to fibrinogen. A monoclonal antibody bound to all three bands, indicating that these were of the same origin. The yield of these components was much higher in the culture supernatant than on washed cells, suggesting that these molecules are essentially extracellular products. In a plasma coagulase test, the 87-kDa band, but not the 19-kDa band, clotted rabbit plasma, demonstrating that the 87-kDa molecule is coagulase. This was further confirmed by the fact that the 87-kDa band binds specifically to prothrombin. It was shown that the 87- and the 19-kDa molecules were present on the cell surface by surface labeling the cells with 125I. In addition, the fact that killed and washed cells could induce plasma clotting demonstrates that staphylococci have coagulase exposed on the surface. It was concluded that cell-bound coagulase has affinity for fibrinogen also in the absence of prothrombin and thus is responsible for the clumping of staphylococci in fibrinogen.     

Location of the coagulase gene in Staphylococcus aureus.

Evidence is presented for the chromosomal location of the coagulase determinant in most strains of Staphylococcus aureus. By the use of a pour-plate technique, transduction of the capacity to produce coagulase to a coagulase-negative mutant of S. aureus was studied. The frequencies of transduction were low unless the transducing phage was exposed to ultraviolet irradiation and the recipient was lysogenised with the transducing phage. Attempts to transfer the coagulase gene from S. aureus to S. epidermidis were not successful.     

Characterization of a humanized monoclonal antibody recognizing clumping factor A expressed by Staphylococcus aureus

We report the humanization and characterization of monoclonal antibody (MAb) T1-2 or tefibazumab, a monoclonal antibody that recognizes clumping factor A expressed on the surface of Staphylococcus aureus. We demonstrate that the binding kinetics of MAb T1-2 is indistinguishable compared to that of its murine parent. Furthermore, MAb T1-2 is shown to enhance the opsonophagocytic uptake of ClfA-coated latex beads, protect against an intravenous challenge in a prophylactic model of rabbit infective endocarditis, and enhance the efficacy of vancomycin therapy in a therapeutic model of established infective endocarditis.     

Contribution of clumping factor B to pathogenesis of experimental endocarditis due to Staphylococcus aureus

Staphylococcus aureus Newman with an insertion mutation in clfB, the gene encoding clumping factor B, only marginally decreased infection rate (P>0.05) in rats with experimental endocarditis. In contrast, clfB complementation on a multicopy plasmid significantly increased infectivity (P<0.05) over the deleted mutants. Although clfB could affect endovascular infection, its importance in experimental endocarditis was limited.     

Characterization of a protective monoclonal antibody recognizing Staphylococcus aureus MSCRAMM protein clumping factor A

The Staphylococcus aureus MSCRAMM (microbial surface components recognizing adhesive matrix molecules) protein clumping factor A (ClfA) has been shown to be a critical virulence factor in several experimental models of infection. This report describes the generation, characterization, and in vivo evaluation of a murine monoclonal antibody (MAb) against ClfA. Flow cytometric analysis revealed that MAb 12-9 recognized ClfA protein expressed by all of the clinical S. aureus strains obtained from a variety of sources. In assays measuring whole-cell S. aureus binding to human fibrinogen, MAb 12-9 inhibited S. aureus binding by over 90% and displaced up to 35% of the previously adherent S. aureus bacteria. Furthermore, a single infusion of MAb 12-9 was protective against an intravenous challenge with a methicillin-resistant strain of S. aureus in a murine sepsis model (P < 0.0001). These data suggest that anti-ClfA MAb 12-9 should be further investigated as a novel immunotherapy for the treatment and prevention of life-threatening S. aureus infections.     

Reassessing the role of Staphylococcus aureus clumping factor and fibronectin-binding protein by expression in Lactococcus lactis.

Since Staphylococcus aureus expresses multiple pathogenic factors, studying their individual roles in single-gene-knockout mutants is difficult. To circumvent this problem, S. aureus clumping factor A (clfA) and fibronectin-binding protein A (fnbA) genes were constitutively expressed in poorly pathogenic Lactococcus lactis using the recently described pOri23 vector. The recombinant organisms were tested in vitro for their adherence to immobilized fibrinogen and fibronectin and in vivo for their ability to infect rats with catheter-induced aortic vegetations. In vitro, both clfA and fnbA increased the adherence of lactococci to their specific ligands to a similar extent as the S. aureus gene donor. In vivo, the minimum inoculum size producing endocarditis in > or =80% of the rats (80% infective dose [ID80]) with the parent lactococcus was > or =10(7) CFU. In contrast, clfA-expressing and fnbA-expressing lactococci required only 10(5) CFU to infect the majority of the animals (P < 0.00005). This was comparable to the infectivities of classical endocarditis pathogens such as S. aureus and streptococci (ID80 = 10(4) to 10(5) CFU) in this model. The results confirmed the role of clfA in endovascular infection, but with a much higher degree of confidence than with single-gene-inactivated staphylococci. Moreover, they identified fnbA as a critical virulence factor of equivalent importance. This was in contrast to previous studies that produced controversial results regarding this very determinant. Taken together, the present observations suggest that if antiadhesin therapy were to be developed, at least both of the clfA and fnbA products should be blocked for the therapy to be effective.     

Genetic variation in Staphylococcus aureus coagulase genes: potential and limits for use as epidemiological marker.

To perform coagulase gene typing, the repeated units encoding hypervariable regions of the Staphylococcus aureus coagulase gene were amplified by the PCR technique; this was followed by AluI restriction enzyme digestion and analysis of restriction fragment length polymorphism (RFLP) patterns. In order to assess the discriminatory power of this typing method, 30 epidemiologically unrelated S. aureus strains which differed by their pulsed-field gel electrophoresis patterns were examined. Although 18 of the 30 strains had unique and unshared AluI RFLP patterns, there were only four observed patterns in the remaining 12 strains. This finding indicated that unrelated strains may share identical AluI RFLP patterns. To elucidate the degree of genetic variation in the C-terminus-encoding loci within the coagulase genes, the PCR products of these 12 strains were subjected to Taq polymerase-mediated sequencing. Sequence analysis confirmed the AluI recognition sites in each of the four RFLP groups and demonstrated that AluI appears to yield the highest RFLP in restriction enzyme analysis. By their DNA sequences the majority of strains sharing common AluI groups could be clearly differentiated from each other and revealed between 93.2 and 98.5% homology. When we determined the nucleotide sequences of two strains after six subcultivations no significant alterations were observed. Because the discriminatory power of the current coagulase gene typing method is not great enough to be used as the sole method to type S. aureus, additional techniques are necessary. Sequence analysis of the repeated unit-encoding region for the typing of S. aureus may be potentially useful as an alternative to other current molecular typing techniques.     

Evaluation of rapid coagulase methods for the identification of Staphylococcus aureus.

Four rapid latex agglutination assays, StaphAurex (Wellcome Diagnostics, Research Triangle Park, N.C.), Bacto Staph (Difco Laboratories, Detroit, Mich.), SeroSTAT (Scott Laboratories, Inc., Fiskeville, R.I.), Veri-Staph (Zeus Technologies, Raritan, N.J.), and two hemagglutination tests, Staphyloslide (BBL Microbiology Systems, Cockeysville, Md.) and Hemastaph (Remel, Lenexa, Kans.), were compared with the conventional slide coagulase, tube coagulase (TC), and thermonuclease (TNase) tests for the identification of Staphylococcus aureus. A total of 118 clinical isolates of S. aureus (52 methicillin resistant), 50 S. epidermidis, 5 S. capitis, 2 S. hominis, 3 S. simulans, 6 S. saprophyticus, and 2 S. warneri were tested. The slide coagulase, TC and TNase tests detected 115 (97.5%), 117 (99.2%), and 118 (100%) of the S. aureus isolates, respectively. All showed 100% specificity. The StaphAurex, Veri-Staph, Staphyloslide, Hemastaph, SeroSTAT, and Bacto Staph assays correctly identified 117 (99.2%), 117 (99.2%), 116 (98.3%), 110 (93.2%), 108 (91.5%), and 107 (90.7%) of the S. aureus isolates, respectively. For methicillin-resistant S. aureus isolates, StaphAurex, Veri-Staph, Staphyloslide, Hemastaph, SeroSTAT, and Bacto Staph showed 1 (2%), 1 (2%), 2 (4%), 7 (13.5%), 7 (13.5%), and 8 (15.4%) false-negative results, respectively. All the commercial agglutination assays demonstrated false-positive results with strains of S. capitis, S. saprophyticus and S. warneri. The overall accuracy of the commercial agglutination assays compared with TC and TNase ranged from 90.7 to 99.2%. We recommend that negative reactions with the rapid commercial test kits for methicillin-resistant Staphylococcus isolates be confirmed with the TC or TNase test.     

Phenotypical and genotypical characterization of epidemic clumping factor-negative, oxacillin-resistant Staphylococcus aureus.

A total of 50 oxacillin-resistant Staphylococcus aureus (ORSA) strains that were clumping factor negative (CFN) and protein A negative by latex agglutination were collected from patients in six different hospitals at different locations in Germany during 1991 and 1992. Antibiograms, bacteriophage typing, and plasmid analysis were performed. The antibiograms showed that, besides oxacillin, all CFN ORSA strains were resistant to gentamicin, clindamycin, erythromycin, ciprofloxacin, and fosfomycin. All these isolates were nontypeable with an international set of phages, and an additional experimental phage set indicated that the strains were phage type 16, 192. Moreover, all isolates possessed a single plasmid of 30 kb, and restriction analysis of those plasmids revealed identical patterns. For genotyping, these 50 isolates were also analyzed by pulsed-field gel electrophoresis (PFGE) and polymerase chain reaction (PCR) of the coagulase and protein A genes and then by restriction enzyme digestion and analysis of restriction fragment length polymorphisms (RFLPs). With 49 strains, electrophoresis of SmaI-digested chromosomal DNA revealed identical PFGE patterns regarding the number and size of the DNA fragments, which could be differentiated from those of clumping factor-positive ORSA strains. Typing for the coagulase gene by PCR revealed PCR products of identical sizes. The AluI restriction digestion patterns of the PCR products were identical. PCR with primers derived from the region of that part of the protein A gene that encodes the immunoglobulin G-binding domains showed a PCR product that was about 170 bp smaller than that of the protein A gene from strains that were positive in the protein A latex agglutination test. Since it is precisely this size that is required in order to encode one immunoglobulin G-binding region, we assume that this is not present in the CFN ORSA strains. The phenotypical and genotypical features identify these very unusual CFN ORSA stains as being of clonal origin.     

Protein A and coagulase expression in epidemic and non-epidemic Staphylococcus aureus.

Strains of Staphylococcus aureus were divided into groups on the basis of antimicrobial sensitivity and epidemiology and tested for protein A expression in a simple microtitre test, which detected the non-immunological binding of immunoglobulin to protein A on whole cells of S aureus. Isolates of the methicillin resistant strain prevalent in south east England (EMRSA) showed a low expression of protein A compared with the other strains of methicillin resistant S aureus (MRSA), other multiple resistant strains, and sensitive strains. Protein A and coagulase expression in 27 strains of MRSA from 15 countries associated with hospital outbreaks were compared with 27 strains of MRSA from 11 countries reported to be sporadic isolates. Twenty four of the 27 outbreak associated MRSA showed low expression of protein A and high expression of coagulase. Conversely, sporadic strains generally gave higher levels of protein A and a wide variety of coagulase reactions. The results suggest that many epidemic strains of MRSA may have phenotypic characteristics that distinguish them from sporadic strains.     

Both complement- and fibrinogen-dependent mechanisms contribute to platelet aggregation mediated by Staphylococcus aureus clumping factor B

Staphylococcus aureus can stimulate activation and aggregation of platelets, which are thought to be factors in the development of infective endocarditis. Previous studies have identified clumping factor A (ClfA) and fibronectin binding proteins A and B (FnBPA and FnBPB) as potent platelet aggregators. These proteins are able to stimulate rapid platelet aggregation by either a fibrinogen- or a fibronectin-dependent process which also requires antibodies specific to each protein. Slower aggregation has been seen in other systems where specific fibrinogen binding ligands are absent and platelet aggregation is mediated by complement and specific antibodies. Bacteria expressing ClfB aggregate platelets with a longer lag time than ClfA or FnBPA and FnBPB. In order to investigate whether ClfB causes platelet aggregation in a complement- or fibrinogen-dependent manner, a non-fibrinogen-binding mutant of ClfB (ClfB Q235A) was constructed. Lactococcus lactis expressing ClfB Q235A was able to stimulate platelet aggregation in platelet-rich plasma without a significant increase in lag time. The requirements for platelet aggregation were investigated using gel-filtered platelets. Fibrinogen and specific anti-ClfB antibodies were found to be sufficient to allow platelet aggregation mediated by the wild-type ClfB protein. It seems that ClfB causes platelet aggregation by a fibrinogen-dependent mechanism. The non-fibrinogen-binding ClfB mutant was unable to stimulate platelet aggregation under these conditions. However, bacteria expressing ClfB Q235A caused platelet aggregation in a complement-dependent manner which required specific anti-ClfB antibodies.     

Molecular typing of Staphylococcus aureus on the basis of coagulase gene polymorphisms.

Staphylocoagulase, a major phenotypic determinant of Staphylococcus aureus, exists in multiple allelic forms, in part because of the existence of gene variants within the 3'-end coding region. This region contains a series of repeating 81-bp DNA sequences which differ both in the number of tandem repeats and the location of AluI restriction sites among different isolates. Utilizing this finding, we developed a novel typing method for S. aureus based on polymerase chain reaction amplification of the variable region of the coagulase gene followed by AluI restriction enzyme digestion and analysis of restriction fragment length polymorphism (RFLP). Among 30 S. aureus isolates studied initially, a total of 10 distinct RFLP patterns were observed. There was excellent correlation of the RFLP patterns with typing of these isolates by multilocus enzyme electrophoresis at 20 chromosomal loci. This coagulase RFLP method was used to analyze an additional 39 S. aureus isolates and successfully traced the source of an outbreak of methicillin-resistant S. aureus infections at a local hospital.