Reliability of latex agglutination tests for identification of Staphylococcus aureus resistant to oxacillin.

Commercial latex agglutination tests (LATs) for the simultaneous detection of clumping factor and protein A are gaining increased acceptance as a means of identifying Staphylococcus aureus. We evaluated two LATs (Accu-Staph; Carr-Scarborough, Decatur, Ga.; Staphaurex; Wellcome, Dartford, England) with particular emphasis on their ability to correctly identify oxacillin-resistant S. aureus. We tested 59 oxacillin-resistant S. aureus, 136 oxacillin-susceptible S. aureus, and 92 coagulase-negative staphylococcal strains with the two LATs and with thermonuclease, slide clumping factor, tube coagulase, and protein A hemagglutination tests. Clumping factor and protein A were present in 96.9 and 82.1% of our S. aureus strains, respectively. Accu-Staph correctly identified 92.8% and Staphaurex correctly identified 91.3% of S. aureus strains. No significant difference in LAT positivity rates, presence of clumping factor, or presence of protein A was found between oxacillin-resistant and -susceptible S. aureus. Overall, there were 31 false-negative LATs for 20 S. aureus strains, 14 with Accu-Staph and 17 with Staphaurex. Ninety-five percent of these strains possessed either clumping factor or protein A or both when these factors were determined independently. There were five false-positive LATs for four strains of coagulase-negative staphylococci (three Staphylococcus epidermidis and one Staphylococcus warneri), four with Accu-Staph and one with Staphaurex. Clumping factor was present in one S. warneri strain. Thus, the specificities of Accu-Staph, Staphaurex, and the clumping factor test were 95.6, 98.9, and 98.9%, respectively. Our results indicated that LATs identify oxacillin-resistant and -susceptible S. aureus equally well; however, they offer no greater sensitivity or specificity than the clumping factor test for identification of S. aureus.     

Novel immunoenzymatic assay for identification of coagulase- and protein A-negative Staphylococcus aureus strains.

A purified monoclonal antibody (MAb) which specifically reacts with Staphylococcus aureus glucosaminidase was obtained. This MAb was utilized to develop an immunoenzymatic assay for the identification of S. aureus strains. The sensitivity of this assay, based on the simultaneous detection of S. aureus glucosaminidase and protein A, was evaluated by analyzing a total of 196 strains, 26 of which did not exhibit one or more of the following properties: protein A, clumping factor, and staphylocoagulase. All strains yielded positive results by the MAb-based immunoenzymatic test. The assay's ability to differentiate between S. aureus and other staphylococci was then analyzed by testing a total of 277 non-S. aureus strains that yielded negative results. Our data demonstrate that this immunoenzymatic assay can be used as a single S. aureus identification criterion, particularly useful for those strains negative for clumping factor, staphylocoagulase, or protein A.     

Comparative performances of six agglutination kits assessed by using typical and atypical strains of Staphylococcus aureus.

Six commercial agglutination tests designed for the identification of Staphylococcus aureus were compared by using a strain collection which included 512 staphylococci representing 33 species (318 isolates of Staphylococcus aureus [including 144 oxacillin resistant], 46 S. epidermidis isolates, 15 S. haemolyticus isolates, 12 S. saprophyticus isolates, 29 S. schleiferi isolates, 30 S. lugdunensis isolates, and 62 other coagulase-negative staphylococci). This group also included a proportion of strains with unusual phenotypes (e.g., 19 coagulase-negative S. aureus isolates, 26 clumping factor-negative S. aureus isolates, and 4 S. aureus isolates each with a double deficiency). The overall sensitivity for identification of typical and atypical S. aureus was high with the Staphaurex Plus test (Murex Biotech) (99.7%), the Pastorex Staph Plus test (Sanofi Diagnostics Pasteur) (99.7%), and the Slidex Staph Plus test (bioMérieux) (100%). The overall rate of specificity was affected by the unusual inclusion in this study of a high proportion of non-S. aureus species, such as S. lugdunensis and S. schleiferi, which express a clumping factor and therefore produce a positive result with the agglutination tests.     

Rapid and reliable identification of Staphylococcus aureus by a latex agglutination test

A latex slide agglutination test detecting clumping factor and protein A simultaneously is recommended for rapid and reliable routine identification of Staphylococcus aureus. Strains (836) of staphylococci isolated from clinical specimens were examined, all S. aureus strains identified by conventional methods were correctly differentiated by the latex test, and no false-positive results occurred with other staphylococci. The reagent is easy to prepare since plasma is the coating material.     

Rapid methods for identification of Staphylococcus aureus when both human and animal staphylococci are tested: comparison with a new immunoenzymatic assay.

A new immunoenzymatic assay (IEA) for the identification of Staphylococcus aureus strains of both human and animal origin was compared with rapid commercial kits. The sensitivities and specificities of the commercial kits varied from 90.2 to 96% and 90.8 to 93.7%, respectively. The IEA did not give any false-negative or false-positive results, while commercial kits gave high percentages of false-positive results among clumping factor-positive non-S. aureus strains. The IEA is particularly useful for isolates for which identification is doubtful, for large-scale epidemiological studies, and for identifying isolates from animals as S. aureus.     

New latex reagent using monoclonal antibodies to capsular polysaccharide for reliable identification of both oxacillin-susceptible and oxacillin-resistant Staphylococcus aureus.

A new latex agglutination test (Pastorex Staph-Plus, Sanofi Diagnostics Pasteur), consisting of a mixture of latex particles coated with fibrinogen and immunoglobulin G for the detection of clumping factor and protein A and latex particles sensitized with monoclonal antibodies directed to Staphylococcus aureus serotype 5 and 8 capsular polysaccharides, was compared with three commercially available rapid agglutination methods for the identification of 220 isolates of S. aureus (61 oxacillin resistant) and 128 isolates of coagulase-negative staphylococci. The sensitivity for identification of S. aureus was high with the Pastorex Staph-Plus test (98.6%) compared with those of the other tests, which ranged from 91.8 to 84.5%. Test sensitivities for the identification of oxacillin-resistant S. aureus were as follows: Pastorex Staph-Plus, 95.1%; Pastorex Staph, 73.8%; Staphyslide, 72.1%; and StaphAurex, 49.2%.     

Role of Protein A in the Serum-Soft Agar Technique

Formation of compact colonies of Staphylococcus aureus in serum-soft agar is mainly a result of a reaction between protein A and the Fc-part of immunoglobulin G and not a clumping factor-fibrinogen reaction.     

Comparison of five agglutination tests for identification of Staphylococcus aureus.

Various commercially produced agglutination kits are widely used for the identification of Staphylococcus aureus. These kits detect the presence of protein A and/or clumping factor on S. aureus. The literature has shown that methicillin-resistant S. aureus (MRSA) isolates which are deficient in both clumping factor and protein A may be misidentified. Two products, Slidex and Staphaurex Plus, utilize specific anti-S. aureus antibodies, potentially giving them greater sensitivity compared to products without these antibodies. We report a prospective study designed to compare the performance characteristics of Fastaph, Slidex, Staphaurex, Staphaurex Plus, Staphyloslide, and the tube coagulase test for the identification of staphylococcal isolates. All discrepant isolates were tested with the Gen-Probe AccuProbe S. aureus test and were identified to the species level with conventional reference biochemicals. A total of 1,193 isolates were tested, including 33 MRSA and 423 methicillin-sensitive S. aureus isolates. The sensitivities and specificities of the tests, respectively, were as follows: Fastaph, 99.1 and 98.9%; Slidex, 99.6 and 96.4%; Staphaurex, 98.9 and 99.9%; Staphaurex Plus, 99.6 and 93.9%; Staphyloslide, 99.1 and 98.9%; and tube coagulase, 99.3 and 100%. Sensitivity was excellent for all of the products tested. The specificities of Fastaph, Staphaurex, and Staphyloslide were excellent, while Staphaurex Plus and Slidex demonstrated less optimal results.     

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.     

Rapid Detection of Epidemic Strains of Methicillin-Resistant Staphylococcus aureus

Fifty methicillin-resistant Staphylococcus aureus (MRSA) initial isolates obtained from patients hospitalized in the orthopedic clinic of the Frankfurt University Hospital and 150 methicillin-sensitive Staphylococcus aureus (MSSA) isolates were investigated in this study to determine whether the Slidex Staph-Kit is capable of differentiating between MRSA and MSSA owing to its unique performance characteristics. The Slidex Staph-Kit is a combined latex hemagglutination test designed to detect clumping factor, protein A, and a specific surface immunogen for S. aureus. Clumping factor-positive strains cause erythrocytes sensitized with fibrinogen to hemagglutinate, thereby resulting in visible red clumps. S. aureus strains deficient in clumping factor agglutinate latex particles sensitized with specific antibodies against surface proteins of S. aureus, thereby resulting in visible white clumps. Our results demonstrate that white clumping has a 99% specificity as well as a 98% positive predictive value for MRSA. Clumping factor-negative MRSA, which have been reported to occur in several countries, are epidemic in the Frankfurt area and account for 80% of all MRSA initial isolates in the orthopedic clinic of the Frankfurt University Hospital. Genotyping of all MRSA isolates by macrorestriction analysis of chromosomal DNA revealed that 83% of clumping factor-negative MRSA are closely related to the “southern-German” epidemic strain. This is the first study demonstrating the Slidex Staph-Kit’s capability for identifying epidemic clumping factor-negative S. aureus strains as methicillin resistant even prior to antimicrobial susceptibility testing.     

Frequency of staphylococcal lysozyme production tested by plate method

Lysozyme production is a frequent property of staphylococcal strains isolated from various sources; all 503 tested strains of Staphylococcus aureus and 13 out of 35 strains of Staphylococcus epidermidis produced an enzyme lysing Micrococcus lysodeikticus as tested by a modified plate method. Lysozyme production by staphylococci is more frequent than the production of free coagulase, clumping factor, staphylokinase, Tween 80 lipase, and HgCl(2) resistance.     

Antibacterial action of extracellular mammalian group IIA phospholipase A2 against grossly clumped Staphylococcus aureus

Fibrinogen-dependent interactions of Staphylococcus aureus are believed to contribute to bacterial virulence by promoting bacterial attachment to fibrinogen-coated surfaces and inducing the formation of bacterial clumps that are likely resistant to phagocytosis. Although S. aureus produces several fibrinogen-binding proteins, the cell wall-associated protein clumping factor (encoded by clfA) appears to be most important in bacterial interactions with immobilized or soluble purified fibrinogen. We have compared bacterial clumping in several strains of S. aureus, including isogenic ClfA+ and ClfA- Newman strains, in the presence of purified rabbit fibrinogen, human plasma, and inflammatory fluid and examined the effect of clumping on bacterial sensitivity to mammalian group IIA phospholipase A2 (PLA2). This enzyme is the major extracellular bactericidal agent in inflammatory fluid active against S. aureus. Both ClfA-dependent and ClfA-independent bacterial clumping was observed, depending on the source and fibrinogen content of the biological fluid. In each case, clumping only partially reduced the antibacterial activity of PLA2, suggesting that this extracellular enzyme can substantially penetrate dense bacterial clumps. Bacterial clumps could be dispersed by added proteases, restoring full antibacterial activity to PLA2. Thus, the extracellular mobilization of group IIA PLA2 during inflammation may provide a mechanism by which the host can control the proliferation and survival of S. aureus even after bacterial clumping.     

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.     

Performance of four slide agglutination methods for identification of Staphylococcus aureus when testing methicillin-resistant staphylococci.

Four commercially available slide agglutination systems for identifying Staphylococcus aureus were compared with the conventional slide (clumping factor) and tube coagulase tests. The systems evaluated included Bacto Staph Latex (Difco Laboratories, Detroit, Mich.), Staphyloslide (BBL Microbiology Systems, Cockeysville, Md.), Mini ID Accu-Staph (Carr-Scarborough Microbiologicals, Inc., Decatur, Ga.), and Staphaurex (Wellcome Diagnostics, Research Triangle Park, N.C.). A total of 338 clinical isolates, including methicillin-resistant S. aureus (n = 149), methicillin-susceptible S. aureus (n = 78), methicillin-resistant, coagulase-negative staphylococci (n = 45), and methicillin-susceptible, coagulase-negative staphylococci (n = 66), were tested by each method. The slide test for clumping factor, the 4-h tube coagulase test, Bacto Staph Latex, Staphyloslide, Mini ID Accu-Staph, and Staphaurex detected 212 (93.4%), 218 (96%), 223 (98.2%), 223 (98.2%), 221 (97.4%), and 224 (98.7%) of the S. aureus (44% methicillin-resistant) isolates, respectively. There were no false-positive results with any of the methods when the 111 strains of coagulase-negative staphylococci were tested. The results of this evaluation suggest that the four slide identification methods tested can provide rapid and accurate identification of methicillin-resistant S. aureus strains.     

Quantitative comparison of clumping factor- and coagulase-mediated Staphylococcus aureus adhesion to surface-bound fibrinogen under flow.

The contributions of clumping factor and coagulase in mediating Staphylococcus aureus adhesion to surface-adsorbed fibrinogen have been quantified by using a new methodology and analysis. The attachment or detachment kinetics of bacteria were directly observed in a radial flow chamber with a well-defined laminar flow field and a spatially varying shear rate and were quantified by recursively scanning the chamber surface and counting cells via automated video microscopy and image analysis with a motorized stage and focus control. Intrinsic rate constants for attachment or detachment were estimated as functions of shear rate for the wild-type Newman strain of S. aureus and for mutants lacking clumping factor, coagulase, or both proteins on surfaces coated with plasma, fibrinogen, or albumin. Clumping factor, but not coagulase, increased the probability of attachment and decreased the probability of detachment of S. aureus on plasma-coated surfaces; however, both clumping factor and, to a lesser extent, coagulase increased the probability of attachment on the purified-fibrinogen-coated surface. All mutants were resistant to detachment on the purified-fibrinogen-coated surface, suggesting the possibility of an additional adhesion mechanism which was independent of coagulase or clumping factor and effective only for fully attached cells. Together, these results suggest that the presence of clumping factor plays the primary role in enhancing adhesion to surfaces with adsorbed fibrinogen, not only by enhancing the probability of cell attachment but also by increasing the strength of the resulting adhesion.     

Comparison of three blood-clotting substances in Staphylococcus aureus strains.

Besides the two well-known blood-clotting substances, coagulase and clumping factor, a third one has been identified from the staphylococci which is a cell surface polysaccharide, is alkali stable, and induces compact-colony formation in serum soft agar. Using some 97 clinical strains of Staphylococcus aureus, we found that in production and activity the substances were distinctly different.     

Use of the Clumping Factor Reaction for the Identification of Encapsulated Strains of Staphylococcus aureus from Human Sources

Using the clumping factor test on 815 strains of Staphylococcus aureus, 775 positive strains were of compact morphology. Of 40 negative strains, 39 were diffuse in serum-soft agar. The test may be used to detect capsulated S. aureus strains.     

Comparison of five tests for identification of Staphylococcus aureus from clinical samples.

Five different laboratory tests for the identification of Staphylococcus aureus were compared. Analyses of 271 presumptive S. aureus strains, supplemented with 59 well-defined methicillin-resistant S. aureus (MRSA) isolates, were performed. Only the Staphaurex Plus (Murex Diagnostics, Dartford, United Kingdom) and the Pastorex Staphplus (Sanofi, Marnes-La-Coquette, France) tests displayed 100% sensitivity. The observed difference with the free-coagulase test (Bacto coagulase plasma; Difco, Detroit, Mich.), a bound-coagulase (clumping factor) test, and the former Staphaurex test (Murex Diagnostics) was caused mainly by the inability of these three tests to identify some MRSA strains correctly. Among Polish MRSA isolates included in the analysis, a group of free-coagulase-negative S. aureus strains was detected. Genetic typing by random amplification of polymorphic DNA revealed that the strains showing aberrant behavior when the different test results were compared belonged to limited number of S. aureus clones.     

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.     

Isolation and Characterization of a sigB Deletion Mutant of Staphylococcus aureus

The sigB gene of Staphylococcus aureus, coding for the alternate sigma factor B, has been deleted by allelic replacement mutagenesis. The mutant grew as well as the parent in vitro, although it was deficient in clumping factor, coagulase, and pigment. In two murine and one rat infection model the mutant showed no reduction in virulence.