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.     

Reagent Strips and Conventional Tests for Acid Production from Mannitol and Coagulase Activity of Staphylococcus: a Comparative Study

A total of 244 Staphylococcus strains were tested simultaneously for acid production from mannitol and for coagulase activity with reagent-impregnated paper strips and with their conventional counterparts. Significant correlation was obtained with 97.9% of the strains for mannitol and with 95% for the coagulase test. The paper strip method is a combined test for both mannitol and coagulase tests, thus making it more convenient and simpler than conventional methods. The results are obtained rapidly within 6 hr by the paper strip method. However, as the paper strip method is designed for the aerobic system, the conventional tests were also carried out under aerobic conditions to compare the results.     

An improved method for the serotyping of free coagulase from Staphylococcus aureus.

The serotyping of free coagulase, one of the most reliable ways to identify strains of Staphylococcus aureus, and widely employed in Japan, has been improved by adding magnetite sand to the reaction mixture. Culture medium supernatant and a type-specific antibody are mixed in a well of a microtiter plate, and plasma-enriched bovine fibrinogen is treated with magnetite sand. The use of tranexamic acid and gum arabic in the reaction mixture also increases the sensitivity of the reaction. Finally, the plate is placed on a magnetic stirrer. If the type of the coagulase corresponds to that of the antibody, no clot formation will occur, and this is easily confirmed by the movement of the sand. Although the amount of reaction mixture required is much less than that for the conventional tube method, our new method is able to detect slight increases in viscosity of the reaction mixture due to fibrin formation even before complete clotting occurs, thus providing very high sensitivity. Clot formation can also be judged by observing a turbid mass of fibrin in the well (Hwang's method), but this approach is a little slower than our method involving immobilization of magnetite sand.     

A simple method for the detection of neurologic disorders associated with CAG repeat expansion using PCR-microtiter plate hybridization

A new screening method was developed for the detection of CAG expanded alleles in patients with hereditary ataxia using polymerase chain reaction-based microtiter plate hybridization (PCR-MPH). The system can be applied to detect pathologic alleles by hybridization with the immobilized (CAG)48 repeat probe derived from the unrelated gene 'ERDA1' except for the CAG repeats. We examined 10 individuals with SCA3, 10 with Huntington disease and 30 normal controls (31 controls for SCA3) using this method. The results showed that a clear discrimination was possible in all cases. We suggest that this system be made available for mass screening of patients with hereditary ataxia disorders. This report is the first to demonstrate that a PCR-MPH system can be successfully applied to DNA size differentiation in addition to base pair mismatches. Also, our design of the probe is unique in that the probe motif stem from the unrelated gene sequence and not from the synthetic oligonucleotides.     

Validity of an enzyme-linked immunosorbent assay with serotype-specific monoclonal antibodies for serotyping human rotavirus in stool specimens

We recently developed a method for serotyping human rotavirus (HRV) by an enzyme-linked immunosorbent assay with HRV serotype-specific neutralizing monoclonal antibodies (ELISA serotyping). In the present study this method was compared with the fluorescent focus neutralization test with serotype-specific rabbit antisera (NT serotyping) in the sensitivity and specificity of the test. Direct serotyping of HRVs which were contained in stool specimens indicated that while only 37% of the samples were successfully serotyped in NT, 78% of the samples could be serotyped in ELISA. Regarding the samples whose serotype could be determined in the two tests, the assigned serotypes were identical in both tests. The results obtained indicated the utility of ELISA serotyping in clinical and epidemiologic studies of HRV infection.     

Molecular cloning and expression of the coagulase gene of Staphylococcus aureus 8325-4

The gene coding for coagulase (coa) was cloned from Staphylococcus aureus 8325-4 in a lambda replacement vector in Escherichia coli. Coagulase (plasma-clotting) activity was measured in lambda coa lysates and an immunoreactive protein of 60 kDa was detected by Western immunoblotting with anti-coagulase serum. This protein comigrated with the major immunoreactive protein in supernatants of S. aureus 8325-4. The coa gene was subcloned in pUC vectors. One recombinant expressed a 60 kDa immunoreactive protein and plasma-clotting activity. A putative beta-galactosidase-coagulase fusion protein and truncated peptides were expressed by variants formed by subcloning. These results are consistent with previously published biochemical data that the prothrombin-binding domain of coagulase is located in the N-terminus of the protein. The cloned coa gene was transferred into S. aureus on a shuttle plasmid. Expression of coagulase was higher in a strain with a mutation in the agr locus, which controls the level of several exoproteins in S. aureus, suggesting that agr normally regulates coagulase expression negatively.     

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.     

The coagulase of Staphylococcus aureus 8325-4. Sequence analysis and virulence of site-specific coagulase-deficient mutants

The sequence of the coagulase gene (coa) from Staphylococcus aureus strain 8325-4 is reported. The deduced amino acid sequence of the coagulase protein is compared with previously reported sequences of coagulases from strains 213 and BB. The secreted mature forms of coagulase proteins are composed of three distinct segments: (i) the N-terminal 150-270 residues, which are c. 50% identical, (ii) a central region with high (greater than 90%) residue identities, and (iii) a C-terminal region composed of repeated 27-amino-acid residue sequences. The variable N-terminal sequences are probably responsible for antigenic differences among coagulases of different serotype. The region of coagulase which binds to prothrombin and activates it to form staphylothrombin is also located in the N-terminal half of the protein. A site-specific substitution mutation in the coa gene, which abolished plasma clotting activity, was isolated by recombinational allele-replacement in strains 8325-4 and M60. The Coa- mutants did not show diminished virulence in subcutaneous and intramammary infections of mice. No evidence for a role for coagulase in virulence of toxigenic or nontoxigenic strains was obtained. This contradicts findings of several groups using Coa- mutants generated by chemical mutagenesis and suggests that the earlier results were obtained with strains that had suffered additional mutations in virulence-related genes.     

Nephelometric assay of staphylococal coagulase

Stutzenberger, Fred J. (Michigan State University, East Lansing), Charles L. San Clemente, and Dharam V. Vadehra. Nephelometric assay of staphylococcal coagulase. J. Bacteriol. 92:1005-1009. 1966.-Clotting of fibrinogen by staphylococcal coagulase was accompanied by an increase in light scattering; this property was used as a basis for a new nephelometric method. Reaction rates, which were now easily and precisely measured, were found to be directly proportional to coagulase concentration, when optimal conditions were maintained. These conditions included pH and concentrations of fibrinogen, coagulase-reacting factor, and sodium chloride in the reaction mixture. A standardized procedure for the assay is outlined, and a unit for the expression of activity is proposed.     

A Yersinia pestis-specific DNA fragment encodes temperature-dependent coagulase and fibrinolysin associated phenotypes

The effect of temperature on coagulase and fibrinolysin expression (Pla) by Yersinia pestis has been implicated in the transmission of plague by fleas. In an attempt to improve our understanding of this process, we have cloned, sequenced and characterized the gene encoding the Pla phenotypes in Y. pestis, and examined its temperature-dependent regulation. The coding region for this gene overlaps a 900bp Y. pestis-specific DNA fragment that we have previously shown to be capable of detecting plague bacilli in fleas. The pla gene contains a single open reading frame encoding 312 amino acids with a predicted molecular weight of 34.7 kD and a putative signal sequence of 20 amino acids. This coding region appears to be sufficient for both coagulase and fibrinolytic activities. In Y. pestis, modulation between coagulase and fibrinolytic activities is temperature-dependent: coagulase activity is most evident at temperatures below 30 degrees C but fibrinolytic activity increases with higher temperatures (greater than 30 degrees C), regardless of the temperature at which the bacteria are grown. Our results lead us to believe that this regulation occurs post-translationally. It is possible that the alternative forms of the Pla protein are essential to 'flea blockage' and subsequent transmission of the plague bacillus to animals.     

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.     

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°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°C for 18 h, and the coagulase reaction is recorded. The plates are then heated at 65°C for 2.5 h, overlaid with toluidine blue-metachromatic diffusion agar, and reincubated at 37°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.     

Use of antiserum-coated latex particles for serotyping Streptococcus pneumoniae

The Quellung reaction provides a standard means for serotyping Streptococcus pneumoniae, but it requires microscopic examination with skillful technique. We have developed an improved agglutination method with anti-rabbit IgG-coated latex particles, which are sensitized with pooled antisera for serotyping/serogrouping S. pneumoniae. Our method is as specific and sensitive as the Quellung test, and much easier to perform.     

Surface antigen structure of Neisseria meningitidis. I. The isolation of a serotypic antigenic complex of N. meningitidis strain B16B6 by direct detergent treatment and its immunochemical characteristics

The method for obtaining a serotyping antigenic complex from N. meningitidis B16B6 by their direct treatment with the mixture of detergents (0.5% sodium desoxycholate and 0,5% cholic acid in the proportion 1 : 1) in 0.5 M KCl solution is proposed. Such treatment has been found to increase the yield of the preparation in terms of protein more than 4 times in comparison with earlier methods for obtaining serotyping antigens. The immunochemical study of the preparation has demonstrated its serological specificity and high immunological activity, not inferior to that of serotyping antigenic preparations from group B meningococci, obtained by the heretofore known methods.     

Elucidation of structural and antigenic properties of pneumococcal serotype 11A, 11B, 11C, and 11F polysaccharide capsules

Despite the emerging impact of serogroup 11 serotypes in Streptococcus pneumoniae epidemiology, the structures of serogroup 11 capsule types have not been fully elucidated, particularly the locations of O-acetyl substitutions. Here, we report the complete structures of the serotype 11B, 11C, and 11F polysaccharides and a revision to the serotype 11A capsular polysaccharide using nuclear magnetic resonance (NMR). All structures shared a linear, tetrasaccharide backbone with a pendant phosphopolyalcohol. Three of four saccharides are conserved in all serotypes. The individual serotype capsules differed in the identity of one saccharide, the pendant phosphopolyalcohol, and the O-acetylation pattern. Though the assigned locations of O-acetate substitutions in this study differed from those of previous reports, our findings were corroborated with strong correlations to serology and genetics. We examined the binding of serotyping sera to serogroup 11 polysaccharides by using flow cytometry and an inhibition-type enzyme-linked immunosorbent assay (ELISA) and found that de-O-acetylation of capsular polysaccharides by mild hydrolysis decreases its immunoreactivity, supporting the crucial role of O-acetylation in the antigenicity of these polysaccharides. Due to strong correlations between polysaccharide structures and capsule biosynthesis genes, we were able to assign target substrates for the O-acetyltransferases encoded by wcwC, wcwR, wcwT, and wcjE. We identified antigenic determinants for serogroup 11 serotyping sera and highlight the idea that conventional serotyping methods are not capable of recognizing all putative variants of S. pneumoniae serogroup 11.     

Characteristics of defective strains of methicillin resistance Staphylococcus aureus that do not produce coagulase or CF

The aim of the study was to estimate frequency of coagulase-negative and CF-negative strains among methicillin-resistant Staphylococcus aureus (MRSA) and to assess their homogeneicity in respect of genotype, phagotype and drug resistance pattern. A total of 186 MRSA strains collected from different hospitals in Gdańsk region were studied. Gens: nuc, mecA, and coa were identified by PCR method. The coagulase tube test for staphylocoagulase and the slide test for clumping factor were used. Coagulase-negative and CF-negative MRSA strains were confirmed by PCR-RFLP method of coa gene; phage typing and drug resistance pattern were evaluated by disc diffusion test. The results of the study showed low frequency of both coagulase-negative and CF-negative MRSA strains (7.25% and 3.76% respectively). Among MRSA population tested the simultaneous occurrence of the strains lacking coagulase and clumping factor was not observed. All coagulase negative MRSA had coagulase gene (coa) and differed from CF-negative strains in respect of coa gene.     

Digoxigenin-labelled inv- and ail-probes for the detection and identification of pathogenic Yersinia enterocolitica in clinical specimens and naturally contaminated pig samples

A non-radioactive colony hybridization method was developed for the rapid detection of Yersinia enterocolitica in primary isolates and for differentiation between pathogenic and non-pathogenic strains. The method is based on, respectively, the presence of the inv -locus in all Yersinia spp. and the presence of the ail -gene in pathogenic Y. enterocolitica only. Hybridization results with ail -probes of 132 strains of Y. enterocolitica were in good agreement with pathogenicity phenotypes as indicated by a tissue culture invasion (TCI) assay and by serotyping. All TCI⁺ strains and only two TCI^- strains were positive by hybridization with ail. Hybridization results with inv - or ail -probes of 150 primary isolates of human, animal or slaughterhouse origin were compared with those of conventional methods to detect and identify Y. enterocolitica. All samples that were positive for Yersinia spp. by cultivation (four of 66) or were positive for pathogenic Y. enterocolitica by cultivation and serotyping (six of 84) were also positive by hybridization with, respectively, the inv - or ail -probe. In three slaughterhouse swab samples, in which Yersinia spp. were not detected by cultivation (2%), strong positive hybridization signals were obtained with the inv - and/or ail -probe. Four other swab samples which were negative by cultivation produced weak positive signals by hybridization with inv - and/or ail - probes. These results indicate that the method can be used for (1) the identification of pathogenic Y. enterocolitica isolates and (2) the detection of Yersinia spp. in primary isolates of naturally contaminated samples.     

Serotyping of dengue viruses by an enzyme-linked immunosorbent assay

We have developed a sandwich enzyme-linked immunosorbent assay for serotyping dengue viruses. In this assay, we used antibody from dengue hemorrhagic fever patients for detection of flavivirus common antigens to confirm virus isolation in C6/36 cells and that from hyperimmune mouse ascitic fluids for serotyping. The anti-dengue antibody was immobilized on microplate wells for capturing of dengue antigens, which were then sandwiched with the same biotinylated antibody. Then the biotin in the solid phase was detected with peroxidase-conjugated streptavidin. We found that all the dengue strains tested were unequivocally identified by this method.     

Effect of High Oxygen Concentration on Virulence of Staphylococcus aureus

Cultures of Staphylococcus aureus were agitated and treated with oxygen or air for a period of 29 days. At intervals of 1, 3, 9, 15, and 29 days, samples of washed cells were tested for coagulase activity and for abscess-producing ability. It was found that aeration with agitation initially increased abscess formation and that the increase was of the same magnitude for oxygen and air. Continued treatment beyond 1 day resulted in a progressive decrease in this activity which was more pronounced in the oxygenated cultures. Throughout the treatment, the bound coagulase activity remained constant. Thus, there appeared to be no quantitative relationship of coagulase titer with virulence as expressed by lesion formation.     

Detection of type-specific antibody to lymphogranuloma venereum serotypes ofChlamydia trachomatis with a microneutralization test

Ten-day monospecific mouse antisera prepared against serotypes L1, L2, and L3 ofChlamydia trachomatis were titrated against homologous and heterologous antigens in the microimmunofluorescence (micro-IF) test and by a new microneutralization method. While broad crossreactivity between serotypes was detected in the micro-IF test, the same antisera demonstrated serotype specific neutralizing activity. The microneutralization test could be used for serotyping new chlamydial isolates when equivocal results have been obtained by the microimmunofluorescence method.