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The fine structure of the capsule of Staphylococcus aureus Smith diffuse was examined by the technique of freeze-substitution and immunoelectron microscopy. The cell surface was covered with a thick layer consisting of fine fibrous structures which were absent from an unencapsulated strain, Smith compact. Anti-teichoic acid antibody did not react with this surface layer but reacted with the surface of strain Smith compact. Anti-capsular antibody, made from the serum of a rabbit immunized with strain Smith diffuse and specific absorption with unencapsulated strain Wood 46, reacted with the fibrous layer of the Smith diffuse strain. Since the anti-teichoic acid antibody did not react with the encapsulated strain Smith diffuse, the capsular layer acts as a barrier to penetration of the anti-teichoic acid antibody through the capsular layer. A portion of a few cell surfaces of the encapsulated strain remained accessible to the anti-teichoic acid antibody. The capsular layer in this portion of the cell surface was thin, and this surface seemed to be a new cell wall surface created by the cell separation.

A total of 89 Staphylococcus aureus strains were tested for [125I]-labelled fibronectin (Fn), type I (Cn-I), type II (Cn-II), type IV (Cn-IV) collagens and laminin (Lm) binding, and nasal carriage isolates (54 strains) demonstrated higher degree of interaction than clinical isolates (35 strains). Strains belonging to nasal carriage group, after preincubation with mucin demonstrated a significant decline in binding to Fn (39.4%), Cn-I (44.7%), Cn-IV (42.0%) and Lm (43.5%) compared with inhibition of binding of clinical isolates to Fn (13.3%), Cn-I (8.0%), Cn-IV (9.8%) and Lm (11.2%). S. aureus strain Nig-6 demonstrated a mucin concentration (in the range 0.01 to 100 mg/ml) dependent decrease of [125I]-labelled serum and connective tissue protein binding. Mucin concentrations of 100, 150, 175 and 250 micrograms/ml when incubated with 10(9) cells, caused 50% displacement of [125I]-labelled Lm, Cn-I, Cn-IV and Fn uptake respectively. Mucin interaction with bacterial cells seems probably important in the pathomechanism of staphylococcal adhesion and colonization.

An immunosorbent column was prepared by coupling a murine monoclonal antibody (MAb) with dual specificity for staphylococcal enterotoxins A (SEA) and E (SEE) to Affi-Gel 10. Purification of both SEA and SEE from culture supernatants was carried out with the immunosorbent column using 0.2 M acetic acid containing 0.15 M NaCl as eluant. The yields obtained were approximately 76% for SEA and 70% for SEE. Purified SEA and SEE were found to be immunologically and electrophoretically homogeneous. Immunoaffinity chromatography using a MAb with dual specificity proved to be valuable in the purification of SEA and SEE, not only from the standpoint of percentage recovery, but also because of the degree of purity and the ease of purification.

For purification of F and S components of the Panton-Valentine leukocidin, an easy three-step method using fast protein liquid chromatography was developed to replace the time-consuming purification procedures previously published. This technique enabled the recovery of 13 and 17 mg of purified F and S, respectively, per litre of culture supernatant. Affinity-purified neutralizing polyclonal antibodies were obtained against each individual component. One hundred and thirty-nine Staphylococcus aureus strains isolated from various clinical samples of hospitalized patients were screened by immunoprecipitation for Panton-Valentine leukocidin (PVL) production. Only 8 strains produced PVL; all originated from injured superficial soft tissues. Contrary to widespread opinion, the 8 PVL-producing strains were never associated with severe infection.

Gas-liquid chromatography of cellular fatty acids is a useful tool for the identification of bacteria. Derivatization of bacterial fatty acids to methyl esters by conventional techniques is usually time-consuming and complicated. A new one-step technique using trimethyl-sulfonium hydroxide allows the direct formation of fatty acid methyl esters within 1-2 min. Some random examples of profiles demonstrate that straight, branched, saturated, unsaturated, hydroxy and cyclopropyl fatty acids match conventional preparations well. The method is a very sensitive one, since only a few colonies are sufficient for preparation of fatty acid methyl esters.

A semi-homogeneous amperometric immunosensor specific to the protein A of Staphylococcus aureus was developed using direct electrochemical detection of phenol produced by alkaline phosphatase from phenyl phosphate. The immunosensor could reliably detect strains of protein A-bearing S. aureus in pure cultures at ca. 10(4) cfu/ml, and at ca. 10(5) cfu/g or ml in various food samples. Due to its semi-homogeneous nature, the system was very simple, easy to operate, and labour-saving. The good correlation between the amperometric current generated by the immunosensor and plate counts illustrated the potential usefulness of this simple system. It proved to be a reliable 24-h detection method for food samples containing very low numbers of protein A-bearing S. aureus after pre-enrichment, as it was able to detect cells that could not directly be enumerated by plate counts.

Human insulin-like growth factor I, IGF-I, was produced in Escherichia coli fused to a synthetic IgG-binding peptide The fusion protein is secreted into the medium during fermentation and was initially purified on an IgG-Sepharose column. After hydroxylamine cleavage, IGF-I was purified to homogeneity. During purification, impurities in the form of modified variants of IGF-I were detected and characterized. The closely related impurities were identified to be a misfolded form of IGF-I, having mismatched disulphide bonds, a form with the single methionine residue in IGF-I oxidized to methionine sulphoxide and a variant in which the methionine residue was substituted by a norleucine residue during protein synthesis. A form proteolytically cleaved between two arginine residue was also detected. These impurities were separated from the major component, native IGF-I, by using reverse-phase h.p.l.c. The modified molecules as well as native IGF-I were characterized both as intact molecules and as fragments, after pepsin digestion, using the techniques of plasma desorption m.s., N-terminal sequencing and amino acid analysis. The oxidized form was 90%, and the norleucine analogue was 70%, as potent as native IGF-I in a biological radioreceptor assay, and the form having mismatched disulphides lacked receptor affinity.

L-[4,5-3H]isoleucine was introduced to label anteiso-fatty acid (AIFA)-containing lipids in Staphylococcus aureus SG 511. After an overnight incubation in peptone broth in the presence of 37 kBq L-[4,5-3H]isoleucine/ml, 8.5-13% of the total radioactivity applied was found to be incorporated into the cells. 22.4-25.6% of the incorporated radioactivity was found in AIFA-containing lipids extracted by chloroform-methanol-water (2:1:0.2, v/v/v) at pH 2. The interphase contained 70-75% of the incorporated radioactivity. Lipoteichoic acid, extracted by phenol-water (80:20, w/v) contained less than 1% of the incorporated radioactivity, as measured after purification by hydrophobic interaction chromatography on octyl sepharose gel. Within 1 h after addition of 10 micrograms/ml penicillin G to exponentially growing cultures of S. aureus, that led to non-lytic death of the cells, 11.9-18.1% of the incorporated L-[4,5-3H]isoleucine label were released. Lipids containing AIFA were excreted to 5.4-8.4% of total incorporated activity; this amount represents more than 1/4 of the labeled cellular lipids.

Ten tested cultures each of Staphylococcus aureus (S. aureus) and of Streptococcus belonging to serological group G bound human IgG to a high extent. Protein A could be solubilized from strain Cowan I of S. aureus by lysozyme, mutanolysine, hydroxylammoniumchloride, hot acid extraction or lysostaphin and subsequently purified by affinity chromatography on human IgG-sepharose. The purified protein A preparation had molecular weights between 29,000 and 63,000 D and inhibited binding of 125I-labeled human IgG to S. aureus Cowan I. Protein G could be solubilized from strain 26540 of the G-streptococci with lysozyme or hot acid extraction and purified by affinity chromatography on human IgG-sepharose. The purified protein G revealed a molecular weight of 67,000 D and inhibited binding of human IgG to the G-streptococci.

The peptidoglycan of Staphylococcus aureus contains relatively short glycan chains and is highly cross-linked via its peptide chains. The material from wild-type (strain H) and mutants H28, H4B and MR-1 was freed from the teichoic-acid-linked component and then hydrolysed by Chalaropsis muramidase to yield disaccharide-repeating units of the glycan with attached peptides either non-cross-linked (monomer) or joined to similar units by one (dimer), two (trimer) or more (oligomer) peptide cross links. The resulting fragments were separated by high-resolution HPLC so that distinguishable components as large as nonamer could be identified. Extrapolation showed that, in S. aureus H, H28 and MR-1, oligomers at least as large as eicosamer formed part of the smooth distribution of oligomer fragments, whereas in strain H4B (PBP4-) the maximum size was around dodecamer. The oligomer distribution profile was related to the polymerization theories of Flory, which allow a distinction to be made between a monomer addition model, whereby each oligomer can only be synthesized by the addition of a single monomer unit to its next lower homologue, and a random addition model, in which an oligomer can be formed by linkage of any combination of its constituent smaller units. In S. aureus close approximation to the random addition model for oligomer synthesis and hence for peptidoglycan cross-linking was observed, both in PBP4+ and PBP4- mutants. The implications for secondary cross-linking in S. aureus cell wall formation are inescapable, although the possibility of an endopeptidase/transpeptidase providing later modification of the peptidoglycan is not completely ruled out.

Although pyrolysis-mass spectrometry (Py-MS) has been used for bacterial taxonomy, many of the mass spectral peaks used for discrimination of organisms have not been correlated to known biomolecules. This work presents the discrimination of five bacterial species based on Py-MS patterns containing only peaks that can be correlated to fatty acids and fatty acid derivatives. These correlations were confirmed by pyrolysis-tandem mass spectrometry of authentic standards and the organisms. The pattern recognition procedures used gave better results when only the fatty acid peaks were used in the analysis than when full spectra were used.

Two proteins derived from the cell wall of Staphylococcus aureus, exhibiting apparent molecular masses of 116 kDa and 145 kDa, were found to bind to human buccal and bovine lactiferous sinus epithelial cells. By using antibodies specific for fibronectin-binding protein of S. aureus of human origin, the 116 kDa protein, but not the 145 kDa protein, was identified as a fibronectin-binding protein. The 145 kDa protein bound to bovine fat globule membranes, human buccal epithelial cells, bovine lactiferous sinus epithelial cells and sheep erythrocytes. The properties of the 145 kDa protein suggest that it is an adhesin with a possible role in the early stages of the development of bovine mastitis.

Among the S. aureus 25/39 are non producing slime and non adherent isolates. These results did not allow a correlation between these properties and pathogenesis. Several different phenotyping systems (biotyping, phage typing, serotyping, antibiotic susceptibility profiling, and plasmid pattern analysis) have been used in an attempt to identify strains of CNS. There is still a need however, for a simple, rapid, and cost effective method of distinguishing true pathogens from simple contaminants. Our results suggest that testing isolates for slime positivity and for adherence property may fulfill this task.

A simple method for the purification of staphylococcal enterotoxins A (SEA), B (SEB), and C2 (SEC2) from fermentor-grown cultures was developed. The toxins were purified by pseudo-affinity chromatography by using the triazine textile dye "Red A" and gave overall yields of 49% (SEA), 44% (SEB), and 53% (SEC2). The purified toxins were homogeneous when analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, but isoelectric focusing of the preparations revealed the microheterogeneity associated with these toxins. The SEA and SEB preparations each consisted of two isoelectric forms with pI values of 7.3 and 6.8 (SEA) and 8.9 and 8.55 (SEB); in contrast, SEC2 contained five different isoelectric forms, with pI values ranging between 7.6 and 6.85. The pattern of elution of the isoelectric forms from the column indicated a cationic-exchange process involved in the binding of toxin to Red A. Such a method forms the basis of a high-yielding, rapid means of purifying the staphylococcal enterotoxins that can easily be adapted to large-scale production.

The composition of polyamines is studied for the first time in representatives of the genus Micrococcus and taxon "conglomeratus", strains Staphylococcus aureus CCM 209, Deinococcus erythromyxa CCM 706 as well as of Erwinia carotovora ATCC 15713 polyamines, which are not extracted by perchloric acid. Considerable amounts of spermine and rarely of spermidine are revealed in cells of Gram positive microorganisms, that differs them from Gram negative bacteria possessing high concentrations of putrescine, spermidine and their derivatives. A procedure is developed for detection of polyamines in cells of Gram positive microorganisms. It is recommended to use the hydrolysis of their cells by 6N HCl for 4 at 120 degrees C or for 8-10h at 100 degrees C with the subsequent electrophoretic separation. Putrescine, as well as comparable with it amount of agmatine and spermidine traces are found in Erwinia carotovora ATCC 15713 cell hydrolyzates, whereas putrescine and agmatine traces are found in perchloric extracts of intact cells. Spermine is not observed in the cells. The binding of polyamines with biopolymers of cells of Gram positive bacteria and their difference by the given character from the Gram negative procaryotes are under discussion.

Solid-state 2H NMR and 31P NMR of 2H-enriched chains and polar head groups, respectively, of dipalmitoylglycerophosphatidylcholine/water dispersions were undertaken to investigate the action of delta-haemolysin from Staphylococcus aureus on biomembranes. When the lipid/toxin molar ratio, Ri, is greater than or equal to 10, the gel-phase 2H powder patterns and the temperature of the gel-fluid phase transition, tc, are unchanged by the presence of the toxin whereas the 31P powder spectra of polar head groups are perturbed. At t greater than tc, a detailed analysis of methylene ordering indicates that delta-haemolysin orders the lipid chains near tc and disorders them for t much greater than tc. These findings are interpreted in terms of peptide location with regard to the membrane and suggest that the position of the toxin depends on the temperature relative to tc. Experiments carried out at Ri = 4 exhibit sharp, isotropic 2H-NMR lines, at t greater than tc, indicating that delta-haemolysin promotes the appearance of very small objects undergoing fast isotropic reorientation which average to zero the quadrupolar interaction. Below tc, one observes gel-phase powder patterns which indicate that the bacterial toxin is unable to form such small structures with ordered dipalmitoylglycerophosphocholine phospholipids. From comparison of the action of delta-haemolysin with that of melittin on same lipids [Dufourc et al. (1986) Biochemistry 25, 6448-6455] it results that both toxins perturb similarly fluid-phase lipids at elevated temperature, but they behave differently with gel-phase lipids, the former toxin being less efficient in membrane restructuring than the latter.

The diagnostic value of counter-current electrophoresis for the detection of Staphylococcus aureus protein A in the material from patients, carriers, and environmental objects is demonstrated: this method identifies 99.8% of S. aureus cultures. Microorganisms of other species and physiologic groups, not incorporating protein A, are undetectable by counter-current electrophoresis.

To observe the possible serological heterogeneity of compact-colony-forming active substance (CCFAS), heat-killed vaccines were prepared by two strains of Staphylococcus aureus, strains SMU 1-46 and SMU 7931, cultured in 0.03 M trishydrochloride-buffered brain heart infusion, pH 8.4. After immunization with the vaccine in rabbits, antibody responses were observed during a period of six weeks after the immunization either by homologous and heterologous organisms using alkaline serum-soft agar technique. The results showed that remarkable antibody production was shown only against homologous strain, but not against heterologous strain. The antibodies were absorbed out only with highly purified preparation of CCFAS extracted from homologous strain and not with heterologous CCFAS. Differences of the major chemical composition of the substances showed that highly purified CCFAS extracted from strain SMU 7931 contained 2.84 and 2.04 times higher amounts of galactose and 2-amino-2-deoxy-D-galacturonic acid than those of CCFAS obtained from strain SMU 1-46.

Infection models were used to clarify the roles of serum and extravascular concentrations in the in-vivo efficacy observed with azithromycin. In-vivo experiments were designed to give serum concentrations well below the MIC and tissue levels generally above the MIC at time of challenge and during the course of infection. The efficacy of azithromycin against a Salmonella enteritidis oral challenge (a tissue-associated infection model) in mice correlated directly with azithromycin liver levels, but not serum concentrations. The significance of extravascular pharmacokinetics was observed in a comparative study of azithromycin and ciprofloxacin against the salmonella challenge. Ciprofloxacin has a greater than 100-fold in-vitro potency advantage over azithromycin against this organism, but azithromycin (5 mg/kg) produced a greater reduction in cfu than ciprofloxacin (100 mg/kg) at the primary site of infection (liver). In another model, extravascular fluid levels, measured by bioassay of implanted paper discs, were compared with plasma levels in relation to control of a localized Staphylococcus aureus infection in rats. Extravascular fluid levels of azithromycin were greater than the MIC of the strain used for five days after a 100 mg/kg dose, while erythromycin levels were less than 20% of the MIC at 30 h after a 200 mg/kg dose. Serum concentrations of both compounds were less than 20% of the MIC at the time of challenge. The antibiotic levels at the site of infection correlated with the reduction of Staph. aureus cfu (99% with azithromycin compared with controls, P less than 0.01; 0% with erythromycin) recovered from inoculated discs. The significance of extravascular concentrations of azithromycin was further supported in other models of localized infections induced with Escherichia coli or a mixture of Staph. aureus and Bacteroides fragilis.

The relationship between capsular polysaccharide types 5 and 8 and esterase electrophoretic types (zymotypes) in 160 French clinical isolates of Staphylococcus aureus was studied. Methicillin-susceptible strains of capsular types 5 and 8 were represented by 11 zymotypes, indicating a high polymorphism. Methicillin-resistant strains were mainly distributed in only two distinct populations. The predominant population was represented by strains of zymotype 6 and capsular type 5, and the second population was represented by strains of zymotype 14 and capsular type 8.

Radioiodinated staphylococcal epidermolytic toxin was found not to bind to erythrocytes, blood leukocytes, trypsin-dispersed keratinocytes, epidermis or whole skin. Moreover the toxin could not be found to bind to murine epithelia by indirect immunofluorescence methods. However, the toxin, measured by radioimmunoassay, could be eluted from the skin of mice undergoing epidermolysis following intraperitoneal injection of toxinogenic Staphylococcus aureus. Furthermore, epidemolysin was measured in the blister fluid of 3 of 5 children with bullous impetigo but not in blister fluid from control patients with other blistering eruptions. Thus epidermolysin has been demonstrated to be present in lesions of the staphylococcal epidermolytic toxin syndrome but its mechanism of action does not involve binding to cells.

The presence of A-protein was sought in strains of S. epidermidis and S. aureus, sensitive or resistant to methicillin. The technique used was that of passive hemagglutination of red cells of sheep, either normal or formolated and sensitised by anti-erythrocyte antibodies. A-protein was not found in any of the 157 strains of S. epidermidis. It was found in 82.5 p. cent of 360 strains of S. aureus. The methicillin resistant strains seemed less rich than the protein A-sensitive strains, which was detected more frequently when formolated red cells were used. The interest of this test for bacteriological diagnosis is discussed.

The teichoic acid of polysaccharide P (poly P) contains glycerol, glucose and phosphate. Hydrofluoric acid and alkali hydrolysates contain glycerol 1-phosphate, glycerol diphosphate, and glucose 1-phosphate, but no glucosyl-glycerol fragments. Glucose and the serological activity of poly P were destroyed by periodate oxidation. Interaction with concanavalin A showed that the glucose is in alpha-configuration and that the hydroxyl groups at positions 3, 4 and 6 are unsubstituted. Most probably, the poly P teichoic acid is a polymer containing a repeating unit in which glycerol 1-phosphate is attached to the 2-position on alpha-D-glucose 1-phosphate.

To study the active site(s) in protein A, partial tryptic digestions of the protein and of intact Staphylococcus aureus were performed. Fragments which bind to the Fc-part of human IgG were isolated by affinity chromatography on IgG-Sepharose 4B and purified by ion-exchange chromatography on phosphocellulose. From a partial tryptic digest of pure protein A at 30 degrees C, pH 8.2 for 30 min we have isolated and characterized six active fragments with molecular weights ranging from 6000 to 8000. Two active fragments, obtained in high yields by digestion at pH 7.2 of intact protein-A-containing bacteria, were shown to be similar to two of the six characterized fragments from the digest of pure protein A. All fragments appeared to have similar amino acid sequences, judged by peptide mapping, specific staining and amino acid analysis; some are very possibly overlapping peptides. Each fragment probably contains only one active site region since all are monovalent in the Fc-reaction when studied with a hemagglutination technique. The maximal molar yield of active fragments obtained from the digestion of pure protein A accounts for about 210% of the amount of protein A used. Thus protein A, suggested to consist of repeating units, should exhibit at least three similar if not identical active regions.

The capsule material of Staphylococcus aureus strain 1193/74 could be separated by precipitation with trichloroacetic acid and ethanol as well as by chromatography on DEAE-cellulose into 13 fractions. All fractions contained saccharides and uronic acids as well as amino acids and appeared in their qualitative composition rather similar. However, in quantitative composition and in chromatographic behaviour a rather high degree of heterogeneity could be observed. No clear cut separation of protein and polysaccharide material could be achieved in any fraction. It is supposed, therefore, that the capsule material does not represent merely an acidic polysaccharide, but contains certain amounts of amino acids or peptides varying in a rather wide range.

Staphylococcal protein A could be extracted in large amounts by simple stirring of the cells with physiological saline (pH 7.8). The amount of protein A obtained by the technique was found to be the same as obtained by lysozyme/lysostaphin techniques (45 micrograms/ml). The protein A extracted by salt-washing technique was of higher molecular weight (71,000) and differing in some biological properties like complement fixation from the protein A prepared by lysostaphin technique.

Concentrated supernates of cultures of 98 strains of Staphylococcus aureus were screened for the production of epidermolytic toxin by (1) biological tests in 3-day-old mice, (2) double-diffusion precipitation tests against specific antiserum, and (3) the appearance of characteristic protein bands on thin-layer-gel isoelectric focusing. Positive results were obtained in all three of these tests with supernates from 11 of these cultures; the same 11 strains, and no others, produced epidermal splitting when newborn mice were challenged with viable organisms. Of the 14 phage-group II strains included in the survey, eight (58%) produced epidermolytic toxin. Three toxinogenic strains belonged to phage groups other than group II. A radial-immunodiffusion test employing antiserum to purified epidermolytic toxin proved satisfactory for measuring amounts of epidermolytic toxin in excess of 200 mug per ml. The results of immunodiffusion tests indicated that six of the 11 positive strains produced two serologically distinct forms of epidermolytic toxin and that the remainder produced only one of these. A striking correlation was observed between the presence of toxin of serotypes i and ii and the occurrence of protein bands i and ii in thin-layer isoelectric-focusing gels. These tests should facilitate the qualitative and quantitative assessment of the production of different serotypes of epidermolytic toxin by S. aureus in future surveys of strains isolated from toxic epidermal necrolysis of Ritter's type and impetigo. The two forms of epidermolytic toxin previously designated ETA (pI=7-0) and ETB (pI=6-0) were detected by preparative isoelectric focusing of sero-type-i toxin. Evidence suggests that studies of the effect of heat should provide a means of investigating the relationship between the different molecular forms of epidermolytic toxin.

Mice actively immunized with capsular polysaccharides extracted from capsular type strains A, B, C, and D, determined by the serum-soft agar technique, were protected against lethal infection by homologous strains, but no animals survived infection by heterologous substance immunization even with at high doses. Passive protective antibody in rabbit antisera prepared using these strains was absorbed out only by homologous capsular polysaccharide in mice. These results indicated that resistance was specific for capsular polysaccharide. The substance contained mainly neutral sugar, small amounts of hexosamine, methyl-pentose, and phosphate although these amounts varied depending on the capsular types strains.

The staphylococcal fibrinogen binding protein of a strain of coagulase-negative Staphylococcus aureus was purified 229 fold in terms of the haemagglutination unit compared to the starting material by affinity chromatography on fibrinogen-Sepharose. By polyacrylamide gel electrophoresis in both reduced and unreduced gels, the protein showed one major band and minor bands with relative molecular masses of 62,000, 61,000, and 59,000, respectively. The isoelectric point was between 10.2 and 10.8 determined by isoelectric focusing in polyacrylamide gel. By the agar diffusion test one band was obtained against anti-whole cell rabbit serum. Amino acid analysis of fibrinogen binding protein showed glycine, glutamic acid, lysine, alanine, aspartic acid, and arginine as the major components. Protein A or teichoic acid extracted from the homologous strain did not show fibrinogen binding activity assayed by haemagglutination and anti-fibrinogen binding activity of anti-whole cell Fab. The amount of the fibrinogen binding protein to absorb the activity of anti-whole cell Fab was decreased to 1/60 of that of the starting material. Sheep red blood cells coated with the fibrinogen binding protein agglutinated in 0.001% (w/v) fibrinogen solution.

Studied was the capacity of 243 S. aureus strains to produce enterotoxin D after they were isolated from food products. It was found that 3.3 per cent of the strains produced enterotoxin D, the simultaneous production of A and D toxins being established in 2.5 per cent of the cases. The detection of an enterotoxin of such antigenic type pointed to its demonstration in cases of food intoxication as well as when hygiene control of food products was carried out.

Mutants of Staphylococcus aureus were isolated which showed changes in pigment composition compared with the parent strain. On the basis of differences in their triterpenoid carotenoid composition they were classified into seven types. In five of these types, there appeared to be a blockage in the biosynthetic pathway which resulted in the absence of some products and accumulation of others. The changes in the other two types appeared to be a consequence of some change in regulation. A scheme for the biosynthesis of triterpenoid carotenoids is presented in which the first C30 intermediate, 4,4'-diapophytoene, is converted via 4,4'-diapophytofluene, 4,4'-diapo-zeta-carotene, 4,4'-diaponeurosporene, 4,4'-diaponeurosporenal, 4,4'-diaponeurosporenoic acid, and glucosyl-diaponeurosporenoate to the major pigment staphyloxanthin.

The validity of quantitative determination of protein A by radial immunodiffusion has been tested using human serum, human IgG and porcine serum. The best results are obtained with porcine serum. Maximal sensitivity is obtained after 48 h of incubation with 15 microliters of porcine serum per 13 ml of gel. For these particular conditions the range of linearity varies between 20 and 300 microgram/ml.

Ca2+-independent protein-modulator (BacM) was found in the culture medium of Staphylococcus aureus. BacM activated calmodulin-dependent cyclic nucleotide phosphodiesterase and Ca2+/Mg2+-ATPase in the same way as calmodulin. BacM was shown to be a proteolytic fragment of the exotoxin secreted by the S. aureus strain under study. The kinetic analyses of the ATPase activation by BacM and CaM were performed. These studies demonstrated that the enzyme molecule contains at least two activator-sensitive sites. Experiments on the ATPase activation by Ca2+ both in the presence and in the absence of BacM and CaM documented that CaM-ATPase and BacM-ATPase complexes can exist at low concentrations of calcium. Analysis of activation curves of ATPase by Ca2+ revealed three Ca2+-binding sites in the enzyme-activator complex.

Mesosomes of Staphylococcus aureus were examined morphologically under the electron microscope. Three different methods of specimen preparation (thin section, freeze-fracture, and negative staining) were used to eliminate artifacts due to sample processing. Mesosomes were rarely seen in intact cells but were quite distinct in autolysed cells incubated in 1.2M sucrose -0.33M acetate buffer (pH 5.8) at 25 degrees C. The phospholipid content increased by 20% of the control as autolysis proceeded. Since the plasma membrane did not show any other significant changes, the development of mesosomes during autolysis was shown to be a real event. Most of the well-developed mesosomes were found at the septum in negatively stained specimens. Initial wall-lysis occurred at this site, suggesting a close relationship between autolysis and the development of mesosomes in S. aureus.

The far-u.v. circular-dichroism spectra of the two epidermolytic toxins was analysed into fractional contributions of 0.09 helix and 0.46 beta-sheet to each toxin structure. Trifluoroethanol perturbation caused an initial increase in dichroic absorption at 205 nm and then a change characterized as a beta-sheet-to-alpha-helix transition. The intense near-u.v. spectra suggested that the toxins have unusually rigid, though different, aromatic-side-chain arrangements.

Immunoglobulin A (IgA) antibodies in human sera with binding specificity for the C-terminal R-D-Ala-D-Ala sequence of the precursor peptide from bacterial cell wall peptidoglycan were detected by an enzyme-linked immunosorbent assay (ELISA). Specificity of the test system was proved by comparing the high binding of specific IgA to albumin-(D-Ala3) as an antigen with the failure to bind to albumin-(L-Ala3), by binding inhibition studies with L-Ala3, D-Ala3, or peptides with structural analogy to peptidoglycan peptide subunit peptides as inhibitors, and by excluding binding of peroxidase-labeled anti-human IgA to immunoglobulin classes others than IgA. Interference of rheumatoid factors of IgA class was excluded by an ELISA for assaying IgA-rheumatoid factor and by the fact that an IgA fraction essentially free of IgG and IgM was isolated from a serum reacting strongly positive in the ELISA for measuring specific IgA to the peptide subunit of peptidoglycan. This isolated IgA again exhibited binding specificity in the ELISA, thus corroborating the existence of specific IgA in human serum to the C-terminal R-D-Ala-D-Ala sequence of peptidoglycan precursor peptide. The existence of IgA antibodies with specificity for bacterial peptidoglycan was further proved by preadsorption of serum to peptidoglycans and subsequent measurement of specific IgA in the ELISA. Screening of human sera for IgA antibodies with specificity for R-D-Ala-D-Ala peptides revealed that specific antibodies directed against this sequence of bacterial cell wall peptidoglycan may be detected in several human sera.

The carotenoids of Staphylococcus aureus 209P have been identified as triterpenoid (C30) carotenoids. The pigments include members of a carotene dehydrogenation sequence with C30 analogs of phytoene, phytofluene, zeta-carotene, and neurosporene. The major xanthophyll present under the cultural conditions used is a carotenoic acid derived from the C30 analog of neurosporene. A cis-isomer of this major xanthophyll has also been characterized, as have four other xanthophylls. Of the latter xanthophylls, two have been identified as the hydroxy and glycosyloxy derivatives of the major xanthophyll, while the remaining two xanthophylls are carotenoid esters. Squalene and menaquinone MK-8 have also been identified in the bacterium.

The role of the major cell wall components of Staphylococcus aureus in the leukopenia, thrombocytopenia, and complement activation associated with S. aureus bacteremia was studied in a guinea pig model. Formalin-killed S. aureus strains HSmR, 52A5, Cowan I, and Cowan EMS and purified peptidoglycan were used. Normal animals given peptidoglycan developed early (5-min) leukopenia, thrombocytopenia, and depletion of C3-C9 hemolytic activity similar to values in animals given killed S. aureus organisms and C4-deficient animals challenged with peptidoglycan. Cobra venom factor-treated animals challenged with peptidoglycan did not develop early leukopenia and thrombocytopenia, but all animal groups persistently had late (greater than 1-hr) leukopenia and thrombocytopenia. This observation suggests that peptidoglycan may play a major role in the early leukopenia and thrombocytopenia associated with S. aureus bacteremia in the guinea pig and that these effects can be mediated by activation of the alternative complement pathway alone. Peptidoglycan also causes a late leukopenia and thrombocytopenia which may occur independently of complement activation.

Toxic shock toxin (TST), also known as pyrogenic exotoxin C (Schlievert et al., J. Infect. Dis. 143:509-516, 1981) and staphylococcal enterotoxin F (Bergdoll et al., Lancet i:1017-1021, 1981), was purified from toxic shock strains of Staphylococcus aureus by preparative isoelectric focusing and by chromatofocusing. Neither method produced an absolutely pure protein as determined by silver staining of sodium dodecyl sulfate-acrylamide gels, although chromatofocusing was the better method of the two. Three molecular weight variants of the protein were found in the two toxic shock syndrome strains that were studied, regardless of the purification method that was used. An isoelectric point of 7.15 and molecular weights of 21,400, 22,100, and 23,200 were determined for the different forms of the protein from electrophoresis data. A sedimentation coefficient of 2.3S was determined by sucrose gradient centrifugation, and a Stokes radius of 2 X 10(-7) cm was determined by gel filtration. An average molecular weight of 18,900 for all of the TST forms was calculated from these data by the Stokes-Einstein equation. A survey for TST in 32 control and 46 toxic shock strains of S. aureus by isoelectric focusing and by agarose gel double immunodiffusion with specific rabbit antiserum revealed that the isoelectric focusing method tends to overestimate the number of TST-positive strains because of the detection of non-TST, neutral staphylococcal proteins. Based on immunodiffusion data, the association of TST with toxic shock strains was found to be 100% in vaginal isolates and 62% in non-vaginal isolates. In the control strains, TST was found in 16% of the vaginal strains and 23% of the non-vaginal strains. The value of this toxin as a marker for toxic shock and its relationship to the pathogenesis of this disease are discussed.

The fine structure of the Staphylococcus aureus cell wall was determined by electron microscopy with the new technique of rapid freezing and substitution fixation. The surface of the cell wall was covered with a fuzzy coat which consisted of fine fibers or an electron-dense mass. Morphological examination of the cell wall, which was treated sequentially with sodium dodecyl sulfate, trypsin, and trichloroacetic acid, revealed that this coat was partially removed by trypsin digestion and was completely removed by trichloroacetic acid extraction but was not affected by sodium dodecyl sulfate treatment, suggesting that the fuzzy coat consists mostly of a complex of teichoic acids and proteins. This was confirmed by the application of the concanavalin A-ferritin technique for teichoic acid and antiferritin immunoglobulin G technique for protein A.

The investigations carried out show that the two main serologic types of phage group II are biochemically different. Contrary to strains of serologic 263-2, staphylococci belonging to type i1-2 do not produce haemoglobinase, are positive in cristal-violet test, produce mostly beta haemolysin, disclose higher lipolytic activity and as a rule possess dark orange pigment. In the first part of this work it was shown, that the serologic types under discussion differ also in respect of the presence of polysaccharide 263, epidemiology, phage typing and resistance to antibiotics. The data mentioned above seem to constitute sufficient base for the subdivision of phage group II into two subunits. It should be stressed that some properties of staphylococci belonging to type i1-2 seem to indicate their possible relation to animal strains of biotype B.

Peptidoglycan (PG) and lipopolysaccharide (LPS) are T cell-independent B cell mitogens and polyclonal activators in mice. The mechanism of in vitro proliferation and polyclonal activation of mouse splenocytes induced by PG from Staphylococcus aureus and LPS from Escherichia coli was further studied by using [3H]thymidine incorporation and protein A hemolytic plaque assays. Concanavalin A-generated suppressor cells suppressed both polyclonal and proliferative responses induced by PG, LPS, and pokeweed mitogen. The suppression of the proliferative responses was similar for all these mitogens, but was significantly less pronounced than the suppression of the polyclonal antibody response. Polyclonal activation induced by LPS was the most susceptible to suppression by concanavalin A-generated suppressor cells, and the suppression was significantly greater than in the PG-induced polyclonal response. Also, PG-induced polyclonal activation was not susceptible to inhibition by polymyxin B, which is an inhibitor of other B cell mitogens and polyclonal activators. For optimal generation of immunoglobulin-secreting cells, PG of LPS had to be present for at least 48 h after the initiation of the cultures. Removal of the mitogens after 4 or 24 h of incubation resulted in a suboptimal response. For effective induction of the proliferative response, the mitogens had to be present in cultures for over 24 h. Polyclonal-activating properties of staphylococcal cell wall components were also compared. PG was by far the most potent inducer of polyclonal antibodies. Teichoic acid was not active as a polyclonal activator, whereas purified cell wall and protein A were very weak inducers of polyclonal antibodies. These studies demonstrate that PG, in addition to LPS, can be a useful probe for studies on polyclonal activation.

Hardware and software for the automatic comparison of densitograms, based on the evaluation of their autocorrelation characteristics, can be used for establishing the characteristics of circulating staphylococcal populations and for their epidemiological marking.