Gel electrophoretic analysis of penicillin-binding proteins of coagulase negative staphylococci.

We analyzed gel electrophoretic banding patterns of penicillin-binding proteins (PBPs) of 16 type strains of coagulase-negative staphylococci (CNS). S. epidermidis, S. haemolyticus, S. saprophyticus, S. hominis, S. xylosus, S. simulans, S. warneri, S. capitis, S. saccharolyticus, S. auricularis, S. caseolyticus, S. gallinarum, S. hycus subsp. hycus, S. cohnii, S. caprae, and S. sciuri subsp. sciuri. The PBP profile of each CNS species was found to be unique and was clearly distinguishable from those of the rest of the species. Together with the previous work of other researchers, this study substantiates the applicability of the PBP profile analysis to the identification of clinical CNS strains.

Immunological detection of penicillin-binding protein 2' in clinical isolates of methicillin-resistant Staphylococcus aureus and Staphylococcus epidermidis.

The additional penicillin-binding protein (PBP 2') that is important in determining intrinsic resistance in methicillin-resistant strains of Staphylococcus aureus (MRSA) has been detected immunologically in strains from a variety of world-wide locations. This additional protein has also been definitively identified both immunologically and as a PBP in methicillin-resistant strains of S. epidermidis (MRSE). The assay described is rapid, specific and sensitive and has been used to detect PBP 2' in S. haemolyticus but not in beta-lactam resistant Streptococci.

Opsonic requirements of Staphylococcus epidermidis.

The opsonic requirements of 18 strains of Staphylococcus epidermidis were compared in pooled normal human serum and in peritoneal dialysate from patients undergoing continuous ambulatory peritoneal dialysis (CAPD). A serum concentration of 2.5% gave optimal opsonisation. The opsonisation of all strains was antibody- and complement-dependent, and there were no significant differences in the pattern of their opsonic requirements. Peritoneal-dialysis effluent from uninfected patients was a poor source of opsonins because of the low levels of immunoglobulin G and of the C3 component of complement it contained. Growth of S. epidermidis in peritoneal-dialysis effluent rather than in broth did not alter its opsonic requirements. Strains from patients undergoing CAPD and suffering from peritonitis were not more resistant to opsonisation and phagocytic killing than those from other sources.

Staphylococcal whole-cell polypeptide analysis: evaluation as a taxonomic and typing tool.

Whole-cell-polypeptide profiles obtained by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) were used in conjunction with the API-Staph technique to identify different strains of Staphylococcus aureus, S. epidermidis, S. saprophyticus and S. capitis. Complete concordance of results from both techniques was achieved with all strains examined. Visual analysis of the polypeptide patterns and comparison by use of the coefficient of Dice showed minor differences in band pattern between strains of the same species but each species produced a pattern distinguishable from that of any other. The results suggest that although SDS-PAGE can be used to identify staphylococcal species, this type of analysis will not readily provide the basis for a typing method.

Effects of extracellular slime produced by Staphylococcus epidermidis on oxidative responses of rabbit alveolar macrophages.

Bacterial slime produced in mass cultures of the RP 12 strain of Staphylococcus epidermidis was extracted with 4 M guanidine-HCl plus 0.05 M sodium acetate and 0.5% CHAPS, concentrated, dialyzed, and subjected to separation on DEAE sephacel columns. Three fractions, I-2A, I-2B, and I-4, were eluted with linear gradients of NaCl. Fractions I-2A and I-2B were alcian blue positive, whereas I-4 was alcian blue negative but the most electronegative fraction. The crude polysaccharide fraction and the three purified fractions were incubated individually for 2.5 or 20 h with normal rabbit alveolar macrophages (AM) to determine their effect on a subsequent PMA-induced oxidative burst. The crude fraction (50-200 micrograms/mL) and I-2B (50-200 micrograms/mL) primed the AM to give approximately a threefold increase in the PMA-induced burst after 2.5 h incubation. In contrast, a 20-h incubation resulted in a 30-40% inhibition of the PMA-induced burst with AM incubated with the same concentrations of the crude, I-2A, or I-2B fractions. Fraction I-4 had no detectable effect. The fractions also were tested to determine if they could elicit an oxidative burst in BCG-immune AM. None of the fractions (up to 500 micrograms/mL) elicited a significant oxidative burst even though BCG-immune AM yielded a PMA-induced burst 100 times that observed with normal resident AM. These data suggest that slime from S. epidermidis can impair the PMA-induced oxidative burst of normal AM during a 20-h incubation period and could explain in part why host defenses are compromised by slime-producing S. epidermidis.

[Automatic analysis of the spectra of extracellular proteins based on their autocorrelation characteristics]. / Avtomaticheskii analiz spektrov vnekletochnykh belkov na baze ikh avtokorreliatsionnykh kharakteristik.

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.

[Comparison of the spectra of membrane proteins in differentiating coagulase-negative staphylococci]. / Sopostavlenie spektrov membrannykh belkov v differentsiatsii koagulazootritsatel'nykh stafilokokkov.

The comparison of the spectra of the membrane proteins of 12 typing strains of coagulase negative staphylococci has revealed differences which probably reflect the distinctive features of membrane proteins, intrinsic to individual bacterial species.

[Extraction of chromosomal DNA from Staphylococcus and Listeria by a rapid method using achromopeptidase]. / Extraction de l'ADN chromosomique de Staphylococcus et de Listeria par une méthode rapide à l'achromopeptidase.

A rapid and simple method for preparation of chromosomal DNA from Gram-positive bacteria is reported. Susceptibility to lysis with Sodium Dodecyl Sulfate (SDS) increases when undergoing treatment with acetone before being digested by bacteriolytic enzymes. Rapid lysis of Staphylococcus and Listeria cells is obtained through a respective treatment by lysozyme with lysostaphine and by lysozyme with achromopeptidase, adding to that the effect of SDS in Tris-Hcl buffer. This procedure of preparing chromosomal DNA provides 1 to 4 mg of DNA out of 1 g of bacterial cells in a day.

Amino acid sequence of a deltalike toxin from Staphylococcus epidermidis.

A deltalike toxin produced by a clinical isolate of Staphylococcus epidermidis was purified, and the amino acid sequence was determined. The toxin molecule consisted of 25 amino acid residues and shared a high degree of molecular homology with delta toxin purified from a Staphylococcus aureus human isolate.

Staphylococcal slime: a cautionary tale.

Slime production by Staphylococcus epidermidis may be important in the adherence to and colonization of biomedical devices, and slime has been proposed to have various effects on the immune system. Attempts were made to isolate, purify, and chemically characterize slime from S. epidermidis cultivated under fluid on tryptic soy broth-agar medium. "Crude slime" from slime-producing strain RP-12 was characterized by a high galactose content. Similar materials in similar yields were isolated from slime-producing strain Kaplan, a non-slime-producing mutant, Kaplan-6A, and sterile medium controls, suggesting that crude slime was derived mainly from the medium. The occurrence of D- and L-galactose and pyruvate and sulfate residues and methylation analysis of these crude slime preparations, monitored by gas-liquid chromatography and mass spectrometry, showed that the agar was the main source of crude slime, suggesting that the preparation was largely an artifact of the growth and isolation procedures. Similar high-galactose-content preparations from both S. epidermidis and Staphylococcus aureus, assumed to be bacterial products and with a variety of biological activities, have been described by other investigators. Growth attached to a solid surface appears to be important for slime production. An accumulation of turned-over cell surface molecules and released macromolecules such as DNA may contribute to slime production. Avoidance of agar and development of a chemically defined medium for slime production are recommended for further studies.

Isolation and characterization of a capsular polysaccharide adhesin from Staphylococcus epidermidis.

We isolated a polysaccharide adhesin from Staphylococcus epidermidis strain RP-62A. The adhesin was composed of a complex mix of monosaccharides (with galactose and glucosamine predominating), bound well to silastic catheter tubing, inhibited adherence of strain RP-62A to catheters, and elicited antibodies that both blocked adherence and stabilized an extracellular structure (visualized by transmission electron microscopy) that appeared to be a capsule. Two of three heterologous, highly adherent strains of coagulase-negative staphylococci also produced this adhesin, and their adherence to catheters was inhibited by both purified adhesin and antibody to adhesin. In contrast, the adherence of one highly adherent and two poorly adherent heterologous strains was unaffected by the RP-62A purified adhesin or antibody, a result suggesting the expression of alternate adhesins by these strains. We conclude that the capsular polysaccharide of some strains of coagulase-negative staphylococci is an important factor in adherence to catheter tubing.

Comparative study of hemolytic substances produced by coagulase-negative Staphylococcus strains.

Hemolytic substances H7, H62, and E56, produced by Staphylococcus haemolyticus 7 and 62 and S. epidermidis 56, respectively, were purified. H7 and H62 are probably similar on the basis of their isoelectric focusing profiles in 8 M urea and complete immunological identity as revealed by immunodiffusion with rabbit anti-H7 and anti-E56 sera. For E56, we observed seven bands instead of three in isoelectric focusing and only partial immunological identity with H7 and H62. However, H7 and E56 were similar with regard to the following characteristics: hemolytic spectra against different erythrocytes, kinetics of erythrocyte lysis, heat stability, and inhibition by phosphatidylcholine. E56 was not active at a temperature lower than or equal to 25 degrees C, and its activity increased more rapidly with increased temperature compared with H7. For both substances, the complexes obtained by molecular filtration on Ultrogel AcA54 and the purified peptides by reverse-phase high-pressure liquid chromatography showed some hemolytic activity. These results suggest that a particular association or the presence of a given peptide could enhance the activity.

Staphylococcus epidermidis extracted slime inhibits the antimicrobial action of glycopeptide antibiotics.

Certain strains of Staphylococcus epidermidis produce a mucoid slime that appears to be an important virulence factor. After crude slime was isolated from selected strains of S. epidermidis, phenol and chloroform were used to remove proteins and lipids. The remaining extract contained a polysaccharide that was seen on SDS gels stained with Stains-all. This extract was an inhibitor of the antimicrobial action of vancomycin, raising the minimum inhibitory concentration (MIC) to vancomycin in all 18 isolates of S. epidermidis. A dose-response curve was seen between the amount of extract added and the degree of resistance, as measured by both MIC and growth curves. A similar effect was noted with MICs of organisms to teicoplanin. Addition of the extract did not change the MIC to LY146032, although a modest effect on growth rate was observed. The extract did not raise the MIC to clindamycin, rifampin, and cefazolin. The extract reversed the synergism seen between vancomycin and gentamicin in the 5 strains tested in time-kill studies. The interference by slime of the antimicrobial effect of vancomycin and teicoplanin may explain why these antibiotics are sometimes ineffective in eradicating foreign body infections due to slime-producing coagulase-negative staphylococci.

Defining Staphylococcus epidermidis cell wall proteins.

Three Staphylococcus epidermidis isolates of differing bacteriophage types were studied to define proteins confined to the cell wall, which were surface exposed and thus available to interact with the host. Three major proteins of 37, 41, and 51 kDa were identified in all whole-cell lysates and cell wall extracts by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis. Two additional proteins of 18 and 25 kDa became evident by using 125I labeling to delineate surface-exposed proteins. A classification scheme using P1 to P5 to delineate the 51-, 41-, 37-, 25- and 18-kDa proteins is proposed. Additionally, murine immune sera were used to identify two immunodominant proteins of 51 and 25 kDa (P1 and P4, respectively).

Comparison of cell wall teichoic acid fractions isolated from three different encapsulated strains of Staphylococcus epidermidis.

Teichoic acid preparations extracted from the cell wall of three serologically different encapsulated strains of Staphylococcus epidermidis, ATCC-31432 (capsular type I), SE-360 (capsular type II) and SE-10 (capsular type III) were purified by DEAE-cellulose and Sephadex G-50 column chromatography. The preparations showed immunological heterogeneity by an agar diffusion test. The chemical properties of the cell wall teichoic acid preparations of capsular types I and III were regarded as N-acetyl-glucosaminyl glycerol-phosphate polymers containing N-acetylglucosamine and phosphate at molar ratios of 0.22-1.0 and 0.33-1.0, respectively. The preparation of capsular type II was assumed to be an alpha-glucosyl glycerol-phosphate polymer containing glucose and phosphate at a molar ratio of 0.49-1.0, and it reacted strongly with concanavalin A. Moreover, alanine, glycine, serine and lysine were shown, among these preparations, to be a common amino acid composition. These results indicate that cell wall teichoic acids obtained from these strains were biochemically and immunologically different from each other.

Studies on coagulase-negative staphylococci in patients undergoing cardiac surgery. Different hypotheses for the origin of multiply-resistant isolates.

Hypotheses for the origin of multiply-resistant coagulase-negative staphylococci from 146 patients undergoing cardiac surgery were tested. All received cephalothin per-operatively. Antibiotic susceptibility testing, phage-typing, bio-typing, and test for Tween-80-splitting enzyme were used to characterize 132 isolates from nose swabs. Seventy-five percent of the pre-operative susceptible isolates were of biotype 1, while biotypes 3 and 4 made up 59% of the post-operative, multiply-resistant isolates. Fifty-three percent of the isolates were typable by phage-typing. Typability of isolates of biotype 1 was high (56%) while almost 75% of biotype 4 were untypable. Susceptible isolates were more often typable than multiply-resistant ones. Of the 146 patients, 105 (72%) carried coagulase-negative staphylococci pre-operatively, only two of them carried multiply-resistant strains. Fifty-nine patients (41%) were colonized with multiply-resistant coagulase-negative staphylococci post-operatively. By combining the results of bio-typing, phage-typing, and test for Tween-splitting enzyme the study made it probable that a maximum of ten patients (6.8%) already carried multiply-resistant strains on admission to the hospital or were carriers of initially susceptible strains which developed multiple-resistance during administration of antibiotics. It therefore seemed likely that most of the patients were deprived of their natural bacterial flora by antibiotic treatment and subsequently colonized post-operatively with multiply-resistant coagulase-negative staphylococci from the environment.

Investigation on extracellular slime substance produced by Staphylococcus epidermidis.

The extracellular slime substance produced by Staphylococcus epidermidis was investigated. Slime production was assessed by bacterial agglutination in the presence of concanavalin A (Con A) or poly-L-lysine and by bacterial adherence to polyethylene. Media for slime production was optimized using these criteria. A phenol-saline extract of crude slime was separated into four fractions on a DEAE-sepharose column. Total protein and sugar content and the monosaccharide constituents were determined. Crude slime and the phenol-saline extract showed a strong precipitation reaction with Con A and poly-L-lysine (double diffusion). Fractions I and II containing mannose as the most abundant sugar reacted with Con A and two other mannose-specific lectins (Lens culinaris, Pisum sativum). This reaction could be inhibited by mannose. Fractions III and IV were precipitated by poly-L-lysine, probably due to a reaction with glucuronic acid which was only present in these fractions. Precoating of polyethylene with crude slime, phenol-saline extract and fractions III and IV resulted in a marked inhibition of attachment of staphylococcal cells. Production of the extracellular slime substance was completely inhibited by subinhibitory concentrations of the glycosylation inhibitor tunicamycin, whereas penicillin had no influence. Extracellular slime substance produced by S. epidermidis seems to be a complex of glycoconjugate character and plays an important role in the attachment to synthetic polymers. The production of slime by staphylococci can be easily determined using mannose specific lectins and poly-L-lysine.

Immunochemical analysis of the extracellular slime substance of Staphylococcus epidermidis.

To analyze immunochemically the extracellular slime substance of Staphylococcus epidermidis, rabbits were repeatedly immunized with the crude slime extract isolated from an adherent, slime-producing clinical Staphylococcus epidermidis strain. Immunoelectron microscopy demonstrated that the target antigens of the resulting antibodies were located in the extracellular slime-like layer of bacterial cells. When these target antigens were characterized by immunoblotting, a variety of antigens were detected, including many with molecular masses higher than 80 kilodaltons and also a predominant one with a molecular mass of 30 kilodaltons. No characteristic differences were observed between the tube adherence test positive and negative Staphylococcus epidermidis isolates. Although there was variation in the number and intensity of high molecular mass antigens, such variations did not correlate with the tube adherence test. Of the 110 Staphylococcus epidermidis isolates studied, 106 (96%) expressed the 30-kilodalton antigen. This component was found in no other Staphylococcus spp. examined in the study. The bacterial component was not only easily detached from bacterial cells but also water-soluble, characteristics implicating a slime-like nature. Further studies are needed to definitively establish the origin and nature of the 30-kilodalton Staphylococcus epidermidis-specific component, and determine its potential benefit as a diagnostic tool.

Characterization of cell envelope proteins of Staphylococcus epidermidis cultured in human peritoneal dialysate.

The cell envelope protein profiles of Staphylococcus epidermidis cultured in used human peritoneal dialysate (HPD) differed markedly from those of cells cultured in nutrient broth. Compared with broth-grown cells, many cell wall proteins were repressed in HPD, although three proteins of 42, 48, and 54 kDa predominated and an iron-repressible 130-kDa protein was induced. Growth in HPD also resulted in expression of two cell membrane proteins of 32 and 36 kDa which were iron repressible. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblot analysis using monospecific polyclonal antisera raised against the 32- and 36-kDa proteins revealed considerable antigenic and molecular mass homology among 12 S. epidermidis isolates from patients with continuous ambulatory peritoneal dialysis-related peritonitis. The 32-kDa antiserum also cross-reacted with a 32-kDa S. aureus cell membrane protein. Immunoblots of S. epidermidis cell walls and membranes were also probed with normal human serum and serum and HPD from continuous ambulatory peritoneal dialysis patients. While the cell wall proteins of S. epidermidis appeared to be relatively poorly immunogenic, the 32- and 36-kDa membrane proteins reacted strongly with antibodies present in each of the body fluids evaluated. These results suggest that the highly conserved 32- and 36-kDa iron-repressible proteins are expressed during growth in vivo and may be involved in iron transport, since all 12 S. epidermidis strains examined also produced iron chelators.