A high molecular weight protein from Staphylococcus intermedius cross-reacts with Staphylococcus aureus enterotoxin antibodies.

Enterotoxin production by Staphylococcus species other than Staphylococcus aureus has been reported. Staphylococcus strains (104 in toto) representing twelve species and subspecies were examined for enterotoxins using a commercial staphylococcal enterotoxin ELISA immunoassay (TECRA, International Bioproducts). Staphylococcus intermedius (24 strains) and S. aureus (7 strains) were positive with this test. Western blots of S. aureus exoproteins demonstrated proteins of approximately 30 kD, consistent with known staphylococcal enterotoxins. The major antigen in all S. intermedius strains, a 75 kD protein, was not analogous to previously described staphylococcal enterotoxins. This protein was unique to S. intermedius. Gel filtration data indicate that the protein is a subunit of a larger protein in vivo. The 75 kD protein cross-reacts with several enterotoxin antibodies. It is unclear whether the protein is a toxin, but its homology with S. aureus enterotoxins may indicate a shared toxic region, or this protein may create false positive results in screening for enterotoxin.

Staphylococcal glycocalyx activates macrophage prostaglandin E2 and interleukin 1 production and modulates tumor necrosis factor alpha and nitric oxide production.

We have examined the effect of staphylococcal glycocalyces on the ability of murine peritoneal macrophages to produce prostaglandin E2 (PGE2) and the inflammatory cytokines interleukin 1 (IL-1) and tumor necrosis factor alpha (TNF-alpha) and to generate nitric oxide. Glycocalyx partially purified under endotoxin-free conditions from defined liquid medium cultures of Staphylococcus lugdunensis or Staphylococcus epidermidis was a strong stimulator of PGE2 and IL-1 production. The addition of 10 to 100 micrograms of glycocalyx per ml induced levels of IL-1 and PGE2 production similar to that induced by 0.1 to 1 micrograms of Escherichia coli lipopolysaccharide (LPS) per ml. In contrast, glycocalyx induced ninefold less TNF-alpha and three- to fourfold less nitrite than LPS. A modulatory effect was suggested by the observation that the amount of TNF-alpha and nitrite generated remained constant whether the macrophages were stimulated with 10 or 100 micrograms of glycocalyx per ml. A selective modulation of macrophage activation was confirmed by the demonstration that costimulation of macrophages with both glycocalyx and LPS resulted in a reduction in TNF-alpha and nitrite generation relative to stimulation with LPS alone even though costimulation had no effect on PGE2 production and increased IL-1 production. Involvement of PGE2 in this modulatory effect was suggested by the ability of indomethacin to augment glycocalyx-stimulated TNF-alpha production and to reverse the inhibitory effect of glycocalyx on LPS induction of TNF-alpha production. However, the inability of indomethacin to reverse the inhibitory effect of glycocalyx on LPS-induced nitric oxide generation suggests that the selective modulation of macrophage function by glycocalyx may be more complex than increased sensitivity to PGE2 feedback inhibition.

Examination of the glycocalyx of four species of Staphylococcus by transmission electron microscopy and image analysis.

The coagulase-negative staphylococci have become important pathogens in human infections involving foreign bodies. The bacterial glycocalyx is a major mediator of attachment of these organisms to medical devices, but the glycocalyx is sometimes difficult to demonstrate. A combination of the techniques of transmission electron microscopy (TEM) and image analysis enabled investigators to reveal the glycocalyx which was previously indiscernible. Eight strains of coagulase-negative staphylococci, including Staphylococcus epidermidis, S. hominis, S. lugdunensis, and S. schleiferi subspecies schleiferi, were grown, treated with anti-staphylococcal serum to stabilize the glycocalyx, and examined by TEM. Image analysis of negatives was then used to enhance the visual images which showed far more glycocalyx than previously seen by TEM alone.

Osteomyelitis experimentally induced with Bacteroides thetaiotaomicron and Staphylococcus epidermidis. Influence of a foreign-body implant.

Experimental osteomyelitis was induced in the rabbit tibia with Staphylococcus epidermidis alone, with Bacteroides thetaiotaomicron alone, and with both bacteria as etiologic agents, in the presence or absence of a foreign-body implant. Animals were monitored by clinical observation and roentgenographic, microbiologic, histologic, immunofluorescent microscopic, and electron microscopic methods. Scanning and transmission electron microscopy showed masses of coccoid and rod-shaped bacteria embedded in a matrix of exopolysaccharide and adhered to bone, marrow, and the foreign-body implant (when present). Of the 58 rabbits receiving an implant, osteomyelitis developed in 48 (83%), and bacteria were recovered by culture from 56 (97%). Of the 31 animals without the implant, osteomyelitis developed in 18 (58%), but no bacteria were recovered by culture. Bacterial recovery appeared to be dependent on the presence of the implant. The rate of induction and the severity of osteomyelitis were enhanced by the presence of the foreign-body implant and by the polymicrobic infection.

Staphylococcal exopolysaccharides inhibit lymphocyte proliferative responses by activation of monocyte prostaglandin production.

The glycocalyx (exopolysaccharides) of Staphylococcus epidermidis has been reported to inhibit a variety of host defense mechanisms. We have examined the inhibitory effects of glycocalyx on the proliferation of human peripheral blood mononuclear cells (PBMC) and the mechanism of this inhibition. Glycocalyx isolated and partially purified under endotoxin-free conditions from defined liquid medium cultures of S. epidermidis and Staphylococcus lugdunensis inhibited the proliferative response of PBMC when added to cultures at 10 to 100 micrograms/ml. Glycocalyx-mediated inhibition of phytohemagglutinin-stimulated proliferation of PBMC required the presence of plastic-adherent peripheral blood monocytes. Culture supernatants of monocytes stimulated with glycocalyx contained a soluble factor that inhibited the proliferation of monocyte-depleted PBMC. This soluble inhibitory factor was not produced in the absence of glycocalyx or in the presence of both glycocalyx and indomethacin. Analysis of the supernatants of cultures of adherent monocytes revealed that glycocalyx from S. epidermidis and from S. lugdunensis could activate monocyte production of prostaglandin E2 (PGE2), human interleukin-1, and tumor necrosis factor alpha. The addition of purified PGE2, at the same levels of PGE2 (greater than or equal to 10(-9) M) generated in the monocyte cultures, to PBMC cultures resulted in a similar inhibition of proliferative responses. It is concluded that, contrary to previous suggestions, the bacterial glycocalyx does not have a direct inhibitory effect on T lymphocytes. However, it does appear that glycocalyx from coagulase-negative staphylococci can activate monocyte PGE2 production and that it is this activity that in turn contributes to the inhibition of T-cell proliferation.

Bacteroides and Staphylococcus glycocalyx: chemical analysis, and the effects on chemiluminescence and chemotaxis of human polymorphonuclear leucocytes.

Glycocalyx (or slime), which is an important virulence factor of many pathogenic bacteria, was isolated from Bacteroides fragilis, Bacteroides thetaiotaomicron and Staphylococcus epidermidis. Organisms were grown for 24 h in a chemically defined, dialysable liquid medium. Bacteria were centrifuged and the supernatant was concentrated and dialysed against distilled water. Total carbohydrate and protein were estimated using standard methods. Thin layer and gas-liquid chromatography of trifluoro acetic acid hydrolysed and non-hydrolysed samples provided evidence for the presence of polysaccharide, the absence of nucleic acids and lipopolysaccharide and for the identification of the individual sugar residues. Glucose, mannose and galactose (B. fragilis), glucose (B. thetaiotaomicron), and glucose and heptose (S. epidermidis) were the sugar residues detected. Uronic acid and hexosamine were detected in all species. Glycocalyx preparations (1 mg/ml) from Bacteroides and Staphylococcus significantly inhibited the chemiluminescence and chemotactic responses of viable human polymorphonuclear leucocytes (PMNL), but were not toxic for PMNL.

Comparison of the pathogenicity of three species of coagulase-negative Staphylococcus in a mouse model with and without a foreign body.

Staphylococcus schleiferi, Staphylococcus lugdunensis, and Staphylococcus epidermidis produce a high incidence of abscesses in a mouse model with an implanted foreign body. We investigated the significance of the foreign body in this process. Fourteen strains of S. schleiferi, S. epidermidis, and S. lugdunensis were tested in our model. A preadhered foreign body was implanted into one mouse group, followed by injection of a test strain. Another group received injection without implant. Abscesses were assessed at 7 days; foreign bodies and infected tissues were cultured. The percent of samples that developed abscesses or were culture positive was compared for each strain. Nearly all mice infected with S. schleiferi developed abscesses and were culture positive. The foreign body made no difference in abscess formation for three of four S. schleiferi but increased the incidence of both organism recovery and abscess for three of five S. epidermidis. The foreign body enhanced abscess formation for four of five S. lugdunensis, with all five strains yielding significantly more culture recovery. Although the pathogenicity of nine strains was increased by the foreign body, five strains yielded high abscess and culture recovery rates that were not enhanced by its presence.

Foreign-body-associated experimental osteomyelitis induced with Bacteroides fragilis and Staphylococcus epidermidis in rabbits.

Bacteroides fragilis and Staphylococcus epidermidis, alone and in combination, were used to induce foreign-body-associated osteomyelitis in a rabbit model. In this model, a catheter, used as a foreign body, was implanted into the medullary cavity of the tibia. Only two of five animals infected with S. epidermidis alone developed culture-positive osteomyelitis, whereas all three animals infected with B. fragilis alone developed osteomyelitis. All six animals infected with both microorganisms developed culture-positive osteomyelitis. Roentgenographic and histologic evaluations confirmed the diagnosis of osteomyelitis. Transmission and scanning electron microscopy showed that when the two microorganisms are involved in a mixed infection, S. epidermidis predominates on the foreign body and B. fragilis predominates in the infected bone and marrow.

Pathogenicity of Staphylococcus lugdunensis, Staphylococcus schleiferi, and three other coagulase-negative staphylococci in a mouse model and possible virulence factors.

Staphylococcus lugdunesis and Staphylococcus schleiferi, two newly described species, have been isolated from numerous types of human infections. We compared the pathogenicity of 30 strains of S. lugdunensis, S. schleiferi, Staphylococcus epidermidis, Staphylococcus warneri, and Staphylococcus hominis, using a mouse model in which a foreign body preadhered with the test strain was implanted subcutaneously, followed by injection of the test strain. All five species of staphylococci produced abscesses. Staphylococcus epidermidis, S. schleiferi, and S. lugdunensis yielded species means of 76-91% abscess formation; 80-100% of the infected foreign bodies and tissues were culture positive. These three species were more virulent than S. warneri or S. hominis, which produced abscesses in 54 and 65% of mice, respectively; only 10-48% of the infected samples were culture positive. Transmission electron microscopy of pure cultures of selected strains showed that all species possessed glycocalyx. All species produced a variety of possible virulence factors, such as alpha and delta hemolysins, as well as the aggressins lipase and esterase. The production of exoenzymes did not always correlate with virulence as demonstrated by abscess formation in mice.

Effect of ciprofloxacin on experimental osteomyelitis in the rabbit tibia, induced with a mixed infection of Staphylococcus epidermidis and Bacteroides thetaiotaomicron.

After induction of experimental polymicrobic osteomyelitis with Staphylococcus epidermidis and Bacteroides thetaiotaomicron (ciprofloxacin MIC, 0.5 micrograms/ml and 4.0 micrograms/ml, respectively), in the presence of a foreign body implant, in a rabbit tibia model, ciprofloxacin was administered to infected animals for 2- and 4-week periods. At necropsy, rabbits in the 2-weeks-treated group had mean ciprofloxacin levels of 5.94 micrograms/ml in serum, 3.63 micrograms/g in marrow, and 1.88 micrograms/g in bone. Rabbits in the 4-weeks-treated group had mean ciprofloxacin levels of 7.77 micrograms/ml in serum, 5.84 micrograms/g in marrow, and 2.01 micrograms/g in bone. Quantitative bacterial plate counts were conducted on weighed samples of infected bone, marrow, and the catheter implant, taken at necropsy from treated and control rabbits. Variable reduction of bacterial numbers was observed in samples from treated animals, as compared to untreated controls. Samples of infected bone, marrow and catheter, showed comparable evidence of osteomyelitis and bacterial colonization in both treated and control animals. Although relatively high tissue levels of ciprofloxacin were attained, little therapeutic effect was observed.

Trospectomycin enhances surface phagocytosis of Bacteroides and Staphylococcus by altering the bacterial glycocalyx.

The influence of trospectomycin on phagocytosis of Bacteriodes thetaiotaomicron, Bacteroides fragilis and Staphylococcus epidermidis was studied in the presence or absence of glycocalyx isolated from these microorganisms. Bacteria were grown with or without 0.25 or 0.5 of the minimal inhibitory concentration (MIC) of trospectomycin, a new chemically synthesized analog of spectinomycin. Surface phagocytosis by human polymorphonuclear leukocytes (PMNL) was determined using a modified fluorochrome assay. Subinhibitory concentrations of trospectomycin significantly enhanced surface phagocytosis of Bacteroides and Staphylococcus. When homologous or heterologous isolated glycocalyx was added to trospectomycin treated bacteria prior to incubation with PMNL, phagocytosis was reduced to levels observed in the untreated bacteria. Addition of glycocalyx to untreated strains produced no significant reduction of phagocytosis. The glycocalyx preparations were free of lipopolysaccharide and did not affect PMNL viability.

The role of glycocalyx in surface phagocytosis of Bacteroides spp., in the presence and absence of clindamycin.

The influence of isolated glycocalyx from Bacteroides thetaiotaomicron and B. fragilis on surface phagocytosis of clindamycin-treated and -untreated homologous and heterologous species was studied. When homologous or heterologous isolated glycocalyx was added to clindamycin-treated B. thetaiotaomicron or B. fragilis before incubation with PMNL, phagocytosis was reduced to levels observed in the untreated control bacteria, but addition of glycocalyx to untreated control strains showed no reduction of phagocytosis. When isolated bacteroides-glycocalyx was added to Staphylococcus aureus or S. epidermidis, phagocytosis of both clindamycin-treated and -untreated bacteria was significantly reduced. The isolated glycocalyx preparations were analysed by thin layer and gas-liquid chromatography; these preparations were free of lipopolysaccharides. The isolated glycocalyx did not affect PMNL viability. Our findings suggest that the glycocalyx is an important virulence factor because it impairs phagocytosis of Bacteroides spp. by PMNL. Clindamycin may enhance opsonophagocytosis of bacteroides by altering the glycocalyx.

Enhancement of opsonophagocytosis of Bacteroides spp. by clindamycin in subinhibitory concentrations.

Radioactively labelled bacteria were incubated overnight in the presence or absence of one-half the MIC of clindamycin, then preopsonized with normal human serum or homologous rabbit antiserum and incubated with human polymorphonuclear leucocytes. Clindamycin in subinhibitory concentrations significantly enhanced the phagocytosis of all four Bacteroides strains. Complement-dependent as well as antibody-dependent phagocytosis was enhanced by clindamycin in one Bacteroides strain. In the other three strains, only antibody dependent phagocytosis was enhanced by clindamycin. Transmission electron microscopy confirmed phagocytosis of the bacteroides.

Ciprofloxacin concentrations in serum, bone and bone marrow of rabbits.

Ciprofloxacin concentrations were determined in serum, bone and bone marrow of rabbits. Four experimental groups of animals were examined: group A (n = 6) received a dosage of 60 mg/kg/day intramuscularly for 4 weeks, groups B (n = 6), C (n = 15) and D (n = 15) received dosages of 120 mg/kg/day subcutaneously for 2 days, 2 weeks, and 4 weeks, respectively. In the kinetic portion of the study, peak serum concentrations of ciprofloxacin measured at the 15 min sampling time were: 2.61 +/- 0.27 micrograms/ml in the 60 mg/kg/day group (group A) and 3.24 +/- 0.78 micrograms/ml in the 120 mg/kg/day group (group B). At necropsy, rabbits in group A had mean ciprofloxacin concentrations of 3.60 +/- 2.27 micrograms/ml in serum, 2.24 +/- 1.19 micrograms/g in marrow and 1.19 +/- 0.44 micrograms/g in bone. Rabbits in group B achieved mean levels of 4.02 +/- 1.23 micrograms/ml in serum, 2.48 +/- 0.79 micrograms/g in marrow, and 1.35 +/- 0.40 micrograms/g in bone. Rabbits in group C achieved mean levels of 5.65 +/- 2.16 micrograms/ml in serum, 3.74 +/- 1.33 micrograms/g in marrow and 1.92 +/- 0.94 micrograms/g in bone. Rabbits in group D achieved mean levels of 7.24 +/- 2.50 micrograms/ml in serum, 4.48 +/- 1.68 micrograms/g in marrow, and 1.93 +/- 0.54 micrograms/g in bone. Differences between mean values for the four experimental groups were not statistically significant.

The Bacteroides glycocalyx as visualized by differential interference contrast microscopy.

The glycocalyx of eight strains representing six species of Bacteroides was examined by differential interference contrast microscopy. Wet mounts in India ink were prepared from bacteria cultured in broth and on an agar medium; the wet mounts were observed by phase-contrast microscopy and differential interference contrast microscopy. With differential interference contrast microscopy, all bacteria demonstrated a glycocalyx, which included capsules surrounding single cells and microcolonies, strands of glycocalyx connecting cells and microcolonies, detached slime, and solid masses of glycocalyx in which innumerable bacteria were enmeshed. Bacteria showed comparable amounts of glycocalyx by visual observation with differential interference contrast microscopy whether grown on plates or in broth. Serial transfers of cultures did not diminish the amount of glycocalyx. Differential interference contrast microscopy proved to be a superior method to phase contrast for examining wet preparations of Bacteroides.

Effect of ciprofloxacin on subcutaneous abscesses induced with Staphylococcus epidermidis and a foreign body implant in the mouse.

Subcutaneous abscesses were induced in mice with Staphylococcus epidermidis strain G19-85 and a foreign body implant. The MIC of ciprofloxacin for this strain was 0.25 microgram/ml. The ciprofloxacin dosage, 120 mg/kg/day, was divided into three injections, administered to the mice subcutaneously at 8 h intervals. Serum concentration kinetics in normal mice (n = 50) were determined. The peak serum level of ciprofloxacin was 3.18 micrograms/ml at the 15 min sampling time; the trough level was 0.53 micrograms/ml at 8 h. Abscesses were found in 96% (n = 49) of the untreated, infected control mice. Three modes of treatment with ciprofloxacin were tested: (1) four prophylactic injections of ciprofloxacin prior to infection reduced abscess formation to 64% (p less than or equal to 0.0002, n = 50). (2) Eleven therapeutic injections, initiated 4 days after infection, reduced abscess formation to 86% (p less than or equal to 0.17, n = 49). (3) One prophylactic injection prior to surgery and five therapeutic injections after infection reduced abscess formation to 43% (p less than or equal to 0.0001, n = 49). Culture results correlated with the abscess formation rates.

Cell wall alterations in staphylococci growing in situ in experimental osteomyelitis.

When cells of both Staphylococcus aureus and Staphylococcus epidermidis are grown in batch culture in nutrient-rich media, their cell walls are regular in thickness, their cell size is within the normal range for each species, and their septation patterns are orderly. When cells of each of these species are examined directly in infected tissue in the rabbit tibia model infection, their cell wall thickness is often much increased and very irregular around the circumference of the cell, their cell size is often increased, and their septation patterns are often severely deranged. All of these alterations in cell wall structure occur in the absence of antibiotics, and we suggest that they may be an expression of phenotypic plasticity in response to altered environmental conditions such as specific nutrient limitations, the presence of antibacterial factors, and growth of the cells on hard surfaces such as rabbit bone or plastic catheters. Some of these specific cell wall alterations are also seen when staphylococcal cells are exposed, in vitro or in vivo, to antibiotics such as clindamycin, but we emphasize that growth in tissue alone is sufficient for their induction.

An electron microscopic study of the effect of clindamycin therapy on bacterial adherence and glycocalyx formation in experimental Staphylococcus aureus osteomyelitis.

After induction of experimental osteomyelitis with Staphylococcus aureus in a rabbit tibia model, clindamycin phosphate (280 mg/kg/day) was used to treat the infected animals for 1, 2 and 3 week periods. Scanning electron microscopy of samples of infected bone tissue taken at necropsy revealed masses of coccoid profiles embedded in a matrix of condensed exopolysaccharide material which adhered to the bone in both infected control animals and in infected animals treated for 1 week with clindamycin phosphate. After 2 and 3 weeks of clindamycin phosphate treatment, the infecting bacteria could not be cultured from tissue samples, and scanning electron microscopy of these samples revealed few coccoid profiles adhering to the bone and marrow. Radiological, microbiological, clinical, histological and electron microscopic findings all indicated recovery from the diseased state with increased length of clindamycin phosphate treatment.

An electron microscopic study of the effect of clindamycin on adherence of Staphylococcus aureus to bone surfaces.

Discs of rabbit tibia, 5 mm thick, were utilized to study the adherence of Staphylococcus aureus to the bone surface in the presence and absence of clindamycin. Bacteria were grown in broth media containing the bone slices and varying concentrations of clindamycin. In the absence of the antibiotic, S. aureus adhered extensively to bone surfaces and formed large microcolonies which were surrounded by an amorphous matrix. In the presence of 0.025 micrograms/ml of clindamycin (0.1 MIC), S. aureus adhered less to bone surfaces, forming smaller and fewer microcolonies. In the presence of 0.0625 micrograms/ml of clindamycin (0.25 MIC), S. aureus adhered to the bone surfaces only sparsely, forming small microcolonies with very little matrix holding them together, and leaving very large areas of the bone surface uncolonized. In the presence of 0.125 micrograms/ml of clindamycin (0.5 MIC), bone surfaces were basically clean, with only one or two cells (no microcolonies) found in crevices and indentations of the bone surface. In the presence of 0.25 micrograms/ml (1 MIC) no bacteria adhered to the bone surfaces.

Differentiation of Campylobacter species by protein banding patterns in polyacrylamide slab gels.

Soluble protein extracts of 37 catalase-positive strains of Campylobacter species were examined by polyacrylamide slab gel electrophoresis (PAGE). Electrophoretic banding patterns showed good correlation with biochemical tests and with available DNA homology data in distinguishing species of Campylobacter but did not differentiate subspecies or biotypes. PAGE patterns indicated that Campylobacter coli is a distinct species. Furthermore, the PAGE patterns indicated that C. jejuni and nalidixic acid-resistant thermophilic Campylobacter species (C. laridis) are each distinct species. The protein banding patterns of C. fetus subsp. venerealis and C. fetus subsp. fetus strains were distinctly different from those of the three thermophilic species.