The effect of growth temperature on Staphylococcus aureus binding to type I collagen.

Many strains of Staphylococcus aureus produce a collagen-binding surface protein that could enable these strains to colonize tissues such as bone. Previous studies indicated that the expression of the collagen receptor varies with growth conditions. We report here that the growth temperature influences the ability of some S. aureus strains to produce this receptor. S. aureus isolates from human, osteomyelitic bone were grown at 37 degrees C and 42 degrees C and tested for agglutination of collagen-coated latex beads. Binding by 42 degrees C grown cells was significantly reduced in five of the seven isolates studied, including a complete loss of collagen binding in three of these isolates. In an 125I-collagen-binding assay, the binding ability of one of these isolates, strain #16, was 20-fold lower if grown at 42 degrees C. Reduced collagen binding by this isolate could be demonstrated after only two cell divisions at 42 degrees C and the cells regained the ability to bind collagen when shifted back to 37 degrees C. Sodium dodecyl sulfate (SDS)-PAGE confirmed the presence of proteins at 117 kDa in strain #16 and 135 kDa in SMH which were absent following growth at 42 degrees C. Chicken IgG, specific for the 117 kDa protein, was found to react in immunoblot assays with these proteins as well as a protein of 135 kDa extracted from S. aureus Cowan 1. The antibody did not react with proteins extracted from non-binding strains. Strains #15 and #21, collagen-binders at both 37 degrees C and 42 degrees C, produced immunoreactive proteins at 110 and 135 kDa, respectively, in lysates from cells grown at both temperatures. Antibody against a recombinant form of a previously characterized collagen receptor was used to confirm cross-reactivity with these novel collagen receptors. These data suggest that the ability to produce the collagen receptor is temperature sensitive in some S. aureus strains associated with osteomyelitis. It is proposed that a better understanding of the environmental effects on collagen receptor production could enhance our understanding of staphylococcal infections in bone and joints.

Binding of a Staphylococcus aureus bone pathogen to type I collagen.

We contrasted the collagen-binding potential of the experimental osteomyelitis pathogen, Staphylococcus aureus strain SMH, to several other strains. These included Cowan 1 (binder), Wood 46 (non-binder) and six capsular variants. These measurements were made using an 125I-collagen binding assay. Formalin-killed S. aureus SMH strongly bound commercial type I iodinated collagen (dissociation constant, Kd = 2 x 10(-9) M). The extent of binding was similar to Cowan 1. Binding was saturable and not inhibited by 100 mM solutions of D-glucose, D-galactose, D-mannose, methyl-alpha-L-fucopyranoside, L-hydroxyproline or L-glycine. D-lactose gave moderate inhibition of binding to collagen, and L-fucose was strongly inhibitory. Trypsinized SMH did not bind collagen. None of four Ruthenium-red-staining staphylococci (encapsulated) avidly bound type I collagen. The encapsulated Smith strain, for example, did not bind to collagen but its capsule-negative variant, Smith compact, showed extensive binding. Three of five non-encapsulated S. aureus were strong collagen binders. These data suggest that the prototype bone pathogen binds to the major protein component of bone's extracellular matrix. Collagen-binding is promoted by protein adhesin(s), not capsule. The latter, in fact, appeared to interfere with this interaction. Binding was inhibited by solutions containing the simple monosaccharide, L-fucose.

Passive protection of rabbits infected with toxic shock syndrome-associated strains of Staphylococcus aureus by monoclonal antibody to toxic shock syndrome toxin 1.

The effectiveness of passive immunization was assessed in an infection model of toxic shock syndrome (TSS) in which monoclonal antibody to TSS toxin 1 (TSST-1) was administered intravenously to rabbits. Previously implanted infection chambers were inoculated with Staphylococcus aureus strains RN4710 and D4508. The former strain carries the TSST-1 gene on plasmid pRN6201; the latter is a TSST-1-negative clinical isolate obtained from a patient with nonmenstrual TSS. Purified monoclonal antibody, MAb 8-5-7 (IgG), was administered in two doses of approximately 1.25 mg each 24 hours before and 24 hours after infection. MAb 8-5-7 provided complete protection against both the TSS-like syndrome and the mortality that occurred in unprotected rabbits infected with strain RN4710 but did not provide complete protection in rabbits infected with strain D4508; three of the five rabbits either displayed signs of illness or died despite treatment. Western-blot analyses of the extracellular proteins produced by strains RN4710 and D4508 that used MAb 8-5-7 as a probe revealed that only TSST-1 produced by RN4710 reacted with the antibody. Thus, if MAb 8-5-7 partially protected animals against infections with strain D4508, the protection appears to have been nonspecific.

Endotoxin enhancement of toxic shock syndrome toxin 1-induced secretion of interleukin 1 by murine macrophages.

The purpose of this study was to determine whether endotoxin could augment toxic shock syndrome toxin 1 (TSST-1)-induced production of interleukin 1 (IL-1) by murine macrophages. Macrophages from C3H/HeJ or C57Bl/6 mice were stimulated with purified TSST-1 alone or in combination with lipopolysaccharide (LPS). A dramatic synergistic thymocyte-proliferative response was induced by supernatants from C57Bl/6 macrophages stimulated with both TSST-1 and LPS. No enhanced response was induced by supernatants from C3H/HeJ macrophages. A portion of the enhanced response induced by C57Bl/6 macrophage supernatants was attributed to synergism between IL-1 and residual TSST-1 in the thymocyte assay. The addition of monoclonal antibody to TSST-1 to the supernatants eliminated the effects of residual TSST-1 in the thymocyte assay and demonstrated a synergistic induction of IL-1. These data (1) show that LPS can enhance macrophage responsiveness to TSST-1; (2) suggest that TSST-1 not only induces IL-1 secretion but also enhances target cell responsiveness to IL-1; and (3) further support the role of IL-1 in the pathogenesis of toxic shock syndrome.

Protection of rabbits in an infection model of toxic shock syndrome (TSS) by a TSS toxin-1-specific monoclonal antibody.

An anti-TSST-1-specific monoclonal antibody (MAb 8-5-7) was tested for its protective capacity in a rabbit infection model to toxic shock syndrome (TSS). The challenge strain of Staphylococcus aureus (RN4710), which contained a plasmid encoding TSS toxin-1, was introduced into previously implanted chambers. Purified monoclonal antibody (1.25 mg of immunoglobulin G) administered parenterally 1 day before and 1 day after initiation of infection provided complete protection against the TSS-like syndrome and the mortality which occurred in unprotected rabbits.

Synergistic induction of interleukin-1 by endotoxin and toxic shock syndrome toxin-1 using rat macrophages.

We studied interleukin-1 (IL-1) secretion by rat peritoneal exudate macrophages stimulated with purified toxic shock syndrome toxin-1 (TSST-1). TSST-1 was observed to be a more potent inducer of IL-1 than was endotoxin. The induction of IL-1 secretion by TSST-1 was not blocked by polymyxin B but could be blocked by monoclonal antibodies directed against TSST-1. Synergistic induction of IL-1 was observed when the cells were stimulated with TSST-1 and endotoxin. The sequence of addition was found to be important for the synergistic response. Enhanced IL-1 production was observed only when macrophages were exposed to endotoxin before or simultaneously with TSST-1. Prior exposure of macrophages to TSST-1 had no enhancing effect on endotoxin-induced IL-1 secretion. We conclude that stimulation of the macrophage by endotoxin enhances the responsiveness of the cells to TSST-1 and may thereby play a role in the pathogenesis of toxic shock syndrome.

Immunological protection of rabbits infected with Staphylococcus aureus isolates from patients with toxic shock syndrome.

Toxic shock syndrome toxin-1 (TSST-1) isolated from the growth medium of Staphylococcus aureus 1169 and 555 was used to immunize male rabbits before infection with either a TSST-1+ or a TSST-1- strain of S. aureus isolated from cases of TSS. None of the immunized rabbits died as a result of the infections, whereas 50% of the nonimmunized rabbits infected with the TSST-1- strain, D4508, and 75% of those infected with the TSST-1+ strain, 555, died. Western blots of crude extracellular protein preparations probed with sera from immunized rabbits indicated that the TSST-1- strain produces a 30,000-molecular-weight protein that cross-reacts with antiserum to TSST-1. Because both organisms caused similar diseases in rabbits, we propose to designate the cross-reacting protein as TSST-2.

Hormonal influence on experimental infections by a toxic shock strain of Staphylococcus aureus.

Subcutaneous infection chambers in rabbits were infected with a strain of Staphylococcus aureus isolated from a patient with toxic shock syndrome. Estrogens (mestranol and 17-beta-estradiol) protected male rabbits and prolonged survival. Neither androgens (testosterone and dihydrotestosterone) nor progesterone affected the susceptibility of intact or ovarihysterectomized female rabbits.

Enhanced susceptibility of male rabbits to infection with a toxic shock strain of Staphylococcus aureus.

Artificial infection chambers in rabbits were infected with a toxic shock strain of Staphylococcus aureus in an attempt to determine the nature of the enhanced virulence of toxic shock strains relative to non-toxic shock strains of staphylococci. The results showed that rabbits immunized with either neutral or acidic proteins were protected from the lethal effects of these infections. Male rabbits were found to be significantly more susceptible to these infections than female rabbits. Castration rendered both sexes equally susceptible to lethal infections. Numerous tissues from all infected rabbits were examined histologically, and most of the pathological findings involved lymphoid tissue. Of special interest was the observation that unprotected male rabbits which died had evidence of lymphoid depletion and that surviving rabbits, both male and female, usually manifested lymphoid hyperplasia. No other pathological response was noted which would characterize these infections, but immunized rabbits had a diminished level of thymic cortex involution that was not different between the sexes.

Malacoplakia associated with vesicoureteral reflux and selective immunoglobulin A deficiency.

A case of malacoplakia involving the lower urinary tract of a young black boy, with associated bilateral vesicoureteral reflux, hydronephrosis and selective immunoglobulin A deficiency is reported. Reflux was caused by the malacoplakia. Reflux and hydronephrosis persisted despite elimination of bacterial infection and malacoplakia by drug therapy. These abnormalities were corrected by a conventional antireflux operation. Malacoplakia appears to be related to immunologic incompetence and diminished levels of intracellular cyclic 3',5' guanine monophosphate. Cholinergic agonists reverse or prevent the pathological changes of malacoplakia.

Characterization of Staphylococcus aureus isolates from patients with toxic shock syndrome, using polyethylene infection chambers in rabbits.

Isolates of Staphylococcus aureus from patients with toxic shock syndrome (TSS) were compared with non-TSS strains of S. aureus with respect to their virulence in rabbits. When the organisms were injected into subcutaneous chambers (perforated polyethylene golf balls) to assess virulence, a rapid mortality was observed with TSS but not with non-TSS strains. Of 16 TSS strains, 11 caused lethal infections in 33 rabbits tested, and none of the 5 control strains caused mortality in 10 rabbits. This evidence of enhanced virulence associated with TSS strains did not appear to be associated with the size of the inoculum. In addition, strains which produced lethal infections appeared to do so despite a reduction in the size of the original inoculum during the first 24 h. All of the TSS strains and none of the non-TSS strains elaborated extracellular protein(s) with a neutral pI when grown in a dialyzed beef heart medium. No other physiological difference was noted between the TSS and non-TSS strains.

Oxacillin-induced lysis of Staphylococcus aureus.

Six clinical isolates of Staphylococcus aureus were compared for their relative susceptibilities to the killing effects of oxacillin. Three of the strains had minimum bactericidal concentrations which were >10 times the minimum bacteriostatic concentration for this antibiotic and were designated tolerant (Tol(+)). The other strains had minimum bactericidal concentrations which were comparable to the minimum bacteriostatic concentration (Tol(-)). Lysis curves of these strains revealed that the Tol(+) strains exhibited a diminished rate of lysis when inhibited by oxacillin. This reduced rate of lysis was reflected also in a reduced rate of viability loss when the cells were exposed to oxacillin. During log growth the uptake of [(14)C]glycerol by Tol(+) cells was 1.5-fold greater than that by Tol(-) cells. Glycerol-labeled cells of each phenotype secreted radioactivity when inhibited by oxacillin. However, the Tol(+) strains released over twice as much label as the Tol(-) strains. No difference in the proportion of lipid secreted by the two phenotypes was found. The behavior of 60 to 65% of the labeled material released by inhibited cells during both sodium dodecyl sulfate gel electrophoresis and Sepharose 6B chromatography corresponded to that of lipoteichoic acid. When the major component of secreted material was added to oxacillin-inhibited Tol(-) strains, an inhibition of the lytic response was observed. These results suggest that oxacillin tolerance in S. aureus could be related to the enhanced secretion of an autolysin inhibitor, such as lipoteichoic acid.

Aminoglycoside modification by gentamicin-resistant isolates of Staphylococcus aureus.

Three clinical isolates of Staphylococcus aureus, which were previously shown to contain a 50S plasmid conferring resistance to several aminoglycosides, were examined for modifying enzymes. Both the wild-type and heat-cured derivatives of the isolates were screened for acetyl-, adenylyl-, and phosphotransferase activities. The substrates were gentamicin, amikacin, and netilmicin; the results indicated that even though all three activites were present, the phosphotransferase reaction was most responsible for resistance to these antibiotics. The absence of any of the modifying activites in cured derivatives of the three isolates supports the conclusion that aminoglycoside resistance in these strains is conferred by a plasmid.

Plasmid-mediated resistance to gentamicin in Staphylococcus aureus.

Two strains isolated from a recent outbreak of infections by gentamicin-resistant Staphylococcus aureus were examined to determine whether genetic control of this resistance is plasmid or chromosomally mediated. Curing techniques indicated a plasmid location in both strains. Physical isolation and characterization of the plasmid deoxyribonucleic acid from one strain revealed that the determinant for gentamicin resistance resides on a 50S plasmid.

Characterization of autolysins from Mycobacterium smegmatis.

This study demonstrates, for the first time, the autolytic enzymes associated with mycobacterial cell walls. Based on the release of radioactivity and ninhydrin-reactive material from isolated cell walls, it was shown that maximum activity occurs during the late log phase of growth and at a buffer pH of about 8.0. Chemical analyses of autolytic digests of isolated cell walls indicated that at least three autolysins are active under the conditions used. These are N-glycolylmuramic acid-L-alanine amidase, an aminopeptidase that releases L-alanine, and an endopeptidase that solubilizes and L-alanyl-D-glutamic acid dippetide. No other endopeptidase, carboxypeptidase, or glycosidase activity was detected.

Susceptibility of clinical isolates of Staphylococcus aureus to killing by oxacillin.

Sixty clinical isolates of Staphylococcus aureus have been screened for their relative susceptibility to the killing action of oxacillin. Only one of these strains was found to be exceptionally resistant to the bactericidal effect of this and other beta-lactam antibiotics. This ability to survive oxacillin inhibition of cell wall synthesis has been called "tolerance". The characteristics of the tolerant organism, which has been designated the Evans strain, in comparison with other isolates of S. aureus indicate that this form of resistance is not apparent from the minimal inhibitory concentration, is not related to an abnormal growth rate, and can be enhanced by treatment with N-methyl-N'-nitro-N-nitrosoguanidine.

Evidence for participation of autolysins in bactericidal action of oxacillin on Staphylococcus aureus.

A comparison of the autolytic enzyme activity in Staphylococcus aureus strains that differ markedly in their rates of lysis and killing after exposure to oxacillin has been made. Log-phase cells of the clinical isolate that is tolerant to oxacillin inhibition were found to contain a level of autolytic enzyme activity comparable to that in a sensitive strain. This autolysin from log-phase cells was recovered after a single freeze-thaw cycle and assayed by using both native and penicillin (un-cross-linked) mureins. These same assays, however, revealed a significant difference in autolysin activity extractable from the two strains if the cells were inhibited by oxacillin. Under these conditions, the S. aureus strain that is susceptible to the killing and lytic effects of oxacillin had considerably more activity on penicillin murein than did the tolerant organism. These results provide evidence that hydrolytic enzymes on the cell surface are required to augment the wall damage initiated by oxacillin and other beta-lactam antibotics to produce a bactericidal effect.