Gary K. Best

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 β-lactam antibotics to produce a bactericidal effect.

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 [14C]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.

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 bones 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.

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.

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.

Lipoteichoic acid inhibition of phagocytosis of Staphylococcus aureus by human polymorphonuclear leukocytes

Abstract Under the influence of cell wall antibiotics, tolerant strains of Staphylococcus aureus have been shown to excrete increased amounts of lipoteichoic acid (LTA) into the growth medium, relative to nontolerant strains. The effect of LTA on the phagocytic and bactericidal rate of isolated neutrophils for S. aureus was studied to determine if the enhanced production of LTA by tolerant strains might account in part for the increased severity of infections due to tolerant organisms. Phagocytosis was measured as leukocyte-associated radioactivity after incubation of neutrophils, human serum, and radiolabeled S. aureus in microtiter wells. The addition of LTA, at final concentrations ranging from 0–5 nmol/ml, to the incubation mixtures resulted in a dose-dependent inhibition of phagocytosis. The decreased uptake of S. aureus by the neutrophils was accompanied by an increase in the number of organisms surviving in the presence of phagocytes. Such an inhibition was not observed when preopsonized bacteria were employed. These results suggest that LTA is able to interfere with the normal rapid opsonization of S. aureus in in vitro phagocytic systems.

Neutrophil function studies in patients with elevated serum IgE levels and recurring Staphylococcus aureus infections

Abstract Leukocyte function tests were performed on seven patients with a history of recurring Staphylococcus aureus infections. Five of the patients had elevated serum immunoglobulin E levels (above 1000 IU/ml) and two had normal IgE levels. Phagocytosis and killing of S. aureus was measured by a microtiter procedure using polymorphonuclear leukocytes isolated by Ficoll centrifugation of dextran-sedimented blood samples. The results revealed that only one of the patients had defective neutrophil function. This patient had the highest serum IgE level of those tested (30,000 IU/ml) and exhibited a diminished bactericidal activity which corresponded with decreased phagocytosis, respiratory burst, and myeloperoxidase activity. None of the reduced function could be corrected by the presence of normal, pooled serum as the source of opsonins. The biochemical basis for this unusual granulocytic defect was not determined, but comparisons were made which showed clear differences from chronic granulomatous disease. Since one of the patients with elevated serum IgE levels manifested an unusual neutrophil defect, additional studies with other such patients may reveal the basis for the recurring S. aureus infection in some of these patients.