David F. Scott

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.

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.

The diurnal variation of peptidyl proline hydroxylase activity in different tissues of the rat.

Abstract The activity of peptidyl proline hydroxylase undergoes diurnal fluctuations in the aorta, heart, liver, lung and skin of the rat. Miximal enzyme activity was noted during the dark period in skin and during the light period in the other tissues examined. Further differences between skin and liver were noted in their responses to adrenalectomy and to hydrocortisone treatment. The data implicate adrenal steroids in the rhythmicity of peptidyl proline hydroxylase in skin but not in liver. Furthermore, this system offeres a model to determine the relationship between glucocorticoid induced alterations of peptidyl proline hydroxylase activity and collagen biosynthesis.

Extracellular Phospholipase A from Streptococcus mutans Strain 6715

J Dent Res 57(2): 194, February 1978. Because a recent report has indicated that phospholipase A (PL-A) causes bone resorption in vitro (RABADJIJA, L. and GOLDHABER, P., J u 250 Dent Res 54:Spec Issue A, 192, 1975), it is of inaterest to determine if microorganisms could influence bone resorption by supplying exogenous 200 PL-A. We report here the presence of PL-A in the spent broth of Streptococcus mutans, strain X150f\ 6715, a known pathogen capable of producing , w caries and bone destruction in experimental anXj 0 imals (FITZGERALD, R.J., ET AL,JADA 76:301, E I00O 1968). ° The bacterial strain (supplied by R.J. Gib< bons, Forsyth Dental Center) was grown anaerobically for 18 hours at 37 C in 10 ml of a dialyzed medium described by CARLSSON, J., ET AL 0 (Arch Oral Biol 14:469, 1969) and then trans0 20 40 60 ferred to nephelometric flasks containing 200 MINUTES ml of the same prewarmed broth. Growth was FIG. -Time course of the conversion of phosphatidyl chofollowed with a Klett Summerson photocololine (PC) to lysophosphatidylcholine (LPC) catalyzed by an rimeter (green filter, 540 nm) and samples were extracellular crude enzyme preparation fronm Streptococcus taken at various stages of growth. The samples mutans 6715.

Two mutations in the locus control region hypersensitivity site-2 (5′HS-2) of haplotype 19 βs chromosomes alter binding oftrans-acting factors

There are five major haplotypes associated with sickle cell anemia (SS). Individuals homozygous for haplotypes 3 (Senegal) and 31 (Saudi Arabian) have high fetal hemoglobin (HbF) levels (15 to 30% of total hemoglobin) whereas individuals homozygous for haplotypes 17 (Cameroon), 19 (Benin), and 20 (Bantu) have low HbF levels (1 to 10%). We previously identified several point mutations in the LCR 5′HS‐2 that were specific for haplotype 19 βs chromosomes (compared to the GenBank HUMHBB reference sequence, T→G at position 8580, A→G at position 8598, and A→T at position 9114). We postulated that one or more of these mutations may alter the binding of specific trans‐acting factors and ultimately affect the expression of HbF in these sickle cell patients. We performed gel mobility shift assays using 32P‐end‐labeled double‐stranded 19mers corresponding to each of the LCR 5′HS‐2 normal (GenBank) and mutant sequences. Nuclear extracts prepared from HeLa and HEL cells were used in our experiments and neither the normal nor mutant sequence at position 8580 bound trans‐acting factors in either nuclear extract. The 8598 mutant increased binding of Sp1; using purified protein and both nuclear extracts. HEL extracts were used to quantify the increase in Sp1 binding to the 8598 mutation and we found an increase in binding of 66 and 47%, respectively, in two shifted bands. The 9114 mutation sharply decreased binding of an unknown trans‐acting factor by 74%. This factor was present in both HeLa and HEL nuclear extracts.

Elevation of prolyl hydroxylase activity in reuber hepatomas

Abstract 1. 1. Prolyl hydroxylase activity (EC 1.14.11.2) was determined in Reuber hepatomas and compared to the level of enzyme activity in liver. 2. 2. Enzyme activity of hepatoma tissue was 2–3.5 times the level in liver tissue.

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.

Inhibition of gene expression by the Gγ 5′ flanking region of the Bantu βs chromosome

βs‐chromosome haplotypes are peculiar to specific regions of Africa and Asia and are associated with the occurrence of different fetal hemoglobin (Hb) levels in sickle cell patients. Among these haplotypes, βs‐chromosomes found in the Senegal and the Arab‐India regions are associated with relatively high levels of HbF expression, whereas those around the Benin, Bantu, and the Cameroon regions show low levels of HbF expression. The roles of 5′HS2 and the 5′ flanking (promoter region) region in the expression of globin genes are well documented. Haplotype specific variations are found in these regions and have been postulated to be involved in the regulation of HbF expression. In this study, we have analyzed the effect of sequence variations in regulatory regions of the Bantu 5′HS2 and 5′ flanking region of the Gγ gene on CAT expression. A diminution was observed in K562 cells when the promoter originated from the Bantu βs chromosome. The decreased expression was independent of the origin of the 5′HS2 sequence—combinations of the Bantu promoter were measured with the Benin, Bantu, or Senegal 5′HS2 sequences in K562 cells. However, expression of the same plasmids in murine erythroleukemic (MEL) cells showed no difference in CAT expression among the various sequence combinations studied. Am. J. Hematol. 59:51–56, 1998.