Junji Sakurada

An exfoliative toxin A-converting phage isolated from Staphylococcus aureus strain ZM.

Exfoliative toxin A (ETA) causes staphylococcal scalded‐skin syndrome in children. The gene for ETA was believed to be coded by the chromosomal DNA. We isolated temperate phages from an ETA‐producing strain, ZM, using a restriction minus strain, 1039, as an indicator. One of the prophages, designated φ‐ZM‐1 mediated lysogenic conversion of ETA. The polymerase chain reaction assay of the eta gene revealed that phage φ‐ZM‐1 carries the structural gene for ETA.

Occurrence of Coagulase Serotype among Staphylococcus aureus Strains Isolated from Healthy Individuals -Special Reference to Correlation with Size of Protein-A Gene-

One‐hundred‐and‐nineteen strains of Staphylococcus aureus isolated from healthy individuals for 3 years between 1991 and 1993 were subjected to an investigation on the producibility of proteins including protein A, coagulase, enterotoxins and toxic‐shock syndrome toxin‐1. Especially, protein A was the center of our interest. Among these strains, 69, 43, 3 and 1 strains were found to have the protein‐A gene containing 5, 4, 3 and 2 IgG‐binding domains, respectively. On the other hand, only one strain was devoid of the protein‐A gene. There were some differences in the profile of the coagulase serotype between the group with 4 IgG‐binding domains and that with 5 IgG‐binding domains. Differences in the profile of toxin production were also observed between the two groups.

Conspicuous Ingestion of Staphylococcus aureus Organisms by Murine Fibroblasts In Vitro

A conspicuous adhesion of Staphylococcus aureus organisms to murine cutaneous fibroblasts and NIH/3T3 cells cultured in vitro and subsequent ingestion of S. aureus organisms by these fibroblasts are described. In the present experimental system, only fibroblasts‐adhering S. aureus organisms were efficiently ingested by fibroblasts unlike S. epidermidis and S. saprophytics. These findings might suggest a correlation between the pathogenesis of S. aureus and its intracellular localization in non‐professional phagocytes such as fibroblasts in a special reference to its higher pathogenicity than those of coagulase negative counterparts.

Intracellular Localization of Staphylococcus aureus within Primary Cultured Mouse Kidney Cells

Staphylococcus aureus Cowan I was incubated with monolayers of cells derived from several portions of mouse kidney, and found to be ingested by all types of the renal cells. Intracellular localization of S. aureus was determined by resistance of intracellular cocci against lysostaphin digestion and confirmed by electron microscopy. From renal medulla, three morphological variants of the hyperosmolarity‐tolerant (HOT) cells were obtained. The rate of cocci‐ingesting cells varied from 16.9% to 93.4% among those of the HOT cells at the end of 3‐hr incubation. From renal cortex, three morphological variants of epithelial cells grew in medium RK‐1. Among them, only the cells on the edge of colony ingested Cowan I, while the epithelial cells on the center of colony ingested few cocci. Transferred from medium RK‐1 to MEM supplemented with 10% FBS, part of the cortical cells changed into fibroblast‐like appearance and obtained the capacity to ingest Cowan I. This result may indicate the correlation between ingesting capacity and cellular morphology. From a glomerulus, epithelial (GE) cells and fibroblast‐like (GF) cells were obtained. The GE cells ingested not only S. aureus Cowan I but Staphylococcus epidermidis and Staphylococcus saprophyticus after 30‐min incubation, while the GF cells, like both of the HOT cells and the cortical cells, ingested only S. aureus. These results suggest a possibility that S. aureus is located within nonprofessional phagocytes during its infection and intracellular coccus plays an important role in its pathogenicity.

Altered Virulence of a Pleiotropic Staphylococcus aureus Mutant with a Low Producibility of Coagulase and Other Factors in Mice

The virulence of a pleiotropic Staphylococcus aureus mutant with an extremely low producibility of coagulase and other factors was investigated in mouse. A mutant strain, designated as CL‐1, showed the same LD50 and the same intrarenal proliferation as its parental strain, when the mutant organisms were inoculated in mice in high doses. The mutant organisms, however, showed a diminished intrarenal proliferation compared with its parental organisms in low doses. This mutant strain expressed a pleiotropic phenotype such as a concomitant reduction in the producibility of coagulase, α‐toxin, and Panton‐Valentine leucocidin. The total effect due to the reduction in producibility of various factors on the virulence of the mutant strain was investigated with studies on the bacterial resistance to the phagocytic activity of leucocytes. A possible role of coagulase and that of some other staphylococcal exoproteins in the pathogenesis of S. aureus were discussed.

Apoptosis Observed in BALB/3T3 Cells Having Ingested Staphylococcus aureus

Staphylococcus aureus was previously shown to be internalized by murine fibroblast. We examined the intracellular events of S. aureus ingested by BALB/3T3 cells. After uptake of strains A191 and A151, isolates from atopic lesion, and a laboratory strain, Cowan I, for 1 hr, BALB/3T3 cells were incubated with 1.25 μ/ml lysostaphin. Laddering of the DNA in multiples of approximately 180 bp occurred within 4 hr following bacterial addition in BALB/3T3 cells infected with A191 and within 18 hr in BALB/3T3 cells infected with A151: histochemical staining by the terminal deoxynucleotidyl transferase‐mediated dUTP‐biotin nick end labeling method revealed that the rate of the fragmentation of nucleic DNA in Cowan I‐infected BALB/3T3 cells at 21 hr following bacterial addition was 0.52 ± 0.25%, significantly higher than that in the control cells. Transmission electron micrographs of BALB/3T3 cells at 4 hr following A191 addition showed that the apoptotic features, including electron‐dense nucleus and plasma membrane blebbing, occurred in some cells in which many staphylococci escaped the endosome and went on to cell division. At the same time, A151 organisms enclosed with endosome membrane were static in the intact BALB/3T3 cells. The significant increase of A191 was confirmed by counting intracellular live bacteria during 2‐ to 6‐hr incubation. These results suggest that internalized S. aureus escapes the endosome, multiplies and induces apoptosis in the fibroblast cell.

Improved Methods for Detection and Serotyping of Coagulase from Staphylococcus aureus

Improved methods for detection and serotyping of staphylocoagulase were concomitantly devised. We devised an improved method for detection of coagulase activity on agarose film in the same manner as single radial immunodiffusion. The amounts of reagents required for detection of coagulase on agarose film were successfully diminished by adding polyethylene glycol (PEG) to the original formula described by Boothby et al. Using microplates in another improved method for coagulase serotyping, the amount of reaction fluid required was considerably less compared with the conventional tube method. PEG was found to be also effective to increase the efficacy of coagulase serotyping. In the presence or absence of anti‐coagulase antisera, culture supernatants of staphylococcal strain grown in brain heart infusion broth were incubated with the reaction fluid containing bovine fibrinogen, rabbit plasma, 6‐amino caproic acid, polyethylene glycol 6,000. Coagulase activity was visualized as a turbid mass formed in the wells. Turbid mass formation due to coagulase activity was type‐specifically inhibited in the presence of type‐specific antisera. Detailed procedures of the methods are precisely described with some preliminary results obtained by the methods.

Different effects of fibronectin on the phagocytosis of Staphylococcus aureus and coagulase-negative staphylococci by murine peritoneal macrophages.

Fibronectin is known to be an important factor in colonization by Staphylococcus aureus of host tissues as well as other extracellular matrix proteins such as collagen and laminin. We investigated the effect of fibronectin on the phagocytosis of the S. aureus Cowan I strain by macrophages and of coagulase‐negative staphylococci (CNS) strains for comparison. Fibronectin‐reduced serum in place of normal serum lowered the phagocytic activity of the macrophages on the Cowan I strain. Purified fibronectin enhanced the phagocytic activity of the strain in a dose‐dependent manner. On the other hand, fibronectin did not show any opsonic effect on the ingestion of CNS strains, though the binding of fibronectin occurred equally well in CNS strains and the Cowan I strain. Fibronectin‐binding protein (FnBP), the specific fibronectin receptor on the surface on S. aureus, was detected in both the Cowan I strain and CNS strains. Polymerase chain reaction confirmed that not only the Cowan I strain, but also CNS strains possessed the FnBP gene. These results indicate that fibronectin shows an opsonic effect on the S. aureus, Cowan I strain but not on CNS strains, and suggest that the binding of fibronectin to FnBP is not sufficient for efficient phagocytosis of the staphylococci strains by macrophages.

Effects of Cytochalasins B and D on Staphylococcus aureus Adherence to and Ingestion by Mouse Renal Cells from Primary Culture

Cytochalasin B (CB) and cytochalasin D (CD), inhibitors of microfilament function of host cell, were examined for their effects on Staphylococcus aureus Cowan I adherence to and ingestion by several types of the hyperosmolarity‐tolerant (HOT) cells obtained from primary culture of mouse kidney. Staphylococcal adherence to the HOT cells with epithelial appearance was extraordinarily enhanced by the treatment of those cells with both 5 μg/ml of CB and CD. In particular, staphylococci adhered to the periphery rather than the center of each cytochalasintreated cell. Staphylococcal ingestion by all types of the HOT cells was markedly inhibited by CD in spite of the enhanced adherence. Contrary to our expectation, inhibition by CB was incomplete, and the enhanced adherence of staphylococci to CB‐treated cells resulted in the enhanced ingestion.

Subcutaneous growth of Staphylococcus aureus concomitantly inoculated with Ehrlich ascites tumor cells

Intratumoral growth of Staphylococcus aureus Cowan I‐derived AP332 was examined by subcutaneous inoculation of cocci in doses ranging from 18 to 1.8 × 105 CFU with Ehrlich ascites tumor cells. Inoculation of 18 CFU AP332 resulted in staphylococcal growth in one of five mice, and the proportion of mice established intratumoral infection increased with the initial inocula. Six other strains of S. aureus also grew in the tumor tissue, and none of the three strains of coagulase‐negative staphylococci grew at all. Ethanol‐killed tumor cells did not promote staphylococcal growth as vigorously as the live tumor cells, especially when the initial inoculum of AP332 was smaller than 104 CFU.