Akemi Usui

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.

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.

Inhibitory effect of bacterial attachment on candidal growth due to adherence with mannose-sensitive pili

A bacterial strain with affinity to Candida albicans was successfully obtained from a natural environment. An uncovered Petri dish containing a suspension of heat‐killed C. albicans cells was allowed to stand in a laboratory for several days. Some bacteria which had adhered to the candidal cells were tested for their ability to agglutinate the cells. A bacterial strain, designated later as CAB‐1, was found to agglutinate candidal cells through bridging by mannose‐sensitive pili. CAB‐1 showed similar bacteriological characteristics to those of Citrobacter freundii by ID test. The adherence of CAB‐1 to candidal cell was precisely presented by scanning electron microscopy. The inhibitory effect of CAB‐1 attachment to candidal cells on the growth of Candida was also preliminarily confirmed.

Efficient Adsorption of Lysostaphin on Bacterial Cells of Lysostaphin-Resistant Staphylococcus aureus Mutant

A simple and efficient method for the purification of staphylolytic endopeptidase (lysostaphin) contained in culture supernatant of Staphylococcus simulans biovar staphylolyticus strain by adsorption of the enzyme on bacterial cells of lysostaphin‐resistant S. aureus mutant was successfully devised. Lysostaphin was sufficiently adsorbed on the heat‐killed mutant cells derived from S. aureus Cowan I and efficiently eluted by 3 M KSCN. Enzyme preparation obtained by a single procedure of the affinity purification was pure enough for practical use. The yield of the enzyme was 25 mg from 1 liter culture and recovery rate was 64%.

Auxiliary Method for Clonal Identification of Staphylococcus aureus by Protein Band Pattern of Released Proteins on SDS-Polyacrylamide Gel

A supportive method for clonal identification of Staphylococcus aureus strains was devised. Culture supernatant obtained by cellophane surface culture was subjected to sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE) without performing any concentration procedure prior to electrophoresis. The combined use of cellophane surface culture and SDS‐PAGE was convenient for determining whether the strains belonged to the same clone or not when conducted in conjunction with other tests for bacteriological characterization.

Conspicuous Growth of Intravenously Inoculated Staphylococcus aureus in Subcutaneously Established Ehrlich Ascites Tumor Tissue of Mice

Intratumoral growth of Staphylococcus aureus was compared with the intrarenal growth, to examine the usefulness of the method as a marker of its pathogenicity. When 5 × 107 CFU/mouse of three derivatives from S. aureus Cowan I with different intrarenal growth were intravenously injected into Ehrlich tumor‐bearing mice, they lodged in the tumor tissue at approximately 103 CFU/0.1 g by 30 min after infection, and grew in the range of 106 CFU/0.1 g to 108 CFU/0.1 g by day 4, regardless of their intrarenal growth capacity. In contrast, S. saprophyticus lodged in both tissues to the same degree as S. aureus, but did not grow at all. The time course of the staphylococcal growth was different between tumor tissue and kidney, suggesting differences in the local responses against S. aureus.