Yoichiro Miyake

Simple Method for Measuring the Antibiotic Concentration Required to Kill Adherent Bacteria

A simple method was developed for measuring the antibiotic susceptibility of bacteria adherent to plastic surfaces. Staphylococcus aureus cells adhered to the bottom of a 96-well plastic tissue culture plate were incubated in serially diluted antibiotic solutions. After a 24-hour-incubation the solutions were removed, and fresh medium without antibiotics was added to each well. The viability of the cells was judged by their growth after a further 24-hour-incubation. The antibiotic concentration required to kill adherent bacteria was far higher than that required for planktonic cells, although we used bactericidal drugs; beta-lactam, quinolone, and aminoglycoside antibiotics. The tolerance demonstrated by adherent cells is likely to play a role in the difficulties encountered in the chemotherapy of biofilm infections.

Cell-surface hydrophobicity of Candida species as determined by the contact-angle and hydrocarbon-adherence methods.

Cell-surface hydrophobicities of six Candida species were studied by two methods: measurement of the contact angle, and partitioning with aqueous-hydrocarbon (n-octane, n-hexadecane and p-xylene) mixtures. C. tropicalis, C. glabrata and C. krusei adhered better to the hydrocarbons than did C. albicans, C. stellatoidea and C. parapsilosis. Contact angles for the less adherent species were smaller than those for the more adherent species. Thus the two methods gave results that were similar overall and indicated that C. tropicalis, C. glabrata and C. krusei have greater cell-surface hydrophobicities than C. albicans, C. stellatoidea and C. parapsilosis.

Antifungal drugs effect adherence of Candida albicans to acrylic surfaces by changing the zeta‐potential of fungal cells

The effect of sub-inhibitory concentrations of antifungal drugs on the adherence of Candida albicans to acrylic surfaces was investigated. Among five antifungals tested, azalomycin F and aculeacin A significantly enhanced the adherence. The zeta-potential of fungal cells was affected by antifungal drugs, whereas no significant change in cell surface hydrophobicity was observed. The relationship obtained between the change in the adherence and that in zeta-potential suggests that the enhanced adherence was caused by decreased electric repulsive forces.

Purification and characterization of neutrophil chemotactic factors of Streptococcus sanguis

Two neutrophil chemotactic factors were isolated from the culture filtrates of Streptococcus sanguis ATCC 10556 and were chemically characterized as N-terminal blocked peptides of low molecular weight. One of the factors consisted of proline, valine, methionine, isoleucine and leucine and the other of methionine, isoleucine, leucine and phenylalanine. In both factors, methionine was detected as the sole N-terminal amino acid, but the amino group was blocked. The removal of N-terminal methionine yielded several N-terminal amino acids, suggesting that S. sanguis produced several N-terminal blocked methionyl peptides, all of which could be chemotactically active.

Ability of enzymes to remove Candida.

Infection by C. albicans is a significant cause of denture stomatitis. Therefore, the results of this study, which demonstrated that yeast lytic enzymes and proteolytic enzymes removed C. albicans from acrylic resin surfaces, suggest that these compounds are potentially useful denture cleansers.

A Long-Term Survey of Methicillin-Resistant Staphylococcus aureus in the Oral Cavity of Children

Methicillin‐resistant Staphylococcus aureus (MRSA), an indigenous bacteria in healthy people, often causes nosocomial infections. If the host human becomes compromised, MRSA can cause a serious infection. The long‐term colonization of MRSA increases this risk. The purpose of this study was to demonstrate the incidence of S. aureus and MRSA colonization in the oral cavities of healthy children, and to examine the stability of identical strains of MRSA over a long‐term period. Fourteen children were examined in two stages (first stage: 1987–88, second stage: 1992–93). Five of the 14 children were negative for S. aureus in both stages, seven children were positive in both stages and two children were positive in only the second stage. The children who were colonized with S. aureus in the first stage always harbored the bacteria in the second stage. Of the seven children that were positive for S. aureus in both stages, three persisted in carrying MRSA. We compared two MRSA strains isolated from the same children in both stages by coagulase typing, antibiogram typing and DNA fingerprinting. In two children, the strains showed the same coagulase types, similar antibiograms and similar DNA fragment profiles. These data strongly suggest that identical strains of MRSA persisted in the oral cavities for more than five years, and that the oral cavity can serve as a reservoir for MRSA with the potential to cause nosocomial infections.

Detection of a staphylococcal endo-β-N-acetylglucosaminidase using polyacrylamide gels

Abstract An activity gel method has been developed for the detection of bacteriolytic enzymes. After gel electrophoresis of the enzymes in the presence of SDS, enzyme activity was seen in the gel as a transparent band resulting from bacteriolysis in the gels containing bacterial cells. Staphylococcal endo- β - N -acetylglucosaminidase, as a model enzyme, was employed to see the analytical advantage of this method relative to the turbidimetric method. This activity gel method detects in the order of 100 pg of staphylococcal endo- β - N -acetylglucosaminidase. The method is simple to use, very sensitive and applicable to renaturable bacteriolytic enzymes.

Electrophoretic studies of extracellular glucosyltransferases and fructosyltransferases from seventeen strains of Streptococcus mutans

Streptococcus mutans was classified by the electrophoretic properties of glucosyltransferases (GTases) and fructosyltransferases (FTases). The cells of serotypes a, d and g did not release extracellular FTases, although those from other serotypes did. The enzymes from cells of serotypes d and g synthesized a good deal of insoluble polysaccharide compared with other serotypes. The enzymes were applied to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and polyacrylamide gel-isoelectric focussing (PAG-IEF). Gels were stained for their activity and protein content. Enzymes belonging to the same serotype gave the same specific pattern on both gels. The seven serotypes could be classified into the following four groups: serotypes d and g, serotype a, serotypes c, e and f, and serotype b. The results agree well with some previous reports based on other methods. The molecular weights of three GTase bands were 156K, 146K and 135K, and of four kinds of FTase bands were 108K, 95K, 80K and 76K. The isoelectric points of main enzymes were 4.25, 4.60, 5.00, 5.55 and 5.70. Those of FTases were 4.25 and 4.60.

Purification of Staphylococcal Exfoliative Toxin by High Pressure Liquid Chromatography

Exfoliative toxin (ET) isolated from a clinical strain of Staphylococcus aureus was purified to homogeneity, using a 3-step HPLC system. NH2-terminal 20 amino residues of purified ET was found to be identical with ETA of S. aureus TA (7), S. aureus TC16 (9) and S. aureus ZM (10), but stability of purified ET was completely different from that of ETA. This purification system gave a high yield of pure ET, which exhibited higher purity than specimens purified by more complicated and time-consuming procedures. It is useful for small-scale purification for the comparative study of ET and easy to scale up for preparative purification.

Rapid purification method of lysostaphin for analysis of cell-wall proteins

Abstract Lysostaphin was purified from a commercially available specimen by using a dye ligand affinity high-pressure liquid chromatography (HPLC). This purification procedure is simple and rapid, with high recovery and is useful for preparing analytical grade-lysostaphin for detailed investigation of the cell-wall proteins of staphylococcus species.