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Biomedical subjects

H Suginaka

Publications and source records attributed to H Suginaka.

At least 37 records · Page 2Linked to original sources
PubMed1997

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.

Carrier State↗
PubMed1997

High incidence of Staphylococcus aureus from dentures and tongues of maxillary resection patients.

The incidence and virulence of Staphylococcus aureus from upper dentures and tongues of maxillary resection and edentulous patients were examined. The incidence rates of S. aureus on dentures and tongues from the 8 maxillary resection patients were significantly higher than those from the 20 maxillary edentulous patients. Some S. aureus strains in both patient groups were resistant to antibiotics, and 1 methicillin-resistant S. aureus strain was isolated in each group. Exfoliative toxin was not produced, whereas diverse types of enterotoxin were detected in 4 of the 8 maxillary resection patients and in 3 of 13 maxillary edentulous patients. These findings show that S. aureus inhabits dentures and tongues of maxillary resection patients more often than those of maxillary edentulous patients and that some strains have potential virulence, suggesting that maxillary resection patients are potentially more exposed to a threat from S. aureus and would be the carriers.

Aged↗
PubMed1997

Cloning and characterization of the fmt gene which affects the methicillin resistance level and autolysis in the presence of triton X-100 in methicillin-resistant Staphylococcus aureus.

In methicillin-resistant Staphylococcus aureus (MRSA) strains, Triton X-100 reduced the oxacillin resistance level, although the degree of reduction varied from strain to strain. To study the responses of MRSA strains to Triton X-100, we isolated a Tn551 insertion mutant of the COL strain that became more susceptible to oxacillin in the presence of 0.02% Triton X-100. The Tn551 insertion of the mutant was transduced back to the parent strain, other MRSA strains (strains KSA8 and NCTC 10443), and methicillin-susceptible strain RN450. All transductants of MRSA strains had reduced levels of resistance to oxacillin in the presence of 0.02% Triton X-100, while those of RN450 did not. Tn551 mutants of KSA8 and NCTC 10443 also had reduced levels of resistance in the absence of 0.02% Triton X-100. The autolysis rates of the transductants in the presence of 0.02% Triton X-100 were significantly increased. Amino acid analysis of peptidoglycan and testing of heat-inactivated cells for their susceptibilities to several bacteriolytic enzymes showed that there were no significant differences between the parents and the respective Tn551 mutants. The Tn551 insertion site mapped at a location different from the previously identified fem and llm sites. Cloning and sequencing showed that Tn551 had inserted at the C-terminal region of a novel gene designated fmt. The putative Fmt protein showed a hydropathy pattern similar to that of S. aureus penicillin-binding proteins and contained two of the three conserved motifs shared by penicillin-binding proteins and beta-lactamases, suggesting that fmt may be involved in cell wall synthesis.

Bacteriolysis↗
PubMed1997

epr, which encodes glycylglycine endopeptidase resistance, is homologous to femAB and affects serine content of peptidoglycan cross bridges in Staphylococcus capitis and Staphylococcus aureus.

Staphylococcus capitis EPK1 produces a glycylglycine endopeptidase, ALE-1 (M. Sugai, T. Fujiwara, T. Akiyama, M. Ohara, H. Komatsuzawa, S. Inoue, and H. Suginaka, J. Bacteriol. 179:1193-1202, 1997), which hydrolyzes interpeptide pentaglycine chains of cell wall peptidoglycan of S. aureus. Characterizations of the enzyme activity and cloning of ale-1 revealed that ALE-1 is very similar to prolysostaphin produced by S. simulans bv. staphylolyticus. Strain EPK1 is resistant to lysis by ALE-1 and by lysostaphin. A gene that renders the cells resistant to glycylglycine endopeptidase (epr) was found 322 bp upstream of and in the opposite orientation to ale-1. The deduced amino acid sequence of epr showed similarities to FemA and FemB, which have been characterized as factors essential for methicillin resistance of S. aureus. Inactivation of either femA or femB causes decreased resistance to methicillin, increased resistance to lysostaphin, and decreased glycine content in the interpeptide chains of peptidoglycan. Therefore, femAB is suggested to be involved in the addition of glycine to pentapeptide peptidoglycan precursor. S. aureus with epr on a multicopy plasmid had phenotypes similar to those of femAB mutants except that it did not alter resistance level to methicillin. These results suggest that epr and femAB belong to the protein family involved in adding amino acids to the pentapeptide peptidoglycan precursor and that epr is involved in the addition of serine to the pentapeptide.

Acetylglucosaminidase↗
PubMed1997

Purification and molecular characterization of glycylglycine endopeptidase produced by Staphylococcus capitis EPK1.

A novel staphylolytic enzyme, ALE-1, acting on Staphylococcus aureus, was purified from a Staphylococcus capitis EPK1 culture supernatant. The optimal pH range for staphylolytic activity was 7 to 9. ALE-1 contains one Zn2+ atom per molecule. Analysis of peptidoglycan fragments released by ALE-1 indicated that the enzyme is a glycylglycine endopeptidase. The effects of various modulators were determined, and we found that o-phenanthroline, iodoacetic acid, diethylpyrocarbonate, and Cu2+ reduced the staphylolytic activity of ALE-1. beta-Casein, elastin, and pentaglycine were poor substrates for ALE-1. Molecular cloning data revealed that ALE-1 is composed of 362 amino acid residues and is synthesized as a precursor protein which is cleaved after Ala at position 35, thus producing a mature ALE-1 of 35.6 kDa. The primary structure of mature ALE-1 is very similar to the proenzyme form of lysostaphin. It has the modular design of an N-terminal domain of tandem repeats of a 13-amino-acid sequence fused to the active site containing C-terminal domain. Unlike lysostaphin, ALE-1 does not undergo processing of the N-terminal repeat domain in broth culture. ale-1 is encoded on the plasmid. Protein homology search suggested that ALE-1 and lysostaphin are members of the novel Zn2+ protease family with a homologous 38-amino-acid-long motif, Tyr-X-His-X(11)-Val-X(12/20)-Gly-X(5-6)-His.

Amino Acid Sequence↗
PubMed1997

Localized perforation of the cell wall by a major autolysin: atl gene products and the onset of penicillin-induced lysis of Staphylococcus aureus.

We investigated the cell surface localization of the atl gene products of Staphylococcus aureus exposed to a lytic concentration (4 MIC) of penicillin G (PCG) by means of immunoelectron microscopy using anti-62-kDa N-acetylmuramyl-L-alanine amidase or anti-51-kDa endo-beta-N-acetylglucosaminidase immunoglobulin G. Protein A-gold conjugates reacting with antigen-antibody complex localized at sites of defects of the cell wall at the nascent cross wall. Anti-62-kDa N-acetylmuramyl-L-alanine amidase or anti-51-kDa endo-beta-N-acetylglucosaminidase immunoglobulin G inhibited the decreased turbidity caused by PCG-induced lysis and the formation of defects in the wall. The autolysis-defective mutant, S. aureus RUSAL2 (atl::Tn551), exposed to 4 MIC of PCG resisted autolysis and formation of the wall defect. These results suggest that activation or deregulation of the atl gene products at localized sites where formation of new cross wall was disturbed by PCG causes small defects in the cell wall in situ, eventually leading to general autolysis.

Antibodies↗
PubMed1996

Isolation and characterization of teichoic acid-lake substance as an adhesin of Staphylococcus aureus to HeLa cells.

A cell wall component that bound to HeLa cells (HeLa cell-binding CWC) was isolated from a clinical isolate of Staphylococcus aureus. The HeLa cell-binding CWC was resistant to heat (100 C, 1 hr) and proteases, did not stain with Coomassie Brilliant Blue R-250 on SDS-PAGE but stained as a broad band with antiserum against the strain on Western blots. These data suggest that the HeLa cell-binding CWC is not a protein, and may be teichoic acid. Purified teichoic acid bound to HeLa cells, whereas fractions without teichoic acid did not. In Western blots, HeLa cell-binding CWC appeared as a broad band of less than 35 kDa, similar to that of purified teichoic acid. These data suggest that the HeLa cell-binding CWC obtained in this study is teichoic acid. Teichoic acid inhibited S. aureus adherence to HeLa cells and bound to the cells time and dose dependently, in a saturable and reversible manner, and therefore appears to be an adhesin of S. aureus to HeLa cells.

Adhesins, Bacterial↗
PubMed1996

An autolysin ring associated with cell separation of Staphylococcus aureus.

atl is a newly discovered autolysin gene in Staphylococcus aureus. The gene product, ATL, is a unique, bifunctional protein that has an amidase domain and a glucosaminidase domain. It undergoes proteolytic processing to generate two extracellular peptidoglycan hydrolases, a 59-kDa endo-beta-N-acetylglucosaminidase and a 62-kDa N-acetylmuramyl-L-alanine amidase. It has been suggested that these enzymes are involved in the separation of daughter cells after cell division. We recently demonstrated that atl gene products are cell associated (unpublished data). The cell surface localization of the atl gene products was investigated by immunoelectron microscopy using anti-62-kDa N-acetylmuramyl-L-alanine amidase or anti-51-kDa endo-beta-N-acetylglucosaminidase immunoglobulin G. Protein A-gold particles reacting with the antigen-antibody complex were found to form a ring structure on the cell surface at the septal region for the next cell division site. Electron microscopic examination of an ultrathin section of the preembedded sample revealed preferential distribution of the gold particles at the presumptive sites for cell separation where the new septa had not been completed. The distribution of the gold particles on the surface of protoplast cells and the association of the gold particles with fibrous materials extending from the cells suggested that some atl gene products were associated with a cellular component extending from the cell membrane, such as lipoteichoic acid. The formation of a ring structure of atl gene products may be required for efficient partitioning of daughter cells after cell division.

Acetylglucosaminidase↗
PubMed1995

Triton X-100 alters the resistance level of methicillin-resistant Staphylococcus aureus to oxacillin.

We used population analysis to examine the effects of Triton X-100 on the level of resistance to oxacillin of 18 methicillin-resistant Staphylococcus aureus. In the presence of 0.02% Triton-X-100, 17 formerly methicillin-resistant strains exhibited enhanced sensitivity to oxacillin. One homogenous isolate, KSAF1 was barely affected by the Triton X-100. Sensitivities of lysostaphin, 51 kDa N-acetylglucosaminidase and 62 kDa N-acetylmuramoyl-L- alanine amidase to heat-inactivated cells were not affected when the bacteria were grown in 0.02% Triton X-100. Our data, together with those of a previous study, suggested that Triton X-100 alters the resistance level of methicillin-resistant S. aureus influencing a factor (s) other than PBPs, bacteriolytic enzymes, or femAB products.

Acetylglucosaminidase↗
PubMed1995

A Staphylococcus aureus autolysin that has an N-acetylmuramoyl-L-alanine amidase domain and an endo-beta-N-acetylglucosaminidase domain: cloning, sequence analysis, and characterization.

The Tn551 insertion site of the autolysis-deficient Staphylococcus aureus mutant RUSAL2 was cloned and used to identify the autolysis gene atl in the parent strain, RN450. The open reading frame for atl was 3768 bp in length, encoding a deduced protein of 1256 amino acids and molecular size of 137,381 Da. The atl gene product is a bifunctional protein that has an amidase domain and an endo-beta-N-acetylglucosaminidase domain which must undergo proteolytic processing to generate the two extracellular lytic enzymes found in the culture broth of S. aureus.

Amino Acid Sequence↗
PubMed1995

Alteration of bacteriolytic enzyme profile of Staphylococcus aureus during growth.

Profiles of cell-associated bacteriolytic activities and those in the culture supernatant of Staphylococcus aureus FDA209P at various stages of growth were analyzed using sodium dodecyl sulfate-polyacrylamide gels containing Micrococcus luteus or S. aureus. In the logarithmic growth phase, the cell-associated bacteriolytic activities extracted with Triton X-100 contained a number of bacteriolytic proteins, the profiles of which were similar to those we reported elsewhere (Sugai, M., Akiyama, T., Komatsuzawa, H., Miyake, Y., and Suginaka, H. (1990) J. Bacteriol., 172, 6494-6498). The proteins include P1, P2, P7, P9, PX, P13, P18 and other minor components. At the stationary growth phase, the bacteriolytic band-profile of the Triton X-100 extract changed dramatically. P1, P7 and P9 disappeared, and the other minor bands had markedly decreased band intensities. On the other hand, P2, PX, P13, and P18 retained their band intensities during the stationary growth phase. The band intensities of P7, P13, PX, and P18 increased in the supernatant during the logarithmic growth phase. These results indicated that the bacteriolytic band-profile changes during growth.

N-Acetylmuramoyl-L-alanine Amidase↗
PubMed1995

Microbiological features of gingivitis in pubertal children.

The subgingival microflora of 42 pubertal children (aged 12-15 years) and 18 young adults (aged 21-25 years) was investigated by anaerobic culture and phase contrast microscopy. Motile rods, spirochetes and Prevotella intermedia were elevated proportionately in pubertal children with gingivitis (median GI > or = 1); however, no statistically significant differences in enumerated organisms on selective media were observed between pubertal children and young adults. These organisms were positively correlated with the index of bleeding on probing and the gingival index of pubertal children. Pubertal children were divided into 3 distinct subject clusters according to the similarity of subgingival microbial features, and the subjects who were then selected for the gingivitis group were distributed into clusters 1 and 2. The proportions of motile rods, P. intermedia and Eikenella corrodens were significantly higher in cluster 2 than in cluster 1. These results suggest that these 2 species and motile rods which differentiated features of clusters, are useful for screening of high-risk subjects for worsening of inflammation.

Actinomyces↗
PubMed1995

In vitro activity of tetracyclines, macrolides, quinolones, clindamycin and metronidazole against periodontopathic bacteria.

We re-evaluated several antibiotics including newer ones, for their in vitro killing activity, as well as their inhibitory activity, against clinical isolates of periodontopathic bacteria. Tetracyclines were active against Porphyromonas gingivalis, and were highly active against Prevotella intermedia, but demonstrated only a low killing activity against Actinobacillus actinomycetemcomitans. Rokitamycin, a new macrolide, and clindamycin were highly active against P. gingivalis and P. intermedia, but showed very weak killing activity against A. actinomycetemcomitans. Quinolones demonstrated excellent bactericidal activity against A. actinomycetemcomitans, and good inhibitory and bactericidal activity against P. gingivalis and P. intermedia. Metronidazole had an activity almost equivalent to quinolones against P. gingivalis and P. intermedia; but it was the least active against A. actinomycetemcomitans.

Aggregatibacter actinomycetemcomitans↗
PubMed1995

Identification of endo-beta-N-acetylglucosaminidase and N-acetylmuramyl-L-alanine amidase as cluster-dispersing enzymes in Staphylococcus aureus.

Two proteins which are capable of dispersing cell clusters of Staphylococcus aureus have been purified from a S. aureus FDA209P culture supernatant. Both of them were found to have bacteriolytic activity. From the elution profile of column chromatography and Western blot (immunoblot) analysis, one of them was identified as a 51-kDa endo-beta-N-acetylglucosaminidase (GL). The other was a 62-kDa protein on the basis of sodium dodecyl sulfate gel electrophoresis. Analysis of the peptidoglycan fragments following treatment with the 62-kDa protein indicated that this protein is an N-acetylmuramyl-L-alanine amidase (AM). In vitro studies of cluster dispersion activities using S. aureus mutant strains Lyt66 or S. aureus Wood46 grown as clusters demonstrated that these two enzymes act synergistically to disperse clusters into single cells. Antiserum against the 51-kDa GL cross-reacted with the 62-kDa AM, and S. aureus FDA209P grown in the presence of anti-51-kDa-GL immunoglobulin G induced giant clusters. Clusters induced by anti-51-kDa GL and by Cibacron blue F3G-A were dispersed by coincubation with the 51-kDa GL and the 62-kDa AM. Western blot analysis demonstrated that the 51-kDa GL and the 62-kDa AM were missing in culture supernatants of S. aureus Lyt66, Wood46, and RUSAL2 (Tn551 autolysin-defective mutant), which grow in clusters. These results strongly suggest that the 51-kDa GL and 62-kDa AM are involved in cell separation of daughter cells after cell division.

Acetylglucosaminidase↗
PubMed1994

Effect of combination of oxacillin and non-beta-lactam antibiotics on methicillin-resistant Staphylococcus aureus.

The in-vitro activity of oxacillin combined with non-beta-lactam antibiotics, bacitracin, vancomycin, enduracidin, tunicamycin, flavomycin, fosfomycin, and cycloserine, was investigated in 23 methicillin-resistant and 15 methicillin-susceptible Staphylococcus aureus strains. In the presence of a non-beta-lactam antibiotic (0.25 MIC), the MICs of oxacillin for methicillin-resistant S. aureus (MRSA) strains and methicillin-susceptible S. aureus (MSSA) strains were lowered. This effect was most marked with MRSA strains, and bacitracin, flavomycin, and tunicamycin increased the susceptibility of MRSA to oxacillin by greater than 200-fold. More than 87% and 77% of MRSA and MSSA strains, respectively, were synergically inhibited by a combination of oxacillin with bacitracin, tunicamycin, flavomycin or cycloserine (fractional inhibitory concentration < or = 0.5). Polyanetholesulfonic acid prevented the lysis of MRSA cells treated with a combination of oxacillin and 0.25 MIC of bacitracin, but did not prevent cell death. The penicillin binding protein (PBP) profile of MRSA was not affected by incubation with 0.25 MIC of non-beta-lactam antibiotics. These results suggest that the increased antibacterial activity of oxacillin in the presence of non-beta-lactam antibiotic is not the consequence of activation of autolysis or of a decrease in bulk PBP synthesis.

Anti-Bacterial Agents↗
PubMed1994

The effect of Triton X-100 on the in-vitro susceptibility of methicillin-resistant Staphylococcus aureus to oxacillin.

The effect of the non-ionic detergent, Triton X-100, on the in-vitro activity of oxacillin against methicillin-resistant (MRSA) and methicillin-susceptible (MSSA) strains of Staphylococcus aureus was investigated. In the presence of Triton X-100, the MICs of oxacillin for both MRSA and MSSA isolates were reduced; this enhancing effect was particularly marked for the MRSA strains. Triton X-100 therefore counteracted the resistance to methicillin encoded by mecA. In the presence of oxacillin at subinhibitory concentrations, Triton X-100 induced the bacteriolysis of MRSA and potentiated the autolysis of these organisms. However, the detergent had no effect on the bacteriolytic enzyme profile or the susceptibility of the bacterial cell wall to bacteriolytic enzymes, nor did it promote the binding of oxacillin to the penicillin-binding protein (PBP) 2A. On the other hand, it stimulated the release from the bacteria of acylated lipoteichoic acid (LTA), a putative endogenous regulator of autolysins. Autolytic enzyme-deficient MRSA mutants were equally as sensitive as the parent strain to the effect of Triton X-100 on susceptibility to oxacillin. These results indicate that the enhanced in-vitro activity of oxacillin against MRSA in the presence of Triton X-100 cannot be accounted for simply by the induction of bacteriolysis following activation of autolytic enzymes by the detergent-stimulated release of LTA.

Autolysis↗
PubMed1994

Isolation and characterization of Staphylococcus aureus mutants which form altered cell clusters.

Staphylococcus aureus FDA 209P produces two extracellular bacteriolytic enzymes, 51-kDa endo-beta-N-acetylglucosaminidase (GL) and 62-kDa N-acetylmuramyl-L-alanine amidase (AM), both of which can disperse cell clusters. To characterize the physiological roles of these enzymes in vivo, mutants with altered autolysin activity were isolated, and their degree of cluster formation in broth culture was assessed. Bacteriolytic activities of GL and AM, produced and secreted from these mutants into the culture fluid and detected with activity gels, coincided well with the degree of cluster formation of the mutants. The mutants with little or no enzyme activity grew in clusters, whereas those with high activity grew as well-separated cocci, suggesting that these enzymes are involved in cell separation of S. aureus in vivo.

Acetylglucosaminidase↗