Reiko Kariyama

Description of a 23.9-Kilobase Chromosomal Deletion Containing a Region Encoding fsr Genes Which Mainly Determines the Gelatinase-Negative Phenotype of Clinical Isolates of Enterococcus faecalis in Urine

ABSTRACT Expression of virulence-related extracellular proteases, gelatinase, and serine protease of Enterococcus faecalis is regulated by a quorum-sensing system encoded by the fsr gene cluster. In this study, a 23.9-kb chromosomal deletion containing the fsr gene cluster region was found to be present in the majority (79%) of gelatinase-negative clinical isolates of E. faecalis from urine.

Urinary Excretion of Anthocyanins in Humans after Cranberry Juice Ingestion

Cranberry, which is rich in polyphenols, including anthocyanins and proanthocyanidins, has been found to have various effects beneficial to human health, including prevention of urinary tract infections. These effects have been associated with polyphenols in the fruit. We investigated the excretion of anthocyanins in human urine after ingestion of cranberry juice. Eleven healthy volunteers consumed 200 ml of cranberry juice containing 650.8 μg total anthocyanins. Urine samples were collected within 24 h before and after consumption. Six of 12 anthocyanins identified in cranberry were quantified in human urine by HPLC coupled with electrospray ionization and tandem mass spectrometry (HPLC–ESI–MS–MS). Among these, peonidin 3-O-galactoside, the second most plentiful anthocyanin in the juice, was found most abundantly in urine within 24 h, corresponding to 41.5 nmol (56.1% of total anthocyanins). The urinary levels of anthocyanins reached a maximum between 3 and 6 h after ingestion, and the recovery of total anthocyanins in the urine over 24 h was estimated to be 5.0% of the amount consumed. This study found high absorption and excretion of cranberry anthocyanins in human urine.

Siamycin Attenuates fsr Quorum Sensing Mediated by a Gelatinase Biosynthesis-Activating Pheromone in Enterococcus faecalis

The expression of two Enterococcus faecalis virulence-related proteases, gelatinase (GelE) and serine protease (SprE), is positively regulated by a quorum-sensing system encoded by the fsr gene cluster. In this system, E. faecalis secretes an autoinducing peptide, gelatinase biosynthesis-activating pheromone (GBAP), which triggers the FsrC-FsrA two-component regulatory system controlling the expression of two transcripts, fsrBDC and gelE-sprE. In the present study, we screened actinomycete metabolites for inhibitors of fsr quorum sensing. E. faecalis was cultured with each actinomycete culture supernatant tested, and the production of gelatinase and the production of GBAP were examined using the first screening and the second screening, respectively. Culture supernatant of Streptomyces sp. strain Y33-1 had the most potent inhibitory effect on both gelatinase production and GBAP production without inhibiting E. faecalis cell growth. The inhibitor in the culture supernatant was identified as a known peptide antibiotic, siamycin I. Siamycin I inhibited both gelatinase production and GBAP production at submicromolar concentrations, and it inhibited E. faecalis cell growth at concentrations above micromolar concentrations. Quantitative analysis of fsrBDC and gelE-sprE transcripts revealed that siamycin I suppressed the expression of both transcripts at a sublethal concentration. Siamycin I attenuated gelatinase production even when an overdose of GBAP was exogenously added to the culture. These results suggested that siamycin I inhibited the GBAP signaling via the FsrC-FsrA two-component regulatory system in a noncompetitive manner. The sublethal concentrations of siamycin I also attenuated biofilm formation. Treatment with siamycin could be a novel means of treating enterococcal infections.

Opr86 Is Essential for Viability and Is a Potential Candidate for a Protective Antigen against Biofilm Formation by Pseudomonas aeruginosa

Pseudomonas aeruginosa is an opportunistic bacterial pathogen that is one of the most refractory to therapy when it forms biofilms in the airways of cystic fibrosis patients. To date, studies regarding the production of an immunogenic and protective antigen to inhibit biofilm formation by P. aeruginosa have been superficial. The previously uncharacterized outer membrane protein (OMP) Opr86 (PA3648) of P. aeruginosa is a member of the Omp85 family, of which homologs have been found in all gram-negative bacteria. Here we verify the availability of Opr86 as a protective antigen to inhibit biofilm formation by P. aeruginosa PAO1 and several other isolates. A mutant was constructed in which Opr86 expression could be switched on or off through a tac promoter-controlled opr86 gene. The result, consistent with previous Omp85 studies, showed that Opr86 is essential for viability and plays a role in OMP assembly. Depletion of Opr86 resulted in streptococci-like morphological changes and liberation of excess membrane vesicles. A polyclonal antibody against Opr86 which showed reactivity to PAO1 cells was obtained. The antibody inhibited biofilm formation by PAO1 and the other clinical strains tested. Closer examination of early attachment revealed that cells treated with the antibody were unable to attach to the surface. Our data suggest that Opr86 is a critical OMP and a potential candidate as a protective antigen against biofilm formation by P. aeruginosa.

Colloid titration of heparin using Cat-Floc (polydiallyldimethyl ammonium chloride) as standard polycation

Abstract The positive polymer, Cat-Floc, was used for colloid titration. Colloid titration with Cat-Floc was not affected by pH between 2 and 12. The polymer combined with heparin, forming a flocculant, and the binding was shown to be stoichiometric by gravimetric analysis of heparin-sulfur. Results showed that heparin could be measured quantitatively by colloid titration with Cat-Floc. The applicability of this method for measurements of other biochemical materials was discussed.

Lipid Composition of Staphylococcus aureus and Its Derived L-forms

Two strains of Staphylococcus aureus (Newman and Tazaki) and their derived L‐forms were cultured in serum‐containing broth and the differences in their lipid compositions were analyzed. Cardiolipin accounted for more than 50% of the total phospholipid phosphorus in L‐forms, but for less than 25% in parent bacteria. The cardiolipin content of L‐forms was very high through all growth phases, although it increased gradually as growth proceeded. Significant amounts of cholesterol and its esters were present in parent strains and L‐forms, all of which incorporated serum cholesterol into the cell membrane. On the other hand, they could be detected in the L‐forms but not in the parent strains when they were cultured in serum‐free broth. To examine the ability of L‐forms to synthesize cholesterol, the cholesterol content of L‐forms cultured in serum‐free broth was compared with that of the medium. The results indicated that staphylococcal L‐forms could synthesize cholesterol and its esters. These differences in lipid composition suggested that modification of membrane lipids may occur as an adaptation‐al change in response to the disappearance of the cell wall.

Flagellar antigens of various species of the genus vibrio and related genera

The antigenicity of purified flagellin prepared from the single polar flagellum of Vibrio parahaemolyticus was found to be common to that of all strains tested, i.e., V. alginolyticus, V. cholerae, V. anguillarum, V. piscium, V. ichthyodermis, Beneckea natriegens, B. campbellii, B. nereida, B. pelagia, and B. neptuna. On the other hand, the antigenicity of purified flagellin prepared from the lateral flagella of V. parahaemolyticus was common to that of V. alginolyticus but not to that of B. campbellii or B. neptuna. Some physicochemical properties of flagellin from the single polar flagellum of V. parahaemolyticus were found to be similar to those of flagellins from other strains, such as those of V. anguillarum and B. neptuna.

Role of psl Genes in Antibiotic Tolerance of Adherent Pseudomonas aeruginosa

ABSTRACT Bacteria attached to a surface are generally more tolerant to antibiotics than their planktonic counterparts, even without the formation of a biofilm. The mechanism of antibiotic tolerance in biofilm communities is multifactorial, and the genetic background underlying this antibiotic tolerance has not yet been fully elucidated. Using transposon mutagenesis, we isolated a mutant with reduced tolerance to biapenem (relative to that of the wild type) from adherent cells. Sequencing analysis revealed a mutation in the pslL gene, which is part of the polysaccharide biosynthesis operon. The Pseudomonas aeruginosa PAO1ΔpslBCD mutant demonstrated a 100-fold-lower survival rate during the exposure of planktonic and biofilm cells to biapenem; a similar phenotype was observed in a mouse infection model and in clinical strains. Transcriptional analysis of adherent cells revealed increased expression of both pslA and pelA, which are directly regulated by bis-(3′,5′)-cyclic dimeric GMP (c-di-GMP). Inactivation of wspF resulted in significantly increased tolerance to biapenem due to increased production of c-di-GMP. The loss of pslBCD in the ΔwspF mutant background abolished the biapenem-tolerant phenotype of the ΔwspF mutant, underscoring the importance of psl in biapenem tolerance. Overexpression of PA2133, which can catalyze the degradation of c-di-GMP, led to a significant reduction in biapenem tolerance in adherent cells, indicating that c-di-GMP is essential in mediating the tolerance effect. The effect of pslBCD on antibiotic tolerance was evident, with 50- and 200-fold-lower survival in the presence of ofloxacin and tobramycin, respectively. We speculate that the psl genes, which are activated by surface adherence through elevated intracellular c-di-GMP levels, confer tolerance to antimicrobials.

Post-operative Infection by Pathogenic Micro-organisms in the Oral Cavity of Patients with Prostatic Carcinoma

The aim of this study was to analyse the change in the oral cavity microflora of 14 patients who had undergone a radical prostatectomy for prostatic carcinoma. The detection of microorganisms in the oral cavity was compared before and after the surgical procedure. Post-operative infection, defined as those patients who had increased Candida species counts and/or pathogenic bacteria only at the postoperative examination, was observed in 10 patients. Six patients showed increased Candida species counts at the post-operative examination compared with the pre-operative examination. In five patients, pathogenic bacterial species were detected at the post-operative examination but not at the pre-operative examination. One patient had detectable pathogenic bacterial species only at the post-operative examination along with increased Candida species counts. Our findings suggest that pre-operative oral hygiene to remove bacterial and Candida species from patients who are scheduled for surgical procedures is important for satisfactory clinical outcomes.

Epidemiology of Chlamydophila caviae-like Chlamydia isolated from urethra and uterine cervix.

In 2000, chlamydial strains OK133 and OK135 were isolated from 2 female patients with cervicitis. These strains were unresponsive to commercially available PCR and LCR test kits for the diagnosis of Chlamydia trachomatis infection, and their phenotypic characteristics were very similar. The OK135 nucleotide sequence in MOMP-VD2 gene closely resembled that of Chlamydophila caviae GPIC. A similar strain was isolated in 2003 from a male patient OKM2 with urethritis, from which the strain SC10-6 was cloned by the plaque purification method. The nucleotide sequence of the entire MOMP gene of SC10-6 was exactly the same as that of OK135. Thus, the strains OK135 and SC10-6, together with OK133, have been called C. caviae-like Chlamydia. We designed primers for nested PCR assay, the product of which showed a single-band 311-bp fragment, to detect C. caviae-like Chlamydia. Of swab specimens obtained from 202 patients from 2003 to 2006 (119 male and 83 female patients), 18 specimens (8.9%) from 14 male and 4 female patients were positive, suggesting that C. caviae-like Chlamydia infection is rather common. Thus far, it has not been determined whether C. caviae-like Chlamydia is pathogenic for humans.