We determined the ferrophilic characteristics of Vibrio vulnificus to evaluate the potential usefulness of iron chelation therapy for the prevention of V. vulnificus infection. Readily available non-transferrin-bound iron (NTBI) is required for the initiation of V. vulnificus growth under in vitro iron-limited conditions and human ex vivo conditions. NTBI aided efficient transferrin-bound iron (TBI) use by V. vulnificus, and the vulnibactin-mediated iron-uptake system was expressed after bacterial growth had been started by NTBI. V. vulnificus required higher NTBI levels for the initiation of growth, produced siderophores at lower levels, and used TBI less efficiently than other bacteria. In addition, the growth of V. vulnificus was inhibited by deferiprone, a clinically available iron chelator. These results show that V. vulnificus is a ferrophilic bacterium that requires higher NTBI levels than other pathogens and that iron chelation therapy might be an effective means of preventing the in vivo growth of V. vulnificus in susceptible patients.

On the basis of the DNA-DNA hybridization patterns and phenotypic characteristics, Fusobacterium nucleatum was classified into five subspecies. Previous studies have suggested that F. nucleatum subsp. vincentii is genetically similar to F. nucleatum subsp. fusiforme. The aim of this study was to investigate the possibility of classifying these two subspecies into a single subspecies by phylogenetic analysis using a single sequence (24,715 bp) concatenated 22 housekeeping genes of eight F. nucleatum strains including type strains of five F. nucleatum subspecies. The phylogenetic analysis indicated that F. nucleatum subsp. vincentii and F. nucleatum subsp. fusiforme were clustered in the same group and each strain of other F. nucleatum subspecies were also separated into the same cluster. These results suggested that F. nucleatum subsp. fusiforme and F. nucleatum subsp. vincentii can be classified into a single subspecies. F. nucleatum subsp. vincentii was early published name; therefore, F. nucleatum subsp. fusiforme Gharbia and Shah 1992 can be regarded as a later synonym of F. nucleatum subsp. vincentii Dzink et al. 1990.

The objective of the study was to investigate the antimicrobial effects of purified single compounds from ethanol-extracted licorice root on Streptococcus mutans. The crude licorice root extract (CLE) was obtained from Glycyrrhiza uralensis, which was subjected to column chromatography to separate compounds. Purified compounds were identified by mass spectrometry and nuclear magnetic resonance. Antimicrobial activities of purified compounds from CLE were evaluated by determining the minimum inhibitory concentration and by performing time-kill kinetics. The inhibitory effects of the compounds on biofilm development were evaluated using crystal violet assay and confocal microscopy. Cell toxicity of substances to normal human gingival fibroblast (NHGF) cells was tested using a methyl thiazolyl tetrazolium assay. Chlorhexidine digluconate (CHX) was used in the control group. Three antimicrobial flavonoids, 1-methoxyficifolinol, licorisoflavan A, and 6,8-diprenylgenistein, were isolated from the CLE. We found that the three flavonoids and CHX had bactericidal effects on S. mutans UA159 at the concentration of ≥4 and ≥1 µg/ml, respectively. The purified compounds completely inhibited biofilm development of S. mutans UA159 at concentrations over 4 μg/ml, which was equivalent to 2 μg/ml of CHX. Confocal analysis showed that biofilms were sparsely scattered in the presence of over 4 μg/ml of the purified compounds. However, the three compounds purified from CLE showed less cytotoxic effects on NHGF cells than CHX at these biofilm-inhibitory concentrations. Our results suggest that purified flavonoids from CLE can be useful in developing oral hygiene products, such as gargling solutions and dentifrices for preventing dental caries.

Cyclic AMP-cAMP receptor protein (CRP) complex plays an essential role in the global regulation of Vibrio vulnificus virulence. We found that growth retardation of V. vulnificus caused by mutation of the crp gene encoding CRP was exacerbated under iron-limited conditions. Accordingly, we investigated the effect of crp mutation on the expression of the vulnibactin-mediated iron-uptake system and the ability of V. vulnificus to utilize transferrin-bound iron, and thus to grow in cirrhotic ascites, a human ex vivo system. The production of vulnibactin was suppressed, and the transcription of the vis and vuuA genes, which encode an enzyme required for vulnibactin synthesis and vulnibactin receptor protein, was also suppressed in the crp mutant. Moreover, the crp mutant could not utilize transferrin-bound iron, and its growth was severely suppressed both on transferrin-bound iron and in cirrhotic ascites. All the defects in the crp mutant were recovered by the in trans complementation of the wild-type crp gene. Putative CRP-binding sequences were found in the regulatory regions of the fur, vis and vuuA genes. These results indicate that crp mutation attenuates the ability to grow on transferrin-bound iron and in a human body fluid by down-regulating the vulnibactin-mediated iron-uptake system.

Pseudomonas aeruginosa is an opportunistic pathogen which is frequently isolated from clinical specimens obtained from burns, surface wounds, the urinary tract, ear and eye infections, and the lungs of patients with cystic fibrosis. 1) P. aeruginosa secretes many extracellular proteins, and of these, several proteases including alkaline protease (AprA) and elastase (LasB) are known to play important roles in the pathogenesis of human infections caused by P. aeruginosa. 2,3)In response to iron deprivation, P. aeruginosa produces two unrelated siderophores, pyoverdin and pyochelin. Both of these siderophores promote the growth of P. aeruginosa when added to media containing transferrins as an iron source. Pyoverdin is considered to be the essential or more effective siderophore because of its higher iron-binding constant (pyoverdin 10 32 and pyochelin 10 5 ). Moreover, a pyochelin-deficient mutant was able to grow in human sera, whereas a pyoverdin-deficient mutant exhibited severely retarded growth, and the addition of pyoverdin was found to restore the retarded growth of pyoverdin-deficient mutants in media containing transferrins. [4][5][6] P. aeruginosa produces several extracellular proteases. Of these, AprA and LasB have been extensively studied. [7][8][9] These two proteases are produced more profoundly in irondeficient conditions than in iron-sufficient conditions, and are able to destroy transferrin, which is a major iron-withholding protein in human body fluids. Because of the high affinity of transferrin for iron, transferrin-bound iron is not freely-available for P. aeruginosa growth. Accordingly, the proteolytic cleavage of transferrin can allow free iron to be released from transferrin, and thus facilitate the pyoverdin-mediated iron-uptake and growth of P. aeruginosa.However, this hypothesis still remains unresolved despite considerable efforts. Some researchers have reported that LasB facilitates pyoverdin-mediated iron-uptake via the proteolytic cleavage of transferrins, 3,7) but others found that LasB had no effect on the iron-uptake from transferrins because no difference was observed between the growths of a LasB-deficient mutant and its wild type strain and LasB was produced after the onset of stationary growth phase in media containing transferrin as an iron source. 9) Similarly, Döring et al. 7) found that AprA had no effect on pyoverdin-mediated iron-uptake from transferrins, but Shigematsu et al.8) more recently reported that AprA can facilitate siderophore-mediated iron-uptake via the proteolytic cleavage of transferrins.Although Shigematsu et al. observed that the growth of an aprA-insertion mutant was suppressed in media containing transferrins versus the wild type strain, they did not directly observe whether the AprA produced during culture could destroy transferrins.8) Rather, they exogenously added purified AprA to culture media in order to induce the proteolytic cleavage of transferrins at culture start. As the aprA-insertional mutation may also cause unexpected changes in the p...