The nucleotide sequence of pR72H cloned from Vibrio parahaemolyticus 93 was determined. We examined all V. parahaemolyticus gene sequences published in the GenBank-EMBL databases for homology and found that no other DNA sequence of V. parahaemolyticus was highly homologous to the sequence reported in this study. A pair of primers, VP33-VP32, derived from a pR72H fragment were selected to detect V. parahaemolyticus. The sensitivity of PCR detection for a pure culture of V. parahaemolyticus was 10 cells from crude bacterial lysates. Furthermore, a detection level of 2.6 fg, equivalent to 1 cell, was obtained by using purified chromosomal DNA as the template. The expected PCR products were obtained from all V. parahaemolyticus strains tested (n ‫؍‬ 124), while no PCR amplicons were found in other vibrios or related genera (n ‫؍‬ 50). High levels (10 6 to 10 10 CFU/ml) of Escherichia coli cells did not affect the PCR assay sensitivity. The presence of 10 8 V. parahaemolyticus cells or 10 9 E. coli cells in the PCR mixtures completely inhibited the PCR. When oyster samples were inoculated with V. parahaemolyticus 93 and cultured in tryptic soy broth containing 3% NaCl for 3 h at 35؇C, an initial sample inoculum level of 9.3 CFU/g was detected in a PCR assay with crude bacterial lysates. The PCR assay with enrichment culturing in salt polymyxin broth was compared with the conventional method for naturally contaminated shellfish and fish samples. We conclude that this PCR assay with enrichment culturing is a good alternative method for the detection of V. parahaemolyticus.

A total of 102 lactic acid bacteria (LAB) were isolated from three different coffee farms in Taiwan. These isolates were classified and identified by the restriction fragment length polymorphism analysis and sequencing of 16S ribosomal DNA. Heterofermentative Leuconostoc, and Weissella species were the most common LAB found in two farms located at an approximate altitude of 800 m. Lactococcus lactis subsp. lactis was the most common LAB found in the remaining farm was located at an approximate altitude of 1,200 m. It is therefore suggested that the altitude and climate may affect the distribution of LAB. On the basis of phylogenetic analysis, two strains included in the genera Enterococcus were considered as two potential novel species or subspecies. In addition, a total of 34 isolates showed the antifungal activity against Aspergillus flavus. Moreover, seven Lactococcus lactis subsp. lactis strains and one Enterococcus faecalis strain were found to have bacteriocin-like inhibitory substance-producing capability. These results suggest that various LAB are associated with fresh coffee cherries in Taiwan. Some of the isolates found in this study showed potential as antifungal agents.

Oxidative stress is one of the major causes of cell death. Using time-lapse confocal recording of live cardiomyocytes, we showed that H2O2 (OH*) caused a marked increase in Na+ and Ca2+ levels in both the cytosol ([Na]cyt, [Ca]cyt) and mitochondria ([Na]m, [Ca]m). The H2O2-induced intracellular Na+ ([Na]i) overload contributed to the H2O2-induced [Ca]cyt/[Ca]m overload via activation of the reverse mode of the Na-Ca exchanger. When myocytes were treated for 40 min with 100 microM H2O2 in normal medium, then returned to H2O2-free medium, the percentage of apoptotic cells increased from 4% at 0 h to 55 and 85% at 4.5 and 16 h, respectively. H2O2-induced apoptosis was completely prevented by using Na-free, but not Ca-free, medium. When a Na+ ionophore cocktail in Ca-free medium was used instead of H2O2 to increase the [Na]i by more than 30 mM without any change in the [Ca]i, cytochrome c release and caspase 3-dependent apoptosis occurred, showing that [Na]i overload per se induced apoptosis. We also showed that the increase in the mitochondrial, but not the cytosolic, Na+ levels resulted in the opening of the permeation transition pore, followed by cytochrome c release. Our findings therefore suggest that H2O2-induced [Na]m overload is an important upstream signal for the apoptotic machinery, and the prevention of [Na]m overload thus represents a particularly attractive target for strategies aimed at preventing oxidative stress-induced cell death.

Lactococcus formosensis sp. nov., a lactic acid bacterium isolated from yan-tsai-shin (fermented broccoli stems) The Institute of Enology and Viticulture, University of Yamanashi, 1-13-1, Kitashin, Kofu, Yamanashi 400-0005, Japan A coccal-shaped organism, designated 516 T , was isolated from yan-tsai-shin (fermented broccoli stems), a traditional fermented food in Taiwan. 16S rRNA gene sequencing results showed that strain 516 T had 98.9 % sequence similarity to that of the type strain Lactococcus garvieae NBRC 100934 T . Comparison of three housekeeping genes, rpoA, rpoB and pheS, revealed that strain 516 T was well separated from Lactococcus garvieae NBRC 100934 T . DNA-DNA hybridization studies indicated that strain 516 T had low DNA relatedness with Lactococcus garvieae NBRC 100934 T (46.1 %). The DNA G+C content of strain 516 T was 38.1 mol% and the major fatty acids were C 16 : 0 (22.7 %), C 19 : 0 cyclo v8c (17.9 %) and summed feature 7 (29.0 %). Based on the evidence, strain 516 T represents a novel species of the genus Lactococcus, for which the name Lactococcus formosensis sp. nov. is proposed. The type strain is 516 T (5NBRC 109475 T 5BCRC 80576 T ).

Synthetic oligonucleotide primers derived from a sequence of the thermostable direct haemolysin (tdh) gene were used in a polymerase chain reaction (PCR) amplification technique to detect this gene in strains of Vibvio pavahaemolyticus. A total of 36 TDH-producing, and 89 TDH-negative Vibvio pavahaemolyticus strains and 46 other vibrios and enteric pathogens were studied. In all, 36 strains of Vibrio pavahaemolyticus from which the tdh gene could be successfully amplified by PCR were found to be TDH-positive in TDH haemolysin assay. No amplification products were obtained from Vibvio pavahaemolyticus strains that were TDH-negative in the haemolysin assay or from other vibrios and enteric pathogens, with the exception of two strains. The PCR results were consistent with DNA hybridization tests. The detection limit for the tdh gene by PCR amplification was 40 pg of total DNA, or broth culture containing 1000 viable cells. Amplification products were confirmed by restriction enzyme digestion and Southern blot hybridization. The PCR method could detect the tdh sequences in stool samples from patients with gastroenteritis caused by V. pavahaemolyticus. This PCR protocol clearly identified TDHproducing strains of V. pavahaemolyticus and provides an alternative to conventional methods for TDH detection by research laboratories, clinical laboratories, regulatory agencies, and the seafood industry.

Aim: To identify and characterize novel bacteriocins from Weissella hellenica 4-7. Methods and Results: Weissella hellenica 4-7, isolated from the traditional Taiwanese fermented food sian-sianzih (fermented clams), was previously found to produce a bacteriocin active against Listeria monocytogenes and some other Gram-positive bacteria. Bacteriocin activity decreased slightly after autoclaving (121°C for 15 min), but was inactivated by protease K and trypsin. Mass spectrometry analysis revealed the bacteriocin mass to be approximately 3205Á6 Da. N-terminal amino acid sequencing yielded a partial sequence, NH 2 -KGFLSWASKATSWLVGP, by Edman degradation. The obtained partial sequence showed high homology with leucocin B-TA33a; however, at least two different residues were observed. No identical peptide or protein was found, and this peptide was therefore considered to be a novel bacteriocin produced by W. hellenica 4-7 and termed weissellicin L. Conclusions: The findings obtained in the current study suggest a novel bacteriocin produced by W. hellenica 4-7. Significance and Impact of the Study: Bacteriocins from Weissella remain rare, and this study is the second report of a bacteriocin produced by W. hellenica.

One coccal strain, designated 0905C15 T , was isolated from fresh cummingcordia, which is the main ingredient of pobuzihi (fermented cummingcordia), a traditional fermented food in Taiwan. 16S rRNA gene sequencing results showed that strain 0905C15 T had 98.22-98.82 % sequence similarity to that of the type strains of four Lactococcus lactis subspecies (L. lactis subsp. lactis BCRC 12312 T , L. lactis subsp. cremoris BCRC 12586 T , L. lactis subsp. hordniae BCRC 80474 T and L. lactis subsp. tructae BCRC 80475 T ). Comparison of two housekeeping genes, recA and rpoB, revealed that strain 0905C15 T was well separated from the reference strains of the genus Lactococcus. DNA-DNA hybridization studies indicated that strain 0905C15 T had low DNA relatedness to the four Lactococcus lactis subspecies (9.7-15.24 %). The DNA G+C content of strain 0905C15 T was 39.6 mol %. Based on the evidence, strain 0905C15 T represents a novel species of the genus Lactococcus, for which the name Lactococcus taiwanensis sp. nov. is proposed. The type strain is 0905C15 T (5NBRC 109049 T 5BCRC 80460 T ).Lactic acid bacteria (LAB) are widely distributed in traditional fermented foods in Taiwan. In our previous study, we isolated a novel species of the genus Lactobacillus named Lactobacillus pobuzihii sp. nov. from pobuzihi (fermented cummingcordia) (Chen et al., 2010). In order to obtain more information on LAB diversity in pobuzihi and fresh cummingcordia, isolation of LAB was performed and a total of 196 LAB strains were isolated (Chen et al., 2013). All isolates were identified based on their phenotypic and phylogenetic characteristics. Phylogenetic analyses, based on 16S rRNA gene sequences, initially placed strain 0905C15 T within the species Lactococcus lactis.

Aim: To isolate and characterize bacteriocin-like inhibitory substance (BLIS)-producing lactic acid bacteria from the intestine of grey mullet. Methods and Results: Inhibitory activity against at least one or more indicator strains was observed in one Enterococcus thailandicus, one Enterococcus faecium and two Lactococcus garvieae strains. Enterococcus faecium B3-8 and Ent. thailandicus B3-22 showed the greatest inhibitory activities against Listeria monocytogenes ATCC 19111 and were therefore further characterized. The results suggested that the inhibitory substances from the two strains showed similar characteristics with respect to sensitivity to heat and proteolytic enzymes. BLIS from Ent. thailandicus B3-22 was characterized by a broader inhibitory spectrum than that from Ent. faecium B3-8. SDS-PAGE revealed that the molecular size of partially purified BLISs from Ent. faecium B3-8 and Ent. thailandicus B3-22 was c. 5 and 3 kDa, respectively. The molecular mass of purified bacteriocin from Ent. thailandicus B3-22 was further determined to be 6319 Da by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). The results indicated that BLIS from Ent. thailandicus B3-22 can effectively inhibit the growth of all tested L. garvieae strains. Conclusions: The findings obtained in this study suggest the potential use of Ent. thailandicus B3-22 as a biocontrol agent against pathogenic L. garvieae in the aquaculture. Significance and Impact of the Study: This is the first report describing the characteristics of BLIS from Ent. thailandicus that showed potential for use as a biocontrol agent in the aquaculture.