Phylogenetics of family Enterobacteriaceae and proposal to reclassify Escherichia hermannii and Salmonella subterranea as Atlantibacter hermannii and Atlantibacter subterranea gen. nov., comb. nov.

Multilocus sequence analysis based on hypervariable housekeeping proteins was utilized to differentiate closely related species in the family Enterobacteriaceae. Of 150 housekeeping proteins, the top 10 hypervariable proteins were selected and concatenated to obtain distance data. Distances between concatenated proteins within the family were 0.9-41.2%, whereas the 16S rRNA and atpD-gyrB-infB-rpoB concatenated sequence (4MLSA) distances were 0.8-6.0% and 0.9-22.1%, respectively. These data indicate that phylogenetic analysis by concatenation of hypervariable proteins is a powerful tool for discriminating species in the family Enterobacteriaceae. To confirm the discriminatory power of the 10 chosen concatenated hypervariable proteins (C10HKP), phylogenetic trees based on C10HKP, 4MLSA, and the 16S rRNA gene were constructed. Comparison of average bootstrap values among C10HKP, 4MLSA and 16S rRNA genes indicated that the C10HKP tree was the most reliable. Location via the C10HKP tree was consistent with existing assignments for almost all species in the family Enterobacteriaceae. However, the C10HKP tree suggested that several species (including Enterobacter massiliensis, Escherichia vulneris, Escherichia hermannii, and Salmonella subterranea) should be reassigned to different clusters than those defined in previous analyses. Furthermore, E. hermannii and S. subterranea appeared to fall onto a branch independent from those occupied by the other Enterobacteriaceae. Therefore, we propose Atlantibacter gen. nov., such that E. hermannii and S. subterranea would be transferred to genus Atlantibacter as Atlantibacter hermannii, comb. nov. and Atlantibacter subterranea. comb. nov., respectively.

A new protocol to detect multiple foodborne pathogens with PCR dipstick DNA chromatography after a six-hour enrichment culture in a broad-range food pathogen enrichment broth.

A quick foodborne pathogen screening method after six-hour enrichment culture with a broad-range food pathogen enrichment broth is described. Pathogenic factors of Salmonella enterica, Shigella spp., enteroinvasive Escherichia coli, and enterohemorrhagic E. coli are amplified with a cocktail primer and rapid polymerase chain reaction (PCR), which finishes amplification in 30 min. The PCR amplicon was differentiated with a dipstick DNA chromatography assay in 5-10 min. Starting from a four- to six-hour enrichment culture, this assay was finished within 45 min. Detection sensitivity of this protocol was less than 2.5 CFU/25 g for S. enterica and 3.3 CFU/25 g for enterohemorrhagic E. coli in spiked ground meat experiments.

Use of blood-free enrichment broth in the development of a rapid protocol to detect Campylobacter in twenty-five grams of chicken meat.

A Food Pathogen Enrichment (FPE) broth, which supports the growth of Campylobacter without lysed blood and CO2, was developed. The FPE broth supports the growth of Campylobacter to the same degree as Bolton and Preston broths. Using the FPE broth, we developed a novel rapid protocol to detect small numbers of Campylobacter in 25g of food. The sensitivity of FPE enrichment and PCR to detect Campylobacter spp. from spiked chicken meat was determined. The detection sensitivities for non-stressed C. jejuni and C. coli from fresh meat ranged from 5.8 to 1.1×10(1)CFU per 25g of chicken meat, and those for freeze-stressed C. jejuni and C. coli from frozen meat ranged from 9.9×10(1) to 2.0×10(2)CFU. The FPE broth enrichment culture (24h) of chicken meat, followed by PCR, resulted in a significantly higher detection score (80% positive) than conventional Bolton enrichment and subsequent colony isolation using mCCDA agar plates (18% positive). Differences between our new protocol and the Bolton enrichment method were due to the overgrowth of many resistant bacteria, especially extended-spectrum beta-lactamase-producing bacteria in the Bolton enrichment broth.

Anthropometric, lifestyle and biomarker assessment of Japanese non-professional ultra-marathon runners.

BACKGROUND: Anthropometric characteristics, lifestyle, and baseline biological markers of Japanese non-professional ultra-marathon runners have not been fully assessed. METHODS: We evaluated anthropometric characteristics, lifestyle, and baseline biological markers of 180 Japanese amateur ultra-marathon runners (144 males [mean age: 50.5 +/- 9.4 (standard deviation) years] and 36 females [48.9 +/- 6.9]), and compared them with those of participants in a community heath check-up program and with the figures in the literature. We furthermore evaluated baseline blood indices according to monthly running distance with analysis of variance adjusted for age, body mass index, smoking and alcohol drinking habits. RESULTS: The ultra-marathon runners demonstrated more favorable values for body mass index and bone density, and the proportion of smoking, and undertaking physical activity (for both sexes), eating breakfast (for males), and having daily bowel movements (for females), while greater proportion of alcohol drinking habit (for both sexes), than the comparison group. Average monthly running distances and standard deviations (km) were 257.2 +/- 128.9 for males and 209.0 +/- 86.2 for females. Male runners possessed beneficial markers, including lowered triglyceride and elevated high-density lipoprotein cholesterol, and their values showed hockey-stick (or inverse hockey-stick) patterns depending on their monthly running distance. Some subjects running more than 300 km/month exhibited signs of an over-reaching/training syndrome, including somewhat lowered hemoglobin, ferritin and white blood cell count, and elevated creatine kinase and lactate dehydrogenase. CONCLUSIONS: Together with a desirable lifestyle, Japanese non-professional ultra-marathon runners with vigorous exercise habit demonstrated a preferable health status according to biological indices.

Establishment of a HPV and p53-mutation-negative human cell line (CA) derived from a squamous carcinoma of the uterine cervix.

OBJECTIVE: Well-characterized human cancer cell lines are important research resources for studying cancer cell biology, as well as for developing new strategies against cancer cell growth and progression. We present a new cell line, CA, established from an invasive non-keratinizing squamous cell carcinoma of the uterine cervix in 36-year-old patient. METHODS: We measured the doubling time of CA cells. To investigate the tumorigenicity of CA, cells were inoculated subcutaneously into the back of nude mice. Several tumor markers were analyzed using culture media by EIA. PCR-based analyses were performed to examine the human papillomavirus (HPV) status and telomerase activity. CA was also screened for p53 mutation using the sequencing technique. RESULTS: The cells show rapid growth in culture with a doubling time of 14.3 h and high migration activity. Monolayer-cultured cells were polygonal, showing a pavement-like arrangement and a tendency to pile up without contact inhibition. Subcutaneous transplantation of the CA cells into nude mice formed solid tumors that were histologically diagnosed as squamous cell carcinoma, whereas no metastasis was observed. Cultured CA cells produced SCC, CEA, TPA, CA125 and SLX. Genetic and molecular analyses revealed high telomerase activity and the absence of HPV DNA. No p53 mutation was observed in this cell line. CONCLUSION: These properties suggest that CA is an aggressive cervical carcinoma cell line and may serve as a useful experimental model for studying HPV role in cervical carcinogenesis.

Induction of sexual co-flocculation of heterothallic fission yeast (Schizosaccharomyces pombe) cells by mating pheromones.

Heterothallic fission yeast (Schizosaccharomyces pombe) cells preincubated with sex pheromone, P- or M-factor of the obverse mating-type cells, in mannose synthetic medium (MSM) results in remarkably increased sexual co-flocculation with obverse mating-type cells almost without time lag, i.e., within 10 min. By contrast, comparable flocculation requires over 1 h if untreated control cells are mixed with obverse mating-type cells. The agglutinin of P cells is more inducible than that of M cells. These pheromonal inductions of sexual co-flocculation are inhibited by the addition of cycloheximide or tunicamycin during preincubation but not by chloramphenicol or hydroxyurea. These results demonstrate that, in addition to (a) the repression of cell division (G1 arrest) and (b) the activation of cell wall autolytic processes (mating-specific elongation of cells: formation of their conjugation tubes), mating pheromones of fission yeast have another important role; (c) to induce sexual co-flocculation (agglutinability). Using our experimental system of preincubation with sexual pheromones, we show that M-agglutinin is heat-stable and its induction is inhibited by tunicamycin, but that P-agglutinin is heat-labile and its induction is only partially inhibited by tunicamycin.