Growth and Survival of Escherichia albertii in Food and Environmental Water at Various Temperatures.

Escherichia albertii is an emerging foodborne pathogen that causes diarrhea. E. albertii has been isolated from various foods, including pork and chicken meat, and environmental waters, such as river water. Although many food poisoning cases have been reported, there have been insufficient analyses of bacterial population behaviors in food and environmental water. In this study, we inoculated 2-5 log CFU of E. albertii into 25 g of pork, chicken meat, Japanese rock oyster, Pacific oyster, and 300 mL of well water and seawater at 4°C, 10°C, 20°C, and 30°C, and analyzed the bacterial population behavior in food and environmental water. After 3 days at 4°C, the population of E. albertii strain EA21 and EA24 in foods maintained approximately 4 log CFU/25 g. After 3 days at 10°C, the population of E. albertii strains in pork and oysters maintained approximately 4 log CFU/25 g, and that in chicken meat increased to approximately 5-6 log CFU/25 g. After 2 days at 20°C, E. albertii strains grew to approximately 6-7 log CFU/25 g in pork and chicken meat, and E. albertii strain EA21 but not EA24 grew to 4.5 log CFU/25 g in Japanese rock oyster, E. albertii strain EA21 but not EA24 slightly grew to 3.1 log CFU/25 g in Pacific oyster. After 1 day at 30°C, E. albertii strains grew to approximately 7-8 log CFU/25 g in chicken meat and pork, grew to approximately 4-6 log CFU/25 g in Japanese rock oyster, and 6-7 log CFU/25 g in Pacific oyster. These results suggest that E. albertii survives without growth below 4°C and grew rapidly at 20°C and 30°C in foods, especially in meat. E. albertii strains did not grow in well water and seawater at 4°C, 10°C, 20°C, and 30°C. The population of E. albertii strains in well water and seawater decreased faster at 30°C than at 4°C, 10°C, and 20°C, suggesting that E. albertii has low viability at 30°C in environmental water.

Proposal of a novel subspecies: Alicyclobacillus hesperidum subspecies aegles.

Alicyclobacillus sp. DSM 11985T was isolated from geothermal soil but had not yet been classified at the species level. The strain produced guaiacol, which is of interest from the viewpoint of food spoilage in the food industry. 16S rRNA gene sequence analysis revealed that strain DSM 11985T was closely related (99.6 % similarity) to Alicyclobacillus hesperidum DSM 12489T. However, strains of A. hesperidum did not produce guaiacol; therefore, we performed the taxonomic characterization of strain DSM 11985T. The results showed that strain DSM 11985T and strains of A. hesperidum showed different phenotypic characteristics in biochemical/physiological tests including guaiacol production. Average nucleotide identity values between strain DSM 11985T and strain DSM 12489T were 95.4-95.9 %, and the in silico DNA-DNA hybridization value using the Genome-to-Genome Distance Calculator between strains DSM 11985T and DSM 12489T was 65.5 %. These values showed that strain DSM 11985T was genetically closely related but separated from strains of A. heparidum. From the above results, a novel subspecies of A. hesperidum, named Alicyclobacillus hesperidum subsp. aegles subsp. nov. is proposed. The type strain is DSM 11985T (=FR-12T=NBRC 113041T).

Antimicrobial Drug-resistance Profile of Vibrio Parahaemolyticus isolated from Japanese Horse Mackerel (Trachurus Japonicus).

This study aimed at investigating antimicrobial resistance (AMR) profile of Vibrio parahaemolyticus (V. parahaemolyticus). The bacteria were isolated from wild-caught and farmed Japanese horse mackerel (Trachurus japonicus), and examined for the antimicrobial drug resistance. Furthermore, the serotype, and the genes of thermostable direct hemolysin (tdh) and cholera toxin transcriptional activator (toxR) of the isolates were investigated by using a serotype testing kit and PCR method. Eighty-eight and 126 V. parahaemolyticus strains were isolated from wild-caught and farmed Japanese horse mackerel, respectively. Ten and 18 distinct serotypes were detected from wild-caught and farmed Japanese horse mackerel. All strains were negative for tdh genes but positive for toxR genes. Resistances to ampicillin (ABP) and to both ABP and fosfomycin (FOM) were observed in 54 and 23 strains from the wild-caught fish, while those resistant strains from farm fish were 112 and 7 strains. Multidrug-resistance to three or four drugs including ABP was observed in one or two strains from the wild-caught fish. These results strongly suggest that the environmental exposure of antimicrobial drugs results in the spread of resistant genes in Japanese horse mackerel. This study highlights the need for monitoring the spread of resistance genes to the human intestinal flora as well as to other bacteria in the environment.

[Discovery of Putative Anti-Kudoa septempunctata Chemical Agents by Comprehensive Assay].

We screened 360 chemicals and discovered that 71 chemicals had anti-Kudoa septempunctata effect. Especially 19 and seven of 71 chemicals were antibiotics and antibacterial agents/disinfectants, respectively. The other 45 chemicals were pesticides, natural toxins, industrial chemicals and medicines for non-infectious diseases. Nineteen antibiotics that possessed anti-Kudoa effect contained four tetracyclines, one steroid, two macrolides, one aminoglycoside, three ß-lactams, one quinolone, two rifamycines, one polyene, one novobiocine, one sulfonamide and two nitroimidazoles. To use these drugs for prevention of Kudoa infection, the further study is need for the determination of effective dose.

[Evaluation of Microbial Spoilage in PET Bottled Soft Drinks by Direct Drinking].

In order to evaluate microbial growth in opened PET bottled soft drinks, inoculation tests were carried out using type and reference strains of various microorganisms. Microorganisms were inoculated into a 500 mL PET bottle containing 250 mL of various soft drinks followed by incubation until 1 week at 4, 25, 35℃ without shaking, and 35℃ with shaking. The microbial counts were measured over time and compared with the results of the previous study "Studies on Contaminants in Soft Drink"2)-4). As a result, similar growth patterns were observed in the combination of tomato juice with Lactobacillus fermentum, sports drink with Candida albicans, and mineral water with Klebsiella pneumoniae. However, in green tea, mixed herb tea, orange juice and coffee with milk, the growth of microorganisms generally tended to be weaker than those of the previous studies. It was considered that components in the soft drinks inhibited the growth of the microorganisms. From the above results, the proliferative properties of type and reference strains in soft drinks were clearly different from the spoiled soft drinks isolates. The results in this study indicated that attention must be paid in the safety evaluation.

[Study of Tea and Coffee on Inactivation of Kudoa septempunctata].

The inhibition of Kudoa septempunctata by green tea extract, black tea extract, and coffee extract were studied. Incubation of about 104 Kudoa spores with green tea extract, black tea extract, or coffee extract at 25℃ for 4 hr reduced the survival ratio of Kudoa to 0%. While coffee extract and green tea extract contain approximately 2 and 1 mM of caffeine, respectively, the incubation of Kudoa spores with 2 and 1 mM of caffeine reduced its survival ratio to 68.2 and 93.3%, respectively. Although green tea extract and black tea extract contain over 1 mM of catechin, incubation with 0.01 mM of catechin was enough to reduce the survival ratio of Kudoa to 20%. These results suggested that green tea extract, black tea extract, and coffee extract have strong inhibitory effects on Kudoa and the effects of green tea extract and black tea extract are mainly manifested through catechin.

Black tea aroma inhibited increase of salivary chromogranin-A after arithmetic tasks.

BACKGROUND: Growing attention has been paid to the effects of food flavor components on alleviating negative brain functions caused by stressful lifestyles. In this study, we investigated the alleviating effect of two kinds of black tea aromas on physical and psychological stress induced by the Uchida-Kraepelin test, based on salivary chromogranin-A (CgA) levels as a stress marker and subjective evaluations (Profile of Mood States). RESULTS: Compared with the water exposure control, inhaling black tea aroma (Darjeeling and Assam in this study) induced lower salivary CgA concentration levels after 30 min of mental stress load tasks. This anti-stress effect of black tea aroma did not differ between the two tea types even though the concentration of the anti-stress components in the Darjeeling tea aroma was higher than that in the Assam aroma. However, Darjeeling tea aroma tended to decrease the tension and/or anxiety score immediately after the first exposure. CONCLUSIONS: Inhaling black tea aroma may diminish stress levels caused by arithmetic mental stress tasks, and Darjeeling tea aroma tended to improve mood before mental stress load.

Green Tea Catechin Metabolites Exert Immunoregulatory Effects on CD4(+) T Cell and Natural Killer Cell Activities.

Tea catechins, such as (-)-epigallocatechin-3-O-gallate (EGCG), have been shown to effectively enhance immune activity and prevent cancer, although the underlying mechanism is unclear. Green tea catechins are instead converted to catechin metabolites in the intestine. Here, we show that these green tea catechin metabolites enhance CD4(+) T cell activity as well as natural killer (NK) cell activity. Our data suggest that the absence of a 4'-hydroxyl on this phenyl group (B ring) is important for the effect on immune activity. In particular, 5-(3',5'-dihydroxyphenyl)-γ-valerolactone (EGC-M5), a major metabolite of EGCG, not only increased the activity of CD4(+) T cells but also enhanced the cytotoxic activity of NK cells in vivo. These data suggest that EGC-M5 might show immunostimulatory activity.

Characteristics of bacterial and fungal growth in plastic bottled beverages under a consuming condition model.

Microbial contamination in unfinished beverages can occur when drinking directly from the bottle. Various microorganisms, including foodborne pathogens, are able to grow in these beverages at room temperature or in a refrigerator. In this study, we elucidated the characteristics of microorganism growth in bottled beverages under consuming condition models. Furthermore, we provide insight into the safety of partially consumed bottled beverages with respect to food hygiene. We inoculated microorganisms, including foodborne pathogens, into various plastic bottled beverages and analysed the dynamic growth of microorganisms as well as bacterial toxin production in the beverages. Eight bottled beverage types were tested in this study, namely green tea, apple juice drink, tomato juice, carbonated drink, sport drink, coffee with milk, isotonic water and mineral water, and in these beverages several microorganism types were used: nine bacteria including three toxin producers, three yeasts, and five moulds. Following inoculation, the bottles were incubated at 35°C for 48 h for bacteria, 25°C for 48 h for yeasts, and 25°C for 28 days for moulds. During the incubation period, the number of bacteria and yeasts and visible changes in mould-growth were determined over time. Our results indicated that combinations of the beverage types and microorganism species correlated with the degree of growth. Regarding factors that affect the growth and toxin-productivity of microorganisms in beverages, it is speculated that the pH, static/shaking culture, temperature, additives, or ingredients, such as carbon dioxide or organic matter (especially of plant origin), may be important for microorganism growth in beverages. Our results suggest that various types of unfinished beverages have microorganism growth and can include food borne pathogens and bacterial toxins. Therefore, our results indicate that in terms of food hygiene it is necessary to consume beverages immediately after opening the bottle.

Microbial contamination associated with consumption and the growth in plastic bottled beverage.

Plastic bottles enable the storage of unfinished beverages, and most of microbial contamination has occurred in the unfinished beverage that was left. Therefore, we investigated microorganisms in various beverages contaminated by pouring and drinking directly by mouth from the bottle, and analyzed the growth of microorganisms in the beverages at room temperature. In the pouring test, microbial growth was detected in 60 of 320 samples, and 13 bacterial strains, 49 mold strains, and 8 yeast strains were isolated. Molds including Cladosporium spp., Tramets spp., Bjerkandera spp., and Penicillium spp. accounted for the majority of isolated microorganisms. In the drinking test, microbial growth was detected in 181 of 352 samples, and 225 bacterial strains, 27 mold strains and 77 yeast strains were isolated. Bacteria including Streptococcus spp. such as S. salivarius and Staphylococcus spp. such as S. aureus accounted for the majority of isolated microorganisms. Enterotoxin-producing S. aureus and Bacillus cereus were also isolated. The pH of the beverage influenced the growth of bacteria. The Brix values of the beverage did not correlate with the growth of microorganisms. These results revealed that various microorganisms including foodborne pathogens were able to grow in numerous types of beverages and that the storage of unfinished beverage in inappropriate condition, such as the storage at room temperature led microorganism to grow easily in beverage. Therefore, it is necessary to consume beverages as soon as possible after opening the bottle.

Sensitive detection of whole-genome differentiation among closely-related species of the genus Fusarium using DNA-DNA hybridization and a microplate technique.

We developed a new system for detection of whole-genome differentiation using DNA-DNA hybridization, and tested its sensitivity with three closely-related Fusarium species. We compared DNA-DNA relatedness to nucleotide sequence homologies of five genetic regions between each of five strains of three Fusarium species. DNA-DNA relatedness by our system was 16.2-86.6%. Sequence homologies of 18S rDNA, rDNA cluster region from ITS1 to 28S rDNA, ß-tub, EF-1α and lys2 were 100.0, 99.0-100.0, 96.7-100.0, 95.1-99.4, and 94.7-100.0%, respectively. Our system could clearly detect differentiation between closely-related fungal species which have very similar morphological-characteristics, and exhibit little diagnoses in nucleotide sequences. Our results suggest that this system is a good tool for identification and phylogenetic analysis of closely-related fungal species.

The first case of Legionella nagasakiensis isolation from hot spring water.

In August, 2010, strain HYMO-6 was isolated from a sample of hot spring water in Aomori, Japan. The 16S rDNA sequences (1,496bp) of this strain (accession number: AB597175) had a similarity of less than 96.6% to other Legionella species, prompting us to hypothesize that this strain might be a novel species belonging to the genus Legionella. However, in March of 2011, it was became clear that the HYMO-6 strain (=JCM 17450 =KCTC 23560 =DSM 24727) was Legionella nagasakiensis CDC-1796-JAP-E(T) (=ATCC BAA-1557(T) =JCM 15315(T)). When this strain was cultured on BCYEα agar at 36°C for 7 d, no long cells were observed. The dominant fatty acids of strain HYMO-6 were 16:1ω7c (32.4%), and the DNA G+C content was 42.0 mol%.

Molecular phylogeny of the higher and lower taxonomy of the Fusarium genus and differences in the evolutionary histories of multiple genes.

BACKGROUND: Species of the Fusarium genus are important fungi which is associated with health hazards in human and animals. The taxonomy of this genus has been a subject of controversy for many years. Although many researchers have applied molecular phylogenetic analysis to examine the taxonomy of Fusarium species, their phylogenetic relationships remain unclear only few comprehensive phylogenetic analyses of the Fusarium genus and a lack of suitable nucleotides and amino acid substitution rates. A previous stugy with whole genome comparison among Fusairum species revealed the possibility that each gene in Fusarium genomes has a unique evolutionary history, and such gene may bring difficulty to the reconstruction of phylogenetic tree of Fusarium. There is a need not only to check substitution rates of genes but also to perform the exact evaluation of each gene-evolution. RESULTS: We performed phylogenetic analyses based on the nucleotide sequences of the rDNA cluster region (rDNA cluster), and the ß-tubulin gene (ß-tub), the elongation factor 1α gene (EF-1α), and the aminoadipate reductase gene (lys2). Although incongruence of the tree topologies between lys2 and the other genes was detected, all genes supported the classification of Fusarium species into 7 major clades, I to VII. To obtain a reliable phylogeny for Fusarium species, we excluded the lys2 sequences from our dataset, and re-constructed a maximum likelihood (ML) tree based on the combined data of the rDNA cluster, ß-tub, and EF-1α. Our ML tree indicated some interesting relationships in the higher and lower taxa of Fusarium species and related genera. Moreover, we observed a novel evolutionary history of lys2. We suggest that the unique tree topologies of lys2 are not due to an analytical artefact, but due to differences in the evolutionary history of genomes caused by positive selection of particular lineages. CONCLUSION: This study showed the reliable species tree of the higher and lower taxonomy in the lineage of the Fusarium genus. Our ML tree clearly indicated 7 major clades within the Fusarium genus. Furthermore, this study reported differences in the evolutionary histories among multiple genes within this genus for the first time.

Genotyping and chlorine-resistance of Methylobacterium aquaticum isolated from water samples in Japan.

For microbial ecological analysis, 14 strains of Methylobacterium aquaticum isolated from water samples were subjected to clustering analysis on the basis of ribotyping and RAPD-PCR tests. The ribopatterns after digestion with EcoRI obtained from 14 strains of M. aquaticum were used to divide the strains into two groups (Groups I and II) with a similarity of 55%. From the analysis of RAPD patterns using primer 208, the 14 strains were divided into 3 groups (A-C) based on a homology of 45% or greater, and from that using primer 272, there were 4 groups (A-D) based on a homology of 50% or greater. The chlorine resistance (99.9% CT values) of these isolates was also experimentally confirmed, and we attempted to define the connection between chlorine resistance and the geno-cluster. The average CT value of group I was 0.89 mg•min/l and the average of group II was 0.69 mg•min/l. No remarkable differences in the CT values for the groups were found.

Evaluation of genetic markers for identifying isolates of the species of the genus Fusarium.

BACKGROUND: Members of the genus Fusarium are well known as one of the most important plant pathogens causing food spoilage and loss worldwide. Moreover, they are associated with human and animal diseases through contaminated foods because they produce mycotoxins. To control fungal hazards of plants, animals and humans, there is a need for a rapid, easy and accurate identification system of Fusarium isolates with molecular methods. RESULTS: To specify genes appropriate for identifying isolates of various Fusarium species, we sequenced the 18S rRNA gene (rDNA), internal transcribed spacer region 1, 5.8S rDNA, 28S rDNA, ß-tubulin gene (ß-tub), and aminoadipate reductase gene (lys2), and subsequently calculated the nucleotide sequence homology with pair-wise comparison of all tested strains and inferred the ratio of the nucleotide substitution rates of each gene. Inter-species nucleotide sequence homology of ß-tub and lys2 ranged from 83.5 to 99.4% and 56.5 to 99.0%, respectively. The result indicated that sequence homologies of these genes against reference sequences in a database have a high possibility of identifying unknown Fusarium isolates when it is more than 99.0%, because these genes had no inter-species pair-wise combinations that had 100% homologies. Other markers often showed 100% homology in inter-species pair-wise combinations. The nucleotide substitution rate of lys2 was the highest among the six genes. CONCLUSION: The lys2 is the most appropriate genetic marker with high resolution for identifying isolates of the genus Fusarium among the six genes we examined in this study.

Identification of Legionella londiniensis isolated from hot spring water samples in Shizuoka, Japan, and cytotoxicity of isolates.

As part of an epidemiological study on legionellosis, we attempted to isolate Legionella spp. from hot spring water and were able to isolate L. londiniensis HYKF-90505 (=JCM 16338), confirming that L. londiniensis inhabits hot spring water in Japan. To investigate the disease potential of L. londiniensis, we examined its ability to grow intracellularly within Acanthamoeba sp. JAC/E1 strain. The isolated HYKF-90505 was able to grow within Acanthamoeba sp. JAC/E1 strain, and we confirmed also that the HYKF-90505 strain showed cytotoxicity for cultured cells such as J774.1 (JCRB0018). However, in a culture of human U937 cells, the bacterial count was not increased by the intracellular growth of the HYKF-90505 strain. Cells infected for 24 h and stained using the Giménez method showed no intracellular growth of the HYKF-90505 strain. Thus, the isolate appears to be weakly pathogenic to humans.

Sporolactobacillus putidus sp. nov., an endospore-forming lactic acid bacterium isolated from spoiled orange juice.

A Gram-positive, endospore-forming, lactic acid bacterium was isolated from spoiled orange juice. The organism, strain QC81-06(T), grew microaerobically or anaerobically at 30-45 degrees C (optimum 35 degrees C) and pH 3.5-5.5 (optimum pH 4.5), and produced acid from various sugars. D-Lactic acid was produced. It contained menaquinone-7 as the major isoprenoid quinone. The G+C content of the genomic DNA was 47.5 mol%. The predominant cellular fatty acids of the strain were iso-C(16 : 0), anteiso-C(15 : 0) and anteiso-C(17 : 0). Phylogenetic analyses based on the 16S rRNA gene and gyrB gene (DNA gyrase B subunit gene) revealed that strain QC81-06(T) clustered with Sporolactobacillus species but the strain was clearly distinct from other Sporolactobacillus species with significant bootstrap values. The levels of 16S rRNA gene and gyrB gene sequence similarities between strain QC81-06(T) and the other strains of the cluster were 96.0-97.0 % and 75.1-77.2 %, respectively. On the basis of these results, strain QC81-06(T) should be classified as a novel Sporolactobacillus species for which the name Sporolactobacillus putidus is proposed. The type strain is strain QC81-06(T) (=DSM 21265(T)=JCM 15325(T)).

Identification of a major polyphenol and polyphenolic composition in leaves of Camellia irrawadiensis.

The polyphenolic composition of Camellia irrawadiensis, which is a closely related species of Camellia sinensis (cultivated tea), was investigated. The most predominant polyphenol, a kind of ellagitannin, was isolated from leaves of C. irrawadiensis. Its structure was established as 1,2-di-O-galloyl-4,6-O-(S)-hexahydroxydiphenoyl-beta-D-glucose (2) on the basis of spectral and chemical evidence. Moreover, the polyphenols [catechins, strictinin (1), compound 2, theogallin, and gallic acid] and two methylxanthines (theobromine and caffeine) in leaves of C. irrawadiensis were determined by HPLC-Photodiode array detector analysis, and were compared to those in C. sinensis and Camellia taliensis. Total catechin content in C. irrawadiensis was lower than that in C. sinensis and C. taliensis. Theobromine content in C. irrawadiensis was higher than that in C. sinensis and C. taliensis. The content of 2 in C. irrawadiensis was 8.4% of dry leaf weight and comprised approximately 60% of the total polyphenols detected, while the compound was not detected in C. sinensis and was reported to be 2.4% in C. taliensis.

Butyricimonas synergistica gen. nov., sp. nov. and Butyricimonas virosa sp. nov., butyric acid-producing bacteria in the family 'Porphyromonadaceae' isolated from rat faeces.

Two bacterial strains, designated MT01(T) and MT12(T), isolated from rat faeces were characterized by using a polyphasic taxonomic approach that included analysis of their phenotypic and biochemical features, cellular fatty acid profiles, menaquinone profiles and phylogeny based on 16S rRNA gene sequences. The 16S rRNA gene sequence analysis showed that these strains were members of the family 'Porphyromonadaceae'. The strains shared 94 % 16S rRNA gene sequence similarity with each other and were related to Odoribacter splanchnicus NCTC 10825(T) (86-87 % sequence similarity). The strains consisted of obligately anaerobic, non-pigmented, non-spore-forming, non-motile, Gram-negative rods. Growth of the strains was inhibited on medium containing 20 % bile. The two strains produced significant levels of butyric and isobutyric acids as end products from glucose. Although the major cellular fatty acid of these two strains and O. splanchnicus JCM 15291(T) was iso-C(15 : 0), strains MT01(T) and MT12(T) showed a higher level of iso-C(15 : 0) (66 and 74 %, respectively) than did O. splanchnicus JCM 15291(T) (48 %). In addition, the ratios of iso-C(15 : 0) to anteiso-C(15 : 0) in whole-cell methanolysates of the two isolates were very much higher than that of O. splanchnicus JCM 15291(T). The major menaquinone of the isolates was MK-10. This menaquinone composition was different from those of other genera of the family 'Porphyromonadaceae', such as Barnesiella (predominant menaquinones: MK-11 and MK-12), Odoribacter (MK-9), Paludibacter (MK-8), Parabacteroides (MK-9 and MK-10), Porphyromonas (MK-9 and MK-10) and Tannerella (MK-10 and MK-11). Menaquinone composition is therefore an important chemotaxonomic characteristic of these micro-organisms. Strains MT01(T) and MT12(T) have DNA G+C contents of 46 mol%. On the basis of these data, strains MT01(T) and MT12(T) represent two novel species of a novel genus, for which the names Butyricimonas synergistica gen. nov., sp. nov. and Butyricimonas virosa sp. nov., respectively, are proposed. The type strains of B. synergistica and B. virosa are MT01(T) (=JCM 15148(T) =CCUG 56610(T)) and MT12(T) (=JCM 15149(T)=CCUG 56611(T)), respectively.