Genotypic characterization of Brochothrix spp. isolated from meat,poultry and fish

Aim: To study genotypic diversity of isolates of Brochothrix thermosphacta recovered from meat, poultry and fish. Methods and Results: A total of 27 bacteria isolated from 19 samples of meat, poultry and fish were identified phenotypically and genotypically using PCR amplification of 16S‐23S rDNA intergenic transcribed spacer (ITS‐PCR), repetitive sequence‐based PCR (rep‐PCR) and 16S rDNA sequencing. Using ITS‐PCR, all bacteria showed the same DNA profile as the reference strains of Br. thermosphacta, allowing typing of the isolates at species level. Using 16S rDNA sequencing, all isolates were identified, at genus and species level, as Br. thermosphacta. Identification as Br. campestris was observed with a lower, but very close, level of similarity. Rep‐PCR was more discriminatory than ITS‐PCR and allowed differentiation of four subgroups among the isolates. Conclusion: Minor genotypic differences among Br. thermosphacta strains from meat, poultry and fish were observed. Significance and Impact of the Study: A rudimentary exploration of genotypic differences of Br. thermosphacta from meat, poultry and fish resulted in preliminary confirmation of the suitability of ITS‐PCR for typing Br. thermosphacta and confirmed the value of rep‐PCR fingerprinting to discriminate between Br. thermosphacta strains.     

小龙虾肠道产木聚糖酶细菌的分离与鉴定

【背景】小龙虾肠道微生物是小龙虾降解纤维素和半纤维素的主要驱动力。【目的】研究肠道内细菌的相对丰度,为揭示肠道微生物在小龙虾纤维素降解过程中的作用提供理论支撑。【方法】采用纯培养法从小龙虾肠道筛选产木聚糖酶细菌,并且对小龙虾肠道细菌进行16S高通量测序。【结果】形态学和16SrRNA基因分子鉴定表明,筛选到的4株产木聚糖酶细菌均属于芽孢杆菌科芽孢杆菌属;结合进一步的生理生化特征鉴定,结果为:菌株Z-3为枯草芽孢杆菌(Bacillus subtilis),菌株Z-4为贝莱斯芽孢杆菌(Bacillus velezensis),菌株Z-29为蜡状芽孢杆菌(Bacillus cereus),菌株Z-30为高地芽孢杆菌(Bacillus altitudinis);16S rRNA基因高通量测序结果表明:在属水平上,小龙虾肠道细菌主要是Candidatus Bacilloplasma、拟杆菌属、弧菌属、不动杆菌属、Dysgonomonas、Tyzzerella3、气单胞菌属和希瓦氏菌属细菌。【结论】小龙虾肠道内细菌资源丰富,且芽孢杆菌属细菌在木质纤维素降解过程中发挥一定功能。     

A novel enterohepatic Helicobacter species 'Helicobacter mastomyrinus' isolated from the liver and intestine of rodents

BACKGROUND: A number of novel Helicobacter species have been isolated from both animals and humans. Many of these helicobacters colonize the lower gastrointestinal tract and hepatobiliary tract and are associated with diseases. METHODS: A spiral-shaped bacterium, with bipolar single-sheathed flagella, was isolated from the liver and cecum of mastomys (the African rodent, Mastomys natalenis), from the feces and ceca of normal mice, and also from the cecum of a mouse with proctitis. 16S ribosomal RNA gene sequence analysis, restriction fragment length polymorphism (RFLP) and fluorophore-enhanced repetitive element polymerase chain reaction (FERP or rep-PCR) analysis were used to classify the organism. RESULTS: The bacterium grew at 37 and 42 degrees C under microaerobic conditions, rapidly hydrolyzed urea, and was catalase and oxidase positive. It did not reduce nitrate to nitrite, and was resistant to cephalothin and nalidixic acid. Like many other enterohepatic Helicobacter species, this organism expressed cytolethal distending toxin and causes cell distention. CONCLUSIONS: The organism was classified as a novel Helicobacter species for which we propose the name 'Helicobacter mastomyrinus'. Although 'H. mastomyrinus', like Helicobacter hepaticus and Helicobacter bilis, colonizes the liver of rodents, the pathogenic potential of this novel helicobacter is unknown.     

Thiocyanate transport across fish intestine (Pleuronectes platessa)

Summary When bathed on both sides with identical chloride-containing salines thein vitro preparation of the plaice intestine maintains a negative (serosa to mucosa) short-circuit current of 107±11 A/cm², a transepithelial potential difference of 5.5±0.6 mV (serosa negative), and a mean mucosal membrane potential of –45.4±0.6 mV. Under these conditions the intracellular chloride activity is 32mm.If chloride in the bathing media is partially, or completely substituted by thiocyanate the measured electrical parameters do not change but transepithelial flux determinations show a reduction in chloride fluxes and the presence of a significant thiocyanate flux. The addition of piretanide (10^–4 m) reduced the short-circuit current and the mucosa-to-serosa fluxes of chloride and thiocyanate; this inhibition is similar to the effect of piretanide on chloride transport in this tissue.The results indicate that thiocyanate is transported in this tissue via the piretanide-sensitive chloride pathway and are compared with the effects of thiocyanate on other tissues reported in the literature.     

Isolation and characterization of dendritic cells and macrophages from the mouse intestine

Within the intestine reside unique populations of innate and adaptive immune cells that are involved in promoting tolerance towards commensal flora and food antigens while concomitantly remaining poised to mount inflammatory responses toward invasive pathogens. Antigen presenting cells, particularly DCs and macrophages, play critical roles in maintaining intestinal immune homeostasis via their ability to sense and appropriately respond to the microbiota. Efficient isolation of intestinal DCs and macrophages is a critical step in characterizing the phenotype and function of these cells. While many effective methods of isolating intestinal immune cells, including DCs and macrophages, have been described, many rely upon long digestions times that may negatively influence cell surface antigen expression, cell viability, and/or cell yield. Here, we detail a methodology for the rapid isolation of large numbers of viable, intestinal DCs and macrophages. Phenotypic characterization of intestinal DCs and macrophages is carried out by directly staining isolated intestinal cells with specific fluorescence-labeled monoclonal antibodies for multi-color flow cytometric analysis. Furthermore, highly pure DC and macrophage populations are isolated for functional studies utilizing CD11c and CD11b magnetic-activated cell sorting beads followed by cell sorting.     

Different stability of posttranslationally modified brain microtubules isolated from cold-temperate fish

Microtubule proteins were isolated by a temperature-dependent assembly-disassembly method from brain tissue of for cold-temperate fish; one fresh water fish (Oncorhynchus mykiss), and three marine fish (Labrus berggylta, Zoarces viviparus andGadus morhua). The -tubulins from all four fish species were acetylated. The -tubulins from the marine fish were composed of a mixture of tyrosinated and detyrosinated tubulin, while the fresh water fish tubulin only reacted with an antibody against detyrosinated tubulin. The isolated microtubules had a similar MAP composition. A 400 kD protein and a MAP2-like protein were found, but MAP1 was missing. All microtubules disassembled upon cooling to 0°C. In spite of these common characteristics, the assembly of microtubules fromLabrus berggylta was inhibited by colchicine and calcium, in contrast to the assembly of microtubules fromOncorhynchus mykiss andZoarces viviparus. For the latter, colchicine was not completely inhibitory even at a concentration as high as 1 mM, and calcium induced the formation of both loosely and densely coiled ribbons. The effects of calcium and colchicine on microtubules fromOncorhynchus mykiss andZoarces viviparus were modulated by either fish or cow MAPs, indicating that the effects are due to intrinsic properties of the fish tubulins and not the MAPs. In view of these findings, our results suggest that there is not correlation between colchicine sensitivity, inability of calcium to inhibit microtubule assembly, and acetylation and detyrosination.     

不同来源株香港海鸥形菌药敏试验结果分析

目的对分离自社区获得性胃肠炎患者及淡水鱼肠道内的香港海鸥形菌(Laribacter hongkongensis,LH)进行药敏试验,为临床合理使用抗菌药物提供依据。方法2005年至2006年杭州市第一人民医院肠道门诊从社区获得性胃肠炎患者中分离到4株及2种淡水鱼体内10株LH菌株,计14株,按美国CLSI推荐的纸片扩散法进行药敏试验。结果药敏结果显示,14株LH菌株对24种常用抗菌药物中的15种药物,分别表现出不同程度的耐药性,耐药率达62.5%;尤其对青霉素、头孢唑啉和头孢哌酮等3种药物,14株LH菌株均为耐药,耐药率为100.0%;其中人体株LHHZ26,对12种药物耐药、1种为中介。LH对3代头孢表现为较高的耐药性,3种头孢药物的耐药率为71.43%~100%;14株LH株对头孢吡肟及头孢哌酮/舒巴坦均为敏感。对氨曲南(14.29%)、庆大霉素(7.14%)、复方新诺明(7.14%)的耐药率较低;喹诺酮类、氯霉素、阿米卡星、美罗培南、亚胺培南等药物均敏感。结论LH菌株对β-内酰胺类药物表现出耐药性,对青霉素类、头孢菌素类药物广泛耐药。建议对LH感染患者喹诺酮类为首选治疗用药。     

Identification of bombesin receptors on isolated muscle cells from human intestine

Smooth muscle cells were isolated separately from the longitudinal and circular muscle layers of human jejunum obtained at surgery and used to determine whether amphibian bombesin-14 and 3 mammalian homologues, GRP-(1-27), GRP-(18-27) and neuromedin B, can cause contraction by acting directly on muscle cells. Circular and longitudinal muscle cells contracted identically in response to bombesin-14 (C50 2 x 10(-12) M). The contractile response was not affected by selective muscarinic, opioid, CCK or serotonin antagonists but was inhibited by the substance P (SP) derivative, [D-Arg1, D-Pro2, D-Trp7,9, Leu11]SP. All 3 mammalian bombesins were less potent than bombesin-14. GRP-(1-27) and GRP-(18-27) were equipotent (C50 4 x 10(-11) M) but 20 times less potent than bombesin-14. Neuromedin B (C50 6 x 10(-12) M) was 3 times less potent than bombesin-14. All bombesins, however, were more potent than other enteric neuropeptides (e.g., tachykinins, opioid peptides). The study demonstrates conclusively the ability of bombesins to cause direct contraction of intestinal smooth muscle cells.     

Uptake of selenite,selenomethionine and selenate by brush border membrane vesicles isolated from rat small intestine

The uptake of selenite, selenate and selenomethionine (SeMet) was performed with brush border membrane vesicles (BBMV) prepared from rats fed selenium-deficient and supplemented diets. At equilibrium (60 min), the uptake of ⁷⁵Se from [⁷⁵Se]selenite ranged from 16.5 to 18.9 nmol mg^-1 protein. There was a curvilinear relationship in the uptake of selenite over a concentration range of 10–1000 m. About 2 nmol mg^-1 protein was obtained with selenomethionine (SeMet) which occurred between 90 and 180 s. In contrast to selenite, there was a linear relationship in the initial uptake of SeMet over a concentration range of 10–1000 m. The uptake of selenate was approximately 50-fold lower than selenite, reaching 350 pmol mg^-1 protein. Dietary selenium level had no effect on the rate of ⁷⁵Se accumulation by BBMV. Dramatic differences are found in the uptake and binding of selenium by BBMV incubated with different selenocompounds.     

Identification and characterization of yeast isolated from indonesian fermented food

Yeast strains with amylolytic activity were isolated from cassavatapé and its precursor,ragi. they were divided into two groups based on their characteristics: group 1, possessing high amylolytic activity and low ethanol yield; and group 2, possessing low amylolytic activity and high ethanol yield. The major strains of the group 1 were identified asEndomyces fibuliger, and those of group 2 were identified asPichia anomala. Based on 18S rDNA analysis, an isolate fromragi that had a high amylolytic activity was thought to be an undescribed species that was related to the basidiomycetous genera.     

Molecular identification and phylogenetic analysis of fungal pathogens isolated from diseased fish in Xinjiang,China

The outbreaks of fungal diseases in cultured fish have been severe in recent years, which is harmful to the healthy and sustainable development of fish farming. In this study, an investigation was conducted for significant fungal infections of 12 species of fish in four regions in Xinjiang, China, to understand the distribution of local fish fungal pathogens. Twenty-six fungal strains with pathogenicity were isolated, and the challenge experiment showed that eight strains from Changji area had high infection rate to fish eggs. Based on internal transcribed spacer sequence data and molecular analysis, the 26 strains were classified into nine different species of six fungal genera. Phylogenetic analysis showed that all strains were divided into two clades, namely Cluster 1 (contains only the genus Mucor) and Cluster 2 (consists of five small branches), and the distribution of strains from the same region was scattered in two clusters. There is no strict host selectivity for these fungi to infect fish. Mucor sp. are the main fungal pathogen of fish in these four regions, whereas Hypophthalmichthys molitrix and Carassius auratus are two types of fish that were susceptible to pathogen. In addition, the environmental adaptability experiments showed that eight highly pathogenic strains have different adaptability to the environment, and their optimum temperature and pH were 25°C and 7.0, respectively, whereas the concentration of NaCl was negatively correlated with the growth of strains. Therefore, these results indicated that the coinfection of multiple fungal pathogens in a culture region should be considered in the future study.     

Blastobotrys serpentis sp. nov., isolated from the intestine of a Trinket snake (Elaphe sp., Colubridae)

Asporogenus yeast strains W113AT and W113B were isolated from the intestine of a dead Trinket snake. The two isolates showed 100% sequence similarity in the D1/D2 domain of the large-subunit (LSU) rRNA gene, internal transcribed spacer (ITS) 1-5.8S rRNA gene-ITS2 region and mitochondrial small-subunit rRNA gene and the cytochrome oxidase II gene sequence and also showed similar phenotypic characteristics. The nearest phylogenetic neighbors of W113AT and W113B based on the sequence of the D1/D2 domain of the LSU rRNA gene were Blastobotrys chiropterorum NRRL Y-17017T and Blastobotrys terrestris NRRL Y-17704T with about 98% similarity. The close affiliation of W113AT and W113B with B. chiropterorum NRRL Y-17017T and B. terrestris NRRL Y-17704T was also evident from the high similarity observed in the nucleotide sequences of the mitochondrial small subunit rRNA (96-97.8%) and the cytochrome oxidase II (95.5-95.6%) genes. In the neighbor-joining phylogenetic trees constructed based on the D1/D2 domain or cytochrome oxidase gene, the isolates clustered with the above-mentioned species. However, the isolates showed a number of differences in their phenotypic properties with B. chiropterorum NRRL Y-17017T and B. terrestris NRRL Y-17704T and hence are regarded as representing a novel member of the genus Blastobotrys, for which the name Blastobotrys serpentis sp. nov. is proposed.     

Specific probiotic characterization of Weissella hellenica DS-12 isolated from flounder intestine

A total of 199 microorganisms were isolated from the intestinal contents of flounder (Paralichthys olivaceus) in a fish farm in Seoul, Korea. Among these strains, DS-12 was selected as a candidate for flounder probiotics because of its excellent exhibition of antimicrobial activity against fish pathogens such as edwardsiella, pasteurella, aeromonas, and vibrio, and initiate growth in 10% NaCl, 10% bile, and in broth at pH 3 for 90 min. This strain was Gram-positive, and catalase-negative coccoid rods that produced gas from glucose and formed more than 90% of lactate as the D(-) isomer. This organism is positioned at a cluster in the genus Weissella on the phylogenetic tree based on 16S rRNA sequences, which were assigned to Weissella hellenica on the basis of DNA-DNA relatedness. However, the type strain of W. hellenica JCM 10103(T) had no antibacterial activity against the fish pathogenic bacteria and was found to be quite different from the DS-12 strain in some sugar fermentation patterns of alpha-methyl-D-glucoside, esculine, cellobiose, melibiose, D-raffinose, and D-turanose, being especially unable to grow at 15 and 35 degrees C in 7% NaCl and 10% bile. The results obtained in the present study demonstrated that the type strain of W. hellenica had no probiotic characteristics, but the strain DS-12 could be used as a specific probiotic for flounder.     

Glycoconjugate characterization in the intestine of Umbrina cirrosa by means of lectin histochemistry

Goblet cells in the intestine of shi drum Umbrina cirrosa showed the presence of glycoconjugates particularly rich in fucose and N-acetylglucosamine residues. They displayed also sialic acid linked to galactosyl(β1→3)N-acetylgalactosamine and to galactosyl(β1→4)N-acetylglucosamine. All the nine horseradish peroxidase-conjugated lectins employed with the only exception of GSA II marked the enterocytes supranuclear region and the cell coat; the cell coat showed a more intense reactivity toward the different lectins, particularly enhanced with the use of fucosyl specific lectins.     

Identification and characterization of chitinolytic bacteria isolated from a freshwater lake

To develop a novel type of biocontrol agent, we focus on bacteria that are characterized by both chitinase activity and biofilm development. Chitinolytic bacteria were isolated from sediments and chitin flakes immersed in the water of a sand dune lake, Sakata, in Niigata, Japan. Thirty-one isolates from more than 5100 isolated strains were examined chitinase activity and biofilm formation. Phylogenetic analysis of these isolates based on the 16S rRNA gene sequences revealed that most isolates belonged to the family Aeromonadaceae, followed by Paenibacillaceae, Enterobacteriaceae, and Neisseriaceae. The specific activity of chitinase of four selected strains was higher than that of a reference strain. The molecular size of one chitinase produced by Andreprevotia was greater than that of typical bacterial chitinases. The dialyzed culture supernatant containing chitinases of the four strains suppressed hyphal growth of Trichoderma reesei. These results indicate that these four strains are good candidates for biocontrol agents.     

Antimicrobial resistance and in vitro gene transfer in bacteria isolated from the ulcers of EUS-affected fish in India

Aims:  The occurrence of drug resistance and plasmid-mediated transferability was investigated in 15 Aeromonas isolates collected from the ulcers of epizootic ulcerative syndrome (EUS)-affected fishes Katla ( Catla catla ), Mrigel ( Cirrhinus mrigala ) and Punti ( Puntius sp.).
Methods and Results:  Disc diffusion assay showed that all the strains were resistant to ampicillin and sensitive to streptomycin. Of the 15 isolates examined, 93·3% isolates were resistant to erythromycin, sulfadiazine and novobiocin, while 66% were resistant to rifampin and 20% to chloramphenicol. All isolates harboured plasmids with sizes ranging from 64 to 23 kbp with a 23-kbp plasmid in common. Plasmids from 11 Aeromonas strains were transferred to Escherichia coli DH5α recipient strain along with the transfer of ampicillin, erythromycin and chloramphenicol resistance determinants with frequencies ranging from 7·0 × 10⁻⁶ to 1·8 × 10⁻⁵ transconjugants per recipient cell.
Conclusions:  The resistance to ampicillin, erythromycin, sulfadiazine, novobiocin and chloramphenicol is prevalent among the bacteria isolated from EUS-affected fish, and resistant determinants of some of these antibiotics have been transferred to the bacteria of other origin.
Significance and Impact of the Study:  The emergence of antibiotic resistance bacteria and gene transfer in vitr o suggests that antibiotics should be used more cautiously to treat Aeromonas infections in aquaculture.