奶牛瘤胃中牛链球菌的分离鉴定

采用厌氧分离技术从奶牛瘤胃中分离出1株细菌,通过对其形态、培养特性、生理生化特性、16S rRNA基因序列测定与同源性分析等研究,确定分离菌株为牛链球菌（Streptococcus bovis）,为进一步研究其对瘤胃发酵的影响奠定了基础。     

The characterization of lactic acid producing bacteria from the rumen of dairy cattle grazing on improved pasture supplemented with wheat and barley grain

Aims:  To identify and characterize the major lactic acid bacteria in the rumen of dairy cattle grazing improved pasture of rye grass and white clover and receiving a maize silage and grain supplement with and without virginiamycin.
Methods and Results:  Eighty-five bacterial isolates were obtained from the rumen of 16 Holstein-Friesian dairy cows. The isolates were initially grouped on the basis of their Gram morphology and by restriction fragment length polymorphism analysis of the PCR amplified 16S rDNA. A more definitive analysis was undertaken by comparing the 16S rDNA sequences. Many of the isolates were closely related to other previously characterized rumen bacteria, including Streptococcus bovis, Lactobacillus vitulinus , Butyrivibrio fibrisolvens , Prevotella bryantii and Selenomonas ruminantium . The in vitro production of l - and/or d -lactate was seen with all but five of the isolates examined, many of which were also resistant to virginiamycin.
Conclusion:  Supplementation of grain with virginiamycin may reduce the risk of acidosis but does not prevent its occurrence in dairy cattle grazing improved pasture.
Significance and Impact of the Study:  This study shows that lactic acid production is caused, not only by various thoroughly researched types of bacteria, but also by others previously identified in the rumen but not further characterized.     

Immunological cross-reactivity between the vaccine and other isolates of Streptococcus bovis and Lactobacillus

Recent studies have showed that immunisation with Streptococcus bovis (Sb-5) and Lactobacillus (LB-27) may confer protection against lactic acidosis in sheep and cattle. The present study was designed to determine the degree of immunological cross-reactivity between Sb-5 and eight other strains of Streptococcus bovis; and between LB-27 and four other isolates of Lactobacillus. The cross-reactivity index (CRI, a low CRI indicates a high degree of immunological cross-reactivity) ranged from 7.3 to 56.1% between the strains of S. bovis (the encapsulated strains with CRIs ranging from 7.3 to 12.4%). For isolates of Lactobacillus the CRIs ranged from 11.5 to 72.2%. The results indicate that all the isolates tested have a certain degree of immunological homology with Sb-5 and LB-27, and suggest that the vaccine may cross-react with a large number of strains of S. bovis and Lactobacillus which may cause lactic acidosis. As most of the S. bovis strains in the rumen are encapsulated, the high degree of homology between Sb-5 and encapsulated S. bovis strains further suggests that the vaccine containing Sb-5 may be effective against a wide range of strains of S. bovis in sheep and cattle.     

Role of Rusa deer Cervus timorensis russa in the cycle of the cattle tick Boophilus microplus in New Caledonia

Two field experiments were conducted to evaluate the efficiency of Rusa deer in the development of the cattle tick Boophilus microplus in comparison with that of steers in the same pastures and under the same conditions of infestation. No difference was noted between a mixed steer/deer herd and a pure steer or pure deer herd in the infestation pattern of each host, suggesting that attachment to the alternative host is mechanical and not affected by the simultaneous presence of the primary host on the pasture. Deer are capable of producing engorged viable females, with weight and reproductive performances similar to or even better than females fed on steers. For moderate levels (1 million larvae per hectare) and high levels (32 million larvae per hectare) of pasture infestation, tick burdens on steers were not very different (e.g. average 1911 and 2681ticks per m² skin, respectively, on day 24). This may be because of saturation of steer skin sites at the moderate larval dose. Deer harboured 2.7–33 times fewer ticks than steers and produce no engorged females at the moderate larval level and 32 times fewer engorged females than steers at the high larval level. Infestation of deer was dosedependent with averages of 12 and 399ticks per m² skin on day 25 at the moderate and high larval levels, respectively. At a high infestation level of the environment, Rusa deer may contribute, but to a limited extent, to infestation of pastures and, consequently, of cattle. However, their role in sustaining a viable tick population requires further investigation.     

Heterologous expression of an endoglucanase gene (endA) from the ruminai anaerobe Ruminococcus flavefaciens 17 in Streptococcus bovis and Streptococcus sanguis

Abstract The heterologous expression of a cloned endoglucanase gene ( endA ) from the ruminai bacterium Ruminococcus flavefaciens 17 was demonstrated in the Streptococcus species S. bovis JB1 and S. sanguis DLL The endA gene was introduced into S. bovis and S. sanguis using the Escherichia coli/Streptococcus shuttle vector pVA838. Expression of the gene was detected by clearing zones around the recombinant colonies on agar plates containing carboxymethylcellulose stained with Congo red. S. bovis JB1 containing the endA gene was capable of utilizing cellotetraose at a faster rate than the parent strain. This is the first demonstration that Streptococcus species can express a gene from a Ruminococcus flavefaciens strain.     

Development of molecular methods for identification of Streptococcus bovis from human and ruminal origins

Streptococcus bovis has been identified as a causative agent in humans for a variety of diseases, including endocarditis, meningitis, and septicemia. Identification of S. bovis strains of human origin in clinical settings has been problematic due to variations in biochemical tests as compared to ruminal strains of S. bovis, and other streptococcal species. DNA-DNA hybridization with chromosomal DNA from various S. bovis strains indicates that strains of human origin are different from those of ruminal origin. Specific probes have been designed from S. bovis 16S rDNA gene sequences that differentiate strains of human and ruminal origin by direct hybridization and PCR analyses. These techniques now allow for rapid identification of S. bovis strains for clinical and other scientific investigations.     

First isolation of Streptococcus downei from human dental plaques

In this study, we isolated four bacterial strains grown on mitis-salivarius sucrose bacitracin agar. The strains had similar biochemical characteristics to biotypes I or II of mutans streptococci. The four isolates were identified as Streptococcus downei by 16S rDNA and dextranase gene (dex) sequencing as well as polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) targeting dex. To our knowledge, this is the first report of the isolation and identification of S. downei from dental plaque in humans. The results suggest that S. downei can inhabit the human oral cavity.     

基于16S rRNA基因序列分析法比较苏尼特双峰驼和阿拉善双峰驼自然发酵酸驼乳的微生物多样性

【目的】传统发酵乳制品是一类未经任何处理自然发酵而成的,其微生态环境未遭破坏,从而乳酸菌的生物学特性和基因多样性得到了很好的保留,具有开发和利用价值。自然发酵酸驼乳常用来治疗多种疾病且效果良好,与其中丰富的乳酸菌资源有着密不可分的联系。然而,目前有关自然发酵酸驼乳微生物菌群及多样性相关研究甚少。因此进一步挖掘内蒙古地区双峰驼自然发酵酸驼乳微生物群落结构和多样性是至关重要的。【方法】本研究采用IlluminaMiseq测序技术,测定了苏尼特和阿拉善双峰驼的自然发酵酸驼乳中微生物16S rRNA V3–V4区序列,并对群落结构和多样性进行了比较分析。【结果】多样性分析表明,苏尼特双峰驼酸驼乳中微生物群落丰富度和种群差异性比阿拉善双峰驼酸驼乳大,细菌多样性也高。在门水平上,苏尼特和阿拉善双峰驼酸驼乳中的菌群均以厚壁菌门(Firmicutes)和变形菌门(Proteobacteria)为主。在属水平上,苏尼特双峰驼酸驼乳主要以乳杆菌属(Lactobacillus)和乳球菌属(Lactococcus)为优势菌群,阿拉善双峰驼酸驼乳以乳杆菌属(Lactobacillus)和醋酸杆菌属(Acetobacter)为优势菌属。此外,肠杆菌属(Enterobacter)、拉乌尔菌属(Raoultella)和明串珠菌属(Leuconostoc)等的含有食源性致病菌和环境污染菌的菌属被检出。综上所述,不同地区不同品种酸驼乳的乳酸菌种类及优势菌群有较大差异,存在显著的地理差异。【结论】通过本研究,不仅对苏尼特和阿拉善双峰驼自然发酵酸驼乳乳酸菌的组成和种类有了明确的认知,为评估发酵酸驼乳微生物群落对消费者身体健康的影响提供了数据基础的同时为今后筛选优势菌群和挖掘新型益生菌奠定基础。     

Phage resistance and altered growth habit in a strain of Streptococcus bovis

Bacteriophage (phi Sb01) of Streptococcus bovis, isolated from pooled rumen fluid of cattle, was a small siphovirus of morphotype B1. It contained double-stranded DNA of length 30.9 kb, which was digested by the restriction endonucleases, EcoRI, HindIII, and PvuII. Bacteria which survived phi Sb01 infection (strain 2BAr) grew in long chains (100-200 cells), ultimately forming large clumps of cells. This growth habit was in distinct contrast to that of the parent host strain which grew predominantly in the form of single cells or diplococci. Strain 2BAr was genetically stable, resistant to phi Sb01 attack, and the observed differences in the growth characteristics of the parent strain and 2BAr indicated that cells of 2BAr were more adherent. In the rumen ecosystem, the selection of phage-resistant bacteria with altered growth characteristics may be a factor in modifying bacterial phenotypes, and thus increasing variability among bacteria which are closely related genetically.     

Production of bacteriocins by Streptococcus bovis strains from Australian ruminants

Aims: To examine the prevalence of bacteriocin production in Streptococcus bovis isolates from Australian ruminants and the feasibility of industrial production of bacteriocin. Methods and Results: Streptococcus bovis strains were tested for production of bacteriocin‐like inhibitory substances (BLIS) by antagonism assay against Lactococcus lactis. BLIS production was associated with source animal location (i.e. proximity of other bacteriocin‐positive source animals) rather than ruminant species/breed or diet. One bacteriocin showing strong inhibitory activity (Sb15) was isolated and examined. Protein sequence, stability and activity spectrum of this bovicin were very similar to bovicin HC5. Production could be increased through serial culturing, and increased productivity could be partially maintained during cold storage of cultures. Conclusions: BLIS production is geographically widely distributed in Eastern Australia, and it appears that the bacteriocin⁺ trait is maintained in animals at the same location. The HC5‐like bacteriocin, originally identified in North America, is also found in Australia. Production of bacteriocin can be increased through serial culturing. Significance and Impact of the Study: The HC5‐like bacteriocins appear to have a broad global distribution. Serial culturing may provide a route towards commercial manufacturing for use in industrial applications, and purified bacteriocin from S. bovis Sb15 could potentially be used to prevent food spoilage or as a feed additive to promote growth in ruminant species.     

The glutamine cyclotransferase reaction of Streptococcus bovis: A novel mechanism of deriving energy from non-oxidative and non-reductive deamination

Abstract Streptococcus bovis deaminated glutamine by a mechanism that did not involve glutaminase. Since pyroglutamate and ammonia were the only end-products, it appeared that glutamine deamination was catalyzed by a cyclotransferase reaction. Stationary S. bovis cells had essentially no intracellular ATP or membrane potential (ΔΨ), however, when they were provided with glutamine, intracellular ATP and ΔΨ increased to 0.52 mM and 158 mV, respectively. When glutamine-energized cells were treated with N , N -dicyclohexylcarbodiimide (DCCD, 150 μM), there was an even greater increase in intracellular ATP (> 5-fold) and the ΔΨ was dissipated. Because toluene-treated cells produced ATP from ADP and P_i, it did not appear that the cell membrane was directly involved in glutamine-dependent ATP generation. The rate of ammonia production was directly proportional to the glutamine concentration, but the stoichiometry of ATP to ammonia was always 1 to 1. Based on these results, it appeared that glutamine was deaminated by glutamine cyclotransferase which was coupled to ATP formation. The membrane bound ATPase then used the ATP to create a ΔΨ.     

PCR amplification of species specific sequences of 16S rDNA and 16S-23S rDNA intergenic spacer region for identification of Streptococcus phocae

Streptococcus phocae, a bacterial pathogen of seals, could reliably be identified by PCR amplification using oligonucleotide primers designed according to species specific segments of the previously sequenced 16S rRNA gene and the 16S-23S rDNA intergenic spacer region of this species. The PCR mediated assay allowed an identification of S. phocae isolated from harbor and gray seals and from Atlantic salmons. No cross-reaction could be observed with 13 different other streptococcal species and subspecies and with Lactococcus garvieae strains investigated for control purposes.     

Restriction fragment length polymorphisms of 16S rDNA and of whole rRNA genes (ribotyping) of Streptococcus iniae strains from the United States and Israel

Streptococcus iniae (junior synonym S. shiloi) isolated from tilapia and trout in Israel and in the United States were subtyped by restriction length polymorphism (RFLP) based on PCR amplified 16S rDNA and by ribotyping. 16S rDNA RFLP discriminated between S. iniae and other fish pathogens but not between S. iniae strains. HindIII and EcoRI ribotypes of S. iniae discriminated American from Israeli strains rejecting the possibility of an epidemiological link between S. iniae infections in the two countries. Israeli strains isolated from tilapia and trout could not be completely differentiated. The S. iniae ATCC 29178^T (T=Type strain) strain, isolated from a freshwater dolphin belonged to a ribotype different from those of all the fish isolates.     

Bacterial competition between a bacteriocin-producing and a bacteriocin-negative strain of Streptococcus bovis in batch and continuous culture

A bacteriocin-producing Streptococcus bovis strain (HC5) outcompeted a sensitive strain (JB1) before it reached stationary phase (pH 6.4), even though it grew 10% slower and cell-free bovicin HC5 could not yet be detected. The success of bacteriocin-negative S. bovis isolates was enhanced by the presence of another sensitive bacterium (Clostridium sticklandii SR). PCR based on repetitive DNA sequences indicated that S. bovis HC5 was not simply transferring bacteriocin genes to S. bovis JB1. When the two S. bovis strains were coinoculated into minimal medium, bacteriocin-negative isolates predominated, and this effect could be explained by the longer lag time (0.5 vs. 1.5 h) of S. bovis HC5. If the glucose concentration of the minimal medium was increased from 2 to 7 mg mL(-1), the effect of lag time was diminished and bacteriocin-producing isolates once again dominated the coculture. When the competition was examined in continuous culture, it became apparent that batch culture inocula were never able to displace a strain that had already reached steady state, even if the inoculum was large. This result indicated that bacterial selection for substrate affinity was even more important than bacteriocin production.     

产氢菌的分离鉴定及其产氢特性研究

从河底污泥中分离到一株产氢菌,通过16S rDNA序列和系统发育分析,鉴定为克雷伯氏菌属,命名为Klebsiella sp. HQ-3.运用单因子试验考察了摇瓶发酵培养基中碳源、氮源、无机盐等因素对克雷伯氏菌HQ-3发酵产氢的影响,并对其产氢特性做了初步研究.结果表明,克雷伯氏HQ-3在葡萄糖浓度为24 g/L,蛋白胨为18 g/ L,KH2PO4为 8g/ L时具有较高产氢量,其最佳产氢初始pH为8.0左右.     

大莲湖池杉林湿地土壤可培养细菌的分离与鉴定

采用牛肉膏蛋白胨培养基培养,从大莲湖池杉林土壤中共分离得到20个菌落形态不同的菌株。通过对这些菌株的形态、培养特征、生理生化特征的研究以及16S rDNA序列分析,初步确定这些菌株分别属于假单胞菌属(Pseudomonas)、芽胞杆菌属(Bacillus)、红球菌属(Rhodococcus)、北里孢菌属(Kitasatosporia)、金黄杆菌属(Chryseobacterium)、不动杆菌属(Acinetobacter)、黄杆菌属(Flavobacterium)、鞘氨醇杆菌属(Sphingobacte-rium)和丛毛单胞菌属(Comamonas)等9个属细菌。其中芽胞杆菌属和不动杆菌属细菌是优势菌,分离到的红球菌属、北里孢菌属、鞘氨醇杆菌属和丛毛单胞菌属细菌在国内湿地土壤中报道较少。     

Alternative strategies of 2-deoxyglucose resistance and low affinity glucose transport in the ruminal bacteria, Streptococcus bovis and Selenomonas ruminantium

Abstract Streptococcus bovis and Selenomonas ruminantium grew in the presence of the glucose analog, 2-deoxyglucose (2-DG), but the cells no longer had high affinity glucose transport. In S. bovis , 2-DG resistance was correlated with a decrease in phosphoenolpyruvate (PEP)-dependent glucose phosphotransferase (PTS) activity. The 2-DG-selected S. bovis cells relied solely upon a low affinity, facilitated diffusion mechanism of glucose transport and a 2-DG-resistant glucokinase (ATP-dependent). The glucokinase activity of S. ruminantium was competitively inhibited by 2-DG, and the 2-DG selected cells continued to use PEP-dependent PTS as a mechanism of glucose transport. In this latter case, the 2-DG selected cells switched from a mannosephosphotransferase (enzyme II) that phosphorylated glucose, mannose, and 2-DG, but not α-methylglucoside to a glucosephosphotransferase (enzyme II) that phosphorylated glucose and α-methylglucoside but not 2-DG or mannose. The glucosephosphotransferase (enzyme II) had a very low affinity for glucose and the transport kinetics were similar to the facilitated diffusion system of S. bovis .