An evaluation of the Oxoid Biochemical Identification System Campy rapid screening test for Campylobacteraceae and Helicobacter spp.

Aims:  To evaluate the Oxoid Biochemical Identification System (OBIS) Campy test (ID0800M) against Campylobacter ; Arcobacter ; and other micro-organisms, with similar colonial morphology, for the detection of l -alanine aminopeptidase ( l -ALA).
Methods and Results:  The KOH and l -ALA (OBIS and Fluka) tests were carried out on every isolate. The procedures were followed as indicated in the OBIS and Fluka kit instructions. A total of 146 strains of 19 species of Campylobacter , seven strains of Arcobacter butzleri , four Arcobacter butzleri -like strains, 42 strains of 10 species of Helicobacter , 96 Gram-negative and 49 Gram-positive clinical isolates were tested. As expected, Campylobacter and Arcobacter strains were negative, while other Gram-negative bacteria were positive for the l -ALA test. An unexpected finding was that Helicobacter strains, although Gram-negative, were all negative for the l -ALA tests suggesting the absence of l -ALA within this genus. This is a novel finding. The absence of l -ALA was confirmed upon comparison with the available full genomic sequences of Helicobacter on the NCBI database.
Conclusions:  The OBIS Campy (ID0800M) test kit proved to be rapid and accurate for the presumptive characterization of Campylobacter and Arcobacter . A novel finding was that Helicobacter species also did not have the l -ALA enzyme.
Significance and Impact of the Study:  The OBIS kit will be useful in diagnostic laboratories for the presumptive diagnosis of Campylobacter , Arcobacter and Helicobacter strains.     

New taxonomic markers for identification of <Emphasis Type="Italic">Rhizobium leguminosarum</Emphasis> and discrimination between closely related species

Rhizobia, producing species-specific exopolysaccharides (EPSs), comprise a very diverse group of soil bacteria that are able to establish nitrogen-fixing symbioses with legumes. Based on the sequences of R. leguminosarum EPS synthesis genes, a sensitive and reliable PCR-based method for identification and subsequent discrimination between Rhizobium species has been developed and tested. For identification of R. leguminosarum, primer sets I–III complementary to sequences of rosR, pssA and pssY genes were proposed. Further sets of primers (IV–VII) were designed for discrimination between R. leguminosarum biovars. The usefulness of the method was examined using a wide range of R. leguminosarum strains isolated from different host plants nodules originating from different regions of Poland. We demonstrate a high discriminating power of primer sets I–III that allow distinguishing R. leguminosarum and two closely related species, R. etli and R. gallicum. This new approach is applicable to identification of R. leguminosarum strains, originating from nodules or soil, where many other closely related bacteria are expected to be present. Based on the nucleotide sequence of rosR and pssA genes, phylogenetic relationships of selected R. leguminosarum isolates were determined. Our results indicate that both rosR and pssA might be useful markers to differentiate and define relationships within a group of R. leguminosarum strains.     

具有降胆固醇功能益生菌的筛选研究

目的通过体外实验从发酵制品及人体肠道内分离筛选出安全高效的降胆固醇乳酸菌,并研究其生理生化特性,为今后功能性乳制品的开发提供优良菌种。方法应用MRS．胆固醇培养基液体发酵法进行目的菌株筛选,并测定其耐酸及耐胆盐等能力。结果筛选出4株具有降胆固醇能力的乳酸菌,其降解率分别为DM8403163．1％、DM8403470．3％、DM850381．1％、DM8605663．1％;同时4株乳酸菌均表现出不同程度的耐酸及耐胆盐能力,其中DM86056能力最强,其他3株乳酸菌次之;经鉴定,DM84031为保加利亚乳杆菌、DM84034为嗜热链球菌、DM8503为詹氏乳杆菌、DN86056为屎肠球菌。结论经上述实验综合比较,DM86056降胆固醇能力比较强且耐酸及耐胆盐等能力也较强,因此有待于将其应用于动物体内进行深入研究。     

Identifying tumor stem-like cells in mouse melanoma cell lines by analyzing the characteristics of side population cells

Increasingly more evidence shows that TSCs possess the characteristics of stem-like cells. However, a link between stem cells and TSCs remains to be shown. We have sorted SP cells and non-SP cells from the B16F10 cell lines by FACS, and then studied their cellular biological characteristics by using a SFCM culture method, proliferative assay in vitro, clone formation assays in soft agar and normal media, tumorigenic assays in C57BL/6 mice, and resistance to chemotherapy assay in vitro, the quantitative detecting expression of ABCG2 and their CD phenotype analysis by a FCM. We detected 0.96% SP cells in the B16F10 cells and found that they had obvious differences in characteristics from non-SP cells. They possessed a marked capacity for self-renewal in soft agar and culture medium, strong tumorigenic potential in C57BL/6 mice, apparent resistance to vinblastin in vitro, upregulated ABCG2 expression, and a high expression of CD44⁺CD133⁺CD24⁺ phenotypes. We conclude that there were a few of SP cells that had the characteristics of tumor stem-like cells which may provide a useful tool and a readily accessible source for further study when specific TSCs markers are unknown.     

拮抗放线菌Y-71的鉴定及其代谢活性物质的性质

本研究从云南采集的腐质土壤样品中分离筛选到一株拮抗放线菌Y-71,通过形态特征、培养特征、生理生化特征、细胞化学组分和基于16SrRNA基因序列的相似性分析等研究,鉴定菌株Y-71为脱叶链霉菌(Streptomyces exfoliatus)。抗菌谱测定及稳定性研究结果表明,菌株Y-71发酵液抑菌谱较广,对革兰氏阳性细菌和革兰氏阴性细菌都有较好的抑制作用;其抑菌活性物质对温度敏感,在70°C处理后抑菌活性丧失;在pH2-8条件下稳定;不能被蛋白酶降解,可初步判定此活性物质不属于蛋白质或肽类物质。本研究为该菌株今后的应用、抗生素的分离提纯奠定了基础。     

内蒙古西部区酸粥中酵母菌的分离鉴定及优势菌分析

从内蒙古地区采集28份酸粥样品,从中分离出40株酵母菌,并对其进行了分子生物学鉴定和生物多样性分析。26S rDNA D1/D2区域 (600bp左右)碱基序列分析结果表明,酸粥中的酵母菌有Issatchenkia orientalis、Saccharomyces cerevisiae、Geotrichum sp.、Candida pararugosa、 Candida parapsilosis、Trichosporon asahii、Trichosporon coremiiforme、Clavispora lusitaniae和Candida tropicalis。经过分析,Issatchenkia orientalis(75%,Frequency percentage)为酸粥中的优势菌。     

传统发酵豆瓣中产毒黄曲霉高效拮抗菌的筛选

从自然发酵的豆瓣中筛选出对产毒黄曲霉菌的生长及其毒素合成均有抑制作用的细菌, 在蚕豆天然培养基(BAM)上利用菌落对峙实验初筛和滤纸片复筛得到1株有较高抑制产毒黄曲霉活性的菌株L4。对L4进行形态学、生理生化特征及16S rRNA序列同源性分析, 鉴定此菌株为枯草芽孢杆菌(Bacillus subtilis)。在抑制黄曲霉生长和黄曲霉毒素B1 (AFB1)合成的研究中表明, 在L4与黄曲霉菌共同培养15 d后, 黄曲霉菌丝产量和黄曲霉毒素B1 产量均比黄曲霉单独培养时显著降低(P < 0.01), AFB1合成受到明显抑制, 抑制率达93.7%。当黄曲霉孢子液与L4发酵上清液1: 1 (V/V)混合后接种在玉米粒上时, 黄曲霉在玉米上的生长和孢子萌发均得到完全抑制。     

Bm-TFF2在毕赤酵母中的表达及鉴定

Bm-TFF2,一种从大蹼铃蟾皮肤分泌物中分离得到的两栖类三叶因子,具有比人三叶因子更强的生物学活性。该研究以Bm-TFF2的cDNA为模板,利用PCR方法扩增Bm-TFF2基因,然后插到含有AOX1启动子和α-因子信号肽序列的表达载体pPIC9K中,采用毕赤酵母表达系统进行分泌表达,并用G418筛选高拷贝整合转化子。SDS-PAGE和Western blotting都检测到Bm-TFF2被分泌性表达于酵母上清。在1%的甲醇诱导表达不同时间后,重组蛋白在72h的表达量最大,可达50mg/L;而不同浓度的饱和硫酸铵沉淀菌液上清时,80%的饱和硫酸铵沉淀量最大。这些结果表明,重组质粒Bm-TFF2-pPIC9K成功构建并在真核细胞中高效表达,这为进一步研究Bm-TFF2的生物学活性及其结构与功能关系奠定了基础。