Urease production and DNA-homology in the species Bifidobacterium suis

Summary Urease production in the species Bifidobacterium suis was studied. The strains examined were strictly homogeneous in their DNA homology relationships. Most strains (74%) possess this enzyme. The presence of urease is therefore of value as additional diagnostic character of this species.     

低pH处理对两歧双歧杆菌KLDS2.0603黏附能力的影响

【目的】确定低pH处理对两歧双歧杆菌KLDS2.0603黏附能力及其表面物理化学性质的影响。【方法】将两歧双歧杆菌KLDS2.0603菌体在不同低pH的PBS溶液中处理一定时间后,采用平板菌落计数法和直接镜检法,测定其经历不同pH的酸性环境后的黏附能力,及其表面疏水性和自动聚集能力。【结果】不同pH的PBS溶液处理后的双歧杆菌菌体,其黏附能力均不同程度下降,除pH 5.0的处理组外,其余处理组均显著低于空白组。此外,经不同pH的PBS溶液处理后,仅pH 3.0和3.5的两处理组,双歧杆菌表面疏水性显著提高。除pH 1.0、1.5和5.0的处理组外,其余处理组的自动聚集能力均显著下降。【结论】低pH的酸性环境会降低两歧双歧杆菌KLDS2.0603的黏附能力,并且双歧杆菌的自动聚集能力和表面疏水性也发生相应变化。除pH 3.0和3.5的处理组外,三者之间呈现一定的正相关性。     

Selection of a Bifidobacterium animalis subsp. lactis strain with a decreased ability to produce acetic acid

We have characterized a new strain, Bifidobacterium animalis subsp. lactis CECT 7953, obtained by random UV mutagenesis, which produces less acetic acid than the wild type (CECT 7954) in three different experimental settings: De Man-Rogosa-Sharpe broth without sodium acetate, resting cells, and skim milk. Genome sequencing revealed a single Phe-Ser substitution in the acetate kinase gene product that seems to be responsible for the strain's reduced acid production. Accordingly, acetate kinase specific activity was lower in the low acetate producer. Strain CECT 7953 produced less acetate, less ethanol, and more yoghourt-related volatile compounds in skim milk than the wild type did. Thus, CECT 7953 shows promising potential for the development of dairy products fermented exclusively by a bifidobacterial strain.     

一株携带质粒的人两歧双歧杆菌的分离与鉴定

目的:分离携带天然质粒的人双歧杆菌.方法:用自制的改良型Blb双歧杆菌选择培养基,从人新鲜粪便分离双歧杆菌,对初步质粒检测阳性的单菌落通过糖发酵试验、(G C)mol%测定和16S rDNA序列分析,进行菌株鉴定.结果:筛选到一株携带天然质粒的人双歧杆菌,编号B200304,在1.0%琼脂糖凝胶上,测得质粒的相对分子质量约为22 kb.通过对该菌株的形态学观察和糖发酵试验等生理生化特征研究,证明该菌株为两歧双歧杆菌(Bifidobacterium bifidum);HPLC法测得其(G C)mol%为55.6,16SrDNA序列分析进一步证实该菌株为两歧双歧杆菌.结论:分离得到一株携带天然质粒的人两歧双歧杆菌新菌株.     

Live/dead state is not the factor influencing adhesion ability of Bifidobacterium animalis KLDS2.0603

Two essential requirements for probiotic bifidobacteria are that they be “live” and have “colonization” ability, following FAO/WHO guideline recommendations. The amount of research on the adhesion ability of bifidobacteria compares poorly with that of other probiotic bacteria, such as lactobacilli. The aim of the present study was to determine how gastrointestinal conditions affect the adhesion ability of bifidobacteria, and to investigate the relationship between the adhesion ability and the live/dead state of bifidobacteria. The adhesion ability of Bifidobacterium animalis KLDS2.0603 that had been subjected to the digestive enzymes, pepsin, trypsin, and proteinase K, was decreased significantly, but these treatments did not significantly change the strain’s survival rates, which were 98.78%, 97.60%, and 97.63% respectively. B. animalis KLDS2.0603 subjected to LiCl retained its adhesion ability but had a lower survival rate (59.28%) than the control group (P<0.01). B. animalis KLDS 2.0603 subjected to sodium metaperiodate exhibited higher adhesion ability than the control group (P<0.01), but the bacterial cells were killed totally. The results of transmission electron microscopy and laser scanning confocal microscopy showed that live/dead state of bifidobacteria was not one of the main factors that affected the adhesion ability of bifidobacteira, and that the substances affecting the adhesion ability of bifidobacteria were on the outer surface layer of the bifidobacterial cells. Our results also indicated that the substances related to the adhesion ability of bifidobacteria are proteinaceous. The above results will help us to understand the adhesion and colonization processes of bifidobacteria in the human gastrointestinal tract.     

适用微生物法检测的农药敏感双歧杆菌菌株筛选研究

目的分离筛选出一株对甲胺磷敏感的双歧杆菌菌株。方法双歧杆菌菌株传代筛选,取一系列浓度(4.0、2.0、1.0、0.5和0.25μg/m L)的甲胺磷农药标准溶液30μL加入含TPY液体培养基的检测管溶液内,双歧杆菌菌株接种到检测管内,37℃厌氧培养12 h,测定检测管液体A值。结果筛选一株甲胺磷敏感的短双歧杆菌菌株LJM-006,微生物抑制法的最佳接种浓度107CFU/m L,最低检测限0.5 mg/L。结论筛选一株甲胺磷敏感的双歧杆菌LJM-006菌株,并可作为微生物法检测甲胺磷残留的菌株。     

Infection in pigs with a rare strain ofS. cholerae suis

Summary In 5 animals out of a herd of pigs slaughtered on account of swine-plagueS. cholerae suis was isolated as a secondary infection. These strains appeared to differ from the diphasicS. cholerae suis. Unlike these they were H₂S-positive.     

一株双歧杆菌质粒聚合酶基因的PCR扩增和鉴定

目的PCR扩增人双歧杆菌天然质粒的聚合酶基因。方法用改良型MRS双歧杆菌选择培养基,从人新鲜粪便分离长双歧杆菌,PCR扩增长双歧杆菌质粒聚合酶(Bifidobacterium plasmid polymerase,BPP)基因,对BPP基因检测阳性的PCR产物通过序列分析,进行鉴定。结果人长双歧杆菌天然质粒的聚合酶基因PCR扩增后,经1.0%琼脂糖凝胶电泳,测得BPP基因的相对分子质量约为1.9 kb。通过BLAST序列比对分析与GenBank中相应基因同源性为96%。结论成功克隆了1株双歧杆菌天然质粒的聚合酶基因,为构建与双歧杆菌宿主质粒相适应的载体奠定了基础。     

Characterisation of a complex restriction/modification system detected in a Bifidobacterium longum strain

 Two type-II restriction endonucleases, BloI and BloII, have been detected in a Bifidobacterium longum strain. BloI is influenced by dam methylation: it cleaves dam ^- but not dam ⁺ DNA. It shows a temperature and pH optimum of 45°C and pH 7.5. Restriction analysis and cloning experiments showed that the recognition sequence is RGATCY and that the enzyme cuts 5′ to the guanine residue. It is an isoschizomer of commercial enzymes, BstYI and XhoII. The second activity is not inhibited by dam methylation. It has a temperature optimum between 25°C and 30°C and shows a broad pH optimum between 4.5 and 7.0. The activity is thermolabile and can be heat-killed by a 5 min incubation at 60°C. Cloning and sequencing experiments revealed that its recognition sequence is CTGCAG and that it cuts 5′ to the second guanine residue in the sequence. This enzyme is the first described isoschizomer of PstI. Received: 22 May 1995/Accepted: 26 July 1995     

Role of extracellular transaldolase from Bifidobacterium bifidum in mucin adhesion and aggregation

The ability of bifidobacteria to establish in the intestine of mammals is among the main factors considered to be important for achieving probiotic effects. The role of surface molecules from Bifidobacterium bifidum taxon in mucin adhesion capability and the aggregation phenotype of this bacterial species was analyzed. Adhesion to the human intestinal cell line HT29 was determined for a collection of 12 B. bifidum strains. In four of them-B. bifidum LMG13195, DSM20456, DSM20239, and A8-the involvement of surface-exposed macromolecules in the aggregation phenomenon was determined. The aggregation of B. bifidum A8 and DSM20456 was abolished after treatment with proteinase K, this effect being more pronounced for the strain A8. Furthermore, a mucin binding assay of B. bifidum A8 surface proteins showed a high adhesive capability for its transaldolase (Tal). The localization of this enzyme on the surface of B. bifidum A8 was unequivocally demonstrated by immunogold electron microscopy experiments. The gene encoding Tal from B. bifidum A8 was expressed in Lactococcus lactis, and the protein was purified to homogeneity. The pure protein was able to restore the autoaggregation phenotype of proteinase K-treated B. bifidum A8 cells. A recombinant L. lactis strain, engineered to secrete Tal, displayed a mucin- binding level more than three times higher than the strain not producing the transaldolase. These findings suggest that Tal, when exposed on the cell surface of B. bifidum, could act as an important colonization factor favoring its establishment in the gut.     

带有氯霉素抗性基因标记的重组布氏杆菌S2的构建及其生物学特性

无法区分免疫接种和自然感染是限制布氏杆菌疫苗应用的主要原因。本研究通过基因同源重组技术,用氯霉素抗性基因(Cmr)替代布氏杆菌弱毒S2株的WbkC基因,筛选获得重组布氏杆菌rS2-WbkC株。试验发现,重组菌rS2-WbkC由原先的光滑型转变成粗糙型。rS2-WbkC在TSA培养基上传25代后仍能稳定表达氯霉素抗性蛋白。小鼠动物试验模型表明,rS2-WbkC与S2有相似的保护性,但rS2比S2具有更高的安全性。rS2-WbkC免疫小鼠后,其抗血清可通过平板凝集试验与S2免疫的血清相区分。本研究为布氏杆菌标记疫苗的研制提供了技术平台。     

Influence of nutrition on the morphology of a strain of Bifidobacterium bifidum

A mucoid variant of Bifidobacterium bifidum was converted from its normal curved rod or bifid form to a highly branched form when grown in a chemically defined minimal medium. Branching could be prevented by the addition of a mixture of dl-alanine, dl-aspartic acid, l(+)-glutamic acid, and dl-serine, but not when any one of these four amino acids was omitted. Although sodium chloride induced pleomorphism, calcium ions were ineffective in suppressing the appearance of these pleomorphic forms. None of the cell wall precursors tested, viz., N-acetyl-d-glucosamine, alpha-epsilon-diaminopimelic acid, and muramic acid, inhibited branching.     

Discovery and characterization of a fructosylated capsule polysaccharide and sialylated lipopolysaccharide in a virulent strain of Actinobacillus suis

We are developing a serotyping system for Actinobacillus suis based on its capsule (K) and lipopolysaccharide O-chain (O) structures. Previously, we have shown that less virulent strains of this swine pathogen express a (1→6)-β-D-glucan as both K- and O-chain polysaccharides and were serologically classified as K:1/O:1. Here, we show that representative A. suis strains with a high (H91-0380; serotype K:2/O:2) and intermediate (C84; serotype K:2/O:1) degree of virulence possess a capsule polysaccharide (K:2) composed of an O-acetylated diglycosyl phosphate repeat decorated with fructose: [→4)-3-O-Ac-β-D-GlcpNAc-(1→3)-[β-D-Fruf-(2→2)]-α-D-Galp-(1→PO(4)(-)→]. In addition, the serotype O:2 lipopolysaccharide was shown to express a sialylated O-chain [→3)-β-D-Galp-(1→4)-[Neu5Ac-(2→3)-α-D-Galp-(1→6)]-β-D-Glcp-(1→6)-β-D-GlcpNAc-(1→]. As (1→6)-β-D-glucan is ubiquitous in the environment, low levels of antibodies in the animals are predicted to prevent disease by K:1/O:1 strains. The greater potential associated with K:2/O:2 and K:2/O:1 strains is most likely due to the absence of (1→6)-β-D-glucan as the K antigen and, in the case of K:2/O:2, the presence of sialic acid in the lipopolysaccharide, a nonulosonic acid known to promote evasion of host recognition.     

Autoaggregation and surface hydrophobicity of bifidobacteria

Thirteen strains of four different Bifidobacterium spp. were observed for their autoaggregation ability and surface hydrophobicity, and correlation between these two traits was determined. Bifidobacteria were classified into high, medium and low autoaggregation strains according to autoaggregation ratio measured from changes in absorbance of media. High autoaggregation strains showed microscopic clustering of cells, whereas low and medium autoaggregation strains showed no such clustering. Autoaggregation ability decreased in high autoaggregation strains but increased in medium and low autoaggregation strains when the assay was performed at higher temperature (37°C compared with 25 and 10°C). Bacterial strains belonging to the high, medium or low autoaggregation group were correlated in terms of their surface hydrophobicity and evaluated based on changes in absorbance of the bacterial suspension before and after extraction with xylene. These results indicate that autoaggregation ability, together with surface hydrophobicity and microscopic image could be used for evaluating the adhesion ability of potential probiotic bifidobacterial strains. Moreover, a synergistic effect of pH and media may be involved in autoaggregation.     

Analysis of polyamine biosynthetic- and transport ability of human indigenous Bifidobacterium

Bifidobacteria are members of the human intestinal microbiota, being numerically dominant in the colon of infants, and also being prevalent in the large intestine of adults. In this study, we measured the concentrations of major polyamines (putrescine, spermidine, and spermine) in cells and culture supernatant of 13 species of human indigenous Bifidobacterium at growing and stationary phase. Except for Bifidobacterium bifidum and Bifidobacterium gallicum, 11 species contained spermidine and/or spermine when grown in Gifu-anaerobic medium (GAM). However, Bifidobacterium scardovii and Bifidobacterium longum subsp. infantis, which contain spermidine when grown in GAM, did not contain spermidine when grown in polyamine-free 199 medium. Of the tested 13 Bifidobacterium species, 10 species showed polyamine transport ability. Combining polyamine concentration analysis in culture supernatant and in cells, with basic local alignment search tool analysis suggested that novel polyamine transporters are present in human indigenous Bifidobacterium.

Abbreviations: Put: putrescine; Spd: spermidine; Spm: spermine; GAM: Gifu anaerobic medium; BHI: brain-heart infusion     

Autoaggregation of Lactobacillus reuteri is mediated by a putative DEAD-box helicase

We have cloned and sequenced a gene from Lactobacillus reuteri that encodes a 56 kDa protein, which mediates autoaggregation of the bacteria. Using an antiserum raised against extracellular proteins from the pig intestinal isolate L. reuteri 1063, we screened a genomic lambda library derived from the same strain. Affinity purification of recombinant protein from the isolated lambda clones showed that one type of clone expressed a protein that efficiently aggregated the parental strain when added to the bacteria. Subcloning and introduction of the corresponding gene, here denoted aggHinto the L. reuteri type strain markedly enhanced aggregation. Furthermore, insertional inactivation of aggH in strain 1063 resulted in an autoaggregation-deficient phenotype. Finally, an affinity-purified and cleaved fusion of AggH protein and the maltose-binding protein, MBP, strongly promoted aggregation of L. reuteri 1063, whereas the uncleaved fusion protein was inactive. Sequencing of aggH revealed that the corresponding protein has extensive sequence homology to the large family of ATP-dependent DEAD-box helicases. These results are intriguing in view of earlier data on the promotion of genetic exchange in Lactobacillus by aggregation.