Identification of Lactobacillus and Bifidobacterium 16S rRNA Gene in Breast Milk of Some Healthy Women in Kinshasa (DR Congo)

Breast milk is important for infant health. Some of its benefits are due to the presence of a specific population of bacteria in the microflora. However, the microbiome of breast milk is influenced by many parameters such as maternal diet, breastfeeding and geographic location. Culture and non-culture methods have been used in studies of this bacterial population worldwide. But in the DR Congo, there was no study reporting the use of culture-independent techniques to characterize the bacterial diversity of human milk. The aim of this study was to identify the bacterial 16S rRNA gene from two genera Lactobacillus and Bifidobacterium. The 16S rRNA gene was also identified from four species: Lactobacillus reuteri, Lactobacillus rhamnosus, Bifidobacterium longum and Bifidobacterium lactis. This analytical cross-sectional study was conducted in Kinshasa. Breast milk from some healthy women was collected from February 2 to 28, 2018. A culture-independent protocol using the classical polymerase chain reaction (PCR) was used to identify the bacterial 16S rRNA gene. The 68 samples of breast milk were collected in a sterile condition. The bacteria-specific ribosomal gene 16S rRNA was detected in 91.18% of Lactobacillus and 32.35% of Bifidobacterium at genus level. At of species level, only Lactobacillus reuteri 16S rRNA gene was identified in 89.71%. The 16S rRNA gene from the other species could not be amplified. There was also an association between educational level and the presence of Bifidobacterium and Lactobacillus 16S rRNA genes in the breast milk (p = 0.008*, p Conclusion: This study demonstrates the presence of the bacterial 16S rRNA gene from Lactobacillus and Bifidobacterium in breast milk at the genus and Lactobacillus reuteri at species level. A further study on the diet, use of antibiotics during pregnancy and lactation practice will provide a better understanding of the microflora of breast milk.

16S rRNA Cloning and Identification of a Strain Isolated from Intestinal Tract of Rex Rabbit

This study was conducted to identify bacterial strain HeNan-001 isolated from intestinal tract of healthy rex rabbit. The strain was identified by gram staining, and OD600 values at different culture time were measured to develop a bacterial growth curve. The metabolic pathways of the bacterium using sugar in fermentation was identified with biochemical tubes. Total RNA of the strain was extracted, and total eDNA was obtained by Oligo （dT） method. Primers were designed using Primer 5.0 hip-software according to the 16S rRNA sequence published in GenBank, through cloning, ligation to vector PMD18T, construction of PMD18T/16S rRNA vector and transformation into DHSα, plasmid was extracted and sequenced, and the sequencing result was compared with sequences in NCBI followed by drawing of phylogenetic tree. The strain was verified to be double-stranded gram-positive cocci, which could ferment glucose, mannose, maltose and sorbitol, producing acids production, but failed to ferment sucrose, serum inulin and β-galactosidase. The phylogenetie tree analysis showed that the isolated bacterium shared the highest homology with an India isolate, and belongs to diplostreptococcus. This study provides significance experimental data and theoretical guidance for further development and utilization of the bacterial strain.

牛支原体的分离鉴定及16S rRNA基因序列分析

为明确甘肃某牛场发生的疑似犊牛支原体肺炎的病原,利用支原体培养基从2份患肺炎的犊牛肺脏病料中分离到2个分离株,分别命名为GT-01和GT-02。通过菌落形态观察、生化试验、特异性PCR鉴定和16SrRNA测序比对对分离株进行鉴定。结果2个分离株在固体培养基上生长的菌落呈典型的"油煎蛋状",不能水解葡萄糖、甘露醇、精氨酸,不消化酪蛋白,PCR能扩增出牛支原体特异的1 911bp目的片段,16SrRNA基因序列与牛支原体国际标准株PG45的同源性分别为99.4%和99.6%。结果表明,2个分离株属于牛支原体。

利用16S rRNA全长序列鉴定植物乳杆菌

从标明含有嗜酸乳杆菌的活菌胶囊中分离到一株乳杆菌Lal菌株,利用16SrRNA全序列分析法和传统分类法对Lal菌株进行分类鉴定表明:Lal菌株属于植物乳杆菌种新株系,该菌株现在已经被中国科学院北京微生物所菌种保藏中心收录保存,编号为ASl.2986。

分析16S rRNA基因部分序列鉴定一株乳酸菌

目的利用聚类分析16S rRNA基因部分序列的方法对乳酸菌进行鉴定。方法在表型鉴定的基础上,应用已发表的16S rRNA基因序列设计引物,扩增L.sp.L5菌株16S rRNA基因,并进行测序,将结果与同属及非同属的乳酸菌进行同源性比较。结果L.sp.L5菌株与同属的乳酸乳球菌同源性为85.5%以上,尤其与乳酸乳球菌叶蝉亚种NCDO 2181T同源性为99.5%,与不同属的肉明串珠菌、漫游球菌、嗜盐四联球菌同源性分别为77.5%、82.8%、79.0%。结论菌株L.sp.L5为乳酸乳球菌。

3株猪源非结核分支杆菌16S rRNA基因序列分析

用PCR方法从3株猪源非结核分支杆菌中扩增16 S rRNA基因5′端片段并进行序列测定。用DNA分析软件将所获得的序列与GenBank中分支杆菌序列相比较,计算种间相似性,构建系统进化树,对菌株进行分类与鉴定。结果表明,3株猪源非结核分支杆菌中浅黄分支杆菌1株,偶发分支杆菌2株。非结核分支杆菌在猪中存在的现象应引起重视,对人畜的影响应进一步研究,以便采取有效的防控措施,保护人类及畜群的健康。

16S rRNA序列分析技术对临床标本中疑难细菌的鉴定

目的应用16S rRNA序列分析技术鉴定临床少见或疑难细菌,提高细菌鉴定的准确性。方法收集临床少见或疑难细菌44株,提取DNA,采用通用引物进行PCR扩增及目的片段基因测序,并将测序结果在核酸数据库中比对分析以确定菌种。结果 44株临床少见或疑难细菌均扩增到16S rRNA目的基因片段并成功测序,40株(90.9%)鉴定到"种",4株(9.1%)鉴定到"属";其中常规方法与测序方法在"属"水平一致的有34株(77.3%),不一致的有10株(22.7%)。结论 16S rRNA序列分析技术可以准确、快速地鉴定临床少见或疑难细菌,可以作为临床细菌鉴定的重要方法之一。

Intestinal toxicity of deoxynivalenol is limited by supplementation with Lactobacillus plantarum JM113 and consequentially altered gut microbiota in broiler chickens

Background: Limited research has focused on the effect of Lactobacillus on the intestinal toxicity of deoxynivalenol(DON).The present study was conducted to investigate the role of Lactobacillus plantarum(L.plantarum) JM113 in protecting against the intestinal toxicity caused by DON.Methods: A total of 144 one-day-old healthy Arbor Acres broilers were randomly distributed into 3 treatments,including the CON(basal diet),the DON(extra 10 mg/kg deoxynivalenol),and the DL(extra 1 × 109 CFU/kg L.plantarum JM113 based on DON group) treatments.The growth performance,organ indexes,intestinal morphology,pancreatic digestive enzymes,intestinal secreted immunoglobulin A(sIgA),jejunal transcriptome,and intestinal microbiota were evaluated.Results: Compared with the CON and DL groups,the DON supplementation altered intestinal morphology,especially in duodenum and jejunum,where villi were shorter and crypts were deeper(P < 0.05).Meanwhile,the significantly decreased mRNA expression of jejunal claudin-1 and occludin(P < 0.05),ileal rBAT and jejunal GLUT1 of 21-day-old broilers(P < 0.05),as well as duodenal PepT1 and ileal rBAT of 42-day-old broilers were identified in the DON group.Moreover,supplementation with L.plantarum JM113 could increase duodenal expression of IL-10 and IL-12 of 21-dayold broilers,ileal s IgA of 42-day-old broilers,and the bursa of Fabricius index of 21-day-old broilers.Further jejunal transcriptome proved that the genes related to the intestinal absorption and metabolism were significantly reduced in the DON group but a significant increase when supplemented with extra L.plantarum JM113.Furthermore,the bacteria related to nutrient utilization,including the Proteobacteria,Escherichia,Cc-115(P < 0.05),Lactobacillus and Prevotella(P < 0.1) were all decreased in the DON group.By contrast,supplementation with L.plantarum JM113 increased the relative abundance of beneficial bacterium,including the Bacteroidetes,Roseburia,Anaerofustis,Anaerostipe,and Ruminococcus bromi(P < 0.05).Specifically,the increased abundance of bacteria in the DL group could be proved by the significantly increased caecal content of propionic acid,n-Butyric acid,and total short-chain fatty acid.Conclusions: L.plantarum JM113 enhanced the digestion,absorption,and metabolic functions of the gut when challenged with DON by reducing the injury to intestinal barriers and by increasing the abundance of beneficial bacterium.

Species identification and phylogenetic analysis of genus Nassarius(Nassariidae)based on mitochondrial genes

Genus Nassarius contains many subgenera, such as Zeuxis, Telasco, Niotha, Varicinassa, Plicarcularia, Nassarius s. str. and Reticunassa. On the basis of morphological characteristics of the shell and radula and sequences of mitochondrial cytochrome oxidase subunit I (COI) and 16S rRNA genes, Nassarius specimens collected from the South China Sea were identified and phylogenetically analyzed. Although Nassarius sp. and Nassarius (Varicinassa) variciferus were morphologically different in their shells, few variations were found among their radular teeth and sequences of mtCOI and mt16S RNA genes. Therefore, Nassarius sp. should be classified as N. (Varicinassa) variciferus. Nassarius (Zeuxis) sp. has only a subtle difference from Nassarius (Zeuxis) algidus on the shell, but it shows obvious differences in radular teeth and DNA sequence, indicating that they are two distinct species. Sequence divergence of mtCOI and mt16S RNA genes within Nassarius species was much lower than that between species, suggesting that these two genes are suitable for Nassarius species identification. Phylogenetic analysis (neighbor-joining and maximum parsimony) based on mtCOI and mt16S rRNA genes revealed the presence of two groups in genus Nassarius and a closest relationship between subgenera Zeuxis and Telasco. Species of subgenus Plicarcularia did not form a single clade. The molecular phylogeny was not congruent with the previous morphological phylogeny. The subgeneric divisions of genus Nassarius appear to be uncertain and unreliable.

Isolation,Identification and Antibiotics Susceptibility Test of Citrobacter freundii from Procambarus clarkia

This experiment was conducted to clarify species and drug resistance of pathogen from the diseased Procambarus clarkia. Pathogenic bacteria from hepatopancreas of the diseased P. clarkia were examined using conventional methods,and then were isolated. The further tests and analysis of the isolated strain were developed,including the regression experiment to P. clarkia,the morphology,physiological and biochemical characteristics,sequence analysis of their 16 S rRNA and gyr B genes,and the susceptibility test to antibiotics. Large colonies with similar morphology and color were obtained. Strain X120523 was identified as Citrobacter freundii,proved to have strong pathogenicity,and was susceptible to quinolones and aminoglycosides.

Molecular Identification of D. nuttalli and D.marginatus

This study was to establish a molecular identification method to distinguish Dermacentor nuttalli(D. nuttalli) and Dermacentor marginatus(D. marginatus),and to study their phylogenetic relationship. The ticks were collected from domestic animals in Xinjiang Uygur Autonomous Region for morphological identification,then their genomic DNAs were isolated for amplifying mitochondrial 16 S rRNA and cytochrome oxidase subunit I gene(COI). The phylogenetic tree was constructed based on the sequencing results of PCR products by employing Mega 5. 0 and Mrbayes 3. 2 for homology analysis. Clustering analysis PCR product for 16 S rRNA from both D. marginatus and D. nuttalli was clustered together with their respective 16 S rRNA sequences previously accessed in GenBank,and that for COI gene as well. These are basically identical with morphological identification results. Our results indicate that morphological identification,combined with molecular markers,would be a simple and accurate method for distinguishing D. nuttalli and D. marginatus.

Identification of Avian-Derived Ingredients in Livestock and Poultry Meat by PCR Technology

In order to establish a quick and specific method which could identify the avian-derived ingredients,this study used 16 S rRNA gene sequence as target site,and designed the specific primers of chicken,pigeon meat and quail meat. The DNA of common livestock and poultry meat( including mutton,beef,pork,rabbit meat,pigeon meat,quail meat,chicken,duck and goose) was used as template. Though PCR amplification and specific detection,a quick determination method was established to identify the avian-derived ingredients. The results showed that the selected primers could identify the ingredients of animal origin effectively and quickly. The method was convenient and concise,and could detect the chicken-derived,pigeon-derived,quail-derived ingredients in livestock and poultry food quickly and accurately.

Isolation,Identification and Susceptibility Analysis of Bacillus pyocyaners from a Canine Pyometra

In order to clarify the causes of the fester disease in companion animals,this study involved isolation,identification and drug susceptibility analysis of pathogenic bacteria from the uterus of an 11-year-old female dog who was admitted to the veterinary hospital of Tianjin Agricultural University.According to the results of colony morphology,smear staining microscopy,physiological and biochemical reaction and 16 S rRNA gene analysis,the bacteria was identified as a Bacillus pyocyaners.The susceptibility analysis showed that the isolate is highly sensitive to aztreonam,cefdinir,cefotaxime,cefepime,ceftazidime,ofloxacin,streptomycin,kanamycin,piperacillin and tobramycin.Moreover,the isolate is moderately sensitive to ceftriaxone,cefuroxime and cefoxitin,but resistant to ampicillin,cefazolin,cefazine and medemycin.The research results provide references for controlling infection caused by B.pyocyaners.

Development and evaluation of a novel multiplex probe array for rapid differential identification of Mycobacterium in clinical specimens

Rapid differential identification of Mycobacterium species is essential for effective diagnosis and management of mycobacteriosis. The aim of this study was to develop a novel multiplex probe array based on the 16S-23S rRNA gene internal transcribed spacer sequence for the genotyping of mycobacteria to the species level. A pair of primers and a set of genus- and species-specific probes were designed from the conserved and polymorphic regions of the 16S rRNA gene, internal transcribed spacer, and 23S rRNA gene sequences of mycobacteria. We used a novel multiplex probe array for identification of 266 clinical specimens obtained from patients with mycobaterial infection. The results showed that the overall specificity and sensitivity of our novel probe array were both 100% for the genus-specific probe and Mycobacterium tuberculosis complex- specific probe. There were 79.3 % （23/29） of nontuberculous mycobacteria which could be identified to the species level directly in the specimens from China. Some intraspecies heterogeneity in M. avium, M. intracellulare, M. chelonae and M. abscessus was observed. With the increase of sequences of internal transcribed spacer and numbers of whole microbial genomes, and further optimization of probes, the multiplex probe army will become a promising tool for the rapid and accurate identification of mycobacteria in ordinary clinical laboratories.

Identification and Characterization of Lactic Acid Bacteria Isolated from Fermented Rice Bran Product

To analyze the microflora in fermented rice bran product, bacterial colonies were grown under various conditions. Although cultivation temperature did not affect the number of bacterial colonies formed on agar plates, twice as many colonies formed under aerobic as under anaerobic conditions. All colonies appearing on the plates showed acid production. Based on 16S rRNA sequence analysis, nearly all of the bacteria in the fermented product were highly similar (>99%) to Lactobacillus johnsonii. In addition, several Bacillus cereus and unidentified Lactobacillus strains that grew only under anaerobic conditions at 30℃?were seen. Random Amplified Polymorphic DNA (RAPD)-PCR analysis showed the amplified patterns of all isolates to differ substantially from the reference strain L. johnsonii. We conclude that L. johnsonii-related strains predominate in fermented rice bran product, and that these bacteria produce lactic acid to decrease the pH of the fermented product. Several novel Lactobacillus strains may also occur in this environment.

奶牛子宫内膜炎化脓隐秘杆菌16SrRNA基因的鉴定与分析

对内蒙古呼和浩特地区奶牛子宫内膜炎化脓隐秘杆菌进行了分离鉴定。经生化实验鉴定的化脓隐秘杆菌利用PCR技术扩增其16S rRNA基因,得到1.5 kb目的片段;将目的片段与T载体连接,测定目的片段的基因序列,与GenBank公布的化脓隐秘杆菌16S rRNA基因序列进行比较分析,其同源率为93%～100%。本试验共分离到化脓隐秘杆菌32株。

昆明犬肠道中乳杆菌的分离鉴定及16S rRNA同源性分析

目的为了进一步开发动物微生态资源。方法用生理生化表型及16S rRNA同源性分析,鉴定从昆明犬肠道内容物中分离到的7株乳杆菌,分别命名为LD1、LD2、LD3、LD4、LD5、LD6和LD7。结果 LD1、LD2、LD4为路氏乳杆菌(Lactobacillus reuteri),LD3、LD5、LD6、LD7为鼠乳杆菌(Lactobacillus murinus)。结论该研究为犬用微生态制剂及益生菌的开发应用奠定基础。

16SrRNA基因与16S-23SrRNA转录单元内间隔区序列分析及其在节旋藻和螺旋藻分类鉴定中的应用

测定了节旋藻属 3个品系和螺旋藻属 1个品系的全长 1 6SrRNA基因和 1 6S 2 3SrRNA转录单元内间隔区序列 (ITS) ,分析了已知的节旋藻、螺旋藻和相关品系的相应序列的同源性 ,构建了系统发生树 ,并评价了这两段DNA序列在节旋藻、螺旋藻种属分类和种质鉴定中的意义。结果表明 :( 1 ) 1 6SrRNA基因序列和ITS序列均可用于节旋藻属和螺旋藻属的属间分类 ,以两序列为基础的系统学分析结果一致 ;( 2 )ITS序列变异程度高于 1 6SrDNA序列 ,适用于节旋藻和螺旋藻属内品系或种质鉴定 ;( 3)节旋藻属可明确界定 ,1 6SrRNA基因序列相似性大于 98% ,ITS序列相似性大于 88% ;( 4 )螺旋藻属某些品系间 1 6SrDNA序列和ITS序列相似性较低 ,与不同属间的序列相似性程度为同一水平。

乳扇制品中乳酸杆菌的分离鉴定及16S rRNA序列同源性分析

目的乳扇是云南大理白族的一种传统乳制品,明确大理乳扇制品中乳杆菌的多样性及优势种群分布,为科学利用奠定基础。方法采用表型鉴定及16S r RNA鉴定方法,对10个家庭作坊的大理乳扇制品中的乳杆菌进行了分离鉴定。结果共分离到50株乳杆菌,通过表型鉴定为8个种,包括植物乳杆菌10株、德氏乳杆菌7株、发酵乳杆菌6株、干酪乳杆菌6株、棒状乳杆菌4株、鼠乳杆菌2株、弯曲乳杆菌3株和食果糖乳杆菌2株;06422和06430两株表型鉴定未能定种,进一步通过16S r RNA鉴定为植物乳杆菌和马酒乳杆菌,06422株与植物乳杆菌L.arizonensin、L.pentosus和L.plantarum P158的同源性分别是100%、100%和99.9%,与乳杆菌属其它种的同源性为83.4%(L.gallinarum)至93.5%(L.brevis),06430株与L.kefiranofaciens.subsp.Kefirgranum的16S r RNA同源性是99.9%,与乳杆菌属其它种的同源性为83.7%(L.plantarum P158)至96.3%(L.acidophilus)。结论大理乳扇制品中有9种乳杆菌,其优势种群为植物乳杆菌、德氏乳杆菌、发酵乳杆菌和干酪乳杆菌等四种。

家养野猪副猪嗜血杆菌的分离鉴定及16S rRNA序列分析

对江西省某家养野猪场临诊疑似副猪嗜血杆菌(Haemophilus parasuis,Hps)感染的病例进行细菌分离鉴定,PCR扩增分离菌株的16SrRNA并进行测序分析,并对分离菌进行细菌形态、生化鉴定和PCR鉴定及序列比对分析。结果显示,获得1株家养野猪源Hps分离株(命名为HPJXYZO1),该分离株与国内外参考菌株序列之间的同源性为93.1%～99.2%,与本实验室江西省家猪源分离株的同源性为84%～92.1%。结果表明,江西省家养野猪中存在Hps感染,分离株与国内外家猪源Hps间的16SrRNA序列差异不大,Hps 16SrRNA核苷酸序列比较稳定,其进化不存在明显的地域相关性。