大熊猫肠道正常菌群的研究

对圈养的１３头大熊猫肠道正常菌群进行了定性及定量检测,发现大熊猫肠道正常菌群与其他动物有较显著差异,厌氧菌的检出率及检出量相对较低,其优势菌为肠杆菌科的大肠埃希氏菌,其次为乳杆菌及粪链球菌。同时,本文还着重探讨了上述结果在大熊猫疾病防治及消化生理上的意义。     

妊娠女性阴道中两种德氏乳杆菌亚种的生物学特性

目的 探讨妊娠女性阴道中德氏乳杆菌的生物学特性，以供临床进一步研究。方法 采用质谱鉴定仪初步鉴定菌种，再用16S rRNA基因和一代测序技术进行确认，同时观察德氏乳杆菌在培养基上的生长特征、染色情况、生化反应和抑菌现象。结果 在妊娠女性阴道中首次分离出德氏乳杆菌印度亚种和sunkii亚种。两种细菌16S rRNA基因扩增产物1 438 bp中存在3个碱基的差异。两种细菌均为革兰阳性兼性厌氧长杆菌。德氏乳杆菌印度亚种菌落边缘不整齐、菌落粗糙，而德氏乳杆菌sunkii亚种菌落边缘整齐微凸起且中间凹陷、菌落湿润，都能产生α-溶血环。两种细菌均能产生精氨酸双水解酶1(ADH1)、精氨酸双水解酶2(ADH2s)、丙氨酸芳胺酶（AlaA）、丙氨酸—苯丙氨酸—脯氨酸芳胺酶（APPA），分解蔗糖（SAC）和D-葡萄糖（dGLU），同时奥普托欣耐受试验（OPTO）呈阳性，过氧化氢试验为阴性，能分解葡萄糖产酸。两种菌都能抑制金黄色葡萄球菌和大肠埃希菌的生长。结论 两种德氏乳杆菌亚种核苷酸序列存在单个碱基差异，直接影响其部分生物学特性。     

圈养大熊猫粪便中微生物多样性的研究

本试验采集了5只圈养成年大熊猫的新鲜粪便,用高通量测序技术,研究了大熊猫粪便中细菌和古菌的结构和组成。试验结果表明:圈养成年大熊猫粪便细菌主要由变形菌门Proteobacteria(74.45%)、厚壁菌门Firmicutes(15.66%)、拟杆菌门Bacteroidetes(4.34%)、蓝藻门Cyanophyta/色球藻纲Chroococcophyceae(4.01%)等组成,其中变形菌门主要以埃希氏杆菌属Esherichia/志贺氏菌属Shigella(49.84%)为主;厚壁菌门主要以梭菌属Clostridium(4.65%)为主;拟杆菌门主要以稳杆菌属Empedobacter(3.51%)为主;蓝藻门全部为未分类的色球藻纲(4.01%)。古菌主要由泉古菌门Crenarchaeota(55.99%)和广古菌门Euryarchaeota(42.33%)组成,其中优势古菌是热变形菌纲Thermoprotei(55.99%)中未分类的属和产甲烷菌属Methanogenium(24.70%)。     

宫颈上皮内瘤变与宫颈微生物群落结构的相关性

目的分析宫颈上皮内瘤变与宫颈微生物群落结构的相关性。方法选取2017年1月至2018年10月于我院就诊的253例女性进行回顾性分析,根据是否患有宫颈上皮内瘤变分为CIN组(86例)及对照组(167例)。采集纳入对象的宫颈菌群并提取细菌基因组DNA,对细菌16S rRNA V3、V4区片段进行PCR扩增,并采用Illuminate Miseq测序平台对PCR产物进行测序,分析两组对象宫颈微生物群落结构,并分析患者宫颈微生物群落结构与宫颈上皮内瘤变的相关性。结果两组对象宫颈微生物群落的Simpson指数及Shannon指数比较差异无统计学意义(t=1.474、1.636,P0.05),而CIN组的Chao指数及ACE指数均高于对照组(t=9.213、10.420,P0.05)。16S rRNA分析显示放线菌是宫颈部位的主要菌群。CIN组女性宫颈微生物群落中放线菌、奇异菌属、放线菌门、阴道奇异菌及奇异变形菌占主要优势(LDA4log10),对照组中杆菌、厚壁菌门等占主要优势。Spearman相关分析示卷曲乳杆菌、惰性乳杆菌、格氏乳杆菌、詹氏乳杆菌与宫颈上皮内瘤变呈负相关,而加特纳菌、阴道奇异菌、普雷沃氏菌、粪球菌与宫颈上皮内瘤变呈正相关(均P0.05)。结论 CIN女性宫颈菌群与健康女性存在明显差异,其中卷曲乳杆菌、惰性乳杆菌、格氏乳杆菌、詹氏乳杆菌与宫颈上皮内瘤变呈负相关,而加特纳菌、阴道奇异菌、普雷沃氏菌、粪球菌与宫颈上皮内瘤变呈正相关。     

乳酸杆菌细胞裂解物对金黄色葡萄球菌、大肠埃希菌的抑制作用

目的探讨乳杆菌DM8909裂解物在体内外对金黄色葡萄球菌、大肠埃希菌的抑制作用。方法通过对乳杆菌超声波破碎制成裂解物,分别用乳杆菌裂解物原液、裂解物稀释液、发酵上清液、乳杆菌活菌制剂进行体内、体外实验,观察乳杆菌各成分对金黄色葡萄球菌、大肠埃希菌的抑制作用。结果德氏乳酸杆菌裂解物对金黄色葡萄球菌、大肠埃希菌的抑制作用与乳杆菌活菌制剂的抑制作用相近。结论德氏乳酸杆菌裂解物在体内外对金黄色葡萄球菌、大肠埃希菌均有较强的抑制作用。     

大熊猫中分离出一株肠道杆菌及其初步鉴定

大熊猫出血性肠炎病原,至今未见报道。对此,1988年5月作者作了初步探索。材料与方法一、标本:采自成都市动物园一疑似患出血性肠炎大熊猫的肛拭子。二、培养基:S—S琼脂平板和伊红美蓝琼脂平板。三、分离培养和鉴定:将肛拭子在床旁分别接种在S—S琼脂平板和伊红美蓝琼脂平板培养基。24小时后挑选可疑菌落作革兰氏染色及荚膜染色、生化试验、动物试验和药敏试验。结果在S—S平板和伊红美蓝平板上生长的细菌几乎是纯种。经形态学、生理学等方面的鉴定,确定为肠道杆菌中克雷伯氏菌族中的产气杆菌。该菌对先锋铋(Cefoperazone),先锋霉素等敏…     

阴道中益生特性乳杆菌的分离与功能评价

目的分离健康女性阴道中的乳杆菌并鉴定其益生特性,为开发治疗妇科疾病的复方益生菌制剂提供新型菌株。方法采集健康女性阴道分泌物并分离筛选乳杆菌,通过16SrDNA序列分析鉴定乳杆菌分离株,并对其产酸性能、产H2O2能力、抑菌能力、产生物膜能力进行检测。结果从50名健康女性阴道内共分离出179株乳杆菌,其中卷曲乳杆菌101株、詹氏乳杆菌42株、格氏乳杆菌26株、植物乳杆菌5株、唾液乳杆菌3株以及干酪乳杆菌2株。179株乳杆菌中有146株具有产酸能力,发酵液pH值的最低的5株菌分别为卷曲乳杆菌J3、卷曲乳杆菌J8、詹氏乳杆菌J87,植物乳杆菌J75以及格氏乳杆菌J35,其pH分别为4.20、4.23、4.24、4.26及4.36;产H2O2弱阳性菌株有87株、阳性有37株、强阳性有9株,这9株菌分别为卷曲乳杆菌J3、卷曲乳杆菌J8、卷曲乳杆菌J20、詹氏乳杆菌J87,詹氏乳杆菌J90、詹氏乳杆菌J15、格氏乳杆菌J11、植物乳杆菌J75、植物乳杆菌J69以及植物乳杆菌J40;能拮抗大肠埃希菌的菌株有115株、拮抗金黄色葡萄球菌的有84株、拮抗白假丝酵母的有52株;经统计,对三者同时有拮抗作用且作用最强的只有6株,分别为卷曲乳杆菌J3、卷曲乳杆菌J50、卷曲乳杆菌J62、詹氏乳杆菌J87、詹氏乳杆菌J16和格氏乳杆菌J66;不同乳杆菌产生物膜能力数值范围在1.0~5.4,卷曲乳杆菌、詹氏乳杆菌、干酪乳杆菌的生物被膜形成能力显著高于其他三种菌(P0.05)。在全部179株菌中,卷曲乳杆菌J3和詹氏乳杆菌J87既具有强的产酸能力和产过氧化氢能力,又有较强抑菌活性,同时产生物膜能力也最强。结论卷曲乳杆菌J3和詹氏乳杆菌J87具有优良的生物学特性,有望成为用于治疗妇科疾病微生态制剂的备选菌株。     

大熊猫肠道杆菌的初步调查

近年来成都动物园大熊猫曾出现类似出血性肠炎的疾病,并有死亡。为搞清病原,作者于1988年11月,对9只大熊猫的粪便作了初步检查。结果如下。材料和方法标本共9份,系采自成都动物园和成都大熊猫繁育研究基地大熊猫的粪便。分别划线接种于S—S平板和伊红美蓝琼脂平板培养基,置37℃孵箱24小时后,观察结果并挑选各种类型的菌落,分别涂片作革兰氏染色,生化反应和血清学鉴定。结果从9份标本中共分离出肠道杆菌31株,其中大肠杆菌20株占65%;变形杆菌4株占13%;枸椽酸杆菌1株占3%;肠杆菌2株占6%;克雷伯氏菌2株占6%;革兰氏阴性杆菌(未鉴定出的)2株占6%…     

健康妇女阴道乳酸杆菌定性与定量研究

本文运用微生态学研究方法,对健康妇女阴道乳酸杆菌的分布情况进行调查。结果表明:健康妇女阴道乳酸杆菌的分离率75.44％,平均活菌数均值(log10~n±SD)7.2810±1.1618。常见的阴道乳酸杆菌种的分离率及活菌对数值为:嗜酸乳杆菌(33.3％,7.6883±1.4076),唾液乳杆菌(10.53％,6.4949±1.4254),乳酸乳杆菌(8.8％,8.0687±1.7102),德氏乳杆菌(7.0％,8.1134±0.6311),赖氏乳杆菌(7.0％,7.3349±0.9052)。揭示这几种乳杆菌可能是健康妇女阴道乳酸杆菌属的重要成员,其生理学意义及种类、数量变化的临床意义,值得进一步研究。     

手机肠杆菌的分离及鉴定

为了解手机肠杆菌的种类及分布情况,对采集的11部手机样本进行细菌的分离纯化、革兰氏染色、氧化酶反应、生化实验和16S rRNA鉴定。结果鉴定出5种肠杆菌,分别为:霍米奇肠杆菌(Enterobacter hormaechei)、蜂房哈夫尼亚氏菌(Hafnia alvei)、迟缓爱德华氏菌(Edwardsiella tarda)、志贺氏痢疾杆菌(Shigella dysenteriae)和嗜线虫致病杆菌(Xenorhabdus nematophila)。优势菌为霍米奇肠杆菌和迟缓爱德华氏菌。     

设计乳杆菌特异性引物并运用菌落PCR技术快速检出和鉴定四川泡菜中的乳杆菌

乳杆菌(Lactobacillus)是益生菌, 也是当前的研究热点之一。研究泡菜等样品中的乳杆菌需要快速的检出方法。根据已完成全基因组测序的14种乳杆菌的16S rDNA序列, 设计一对乳杆菌特异性引物。PCR检测结果表明该引物对乳杆菌和明串珠菌能扩增出800 bp的片段, 对表皮葡萄球菌、乳酸乳球菌和枯草芽胞杆菌却没有扩增条带, 具有一定的乳杆菌特异性。结合MRS乳杆菌半选择培养基和革兰氏染色, 运用菌落PCR技术, 可以快速高效地检出四川泡菜中的乳杆菌。再通过对PCR扩增片段测序, 可以将乳杆菌鉴定到种。从16份四川泡菜样品中检出了15株乳杆菌, 其中14株被鉴定为植物乳杆菌, 1株需进一步鉴定才能确定种。该方法可以检出乳杆菌新种。     

Detection of extracellular bound proteinase in EPS-producing lactic acid bacteria cultures on skim milk agar

AIMS: Skim milk agar was developed to investigate extracellular cell-bound proteinase in yogurt cultures, Streptococcus thermophilus and Lactobacillus bulgaricus. METHODS AND RESULTS: The Lact. bulgaricus cultures produced more extracellular cell-bound proteinase than did Strep. thermophilus cultures. Strong positive correlations between the size of the exopolysaccharide (EPS) layer and extracellular cell-bound proteinase were found for both Streptococcus and Lactobacillus cultures. CONCLUSION: Strong positive linear relationships existed between the EPS size and colony size and the diameter of clear zone and colony size for Streptococcus cultures, whereas weak positive linear relationships were observed for Lactobacillus cultures. SIGNIFICANCE AND IMPACT OF THE STUDY: These data are useful to validate the relationship between extracellular proteinase and the EPS size of LAB. Also, a convenient medium to detect the presence of extracellular cell-bound proteinase of LAB is valuable for dairy industries.     

A note on the use of a plasmid as a DNA probe in the detection of a Lactobacillus fermentum strain in porcine stomach contents.

A plasmid (about 50 kb) was used as a DNA probe to enumerate, by colony hybridization, a strain of Lactobacillus fermentum in the stomach contents of eight piglets. The population sizes obtained by colony hybridization were in agreement with estimated levels calculated on the basis of plasmid profiling of colonies isolated at random from the total lactobacillus population.     

A note on the use of a plasmid as a DNA probe in the detection of a Lactobacillus fermentum strain in porcine stomach contents

A plasmid (about 50 kb) was used as a DNA probe to enumerate, by colony hybridization, a strain of Lactobacillus fermentum in the stomach contents of eight piglets. The population sizes obtained by colony hybridization were in agreement with estimated levels calculated on the basis of plasmid profiling of colonies isolated at random from the total lactobacillus population.     

The proliferative capacity of mouse fibrosarcoma cells that survived x-irradiation

Delayed reproductive death, the appearance of colonies with a reduced cell density (impaired colonies) and the number of giant cells per colony were investigated in murine fibrosarcoma cells after irradiation with 3 to 9 Gy of x-rays. Radiation survivors were replated after reaching confluence, which occurred after 13 to 15 doublings; this procedure was repeated three times. The replating efficiency decreased in a dose-dependent manner, the survivors of 9 Gy achieving only 30% of the plating efficiency of unirradiated cells. After the third replating, i.e. after 40 to 45 doublings, the plating efficiency of the survivors approached that of the controls. The median colony size of the survivors showed a similar dose-dependent decrease, which was pronounced after the first replating but still remained significant after the third replating. The fraction of impaired colonies was increased to more than 30% in 9-Gy survivors, and though abating, the increase was still significant even after the third replating. Evidence of residual damage was also provided by the presence of giant cells. For instance, after 6 Gy irradiation and 13 to 15 doublings, the proportion of colonies with giant cells was 60%, decreasing only to 45% after 40 to 45 doublings. The number of giant cells per colony was 1.4 in colonies arising immediately after 6 Gy, decreasing to 0.9 after the third replating. These results suggest that the proliferative capacity of surviving cells is depressed even longer than their clonogenic capacity.     

EZAL-MY96乳酸菌种菌株分离鉴定

目的：对引进法国罗地亚EZAL-MY96直投式酸奶发酵剂(DVS)进行菌株分离鉴定。方法：利用半选择性培养基进行发酵剂菌株分离,并采用糖发酵实验和RAPD指纹图谱进行鉴定。结果：EZAL-MY96中球菌的平板菌落与光镜显微观察、糖发酵实验和RAPD指纹图谱均只有1种情况,而杆菌均有2种情况。结论：从EZAL-MY96菌种中共分离到3株菌,其中1株鉴定为嗜热链球菌,1株为德氏乳杆菌保加利亚亚种,另1株为未知的乳杆菌。     

乳杆菌抑制金黄色葡萄球菌对HeLa细胞的粘附

对弯曲乳杆菌Lactobacillus crispatus T79-3和T90-1、詹氏乳杆菌Lactobacillus jensenii T118-3和T231-1四株乳杆菌对金黄色葡萄球菌生长的抑制效果以及抑菌成分进行了分析,比较乳杆菌排除、竞争、置换3种不同作用方式对金黄色葡萄球菌粘附HeLa细胞的抑制作用。结果表明4株乳杆菌皆能抑制金黄色葡萄球菌的生长及其粘附HeLa细胞的能力,分析发现4株乳杆菌发挥抑制作用的主要成分是有机酸,同时比较分析乳杆菌3种不同作用方式发现它们对金黄色葡萄球菌粘附HeLa细胞的抑制效果不同,其中,排除作用方式效果最好。另外,乳杆菌对金黄色葡萄球菌粘附HeLa细胞的抑制作用具有浓度依赖性,随着乳杆菌浓度增大,抑制作用增强并逐渐达到饱和。4株乳杆菌中,T79-3粘附能力最强,对金黄色葡萄球菌的抑制作用最强,排除作用方式抑制金黄色葡萄球菌粘附HeLa细胞作用效果较好,提示乳杆菌T79-3有可能作为益生菌防治妇女泌尿生殖道感染。     

Comparison of two selective media for the detection and enumeration of Lactobacilli in human faeces

The enumeration of faecal bacteria is an important requirement for many studies of bowel health. One approach is the use of selective culture media for the culture and identification of genera or species from faeces. This study compares the culture of Lactobacilli from dilution series of faecal samples from six healthy human volunteers on two commonly used media, LAMVAB and Rogosa agar. Colonies were counted after a 72-h anaerobic incubation at 37 degrees C, and colony morphology recorded by a single observer. DNA was isolated from a representative number of colonies and genus-specific PCR, single-stranded conformation polymorphism (SSCP) and DNA sequencing performed. Total colony counts ranged from <3.00 to 7.48 log(10) cfu/g of faeces for LAMVAB and 5.09 to 7.66 log(10) cfu/g for Rogosa. For each subject, the total colony count was higher on Rogosa than that obtained with LAMVAB agar. SSCP analysis and DNA sequencing indicated that colony morphology was not an accurate predictor of genus identity. Growth of two species, Lactobacillus acidophilus and Lactobacillus gasseri, was not supported on LAMVAB medium. Rogosa agar was more likely to support growth of non-Lactobacillus species. Therefore, neither medium gave a fully accurate representation of the Lactobacilli species present in human faecal samples.     

Dispersal and group formation dynamics in a rare and endangered temperate forest bat (Nyctalus lasiopterus,Chiroptera: Vespertilionidae)

For elusive mammals like bats, colonization of new areas and colony formation are poorly understood, as is their relationship with the genetic structure of populations. Understanding dispersal and group formation behaviors is critical not only for a better comprehension of mammalian social dynamics, but also for guiding conservation efforts of rare and endangered species. Using nuclear and mitochondrial markers, we studied patterns of genetic diversity and differentiation among and within breeding colonies of giant noctule bats (Nyctalus lasiopterus), their relation to a new colony still in formation, and the impact of this ongoing process on the regionwide genetic makeup. Nuclear differentiation among colonies was relatively low and mostly nonsignificant. Mitochondrial variation followed this pattern, contrasting with findings for other temperate bat species. Our results suggest that this may indicate a recent population expansion. On average, female giant noctules were not more closely related to other colony members than to foreign individuals. This was also true for members of the newly forming colony and those of another, older group sampled shortly after its formation, suggesting that contrary to findings for other temperate bats, giant noctule colonies are not founded by relatives. However, mother–daughter pairs were found in the same populations more often than expected under random dispersal. Given this indication of philopatry, the lack of mitochondrial differentiation among most colonies in the region is probably due to the combination of a recent population expansion and group formation events.     

Rapid confirmation of Listeria monocytogenes isolated from foods by a colony blot assay using a digoxigenin-labeled synthetic oligonucleotide probe.

An oligodeoxyribonucleotide probe based on the sequence of a 321-bp internal fragment of the msp gene encoding a major secreted polypeptide of Listeria monocytogenes was labeled with digoxigenin by using terminal deoxynucleotidyl transferase. The specificity of the digoxigenin-labeled probe was determined by dot blot assays. The probe reacted with all strains of L. monocytogenes tested (12 of 12 strains representing five serotypes). The probe did not react with any other Listeria species or with other gram-positive bacteria (Brochothrix, Erysipelothrix, Corynebacterium, Rhodococcus, Lactobacillus, Leuconostoc, Bacillus, Staphylococcus, and Streptococcus). The probe was used to develop a colony blot assay for the rapid confirmation of L. monocytogenes on Listeria-selective agars which had been streaked with food enrichment cultures. Forty-eight food samples were tested by conventional culture and DNA colony blot assay. The sensitivity and specificity of the DNA colony blot were 100 and 97%, respectively.