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16S rDNA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（P<0.05）。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。     

女性阴道中产细菌素的乳杆菌筛选与鉴定

目的对从60例健康女性阴道中筛选出产生细菌素的优势乳杆菌进行鉴定,并为研制开发微生态制剂提供优良可靠菌种。方法利用牛津杯法筛选出19株乳杆菌,其菌株发酵乳酸量高并且产生细菌素。对19株乳杆菌进行了多项理化鉴定。结果19株乳杆菌分别为：格氏乳杆菌9株,唾液乳杆菌1株,卷曲乳杆菌9株。结论筛选的19株乳杆菌是健康女性阴道中的优势有益菌,具有较强的产酸能力,都产生细菌素,其中16株产生过氧化氢,某些菌株具有较高的生产应用价值。     

应用rep-PCR分型技术筛选潜在治疗性乳杆菌

分离鉴定阴道弯曲乳酸杆菌并对其进行基因分型分析和产H2O2能力测定,初步筛选具有潜在防治女性生殖道感染的弯曲乳酸杆菌菌株。将健康妇女阴道分泌物接种到de Man,Rogosa and Sharpe (MRS) 培养基,分离培养乳酸杆菌。通过16S rRNA序列进行乳酸杆菌分类鉴定,重复序列片段PCR扩增方法进行弯曲乳酸杆菌的基因分型,并进一步采用pH直接测酸法和辣根过氧化物酶催化四甲基联苯胺与过氧化氢反应显色法检测了10株弯曲乳酸杆菌产酸和产H2O2能力。经过序列比对鉴定,共得到65株乳酸杆菌。其中,弯曲乳杆菌Lactobacillus crispatus 19株,詹氏乳杆菌Lactobacillus jensenii 17株,发酵乳杆菌Lactobacillus fermentum 12株;rep-PCR分型发现不同种类的乳酸杆菌和同一种弯曲乳酸杆菌均表现为不同的带型指纹图;10株弯曲乳酸杆菌均产酸,其中T22-3和T29-5两株弯曲乳酸杆菌产H2O2量最高。结果表明个体阴道内乳酸杆菌分布具有差异,弯曲乳酸杆菌具有种内多样性,产H2O2丰富的T22-3和T29-5两株弯曲乳酸杆菌有可能作为防治女性生殖道感染的有益菌株。     

鼠李糖乳杆菌-格氏乳杆菌联用对阴道致病菌的抑制作用

目的本文通过提取细菌基因组进行16S rDNA PCR扩增和测序,分析菌株的进化树分支,鉴定一株乳酸菌菌株RD-0060并检测RD-0060与已有菌株RD-0046联用的抑菌能力和细胞粘附能力。方法结合现有菌株RD-0046(格氏乳杆菌,Lactobacillus gasseri),采用牛津杯法研究RD-0060单菌、RD-0060和RD-0046联用抑制致病菌的能力。通过共培养细菌和阴道上皮细胞VK2/E6E7,研究RD-0060单菌和RD-0060/RD-0046二联菌粘附能力。结果 RD-0060为鼠李糖乳杆菌(Lactobacillus rhamnosus),具有抑制阿托波菌、阴道加德纳菌和常见好氧型病菌的功能,对阴道上皮细胞也有较强的粘附能力;RD-0060和RD-0046二联菌的抑菌效果和细胞粘附能力比单菌株更强。结论鼠李糖乳杆菌和格氏乳杆菌联用能显著抑制阴道致病菌生长,并且能够大量粘附阴道细胞,而且两菌株联用有协同效果,具有良好的临床应用和开发前景。     

乳杆菌阴道分离株16S rDNA鉴定及优势菌株产H_2O_2和自凝集能力分析

目的从阴道分泌物标本分离鉴定乳杆菌,分析与健康相关优势菌种产过氧化氢(H2O2)和自凝集能力,揭示菌株特异性的潜在益生特性。方法利用MRS固体培养基从41例阴道分泌物标本中分离单菌落,纯培养后提取细菌基因组DNA,PCR扩增16S rDNA序列,依据序列一致性确定细菌种属。利用TMB-HRP-MRS培养基测定优势乳杆菌菌株产H2O2能力,并测定其自凝集能力。结果获得155个细菌分离株,分属乳杆菌属104株(67.1%)、肠球菌属21株(13.5%)、链球菌属28株(18.1%)、葡萄球菌属1株(0.6%)和双歧杆菌属1株(0.6%)。各菌属在无或有妇科临床症状患者标本中的构成比例差异有统计学意义(χ2=7.4918,P=0.0236),乳杆菌在前者检出率为83.3%,显著高于其在后者47.1%的检出率(χ2=4.4879,P=0.0341)。乳杆菌中卷曲乳杆菌分离株所占比例最高(67.3%,70/104)。来自不同标本的22株卷曲乳杆菌中77.3%菌株产H2O2,且这些菌株具有强弱不同的自凝集能力(27.5%~97.3%)。结论乳杆菌属特别是卷曲乳杆菌种是健康阴道环境中优势菌属,其产H2O2和自凝集能力存在明显菌株特异性,有2株卷曲乳杆菌此两特性均很强可供进一步研发。     

细菌性阴道病患者阴道分泌物16SrDNA序列分析

目的分析健康妇女及细菌性阴道病(Bacterial vaginosis,BV)患者阴道分泌物16S rDNA序列。方法提取20例健康妇女及40例BV患者阴道分泌物标本中的总DNA,针对细菌16S rDNA保守区设计通用引物进行PCR扩增、克隆、测序,将获得的16S rDNA序列与美国国立生物技术信息中心(NCBI)数据库中的发表序列进行比对,分析克隆群中细菌种类和比例。结果通过阴道分泌物16S rDNA序列分析,发现健康妇女阴道分泌物中以卷曲乳酸杆菌(Lactobacillus crispatus),惰性乳酸杆菌(Lactobacillus iners),加氏乳酸杆菌(Lactobacillus gasseri)为优势菌种,而BV患者阴道菌群种类繁多,以加德纳菌属(Gardnerella)和奇异菌属(Atopobium vaginae)克隆子占较大比例,仅4例患者可见卷曲乳酸杆菌(Lactobacillus crispatus),其他患者均未见有乳酸杆菌克隆子且奇异菌属阴道病患者甲硝唑治疗疗效较差。结论健康妇女和BV患者阴道分泌物菌群种类有较大区别,BV患者在治疗前进行16S rDNA序列分析检测有较大的临床意义。     

应用特异PCR快速鉴定微生物肥料中4种乳酸菌

【目的】植物乳杆菌(Lactobacillus plantarum)、鼠李糖乳杆菌(L.rhamnosus)、嗜酸乳杆菌(L.acidophilus)和德氏乳杆菌(L.delbrueckii)是微生物肥料生产中常用的乳酸菌,它们表型特征相似,若采用传统方法鉴定则费时费力,为准确、快速地鉴定这些种,建立种特异PCR方法。【方法】利用NCBI中Primer-BLAST(引物设计和特异性检验工具),以GenBank数据库中上述菌种的recA和gyrB为靶基因,设计和筛选种特异性引物从而建立相应特异PCR鉴定方法。【结果】经过乳杆菌属(Lactobacillus)、乳球菌属(Lactococcus)、片球菌属(Pediococcus)、芽孢杆菌属(Bacillus)、类芽孢杆菌属(Paenibacillus)、短芽孢杆菌属(Brevibacillus)和假单胞菌属(Pseudomonas)7个属24个种共40株标准菌株的实验验证,4个目标种分别扩增出唯一的目的产物,而其他种均无目的扩增产物。采用建立的4种特异PCR方法对产品中分离的16株乳杆菌进行鉴定,结果与16S rDNA序列分析、Biolog鉴定结果一致。【结论】建立的特异PCR鉴定方法均具有较高的种内通用性和种间特异性,可快速、准确的用于微生物制剂中植物乳杆菌、德氏乳杆菌、鼠李糖乳杆菌、嗜酸乳杆菌的检测和鉴定,具有较好的应用前景。     

女性终末期肾病月经异常患者性激素周期性改变与 子宫内膜乳杆菌相关性分析

目的研究女性终末期肾病月经异常患者性激素周期性改变与子宫内膜乳杆菌的相关性。方法选择2016年5月-2018年6月在本院诊治的126例ESRD女性患者为研究对象,其中月经失调患者66例。将ESRD患者中非月经异常患者设为月经正常组60例,月经周期延长患者为延长组36例,周期缩短患者为缩短组30例。收集患者临床资料,检测患者子宫内膜分泌物微生物组成。结果缩短组患者卵泡期、排卵期、黄体期E2水平明显低于正常组。延长组和缩短组卷曲乳杆菌(Lactobacillus crispatus)、惰性乳杆菌(Lactobacillus iners)、阴道乳杆菌(Lactobacillus vaginalis)检出率明显低于正常组,缩短组加氏乳杆菌(Lactobacillus gasseri)检出率明显低于正常组。卷曲乳杆菌(Lactobacillus crispatus)、惰性乳杆菌(Lactobacillus iners)、詹氏乳杆菌(Lactobacillus jensenii)、阴道乳杆菌(Lactobacillus vaginalis)与月经异常存在相关性。结论大部分ESRD女性患者存在月经失调和性激素水平异常,同时伴随子宫内膜微生物的改变。     

具高抗氧化能力乳酸菌菌株的筛选与鉴定

目的筛选抗氧化能力强的乳酸菌菌株并进行菌种鉴定。方法通过清除DPPH自由基、清除超氧阴离子自由基、还原能力等实验,筛选出高抗氧化能力的乳酸菌菌株。运用16SrDNA序列分析方法,对抗氧化能力强的菌株进行鉴定。结果本研究筛选得到的抗氧化能力较强的菌株有长双歧杆菌NQ1501、La8、La5和保加利亚乳杆菌NQ2508,经16SrDNA鉴定和系统进化分析,La5鉴定为发酵乳杆菌(Lactobacillus fermentum),La8鉴定为鼠李糖乳杆菌(Lactobacillus rhamnosus)。结论本研究筛选出四株具有高抗氧化活性的乳酸菌,分别为长双歧杆菌NQ1501、鼠李糖乳杆菌La8、发酵乳杆菌La5和保加利亚乳杆菌NQ2508。     

ICR小鼠肠道中乳酸菌组成的克隆文库研究

目的对健康雄性ICR小鼠肠道内乳酸菌的组成结构进行研究。方法收集10只健康雄性ICR小鼠的新鲜粪便样品,提取粪便样品中微生物的总DNA,采用乳酸菌类群特异性引物（Lac1）和细菌通用性引物（1391r）的组合扩增16SrRNA基因并构建乳酸菌特异性克隆文库,研究小鼠肠道内各种乳酸菌的组成和比例。结果克隆文库的分析结果表明罗伊乳杆菌（Lactobacillus reuteri）和约氏乳杆菌（Lactobacillus johnsonii）为ICR小鼠肠道内的优势种,其他还包括鼠乳杆菌（Lactobacillus murinus）、阴道乳杆菌（Lactobacillus vaginalis）、肠乳杆菌（Lacto-bacillus intestinalis）等乳酸菌以及一个潜在的乳酸菌新种。结论健康ICR小鼠肠道内乳酸菌的多样性较高;L.re-uteri种可能具有较高的菌株水平多样性。     

Genetic diversity of vaginal lactobacilli from women in different countries based on 16S rRNA gene sequences

AIMS: Lactobacilli are widely distributed in food and the environment, and some colonize the human body as commensal bacteria. The aim of this study was to determine the species of lactobacilli that colonize the vagina and compare them with those found in food and the environment. METHODS AND RESULTS: Thirty-five Lactobacillus strains from women from seven countries were isolated, and sequences from 16S rRNA genes were determined and compared with existing data in GenBank. A phylogenetic tree was achieved using the Neighbour-Joining method based on the analysis of 1465 nucleotides. The results showed that most vaginal isolates were L. crispatus, L. jensenii and L. gasseri. Some were L. vaginalis, L. fermentum, L. mucosae, L. paracasei and L. rhamnosus. Two isolates from a native American woman displayed distinct branches, indicating novel phylotypes. Few vaginal isolates matched food or environmental Lactobacillus species. CONCLUSIONS: Most women worldwide were colonized by three common Lactobacillus species: L. crispatus, L. jensenii and L. gasseri. SIGNIFICANCE AND IMPACT OF THE STUDY: Knowledge of vaginal Lactobacillus species richness and distribution in women worldwide may lead to the design of better probiotic products as bacterial replacement therapy.     

Molecular identification of vaginal lactobacilli isolated from Bulgarian women

Lactobacilli play an important role in maintaining the vaginal health of women. The development of suitable bacterial replacement therapies for the treatment of vaginosis requires knowledge of the vaginal lactobacilli species representation. The aim of this study was to identify at the species level vaginal Lactobacillus isolates obtained from Bulgarian women in childbearing age by using different molecular methods. Twenty-two strains of lactobacilli isolated from vaginal samples were identified and grouped according to their genetic relatedness. A combined approach, which included amplified ribosomal DNA restriction analysis (ARDRA), ribotyping and polymerase chain reaction (PCR) with species-specific oligonucleotide primers was applied. All vaginal isolates were grouped into 5 clusters in␣comparison with a set of 21 reference strains based␣on the initial ARDRA results, which was then confirmed by ribotyping. Finally, the strains were subjected to PCR analysis with eight different species-specific primer pairs, which allowed most of␣them to be classified as belonging to one of␣the␣following species: Lactobacillus crispatus, Lactobacillus fermentum, Lactobacillus gasseri, Lactobacillus helveticus and Lactobacillus plantarum. In conclusion, this study suggests that the most straightforward identification strategy for vaginal lactobacilli would be grouping by ARDRA or ribotyping, followed by PCR specific primers identification at species level.     

Hydrogen peroxide produced by Lactobacillus species as a regulatory molecule for vaginal microflora

A total of 33 strains of Lactobacillus belonging to 9 species, isolated from vagina, were tested for production of hydrogen peroxide. We observed that the following species: L. delbrueckii, L. acidophilus, L. crispatus, L. johnsonii and L. gasseri dominated over other species in secretion of hydrogen peroxide to the growth medium. Concentration of this substance amounted from 0.05 to 1.06 mM (in case of strong aeration the concentration increased up to 1.8 mM). Moreover, killing properties of the pure hydrogen peroxide exerted toward Escherichia coli and Candida albicans were less prominent than these of the supernatants of cultures of Lactobacillus strains producing H2O2.     

β-Galactosidase and 6-phospho-β-galactosidase activities in strains of the Lactobacillus acidophilus complex

β-Galactosidase (β-gal) and 6-phospho-β-galactosidase (P-β-gal) activities were measured in a total of 34 strains from Lactobacillus acidophilus, Lactobacillus crispatus, Lactobacillus amylovorus, Lactobacillus gasseri and Lactobacillus johnsonii. The Lact. gasseri strains have P-β-gal but little or no β-gal activities. The strains from other species have β-gal but only very little P-β-gal activities.     

Rapid identification of 11 human intestinal Lactobacillus species by multiplex PCR assays using group- and species-specific primers derived from the 16S-23S rRNA intergenic spacer region and its flanking 23S rRNA

Rapid and reliable two-step multiplex polymerase chain reaction (PCR) assays were established to identify human intestinal lactobacilli; a multiplex PCR was used for grouping of lactobacilli with a mixture of group-specific primers followed by four multiplex PCR assays with four sorts of species-specific primer mixtures for identification at the species level. Primers used were designed from nucleotide sequences of the 16S-23S rRNA intergenic spacer region and its flanking 23S rRNA gene of members of the genus Lactobacillus which are commonly isolated from human stool specimens: Lactobacillus acidophilus, Lactobacillus crispatus, Lactobacillus delbrueckii (ssp. bulgaricus and ssp. lactis), Lactobacillus fermentum, Lactobacillus gasseri, Lactobacillus jensenii, Lactobacillus paracasei (ssp. paracasei and ssp. tolerans), Lactobacillus plantarum, Lactobacillus reuteri, Lactobacillus rhamnosus and Lactobacillus salivarius (ssp. salicinius and ssp. salivarius). The established two-step multiplex PCR assays were applied to the identification of 84 Lactobacillus strains isolated from human stool specimens and the PCR results were consistent with the results from the DNA-DNA hybridization assay. These results suggest that the multiplex PCR system established in this study is a simple, rapid and reliable method for the identification of common Lactobacillus isolates from human stool samples.     

Use of Group-Specific and RAPD-PCR Analyses for Rapid Differentiation of <Emphasis Type="Italic">Lactobacillus</Emphasis> Strains from Probiotic Yogurts

The increasing interest in probiotic lactobacilli implicates the requirement of techniques that allow a rapid and reliable identification of these organisms. In this study, group-specific PCR and RAPD-PCR analyses were used to identify strains of the Lactobacillus casei and Lactobacillus acidophilus groups most commonly used in probiotic yogurts. Group-specific PCR with primers for the L. casei and L. acidophilus groups, as well as L. gasseri/johnsonii, could differentiate between 20 Lactobacillus strains isolated from probiotic yogurts and assign these into the corresponding groups. For identification of these strains to species or strain level, RAPD profiles of the 20 Lactobacillus strains were compared with 11 reference strains of the L. acidophilus and L. casei group. All except one strain could be attributed unambigously to the species L. acidophilus, L. johnsonii, L. crispatus, L. casei, and L. paracasei. DNA reassociation analysis confirmed the classification resulting from the RAPD-PCR.     

Adherence of clinically isolated lactobacilli to human cervical cells in competition with Neisseria gonorrhoeae

Lactobacilli are normal inhabitants of our microbiota and are known to protect against pathogens. Neisseria gonorrhoeae is a human specific pathogenic bacterium that colonises the urogenital tract where it causes gonorrhoea. In this study we analysed early interactions between lactobacilli and gonococci and investigated how they compete for adherence to human epithelial cervical cells. We show that lactobacilli adhere at various levels and that the number of adherent bacteria does not correlate to the level of protection against gonococcal infection. Protection against gonococcal adhesion varied between Lactobacillus species. Lactobacillus crispatus, Lactobacillus gasseri and Lactobacillus reuteri were capable of reducing gonococcal adherence while Lactobacillus rhamnosus was not. Lactobacillus strains of vaginal origin had the best capacity to remain attached to the host cell during gonococcal adherence. Further, we show that gonococci and lactobacilli interact with each other with resultant lactobacilli incorporation into the gonococcal microcolony. Hence, gonococci bind to colonised lactobacilli and this complex frequently detaches from the epithelial cell surface, resulting in reduced bacterial colonisation. Also, purified gonococcal pili are capable of removing adherent lactobacilli from the cell surface. Taken together, we reveal novel data regarding gonococcal and lactobacilli competition for adherence that will benefit future gonococcal prevention and treatments.     

Inhibition of Staphylococcus aureus by H2O2-producing Lactobacillus gasseri isolated from the vaginal tract of cattle

Infectious diseases are the major reproductive complication during postpartum. The bacteria that colonize the postpartum uterus of cattle are specific as well as opportunistic pathogens that include Staphylococcus aureus. The antibiotics and antiseptic agents used in the treatment of postpartum acute infections induce residues in foods, spread of bacterial resistance, increase in financial costs and failures in defense mechanisms of the host. Preventive treatment with probiotic products could decrease the use of antibiotics in dairy farming systems. Lactobacilli are present in the vaginal microflora of healthy cows. They can prevent pathogen colonization by mechanisms such as the production of antagonistic substances as lactic acid, H2O2, or bacteriocins. The aim of the present study was the selection of H2O2 generating lactobacilli from a group of 72 strains isolated from the vagina of cattle. Both Lactobacillus gasseri CRL1421 and Lactobacillus gasseri CRL1412, which share some probiotic properties, produce H2O2, detected by the plate colorimetric method. They were chosen to study the kinetics of H2O2 production under different culture conditions. Both microorganisms produced greater amounts of H2O2 in aerated than in static cultures. As L. gasseri CRL1421 had a greater capacity to generate H2O2, associative cultures with this strain and S. aureus were conducted. A significant decrease in the growth of the pathogen was detected after culture for 6h, this effect being greater under aerated conditions. The addition of catalase to mixed cultures partially abolished the inhibition, an effect that could be attributed to the combined action of H2O2 and other antagonistic metabolites. The simultaneous addition of catalase and NaOH to these cultures restored S. aureus growth. This observation suggests that the inhibition was produced by the combination of H2O2 and lactic acid, both released by the lactobacilli. Electron microphotographs showed the damage caused by the lactobacilli supernatant on the pathogenic cells. The treatment of S. aureus with lactic acid and hydrogen peroxide evidenced that each metabolite produced a different type of morphological damage. The number of viable cells obtained agrees with the electron microscopy observations. The results support the idea that L. gasseri CRL1421 could be successfully included in a probiotic product to prevent S. aureus infection in cows.