16S rDNA技术研究新生腹泻仔猪粪样细菌区系的多样性变化

用PCR/DGGE技术跟踪一窝5头新生腹泻仔猪自然康复、补饲、断奶过程中粪样细菌区系的演变,构建3头仔猪42日龄粪样的16S rDNA克隆库,分析匹配于DGGE优势谱带23个克隆的16S rDNA序列。结果表明,DGGE图谱由简单(2日龄)到复杂(10日龄),再回复简单(16日龄)到复杂(断奶),最后趋于稳定。2、16日龄DGGE图谱最简单、相似,最优势谱带为大肠杆菌;10日龄(补饲后3天)图谱复杂,大肠杆菌存在但不是最优势谱带,补饲前后图谱的相似性低,补饲导致了粪样细菌区系结构的显著变化;断奶前(27日龄)和后(35、42日龄)图谱复杂,优势谱带、图谱相似性均趋向稳定。序列分析表明,23个克隆中除5个与未知细菌最相似外,其余最相似菌分属于肠球菌(Enterococcus),链球菌(Streptococcus),梭菌(Clostridium),消化链球菌(Peptostreptococcus)和乳酸杆菌(Lactobacillus)。     

鲎素抗菌肽饲喂小鼠对小鼠肠道微生物菌群的影响

目的应用PCR和DGGE技术分析饲喂鲎素对小鼠粪样细菌区系变化的影响。方法将健康21日龄清洁级KM小鼠,随机分为4个处理,分别饲喂生理盐水、合成鲎素、抗生素和益生菌。粪样细菌16SrDNA的V6-V8可变区经PCR扩增,扩增产物经DGGE电泳后再进行相似性分析。切下9条DGGE胶中特异性条带,PCR扩增,TA克隆,测序,鉴定细菌种属。结果对照组DGGE电泳条带较多,在不同时期肠道菌群相似性均较高,相似性在73.5%～76.7%;灌胃合成鲎素和益生菌悬液组,DGGE电泳条带数和对照组比略有减少,不同处理时期肠道菌群相似性在68.1%～69.4%;灌胃抗生素组,DGGE电泳条带数明显减少,不同处理时期肠道菌群相似性在51.4%～63.0%。结论断奶小鼠在灌胃鲎素和抗生素后,肠道微生物区系和对照比发生了改变,最终形成特定的微生物区系;DGGE中特异条带的鉴定表明,灌胃鲎素可以促进粪链球菌和乳酸杆菌的生长,提高数量。     

益生菌Lactobacillus amylovorus S1对仔猪后肠菌群的影响

结合PCR/DGGE(Denaturinggradientgelelectrophoresis,变性梯度凝胶电泳)和16SrDNA序列分析技术,研究添加益生菌LactobacillusamylovorusS1后仔猪从7至35日龄(断奶后两周)后肠菌群的变化。6窝新生仔猪被随机分成两组:对照组和处理组,处理组仔猪于7、9和11日龄口服L.amylovorusS1菌液(活菌数5×109CFU/mL)。分别于7、14、21、24和35日龄,每窝随机屠宰一头仔猪,收集肠道样品。比较不同日龄仔猪后肠菌群DGGE图谱表明,断奶后图谱中多数高GC含量细菌条带消失,至断奶后两周又逐渐出现。序列分析显示,这些高GC含量细菌主要为乳酸杆菌。统计分析表明,仔猪口服益生菌S1对其盲肠和结肠菌群的多样性指数无显著影响。通过比较处理组和对照组图谱发现,处理组14日龄出现一特异条带,与其匹配的序列的最相似已知菌为Clostridiumdisporicum,相似性为95%;而35日龄对照组有一特异优势条带,该条带被鉴定为猪链球菌(Streptococcussuis),相似性为99%。     

应用16S rRNA基因技术研究仔猪结肠梭菌Ⅳ群变化

【目的】研究从7日龄到35日龄(断奶后两周)仔猪结肠中梭菌Ⅳ群(柔嫩梭菌群,Clostridium Leptumgroup)结构以及数量的变化,探究该菌群与结肠中丁酸浓度的关系。【方法】选取3窝新生仔猪,分别在7、14、21(断奶)、24和35日龄每窝随机屠宰1头,收集结肠内容物,利用气相色谱测定挥发性脂肪酸(Volatilefatty acid,VFA);提取结肠内容物总细菌DNA,利用基于16S rRNA基因的变性梯度凝胶电泳(Denaturinggradient gel electrophoresis,DGGE)和Real-time PCR技术定性、定量分析梭菌Ⅳ群。【结果】对仔猪结肠中梭菌Ⅳ群DGGE图谱进行相似性分析显示,7日龄仔猪样品归于一簇,数值达90%以上,而与其他日龄的样品间相似性较低,断奶前(21日龄)和断奶后3天菌群则没有显著变化。Real-time PCR定量分析显示,仔猪结肠总细菌拷贝数在断奶后3天显著降低,这一趋势与结肠中的总挥发性脂肪酸和丁酸浓度变化相似,而梭菌Ⅳ群拷贝数变化则不显著。克隆和测序分析表明,仔猪结肠梭菌Ⅳ群中的优势菌为Faecalibacteriumprausnitzii,Subdoligranulum variabile以及一些未培养细菌。【结论】7日龄至14日龄仔猪结肠中梭菌Ⅳ群发生改变,但断奶对其影响不大;该菌群和结肠丁酸浓度关系还需进一步研究。     

益生菌Lactobacillus amylovorus S1对仔猪后肠菌群的影响

结合PCR/DGGE (Denaturing gradient gel electrophoresis,变性梯度凝胶电泳)和16S rDNA序列分析技术,研究添加益生菌Lactobacillus amylovorus S1后仔猪从7至35日龄（断奶后两周）后肠菌群的变化。6窝新生仔猪被随机分成两组：对照组和处理组,处理组仔猪于7、9和11日龄口服L. amylovorus S1菌液（活菌数5×10.9 CFU/mL）。分别于7、14、21、24和35日龄,每窝随机屠宰一头仔猪,收集肠道样品。比较不同日龄仔猪后肠菌群DGGE图谱表明,断奶后图谱中多数高GC含量细菌条带消失,至断奶后两周又逐渐出现。序列分析显示,这些高GC含量细菌主要为乳酸杆菌。统计分析表明,仔猪口服益生菌S1对其盲肠和结肠菌群的多样性指数无显著影响。通过比较处理组和对照组图谱发现,处理组14日龄出现一特异条带,与其匹配的序列的最相似已知菌为Clostridium disporicum,相似性为95％;而35日龄对照组有一特异优势条带,该条带被鉴定为猪链球菌（Streptococcus suis）,相似性为99％。     

健康与腹泻仔猪粪样中挥发性脂肪酸和菌群区系的比较

[目的]本试验通过对比健康与腹泻仔猪粪样挥发性脂肪酸( Volatile fatty acid,VFA)以及菌群的差异,初步探讨腹泻对仔猪后肠环境的影响.[方法]采集腹泻和健康仔猪粪样,气相色谱测定挥发性脂肪酸浓度;提取粪样总细菌核酸,利用变性梯度凝胶电泳(Denaturing gradient gel electrophoresis,DGGE)和Real-time PCR技术定性、定量分析总细菌和梭菌Ⅳ菌群.[结果]与健康仔猪相比,腹泻仔猪粪样中乙酸含量有升高的趋势,支链脂肪酸(Branched chain fatty acid,BCFA)含量显著降低(p＜0.05),总VFA、丙酸和丁酸含量有降低的趋势,但差异均不显著;乙酸占总挥发酸比例显著升高(p＜0.05),而丙酸及BCFA占总挥发酸比例则显著降低(p＜0.05),丁酸占总挥发酸比例有降低趋势.PCR-DGGE指纹技术分析表明:腹泻发生后,仔猪粪样中总细菌和梭菌Ⅳ菌群在DGGE图谱上并没有特异性的条带消失或出现,但相似性分析显示,腹泻仔猪样品趋于归类于同一簇.Real-time PCR定量分析显示:仔猪腹泻后,粪样中的总细菌和梭菌Ⅳ菌群的数量显著下降(p＜0.05);而大肠杆菌和乳酸杆菌的数量变化不显著.[结论]与健康仔猪相比,腹泻仔猪粪样挥发性脂肪酸组成发生改变,并且微生物区系中的一些菌群也发生变化.     

基于18S rRNA基因的PCR/DGGE技术研究山羊瘤胃原虫动态变化

应用PCR/DGGE技术对山羊采食前后瘤胃原虫的多样性随时间的变化进行了研究.采食前和采食后1、2、4和8h分别采集瘤胃内容物样品,对瘤胃原虫特异性的18SrRNA基因经PCR扩增、DGGE电泳分析.结果显示,采食前后DGGE图谱由10条左右清晰可辨的谱带组成,图谱上的泳带反映了瘤胃内的优势原虫,泳带数量和位置的复杂性说明了瘤胃原虫的多样性.并对其中两个优势条带切胶测序,基因序列分析分属于毛口目和内毛目.瘤胃原虫区系相似性分析,采食前为90.5%～94.1%;采食后相似性发生改变,且存在个体差异.多样性指数由采食前的O.82逐渐上升到采食后8h的O.90(P>0.05).DGGE指纹技术有效地反映了不同样品中原虫优势种的组成,并可通过此技术跟踪研究瘤胃原虫区系在不同条件下的动态变化.     

PCR-DGGE法在慢性胃炎病人舌苔菌群结构研究中的应用

在早间未刷牙和进食的情况下,刮取胃炎病人与正常人舌苔,去除杂质,分离菌体,采用酚/氯仿法抽提细菌基因组DNA,并对其中16S rDNA V3可变区进行聚合酶链式反应(PCR)扩增和变性梯度凝胶电泳(DGGE)测定.用Bionumerics软件对DGGE分子指纹图谱进行舌苔菌群结构相似性分析.实验结果表明,采用该方法成功地扩增出16S rDNA V3区片段,为230 bp.DGGE分子指纹图谱结果表明,正常人的舌苔菌群最高相似性为0.74,胃炎病人的舌苔菌群的最高相似性为0.52,正常人的舌苔菌群与胃炎病人的舌苔菌群相似性最高为0.38,即胃炎病人舌苔菌群结构发生了变化.     

新疆一号冰川土壤细菌多样性的研究

应用变性梯度凝胶电泳（DGGE）技术分离PCR扩增的16SrDNA来研究土壤微生物的多样性。直接从新疆一号冰川不同海拔高度的土壤样品中提取总DNA。用两套细菌通用引物分别扩增16SrDNA的V3和V6／V9高变区的特异性片段,PCR产物进行DGGE分析。PCR—DGGE图谱表明,PCR产物经DGGE检测到的条带清晰且分离效果好。结果表明,PCR—DGGE是一种快速研究微生物群落结构的有效方法。     

杉木连栽土壤微生物多样性的比较研究

分析了位于湖南会同广坪镇1～4代人工杉木林根际土壤微生物数量变化情况,结果表明,随杉木连栽代数的增加,根际土壤中三大类群微生物数量发生显著变化,细菌和放线菌数量明显减少,真菌数量显著增加.利用PCR和DGGE技术分析了1～4代杉木林根际土壤细菌区系和真菌区系.结果表明,细菌生物多样性在不同连栽代数杉木根际土壤中变化不明显,各代杉木土壤之间细菌的遗传相似性为87%.而真菌随连栽代数的增加,DGGE图谱带逐渐减少,真菌生物多样性降低,各代杉木土壤之间真菌的遗传相似性也较低,仅为45%.对各代杉木土壤主要真菌类群的分析表明,随连栽代数的增加,病原真菌及产毒真菌增加显著.     

Analysis of 16S rDNA reveals bacterial shift during in vitro fermentation of fermentable carbohydrate using piglet faeces as inoculum

In vitro fermentation of sugar beet pulp (SBP) was carried out to determine which bacterial species would be enriched by use of this carbohydrate source. Faeces from four weaning piglets as a source of inoculum was also compared. The microbial diversity of the prominent bacteria before and after this in vitro fermentation was analysed using denaturing gradient gel electrophoresis (DGGE) of PCR amplicons of 16S rDNA. Before fermentation, the DGGE profiles showed differences between cultures inoculated with faeces from different piglets, though some bands were common to all piglets. After fermentation of SBP, three dominant bands appeared, of which two bands appeared in all samples and one for both replicates of one piglet. Sequences of the corresponding 16S rDNA of two bands showed 92% similarity to Eubacterium eligens and 96% similarity to Lachnospira pectinoschiza, and that of the third band 95% to L. pectinoschiza.     

16S ribosomal RNA-based methods to monitor changes in the hindgut bacterial community of piglets after oral administration of Lactobacillus sobrius S1

16S ribosomal RNA (rRNA) gene based PCR/denaturing gradient gel electrophoresis (DGGE) and real-time PCR were used to monitor the changes in the composition of microbiota in the hindgut of piglets after oral administration of Lactobacillus sobrius S1. Six litters of neonatal piglets were divided randomly into control group and treatment group. At 7, 9, and 11 days of age, piglets in the treatment group orally received a preparation of L. sobrius S1. At 7, 14, 21(weaning), 24, and 35 days of age, one piglet from each litter was sacrificed and digesta samples of hindgut were collected. DGGE analysis of 16S rRNA gene V6-V8 region for all bacteria showed that several populations present in the hindgut of piglets, represented by far-migrating bands, disappeared after weaning. Most of these bands corresponded to Lactobacillus spp. as revealed by sequence analysis. Quantitative real-time PCR specific for lactobacilli further demonstrated that the number of lactobacilli population tended to decrease after the piglets were weaned. Drastic changes of L. amylovorus and L. sobrius in total Lactobacillus populations were also observed in the colon of piglets around weaning, as monitored by 16S rRNA gene V2-V3 region based Lactobacillus-specific PCR-DGGE. Species-specific real-time PCR also revealed that the population of L. sobrius declined apparently in the colon of piglets after weaning. No remarkable changes in the overall microbial community in the hindgut were found between control and treatment groups. However, comparison of DGGE profiles between the two groups revealed a specific band related to Clostridium disporicum that was found in treatment group on day 14. On day 35, a specific band appeared only in the control group, representing a population most closely related to Streptococcus suis (99%). Real-time PCR showed that L. sobrius 16S rRNA gene copies in treatment group were relatively higher than in the control group (10(8.45) vs. 10(6.83)) on day 35, but no significant difference was observed between the two groups.     

Changes in the diversity of pig ileal lactobacilli around weaning determined by means of 16S rRNA gene amplification and denaturing gradient gel electrophoresis

Our study aimed to provide a comprehensive characterization of changes in porcine intestinal Lactobacillus populations around the time of weaning based on 16S rRNA gene amplification and denaturing gradient gel electrophoresis (DGGE). DNA was extracted from the ileal contents of piglets at weaning (28 days of age) and after 1, 2, 5 and 11 days. PCR amplicons (V2-V3 fragments of 16S rRNA genes) were separated using DGGE. Predominant bands were excised and sequenced after reamplification. A band corresponding to Lactobacillus salivarius was present 1 and 2 days post-weaning (pw), while Lactobacillus crispatus was detected only 1 and 11 days pw. Lactobacillus sobrius gave the most dominant band in all animals. The number of bands decreased from 13+/-3 at weaning to 9+/-1 at 5 days pw, but the species richness had recovered by 11 days pw. The similarity of profiles between sampling days was high for 1 and 2 days pw (>91%), but was low for 5 and 11 days pw (<59%). The diversity of the profiles was lower 5 days pw, based on the Shannon diversity index (0.83+/-0.076 vs. 1.02+/-0.127 at weaning, P=0.042), but had recovered to preweaning values by 11 days pw. The application of group-specific DGGE showed that the Lactobacillus community within the porcine ileum undergoes dramatic, partly reversible changes as a consequence of weaning.     

Specific response of a novel and abundant Lactobacillus amylovorus-like phylotype to dietary prebiotics in the guts of weaning piglets

Using 16S rRNA gene-based approaches, we analyzed the responses of ileal and colonic bacterial communities of weaning piglets to dietary addition of four fermentable carbohydrates (inulin, lactulose, wheat starch, and sugar beet pulp). An enriched diet and a control diet lacking these fermentable carbohydrates were fed to piglets for 4 days (n = 48), and 10 days (n = 48), and the lumen-associated microbiota were compared using denaturing gradient gel electrophoresis (DGGE) analysis of amplified 16S rRNA genes. Bacterial diversities in the ileal and colonic samples were measured by assessing the number of DGGE bands and the Shannon index of diversity. A higher number of DGGE bands in the colon (24.2 +/- 5.5) than in the ileum (9.7 +/- 4.2) was observed in all samples. In addition, significantly higher diversity, as measured by DGGE fingerprint analysis, was detected in the colonic microbial community of weaning piglets fed the fermentable-carbohydrate-enriched diet for 10 days than in the control. Selected samples from the ileal and colonic lumens were also investigated using fluorescent in situ hybridization (FISH) and cloning and sequencing of the 16S rRNA gene. This revealed a prevalence of Lactobacillus reuteri in the ileum and Lactobacillus amylovorus-like populations in the ileum and the colon in the piglets fed with fermentable carbohydrates. Newly developed oligonucleotide probes targeting these phylotypes allowed their rapid detection and quantification in the ileum and colon by FISH. The results indicate that addition of fermentable carbohydrates supports the growth of specific lactobacilli in the ilea and colons of weaning piglets.     

Effects of dietary alpha-ketoglutarate on bacteria profiles in the faeces of lactating sows and their suckling piglets

ABSTRACT

The aim of the study was to investigate the effects of dietary alpha-ketoglutarate (AKG) on the faecal bacteria composition of suckling piglets after supplementation of AKG to the diet of lactating sows. After farrowing, the sows were assigned to either a normal lactation diet (control group, n = 12) or a diet supplemented with 0.25% AKG (AKG group, n = 12) based on body weight (BW) and parity. During the 21-d suckling period, BW and diarrhoea occurrences of piglets were recorded daily, while faeces were sampled weekly from sows and piglets. The levels of pH, ammonia, short-chain fatty acids (SCFA) and lactate in the faeces of piglets were determined. In particular, bacteria profiles in faeces of sows and their suckling piglets were examined by Illumina sequencing. The results showed that the AKG diet altered the faecal bacteria composition in sows during the 21-d lactation period, leading to increases (p < 0.05) in the abundances of genera Prevotella, Lactobacillus, Bacteroides and Methanobrevibacter, but decreases (p < 0.05) in the abundances of genera Oscillospira and Dorea. AKG supplement to the sows during lactation indirectly enhanced (p < 0.05) bacterial richness and SCFA levels (especially, acetate) in the faeces of piglets during the 21-d suckling period. It is suggested that maternal AKG supplementation alters the composition of faecal bacteria in the sows, and increases the faecal bacteria richness and acetate levels in the piglets, which might be associated with an enhanced growth performance of piglets.     

Specific Response of a Novel and Abundant Lactobacillus amylovorus-Like Phylotype to Dietary Prebiotics in the Guts of Weaning Piglets

Using 16S rRNA gene-based approaches, we analyzed the responses of ileal and colonic bacterial communities of weaning piglets to dietary addition of four fermentable carbohydrates (inulin, lactulose, wheat starch, and sugar beet pulp). An enriched diet and a control diet lacking these fermentable carbohydrates were fed to piglets for 4 days (n = 48), and 10 days (n = 48), and the lumen-associated microbiota were compared using denaturing gradient gel electrophoresis (DGGE) analysis of amplified 16S rRNA genes. Bacterial diversities in the ileal and colonic samples were measured by assessing the number of DGGE bands and the Shannon index of diversity. A higher number of DGGE bands in the colon (24.2 ± 5.5) than in the ileum (9.7 ± 4.2) was observed in all samples. In addition, significantly higher diversity, as measured by DGGE fingerprint analysis, was detected in the colonic microbial community of weaning piglets fed the fermentable-carbohydrate-enriched diet for 10 days than in the control. Selected samples from the ileal and colonic lumens were also investigated using fluorescent in situ hybridization (FISH) and cloning and sequencing of the 16S rRNA gene. This revealed a prevalence of Lactobacillus reuteri in the ileum and Lactobacillus amylovorus-like populations in the ileum and the colon in the piglets fed with fermentable carbohydrates. Newly developed oligonucleotide probes targeting these phylotypes allowed their rapid detection and quantification in the ileum and colon by FISH. The results indicate that addition of fermentable carbohydrates supports the growth of specific lactobacilli in the ilea and colons of weaning piglets.     

Effect of the probiotic Enterococcus faecium NCIMB10415 on cell numbers of total Enterococcus spp., E. faecium and E. faecalis in the intestine of piglets

Sows and their piglets were fed a diet supplemented with or without the probiotic E. faecium NCIMB10415 (also known as SF68). Piglets were sacrificed 14, 28, 35 and 56 days after birth and DNA from intestinal segments was extracted and purified. A real time PCR assay was used to distinguish Enterococcus spp. (16s rDNA based), E. faecium (Efaafm gene), E. faecalis (Efaafs gene) as well as the probiotic strain (unique plasmid sequence). Extracts of autoclaved sow feces inoculated with E. faecium and E. faecalis cultures were used to calibrate real time PCR results. The probiotic strain was detected in 14 day old suckling piglets before the piglets had access to the starter diet. In piglets of the probiotic group, probiotic E. faecium cell counts were always a significant proportion of total E. faecium cells in stomach digesta (4-20%), however only a small fraction of the total Enterococcus spp. cell number on day 14 and 28 in all intestinal segments (0.1-0.7%). Compared to control samples, the probiotic E. faecium strain significantly (p < or = 0.05) decreased the amount of total Enterococcus spp. and E. faecalis cells in the colon of 14 day old suckling piglets as well as in jejunum and colon samples one week after weaning. E. faecium cell counts were not modified on any sampling day or intestinal segment. This study showed that the presence of probiotic E. faecium NCIMB10415 coincided with reduced total E. faecalis, but not total E. faecium cell numbers in the intestine of piglets. In view of unchanged cell numbers and ratios in sow feces, modifications must have taken place within the intestine of suckling piglets.