Application of a single‐colony coculture technique to the isolation of hitherto unculturable gut bacteria

Molecular studies have led to postulation of a relationship between gut microbiota and certain diseases. However, because studies of hitherto uncultured species in vivo are essential for characterizing the biology and pathogenic properties of gut bacteria, techniques for culturing and isolating such bacteria must be developed. Here, a technique is described that partially overcomes the obstacles that prevent detection of interbacterial communication in vitro and are thus responsible for the failure to culture certain bacterial species. For this purpose, a ring with a membrane filter at the bottom was designed and a relatively simple nutrient medium was used instead of conventional media. Gut bacteria were cocultivated in soft agar separated by the membrane filter to simulate interbacterial communication in vitro. Use of this soft agar coculture technique led to the successful isolation of hitherto uncultured bacteria and the demonstration of multistage interbacterial communication among gut bacteria in vitro. Cultivation and isolation of single colonies of bacteria that require other bacteria for growth will enhance efforts to better understand the physiological and pathogenic roles of gut microbiota.     

Effects of Lactobacillus acidophilus 43121 and a mixture of Lactobacillus casei and Bifidobacterium longum on the serum cholesterol level and fecal sterol excretion in hypercholesterolemia-induced pigs

The hypocholesterolemic effects of Lactobacillus acidophilus 43121 (43121) and a mixture of Lactobacillus casei and Bifidobacterium longum (MIX) were studied in hypercholesterolemia-induced pigs. Serum total cholesterol was decreased by supplementation of either 43121 or MIX, although, high-density lipoprotein cholesterol was not changed. The hypocholesterolemic effect of 43121 and MIX was mainly due to bile acid dehydroxylation, this effect being supplementation-time dependent.     

In vitro fermentation of rice bran combined with Lactobacillus acidophilus 14 150B or Bifidobacterium longum 05 by the canine faecal microbiota

The fermentability of rice bran (RB), alone or in combination with one of two probiotics, by canine faecal microbiota was evaluated in stirred, pH-controlled, anaerobic batch cultures. RB enhanced the levels of bacteria detected by probes Bif164 (bifidobacteria) and Lab158 (lactic acid bacteria); however, addition of the probiotics did not have a significant effect on the predominant microbial counts compared with RB alone. RB sustained levels of Bifidobacterium longum 05 throughout the fermentation; in contrast, Lactobacillus acidophilus 14 150B levels decreased significantly after 5-h fermentation. RB fermentation induced changes in the short-chain fatty acid (SCFA) profile. However, RB combined with probiotics did not alter the SCFA levels compared with RB alone. Denaturing gradient gel electrophoresis analysis of samples obtained at 24 h showed a treatment effect with RB, which was not observed in the RB plus probiotic systems. Overall, the negative controls displayed lower species richness than the treatment systems and their banding profiles were distinct. This study illustrates the ability of a common ingredient found in pet food to modulate the canine faecal microbiota and highlights that RB may be an economical alternative to prebiotics for use in dog food.     

A comparative study of the preventative effects exerted by three probiotics, Bifidobacterium lactis, Lactobacillus casei and Lactobacillus acidophilus, in the TNBS model of rat colitis

AIMS: The intestinal anti-inflammatory effects of three probiotics with immunomodulatory properties, Lactobacillus casei, Lactobacillus acidophilus and Bifidobacterium lactis, were evaluated and compared in the trinitrobenzenesulphonic acid (TNBS) model of rat colitis. METHODS AND RESULTS: Colitis was induced in rats by intracolonic administration of 10 mg of TNBS dissolved in 0.25 ml of 50% ethanol. Each probiotic was administered orally (5x10(8) CFU suspended in 0.5 ml of skimmed milk) for 3 weeks, starting 2 weeks before the administration of TNBS. Colonic damage was evaluated histologically and biochemically 1 week after TNBS instillation. The results obtained revealed that all probiotics assayed showed intestinal anti-inflammatory effects, macroscopically evidenced by a significant reduction in the colonic weight/length ratio. Only B. lactis showed a lower incidence of diarrhoea in comparison with untreated rats. Biochemically, all probiotics restored colonic glutathione levels, depleted as a consequence of the oxidative stress of the inflammatory process. Bifidobacterium lactis treatment reduced colonic tumour necrosis factor (TNF)-alpha production, and inducible nitric oxide synthase (iNOS) and cyclo-oxygenase-2 (COX-2) expression; L. acidophilus administration reduced colonic leukotriene B4 production and iNOS expression and L. casei intake was associated with a decrease in colonic COX-2 expression. CONCLUSION: The three probiotics assayed have shown intestinal anti-inflammatory activity in the TNBS model of rat colitis, although each probiotic shows its own anti-inflammatory profile. SIGNIFICANCE AND IMPACT OF THE STUDY: These probiotics could be considered as potential adjuvants in the treatment of inflammatory bowel disease, although more studies are required in order to demonstrate their efficacy in humans.     

Role of autoinducer-2 on the adhesion ability of Lactobacillus acidophilus

Aims:  Lactobacilli adhere to the intestinal epithelium and this intimate association likely promotes retention in the gastrointestinal tract and communication with the immune system. The aim of this study was to investigate whether or not the quorum-sensing signalling molecule, autoinducer (AI)-2, was produced by Lactobacillus acidophilus and affected adherence to intestinal epithelial cells.
Methods:  Microarray analysis of concentrated cells of L. acidophilus NCFM revealed several genes involved in a classic stress response and potentially adhesion. Putative genes linked to the synthesis of the interspecies signalling molecule, AI-2, were overexpressed. Examination of the NCFM genome revealed the complete pathway for AI-2 synthesis. AI-2 activity from NCFM was detected using the Vibrio harveyi BB170 assay system. Using site-specific integration, an isogenic mutation was created in luxS and the resulting mutant did not produce AI-2. In addition to some minor metabolic effects, the luxS mutation resulted in 58% decrease in adherence to Caco-2 cells.
Conclusion:  L. acidophilus NCFM encodes the genes for synthesis of the quorum-sensing signal, AI-2, and produces this molecule during planktonic growth.
Significance and Impact of the Study:  The ability to produce AI-2 affects the ability of L. acidophilus to attach to intestinal epithelial cells.     

Impact of selected Lactobacillus and Bifidobacterium species on Listeria monocytogenes infection and the mucosal immune response

The pathogenesis of Listeria monocytogenes depends on its ability to attach to and invade the gastrointestinal epithelium and subsequently withstand the host immune response. Despite a thorough understanding of the intracellular phase of infection, relatively little is known about how the pathogen behaves in the gastrointestinal tract and whether it is affected by the presence of host commensal microbiota. Lactobacillus and Bifidobacterium are two important genera of the human gut microbiota proposed to possess probiotic effects. Here we demonstrate that probiotic bacteria significantly inhibit subsequent listerial infection in an in vitro C2Bbe1 epithelial cell model. In the case of Lactobacilli, inhibition was due to a combination of acid production and secretion of an as yet unidentified protein. In the case of Bifidobacterium, inhibition was attributable to an extracellular proteinaceous secreted compound. In addition, we observed a significant reduction in interleukin-8 and an increase in IL-10 cytokines secreted from epithelial cells following probiotic pretreatment and subsequent infection with Listeria. A reduction in the infection of epithelial cells and an altered mucosal immune response suggests that probiotic bacteria could be of therapeutic benefit against listerial infection. This study infers a role for probiotic bacteria as an antagonist of Li. monocytogenes infection.     

Membrane fatty acid composition and fluidity are involved in the resistance to freezing of Lactobacillus buchneri R1102 and Bifidobacterium longum R0175

Determinations of membrane fatty acid composition and fluidity were used together with acidification activity and viability measurements to characterize the physiological state after freezing of Lactobacillus buchneri R1102 and Bifidobacterium longum R0175 cells harvested in the exponential and stationary growth phases. For both strains, lower membrane fluidity was achieved in cells harvested in the stationary growth phase. This change was linked to a lower unsaturated-to-saturated fatty acid ratio for both strains and a higher cyclic-to-saturated fatty acid ratio for L. buchneri R1102 alone. These membrane properties were linked to survival and to maintenance of acidification activity of the cells after freezing, which differed according to the strain and the growth phase. Survival of B. longum R0175 was increased by 10% in cells with low membrane fluidity and high relative saturated fatty acid contents, without any change in acidification activity. Acidification activity was more degraded (70 min) in L. buchneri R1102 cells displaying low membrane fluidity and high saturated and cyclic fatty acid levels. Finally, this study showed that membrane modifications induced by the growth phase differed among bacterial strains in terms of composition. By lowering membrane fluidity, these modifications could be beneficial for survival of B. longum R0175 during the freezing process but detrimental for maintenance of acidification activity of L. buchneri R1102.     

Rapid detection of human fecal Eubacterium species and related genera by nested PCR method

PCR procedures based on 16S rDNA gene sequence specific for seven Eubacterium spp. and Eggerthella lenta that predominate in the human intestinal tract were developed, and used for direct detection of these species in seven human feces samples. Three species of Eggerthella lenta, Eubacterium rectale, and Eubacterium eligens were detected from seven fecal samples. Eubacterium biforme was detected from six samples. It was reported that E. rectale, E. eligens, and E. biforme were difficult to detect by traditional culture method, but the nested PCR method is available for the detection of these species. This result shows that the nested PCR method utilizing a universal primer pair, followed by amplification with species-specific primers, would allow rapid detection of Eubacterium species in human feces.     

Predominant genera of fecal microbiota in children with atopic dermatitis are not altered by intake of probiotic bacteria Lactobacillus acidophilus NCFM and Bifidobacterium animalis subsp. lactis Bi-07

The effect of probiotic bacteria Lactobacillus acidophilus NCFM and Bifidobacterium lactis Bi-07 on the composition of the Lactobacillus group, Bifidobacterium and the total bacterial population in feces from young children with atopic dermatitis was investigated. The study included 50 children randomized to intake of one of the probiotic strain or placebo. Microbial composition was characterized by denaturing gradient gel electrophoresis, quantitative PCR and, in a subset of subjects, by pyrosequencing of the 16S rRNA gene. The core population of the Lactobacillus group was identified as Lactobacillus gasseri, Lactobacillus fermentum, Lactobacillus oris, Leuconostoc mesenteroides, while the bifidobacterial community included Bifidobacterium adolescentis, Bifidobacterium bifidum, Bifidobacterium longum and Bifidobacterium catenulatum. The fecal numbers of L. acidophilus and B. lactis increased significantly after intervention, indicating survival of the ingested bacteria. The levels of Bifidobacterium correlated positively (P=0.03), while the levels of the Lactobacillus group negatively (P=0.01) with improvement of atopic eczema evaluated by the Severity Scoring of Atopic Dermatitis index. This correlation was observed across the whole study cohort and not attributed to the probiotic intake. The main conclusion of the study is that administration of L. acidophilus NCFM and B. lactis Bi-07 does not affect the composition and diversity of the main bacterial populations in feces.     

Orally supplemented Lactobacillus acidophilus strain L‐92 inhibits passive and active cutaneous anaphylaxis as well as 2,4‐dinitroflurobenzene and mite fecal antigen induced atopic dermatitis‐like skin lesions in mice

Oral supplementation of lactic acid bacteria is a potential approach to the prevention and manipulation of allergic diseases such as atopic dermatitis. Our previous report showed that heat‐killed Lactobacillus acidophilus strain L‐92 (L‐92) possessed anti‐allergic properties, although its physiological function in atopic dermatitis has largely remained undefined. To evaluate the anti‐allergic efficacy of L‐92, we used four experimental animal models with the major features of atopic dermatitis and compared the results to those of clinically active drugs. ICR mice were passively sensitized by anti‐dinitrophenyl mouse monoclonal IgE for passive cutaneous anaphylaxis (PCA), and BALB/c mice were actively sensitized by ovalbumin for active cutaneous anaphylaxis (ACA). Allergic reaction was induced by repeated exposure to 2,4‐dinitroflurobenzene (DNFB) and mite (Dermatophagoides farinae) fecal allergen, in BALB/c and NC/Nga mice, respectively. Orally administrated L‐92 significantly inhibited the vascular permeability increase in both PCA and ACA, and the elevation of ovalbumin‐specific IgE titer in ACA. Moreover, repeated applications of DNFB and mite fecal antigen onto the BALB/c and NC/Nga mouse ear, respectively, caused clinical symptoms similar to atopic dermatitis such as ear swelling, scratching behavior and elevation of total serum IgE levels that were also moderately suppressed by L‐92. In addition, L‐92 treated mice exhibited lower levels of mast cells, eosinophil infiltration and Th1/Th2 cytokine expression. Our results, therefore, suggest that oral administration of L‐92 might be useful for alleviating allergic symptoms.     

粪便微生物宏基因组来源L-天冬酰胺酶的性质表征及应用研究

l-天冬酰胺酶是氨基酸代谢的关键酶,广泛应用于食品和医药领域,肠道菌群及其产生的l-天冬酰胺酶与宿主健康和疾病关系密切。【目的】获取肠道微生物来源的新型l-天冬酰胺酶,并对其进行性质表征和应用研究。【方法】以西黑冠长臂猿粪便微生物宏基因组为模板,克隆l-天冬酰胺酶基因,并在大肠杆菌中表达;对表达出的酶进行酶学性质研究,并用于处理薯条和癌细胞。【结果】克隆获得l-天冬酰胺酶基因NCasn5,全长996 bp,重组酶NCasn5分子量大小为37.296 kDa,最适pH为8.0,最适温度为60 ℃,K_m和V_max值分别为(3.33±0.21) mmol/L和(836.30±13.91)µmol/(min·mg),37 ℃体外血清半衰期约69 h。NCasn5能降低薯条中69.35%的丙烯酰胺含量,抑制人肝癌细胞QGY-7703和人恶性黑色素瘤细胞A-375细胞的生长。【结论】本研究获得的新型l-天冬酰胺酶,具有良好的热稳定性和较长的血清半衰期,不仅无谷氨酰胺酶活性,还能减少油炸薯条中丙烯酰胺的含量,也能诱导癌细胞QGY-7703和A-375凋亡,在食品加工及医药领域具有潜在的应用价值。     

Effects of green tea consumption on human fecal microbiota with special reference to Bifidobacterium species

Green tea is one of the most popular beverages in the world. Its beneficial health effects and components have been extensively reviewed. However, little is known about the influence of green tea consumption on the human intestinal microbiota (HIM), which plays a crucial role in human health. Ten volunteers who did not usually consume green tea, drank it for 10 days and then stopped drinking it for 7 days. Their fecal samples were collected at three time points: before beginning the 10‐day green‐tea regime, at the conclusion of that 10 days, and 7 days after stopping the regime. Their fecal samples were analyzed by terminal restriction fragment length polymorphism with specific primer‐restriction enzyme systems for HIM and by using a real‐time PCR method for the Bifidobacterium species. Although the HIM of each subject was relatively stable, the proportion of Bifidobacterium species played an important role in the classification of their fecal microbiota. Although there were inter‐individual differences in the Bifidobacterium species, an overall tendency for the proportion of bifidobacteria to increase because of green tea consumption was noted. However, little change was observed in the composition of Bifidobacterium species in each sample. This suggests that the change in proportion was induced, not by an inter‐species transition, but by an intra‐species increase and/or decrease. In conclusion, green tea consumption might act as a prebiotic and improve the colon environment by increasing the proportion of the Bifidobacterium species.     

Starvation induces physiological changes that act on the cryotolerance of Lactobacillus acidophilus RD758

The relationship between lactose starvation and cryotolerance was investigated in Lactobacillus acidophilus RD758. Cryotolerance was measured from the acidification activity of cells recovered after 18-h lactose starvation. It was compared to that of nonstarved cells, both of them in a stationary phase and in the same medium. This measurement allowed quantifying the initial acidification activity before freezing, as well as the loss of acidification activity during freezing and the rate of loss during frozen storage. Even if initial acidification activity was similar for nonstarved and starved bacteria, the latter displayed a significantly better resistance to freezing and frozen storage at -20°C. To investigate the mechanisms that triggered these cryotolerance phenomena, the membrane fatty acid composition was determined by gas chromatography, and the proteome was established by 2-D electrophoresis, for starved and nonstarved cells. The main outcome was that the improved cryotolerance of starved cells was ascribed to two types of physiological responses as a result of starvation. The first one corresponded to an increased synthesis of unsaturated, cyclic, and branched fatty acids, to the detriment of saturated fatty acids, thus corresponding to enhanced membrane fluidity. The second response concerned the upregulation of proteins involved in carbohydrate and energy metabolisms and in pH homeostasis, allowing the cells to be better prepared for counteracting the stress they encountered during subsequent cold stress. These two phenomena led to a cross-protection phenomenon, which allowed better cryotolerance of Lb. acidophilus RD758, following cellular adaptation by starvation.     

粪便样本不同保存方式对肠道菌群测序结果的影响

[目的]本文探究了3种室温保存剂和-80℃C冷冻保存对粪便样本中菌群结构的影响,为大规模、标准化的采样提供参考.[方法]本研究采集了5名健康志愿者的新鲜粪便作为测试样本,采用4种不同的保存方式保存:DETs室温保存、GITC室温保存、RNAlater室温保存和-80℃冷冻保存,在保存0、1、3、7、14、28 d后,采...     

粪菌移植研究的文献计量学和可视化分析

【背景】粪菌移植是近年医学领域研究的热点,不但能够治疗消化系统疾病,而且在神经及精神系统、心血管系统相关疾病的治疗中均有不错的疗效,有着广阔的应用前景。【目的】掌握国内外粪菌移植的研究现状、热点及发展趋势,为相关领域科研工作者的研究提供参考。【方法】基于Web of Science核心数据库,通过CiteSpace对2011-2021年的年度发文量、作者、国家、期刊、被引情况和关键词等进行可视化分析。【结果】筛选后共纳入4 905篇文献,目前全球粪菌移植研究的文献数量呈快速增长趋势;美国和中国是发文量最多的国家。中国学者的总发文量虽然位居世界第二,但中心度和篇均被引频次较低,说明受关注程度及学术影响力不足,在发文质量上还有待提高;Gastroenterology是国内外学者发文量最多的期刊,Frontiers in Microbiology是中国学者发文量最多的期刊;粪菌移植呈现出多学科交叉的发展特点;粪菌移植目前的研究热点主要与肠内疾病(炎症性肠病、艰难梭菌感染)和肠外疾病(如抑郁、冠状动脉粥样硬化等)有关;粪菌移植在未成年人中的应用、对胰岛素敏感度的影响、测序技术在肠道菌群的应用及...     

戊糖片球菌368对猪生长性能、粪菌结构和代谢产物的影响

[背景]随着"禁抗令"的推行,如何寻找安全的抗生素替代品迫在眉睫.[目的]研究戊糖片球菌368对育肥猪的影响.[方法]选用20头育肥猪,随机分为2组,每组2个重复,每个重复5头.对照组饲喂基础饲粮,试验组饲喂基础饲粮+戊糖片球菌368(1010CFU/kg),试验期28 d.[结果]试验期内,试验组和对照组的平均日增重...     

Influence of freeze-drying and spray-drying preservation methods on survivability rate of different types of protectants encapsulated Lactobacillus acidophilus FTDC 3081

ABSTRACT

The aims of this study were to compare the effectiveness of different drying methods and to investigate the effects of adding a series of individual protectant such as skim milk, sucrose, maltodextrin, and corn starch for preserving Lactobacillus acidophilus FTDC 3081 cells during spray and freeze-drying and storage at different temperatures. Results showed a remarkable high survival rate of 70–80% immediately after spray- and freeze-drying in which the cell viability retained at the range of 10⁹ to 10¹⁰ CFU/mL. After a month of storage, maltodextrin showed higher protective ability on both spray- and freeze-dried cells as compared to other protective agents at 4°C, 25°C, and 40°C. A complete loss in viability of spray-dried L. acidophilus FTDC 3081 was observed after a month at 40°C in the absence of protective agent.     

In vitro fermentation of sugar beet arabino-oligosaccharides by fecal microbiota obtained from patients with ulcerative colitis to selectively stimulate the growth of Bifidobacterium spp. and Lactobacillus spp

The potential prebiotic properties of arabino-oligosaccharides (AOS) derived from sugar beet pulp was studied using mixed cultures of human fecal bacteria from patients with ulcerative colitis (UC), in remission or with active disease, and in healthy controls. These results were compared to those for fructo-oligosaccharides (FOS), which are known to have a prebiotic effect. Fermentation studies were carried out using a small-scale static batch system, and changes in the fecal microbial communities and metabolites were monitored after 24 h by quantitative real-time PCR and short-chain fatty acid analysis. With a few minor exceptions, AOS affected the communities similarly to what was seen for FOS. Quantitative real-time PCR revealed that Bifidobacterium spp. and Lactobacillus spp. were selectively increased after fermentation of AOS or FOS by fecal microbiota derived from UC patients. The stimulation of growth of Lactobacillus spp. and Bifidobacterium spp. was accompanied by a high production of acetate and hence a decrease of pH. The fermentation of AOS may help improve the inflammatory conditions in UC patients through stimulation of bacteria eliciting anti-inflammatory responses and through production of acetate. AOS may therefore represent a new prebiotic candidate for reduction of the risk of flare-ups in UC patients. However, human trials are needed to confirm a health-promoting effect.     

The effect of bile salts on survival and morphology of a potential probiotic strain Lactobacillus acidophilus M92

Bile tolerance is an important criterion in the selection of microbial strains for probiotic use. The survival and morphological changes of a potential probiotic strain, Lactobacillus acidophilus M92, in the presence of bile salts were examined. Lactobacillus acidophilus M92 has shown a satisfactory degree of tolerance against oxgall and individual bile salts tested, especially to taurocholate. The higher resistance of L. acidophilus M92 against taurine-conjugated bile salts relative to deconjugated and glycine-conjugated bile salts was attributed to its reaction to the stronger acidity of the former. Furthermore, bile salt hydrolase (BSH) was active when L. acidophilus M92 was grown in the presence of sodium taurocholate. The rate of BSH activity was highest at the exponential growth phase. It was hypothesised that BSH activity may be important for the bile salt resistance of this strain. The colonial and cellular morphology may also be a valuable parameter in the selection of bile salt-resistant Lactobacillus strains for probiotic use. Smooth (S) and rough (R) colonies, appeared in the original L. acidophilus M92 bacterial culture and demonstrated a different degree of bile tolerance. Rough colonies were more sensitive to bile salts than smooth ones. The R colony cells assumed a round form, probably induced by gaps in the cell wall caused by the cytotoxicity of glycodeoxycholate. This revised version was published online in July 2006 with corrections to the Cover Date.     

Captive environment influences the composition and diversity of fecal microbiota in Indo-Pacific bottlenose dolphins,Tursiops aduncus

Captive environments impact the microbiota of captive animals; however, the comparison of microbiota between wild and captive dolphins has been poorly investigated. To explore the impact of a captive environment, we characterized the fecal microbiota of nine wild and four captive Indo-Pacific bottlenose dolphins, Tursiops aduncus, using a next-generation sequencing and revealed differences in the fecal microbiota between the analyzed groups. Statistical differences in abundances of the phyla Firmicutes and Proteobacteria were found between the wild and captive dolphins. Thirty-six genera (22.9% of the total genera detected in all dolphins) were shared between the groups, whereas 79 (50.3%) and 42 (26.8%) genera were found only in the wild or captive dolphins, respectively. Several pathogenic bacterial genera, including Morganella and Mycoplasma, were detected only in the captive dolphins, and the genus Lactobacillus was found only in the wild dolphins. LefSe and SIMPER analyses revealed that the genus Clostridium sensu stricto 1 was significantly more abundant in the captive dolphins than in the wild ones and contributed the most to the dissimilarity of fecal microbiota between the groups. Our results indicate that the captive environment impacts the fecal microbiota of dolphins and reinforces the importance of monitoring potentially pathogenic bacteria in captivity.