Cultivable bacterial diversity from the human colon

Knowledge of the composition of the colonic microbiota is important for our understanding of how the balance of these microbes is influenced by diet and the environment, and which bacterial groups are important in maintaining gut health or promoting disease. Molecular methodologies have advanced our understanding of the composition and diversity of the colonic microbiota. Importantly, however, it is the continued isolation of bacterial representatives of key groups that offers the best opportunity to conduct detailed metabolic and functional studies. This also permits bacterial genome sequencing which will accelerate the linkage to functionality. Obtaining new human colonic bacterial isolates can be challenging, because most of these are strict anaerobes and many have rather exact nutritional and physical requirements. Despite this many new species are being isolated and described that occupy distinct niches in the colonic microbial community. This review focuses on these under-studied yet important gut anaerobes.     

Understanding the effects of diet on bacterial metabolism in the large intestine

Recent analyses of ribosomal RNA sequence diversity have demonstrated the extent of bacterial diversity in the human colon, and have provided new tools for monitoring changes in the composition of the gut microbial community. There is now an excellent opportunity to correlate ecological niches and metabolic activities with particular phylogenetic groups among the microbiota of the human gut. Bacteria that associate closely with particulate material and surfaces in the gut include specialized primary degraders of insoluble substrates, including resistant starch, plant structural polysaccharides and mucin. Butyrate-producing bacteria found in human faeces belong mainly to the clostridial clusters IV and XIVa. In vitro and in vivo evidence indicates that a group related to Roseburia and Eubacterium rectale plays a major role in mediating the butyrogenic effect of fermentable dietary carbohydrates. Additional cluster XIVa species can convert lactate to butyrate, while some members of the clostridial cluster IX convert lactate to propionate. The metabolic outputs of the gut microbial community depend not only on available substrate, but also on the gut environment, with pH playing a major role. Better understanding of the colonic microbial ecosystem will help to explain and predict the effects of dietary additives, including nondigestible carbohydrates, probiotics and prebiotics.     

Abundance and diversity of mucosa-associated hydrogenotrophic microbes in the healthy human colon

Hydrogenotrophic microbiota have a significant impact on colonic health; however, little is known about their diversity and ecology in situ. Here, molecular-based methods and multivariate analyses were used to examine the abundance and diversity of mucosa-associated hydrogenotrophic microbes in 90 biopsies collected from right colon, left colon and rectum of 25 healthy subjects. Functional genes of all three hydrogenotrophic groups were detected in at least one colonic region of all subjects. Methanogenic archaea (MA) constituted approximately one half of the hydrogenotrophic microbiota in each colonic region. Sulfate-reducing bacteria (SRB) were more abundant than acetogens in right colon, while acetogens were more abundant than SRB in left colon and rectum. MA genotypes exhibited low diversity, whereas SRB genotypes were diverse and generally similar across the three regions within subject but significantly variable among subjects. Multivariate cluster analysis defined subject-specific patterns for the diversity of SRB genotypes; however, neither subject- nor region-specific clusters were observed for the abundance of hydrogenotrophic functional genes. Sequence analyses of functional gene clones revealed that mucosa-associated SRB were phylogenetically related to Desulfovibrio piger, Desulfovibrio desulfuricans and Bilophila wadsworthia; whereas MA were related to Methanobrevibacter spp., Mb. smithii and the order Methanomicrobiales. Together these data demonstrate for the first time that the human colonic mucosa is persistently colonized by all three groups of hydrogenotrophic microbes, which exhibit segmental and interindividual variation in abundance and diversity.     

The oral biome in the aetiology and management of dental disease: Current concepts and ethical considerations

Our understanding of the complexity of the oral biome and of the role of the various constituent bacteria in the aetiology of dental disease is growing. Probiotics and their relationship with prebiotics, as well as other microbiome‐based interventions, could be useful in preventing and treating dental disease and in promoting oral health. However, given the promise and early stage of this treatment approach, there are also a number of ethical, social and regulatory issues associated with innovative probiotic therapy. In this article, a brief update is given on contemporary theories of the aetiology and management of the two commonest dental diseases, and on the roles of pre‐ and probiotics and oral biome transplant in the management of these diseases. The focus is primarily on four core issues: informed consent, risk–benefit assessment, how to determine suitable healthy donors, and commercialization and regulation. We discuss the safety and benefits of oral probiotics, not only concerning the products and quality control during their manufacture, but also regarding the depth of public knowledge about this topic. We point out that the requirement of listing ingredients honestly might be insufficient, and that the prevalent rhetoric of ‘natural’ and ‘organic’ as well as some health claims in the translational, innovative probiotic industry and markets are themselves misleading and should be carefully scrutinized. Finally, we suggest an ethical imperative to find a balance between scientific research and industry, and public health in the regulation of probiotics.     

Bacterial Production in the Recently Flooded Sep Reservoir: Diel Changes in Relation to Dissolved Carbohydrates and Combined Amino Acids

The spatial distribution of bacterial abundance and production were measured every 4 h in a recently flooded oligo-mesotrophic reservoir (the Sep Reservoir, Puy-De-Dôme, France), in relation to concentrations of dissolved carbohydrates and combined amino acids. The concentration of dissolved organic matter (DOM) components in the recently flooded Sep Reservoir were higher than those measured in other lakes of similar trophic status. Short-term variations in the bacterial production in this new reservoir appeared cyclical and endogenous to bacterial communities. These results highlight the need for the evaluation of diel changes in bacterial production, if estimation of the daily production rate of bacteria is to be done accurately for a reliable model of carbon flow through bacterioplankton and ultimately through aquatic microbial food webs. Bacterial growth, measured over time and space, did not appear exclusively governed by DOM components from phytoplankton primary production.     

长期施肥对稻田土壤细菌、古菌多样性和群落结构的影响

稻田土壤是“迷失碳”的重要吸纳场所之一,也是温室气体(CH4和N2O等)的重要排放源.大气温室气体的动态变化与土壤碳氮转化的微生物过程紧密相关.以湖南桃江国家级稻田肥力变化长期定位试验点为平台,采用PCR-克隆测序和实时荧光定量PCR技术,研究不施肥(CK)、施氮磷钾肥(NPK)和氮磷钾肥+秸秆还田(NPKS)3种长期施肥制度(＞25 a)对稻田土壤细菌和古菌群落结构及数量的影响.细菌和古菌16S rRNA基因文库分析结果表明:稻田土壤细菌主要类群为变形菌、酸杆菌、绿弯菌,而古菌主要为泉古菌和广古菌.长期施肥导致土壤细菌和古菌种群结构产生明显差异,与CK相比,NPK和NPKS处理稻田土壤的变形菌、酸杆菌和泉古菌相对丰度增加.LIBSHUFF软件分析结果也表明,16S rRNA基因文库在CK、NPK及NPKS处理间存在显著差异.3种施肥处理的稻田土壤细菌16S rRNA基因拷贝数为每克干土0.58× 1010～1.06×1010个,古菌为每克干土1.16×106 ～ 1.72×106个.施肥(NPK和NPKS)后,细菌和古菌的多样性和数量增加,且NPKS＞NPK.说明长期施肥显著影响土壤细菌和古菌群落结构、多样性及数量.     

Carbon catabolite repression in pectin digestion by the phytopathogen Dickeya dadantii

The catabolism of pectin from plant cell walls plays a crucial role in the virulence of the phytopathogen Dickeya dadantii. In particular, the timely expression of pel genes encoding major pectate lyases is essential to circumvent the plant defense systems and induce massive pectinolytic activity during the maceration phase. Previous studies identified the role of a positive feedback loop specific to the pectin-degradation pathway, whereas the precise signals controlling the dynamics of pectate lyase expression were unclear. Here, we show that the latter is controlled by a metabolic switch involving both glucose and pectin. We measured the HPLC concentration profiles of the key metabolites related to these two sources of carbon, cAMP and 2-keto-3-deoxygluconate, and developed a dynamic and quantitative model of the process integrating the associated regulators, cAMP receptor protein and KdgR. The model describes the regulatory events occurring at the promoters of two major pel genes, pelE and pelD. It highlights that their activity is controlled by a mechanism of carbon catabolite repression, which directly controls the virulence of D. dadantii. The model also shows that quantitative differences in the binding properties of common regulators at these two promoters resulted in a qualitatively different role of pelD and pelE in the metabolic switch, and also likely in conditions of infection, justifying their evolutionary conservation as separate genes in this species.     

Protists have divergent effects on bacterial diversity along a productivity gradient

Productivity and predation are thought to be crucial drivers of bacterial diversity. We tested how the productivity–diversity of a natural bacterial community is modified by the presence of protist predators with different feeding preferences. In the absence of predators, there was a unimodal relationship between bacterial diversity and productivity. We found that three protist species (Bodo, Spumella and Cyclidium) had widely divergent effects on bacterial diversity across the productivity gradient. Bodo and Cyclidium had little effect on the shape of the productivity–diversity gradient, while Spumella flattened the relationship. We explain these results in terms of the feeding preferences of these predators.     

Metabolic and functional properties of lactic acid bacteria in the gastro-intestinal ecosystem: a comparative in vitro study between bacteria of intestinal and fermented food origin

Metabolic and functional properties of probiotic lactic acid bacteria (LAB) in the human gastro-intestinal ecosystem may be related to certain beneficial health effects. In this study, lactobacilli of either intestinal or fermented food origin were compared in their capability to survive low pH and bile, in their metabolic activity in the presence of bile salts and mucins, as well as in their potential to attach to enterocyte-like CaCO-2 cells. Food fermenting bacteria especially strains of the species Lactobacillus plantarum showed high tolerance to the consecutive exposure to hydrochloric acid (pH 1.5-2.5) and cholic acid (10 mM). Growth in and deconjugation of glycocholic (5 mM) and taurocholic acids (5 mM), as demonstrated for all lactobacilli of intestinal origin, was detected for food fermenting strains of the species L. plantarum, but not L. paracasei and L. sakei. Degradation of mucins was not observed for lactobacilli. Adhesion to the intestinal epithelial cell line CaCO-2 was demonstrated for several food fermenting bacterial strains in vitro. Soluble factors in the spent culture supernatants from intestinal and fermented food lactobacilli but not staphylococci cross reacted and synergized with cell wall components to promote adhesion to CaCO-2 cells. A competitive role of fecal bacteria on the adhesion of lactobacilli to CaCO-2 cells was demonstrated. In conclusion we have shown that metabolic and functional properties of intestinal lactobacilli are also found in certain bacteria of fermented food origin.     

沙埋对干旱沙区真藓结皮层细菌群落结构和多样性的影响

作为干旱沙区常见干扰之一的沙埋显著影响着生物结皮的结构和功能,但其内在的生物学机理还不清楚。利用高通量测序技术,通过对0(对照)、0.5(浅层)、2和10mm(深层)沙埋处理后的腾格里沙漠东南缘沙坡头地区真藓(Bryum argenteum)结皮层细菌群落物种组成与丰度的测定,研究了沙埋对真藓结皮层细菌群落结构和多样性的影响。结果表明:(1)共检测到沙坡头地区真藓结皮层细菌38门106纲181属,以放线菌、变形菌、蓝藻、浮霉菌、拟杆菌和酸杆菌等为主(占细菌群落的78.4%—83.0%);(2)PCA分析表明沙埋导致该地区真藓结皮层细菌群落结构组成发生明显改变。无沙埋时,真藓结皮层细菌群落中相对丰度最高的是蓝藻(18.6%),随着沙埋厚度的增加,依次变为变形菌(21.5%,沙埋厚度0.5mm)、浮霉菌(21.5%,沙埋厚度2mm)和放线菌(23.3%,沙埋厚度10mm);浅层沙埋显著增加了真藓结皮层细菌群落中光合菌、固氮菌和产菌丝体细菌等关键功能菌的丰度,但深层沙埋降低了它们的丰度;(3)沙埋显著增加了真藓结皮层细菌群落多样性(P0.05)和物种丰富度(P0.05),0.5mm沙埋后的细菌群落丰富度指数最高,2mm沙埋后的结皮层细菌群落多样性指数最高。揭示了沙埋对干旱沙区真藓结皮层细菌群落结构与多样性的影响,为深入理解沙埋对沙区生物结皮结构和生态功能影响的生物学机制提供了一定的理论依据。     

PCR-DGGE analysis reveals a distinct diversity in the bacterial population attached to the rumen epithelium

Bacteria attached to the rumen epithelium (or epimural community) are not well characterised and their role in rumen functioning is not totally understood. There is just one published report of a clone library from one cow that suggests that this epimural community differs from the bacteria associated with the rumen digestive contents. However, this time-consuming approach is not adapted for examining microbial population changes in groups of animals. In in vivo studies, when samples from several animals have to be analysed simultaneously, a simpler technique has to be used. In this study, a genetic fingerprinting technique, polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE), was used to characterise the structure of the bacterial population attached to the rumen epithelium. This community was compared with that present in the solid and liquid phases of rumen content under two contrasting diets. Rumen samples were obtained from four forage-fed and four high-concentrate-fed (80 : 20, wheat grain : hay) 5-month-old lambs. After slaughter, samples from five epithelial sites and the solid and liquid digesta phases were taken for DNA extraction and analysis. Bacterial communities were profiled by PCR-DGGE using bacterial-specific 16S rDNA primers. Analysis of the fingerprint revealed that the epithelial community differed from those of rumen content in both diets. As expected, the nature of the feed influenced the bacterial communities from the solid and liquid rumen phases but no diet effect was observed in the rumen epithelial profiles suggesting a strong host effect on this bacterial population. Additionally, no differences were observed among the five epithelial sampling sites taken from each animal. The profile of the bacterial population attached to the rumen epithelium presented a high inter-animal variation, whether this difference has an influence in the function of this community remains to be determined.     

The effects of inulin,dried Jerusalem artichoke tuber and a multispecies probiotic preparation on microbiota ecology and immune status of the large intestine in young pigs

The study aimed at determining the effect of two types of prebiotics and a multispecies probiotic on microbiota activity and composition, as well as mucosal immunity in the large intestine of young pigs. In total 48 piglets were divided into 6 groups (n = 8), which received from day 10 of life probiotic-unsupplemented (PU) or probiotic-supplemented (PS) diets. Probiotics were added at 0.5 g/kg diet and contained: Lactococcus lactis, Carnobacterium divergens, Lactobacillus casei, Lactobacillus plantarum and Saccharomyces cerevisiae. The PU and PS diets were formulated without prebiotic addition (control) or with addition of 2% of inulin from chicory root (IN) or 4% of dried Jerusalem artichoke tubers (DJA). After 40 days of feeding, digesta and tissue samples were taken from the caecum and three sections of the colon for analyses of microbiota activity and composition, secretory immunoglobulin A (sIgA) and intraepithelial lymphocytes (IEL). IN diets decreased the caecal digesta pH and β-glucosidase activity but increased propionic, valeric and total short chain fatty acid (SCFA) concentrations compared to control diets. Feeding DJA diets increased caecal valeric acid level, decreased the concentration of isoacids in the colon, reduced β-glucosidase and β-glucuronidase activity in the middle colon and increased Bifidobacterium spp. populations in the proximal and distal colon. PS diets increased the caecal acetic acid and total SCFA level, and Clostridium spp. populations in the distal colon. Neither probiotic nor prebiotics affected sIgA level or IEL number in the large intestine. In conclusion, DJA modified the microbiota ecology in the large intestine of young pigs to a greater extent than IN and the applied probiotic did not enhance effects of prebiotics.     

Speedy speciation in a bacterial microcosm: new species can arise as frequently as adaptations within a species

Microbiologists are challenged to explain the origins of enormous numbers of bacterial species worldwide. Contributing to this extreme diversity may be a simpler process of speciation in bacteria than in animals and plants, requiring neither sexual nor geographical isolation between nascent species. Here, we propose and test a novel hypothesis for the extreme diversity of bacterial species—that splitting of one population into multiple ecologically distinct populations (cladogenesis) may be as frequent as adaptive improvements within a single population''s lineage (anagenesis). We employed a set of experimental microcosms to address the relative rates of adaptive cladogenesis and anagenesis among the descendants of a Bacillus subtilis clone, in the absence of competing species. Analysis of the evolutionary trajectories of genetic markers indicated that in at least 7 of 10 replicate microcosm communities, the original population founded one or more new, ecologically distinct populations (ecotypes) before a single anagenetic event occurred within the original population. We were able to support this inference by identifying putative ecotypes formed in these communities through differences in genetic marker association, colony morphology and microhabitat association; we then confirmed the ecological distinctness of these putative ecotypes in competition experiments. Adaptive mutations leading to new ecotypes appeared to be about as common as those improving fitness within an existing ecotype. These results suggest near parity of anagenesis and cladogenesis rates in natural populations that are depauperate of bacterial diversity.     

Evaluation of five antibiotics on larval gut bacterial diversity of Plutella xylostella (Lepidoptera: Plutellidae)

Larvae of the diamondback moth, Plutella xylostella L. (Lepidoptera: Plutellidae), have rich microbial communities inhabiting the gut, and these bacteria contribute to the fitness of the pest. In this study we evaluated the effects of five antibiotics (rifampicin, ampicillin, tetracycline, streptomycin sulfate and chloramphenicol) on the gut bacterial diversity of P. xylostella larvae. We screened five different concentrations for each antibiotic in a leaf disc assay, and found that rifampicin and streptomycin sulfate at 3 mg/mL significantly reduced the diversity of the bacterial community, and some bacterial species could be rapidly eliminated. The number of gut bacteria in the rifampicin group and streptomycin sulfate group decreased more rapidly than the others. With the increase of antibiotic concentration, the removal efficiency was improved, whereas toxic effects became more apparent. All antibiotics reduced larval growth and development, and eventually caused high mortality, malformation of the prepupae, and hindered pupation and adult emergence. Among the five antibiotics, tetracycline was the most toxic and streptomycin sulfate was a relatively mild one. Some dominant bacteria were not affected by feeding antibiotics alone. Denaturing gradient gel electrophoresis graph showed that the most abundant and diverse bacteria in P. xylostella larval gut appeared in the cabbage feeding group, and diet change and antibiotics intake influenced gut flora abundance. Species diversity was significantly reduced in the artificial diet and antibiotics treatment groups. After feeding on the artificial diet with rifampicin, streptomycin sulfate and their mixture for 10 days, larval gut bacteria could not be completely removed as detected with the agarose gel electrophoresis method.     

Evidence for the evolutionary relatedness of the proteins of the bacterial phosphoenolpyruvate:sugar phosphotransferase system

The phosphoenolpyruvate:sugar phosphotransferase system (PTS) found in enteric bacteria is a complex enzyme system consisting of a non-sugar-specific phosphotransfer protein called Enzyme I, two small non-sugar-specific phosphocarrier substrates of Enzyme I, designated HPr and FPr, and at least 11 sugar-specific Enzymes II or Enzyme II-III pairs which are phosphorylated at the expense of phospho-HPr or phospho-FPr. In this communication, evidence is presented which suggests that these proteins share a common evolutionary origin and that a fructose-specific phosphotransferase may have been the primordial ancestor of them all. The evidence results from an evaluation of 1) PTS protein sequence data; 2) structural analysis of operons encoding proteins of the PTS; 3) genetic regulatory mechanisms controlling expression of these operons; 4) enzymatic characteristics of the PTS systems; 5) immunological cross reactivities of these proteins; 6) comparative studies of phosphotransferase systems from evolutionarily divergent bacteria; 7) the nature of the phosphorylated protein intermediates; 8) molecular weight comparisons among the different Enzymes II and Enzyme II-III pairs; and 9) interaction studies involving different PTS protein constituents. The evidence leads to a unifying theory concerning the evolutionary origin of the system, explains many structural, functional, and regulatory properties of the phosphotransferase system, and leads to specific predictions which should guide future research concerned with genetic, biochemical, and physiological aspects of the system.     

泥浸汁对太湖沉积物中的好氧可培养细菌多样性的影响

【目的】研究添加泥浸汁与否对太湖沉积物中可培养细菌的影响。【方法】采用R2A培养基和添加泥浸汁R2A培养基对沉积物中细菌进行分离培养,16S r RNA基因系统发育分析比较种群结构。【结果】培养基中添加泥浸汁,可使可培养细菌的种类数量增加到1.6倍。16S r RNA基因序列分析表明,培养的优势细菌类群存在明显差别。R2A培养基上生长的细菌主要为厚壁菌门(52%)、放线菌门(24%)、变形菌门(20%)和拟杆菌门(4%),其中大部分细菌与芽孢杆菌属、假单胞菌属、节杆菌属等关系密切;而添加泥浸汁的R2A培养基上生长的细菌则主要为变形菌门(40%)、放线菌门(35%)、厚壁菌门(22.5%)和拟杆菌门(2.5%),与鞘脂单胞菌属、芽孢杆菌属、副球菌属、节杆菌属等关系密切。【结论】添加泥浸汁原始营养因子可提高沉积物中可培养细菌的多样性,提高菌种可培养效率。