Monitoring of antibiotic-induced alterations in the human intestinal microflora and detection of probiotic strains by use of terminal restriction fragment length polymorphism

Terminal restriction fragment length polymorphism (T-RFLP) was investigated as a tool for monitoring the human intestinal microflora during antibiotic treatment and during ingestion of a probiotic product. Fecal samples from eight healthy volunteers were taken before, during, and after administration of clindamycin. During treatment, four subjects were given a probiotic, and four subjects were given a placebo. Changes in the microbial intestinal community composition and relative abundance of specific microbial populations in each subject were monitored by using viable counts and T-RFLP fingerprints. T-RFLP was also used to monitor specific bacterial populations that were either positively or negatively affected by clindamycin. Some dominant bacterial groups, such as Eubacterium spp., were easily monitored by T-RFLP, while they were hard to recover by cultivation. Furthermore, the two probiotic Lactobacillus strains were easily tracked by T-RFLP and were shown to be the dominant Lactobacillus community members in the intestinal microflora of subjects who received the probiotic.     

Characterization of Microbial Communities Found in the Human Vagina by Analysis of Terminal Restriction Fragment Length Polymorphisms of 16S rRNA Genes

To define and monitor the structure of microbial communities found in the human vagina, a cultivation-independent approach based on analyses of terminal restriction fragment length polymorphisms (T-RFLP) of 16S rRNA genes was developed and validated. Sixteen bacterial strains commonly found in the human vagina were used to construct model communities that were subsequently used to develop efficient means for the isolation of genomic DNA and an optimal strategy for T-RFLP analyses. The various genera in the model community could best be resolved by digesting amplicons made using bacterial primers 8f and 926r with HaeIII; fewer strains could be resolved using other primer-enzyme combinations, and no combination successfully distinguished certain species of the same genus. To demonstrate the utility of the approach, samples from five women that had been collected over a 2-month period were analyzed. Differences and similarities among the vaginal microbial communities of the women were readily apparent. The T-RFLP data suggest that the communities of three women were dominated by a single phylotype, most likely species of Lactobacillus. In contrast, the communities of two other women included numerically abundant populations that differed from Lactobacillus strains whose 16S rRNA genes had been previously determined. The T-RFLP profiles of samples from all the women were largely invariant over time, indicating that the kinds and abundances of the numerically dominant populations were relatively stable throughout two menstrual cycles. These findings show that T-RFLP of 16S rRNA genes can be used to compare vaginal microbial communities and gain information about the numerically dominant populations that are present.     

Lactobacillus paracasei subsp. paracasei LC01 positively modulates intestinal microflora in healthy young adults

Lactobacillus paracasei subsp. paracasei LC01 (LC01) can tolerate intestinal stresses and has antioxidant activity. To evaluate the effect of the bacterium on human intestinal microflora, a randomized, double-blind, placebo-controlled human trial was carried out. Fifty-two healthy adult volunteers were randomized equally to two groups. One group consumed 12% (wt/vol) skimmed milk supplemented with 10¹⁰ CFU of LC01 each day for the 4-week treatment period, and then consumed placebo in the next treatment period, separated by a 2-week washout. The other group followed the reverse order. Group-specific real-time PCR and biochemical analyses was used to determine the intestinal bacterial composition of fecal samples collected at the end of every period, and the concentration of short-chain fatty acids and ammonia. A significant inhibition in fecal Escherichia coli and increase in Lactobacillus, Bifidobacterium, and Roseburia intestinalis were observed after consumption of LC01. Acetic acid and butyric acid were significantly higher in the probiotic stage and fecal ammonia was significantly lower. The results indicated a modulation effect of LC01 on the intestinal microflora of young adults, suggesting a beneficial effect on bowel health. LC01 may have potential value as a probiotic.     

Dynamics of a Pig Slurry Microbial Community during Anaerobic Storage and Management

The microbial community of a pig slurry on a farm was monitored for 6 months using both molecular and cultural approaches. Sampling was carried out at all the different stages of effluent handling, from the rearing build-up to slurry spreading. Total DNA of each sample was extracted and analyzed by PCR-single-strand conformation polymorphism (SSCP) analysis using primers targeting the 16S rRNA genes from the archaeal and bacterial domains and also the Eubacterium-Clostridium, Bacillus-Streptococcus-Lactobacillus, and Bacteroides-Prevotella groups. A comparison of the SSCP profiles showed that there were rapid changes in the dominant bacterial community during the first 2 weeks of anaerobic storage and that the community was relatively stable thereafter. Several bacterial populations, identified as populations closely related to uncultured Clostridium and Porphyromonas and to Lactobacillus and Streptococcus cultured species commonly isolated from pig feces, remained present and dominant from the rearing build-up to the time of spreading. Enumeration of fecal indicators (enterococci and Escherichia coli) performed in parallel using cultural methods revealed the same trends. On the other hand, the archaeal community adapted slowly during pig slurry storage, and its diversity increased. A shift between two hydrogenotrophic methanogenic Methanobrevibacter populations from the storage pit to the pond was observed. Microorganisms present in pig slurry at the time of spreading could not be detected in soil after spreading by either molecular or cultural techniques, probably because of the detection limit inherent in the two techniques.     

Alteration of the Canine Small-Intestinal Lactic Acid Bacterium Microbiota by Feeding of Potential Probiotics

Five potentially probiotic canine fecal lactic acid bacterium (LAB) strains, Lactobacillus fermentum LAB8, Lactobacillus salivarius LAB9, Weissella confusa LAB10, Lactobacillus rhamnosus LAB11, and Lactobacillus mucosae LAB12, were fed to five permanently fistulated beagles for 7 days. The survival of the strains and their potential effects on the indigenous intestinal LAB microbiota were monitored for 17 days. Denaturing gradient gel electrophoresis (DGGE) demonstrated that the five fed LAB strains survived in the upper gastrointestinal tract and modified the dominant preexisting indigenous jejunal LAB microbiota of the dogs. When the LAB supplementation was ceased, DGGE analysis of jejunal chyme showed that all the fed LAB strains were undetectable after 7 days. However, the diversity of the intestinal indigenous microbiota of the dogs, as characterized from jejunal chyme plated on Lactobacillus selective medium without acetic acid, was reduced and did not return to the original level during the study period. In all but one dog, an indigenous Lactobacillus acidophilus strain emerged as the dominant LAB strain. In conclusion, strains LAB8 to LAB12 have potential as probiotic strains for dogs as they survive in and dominate the jejunal LAB microbiota during feeding and have the ability to modify the intestinal microbiota.     

Community Structure of Actively Growing Bacterial Populations in Plant Pathogen Suppressive Soil

The bacterial community in soil was screened by using various molecular approaches for bacterial populations that were activated upon addition of different supplements. Plasmodiophora brassicae spores, chitin, sodium acetate, and cabbage plants were added to activate specific bacterial populations as an aid in screening for novel antagonists to plant pathogens. DNA from growing bacteria was specifically extracted from the soil by bromodeoxyuridine immunocapture. The captured DNA was fingerprinted by terminal restriction fragment length polymorphism (T-RFLP). The composition of the dominant bacterial community was also analyzed directly by T-RFLP and by denaturing gradient gel electrophoresis (DGGE). After chitin addition to the soil, some bacterial populations increased dramatically and became dominant both in the total and in the actively growing community. Some of the emerging bands on DGGE gels from chitin-amended soil were sequenced and found to be similar to known chitin-degrading genera such as Oerskovia, Kitasatospora, and Streptomyces species. Some of these sequences could be matched to specific terminal restriction fragments on the T-RFLP output. After addition of Plasmodiophora spores, an increase in specific Pseudomonads could be observed with Pseudomonas-specific primers for DGGE. These results demonstrate the utility of microbiomics, or a combination of molecular approaches, for investigating the composition of complex microbial communities in soil.     

Robustness of Gut Microbiota of Healthy Adults in Response to Probiotic Intervention Revealed by High-Throughput Pyrosequencing

Probiotics are live microorganisms that potentially confer beneficial outcomes to host by modulating gut microbiota in the intestine. The aim of this study was to comprehensively investigate effects of probiotics on human intestinal microbiota using 454 pyrosequencing of bacterial 16S ribosomal RNA genes with an improved quantitative accuracy for evaluation of the bacterial composition. We obtained 158 faecal samples from 18 healthy adult Japanese who were subjected to intervention with 6 commercially available probiotics containing either Bifidobacterium or Lactobacillus strains. We then analysed and compared bacterial composition of the faecal samples collected before, during, and after probiotic intervention by Operational taxonomic units (OTUs) and UniFrac distances. The results showed no significant changes in the overall structure of gut microbiota in the samples with and without probiotic administration regardless of groups and types of the probiotics used. We noticed that 32 OTUs (2.7% of all analysed OTUs) assigned to the indigenous species showed a significant increase or decrease of ≥10-fold or a quantity difference in >150 reads on probiotic administration. Such OTUs were found to be individual specific and tend to be unevenly distributed in the subjects. These data, thus, suggest robustness of the gut microbiota composition in healthy adults on probiotic administration.     

Diversity and Succession of the Intestinal Bacterial Community of the Maturing Broiler Chicken

The diversity of bacterial floras in the ilea and ceca of chickens that were fed a vegetarian corn-soy broiler diet devoid of feed additives was examined by analysis of 1,230 partial 16S rRNA gene sequences. Nearly 70% of sequences from the ileum were related to those of Lactobacillus, with the majority of the rest being related to Clostridiaceae (11%), Streptococcus (6.5%), and Enterococcus (6.5%). In contrast, Clostridiaceae-related sequences (65%) were the most abundant group detected in the cecum, with the other most abundant sequences being related to Fusobacterium (14%), Lactobacillus (8%), and Bacteroides (5%). Statistical analysis comparing the compositions of the different 16S rRNA libraries revealed that population succession occurred during some sampling periods. The significant differences among cecal libraries at 3 and 7 days of age, at 14 to 28 days of age, and at 49 days of age indicated that successions occurred from a transient community to one of increasing complexity as the birds aged. Similarly, the ileum had a stable bacterial community structure for birds at 7 to 21 days of age and between 21 to 28 days of age, but there was a very unique community structure at 3 and 49 days of age. It was also revealed that the composition of the ileal and cecal libraries did not significantly differ when the birds were 3 days old, and in fact during the first 14 days of age, the cecal microflora was a subset of the ileal microflora. After this time, the ileum and cecum had significantly different library compositions, suggesting that each region developed its own unique bacterial community as the bird matured.     

DGGE and T-RFLP Analysis of Bacterial Succession during Mushroom Compost Production and Sequence-aided T-RFLP Profile of Mature Compost

The amount of button mushroom (Agaricus bisporus) harvested from compost is largely affected by the microbial processes taking place during composting and the microbes inhabiting the mature compost. In this study, the microbial changes during the stages of this specific composting process were monitored, and the dominant bacteria of the mature compost were identified to reveal the microbiological background of the favorable properties of the heat-treated phase II mushroom compost. 16S ribosomal deoxyribonucleic acid (rDNA)-based denaturing gradient gel electrophoresis (DGGE) and terminal restriction fragment length polymorphism (T-RFLP) molecular fingerprinting methods were used to track the succession of microbial communities in summer and winter composting cycles. DNA from individual DGGE bands were reamplified and subjected to sequence analysis. Principal component analysis of fingerprints of the composting processes showed intensive changes in bacterial community during the 22-day procedure. Peak temperature samples grouped together and were dominated by Thermus thermophilus. Mature compost patterns were almost identical by both methods (DGGE, T-RFLP). To get an in-depth analysis of the mature compost bacterial community, the sequence data from cultivation of the bacteria and cloning of environmental 16S rDNA were uniquely coupled with the output of the environmental T-RFLP fingerprints (sequence-aided T-RFLP). This method revealed the dominance of a supposedly cellulose-degrading consortium composed of phylotypes related to Pseudoxanthomonas, Thermobifida, and Thermomonospora.     

An In Vitro Study of the Probiotic Potential of a Bile-Salt-Hydrolyzing Lactobacillus fermentum Strain, and Determination of Its Cholesterol-Lowering Properties

This study evaluated the use of a bile-salt-hydrolyzing Lactobacillus fermentum strain as a probiotic with potential hypocholesterolemic properties. The effect of L. fermentum on representative microbial populations and overall metabolic activity of the human intestinal microbiota was investigated using a three-stage continuous culture system. Also, the use of galactooligosaccharides as a prebiotic to enhance growth and/or activity of the Lactobacillus strain was evaluated. Administration of L. fermentum resulted in a decrease in the overall bifidobacterial population (ca. 1 log unit). In the in vitro system, no significant changes were observed in the total bacterial, Lactobacillus, Bacteroides, and clostridial populations through L. fermentum supplementation. Acetate production decreased by 9 to 27%, while the propionate and butyrate concentrations increased considerably (50 to 90% and 52 to 157%, respectively). A general, although lesser, increase in the production of lactate was observed with the administration of the L. fermentum strain. Supplementation of the prebiotic to the culture medium did not cause statistically significant changes in either the numbers or the activity of the microbiota, although an increase in the butyrate production was seen (29 to 39%). Results from this in vitro study suggest that L. fermentum KC5b is a candidate probiotic which may affect cholesterol metabolism. The short-chain fatty acid concentrations, specifically the molar proportion of propionate and/or bile salt deconjugation, are probably the major mechanism involved in the purported cholesterol-lowering properties of this strain.     

Analysis of the Fecal Microflora of Human Subjects Consuming a Probiotic Product Containing Lactobacillus rhamnosus DR20

The composition of the fecal microflora of 10 healthy subjects was monitored before (6-month control period), during (6-month test period), and after (3-month posttest period) the administration of a milk product containing Lactobacillus rhamnosus DR20 (daily dose, 1.6 × 10⁹ lactobacilli). Monthly fecal samples were examined by a variety of methods, including bacteriological culture analysis, fluorescent in situ hybridization with group-specific DNA probes, denaturing gradient gel electrophoresis of the V2-V3 region of 16S rRNA genes amplified by PCR, gas-liquid chromatography, and bacterial enzyme activity analysis. The composition of the Lactobacillus population of each subject was analyzed by pulsed-field gel electrophoresis of bacterial DNA digests in order to differentiate between DR20 and other strains present in the samples. Representative isolates of lactobacilli were identified to the species level by sequencing the V2-V3 region of their 16S rRNA genes and comparing the sequences obtained (BLAST search) to sequences in the GenBank database. DR20 was detected in the feces of all of the subjects during the test period, but at different frequencies. The presence of DR20 among the numerically predominant strains was related to the presence or absence of a stable indigenous population of lactobacilli during the control period. Strain DR20 did not persist at levels of >10² cells per g in the feces of most of the subjects after consumption of the product ceased; the only exception was one subject in which this strain was detected for 2 months during the posttest period. We concluded that consumption of the DR20-containing milk product transiently altered the Lactobacillus and enterococcal contents of the feces of the majority of consumers without markedly affecting biochemical or other bacteriological factors.     

Bacterial Communities in Semen from Men of Infertile Couples: Metagenomic Sequencing Reveals Relationships of Seminal Microbiota to Semen Quality

Some previous studies have identified bacteria in semen as being a potential factor in male infertility. However, only few types of bacteria were taken into consideration while using PCR-based or culturing methods. Here we present an analysis approach using next-generation sequencing technology and bioinformatics analysis to investigate the associations between bacterial communities and semen quality. Ninety-six semen samples collected were examined for bacterial communities, measuring seven clinical criteria for semen quality (semen volume, sperm concentration, motility, Kruger''s strict morphology, antisperm antibody (IgA), Atypical, and leukocytes). Computer-assisted semen analysis (CASA) was also performed. Results showed that the most abundant genera among all samples were Lactobacillus (19.9%), Pseudomonas (9.85%), Prevotella (8.51%) and Gardnerella (4.21%). The proportion of Lactobacillus and Gardnerella was significantly higher in the normal samples, while that of Prevotella was significantly higher in the low quality samples. Unsupervised clustering analysis demonstrated that the seminal bacterial communities were clustered into three main groups: Lactobacillus, Pseudomonas, and Prevotella predominant group. Remarkably, most normal samples (80.6%) were clustered in Lactobacillus predominant group. The analysis results showed seminal bacteria community types were highly associated with semen health. Lactobacillus might not only be a potential probiotic for semen quality maintenance, but also might be helpful in countering the negative influence of Prevotella and Pseudomonas. In this study, we investigated whole seminal bacterial communities and provided the most comprehensive analysis of the association between bacterial community and semen quality. The study significantly contributes to the current understanding of the etiology of male fertility.     

基于16S rRNA基因测序技术分析猪链球菌2型感染BALB/c小鼠粪便样本中微生物的变化

【目的】通过研究粪便样本的微生物特点间接探讨猪链球菌2型感染BALB/c小鼠后肠道微生物的变化。【方法】本研究采用IlluminaMiSeq测序技术,测定了健康小鼠和猪链球菌2型感染小鼠粪便中微生物16S rRNA V3-V4区序列,并对群落结构和多样性进行了比较分析。【结果】多样性分析表明,感染组和对照组小鼠的粪便中微生物多样性和群落组成均不相同,存在较大的差异,感染组展示了较高的物种丰度和种群差异性。在门水平上,相对于对照组,感染组增加厚壁菌门和拟杆菌门等有益微生物比例,以提高机体免疫力,但也同时增加了变形杆菌等条件致病菌的比例,增加了疾病发生的可能性。在科水平上,感染组和对照组优势菌均含有瘤胃菌科,但其所占细菌总序列的比例存在较大的差异,分别占感染组和对照组细菌总序列的36.58%和11.02%。在属水平上,感染组和对照组的优势菌属类别及占总微生物比例存在显著的差异。感染组主要以瘤胃菌科UCG-002和乳酸杆菌属为优势菌属,对照组以螺旋体科GWE2-31-10和密螺旋体属2为优势菌属。综上,BALB/c小鼠感染猪链球菌2型后存在肠道微生态失调。【结论】通过本研究,不仅对猪链球菌2型感染后小鼠的粪便微生物多样性和组成种类有了一定的认识,而且为今后筛选有益微生物、通过调整微生物治疗猪链球菌2型感染提供了依据。     

Intestinal microbiota of gibel carp (Carassius auratus gibelio) and its origin as revealed by 454 pyrosequencing

The intestinal microbiota has received increasing attention, as it influences growth, feed conversion, epithelial development, immunity as well as the intrusion of pathogenic microorganisms in the intestinal tract. In this study, pyrosequencing was used to explore the bacterial community of the intestine in gibel carp (Carassius auratus gibelio), and the origin of these microorganisms. The results disclosed great bacterial diversities in the carp intestines and cultured environments. The gibel carp harbored characteristic intestinal microbiota, where Proteobacteria were predominant, followed by Firmicutes. The analysis on the 10 most abundant bacterial operational taxonomic units (OTUs) revealed a majority of Firmicutes in the intestinal content (by decreasing order: Veilonella sp., Lachnospiraceae, Lactobacillales, Streptococcus sp., and Lactobacillus sp.). The second most abundant OTU was Rothia sp. (Actinobacteria). The most likely potential probiotics (Lactobacillus sp., and Bacillus sp.) and opportunists (Aeromonas sp., and Acinetobacter sp.) were not much abundant. Bacterial community comparisons showed that the intestinal community was closely related to that of the sediment, indicating the importance of sediment as source of gut bacteria in gibel carp. However, 37.95 % of the OTUs detected in feed were retrieved in the intestine, suggesting that food may influence markedly the microbiota of gibel carp, and therefore may be exploited for oral administration of probiotics.     

In vitro screening of probiotic lactic acid bacteria and prebiotic glucooligosaccharides to select effective synbiotics

Probiotics and prebiotics have been demonstrated to positively modulate the intestinal microflora and could promote host health. Although some studies have been performed on combinations of probiotics and prebiotics, constituting synbiotics, results on the synergistic effects tend to be discordant in the published works. The first aim of our study was to screen some lactic acid bacteria on the basis of probiotic characteristics (resistance to intestinal conditions, inhibition of pathogenic strains). Bifidobacterium was the most resistant genus whereas Lactobacillus farciminis was strongly inhibited. The inhibitory effect on pathogen growth was strain dependent but lactobacilli were the most effective, especially L. farciminis. The second aim of the work was to select glucooligosaccharides for their ability to support the growth of the probiotics tested. We demonstrated the selective fermentability of oligodextran and oligoalternan by probiotic bacteria, especially the bifidobacteria, for shorter degrees of polymerisation and absence of metabolism by pathogenic bacteria. Thus, the observed characteristics confer potential prebiotic properties on these glucooligosaccharides, to be further confirmed in vivo, and suggest some possible applications in synbiotic combinations with the selected probiotics. Furthermore, the distinctive patterns of the different genera suggest a combination of lactobacilli and bifidobacteria with complementary probiotic effects in addition to the prebiotic ones. These associations should be further evaluated for their synbiotic effects through in vitro and in vivo models.     

Community Structure of Denitrifiers, Bacteria, and Archaea along Redox Gradients in Pacific Northwest Marine Sediments by Terminal Restriction Fragment Length Polymorphism Analysis of Amplified Nitrite Reductase (nirS) and 16S rRNA Genes

Steep vertical gradients of oxidants (O₂ and NO₃⁻) in Puget Sound and Washington continental margin sediments indicate that aerobic respiration and denitrification occur within the top few millimeters to centimeters. To systematically explore the underlying communities of denitrifiers, Bacteria, and Archaea along redox gradients at distant geographic locations, nitrite reductase (nirS) genes and bacterial and archaeal 16S rRNA genes (rDNAs) were PCR amplified and analyzed by terminal restriction fragment length polymorphism (T-RFLP) analysis. The suitablility of T-RFLP analysis for investigating communities of nirS-containing denitrifiers was established by the correspondence of dominant terminal restriction fragments (T-RFs) of nirS to computer-simulated T-RFs of nirS clones. These clones belonged to clusters II, III, and IV from the same cores and were analyzed in a previous study (G. Braker, J. Zhou, L. Wu, A. H. Devol, and J. M. Tiedje, Appl. Environ. Microbiol. 66:2096–2104, 2000). T-RFLP analysis of nirS and bacterial rDNA revealed a high level of functional and phylogenetic diversity, whereas the level of diversity of Archaea was lower. A comparison of T-RFLPs based on the presence or absence of T-RFs and correspondence analysis based on the frequencies and heights of T-RFs allowed us to group sediment samples according to the sampling location and thus clearly distinguish Puget Sound and the Washington margin populations. However, changes in community structure within sediment core sections during the transition from aerobic to anaerobic conditions were minor. Thus, within the top layers of marine sediments, redox gradients seem to result from the differential metabolic activities of populations of similar communities, probably through mixing by marine invertebrates rather than from the development of distinct communities.     

Importance of microbial defence systems to bile salts and mechanisms of serum cholesterol reduction

An important feature of the intestinal microbiota, particularly in the case of administered probiotic microorganisms, is their resistance to conditions in the gastrointestinal tract, particularly tolerance to and growth in the presence of bile salts. Bacteria can use several defence mechanisms against bile, including special transport mechanisms, the synthesis of various types of surface proteins and fatty acids or the production of exopolysaccharides. The ability to enzymatically hydrolyse bile salts occurs in a variety of bacteria. Choloylglycine hydrolase (EC 3.5.1.24), a bile salt hydrolase, is a constitutive intracellular enzyme responsible for the hydrolysis of an amide bond between glycine or taurine and the steroid nucleus of bile acids. Its presence was demonstrated in specific microorganisms from several bacterial genera (Lactobacillus spp., Bifidobacterium spp., Clostridium spp., Bacteroides spp.). Occurrence and gene arrangement encoding this enzyme are highly variable in probiotic microorganisms. Bile salt hydrolase activity may provide the possibility to use the released amino acids by bacteria as sources of carbon and nitrogen, to facilitate detoxification of bile or to support the incorporation of cholesterol into the cell wall. Deconjugation of bile salts may be directly related to a lowering of serum cholesterol levels, from which conjugated bile salts are synthesized de novo. Furthermore, the ability of microorganisms to assimilate or to bind ingested cholesterol to the cell wall or to eliminate it by co-precipitation with released cholic acid was also documented. Some intestinal microflora produce cholesterol reductase that catalyses the conversion of cholesterol to insoluble coprostanol, which is subsequently excreted in faeces, thereby also reducing the amount of exogenous cholesterol.     

Axial Dynamics, Stability, and Interspecies Similarity of Bacterial Community Structure in the Highly Compartmentalized Gut of Soil-Feeding Termites (Cubitermes spp.)

The highly compartmentalized gut of soil-feeding termites is characterized by pronounced axial dynamics in physicochemical conditions and microbial processes. In a companion paper (D. Schmitt-Wagner, M. W. Friedrich, B. Wagner, and A. Brune, Appl. Environ. Microbiol. 69:6007-6017, 2003), we demonstrated that the variety of physicochemical conditions in the different gut compartments of Cubitermes spp. is reflected in the diversity of the respective intestinal microbial communities. Here, we used molecular fingerprints of 16S rRNA genes of the bacterial community, obtained by terminal restriction fragment length polymorphism (T-RFLP) analysis, to describe the axial dynamics of the bacterial community structure in the different gut sections. Comparison of the T-RFLP profiles with the predicted terminal restriction fragments of the clones in clone libraries of the gut segments in Cubitermes orthognathus confirmed that all hindgut sections harbored distinct bacterial communities. Morisita indices of community similarity, calculated by comparing the different patterns, revealed large differences between the bacterial communities of soil, gut, and nest material and also among the individual gut sections. By contrast, comparison of the homologous gut segments of different Cubitermes species indicated that the three termite species investigated possessed a similar, gut-specific microbiota that remained comparatively stable even during several months of maintenance in the laboratory.     

Gastric acid reduction leads to an alteration in lower intestinal microflora

To clarify the alterations in lower intestinal microflora induced by gastric acid reduction, the dynamics of 12 major genera or groups of bacteria comprising the microflora in feces and colonic contents were examined by quantitative real-time PCR in proton pump inhibitor-treated rats and in asymptomatic human subjects with hypochlorhydria. In both rat and human experiments, most genera or groups of intestinal microflora (facultative and obligate anaerobes) proliferated by gastric acid reduction, and marked and significant increases in the Lactobacilli group and Veillonella, oropharyngeal bacteria, were observed. In rats, potent gastric acid inhibition led to a marked and significant increase of intestinal bacteria, including the Bacteroidesfragilis group, while Bifidobacterium, a beneficial bacterial species, remained at a constant level. These results strongly indicate that the gastric acid barrier not only controls the colonization and growth of oropharyngeal bacteria, but also regulates the population and composition of lower intestinal microflora.     

Probiotic properties of Lactobacillus isolates originating from porcine intestine and feces

In this study, a total of 94 lactic acid bacterial (LAB) isolates of porcine small intestinal and fecal origin were screened for their probiotic properties. The aim was to evaluate whether their isolation site and putative species identity play a role in these characteristics and whether either of these can be used as a predictive factor for the probiotic potential of bacterial isolates. The isolates were preliminarily identified by partial 16S rRNA gene sequencing and characterized in vitro for their pH and bile tolerance, adhesion capacity towards porcine enterocytes isolated from five intestinal sites and for antimicrobial activity towards five indicator pathogens. The interdependence of these characteristics was statistically evaluated. The isolates tolerated low pH and bile well. Adherence to the enterocytes of different origins did not correlate with the strain isolation site. In general, higher adherence was observed to colon cells in comparison to the small intestinal enterocytes. Culture filtrates of the isolates caused a decrease of up to three orders of magnitude in the intestinal pathogen cell numbers. The inhibition was mostly due to lactic and other organic acids. The predominating phylotypes identified were Lactobacillus reuteri and Lactobacillus salivarius, of which the former generally had the best adhesion capacity, whereas the latter appeared to be the best inhibitor. Based on the results, several strains of the pig Lactobacillus isolates tested may function as promising candidates for use in probiotic products. However, it was not possible to use the isolation site or the species identity of the isolates as reliable preliminary screening factors.