Characteristics of microorganisms colonizing human intestine

Introduction of novel methods of microbial diagnostics has considerably broadened our conceptions on the qualitative and quantitative variety of microorganisms inhabiting human gastrointestinal tract. In this review morphological and functional properties of obligate anaerobic bacteria (bifidobacteria, lactobacilli, eubacteria, peptostreptococci, clostridia, bacteroids, fusobacteria) and facultative anaerobic microorganisms (enterobacteria, staphylococci, streptococci, yeast-like fungi of the genus Candida) capable of colonizing human intestine are briefly characterized.     

Diversity,ecology and intestinal function of bifidobacteria

The human gastrointestinal tract represents an environment which is a densely populated home for a microbiota that has evolved to positively contribute to host health. At birth the essentially sterile gastrointestinal tract (GIT) is rapidly colonized by microorganisms that originate from the mother and the surrounding environment. Within a short timeframe a microbiota establishes within the (breastfed) infant's GIT where bifidobacteria are among the dominant members, although their numerical dominance disappears following weaning. The numerous health benefits associated with bifidobacteria, and the consequent commercial relevance resulting from their incorporation into functional foods, has led to intensified research aimed at the molecular understanding of claimed probiotic attributes of this genus. In this review we provide the current status on the diversity and ecology of bifidobacteria. In addition, we will discuss the molecular mechanisms that allow this intriguing group of bacteria to colonize and persist in the GIT, so as to facilitate interaction with its host.     

Diversity, vitality and activities of intestinal lactic acid bacteria and bifidobacteria assessed by molecular approaches

While lactic acid bacteria and bifidobacteria have been scientifically important for over a century, many of these are marketed today as probiotics and have become a valuable and rapidly expanding sector of the food market that is leading functional foods in many countries. The human gastro-intestinal tract with its various compartments and complex microbiota is the primary target of most of these functional foods containing lactic acid bacteria and bifidobacteria (LAB&B). In addition, their use as vectors for delivery of molecules with therapeutic value to the host via the intestinal tract is being studied. This review focuses on molecular approaches for the investigation of the diversity of lactic acid bacteria and bifidobacteria in the human intestine, as well as tracking of probiotic bacteria within this complex ecosystem. Moreover, methodologies to determine the viability of the lactic acid bacteria and bifidobacteria and molecular approaches to study the mechanisms by which they adapt, establish and interact with the human host via the digestive tract, are described.     

Gastrointestinal flora of cotton rats

The gastrointestinal (GI) flora of cotton rats was examined. No lactobacilli were detected in any part of the GI tract. Anaerobes, including Peptococcaceae, Bacteroidaceae, bifidobacteria and eubacteria, were the predominant bacteria in the stomach, small intestine, caecum and faeces. Aerobes and facultative anaerobes, including Enterobacteriaceae and streptococci, were detected at low numbers and very low frequency of occurrence in all parts of the GI tract. Sixty-one isolates of bifidobacteria were recovered from the stomach, small intestine, caecum and faeces of cotton rats. They were identified as Bifidobacterium animalis, B. pseudolongum biovar a and b. The study showed that the GI flora of cotton rats seem to be very different from the GI flora in other rodents.     

Fecal microflora of Greek healthy neonates

This study aimed to explore, in our geographical region, the development of intestinal microflora and the colonization patterns of lactic acid bacteria and bifidobacteria during the first three months of life and to investigate the effect of the mode of delivery. Fecal specimens from 82 healthy, full-term infants were collected prospectively 4, 30 and 90 days after delivery and subcultured on nonselective and selective media. Identification of isolates was performed by microbiological and molecular methods. For the delivery effect, two groups of vaginally or caesarean-delivered exclusively breast-fed infants were studied. Despite the early high total counts of aerobes and anaerobes, colonization of lactobacilli and bifidobacteria was overall limited until 3 months of age. Furthermore, caesarean-delivered infants were less often colonized with lactobacilli at day 4 (4% vs. 59%, p = 0.000) and with bifidobacteria at day 4 (0% vs. 23%, p = 0.015) and 30 (0% vs. 35%, p = 0.042) compared to vaginally delivered ones. No bacterial populations differences were detected to compare colonized infants. Identification results indicated the predominance of Lactobacillus rhamnosus, Lactobacillus johnsonii and Lactobacillus paracasei species in neonatal gut microflora up to the first month of life and diversity of Lactobacillus species in vaginally delivered, colonized newborns, at fourth day. Furthermore, Bifidobacterium longum and Bifidobacterium breve were the most frequently detected Bifidobacterium species in vaginally delivered, breast-fed infants. In conclusion our study revealed a restricted colonization pattern of lactic acid bacteria in Greek infants and a delay in the development of Lactobacillus and Bifidobacterium spp. microbiota after caesarean section. Further analysis of potential consequences of these findings is required.     

Behaviour of the pharmaceutical probiotic preparation VSL#3 in human ileostomy effluent containing its own natural elements

The pharmaceutical probiotic VSL#3 (300 billion cfu/g lactic acid bacteria & bifidobacteria) was inoculated into human ileostomy effluent (HIE) to assess its behaviour vs the ileo-caecal tract. Separately, yogurt bacteria (yog) and bifidobacteria (Bif) present in VSL#3 were also inoculated into HIE. During 37 degrees C incubation (anaerobic condition) at zero, six and 24 hours, both cell growth in control HIE and indigenous Bif growth in HIE+yog were observed. Cells remained viable and metabolically active as shown by the increase in L(+) lactic acid in HIE+VSL#3 and HIE+yog and the pH decreased (approx. 5.5 compared with the 6.2 of control HIE). Total SCFA Short Chain Fatty Acids decreased in HIE+yog and HIE+VSL#3 at 6h and in all cultures at 24h; butyric acid decreased in HIE+Bif and HIE+VSL#3. Generally in vivo the bacteria remain in the ileo-caecal tract no longer than six h, therefore VSL#3 bacteria seem able to pass this barrier viably, colonizing the large bowel.     

Effects of age and starvation on the gastrointestinal microflora and the heat resistance of fecal bacteria in rats.

The microflora of the gastrointestinal tract in rats 1 day to 100 weeks old, of the cecal contents and wall in starved rats, and of heat-treated feces of normal rats was determined by cultural examination. Streptococci, staphylococci, lactobacilli, actinobacilli, and coliforms colonized the tract during the 1st week of life. Bacteroidaceae, veillonellae, catenabacteria (composed of eubacteria and anaerobic lactobacilli), clostridia, bifidobacteria, anaerobic gram-positive cocci, fusiform bacteria, curved rods, and spirochetes appeared when the rats were 2 to 4 weeks old. Yeasts were slower in colonizing the tract than any other organism. Dramatic changes occurred in the microflora of rats 2 to 4 weeks of age. There was a time lag between the changes in enterococcal and coliform populations. The enterococcal population was depressed over a period from 2 to 6 weeks of age. Bifidobacteria showed a larger population at 4 to 9 weeks than at any other age. The microflora of the stomach was the same as that of the small intestine, with some exceptions. It differed markedly from that of the cecum. The ratio of total aerobic count to anaerobic count gradually increased in the stomach, but decreased in the cecum, with advance in age. The microorganisms distributed in the tract could be divided roughly into 3 types. The population of each organism, except spirochetes, in the cecal wall was approximately 1/1,000 of that in the cecal contents. One of the 2 types of spirochetes was found only in the cecal wall and in a high incidence, forming a large population. In rats starved for 48 hr, coliforms, Proteus spp., anaerobic gram-positive cocci, Clostridia, and some bacteroidaceae showed an increase in population in the cecum, but lactobacilli, veillonellae, and spirochetes decreased. The major organisms cultured from the heat-treated feces were fusiform and curved bacteria, some members of Bacillus, minor anaerobic cocci, and straight rods.     

Evidence for the Colonization of Lactic Acid Bacteria in the Gastrointestinal Tract of Suckling Mink

Lactic acid bacteria are considered indigenous members of the gastrointestinal microflora in a number of animal species (Savage 1977a). Some intestinal strains of lactobacilli and streptococci are aWe to adhere to stratified squamous epithelium of some animals (Tannock et al. 1987), in the non-secreting part of the stomach of piglets (Barrow et al. 1980, Fuller et al. 1978) and rodents (Tannock et al. 1982), and in the crop of poultry (Fuller 1978). The presence of lactic acid bacteria in the digestive tract is believed to be of beneficial value to the host animal (Fuller 1989). The production of organic acids in the stomach or the crop helps maintaining a low pH which may be important for inhibiting the colonization of potentially pathogenic bacteria, particularly in the newborn animal (Barrow et al 1980, Fuller 1977, Fuller 1978). The adhesion of lactobacilli to squamous epithelium is host specific: strains capable of adhering to the epithelium of piglets are usually not able to adhere in rodents or poultry and vice versa (Fuller 1978, Lin & Savage 1984, Tannock et al 1982). Adhesion of lactic acid bacterial strains to other epithelia than stratified squamous epithelium has been reported. Thus, the attachment of lactobacilli to cells from the secreting epithelium of the murine stomach (Kotarski & Savage 1979), to intestinal cells of humans (Goldin & Gorbach 1987), and to columnar epithelial cells of piglets and calves (Mäyrä-Mäkinen et al 1983) has been demonstrated using in vitro methods. In another study the in vivo attachment of Enterococcus faecium to duodenal epithelium of gnotobi-otic chickens was demonstrated (Fuller et al 1981). Recent research indicated that in adult mink lactic acid bacteria are not indigenous members of the intestinal flora, and they do not attach to epithelium in any part of the gastrointestinal tract (Federsen & Jørgensen 1992). The present paper presents evidence that Gram positive cocci may colonize the gut of suckling mink kits and attach to the gut mucosa.     

Intestinal colonization of laboratory rats with Oxalobacter formigenes.

Six strains of Oxalobacter formigenes (anaerobic oxalate-degrading bacteria) were examined for their ability to colonize the gastrointestinal tracts of adult laboratory rats. These rats did not harbor O. formigenes. Strain OxCR6, isolated from the cecal contents of a laboratory rat that was naturally colonized by oxalate-degrading bacteria, colonized the ceca and colons of adult rats fed a diet that contained 4.5% sodium oxalate. Five days after rats were inoculated intragastrically with 10(9) viable cells of strain OxCR6, oxalate degradation rates in cecal and colonic contents increased by 19 and 40 times, respectively. Viable counts of strain OxCR6 from these rats averaged 10(8)/g (dry weight) of cecal contents. Strain OxCR6 was not detected in the cecal contents of inoculated rats fed diets that contained less than 3.0% sodium oxalate. Strains of O. formigenes isolated from the cecal contents of swine, guinea pigs, and wild rats and from human feces also colonized the ceca of laboratory rats; a ruminal strain failed to colonize the rat cecum.     

Variable response to exogenous Lactobacillus acidophilus NCFM consumed in different delivery vehicles

AIMS: To study the effects of the delivery vehicle for Lactobacillus acidophilus on the human faecal microbiota. Our hypotheses were that (i) the delivery vehicle would influence faecal lactobacilli numbers and (ii) consumption of Lact. acidophilus would influence the populations of Bifidobacterium and hydrogen sulphide-producing bacteria. METHODS AND RESULTS: Ten subjects each received Lact. acidophilus with skim milk or water. Lactobacillus, Bifidobacterium and hydrogen sulphide-producing bacterial populations were analysed before, during and after each treatment. Regardless of the vehicle, faecal lactobacilli populations changed during treatment. Bifidobacteria and the hydrogen sulphide-producing bacteria underwent no statistically significant population changes. Intra- and intersubject variability was observed. CONCLUSIONS: The vehicle in which Lact. acidophilus was delivered did not influence faecal lactobacilli numbers. Consumption of Lact. acidophilus did not influence the populations of Bifidobacterium and hydrogen sulphide-producing bacteria. The lactobacilli populations of subjects were variable. The fed lactobacilli did not appear to colonize the gastrointestinal tract. SIGNIFICANCE AND IMPACT OF THE STUDY: We provide evidence that (i) there was no collective advantage to using skim milk as a delivery vehicle vs water; (ii) exogenous Lact. acidophilus did not affect endogenous bifidobacteria or hydrogen sulphide-producing bacteria; (iii) data should be carefully examined before pooling for analysis and (iv) continuous feeding was required to maintain an elevated lactobacilli population.     

Avoparcin and vancomycin: useful antibiotics for the isolation of brewery lactic acid bacteria

Vancomycin (30 μg) and avoparcin (20 μg) have no effect on growth of hetero-fermentative lactobacilli, mesophilic homofermentative lactobacilli, Lactobacillus salivarius and bacteria of the genera Leuconostoc and Pediococcus. The growth of thermophilic homofermentative lactobacilli (with the exception of Lacto salivarius ), enteric and lactic streptococci and all other Gram-positive bacteria encountered in breweries (and likely to be encountered in foods) is inhibited by these antibiotics. The recovery of sub-lethally stressed cells is slightly reduced by the presence of either antibiotic. Vancomycin and avoparcin are useful selective agents for the detection of lactic acid bacteria in the brewing industry and may also be of use, for quality control purposes, in the distilling, wine, cider, dairy and meat industries. These antibiotics may also find applications in differentiation of pediococci from streptococci, and in rapid identification of lactobacilli.     

Gnotobiotic Silage

Selected strains of lactic acid bacteria isolated from grass silage were found to flourish when inoculated into irradiation-sterilized forage under gnotobiotic conditions. The acid content and pH of these silages resembled naturally fermented silage. Inoculation of gnotobiotic silage with Clostridium sporogenes and C. tyrobutyricum failed to cause any noticeable deterioration of silage quality.     

The inhibitory activity of Lactobacillus spp. isolated from breast milk on gastrointestinal pathogenic bacteria of nosocomial origin

Milk acts as a mean for transporting many essential substances from the mother to the child. In human beings, milk includes several predominant bacteria, such as staphylococci, streptococci, micrococci, lactobacilli, enterococci, lactococci and bifidobacteria. Besides, its intake favors the predominance of bifidobacteria and lactobacilli in the child’s intestinal microbiota. The present work explores the isolation and selection of lactobacilli strains with probiotic potential, focusing in their degree of hydrophobicity and antagonism against important gastrointestinal nosocomial pathogens. 98 lactobacilli were isolated from 48 breast milk samples, with most strains belonging to the obligately homofermentative group (36.7%). 63% of the isolated strains showed a high degree of hydrophobicity when tested on three solvents and were selected for detecting antimicrobial activity against gastrointestinal pathogens, including Escherichia coli, Shigella spp, Pseudomonas spp and Salmonella spp strains. When applying the agar diffusion test, many isolated strains presented inhibitory activity against pathogenic strains. We observed that: Salmonella enteriditis was the most inhibited pathogen, and the strains with the most inhibitory power were AR2 and O1 (both highly hydrophobic lactic acid bacteria), which showed an opposing effect against all nosocomial pathogens tested. Although more in vitro, in vivo or clinical data would be needed before any conclusion on the probiotic properties of the strains can be drawn, our results demonstrate that some of the tested strains may have good probiotic potential for their inclusion in products targeting infants.     

Specific identification and targeted characterization of Bifidobacterium lactis from different environmental isolates by a combined multiplex-PCR approach

The species Bifidobacterium lactis, with its main representative strain Bb12 (DSM 10140), is a yoghurt isolate used as a probiotic strain and is commercially applied in different types of yoghurts and infant formulas. In order to ensure the genetic identity and safety of this bacterial isolate, species- and strain-specific molecular tools for genetic fingerprinting must be available to identify isolated bifidobacteria or lactic acid bacteria from, e.g., various clinical environments of relevance in medical microbiology. Two opposing rRNA gene-targeted primers have been developed for specific detection of this microorganism by PCR. The specificity of this approach was evaluated and verified with DNA samples isolated from single and mixed cultures of bifidobacteria and lactobacilli (48 isolates, including the type strains of 29 Bifidobacterium and 9 Lactobacillus species). Furthermore, we performed a Multiplex-PCR using oligonucleotide primers targeting a specific region of the 16S rRNA gene for the genus Bifidobacterium and a conserved eubacterial 16S rDNA sequence. The specificity and sensitivity of this detection with a pure culture of B. lactis were, respectively, 100 bacteria/ml after 25 cycles of PCR and 1 to 10 bacteria/ml after a 50-cycle nested-PCR approach.     

Enrichment of an alkaline-adapted association of streptococci and lactobacilli

By gradually increasing the starting pH during subsequent enrichment test runs, an association of lactic acid bacteria adapted readily to alkaline pH values. An association of homofermentative lactobacilli and streptococci capable of initiating growth at pH 10˙0 and rapidly producing lactic acid (3˙11 g/l/d) down to a final pH of ca 4˙0 was thus obtained. The streptococci present in the enriched association were capable of growing at pH 9˙5, whereas the lactobacilli were only capable of surviving at such high pH values.     

Establishment and development of lactic acid bacteria and bifidobacteria microbiota in breast-milk and the infant gut

The initial establishment of lactic acid bacteria (LAB) and bifidobacteria in the newborn and the role of breast-milk as a source of these microorganisms are not yet well understood. The establishment of these microorganisms during the first 3 months of life in 20 vaginally delivered breast-fed full-term infants, and the presence of viable Bifidobacterium in the corresponding breast-milk samples was evaluated. In 1 day-old newborns Enterococcus and Streptococcus were the microorganisms most frequently isolated, from 10 days of age until 3 months bifidobacteria become the predominant group. In breast-milk, Streptococcus was the genus most frequently isolated and Lactobacillus and Bifidobacterium were also obtained. Breast-milk contains viable lactobacilli and bifidobacteria that might contribute to the initial establishment of the microbiota in the newborn.     

Establishment of specific pathogen-free (SPF) rat colonies using gnotobiotic techniques.

Gnotobiotic Wistar rats were produced using gnotobiotic techniques, which were established in the production of a SPF mouse colony, in order to establish a barrier-sustained colony. One strain of Escherichia coli, 28 strains of Bacteriodaceae (B-strains), three strains of Lactobacillus (L-strains) and a chloroform-treated fecal suspension (CHF, Clostridium mixture) were prepared from conventional Wistar rats as the microflora source. Two groups of limited-flora rats, E. coli plus B-strains and E. coli plus CHF, were produced. After confirmation that Clostridium difficile was not detected in the CHF-inoculated rats, two groups of limited-flora rats were transferred to an isolator and housed together in a cage. These rats were then orally inoculated with L-strains. The gnotobiotic rats showed colonization resistance to Pseudomonas aeruginosa, and the number of E. coli in the feces was 10(5) to 10(6)/g. The gnotobiotic rats were transferred to a barrier room as a source of intestinal flora for SPF colonies. In the SPF rats, basic cecal flora was mainly composed of Bacteroidaceae, clostridia, fusiform-shaped bacteria and lactobacilli, and did not change over a long period. Their flora became similar to that of conventional rats.     

Distribution of indigenous Lactobacilli in the digestive tract of conventional and gnotobiotic rats.

Distribution of indigenous lactobacilli in the gastrointestinal tracts of rats was investigated at the species level. The indigenous lactobacilli isolated from conventional rats were divided into three groups, Lactobacillus acidophilus and its related strains, L. fermentum, and L. murini. Localization of the Lactobacillus groups in the gastrointestinal tracts could be distinguished clearly based on arabinose and glucose fermentation reaction of isolates from each part of the gastrointestinal tract. Group I (L. acidophilus and the related strains) and Group II (L. fermentum) were the major populations of lactobacilli on the walls of the non-glandular part and in the contents of the stomachs of both conventional and gnotobiotic rats. Group I predominated in all the parts of the digestive tract of conventional rats, whereas Group II was in the minority in the lower part of the gastrointestinal tracts of both groups of rats. Group III (L. murini) was the predominant population of Lactobacillus in the lower part of small intestine of conventional rats and in all parts of the gastrointestinal tracts of gnotobiotic rats except for the wall of the non-glandular part of the stomach.     

Gnotobiotic Silage

Selected strains of lactic acid bacteria isolated from grass silage were found to flourish when inoculated into irradiation-sterilized forage under gnotobiotic conditions. The acid content and pH of these silages resembled naturally fermented silage. Inoculation of gnotobiotic silage with Clostridium sporogenes and C. tyrobutyricum failed to cause any noticeable deterioration of silage quality.     

Role of indigenous lactobacilli in gastrin-mediated acid production in the mouse stomach

It is known that the stomach is colonized by indigenous lactobacilli in mice. The aim of this study was to examine the role of such lactobacilli in the development of the stomach. For a DNA microarray analysis, germ-free BALB/c mice were orally inoculated with 10(9) CFU lactobacilli, and their stomachs were excised after 10 days to extract RNA. As a result, lactobacillus-associated gnotobiotic mice showed dramatically decreased expression of the gastrin gene in comparison to germ-free mice. The mean of the log(2) fold change in the gastrin gene was -4.3. Immunohistochemistry also demonstrated the number of gastrin-positive (gastrin(+)) cells to be significantly lower in the lactobacillus-associated gnotobiotic mice than in the germ-free mice. However, there was no significant difference in the number of somatostatin(+) cells in these groups of mice. Consequently, gastric acid secretion also decreased in the mice colonized by lactobacilli. In addition, an increase in the expression of the genes related to muscle system development, such as nebulin and troponin genes, was observed in lactobacillus-associated mice. Moreover, infection of germ-free mice with Helicobacter pylori also showed the down- and upregulation of gastrin and muscle genes, respectively, in the stomach. These results thus suggested that indigenous lactobacilli in the stomach significantly affect the regulation of gastrin-mediated gastric acid secretion without affecting somatostatin secretion in mice, while H. pylori also exerts such an effect on the stomach.