Comparison of media for the detection of bifidobacteria, lactobacilli and total anaerobes from faecal samples.

The interest in functional foods, probiotics and prebiotics requires a proper method to determine specific bacterial groups in the intestinal flora, especially bifidobacteria and lactobacilli. Three media for lactobacilli (MRS, Rogosa, LAMVAB), three media for bifidobacteria (RB, NPNL, Beerens medium) and nine media for total anaerobes have been tested for selectivity and recovery. For total anaerobes Faecal Reinforced Clostridial Agar (FRCA) showed the highest cfu/g, followed by Columbia Blood Agar and BHI Blood agar. There were no significant differences between the media tested. Reduced physiological salt solution was found to be the best dilution medium. For bifidobacteria and lactobacilli samples of human faeces, cat faeces and pig ileal contents were used. Bifidobacteria could reliably be determined on all three media tested in human faeces, but not on pig ileal contents or cat faeces. Absolute counts were highest in human samples. No lactobacilli could be isolated on MRS in either sample, none of the colonies in the countable plates were lactobacilli. For Rogosa over 90% of the colony types observed in human samples were not lactobacilli. For cat faeces this was 58%, but no false positives were observed in the pig ileal samples. For LAMVAB the percentages of false positive colony types were 9, 14 and 0% for human, cat and pig samples. It can be concluded that for bifidobacteria RB and Beerens medium show comparable results, and can be used to quantify bifidobacteria in human faeces, but none of the media tested is suitable for reliably counting bifidobacteria from pig and cat samples. For lactobacilli LAMVAB shows the highest sensitivity.     

Probiogenomics as a tool to obtain genetic insights into adaptation of probiotic bacteria to the human gut

Bifidobacteria and lactobacilli are widely exploited as health-promoting bacteria in many functional foods. However, the molecular mechanisms as to how these bacteria positively impact on host health are far from completely understood. For this reason these microorganisms represent a growing area of interest with respect to their genomics, molecular biology and genetics. Recent genome sequencing of a large number of strains of bifidobacteria and lactobacilli has allowed access to the complete genetic makeup of representative members of these bacteria. Here, we will discuss how the analysis of genomic data has helped us to understand the mechanisms by which these bacteria adapt to the specific environment of the gastrointestinal tract, while also revealing genetic functions that mediate specific host-microbe interactions.     

Generation of NO by probiotic bacteria in the gastrointestinal tract

Probiotic bacteria elicit a number of beneficial effects in the gut but the mechanisms for these health promoting effects are not entirely understood. Recent in vitro data suggest that lactobacilli can utilise nitrate and nitrite to generate nitric oxide, a gas with immunomodulating and antibacterial properties. Here we further characterised intestinal NO generation by bacteria. In rats, dietary supplementation with lactobacilli and nitrate resulted in a 3-8 fold NO increase in the small intestine and caecum, but not in colon. Caecal NO levels correlated to nitrite concentration in luminal contents. In neonates, colonic NO levels correlated to the nitrite content of breast milk and faeces. Lactobacilli and bifidobacteria isolated from the stools of two neonates, generated NO from nitrite in vitro, whereas S. aureus and E. coli rapidly consumed NO. We here show that commensal bacteria can be a significant source of NO in the gut in addition to the mucosal NO production. Intestinal NO generation can be stimulated by dietary supplementation with substrate and lactobacilli. The generation of NO by some probiotic bacteria can be counteracted by rapid NO consumption by other strains. Future studies will clarify the biological role of the bacteria-derived intestinal NO in health and disease.     

Search of promising strains of bifidobacteria and lactobacillus for the development of new biopreparations

For the first time the species composition of bifidobacteria and lactobacilli in clinically healthy young children has been studied. As revealed in this study, the dominating species of bifidobacteria are B. longum, B. adolescentis and B. infantis, while the dominating species of lactobacilli are Lactobacillus acidophilus and L. rhamnosus. In 83 isolated cultures of bifidobacteria and 34 isolated species of lactobacilli the activity of acid formation and the antagonistic activity with respect to Escherichia coli, Klebsiella pneumoniae, Staphylococcus aureus, Enterococcus faecalis, Clostridium perfringens have been tested. B. longum strain 58B, B. infantis strain 37B, L. rhamnosus strain 12L and L. acidophilus strain 27L, typical for children of this age group, having good antagonistic activity and pronounced acid-forming properties, have been selected. These strains hold good promise to be used as the basis for the development of a complex probiotic preparation for correcting intestinal microflora in young children.     

Bifidobacterium and Lactobacillus DNA in the human placenta

Aims:  Bifidobacteria and lactobacilli are part of the human normal intestinal microbiota and may possibly be transferred to the placenta. It was hypothesized that intestinal bacteria or their components are present in the placenta and that the foetus may be exposed to them. We investigated the presence of bifidobacteria and lactobacilli and their DNA in the human placenta.
Methods and Results:  We studied 34 human placentae (25 vaginal and nine caesarean deliveries) for the presence Bifidobacterium spp. and Lactobacillus rhamnosus. Cultivation was used for the detection of viable cells and genus and species-specific PCR for the detection of DNA. No bifidobacteria or lactobacilli were found by cultivation. Bifidobacterial DNA was detected in 33 and L. rhamnosus DNA in 31 placenta samples.
Conclusions:  DNA from intestinal bacteria was found in most placenta samples. The results suggest that horizontal transfer of bacterial DNA from mother to foetus may occur via placenta.
Significance and Impact of the Study:  Bacterial DNA contains unmethylated CpG oligodeoxynucleotide motifs which induce immune effects. Specific CpG motifs activate Toll-like receptor 9 and subsequently trigger Th-1-type immune responses. Although the newborn infant is considered immunologically immature, exposure by bacterial DNA may programme the infant's immune development during foetal life earlier than previously considered.     

Use of antibiotic-resistant Bifidobacterium in treating ulcerative-necrotic enterocolitis in young children

Ulceronecrotic colitis in premature children is accompanied by the development of pronounced intestinal dysbacteriosis characterized by a sharp increase in the number of Escherichia, enterococci, staphylococci and the appearance of opportunistic enterobacteria (Klebsiella, Serratia, Citrobacter, Proteus) in large amounts. Clinical convalescence was observed in 2 weeks in children receiving antibiotic-resistant bifidobacteria with antibiotics and in 3-4 weeks in children receiving commercial bifidobacteria with antibiotics, while children treated only with antibiotics showed no signs of clinical convalescence during the whole course of treatment. After the course of treatment the most effective recovery of the intestinal microflora was observed in the group of patients who had received antibiotics in combination with antibiotic-resistant bifidobacteria. It was manifested by a decrease in the number of Escherichia, Klebsiella, Proteus, Serratia, staphylococci and enterococci simultaneously with an increase in the number of endogenous lactobacilli and bifidobacteria. In those children who had received commercial bifidobacteria in combination with antibiotics or had been treated only with antibiotics the process of the recovery of the intestinal microflora was considerably less pronounced than in the patients of the first group; a decrease in the number of Escherichia, Klebsiella, Proteus and Serratia occurred to a lesser extent, and at the same time an essential increase in the number of enterococci and staphylococci was observed. The level of endogenous lactobacilli and bifidobacteria was considerably reduced. Antibiotic-resistant bifidobacteria actively took in the intestine of the patients. On day 5 after the course of treatment was over their level was (4.3 +/- 0.4) X 10(4) cells/g feces.     

Studies on Streptococci

The population levels of bacteria in the contents and the walls of the gastrointestinal tract of gnotobiotic rats inoculated with lactic acid bacteria (streptococci, lactobacilli, and bifidobacteria) from humans and rats were determined. Lactobacilli as well as streptococci isolated from rats colonized in the digestive tracts of the gnotobiotic rats at a high population level, characteristically highest in the stomach. On the other hand, in the rats inoculated with human lactic acid bacteria, streptococci were dominant in the lower tract. The human lactobacilli or bifidobacteria did not colonize when the organisms in each genus were inoculated together with streptococci. However, when all three genera were inoculated together, lactobacilli and bifidobacteria colonized. Observations on the species of streptococci showed that the intestinal type of streptococci was found to colonize at a high population level, but the oral type was not. Strains of the same genus of lactic acid bacteria from humans and from rats showed different colonization patterns.     

马齿苋多糖对溃疡性结肠炎小鼠肠道菌群及血内毒素的影响

目的应用马齿苋多糖对溃疡性结肠炎小鼠进行调整治疗,达到从微生态学角度防治溃疡性结肠炎。方法应用DSS制备溃疡性结肠炎小鼠模型,随机分成2组：正常对照组、模型组,造模成功后模型组再分为自然恢复组、马齿苋多糖治疗组。分别于造模后、给药7 d后处死小鼠,进行肠道菌群检测、血内毒素含量测定。结果 DSS造模后模型组小鼠肠道菌群失调,外周血内毒素含量升高。马齿苋多糖治疗7 d后治疗组小鼠肠道双歧杆菌和乳酸杆菌数量明显上升,外周血内毒素含量明显下降。结论马齿苋多糖可以提高双歧杆菌和乳酸杆菌数量,降低外周血内毒素含量,调节肠道微生态失调,对溃疡性结肠炎发挥了一定的治疗作用。     

Analysis of fecal populations of bifidobacteria and lactobacilli and investigation of the immunological responses of their human hosts to the predominant strains.

The bifidobacterial and lactobacillus populations of fecal samples collected from 10 human subjects were studied. The numbers of bifidobacteria were similar in the fecal samples of all of the subjects, but lactobacillus numbers varied, even between samples collected from the same individual. Analysis of the composition of the bacterial populations by ribotyping and pulsed-field gel electrophoresis to differentiate between strains showed that, at least for the numerically predominant strains, each subject harbored a unique collection of bifidobacteria and lactobacilli. Predominant bifidobacterial and lactobacillus strains detected in the feces of each subject were used in immunological assays (lymphocyte transformation, serum antibody titers) to determine the influence of the bacteria on the immune system of their host. Immunoglobulin G antibodies reactive with lactobacilli were detected at high concentrations; antibodies reactive with bifidobacteria were present at lower concentrations. The antibodies appeared to be genus specific rather than strain specific. The results of the study emphasized the complexity of the relationship that exists between the intestinal microflora and the human host.     

Intestinal microflora and homeostasis of the mucosal immune response: implications for probiotic bacteria?

The intestinal microflora can be considered a postnatally acquired organ that is composed of a large diversity of bacteria that perform important functions for the host and can be modulated by environmental factors, such as nutrition. Specific components of the intestinal microflora, including lactobacilli and bifidobacteria, have been associated with beneficial effects on the host, such as promotion of gut maturation and integrity, antagonisms against pathogens and immune modulation. Beyond this, the microflora seems to play a significant role in the maintenance of intestinal immune homeostasis and prevention of inflammation. The contribution of the intestinal epithelial cell in the first line of defense against pathogenic bacteria and microbial antigens has been recognized. However, the interactions of intestinal epithelial cells with indigenous bacteria are less well understood. This review will summarize the increasing scientific attention to mechanisms of the innate immune response of the host towards different components of the microflora, and suggest a potential role for selected probiotic bacteria in the regulation of intestinal inflammation.     

Denaturing gradient gel electrophoresis (DGGE)-based monitoring of intestinal lactobacilli and bifidobacteria of pigs during a feeding trial

The aim of this study was to determine the influence of different feeding strategies on the gut microbiota of organic growing-finishing pigs. A total of 76 pigs were allocated to four different dietary treatments (control, probiotics, maize silage and grass silage). Effects of the applied probiotic preparation on the composition of the intestinal and faecal microbiota were monitored. By using a DGGE (denaturing gradient gel electrophoresis)-based methodology, fingerprints of the intestinal microbiota were obtained. The total microbial DNA was isolated from faecal and colon samples and amplified with PCR using different primer sets to detect bifidobacteria and lactobacilli. PCR products were separated using DGGE and the resulting profiles were compared with the findings of the other dietary treatments. Bands were excised from the gels and sequenced for further identification. Particularly two different DGGE profiles of bifidobacteria were observed, while lactobacilli showed larger variety within the dietary treatments.     

Dermal bioactives from lactobacilli and bifidobacteria

Lactobacilli and bifidobacteria are the most common genera of probiotics with documented potentials on gut health. Recent studies have suggested that such potentials can be extended beyond gut well-being, such as that of dermal health. Our present study aimed to evaluate the production of bioactives that are essential for skin defense, such as lipoteichoic acid, peptidoglycan, hyaluronic acid, sphingomyelinase, lactic acid, acetic acid, and diacetyl, from lactobacilli and bifidobacteria grown in milk. All strains studied showed the presence of LTA in the cell wall fraction, with higher amounts from Lactobacillus rhamnosus FTDC 8313 and Bifidobacterium longum BL 8643 than other strains studied. Meanwhile, all strains studied showed equal concentrations of cell wall peptidoglycan. Our results showed that all strains studied were capable of producing hyaluronic acid, with higher production by lactobacilli than bifidobacteria. Production of diacetyl was more prevalent from strains of lactobacilli, while bifidobacteria produced higher amounts of acetic acid. Strains of lactobacilli and bifidobacteria studied also produced acid and neutral sphingomyelinase, an enzyme that generates ceramides and subsequent development of physical barriers in the stratum corneum. Our current findings show that bioactive and inhibitive extracts are produced from the fermentation of lactobacilli and bifidobacteria in milk, with potentials for dermal applications.     

Applyed aspects of molecular biology of Bifidobacteria and Lactobacillus

The data obtained in the molecular genetic studies of the representatives of the genera Bifidobacterium and Lactobacillus are given. At present the genomes of Bifidobacterium longum, Lactobacillus acidophilus and Lactobacillus plantarum sized 2,256.640, 1,993.564 and 3,308.274 base pairs respectively, were completely sequenced. The use of different methods of gene indication and gene typing essentially widened the existing notions of the species variety of these bacteria, made it possible to change the classification of known species and to find out new ones, vegetating in different biotopes of the human body. Molecular genetic approaches also ascertained the taxonomic position of production strains of lactic-acid bacteria used in food industry, helped determine the kinetics of the passage of probiotic strains of bifidobacteria and lactobacilli along the gastrointestinal tract, as well as determine the duration of their preservation in the intestine after oral administration. In addition, the data on the evaluation of the therapeutic effect of some preparations, probiotics and synbiotics, with determination of the population level of endogenic and orally introduced exogenic bifidobacteria and lactobacilli are presented.     

Does the intestinal bifidobacterial colonisation affect bacterial translocation?

The aim of this work was to investigate the possible role of the intestinal anaerobic flora (especially bifidobacteria) in regulating bacterial translocation (BT) which can be defined as the passage of intestinal microbes through the mucosa to internal organs. Default in BT regulation concurs with pathogenesis of sepsis in various human conditions, such as acute pancreatitis, cirrhosis, necrotising enterocolitis or multiple organ failure. The intestinal flora was studied in human flora associated mice (HF mice) and BT was quantified in Peyer's patches (PP), blood, spleen, liver and lungs. HF mice displayed a heterogenic intestinal colonisation with bifidobacteria. High colonisation of both caecum and colon by bifidobacteria led to a poorer bacterial contamination of blood, liver and lungs. Moreover, ileal, caecal and colonic bifidobacterial counts negatively correlated with the bacterial dissemination (number of contaminated organs per mouse). In contrast, Bacteroides fragilis group counts positively correlated with bacteraemia, lungs contamination or bacterial dissemination. Additionally, clostridia localised in the colon affected bacterial uptake by PP and lungs contamination as indicated by positive correlations between bacterial populations in these respective locations. These results indicate that bifidobacteria, when established in high counts, reduced BT to liver, blood and lungs, whereas B. fragilis group favoured the bacterial passage. Clostridia established in the distal ileum also seemed to favour BT to lungs. The manipulation of the bacterial flora to optimise the regulatory effect on BT should therefore focus on the selective promotion of bifidobacteria and avoid an increase in potentially detrimental populations such as B. fragilis group and clostridia.     

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.     

Indigenous bacteria and bacterial metabolic products in the gastrointestinal tract of broiler chickens

The gastrointestinal tract is a dynamic ecosystem containing a complex microbial community. In this paper, the indigenous intestinal bacteria and the microbial fermentation profile particularly short chain fatty acids (SCFA), lactate, and ammonia concentrations are reviewed. The intestinal bacterial composition changes with age. The bacterial density of the small intestine increases with age and comprises of lactobacilli, streptococci, enterobacteria, fusobacteria and eubacteria. Strict anaerobes (anaerobic gram-positive cocci, Eubacterium spp., Clostridium spp., Lactobacillus spp., Fusobacterium spp. and Bacteroides) are predominating caecal bacteria in young broilers. Data from culture-based studies showed that bifidobacteria could not be isolated from young birds, but were recovered from four-week-old broilers. Caecal lactobacilli accounted for 1.5-24% of the caecal bacteria. Gene sequencing of caecal DNA extracts showed that the majority of bacteria belonged to Clostridiaceae. Intestinal bacterial community is influenced by the dietary ingredients, nutrient levels and physical structure of feed. SCFA and other metabolic products are affected by diet formulation and age. Additional studies are required to know the bacterial metabolic activities together with the community analysis of the intestinal bacteria. Feed composition and processing have great potential to influence the activities of intestinal bacteria towards a desired direction in order to support animal health, well-being and microbial safety of broiler meat.     

Impact of colostomy on intestinal microflora and bacterial translocation in young rats fed with heat-killed Lactobacillus acidophilus strain LB

A rat animal model of left colostomy was found to significantly impair the growth curve of rats. Assessment of the intestinal flora showed that colostomy mostly affects the cecal but not colonic microflora. Generally, the number of enterococci was increased in both ileum and cecum; cecal lactobacilli also rose, accounting for a promotion of lactic acid bacteria in colostomised rats. No significant differences between colostomised, laparotomised and control rats could be observed for the translocation of intestinal bacteria to internal organs of rats (i.e. spleen, kidneys, lungs or liver), whatever their diet. Heat-killed Lactobacillus acidophilus strain LB administration (dead probiotic bacteria) tended to exhibit a stimulatory effect on bifidobacteria, probably affecting the culture-medium fermentation substances included in the pharmaceutical product. This effect was abolished by laparotomy and colostomy. A trend towards a probiotic-like effect, not susceptible to colostomy, was also witnessed as counts of lactobacilli tended to increase in both cecum and colon of all animals fed with L. acidophilus LB.     

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.