Identification of lactobacilli and bifidobacteria

Selective culture media and phenotypic tests enable lactobacilli to be differentiated from morphologically similar bacteria. The accurate identification of Lactobacillus species can be accomplished by reference to 16S rRNA gene sequences. Species-specific, PCR primers that target the 16S-23S rRNA spacer region are available for a limited number of Lactobacillus species. Molecular methods for the comprehensive identification of Bifidobacterium species are not yet available. Only DNA-DNA reassociation provides a reliable means of species identification for this genus at present. Bifidobacteria can be differentiated from morphologically similar bacteria by the use of genus-specific, PCR primers or oligonucleotide probes.     

Genetic modification of intestinal lactobacilli and bifidobacteria

Lactobacilli and bifidobacteria are important members of the gastrointestinal microflora of man and animals. There is a substantial and growing body of evidence that these microbes provide benefits to the host in which they reside. Understanding the roles of these two groups of bacteria in the intestine continues to be a significant challenge. To this end, genetic characterisation and manipulation of intestinal lactobacilli and bifidobacteria is essential to define their contributions to the intestinal microflora, and to potentially exploit any beneficial or unique properties. This review will describe the tools and strategies currently available for the genetic manipulation of lactobacilli and bifidobacteria. Additionally, the ramifications and opportunities that may arise as a result of the genetic manipulation of probiotic lactobacilli and bifidobacteria will be addressed.     

Extensive intra-phylotype diversity in lactobacilli and bifidobacteria from the honeybee gut

Background

In the honeybee Apis mellifera, the bacterial gut community is consistently colonized by eight distinct phylotypes of bacteria. Managed bee colonies are of considerable economic interest and it is therefore important to elucidate the diversity and role of this microbiota in the honeybee. In this study, we have sequenced the genomes of eleven strains of lactobacilli and bifidobacteria isolated from the honey crop of the honeybee A. mellifera.

Results

Single gene phylogenies confirmed that the isolated strains represent the diversity of lactobacilli and bifidobacteria in the gut, as previously identified by 16S rRNA gene sequencing. Core genome phylogenies of the lactobacilli and bifidobacteria further indicated extensive divergence between strains classified as the same phylotype. Phylotype-specific protein families included unique surface proteins. Within phylotypes, we found a remarkably high level of gene content diversity. Carbohydrate metabolism and transport functions contributed up to 45% of the accessory genes, with some genomes having a higher content of genes encoding phosphotransferase systems for the uptake of carbohydrates than any previously sequenced genome. These genes were often located in highly variable genomic segments that also contained genes for enzymes involved in the degradation and modification of sugar residues. Strain-specific gene clusters for the biosynthesis of exopolysaccharides were identified in two phylotypes. The dynamics of these segments contrasted with low recombination frequencies and conserved gene order structures for the core genes. Hits for CRISPR spacers were almost exclusively found within phylotypes, suggesting that the phylotypes are associated with distinct phage populations.

Conclusions

The honeybee gut microbiota has been described as consisting of a modest number of phylotypes; however, the genomes sequenced in the current study demonstrated a very high level of gene content diversity within all three described phylotypes of lactobacilli and bifidobacteria, particularly in terms of metabolic functions and surface structures, where many features were strain-specific. Together, these results indicate niche differentiation within phylotypes, suggesting that the honeybee gut microbiota is more complex than previously thought.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1476-6) contains supplementary material, which is available to authorized users.     

Antagonistic activities of lactobacilli and bifidobacteria against microbial pathogens

The gastrointestinal tract is a complex ecosystem that associates a resident microbiota and cells of various phenotypes lining the epithelial wall expressing complex metabolic activities. The resident microbiota in the digestive tract is a heterogeneous microbial ecosystem containing up to 1 x 10(14) colony-forming units (CFUs) of bacteria. The intestinal microbiota plays an important role in normal gut function and maintaining host health. The host is protected from attack by potentially harmful microbial microorganisms by the physical and chemical barriers created by the gastrointestinal epithelium. The cells lining the gastrointestinal epithelium and the resident microbiota are two partners that properly and/or synergistically function to promote an efficient host system of defence. The gastrointestinal cells that make up the epithelium, provide a physical barrier that protects the host against the unwanted intrusion of microorganisms into the gastrointestinal microbiota, and against the penetration of harmful microorganisms which usurp the cellular molecules and signalling pathways of the host to become pathogenic. One of the basic physiological functions of the resident microbiota is that it functions as a microbial barrier against microbial pathogens. The mechanisms by which the species of the microbiota exert this barrier effect remain largely to be determined. There is increasing evidence that lactobacilli and bifidobacteria, which inhabit the gastrointestinal microbiota, develop antimicrobial activities that participate in the host's gastrointestinal system of defence. The objective of this review is to analyze the in vitro and in vivo experimental and clinical studies in which the antimicrobial activities of selected lactobacilli and bifidobacteria strains have been documented.     

Antagonistic activity of probiotic lactobacilli and bifidobacteria against entero- and uropathogens

AIM: To develop in vitro assays for comparing the antagonistic properties and anti-oxidative activity of probiotic Lactobacillus and Bifidobacterium strains against various entero- and urinary pathogens. METHODS AND RESULTS: The antagonistic activity of five probiotic lactobacilli (Lactobacillus rhamnosus GG, Lactobacillus fermentum ME-3, Lactobacillus acidophilus La5, Lactobacillus plantarum 299v and Lactobacillus paracasei 8700:2) and two bifidobacteria (Bifidobacterium lactis Bb12, Bifidobacterium longum 46) against six target pathogens was estimated using different assays (solid and liquid media, anaerobic and microaerobic cultivation) and ranked (low, intermediate and high). Bacterial fermentation products were determined by gas chromatography, and the total anti-oxidative activity of probiotics was measured using linolenic acid test. Pyelonephritic Escherichia coli was highly suppressed by GG and both bifidobacteria strains. Lactobacilli strains 8700:2, 299v and ME-3 were the most effective against Salmonella enterica ssp. enterica in microaerobic while ME-3 and both bifidobacteria expressed high activity against Shigella sonnei in anaerobic milieu. Lact. paracasei, Lact. rhamnosus and Lact. plantarum strains showed intermediate antagonistic activity against Helicobacter pylori under microaerobic conditions on solid media. The highest anti-oxidative activity was characteristic for Lact. fermentum ME-3 (P < 0.05). No efficient antagonist against Clostridium difficile was found. The positive correlations between the pH, lactic acid production and anti-microbial activity for all tested probiotics were assessed. CONCLUSIONS: Developed experimental assays enable to compare the anti-microbial and -oxidative activity of Lactobacillus and/or Bifidobacterium probiotics, which have been claimed to possess the ability of suppressing the growth of various enteric and urinary pathogens. SIGNIFICANCE AND IMPACT OF THE STUDY: Screening Lactobacillus and Bifidobacterium sp. strains according to their activity in various environmental conditions could precede the clinical efficacy studies for adjunct treatment with probiotics in cure of different gastrointestinal and urinary tract infections.     

Mechanism of growth inhibition by free bile acids in lactobacilli and bifidobacteria

The effects of the free bile acids (FBAs) cholic acid (CA), deoxycholic acid (DCA), and chenodeoxycholic acid on the bioenergetics and growth of lactobacilli and bifidobacteria were investigated. It was found that these FBAs reduced the internal pH levels of these bacteria with rapid and stepwise kinetics and, at certain concentrations, dissipated DeltapH. The bile acid concentrations that dissipated DeltapH corresponded with the MICs for the selected bacteria. Unlike acetate, propionate, and butyrate, FBAs dissipated the transmembrane electrical potential (DeltaPsi). In Bifidobacterium breve JCM 1192, the synthetic proton conductor pentachlorophenol (PCP) dissipated DeltapH with a slow and continuous kinetics at a much lower concentration than FBAs did, suggesting the difference in mode of action between FBAs and true proton conductors. Membrane damage assessed by the fluorescence method and a viability decrease were also observed upon exposure to CA or DCA at the MIC but not to PCP or a short-chain fatty acid mixture. Loss of potassium ion was observed at CA concentrations more than 2 mM (0.4x MIC), while leakage of other cellular components increased at CA concentrations more than 4 mM (0.8 x MIC). Additionally, in experiments with membrane phospholipid vesicles extracted from Lactobacillus salivarius subsp. salicinius JCM 1044, CA and DCA at the MIC collapsed the DeltapH with concomitant leakage of intravesicular fluorescent pH probe, while they did not show proton conductance at a lower concentration range (e.g., 0.2x MIC). Taking these observations together, we conclude that FBAs at the MIC disturb membrane integrity and that this effect can lead to leakage of proton (membrane DeltapH and DeltaPsi dissipation), potassium ion, and other cellular components and eventually cell death.     

Occurrence of bifidobacteria and lactobacilli in digestive tract of some freshwater fishes

Distal parts of the fish intestine were analyzed for presence of bifidobacteria and lactobacilli using selective agars. Seventy seven samples from Cyprinus carpio, Oncorhynchus mykiss, Carassius auratus, Tinca tinca, Perca fluviatilis, Rutilus rutilus, Scardinius erythrophthalmus, Oreochromis niloticus, and Squalius cephalus were collected randomly throughout years 2008 and 2009. Bifidobacteria were detected in 5 samples from 4 fish species at counts 2.18–4.29 log CFU/g, lactobacilli were present in 6 fish species at counts 1.21–3.65 log CFU/g. Seven bifidobacterial isolates were identified to the species level using biochemical tests and by sequencing of 16S rRNA gene. Three strains belonged to species B. longum, two isolates were identified as B. dentium, one strain as B. asteroides and one isolate was not determined to the species level by employed methods. As identified bifidobacterial species are considered to be of human, animal or honeybee origin, they probably derived as contamination from sewage or other sources. After further more detail testing, the possible use of isolated bifidobacteria as probiotics is promising since they were able to pass through the digestive tract successfully.     

Advancing mechanistic understanding and bioengineering of probiotic lactobacilli and bifidobacteria by genome editing

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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.     

Resistant starch reduces large intestinal pH and promotes fecal lactobacilli and bifidobacteria in pigs

Dietary resistant starch (RS) may have prebiotic properties but its effects on fermentation and the microbial population are inconsistent. This meta-analysis aimed to quantify the relationship between RS type 2 (RS2) and intestinal short-chain fatty acids (SCFA) and pH as well as certain key bacterial taxa for intestinal health in pigs. From the 24 included articles with sufficient information about the animal, and dietary and physiological measurements published between 2000 and 2017, individual sub-data sets for fermentation metabolites, pH, bacterial abundances and apparent total tract digestibility were built and used to parameterize prediction models on the effect of RS2, accounting for inter- and intra-study variability. In addition, the effect of pig’s BW at the start of the experiment and duration of the experimental period on response variables were also evaluated using backward elimination analysis. Dietary RS levels ranged from 0% to 78.0% RS, with median and mean RS levels of 28.8% and 23.0%, respectively. Negative relationships could be established between dietary RS and pH in the large intestine (P<0.05), with a stronger effect in the mid and distal colon, and feces (R²=0.64 to 0.81; P<0.001). A dietary level of 15% RS would lower the pH in the proximal, mid-, distal colon and feces by 0.2, 0.6, 0.4 and 0.6 units, respectively. Increasing RS levels, however, did not affect SCFA concentrations in the hindgut, but enhanced the molar proportion of propionate in mid-colon and reduced those of acetate in mid-colon and of butyrate in mid- and distal colon (R²=0.46 to 0.52; P<0.05). Backward elimination indicated an age-related decrease in mid-colonic propionate proportion and increase in mid- and distal colonic butyrate proportion (P<0.05), thereby modulating RS2 effects. In feces, increasing RS levels promoted fecal lactobacilli (R²=0.46; P<0.01) and bifidobacteria (R²=0.57; P<0.01), whereby the slope showed the need for a minimal RS level of 10% for a 0.5 log unit-increase in their abundance. Best-fit equations further supported that a longer experimental period increased fecal lactobacilli but decreased fecal bifidobacteria (P<0.05). In conclusion, dietary RS2 seems to effectively decrease digesta pH throughout the large intestine and increase lactic acid-producing bacteria in feces of pigs which may limit the growth of opportunistic pathogens in the hindgut. To achieve these physiologically relevant changes, dietary RS should surpass 10% to 15%.     

Utilisation of steviol glycosides from Stevia rebaudiana (Bertoni) by lactobacilli and bifidobacteria in in vitro conditions

In the current study, eight strains of bifidobacteria and seven strains of lactobacilli were tested for their ability to grow in the presence of rebaudioside A and steviol glycosides from the sweetener Natusweet M001 originating from herb Stevia rebaudiana (Bertoni). Stevia is gaining popularity as a natural, non-caloric sugar substitute, and recently, it was allowed as a food additive by European Union too. Utilisation of steviol glycosides by intestinal microbiota suggests that they might have potential prebiotic effect. Based on the evaluation of bacterial density and pH values in our in vitro study, it was found that lactobacilli and bifidobacteria tested were able to utilise steviol glycosides as a carbon source only to a very limited extent. All strains tested showed significantly lower change in the absorbance A₅₄₀ (P?<?0.05) and pH decrease of the growth media as compared with the positive controls (medium containing glucose as a carbon source and de Man Rogosa Sharpe broth). We concluded that a suggested prebiotic effect was not confirmed either in the case of rebaudioside A or in the case of the sweetener Natusweet M001 containing a mixture of steviol glycosides.     

Stationary-phase acid and heat treatments for improvement of the viability of probiotic lactobacilli and bifidobacteria

AIMS: To investigate whether sublethal treatments of stationary-phase probiotic cultures enhance their survival during lethal treatments and to adapt these treatments to the fermenter-scale production of probiotic cultures. METHODS AND RESULTS: Conditions for acid and heat pretreatments were screened for three Lactobacillus and two Bifidobacterium strains. Strains were sublethally treated both at laboratory scale and at fermenter scale in a strain-specific manner and exposed to a subsequent lethal treatment. At laboratory scale viability improvement was detected in each strain. However, improvement was more pronounced in the Lactobacillus than in the Bifidobacterium strains. At fermenter scale three strains were tested: for the two Lactobacillus strains a marked improvement in viability was obtained whereas for the Bifidobacterium strain the improvement was either minor or not detected. CONCLUSIONS: Development of treatments for viability enhancement of probiotic strains is feasible, but strain-specific optimization is necessary to obtain notable improvements. SIGNIFICANCE AND IMPACT OF THE STUDY: Strain-specific treatments were developed for the viability enhancement of stationary-phase probiotic cells both at laboratory and fermenter scale. These results can be utilised in the production of probiotic cultures with improved viability.     

Molecular approaches for the detection and identification of bifidobacteria and lactobacilli in the human gastrointestinal tract

In this review an overview of various molecular techniques and their application for the detection and identification of bifidobacteria and lactobacilli in the gastrointestinal (GI) tract is presented. The techniques include molecular typing techniques such as amplified ribosomal DNA restriction analysis (ARDRA), randomly amplified polymorphic DNA (RAPD), pulsed field gel electrophoresis (PFGE), ribotyping and community profiling techniques such as PCR coupled to temperature and denaturing gradient gel electrophoresis (PCR-TGGE and PCR-DGGE, respectively). Special attention is given to oligonucleotide probes and primers that target the ribosomal RNA (rRNA) sequences and their use in PCR and different hybridisation techniques such as DNA microarrays and fluorescent in situ hybridisation (FISH). In addition, recent findings based on the molecular studies of bifidobacteria and lactobacilli in the GI-tract are reviewed.     

The primary screening of bifidobacteria and lactobacilli strains to develop effective probiotic preparations based on them

10 Bifidobacterium strains and 10 Lactobacillus strains were studied for their antagonistic activity with respect to Escherichia coli, Klebsiella ozaenae, Staphylococcus aureus, Enterococcus faecalis, Pseudomonas aeruginosa and for their sensitivity to antibiotics, widely used in clinical practice. L. acidophilus strain 5/4, L. acidophilus strain 18/4, B. adolescentis strain UX, B. longum strain 44 exhibited the highest antagonistic activity and the highest degree of antibiotic resistance. The restriction analysis of the chromosomal DNA of these strains was then made and their plasmid content was studied, making it possible to recognize these strains in future in the course of in vivo experiments.     

Enzyme-assisted extraction of bioactives from plants

Demand for new and novel natural compounds has intensified the development of plant-derived compounds known as bioactives that either promote health or are toxic when ingested. Enhanced release of these bioactives from plant cells by cell disruption and extraction through the cell wall can be optimized using enzyme preparations either alone or in mixtures. However, the biotechnological application of enzymes is not currently exploited to its maximum potential within the food industry. Here, we discuss the use of environmentally friendly enzyme-assisted extraction of bioactive compounds from plant sources, particularly for food and nutraceutical purposes. In particular, we discuss an enzyme-assisted extraction of stevioside from Stevia rebaudiana, as an example of a process of potential value to the food industry.     

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.