Differences between the normal vaginal bacterial community of baboons and that of humans

Humans and baboons (Papio spp.) share considerable anatomical and physiological similarities in their reproductive tracts. Given the similarities, it is reasonable to expect that the normal vaginal microbial composition (microbiota) of baboons would be similar to that of humans. We have used a 16S rRNA phylogenetic approach to assess the composition of the baboon vaginal microbiota in a set of nine animals from a captive facility and six from the wild. Results show that although Gram‐positive bacteria dominate in baboons as they do in humans, there are major differences between the vaginal microbiota of baboons and that of humans. In contrast to humans, the species of Gram‐positive bacteria (Firmicutes) were taxa other than Lactobacillus species. In addition, some groups of Gram‐negative bacteria that are not normally abundant in humans were found in the baboon samples. A further level of difference was also seen even within the same bacterial phylogenetic group, as baboon strains tended to be more phylogenetically distinct from human strains than human strains were with each other. Finally, results of our analysis suggests that co‐evolution of microbes and their hosts cannot account for the major differences between the microbiota of baboons and that of humans because divergences between the major bacterial genera were too ancient to have occurred since primates evolved. Instead, the primate vaginal tracts appear to have acquired discrete subsets of bacteria from the vast diversity of bacteria available in the environment and established a community responsive to and compatible with host species physiology. Am. J. Primatol. 73:119–126, 2011. © 2010 Wiley‐Liss, Inc.     

Development and Application of Random DIG-Labelled Probes Based on Total Genomic Lactobacillus DNA

Summary Nine Lactobacillus-specific and non-isotopically (digoxygenin) labelled probes were developed on the basis of Lactobacillus total chromosomal DNA. Their specificity and applicability for Lactobacillus discrimination was proven by DNA–DNA hybridization to reference strains from the American Type Culture Collection (ATCC). The DNA probes were divided into three groups depending on the ability to hybridize to DNA from the same and/or from a group of related Lactobacillus strains. They were assayed in the species-specific detection of vaginal strains from the genus Lactobacillus. Six DNA probes were successfully applied for characterization of 21 newly isolated vaginal Lactobacilli. The species affliation of some isolates was determined. The developed DNA probes were evaluated for usage as a qualitative hybridization test for detection of Lactobacillus species in mixed cultures, obtained directly from vaginal samples without strain isolation.     

Isolation of lactobacilli with probiotic properties from the human stomach

Aims: Recent evidence suggests that the human gastric microbiota is much more diverse than previously thought. The aim of this study was to assess the potential for isolating lactobacilli from the human stomach. Methods and Results: Lactobacilli were selectively cultured from gastric biopsies from 12 patients undergoing routine endoscopy. Lactobacilli were present in four of 12 biopsies. We isolated, in total 10 different strains representing five species (Lactobacillus gasseri, L. fermentum, L. vaginalis, L. reuteri and L. salivarius). The 10 isolates varied greatly in their ability to inhibit the growth of two Gram‐positive bacteria and two Gram‐negative bacteria. Furthermore, the acid and bile resistance profiles of the 10 isolates spanned a wide range. Conclusions: Five different Lactobacillus species were cultured from human gastric biopsies for the first time. Significance and Impact of the Study: Diverse Lactobacillus species are more prevalent in the human stomach than previously recognized, representing an untapped source of bacteria with beneficial probiotic and/or biotechnological properties.     

Vaginotypes of the human vaginal microbiome

The human vaginal environment harbours a community of bacteria that plays an important role in maintaining vaginal health and in protecting this environment from various urogenital infections. This bacterial population, also known as vaginal microbiota, has been demonstrated to be dominated by members of the Lactobacillus genus. Several studies employing 16S rRNA gene-based amplicon sequencing have classified the vaginal microbiota into five distinct community state types (CSTs) or vaginotypes. To deepen our understanding of the vaginal microbiota we performed an in-depth meta-analysis of 1312 publicly available datasets concerning healthy vaginal microbiome information obtained by metagenomics sequencing. The analysis confirmed the predominance of taxa belonging to the Lactobacillus genus, followed by members of the genera Gardnerella, Vibrio and Atopobium. Moreover, the statistical robustness offered by this meta-analysis allowed us to disentangle the species-level composition of dominant and accessory taxa constituting each vaginotype and to revisit and refine the previously proposed CST classification. In addition, a functional characterization of the metagenomic datasets revealed particular genetic features associated with each assigned vaginotype.     

Screening and characterization of Lactobacillus strains producing large amounts of exopolysaccharides

A total of 182 Lactobacillus strains were screened for production of extracellular polysaccharides (EPS) by a new method: growth in liquid media with high sugar concentrations. Sixty EPS-positive strains were identified; 17 strains produced more than 100 mg/l soluble EPS. Sucrose was an excellent substrate for abundant EPS synthesis. The ability to produce glucans appears to be widespread in the genus Lactobacillus. The monosaccharide composition of EPS produced by Lactobacillus reuteri strain LB 121 varied with the growth conditions (solid compared to liquid medium) and the sugar substrates (sucrose or raffinose) supplied in the medium. Strain LB 121 produced both a glucan and a fructan on sucrose, but only a fructan on raffinose. This is the first report of fructan production by a Lactobacillus species. EPS production increased with increasing sucrose concentrations and involved extracellular sucrase-type enzymes. Received: 20 March 1998 / Received revision: 12 August 1998 / Accepted: 12 August 1998     

The Host as the Driver of the Microbiota in the Gut and External Environment of Drosophila melanogaster

Most associations between animals and their gut microbiota are dynamic, involving sustained transfer of food-associated microbial cells into the gut and shedding of microorganisms into the external environment with feces, but the interacting effects of host and microbial factors on the composition of the internal and external microbial communities are poorly understood. This study on laboratory cultures of the fruit fly Drosophila melanogaster reared in continuous contact with their food revealed time-dependent changes of the microbial communities in the food that were strongly influenced by the presence and abundance of Drosophila. When germfree Drosophila eggs were aseptically added to nonsterile food, the microbiota in the food and flies converged to a composition dramatically different from that in fly-free food, showing that Drosophila has microbiota-independent effects on the food microbiota. The microbiota in both the flies that developed from unmanipulated eggs (bearing microorganisms) and the associated food was dominated by the bacteria most abundant on the eggs, demonstrating effective vertical transmission via surface contamination of eggs. Food coinoculated with a four-species defined bacterial community of Acetobacter and Lactobacillus species revealed the progressive elimination of Lactobacillus from the food bearing few or no Drosophila, indicating the presence of antagonistic interactions between Acetobacter and Lactobacillus. Drosophila at high densities ameliorated the Acetobacter/Lactobacillus antagonism, enabling Lactobacillus to persist. This study with Drosophila demonstrates how animals can have major, coordinated effects on the composition of microbial communities in the gut and immediate environment.     

The inconstant gut microbiota of Drosophila species revealed by 16S rRNA gene analysis

The gut microorganisms in some animals are reported to include a core microbiota of consistently associated bacteria that is ecologically distinctive and may have coevolved with the host. The core microbiota is promoted by positive interactions among bacteria, favoring shared persistence; its retention over evolutionary timescales is evident as congruence between host phylogeny and bacterial community composition. This study applied multiple analyses to investigate variation in the composition of gut microbiota in drosophilid flies. First, the prevalence of five previously described gut bacteria (Acetobacter and Lactobacillus species) in individual flies of 21 strains (10 Drosophila species) were determined. Most bacteria were not present in all individuals of most strains, and bacterial species pairs co-occurred in individual flies less frequently than predicted by chance, contrary to expectations of a core microbiota. A complementary pyrosequencing analysis of 16S rRNA gene amplicons from the gut microbiota of 11 Drosophila species identified 209 bacterial operational taxonomic units (OTUs), with near-saturating sampling of sequences, but none of the OTUs was common to all host species. Furthermore, in both of two independent sets of Drosophila species, the gut bacterial community composition was not congruent with host phylogeny. The final analysis identified no common OTUs across three wild and four laboratory samples of D. melanogaster. Our results yielded no consistent evidence for a core microbiota in Drosophila. We conclude that the taxonomic composition of gut microbiota varies widely within and among Drosophila populations and species. This is reminiscent of the patterns of bacterial composition in guts of some other animals, including humans.     

Assessment and comparison of probiotic potential of four Lactobacillus species isolated from feces samples of Iranian infants

The probiotic potential of Lactobacillus species isolated from infant feces was investigated. For this study, the antibiotic susceptibility, tolerance in gut‐related conditions, antimicrobial activity, and ability to adhere to a human colorectal adenocarcinoma cell line (Caco‐2 cells) of four common Lactobacillus species (Lactobacillus paracasei [n = 15], Lactobacillus rhamnosus [n = 45], Lactobacillus gasseri [n = 20] and Lactobacillus fermentum [n = 18]) were assessed. Most isolates that which were sensitive to imipenem, ampicillin, gentamycin, erythromycin and tetracycline were selected for other tests. L. gasseri isolates had the greatest sensitivity to gastric and intestinal fluids (<10% viability). L. fermentum (FH5, FH13 and FH18) had the highest adhesion to Caco‐2 cells. The lowest antibacterial activity against pathogenic bacteria was shown by L. gasseri strains in spot tests. Furthermore, non‐adjusted cell‐free culture supernatants with low pH had greater antimicrobial activity, which was related to organic acid. The results showed that some isolates of L. rhamnosus and L. fermentum are suitable for use as a probiotic.     

Live and heat-inactivated lactobacilli from feces inhibit <Emphasis Type="Italic">Salmonella typhi</Emphasis> and <Emphasis Type="Italic">Escherichia coli</Emphasis> adherence to caco-2 cells

A quantitative approach has been proposed to evaluate the competitive inhibition of Escherichia coli and Salmonella typhi by live and heat-inactivated laboratory isolated Lactobacillus sp. on adhesion to monolayer of Caco-2 cells. Three species of Lactobacillus (L. casei, L. acidophilus, L. agilis) isolated from human neonate feces and two commercial probiotic strains (L. casei, L. acidophilus) have been compared for probiotic activity. All lactobacilli were able to attach to the Caco-2 cells, however, the degree of adhesion was bacterial strain-dependent. The adhesion indices of the two commercial probiotic strains were not significantly different from the values obtained for the other two similar fecal strains (p > 0.01). The inhibition of attachment of the pathogenic bacteria by inactivated cells of fecal L. acidophilus was examined and compared to the results of live bacteria. The inhibition pattern was similar for live and heat-inactivated L. acidophilus (p > 0.01). The number of attached pathogenic bacteria to the Caco-2 cells decreased when the number of L. acidophilus increased from 10⁶ to 10⁹ CFU/mL. The heat-inactivated L. acidophilus displayed similar probiotic activity compared to the live bacteria.     

Intake of Meat Proteins Substantially Increased the Relative Abundance of Genus Lactobacillus in Rat Feces

Diet has been shown to have a critical influence on gut bacteria and host health, and high levels of red meat in diet have been shown to increase colonic DNA damage and thus be harmful to gut health. However, previous studies focused more on the effects of meat than of meat proteins. In order to investigate whether intake of meat proteins affects the composition and metabolic activities of gut microbiota, feces were collected from growing rats that were fed with either meat proteins (from beef, pork or fish) or non-meat proteins (casein or soy) for 14 days. The resulting composition of gut microbiota was profiled by sequencing the V4-V5 region of the 16S ribosomal RNA genes and the short chain fatty acids (SCFAs) were analyzed using gas chromatography. The composition of gut microbiota and SCFA levels were significantly different between the five diet groups. At a recommended dose of 20% protein in the diet, meat protein-fed rats had a higher relative abundance of the beneficial genus Lactobacillus, but lower levels of SCFAs and SCFA-producing bacteria including Fusobacterium, Bacteroides and Prevotella, compared with the soy protein-fed group. Further work is needed on the regulatory pathways linking dietary protein intake to gut microbiota.     

Surface displaced alfa-enolase of Lactobacillus plantarum is a fibronectin binding protein

Background  

Lactic acid bacteria of the genus Lactobacillus and Bifidobacterium are one of the most important health promoting groups of the human intestinal microbiota. Their protective role within the gut consists in out competing invading pathogens for ecological niches and metabolic substrates. Among the features necessary to provide health benefits, commensal microorganisms must have the ability to adhere to human intestinal cells and consequently to colonize the gut. Studies on mechanisms mediating adhesion of lactobacilli to human intestinal cells showed that factors involved in the interaction vary mostly among different species and strains, mainly regarding interaction between bacterial adhesins and extracellular matrix or mucus proteins. We have investigated the adhesive properties of Lactobacillus plantarum, a member of the human microbiota of healthy individuals.     

Alterations in the diversity and composition of mice gut microbiota by lytic or temperate gut phage treatment

Phages, the most abundant species in the mammalian gut, have numerous advantages as biocontrol agent over antibiotics. In this study, mice were orally treated with the lytic gut phage PA13076 (group B), the temperate phage BP96115 (group C), no phage (group A), or streptomycin (group D) over 31 days. At the end of the experiment, fecal microbiota diversity and composition was determined and compared using high-throughput sequencing of the V3–V4 hyper-variable region of the 16S rRNA gene and virus-like particles (VLPs) were quantified in feces. There was high diversity and richness of microbiota in the lytic and temperate gut phage-treated mice, with the lytic gut phage causing an increased alpha diversity based on the Chao1 index (p < 0.01). However, the streptomycin treatment reduced the microbiota diversity and richness (p = 0.0299). Both phage and streptomycin treatments reduced the abundance of Bacteroidetes at the phylum level (p < 0.01) and increased the abundance of the phylum Firmicutes. Interestingly, two beneficial genera, Lactobacillus and Bifidobacterium, were enhanced by treatment with the lytic and temperate gut phage. The abundance of the genus Escherichia/Shigella was higher in mice after temperate phage administration than in the control group (p < 0.01), but lower than in the streptomycin group. Moreover, streptomycin treatment increased the abundance of the genera Klebsiella and Escherichia/Shigella (p < 0.01). In terms of the gut virome, fecal VLPs did not change significantly after phage treatment. This study showed that lytic and temperate gut phage treatment modulated the composition and diversity of gut microbiota and the lytic gut phage promoted a beneficial gut ecosystem, while the temperate phage may promote conditions enabling diseases to occur.

     

Antibody-supported screening of alcohol dehydrogenases

Polyclonal antibodies raised against purified (R)-specific alcohol dehydrogenase of Lactobacillus kefir were used in Western blot analyses to search for structurally or immunologically related proteins. No immunochemical reactions were found with commercially available alcohol dehydrogenases (from yeast, horse liver and Thermoanaerobium brockii), but screening among the genus Lactobacillus revealed that each strain of a subgroup of Betabacterium gave positive results whereas strains of the other subgroups of Lactobacillus were found to be inactive. However, enzymatic assays with these antibody-positive strains showed, that besides L. kefir itself, only the strains of L. brevis possess alcohol dehydrogenase activity with acetophenone and NADPH as substrates.     

Characterization of the beneficial properties of lactobacilli isolated from bullfrog (<Emphasis Type="Italic">Rana catesbeiana</Emphasis>) hatchery

The present work addresses the isolation and partial identification of the microbial population of a R. catesbeiana hatchery in spring and summer as well as some beneficial properties of Lactobacillus strains isolated in different seasons and hatchery areas. The bacterial population was grouped into the following taxa: Lactobacillus spp., Pediococcus spp., Enterococcus faecalis and Ent. faecium, and Enterobacteriaceae (Enterobacter spp., Escherichia coli) while Pseudomonas aeruginosa and Staphylococcus epidermidis were isolated from frogs displaying red-leg syndrome. The Lactobacillus plantarum and L. curvatus strains isolated showed to inhibit the growth of red-leg syndrome associated pathogens and food-borne bacteria by organic acids. While L. plantarum CRL 1606 also inhibited red-leg syndrome related pathogens by hydrogen peroxide, meat spoilage bacteria were only inhibited by acidity. However, by using a MRS medium added with tetramethyl-benzidine and peroxidase, a high percentage of H₂O₂-producing lactobacilli were detected. The surface properties of Lactobacillus strains showed that a few strains were able to agglutinate ABO human erythrocytes, while the highest number of strains had a low to medium degree of hydrophobicity. This paper constitute the first study related to the beneficial properties of Lactobacillus isolated from a bullfrog hatchery, as well as the selection criteria applied to a group of strains, which could help to control or prevent bacterial infectious diseases in raniculture.     

Electrophoretic patterns of esterases and lactate-and l-malate-dehydrogenases from Flavobacterium species

Esterases and lactate-(LDH) and l-malate-(MDH) dehydrogenases of 64 strains of 13 species of Flavobacterium (particularly 46 strains of F. meningosepticum belonging to 15 different serotypes) were analyzed by horizontal polyacrylamide-agarose gel electrophoresis. Twenty-five esterase types were detected; these enzymes were distinguished by their spectra of hydrolytic activity towards five synthetic substrates (hydrolytic type) and their electrolytic mobilities (electrophoretic type). No variant of LDH was detected in all the strains of Flavobacterium. No variant of MDH was detected in three species: F. aquatile, F. branchiophilum, and F. breve and in serotype I, J and L. of F. meningosepticum. These results within the genus Flavobacterium permit precise identification, which may be useful for characterization of strains kept in collections as well as for epidemiological studies.     

Influence of intrapartum antibiotic prophylaxis against group B Streptococcus on the early newborn gut composition and evaluation of the anti-Streptococcus activity of Bifidobacterium strains

Several factors are known to influence the early colonization of the gut in newborns. Among them, the use of antibiotics on the mother during labor, referred to as intrapartum antibiotic prophylaxis (IAP), has scarcely been investigated, although this practice is routinely used in group B Streptococcus (GBS)-positive women. This work is therefore aimed at verifying whether IAP can influence the main microbial groups of the newborn gut microbiota at an early stage of microbial establishment. Fifty-two newborns were recruited: 26 born by mothers negative to GBS (control group) and 26 by mothers positive to GBS and subjected to IAP with ampicillin (IAP group). Selected microbial groups (Lactobacillus spp., Bidobacterium spp., Bacteroides fragilis, Clostridium difficile, and Escherichia coli) were quantified with real-time PCR on DNA extracted from newborn feces. Further analysis was performed within the Bidobacterium genus by using DGGE after amplification with genus-specific primers. Results obtained showed a significant decrease of the bifidobacteria counts after antibiotic treatment of the mother. Bifidobacteria were found to be affected by IAP not only quantitatively but also qualitatively. In fact, IAP determined a decrement in the frequency of Bidobacterium breve, Bidobacterium bifidum, and Bidobacterium dentium with respect to the control group. Moreover, this study has preliminarily evaluated that some bifidobacterial strains, previously selected for use in infants, have antibacterial properties against GBS and are therefore potential candidates for being applied as probiotics for the prevention of GBS infections.     

Evidence for in vitro anti-genotoxicity of cheese non-starter lactobacilli

The inhibition of direct acting DNA reactive agents by 63 non-starter lactobacilli isolated from raw ewes milk cheeses was examined by short-term assay (SOS-Chromotest) and compared with already characterized starter lactobacilli. The screening revealed strains active against the nitroarene 4-nitroquinoline-1-oxide (NQO) and the alkylating agent N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) in different species of the genus Lactobacillus (L. rhamnosus, L. casei, L. plantarum, L. brevis, Lactobacillus spp.). It was proved that the anti-genotoxicity was strain-dependent, and always associated with spectroscopic modification of genotoxins. The frequency of strains inhibiting nitroarene genotoxicity was comparable for non-starter and starter lactobacilli, whereas inhibition of the alkylating agent was largely predominant in non-starter isolates. Seventeen strains presented inhibitory activity against both genotoxins. DNA RAPD-PCR performed with M13, Pro-Up and RPO2 primers on the lactobacilli under examination showed genetic diversity in these strains. The non-starter isolates clustered in seven groups and the strains presenting a high degree of activity against 4-nitroquinoline-1-oxide clustered in a single group with a similarity around 75%. Interestingly, the strains with anti-genotoxic properties also showed acid-bile tolerance, indicating that the autochthonous lactobacilli which survive cheese ripening may also reach the gut as viable cells and could prevent genotoxin DNA damage to enterocytes, as is desirable for probiotic bacteria.     

Functional proteomics within the genus Lactobacillus

Lactobacillus are mainly used for the manufacture of fermented dairy, sourdough, meat, and vegetable foods or used as probiotics. Under optimal processing conditions, Lactobacillus strains contribute to food functionality through their enzyme portfolio and the release of metabolites. An extensive genomic diversity analysis was conducted to elucidate the core features of the genus Lactobacillus, and to provide a better comprehension of niche adaptation of the strains. However, proteomics is an indispensable “omics” science to elucidate the proteome diversity, and the mechanisms of regulation and adaptation of Lactobacillus strains. This review focuses on the novel and comprehensive knowledge of functional proteomics and metaproteomics of Lactobacillus species. A large list of proteomic case studies of different Lactobacillus species is provided to illustrate the adaptability of the main metabolic pathways (e.g., carbohydrate transport and metabolism, pyruvate metabolism, proteolytic system, amino acid metabolism, and protein synthesis) to various life conditions. These investigations have highlighted that lactobacilli modulate the level of a complex panel of proteins to growth/survive in different ecological niches. In addition to the general regulation and stress response, specific metabolic pathways can be switched on and off, modifying the behavior of the strains.     

Effects of Age and Strain on the Microbiota Colonization in an Infant Human Flora-Associated Mouse Model

The establishment of human flora-associated animal models allows the in vivo manipulation of host, microbial, and environmental parameters to influence the gut microbial community. However, it is difficult to simulate infant gut microbiota in germ-free animals because of the variation and dynamic state of infant microbial communities. In this study, the effects of age and strain on intestinal microbiota were observed in an infant human flora-associated (IHFA) mouse model. To establish an IHFA model, postnatal day (PND) 1 germ-free mice (Kunming, n = 10; BALB/c, n = 10) were infected with feces from a breast-fed infant. Microbiota in the feces of BALB/c mice (at PND 7, 14, and 21), and Kunming mice (at PND 14) were analyzed by PCR-denaturing gradient gel electrophoresis. Bifidobacteria and lactobacilli levels in the feces of BALB/c and Kunming mice (PND 7/14/21) were detected by quantitative real-time PCR. The Dice similarity coefficient (Cs) for the fecal microbiota of IHFA mice in comparison with the HD donor sample was higher for BALB/c mice than for Kunming mice (P < 0.05). In addition, the DCs at PND 7 were lower than those at PND 14 and PND 21 in both mouse strains (P < 0.05). The Bifidobacteria and Lactobacillus species colonizing the BALB/c mice were similar to those in the Kunming mice (at PND 7/14/21). The bifidobacteria counts increased with age in both mouse strains, whereas the lactobacilli counts decreased with age in both strains. These results suggest that both age and strain influence microbiota patterns in the IHFA mouse model.     

Genetic diversity of the genus <Emphasis Type="Italic">Lactobacillus</Emphasis> bacteria from the human gastrointestinal microbiome

The species and strain genetic diversity of bacterial cultures belonging to the genus Lactobacillus, which were isolated from the gastrointestinal microbiome of the human population living in the former Soviet Union in the years 1960–1980, was studied. The bacteria demonstrated probiotic characteristics. Phylogenetic analysis of sequences of the gene coding for 16S rRNA detected earlier by us, showed that the gene found in bacteria isolated from the intestinal content of healthy adultsand represented by species L. plantarum, L. helveticus, L. casei/paracasei, L. rhamnosus, and L. fermentum has high homology (97–100%) with this gene in type representatives of the species. The genotypic and strain diversity of cultures was studied using RAPD-PCR and nonspecific primers. This method with the use of the ERIC-1 primer gave reliable and reproducible results as compared that using with M13 and MSP primers and allowed the identification of examined bacteria belonging to the genus Lactobacillus at the level of species and certification at the strain level.