Elisabeth Neumann

Identification to the species level of Lactobacillus isolated in probiotic prospecting studies of human, animal or food origin by 16S-23S rRNA restriction profiling

BackgroundThe accurate identification of Lactobacillus and other co-isolated bacteria during microbial ecological studies of ecosystems such as the human or animal intestinal tracts and food products is a hard task by phenotypic methods requiring additional tests such as protein and/or lipids profiling.ResultsBacteria isolated in different probiotic prospecting studies, using de Man, Rogosa and Sharpe medium (MRS), were typed at species level by PCR amplification of 16S-23S rRNA intergenic spacers using universal primers that anneal within 16S and 23S genes, followed by restriction digestion analyses of PCR products. The set of enzymes chosen differentiates most species of Lactobacillus genus and also co-isolated bacteria such as Enterococcus, Streptococcus, Weissella, Staphylococcus, and Escherichia species. The in silico predictions of restriction patterns generated by the Lactobacillus shorter spacers digested with 11 restriction enzymes with 6 bp specificities allowed us to distinguish almost all isolates at the species level but not at the subspecies one. Simultaneous theoretical digestions of the three spacers (long, medium and short) with the same set of enzymes provided more complex patterns and allowed us to distinguish the species without purifying and cloning of PCR products.ConclusionLactobacillus isolates and several other strains of bacteria co-isolated on MRS medium from gastrointestinal ecosystem and fermented food products could be identified using DNA fingerprints generated by restriction endonucleases. The methodology based on amplified ribosomal DNA restriction analysis (ARDRA) is easier, faster and more accurate than the current methodologies based on fermentation profiles, used in most laboratories for the purpose of identification of these bacteria in different prospecting studies.

Monoassociation with Lactobacillus acidophilus UFV-H2b20 stimulates the immune defense mechanisms of germfree mice

Probiotics are formulations containing live microorganisms or microbial stimulants that have some beneficial influence on the maintenance of a balanced intestinal microbiota and on the resistance to infections. The search for probiotics to be used in prevention or treatment of enteric infections, as an alternative to antibiotic therapy, has gained significant impulse in the last few years. Several studies have demonstrated the beneficial effects of lactic acid bacteria in controlling infection by intestinal pathogens and in boosting the hosts nonspecific immune response. Here, we studied the use of Lactobacillus acidophilus UFV-H2b20, a lactic acid bacterium isolated from a human newborn from Viçosa, Minas Gerais, Brazil, as a probiotic. A suspension containing 10(8) cells of Lactobacillus acidophilus UFV-H2b20 was inoculated into groups of at least five conventional and germfree Swiss mice to determine its capacity to stimulate the host mononuclear phagocytic activity. We demonstrate that this strain can survive the stressing conditions of the intestinal tract in vivo. Moreover, the monoassociation of germfree mice with this strain for seven days improved the hosts macrophage phagocytic capacity, as demonstrated by the clearance of a Gram-negative bacterium inoculated intravenously. Monoassociated mice showed an undetectable number of circulating E. coli, while 0.1% of the original inoculum was still present in germfree animals. Mice treated with viable or heat-killed Lactobacillus acidophilus UFV-H2b20 presented similarly improved clearance capacity when compared with germfree controls. In addition, monoassociated mice had twice the amount of Kupffer cells, which are responsible for the clearance of circulating bacteria, compared to germfree controls. These results suggest that the L. acidophilus strain used here stimulates a nonspecific immune response and is a strong candidate to be used as a probiotic.

Genetic transformation of novel isolates of chicken Lactobacillus bearing probiotic features for expression of heterologous proteins: a tool to develop live oral vaccines

BackgroundThe use of lactic acid bacteria as vehicles to delivery antigens to immunize animals is a promising issue. When genetically modified, these bacteria can induce a specific local and systemic immune response against selected pathogens. Gastric acid and bile salts tolerance, production of antagonistic substances against pathogenic microorganisms, and adhesive ability to gut epithelium are other important characteristics that make these bacteria useful for oral immunization.ResultsBacteria isolated on de Man, Rogosa and Sharpe medium (MRS) from different gastrointestinal portions of broiler chicks were evaluated for their resistance to artificial gastric acid and bile salts, production of hydrogen peroxide, and cell surface hydrophobicity. Thirty-eight isolates were first typed at species level by PCR amplification of 16S-23S rRNA intergenic spacers using universal primers that anneal within 16S and 23S genes, followed by restriction digestion analyses of PCR amplicons (PCR-ARDRA). An expression cassette was assembled onto the pCR2.1-Topo vector by cloning the promoter, leader peptide, cell wall anchor and terminator sequences derived from the laminin binding S-layer protein gene of L. crispatus strain F5.7 (lbs gene). A sequence encoding the green fluorescent protein (GFP) was inserted as reporter gene, and an erythromycin resistance gene was added as selective marker. All constructs were able to express GFP in the cloning host E. coli XL1-Blue and different Lactobacillus strains as verified by FACS and laser scanning confocal microscopy.ConclusionLactobacillus isolated from gastrointestinal tract of broiler chickens and selected for probiotic characteristics can be genetically modified by introducing an expression cassette into the lbs locus. The transformed bacteria expressed on its cell wall surface different fluorescent proteins used as reporters of promoter function. It is possible then that similar bacterial model expressing pathogen antigens can be used as live oral vaccines to immunize broilers against infectious diseases.

Selection of lactic acid bacteria from Brazilian kefir grains for potential use as starter or probiotic cultures

Brazilian kefir is a homemade fermented beverage that is obtained by incubating milk or a brown sugar solution with kefir grains that contribute their different microbiological compositions. It is highly important to isolate and characterize microorganisms from Brazilian kefir grains to obtain starter cultures for the industrial production of a standardized commercial kefir. Thus, the present study aimed to isolate lactic acid bacteria from eight kefir grains that were propagated in milk or sugar solutions from five different locations in Brazil and to select Lactobacillus isolates based on desirable in vitro probiotic properties. One hundred eight isolates from both substrates were identified by amplified ribosomal DNA restriction analysis and/or 16S rRNA gene sequencing and were determined to belong to the following 11 species from the genera: Lactococcus, Leuconostoc, Lactobacillus (L.), and Oenococcus. Leuconostoc mesenteroides, Lactobacillus kefiri, and Lactobacillus kefiranofaciens were isolated only from milk grains, whereas Lactobacillus perolens, Lactobacillus parafarraginis, Lactobacillus diolivorans, and Oenococcus oeni were isolated exclusively from sugar water grains. When the microbial compositions of four kefir grains were evaluated with culture-independent analyses, L. kefiranofaciens was observed to predominant in milk grains, whereas Lactobacillus hilgardii was most abundant in sugar water kefir. Unfortunately, L. hilgardii was not isolated from any grain, although this bacteria was detected with a culture-independent methodology. Fifty-two isolated Lactobacilli were tested for gastric juice and bile salt tolerance, antagonism against pathogens, antimicrobial resistance, and surface hydrophobicity. Three Lactobacillus strains (L. kefiranofaciens 8U, L. diolivorans 1Z, and Lactobacillus casei 17U) could be classified as potential probiotics. In conclusion, several lactic acid bacteria that could be used in combination with yeasts as starter cultures for both milk kefir and sugar water kefir were characterized, and the functional properties of several of the lactobacilli isolated from the kefir grains were suggestive of their possible use as probiotics in both kefir and other dairy products.

Probiotics protect mice against experimental infections.

Objectives Our group has concerned itself with the study of the effect of probiotics on the resistance to infections using experimental models. Here, we will focus on evidence that the UFV-H2b20 strain of Lactobacillus delbrueckii var. bulgaricus may be considered a probiotic and has protective effects on mice against a variety of bacterial infections. Methods Germ-free, monoassociated, and conventional mice were used. Mice were treated with probiotics and challenged with Escherichia coli, Salmonella enterica serovar Typhimurium, or Listeria monocytogenes, and the outcome of infection was measured as mortality, quantification of bacteria in target organs, and systemic of local cytokine production. Results L. delbrueckii increased clearance of E. coli and production of systemic inflammatory cytokines. This strain also protected monoassociated and conventional mice against infection with S. enterica serovar Typhimurium. Finally, monoassociated mice were more resistant to L. monocytogenes as measured by mortality and the number of bacteria in spleen and liver. In addition, monoassociated mice challenged with L. monocytogenes showed increased production of inflammatory cytokines (interferon-γ and tumor necrosis factor-α) and nitric oxide. Interestingly, interleukin-10 levels were not altered by monoassociation or infection. Conclusions L. delbrueckii UFV-H2b20 protects mice against infection, apparently by eliciting the up-regulation of production of inflammatory cytokines.

In vitro assessment of functional properties of lactic acid bacteria isolated from faecal microbiota of healthy dogs for potential use as probiotics

Lactic acid bacteria were isolated and identified in the faeces of Chinese Crested and Yorkshire terrier pups and their probiotic features were investigated in vitro. Thirty seven isolates were identified as Lactobacillus or Enterococcus. Out of these isolates, 31 were lactic acid bacteria (LAB) and belonged to the species Lactobacillus reuteri (16/37; 43.3%), Lactobacillus animalis (7/37; 18.9%), Lactobacillus acidophilus (3/37; 8.1%), Lactobacillus sanfranciscensis (2/37; 5.4%), Lactobacillus murinus (2/37; 5.4%), and Lactobacillus paraplantarum (1/37; 2.7%), while six other LAB isolates were Enterococcus spp. (6/37; 16.2%). Strains were tested for resistance to gastric acidity (pH 2.5 for 3 h) and bile salts (0.3% ox gall), cell surface hydrophobicity by microbial adhesion to solvents, antagonism against pathogenic bacteria (Staphylococcus aureus, Enterococcus faecalis, Bacillus cereus, Pseudomonas aeruginosa, Escherichia coli, Salmonella enterica serovar Typhimurium and Listeria monocytogenes), production of hydrogen peroxide, and antibiotic susceptibility. Thirty four strains were highly resistant to acidic conditions with slight (18 strains) to moderate (16 strains) growth inhibition by bile salts. Seven isolates had highly hydrophobic cellular surfaces and 28 strains exhibited strong antagonism against the bacterial pathogens tested, although 8 isolates tested against Leptospira interrogans had no effect on pathogen growth. All isolates produced low rates of hydrogen peroxide. Based on these results, two Lactobacillus strains showed promising probiotic-related features and merit investigation as probiotics for dogs.

Lactobacillus species identification by amplified ribosomal 16S-23S rRNA restriction fragment length polymorphism analysis.

Lactic acid bacteria strains are commonly used for animal and human consumption due to their probiotic properties. One of the major genera used is Lactobacillus, a highly diverse genus comprised of several closely related species. The selection of new strains for probiotic use, especially strains of Lactobacillus, is the focus of several research groups. Accurate identification to species level is fundamental for research on new strains, as well as for safety assessment and quality assurance. The 16S-23S internal transcribed spacer (ITS-1) is a deeply homologous region among prokaryotes that is commonly used for identification to the species level because it is able to acquire and accumulate mutations without compromising general bacterial metabolism. In the present study, 16S-23S ITS regions of 45 Lactobacillus species (48 strains) were amplified and subjected to independent enzymatic digestions, using 12 restriction enzymes that recognise six-base sequences. Twenty-nine species showed unique restriction patterns, and could therefore be precisely identified solely by this assay (64%). This approach proved to be reproducible, allowing us to establish simplified restriction patterns for each evaluated species. The restriction patterns of each species were similar among homologous strains, and to a large extent reflected phylogenetic relationships based on 16S rRNA sequences, demonstrating the promising nature of this region for evolutionary studies.

Safety and protective effectiveness of two strains of Lactobacillus with probiotic features in an experimental model of salmonellosis.

Two strains of Lactobacillus, previously isolated from bovine faeces and tested in vitro for properties desired in probiotics, were evaluated for their in vivo effectiveness in protecting against experimental salmonellosis. L. salivarius L38 and L. acidophilus L36 previously demonstrated the ability to successfully colonize the gastrointestinal tract of germ-free mice and stimulate the immune system associated with the intestinal mucosa. L38- or L36-feeding showed no detrimental effect on the general health indicators and did not induce changes in normal architecture of liver and small intestine, indicating that the use of these strains is apparently safe. In control animals fed L38 strain, several cytokines had augmented mRNA levels that can be associated with a homeostatic state of intestinal mucosa, while L36 had less diverse regulation. IgA production and secretion in the intestinal lumen induced by infection was abrogated by pretreating with both lactobacilli. In addition, liver and small intestine histological scores and, translocation of Salmonella cells to liver and spleen, indicated that these strains did not confer protection against the infection. So, the IL-12:IL-18➔IFN-γ axis, essential for an effective immune response against Salmonella, was not favored with L38 or L36 strains. However, increased expression of IL-10 in different portions of the gastrointestinal tract of L38-fed animals is indicative of anti-inflammatory effect to be explored furthermore.

Effect of intestinal colonisation by two Lactobacillus strains on the immune response of gnotobiotic mice

The effect of intestinal colonisation on the immune system was investigated in germ-free mice monoassociated with Lactobacillus strains isolated from calf faeces. Single doses of Lactobacillus acidophilus L36 or Lactobacillus salivarius L38 were administered to germ-free mice by intragastric gavage. Ten days later, the mice were euthanised. Gene expression levels of interleukin 5 (IL-5), IL-6, IL-10, IL-12b, IL-17a, gamma interferon (IFN-γ), transforming growth factor beta 1 (TGF-β1), and tumour necrosis factor alpha (TNF-α) were quantified in segments of the small and large intestines by real time quantitative polymerase chain reaction. All the mice were colonised rapidly after Lactobacillus administration with intestinal counts ranging from 6.53 to 8.26 log cfu/g. L. acidophilus L36 administration increased the expression of cytokines involved with the Th2 (IL-5, IL-6 and TGF-β1) and Th17 (IL-17a, TNF-α and IL-6) inflammatory response, whereas L. salivarius L38 appeared to stimulate a pattern of less diversified cytokines in the intestine. Intragastric gavage of L. acidophilus L36 and L. salivarius L38 induced similar levels of colonisation in the digestive tracts of germ-free mice but stimulated different immune responses in the intestinal mucosa. The different immunomodulation patterns might facilitate the potential use of these lactobacilli as probiotics to treat distinct pathological conditions, for example protection against Citrobacter rodentium infection by stimulating IL-17 production.

Transfer of antibiotic resistance determinants between lactobacilli isolates from the gastrointestinal tract of chicken

The aim of this study was to assess the potential horizontal transfer of genetic traits for antibiotic resistance between lactobacilli isolated from the chicken gut, both in vitro and in vivo. Thirty-seven Lactobacillus spp. strains isolated from the gizzard, small and large intestines and caeca of free-range broiler chickens showed multi-drug resistance as assessed by disc diffusion assays. The minimum inhibitory concentration (MIC) for vancomycin, tetracycline, erythromycin and chloramphenicol was determined in De Man, Rogosa and Sharpe broth in a microplate assay. Almost all the lactobacilli isolates were resistant to vancomycin (except strains belonging to the Lactobacillus acidophilus group) and to tetracycline (MIC≥128 μg/ml). Only five strains were resistant to erythromycin, and six to chloramphenicol. The transfer rate in filter mating experiments performed using L. acidophilus strain 4M14E (EmR), Lactobacillus vaginalis strain 5M14E (CmR), Lactobacillus salivarius strain 5C14C (EmR), and the 4G14L and 3C14C strains of Lactobacillus reuteri (CmR) showed a frequency of approximately 1×104 cfu/ml of double-resistant transconjugants for the different combinations. The exception was the L. salivarius 5C14C (EmR) and L. vaginalis 5M14E (CmR) mating combination, which produced no transconjugants. In vivo experiments performed in gnotobiotic mice by mating L. acidophilus 4M14E (EmR) with L. reuteri 3C14C (CmR), L. reuteri 4G14L (CmR) or L. vaginalis 5M14E (CmR) resulted in transconjugants at 3.95±0.29, 3.16±0.33, and 4.55±1.52 log10 cfu/g of faeces, respectively. Taken together, these data suggest that genetic exchange may occur between native bacterial strains within the gastrointestinal tract of chickens, which might maintain a dynamic gene pool conferring antibiotic resistance upon indigenous microbiota components, even in the absence of the pathogens. This possibility must be taken into account as a complementary criterion when lactobacilli are screened for probiotic use.