Real-Time PCR Assays for the Specific Identification of Probiotic Strains Lactobacillus gasseri BNR17 and Lactobacillus reuteri LRC (NCIMB 30242)

The broad spectrum of health benefits attributed to probiotics has contributed to a rapid increase in the value of the probiotic market. Probiotic health benefits can be strain specific. Thus, strain-level identification of probiotic strains is of paramount importance to ensure probiotic efficacy. Both Lactobacillus gasseri BNR17 and Lactobacillus reuteri LRC (NCIMB 30242) strains have clinically proven health benefits; however, no assays were developed to enable strain-level identification of either of these strains. The objective of this study is to develop strain-specific PCR-based methods for Lactobacillus gasseri BNR17 and Lactobacillus reuteri LRC strains, and to validate these assays according to the guidelines for validating qualitative real-time PCR assays. Using RAST (Rapid Annotation using Subsystem Technology), unique sequence regions were identified in the genome sequences of both strains. Probe-based assays were designed and validated for specificity, sensitivity, efficiency, repeatability, and reproducibility. Both assays were specific to target strain with 100% true positive and 0% false positive rates. Reaction efficiency for both assays was in the range of 90 to 108% with R square values > 0.99. Repeatability and reproducibility were evaluated using five samples at three DNA concentrations each and relative standard deviation was < 4% for repeatability and < 8% for reproducibility. Both of the assays developed and validated in this study for the specific identification of Lactobacillus gasseri BNR17 and Lactobacillus reuteri LRC strains are specific, sensitive, and precise. These assays can be applied to evaluate and ensure compliance in probiotic products.

     

Identification of One Novel Candidate Probiotic Lactobacillus plantarum Strain Active against Influenza Virus Infection in Mice by a Large-Scale Screening

In this study, we developed a large-scale screening of bacterial strains in order to identify novel candidate probiotics with immunomodulatory properties. For this, 158 strains, including a majority of lactic acid bacteria (LAB), were screened by two different cellular models: tumor necrosis factor alpha (TNF-α)-activated HT-29 cells and peripheral blood mononuclear cells (PBMCs). Different strains responsive to both models (pro- and anti-inflammatory strains) were selected, and their protective effects were tested in vivo in a murine model of influenza virus infection. Daily intragastric administrations during 10 days before and 10 days after viral challenge (100 PFU of influenza virus H1N1 strain A Puerto Rico/8/1934 [A/PR8/34]/mouse) of Lactobacillus plantarum CNRZ1997, one potentially proinflammatory probiotic strain, led to a significant improvement in mouse health by reducing weight loss, alleviating clinical symptoms, and inhibiting significantly virus proliferation in lungs. In conclusion, in this study, we have combined two cellular models to allow the screening of a large number of LAB for their immunomodulatory properties. Moreover, we identified a novel candidate probiotic strain, L. plantarum CNRZ1997, active against influenza virus infection in mice.     

Congruent Strain Specific Intestinal Persistence of Lactobacillus plantarum in an Intestine-Mimicking In Vitro System and in Human Volunteers

Background

An important trait of probiotics is their capability to reach their intestinal target sites alive to optimally exert their beneficial effects. Assessment of this trait in intestine-mimicking in vitro model systems has revealed differential survival of individual strains of a species. However, data on the in situ persistence characteristics of individual or mixtures of strains of the same species in the gastrointestinal tract of healthy human volunteers have not been reported to date.

Methodology/Principal Findings

The GI-tract survival of individual L. plantarum strains was determined using an intestine mimicking model system, revealing substantial inter-strain differences. The obtained data were correlated to genomic diversity of the strains using comparative genome hybridization (CGH) datasets, but this approach failed to discover specific genetic loci that explain the observed differences between the strains. Moreover, we developed a next-generation sequencing-based method that targets a variable intergenic region, and employed this method to assess the in vivo GI-tract persistence of different L. plantarum strains when administered in mixtures to healthy human volunteers. Remarkable consistency of the strain-specific persistence curves were observed between individual volunteers, which also correlated significantly with the GI-tract survival predicted on basis of the in vitro assay.

Conclusion

The survival of individual L. plantarum strains in the GI-tract could not be correlated to the absence or presence of specific genes compared to the reference strain L. plantarum WCFS1. Nevertheless, in vivo persistence analysis in the human GI-tract confirmed the strain-specific persistence, which appeared to be remarkably similar in different healthy volunteers. Moreover, the relative strain-specific persistence in vivo appeared to be accurately and significantly predicted by their relative survival in the intestine-mimicking in vitro assay, supporting the use of this assay for screening of strain-specific GI persistence.     

Putative and Unique Gene Sequence Utilization for the Design of Species Specific Probes as Modeled by Lactobacillus plantarum

The concept of utilizing putative and unique gene sequences for the design of species specific probes was tested. The abundance profile of assigned functions within the Lactobacillus plantarum genome was used for the identification of the putative and unique gene sequence, csh. The targeted gene (csh) was used as the template for PCR amplification and construction of a non-radioactive DIG labeled probe. The csh derived probe aided in the preliminary and rapid identification of L. plantarum from mixed cultures by colony hybridization. The method described here for the rapid identification of L. plantarum can also be applied for the rapid detection of other bacteria if a unique gene sequence can be identified from its complete genome sequence.     

Differentiation of Lactobacillus Species by Molecular Typing

A total of 64 type, reference, clinical, health food, and stock isolates of microaerophilic Lactobacillus species were examined by restriction fragment length polymorphisms. Of particular interest were members of six of the eight species most commonly recovered from the vaginas of healthy premenopausal women, namely, Lactobacillus jensenii, L. casei, L. rhamnosus, L. acidophilus, L. plantarum, and L. fermentum. Six main groupings were identified on the basis of ribotyping. This technique was able to classify fresh isolates to the species level. In the case of the ribotype A grouping for L. rhamnosus, differences between strains were evident by chromosome typing (chromotyping). Many isolates did not possess plasmids. Six L. rhamnosus strains isolated from four different health food products appeared to be identical to L. rhamnosus ATCC 21052. The molecular typing system is useful for identifying and differentiating Lactobacillus isolates. Studies of strains of potential importance to the urogenital flora should include molecular characterization as a means of comparing genetic traits with those of strains whose characteristics associated with colonization and antagonism against pathogens have been defined.Lactobacilli colonizing human tissues have long been considered important for the maintenance of a healthy gastrointestinal tract (4) and urogenital tract (20, 21, 22, 25). The disruption of the lactobacillus flora has been associated with many urogenital infections, and as these afflict over 150 million women worldwide each year, this area of study is an important one. Indeed, many patients resort to taking health food products containing lactobacilli as a means of trying to maintain a healthy intestinal (and in some cases vaginal) flora.The typing of lactobacilli has generally been conducted by cell and colony morphology and biochemical tests. These techniques type bacteria based on their ability to ferment sugars and produce acids such as lactic acid and acetic acid (9). Unfortunately, these typing methods are not completely accurate, and strains which show intermediate characteristics are frequently encountered (9, 33).Many studies emphasize that the classification of lactobacilli is unsatisfactory and does not reflect the real phylogenetic relatedness of different strains and species (6, 19, 30). Several new genetic and chemotaxonomic approaches have been used during the last 14 years with an aim of improving the classification and identification of lactobacilli: for example, analysis of plasmid content (18), sodium dodecyl sulfate-polyacrylamide gel electrophoresis patterns of whole-cell protein (19) and of total soluble cell protein (8), sequencing of rRNA (5, 6, 19), restriction endonuclease fingerprinting (14, 30), and DNA-DNA hybridization (6, 17, 19, 27). All of these approaches have improved the taxonomic knowledge of the generic and suprageneric relationships of lactobacilli. However, no analysis of urogenital or health food isolates of lactobacilli by molecular typing has been reported.Plasmid typing of Lactobacillus strains has been suggested as a taxonomic tool in a number of reports (27, 31), but there is evidence to suggest that it is not very effective (2, 12, 15, 28), since plasmids can be absent or unstable.Chromosome typing (restriction endonuclease fingerprinting of chromosomal DNA) (chromotyping) has been applied to the discrimination of strains of lactobacilli and has been found more specific and reproducible than plasmid content analysis (14). However, one of the disadvantages of chromotyping is that comparing electrophoretic patterns consisting of up to 100 bands is difficult. Hence, chromotyping alone may not be widely used for typing large numbers of strains.A sensitive but rapid method for species differentiation involves the use of ribotyping (1, 27), which combines Southern hybridization of chromosomal DNA fingerprints with the use of Escherichia coli rRNA probes, thereby discriminating between various species and individual strains of lactobacilli. The method involves the separation and identification of the rRNA genes present within the bacterial genome, which exist in various copy numbers (10, 13), making it possible to delineate species based on differences in the restriction fragment length polymorphisms of the rRNA genes. Of particular importance is the fact that specific regions of the rRNA genes have remained well conserved because of their functional importance, thus allowing the detection of a broad range of bacteria with 16S and 23S rRNA of E. coli as probes.The aim of this study was to develop a method to test the efficacy of ribotyping of Lactobacillus type and reference strains and to use this method to characterize a number of clinical, health food, and laboratory isolates.     

Identification of Antioxidants Produced by Lactobacillus plantarum

We identified two compounds that demonstrated 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity from cultures of Lactobacillus plantarum. Spectroscopic analyses proved these compounds to be L-3-(4-hydroxyphenyl) lactic acid (HPLA) and L-indole-3-lactic acid (ILA). The respective EC₅₀ values for HPLA and ILA were 36.6 ± 4.3 mM and 13.4 ± 1.0 mM.     

Exopolysaccharide Production and Ropy Phenotype Are Determined by Two Gene Clusters in Putative Probiotic Strain Lactobacillus paraplantarum BGCG11

Lactobacillus paraplantarum BGCG11, a putative probiotic strain isolated from a soft, white, artisanal cheese, produces a high-molecular-weight heteropolysaccharide, exopolysaccharide (EPS)-CG11, responsible for the ropy phenotype and immunomodulatory activity of the strain. In this study, a 26.4-kb region originating from the pCG1 plasmid, previously shown to be responsible for the production of EPS-CG11 and a ropy phenotype, was cloned, sequenced, and functionally characterized. In this region 16 putative open reading frames (ORFs), encoding enzymes for the production of EPS-CG11, were organized in specific loci involved in the biosynthesis of the repeat unit, polymerization, export, regulation, and chain length determination. Interestingly, downstream of the eps gene cluster, a putative transposase gene was identified, followed by an additional rfb gene cluster containing the rfbACBD genes, the ones most probably responsible for dTDP-l-rhamnose biosynthesis. The functional analysis showed that the production of the high-molecular-weight fraction of EPS-CG11 was absent in two knockout mutants, one in the eps and the other in the rfb gene cluster, as confirmed by size exclusion chromatography analysis. Therefore, both eps and rfb genes clusters are prerequisites for the production of high-molecular-weight EPS-CG11 and for the ropy phenotype of strain L. paraplantarum BGCG11.     

Properties of the Probiotic Strain Lactobacillus plantarum 8-RA-3 Grown in a Biofilm by Solid Substrate Cultivation Method

The biofilm formation took place in 48?h within the solid substrate cultivation of Lactobacillus plantarum 8-RA-3 strain on the wheat bran saturated with the MRS medium. The drying of the bran fermented by lactobacilli resulted in a decrease in the number of colony-forming units (CFU) from 23.0?×?10⁸ to 6.9?×?10⁵?CFU/g in daily samples and to less than 10⁴?CFU/g in 2- and 3-day samples. However, according to the fluorescence-based live/dead assay data, more than 40?% of the non-cultured bacteria were viable. As a result of mice kept on a diet with the introduction of bran fermented by Lact. plantarum 8-RA-3 for 72?h into the fodder, a recovery of normal level of intestinal lactobacilli, inhibited by administration of antibiotic was noted. The strain genetically identical to the Lact. plantarum 8-RA-3 was isolated from the feces of these mice. The results indicate that solid substrate cultivated Lact. plantarum 8-RA-3 strain formed a biofilm. Once dried and transferred into a non-cultured state, biofilm cells retained its viability and biological activity.     

Intraspecific Genotypic Characterization of Lactobacillus rhamnosus Strains Intended for Probiotic Use and Isolates of Human Origin

A set of 118 strains of the species Lactobacillus rhamnosus was collected, including probiotic strains, research strains with potential probiotic properties, food starter cultures, and human isolates. The majority of the strains were collected from companies, hospitals, or culture collections or were obtained after contacting authors who reported clinical case studies in the literature. The present work aimed to reveal the genotypic relationships between strains of these diverse sources. All strains were initially investigated using fluorescent amplified fragment length polymorphism (FAFLP) with three different primer combinations. Numerical analysis of FAFLP data allowed (i) confirmation of the identification of all strains as members of L. rhamnosus and (ii) delineation of seven stable intraspecific FAFLP clusters. Most of these clusters contained both (potentially) probiotic strains and isolates of human origin. For each of the clusters, strains of different sources were selected for pulsed-field gel electrophoresis (PFGE) of macrorestriction fragments obtained with the enzymes NotI and AscI. Analysis of PFGE data indicated that (i) some (potentially) probiotic strains were indistinguishable from other probiotic strains, suggesting that several companies may use duplicate cultures of the same probiotic strain, and (ii) in a number of cases human isolates from sterile body sites were indistinguishable from a particular probiotic strain, suggesting that some of these isolates may be reisolations of commercial strains.     

Degradation of Raw Starch by a Wild Amylolytic Strain of Lactobacillus plantarum

Lactobacillus plantarum A6, isolated from fermented cassava, can break down cassava raw starch that has not been subjected to preliminary physicochemical treatment. When the pH was kept at 6, the microorganism cultured in a bioreactor excreted a high α-amylase activity (60 U/ml). Synthesis of the enzyme occurred during the stationary phase and resulted in full hydrolysis of the cassava starch granules. This gave 41 g of lactic acid from 45 g of raw starch after 3 days of fermentation. Enzymatic attack was evident under scanning electron microscopy in the rougher appearance of the surface of starch granules and in the presence of large cavities in some of them. In contrast, when the pH was not regulated, only a small amount of α-amylase activity was produced (2 U/ml) and no decrease in the starch content of the medium was observed. However, under scanning electron microscopy, some granules displayed a rougher surface, which might have been the result of weak enzymatic attack.     

Persistence of Colonization of Human Colonic Mucosa by a Probiotic Strain, Lactobacillus rhamnosus GG, after Oral Consumption

Lactobacillus rhamnosus GG is one of the most thoroughly studied probiotic strains. Its advantages in the treatment of gastrointestinal disorders are well documented. The aim of the present study was to demonstrate with colonic biopsies the attachment of strain GG to human intestinal mucosae and the persistence of the attachment after discontinuation of GG administration. A whey drink fermented with strain GG was fed to human volunteers for 12 days. Fecal samples were collected before, during, and after consumption. L. rhamnosus GG-like colonies were detected in both fecal and colonic biopsy samples. Strain GG was identified by its characteristic colony morphology, a lactose fermentation test, and PCR. This study showed that strain GG was able to attach in vivo to colonic mucosae and, although the attachment was temporary, to remain for more than a week after discontinuation of GG administration. The results demonstrate that the study of fecal samples alone is not sufficient in evaluating colonization by a probiotic strain.     

Functional and Probiotic Attributes of an Indigenous Isolate of Lactobacillus plantarum

Background

Probiotic microorganisms favorably alter the intestinal microflora balance, promote intestinal integrity and mobility, inhibit the growth of harmful bacteria and increase resistance to infection. Probiotics are increasingly used in nutraceuticals, functional foods or in microbial interference treatment. However, the effectiveness of probiotic organism is considered to be population-specific due to variation in gut microflora, food habits and specific host-microbial interactions. Most of the probiotic strains available in the market are of western or European origin, and a strong need for exploring new indigenous probiotic organisms is felt.

Methods and Findings

An indigenous isolate Lp9 identified as Lactobacillus plantarum by molecular-typing methods was studied extensively for its functional and probiotic attributes, viz., acid and bile salt tolerance, cell surface hydrophobicity, autoaggregation and Caco-2 cell-binding as well as antibacterial and antioxidative activities. Lp9 isolate could survive 2 h incubation at pH 1.5–2.0 and toxicity of 1.5–2.0% oxgall bile. Lp9 could deconjugate major bile salts like glycocholate and deoxytaurocholate, indicating its potential to cause hypocholesterolemia. The isolate exhibited cell-surface hydrophobicity of ∼37% and autoaggregation of ∼31%. Presence of putative probiotic marker genes like mucus-binding protein (mub), fibronectin-binding protein (fbp) and bile salt hydrolase (bsh) were confirmed by PCR. Presence of these genes suggested the possibility of specific interaction and colonization potential of Lp9 isolate in the gut, which was also suggested by a good adhesion ratio of 7.4±1.3% with Caco-2 cell line. The isolate demonstrated higher free radical scavenging activity than standard probiotics L. johnsonii LA1 and L. acidophilus LA7. Lp9 also exhibited antibacterial activity against E. coli, L. monocytogenes, S. typhi, S. aureus and B. cereus.

Conclusion

The indigenous Lactobacillus plantarum Lp9 exhibited high resistance against low pH and bile and possessed antibacterial, antioxidative and cholesterol lowering properties with a potential for exploitation in the development of indigenous functional food or nutraceuticals.     

Complete Genome Sequence of Lactobacillus fermentum CECT 5716, a Probiotic Strain Isolated from Human Milk

Lactobacillus fermentum is a heterofermentative lactic acid bacterium and is frequently isolated from mucosal surfaces of healthy humans. Lactobacillus fermentum CECT 5716 is a well-characterized probiotic strain isolated from human milk and, at present, is used in commercial infant formulas. Here, we report the complete and annotated genome sequence of this strain.Breast milk is the best food for neonates because it provides a unique combination of nutrients and bioactive compounds, ensuring correct growth and development of the infant. In addition, it also contains probiotic bacteria (4, 5). In a previous study, we isolated Lactobacillus fermentum CECT 5716 from such biological fluid (3). Subsequent studies revealed that this strain was a good probiotic candidate since it reached high survival rates when exposed to gastrointestinal tract-like conditions, showed a strong adherence to intestinal cells, stimulated the expression of mucin-encoding genes, produced antimicrobial compounds, and displayed in vivo and in vitro immunomodulatory and antibacterial properties against pathogenic bacteria (1, 5, 7). L. fermentum CECT 5716 showed a beneficial effect in a murine model of intestinal inflammation, reducing the inflammatory response and the intestinal damage (2). In addition, consumption of this strain enhances the response to influenza vaccination in healthy volunteers and reduces the incidence of influenza-like illness (8).In order to interrogate the genome sequence of Lactobacillus fermentum CECT 5716 with regard to its probiotic properties, the complete genome sequence was determined by a whole-genome shotgun strategy using 454 pyrosequencing technology (454 Life Sciences, Banford, CT). The initial draft assembly provided by 454 Life Sciences was based on 193,362 pyrosequencing reads with an average read length of 250 nucleotides which assembled into 1,343 contigs. Sequence reads were assembled automatically with the Life Sciences GS FLX (Newbler) program. The genome sequence of Lactobacillus fermentum IFO 3956 (6) was used to order these contigs into large scaffolds. The assembling process was relatively complex due to the 83 transposase-encoding regions that were found in the CECT 5716 genome.The complete genome of Lactobacillus fermentum CECT 5716 consists of a circular chromosome of 2,100,449 bp, with a GC content of 51.49%, and has no plasmid. Its chromosome contains 1,109 predicted protein-encoding genes, 54 tRNA-encoding genes, and 20 rRNA-encoding genes. The comparison of the CECT 5716 and IFO 3956 genomes revealed that they were highly similar, with the exception of 16 protein-encoding genes that are present in CECT 5716 but not in IFO 3956. Among them, there are putative enzymes involved in the metabolism of purines (allantoinase, GMP oxidoreductase, GMP synthase), amino acids (serine-pyruvate transaminase, 3 glutamate synthases), lipids (acyltransferase), and carbohydrates (mannose-6-phosphate isomerase).     

Effects of Probiotic Lactobacillus acidophilus Strain INMIA 9602 Er 317/402 and Putative Probiotic Lactobacilli on DNA Damages in the Small Intestine of Wistar Rats In Vivo

Probiotics and Antimicrobial Proteins - Double-strand breaks in the DNA of the small intestine in male Wistar rats were studied using a neutral comet assay after 7&nbsp;days of feeding with a...     

Identification and characterization of antibiotic resistance genes in Lactobacillus reuteri and Lactobacillus plantarum

Aims:  The study aimed to identify the resistance genes mediating atypical minimum inhibitory concentrations (MICs) for tetracycline, erythromycin, clindamycin and chloramphenicol within two sets of representative strains of the species Lactobacillus reuteri and Lactobacillus plantarum and to characterize identified genes by means of gene location and sequencing of flanking regions.
Methods and Results:  A tet (W) gene was found in 24 of the 28 Lact. reuteri strains with atypical MIC for tetracycline, whereas four of the six strains with atypical MIC for erythromycin were positive for erm (B) and one strain each was positive for erm (C) and erm (T). The two Lact. plantarum strains with atypical MIC for tetracycline harboured a plasmid-encoded tet (M) gene. The majority of the tet (W)-positive Lact. reuteri strains and all erm -positive Lact. reuteri strains carried the genes on plasmids, as determined by Southern blot and a real-time PCR method developed in this study.
Conclusions:  Most of the antibiotic-resistant strains of Lact. reuteri and Lact. plantarum harboured known plasmid-encoded resistance genes. Examples of putative transfer machineries adjacent to both plasmid- and chromosome-located resistance genes were also demonstrated.
Significance and Impact of the Study:  These data provide some of the knowledge required for assessing the possible risk of using Lact. reuteri and Lact. plantarum strains carrying antibiotic resistance genes as starter cultures and probiotics.     

Enhancement in Iron Absorption on Intake of Chemometrically Optimized Ratio of Probiotic Strain Lactobacillus plantarum 299v with Iron Supplement Pearl Millet

Biological Trace Element Research - This research article aims to establish the intake ratio of probiotic Lactobacillus plantarum 299v with iron supplement pearl millet by central composite design...     

Impact of Environmental and Genetic Factors on Biofilm Formation by the Probiotic Strain Lactobacillus rhamnosus GG

Lactobacillus rhamnosus GG (ATCC 53103) is one of the clinically best-studied probiotic organisms. Moreover, L. rhamnosus GG displays very good in vitro adherence to epithelial cells and mucus. Here, we report that L. rhamnosus GG is able to form biofilms on abiotic surfaces, in contrast to other strains of the Lactobacillus casei group tested under the same conditions. Microtiter plate biofilm assays indicated that in vitro biofilm formation by L. rhamnosus GG is strongly modulated by culture medium factors and conditions related to the gastrointestinal environment, including low pH; high osmolarity; and the presence of bile, mucins, and nondigestible polysaccharides. Additionally, phenotypic analysis of mutants affected in exopolysaccharides (wzb), lipoteichoic acid (dltD), and central metabolism (luxS) showed their relative importance in biofilm formation by L. rhamnosus GG.