Acidophilus Complex Research Articles

In vitro assessment of probiotic potential and safety of two Lactobacillus gasseri strains isolated from mother's breast milk and infant's feces in Vietnam

Probiotics have multiple beneficial effects on human health, including modulation of the immune system, inhibition of enteric pathogens, and prevention of metabolic disorders. Lactic acid bacteria (LAB) are the most common bacterial strains used for probiotic production. Among those, species belonging to the Lactobacillus acidophilus complex, especially L. gasseri, were well characterized and reported for probiotics. This study evaluated the potential probiotic characteristics of two L. gasseri strains (designed as VTCC 12791 and VTCC 12792) isolated from a pair of mother's breast milk and infant's feces. Both strains completely survived in the simulated gastric juice, highly tolerated the simulated intestinal juice (81-84%), strongly adhered to the HT-29 cell (23.5% for VTCC 12791 strain and 5.5% for VTCC 12792 strain), and had antagonistic activity against food-borne pathogens of Aeromonas dhakensis, Vibrio vulnificus, and Listeria monocytogenes, which demonstrated better beneficial properties than those of the commercial reference Lacticaseibacillus casei Shirota (LcS) strain. Additionally, both strains exhibited xanthine oxidase inhibitory activity (approximately 24%), which can lead to reducing serum uric acid. Regarding the safety aspect, the strains were sensitive to several antibiotics of ampicillin, vancomycin, tetracycline, chloramphenicol, erythromycin, and possessed alpha-hemolytic activity, which were similar to those of LcS strain. Our results suggested that L. gasseri VTCC 12791 and VTCC 12792 are potential candidates for producing probiotics and functional foods. Further in vivo studies are needed to prove their potential health benefits, especially in the treatment of hyperuricemia.

Conserved Genome Organization and Core Transcriptome of the Lactobacillus acidophilus Complex.

The Lactobacillus genus encompasses a genetically and functionally diverse group of species, and contains many strains widely formulated in the human food supply chain as probiotics and starter cultures. Within this genetically expansive group, there are several distinct clades that have high levels of homology, one of which is the Lactobacillus acidophilus group. Of the uniting features, small genomes, low GC content, adaptation to dairy environments, and fastidious growth requirements, are some of the most defining characteristics of this group. To better understand what truly links and defines this clade, we sought to characterize the genomic organization and content of the genomes of several members of this group. Through core genome analysis we explored the synteny and intrinsic genetic underpinnings of the L. acidophilus clade, and observed key features related to the evolution and adaptation of these organisms. While genetic content is able to provide a large map of the potential of each organism, it does not always reflect their functionality. Through transcriptomic data we inferred the core transcriptome of the L. acidophilus complex to better define the true metabolic capabilities that unite this clade. Using this approach we have identified seven small ORFs that are both highly conserved and transcribed in diverse members of this clade and could be potential novel small peptide or untranslated RNA regulators. Overall, our results reveal the core features of the L. acidophilus complex and open new avenues for the enhancement and formulation and of next generation probiotics and starter cultures.

A novel real-time PCR assay for highly specific detection and quantification of vaginal lactobacilli

PCR detection and quantification of vaginal lactobacilli remains problematic because of the high level of genetic heterogeneity and taxonomic complexity within the genus Lactobacillus. The aim of the present study was to identify conserved sequences among the genomes of major species of vaginal lactobacilli that could be used for the development of a PCR-based method for quantitative determination of vaginal microbiota-specific lactobacilli. Comparative analysis of the genomes of several species of vaginal lactobacilli allowed us to identify conserved regions in the rplK gene, which encodes ribosomal protein L11, and to design group-specific PCR primers and a probe for selected species from the L. acidophilus complex, including major vaginal lactobacilli Lactobacillus crispatus, L. gasseri, L. iners and L. jensenii as well as other species that are less common in vaginal microbiota. The applicability of the new assay in routine diagnostic testing was evaluated using a set of clinical samples. The assay was able to detect and quantify vagina-associated lactobacilli within a wide range of initial DNA template concentrations, indicating promising potential for clinical applications.

Positive selection on D-lactate dehydrogenases of Lactobacillus delbrueckii subspecies bulgaricus.

Lactobacillus delbrueckii has been widely used for yogurt fermentation. It has genes encoding both D- and L-type lactate dehydrogenases (LDHs) that catalyse the production of L(+) or D(-) stereoisomer of lactic acid. D-lactic acid is the primary lactate product by L. delbrueckii, yet it cannot be metabolised by human intestine. Since it has been domesticated for long time, an interesting question arises regarding to whether the selection pressure has affected the evolution of both L-LDH and D-LDH genes in the genome. To answer this question, in this study the authors first investigated the evolution of these two genes by constructing phylogenetic trees. They found that D-LDH-based phylogenetic tree could better represent the phylogenetic relationship in the acidophilus complex than L-LDH-based tree. They next investigated the evolutions of LDH genes of L. delbrueckii at amino acid level, and found that D-LDH gene in L. delbrueckii is positively selected, possibly a consequence of long-term domestication. They further identified four amino acids that are under positive selection. One of them, V261, is located at the centre of three catalytic active sites, indicating likely functional effects on the enzyme activity. The selection from the domestication process thus provides direction for future engineering of D-LDH.

Peptidoglycan Hydrolases as Species-Specific Markers to Differentiate Lactobacillus helveticus from Lactobacillus gallinarum and Other Closely Related Homofermentative Lactobacilli

We propose a new method that allows accurate discrimination of Lactobacillus helveticus from other closely related homofermentative lactobacilli, especially Lactobacillus gallinarum. This method is based on the amplification by PCR of two peptidoglycan hydrolytic genes, Lhv_0190 and Lhv_0191. These genes are ubiquitous and show high homology at the intra-species level. The PCR method gave two specific PCR products, of 542 and 747 bp, for 25 L. helveticus strains coming from various sources. For L. gallinarum, two amplicons were obtained, the specific 542 bp amplicon and another one with a size greater than 1,500 bp. No specific PCR products were obtained for 12 other closely related species of lactobacilli, including the L. acidophilus complex, L. delbrueckii, and L. ultunensis. The developed PCR method provided rapid, precise, and easy identification of L. helveticus. Moreover, it enabled differentiation between the two closely phylogenetically related species L. helveticus and L. gallinarum.

Genomic and phenotypic evidence for probiotic influences ofLactobacillus gasserion human health

Certain lactic acid bacteria (LAB) have the capacity to occupy mucosal niches of humans, including the oral cavity, gastrointestinal tract, and vagina. Among commensal, LAB are species of the acidophilus complex, which have proven to be a substantial reservoir for microorganisms with probiotic attributes. Specifically, Lactobacillus gasseri is an autochthonous microorganism which has been evaluated for probiotic activity based on the availability of genome sequence and species-specific adaptation to the human mucosa. Niche-related characteristics of L. gasseri contributing to indigenous colonization include tolerance of low pH environments, resistance to bile salts, and adhesion to the host epithelium. In humans, L. gasseri elicits various health benefits through its antimicrobial activity, bacteriocin production, and immunomodulation of the innate and adaptive systems. The genomic and empirical evidence supporting use of L. gasseri in probiotic applications is substantiated by clinical trial data displaying maintenance of vaginal homeostasis, mitigation of Helicobacter pylori infection, and amelioration of diarrhea.

Development of a Tiered Multilocus Sequence Typing Scheme for Members of the Lactobacillus acidophilus Complex

Members of the Lactobacillus acidophilus complex are associated with functional foods and dietary supplements because of purported health benefits they impart to the consumer. Many characteristics of these microorganisms are reported to be strain specific. Therefore, proper strain typing is essential for safety assessment and product labeling, and also for monitoring strain integrity for industrial production purposes. Fifty-two strains of the L. acidophilus complex (L. acidophilus, L. amylovorus, L. crispatus, L. gallinarum, L. gasseri, and L. johnsonii) were genotyped using two established methods and compared to a novel multilocus sequence typing (MLST) scheme. PCR restriction fragment length polymorphism (PCR-RFLP) analysis of the hsp60 gene with AluI and TaqI successfully clustered 51 of the 52 strains into the six species examined, but it lacked strain-level discrimination. Random amplified polymorphic DNA PCR (RAPD-PCR) targeting the M13 sequence resulted in highly discriminatory profiles but lacked reproducibility. In this study, an MLST scheme was developed using the conserved housekeeping genes fusA, gpmA, gyrA, gyrB, lepA, pyrG, and recA, which identified 40 sequence types that successfully clustered all of the strains into the six species. Analysis of the observed alleles suggests that nucleotide substitutions within five of the seven MLST loci have reached saturation, a finding that emphasizes the highly diverse nature of the L. acidophilus complex and our unconventional application of a typically intraspecies molecular typing tool. Our MLST results indicate that this method could be useful for characterization and strain discrimination of a multispecies complex, with the potential for taxonomic expansion to a broader collection of Lactobacillus species.

Enzymology and Structure of the GH13_31 Glucan 1,6-α-Glucosidase That Confers Isomaltooligosaccharide Utilization in the Probiotic Lactobacillus acidophilus NCFM

Isomaltooligosaccharides (IMO) have been suggested as promising prebiotics that stimulate the growth of probiotic bacteria. Genomes of probiotic lactobacilli from the acidophilus group, as represented by Lactobacillus acidophilus NCFM, encode α-1,6 glucosidases of the family GH13_31 (glycoside hydrolase family 13 subfamily 31) that confer degradation of IMO. These genes reside frequently within maltooligosaccharide utilization operons, which include an ATP-binding cassette transporter and α-glucan active enzymes, e.g., maltogenic amylases and maltose phosphorylases, and they also occur separated from any carbohydrate transport or catabolism genes on the genomes of some acidophilus complex members, as in L. acidophilus NCFM. Besides the isolated locus encoding a GH13_31 enzyme, the ABC transporter and another GH13 in the maltooligosaccharide operon were induced in response to IMO or maltotetraose, as determined by reverse transcription-PCR (RT-PCR) transcriptional analysis, suggesting coregulation of α-1,6- and α-1,4-glucooligosaccharide utilization loci in L. acidophilus NCFM. The L. acidophilus NCFM GH13_31 (LaGH13_31) was produced recombinantly and shown to be a glucan 1,6-α-glucosidase active on IMO and dextran and product-inhibited by glucose. The catalytic efficiency of LaGH13_31 on dextran and the dextran/panose (trisaccharide) efficiency ratio were the highest reported for this class of enzymes, suggesting higher affinity at distal substrate binding sites. The crystal structure of LaGH13_31 was determined to a resolution of 2.05 Å and revealed additional substrate contacts at the +2 subsite in LaGH13_31 compared to the GH13_31 from Streptococcus mutans (SmGH13_31), providing a possible structural rationale to the relatively high affinity for dextran. A comprehensive phylogenetic and activity motif analysis mapped IMO utilization enzymes from gut microbiota to rationalize preferential utilization of IMO by gut residents.

Phylogenomic reconstruction of lactic acid bacteria: an update

BackgroundLactic acid bacteria (LAB) are important in the food industry for the production of fermented food products and in human health as commensals in the gut. However, the phylogenetic relationships among LAB species remain under intensive debate owing to disagreements among different data sets.ResultsWe performed a phylogenetic analysis of LAB species based on 232 genes from 28 LAB genome sequences. Regardless of the tree-building methods used, combined analyses yielded an identical, well-resolved tree topology with strong supports for all nodes. The LAB species examined were divided into two groups. Group 1 included families Enterococcaceae and Streptococcaceae. Group 2 included families Lactobacillaceae and Leuconostocaceae. Within Group 2, the LAB species were divided into two clades. One clade comprised of the acidophilus complex of genus Lactobacillus and two other species, Lb. sakei and Lb. casei. In the acidophilus complex, Lb. delbrueckii separated first, while Lb. acidophilus/Lb. helveticus and Lb. gasseri/Lb. johnsonii were clustered into a sister group. The other clade within Group 2 consisted of the salivarius subgroup, including five species, Lb. salivarius, Lb. plantarum, Lb. brevis, Lb. reuteri, Lb. fermentum, and the genera Pediococcus, Oenococcus, and Leuconostoc. In this clade, Lb. salivarius was positioned most basally, followed by two clusters, one corresponding to Lb. plantarum/Lb. brevis pair and Pediococcus, and the other including Oenococcus/Leuconostoc pair and Lb. reuteri/Lb. fermentum pair. In addition, phylogenetic utility of the 232 genes was analyzed to identify those that may be more useful than others. The genes identified as useful were related to translation and ribosomal structure and biogenesis (TRSB), and a three-gene set comprising genes encoding ultra-violet resistance protein B (uvrB), DNA polymerase III (polC) and penicillin binding protein 2B (pbpB).ConclusionsOur phylogenomic analyses provide important insights into the evolution and diversification of LAB species, and also revealed the phylogenetic utility of several genes. We infer that the occurrence of multiple, independent adaptation events in LAB species, have resulted in their occupation of various habitats. Further analyses of more genes from additional, representative LAB species are needed to reveal the molecular mechanisms underlying adaptation of LAB species to various environmental niches.

Extensive horizontal transfer of core genome genes between two Lactobacillus species found in the gastrointestinal tract.

BackgroundWhile genes that are conserved between related bacterial species are usually thought to have evolved along with the species, phylogenetic trees reconstructed for individual genes may contradict this picture and indicate horizontal gene transfer. Individual trees are often not resolved with high confidence, however, and in that case alternative trees are generally not considered as contradicting the species tree, although not confirming it either. Here we conduct an in-depth analysis of 401 protein phylogenetic trees inferred with varying levels of confidence for three lactobacilli from the acidophilus complex. At present the relationship between these bacteria, isolated from environments as diverse as the gastrointestinal tract (Lactobacillus acidophilus and Lactobacillus johnsonii) and yogurt (Lactobacillus delbrueckii ssp. bulgaricus), is ambiguous due to contradictory phenotypical and 16S rRNA based classifications.ResultsAmong the 401 phylogenetic trees, those that could be reconstructed with high confidence support the 16S-rRNA tree or one alternative topology in an astonishing 3:2 ratio, while the third possible topology is practically absent. Lowering the confidence threshold for trees to be taken into consideration does not significantly affect this ratio, and therefore suggests that gene transfer may have affected as much as 40% of the core genome genes. Gene function bias suggests that the 16S rRNA phylogeny of the acidophilus complex, which indicates that L. acidophilus and L. delbrueckii ssp. bulgaricus are the closest related of these three species, is correct. A novel approach of comparison of interspecies protein divergence data employed in this study allowed to determine that gene transfer most likely took place between the lineages of the two species found in the gastrointestinal tract.ConclusionThis case-study reports an unprecedented level of phylogenetic incongruence, presumably resulting from extensive horizontal gene transfer. The data give a first indication of the large extent of gene transfer that may take place in the gastrointestinal tract and its accumulated effect. For future studies, our results should encourage a careful weighing of data on phylogenetic tree topology, confidence and distribution to conclude on the absence or presence and extent of horizontal gene transfer.

Cell growth and bacteriocin production of probiotic Lactobacillus strains in different media

Growth, metabolism, and bacteriocin production by seven Lactobacillus strains including five commercial probiotic strains were studied during fermentation in MRS medium and milk medium at constant pH 6.5. These strains were Lactobacillus acidophilus ACC, L. acidophilus IBB 801, L. casei Imunitas, L. casei YIT 9029, L. gasseri K7, L. johnsonii La1, and L. rhamnosus GG. Although the L. casei complex strains grew to higher cell levels than the L. acidophilus complex strains in MRS medium, monitored bacteriocin titres were higher for the L. acidophilus complex strains. L. johnsonii La1 and L. gasseri K7 grew in milk medium only when yeast extract was added. Addition of yeast extract (0.3–1.0% w/v) to milk medium enhanced both growth and bacteriocin production for all strains. Bacteriocin production was clearly observed in yeast extract supplemented milk medium for L. acidophilus IBB 801, L. johnsonii La1, and L. gasseri K7. L. acidophilus IBB 801, the only strain of dairy origin, displayed the best growth (10.5 log CFU mL−1) and bacteriocin production (3200 AU mL−1). These findings demonstrated that probiotic lactobacilli from an intestinal origin are difficult to cultivate in milk.

Comparison of selective and differential media for the accurate enumeration of strains of Lactobacillus acidophilus, Bifidobacterium spp. and Lactobacillus casei complex from commercial yoghurts

Various selective and differential media were evaluated for their suitability to provide reliable counts of Lactobacillus acidophilus, Bifidobacterium spp. and L. casei from nine different commercial probiotic yoghurts. Selective media reported to develop only single type of colonies, demonstrated two types of colonies with some yoghurts, making it difficult to conclusively enumerate the probiotic colonies. Similarly, colony characteristics on the differential media were found too subjective to suitably distinguish the probiotic colonies. Counts of each probiotic strain from the same yoghurt sample also varied on the different media. Except for LC agar, no selective or differential medium provided reliable counts of probiotic bacteria in all yoghurts. This study demonstrates the urgent need to develop reliable techniques to accurately enumerate probiotic bacteria in commercial yoghurts.

Distribution of theLactobacillus acidophilusComplex in Human Fecal Specimens Examined by Restriction Fragment Length Polymorphism of the 16S rRNA Genes

Fresh fecal samples from healthy volunteers were examined for their content of the Lactobacillus acidophilus complex (LAC). A two-step isolation method for fecal lactobacilli was developed and employed in this study. Isolates of lactobacilli were identified according to their restriction fragment length polymorphism (RFLP) of genes coding for ribosomal RNA. Our results suggested that all samples contain lactobacilli and the most dominant species of human fecal lactobacilli is L. paracasei. In addition, several strains of LAC were recovered from specimens of volunteers. Among six LAC species, namely, A1: L. acidophilus, A2: L. crispatus, A3: L. amylovorus, A4: L. gallinarum, B1: L. gasseri, and B2: L. johnsonii, isolates classified into cluster B were recovered from 6 out of 15 volunteers; however isolates of cluster A were recovered from 3 of 15 volunteers. Four of 15 volunteers had strains of L. gasseri, 3 of them had strains of L. johnsonii, and strains of L. amylovorus were also isolated from specimens of 3 of 15 volunteers. These findings suggest that group B of LAC is the predominant cluster of LAC found in human feces and L. amylovorus was the only species of cluster A found in this study. No volunteer with blood type O had LAC in the fecal Lactobacillus flora.

Identification of randomly selected colonies of lactobacilli from normal vaginal fluid by pyrosequencing of the 16S rDNA variable V1 and V3 regions

The present study aimed to characterize lactobacilli in vaginal fluid from 23 adult healthy women by using high-throughput DNA sequencing for identification of a large number of randomly selected colonies appearing on Rogosa and blood agar. The typing method was based on broad-range PCR of 16S rRNA gene variable regions V1 and V3, pyrosequencing, and classification of the fragments by alignment with NCBI-catalogued sequences and type strain sequences. Four major groups of sequences were found among the 402 isolates clearly corresponding to Lactobacillus crispatus, Lactobacillus gasseri, Lactobacillus iners and Lactobacillus jensenii when compared to the sequences obtained for type strains. Our results indicate that pyrosequencing of 16S rRNA gene fragments as used here is a fast and reliable method well suited for identification to the species level, even within the Lactobacillus acidophilus complex.

Vaginal lactobacillus flora of healthy Swedish women.

Species of the Lactobacillus acidophilus complex are generally considered to constitute most of the vaginal Lactobacillus flora, but the flora varies between studies. However, this may be due to difficulties in identifying the closely related species within the L. acidophilus complex by using traditional methods and to variations in the vaginal status of the participants. Two hundred two isolates from the vaginal fluids of 23 Swedish women without bacterial vaginosis, as defined by the criteria of Nugent et al. (R. P. Nugent, M. A. Krohn, and S. L. Hillier, J. Clin. Microbiol. 29:297-301, 1991), were typed by randomly amplified polymorphic DNA (RAPD) analysis and identified to the species level by temporal temperature gradient gel electrophoresis, multiplex PCR, and 16S ribosomal DNA sequencing. The vaginal flora of most participants was dominated by a single RAPD type, but five of them harbored two RAPD types representing two different species or strains. The most frequently occurring species were Lactobacillus crispatus, Lactobacillus gasseri, Lactobacillus iners, and Lactobacillus jensenii. L. iners has not previously been reported as one of the predominant Lactobacillus species in the vagina.

Use of the DNA sequence of variable regions of the 16S rRNA gene for rapid and accurate identification of bacteria in theLactobacillus acidophiluscomplex

The Lactobacillus acidophilus complex includes Lact. acidophilus, Lactobacillus amylovorus, Lactobacillus crispatus, Lactobacillus gallinarum, Lactobacillus gasseri and Lactobacillus johnsonii. The objective of this work was to develop a rapid and definitive DNA sequence-based identification system for unknown isolates of the Lact. acidophilus complex. A approximately = 500 bp region of the 16S rRNA gene, which contained the V1 and V2 variable regions, was amplified from the isolates by the polymerase chain reaction. The sequence of this region of the 16S rRNA gene from the type strains of the Lact. acidophilus complex was sufficiently variable to allow for clear differentiation amongst each of the strains. As an initial step in the characterization of potentially probiotic strains, this technique was successfully used to identify a variety of unknown human intestinal isolates. The approach described here represents a rapid and definitive method for the identification of Lact. acidophilus complex members.

Molecular identification of potentially probiotic lactobacilli.

The rRNA-targeted oligonucleotide probes are useful for the identification of Lactobacillus acidophilus, L. gasseri, L. johnsonii, L. crispatus, and L. amylovorus. However, the oligonucleotide probe designed for L. helveticus hybridized with nucleic acids of type strains of L. gallinarum and L. helveticus. Hence, the similarity among the 73 strains of lactobacilli was evaluated on the basis of their randomly amplified polymorphic DNA (RAPD) profiles derived from five single-primer reactions. These strains were grouped into seven clusters at a similarity level of 30%, which corresponded to six separate species of the L. acidophilus complex (L. johnsonii, L. gallinarum, L. amylovorus, L. crispatus, L. acidophilus, and L. gasseri, respectively) and L. helveticus. For the first time, strains of L. gallinarum were characterized by RAPD and PFGE analyses. The genome length in that species was estimated to be near 1.45 Mb with the summation of ApaI fragments, and near 1.95 Mb with the summation of SmaI fragments.

β-Galactosidase and 6-phospho-β-galactosidase activities in strains of the Lactobacillus acidophilus complex

β-Galactosidase (β-gal) and 6-phospho-β-galactosidase (P-β-gal) activities were measured in a total of 34 strains from Lactobacillus acidophilus, Lactobacillus crispatus, Lactobacillus amylovorus, Lactobacillus gasseri and Lactobacillus johnsonii. The Lact. gasseri strains have P-β-gal but little or no β-gal activities. The strains from other species have β-gal but only very little P-β-gal activities.