Multilocus sequence typing of Lactobacillus casei reveals a clonal population structure with low levels of homologous recombination

Robust genotyping methods for Lactobacillus casei are needed for strain tracking and collection management, as well as for population biology research. A collection of 52 strains initially labeled L. casei or Lactobacillus paracasei was first subjected to rplB gene sequencing together with reference strains of Lactobacillus zeae, Lactobacillus rhamnosus, and other species. Phylogenetic analysis showed that all 52 strains belonged to a single compact L. casei-L. paracasei sequence cluster, together with strain CIP107868 (= ATCC 334) but clearly distinct from L. rhamnosus and from a cluster with L. zeae and CIP103137(T) (= ATCC 393(T)). The strains were genotyped using amplified fragment length polymorphism, multilocus sequence typing based on internal portions of the seven housekeeping genes fusA, ileS, lepA, leuS, pyrG, recA, and recG, and tandem repeat variation (multilocus variable-number tandem repeats analysis [MLVA] using nine loci). Very high concordance was found between the three methods. Although amounts of nucleotide variation were low for the seven genes (pi ranging from 0.0038 to 0.0109), 3 to 12 alleles were distinguished, resulting in 31 sequence types. One sequence type (ST1) was frequent (17 strains), but most others were represented by a single strain. Attempts to subtype ST1 strains by MLVA, ribotyping, clustered regularly interspaced short palindromic repeat characterization, and single nucleotide repeat variation were unsuccessful. We found clear evidence for homologous recombination during the diversification of L. casei clones, including a putative intragenic import of DNA into one strain. Nucleotides were estimated to change four times more frequently by recombination than by mutation. However, statistical congruence between individual gene trees was retained, indicating that recombination is not frequent enough to disrupt the phylogenetic signal. The developed multilocus sequence typing scheme should be useful for future studies of L. casei strain diversity and evolution.     

Genomic Adaptation of the Lactobacillus casei Group

Lactobacillus casei, L. paracasei, and L. rhamnosus form a closely related taxonomic group (Lactobacillus casei group) within the facultatively heterofermentative lactobacilli. Here, we report the complete genome sequences of L. paracasei JCM 8130 and L. casei ATCC 393, and the draft genome sequence of L. paracasei COM0101, all of which were isolated from daily products. Furthermore, we re-annotated the genome of L. rhamnosus ATCC 53103 (also known as L. rhamnosus GG), which we have previously reported. We confirmed that ATCC 393 is distinct from other strains previously described as L. paracasei. The core genome of 10 completely sequenced strains of the L. casei group comprised 1,682 protein-coding genes. Although extensive genome-wide synteny was found among the L. casei group, the genomes of ATCC 53103, JCM 8130, and ATCC 393 contained genomic islands compared with L. paracasei ATCC 334. Several genomic islands, including carbohydrate utilization gene clusters, were found at the same loci in the chromosomes of the L. casei group. The spaCBA pilus gene cluster, which was first identified in GG, was also found in other strains of the L. casei group, but several L. paracasei strains including COM0101 contained truncated spaC gene. ATCC 53103 encoded a higher number of proteins involved in carbohydrate utilization compared with intestinal lactobacilli, and extracellular adhesion proteins, several of which are absent in other strains of the L. casei group. In addition to previously fully sequenced L. rhamnosus and L. paracasei strains, the complete genome sequences of L. casei will provide valuable insights into the evolution of the L. casei group.     

Strain identification of probiotic Lactobacillus casei-related isolates with randomly amplified polymorphic DNA and pulsed-field gel electrophoresis methods

Typing of reference strains and isolates identified as Lactobacillus casei, Lactobacillus paracasei or Lactobacillus rhamnosus was carried out using randomly amplified polymorphic DNA (RAPD) and pulsed-field gel electrophoresis (PFGE) analyses. Strains of L. paracasei were mainly grouped in the same cluster as those of L. casei. The RAPD fingerprints of strains ATCC 393 and ATCC 15820 differ from those of the L. rhamnosus and L. paracasei/casei strains further supporting classification of these strains as a separate group. The RAPD profiling could be used for classification and discrimination of isolates belonging to the L. casei group.     

Polyphasic characterization of a novel species in the Lactobacillus casei group from cow manure of Taiwan: Description of L. chiayiensis sp. nov.

Two Gram-stain-positive, rod-shaped, non-motile, catalase-negative and facultative anaerobic strains, NCYUAS^T and BCRC 18859 (=NRIC 1947), were isolated from cow manure of Taiwan and coconut juice of Philippines, respectively. Comparative sequence analysis of 16S rRNA gene revealed that the novel strains were members of the genus Lactobacillus. These two strains had 100% of 16S rRNA gene sequence similarity and 98.6% of average nucleotide identity (ANI) value based on whole genome sequences. On the basis of 16S rRNA gene sequence similarity, the type strains of Lactobacillus casei (99.6% similarity), Lactobacillus paracasei subsp. paracasei (99.1%), L. paracasei subsp. tolerans (99.1%), Lactobacillus rhmnosus (99.0%) and ‘Lactobacillus zeae’ (99.7%) were the closest neighbors to these novel strains. The results of phenotypic and chemotaxonomic characterization, multilocus sequence analysis (MLSA) based on the sequences of three housekeeping genes (dnaK, pheS and yycH), whole-genome sequence (WGS)-based comparison by ANI and in silico DNA–DNA hybridization (isDDH), species-specific PCR and whole-cell MALDI-TOF MS spectral pattern analyses demonstrated that the novel two strains represented a single, novel species within the L. casei group, for which the name Lactobacillus chiayiensis sp. nov., is proposed. The type strain is NCYUAS^T (=BCRC 81062^T = NBRC 112906^T).     

Characterization of the Lactobacillus casei group based on the profiling of ribosomal proteins coded in S10-spc-alpha operons as observed by MALDI-TOF MS

The taxonomy of the members of the Lactobacillus casei group is complicated because of their phylogenetic similarity and controversial nomenclatural status. In this study, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) of ribosomal proteins coded in the S10-spc-alpha operon, termed S10-GERMS, was applied in order to classify 33 sample strains belonging to the L. casei group. A total of 14 types of ribosomal protein genes coded in the operon were first sequenced from four type strains of the L. casei group (L. casei JCM 1134^T, L. paracasei subsp. paracasei JCM 8130^T, L. paracasei subsp. tolerans JCM 1171^T, and L. rhamnosus JCM 1136^T) together with L. casei JCM 11302, which is the former type strain of ‘L. zeae’. The theoretical masses of the 14 types of ribosomal proteins used as biomarkers were classified into five types and compiled into a ribosomal protein database. The observed ribosomal proteins of each strain, identified by MALDI-TOF MS, were categorized into types based on their masses, summarized as ribosomal protein profiles, and they were used to construct a phylogenetic tree. The 33 sample strains, together with seven genome-sequenced strains, could be classified into four major clusters, which coincided precisely with the taxa of the (sub)species within the L. casei group. Three “ancient” strains, identified as L. acidophilus and L. casei, were correctly re-identified as L. paracasei subsp. paracasei by S10-GERMS. S10-GERMS would thus appear to be a powerful tool for phylogenetic characterization, with considerable potential for management of culture collections.     

Consensus-Degenerate Hybrid Oligonucleotide Primers for Amplification of Priming Glycosyltransferase Genes of the Exopolysaccharide Locus in Strains of the Lactobacillus casei Group

A primer design strategy named CODEHOP (consensus-degenerate hybrid oligonucleotide primer) for amplification of distantly related sequences was used to detect the priming glycosyltransferase (GT) gene in strains of the Lactobacillus casei group. Each hybrid primer consisted of a short 3′ degenerate core based on four highly conserved amino acids and a longer 5′ consensus clamp region based on six sequences of the priming GT gene products from exopolysaccharide (EPS)-producing bacteria. The hybrid primers were used to detect the priming GT gene of 44 commercial isolates and reference strains of Lactobacillus rhamnosus, L. casei, Lactobacillus zeae, and Streptococcus thermophilus. The priming GT gene was detected in the genome of both non-EPS-producing (EPS⁻) and EPS-producing (EPS⁺) strains of L. rhamnosus. The sequences of the cloned PCR products were similar to those of the priming GT gene of various gram-negative and gram-positive EPS⁺ bacteria. Specific primers designed from the L. rhamnosus RW-9595M GT gene were used to sequence the end of the priming GT gene in selected EPS⁺ strains of L. rhamnosus. Phylogenetic analysis revealed that Lactobacillus spp. form a distinctive group apart from other lactic acid bacteria for which GT genes have been characterized to date. Moreover, the sequences show a divergence existing among strains of L. rhamnosus with respect to the terminal region of the priming GT gene. Thus, the PCR approach with consensus-degenerate hybrid primers designed with CODEHOP is a practical approach for the detection of similar genes containing conserved motifs in different bacterial genomes.     

Isolation and phenotypic characterization of Lactobacillus casei and Lactobacillus paracasei bacteriophage-resistant mutants

Aims: To isolate and characterize bacterial strains derived from Lactobacillus casei and Lactobacillus paracasei strains and resistant to phage MLC‐A. Methods and Results: Two of nine assayed strains rendered resistant mutants with recovery efficiencies of 83% (Lact. paracasei ATCC 27092) and 100% (Lact. casei ATCC 27139). DNA similarity coefficients (RAPD–PCR) confirmed that no significant genetic changes occurred while obtaining resistant mutants. Neither parent nor mutant strains spontaneously released phages. Phage‐resistant mutants were tested against phages PL‐1, J‐1, A2 and MLC‐A8. Lactobacillus casei ATCC 27092 mutants showed, overall, lower phage resistance than Lact. paracasei ATCC 27092 ones, but still higher than that of the parent strain. Lactobacillus paracasei ATCC 27092 mutants moderately adsorbed phage MLC‐A only in calcium presence, although their parent strain successfully did it with or without calcium. Adsorption rates for Lact. casei ATCC 27139 and its mutants were highly influenced by calcium. Again, phage adsorption was higher on the original strain. Conclusions: Several isolates derived from two Lact. casei and Lact. paracasei strains showed resistance to phage MLC‐A but also to other Lact. casei and Lact. paracasei phages. Significance and Impact of the Study: This study highlights isolation of spontaneous bacteriophage‐resistant mutants from Lact. casei and Lact. paracasei as a good choice for use in industrial rotation schemes.     

Human milk and mucosal lacto- and galacto-<Emphasis Type="Italic">N</Emphasis>-biose synthesis by transgalactosylation and their prebiotic potential in <Emphasis Type="Italic">Lactobacillus</Emphasis> species

Lacto-N-biose (LNB) and galacto-N-biose (GNB) are major building blocks of free oligosaccharides and glycan moieties of glyco-complexes present in human milk and gastrointestinal mucosa. We have previously characterized the phospho-β-galactosidase GnbG from Lactobacillus casei BL23 that is involved in the metabolism of LNB and GNB. GnbG has been used here in transglycosylation reactions, and it showed the production of LNB and GNB with N-acetylglucosamine and N-acetylgalactosamine as acceptors, respectively. The reaction kinetics demonstrated that GnbG can convert 69 ± 4 and 71 ± 1 % of o-nitrophenyl-β-d-galactopyranoside into LNB and GNB, respectively. Those reactions were performed in a semi-preparative scale, and the synthesized disaccharides were purified. The maximum yield obtained for LNB was 10.7 ± 0.2 g/l and for GNB was 10.8 ± 0.3 g/l. NMR spectroscopy confirmed the molecular structures of both carbohydrates and the absence of reaction byproducts, which also supports that GnbG is specific for β1,3-glycosidic linkages. The purified sugars were subsequently tested for their potential prebiotic properties using Lactobacillus species. The results showed that LNB and GNB were fermented by the tested strains of L. casei, Lactobacillus rhamnosus (except L. rhamnosus strain ATCC 53103), Lactobacillus zeae, Lactobacillus gasseri, and Lactobacillus johnsonii. DNA hybridization experiments suggested that the metabolism of those disaccharides in 9 out of 10 L. casei strains, all L. rhamnosus strains and all L. zeae strains tested relies upon a phospho-β-galactosidase homologous to GnbG. The results presented here support the putative role of human milk oligosaccharides for selective enrichment of beneficial intestinal microbiota in breast-fed infants.     

Molecular Discrimination of Lactobacilli Used as Starter and Probiotic Cultures by Amplified Ribosomal DNA Restriction Analysis

Lactic acid bacteria such as Lactobacillus helveticus, L. delbrueckii subsp. delbrueckii, L. delbrueckii subsp. lactis, L. delbrueckii subsp. bulgaricus, L. acidophilus, and L. casei related taxa which are widely used as starter or probiotic cultures can be identified by amplified ribosomal DNA restriction analysis (ARDRA). The genetic discrimination of the related species belonging to these groups was first obtained by PCR amplifications by using group-specific or species-specific 16S rDNA primers. The numerical analysis of the ARDRA patterns obtained by using CfoI, HinfI, Tru9I, and ScrFI was an efficient typing tool for identification of species of the L. acidophilus and L. casei complex. ARDRA by using CfoI was a reliable method for differentiation of L. delbrueckii subsp. bulgaricus and L. delbrueckii subsp. lactis. Finally, strains ATCC 393 and ATCC 15820 exhibited unique ARDRA patterns with CfoI and Tru9I restriction enzymes as compared with the other strains of L. casei, L. paracasei, and L. rhamnosus. Received: 30 August 2000 / Accepted: 2 October 2000     

Study of Adhesion and Survival of Lactobacilli and Bifidobacteria on Table Olives with the Aim of Formulating a New Probiotic Food

With the aim of developing new functional foods, a traditional product, the table olive, was used as a vehicle for incorporating probiotic bacterial species. Survival on table olives of Lactobacillus rhamnosus (three strains), Lactobacillus paracasei (two strains), Bifidobacterium bifidum (one strain), and Bifidobacterium longum (one strain) at room temperature was investigated. The results obtained using a selected olive sample demonstrated that bifidobacteria and one strain of L. rhamnosus (Lactobacillus GG) showed a good survival rate, with a recovery of about 10⁶ CFU g⁻¹ after 30 days. The Lactobacillus GG population remained unvaried until the end of the experiment, while a slight decline (to about 10⁵ CFU g⁻¹) was observed for bifidobacteria. High viability, with more than 10⁷ CFU g⁻¹, was observed throughout the 3-month experiment for L. paracasei IMPC2.1. This strain, selected for its potential probiotic characteristics and for its lengthy survival on olives, was used to validate table olives as a carrier for transporting bacterial cells into the human gastrointestinal tract. L. paracasei IMPC2.1 was recovered from fecal samples in four out of five volunteers fed 10 to 15 olives per day carrying about 10⁹ to 10¹⁰ viable cells for 10 days.     

Protective effect of Lactobacillus casei strain Shirota against lethal infection with multi-drug resistant Salmonella enterica serovar Typhimurium DT104 in mice

Aims: The anti‐infectious activity of lactobacilli against multi‐drug resistant Salmonella enterica serovar Typhimurium DT104 (DT104) was examined in a murine model of an opportunistic antibiotic‐induced infection. Methods and Results: Explosive intestinal growth and subsequent lethal extra‐intestinal translocation after oral infection with DT104 during fosfomycin (FOM) administration was significantly inhibited by continuous oral administration of Lactobacillus casei strain Shirota (LcS), which is naturally resistant to FOM, at a dose of 10⁸ colony‐forming units per mouse daily to mice. Comparison of the anti‐Salmonella activity of several Lactobacillus type strains with natural resistance to FOM revealed that Lactobacillus brevis ATCC 14869^T, Lactobacillus plantarum ATCC 14917^T, Lactobacillus reuteri JCM 1112^T, Lactobacillus rhamnosus ATCC 7469^T and Lactobacillus salivarius ATCC 11741^T conferred no activity even when they obtained the high population levels almost similar to those of the effective strains such as LcS, Lact. casei ATCC 334^T and Lactobacillus zeae ATCC 15820^T. The increase in concentration of organic acids and maintenance of the lower pH in the intestine because of Lactobacillus colonization were correlated with the anti‐infectious activity. Moreover, heat‐killed LcS was not protective against the infection, suggesting that the metabolic activity of lactobacilli is important for the anti‐infectious activity. Conclusion: These results suggest that certain lactobacilli in combination with antibiotics may be useful for prophylaxis against opportunistic intestinal infections by multi‐drug resistant pathogens, such as DT104. Significance and Impact of the Study: Antibiotics such as FOM disrupt the metabolic activity of the intestinal microbiota that produce organic acids, and that only probiotic strains that are metabolically active in vivo should be selected to prevent intestinal infection when used clinically in combination with certain antibiotics.     

Comparative evaluation of automated ribotyping and RAPD-PCR for typing of <Emphasis Type="Italic">Lactobacillus</Emphasis> spp. occurring in dental caries

A group of 67 Lactobacillus spp. strains containing Lactobacillus casei/paracasei, Lactobacillus fermentum, Lactobacillus gasseri, Lactobacillus plantarum, Lactobacillus rhamnosus and Lactobacillus salivarius species isolated from early childhood caries and identified to the species level in a previous study (?vec et al., Folia Microbiol 54:53–58, 2009) was characterized by automated ribotyping performed by the RiboPrinter^® microbial characterization system and by randomly amplified polymorphic DNA fingerprinting (RAPD-PCR) with M13 primer to evaluate these techniques for characterization of lactobacilli associated with dental caries. Ribotyping revealed 55 riboprints among the analysed group. The automatic identification process performed by the RiboPrinter system identified 18 strains to the species level, however cluster analysis divided obtained ribotype patterns into individual clusters mostly corresponding to the species assignment of particular strains. RAPD-PCR fingerprints revealed by the individual Lactobacillus spp. showed higher variability than the ribotype patterns and the fingerprint profiles generated by the analysed species were distributed among one to four clusters. In conclusion, ribotyping is shown to be more convenient for the identification purposes while RAPD-PCR fingerprinting results indicate this method is a better tool for typing of Lactobacillus spp. strains occurring in dental caries.     

The <Emphasis Type="Italic">dnaK</Emphasis> gene as a molecular marker for the classification and discrimination of the <Emphasis Type="Italic">Lactobacillus casei</Emphasis> group

It is hard to accurately identify specific species of the Lactobacillus casei group using phenotypic techniques alone. Some strains of this species group are considered to be probiotic and are widely applied in the food industry. In this study, we compared the use of two phylogenetic markers, the 16S rRNA and dnaK genes, for species discrimination of the members of the L. casei group using sequencing and RFLP. The results showed that L. casei, Lactobacillus paracasei, Lactobacillus zeae and Lactobacillus rhamnosus could be clearly distinguished based on the dnaK gene. The average sequence similarity for the dnaK gene (87.8%) among type strains was significantly less than that of the 16S rRNA sequence (99.1%). Therefore, the dnaK gene can be proposed as an additional molecular phylogenetic marker for L. casei that provides higher resolution than 16S rRNA. Species-specific RFLP profiles of the Lactobacillus strains were obtained with the enzyme ApoI. Our data indicate that the phylogenetic relationships between these strains are easily resolved using sequencing of the dnaK gene or RFLP assays.     

Survey on proteolytic activity and diversity of proteinase genes in mesophilic lactobacilli

Lactocepins or CEPs are large cell wall bound extracellular proteinases of lactic acid bacteria, involved in protein breakdown and utilization. They are responsible for many health-promoting traits of food products fermented with these organisms, but also essential for probiotic effects of certain strains. Different mesophilic strains selected within the species Lactobacillus zeae, Lb. casei, Lb. rhamnosus, and Lb. plantarum were analyzed for their proteolytic activity towards main fractions of milk proteins—caseins and whey proteins. The strains showing excellent proteolytic features were further examined for presence of corresponding proteinase gene(s). It was found that Lb. zeae LMG17315 possessed catalytic domains of three distinct proteinase genes, unique feature in Lb. casei group, which are similar but not identical to previously characterized prtP and prtR genes. Lb. casei neotype strain ATCC393 was also analysed and based on obtained results its reclassification in taxon Lb. zeae is supported. In addition, we report catalytic domain of prtR-type gene in Lb. plantarum LMG9208, which is first such report in this species, and it is first time that this gene is reported outside Lb. casei group.     

Characterization of the Properties of Human- and Dairy-Derived Probiotics for Prevention of Infectious Diseases in Fish

The present study aimed to investigate the potential probiotic properties of six lactic acid bacteria (LAB) intended for human use, Lactobacillus rhamnosus ATCC 53103, Lactobacillus casei Shirota, Lactobacillus bulgaricus, L. rhamnosus LC 705, Bifidobacterium lactis Bb12, and Lactobacillus johnsonii La1, and one for animal use, Enterococcus faecium Tehobak, for use as a fish probiotic. The strains for human use were specifically chosen since they are known to be safe for human use, which is of major importance because the fish are meant for human consumption. The selection was carried out by five different methods: mucosal adhesion, mucosal penetration, inhibition of pathogen growth and adhesion, and resistance to fish bile. The adhesion abilities of the seven LAB and three fish pathogens, Vibrio anguillarum, Aeromonas salmonicida, and Flavobacterium psychrophilum, were determined to mucus from five different sites on the surface or in the gut of rainbow trout. Five of the tested LAB strains showed considerable adhesion to different fish mucus types (14 to 26% of the added bacteria). Despite their adhesive character, the LAB strains were not able to inhibit the mucus binding of A. salmonicida. Coculture experiments showed significant inhibition of growth of A. salmonicida, which was mediated by competition for nutrients rather than secretion of inhibitory substances by the probiotic bacteria as measured in spent culture liquid. All LAB except L. casei Shirota showed tolerance against fish bile. L. rhamnosus ATCC 53103 and L. bulgaricus were found to penetrate fish mucus better than other probiotic bacteria. Based on bile resistance, mucus adhesion, mucus penetration, and suppression of fish pathogen growth, L. rhamnosus ATCC 53103 and L. bulgaricus can be considered for future in vivo challenge studies in fish as a novel and safe treatment in aquaculture.     

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.     

Comparison of Fructose-1,6-Bisphosphatase Gene (<Emphasis Type="Italic">fbp</Emphasis>) Sequences for the Identification of <Emphasis Type="Italic">Lactobacillus rhamnosus</Emphasis>

Comparative analysis of fructose-1,6-bisphosphatase gene (fbp) sequences was evaluated for the differentiation of reference and clinical strains of Lactobacillus rhamnosus. The sequences of 1,971 nucleotides of the fbp gene were determined on both DNA strands for 21 L. rhamnosus strains, representing reference, probiotic, and clinical strains. No PCR amplification of the fbp gene was observed for other species of the Lactobacillus casei complex (L. casei and L. zeae) or strains of Lactobacillus acidophilus, Streptococcus thermophilus, and Escherichia coli. Phylogenetic analysis of the fbp putative amino acid sequences of L. rhamnosus strains by the neighbor-joining method showed clear distinct positions of this species. The phylogenetic tree, derived from fbp nucleotide sequences, showed four clear divisions between strains of L. rhamnosus. From a taxonomic point of view, our results confirm for the first time that fbp gene sequences have high discriminating power for strains of L. rhamnosus that are difficult to differentiate.     

Inhibitory effect of oral Lactobacillus against oral pathogens

Aims: To determine the inhibitory effect of oral Lactobacillus against putative oral pathogens. Methods and Results: Total 357 strains comprising 10 species of oral Lactobacillus, Lactobacillus fermentum (195), Lactobacillus salivarius (53), Lactobacillus casei (20), Lactobacillus gasseri (18), Lactobacillus rhamnosus (14), Lactobacillus paracasei (12), Lactobacillus mucosae (12), Lactobacillus oris (12), Lactobacillus plantarum (11) and Lactobacillus vaginalis (10) were used as producer strains. Inhibitory effect against a panel of indicators, periodontitis‐ and caries‐related pathogens, was assessed. Most oral Lactobacillus was able to inhibit the growth of both periodontitis‐ and caries‐related pathogens. The strongest inhibitory activity was associated with Lact. paracasei, Lact. plantarum, Lact. rhamnosus, Lact. casei and Lact. salivarius. Lactobacillus SD1–SD6, representing the six species with the strong inhibitory effect, inhibited growth of Streptococcus mutans ATCC 25175 in the biofilm model. Also, it was demonstrated that growth of Strep. mutans was inhibited in a mixture with Lact. paracasei SD1. The inhibition was enhanced in acidic condition and 5% glucose. Conclusions: The results have shown that oral Lactobacillus SD1–SD6 showed a strong inhibitory effect against Strep. mutans and Streptococcus sobrinus, as well as, Gram‐negative periodontal pathogens Porphyromonas gingivalis and Aggregatibacter actinomycetemcomitans. Significance and Impact of the Study: The results indicated that Lactobacillus may be of benefit as probiotics for the prevention of oral diseases.     

Quantitative Real-Time PCR Analysis of Fecal Lactobacillus Species in Infants Receiving a Prebiotic Infant Formula

The developing intestinal microbiota of breast-fed infants is considered to play an important role in the priming of the infants' mucosal and systemic immunity. Generally, Bifidobacterium and Lactobacillus predominate the microbiota of breast-fed infants. In intervention trials it has been shown that lactobacilli can exert beneficial effects on, for example, diarrhea and atopy. However, the Lactobacillus species distribution in breast-fed or formula-fed infants has not yet been determined in great detail. For accurate enumeration of different lactobacilli, duplex 5′ nuclease assays, targeted on rRNA intergenic spacer regions, were developed for Lactobacillus acidophilus, Lactobacillus casei, Lactobacillus delbrueckii, Lactobacillus fermentum, Lactobacillus paracasei, Lactobacillus plantarum, Lactobacillus reuteri, and Lactobacillus rhamnosus. The designed and validated assays were used to determine the amounts of different Lactobacillus species in fecal samples of infants receiving a standard formula (SF) or a standard formula supplemented with galacto- and fructo-oligosaccharides in a 9:1 ratio (OSF). A breast-fed group (BF) was studied in parallel as a reference. During the 6-week intervention period a significant increase was shown in total percentage of fecal lactobacilli in the BF group (0.8% ± 0.3% versus 4.1% ± 1.5%) and the OSF group (0.8% ± 0.3% versus 4.4% ± 1.4%). The Lactobacillus species distribution in the OSF group was comparable to breast-fed infants, with relatively high levels of L. acidophilus, L. paracasei, and L. casei. The SF-fed infants, on the other hand, contained more L. delbrueckii and less L. paracasei compared to breast-fed infants and OSF-fed infants. An infant milk formula containing a specific mixture of prebiotics is able to induce a microbiota that closely resembles the microbiota of BF infants.