Identification and characterization of the novel LysM domain-containing surface protein Sep from Lactobacillus fermentum BR11 and its use as a peptide fusion partner in Lactobacillus and Lactococcus

Examination of supernatant fractions from broth cultures of Lactobacillus fermentum BR11 revealed the presence of a number of proteins, including a 27-kDa protein termed Sep. The amino-terminal sequence of Sep was determined, and the gene encoding it was cloned and sequenced. Sep is a 205-amino-acid protein and contains a 30-amino-acid secretion signal and has overall homology (between 39 and 92% identity) with similarly sized proteins of Lactobacillus reuteri, Enterococcus faecium, Streptococcus pneumoniae, Streptococcus agalactiae, and Lactobacillus plantarum. The carboxy-terminal 81 amino acids of Sep also have strong homology (86% identity) to the carboxy termini of the aggregation-promoting factor (APF) surface proteins of Lactobacillus gasseri and Lactobacillus johnsonii. The mature amino terminus of Sep contains a putative peptidoglycan-binding LysM domain, thereby making it distinct from APF proteins. We have identified a common motif within LysM domains that is shared with carbohydrate binding YG motifs which are found in streptococcal glucan-binding proteins and glucosyltransferases. Sep was investigated as a heterologous peptide expression vector in L. fermentum, Lactobacillus rhamnosus GG and Lactococcus lactis MG1363. Modified Sep containing an amino-terminal six-histidine epitope was found associated with the cells but was largely present in the supernatant in the L. fermentum, L. rhamnosus, and L. lactis hosts. Sep as well as the previously described surface protein BspA were used to express and secrete in L. fermentum or L. rhamnosus a fragment of human E-cadherin, which contains the receptor region for Listeria monocytogenes. This study demonstrates that Sep has potential for heterologous protein expression and export in lactic acid bacteria.     

Peptide Surface Display and Secretion Using Two LPXTG-Containing Surface Proteins from Lactobacillus fermentum BR11

A locus encoding two repetitive proteins that have LPXTG cell wall anchoring signals from Lactobacillus fermentum BR11 has been identified by using an antiserum raised against whole L. fermentum BR11 cells. The first protein, Rlp, is similar to the Rib surface protein from Streptococcus agalactiae, while the other protein, Mlp, is similar to the mucus binding protein Mub from Lactobacillus reuteri. It was shown that multiple copies of mlp exist in the genome of L. fermentum BR11. Regions of Rlp, Mlp, and the previously characterized surface protein BspA were used to surface display or secrete heterologous peptides in L. fermentum. The peptides tested were 10 amino acids of the human cystic fibrosis transmembrane regulator protein and a six-histidine epitope (His₆). The BspA promoter and secretion signal were used in combination with the Rlp cell wall sorting signal to express, export, and covalently anchor the heterologous peptides to the cell wall. Detection of the cell surface protein fusions revealed that Rlp was a significantly better surface display vector than BspA despite having lower cellular levels (0.7 mg per liter for the Rlp fusion compared with 4 mg per liter for the BspA fusion). The mlp promoter and encoded secretion signal were used to express and export large (328-kDa at 10 mg per liter) and small (27-kDa at 0.06 mg per liter) amino-terminal fragments of the Mlp protein fused to the His₆ and CFTR peptides or His₆ peptide, respectively. Therefore, these newly described proteins from L. fermentum BR11 have potential as protein production and targeting vectors.     

Strain-Dependent Augmentation of Tight-Junction Barrier Function in Human Primary Epidermal Keratinocytes by Lactobacillus and Bifidobacterium Lysates

In this study, we investigated whether probiotic lysates can modify the tight-junction function of human primary keratinocytes. The keratinocytes were grown on cell culture inserts and treated with lysates from Bifidobacterium longum, Lactobacillus plantarum, Lactobacillus reuteri, Lactobacillus fermentum, or Lactobacillus rhamnosus GG. With the exception of L. fermentum (which decreased cell viability), all strains markedly enhanced tight-junction barrier function within 24 h, as assessed by measurements of transepithelial electrical resistance (TEER). However, B. longum and L. rhamnosus GG were the most efficacious, producing dose-dependent increases in resistance that were maintained for 4 days. These increases in TEER correlated with elevated expression of tight-junction protein components. Neutralization of Toll-like receptor 2 abolished both the increase in TEER and expression of tight-junction proteins induced by B. longum, but not L. rhamnosus GG. These data suggest that some bacterial strains increase tight-junction function via modulation of protein components but the different pathways involved may vary depending on the bacterial strain.     

Improvement of LysM-Mediated Surface Display of Designed Ankyrin Repeat Proteins (DARPins) in Recombinant and Nonrecombinant Strains of Lactococcus lactis and Lactobacillus Species

Safety and probiotic properties make lactic acid bacteria (LAB) attractive hosts for surface display of heterologous proteins. Protein display on nonrecombinant microorganisms is preferred for therapeutic and food applications due to regulatory requirements. We displayed two designed ankyrin repeat proteins (DARPins), each possessing affinity for the Fc region of human IgG, on the surface of Lactococcus lactis by fusing them to the Usp45 secretion signal and to the peptidoglycan-binding C terminus of AcmA, containing lysine motif (LysM) repeats. Growth medium containing a secreted fusion protein was used to test its heterologous binding to 10 strains of species of the genus Lactobacillus, using flow cytometry, whole-cell enzyme-linked immunosorbent assay (ELISA), and fluorescence microscopy. The fusion proteins bound to the surfaces of all lactobacilli; however, binding to the majority of bacteria was only 2- to 5-fold stronger than that of the control. Lactobacillus salivarius ATCC 11741 demonstrated exceptionally strong binding (32- to 55-fold higher than that of the control) and may therefore be an attractive host for nonrecombinant surface display. Genomic comparison of the species indicated the exopolysaccharides of Lb. salivarius as a possible reason for the difference. Additionally, a 15-fold concentration-dependent increase in nonrecombinant surface display on L. lactis was demonstrated by growing bacteria with sublethal concentrations of the antibiotics chloramphenicol and erythromycin. Nonrecombinant surface display on LAB, based on LysM repeats, was optimized by selecting Lactobacillus salivarius ATCC 11741 as the optimal host and by introducing antibiotics as additives for increasing surface display on L. lactis. Additionally, effective display of DARPins on the surfaces of nonrecombinant LAB has opened up several new therapeutic possibilities.     

Antimicrobial susceptibility of microflora from ovine cheese

Strains identified in ovine cheese and bryndza by matrix-assisted laser desorption/ionization time-of-flight analysis belonged to ten species of non-enterococcal lactic acid bacteria and included Lactobacillus casei/Lactobacillus paracasei, Lactobacillus plantarum, Lactobacillus rhamnosus, Lactobacillus helveticus, Lactobacillus delbrueckii, Lactobacillus fermentum, Lactobacillus brevis, Lactococcus lactis, Pediococcus pentosaceus and Pediococcus acidilactici. The susceptibility toward antibiotics was determined in lactobacilli, lactococci and pediococci and also in Escherichia coli for comparison. Analysis of L. fermentum and pediococci revealed the presence of non-wild-type epidemiological cut-offs in streptomycin, clindamycin or gentamicin. E. coli were resistant to ampicillin, tetracycline, enrofloxacin and florfenicol. No extended spectrum β-lactamases were detected.     

Identification of Collagen-Binding Proteins in Lactobacillus spp. with Surface-Enhanced Laser Desorption/Ionization–Time of Flight ProteinChip Technology

Biosurfactants produced by Lactobacillus fermentum RC-14, L. rhamnosus GR-1 and 36, and L. casei Shirota were found to contain proteins that bind to both collagen types III and VI, as determined by surface-enhanced laser desorption/ionization (SELDI)–time of flight mass spectrometry. Both collagen types III and VI immobilized on SELDI preactivated ProteinChip arrays detected several different sizes (2 to 48 kDa) of collagen-binding proteins. Overall, the RC-14-produced biosurfactant contained the greatest number of collagen-binding proteins (RC-14 > GR-1 > 36 > Shirota), including the mature form of a previously cloned 29-kDa collagen-binding protein (referred to in its mature 26-kDa form). Although biosurfactants isolated from L. casei Shirota and L. rhamnosus 36 and GR-1 also contain several collagen-binding proteins, they do not contain the 26-kDa collagen-binding protein. Together, these results demonstrate the utility of the SELDI system as a means of rapidly characterizing clinically important but complex biosurfactant solutions.     

Effect of Two Probiotic Strains of Lactobacillus on In Vitro Adherence of Listeria monocytogenes,Streptococcus agalactiae,and Staphylococcus aureus to Vaginal Epithelial Cells

The lactobacilli probiotics maintain a normal vaginal biota and prevent disease recurrence. This microorganisms form a pellicle on the vaginal epithelium that acts as a biologic barrier against colonization by pathogenic bacteria. In this paper were realized assays of exclusion, competition, and displacement. For these test, vaginal epithelial cells, two strains of lactobacilli and pathogenic bacteria (Staphylococcus aureus, Streptococcus agalactiae and Listeria monocytogenes) were used. The lactobacilli strains showed a great capacity of adherence, with a mean of 83.5 ± 26.67 Lactobacillus fermentum cells and 56.2 ± 20.87 Lactobacillus rhamnosus cells per vaginal epithelial cells. L. fermentum and L. rhamnosus were able to reduce the adherence of S. aureus, S. agalactiae and L. monocytogenes in a significant level in this assay (P < 0.01). The lactobacilli used in this study protect the vaginal epithelium through a series of barriers and interference mechanisms. The aim of present study was to assess the ability of vaginal Lactobacillus strains, selected for their probiotic properties, to block the adherence of pathogenic microorganisms in vitro by displacement, competition, and exclusion mechanisms.     

Characterization of a Novel LysM Domain from Lactobacillus fermentum Bacteriophage Endolysin and Its Use as an Anchor To Display Heterologous Proteins on the Surfaces of Lactic Acid Bacteria

The endolysin Lyb5, from Lactobacillus fermentum temperate bacteriophage φPYB5, showed a broad lytic spectrum against Gram-positive as well as Gram-negative bacteria. Sequence analysis revealed that the C terminus of the endolysin Lyb5 (Ly5C) contained three putative lysin motif (LysM) repeat regions, implying that Ly5C was involved in bacterial cell wall binding. To investigate the potential of Ly5C for surface display, green fluorescent protein (GFP) was fused to Ly5C at its N or C terminus and the resulting fusion proteins were expressed in Escherichia coli. After being mixed with various cells in vitro, GFP was successfully displayed on the surfaces of Lactococcus lactis, Lactobacillus casei, Lb. brevis, Lb. plantarum, Lb. fermentum, Lb. delbrueckii, Lb. helveticus, and Streptococcus thermophilus cells. Increases in the fluorescence intensities of chemically pretreated L. lactis and Lb. casei cells compared to those of nonpretreated cells suggested that the peptidoglycan was the binding ligand for Ly5C. Moreover, the pH and concentration of sodium chloride were optimized to enhance the binding capacity of GFP-Ly5C, and high-intensity fluorescence of cells was observed under optimal conditions. All results suggested that Ly5C was a novel anchor for constructing a surface display system for lactic acid bacteria (LAB). To demonstrate the applicability of the Ly5C-mediated surface display system, β-galactosidase (β-Gal) from Paenibacillus sp. strain K1, replacing GFP, was functionally displayed on the surfaces of LAB cells via Ly5C. The success in surface display of GFP and β-Gal opened up the feasibility of employing the cell wall anchor of bacteriophage endolysin for surface display in LAB.Surface display of heterologous proteins or peptides on bacteria is potentially important in several areas of biotechnology, including development of live vaccine delivery systems, diagnostics, whole-cell absorbents, and novel biocatalysts (11). Lactic acid bacteria (LAB) have the status of being generally recognized as safe (GRAS), making them certainly more useful in food and medical applications than other bacterial species. The development of cell surface display systems for LAB has recently become one of the most active research areas. Most of the cell surface display systems for LAB reported thus far have made use of the C terminus of a cell wall-anchoring protein via an LPXTG motif (8, 12, 19, 24). This anchoring mechanism requires processing by a sortase for covalent anchoring of the protein to the cell wall peptidoglycan (15). Various anchoring proteins, such as membrane-spanning protein PgsA (16) and S-layer protein (3), have also been exploited for surface display. However, heterologous proteins have been anchored to the producer cells, and the use of genetically modified organisms is less desirable or at least still being debated. Surface display of heterologous proteins on genetically unmodified Gram-positive bacteria has been successfully carried out using the peptidoglycan binding lysin motif (LysM) domain of the major autolysin AcmA of Lactococcus lactis (1, 2, 4, 18, 28).LysM was first discovered in the lysozyme of Bacillus phage φ29 as a C-terminal repeat composed of 44 amino acids separated by 7 amino acids (6). LysM is a common module found in more than 4,000 proteins of both prokaryotes and eukaryotes (6). Many bacterial proteins containing LysM are peptidoglycan hydrolases, such as p60 (20), Sep (26), LytF (31), AcmA (5), and Mur (7). The best-characterized LysM-containing protein is the N-acetylglucosaminidase AcmA of L. lactis subsp. cremoris MG1363. AcmA is the major autolysin and is required for cell separation and cell lysis during the stationary phase of L. lactis (5). It contains three domains: the N-terminal signal peptide, an active domain, and a C-terminal peptidoglycan anchor (cA) which consists of three LysM repeats (22). Several functional proteins, including malaria parasite surface antigen, β-lactamase, α-amylase, and viral capsid proteins, have been noncovalently bound to cell walls of AcmA-producing and non-AcmA-producing L. lactis as well as several other Gram-positive bacteria via cA (4, 17, 18, 23, 25).Endolysins from bacteriophages are cell wall hydrolases involved in cell lysis to release the progeny particles from the host cells (9, 30). Most endolysins lack a signal peptide and are translocated across the membrane by the aid of the holin protein. This protein typically contains an N-terminal catalytic domain and a C-terminal cell wall binding domain (33). The endolysins Ply118 and Ply500 of a Listeria monocytogenes phage share a unique C-terminal cell wall binding domain which establishes specific recognition of and high-affinity binding to bacterial cell wall carbohydrates (13). The temperate bacteriophage φPYB5, isolated from the Lactobacillus fermentum YB5 strain, has a hexagonal head, noncontractile tails, and several fibers and belongs to Bradley''s group B as defined by the International Committee on Taxonomy of Viruses (32). The sequence of the endolysin gene lyb5 from the genome of φPYB5 has been deposited in GenBank under accession number {"type":"entrez-nucleotide","attrs":{"text":"EF531306","term_id":"145687952","term_text":"EF531306"}}EF531306, and the gene product has been successfully expressed in Escherichia coli and has shown a broad lytic spectrum (30).Here, we generated a fusion of green fluorescent protein (GFP) to the C terminus of Lyb5 (Ly5C) to construct a surface display system for LAB. The GFP was bound to the surfaces of various LAB cells by the aid of Ly5C. Moreover, by using the system constructed, β-galactosidase (β-Gal) was functionally displayed on the surfaces of LAB cells and retained its activity.     

Flow Cytometric Testing of Green Fluorescent Protein-Tagged Lactobacillus rhamnosus GG for Response to Defensins

Lactobacillus rhamnosus GG is of general interest as a probiotic. Although L. rhamnosus GG is often used in clinical trials, there are few genetic tools to further determine its mode of action or to develop it as a vehicle for heterologous gene expression in therapy. Therefore, we developed a reproducible, efficient electroporation procedure for L. rhamnosus GG. The best transformation efficiency obtained was 10⁴ transformants per μg of DNA. We validated this protocol by tagging L. rhamnosus GG with green fluorescent protein (GFP) using the nisin-controlled expression (NICE) system. Parameters for overexpression were optimized, which allowed expression of gfp in L. rhamnosus GG upon induction with nisin. The GFP⁺ strain can be used to monitor the survival and behavior of L. rhamnosus GG in vivo. Moreover, implementation of the NICE system as a gene expression switch in L. rhamnosus GG opens up possibilities for improving and expanding the performance of this strain. The GFP-labeled strain was used to demonstrate that L. rhamnosus GG is sensitive to human beta-defensin-2 but not to human beta-defensin-1.     

Production of a Heterologous Nonheme Catalase by Lactobacillus casei: an Efficient Tool for Removal of H2O2 and Protection of Lactobacillus bulgaricus from Oxidative Stress in Milk

Lactic acid bacteria (LAB) are generally sensitive to H₂O₂, a compound that they can paradoxically produce themselves, as is the case for Lactobacillus bulgaricus. Lactobacillus plantarum ATCC 14431 is one of the very few LAB strains able to degrade H₂O₂ through the action of a nonheme, manganese-dependent catalase (hereafter called MnKat). The MnKat gene was expressed in three catalase-deficient LAB species: L. bulgaricus ATCC 11842, Lactobacillus casei BL23, and Lactococcus lactis MG1363. While the protein could be detected in all heterologous hosts, enzyme activity was observed only in L. casei. This is probably due to the differences in the Mn contents of the cells, which are reportedly similar in L. plantarum and L. casei but at least 10- and 100-fold lower in Lactococcus lactis and L. bulgaricus, respectively. The expression of the MnKat gene in L. casei conferred enhanced oxidative stress resistance, as measured by an increase in the survival rate after exposure to H₂O₂, and improved long-term survival in aerated cultures. In mixtures of L. casei producing MnKat and L. bulgaricus, L. casei can eliminate H₂O₂ from the culture medium, thereby protecting both L. casei and L. bulgaricus from its deleterious effects.     

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.     

System Using Tandem Repeats of the cA Peptidoglycan-Binding Domain from Lactococcus lactis for Display of both N- and C-Terminal Fusions on Cell Surfaces of Lactic Acid Bacteria

Here, we established a system for displaying heterologous protein to the C terminus of the peptidoglycan-binding domain (cA domain) of AcmA (a major autolysin from Lactococcus lactis). Western blot and flow cytometric analyses revealed that the fusion proteins (cA-AmyA) of the cA domain and α-amylase from Streptococcus bovis 148 (AmyA) are efficiently expressed and successfully displayed on the surfaces of L. lactis cells. AmyA was also displayed on the cell surface while retaining its activity. Moreover, with an increase in the number of cA domains, the quantity of cA-AmyA fusion proteins displayed on the cell surface increased. When three repeats of the cA domain were used as an anchor protein, 82% of α-amylase activity was detected on the cells. The raw starch-degrading activity of AmyA was significantly higher when AmyA was fused to the C terminus of the cA domain than when it was fused to the N terminus. In addition, cA-AmyA fusion proteins were successfully displayed on the cell surfaces of Lactobacillus plantarum and Lactobacillus casei.     

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.     

Lactobacillus bulgaricus Proteinase Expressed in Lactococcus lactis Is a Powerful Carrier for Cell Wall-Associated and Secreted Bovine β-Lactoglobulin Fusion Proteins

Lactic acid bacteria have a good potential as agents for the delivery of heterologous proteins to the gastrointestinal mucosa and thus for the reequilibration of inappropriate immune responses to food antigens. Bovine β-lactoglobulin (BLG) is considered a major allergen in cow's milk allergy. We have designed recombinant Lactococcus lactis expressing either full-length BLG or BLG-derived octapeptide T6 (IDALNENK) as fusions with Lactobacillus bulgaricus extracellular proteinase (PrtB). In addition to constructs encoding full-length PrtB for the targeting of heterologous proteins to the cell surface, we generated vectors aiming at the release into the medium of truncated PrtB derivatives lacking 100 (PrtB, PrtB-BLG, and PrtB-T6) or 807 (PrtBΔ) C-terminal amino acids. Expression of recombinant products was confirmed using either anti-PrtB, anti-BLG, or anti-peptide T6 antiserum. All forms of the full-length and truncated recombinant products were efficiently translocated, irrespective of the presence of eucaryotic BLG sequences in the fusion proteins. L. lactis expressing PrtB-BLG yielded up to 170 μg per 10⁹ CFU in the culture supernatant and 9 μg per 10⁹ CFU at the bacterial cell surface within 14 h. Therefore, protein fusions relying on the use of PrtB gene products are adequate for concomitant cell surface display and secretion by recombinant L. lactis and thus may ensure maximal bioavailability of the eucaryotic antigen in the gut-associated lymphoid tissue.     

Detection and localization of single LysM-peptidoglycan interactions

The lysin motif (LysM) is a ubiquitous protein module that binds peptidoglycan and structurally related molecules. Here, we used single-molecule force spectroscopy (SMFS) to measure and localize individual LysM-peptidoglycan interactions on both model and cellular surfaces. LysM modules of the major autolysin AcmA of Lactococcus lactis were bound to gold-coated atomic force microscopy tips, while peptidoglycan was covalently attached onto model supports. Multiple force curves recorded between the LysM tips and peptidoglycan surfaces yielded a bimodal distribution of binding forces, presumably reflecting the occurrence of one and two LysM-peptidoglycan interactions, respectively. The specificity of the measured interaction was confirmed by performing blocking experiments with free peptidoglycan. Next, the LysM tips were used to map single LysM interactions on the surfaces of L. lactis cells. Strikingly, native cells showed very poor binding, suggesting that peptidoglycan was hindered by other cell wall constituents. Consistent with this notion, treatment of the cells with trichloroacetic acid, which removes peptidoglycan-associated polymers, resulted in substantial and homogeneous binding of the LysM tip. These results provide novel insight into the binding forces of bacterial LysMs and show that SMFS is a promising tool for studying the heterologous display of proteins or peptides on bacterial surfaces.     

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

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

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.     

Probiotic Strains Influence on Infant Microbiota in the In Vitro Colonic Fermentation Model GIS1

The main goal of our study was to evaluate the effect of the individual administration of five lyophilized lactic acid bacteria strains (Lactobacillus fermentum 428ST, Lactobacillus rhamnosus E4.2, Lactobacillus plantarum FCA3, Lactobacillus sp. 34.1, Weissella paramesenteroides FT1a) against the in vitro simulated microbiota of the human colon using the GIS1 system. The influence on the metabolic activity was also assessed by quantitative determination of proteins and polysaccharides at each segment of human colon. The obtained results indicated that the lactic acid bacteria L. rhamnosus E4.2 and W. paramesenteroides FTa1 had better efficiency in synthesising exopolysaccharides and also a better probiotic potential and therefore could be recommended for use in probiotics products or food industry.     

Stable integration and expression of heterologous genes in several lactobacilli using an integration vector constructed from the integrase and attP sequences of phage ΦAT3 isolated from Lactobacillus casei ATCC 393

An integration vector capable of stably integrating and maintaining in the chromosomes of several lactobacilli over hundreds of generations has been constructed. The major integration machinery used is based on the ΦAT3 integrase (int) and attP sequences determined previously. A novel core sequence located at the 3′ end of the tRNA^leu gene is identified in Lactobacillus fermentum ATCC 14931 as the integration target by the integration vector though most of such sequences found in other lactobacilli are similar to that determined previously. Due to the lack of an appropriate attB site in Lactococcus lactis MG1363, the integration vector is found to be unable to integrate into the chromosome of the strain. However, such integration can be successfully restored by cotransforming the integration vector with a replicative one harboring both attB and erythromycin resistance sequences into the strain. Furthermore, the integration vector constructed carries a promoter region of placT from the chromosome of Lactobacillus rhamnosus TCELL-1 which is used to express green fluorescence and luminance protein genes in the lactobacilli studied.