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Clustered regularly interspaced short palindromic repeats (CRISPRs) and CRISPR-associated (Cas) proteins form adaptive immune systems that occur in many bacteria and most archaea. In addition to protecting bacteria from phages and other invasive mobile genetic elements, CRISPR-Cas molecular machines can be repurposed as tool kits for applications relevant to the food industry. A primary concern of the food industry has long been the proper management of food-related bacteria, with a focus on both enhancing the outcomes of beneficial microorganisms such as starter cultures and probiotics and limiting the presence of detrimental organisms such as pathogens and spoilage microorganisms. This review introduces CRISPR-Cas as a novel set of technologies to manage food bacteria and offers insights into CRISPR-Cas biology. It primarily focuses on the applications of CRISPR-Cas systems and tools in starter cultures and probiotics, encompassing strain-typing, phage resistance, plasmid vaccination, genome editing, and antimicrobial activity.

The Klaenhammer group at North Carolina State University pioneered genomic applications in food microbiology and beneficial lactic acid bacteria used as starter cultures and probiotics. Dr. Todd Klaenhammer was honored to be the first food scientist elected to the National Academy of Sciences (2001). The program was recognized with the highest research awards presented by the American Dairy Science Association (Borden Award 1996), the Institute of Food Technologists (Nicholas Appert Medal, 2007), and the International Dairy Federation (Eli Metchnikoff Award in Biotechnology, 2010) as well as with the Outstanding Achievement Award from the University of Minnesota (2001) and the Oliver Max Gardner Award (2009) for outstanding research across the 16-campus University of North Carolina system. Dr. Klaenhammer is a fellow of the American Association for the Advancement of Science, the American Dairy Science Association, and the Institute of Food Technology. Over his career, six of his PhD graduate students were awarded the annual Kenneth Keller award for the outstanding PhD dissertation that year in the College of Agriculture and Life Sciences. He championed the use of basic microbiology and genomic approaches to set a platform for translational applications of beneficial microbes in foods and their use in food preservation and probiotics and as oral delivery vehicles for vaccines and biotherapeutics. Dr. Klaenhammer was also a founding and co-chief editor of the Annual Review of Food Science and Technology.

Clostridium botulinum and Bacillus anthracis produce potent toxins that cause severe disease in humans. New and improved vaccines are needed for both of these pathogens. For mucosal vaccine delivery using lactic acid bacteria, chromosomal expression of antigens is preferred over plasmid-based expression systems, as chromosomal expression circumvents plasmid instability and the need for antibiotic pressure. In this study, we constructed three strains of Lactobacillus acidophilus NCFM expressing from the chromosome (i) the nontoxic host receptor-binding domain of the heavy chain of Clostridium botulinum serotype A neurotoxin (BoNT/A-Hc), (ii) the anthrax protective antigen (PA), and (iii) both the BoNT/A-Hc and the PA. The BoNT/A-Hc vaccine cassette was engineered to contain the signal peptide from the S-layer protein A from L. acidophilus and a dendritic-cell-targeting peptide. A chromosomal region downstream of lba0889 carrying a highly expressed enolase gene was selected for insertion of the vaccine cassettes. Western blot analysis confirmed the heterologous expression of the two antigens from plasmid and chromosome locations. Stability assays demonstrated loss of the vaccine cassettes from expression plasmids without antibiotic maintenance. RNA sequencing showed high expression of each antigen and that insertion of the vaccine cassettes had little to no effect on the transcription of other genes in the chromosome. This study demonstrated that chromosomal integrative recombinant strains are promising vaccine delivery vehicles when targeted into high-expression chromosomal regions. Levels of expression match high-copy-number plasmids and eliminate the requirement for antibiotic selective maintenance of recombinant plasmids. IMPORTANCE Clostridium botulinum and Bacillus anthracis produce potent neurotoxins that pose a biochemical warfare concern; therefore, effective vaccines against these bacteria are required. Chromosomal expression of antigens is preferred over plasmid-based expression systems since expressing antigens from a chromosomal location confers an advantage to the vaccine strains by eliminating the antibiotic maintenance required for plasmids and negates issues with plasmid instability that would result in loss of the antigen. Lactic acid bacteria, including Lactobacillus acidophilus , have shown potential for mucosal vaccine delivery, as L. acidophilus is bile and acid tolerant, allowing transit through the gastrointestinal tract where cells interact with host epithelial and immune cells, including dendritic cells. In this study, we successfully expressed C. botulinum and B. anthracis antigens in the probiotic L. acidophilus strain NCFM. Both antigens were highly expressed individually or in tandem from the chromosome of L. acidophilus .

Autolysins, also known as peptidoglycan hydrolases, are enzymes that hydrolyze specific bonds within bacterial cell wall peptidoglycan during cell division and daughter cell separation. Within the genome of Lactobacillus acidophilus NCFM, there are 11 genes encoding proteins with peptidoglycan hydrolase catalytic domains, 9 of which are predicted to be functional. Notably, 5 of the 9 putative autolysins in L. acidophilus NCFM are S-layer-associated proteins (SLAPs) noncovalently colocalized along with the surface (S)-layer at the cell surface. One of these SLAPs, AcmB, a β- N -acetylglucosaminidase encoded by the gene lba0176 ( acmB ), was selected for functional analysis. In silico analysis revealed that acmB orthologs are found exclusively in S-layer- forming species of Lactobacillus . Chromosomal deletion of acmB resulted in aberrant cell division, autolysis, and autoaggregation. Complementation of acmB in the Δ acmB mutant restored the wild-type phenotype, confirming the role of this SLAP in cell division. The absence of AcmB within the exoproteome had a pleiotropic effect on the extracellular proteins covalently and noncovalently bound to the peptidoglycan, which likely led to the observed decrease in the binding capacity of the Δ acmB strain for mucin and extracellular matrices fibronectin, laminin, and collagen in vitro . These data suggest a functional association between the S-layer and the multiple autolysins noncovalently colocalized at the cell surface of L. acidophilus NCFM and other S-layer-producing Lactobacillus species. IMPORTANCE Lactobacillus acidophilus is one of the most widely used probiotic microbes incorporated in many dairy foods and dietary supplements. This organism produces a surface (S)-layer, which is a self-assembling crystalline array found as the outermost layer of the cell wall. The S-layer, along with colocalized associated proteins, is an important mediator of probiotic activity through intestinal adhesion and modulation of the mucosal immune system. However, there is still a dearth of information regarding the basic cellular and evolutionary function of S-layers. Here, we demonstrate that multiple autolysins, responsible for breaking down the cell wall during cell division, are associated with the S-layer. Deletion of the gene encoding one of these S-layer-associated autolysins confirmed its autolytic role and resulted in reduced binding capacity to mucin and intestinal extracellular matrices. These data suggest a functional association between the S-layer and autolytic activity through the extracellular presentation of autolysins.

Bacterial surface layers (S-layers) are crystalline arrays of self-assembling proteinaceous subunits called S-layer proteins (Slps) that comprise the outermost layer of the cell envelope. Many additional proteins that are associated with or embedded within the S-layer have been identified in Lactobacillus acidophilus NCFM, an S-layer-forming bacterium that is widely used in fermented dairy products and probiotic supplements. One putative S-layer-associated protein (SLAP), LBA0191, was predicted to mediate adhesion to fibronectin based on the in silico detection of a fibronectin-binding domain. Fibronectin is a major component of the extracellular matrix (ECM) of intestinal epithelial cells. Adhesion to intestinal epithelial cells is considered an important trait for probiotic microorganisms during transit and potential association with the intestinal mucosa. To investigate the functional role of LBA0191 (designated FbpB) in L. acidophilus NCFM, an fbpB -deficient strain was constructed. The L. acidophilus mutant with a deletion of fbpB lost the ability to adhere to mucin and fibronectin in vitro . Homologues of fbpB were identified in five additional putative S-layer-forming species, but no homologues were detected in species outside the L. acidophilus homology group.

This investigation assessed the potential of isolating novel probiotics from mothers and their infants. A subset of 21 isolates among 126 unique bacteria from breast milk and infant stools from 15 mother-infant pairs were examined for simulated GI transit survival, adherence to Caco-2 cells, bacteriocin production, and lack of antibiotic resistance. Of the 21 selected isolates a Lactobacillus crispatus isolate and 3 Lactobacillus gasseri isolates demonstrated good profiles of in vitro GI transit tolerance and Caco-2 cell adherence. Bacteriocin production was observed only by L. gasseri and Enterococcus faecalis isolates. Antibiotic resistance was widespread, although not universal, among isolates from infants. Highly similar isolates (≥ 97% similarity by barcode match) of Bifidobacterium longum subsp. infantis (1 match), Lactobacillus fermentum (2 matches), Lactobacillus gasseri (6 matches), and Enterococcus faecalis (1 match) were isolated from 5 infant-mother pairs. Antibiotic resistance profiles between these isolate matches were similar, except in one case where the L. gasseri isolate from the infant exhibited resistance to erythromycin and tetracycline, not observed in matching mother isolate. In a second case, L. gasseri isolates differed in resistance to ampicillin, chloramphenicol and vancomycin between the mother and infant. In this study, gram positive bacteria isolated from mothers' breast milk as well as their infants exhibited diversity in GI transit survival and acid inhibition of pathogens, but demonstrated limited ability to produce bacteriocins. Mothers and their infants offer the potential for identification of probiotics; however, even in the early stages of development, healthy infants contain isolates with antibiotic resistance.

Lactobacillus acidophilus NCFM is a well-characterized probiotic microorganism, supported by a decade of genomic and functional phenotypic investigations. L. acidophilus deficient in lipoteichoic acid (LTA), a major immunostimulant in Gram-positive bacteria, has been shown to shift immune system responses in animal disease models. However, the pleiotropic effects of removing LTA from the cell surface in lactobacilli are unknown. In this study, we surveyed the global transcriptional and extracellular protein profiles of two strains of L. acidophilus deficient in LTA. Twenty-four differentially expressed genes specific to the LTA-deficient strains were identified, including a predicted heavy metal resistance operon and several putative peptidoglycan hydrolases. Cell morphology and manganese sensitivity phenotypes were assessed in relation to the putative functions of differentially expressed genes. LTA-deficient L. acidophilus exhibited elongated cellular morphology and their growth was severely inhibited by elevated manganese concentrations. Exoproteomic surveys revealed distinct changes in the composition and relative abundances of several extracellular proteins and showed a bias of intracellular proteins in LTA-deficient strains of L. acidophilus. Taken together, these results elucidate the impact of ltaS deletion on the transcriptome and extracellular proteins of L. acidophilus, suggesting roles of LTA in cell morphology and ion homeostasis as a structural component of the Gram positive cell wall.

α-Glucans from bacterial exo-polysaccharides or diet, e.g., resistant starch, legumes and honey are abundant in the human gut and fermentation of resistant fractions of these α-glucans by probiotic lactobacilli and bifidobacteria impacts human health positively. The ability to degrade polymeric α-glucans is confined to few strains encoding extracellular amylolytic activities of glycoside hydrolase (GH) family 13. Debranching pullulanases of the subfamily GH13_14 are the most common extracellular GH13 enzymes in lactobacilli, whereas corresponding enzymes are mainly α-amylases and amylopullulanases in bifidobacteria. Extracellular GH13 enzymes from both genera are frequently modular and possess starch binding domains, which are important for efficient catalysis and possibly to mediate attachment of cells to starch granules. α-1,6-Linked glucans, e.g., isomalto-oligosaccharides are potential prebiotics. The enzymes targeting these glucans are the most abundant intracellular GHs in bifidobacteria and lactobacilli. A phosphoenolpyruvate-dependent phosphotransferase system and a GH4 phospho-α-glucosidase are likely involved in metabolism of isomaltose and isomaltulose in probiotic lactobacilli based on transcriptional analysis. This specificity within GH4 is unique for lactobacilli, whereas canonical GH13 31 α-1,6-glucosidases active on longer α-1,6-gluco-oligosaccharides are ubiquitous in bifidobacteria and lactobacilli. Malto-oligosaccharide utilization operons encode more complex, diverse, and less biochemically understood activities in bifidobacteria compared to lactobacilli, where important members have been recently described at the molecular level. This review presents some aspects of α-glucan metabolism in probiotic bacteria and highlights vague issues that merit experimental effort, especially oligosaccharide uptake and the functionally unassigned enzymes, featuring in this important facet of glycan turnover by members of the gut microbiota.

Commercial probiotic strains for food or supplement use can be altered in different ways for a variety of purposes. Production conditions for the strain or final product may be changed to address probiotic yield, functionality, or stability. Final food products may be modified to improve flavor and other sensory properties, provide new product formats, or respond to market opportunities. Such changes can alter the expression of physiological traits owing to the live nature of probiotics. In addition, genetic approaches may be used to improve strain attributes. This review explores whether genetic or phenotypic changes, by accident or design, might affect the efficacy or safety of commercial probiotics. We highlight key issues important to determining the need to re-confirm efficacy or safety after strain improvement, process optimization, or product formulation changes. Research pinpointing the mechanisms of action for probiotic function and the development of assays to measure them are greatly needed to better understand if such changes have a substantive impact on probiotic efficacy.

Summary Glycogen metabolism contributes to energy storage and various physiological functions in some prokaryotes, including colonization persistence. A role for glycogen metabolism is proposed on the survival and fitness of Lactobacillus acidophilus, a probiotic microbe, in the human gastrointestinal environment. L. acidophilus NCFM possesses a glycogen metabolism (glg) operon consisting of glgBCDAP-amy-pgm genes. Expression of the glg operon and glycogen accumulation were carbon source- and growth phase-dependent, and were repressed by glucose. The highest intracellular glycogen content was observed in early log-phase cells grown on trehalose, which was followed by a drastic decrease of glycogen content prior to entering stationary phase. In raffinose-grown cells, however, glycogen accumulation gradually declined following early log phase and was maintained at stable levels throughout stationary phase. Raffinose also induced an overall higher temporal glg expression throughout growth compared with trehalose. Isogenic ΔglgA (glycogen synthase) and ΔglgB (glycogen-branching enzyme) mutants are glycogen-deficient and exhibited growth defects on raffinose. The latter observation suggests a reciprocal relationship between glycogen synthesis and raffinose metabolism. Deletion of glgB or glgP (glycogen phosphorylase) resulted in defective growth and increased bile sensitivity. The data indicate that glycogen metabolism is involved in growth maintenance, bile tolerance and complex carbohydrate utilization in L. acidophilus.

In recent years, a plethora of studies have demonstrated the paramount physiological importance of the gut microbiota on various aspects of human health and development. Particular focus has been set on probiotic members of this community, the best studied of which are assigned into the Lactobacillus and Bifidobacterium genera. Effects such as pathogen exclusion, alleviation of inflammation and allergies, colon cancer, and other bowel disorders are attributed to the activity of probiotic bacteria, which selectively ferment prebiotics comprising mainly non-digestible oligosaccharides. Thus, glycan metabolism is an important attribute of probiotic action and a factor influencing the composition of the gut microbiota.In the quest to understand the molecular mechanism of this selectivity for certain glycans, we have explored the routes of uptake and utilization of a variety of oligosaccharides differing in size, composition, and glycosidic linkages. A combination of “omics” technologies bioinformatics...

Analysis of global temporal gene expression by human intestinal cells when exposed to Lactobacillus acidophilus revealed induction of immune-related pathways and NF-κB target genes after a 1-h exposure, compared to a 4- or 8-h exposure. Additionally, an L. acidophilus derivative expressing covalently bound flagellin resulted in increased induction of il8 , cxc1 , and cxcl2 compared to the parent L. acidophilus .

The organisation of genes conferring utilisation of raffinose family oligosaccharides (RFOs) has been analysed in several probiotic bacteria from the Bifidobacterium and Lactobacillus genera. Glycoside hydrolase family 36 (GH36) α-galatosidase encoding genes occur together with sugar transport systems of the glycoside–pentoside–hexuronide cation symporter family (GPH), sugar phosphotransferase systems (PTSs) or ATP-binding cassette systems (ABCs) highlighting the diversity of RFO uptake. The GH36 genes are often clustered together with sucrose hydrolases or phosphorylases ensuring the degradation of RFO to monosaccharides. Differential proteomics and transcriptomics data from our laboratories implicated ABC transporters in the uptake of RFO in both Lactobacillus acidophilus NCFM and Bifidobacterium animalis subsp. lactis Bl-04. Interestingly, only one of three GH36 encoding genes in B. animalis subsp. lactis Bl-04 was upregulated upon growth on RFO, suggesting that the other two gene products may have dif...

Lactobacillus acidophilus NCFM is a probiotic microbe that survives passage through the human gastrointestinal tract and interacts with the host epithelium and mucosal immune cells. The potential for L. acidophilus to express antigens at mucosal surfaces has been investigated with various antigens and plasmid expression vectors. Plasmid instability and antibiotic selection complicate the possibility of testing these constructs in human clinical trials. Integrating antigen encoding genes into the chromosome for expression is expected to eliminate selection requirements and provide genetic stability. In this work, a reporter gene encoding a β-glucuronidase (GusA3) was integrated into four intergenic chromosomal locations. The integrants were tested for genetic stability and GusA3 activity. Two locations were selected for insertion downstream of constitutively highly expressed genes, one downstream of slpA (LBA0169), encoding a highly expressed surface-layer protein, and one downstream of phosphopyruvate hydratase (LBA0889), a highly expressed gene with homologs in other lactic acid bacteria. An inducible location was selected downstream of lacZ (LBA1462), encoding a β-galactosidase. A fourth location was selected in a low-expression region. The expression of gusA3 was evaluated from each location by measuring GusA3 activity on 4-methyl-umbelliferyl-β- d -glucuronide (MUG). GusA3 activity from both highly expressed loci was more than three logs higher than the gusA3 -negative parent, L. acidophilus NCK1909. GusA3 activity from the lacZ locus was one log higher in cells grown in lactose than in glucose. The differences in expression levels between integration locations highlights the importance of rational targeting with gene cassettes intended for chromosomal expression.

Display of heterologous antigens on the cell surface is considered a useful technique for vaccine delivery by recombinant lactobacilli. In this study, two recombinant Lactobacillus acidophilus derivatives displaying Salmonella flagellin (FliC) were constructed using different anchor motifs. In one instance, the FliC protein was fused to the C-terminal region of a cell envelope proteinase (PrtP) and was bound to the cell wall by electrostatic bonds. In the other case, the same antigen was conjugated to the anchor region of mucus binding protein (Mub) and was covalently associated with the cell wall by an LPXTG motif. These two recombinant L. acidophilus cell surface displays resulted in dissimilar maturation and cytokine production by human myeloid dendritic cells. The surface-associated antigen was highly sensitive to simulated gastric and small intestinal juices. By supplementation with bicarbonate buffer and soybean trypsin inhibitor, the cell surface antigen was protected from proteolytic enzymes during gastric challenge in vitro . The protective reagents also increased the viability of the L. acidophilus cells upon challenge with simulated digestive juices. These results demonstrate the importance of protecting cells and their surface-associated antigens during oral immunization.

The trillions of microbes residing within the intestine induce critical signals that either regulate or stimulate host immunity via their bacterial products. To better understand the immune regulation elicited by lipoteichoic acid (LTA)-deficient Lactobacillus acidophilus NCFM in steady state and induced inflammation, we deleted phosphoglycerol transferase gene, which synthesizes LTA in L. acidophilus NCFM. In vitro and in vivo experiments were conducted in order to compare the immune regulatory properties of the L. acidophilus strain deficient in LTA (NCK2025) with its wild-type parent (NCK56) in C57BL/6, C57BL/6 recombination-activation gene 1-deficient (Rag1 -/-) and C57BL/6 Rag1-/-IL-10-/- mice. We demonstrate that NCK2025 significantly activates the phosphorylation of Erk1/2 but downregulates the phosphorylation of Akt1, cytosolic group IV PLA2 and p38 in mouse dendritic cells. Similarly, mice treated orally with NCK2025 exhibit decreased phosphorylation of inflammatory signals (Akt1, cytosolic group IV PLA2 or P38) but upregulate Erk1/2-phosphorylation in colonic epithelial cells in comparison with mice treated with NCK56. In addition, regulation of pathogenic CD4+ T cell induced colitis by NCK2025 was observed in Rag1 -/- but not Rag1-/-IL-10 -/- mice suggests a critical role of IL-10 that may be tightly regulated by Erk1/2 signaling. These data highlight the immunosuppressive properties of NCK2025 to deliver regulatory signals in innate cells, which results in the mitigation of T-cell-induced colitis in vivo.

Imbalance in the regulatory immune mechanisms that control intestinal cellular and bacterial homeostasis may lead to induction of the detrimental inflammatory signals characterized in humans as inflammatory bowel disease. Induction of proinflammatory cytokines (i.e., IL-12) induced by dendritic cells (DCs) expressing pattern recognition receptors may skew naive T cells to T helper 1 polarization, which is strongly implicated in mucosal autoimmunity. Recent studies show the ability of probiotic microbes to treat and prevent numerous intestinal disorders, including Clostridium difficile -induced colitis. To study the molecular mechanisms involved in the induction and repression of intestinal inflammation, the phosphoglycerol transferase gene that plays a key role in lipoteichoic acid (LTA) biosynthesis in Lactobacillus acidophilus NCFM (NCK56) was deleted. The data show that the L. acidophilus LTA-negative in LTA (NCK2025) not only down-regulated IL-12 and TNFα but also significantly enhanced IL-10 in DCs and controlled the regulation of costimulatory DC functions, resulting in their inability to induce CD4 + T-cell activation. Moreover, treatment of mice with NCK2025 compared with NCK56 significantly mitigated dextran sulfate sodium and CD4 + CD45RB high T cell-induced colitis and effectively ameliorated dextran sulfate sodium-established colitis through a mechanism that involves IL-10 and CD4 + FoxP3 + T regulatory cells to dampen exaggerated mucosal inflammation. Directed alteration of cell surface components of L. acidophilus NCFM establishes a potential strategy for the treatment of inflammatory intestinal disorders.

Recent research efforts have focused on understanding the interactions of probiotic bacteria with commensal gut bacteria and the human host as a means to determine mechanisms of probiotic functionality that contribute to their beneficial attributes. Our growing understanding of the intrinsic interactions between probiotic and commensal bacteria and between the milieu of bacteria and the host tissues of the gastrointestinal tract (GIT) has been facilitated by the use of ‘omic’ technologies. Surveys of bacterial inhabitants in the GIT using sequencing technologies have demonstrated the complexities of this human organ which varies between different populations and individuals, such as diet. In addition, transcriptomics have rapidly facilitated an insight into the complex communication between bacteria (commensal and probiotic) and the GIT. This review outlines the recent important advances in this exciting area of research, which has led to a greater understanding of the critical interface between gut microbiota, probiotic bacteria and the host.

Prophage Lrm1 was induced with mitomycin C from an industrial Lactobacillus rhamnosus starter culture, M1. Electron microscopy of the lysate revealed relatively few intact bacteriophage particles among empty heads and disassociated tails. The defective Siphoviridae phage had an isometric head of approximately 55 nm and noncontractile tail of about 275 nm with a small baseplate. In repeated attempts, the prophage could not be cured from L. rhamnosus M1, nor could a sensitive host be identified. Sequencing of the phage Lrm1 DNA revealed a genome of 39,989 bp and a G+C content of 45.5%. A similar genomic organization and mosaic pattern of identities align Lrm1 among the closely related Lactobacillus casei temperate phages A2, ΦAT3, and LcaI and with L. rhamnosus virulent phage Lu-Nu. Of the 54 open reading frames (ORFs) identified, all but 8 shared homology with other phages of this group. Five unknown ORFs were identified that had no homologies in the databases nor predicted functions. Notably, Lrm1 encodes a putative endonuclease and a putative DNA methylase with homology to a methylase in Lactococcus lactis phage Tuc2009. Possibly, the DNA methylase, endonuclease, or other Lrm1 genes provide a function crucial to L. rhamnosus M1 survival, resulting in the stability of the defective prophage in its lysogenic state. The presence of a defective prophage in an industrial strain could provide superinfection immunity to the host but could also contribute DNA in recombination events to produce new phages potentially infective for the host strain in a large-scale fermentation environment.

The lactic acid bacteria (LAB) are one of the most industrially important groups of bacteria. These organisms are used in a variety of ways, including food production, health improvement and production of macromolecules, enzymes and metabolites. The genome sequencing of 20 LAB provides an expanded view of their genetic and metabolic capacities and enables researchers to perform functional and comparative genomic studies. This review highlights some of the findings from these analyses in the context of the numerous roles the LAB play.

Invariant and highly conserved amino acids within a primase consensus sequence were targeted by site-specific mutations within the putative primase of Streptococcus thermophilus phage kappa3. PCR products containing the desired mutation(s) within putative ATPase/helicase and/or oligomerization domains of the kappa3-encoded primase gene were cloned into a high-copy-number vector and expressed in S. thermophilus NCK1125. The majority of the plasmid constructs failed to alter phage sensitivity; however, four of the constructs conferred strong phage resistance upon the host. Expression of the K238(A/T) and RR340-341AA mutant proteins in trans suppressed the function of the native phage primase protein in a dominant negative fashion via a proposed subunit poisoning mechanism. These constructs completely inhibited phage genome synthesis and reduced the efficiencies of plaquing and centre of infection formation by more than 9 and 3.5 logs, respectively. Amber mutations introduced upstream of the transdominant RR340-341AA and K238(A/T) mutations restored phage genome replication and sensitivity of the host, indicating that translation was required to confer phage resistance. Introduction of an E437A mutation in a putative oligomerization domain located downstream of the transdominant K238T mutation also completely suppressed phage resistance. This study appears to represent the first use of transdominant proteins to inhibit phages that are disruptive to cultures used in industrial fermentations.

Surface layer proteins of probiotic lactobacilli are theoretically efficient epitope-displaying scaffolds for oral vaccine delivery due to their high expression levels and surface localization. In this study, we constructed genetically modified Lactobacillus acidophilus strains expressing the membrane proximal external region (MPER) from human immunodeficiency virus type 1 (HIV-1) within the context of the major S-layer protein, SlpA. Intragastric immunization of mice with the recombinants induced MPER-specific and S-layer protein-specific antibodies in serum and mucosal secretions. Moreover, analysis of systemic SlpA-specific cytokines revealed that the responses appeared to be Th1 and Th17 dominant. These findings demonstrated the potential use of the Lactobacillus S-layer protein for development of oral vaccines targeting specific peptides.

A large number of studies have investigated gastrointestinal microbiota and changes in the gastrointestinal community. However, a concern in these studies is how best to assess changes in gastrointestinal community structure. This paper presents two different human trials where the fecal terminal restriction fragment length polymorphism data sets were analyzed to search for treatment effects. Principle components analysis and cluster analysis based on grouped data are compared with analysis of data by subject using distance coefficients. Comparison with baseline within an individual before grouping by treatment provided a clearer indication of treatment effects than did an evaluation of data grouped before analysis. In addition, a large within-subject sample size and multiple baseline samples are necessary to accurately analyze treatment effects.

Bacteriophages (phages) have the potential to interfere with any industry that produces bacteria as an end product or uses them as biocatalysts in the production of fermented products or bioactive molecules. Using microorganisms that drive food bioprocesses as an example, this review will describe a set of genetic tools that are useful in the engineering of customized phage-defence systems. Special focus will be given to the power of comparative genomics as a means of streamlining target selection, providing more widespread phage protection, and increasing the longevity of these industrially important bacteria in the bioprocessing environment.

Lactobacilli are major inhabitants of the normal microflora of the gastrointestinal tract, and some select species have been used extensively as probiotic cultures. One potentially important property of these organisms is their ability to interact with epithelial cells in the intestinal tract, which may promote retention and host-bacterial communication. However, the mechanisms by which they attach to intestinal epithelial cells are unknown. The objective of this study was to investigate cell surface proteins in Lactobacillus acidophilus that may promote attachment to intestinal tissues. Using genome sequence data, predicted open reading frames were searched against known protein and protein motif databases to identify four proteins potentially involved in adhesion to epithelial cells. Homologous recombination was used to construct isogenic mutations in genes encoding a mucin-binding protein, a fibronectin-binding protein, a surface layer protein, and two streptococcal R28 homologs. The abilities of the mutants to adhere to intestinal epithelial cells were then evaluated in vitro. Each strain was screened on Caco-2 cells, which differentiate and express markers characteristic of normal small-intestine cells. A significant decrease in adhesion was observed in the fibronectin-binding protein mutant (76%) and the mucin-binding protein mutant (65%). A surface layer protein mutant also showed reduction in adhesion ability (84%), but the effect of this mutation is likely due to the loss of multiple surface proteins that may be embedded in the S-layer. This study demonstrated that multiple cell surface proteins in L. acidophilus NCFM can individually contribute to the organism's ability to attach to intestinal cells in vitro.

Analysis of the automated computer annotation of the early draft phase genome of Lactobacillus acidophilus NCFM revealed the previously discovered S-layer gene slpA and an additional partial ORF with weak similarities to S-layer proteins. The entire gene was sequenced to reveal a 1799-bp gene coding for 599 amino acids with a calculated molecular mass of 64.8 kDa. No transcription or translation signals could be determined in close proximity to the 5'-region. However, a strong putative terminator with a free energy of -16.84 kcal/mol was identified directly downstream of the gene. A PSI-Blast analysis showed similarities to members of S-layer proteins, cell-wall associated proteinases and hexosyl-transferases. Calculation of an unrooted phylogenetic tree with other examples of S-layer proteins and proteinases placed the deduced protein separately from both groups. A derivative of L. acidophilus NCFM was constructed by targeted integration into the gene. SDS-PAGE analysis of non-covalently linked proteins of the cell wall of the mutant, compared to the wild type, revealed the loss of a cell-surface protein. Phenotypic analyses of the mutant revealed significant changes in cell morphology, altered responses to various environmental stresses, and lowered cell adhesion. Based on the in silico and functional analyses, we ascertained that this protein plays a role in cell-wall processing during the growth and cell-cell separation and designated the gene as cell-division protein, cdpA.

Amino acid decarboxylation-antiporter reactions are one of the most important systems for maintaining intracellular pH between physiological limits under acid stress. We analyzed the Lactobacillus acidophilus NCFM complete genome sequence and selected four open reading frames with similarities to genes involved with decarboxylation reactions involved in acid tolerance in several microorganisms. Putative genes encoding an ornithine decarboxylase, an amino acid permease, a glutamate γ-aminobutyrate antiporter, and a transcriptional regulator were disrupted by insertional inactivation. The ability of L. acidophilus to survive low-pH conditions, such as those encountered in the stomach or fermented dairy foods, was investigated and compared to the abilities of early- and late-stationary-phase cells of the mutants by challenging them with a variety of acidic conditions. All of the integrants were more sensitive to low pH than the parental strain. Interestingly, each integrant also exhibited an adaptive acid response during logarithmic growth, indicating that multiple mechanisms are present and orchestrated in L. acidophilus in response to acid challenge.

In living organisms, exposure to oxygen provokes oxidative stress. A widespread mechanism for protection against oxidative stress is provided by the antioxidant enzymes: superoxide dismutases (SODs) and hydroperoxidases. Generally, these enzymes are not present in Lactobacillus spp. In this study, we examined the potential advantages of providing a heterologous SOD to some of the intestinal lactobacilli. Thus, the gene encoding the manganese-containing SOD ( sodA ) was cloned from Streptococcus thermophilus AO54 and expressed in four intestinal lactobacilli. A 1.2-kb PCR product containing the sodA gene was cloned into the shuttle vector pTRK563, to yield pSodA, which was functionally expressed and complemented an Escherichia coli strain deficient in Mn and FeSODs. The plasmid, pSodA, was subsequently introduced and expressed in Lactobacillus gasseri NCK334, Lactobacillus johnsonii NCK89 , Lactobacillus acidophilus NCK56, and Lactobacillus reuteri NCK932. Molecular and biochemical analyses confirmed the presence of the gene ( sodA ) and the expression of an active gene product (MnSOD) in these strains of lactobacilli. The specific activities of MnSOD were 6.7, 3.8, 5.8, and 60.7 U/mg of protein for L. gasseri , L. johnsonii , L. acidophilus , and L. reuteri , respectively. The expression of S. thermophilus MnSOD in L. gasseri and L. acidophilus provided protection against hydrogen peroxide stress. The data show that MnSOD protects cells against hydrogen peroxide by removing O 2 ·− and preventing the redox cycling of iron. To our best knowledge, this is the first report of a sodA from S. thermophilus being expressed in other lactic acid bacteria.

Listeria monocytogenes contamination of ready-to-eat foods has been implicated in numerous outbreaks of food-borne listeriosis. However, the health hazards posed by L. monocytogenes detected in foods may vary, and speculations exist that strains actually implicated in illness may constitute only a fraction of those that contaminate foods. In this study, examination of 34 serogroup 4 (putative or confirmed serotype 4b) isolates of L. monocytogenes obtained from various foods and food-processing environments, without known implication in illness, revealed that many of these strains had methylation of cytosines at GATC sites in the genome, rendering their DNA resistant to digestion by the restriction endonuclease Sau3AI. These strains also harbored a gene cassette with putative restriction-modification system genes as well as other, genomically unlinked genetic markers characteristic of the major epidemic-associated lineage of L. monocytogenes (epidemic clone I), implicated in numerous outbreaks in Europe and North America. This may reflect a relatively high fitness of strains with these genetic markers in foods and food-related environments relative to other serotype 4b strains and may partially account for the repeated involvement of such strains in human food-borne listeriosis. Food contamination by Listeria monocytogenes has been implicated in numerous outbreaks and sporadic cases of human illness. Most commonly implicated in listeriosis are highly processed, ready-to-eat (RTE) foods that are kept refrigerated for various periods of time. At risk for listeriosis are people in the extremes of age, pregnant women and their fetuses, cancer patients, and others experiencing immunosuppression (13, 24, 35, 38). Listeriosis can have severe symptoms (septicemia, meningitis, and stillbirths) and a high mortality rate (20 to 30%). Hence, regulations exist in numerous nations concerning the density (e.g., 1 CFU/25 g) of cells of the etiologic agent permissible in RTE foods. Such regulations are based on the hypothesis that any L. monocytogenes strain that can be detected in RTE foods has the potential to pose serious hazards to human health. The potential hazard posed by listerial contamination of RTE foods can be influenced by the number of cells at the point of consumption, which would depend on conditions of storage, type of food matrix and its impact on growth, presence of competing microflora and antimicrobial agents, etc. In addition, the strain type of L. monocytogenes involved may be of importance. It is likely, based on studies with other bacterial pathogens, that some strains and strain clusters (clonal groups) within the species might be more pathogenic than others. Speculations have been formulated that only a fraction of the strains of L. monocytogenes found in foods may be capable of causing human illness (20). There is indeed evidence that the repertoire of strains ca

The putative primase gene and other genes associated with the Sfi21-prototype genome replication module are highly conserved in Streptococcus thermophilus bacteriophages. Expression of antisense RNAs complementary to the putative primase gene ( pri3.1 ) from S. thermophilus phage κ3 provided significant protection from κ3 and two other Sfi21-type phages. Expression of pri3.10-AS , an antisense RNA that covered the entire primase gene, reduced the efficiency of plaquing (EOP) of κ3 to 3 × 10 −3 and reduced its burst size by 20%. Mutant phages capable of overcoming antisense inhibition were not recovered. Thirteen primase-specific antisense cassettes of different lengths (478 to 1,512 bp) were systematically designed to target various regions of the gene. Each cassette conferred some effect, reducing the EOP to between 0.8 and 3 × 10 −3 . The largest antisense RNAs (1.5 kb) were generally found to confer the greatest reductions in EOP, but shorter (0.5 kb) antisense RNAs were also effective, especially when directed to the 5′ region of the gene. The impacts of primase-targeted antisense RNAs on phage development were examined. The expression of pri3.10-AS resulted in reductions in target RNA abundance and the number of phage genomes synthesized. Targeting a key genome replication function with antisense RNA provided effective phage protection in S. thermophilus.

We analyzed the tuf gene, encoding elongation factor Tu, from 33 strains representing 17 Lactobacillus species and 8 Bifidobacterium species. The tuf sequences were aligned and used to infer phylogenesis among species of lactobacilli and bifidobacteria. We demonstrated that the synonymous substitution affecting this gene renders elongation factor Tu a reliable molecular clock for investigating evolutionary distances of lactobacilli and bifidobacteria. In fact, the phylogeny generated by these tuf sequences is consistent with that derived from 16S rRNA analysis. The investigation of a multiple alignment of tuf sequences revealed regions conserved among strains belonging to the same species but distinct from those of other species. PCR primers complementary to these regions allowed species-specific identification of closely related species, such as Lactobacillus casei group members. These tuf gene-based assays developed in this study provide an alternative to present methods for the identification for lactic acid bacterial species. Since a variable number of tuf genes have been described for bacteria, the presence of multiple genes was examined. Southern analysis revealed one tuf gene in the genomes of lactobacilli and bifidobacteria, but the tuf gene was arranged differently in the genomes of these two taxa. Our results revealed that the tuf gene in bifidobacteria is flanked by the same gene constellation as the str operon, as originally reported for Escherichia coli . In contrast, bioinformatic and transcriptional analyses of the DNA region flanking the tuf gene in four Lactobacillus species indicated the same four-gene unit and suggested a novel tuf operon specific for the genus Lactobacillus .

The inaugural meeting of the International Scientific Association for Probiotics and Prebiotics (ISAPP) was held May 3 to May 5 2002 in London, Ontario, Canada. A group of 63 academic and industrial scientists from around the world convened to discuss current issues in the science of probiotics and prebiotics. ISAPP is a non-profit organization comprised of international scientists whose intent is to strongly support and improve the levels of scientific integrity and due diligence associated with the study, use, and application of probiotics and prebiotics. In addition, ISAPP values its role in facilitating communication with the public and healthcare providers and among scientists in related fields on all topics pertinent to probiotics and prebiotics. It is anticipated that such efforts will lead to development of approaches and products that are optimally designed for the improvement of human and animal health and well being. This article is a summary of the discussions, conclusions, and recommendations made by 8 working groups convened during the first ISAPP workshop focusing on the topics of: definitions, intestinal flora, extra-intestinal sites, immune function, intestinal disease, cancer, genetics and genomics, and second generation prebiotics.

Lactic acid bacterial strains were isolated from brines sampled after 7 days of an industrial sauerkraut fermentation, and six strains were selected on the basis of susceptibility to bacteriophages. Bacterial growth in cabbage juice was monitored, and the fermentation end products were identified, quantified, and compared to those of Leuconostoc mesenteroides . Identification by biochemical fingerprinting, endonuclease digestion of the 16S-23S intergenic transcribed spacer region, and sequencing of variable regions V1 and V2 of the 16S rRNA gene indicated that the six selected sauerkraut isolates were Leuconostoc fallax strains. Random amplification of polymorphic DNA fingerprints indicated that the strains were distinct from one another. The growth and fermentation patterns of the L. fallax isolates were highly similar to those of L. mesenteroides . The final pH of cabbage juice fermentation was 3.6, and the main fermentation end products were lactic acid, acetic acid, and mannitol for both species. However, none of the L. fallax strains exhibited the malolactic reaction, which is characteristic of most L. mesenteroides strains. These results indicated that in addition to L. mesenteroides , a variety of L. fallax strains may be present in the heterofermentative stage of sauerkraut fermentation. The microbial ecology of sauerkraut fermentation appears to be more complex than previously indicated, and the prevalence and roles of L. fallax require further investigation.

This paper presents the first report of bacteriophage isolated from commercial vegetable fermentations. Nine phages were isolated from two 90-ton commercial sauerkraut fermentations. These phages were active against fermentation isolates and selected Leuconostoc mesenteroides and Lactobacillus plantarum strains, including a starter culture. Phages were characterized as members of the Siphoviridae and Myoviridae families. All Leuconostoc phages reported previously, primarily of dairy origin, belonged to the Siphoviridae family.

On November 2–3, 2009 an international group of scientists representing multiple disciplines gathered to consider the current state of our understanding of the symbiotic and beneficial relationships between microbes and humans and to define the challenges, gaps in knowledge, and opportunities that this exciting field of study now offers. A number of adjectives come to mind when describing the subject of microbes and health, ranging from ancient and historic , to integrative and interdisciplinary , to timely and pressing . Coexistence and coevolution with microbes has been a theme of life on Earth for all metazoans past and present. Historically, the discovery that microbes are an integral part of us was made as soon as Antonie van Leeuwenhoek peered through his microscope and examined dental plaque sampled from himself and others (without institutional review board approval!). His sense of awe and his early appreciation of the diversity our microbial partners were evident in the words he chose for a letter written to the Royal Society of London in September 1683: > “Though my teeth are kept usually very clean, nevertheless, when I view them in a magnifying glass, I find growing between them a little white matter as thick as wetted flower: in this substance though I could not perceive any motion, I judged there might probably be living creatures. I therefore took some of this flower and mixed it either with pure rain water wherein were no animals, or else with some of my spittle (having no air bubbles to cause a motion in it) and then to my great surprise perceived that the aforesaid matter maintained very many small living animals, which moved themselves very extravagantly. ….The spittle of an old man that had lived soberly, had no animals in it; but the substance upon and between his teeth … [↵][1]1To whom correspondence should be addressed. E-mail: jgordon{at}wustl.edu. [1]: #xref-corresp-1-1

Recombinant phages are generated when Lactococcus lactis subsp. lactis harboring plasmids encoding the abortive type (Abi) of phage resistance mechanisms is infected with small isometric phages belonging to the P335 species. These phage variants are likely to be an important source of virulent new phages that appear in dairy fermentations. They are distinguished from their progenitors by resistance to Abi defenses and by altered genome organization, including regions of L. lactis chromosomal DNA. The objective of this study was to characterize four recombinant variants that arose from infection of L. lactis NCK203 (Abi + ) with phage φ31. Hin dIII restriction maps of the variants (φ31.1, φ31.2, φ31.7, and φ31.8) were generated, and these maps revealed the regions containing recombinant DNA. The recombinant region of phage φ31.1, the variant that occurred most frequently, was sequenced and revealed 7.8 kb of new DNA compared with the parent phage, φ31. This region contained numerous instances of homology with various lactococcal temperate phages, as well as homologues of the lambda recombination protein BET and Escherichia coli Holliday junction resolvase Rus, factors which may contribute to efficient recombination processes. A sequence analysis and phenotypic tests revealed a new origin of replication in the φ31.1 DNA, which replaced the φ31 origin. Three separate Hin dIII fragments, accounting for most of the recombinant region of φ31.1, were separately cloned into gram-positive suicide vector pTRK333 and transformed into NCK203. Chromosomal insertions of each plasmid prevented the appearance of different combinations of recombinant phages. The chromosomal insertions did not affect an inducible prophage present in NCK203. Our results demonstrated that recombinant phages can acquire DNA cassettes from different regions of the chromosome in order to overcome Abi defenses. Disruption of these regions by insertion can alter the types and diversity of new phages that appear during phage-host interactions.

Probiotics might reduce gut-derived microbial lipopolysaccharide (LPS) by restoring bowel flora in patients with alcoholic hepatitis (AH). We evaluated the therapeutic effects of probiotics in patients with AH.Between September 2010 and April 2012, 117 patients (probiotics 60 and placebo 57) were prospectively randomized to receive the 7 days of cultured Lactobacillus subtilis/Streptococcus faecium (1500 mg/day) or placebo. All patients were hospitalized and were not permitted to consume alcohol for the 7 days of the study. Liver function test, proinflammatory cytokines, LPS, and colony-forming units by stool culture were examined and compared after therapy.In both groups, the mean levels of aspartate aminotransferase/alanine aminotransferase, alkaline phosphatase, γ-glutamyl transpeptidase, bilirubin, and prothrombin time were significantly improved after 7 days of abstinence. In the probiotics group (baseline and after), albumin (3.5 ± 0.7 and 3.7 ± 0.6 g/dl, P=0.038) and tumor necrosis factor-α (121 ± 244 and 71 ± 123 pg/ml, P=0.047) showed differences. In addition, the number of colony-forming units of Escherichia coli was significantly reduced (435 ± 287 and 168 ± 210, P=0.002). In the placebo group, the level of LPS (1.7 ± 2.8 and 2.0 ± 2.7 EU/ml) was significantly increased. In the intergroup comparison, significant differences in the levels of tumor necrosis factor-α (P=0.042) and LPS (P=0.028) were observed between the groups.Immediate abstinence is the most important treatment for patients with AH. In addition, 7 days of oral supplementation with cultured L. subtilis/S. faecium was associated with restoration of bowel flora and improvement of LPS in patients with AH.

Lactococcus lactis is industrially important microorganism used in many dairy fermentations. Numerous genes and gene expression signals from this organism have now been identified and characterized. Recently, several naturally occurring, inducible gene-expression systems have also been described in L. lactis. The main features of these systems can be exploited to design genetically engineered expression cassettes for controlled production of various proteins and enzymes. Novel gene-expression systems in Lactococcus have great potential for development of industrial cultures with desirable metabolic traits for a variety of bioprocessing applications.

Lactacin F is a two-component class II bacteriocin produced by Lactobacillus johnsonii VPI 11088. The laf operon is composed of the bacteriocin structural genes, lafA and lafX, and a third open reading frame, ORFZ. Two strategies were employed to study the function of ORFZ. This gene was disrupted in the chromosome of NCK64, a lafA729 lafX ORFZ derivative of VPI 11088. A disruption cassette consisting of ORFZ interrupted with a cat gene was cloned into pSA3 and introduced into NCK64. Manipulation of growth temperatures and antibiotic selection resulted in homologous recombination which disrupted the chromosomal copy of ORFZ with the cat gene. This ORFZ mutation resulted in loss of immunity to lactacin F but had little effect on production of LafX, which is not bactericidal without LafA. Expression of ORFZ in this ORFZ- background rescued the immune phenotype. Expression of ORFZ in a bacteriocin-sensitive derivative of VPI 11088 also reestablished immunity. These data indicate that ORFZ, renamed lafI, encodes the immunity factor for the lactacin F system. The sensitivity of various Lactobacillus strains to lactacin F was further evaluated. Lactacin F inhibited 11 strains including several members of the A1, A2, A3, A4, B1, and B2 L. acidophilus homology groups. Expression of lafI in bacteriocin-sensitive strains L. acidophilus ATCC 4356, L. acidophilus NCFM/N2, L. fermentum NCDO1750, L. gasseri ATCC 33323, and L. johnsonii ATCC 33200 provided immunity to lactacin F. Furthermore, it was shown that lactacin F production by VPI 11088 could be used to select for L. fermentum NCDO1750 transformants containing the recombinant plasmid encoding LafI. The data demonstrate that lafI is functional in heterologous hosts, suggesting that it may be a suitable food-grade genetic marker for use in lactobacillus species.

Antibiotic treatment to treat specific infections has the potential to effectively target the offending microbe as well as other microbes that colonize sites within a host. Antibiotic-associated diarrhea (AAD) is a classic example resulting from disruption of host microbial communities; 20% of patients with AAD are likely to become colonized with Clostridium difficile. Restoration of a “normal” microbial community within the host using probiotic bacteria is one approach to circumvent AAD and C. difficile infection. The goals of this study were to assess the interactions between Streptococcus thermophilus, a potential probiotic organism and C. difficile using both in vitro and in vivo systems. Exposure of C. difficile to filtered supernatants from S. thermophilus showed a dose-dependent, bactericidal effect due to lactic acid. Additional studies show that levels of lactic acid (10 mM) that did not inhibit bacterial growth had the potential to decrease tcdA expression and TcdA release into the extracellular milieu. In vivo, treatment with viable S. thermophilus significantly increased luminal levels of lactate in the cecum compared with UV-irradiated S. thermophilus. In the context of infection with C. difficile, mice treated with viable S. thermophilus exhibited 46% less weight loss compared with untreated controls; moreover, less pathology, diarrhea, and lower detectable toxin levels in cecal contents were evident more often in S. thermophillus treated mice. A significant, inverse correlation (Spearman r = -0.942, p = 0.017) between the levels of luminal lactate and abundance of C. difficile were noted suggesting that lactate produced by S. thermophilus is a factor impacting the progression of C. difficile infection in the murine system.

Background The cellular and molecular mechanisms of inflammatory bowel disease are not fully understood; however, data indicate that uncontrolled chronic inflammation induced by bacterial gene products, including lipoteichoic acid (LTA), may trigger colonic inflammation resulting in disease pathogenesis. LTA is a constituent glycolipid of Gram-positive bacteria that shares many inflammatory properties with lipopolysaccharide and plays a critical role in the pathogenesis of severe inflammatory responses via Toll-like receptor 2. Accordingly, we elucidate the role of LTA in immune stimulation and induced colitis in vivo. Methods To better understand the molecular mechanisms utilized by the intestinal microbiota and their gene products to induce or subvert inflammation, specifically the effect(s) of altered surface layer protein expression on the LTA-mediated pro-inflammatory response, the Lactobacillus acidophilus s urface l ayer p rotein (Slp) genes encoding SlpB and SlpX were deleted resulting in a SlpB- and SlpX- mutant that continued to express SlpA (assigned as NCK2031). Results Our data show profound activation of dendritic cells by NCK2031, wild-type L. acidophilus (NCK56), and purified Staphylococcus aureus-LTA. In contrary to the LTA-deficient strain NCK2025, the LTA-expressing strains NCK2031 and NCK56, as well as S. aureus-LTA, induce pro-inflammatory innate and T cell immune responses in vivo. Additionally, neither NCK2031 nor S. aureus-LTA supplemented in drinking water protected mice from DSS-colitis, but instead, induced significant intestinal inflammation resulting in severe colitis and tissue destruction. Conclusions These findings suggest that directed alteration of two of the L. acidophilus NCFM-Slps did not ameliorate LTA-induced pro-inflammatory signals and subsequent colitis.

Aim: To investigate the mechanism(s) by which the intestinal commensal microbe Lactobacillus acidophilus can affect host immunity, we studied the role of a component of the cell wall, lipoteichoic acid, in colitis. Materials & methods: Colitis was induced by the intraperitoneal injection of pathogenic CD4+CD25-CD45RBhi T cells into Rag1-/- mice. The parental strain, NCK56, or the lipoteichoic acid-deficient strain, NCK2025, was then administered orally. Fluorescent microscopy was employed to examine resulting cell populations and their cytokine production in the colon. Results: NCK2025 enhanced IL-10 production by dendritic cells and macrophages. Increased numbers of regulatory dendritic cells coincided with the induction of activated FoxP3+ Tregs. Conclusion: These results suggest that the oral administration of the genetically modified strain NCK2025 may be an effective immunotherapeutic approach that reprograms the immune response in colonic inflammatory conditions.

A quantitative assay performed with bacterial cells labelled with [3H]thymidine was used to investigate factors involved in the adherence of human isolates Lactobacillus acidophilus BG2FO4 and NCFM/N2 and Lactobacillus gasseri ADH to human Caco-2 intestinal cells. For all three strains, adherence was concentration dependent, greater at acidic pH values, and significantly greater than adherence of a control dairy isolate, Lactobacillus delbrueckii subsp. bulgaricus 1489. Adherence of L. acidophilus BG2FO4 and NCFM/N2 was decreased by protease treatment of the bacterial cells, whereas adherence of L. gasseri ADH either was not affected or was enhanced by protease treatment. Putative surface layer proteins were identified on L. acidophilus BG2FO4 and NCFM/N2 cells but were not involved in adherence. Periodate oxidation of bacterial cell surface carbohydrates significantly reduced adherence of L. gasseri ADH, moderately reduced adherence of L. acidophilus BG2FO4, and had no effect on adherence of L. acidophilus NCFM/N2. These results indicate that Lactobacillus species adhere to human intestinal cells via mechanisms which involve different combinations of carbohydrate and protein factors on the bacterial cell surface. The involvement of a secreted bridging protein, which has been proposed as the primary mediator of adherence of L. acidophilus BG2FO4 in spent culture supernatant (M.-H. Coconnier, T. R. Klaenhammer, S. Kernéis, M.-F. Bernet, and A. L. Servin, Appl. Environ. Microbiol. 58:2034-2039, 1992), was not confirmed in this study. Rather, a pH effect on Caco-2 cells contributed significantly to the adherence of this strain in spent culture supernatant.(ABSTRACT TRUNCATED AT 250 WORDS)

SummaryA laboratory scale bioreactor was used for continuous acidification and inoculation of milk with a proteinase-negative, lactose-fermenting strain,Lactococcus lactissubsp.lactisC2S. Calcium alginate-entrapped cells were immobilized on a spiral stainless steel mesh incorporated into a column bioreactor and used to acidify and inoculate reconstituted skim milk. Characteristics of the immobilized cell bioreactor (ICB) were compared with those of a free cell bioreactor (FCB) during challenge with a virulent phage. Steady state biomass and lactate productivities were respectively 25-fold and 12-fold larger with the ICB than with the FCB. The ICB and the FCB were inoculated with the prolate phage c2 at multiplicities of infection of 0·25 and 0·02 respectively. Within 90 min of the infection, the FCB viable cell concentration dropped by five orders of magnitude and never recovered, while the plaque forming units/ml increased dramatically. In the ICB, released cells decreased immediately after infection, but subsequently increased, while the plaque forming units/ml steadily declined, indicating that phage were being washed out of the bioreactor. Productivity of FCB decreased to zero, whereas productivity of the ICB only decreased ∼ 60% and subsequently recovered to its initial steady state value.