SummaryThe use of lactobacilli as probiotics in swine has been gaining attention due to their ability to improve growth performance and carcass quality, prevent gastrointestinal infection and most importantly, their 'generally recognized as safe' status. Previous studies support the potential of lactobacilli to regulate host immune systems, enhance gut metabolic capacities and maintain balance in the gut microbiota. Research on swine gut microbiota has revealed complex gut microbial community structure and showed the importance of Lactobacillus to the host's health. However, the species-and strain-specific characteristics of lactobacilli that confer probiotic benefits are still not well understood. The diversity of probiotic traits in a complex gut ecosystem makes it challenging to infer the relationships between specific functions of Lactobacillus sp. and host health. In this review, we provide an overview of how lactobacilli play a pivotal role in the swine gut ecosystem and identify key characteristics that influence gut microbial community structure and the health of pigs. In addition, based on recent and ongoing meta-omics and omics research on the gut microbiota of pigs, we suggest a workflow combining culture-dependent and cultureindependent approaches for more effective selection of probiotic lactobacilli.

Aims: In this report, we characterized the probiotic potential of Lactobacillus mucosae LM1, focusing on its in vitro mucin-adhesion abilities. Methods and Results: Screening assays were used to evaluate LM1. Previous studies on Lact. mucosae species have been performed, but few have examined the ability of this species to adhere to and colonize the intestinal mucosa. Thus, adhesion, aggregation and pathogen inhibition assays of LM1 along with microbial adhesion to solvents (MATS) assay were carried out in comparison with another putative probiotic, Lactobacillus johnsonii PF01, and the commercial strain, Lactobacillus rhamnosus GG. Based on MATS assay, the cell surfaces of the lactobacilli strains were found to be hydrophobic and highly electron-donating, but the average hydropathy (GRAVY) index of predicted surface-exposed proteins in the LM1 genome indicated that most were hydrophilic. LM1 showed the highest adhesion, aggregation and hydrophobicity among the strains tested and significantly inhibited the adhesion of Escherichia coli K88 and Salmonella enterica serovar Typhimurium KCCM 40253. Correlations among adhesion, aggregation and hydrophobicity, as well as between coaggregation and displacement of E. coli, were observed. Conclusions: Increased adhesion may not always correlate with increased pathogen inhibition due to various strain-specific mechanisms. Nevertheless, LM1 has promising probiotic properties that can be explored further using a genomics approach. Significance and Impact of the Study: Our data on adhesion of LM1 strain showed a significant correlation between adhesion, hydrophobicity of cell surface and autoaggregation. This study gives basic knowledge for the elucidation of the adhesion mechanism of Lactobacillus sp. and prediction of its adherence in specific host models.

Aims: To clone, characterize and compare the bile salt hydrolase (BSH) genes of Lactobacillus johnsonii PF01. Methods and Results: The BSH genes were amplified by polymerase chain reaction (PCR) using specific oligonucleotide primers, and the products were inserted into the pET21b expression vector. Escherichia coli BLR (DE3) cells were transformed with pET21b vectors containing the BSH genes and induced using 0Á1 mmol l À1 isopropylthiolgalactopyranoside. The overexpressed BSH enzymes were purified using a nickel-nitrilotriacetic acid (Ni 2+ -NTA) agarose column and their activities characterized. BSH A hydrolysed tauro-conjugated bile salts optimally at pH 5Á0 and 55°C, whereas BSH C hydrolysed glycoconjugated bile salts optimally at pH 5Á0 and 70°C. The enzymes had no preferential activities towards a specific cholyl moiety. Conclusions: BSH enzymes vary in their substrate specificities and characteristics to broaden its activity. Despite the lack of conservation in their putative substrate-binding sites, these remain functional through motif conservation. Significance and Impact of the Study: This is to our knowledge the first report of isolation of BSH enzymes from a single strain, showing hydrolase activity towards either glyco-conjugated or tauro-conjugated bile salts. Future structural homology studies and site-directed mutagenesis of sites associated with substrate specificity may elucidate specificities of BSH enzymes.

e Lactobacillus mucosae LM1, isolated from stool samples of a healthy piglet, displays good in vitro mucin adhesion and antimicrobial activity against pathogenic bacteria. To elucidate its antimicrobial effects and to find its epithelial cell and mucin adhesion genes, the genomic sequence of L. mucosae LM1 was investigated.L actobacillus mucosae, found in the mammalian gastrointestinal tract, has been shown to have the ability to adhere to mucosal surfaces (2,3,9). Previous reports have identified L. mucosae as having the ability to attach tightly to the epithelium of the human intestine and to produce antimicrobials and a biofilm when exposed to the physiological conditions of the gut (4). The adhesion of lactobacilli to gastrointestinal mucus makes them a good choice for probiotics since it increases their ability to colonize the gut efficiently, modulate the intestinal immune system, and inhibit pathogenic bacteria (1, 4, 9).L. mucosae LM1 was isolated from stool samples from a healthy piglet (6). Preliminary trials regarding the adhesion and antibacterial activity of L. mucosae LM1 demonstrated good mucin-binding activity in vitro and antibacterial activity against pathogenic bacteria. The genome of L. mucosae LM1 was determined using a Roche 454 GS FLX sequencer and Illumina GA IIx platform. All reads were assembled into 55 contigs by de novo assembly. The initial draft assembly was prepared from the libraries of 22,092,187 reads (950ϫ coverage) using Newbler Assembler 2.3 (Roche), CLC Genomics Workbench 4.8 (CLCbio), and CodonCode Aligner (CodonCode Co.). A functional annotation was performed by the Rapid Annotation using Subsystem Technology (RAST) server and BLASTP-based comparisons with the KEGG and COG databases.The draft genome of L. mucosae LM1 included 2,213,697 bp with a 45.87% GϩC content, 2,039 protein-coding genes, and 56 tRNA-encoding genes. Functions were assigned to 64.6% (1,318) of the total coding sequences; 8.7% (428) were found to be hypothetical proteins that are unique to this strain. A phylogenetic tree produced from the 16S rRNA genes revealed that strain LM1 is most closely related to L. mucosae CCUG 43169 (8). Likewise, 16S rRNA analysis showed strong homology to other Lactobacillus species with completed genomes, including Lactobacillus reuteri DSM 20016 (NCBI reference NC_009513.1), with 94% similarity, and Lactobacillus fermentum IF03956 (GenBank reference AP008937.1), with 95% similarity. The 16S rRNA gene sequence was extracted from whole-genome shotgun assemblies derived from the EzTaxon-e database (5).An analysis of the L. mucosae LM1 genome revealed that LM1 has a specific mucus-binding protein (mub) gene (LBLM1_04370), which showed 95% coverage and 93% similarity to the best-matched L. reuteri mub gene. The mucus-binding activity induced by this mub gene has antimicrobial effects through cell surface protection (7,8). Moreover, the L. mucosae LM1 genome includes a putative ABC transporter and adhesin-like protein (LBLM1_10110) with significant homology (100% coverage and 93...

Lactobacillus mucosae is currently of interest as putative probiotics due to their metabolic capabilities and ability to colonize host mucosal niches. L. mucosae LM1 has been studied in its functions in cell adhesion and pathogen inhibition, etc. It demonstrated unique abilities to use energy from carbohydrate and non-carbohydrate sources. Due to these functions, we report the first complete genome sequence of an L. mucosae strain, L. mucosae LM1. Analysis of the pan-genome in comparison with closely-related Lactobacillus species identified a complete glycogen metabolism pathway, as well as folate biosynthesis, complementing previous proteomic data on the LM1 strain. It also revealed common and unique niche-adaptation genes among the various L. mucosae strains. The aim of this study was to derive genomic information that would reveal the probable mechanisms underlying the probiotic effect of L. mucosae LM1, and provide a better understanding of the nature of L. mucosae sp.

Lactobacillus mucosae is a natural resident of the gastrointestinal tract of humans and animals and a potential probiotic bacterium. To understand the global protein expression profile and metabolic features of L. mucosae LM1 in the early stationary phase, the QExactiveTM Hybrid Quadrupole-Orbitrap Mass Spectrometer was used. Characterization of the intracellular proteome identified 842 proteins, accounting for approximately 35% of the 2,404 protein-coding sequences in the complete genome of L. mucosae LM1. Proteome quantification using QExactiveTM Orbitrap MS detected 19 highly abundant proteins (> 1.0% of the intracellular proteome), including CysK (cysteine synthase, 5.41%) and EF-Tu (elongation factor Tu, 4.91%), which are involved in cell survival against environmental stresses. Metabolic pathway annotation of LM1 proteome using the Kyoto Encyclopedia of Genes and Genomes (KEGG) database showed that half of the proteins expressed are important for basic metabolic and biosynthetic processes, and the other half might be structurally important or involved in basic cellular processes. In addition, glycogen biosynthesis was activated in the early stationary phase, which is important for energy storage and maintenance. The proteogenomic data presented in this study provide a suitable reference to understand the protein expression pattern of lactobacilli in standard conditions.

Bacterial lectins are carbohydrate-binding adhesins that recognize glycoreceptors in the gut mucus and epithelium of hosts. In this study, the contribution of lectin-like activities to adhesion of Lactobacillus mucosae LM1 and Lactobacillus johnsonii PF01, which were isolated from swine intestine, were compared to those of the commercial probiotic Lactobacillus rhamnosus GG. Both LM1 and PF01 strains have been reported to have good adhesion ability to crude intestinal mucus of pigs. To confirm this, we quantified their adhesion to porcine gastric mucin and intestinal porcine enterocytes isolated from the jejunum of piglets (IPEC-J2). In addition, we examined their carbohydrate-binding specificities by suspending bacterial cells in carbohydrate solutions prior to adhesion assays. We found that the selected carbohydrates affected the adherences of LM1 to IPEC-J2 cells and of LGG to mucin. In addition, compared to adhesion to IPEC-J2 cells, adhesion to mucin by both LM1 and LGG was characterized by enhanced specific recognition of glycoreceptor components such as galactose, mannose, and N-acetylglucosamine. Hydrophobic interactions might make a greater contribution to adhesion of PF01. A similar adhesin profile between a probiotic and a pathogen, suggest a correlation between shared pathogen-probiotic glycoreceptor recognition and the ability to exclude enteropathogens such as Escherichia coli K88 and Salmonella Typhimurium KCCM 40253. These findings extend our understanding of the mechanisms of the intestinal adhesion and pathogen-inhibition abilities of probiotic Lactobacillus strains.

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