Lactobacillus acidophilus NCFM is a probiotic bacterium that has been produced commercially since 1972. The complete genome is 1,993,564 nt and devoid of plasmids. The average GC content is 34.71% with 1,864 predicted ORFs, of which 72.5% were functionally classified. Nine phage-related integrases were predicted, but no complete prophages were found. However, three unique regions designated as potential autonomous units (PAUs) were identified. These units resemble a unique structure and bear characteristics of both plasmids and phages. Analysis of the three PAUs revealed the presence of two R͞M systems and a prophage maintenance system killer protein. A spacers interspersed direct repeat locus containing 32 nearly perfect 29-bp repeats was discovered and may provide a unique molecular signature for this organism. In silico analyses predicted 17 transposase genes and a chromosomal locus for lactacin B, a class II bacteriocin. Several mucus-and fibronectin-binding proteins, implicated in adhesion to human intestinal cells, were also identified. Gene clusters for transport of a diverse group of carbohydrates, including fructooligosaccharides and raffinose, were present and often accompanied by transcriptional regulators of the lacI family. For protein degradation and peptide utilization, the organism encoded 20 putative peptidases, homologs for PrtP and PrtM, and two complete oligopeptide transport systems. Nine two-component regulatory systems were predicted, some associated with determinants implicated in bacteriocin production and acid tolerance. Collectively, these features within the genome sequence of L. acidophilus are likely to contribute to the organisms' gastric survival and promote interactions with the intestinal mucosa and microbiota.adhesion ͉ stress response ͉ proteolytic system ͉ sugar metabolism ͉ in silico analysis

Previous studies on the differences in gut microbiota between exclusively breastfed (EBF) and non-EBF infants have provided highly variable results. Here we perform a meta-analysis of seven microbiome studies (1825 stool samples from 684 infants) to compare the gut microbiota of non-EBF and EBF infants across populations. In the first 6 months of life, gut bacterial diversity, microbiota age, relative abundances of Bacteroidetes and Firmicutes, and predicted microbial pathways related to carbohydrate metabolism are consistently higher in non-EBF than in EBF infants, whereas relative abundances of pathways related to lipid metabolism, vitamin metabolism, and detoxification are lower. Variation in predicted microbial pathways associated with non-EBF infants is larger among infants born by Caesarian section than among those vaginally delivered. Longer duration of exclusive breastfeeding is associated with reduced diarrhea-related gut microbiota dysbiosis. Furthermore, differences in gut microbiota between EBF and non-EBF infants persist after 6 months of age. Our findings elucidate some mechanisms of short and long-term benefits of exclusive breastfeeding across different populations.

In silico genome analysis of Lactobacillus acidophilus NCFM coupled with gene expression studies have identified putative genes and regulatory networks that are potentially important to this organism's survival, persistence, and activities in the gastrointestinal tract. Correlation of key genotypes to phenotypes requires an efficient gene replacement system. In this study, use of the upp-encoded uracil phosphoribosyltransferase (UPRTase) of L. acidophilus NCFM was explored as a counterselection marker to positively select for recombinants that have resolved from chromosomal integration of pORI-based plasmids. An isogenic mutant carrying a upp gene deletion was constructed and was resistant to 5-fluorouracil (5-FU), a toxic uracil analog that is also a substrate for UPRTase. A 3.0-kb pORI-based counterselectable integration vector bearing a upp expression cassette, pTRK935, was constructed and introduced into the ⌬upp host harboring the pTRK669 helper plasmid. Extrachromosomal replication of pTRK935 complemented the mutated chromosomal upp allele and restored sensitivity to 5-FU. This host background provides a platform for a two-step plasmid integration and excision strategy that can select for plasmid-free recombinants with either the wild-type or mutated allele of the targeted gene in the presence of 5-FU. The efficacy of the system was demonstrated by in-frame deletion of the slpX gene (LBA0512) encoding a novel 51-kDa secreted protein associated with the S-layer complex of L. acidophilus. The resulting ⌬slpX mutant exhibited lower growth rates, increased sensitivity to sodium dodecyl sulfate, and greater resistance to bile. Overall, this improved gene replacement system represents a valuable tool for investigating the mechanisms underlying the probiotic functionality of L. acidophilus.Lactobacillus acidophilus NCFM is a commercially established probiotic bacterium that is widely used in dietary supplements and in milk and fermented dairy products (47). Originally a human intestinal isolate from the 1970s (8), this strain has since been examined extensively for various desirable traits. Due to the importance of understanding the molecular mechanisms involved in probiotic functions, the complete genome sequence of L. acidophilus NCFM was determined (2). The NCFM genome sequence serves as a blueprint for in silico identification of candidate gene loci and gene regulatory networks that may play essential roles in the survival and host interactions of this microorganism in the gastrointestinal (GI) tract, including genes involved in acid tolerance (5), bile tolerance (38, 42), adherence factors (20), environmental sensing and response (7), prebiotic sugar utilization (9), polysaccharide biosynthesis, oxalate degradation (6), and bacteriocin production (22). In addition, ongoing microarray gene expression studies have revealed specific gene sets of interest that are being investigated further. The fundamental approach for establishing functional roles for important gene features involves inactivation of the genes ...

The transport and catabolic machinery involved in carbohydrate utilization by Lactobacillus acidophilus was characterized genetically by using whole-genome cDNA microarrays. Global transcriptional profiles were determined for growth on glucose, fructose, sucrose, lactose, galactose, trehalose, raffinose, and fructooligosaccharides. Hybridizations were carried out by using a roundrobin design, and microarray data were analyzed with a two-stage mixed model ANOVA. Differentially expressed genes were visualized by hierarchical clustering, volcano plots, and contour plots. Overall, only 63 genes (3% of the genome) showed a >4-fold induction. Specifically, transporters of the phosphoenolpyruvate: sugar transferase system were identified for uptake of glucose, fructose, sucrose, and trehalose, whereas ATP-binding cassette transporters were identified for uptake of raffinose and fructooligosaccharides. A member of the LacS subfamily of galactosidepentose hexuronide translocators was identified for uptake of galactose and lactose. Saccharolytic enzymes likely involved in the metabolism of monosaccharides, disaccharides, and polysaccharides into substrates of glycolysis were also found, including enzymatic machinery of the Leloir pathway. The transcriptome appeared to be regulated by carbon catabolite repression. Although substrate-specific carbohydrate transporters and hydrolases were regulated at the transcriptional level, genes encoding regulatory proteins CcpA, Hpr, HprK͞P, and EI were consistently highly expressed. Genes central to glycolysis were among the most highly expressed in the genome. Collectively, microarray data revealed that coordinated and regulated transcription of genes involved in sugar uptake and metabolism is based on the specific carbohydrate provided. L. acidophilus's adaptability to environmental conditions likely contributes to its competitive ability for limited carbohydrate sources available in the human gastrointestinal tract.ATP-binding cassette ͉ carbon catabolite repression ͉ fructooligosaccharide ͉ galactoside-pentose hexuronide

BackgroundAdvancements in Next Generation Sequencing (NGS) technologies regarding throughput, read length and accuracy had a major impact on microbiome research by significantly improving 16S rRNA amplicon sequencing. As rapid improvements in sequencing platforms and new data analysis pipelines are introduced, it is essential to evaluate their capabilities in specific applications. The aim of this study was to assess whether the same project-specific biological conclusions regarding microbiome composition could be reached using different sequencing platforms and bioinformatics pipelines.ResultsChicken cecum microbiome was analyzed by 16S rRNA amplicon sequencing using Illumina MiSeq, Ion Torrent PGM, and Roche 454 GS FLX Titanium platforms, with standard and modified protocols for library preparation. We labeled the bioinformatics pipelines included in our analysis QIIME1 and QIIME2 (de novo OTU picking [not to be confused with QIIME version 2 commonly referred to as QIIME2]), QIIME3 and QIIME4 (open reference OTU picking), UPARSE1 and UPARSE2 (each pair differs only in the use of chimera depletion methods), and DADA2 (for Illumina data only). GS FLX+ yielded the longest reads and highest quality scores, while MiSeq generated the largest number of reads after quality filtering. Declines in quality scores were observed starting at bases 150–199 for GS FLX+ and bases 90–99 for MiSeq. Scores were stable for PGM-generated data. Overall microbiome compositional profiles were comparable between platforms; however, average relative abundance of specific taxa varied depending on sequencing platform, library preparation method, and bioinformatics analysis. Specifically, QIIME with de novo OTU picking yielded the highest number of unique species and alpha diversity was reduced with UPARSE and DADA2 compared to QIIME.ConclusionsThe three platforms compared in this study were capable of discriminating samples by treatment, despite differences in diversity and abundance, leading to similar biological conclusions. Our results demonstrate that while there were differences in depth of coverage and phylogenetic diversity, all workflows revealed comparable treatment effects on microbial diversity. To increase reproducibility and reliability and to retain consistency between similar studies, it is important to consider the impact on data quality and relative abundance of taxa when selecting NGS platforms and analysis tools for microbiome studies.Electronic supplementary materialThe online version of this article (10.1186/s12866-017-1101-8) contains supplementary material, which is available to authorized users.

The atypical antipsychotic risperidone (RSP) is often associated with weight gain and cardiometabolic side effects. The mechanisms for these adverse events are poorly understood and, undoubtedly, multifactorial in etiology. In light of growing evidence implicating the gut microbiome in the host's energy regulation and in xenobiotic metabolism, we hypothesized that RSP treatment would be associated with changes in the gut microbiome in children and adolescents. Thus, the impact of chronic (>12 months) and short-term use of RSP on the gut microbiome of pediatric psychiatrically ill male participants was examined in a cross-sectional and prospective (up to 10 months) design, respectively. Chronic treatment with RSP was associated with an increase in body mass index (BMI) and a significantly lower ratio of Bacteroidetes:Firmicutes as compared with antipsychotic-naïve psychiatric controls (ratio=0.15 vs 1.24, respectively; P<0.05). Furthermore, a longitudinal observation, beginning shortly after onset of RSP treatment, revealed a gradual decrease in the Bacteroidetes:Firmicutes ratio over the ensuing months of treatment, in association with BMI gain. Lastly, metagenomic analyses were performed based on extrapolation from 16S ribosomal RNA data using the software package, Phylogenetic Investigation of Communities by Reconstruction of Unobserved States (PICRUSt). Those data indicate that gut microbiota dominating the RSP-treated participants are enriched for pathways that have been implicated in weight gain, such as short-chain fatty acid production.

This study presents the complete genome sequence of Lactobacillus gasseri ATCC 33323, a neotype strain of human origin and a native species found commonly in the gastrointestinal tracts of neonates and adults. The plasmid-free genome was 1,894,360 bp in size and predicted to encode 1,810 genes. The GC content was 35.3%, similar to the GC content of its closest relatives, L. johnsonii NCC 533 (34%) and L. acidophilus NCFM (34%). Two identical copies of the prophage LgaI (40,086 bp), of the Sfi11-like Siphoviridae phage family, were integrated tandomly in the chromosome. A number of unique features were identified in the genome of L. gasseri that were likely acquired by horizontal gene transfer and may contribute to the survival of this bacterium in its ecological niche. L. gasseri encodes two restriction and modification systems, which may limit bacteriophage infection. L. gasseri also encodes an operon for production of heteropolysaccharides of high complexity. A unique alternative sigma factor was present similar to that of B. caccae ATCC 43185, a bacterial species isolated from human feces. In addition, L. gasseri encoded the highest number of putative mucusbinding proteins (14) among lactobacilli sequenced to date. Selected phenotypic characteristics that were compared between ATCC 33323 and other human L. gasseri strains included carbohydrate fermentation patterns, growth and survival in bile, oxalate degradation, and adhesion to intestinal epithelial cells, in vitro. The results from this study indicated high intraspecies variability from a genome encoding traits important for survival and retention in the gastrointestinal tract.

Colorectal cancer (CRC) is the third most common cancer in the world and the second leading cause of cancer deaths in the US and Spain. The molecular mechanisms involved in the etiology of CRC are not yet elucidated due in part to the complexity of the human gut microbiota. In this study, we compared the microbiome composition of 90 tumor and matching adjacent tissue (adjacent) from cohorts from the US and Spain by 16S rRNA amplicon sequencing in order to determine the impact of the geographic origin on the CRC microbiome. Data showed a significantly (P < 0.05) higher Phylogenetic Diversity ( Predicted metagenome functional content from 16S rRNA surveys showed that bacterial motility proteins and proteins involved in flagellar assembly were over represented in adjacent tissues of both cohorts, while pathways involved in fatty acid biosynthesis, the MAPK signaling pathway, and bacterial toxins were over represented in tumors. Our study suggests that microbiome compositional and functional dissimilarities by geographic location should be taken in consideration when approaching CRC therapeutic options.