April 21, 2020  |  

Antibiotic susceptibility of plant-derived lactic acid bacteria conferring health benefits to human.

Lactic acid bacteria (LAB) confer health benefits to human when administered orally. We have recently isolated several species of LAB strains from plant sources, such as fruits, vegetables, flowers, and medicinal plants. Since antibiotics used to treat bacterial infection diseases induce the emergence of drug-resistant bacteria in intestinal microflora, it is important to evaluate the susceptibility of LAB strains to antibiotics to ensure the safety and security of processed foods. The aim of the present study is to determine the minimum inhibitory concentration (MIC) of antibiotics against several plant-derived LAB strains. When aminoglycoside antibiotics, such as streptomycin (SM), kanamycin (KM), and gentamicin (GM), were evaluated using LAB susceptibility test medium (LSM), the MIC was higher than when using Mueller-Hinton (MH) medium. Etest, which is an antibiotic susceptibility assay method consisting of a predefined gradient of antibiotic concentrations on a plastic strip, is used to determine the MIC of antibiotics world-wide. In the present study, we demonstrated that Etest was particularly valuable while testing LAB strains. We also show that the low susceptibility of the plant-derived LAB strains against each antibiotic tested is due to intrinsic resistance and not acquired resistance. This finding is based on the whole-genome sequence information reflecting the horizontal spread of the drug-resistance genes in the LAB strains.

April 21, 2020  |  

Complete genome sequence of Bacillus velezensis JT3-1, a microbial germicide isolated from yak feces

Bacillus velezensis JT3-1 is a probiotic strain isolated from feces of the domestic yak (Bos grunniens) in the Gansu province of China. It has strong antagonistic activity against Listeria monocytogenes, Staphylococcus aureus, Escherichia coli, Salmonella Typhimurium, Mannheimia haemolytica, Staphylococcus hominis, Clostridium perfringens, and Mycoplasma bovis. These properties have made the JT3-1 strain the focus of commercial interest. In this study, we describe the complete genome sequence of JT3-1, with a genome size of 3,929,799 bp, 3761 encoded genes and an average GC content of 46.50%. Whole genome sequencing of Bacillus velezensis JT3-1 will lay a good foundation for elucidation of the mechanisms of its antimicrobial activity, and for its future application.

April 21, 2020  |  

Single-Scaffold Genome Sequence of Probiotic Strain Bifidobacterium breve BR03 (DSM 16604), Obtained by Combining Hybrid Sequencing and Optical Mapping.

Bifidobacterium breve BR03 (DSM 16604) is known for its health-promoting activity. We present a single-scaffold genome obtained by using a hybrid approach combining long- and short-read sequencing techniques integrated by an optical map. This approach could be set as an industry standard for probiotic strain characterization.Copyright © 2019 Fracchetti et al.

April 21, 2020  |  

Complete Genome Sequence of Lactic Acid Bacterium Pediococcus acidilactici Strain ATCC 8042, an Autolytic Anti-bacterial Peptidoglycan Hydrolase Producer

Pediococcus acidilactici is a probiotic bacterium that is industrially utilized in the food industry and antibiotics development. Here, we determine the complete nucleotide sequence of the genome of Pediococcus acidilactici ATCC 8042. The genome was sequenced by the PacBio RSII to generate a single contig consisting of circular chromosome sequence. Illumina MiniSeq sequencing platform and Sanger sequencing method were additionally utilized to correct errors resulting from the long-read sequencing platform. The sequence consists of 2,009,598 bp with a G + C content of 42.1% and contains 1,865 protein-coding sequences. Based on the sequence information, we could confirm and predict the presence of four peptidoglycan hydrolases by HyPe software. This work, therefore, provides the complete genomic information of P. acidilactici ATCC 8042 with a profitable potential of genome-scale comprehension of anti-pathogenic activity, which can be applied in nutraceutical and pharmaceutical biotechnology field.

April 21, 2020  |  

Comparative genomic analysis of Lactobacillus mucosae LM1 identifies potential niche-specific genes and pathways for gastrointestinal adaptation.

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. Copyright © 2017 Elsevier Inc. All rights reserved.

April 21, 2020  |  

Distribution and antimicrobial activity of lactic acid bacteria from raw camel milk.

Consumer demand for natural pathogen-control agents for substitution of synthetic food preservatives and traditional antibiotics is increasing. This study aimed to reveal the distribution of lactic acid bacteria (LAB) in raw camel milk and to characterize their antimicrobial traits. The genetic identification by 16S rRNA sequencing of 58 LAB isolates showed the predominance of Enterococcus (24.2%), Lactococcus (22.4%) and Pediococcus (20.7%) genera in raw camel milk. These genera exhibited inhibitory activity against a broad spectrum of Gram-positive and Gram-negative bacteria including multidrug-resistant Salmonella. Among these LAB, two isolates-identified as Pediococcus pentosaceus CM16 and Lactobacillus brevis CM22-were selected for their strong bacteriocinogenic anti-listerial activity estimated at 1600 and 800 AU/mL, respectively. The bacteriocins produced were partially purified by ammonium sulphate precipitation and gel filtration and then biochemically characterized. The proteinaceous nature of bacteriocins was confirmed by the susceptibility to enzymes. These bacteriocins showed significant technological characteristics such as heat-resistance, and stability over a wide range of pH (2.0-10.0). In conclusion, these results indicated that Pediococcus pentosaceus CM16 and Lactobacillus brevis CM22 could be useful as potential probiotics. Moreover, their partially purified bacteriocins may play an important role as food preservatives and feed additives. To our knowledge, this is the first report describing the distribution of LAB population in raw camel milk and the characterization of their bacteriocins from the Arabian Peninsula of western Asia.

April 21, 2020  |  

A Newly Isolated Bacillus subtilis Strain Named WS-1 Inhibited Diarrhea and Death Caused by Pathogenic Escherichia coli in Newborn Piglets.

Bacillus subtilis is recognized as a safe and reliable human and animal probiotic and is associated with bioactivities such as production of vitamin and immune stimulation. Additionally, it has great potential to be used as an alternative to antimicrobial drugs, which is significant in the context of antibiotic abuse in food animal production. In this study, we isolated one strain of B. subtilis, named WS-1, from apparently healthy pigs growing with sick cohorts on one Escherichia coli endemic commercial pig farm in Guangdong, China. WS-1 can strongly inhibit the growth of pathogenic E. coli in vitro. The B. subtilis strain WS-1 showed typical Bacillus characteristics by endospore staining, biochemical test, enzyme activity analysis, and 16S rRNA sequence analysis. Genomic analysis showed that the B. subtilis strain WS-1 shares 100% genomic synteny with B. subtilis with a size of 4,088,167 bp. Importantly, inoculation of newborn piglets with 1.5 × 1010 CFU of B. subtilis strain WS-1 by oral feeding was able to clearly inhibit diarrhea (p < 0.05) and death (p < 0.05) caused by pathogenic E. coli in piglets. Furthermore, histopathological results showed that the WS-1 strain could protect small intestine from lesions caused by E. coli infection. Collectively, these findings suggest that the probiotic B. subtilis strain WS-1 acts as a potential biocontrol agent protecting pigs from pathogenic E. coli infection. Importance: In this work, one B. subtilis strain (WS-1) was successfully isolated from apparently healthy pigs growing with sick cohorts on one E. coli endemic commercial pig farm in Guangdong, China. The B. subtilis strain WS-1 was identified to inhibit the growth of pathogenic E. coli both in vitro and in vivo, indicating its potential application in protecting newborn piglets from diarrhea caused by E. coli infections. The isolation and characterization will help better understand this bacterium, and the strain WS-1 can be further explored as an alternative to antimicrobial drugs to protect human and animal health.

April 21, 2020  |  

Whole Genome Analysis of Lactobacillus plantarum Strains Isolated From Kimchi and Determination of Probiotic Properties to Treat Mucosal Infections by Candida albicans and Gardnerella vaginalis.

Three Lactobacillus plantarum strains ATG-K2, ATG-K6, and ATG-K8 were isolated from Kimchi, a Korean traditional fermented food, and their probiotic potentials were examined. All three strains were free of antibiotic resistance, hemolysis, and biogenic amine production and therefore assumed to be safe, as supported by whole genome analyses. These strains demonstrated several basic probiotic functions including a wide range of antibacterial activity, bile salt hydrolase activity, hydrogen peroxide production, and heat resistance at 70°C for 60 s. Further studies of antimicrobial activities against Candida albicans and Gardnerella vaginalis revealed growth inhibitory effects from culture supernatants, coaggregation effects, and killing effects of the three probiotic strains, with better efficacy toward C. albicans. In vitro treatment of bacterial lysates of the probiotic strains to the RAW264.7 murine macrophage cell line resulted in innate immunity enhancement via IL-6 and TNF-a production without lipopolysaccharide (LPS) treatment and anti-inflammatory effects via significantly increased production of IL-10 when co-treated with LPS. However, the degree of probiotic effect was different for each strain as the highest TNF-a and the lowest IL-10 production by the RAW264.7 cell were observed in the K8 lysate treated group compared to the K2 and K6 lysate treated groups, which may be related to genomic differences such as chromosome size (K2: 3,034,884 bp, K6: 3,205,672 bp, K8: 3,221,272 bp), plasmid numbers (K2: 3, K6 and K8: 1), or total gene numbers (K2: 3,114, K6: 3,178, K8: 3,186). Although more correlative inspections to connect genomic information and biological functions are needed, genomic analyses of the three strains revealed distinct genomic compositions of each strain. Also, this finding suggests genome level analysis may be required to accurately identify microorganisms. Nevertheless, L. plantarum ATG-K2, ATG-K6, and ATG-K8 demonstrated their potential as probiotics for mucosal health improvement in both microbial and immunological contexts.

October 23, 2019  |  

Gene editing and genetic engineering approaches for advanced probiotics: A review.

The applications of probiotics are significant and thus resulted in need of genome analysis of probiotic strains. Various omics methods and systems biology approaches enables us to understand and optimize the metabolic processes. These techniques have increased the researcher’s attention towards gut microbiome and provided a new source for the revelation of uncharacterized biosynthetic pathways which enables novel metabolic engineering approaches. In recent years, the broad and quantitative analysis of modified strains relies on systems biology tools such as in silico design which are commonly used methods for improving strain performance. The genetic manipulation of probiotic microorganisms is crucial for defining their role in intestinal microbiota and exploring their beneficial properties. This review describes an overview of gene editing and systems biology approaches, highlighting the advent of omics methods which allows the study of new routes for studying probiotic bacteria. We have also summarized gene editing tools like TALEN, ZFNs and CRISPR-Cas that edits or cleave the specific target DNA. Furthermore, in this review an overview of proposed design of advanced customized probiotic is also hypothesized to improvise the probiotics.

September 22, 2019  |  

The effects of probiotics administration on the milk production, milk components and fecal bacteria microbiota of dairy cows

Probiotics administration can improve host health. This study aims to determine the effects of probiotics (Lactobacillus casei Zhang and Lactobacillus plantarum P-8) administration on milk production, milk functional components, milk composition, and fecal microbiota of dairy cows. Variations in the fecal bacteria microbiota between treatments were assessed based on 16S rRNA profiles determined by PacBio single molecule real-time sequencing technology. The probiotics supplementation significantly increased the milk production and the contents of milk immunoglobulin G (IgG), lactoferrin (LTF), lysozyme (LYS) and lactoperoxidase (LP), while the somatic cell counts (SCC) significantly decreased (P < 0.01). However, no significant difference was found in the milk fat, protein and lactose contents (P > 0.05). Although the probiotics supplementation did not change the fecal bacteria richness and diversity, significantly more rumen fermentative bacteria (Bacteroides, Roseburia, Ruminococcus, Clostridium, Coprococcus and Dorea) and beneficial bacteria (Faecalibacterium prausnitzii) were found in the probiotics treatment group. Meanwhile, some opportunistic pathogens e.g. Bacillus cereus, Cronobacter sakazakii and Alkaliphilus oremlandii, were suppressed. Additionally, we found some correlations between the milk production, milk components and fecal bacteria. To sum up, our study demonstrated the beneficial effects of probiotics application in improving the quality and quantity of cow milk production.

September 22, 2019  |  

Assessment of the physicochemical properties and bacterial composition of Lactobacillus plantarum and Enterococcus faecium-fermented Astragalus membranaceus using single molecule, real-time sequencing technology.

We investigated if fermentation with probiotic cultures could improve the production of health-promoting biological compounds in Astragalus membranaceus. We tested the probiotics Enterococcus faecium, Lactobacillus plantarum and Enterococcus faecium?+?Lactobacillus plantarum and applied PacBio single molecule, real-time sequencing technology (SMRT) to evaluate the quality of Astragalus fermentation. We found that the production rates of acetic acid, methylacetic acid, aethyl acetic acid and lactic acid using E. faecium?+?L. plantarum were 1866.24?mg/kg on day 15, 203.80?mg/kg on day 30, 996.04?mg/kg on day 15, and 3081.99?mg/kg on day 20, respectively. Other production rates were: polysaccharides, 9.43%, 8.51%, and 7.59% on day 10; saponins, 19.6912?mg/g, 21.6630?mg/g and 20.2084?mg/g on day 15; and flavonoids, 1.9032?mg/g, 2.0835?mg/g, and 1.7086?mg/g on day 20 using E. faecium, L. plantarum and E. faecium?+?L. plantarum, respectively. SMRT was used to analyze microbial composition, and we found that E. faecium and L. plantarum were the most prevalent species after fermentation for 3 days. E. faecium?+?L. plantarum gave more positive effects than single strains in the Astragalus solid state fermentation process. Our data demonstrated that the SMRT sequencing platform is applicable to quality assessment of Astragalus fermentation.

September 22, 2019  |  

The microbiota of freshwater fish and freshwater niches contain omega-3 producing Shewanella species.

Approximately 30 years ago, it was discovered that free-living bacteria isolated from cold ocean depths could produce polyunsaturated fatty acids (PUFA) such as eicosapentaenoic acid (EPA) (20:5n-3) or docosahexaenoic acid (DHA) (22:6n-3), two PUFA essential for human health. Numerous laboratories have also discovered that EPA- and/or DHA-producing bacteria, many of them members of the Shewanella genus, could be isolated from the intestinal tracts of omega-3 fatty acid-rich marine fish. If bacteria contribute omega-3 fatty acids to the host fish in general or if they assist some bacterial species in adaptation to cold, then cold freshwater fish or habitats should also harbor these producers. Thus, we undertook a study to see if these niches also contained omega-3 fatty acid producers. We were successful in isolating and characterizing unique EPA-producing strains of Shewanella from three strictly freshwater native fish species, i.e., lake whitefish (Coregonus clupeaformis), lean lake trout (Salvelinus namaycush), and walleye (Sander vitreus), and from two other freshwater nonnative fish, i.e., coho salmon (Oncorhynchus kisutch) and seeforellen brown trout (Salmo trutta). We were also able to isolate four unique free-living strains of EPA-producing Shewanella from freshwater habitats. Phylogenetic and phenotypic analyses suggest that one producer is clearly a member of the Shewanella morhuae species and another is sister to members of the marine PUFA-producing Shewanella baltica species. However, the remaining isolates have more ambiguous relationships, sharing a common ancestor with non-PUFA-producing Shewanella putrefaciens isolates rather than marine S. baltica isolates despite having a phenotype more consistent with S. baltica strains. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

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