Association between host genetics of sheep and the rumen microbial composition.

A synergy between the rumen microbiota and the host genetics has created a symbiotic relationship, beneficial to the host's health. In this study, the association between the host genetics and rumen microbiome of Damara and Meatmaster sheep was investigated. The composition of rumen microbiota was estimated through the analysis of the V3-V4 region of the 16S rRNA gene, while the sheep blood DNA was genotyped with Illumina OvineSNP50 BeadChip and the genome-wide association (GWA) was analyzed. Sixty significant SNPs dispersed in 21 regions across the Ovis aries genome were found to be associated with the relative abundance of seven genera: Acinetobacter, Bacillus, Clostridium, Flavobacterium, Prevotella, Pseudomonas, and Streptobacillus. A total of eighty-four candidate genes were identified, and their functional annotations were mainly associated with immunity responses and function, metabolism, and signal transduction. Our results propose that those candidate genes identified in the study may be modulating the composition of rumen microbiota and further indicating the significance of comprehending the interactions between the host and rumen microbiota to gain better insight into the health of sheep.

Extracts of Terminalia sericea Enhance Cell Migratory Activity of Endothelial Hybrid and Fibroblast Cells In Vitro.

Terminalia sericea is a plant that has been used amongst others medicinally to treat wounds. The aim of this study was to assess the in vitro wound healing ability of T. sericea. Hot water, methanol, ethyl acetate, and hexane extracts were prepared. Thin layer chromatography (TLC) and ultra-performance liquid chromatography time of flight mass spectrometry (UPLC-TOF-MS) were used to determine the phytochemical classes and genus specific compounds present in the plant. Cytotoxicity was assessed in the SC-1 fibroblast and EA.hy926 endothelial hybrid cell lines using the sulforhodamine B assay. The effect of the extracts on cellular migration in both cell lines was assessed using the scratch assay. The major phytochemical classes detected in the extracts using TLC were alkaloids, coumarins, flavonoids, glycosides, phenolics, saponins, sterols, and terpenoids. The genus-specific compounds punicalagin, sericoside, anolignan B, and arjunic acid were identified in the extracts by means of UPLC-QTOF-MS. Cytotoxicity was not observed after 24 h of exposure and a generally low cytotoxic trend was noted after 72 h. A significant (p < 0.05) enhancement of cell migration in both cell lines was noted in the scratch assay. The wound healing ability of T. sericea is mainly attributed to the migratory and proliferative activity of the extracts responsible for the acceleration of wound closure. Isolation and individualized testing of the active compounds is warranted.

Elucidating the Mechanisms of Cell-to-Cell Crosstalk in Probiotics Co-culture: A Proteomics Study of Limosilactobacillus reuteri ZJ625 and Ligilactobacillus salivarius ZJ614.

Limosilactobacillus reuteri ZJ625 and Ligilactobacillus salivarius ZJ614 are potential probiotic bacteria with improved benefits when administered to the host as a multi-strain preparation. To elucidate the mechanisms of cell-to-cell crosstalk between these two strains, we studied their intracellular and extracellular proteomes in co-culture by liquid-chromatography mass-spectrometry (LC-MS) using Dionex Nano-RSLC and fusion mass spectrometer. The experiment consisted of five biological replicates, and samples were collected during the mid-exponential growth phase. The quantitative proteomic profiles revealed several differentially expressed proteins (DEPs), which are down- or up-regulated between and within groups for both the intracellular and extracellular proteomes. These DEPs include proteins synthesising autoinducer-2, a sensor compound for cell-to-cell bacterial crosstalk during quorum sensing in mixed culture. Other important DEPs identified include enolase, phosphoglycerate kinase, and l-lactate dehydrogenase, which play roles in carbohydrate metabolism. Proteins associated with transcription, ATP production and transport across the membrane, DNA repair, and those with the potential to bind to the host epithelium were also identified. The post-translational modifications associated with the proteins include oxidation, deamidation, and ammonia loss. Importantly, this study revealed a significant expression of S-ribosylhomocysteine lyase (luxS) involved in synthesising autoinducer-2 that plays important roles in quorum sensing, aiding bacterial cell-to-cell crosstalk in co-cultures. The proteome of L. salivarius ZJ614 was most affected when co-cultured with L. reuteri ZJ625. In contrast, omitting some medium components from the defined medium exerted more effects on L. reuteri ZJ625 than L. salivarius ZJ614.

'Multi-omics' data integration: applications in probiotics studies.

The concept of probiotics is witnessing increasing attention due to its benefits in influencing the host microbiome and the modulation of host immunity through the strengthening of the gut barrier and stimulation of antibodies. These benefits, combined with the need for improved nutraceuticals, have resulted in the extensive characterization of probiotics leading to an outburst of data generated using several 'omics' technologies. The recent development in system biology approaches to microbial science is paving the way for integrating data generated from different omics techniques for understanding the flow of molecular information from one 'omics' level to the other with clear information on regulatory features and phenotypes. The limitations and tendencies of a 'single omics' application to ignore the influence of other molecular processes justify the need for 'multi-omics' application in probiotics selections and understanding its action on the host. Different omics techniques, including genomics, transcriptomics, proteomics, metabolomics and lipidomics, used for studying probiotics and their influence on the host and the microbiome are discussed in this review. Furthermore, the rationale for 'multi-omics' and multi-omics data integration platforms supporting probiotics and microbiome analyses was also elucidated. This review showed that multi-omics application is useful in selecting probiotics and understanding their functions on the host microbiome. Hence, recommend a multi-omics approach for holistically understanding probiotics and the microbiome.

Genome Assembly of a Putative Plant Growth-Stimulating Bacterial Sweet Pepper Fruit Isolate, Enterobacter hormaechei SRU4.4.

Here, we report the draft genome sequence of Enterobacter hormaechei SRU4.4. This bacterium (genome size = 4,440,516 bp; coding sequences = 4,100; G+C content = 56%) encodes for genes attributed to plant growth promotion (PGP).

De novo Genome Assessment of Serratia marcescens SGT5.3, a Potential Plant Growth-Promoting Bacterium Isolated from the Surface of Capsicum annuum Fruit.

Serratia marcescens SGT5.3, a potential plant growth-promoting strain with a wide range of functions, was isolated from the surface of Capsicum annuum fruit. Here, we report the whole-genome sequence of this bacterium. Gene prediction revealed various functional genes potentially involved in plant growth promotion and development.

Formulation of Chemically Defined Media and Growth Evaluation of Ligilactobacillus salivarius ZJ614 and Limosilactobacillus reuteri ZJ625.

Lactic acid bacteria are increasingly becoming important dietary supplements due to their health benefits when consumed in adequate quantity. The increasing attention on these important microbes has necessitated an in-depth understanding of their physiological processes, such as nutritional requirements and growth patterns, to better harness their probiotic potentials. This study was carried out to determine the nutritional requirements for the growth of L. salivarius ZJ614 and L. reuteri ZJ625 from a chemically defined medium and evaluate growth kinetics by fitting different sigmoidal growth models. The complete CDM contains 49 nutritional ingredients such as glucose, Tween 80®, mineral salts, buffers, amino acids, vitamins, and nucleotides at defined concentrations. In addition, the minimal nutritional requirements of the isolates were determined in a series of single-omission experiments (SOEs) to compose the MDM. Growth curve data were generated by culturing in an automated 96-well micro-plate reader at 37°C for 36 h, and photometric readings (optical density: OD600) were taken. The data were summarized in tables and charts using Microsoft Excel, while growth evaluation was carried out using open-source software (Curveball) on Python. The results revealed that omission of the amino acids, vitamins, and nucleotides groups resulted in 2.0, 20.17, and 60.24% (for L. salivarius ZJ614) and 0.95, 42.7, and 70.5% (for L. reuteri ZJ625) relative growths, respectively. Elimination of the individual CDM components also indicates varying levels of growth by the strains. The growth curve data revealed LogisticLag2 and Baranyi-Roberts models as the best fits for L. reuteri ZJ625 and L. salivarius ZJ614, respectively. All the strains showed appreciable growth on the CDM and MDM as observed in de Man-Rogosa-Sharpe (MRS) broth. We also described the growth kinetics of L. reuteri ZJ625 and L. salivarius ZJ614 in the CDM, and the best models revealed the estimated growth parameters.

Effects of Lactobacillus rhamnosus and Enterococcus faecalis Supplementation as Direct-Fed Microbials on Rumen Microbiota of Boer and Speckled Goat Breeds.

The effects on rumen microbial communities of direct-fed probiotics, Lactobacillus rhamnosus and Enterococcus faecalis, singly and in combination as feed supplements to both the Boer and Speckled goats were studied using the Illumina Miseq platform targeting the V3-V4 region of the 16S rRNA microbial genes from sampled rumen fluid. Thirty-six goats of both the Boer and Speckled were divided into five experimental groups: (T1) = diet + Lactobacillus rhamnosus; (T2) = diet + Enterococcus faecalis; (T3) = diet + Lactobacillus rhamnosus + Enterococcus faecalis; (T4, positive control) = diet + antibiotic and (T5, negative control) = diet without antibiotics and without probiotics. Our results revealed that Bacteroidetes, Firmicutes, TM7, Proteobacteria, and Euryarchaeota dominate the bacterial communities. In our observations, Lactobacillus rhamnosus and Enterococcus faecalis supplements reduced the archaeal population of Methanomassiliicocca in the T1, T2 and T3 groups, and caused an increase in the T4 group. Chlamydiae were present only in the T5 group, suggesting that probiotic and antibiotic inhibit the growth of pathogens in the rumen. We inferred, based on our results, that Lactobacillus rhamnosus and Enterococcus faecalis favour the survival of beneficial microbial communities in the goats' rumen. This may lead to an overall improved feed efficacy and growth rate.

Epiphytic Bacteria from Sweet Pepper Antagonistic In Vitro to Ralstonia solanacearum BD 261, a Causative Agent of Bacterial Wilt.

Biological control of plant pathogens, particularly using microbial antagonists, is posited as the most effective, environmentally-safe, and sustainable strategy to manage plant diseases. However, the roles of antagonists in controlling bacterial wilt, a disease caused by the most devastating and widely distributed pathogen of sweet peppers (i.e., R. solanacearum), are poorly understood. Here, amplicon sequencing and several microbial function assays were used to depict the identities and the potential antagonistic functions of bacteria isolated from 80 red and green sweet pepper fruit samples, grown under hydroponic and open soil conditions, with some plants, fungicide-treated while others were untreated. Amplicon sequencing revealed the following bacterial strains: Bacillus cereus strain HRT7.7, Enterobacter hormaechei strain SRU4.4, Paenibacillus polymyxa strain SRT9.1, and Serratia marcescens strain SGT5.3, as potential antagonists of R. solanacearum. Optimization studies with different carbon and nitrogen sources revealed that maximum inhibition of the pathogen was produced at 3% (w/v) starch and 2,5% (w/v) tryptone at pH 7 and 30 °C. The mode of action exhibited by the antagonistic isolates includes the production of lytic enzymes (i.e., cellulase and protease enzymes) and siderophores, as well as solubilization of phosphate. Overall, the results demonstrated that the maximum antimicrobial activity of bacterial antagonists could only be achieved under specific environmental conditions (e.g., available carbon and nitrogen sources, pH, and temperature levels), and that bacterial antagonists can also indirectly promote crop growth and development through nutrient cycling and siderophore production.

Distribution of Important Probiotic Genes and Identification of the Biogenic Amines Produced by Lactobacillus acidophilus PNW3.

The genome of Lactobacillus acidophilus PNW3 was assessed for probiotic and safety potentials. The genome was completely sequenced, assembled using SPAdes, and thereafter annotated with NCBI prokaryotic genome annotation pipeline (PGAP) and rapid annotation using subsystem technology (RAST). Further downstream assessment was determined using appropriate bioinformatics tools. The production of biogenic amines was confirmed through HPLC analysis and the evolutionary trend of the strain was determined through the Codon Tree pipeline. The strain was predicted as a non-human pathogen. A plethora of encoding genes for lactic acids and bioactive peptides production, adhesion molecules, resistance to the harsh gut environmental conditions, and improvement of the host metabolism, which are putative for important probiotic functionalities, were located at different loci within the genome. A bacteriocin predicted to be helveticin J was identified as one of the secondary metabolites. The maximum zone of inhibition exhibited by the crude bacteriocin against STEC E. coli O177 was 21.7 ± 0.58 mm and 24.3 ± 1.15 mm after partial purification (250 µg/mL). Three coding sequences were identified for insertion sequences and one for the CRISPR-Cas fragment. The protein-encoding sequence for Ornithine decarboxylase was found within the genome. L. acidophilus PNW3 presents important features categorizing it as a viable and safe probiotic candidate, though further safety investigations are necessary. The application of probiotics in livestock-keeping would ensure improved public health and food security.

Bacterial communities associated with the surface of fresh sweet pepper (Capsicum annuum) and their potential as biocontrol.

Fresh produce vegetables are colonized by different bacterial species, some of which are antagonistic to microbes that cause postharvest losses. However, no comprehensive assessment of the diversity and composition of bacteria inhabiting surfaces of fresh pepper plants grown under different conditions has been conducted. In this study, 16S RNA amplicon sequencing was used to reveal bacterial communities inhabiting the surfaces of red and green pepper (fungicides-treated and non-fungicides-treated) grown under hydroponic and open field conditions. Results revealed that pepper fruit surfaces were dominated by bacterial phylum Proteobacteria, Firmicutes, Actinobacteria, and, Bacteroidetes. The majority of the bacterial operation taxonomic units (97% similarity cut-off) were shared between the two habitats, two treatments, and the two pepper types. Phenotypic predictions (at phylum level) detected a high abundance of potentially pathogenic, biofilm-forming, and stress-tolerant bacteria on samples grown on open soils than those from hydroponic systems. Furthermore, bacterial species of genera mostly classified as fungal antagonists including; Acinetobacter, Agrobacterium, and Burkholderia were the most abundant on the surfaces. These results suggest that peppers accommodate substantially different bacterial communities with antagonistic activities on their surfaces, independent of employed agronomic strategies and that the beneficial bacterial strains maybe more important for peppers established on open fields, which seems to be more vulnerable to abiotic and biotic stresses.

Integrated genome-based probiotic relevance and safety evaluation of Lactobacillus reuteri PNW1.

This study evaluates whole-genome sequence of Lactobacillus reuteri PNW1 and identifies its safety genes that may qualify it as a putative probiotic. It further extracted the bacteriocin produced by the strain and tested its effectiveness against pathogenic STEC E. coli O177. The genomic DNA was sequenced on illuminal Miseq instrument and the sequenced data was assessed for quality reads before assembled with SPAdes. The draft assembly was annotated with Prokaryotic Genome Annotation Pipeline (PGAP) and Rapid Annotations using Subsystems Technology (RAST). Further downstream analyses were carried out using appropriate bioinformatic tools. Production of biogenic amines was biochemically confirmed through HPLC analysis. The assembled genome was 2,430,215 bp long in 420 contigs with 39% G+C content. Among all known genes, putatively responsible for the production of toxic biochemicals, only arginine deiminase (EC3.5.3.6) was spotted. Coding sequences (CDS) putative for D-lactate dehydrogenase (EC1.1.1.28), L-lactate dehydrogenase (EC1.1.1.27) and bacteriocin helveticin J were found within the genome together with plethora of other probiotic important genes. The strain harbours only resistant genes putative for Lincosamide (lnuC) and Tetracycline resistant genes (tetW). There was no hit found for virulence factors and probability of the strain being a human pathogen was zero. Two intact prophage regions were detected within the genome of L. reuteri PNW1 and nine CDS were identified for insertion sequence by OASIS which are belong to seven different families. Five putative CDS were identified for the CRISPR, each associated with Cas genes. Maximum zone of inhibition exhibited by the bacteriocin produced L. reuteri PNW1 is 20.0±1.00 mm (crude) and 23.3±1.15 mm (at 0.25 mg/ml) after being partially purified. With the strain predicted as non-human pathogen, coupled with many other identified desired features, L. reuteri PNW1 stands a chance of making good and safe candidates for probiotic, though further in-vivo investigations are still necessary.

Antibiogram and beta-lactamase genes among cefotaxime resistant E. coli from wastewater treatment plant.

BACKGROUND: The World Health Organization (WHO) recently classified Enterobacteriaceae resistance to third-generation cephalosporin into the group of pathogens with critical criteria for future research. METHODS: A study to assess the antibiogram and beta-lactamase genes among the cefotaxime resistant E. coli (CREc) from a South African wastewater treatment plant (WWTP) was conducted using standard phenotypic and molecular biology characterization methods. RESULTS: Approximate total E. coli (TEc) concentration (log10 CFU/mL) ranged between 5.7 and 6.8 among which cefotaxime resistant E. coli were between 1.8 and 4.8 (log10 CFU/mL) for cefotaxime antibiotic concentration of 4 and 8 mg/L in the influent samples. Effluent samples, heavily influenced by the chlorination had only 0.3 log10 CFU/mL of TEc. Fifty-one cefotaxime resistant isolates were selected out of an overall of 75 isolates, and subjected to a new round of testing, with a follow up of 36 and 48 isolates for both colistin and gentamicin, respectively as guided by initial results. Selected CREc exhibited resistance to amoxicillin-clavulanic acid (35.3%; n = 51), colistin sulphate (76.5%; n = 36), ciprofloxacin (47.1%; n = 51), gentamicin (87.5%; n = 48) and intermediate-resistance to meropenem (11.8%; n = 51). Extended spectrum-beta-lactamase genes detected, viz.: blaCTX-M (52.6%; n = 38) and blaTEM (84.2%; n = 38) and concurrent blaCTX-M + blaTEM (36.8%; n = 38), but no blaSHV was detected. Carbapenem resistance genes, blaKPC-2 (15.8%; n = 38), blaOXA-1 (57.9%; n = 38), blaNDM-1 (15.8%; n = 38) were also detected. Approximately, 10.5 - 36.8% (n = 38) co-occurrence of two or more beta-lactamase genes was detected in some isolates. Out of the selected number (n = 30), 7(23.3%) were enterotoxigenic E. coli (ETEC), 14 (46.7%) were Enteroaggregative E. coli (EAEC), but no enteropathogenic E. coli (EPEC) was detected. CONCLUSION: Resistance to cefotaxime and the presence of a wide range of beta-lactamase genes exposed the potential risks associated with these pathogens via occupational and domestic exposure during the reuse of treated wastewater.

Analysis of bacteriological pollution and the detection of antibiotic resistance genes of prevailing bacteria emanating from pig farm seepage.

Management and disposal of pig farm seepage constitute a serious environmental challenge, and seepage discharge from agricultural waste-water is considered to be one of the greatest contributors of organic substances, bacterial pathogens, and antibiotic resistance genes into the environment. The objectives of this study were to assess the level of bacteriological pollution and to identify the resident antibiotic-resistant genes of culturable bacteria from a studied pig farm seepage. Enumeration of the viable bacterial cell of plated bacteria suspensions (10-1 to 10-8  cfu/mL) was performed; also, identification of pure bacterial colonies was done using an API 20E bacterial identification kit. CLSI guidelines for antimicrobial susceptibility testing were adopted to determine the antibiotic susceptibility/resistance of the cultured bacterial isolates. Identification of resident-resistant genes was done using molecular biology procedures. The results on viable cells in seepage samples ranged from 4.30 × 102 to 1.29 × 109  cfu/mL. Pseudomonas luteola, Enterococcus vulneris, Salmonella choleraesuis spp arizonae, Escherichia coli, Enterobacter cloacae, Proteus mirabillis etc. were isolated from the pig farm soil samples. Almost all of the cultured isolates were resistant to Penicillin G, Vancomycin, Oxytetracycline, Spectinomycin, and Lincomycin. The most frequent resistant genes detected in the isolates were Van A, Van B, InuA, aph (3")-llla, blaTEM, Otr A, and Otr B. It was inferred from the study that Pig farm seepage has the ability to cause bacterial pollution that may negatively impact the natural environment, by introducing bacteria pathogens that harbor antibiotic-resistant genes.