Probiotic Potential of Bacillus Subtilis Strain I3: Antagonistic Activity Against Chalkbrood Pathogen and Pesticide Degradation for Enhancing Honeybee Health.

To explore the potential of probiotic candidates beneficial for honeybee health through the modulation of the gut microbiome, bee gut microbes were isolated from bumblebee (Bombus terrestris) and honeybee (Apis mellifera) using diverse media and cultural conditions. A total of 77 bee gut bacteria, classified under the phyla Proteobacteria, Firmicutes, and Actinobacteria, were identified. The antagonistic activity of the isolates against Ascosphaera apis, a fungal pathogen responsible for chalkbrood disease in honeybee larvae, was investigated. The highest growth inhibition percentage against A. apis was demonstrated by Bacillus subtilis strain I3 among the bacterial strains. The presence of antimicrobial peptide genes in the I3 strain was detected using PCR amplification of gene fragments encoding surfactin and fengycin utilizing specific primers. The export of antimicrobial peptides by the I3 strain into growth medium was verified using liquid chromatography coupled with mass spectroscopy. Furthermore, the strain's capabilities for degrading pesticides, used for controlling varroa mites, and its spent growth medium antioxidant activity were substantiated. The survival rate of honeybees infected with (A) apis was investigated after feeding larvae with only medium (fructose + glucose + yeast extract + royal jelly), (B) subtilis I3 strain, A. apis with medium and I3 strain + A. apis with medium. Honeybees receiving the I3 strain + A. apis exhibited a 50% reduction in mortality rate due to I3 strain supplementation under experimental conditions, compared to the control group. In silico molecular docking revealed that fengycin hydrolase from I3 strain effectively interacted with tau-fluvalinate, suggesting its potential in bee health and environmental protection. Further studies are needed to confirm the effects of the I3 strain in different populations of honey bees across several regions to account for genetic and environmental variations.

Cotton microbiome profiling and Cotton Leaf Curl Disease (CLCuD) suppression through microbial consortia associated with Gossypium arboreum.

The failure of breeding strategies has caused scientists to shift to other means where the new approach involves exploring the microbiome to modulate plant defense mechanisms against Cotton Leaf Curl Disease (CLCuD). The cotton microbiome of CLCuD-resistant varieties may harbor a multitude of bacterial genera that significantly contribute to disease resistance and provide information on metabolic pathways that differ between the susceptible and resistant varieties. The current study explores the microbiome of CLCuD-susceptible Gossypium hirsutum and CLCuD-resistant Gossypium arboreum using 16 S rRNA gene amplification for the leaf endophyte, leaf epiphyte, rhizosphere, and root endophyte of the two cotton species. This revealed that Pseudomonas inhabited the rhizosphere while Bacillus was predominantly found in the phyllosphere of CLCuV-resistant G. arboreum. Using salicylic acid-producing Serratia spp. and Fictibacillus spp. isolated from CLCuD-resistant G. arboreum, and guided by our analyses, we have successfully suppressed CLCuD in the susceptible G. hirsutum through pot assays. The applied strains exhibited less than 10% CLCuD incidence as compared to control group where it was 40% at 40 days post viral inoculation. Through detailed analytics, we have successfully demonstrated that the applied microbes serve as a biocontrol agent to suppress viral disease in Cotton.

Assessing potential impact of gut microbiome disruptions on the environmental stress resilience of indoor-reared Bombus terrestris.

Bumblebees are crucial for both natural ecosystems and agriculture, but their decline in distribution and abundance over the past decade is alarming. The global importance of bumblebees in natural ecosystems and agricultural food production cannot be overstated. However, the reported decline over the past decade has led to a surge of interest in understanding and addressing bumblebee population decline. Hence, we aimed to detect disruptions in the gut microbiome of male and worker bumblebees reared indoor and outdoor to assess potential resilience to environmental stress. Using the Illumina MiSeq platform for 16s rRNA amplicon sequencing, we analyzed the gut microbiome of male and worker bees that were raised indoors (designated as the IM and IW group) and those that were raised outdoors (also designated as the OM and OW group). Our results show presence of core bacteria Neisseriaceae, Orbaceae, Lactobacillaceae and Bifidobacteriaceae from indoor reared worker bees. However, a higher abundance of Bifidobacterium and absence of Fructobacillus from indoor reared worker bees was also observed. Indoor-reared male bees had lower diversity and fewer observed OTUs compared to outdoor-reared male bees. Additionally, the relative abundance of Actinobacteriota, Bacteroidota, and Firmicutes was significantly lower in indoor-reared males, while Proteobacteria was significantly increased. Despite this, we did not observe any dysbiosis in the gut microbiota of indoor-reared bumblebees when comparing the role of the gut symbionts among the groups. These results suggest that indoor-reared Bombus terrestris may be resilient to environmental stress when used as outdoor pollinators.

Fecal Microbiota Transplants for Inflammatory Bowel Disease Treatment: Synthetic- and Engineered Communities-Based Microbiota Transplants Are the Future.

The human intestine harbors a huge number of diverse microorganisms where a variety of complex interactions take place between the microbes as well as the host and gut microbiota. Significant long-term variations in the gut microbiota (dysbiosis) have been associated with a variety of health conditions including inflammatory bowel disease (IBD). Conventional fecal microbiota transplantations (FMTs) have been utilized to treat IBD and have been proved promising. However, various limitations such as transient results, pathogen transfer, storage, and reproducibility render conventional FMT less safe and less sustainable. Defined synthetic microbial communities (SynCom) have been used to dissect the host-microbiota-associated functions using gnotobiotic animals or in vitro cell models. This review focuses on the potential use of SynCom in IBD and its advantages and relative safety over conventional FMT. Additionally, this review reinforces how various technological advances could be combined with SynCom to have a better understanding of the complex microbial interactions in various gut inflammatory diseases including IBD. Some technological advances including the availability of a gut-on-a-chip system, intestinal organoids, ex vivo intestinal cultures, AI-based refining of the microbiome structural and functional data, and multiomic approaches may help in making more practical in vitro models of the human host. Additionally, an increase in the cultured diversity from gut microbiota and the availability of their genomic information would further make the design and utilization of SynCom more feasible. Taken together, the combined use of the available knowledge of the gut microbiota in health and disease and recent technological advances and the development of defined SynCom seem to be a promising, safe, and sustainable alternative to conventional FMT in treating IBD.

Alteration of Bacterial Wilt Resistance in Tomato Plant by Microbiota Transplant.

Plant-associated microbiota plays an important role in plant disease resistance. Bacterial wilt resistance of tomato is a function of the quantitative trait of tomato plants; however, the mechanism underlying quantitative resistance is unexplored. In this study, we hypothesized that rhizosphere microbiota affects the resistance of tomato plants against soil-borne bacterial wilt caused by Ralstonia solanacearum. This hypothesis was tested using a tomato cultivar grown in a defined soil with various microbiota transplants. The bacterial wilt-resistant Hawaii 7996 tomato cultivar exhibited marked suppression and induction of disease severity after treatment with upland soil-derived and forest soil-derived microbiotas, respectively, whereas the transplants did not affect the disease severity in the susceptible tomato cultivar Moneymaker. The differential resistance of Hawaii 7996 to bacterial wilt was abolished by diluted or heat-killed microbiota transplantation. Microbial community analysis revealed the transplant-specific distinct community structure in the tomato rhizosphere and the significant enrichment of specific microbial operational taxonomic units (OTUs) in the rhizosphere of the upland soil microbiota-treated Hawaii 7996. These results suggest that the specific transplanted microbiota alters the bacterial wilt resistance in the resistant cultivar potentially through a priority effect.

Culturing Simpler and Bacterial Wilt Suppressive Microbial Communities from Tomato Rhizosphere.

Plant phenotype is affected by a community of associated microorganisms which requires dissection of the functional fraction. In this study, we aimed to culture the functionally active fraction of an upland soil microbiome, which can suppress tomato bacterial wilt. The microbiome fraction (MF) from the rhizosphere of Hawaii 7996 treated with an upland soil or forest soil MF was successively cultured in a designed modified M9 (MM9) medium partially mimicking the nutrient composition of tomato root exudates. Bacterial cells were harvested to amplify V3 and V4 regions of 16S rRNA gene for QIIME based sequence analysis and were also treated to Hawaii 7996 prior to Ralstonia solanacearum inoculation. The disease progress indicated that the upland MM9 1st transfer suppressed the bacterial wilt. Community analysis revealed that species richness was declined by successive cultivation of the MF. The upland MM9 1st transfer harbored population of phylum Proteobacteria (98.12%), Bacteriodetes (0.69%), Firmicutes (0.51%), Actinobacteria (0.08%), unidentified (0.54%), Cyanobacteria (0.01%), FBP (0.001%), OD1 (0.001%), Acidobacteria (0.005%). The family Enterobacteriaceae of Proteobacteria was the dominant member (86.76%) of the total population of which genus Enterobacter composed 86.76% making it a potential candidate to suppress bacterial wilt. The results suggest that this mixed culture approach is feasible to harvest microorganisms which may function as biocontrol agents.

Rhizosphere microbiome structure alters to enable wilt resistance in tomato.

Tomato variety Hawaii 7996 is resistant to the soil-borne pathogen Ralstonia solanacearum, whereas the Moneymaker variety is susceptible to the pathogen. To evaluate whether plant-associated microorganisms have a role in disease resistance, we analyzed the rhizosphere microbiomes of both varieties in a mesocosm experiment. Microbiome structures differed between the two cultivars. Transplantation of rhizosphere microbiota from resistant plants suppressed disease symptoms in susceptible plants. Comparative analyses of rhizosphere metagenomes from resistant and susceptible plants enabled the identification and assembly of a flavobacterial genome that was far more abundant in the resistant plant rhizosphere microbiome than in that of the susceptible plant. We cultivated this flavobacterium, named TRM1, and found that it could suppress R. solanacearum-disease development in a susceptible plant in pot experiments. Our findings reveal a role for native microbiota in protecting plants from microbial pathogens, and our approach charts a path toward the development of probiotics to ameliorate plant diseases.

Biochemical and Structural Basis of Triclosan Resistance in a Novel Enoyl-Acyl Carrier Protein Reductase.

Enoyl-acyl carrier protein reductases (ENR), such as FabI, FabL, FabK, and FabV, catalyze the last reduction step in bacterial type II fatty acid biosynthesis. Previously, we reported metagenome-derived ENR homologs resistant to triclosan (TCL) and highly similar to 7-α hydroxysteroid dehydrogenase (7-AHSDH). These homologs are commonly found in Epsilonproteobacteria, a class that contains several human-pathogenic bacteria, including the genera Helicobacter and Campylobacter Here we report the biochemical and predicted structural basis of TCL resistance in a novel 7-AHSDH-like ENR. The purified protein exhibited NADPH-dependent ENR activity but no 7-AHSDH activity, despite its high homology with 7-AHSDH (69% to 96%). Because this ENR was similar to FabL (41%), we propose that this metagenome-derived ENR be referred to as FabL2. Homology modeling, molecular docking, and molecular dynamic simulation analyses revealed the presence of an extrapolated six-amino-acid loop specific to FabL2 ENR, which prevented the entry of TCL into the active site of FabL2 and was likely responsible for TCL resistance. Elimination of this extrapolated loop via site-directed mutagenesis resulted in the complete loss of TCL resistance but not enzyme activity. Phylogenetic analysis suggested that FabL, FabL2, and 7-AHSDH diverged from a common short-chain dehydrogenase reductase family. This study is the first to report the role of the extrapolated loop of FabL2-type ENRs in conferring TCL resistance. Thus, the FabL2 ENR represents a new drug target specific for pathogenic Epsilonproteobacteria.

Specific and Sensitive Primers Developed by Comparative Genomics to Detect Bacterial Pathogens in Grains.

Accurate and rapid detection of bacterial plant pathogen is the first step toward disease management and prevention of pathogen spread. Bacterial plant pathogens Clavibacter michiganensis subsp. nebraskensis (Cmn), Pantoea stewartii subsp. stewartii (Pss), and Rathayibacter tritici (Rt) cause Goss's bacterial wilt and blight of maize, Stewart's wilt of maize and spike blight of wheat and barley, respectively. The bacterial diseases are not globally distributed and not present in Korea. This study adopted comparative genomics approach and aimed to develop specific primer pairs to detect these three bacterial pathogens. Genome comparison among target pathogens and their closely related bacterial species generated 15-20 candidate primer pairs per bacterial pathogen. The primer pairs were assessed by a conventional PCR for specificity against 33 species of Clavibacter, Pantoea, Rathayibacter, Pectobacterium, Curtobacterium. The investigation for specificity and sensitivity of the primer pairs allowed final selection of one or two primer pairs per bacterial pathogens. In our assay condition, a detection limit of Pss and Cmn was 2 pg/µl of genomic DNA per PCR reaction, while the detection limit for Rt primers was higher. The selected primers could also detect bacterial cells up to 8.8 × 103 cfu to 7.84 × 104 cfu per gram of grain seeds artificially infected with corresponding bacterial pathogens. The primer pairs and PCR assay developed in this study provide an accurate and rapid detection method for three bacterial pathogens of grains, which can be used to investigate bacteria contamination in grain seeds and to ultimately prevent pathogen dissemination over countries.

Distribution of triclosan-resistant genes in major pathogenic microorganisms revealed by metagenome and genome-wide analysis.

The substantial use of triclosan (TCS) has been aimed to kill pathogenic bacteria, but TCS resistance seems to be prevalent in microbial species and limited knowledge exists about TCS resistance determinants in a majority of pathogenic bacteria. We aimed to evaluate the distribution of TCS resistance determinants in major pathogenic bacteria (N = 231) and to assess the enrichment of potentially pathogenic genera in TCS contaminated environments. A TCS-resistant gene (TRG) database was constructed and experimentally validated to predict TCS resistance in major pathogenic bacteria. Genome-wide in silico analysis was performed to define the distribution of TCS-resistant determinants in major pathogens. Microbiome analysis of TCS contaminated soil samples was also performed to investigate the abundance of TCS-resistant pathogens. We experimentally confirmed that TCS resistance could be accurately predicted using genome-wide in silico analysis against TRG database. Predicted TCS resistant phenotypes were observed in all of the tested bacterial strains (N = 17), and heterologous expression of selected TCS resistant genes from those strains conferred expected levels of TCS resistance in an alternative host Escherichia coli. Moreover, genome-wide analysis revealed that potential TCS resistance determinants were abundant among the majority of human-associated pathogens (79%) and soil-borne plant pathogenic bacteria (98%). These included a variety of enoyl-acyl carrier protein reductase (ENRs) homologues, AcrB efflux pumps, and ENR substitutions. FabI ENR, which is the only known effective target for TCS, was either co-localized with other TCS resistance determinants or had TCS resistance-associated substitutions. Furthermore, microbiome analysis revealed that pathogenic genera with intrinsic TCS-resistant determinants exist in TCS contaminated environments. We conclude that TCS may not be as effective against the majority of bacterial pathogens as previously presumed. Further, the excessive use of this biocide in natural environments may selectively enrich for not only TCS-resistant bacterial pathogens, but possibly for additional resistance to multiple antibiotics.

Identification of a Gene Involved in the Negative Regulation of Pyomelanin Production in Ralstonia solanacearum.

Ralstonia solanacearum causes bacterial wilt in a wide variety of host plant species and produces a melanin-like blackish-brown pigment in stationary phase when grown in minimal medium supplemented with tyrosine. To study melanin production regulation in R. solanacearum, five mutants exhibiting overproduction of melanin-like pigments were selected from a transposon (Tn) insertion mutant library of R. solanacearum SL341. Most of the mutants, except one (SL341T), were not complemented by the original gene or overproduced melanins. SL341T showed Tn insertion in a gene containing a conserved domain of eukaryotic transcription factor. The gene was annotated as a hypothetical protein, given its weak similarity to any known proteins. Upon complementation with its original gene, the mutant strains reverted to their wild-type phenotype. SL341T produced 3-folds more melanin at 72 h post-incubation compared with wild-type SL341 when grown in minimal medium supplemented with tyrosine. The chemical analysis of SL341T cultural filtrate revealed the accumulation of a higher amount of homogentisate, a major precursor of pyomelanin, and a lower amount of dihydroxyphenylalanine, an intermediate of eumelanin, compared with SL341. The expression study showed a relatively higher expression of hppD (encoding hydroxyphenylpyruvate dioxygenase) and lower expression of hmgA (encoding homogentisate dioxygenase) and nagL (encoding maleylacetoacetate isomerase) in SL341T than in SL341. SL341 showed a significantly higher expression of tyrosinase gene compared with SL341T at 48 h post-incubation. These results indicated that R. solanacearum produced both pyomelanin and eumelanin, and the novel hypothetical protein is involved in the negative regulation of melanin production.