Earthworms can redistribute soil microbiota, and thus might affect the profile of virulence factor genes (VFGs) which are carried by pathogens in soils. Nevertheless, the knowledge of VFG profile in the earthworm guts and its interaction with earthworm gut microbiome is still lacking. Herein, we characterized earthworm gut and soil microbiome and VFG profiles in natural and agricultural ecosystems at a national scale using metagenomics. VFG profiles in the earthworm guts significantly differed from those in the surrounding soils, which was mainly driven by variations of bacterial communities. Furthermore, the total abundance of different types of VFGs in the earthworm guts was about 20-fold lower than that in the soils due to the dramatic decline (also by approximately 20-fold) of VFG-carrying bacterial pathogens in the earthworm guts. Additionally, five VFGs related to nutritional/metabolic factors and stress survival were identified as keystones merely in the microbe-VFG network in the earthworm guts, implying their pivotal roles in facilitating pathogen colonization in earthworm gut microhabitats. These findings suggest the potential roles of earthworms in reducing risks related to the presence of VFGs in soils, providing novel insights into earthworm-based bioremediation of VFG contamination in terrestrial ecosystems.

The previous studies revealed that the type VI secretion system (T6SS) has an essential role in bacterial competition and virulence in many gram-negative bacteria. However, the role of T6SS in virulence in Pectobacterium atrosepticum remains controversial. We examined a closely related strain, PccS1, and discovered that its T6SS comprises a single copy cluster of 17 core genes with a higher identity to homologs from P. atrosepticum. Through extensive phenotypic and functional analyses of over 220 derivatives of PccS1, we found that three of the five VgrGs could be classified into group I VgrGs. These VgrGs interacted with corresponding DUF4123 domain proteins, which were secreted outside of the membrane and were dependent on either T6SS or T4SS. This interaction directly governed virulence and competition. Meanwhile, supernatant proteomic analyses with stains defective in T6SS or/and T4SS confirm that effectors, such as FhaB, were secreted redundantly to control the virulence and suppress host callose-deposition in the course of infection. Notably, this redundant secretion mechanism between T6SS and T4SS is believed to be the first of its kind in bacteria.

Respiratory diseases due to viral or bacterial agents, either alone or in combination, cause substantial economic burdens to the swine industry worldwide. Rapid and reliable detection of causal pathogens is crucial for effective epidemiological surveillance and disease management. This research aimed to employ the multiplex ligation-dependent probe amplification (MLPA) assay for simultaneous detection of seven distinct pathogens causing respiratory problems in swine, porcine reproductive and respiratory syndrome virus (PRRSV), swine influenza virus (SIV), porcine respiratory coronavirus (PRCV), porcine circovirus type 2 (PCV2), Pasteurella multocida, Actinobacillus pleuropneumoniae, and Glässerella parasuis. The results indicated no probe cross-reactivity among the seven target agents with other swine pathogens. The detection limits ranged from 5 to 34 copies per assay for the target organisms. The MLPA assay was evaluated with 88 samples and compared to real-time or multiplex PCR for the target pathogens. The MLPA assay demonstrated high relative test sensitivities (100 %) and reasonable to good relative specificities at 62.5 %, 95.1 %, 86.8 %, and 97.6 % for PRRSV, P. multocida, G. parasuis, and PCV2, respectively, relative to comparator PCR assays. In 71 samples where MLPA and comparator PCR assays matched exactly, infections were detected in 64 samples (90.1 %), with PRRSV being the most commonly found virus and 50.7 % of the samples showing co-infection with two to five of the pathogens. This approach serves as a valuable tool for conducting differential diagnoses and epidemiological investigations of pathogen prevalence within swine populations.

Household-related microbiome is closely related with human health. However, the knowledge about profiles of antibiotic resistance genes (ARGs) and virulence factor genes (VFGs) which are carried by microbes inside homes and their temporal dynamics are rather limited. Here we monitored the seasonal changes of bacterial community (especially pathogenic bacteria), ARGs, and VFGs in household dust samples during two years. Based on metagenomic sequencing, the dust-related bacterial pathogenic community, ARGs, and VFGs all harbored the lowest richness in spring among four seasons. Their structure (except that of VFGs) also exhibited remarkable differences among the seasons. The structural variations of ARGs and VFGs were almost explained by mobile genetic elements (MGEs), bacterial pathogens, and particulate matter-related factors, with MGEs explaining the most. Moreover, the total normalized abundance of ARGs or VFGs showed no significant change across the seasons. Results of metagenomic binning and microbial network both showed that several pathogenic taxa (e.g., Ralstonia pickettii) were strongly linked with numerous ARGs (mainly resistant to multidrug) and VFGs (mainly encoding motility) simultaneously. Overall, these findings underline the significance of MGEs in structuring ARGs and VFGs inside homes along with seasonal variations, suggesting that household dust is a neglected reservoir for ARGs and VFGs.

Worldwide crop yields are threatened by persistent pathogenic bacteria that cause significant damage and jeopardize global food security. Chemical pesticides have shown limited effectiveness in protecting crops from severe yield loss. To address this obstacle, there is a growing need to develop environmentally friendly bactericides with broad-spectrum and sustained protection against persistent crop pathogens. Here, we present a method for preparing a nanocomposite that combines antimicrobial peptides (AMPs) and bimetallic Cu-Ag nanoparticles anchored onto multiwalled carbon nanotubes (MWCNTs). The nanocomposite exhibited dual antibacterial activity by disrupting bacterial cell membranes and splicing nucleic acids. By functionalizing MWCNTs with small AMPs (sAMPs), we achieved enhanced stability and penetration of the nanocomposite, and improved loading capacity of the Cu-Ag nanoparticles. The synthesized MWCNTs&CuNCs@AgNPs@P nanocomposites demonstrated broad-spectrum lethality against both Gram-positive and Gram-negative bacterial pathogens. Glasshouse pot trials confirmed the efficacy of the nanocomposites in protecting rice crops against bacterial leaf blight and tomato crops against bacterial wilt. These findings highlight the excellent antibacterial properties of the MWCNTs&CuNCs@AgNPs@P nanocomposite and its potential to replace chemical pesticides, offering significant advantages for agricultural applications.

BACKGROUND: Traditional methods for detecting insect-borne bacterial pathogens are time-consuming and require specialized laboratory facilities, limiting their applicability in areas without access to such resources. Consequently, rapid and efficient detection methods for insect-borne bacterial diseases have become a pressing need in disease prevention and control.
METHODS: We aligned the ribosomal 16S rRNA sequences of seven bacterial species (Staphylococcus aureus, Shigella flexneri, Aeromonas caviae, Vibrio vulnificus, Salmonella enterica, Proteus vulgaris, and Yersinia enterocolitica) by DNASTAR Lasergene software. Using DNASTAR Lasergene and Primer Premier software, we designed universal primers RLB-F and RLB-R, two species-specific probes for each pathogen, and a universal probe (catch-all). The PCR products of seven standard strains were hybridized with specific oligonucleotide probes fixed on the membrane for specific experimental procedures. To evaluate the sensitivity of PCR-RLB, genomic DNA was serially diluted from an initial copy number of 1010 to 100 copies/μl in distilled water. These dilutions were utilized as templates for the PCR-RLB sensitivity analysis. Simultaneous detection of seven fly-borne bacterial pathogens from field samples by the established PCR-RLB method was conducted on a total of 1060 houseflies, collected from various environments in Lanzhou, China.
RESULTS: The established PCR-RLB assay is capable of detecting bacterial strains of about 103 copies/μl for S. aureus, 103 copies/μl for S. flexneri, 105 copies/μl for A. caviae, 105 copies/μl for V. vulnificus, 100 copies/μl for S. enterica, 105 copies/μl for P. vulgaris, and 100 copies/μl for Y. enterocolitica. The results demonstrate that the detection rate of the established PCR-RLB method is higher (approximately 100 times) compared to conventional PCR. This method was applied to assess the bacterial carrier status of flies in various environments in Lanzhou, China. Among the seven bacterial pathogens carried by flies, S. enterica (34.57%), S. flexneri (32.1%), and Y. enterocolitica (20.37%) were found to be the predominant species.
CONCLUSIONS: Overall, this research shows that the rapid and efficient PCR-RLB detection technology could be a useful for surveillance and therefore effective prevention and control the spread of insect-borne diseases. Meanwhile, the experimental results indicate that urban sanitation and vector transmission sources are important influencing factors for pathogen transmission.

UNASSIGNED: The gastrointestinal tract and oral cavity of animal species harbor complex microbial communities, the composition of which is indicative of the behavior, co-evolution, diet, and immune system of the host.
UNASSIGNED: This study investigated the microbial composition in snakes from varying altitudinal ranges by assessing the fecal and oral bacterial communities in Protobothrops mucrosquamatus, Elaphe dione, and Gloydius angusticeps from Sichuan Province, China, using metagenomic sequencing.
UNASSIGNED: It was revealed that Bacteroidetes, Proteobacteria, Firmicutes, and Fusobacteria were the core microbial phyla in fecal samples across all three species, while Proteobacteria, Bacteroidetes, Actinobacteria, and Firmicutes were the core microbial phyla in oral samples across all three species. Notably, the dominance of Armatimonadetes was documented for the first time in the feces of all three species. Comparative analysis of the microbiomes of the three species indicated distinct microbiological profiles between snakes living at low- and high-altitude regions. Furthermore, 12 to 17 and 22 to 31 bacterial pathogens were detected in the oral and fecal samples, respectively, suggesting that snakes may serve as a novel reservoir for emerging diseases. Overall, this study provides a comparative analysis of the fecal and oral microbiomes in three snake species. Future investigations are anticipated to further elucidate the influence of age, genetics, behavior, diet, environment, ecology, and evolution on the gut and oral microbial communities of snakes.

The discharge from pig farms presents significant challenges to the environment and human health, specifically regarding the dissemination of antimicrobial resistance (AMR). Fermentation bed culture has emerged as an increasingly popular and environmentally friendly pig farming model in China, as it minimizes the release of harmful substances into the environment. However, there remains a limited understanding of the occurrence and dynamics of microbiome and antibiotic resistome in fermentation bed culture. Herein, we collected fermentation bed materials (FBM) from four fermentation bed culture pig farms with varying service ages and investigated their bacterial communities, antibiotic resistance genes (ARGs), mobile genetic elements (MGEs), metal resistance genes (MRGs) and potential antibiotic-resistant bacterial hosts through metagenomics. Pseudomonadota, Actinomycetota, Bacteroidota and Bacillota were identified as the dominant phyla present in the FBM. In total, we detected 258 unique ARGs in the FBM samples, with 79 core ARGs shared by all FBM samples, accounting for 95 % of the total ARG abundance. Our results revealed significant variations in microbial communities and ARG profiles across varying service ages of FBM. Compared to long-term FBW, short-term FBM exhibited higher numbers and abundances of ARGs, MRGs and MGEs, along with higher levels of potential bacterial pathogens and high-risk ARGs. Further analysis of metagenome-assembled genome (MAG) indicated that the putative hosts of ARGs primarily belonged to Pseudomonadota, Actinomycetota and Bacillota. Alarmingly, among the 80 recovered ARG-carrying MAGs, 23 MAGs encoded multi-resistance, including clinically significant species that require urgent attention. Overall, this study provided valuable insights into the temporal patterns of antibiotic resistome and bacterial communities within FBM, enhancing our understanding of FBM in pig farming. The findings could potentially contribute to the development of effective strategies for evaluating and regulating fermentation bed culture practices in pig farming.

Although thaumatin-like proteins (TLPs) are involved in resistance to a variety of fungal diseases, whether the TLP5 and TLP6 genes in tomato plants (Solanum lycopersicum) confer resistance to the pathogenesis of soil-borne diseases has not been demonstrated. In this study, five soil-borne diseases (fungal pathogens: Fusarium solani, Fusarium oxysporum, and Verticillium dahliae; bacterial pathogens: Clavibacter michiganense subsp. michiganense and Ralstonia solanacearum) were used to infect susceptible \"No. 5\" and disease-resistant \"S-55\" tomato cultivars. We found that SlTLP5 and SlTLP6 transcript levels were higher in susceptible cultivars treated with the three fungal pathogens than in those treated with the two bacterial pathogens and that transcript levels varied depending on the pathogen. Moreover, the SlTLP5 and SlTLP6 transcript levels were much higher in disease-resistant cultivars than in disease-susceptible cultivars, and the SlTLP5 and SlTLP6 transcript levels were higher in cultivars treated with the same fungal pathogen than in those treated with bacterial pathogens. SlTLP6 transcript levels were higher than SlTLP5. SlTLP5 and SlTLP6 overexpression and gene-edited transgenic mutants were generated in both susceptible and resistant cultivars. Overexpression and knockout increased and decreased resistance to the five diseases, respectively. Transgenic plants overexpressing SlTLP5 and SlTLP6 inhibited the activities of peroxidase (POD), superoxide dismutase (SOD), ascorbate peroxidase (APX), and catalase (CAT) after inoculation with fungal pathogens, and the activities of POD, SOD, and APX were similar to those of fungi after infection with bacterial pathogens. The activities of CAT were increased, and the activity of β-1,3-glucanase was increased in both the fungal and bacterial treatments. Overexpressed plants were more resistant than the control plants. After SlTLP5 and SlTLP6 knockout plants were inoculated, POD, SOD, and APX had no significant changes, but CAT activity increased and decreased significantly after the fungal and bacterial treatments, contrary to overexpression. The activity of β-1,3-glucanase decreased in the treatment of the five pathogens, and the knocked-out plants were more susceptible to disease than the control. In summary, this study contributes to the further understanding of TLP disease resistance mechanisms in tomato plants.

Microplastics can be colonized by microorganisms and form plastisphere. However, knowledge of bacterial community succession and the enrichment of antibiotic resistance genes (ARGs) and pathogens on microplastics in aquaculture environments is limited. Here, we conducted a 30-day continuous exposure experiment at an oyster farm. Results showed that the alpha-diversity of communities on most microplastics continuously increased and was higher than in planktonic communities after 14 days. Microplastics could selectively enrich certain bacteria from water which can live a sessile lifestyle and promote colonization by other bacteria. The composition and function of plastisphere communities were distinct from those in the surrounding water and influenced by polymer type and exposure time. Microplastics can enrich ARGs (sul1, qnrS and blaTEM) and harbor potential pathogens (e.g., Pseudomonas aeruginosa). Therefore, microplastic pollution may pose a critical threat to aquaculture ecosystems and human health. Our study provides further insight into the ecological risks of microplastics.