Synergistic antimicrobial activity of alpha-linolenic acid in combination with tetracycline or florfenicol against multidrug-resistant Salmonella typhimurium.

Salmonella is a major foodborne pathogen that can be transmitted from livestock and poultry to humans through the food chain. Due to the widespread use of antibiotics, antibiotic resistance Salmonella has become an important factor threatening food safety. Combining antibiotic and non-antibiotic agents is a promising approach to address the widespread emergence of antibiotic-resistant pathogens. In this study, we investigated the antibiotic resistance profile and molecular characterization of different serotypes of Salmonella isolated from large-scale egg farms using drug susceptibility testing and whole genome sequencing. The synergistic effect of alpha-linolenic acid (ALA) with antibiotics was evaluated using the checkerboard test and time-kill curve. The molecular mechanism of α-linolenic acid synergism was explored using biochemical assays, pull-down assays, and molecular docking. In vivo efficacy of ALA in combination with florfenicol (FFC) or tetracycline (TET) against multidrug-resistant (MDR) Salmonella enterica subsp. enterica serovar typhimurium was also investigated using a mouse model. We found that ALA reduced the minimum inhibitory concentration (MIC) of tetracycline and florfenicol in all strains tested. When ALA (512 mg/L) was combined with florfenicol (32 mg/L) or tetracycline (16 mg/L), we observed disruption of cell membrane integrity, increased outer membrane permeability, lowered cell membrane potential, and inhibition of proton-drive-dependent efflux pumps. The synergistic treatment also inhibited biofilm production and promoted oxidative damage. These changes together led to an increase in bacterial antibiotic susceptibility. The improved efficacy of ALA combination treatment with antibiotics was validated in the mouse model. Molecular docking results indicate that ALA can bind to membrane proteins via hydrogen bonding. Our findings demonstrated that combined treatment using ALA and antibiotics is effective in preventing infections involving MDR bacteria. Our results are of great significance for the scientific and effective prevention and control of antibiotic resistance Salmonella, as well as ensuring food safety.

Virus-like Particles vaccine based on co-expression of G5 Porcine rotavirus VP2-VP6-VP7 induces a powerful immune protective response in mice.

Porcine rotavirus (PoRV), a member of the Reoviridae family, constitutes a principal etiological agent of acute diarrhea in piglets younger than eight weeks of age, and it is associated with considerable morbidity and mortality within the swine industry. The G5 genotype rotavirus strain currently predominates in circulation. To develop a safe and effective porcine rotavirus vaccine, we generated an insect cell-baculovirus expression system, and successfully expressed these three viral proteins and assembled them into virus-like particles (VLPs) co-displaying VP2, VP6, and VP7. Transmission electron microscopy (TEM) analysis revealed that the VP2-VP6-VP7 VLPs exhibited a "wheeled" morphology resembling that of native rotavirus particles, with an estimated diameter of approximately 65 nm. To evaluate the immunogenicity and protective efficacy of these VP2-VP6-VP7 VLPs, we immunized BALB/C mice with four escalating doses of the VLPs, ranging from 5 to 40 µg of VLP protein per dose. ELISA-based assessments of PoRV-specific antibodies and T cell cytokines, including IL-4, IL-2, and IFN-γ, demonstrate that immunization with VP2-VP6-VP7 VLPs can effectively elicit both humoral and cellular immune responses in mice, resulting in a notable induction of neutralizing antibodies. On days 4, 6, 8, and 10 post-infection (dpi), the VLP-vaccinated group exhibited significantly reduced levels of PoRV RNA copy numbers when compared to the PBS controls. Histological examination of the duodenum, ileum, and kidneys revealed that VP2-VP6-VP7 VLPs provided effective protection against PoRV induced intestinal injury. Collectively, these findings indicate that the VLPs generated in this study possess strong immunogenicity and suggest the considerable promise of the VLP-based vaccine candidate in the prevention and containment of Porcine Rotavirus infections.

Host restriction factor Rab11a limits porcine epidemic diarrhea virus invasion of cells via fusion peptide-mediated membrane fusion.

Porcine epidemic diarrhea virus (PEDV) causes enormous economic losses to the pork industry, and its extensive cell tropism poses a substantial challenge to public health and safety. However, the invasion mechanisms and relevant host factors of PEDV remain poorly understood. In this study, we identified 422 differentially expressed genes related to PEDV infection through transcriptome analysis. Among these, Annexin A2 (ANXA2), Prohibitin-2 (PHB2), and Caveolin-2 (CAV2) were identified through screening and verifying as having a specific interaction with the PEDV S protein, and positive regulation of PEDV internalization was validated by siRNA and overexpression tests. Subsequently, using host membrane protein interaction networks and co-immunoprecipitation analysis, we found that ANXA2 PHB2 or CAV2 directly interact with Rab11a. Next, we constructed a pseudovirus model (LV-PEDV S-GFP) to further confirm that the downregulation of Rab11a could promote PEDV invasion. In detail, ANXA2, PHB2, or CAV2 promoted PEDV invasion via downregulating Rab11a. Furthermore, we showed that the S-protein fusion peptide (FP) was sufficient for S-protein interaction with ANXA2, PHB2, CAV2, and Rab11a, and the addition of exogenous GTP could regulate the efficiency of PEDV invasion. Collectively, ANXA2, PHB2, or CAV2 influenced the membrane fusion of PEDV with host cells through the host restriction factor Rab11a. This study could be targeted for future research to develop strategies for the control of PEDV.

Host restriction factor Rab11a limits Porcine deltacoronavirus invasion of cells via fusion peptide-mediated membrane fusion.

Porcine deltacoronavirus (PDCoV) poses a serious threat to pork industry and has the potential for cross-species transmission. Yet, the invasion mechanisms and host factors involved are still unknown. In the present work, using siRNA interference and co-immunoprecipitation, we identified Annexin A2 (ANXA2), Prohibitin-2 (PHB2), or Caveolin-2 (CAV2) as host factors positively regulating the internalization of PDCoV. We further found that Rab11a co-localized with PDCoV S and inhibited PDCoV internalization. Subsequently, a pseudoviral infection model (LV-PDCoV S-GFP) was constructed, and ANXA2 or CAV2 promoted PDCoV invasion by downregulating Rab11a. Our results also indicated that ANXA2, CAV2, and Rab11a interact with the S protein via S-FP, thereby regulating virus-host membrane fusion. Through LV-PDCoV S-GFP infection, we found that Rab11a may act as a host restriction factor, and it could regulate the invasion efficiency of PDCoV by adding of exogenous GTP. These findings revealed that Rab11a was an exciting target to restrict fusion of PDCoV with host cell membranes. AVAILABILITY OF DATA AND MATERIAL: Not applicable.

Isolation and characterization of porcine epidemic diarrhea virus with mutations in the spike gene in China.

Porcine epidemic diarrhea (PED) caused by porcine epidemic diarrhea virus (PEDV) has caused enormous economic losses to the global swine industry. Due to frequent mutations in the spike (S) gene of PEDV, commercial vaccines used today are gradually losing their protective efficacy against variants. It's significant to monitor the S gene of PEDV variants and understand its evolutionary trend. In this study, we report four novel PEDV strains isolated from Sichuan, Guangdong and Shanxi Provinces and determined their S gene sequences. Phylogenetic analysis showed that they all belong to GII genotype. Amino acid alignment revealed a unique mutation pattern. We also predicted their three-dimensional structures and continuous B-cell epitopes and compared them to those of the vaccine strain. Our study provides references for understanding the evolution of S gene and antigenic change of S protein, which are of great significance for formulating the prevention and control of PEDV.

Bacterial membrane vesicles from swine farm microbial communities harboring and safeguarding diverse functional genes promoting horizontal gene transfer.

Antibiotic resistance (AMR) poses a significant global health challenge, with swine farms recognized as major reservoirs of antibiotic resistance genes (ARGs). Recently, bacterial membrane vesicles (BMVs) have emerged as novel carriers mediating horizontal gene transfer. However, little is known about the ARGs carried by BMVs in swine farm environments and their transfer potential. This study investigated the distribution, sources, and microbiological origins of BMVs in three key microbial habitats of swine farms (feces, soil, and fecal wastewater), along with the ARGs and mobile genetic elements (MGEs) they harbor. Characterization of BMVs revealed particle sizes ranging from 20 to 500 nm and concentrations from 108 to 1012 particles/g, containing DNA and proteins. Metagenomic sequencing identified BMVs predominantly composed of members of the Proteobacteria phyla, including Pseudomonadaceae, Moraxellaceae, and Enterobacteriaceae, carrying diverse functional genes encompassing resistance to 14 common antibiotics and 74,340 virulence genes. Notably, multidrug resistance, tetracycline, and chloramphenicol resistance genes were particularly abundant. Furthermore, BMVs harbored various MGEs, primarily plasmids, and demonstrated the ability to protect their DNA cargo from degradation and facilitate horizontal gene transfer, including the transmission of resistance genes. In conclusion, this study reveals widespread presence of BMVs carrying ARGs and potential virulence genes in swine farm feces, soil, and fecal wastewater. These findings not only provide new insights into the role of extracellular DNA in the environment but also highlight concerns regarding the gene transfer potential mediated by BMVs and associated health risks.

The hidden threat: Comprehensive assessment of antibiotic and disinfectant resistance in commercial pig slaughterhouses.

The presence of antibiotic resistance genes (ARGs), disinfectant resistance genes (DRGs), and pathogens in animal food processing environments (FAPE) poses a significant risk to human health. However, knowledge of the contamination and risk profiles of a typical commercial pig slaughterhouse with periodic disinfectant applications is limited. By creating the overall metagenomics-based behavior and risk profiles of ARGs, DRGs, and microbiomes in a nine-section pig slaughterhouse, an important FAPE in China. A total of 454 ARGs and 84 DRGs were detected in the slaughterhouse with resistance genes for aminoglycosides and quaternary ammonium compounds, respectively. The entire slaughtering chain is a hotspot for pathogens, including 83 human pathogenic bacteria (HPB), with 47 core HPB. In addition, 68 high-risk ARGs were significantly correlated with 55 HPB, 30 of which were recognized as potential bacteria co-resistant to antibiotics and disinfectants, confirm a three-fold risk of ARGs, DRGs, and pathogens prevailing throughout the chain. Pre-slaughter pig house (PSPH) was the major risk source for ARGs, DRGs, and HPB. Moreover, 75 Escherichia coli and 47 Proteus mirabilis isolates showed sensitivity to potassium monopersulfate and sodium hypochlorite, suggesting that slaughterhouses should use such related disinfectants. By using whole genome multi-locus sequence typing and single nucleotide polymorphism analyses, genetically closely related bacteria were identified across distinct slaughter sections, suggesting bacterial transmission across the slaughter chain. Overall, this study underscores the critical role of the PSPH section as a major source of HPB, ARGs, and DRGs contamination in commercial pig slaughterhouses. Moreover, it highlights the importance of addressing clonal transmission and cross-contamination of antibiotic- and disinfectant-resistant bacteria within and between slaughter sections. These issues are primarily attributed to the microbial load carried by animals before slaughter, carcass handling, and content exposure during visceral treatment. Our findings provide valuable insights for One Health-oriented slaughterhouse management practices.

Inappropriate use of antibiotic enhances antibiotic resistance dissemination in ESBL-EC: Role of ydcz in outer membrane vesicles biogenesis and protein transport.

Extended-spectrumß-lactam producing Escherichia coli (ESBL-EC) readily colonizes live poultry and serves as a major source of contamination in retail chicken meat, posing significant threats to public health. This study aims to investigate the impact of inappropriate antibiotic use on the dissemination and exacerbation of antibiotic resistance in ESBL-EC and explore the underlying molecular mechanisms. Through experimental analysis, we propose a hypothesis that inappropriate antibiotic use may exacerbate resistance by affecting vesicle formation and protein secretion. Experimental results demonstrate that under the influence of amoxicillin, the concentration of proteins secreted in outer membrane vehicles (OMVs) by ESBL-EC significantly increases, along with a significant upregulation in the expression of the CTX-M-55-type Extended-spectrum beta-lactamase (CTX-M-55). Proteomic analysis and differential gene knockout experiments identified the key protein YdcZ, associated with OMVs formation and protein transportation in ESBL-EC under amoxicillin treatment. Further investigations reveal direct interactions between YdcZ and other proteins (YdiH and BssR). Upon ydcz gene knockout, a significant decrease in protein concentration within OMVs is observed, accompanied by a noticeable reduction in protection against sensitive bacteria. These findings suggest a critical role of YdcZ in regulating the process of protein transportation to OMVs in ESBL-EC under the influence of amoxicillin. In summary, our research uncovers the significant role of inappropriate antibiotic use in promoting the secretion of OMVs by ESBL-EC, aiding the survival of antibiotic-sensitive bacteria in the vicinity of infection sites. These findings provide new insights into the mechanisms underlying antibiotic-induced bacterial resistance dissemination and offer novel avenues for exploring prevention and control strategies against bacterial resistance propagation.

Dietary α-linolenic acid supplementation enhances resistance to Salmonella Typhimurium challenge in chickens by altering the intestinal mucosal barrier integrity and cecal microbes.

Salmonella is an important foodborne pathogen. Given the ban on the use of antibiotics during the egg-laying period in China, finding safe and effective alternatives to antibiotics to reduce Salmonella enterica subsp. enterica serovar Typhimurium (S. Typhimurium) infections in chickens is essential for the prevention and control of this pathogen and the protection of human health. Numerous studies have shown that unsaturated fatty acids have a positive effect on intestinal inflammation and resistance to infection by intestinal pathogens. Here we investigated the protective effect of α-linolenic acid (ALA) against S. Typhimurium infection in chickens and further explored its mechanism of action. We added different proportions of ALA to the feed and observed the effect of ALA on S. Typhimurium colonization using metagenomic sequencing technology and physiological index measurements. The role of gut flora on S. Typhimurium colonization was subsequently verified by fecal microbiota transplantation (FMT). We found that ALA protects chickens from S. Typhimurium infection by reducing intestinal inflammation through remodeling the gut microbiota, up-regulating the expression of ileocecal barrier-related genes, and maintaining the integrity of the intestinal epithelium. Our data suggest that supplementation of feed with ALA may be an effective strategy to alleviate S. Typhimurium infection in chickens.

Establishment of a Simple, Sensitive, and Specific Salmonella Detection Method Based on Recombinase-Aided Amplification Combined with dsDNA-Specific Nucleases.

Salmonella is a common foodborne pathogen that can cause food poisoning, posing a serious threat to human health. Therefore, quickly, sensitively, and accurately detecting Salmonella is crucial to ensuring food safety. For the Salmonella hilA gene, we designed Recombinase-aided amplification (RAA) primers and dsDNA-specific nuclease (DNase) probes. The ideal primer and probe combination was found when conditions were optimized. Under UV light, a visual Salmonella detection technique (RAA-dsDNase) was developed. Additionally, the RAA-dsDNase was modified to further reduce pollution hazards and simplify operations. One-pot RAA-dsDNase-UV or one-pot RAA-dsDNase-LFD was developed as a Salmonella detection method, using UV or a lateral flow dipstick (LFD) for result observation. Among them, one-pot RAA-dsDNase and one-pot RAA-dsDNase-LFD had detection times of 50 min and 60 min, respectively, for detecting Salmonella genomic DNA. One-pot RAA-dsDNase-UV had a detection limit of 101 copies/µL and 101 CFU/mL, while one-pot RAA-dsDNase-LFD had a sensitivity of 102 copies/µL and 102 CFU/mL. One-pot RAA-dsDNase-UV and one-pot RAA-dsDNase-LFD assays may identify 17 specific Salmonella serovars witho ut causing a cross-reaction with the remaining 8 bacteria, which include E. coli. Furthermore, Salmonella in tissue and milk samples has been reliably detected using both approaches. Overall, the detection method developed in this study can quickly, sensitively, and accurately detect Salmonella, and it is expected to become an important detection tool for the prevention and control of Salmonella in the future.