Clostridium perfringens is a well-known pathogen that induces foodborne illness worldwide. In this study, the presumed endolysin gene Lys1472 from C. perfringens 2-2 was cloned and expressed in E. coli. Lys1472 has a catalytic domain homologous to that of GH25_muramidase and two SH3B binding domains. Lys1472 displayed excellent lytic activity against C. perfringens with a lytic rate of 100% and was stable in optimum conditions at temperatures (-20 similar to 50 degrees C), pH ranges (4-10), and NaCl (0-500 mM). All the tested metal ions slightly decreased the lytic activity of Lys1472, which was completely inactivated under divalent conditions of Zn2+ and Fe2+. Lys1472 treatment improved the survival rate of the larvae to 85% at 24 h Lys1472 treatment inhibited C. perfringens growth on chicken breast, yielding a 3-log reduction at 0, 3, 6 and 12 h, at 4 degrees C. Lys1472 treatment inhibited C. perfringens growth on chicken breast, resulting in a 3-log reduction at 0, 3, and 6 h, at 25 degrees C. The results of this study suggest that Lys1472 could serve as an alternative for controlling and treating foodborne C. perfringens contamination.

Tetracyclines are highly susceptible to photolysis, causing pollution in water, land and other ecosystems, as well as posing threats to human health. In this study, we found that only tigecycline and minocycline reduce the antibacterial activity in light, and this inferiority has severely hindered their scope of clinical use. By strategically regulating the photolysis pathway, we identified chalcanthite, which primarily contains Cu2+. By conducting antibiotic susceptibility test and electron microscopy analysis, Cu2+ salts (including copper sulfate, copper chloride, and copper gluconate) were confirmed to restore the antibacterial activity of tigecycline under light exposure against all tested bacterial strains. Spectroscopic characterization combined with quantum chemical calculations elucidated the molecular mechanism by which Cu2+ selectively modulates the photolytic degradation pathway of tigecycline and structural determinants (dimethylamino groups on the D ring). Multi-omics technologies revealed the mechanism underlying the Cu2+-mediated (non-antibacterial) inhibition of bacterial ferric citrate transporter. Animal infection models were developed using diverse drug-resistant pathogens to validate the in vivo therapeutic efficacy of copper gluconate-tigecycline combination therapy under photic conditions. These findings revealed effective adjuvants that facilitate the development of topical tigecycline formulations, and the establishment of a scientific foundation for synthesizing photostable tetracycline-based antibiotics through structural optimization.

Clostridium perfringens (C. perfringens) is an important zoonotic pathogen. The diseases such as necrotic enteritis (NE), enterotoxemia, gas gangrene and food poisoning caused by its infection seriously threaten the lives of both humans and animals. However, under the severe situation of antibiotic resistance, the development of new antibacterial strategies or drugs deserves great attention. In this study, we selected the virulence factor Type IV pili (TFP) of C. perfringens as the target for drug screening. The gliding motility, biofilm formation, cell adhesion and antibacterial activity of the natural compound isoxanthohumol (IXN) against C. perfringens were determined. Transmission electron microscopy (TEM), TFP gene transcription analysis and Western blot were used to detect the expression of PilA pilin. The therapeutic effect of IXN on C. perfringens infection was demonstrated through a mouse gas gangrene model. It was confirmed that IXN inhibits the function of TFP by down-regulating TFP-encoding genes and two-component regulatory genes. In conclusion, our study shows that IXN has the potential to inhibit the function of TFP in C. perfringens and for anti-infection applications.

The prevalence of antimicrobial resistance is increasing among gram-positive bacteria, particularly the resistance of methicillin-resistant Staphylococcus aureus (MRSA) to β-lactam antibiotics. Checkerboard minimum inhibitory concentration (MIC) assays, time-killing assays, enzyme inhibition assays, hemolysis tests, and cytotoxicity analyses were performed to confirm the synergistic effects of tunicamycin (TUN) in combination with β-lactam antibiotics at levels without potential cytotoxicity. TUN at ≤ 1 μg/mL decreased the MICs of cephalosporins or penicillins against MRSA strain USA300, which became sensitive. TUN showed synergistic effects with cefuroxime sodium (FIC index ≤ 0.19) against tested gram-positive bacterial strains of both human clinical and animal origin. Time-killing assays indicated that the combination treatment eliminated MRSA 3–9 h after inoculation. Furthermore, we confirmed the better synergy of specific β-lactam antibiotics at potentially cytotoxicity-free concentrations in combination with TUN against β-lactam-resistant gram-positive microorganisms. This study provides an experimental basis for combination treatment with TUN and β-lactam antibiotics with specific chemical structures for the clinical treatment of gram-positive bacterial infections.

Clostridium perfringens ( C. perfringens ) induces severe clinical manifestations including necrotic enteritis (NE), enterotoxemia, foodborne illness, and gas gangrene, posing substantial threats to public health and livestock industries. Our study focused on the type IV pilus (TFP) of C. perfringens to elucidate the molecular mechanisms underlying natural compound-mediated bacterial inhibition. Firstly, we identified galangin as a candidate through phenotypic screening of TFP-mediated gliding motility and biofilm formation. Subsequently, the in vitro evaluations encompassed bacterial growth kinetics, minimum inhibitory concentration (MIC) determination, cytotoxicity profiling, and cell adhesion assays. Then, the NE-broilers model was established to assess galangin’s therapeutic potential, incorporating qPCR analysis of virS / virR two-component regulatory genes and TFP-encoding genes ( pilA , pilC , pilD , pilT and pilM ), complemented by transmission electron microscopy (TEM) for TFP ultrastructural characterization. Galangin(8 μg/mL) significantly attenuated bacterial motility (29.79%) and biofilm formation (24.85%). It reduced Caco-2 cell adhesion by 45.72% ( p<0.05 ) while maintaining cellular viability. In vivo administration (20 mg/kg galangin) enhanced broiler average weight (galangin group 99.86 g vs 52.68 g Infection group) and improved feed conversion ratio (3.09 vs 5.76). Histopathological analysis demonstrated reduction in ileal lesion, accompanied by 0.78-log CFU/g bacterial load decrease and normalized inflammatory cytokine levels (IL-1β 38.92%, TNF-α 31.53%, IL-6 31.13%). TEM revealed complete TFP structural disintegration, while qPCR confirmed significant downregulation of virR / virS ( virR :1.89-fold, virS :1.34-fold) and TFP genes ( pilA :1.27-fold, pilD :1.43-fold, pilT :2.11-fold, pilM :1.95-fold). This study establishes galangin as a novel TFP-targeted anti-virulence agent against C. perfringens , providing mechanistic insights and therapeutic validation for NE prevention.

Pseudomonas aeruginosa is a common opportunistic pathogen. The potential efficacy of phage therapy has attracted the attention of researchers, but efficient gene-editing tools are lacking, limiting the study of their biological properties. Here, we designed a type V CRISPR-Cas12a system for the gene editing of P. aeruginosa phages. We first evaluated the active cutting function of the CRISPR-Cas12a system in vitro and discovered that it had a higher gene-cutting efficiency than the type II CRISPR-Cas9 system in three different P. aeruginosa phages. We also demonstrated the system's ability to precisely edit genes in Escherichia coli phages, Salmonella phages, and P. aeruginosa phages. Using the aforementioned strategies, non-essential P. aeruginosa phage genes can be efficiently deleted, resulting in a reduction of up to 5,215 bp (7.05%). Our study has provided a rapid, efficient, and time-saving tool that accelerates progress in phage engineering.

Phage therapy is gaining momentum as an alternative to antibiotics in the treatment of salmonellosis caused by Salmonella. In this study, a novel Salmonella phage, vB_SalS_JNS02, was isolated successfully from poultry farms in Shandong, China. The biological characteristics of vB_SalS_JNS02 were analysed, which revealed a short latent period of approximately 10 min and a burst size of 110 PFU/cell. Moreover, vB_SalS_JNS02 exhibited remarkable stability across a wide pH range (pH 3-12) and temperatures ranging from 30 to 80 °C. Genome sequencing analysis provided valuable insights into the genetic composition of vB_SalS_JNS02, which consists of a double-stranded DNA genome that spans 42,450 base pairs and has a G + C content of 49.4%. Of significant importance, the genomic sequence of vB_SalS_JNS02 did not contain any genes related to lysogenicity, virulence, or antibiotic resistance. The phage's efficacy was evaluated in a larval challenge study. Treatment with the phage resulted in increased survival of Galleria mellonella larvae (100%, 70%, and 85%) (MOI 0.1) in the prophylactic treatment, co-infection treatment, and remedial treatment experiments, respectively. Another in vivo experiment investigated the potential application of the phage in broiler chickens and revealed that a single oral dose of vB_SalS_JNS02 (108 PFU/mL, 100 µL/chick) administered 3 h after S. enteritidis oral administration provided effective protection. The introduction of bacteriophage not only enhances the production of secretory immunoglobulin A (sIgA), but also induces alterations in the composition of the gut microbial community. Phage therapy increases the relative abundance of beneficial bacteria, which helps to maintain intestinal barrier homeostasis. However, it is unable to fully restore the disrupted intestinal microbiome caused by S. enteritidis infection. Importantly, no significant adverse effects were observed in the animal subjects following oral administration of the phage, and our findings highlight vB_SalS_JNS02 is a hopeful candidate as a promising tool to target Salmonella infections in poultry.

C. perfringens is a zoonotic pathogen that causes NE, enterotoxemia, food poisoning and gas gangrene in animals and humans and thus seriously endangers public safety and the development of animal husbandry. Overcoming this health risk requires new approaches for antibiotic discovery and the screening of unique bacterial targets. In this work, we identified an active natural compound inhibitor targeting C. perfringens TFP. Based on the TFP-mediated gliding motility phenotype, we screened of numerous natural compounds and identified galangin as a nonantibacterial compound that inhibits C. perfringens cell adhesion and other functions. Galangin inhibits the formation of TFP by reducing the transcription of related genes, such as pilA, pilC, pilT, and pilM, disrupting the pathogenicity of C. perfringens mediated by TFP. The cell adhesion test and broiler model showed that galangin significantly inhibited C. perfringensvirulence in vivo and in vitro and exerted a comprehensive protective effect on infected broilers. Inhibition of TFP function is an effective strategy for the development of drugs targeting C. perfringensinfection. Our evidence proves that galangin can inhibit C. perfringensTFP in vivo and in vitro.

This study aims to study the inhibitory effect of platycladi cacumen granules(PCG)on the function of type Ⅳ pili of C.perfringens and its action mechanism.The gliding motility and biofilm formation assay were firstly determined in vitro.Then,the effect of PCG on the adhesion of C.perfringens to Caco-2 cells were detected.Finally,the mRNA expression levels of TFP related genes were measured by qRT-PCR,and the C.perfringens TFP morphology was evaluated by transmission electron microscopy(TEM).The results showed that PCG markedly reduced the gli-ding motility and biofilm formation at the concentration of 1.3 g/L.Meanwhile,PCG significantly inhibited the adhesion of C.perfringens to Caco-2 cells,and there was no obvious cytotoxicity within the effective concentration range.The results of qRT-PCR and TEM showed that PCG could reduce the transcription level of TFP related genes(virR,virS,pilA,pilD,pilT,pilC and pilM),destroy the TFP morphology,and inhibit the TFP-mediated functions.

The purpose was to screen type III secretory system (T3SS) inhibitors of Salmonella enterica serovar Typhimurium (S. Typhimurium) from natural compounds. The pharmacological activities and action mechanisms of candidate compounds in vivo and in vitro were systematically studied and analyzed. Using a SipA-β-lactamase fusion reporting system, we found that quercitrin significantly blocked the translocation of SipA into eukaryotic host cells without affecting the growth of bacteria. Adhesion and invasion assay showed that quercitrin inhibited S. Typhimurium invasion into host cells and reduced S. Typhimurium mediated host cell damage. β-galactosidase activity detection and Western blot analysis showed that quercitrin significantly inhibited the expression of SPI-1 genes (hilA and sopA) and effectors (SipA and SipC). The results of animal experiments showed that quercitrin significantly reduced colony colonization and alleviated the cecum pathological injury of the infected mice. Small molecule inhibitor quercitrin directly inhibited the function of T3SS and provided a potential antibiotic alternative against S. Typhimurium infection. Importance: T3SS plays a crucial role in the bacterial invasion and pathogenesis of S. Typhimurium. Compared with conventional antibiotics, small molecules could inhibit the virulence factors represented by S. Typhimurium T3SS. They have less pressure on bacterial vitality and a lower probability of producing drug resistance. Our results provide strong evidence for the development of novel inhibitors against S. Typhimurium infection.

Host-directed therapy (HDT) is an emerging novel approach for treating multidrug-resistant Staphylococcus aureus (S. aureus) infection. Functioning as the indispensable specific cellular receptor for α-toxin (Hla), a-disintegrin and metalloproteinase 10 (ADAM10) is exploited to accelerate S. aureus infection through diverse mechanisms. The extraordinary contribution of ADAM10 to S. aureus pathogenesis renders it an attractive HDT target for combating S. aureus infection. Our study is the first to demonstrate the indispensable role of ADAM10 in S. aureus-induced necroptosis, and it enhances our knowledge of the role of ADAM10 in S. aureus infection. Using a fluorogenic substrate assay, we further identified kaempferol as a potent ADAM10 inhibitor that effectively protected mice from S. aureus infection by suppressing Hla-mediated barrier disruption and necroptosis. Collectively, our work presents a novel host-directed therapeutic strategy for using the promising candidate kaempferol to treat S. aureus infection and other diseases relevant to the disordered upregulation of ADAM10.

As a common intracellular facultative anaerobic Gram-positive bacterium, Listeria monocytogenes (L. monocytogenes) exhibits strong resistance to extreme environments, such as low temperature and a wide range of pH values, causing contamination in food production and processing. Sortase A (SrtA) and listeriolysin O (LLO), two crucial virulence factors of L. monocytogenes, are widely recognized as potential targets for the development of anti-L. monocytogenes infection drugs. In this study, we found that genistin simultaneously inhibits the peptidase activity of SrtA and the hemolytic activity of LLO without affecting the growth of L. monocytogenes, alleviating concerns about developing resistance. Furthermore, we demonstrated that genistin reduces L. monocytogenes biofilm formation and invasion of human colorectal cancer (Caco-2) cells. Subsequent mechanistic studies revealed that genistin inhibited LLO-mediated Caco-2 cell damage by blocking LLO oligomerization. Fluorescence quenching assay revealed the potential binding mode of SrtA and LLO to genistin. Genistin might bind to the active pocket of SrtA through residues Leu33, Asn29, and Met40, interacting with D1 domain of LLO involved in oligomerization and pore formation through residues Asn259. Studies in infection models revealed that genistin reduces mortality and pathological damage in mice infected with L. monocytogenes. These results indicate that genistin is a promising anti-virulence agent that could be considered an alternative candidate for the treatment of L. monocytogenes infection.

Listeria monocytogenes is a ubiquitous Gram-positive foodborne pathogen that is responsible for listeriosis in both humans and several animal species. The bacterium secretes a pore-forming cholesterol-dependent cytolysin, listeriolysin O (LLO), a major virulence factor involved in the activation of cellular processes. The ability of LLO to lyse erythrocytes is a measure of LLO activity. We used hemolytic activity assay to screen the LLO inhibitors. Acacetin was found to be an LLO inhibitor, which is a di-hydroxy and mono-methoxy flavone present in various plants, including Black locust, Damiana, and Silver birch. As the features of acacetin are of low toxicity and have less acquired resistance, it comes to a hotspot in drug development. In our study, we report that acacetin antagonized the hemolytic activity of L. monocytogenes culture supernatants and purified LLO by directly interfering with the formation of oligomers without inhibiting the bacterial growth and the expression of LLO. Acacetin also relieved the injury of alveolar epithelial cells by inhibiting LLO activity. Further, acacetin significantly promoted the clearance of L. monocytogenes and alleviated the histopathological damage, thereby raising survival rate, which conferred mice with effective protection against L. monocytogenes infection. Using molecular docking and dynamics simulation, we further proved the mechanism of acacetin antagonizing LLO pore-forming activity by direct binding to the second membrane-inserting helix bundle (HB2) of LLO domain 3. These data suggested that acacetin recedes the virulence of L. monocytogenes both in vivo and in vitro, and this study provided a promising candidate and potential alternative for the prevention and treatment of L. monocytogenes infections.

中国在教育领域一直深化改革蓬勃发展。随着时代的进步及现代化进程的推进，对各个行业高层次人才需求的不断增长，曾经容易被忽略的冷门专业如今成为社会就业前景明朗的热门。笔者结合中国高等教育兽医理学与动物毒理学的教学现状，从教学内容、教学方式、科研成果及对现代化畜牧业的影响入手进行分析，以期为教学改革提供理论参考。

The emergence and spread of the mcr-1 gene and its mutants has immensely compromised the efficient usage of colistin for the treatment of drug-resistant Gram-negative bacterial infection in clinical settings. However, there are currently no clinically available colistin synergis. Here we identify artemisinin derivatives, such as dihydroartemisinin (DHA), that produces a synergistic antibacterial effect with colistin against the majority of Gram-negative bacteria (FIC < 0.5) without induced resistance, particularly those carrying the mcr-1 gene. Mechanism analysis reveals the direct engagement of DHA with the active center of MCR-1 to inhibit the activity of MCR-1. Meanwhile, the results from transcriptome and electron microscope analysis show that DHA could also simultaneously affect the flagellar assembly and the energy metabolism of bacteria. Moreover, in the mouse infection models of Gram-negative bacteria, combination therapy shows remarkable treatment benefits, as shown by an improved survival rate, reduced morbidity, alleviated pathological injury and decreased bacterial loading. Due to the generally safe profile of specialized malaria medication administration in humans, artemisinin derivatives are a promising class of multi-target inhibitors on bacterial resistance and virulence that can be used to extend the usage life of colistin and to tackle the inevitability of serious bacterial infection with colistin.

Background:Listeria monocytogenes (L. monocytogenes), as a pandemic foodborne pathogen, severely threatens food security and public health care worldwide, which evolves multiple bacterial virulence factors (such as listeriolysin O, LLO) for manipulating the immune response of L. monocytogenes-host interactions.Methods:Hemolysis assay was employed to screen a potential LLO inhibitor and the underlying mechanisms were investigated using molecular dynamics (MD) simulation and oligomerization assay. The effects of candidates on immune response were examined by qRT-PCR and immunoblotting analysis. Histological analysis, ELISA assay and biochemistry detection were conducted to assess in vivo efficacy of candidates.Results:In the present study, natural terpenoid atractylodin was characterized as an alternative drug candidate for the treatment of L. monocytogenes by the regulation of LLO function and host Nrf2/NLRP3 signaling pathway. Notably, in vivo infection model by L. monocytogenes also highlighted that atractylodin treatment provided effective therapeutic benefits, as evidenced by decreased bacterial burden and diminished inflammation. Congruently, the survival rate of L. monocytogenes-infection mice increased significantly from 10.0% to 40.0% by atractylodin treatment.Conclusion:Collectively, our study showed for the first time that atractylodin has tremendous potential to attenuate L. monocytogenes pathogenicity by blocking LLO pore formation and mediating the suppression of inflammation and oxidative stress, providing a promising therapeutic strategy and broadening the applications of atractylodin against L. monocytogenes infection.

通过滑动运动和细胞黏附试验,研究羌活油对沙门菌体外致病性的影响。随后,选择6～8周龄雌性BALB/c小鼠灌胃攻菌建立沙门菌感染小鼠模型,并给予一定剂量的羌活油治疗。通过感染小鼠存活率、靶器官（肝脏和脾脏）菌落定植数、盲肠组织病理学和炎性细胞因子（IL-1β和TNF-α）含量等指标评价羌活油对沙门菌感染小鼠的保护作用。体外试验显示,羌活油在质量浓度为0.032 g/L时,显著抑制沙门菌的滑动运动水平,显著降低沙门菌黏附至HeLa细胞,且在有效浓度范围内不影响细菌生长和无明显细胞毒性;100 mg/kg体质量的羌活油显著提高沙门菌感染小鼠的存活率,缓解感染小鼠盲肠组织病理损伤,降低感染小鼠靶器官（肝脏和脾脏）菌落定植数和盲肠组织炎性细胞因子水平。结果表明,羌活油是一种治疗沙门菌感染的潜在药物。

Salmonella enterica serovar Typhimurium (S. Typhimurium) is a zoonotic pathogen that can cause food poisoning and diarrhea in both humans and animals worldwide. The Salmonella pathogenicity island (SPI) genes encoded type III secretion system (T3SS) is important for S. Typhimurium invasion and replication in host cells. Due to the increasing problem of antibiotic resistance, antibiotic treatment for clinical Salmonella infection has gradually been limited. Anti-virulence inhibitors are a promising alternative to antibiotics because they do not easily induce bacterial antibiotic resistance. Here, we systematically evaluated the therapeutic effect of tannic acid (TA) on Salmonella-infected mice and elucidated its anti-infection mechanism. TA treatment improved the survival rate of S. Typhimurium-infected mice and alleviated cecum pathological lesions. In addition, TA inhibited S. Typhimurium invasion to HeLa cells without affecting their growth. Further studies showed that TA could inhibit the expression of sipA and sipB. This inhibition may be implemented by inhibiting the transcription of key regulatory and structural genes of the T3SS. This study provides an alternative anti-virulence strategy for Salmonella infection treatment.

Streptococcus pneumoniae is an important pathogen that causes otitis media, pneumonia, meningitis and bacteremia. As an important virulence factors of S. pneumoniae, pneumolysin (PLY) can penetrate cell membranes and lead to cell lysis and inflammation, which is one of the main causes of infection and damage of S. pneumoniae. Therefore, using pneumolysin as a target to study its inhibitors can provide a new treatment strategy for pneumococcal disease. This study analyzed the inhibitory effect of the natural compound hederagenin on PLY in vitro. The results show that hederagenin has great potential as a new strategy for the treatment of pneumococcal diseases.

New therapeutic strategies for clinical Salmonella enterica serovar Typhimurium (S. Typhimurium) infection are urgently needed due to the generation of antibiotic-resistant bacteria. Inhibition of bacterial virulence has been increasingly regarded as a potential and innovative strategy for the development of anti-infection drugs. Salmonella pathogenicity island (SPI)-encoded type III secretion system (T3SS) represents a key virulence factor in S. Typhimurium, and active invasion and replication in host cells is facilitated by the secretion of T3SS effector proteins. In this study, we found that harmine could inhibit T3SS secretion; thus, its potential anti-S. Typhimurium infection activity was elucidated. Harmine inhibits the secretion and expression of T3SS effector proteins and consequently attenuates the S. Typhimurium invasion function of HeLa cells. This inhibition may be implemented by reducing the transcription of pathogenesis-related SPI-1 transcriptional activator genes hilD, hilC, and rtsA. Harmine improves the survival rate and bacterial loads of mice infected with S. Typhimurium. In summary, harmine, an effective T3SS inhibitor, could be a leading compound for the development of treatments for Salmonella infection.