Exploring the destructive synergy between IL-33 and Suilysin hemolysis on blood-brain barrier stability.

Streptococcus suis type 2 (SS2) is a zoonotic pathogen capable of eliciting meningitis, presenting significant challenges to both the swine industry and public health. Suilysin (Sly), one of SS2 most potent virulence determinants, releases a surfeit of inflammatory agents following red blood cell lysis. Notably, while current research on Sly role in SS2-induced meningitis predominantly centers on its interaction with the blood-brain barrier (BBB), the repercussions of Sly hemolytic products on BBB function have largely been sidestepped. In this vein, our study delves into the ramifications of Sly-induced hemolysis on BBB integrity. We discern that Sly hemolytic derivatives exacerbate the permeability of Sly-induced in vitro BBB models. Within these Sly hemolytic products, Interleukin-33 (IL-33) disrupts the expression and distribution of Claudin-5 in brain microvascular endothelial cells, facilitating the release of Interleukin-6 (IL-6) and Interleukin-8 (IL-8), thereby amplifying BBB permeability. Preliminary mechanistic insights suggest that IL-33-driven expression of IL-6 and IL-8 is orchestrated by the p38-mitogen-activated protein kinase signaling, whereas matrix metalloproteinase 9 mediates IL-33-induced suppression of Claudin-5. To validate these in vitro findings, an SS2-infected mouse model was established, and upon intravenous administration of growth stimulation expressed gene 2 (ST2) antibodies, in vivo results further underscored the pivotal role of the IL-33/ST2 axis during SS2 cerebral invasion. In summation, this study pioneerly illuminates the involvement of Sly hemolytic products in SS2-mediated BBB compromise and spotlights the instrumental role and primary mechanism of IL-33 therein. These insights enrich our comprehension of SS2 meningitis pathogenesis, laying pivotal groundwork for therapeutic advancements against SS2-induced meningitis.IMPORTANCEThe treatment of meningitis caused by Streptococcus suis type 2 (SS2) has always been a clinical challenge. Elucidating the molecular mechanisms by which SS2 breaches the blood-brain barrier (BBB) is crucial for the development of meningitis therapeutics. Suilysin (Sly) is one of the most important virulence factors of SS2, which can quickly lyse red blood cells and release large amounts of damage-associated molecular patterns, such as hemoglobin, IL-33, cyclophilin A, and so on. However, the impact of these hemolytic products on the function of BBB is unknown and ignored. This study is the first to investigate the effect of Sly hemolytic products on BBB function. The data are crucial for the study of the pathogenesis of SS2 meningitis and can provide an important reference for the development of meningitis therapeutics.

Brain Immune Cell Infiltration and Serum Metabolomic Characteristics Reveal that Lauric Acid Promotes Immune Cell Infiltration in Brain and Streptococcus suis Meningitis in Mice.

Although naturally Streptococcus suis serotype 2 (SS2) causes meningitis resulting in death or sequela of neurological symptoms in pigs and humans, severely threatening public health in the world, it has been difficult to build up and confirm experimental meningitis mouse models with obvious neurological syndrome for about two decades, which strongly hampers the in-depth study on the control measures and mechanisms of SS2-induced meningitis. In this study, a typical meningitis mouse model of SS2 was successfully established, as confirmed by the behavioral indicators of balance beam test, suspension test, and gait analysis. With bacteria gathering in the brain, distinguishable unique features including meningeal thickening, vacuolization of the Nissl body, brain barrier damage, glial cell activation, and more infiltration of T cells, macrophages, and DCs are observed in SS2 meningitis mice with typical neurological signs. Some meningitis mice were also accompanied by identical nephritis, ophthalmia, and cochlearitis. Investigation of the metabolic features demonstrated the downregulated cholic acid and upregulated 2-hydroxyvaleric acid, tetrahydrocortisone, nicotinic acid, and lauric acid in blood serum of mice and piglets with meningitis. And feeding trials show that lauric acid can promote meningitis by promoting the infiltration of immune cells into brain. These findings demonstrated that infection of ICR (improved castle road) mice with SS2 was able to induce typical meningitis accompanied by immune cell infiltration and lauric acid upregulation. These data provide a basis for the deep study of SS2 meningitis.

Rapid and facile detection of largemouth bass ranavirus with CRISPR/Cas13a.

Largemouth bass ranavirus (LMBV) is an epidemic disease that seriously jeopardizes the culture of largemouth bass（Micropterus salmoides）, and it has a very high incidence in largemouth bass. Once an outbreak occurs, it may directly lead to the failure of the culture, resulting in substantial economic losses, but there is no effective vaccine or special effective drug yet. Consequently, it is important to establish an accurate, sensitive, convenient and specific detection approach for preventing LMBV infection. The recombinant enzyme-assisted amplification (RAA) technology was used in combination with clustered regularly interspaced short palindromic repeats (CRISPR), and associated protein 13a (CRISPR/Cas13a) to detect LMBV. We designed RAA primers and CRISPR RNA (crRNA) that targeted the conserved region in the LMBV main capsid protein (MCP) gene, amplified sample nucleic acids using the RAA technology, performed CRISPR/Cas13a fluorescence detection and evaluated the sensitivity and specificity of the established method with qPCR as a control method. This technique was able to determine the results by collecting fluorescence signals, visualizing fluorescence by UV excitation and combining with lateral flow strips (LFS). The sensitivity and specificity of the established method were consistent with the qPCR method. Besides, it was performed at a constant temperature of 37 °C and the sensitivity of the reaction system was 3.1 × 101 copies/µL, with no cross-reactivity with other common aquatic pathogens. Further, the positive detection rate of the proposed method in 32 clinical samples was consistent with that of qPCR. In conclusion, our established RAA-CRISPR/Cas13 method for detecting LMBV is sensitive, simple and specific, which is applicable in the rapid on-site detection and epidemiological monitoring of LMBV.

Enolase of Streptococcus suis serotype 2 promotes biomolecular condensation of ribosomal protein SA for HBMECs apoptosis.

BACKGROUND: Ribosomal protein SA (RPSA) of human brain microvascular endothelial cells (HBMECs) can transfer from the cytosol to the cell surface and act as a receptor for some pathogens, including Streptococcus suis serotype 2 (SS2), a zoonotic pathogen causing meningitis in pigs and humans. We previously reported that SS2 virulence factor enolase (ENO) binds to RPSA on the cell surface of HBMECs and induces apoptosis. However, the mechanism that activates RPSA translocation to the cell surface and induces ENO-mediated HBMEC apoptosis is unclear. RESULTS: Here, we show that RPSA localization and condensation on the host cell surface depend on its internally disordered region (IDR). ENO binds to the IDR of RPSA and promotes its interaction with RPSA and vimentin (VIM), which is significantly suppressed after 1,6-Hexanediol (1,6-Hex, a widely used tool to disrupt phase separation) treatment, indicating that ENO incorporation and thus the concentration of RPSA/VIM complexes via co-condensation. Furthermore, increasing intracellular calcium ions (Ca2+) in response to SS2 infection further facilitates the liquid-like condensation of RPSA and aggravates ENO-induced HBMEC cell apoptosis. CONCLUSIONS: Together, our study provides a previously underappreciated molecular mechanism illuminating that ENO-induced RPSA condensation activates the migration of RPSA to the bacterial cell surface and stimulates SS2-infected HBMEC death and, potentially, disease progression. This study offers a fresh avenue for investigation into the mechanism by which other harmful bacteria infect hosts via cell surfaces' RPSA.

Identification and application of an endophytic fungus Arcopilus aureus from Panax notoginseng against crop fungal disease.

Endophytic fungi are important microbial resources for developing novel antibacterial and antifungal drugs to prevent and control crop diseases. Panax notoginseng has been used as a Chinese medicinal herb for a long time, as it has various bioactivities. However, information on endophytic fungi isolated from Panax notoginseng is rare. In this study, an endophytic fungus known as SQGX-6, which was later identified as the golden hair fungus Arcopilus aureus, was isolated from Panax notoginseng. SQGX-6 was extracted using ethyl acetate, and the active components of the fungus were identified using ultra-performance liquid chromatography-mass spectrometry (UHPLC-MS). The antifungal and antioxidant activities of the extract were determined and evaluated in vitro and in vivo. SQGX-6 and its extract inhibited the growth of Corn stalk rot (Fusarium graminearum), Corn southern leaf blight (Helminthosporium maydis), and Tomato gray mold (Botrytis cinerea) in vitro. The free radical scavenging rates for 2,2-Diphenyl-1-pyridinyl hydrazide (DPPH) radical scavenging activity, 3-Ethylbenzothiazoline-6-Sulfonic Acid Radical scavenging (ABTS) activity were also downregulated by the SQGX-6 extract. In vivo, the SQGX-6 extract inhibited the mycelial growth rates of the three aforementioned fungi and downregulated malondialdehyde (MDA) content and upregulated peroxidase (POD) and phenylalanine ammonia-lyase (PAL) content in fruits, leading to significant reduction in damage to cherry tomatoes caused by Botrytis cinerea. UHPLC-MS was performed to identify various active substances, including Alkaloids, Azoles, Benzofurans, Coumarins, Flavonoids, Organic acids, Phenols, and plant growth regulators contained in the extract. These results suggested that the endophytic fungus SQGX-6 of Panax notoginseng and its extract have excellent antifungal and antioxidant activities, and thus, it is an important microbial resource for the developing novel drugs against plant fungal infections.

The pig intestinal phageome is an important reservoir and transfer vector for virulence genes.

Bacteriophages (phages) can significantly influence the composition and functions of their host communities, and enhance host pathogenicity via the transport of phage-encoded virulence genes. Phages are the main component of animal gut viruses, however, there are few reports on the piglet gut phageome and its contribution to virulence genes. Here, a total of 185 virulence genes from 59,955 predicted genes of gut phages in weaned piglets were identified, with 0.688 % of the phage contigs coding for at least one virulence gene. The virulence gene pblA was the most abundant, with various virulence genes significantly correlated with gut phages and their encoded mobile gene element (MGE) genes. Importantly, multiple virulence genes and MGE genes coexist in some phage sequences, and up to 12 virulence genes were detected in a single phage sequence, greatly increasing the risk of phage-mediated transmission of virulence genes into the bacterial genome. In addition, diarrhoea has driven changes in the composition and structure of phage and bacterial communities in the intestinal tract of weaned piglets, significantly increasing the abundance of phage contigs encoding both virulence genes and MGE genes in faecal samples, which potentially increases the risk of phage-mediated virulence genes being transfected into the gut bacterial genome. In summary, this study expands our understanding of the gut microbiome of piglets, advances our understanding of the potential role of phages in driving host pathogenesis in the gut system, and provides new insights into the sources of virulence genes and genetic evolution of bacteria in pig farm environments.

Tracheal epithelial cell-exosome-derived MiR-21-5p inhibits alveolar macrophage pyroptosis to resist pulmonary bacterial infection through PIK3CD-autophagy pathway.

AIMS: Structural cells play an important role in regulating immune cells during infection. Our aim was to determine whether structural porcine tracheal epithelial cells (PTECs) can regulate alveolar macrophages (AMs) to prevent bacterial pneumonia, explore the underlying mechanism(s) and therapeutic target. MATERIALS AND METHODS: Actinobacillus pleuropneumoniae (APP) was used as the model strain for infection studies. Small RNA sequencing was used to identify differentially abundant exosome-derived miRNAs. The role of PTECs exosome-derived miR-21-5p in regulating AMs autophagy, pyroptosis, reactive oxygen species (ROS) was determined using RT-qPCR, western-blotting, flow cytometry, immunohistochemistry. Luciferase reporter assays were conducted to identify potential binding targets of miR-21-5p. The universality of miR-21-5p action on resistance to bacterial pulmonary infection was demonstrated using Klebsiella pneumoniae or Staphylococcus aureus in vitro and in vivo infection models. KEY FINDINGS: MiR-21-5p was enriched in PETCs-derived exosomes, which protected AMs against pulmonary bacterial infection. Mechanistically, miR-21-5p targeted PIK3CD, to promote autophagy of AMs, which reduced the pyroptosis induced by APP infection via inhibiting the over-production of ROS, which in turn suppressed the over-expression of pro-inflammatory cytokines, and increased bacterial clearance. Importantly, the protective effect and mechanism of miR-21-5p were universal as they also occurred upon challenge with Klebsiella pneumoniae and Staphylococcus aureus. SIGNIFICANCE: Our data reveals miR-21-5p can promote pulmonary resistance to bacterial infection by inhibiting pyroptosis of alveolar macrophages through the PIK3CD-autophagy-ROS pathway, suggesting PIK3CD may be a potential therapeutic target for bacterial pneumonia.

O-acetyl-homoserine sulfhydrylase deficient Streptococcus suis serotype 2 strain SC19 becomes an avirulent strain and provides immune protection against homotype infection in mice.

O-acetyl-homoserine sulfhydrylase (OAHS) is a pyridoxal 5'-phosphate-dependent enzyme involved in microbial methionine biosynthesis, which catalyzes the conversion of o-acetyl-homoserine (OAH) to homocysteine. In our previous study, we found that OAHS of Streptococcus suis serotype 2 (SS2) can interact with the porcine blood-brain barrier (BBB) model, but whether OAHS regulates the penetration of BBB during SS2 infection is still unclear. To explore the role of OAHS in SS2 infection, OAHS-deficient SS2 mutant strain (SC19-ΔOAHS) and gene complemental strain (SC19-cΔOAHS) were constructed. Compared to the parent strain, with the loss of oahs, the chain length of SC19-ΔOAHS was shortened, the virulence was significantly reduced, the survival rate of mice infected with SC19-ΔOAHS was obviously increased accompanied by the relieved clinical symptoms. And the survival ability of SC19-ΔOAHS in whole blood was also remarkably decreased. Interestingly, the adhesion of SC19-ΔOAHS to endothelial cells was markedly increased, but the deficiency of OAHS significantly inhibited the strain penetrating BBB both in vivo and in vitro. Most of these phenomena can be reversed by the complemental strain (SC19-cΔOAHS). Further study showed that the deficiency of OAHS severely reduced SC19-induced endothelial cell apoptosis, tight junctions (TJs) protein impairment and the expression of SS2 virulence factor Enolase (Eno), involved in the destruction of BBB. Additionally, SC19-ΔOAHS immunized mice were able to resist SC19 or JZLQ022 infection. In conclusion, we confirmed that OAHS promoted the pathogenicity by enhancing host's BBB permeability and immune escape, and SC19- ΔOAHS is a potential live vaccine.

Rapid detection of grass carp reovirus type 1 using RPA-based test strips combined with CRISPR Cas13a system.

Introduction: Due to the existence of grass carp reovirus (GCRV), grass carp hemorrhagic disease occurs frequently, and its high pathogenicity and infectivity are great challenges to the aquaculture industry. As a highly pathogenic pathogen, the outbreak of hemorrhagic disease often causes tremendous economic losses. Therefore, it is important to rapidly and accurately detect GCRV on site to control timely. Methods: In this study, recombinant enzyme amplification (RPA) combined with clustered regularly interspaced short palindromic repeats (CRISPR)/Cas13a system was employed to establish a method to detect the vp7 gene of grass carp reovirus type 1. This method can be adopted for judging the results by collecting fluorescence signal, ultraviolet excitation visual fluorescence and test strip. Results: Combined with the RPA amplification experiment, the detection limit of the RPA-CRISPR method can reach 7.2 × 101 copies/µL of vp7 gene per reaction, and the detection process can be completed within 1 h. In addition, this method had no cross-reaction with the other 11 common aquatic pathogens. Then, the performance of the RPA-CRISPR/Cas13a detection method was evaluated by comparing it with the real-time fluorescence quantitative PCR detection method of clinical samples. The results of RPA-CRISPR/Cas13a detection were shown to be in consistence with the results obtained from the real-time fluorescence quantitative PCR detection. The coincidence rate of this method with 26 GCRV clinical samples was 92.31%. Discussion: In summary, this method has high sensitivity, specificity and on-site practicability for detecting GCRV type 1, and has great application potential in on-site GCRV monitoring.

High-dimensional analysis reveals an immune atlas and novel neutrophil clusters in the lungs of model animals with Actinobacillus pleuropneumoniae-induced pneumonia.

Due to the increase in bacterial resistance, improving the anti-infectious immunity of the host is rapidly becoming a new strategy for the prevention and treatment of bacterial pneumonia. However, the specific lung immune responses and key immune cell subsets involved in bacterial infection are obscure. Actinobacillus pleuropneumoniae (APP) can cause porcine pleuropneumonia, a highly contagious respiratory disease that has caused severe economic losses in the swine industry. Here, using high-dimensional mass cytometry, the major immune cell repertoire in the lungs of mice with APP infection was profiled. Various phenotypically distinct neutrophil subsets and Ly-6C+ inflammatory monocytes/macrophages accumulated post-infection. Moreover, a linear differentiation trajectory from inactivated to activated to apoptotic neutrophils corresponded with the stages of uninfected, onset, and recovery of APP infection. CD14+ neutrophils, which mainly increased in number during the recovery stage of infection, were revealed to have a stronger ability to produce cytokines, especially IL-10 and IL-21, than their CD14- counterparts. Importantly, MHC-II+ neutrophils with antigen-presenting cell features were identified, and their numbers increased in the lung after APP infection. Similar results were further confirmed in the lungs of piglets infected with APP and Klebsiella pneumoniae infection by using a single-cell RNA-seq technique. Additionally, a correlation analysis between cluster composition and the infection process yielded a dynamic and temporally associated immune landscape where key immune clusters, including previously unrecognized ones, marked various stages of infection. Thus, these results reveal the characteristics of key neutrophil clusters and provide a detailed understanding of the immune response to bacterial pneumonia.

Potential of phage EF-N13 as an alternative treatment strategy for mastitis infections caused by multidrug-resistant Enterococcus faecalis.

Bovine mastitis is the most common and costly disease affecting dairy cattle throughout the world. Enterococcus faecalis is one of the environmental origin mastitis-causing pathogens. The treatment of bovine mastitis is primarily based on antibiotics. Due to the negative impact of developing antibiotic resistance and adverse effects on soil and water environments, the trend toward use of nonantibiotic treatments is increasing. Phages may represent a promising alternative treatment strategy. However, it is unknown whether phages have therapeutic effects on E. faecalis-induced mastitis. Thus, the objective of this study was to investigate the degree of protection conferred by a phage during murine mastitis caused by multidrug-resistant E. faecalis. Enterococcus faecalis was isolated from the milk of dairy cows with mastitis, and a phage was isolated using the E. faecalis isolates as hosts. The bactericidal ability of the phage against E. faecalis and the ability to prevent biofilm formation were determined in vitro. The therapeutic potential of the phage on murine mastitis was evaluated in vivo. We isolated 14 strains of E. faecalis from the milk of cows with mastitis, all of which exhibited multidrug resistance, and most (10/14) could form strong biofilms. Subsequently, a new phage (EF-N13) was isolated using the multidrug-resistant E. faecalis N13 (isolated from mastitic milk) as the host. The phage EF-N13 belongs to the family Myoviridae, which has short latent periods (5 min) and high bursts (284 pfu/cell). The genome of EF-N13 lacked bacterial virulence-, antibiotic resistance-, and lysogenesis-related genes. Furthermore, bacterial loading in the raw milk medium was significantly reduced by EF-N13 and was unaffected by potential IgG antibodies. In fact, EF-N13 could effectively prevent the formation of biofilm by multidrug-resistant E. faecalis. All of these characteristics suggest that EF-N13 has potential as mastitis therapy. In vivo, 1 × 105 cfu/gland of multidrug-resistant E. faecalis N13 resulted in mastitis development within 24 h. A single dose of phage EF-N13 (1 × 104, 1 × 105, or 1 × 106 pfu/gland) could significantly decrease bacterial counts in the mammary gland at 24 h postinfection. Histopathological observations demonstrated that treatment with phage EF-N13 effectively alleviated mammary gland inflammation and damage. This effect was confirmed by the lower levels of proinflammatory cytokines IL-6, IL-1ß, and tumor necrosis factor-α in the mammary gland treated with phage EF-N13 compared with those treated with phosphate-buffered saline. Overall, the data underscored the potential of phage EF-N13 as an alternative therapy for bovine mastitis caused by multidrug-resistant E. faecalis.

Adh Promotes Actinobacillus pleuropneumoniae Survival in Porcine Alveolar Macrophages by Inhibiting CHAC2-Mediated Respiratory Burst and Inflammatory Cytokine Expression.

Actinobacillus pleuropneumoniae (A. pleuropneumoniae) causes porcine pleuropneumonia that seriously endangers pig's health. Adh, located in the head region of trimeric autotransporter adhesion of A. pleuropneumoniae, affects bacterial adhesion and pathogenicity. However, how Adh mediates A. pleuropneumoniae immune invasion is still unclear. Here, we established the A. pleuropneumoniae strain L20 or L20 ΔAdh-infected porcine alveolar macrophages (PAM) model, and applied protein overexpression, RNA interference, qRT-PCR, Western blot and immunoflourescence techniques to dissect the effects of Adh on PAM during A. pleuropneumoniae infection. We found that Adh could increase the A. pleuropneumoniae adhesion and intracellular survival in PAM. Gene chip analysis of piglet lungs further showed that Adh significantly induced cation transport regulatory-like protein 2 (CHAC2) expression, whose overexpression suppressed the phagocytic capacity of PAM. Furthermore, CHAC2 overexpression dramatically increased glutathione (GSH) expression, decreased reactive oxygen species (ROS), and promoted A. pleuropneumoniae survival in PAM, while the knockdown of CHAC2 reversed these phenomena. Meanwhile, CHAC2 silence activated the NOD1/NF-κB pathway, resulting in an increase in IL-1ß, IL-6, and TNF-α expression, whereas this effect was weakened by CHAC2 overexpression and addition of NOD1/NF-κB inhibitor ML130. Moreover, Adh enhanced the secretion of LPS of A. pleuropneumoniae, which regulated the expression of CHAC2 via TLR4. In conclusion, through a LPS-TLR4-CHAC2 pathway, Adh inhibits respiratory burst and inflammatory cytokines expression to promote A. pleuropneumoniae survival in PAM. This finding may provide a novel target for the prevention and treatment of A. pleuropneumoniae.

Untargeted metabolomics reveals alternations in metabolism of bovine mammary epithelial cells upon IFN-γ treatment.

BACKGROUND: IFN-γ is a pleiotropic cytokine that has been shown to affect multiple cellular functions of bovine mammary epithelial cells (BMECs) including impaired milk fat synthesis and induction of malignant transformation via depletion of arginine, one of host conditionally essential amino acids. But the molecular mechanisms of these IFN-γ induced phenotypes are still unknown. METHODS: BMECs were treated with IFN-γ for 6 h, 12 h, and 24 h. The metabolomic profiling in BMECs upon IFN-γ induction were assessed using untargeted ultra-performance liquid chromatography-mass spectrometry (UPLC-MS) metabolomic analysis. Key differentially expressed metabolites (DEMs) were quantified by targeted metabolomics. RESULTS: IFN-γ induction resulted in significant differences in the contents of metabolites. Untargeted analysis identified 221 significantly DEMs, most of which are lipids and lipid-like molecules, organic acids and derivatives, organ heterocyclic compounds and benzenoids. According to Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis, DEMs were enriched in fructose and mannose metabolism, phosphotransferase system (PTS), ß-alanine metabolism, arginine and proline metabolism, methane metabolism, phenylalanine metabolism, and glycolysis/gluconeogenesis. Quantification of selected key DEMs by targeted metabolomics showed significantly decreased levels of D-(-)-mannitol, argininosuccinate, and phenylacetylglycine (PAG), while increased levels in S-hydroxymethylglutathione (S-HMG) and 2,3-bisphospho-D-glyceric acid (2,3-BPG). CONCLUSIONS: These results provide insights into the metabolic alterations in BMECs upon IFN-γ induction and indicate potential theoretical basis for clarifying IFN-γ-induced diseases in mammary gland.

Metagenomics analysis reveals potential pathways and drivers of piglet gut phage-mediated transfer of ARGs.

The growing prevalence of antibiotic-resistant pathogens has led to a better understanding of the underlying processes that lead to this expansion. Intensive pig farms are considered one of the hotspots for antibiotic resistance gene (ARG) transmission. Phages, as important mobile carriers of ARGs, are widespread in the animal intestine. However, our understanding of phage-associated ARGs in the pig intestine and their underlying drivers is limited. Here, metagenomic sequencing and analysis of viral DNA and total DNA of different intestinal (ileum, cecum and feces) contents in healthy piglets and piglets with diarrhea were separately conducted. We found that phages in piglet ceca are the main repository for ARGs and mobile genetic element (MGE) genes. Phage-associated MGEs are important factors affecting the maintenance and transfer of ARGs. Interestingly, the colocalization of ARGs and MGE genes in piglet gut phages does not appear to be randomly selected but rather related to a specific phage host (Streptococcus). In addition, in the feces of piglets with diarrhea, the abundance of phages carrying ARGs and MGE genes was significantly increased, as was the diversity of polyvalent phages (phages with broad host ranges), which would facilitate the transfection and wider distribution of ARGs in the bacterial community. Moreover, the predicted host spectrum of polyvalent phages in diarrheal feces tended to be potential enteropathogenic genera, which greatly increased the risk of enteropathogens acquiring ARGs. Notably, we also found ARG-homologous genes in the sequences of piglet intestinal mimiviruses, suggesting that the piglet intestinal mimiviruses are a potential repository of ARGs. In conclusion, this study greatly expands our knowledge of the piglet gut microbiome, revealing the underlying mechanisms of maintenance and dissemination of piglet gut ARGs and providing a reference for the prevention and control of ARG pollution in animal husbandry.

Development and application of an indirect ELISA and nested PCR for the epidemiological analysis of Klebsiella pneumoniae among pigs in China.

Introduction: Klebsiella pneumoniae (K. pneumoniae) is an important opportunistic and zoonotic pathogen which is associated with many diseases in humans and animals. However, the pathogenicity of K. pneumoniae has been neglected and the prevalence of K. pneumoniae is poorly studied due to the lack of rapid and sensitive diagnosis techniques. Methods: In this study, we infected mice and pigs with K. pneumoniae strain from a human patient. An indirect ELISA was established using the KHE protein as the coating protein for the detection of K. pneumoniae specific antibody in clinical samples. A nested PCR method to detect nuclei acids of K. pneumoniae was also developed. Results: We showed that infection with K. pneumoniae strain from a human patient led to mild lung injury of pigs. For the ELISA, the optimal coating concentration of KHE protein was 10 µg/mL. The optimal dilutions of serum samples and secondary antibody were 1:100 and 1:2500, respectively. The analytical sensitivity was 1:800, with no cross-reaction between the coated antigen and porcine serum positive for antibodies against other bacteria. The intra-assay and inter-assay reproducibility coefficients of variation are less than 10%. Detection of 920 clinical porcine serum samples revealed a high K. pneumoniae infection rate by established indirect ELISA (27.28%) and nested PCR (19.13%). Moreover, correlation analysis demonstrated infection rate is positively correlated with gross population, Gross Domestic Product (GDP), and domestic tourists. Discussion: In conclusion, K. pneumoniae is highly prevalent among pigs in China. Our study highlights the role of K. pneumoniae in pig health, which provides a reference for the prevention and control of diseases associated with K. pneumoniae.

Recent advances on the regulation of bacterial biofilm formation by herbal medicines.

Biofilm formation is a fundamental part of life cycles of bacteria which affects various aspects of bacterial-host interactions including the development of drug resistance and chronic infections. In clinical settings, biofilm-related infections are becoming increasingly difficult to treat due to tolerance to antibiotics. Bacterial biofilm formation is regulated by different external and internal factors, among which quorum sensing (QS) signals and nucleotide-based second messengers play important roles. In recent years, different kinds of anti-biofilm agents have been discovered, among which are the Chinese herbal medicines (CHMs). CHMs or traditional Chinese medicines have long been utilized to combat various diseases around the world and many of them have the ability to inhibit, impair or decrease bacterial biofilm formation either through regulation of bacterial QS system or nucleotide-based second messengers. In this review, we describe the research progresses of different chemical classes of CHMs on the regulation of bacterial biofilm formation. Though the molecular mechanisms on the regulation of bacterial biofilm formation by CHMs have not been fully understood and there are still a lot of work that need to be performed, these studies contribute to the development of effective biofilm inhibitors and will provide a novel treatment strategy to control biofilm-related infections.

The two-component system CpxA/CpxR is critical for full virulence in Actinobacillus pleuropneumoniae.

Actinobacillus pleuropneumoniae, a major bacterial porcine respiratory tract pathogen causing pig pleuropneumonia, has resulted in high economic losses worldwide. The mutation of the two-component system CpxAR strongly impacted the virulence of A. pleuropneumoniae, but the underlying regulatory mechanism remained unclear. Here, we found that CpxAR positively regulated the cpxDCBA gene cluster involved in polysaccharide capsule export. A capsular layer was confirmed in wild-type cells by transmission electron microscopy, whereas cpxAR and cpxD mutants were non-capsulated. The mutants for polysaccharide capsule export gene cpxD exhibited non-capsulated and were strongly impaired in virulence for mice, indicating a major role of CPS export system in virulence. We then demonstrated that CpxR directly regulated the transcription of the CPS export gene cluster cpxDCBA. Taken together, our data suggested that CpxAR is a key modulator of capsule export that facilitates A. pleuropneumoniae survival in the host.

LAP3 contributes to IFN-γ-induced arginine depletion and malignant transformation of bovine mammary epithelial cells.

BACKGROUND: IFN-γ has been traditionally recognized as an inflammatory cytokine that involves in inflammation and autoimmune diseases. Previously we have shown that sustained IFN-γ induced malignant transformation of bovine mammary epithelial cells (BMECs) via arginine depletion. However, the molecular mechanism underlying this is still unknown. METHODS: In this study, the amino acids contents in BMECs were quantified by a targeted metabolomics method. The acquisition of differentially expressed genes was mined from RNA-seq dataset and analyzed bioinformatically. Quantitative reverse transcription polymerase chain reaction (qRT-PCR), enzyme-linked immunosorbent assay (ELISA), western blotting, and immunohistochemistry (IHC) assay were performed to detect gene mRNA and protein expression levels. CCK-8 and would healing assays were used to detect cell proliferation and migration abilities, respectively. Cell cycle phase alternations were analyzed by flow cytometry. RESULTS: The targeted metabolomics analysis specifically discovered IFN-γ induced arginine depletion through accelerating arginine catabolism and inhibiting arginine anabolism in BMECs. Transcriptome analysis identified leucine aminopeptidase 3 (LAP3), which was regulated by p38 and ERK MAPKs, to downregulate arginine level through interfering with argininosuccinate synthetase (ASS1) as IFN-γ stimulated. Moreover, LAP3 also contributed to IFN-γ-induced malignant transformation of BMECs by upregulation of HDAC2 (histone deacetylase 2) expression and promotion of cell cycle proteins cyclin A1 and D1 expressions. Arginine supplementation did not affect LAP3 and HDAC2 expressions, but slowed down cell cycle process of malignant BMECs. In clinical samples of patients with breast cancer, LAP3 was confirmed to be upregulated, while ASS1 was downregulated compared with healthy control. CONCLUSIONS: These results demonstrated that LAP3 mediated IFN-γ-induced arginine depletion to malignant transformation of BMECs. Our findings provide a potential therapeutic target for breast cancer both in humans and dairy cows.

The Combination of Phages and Faecal Microbiota Transplantation Can Effectively Treat Mouse Colitis Caused by Salmonella enterica Serovar Typhimurium.

Salmonella enterica serovar Typhimurium (S. Typhimurium) is one of the common causes of human colitis. In the present study, two lytic phages vB_SenS-EnJE1 and vB_SenS-EnJE6 were isolated and the therapeutic effect of the combination of phages and faecal microbiota transplantation (FMT) on S. Typhimurium-induced mouse colitis was investigated. The characteristics and genome analysis indicated that they are suitable phages for phage therapy. Results showed that vB_SenS-EnJE1 lysis 41/54 Salmonella strains of serotype O4, and vB_SenS-EnJE6 lysis 46/54 Salmonella strains of serotypes O4 and O9. Severe inflammatory symptoms and disruption of the intestinal barrier were observed in S. Typhimurium -induced colitis. Interestingly, compared with a single phage cocktail (Pc) or single FMT, the combination of Pc and FMT (PcFMT) completely removed S. Typhimurium after 72 h of treatment, and significantly improved pathological damage and restored the intestinal barrier. Furthermore, PcFMT effectively restored the intestinal microbial diversity, especially for Firmicutes/Bacteroidetes [predominantly bacterial phyla responsible for the production of short-chain fatty acids (SCFA)]. Additionally, we found that PcFMT treatment significantly increased the levels of SCFA. All these data indicated that the combination of phages and FMT possesses excellent therapeutic effects on S. Typhimurium -induced intestinal microbiota disorder diseases. Pc and FMT played roles in "eliminating pathogens" and "strengthening vital qi," respectively. This study provides a new idea for the treatment of intestinal microbiota disorder diseases caused by specific bacterial infections.

A phage cocktail combined with the enteric probiotic Lactobacillus reuteri ameliorated mouse colitis caused by S. typhimurium.

Salmonella enterica serovar Typhimurium (S. typhimurium) is one of the most important foodborne pathogens that causes colitis in humans. In this study, we compared the effects of a therapeutic treatment using a phage cocktail (Pc) in combination or not with Lactobacillus reuteri (L. reuteri) in an S. typhimurium-induced colitis murine model. An oral administration of 4 × 108 CFU per mouse of S. typhimurium resulted in intestinal barrier disruption and severe inflammatory symptoms. S. typhimurium in the colon of the mice treated with the Pc and L. reuteri (PcLR) combination were completely removed compared to those in the single Pc or single L. reuteri treatment groups. Furthermore, compared with the infected group, the intestinal barrier and colonic pathological damage were significantly improved in the PcLR-treated group. Additionally, the short-chain fatty acid (SCFA) levels in the feces of the mice in the PcLR treatment group were significantly increased compared to those in the feces of the mice in the infected group. In addition, the combination of Pc with acetate and reuterin released by L. reuteri (PcReAc) can also achieve the same effect as PcLR treatment. Thus, these results indicated that the acetate and reuterin released by L. reuteri play an important role in the treatment. The extraordinary therapeutic effects of PcLR and PcReAc depend on the specific bactericidal activity of Pc and the broad-spectrum bactericidal activity and immunomodulation of L. reuteri (or acetate and reuterin) in the host. This study provides a new concept for the treatment of inflammatory diseases caused by intestinal pathogens.