Changjiang Sun

Clostridium perfringens is one of the critical causative agents causing diarrhea in piglets, with significant economic losses to the pig industry. Under normal gut microbiota homeostasis and well-managed barns, diarrhea caused by C. perfringens could be controlled. Some reports show that probiotics, such as Bacillus subtilis, are beneficial in preventing necrotic enteritis (NE) in chickens, but few reports on piglets. Clostridium perfringens was found in the piglets’ diarrhea with intestinal microbiota dysbiosis in our survey. Bacillus subtilis G2B9-Q, which was isolated from the feces of healthy pigs, was found to have anti-Clostridium activity after screening. Clostridium perfringens was used to challenge mice by intraperitoneal injection for modeling to evaluate the anti-infective activity of cell-free supernatant (CFS) of B. subtilis G2B9-Q and different concentrations of B. subtilis G2B9-Q by oral administration. The results showed that G2B9-Q can mitigate intestinal lesions caused by C. perfringens infection, reduce inflammatory reactions, and modulate intestinal microbiota. The CFS of G2B9-Q can alleviate the pathological damage of intestinal tissues caused by C. perfringens infection, reduce the concentration of TNF-α and IL-10 in the sera of mice, as well as the relative expression levels of alpha toxin (CPA), perfringolysin O (PFO) toxin, IL-10, IL-22, and TNF-α in the jejunum and colon tissues, and alleviate the changes in gut microbiota structure caused by C. perfringens infection, which showed better therapeutic effects and indicated that the metabolites of G2B9-Q are essential mediators for their beneficial effects. Therefore, the CFS of G2B9-Q could potentially replace antibiotics in treating C. perfringens infection.

Porcine circovirus disease (PCVD) caused by porcine circovirus type 2 (PCV2) is widely distributed in pig farms. Up until now, nine genotypes of PCV2, PCV2a to 2i, have been identified in diseased pigs worldwide. This study analyzed 302 samples collected in the Jilin Province of China from 2016 to 2021, followed by genetic analysis of the PCV2 isolates. Meanwhile, the antigen epitopes, amino acid mutations, 3D structure of the PCV2 isolates and commercially available vaccine strains were evaluated and compared. The results showed that the predominant genotypes of PCV2 were PCV2b, followed by PCV2e and PCV2d in Jilin Province during 2016–2021. Although mutations were detected in the isolates, no recombination occurred in the PCV2 isolates, indicating a stable genotype of PCV2 in Jilin Province during these years. Moreover, the B cell epitopes in the Cap and Rep proteins of eighteen PCV2 isolates and T cell epitopes in the Cap of the isolates were changed compared to three currently used vaccine strains. The mutations in the Cap and Rep proteins did not affect their spatial conformation. Therefore, bivalent or multivalent vaccines with different genotypes of PCV2 might improve the protective effect of vaccines.

Aerococcus viridans is an opportunistic pathogen that is clinically associated with various human and animal diseases. In this study, the first identified A. viridans phage, vB_AviM_AVP (abbreviated as AVP), was isolated and studied. AVP belongs to the family Myoviridae. AVP harbors a double-stranded DNA genome with a length of 133,806 bp and a G + C content of 34.51%. The genome sequence of AVP showed low similarity (<1% identity) to those of other phages, bacteria, or other organisms in the database. Among 165 predicted open reading frames (ORFs), there were only 69 gene products exhibiting similarity (≤65% identity) to proteins of known functions in the database. In addition, the other 36 gene products did not match any viral or prokaryotic sequences in any publicly available database. On the basis of the putative functions of the ORFs, the genome of AVP was divided into three modules: nucleotide metabolism and replication, structural components, and lysis. A phylogenetic analysis of the terminase large subunits and capsid proteins indicated that AVP represents a novel branch of phages. The observed characteristics of AVP indicate that it represents a new class of phages.

Staphylococcus aureus (S. aureus) is a common and dangerous pathogen that causes various infectious diseases. Skin damage, such as burn wounds, are at high risk of Staphylococcus aureus colonization and infection, which increases morbidity and mortality. The phage lysin LysGH15 exhibits highly efficient lytic activity against methicillin-resistant S. aureus (MRSA) and methicillin-susceptible S. aureus (MSSA) strains. Apigenin (api) significantly decreases haemolysis of rabbit erythrocytes caused by S. aureus and shows anti-inflammatory function. LysGH15 and api were added to Aquaphor to form an LysGH15-api-Aquaphor (LAA) ointment. The LAA ointment simultaneously exhibited bactericidal activity against S. aureus and inhibited haemolysis. In an LAA-treated mouse model of an MRSA-infected skin wound, the mean bacterial colony count decreased to approximately 10² CFU/mg at 18 h after treatment (and the bacteria became undetectable at 96 h), whereas the mean count in untreated mice was approximately 10⁵ CFU/mg of tissue. The LAA ointment also reduced the levels of pro-inflammatory cytokines (TNF-α, IL-1β, and IFN-γ) and accelerated wound healing in the mouse model. These data demonstrate the potential efficacy of a combination of LysGH15 and api for use as a topical antimicrobial agent against S. aureus.

In recent years, after the emergence of a large number of multidrug-resistant bacteria, phages and phage-associated products for the prevention and control of bacterial disease have revealed prominent advantages as compared with antibiotics. However, bacteria are susceptible to becoming phage-resistant, thus severely limiting the application of phage therapy. In this study, Escherichia coli cells were incubated with lytic bacteriophages to obtain mutants that were resistant to the lytic phages. Then, bacteriophages against the phage-resistant variants were isolated and subsequently mixed with the original lytic phage to prepare a novel phage cocktail for bactericidal use. The data showed that our phage cocktail not only had notable bactericidal effects, including a widened host range and rapid lysis, but also decreased the generation and mutation frequency of phage-resistant strains in vitro. In addition, we tested our cocktail in a murine bacteremia model. The results suggested that compared with the single phage, fewer phage-resistant bacteria appeared during the treatment of phage cocktail, thus prolonging the usable time of the phage cocktail and improving its therapeutic effect in phage applications. Importantly, our preparation method of phage cocktail was proved to be generalizable. Because the bacteriophage against the phage-resistant strain is an ideal guard that promptly attacks potential phage resistance, this guard-killer dual-function phage cocktail provides a novel strategy for phage therapy that allows the natural ecology to be sustained.

Bacteriophage endolysin is one of the most promising antibiotic substitutes, but in Gram-negative bacteria, the outer membrane prevents the lysin from hydrolyzing peptidoglycans and blocks the development of lysin applications. The prime strategy for new antibiotic substitutes is allowing lysin to access the peptidoglycan from outside of the bacteria by reformation of the lysin. In this study, the novel Escherichia coli (E. coli) phage lyase lysep3, which lacks outside-in catalytic ability, was fused with the N-terminal region of the Bacillus amyloliquefaciens lysin including its cell wall binding domain D8 through the best manner of protein fusion based on the predicted tertiary structure of lysep3-D8 to obtain an engineered lysin that can lyse bacteria from the outside. Our results showed that lysep3-D8 could lyse both Gramnegative and Gram-positive bacteria, whereas lysep3 and D8 have no impact on bacterial growth. The MIC of lysep3-D8 on E. coli CVCC1418 is 60 μg/ml; lysep3-D8 can inhibit the growth of bacteria up to 12 h at this concentration. The bactericidal spectrum of lysep3-D8 is broad, as it can lyse of all of 14 E. coli strains, 3 P. aeruginosa strains, 1 Acinetobacter baumannii strain, and 1 Streptococcus strain. Lysep3-D8 has sufficient bactericidal effects on the 14 E. coli strains tested at the concentration of 100 μg/ml. The cell wall binding domain of the engineered lysin can destroy the integrity of the outer membrane of bacteria, thus allowing the catalytic domain to reach its target, peptidoglycan, to lyse the bacteria. Lysep3-D8 can be used as a preservative in fodder to benefit the health of animals. The method we used here proved to be a successful exploration of the reformation of phage lysin.

Bacteriophages represent one prospect for preventing and treating multi-drug-resistant Escherichia coli. In this study, we have isolated a novel E. coli-specific bacteriophage and characterised its biological properties. vB_EcoM-ep3 has a broad host range and was able to lyse 9 out of 15 clinical isolates of multi-drug-resistant pathogenic E. coli from chickens. The optimal multiplicity of infection for vB_EcoM-ep3 in host bacteria was 0.01. vB_EcoM-ep3 was thermostable at temperatures below 50 °C for up to 60 min. Electron microscopy demonstrated that vB_EcoM-ep3 belongs to Myoviridae. The vB_EcoM-ep3 genome contained 42,351 pairs of nucleotides with a GC content of 53.35 %. There were 52 predicted open reading frames that appeared to overlap and have a modular structure. Phylogenetic analysis indicates that the closest evolutionary relative to vB_EcoM-ep3 is the previously reported E. coli phage vB_EcoM_ECO1230-10. However, there was no homology between reported E. coli phage lysins and the vB_EcoM-ep3 lysin gene. Lysep3 was 58 % similar to the Pseudomonas phage PPpW-3 lysin despite showing no similarities at the gene sequence level. And Lysep3 has good lysis activity.

The Mycobacterium tuberculosis H37Rv and BCG effects on the host cell transcriptional profile consider a main research point. In the present study the transcriptome profiling analysis of RAW264.7 either infected with Mycobacterium tuberculosis H37Rv or BCG have been reported using Solexa/Illumina digital gene expression (DGE). The DGE analysis showed 1,917 different expressed genes between the BCG and H37Rv group. In addition, approximately 5% of the transcripts appeared to be predicted genes that have never been described before. KEGG Orthology (KO) annotations showed more than 71% of these transcripts are possibly involved in approximately 210 known metabolic or signaling pathways. The gene of the 28 pathways about pathogen recognition receptors and Mycobacterium tuberculosis interaction with macrophages were analyzed using the CLUSTER 3.0 available, the Tree View tool and Gene Orthology (GO). Some genes were randomly selected to confirm their altered expression levels by quantitative real-time PCR (qRT-PCR). The present study used DGE from pathogen recognition receptors and Mycobacterium tuberculosis interaction with macrophages to understand the interplay between Mycobacterium tuberculosis and RAW264.7. Meanwhile find some important host protein which was affected by Mycobacterium tuberculosis to provide evidence for the further improvement of the present efficacy of existing Mycobacterium tuberculosis therapy and vaccine.