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.

Biological characterization of the phage lysin AVPL and its efficiency against Aerococcus viridans-induced mastitis in a murine model.

Aerococcus viridans (A. viridans) is an important opportunistic zoonotic pathogen that poses a potential threat to the animal husbandry industry, such as cow mastitis, due to the widespread development of multidrug-resistant strains. Phage lysins have emerged as a promising alternative antibiotic treatment strategy. However, no lysins have been reported to treat A. viridans infections. In this study, the critical active domain and key active sites of the first A. viridans phage lysin AVPL were revealed. AVPL consists of an N-terminal N-acetylmuramoyl-L-alanine amidase catalytic domain and a C-terminal binding domain comprising two conserved LysM. H40, N44, E52, W68, H147, T157, F60, F64, I77, N92, Q97, H159, V160, D161, and S42 were identified as key sites for maintaining the activity of the catalytic domain. The LysM motif plays a crucial role in binding AVPL to bacterial cell wall peptidoglycan. AVPL maintains stable activity in the temperature range of 4-45°C and pH range of 4-10, and its activity is independent of the presence of metal ions. In vitro, the bactericidal effect of AVPL showed efficient bactericidal activity in milk samples, with 2 µg/mL of AVPL reducing A. viridans by approximately 2 Log10 in 1 h. Furthermore, a single dose (25 µg) of lysin AVPL significantly reduces bacterial load (approximately 2 Log10) in the mammary gland of mice, improves mastitis pathology, and reduces the concentration of inflammatory cytokines (TNF-α, IL-1ß, and IL-6) in mammary tissue. Overall, this work provides a novel alternative therapeutic drug for mastitis induced by multidrug-resistant A. viridans. IMPORTANCE: A. viridans is a zoonotic pathogen known to cause various diseases, including mastitis in dairy cows. In recent years, there has been an increase in antibiotic-resistant or multidrug-resistant strains of this pathogen. Phage lysins are an effective approach to treating infections caused by multidrug-resistant strains. This study revealed the biological properties and key active sites of the first A. viridans phage lysin named AVPL. AVPL can effectively kill multidrug-resistant A. viridans in pasteurized whole milk. Importantly, 25 µg AVPL significantly alleviates the symptoms of mouse mastitis induced by A. viridans. Overall, our results demonstrate the potential of lysin AVPL as an antimicrobial agent for the treatment of mastitis caused by A. viridans.

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.

Aerococcus viridans Phage Lysin AVPL Had Lytic Activity against Streptococcus suis in a Mouse Bacteremia Model.

Streptococcus suis (S. suis) is a swine pathogen that can cause sepsis, meningitis, endocarditis, and other infectious diseases; it is also a zoonotic pathogen that has caused a global surge in fatal human infections. The widespread prevalence of multidrug-resistant S. suis strains and the decline in novel antibiotic candidates have necessitated the development of alternative antimicrobial agents. In this study, AVPL, the Aerococcus viridans (A. viridans) phage lysin, was found to exhibit efficient bactericidal activity and broad lytic activity against multiple serotypes of S. suis. A final concentration of 300 µg/mL AVPL reduced S. suis counts by 4-4.5 log10 within 1 h in vitro. Importantly, AVPL effectively inhibited 48 h S. suis biofilm formation and disrupted preformed biofilms. In a mouse model, 300 µg/mouse AVPL protected 100% of mice from infection following the administration of lethal doses of multidrug-resistant S. suis type 2 (SS2) strain SC19, reduced the bacterial load in different organs, and effectively alleviated inflammation and histopathological damage in infected mice. These data suggest that AVPL is a valuable candidate antimicrobial agent for treating S. suis infections.

Phage resistance mutation triggered by OmpC deficiency in Klebsiella pneumoniae induced limited fitness costs.

Outer membrane proteins (OMPs) play an important role in bacterial fitness costs. Derived from the interaction between Klebsiella pneumoniae K7 and phage GH-K3, K7RB is an outer membrane porin-deficient phage-resistant mutant strain triggered by ompC712 deletion, exhibits expression inhibition of OmpC, OmpN, KPN_02430 and OmpF, but its fitness costs and regulatory mechanism remains unknown. In this study, compared with K7, K7RB showed almost unaffected growth rate, slightly decreased virulence, and increased resistance to some antibiotics. Transcriptome analysis showed that the pathways of glycerolipid metabolism and nitrogen metabolism in K7RB were significantly inhibited, while the transcription of permeases belonging to ABC transporters tended to be active, nutrient uptakes such as citrate and phenylalanine were also enhanced. However, transcriptional up-regulation in K7RB was inhibited by overexpression of OmpC, OmpN, KPN_02430 and OmpF in general. Overexpression of OmpN, KPN_02430 and OmpF, respectively, restoring the sensitivity of strains to antibiotics to varying degrees, while OmpC overexpression aggravated the bacterial drug-resistance especially to ß-lactam antibiotics. Besides, unlike OmpC and OmpF, overexpression of OmpN and KPN_02430 reduced bacterial virulence. In brief, by revealing the limited fitness costs of phage-resistant mutant K. pneumoniae with porin-deficiency, our study providing a reference for the design and development of drugs to inhibit the ways of bacterial metabolic rewiring and to increase fitness costs.

Oral vaccination with recombinant Lactobacillus casei expressing Aha1 fused with CTB as an adjuvant against Aeromonas veronii in common carp (Cyprinus carpio).

Aeromonas veronii (A. veronii) is a pathogenic that can infect human, animal and aquatic organisms, in which poses a huge threat to the health of many aquatic organisms such as Cyprinus carpio. In this study, Lactobacillus casei (L. casei) strain CC16 was used as antigen deliver carrier and fused with cholera toxin B subunit (CTB) as an adjuvant to construct the recombinant L. casei pPG-Aha1/Lc CC16(surface-displayed) and pPG-Aha1-CTB/Lc CC16(surface-displayed) expressing Aha1 protein of A. veronii, respectively. And the immune responses in Cyprinus carpio by oral route was explored. Our results demonstrated that the recombinant strains could stimulate high serum specific antibody immunoglobulin M (IgM) and induce a stronger acid phosphatase (ACP), alkaline phosphatase (AKP), C3, C4, lysozyme (LZM), Lectin and superoxide dismutase (SOD) activity in Cyprinus carpio compared with control groups. Meanwhile, the expression of Interleukin-10 (IL-10), Interleukin-1ß (IL-1ß), Tumor Necrosis Factor-α (TNF-α), immunoglobulin Z1 (IgZ1) and immunoglobulin Z2 (IgZ2) in the tissues were significantly upregulated compared with Lc-pPG or PBS groups, indicating that humoral and cell immune response were triggered. Additionally, recombinant L. casei could survive and colonize in fish intestine. Significantly, recombinant L. casei provides immune protection against A. veronii infection, which Cyprinus carpio received pPG-Aha1-CTB/Lc CC16 (64.29%) and pPG-Aha1/Lc CC16 (53.57%) had higher survival rates compared with the controls. Thus, we demonstrated that recombinant pPG-Aha1/Lc CC16 and pPG-Aha1-CTB/Lc CC16 may be the promising strategy for the development of an oral vaccine against A. veronii.

Therapeutic Efficacy of Phage PIZ SAE-01E2 against Abortion Caused by Salmonella enterica Serovar Abortusequi in Mice.

Salmonella enterica subsp. enterica serovar Abortusequi is a frequently reported pathogen causing abortion in mares. In this study, the preventive and therapeutic effects of phage PIZ SAE-01E2 against S Abortusequi in a mouse model of abortion were investigated. Phage PIZ SAE-01E2 was stable at different temperatures (4 to 70°C) and pH values (pH 4 to 10) and could lyse the majority of the Salmonella serogroup O:4 and O:9 strains tested (25/28). There was no lysogeny-related, toxin, or antibiotic resistance-related gene in the genome of PIZ SAE-01E2. All of these characteristics indicate that PIZ SAE-01E2 has the potential for use in phage therapy. In in vivo experiments, 2 × 103 CFU/mouse of S Abortusequi ATCC 9842 was sufficient to lead to murine abortion (gestational day 14.5) within 48 h. A single intraperitoneal inoculation of PIZ SAE-01E2 (108 PFU/mouse, multiplicity of infection = 105) 1 h before or after S Abortusequi challenge provided effective protection to all pregnant mice (10/10). After 24 h of treatment with phage PIZ SAE-01E2, the bacterial loads in both the placenta and the uterus of the infected mice were significantly decreased (<102 CFU/g) compared to those in the placenta and the uterus of the mice in the control group (>106 CFU/g). In addition, the levels of inflammatory cytokines in the placenta and blood of the mice in the phage administration groups were significantly reduced (P < 0.05) compared to those in the placenta and blood of the mice in the control group. Altogether, these findings indicate that PIZ SAE-01E2 shows the potential to block abortions induced by S Abortusequi in vivoIMPORTANCES Abortusequi is an important pathogen that can induce abortions in mares. Although S Abortusequi has been well controlled in Europe and the United States due to strict breeding and health policies, it is still widespread in African and Asian countries and has proven difficult to control. In China, abortions caused by S Abortusequi have also been reported in donkeys. So far, there is no commercial vaccine. Thus, exploiting alternative efficient and safe strategies to control S Abortusequi infection is essential. In this study, a new lytic phage, PIZ SAE-01E2, infecting S Abortusequi was isolated, and the characteristics of PIZ SAE-01E2 indicated that it has the potential for use in phage therapy. A single intraperitoneal inoculation of PIZ SAE-01E2 before or after S Abortusequi challenge provided effective protection to all pregnant mice. Thus, PIZ SAE-01E2 showed the potential to block abortions induced by S Abortusequi in vivo.

Biological properties and genomics analysis of vB_KpnS_GH-K3, a Klebsiella phage with a putative depolymerase-like protein.

Bacteriophages have been recently revisited as an alternative biocontrol tool due to the limitations of antibiotic treatment. In this study, we reported on the biological characteristics and genomic information of vB_KpnS_GH-K3 (abbreviated as GH-K3), a Klebsiella phage of the Siphoviridae family, which was previously isolated from a hospital sewage system. One-step growth curve analysis indicated that the burst size of GH-K3 was 291 PFU/cell. GH-K3 maintained a stable titer in a broad range of pH values (6-10) and temperature (up to 50 °C). Based on bioinformatics analysis, GH-K3 comprises of 49,427 bp containing a total of 77 open reading frames (ORFs), which share high degree of nucleotide similarity and close evolutionary relationships with at least 12 other Klebsiella phages. Of note, GH-K3 gp32 was identified as a unique ORF. The major segment of gp32 sequence at the C-terminus (residues 351-907) was found highly variable as determined by its mismatch with the nucleotide and protein sequences available at NCBI database. Furthermore, HHpred analysis indicated that GH-K3 gp32 contains three domains (PDB ID: 5W6S_A, 3GQ8_A and 1BHE_A) similar to depolymerase (depoKP36) of Klebsiella phage KP36 suggestive of a potential depolymerase activity during host receptor-binding in the processes of phage infection. Altogether, the current data revealed a novel putative depolymerase-like protein which is most likely to play an important role in phage-host interaction.

Antibacterial Effects of Phage Lysin LysGH15 on Planktonic Cells and Biofilms of Diverse Staphylococci.

Treatment of infections caused by staphylococci has become more difficult because of the emergence of multidrug-resistant strains as well as biofilm formation. In this study, we observed the ability of the phage lysin LysGH15 to eliminate staphylococcal planktonic cells and biofilms formed by Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus haemolyticus, and Staphylococcus hominis All these strains were sensitive to LysGH15, showing reductions in bacterial counts of approximately 4 log units within 30 min after treatment with 20 µg/ml of LysGH15, and the MICs ranged from 8 µg/ml to 32 µg/ml. LysGH15 efficiently prevented biofilm formation by the four staphylococcal species at a dose of 50 µg/ml. At a higher dose (100 µg/ml), LysGH15 also showed notable disrupting activity against 24-h and 72-h biofilms formed by S. aureus and coagulase-negative species. In the in vivo experiments, a single intraperitoneal injection of LysGH15 (20 µg/mouse) administered 1 h after the injection of S. epidermidis at double the minimum lethal dose was sufficient to protect the mice. The S. epidermidis cell counts were 4 log units lower in the blood and 3 log units lower in the organs of mice 24 h after treatment with LysGH15 than in the untreated control mice. LysGH15 reduced cytokine levels in the blood and improved pathological changes in the organs. The broad antistaphylococcal activity exerted by LysGH15 on planktonic cells and biofilms makes LysGH15 a valuable treatment option for biofilm-related or non-biofilm-related staphylococcal infections.IMPORTANCE Most staphylococcal species are major causes of health care- and community-associated infections. In particular, Staphylococcus aureus is a common and dangerous pathogen, and Staphylococcus epidermidis is a ubiquitous skin commensal and opportunistic pathogen. Treatment of infections caused by staphylococci has become more difficult because of the emergence of multidrug-resistant strains as well as biofilm formation. In this study, we found that all tested S. aureus, S. epidermidis, Staphylococcus haemolyticus, and Staphylococcus hominis strains were sensitive to the phage lysin LysGH15 (MICs ranging from 8 to 32 µg/ml). More importantly, LysGH15 not only prevented biofilm formation by these staphylococci but also disrupted 24-h and 72-h biofilms. Furthermore, the in vivo efficacy of LysGH15 was demonstrated in a mouse model of S. epidermidis bacteremia. Thus, LysGH15 exhibits therapeutic potential for treating biofilm-related or non-biofilm-related infections caused by diverse staphylococci.

A Smooth-Type, Phage-Resistant Klebsiella pneumoniae Mutant Strain Reveals that OmpC Is Indispensable for Infection by Phage GH-K3.

Bacteriophage can be used as an alternative or complementary therapy to antibiotics for treating multidrug-resistant bacterial infections. However, the rapid emergence of resistant host variants during phage treatment has limited its therapeutic applications. In this study, a potential phage-resistant mechanism of Klebsiella pneumoniae was revealed. After phage GH-K3 treatment, a smooth-type colony, named K7RB, was obtained from the K. pneumoniae K7 culture. Treatment with IO4- and/or proteinase K indicated that polysaccharides of K7 played an important role in phage recruitment, and protein receptors on K7 were essential for effective infection by GH-K3. Differences in protein expression between K7 and K7RB were quantitatively analyzed by liquid chromatography-tandem mass spectrometry. Among differentially expressed proteins, OmpC, OmpN, KPN_02430, and OmpF were downregulated significantly in K7RBtrans-Complementation of OmpC in K7RB conferred rapid adsorption and sensitivity to GH-K3. In contrast, a single-base deletion mutation of ompC in K7, which resulted in OmpC silencing, led to lower adsorption efficiency and resistance to GH-K3. These assays proved that OmpC is the key receptor-binding protein for GH-K3. In addition, the native K. pneumoniae strains KPP14, KPP27, and KPP36 showed low or no sensitivity to GH-K3. However, these strains became more sensitive to GH-K3 after their native receptors were replaced by OmpC of K7, suggesting that OmpCK7 was the most suitable receptor for GH-K3. This study revealed that K7RB became resistant to GH-K3 due to gene mutation of ompC and that OmpC of K7 is essential for effective infection by GH-K3.IMPORTANCE With increased incidence of multidrug-resistant (MDR) bacterial strains, phages have regained attention as promising potential antibacterial agents. However, the rapid emergence of resistant variants during phage treatment has limited the therapeutic applications of phage. According to our trans-complementation, ompC mutation, and phage adsorption efficiency assays, we identified OmpC as the key receptor-binding protein (RBP) for phage GH-K3, which is essential for effective infection. This study revealed that the phage secondary receptor of K. pneumoniae, OmpC, is the essential RBP not only for phage infecting Gram-negative bacteria, such as Escherichia coli and Salmonella, but also for K. pneumoniae.

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.

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.

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.

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 novel lysin Ply1228 provides efficient protection against Streptococcus suis type 2 infection in a murine bacteremia model.

Streptococcus suis is an important zoonotic pathogen that is difficult to control with antibiotics due to the widespread development of multidrug-resistant strains. Phage lysin is considered a potential therapeutic agent to combat S. suis. In this study, the novel lysin Ply1228 derived from the prophage of S. suis type 12 was identified. Bioinformatics analysis showed that Ply1228 contains a CHAP catalytic domain, which is a binding domain composed of a CW-7 binding motif and an amidase-2 catalytic domain. The CHAP catalytic domain is essential for the bactericidal function of lysin Ply1228 and does not depend on the presence of Ca2+. C34 and H99 of the CHAP domain were identified as the key active sites. The CW-7 binding motif plays a key binding role in Ply1228. Ply1228 can specifically lyse S. suis, including types 2, 3, 7, 9, 10, 12, 14, and 27. Within 10 min, Ply1228 killed 4 log of the S. suis population, which had a starting concentration of approximately 107 CFU/mL. In addition, Ply1228 showed favourable thermal and pH stability. The therapeutic effect of Ply1228 was further investigated in a mouse model of S. suis bacteremia. The administration of the lysin Ply1228 (200 µg/mouse) 1 h after the intraperitoneal injection of 2 × MLD of SS2 strain SC225 was sufficient to protect the mice (P < 0.0001) and significantly reduced the bacterial loads in the blood and organs (livers, spleens, lungs and kidneys). The levels of inflammation and histopathological damage in infected mice were effectively relieved after the Ply1228 treatment. These results indicate that Ply1228 might represent a new enzybiotic candidate for S. suis infection.

vB-ApyS-JF1, the First Trueperella pyogenes Phage, Shows Potential as an Alternative Treatment Strategy for Trueperella pyogenes Infections.

Trueperella pyogenes (T. pyogenes) is an important opportunistic animal pathogen that causes huge economic losses to the animal husbandry industry. The emergence of bacterial resistance and the unsatisfactory effect of the vaccine have prompted investigators to explore alternative strategies for controlling T. pyogenes infection. Due to the ability of phages to kill multidrug-resistant bacteria, the use of phage therapy to combat multidrug-resistant bacterial infections has attracted attention. In this study, a T. pyogenes phage, vB-ApyS-JF1 (JF1), was isolated from sewage samples, and its whole genome and biological characteristics were elucidated. Moreover, the protective effect of phage JF1 on a mouse bacteremic model caused by T. pyogenes was studied. JF1 harbors a double-stranded DNA genome with a length of 90,130 bp (30.57% G + C). The genome of JF1 lacked bacterial virulence-, antibiotic resistance- and lysogenesis-related genes. Moreover, the genome sequence of JF1 exhibited low coverage (<6%) with all published phages in the NCBI database, and a phylogenetic analysis of the terminase large subunits and capsid indicated that JF1 was evolutionarily distinct from known phages. In addition, JF1 was stable over a wide range of pH values (3 to 11) and temperatures (4 to 50°C) and exhibited strong lytic activity against T. pyogenes in vitro. In murine experiments, a single intraperitoneal administration of JF1 30 min post-inoculation provided 100% protection for mice against T. pyogenes infection. Compared to the phosphate-buffered saline (PBS) treatment group, JF1 significantly (P < 0.01) reduced the bacterial load in the blood and tissues of infected mice. Meanwhile, treatment with phage JF1 relieved the pathological symptoms observed in each tissue. Furthermore, the levels of the inflammatory cytokines tumour necrosis factor-α (TNF-α), interferon-γ (IFN-γ), and interleukin-6 (IL-6) in the blood of infected mice were significantly (P < 0.01) decreased in the phage-treated group. Taken together, these results indicate that phage JF1 demonstrated great potential as an alternative therapeutic treatment against T. pyogenes infection.

Caveolae/rafts protect human cerebral microvascular endothelial cells from Streptococcus suis serotype 2 α-enolase-mediated injury.

Streptococcus suis serotype 2 (SS2) is an important zoonotic pathogen that causes meningitis. The ubiquitously expressed 40S ribosome protein SA (RPSA) is a multifunctional protein involved in the pathogenesis of multiple pathogens, especially those causing meningitis. However, the role of RPSA in SS2-induced meningitis is not clear. In this study, immunofluorescence staining revealed that SS2 infection promoted the intracellular transfer of RPSA to the surface of human cerebral microvascular endothelial cells (HCMECs). Moreover, SS2 infection promoted the accumulation of caveolin 1 (CAV1) and the formation of membrane bulges where RPSA enveloped CAV1 on the cell surface. SS2 infection also caused dynamic changes in the localization of RPSA and CAV1 on the cell surface which could be eliminated by disruption of caveolae/rafts by addition of methyl-ß-cyclodextrin (MßCD). Co-immunoprecipitation analysis demonstrated that α-enolase (ENO), a key virulence factor of SS2, interacted with RPSA, and promoted the interaction between RPSA and CAV1. Immunofluorescence staining, western blotting and flow cytometry analyses showed that damaged caveolae/rafts significantly enhanced ENO adhesion to HCMECs, promoted the "destruction" of RPSA by ENO, and enhanced the toxic effect of ENO on HCMECs. Importantly, these effects could be relieved upon the addition of cholesterol. We conclude that caveolae/rafts weaken the toxic effect of SS2 ENO on RPSA-mediated events in HCMECs. Our study has led to better understanding of the roles of RPSA and caveolae/rafts upon SS2 infection, and a new pathological role for RPSA in infection.

Combination Therapy of Phage vB_KpnM_P-KP2 and Gentamicin Combats Acute Pneumonia Caused by K47 Serotype Klebsiella pneumoniae.

Klebsiella pneumoniae (K. pneumoniae) is an important nosocomial and community acquired opportunistic pathogen which causes various infections. The emergence of multi-drug resistant (MDR) K. pneumoniae and carbapenem-resistant hypervirulent K. pneumoniae (CR-hvKP) has brought more severe challenge to the treatment of K. pneumoniae infection. In this study, a novel bacteriophage that specifically infects K. pneumoniae was isolated and named as vB_KpnM_P-KP2 (abbreviated as P-KP2). The biological characteristics of P-KP2 and the bioinformatics of its genome were analyzed, and then the therapeutic effect of P-KP2 was tested by animal experiments. P-KP2 presents high lysis efficiency in vitro. The genome of P-KP2 shows homology with nine phages which belong to "KP15 virus" family and its genome comprises 172,138 bp and 264 ORFs. Besides, P-KP2 was comparable to gentamicin in the treatment of lethal pneumonia caused by K. pneumoniae W-KP2 (K47 serotype). Furthermore, the combined treatment of P-KP2 and gentamicin completely rescued the infected mice. Therefore, this study not only introduces a new member to the phage therapeutic library, but also serves as a reference for other phage-antibiotic combinations to combat MDR pathogens.

The Yersinia Phage X1 Administered Orally Efficiently Protects a Murine Chronic Enteritis Model Against Yersinia enterocolitica Infection.

Yersinia enterocolitica is generally considered an important food-borne pathogen worldwide, especially in the European Union. A lytic Yersinia phage X1 (Viruses; dsDNA viruses, no RNA stage; Caudovirales; and Myoviridae) was isolated. Phage X1 showed a broad host range and could effectively lyse 27/51 Y. enterocolitica strains covering various serotypes that cause yersiniosis in humans and animals (such as serotype O3 and serotype O8). The genome of this phage was sequenced and analyzed. No toxin, antibiotic-resistance or lysogeny related modules were found in the genome of phage X1. Studies of phage stability confirmed that X1 had a high tolerance toward a broad range of temperatures (4-60°C) and pH values (4-11) for 1 h. The ability to resist harsh acidic conditions and enzymatic degradation in vitro demonstrated that phage X1 is suitable for oral administration, and in particular, that this phage can pass the stomach barrier and efficiently reach the intestine in vivo without losing infectious ability. The potential of this phage against Y. enterocolitica infection in vitro was studied. In animal experiments, a single oral administration of phage X1 at 6 h post infection was sufficient to eliminate Y. enterocolitica in 33.3% of mice (15/45). In addition, the number of Y. enterocolitica strains in the mice was also dramatically reduced to approximately 103 CFU/g after 18 h compared with 107 CFU/g in the mice without phage treatment. Treatment with phage X1 showed significant improvement by intestinal histopathologic observations. Moreover, proinflammatory cytokine levels (IL-6, TNF-α, and IL-1ß) were significantly reduced (P < 0.05). These results indicate that phage X1 is a promising candidate to control infection by Y. enterocolitica in vivo.

Bacteriophage Protects Against Aerococcus viridans Infection in a Murine Mastitis Model.

Bovine mastitis, an inflammatory disease that occurs frequently in early lactation or the dry period, is primarily caused by bacterial infections. There is growing evidence that Aerococcus viridans (A. viridans) is becoming an important cause of bovine mastitis. The treatment of bovine mastitis is primarily based on antibiotics, which not only leads to a large economic burden but also the development of antibiotic resistance. On the other hand, bacteriophages present a promising alternative treatment strategy. The object of this study was to evaluate the potential of a previously isolated A. viridans phage vB_AviM_AVP (AVP) as an anti-mastitis agent in an experimental A. viridans-induced murine mastitis model. A. viridans N14 was isolated from the milk of clinical bovine mastitis and used to establish a mastitis model in mice. We demonstrated that administration of phage AVP significantly reduced colony formation by A. viridans and alleviated damage to breast tissue. In addition, reduced inflammation was indicated by decreased levels of inflammatory cytokines (TNF-α, IL-1ß, and IL-6) and myeloperoxidase (MPO) activity in the phage-treated group compared to those in the phosphate buffered saline (PBS)-treated group. To the best of our knowledge, this report is the first to show the potential use of phages as a treatment for A. viridans-induced mastitis.