Staphylococcus aureus (S. aureus) is a Gram-positive pathogen causing a variety of infections in humans and animals. Extensive use of antibiotics has led to the emergence of methicillin-resistant S. aureus (MRSA). As an alternative antibacterial agent against drug-resistant S. aureus, a lytic phage, designated SLPW, was isolated from fecal sewage in a pig farm. The SLPW was morphologically classified under Podoviridae and contains a double-stranded DNA genome. The genome of SLPW was 17,861 bp (29.35% G+C) containing 20 open reading frames and lacked regions encoding lysogeny-related integrase gene and cI repressor gene. Phage SLPW showed a broad host range and high efficiency of plating against various types of S. aureus. One-step growth curve showed a short latency period (10 min) and a long lytic period (120 min). Phage SLPW remained stable under a wide range of temperatures or pH and was almost unaffected in chloroform or ultraviolet light. Further, it efficiently lysed MRSA strains in vitro and in vivo. Intraperitoneal phage administration at 1 h post-infection cured the mice and reduced the bacterial expression of inflammatory cytokines in mice. Specifically, the phage SLPW displayed a wide antibacterial spectrum. It was therapeutically effective against intra-abdominal infection in mice harboring different multilocus sequence typing (MLST) types of S. aureus strains. Therefore, phage SLPW is a potential therapeutic agent against MRSA infections.

Stimulator of IFN genes (STING) is an adaptor that functions downstream of retinoic acid–inducible gene I (RIG-I) in mammalian cells; however, RIG-I is absent in chickens. We identified chicken STING (chSTING) as a critical mediator of virus-triggered type I IFN signaling in RIG-I–null chicken cells. Overexpression of chSTING in DF-1 cells inhibited Newcastle disease virus and avian influenza virus (AIV) viral replication and activated IRF-7 and NF-κB to induce expression of type I IFNs. Knockdown of endogenous chSTING abolished virus-triggered activation of IRF-7 and IFN-β and increased viral yield. chSTING was a critical component in the virus-triggered IRF-7 activation pathway and the cellular antiviral response. chSTING predominantly localized to the outer membrane of the endoplasmic reticulum and was also found in the mitochondrial membrane. Furthermore, knockdown of chSTING blocked polyinosinic-polycytidylic acid–, poly(deoxyadenylic-deoxythymidylic) acid–, and melanoma differentiation–associated gene 5 (MDA5)-stimulated induction of IFN-β. Coimmunoprecipitation experiments indicated that chicken MDA5 could interact with chSTING, and this interaction was enhanced by ectopically expressed chicken mitochondrial antiviral-signaling protein. Together, these results indicated that chSTING is an important regulator of chicken innate immune signaling and might be involved in the MDA5 signaling pathway in chicken cells. These results help with understanding the biological role of STING in innate immunity during evolution.

Streptococcus suis serotype 2 (SS2) is an important zoonotic pathogen that causes serious diseases in pigs and humans. GdpP protein is a recently discovered specific phosphodiesterase that degrades cyclic diadenosine monophosphate (c-di-AMP). It is widely distributed among the firmicutes phylum and altered expression of GdpP is associated with several phenotypes in various bacterial strains. We investigated the role of GdpP in physiology and virulence in SS2. An in-frame mutant of gdpP was constructed using homologous recombination and bacterial growth, biofilm formation, hemolytic activity, cell adherence and invasion, expression of virulence factors, and virulence were evaluated. Disruption of gdpP increased intracellular c-di-AMP level and affected growth and increased biofilm formation of SS2. Simultaneously, the gdpP mutant strain exhibited a significant decrease in hemolytic activity and adherence to and invasion of HEp-2 cells compared with the parental strain. Quantitative reverse transcriptase polymerase chain reaction indicated significantly reduced expression of the known virulence genes cps2, sly, fpbs, mrp, ef and gdh in the gdpP mutant. In murine infection models, the gdpP mutant strain was attenuated, and impaired bacterial growth was observed in specific organs. All these findings revealed a significant contribution of gdpP and its substrate (c-di-AMP) to the biology and virulence of SS2.

Bacterial biofilms are crucial to the pathogenesis of many important infections and are difficult to eradicate. Streptococcus suis is an important pathogen of pigs, and here the biofilm-forming ability of 32 strains of this species was determined. Significant biofilms were completely formed by 10 of the strains after 60 h of incubation, with exopolysaccharide production in the biofilm significantly higher than that in the corresponding planktonic cultures. S. suis strain SS2-4 formed a dense biofilm, as revealed by scanning electron microscopy, and in this state exhibited increased resistance to a number of antibiotics (ampicillin, amoxicillin, ciprofloxacin, kanamycin, and rifampin) compared to that of planktonic cultures. A bacteriophage lysin, designated LySMP, was used to attack biofilms alone and in combination with antibiotics and bacteriophage. The results demonstrated that the biofilms formed by S. suis, especially strains SS2-4 and SS2-H, could be dispersed by LySMP and with >80% removal compared to a biofilm reduction by treatment with either antibiotics or bacteriophage alone of less than 20%; in addition to disruption of the biofilm structure, the S. suis cells themselves were inactivated by LySMP. The efficacy of LySMP was not dose dependent, and in combination with antibiotics, it acted synergistically to maximize dispersal of the S. suis biofilm and inactivate the released cells. These data suggest that bacteriophage lysin could form part of an effective strategy to treat S. suis infections and represents a new class of antibiofilm agents.

IFN regulatory factor (IRF) 3 has been identified as the most critical regulator of both RNA and DNA virus–induced IFN production in mammals. However, ambiguity exists in research on chicken IRFs; in particular IRF3 seems to be missing in chickens, making IFN regulation in chickens unclear. In this study, we comprehensively investigated the potential IFN-related IRFs in chickens and showed that IRF7 is the most critical IFN-β regulator in chickens. With a chicken IRF7 (chIRF7) knockout DF-1 cell line, we conducted a series of experiments to demonstrate that chIRF7 is involved in both chicken STING (chSTING)- and chicken MAVS (chMAVS)-mediated IFN-β regulation in response to DNA and RNA viral infections, respectively. We further examined the mechanisms of chIRF7 activation by chSTING. We found that chicken TBK1 (chTBK1) is indispensable for chIRF7 activation by chSTING as well as that chSTING interacts with both chIRF7 and chTBK1 to function as a scaffold in chIRF7 activation by chTBK1. More interestingly, we discovered that chSTING mediates the activation of chIRF7 through a conserved SLQxSyS motif. In short, we confirmed that although IRF3 is missing in chickens, they employ IRF7 to reconstitute corresponding IFN signaling to respond to both DNA and RNA viral infections. Additionally, we uncovered a mechanism of chIRF7 activation by chSTING. The results will enrich and deepen our understanding of the regulatory mechanisms of the chicken IFN system.