Exploiting Broad-Spectrum Chimeric Lysin to Cooperate with Mupirocin against Staphylococcus aureus-Induced Skin Infections and Delay the Development of Mupirocin Resistance.

Staphylococcus aureus often leads to severe skin infections. However, S. aureus is facing a crisis of antibiotic resistance. The combination of phage and antibiotics is effective for drug-resistant S. aureus infections. Therefore, it is worth exploiting novel antibacterial agents to cooperate with antibiotics against S. aureus infections. Herein, a novel chimeric lysin ClyQ was constructed, which was composed of a cysteine- and histidine-dependent amidohydrolase/peptidase (CHAP) catalytic domain from S. aureus phage lysin LysGH15 and cell wall-binding domain (CBD) from Enterococcus faecalis phage lysin PlyV12. ClyQ had an exceptionally broad host range targeting streptococci, staphylococci, E. faecalis, and E. rhusiopathiae. ClyQ combined with mupirocin (2.64 log reduction) was more effective at treating S. aureus skin infections than ClyQ (0.46 log reduction) and mupirocin (2.23 log reduction) alone. Of equal importance, none of S. aureus ATCC 29213 or S3 exposed to ClyQ developed resistance, and the combination of ClyQ and mupirocin delayed the development of mupirocin resistance. Collectively, chimeric lysin ClyQ enriches the reservoirs for treating S. aureus infections. Our findings may provide a way to alleviate the current antibiotic resistance crisis. IMPORTANCE Staphylococcus aureus, as an Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species (ESKAPE) pathogen, can escape the elimination of existing antibiotics. At present, phages and phage lysins against S. aureus infections are considered alternative antibacterial agents. However, the development of broad-spectrum chimeric phage lysins to cooperate with antibiotics against S. aureus infections remains at its initial stage. In this study, we found that the broad-host-range chimeric lysin ClyQ can synergize with mupirocin to treat S. aureus skin infections. Furthermore, the development of S. aureus resistance to mupirocin is delayed by the combination of ClyQ and mupirocin in vitro. Our results bring research attention toward the development of chimeric lysin that cooperates with antibiotics to overcome bacterial infections.

Temperate phage influence virulence and biofilm-forming of Salmonella Typhimurium and enhance the ability to contaminate food product.

Salmonella is a food-borne zoonotic pathogen that threatens food safety and public health security. Temperate phages can influence bacterial virulence and phenotype and play an important role in bacterial evolution. However, most studies on Salmonella temperate phages focus on prophage induced by bacteria, with few reports on Salmonella temperate phages isolated in the environment. Moreover, whether temperate phages drive bacterial virulence and biofilm formation in food and animal models remains unknown. In this study, Salmonella temperate phage vB_Sal_PHB48 was isolated from sewage. TEM and phylogenetic analysis indicated that phage PHB48 belongs to the Myoviridae family. Additionally, Salmonella Typhimurium integrating PHB48 was screened and designated as Sal013+. Whole genome sequencing revealed that the integration site was specific and we confirmed that the integration of PHB48 did not change the O-antigen and coding sequences of Sal013. Our in vitro and in vivo studies showed that the integration of PHB48 could significantly enhance the virulence and biofilm formation of S. Typhimurium. More importantly, the integration of PHB48 significantly improved the colonization and contamination ability of bacteria in food samples. In conclusion, we isolated Salmonella temperate phage directly from the environment and systematically clarified that PHB48 enhanced the virulence and biofilm-forming ability of Salmonella. In addition, we found that PHB48 increased the colonization and contamination ability of Salmonella in food samples. These results indicated that the highly pathogenic Salmonella induced by temperate phage was more harmful to food matrices and public health security. Our results could enhance the understanding of the evolutionary relationship between bacteriophages and bacteria, and raise public awareness of large-scale outbreaks resulting from Salmonella virulence enhancement in food industry.

Streptococcus suis prophage lysin as a new strategy for combating streptococci-induced mastitis and Streptococcus suis infection.

OBJECTIVES: The genus Streptococcus contains species of important zoonotic pathogens such as those that cause bovine mastitis. Unfortunately, many Streptococcus species have developed antibiotic resistance. Phage lysins are considered promising alternatives to antibiotics because it is difficult for bacteria to develop lysin resistance. However, there remains a lack of phage lysin resources for the treatment of streptococci-induced mastitis. METHODS: We identified the prophage lysin Lys0859 from the genome of the Streptococcus suis SS0859 strain. Lys0859 was subsequently characterized to determine its host range, MIC, bactericidal activity in milk, and ability to clear biofilms in vitro. Finally, to determine the effects of Lys0859 on the treatment of both bovine mastitis and S. suis infection in vivo, we established models of Streptococcus agalactiae ATCC 13813-induced mastitis and S. suis serotype 2 SC19 systemic infection. RESULTS: Our results demonstrate that Lys0859 possesses broad-spectrum lytic activity against Streptococcus and Staphylococcus species isolated from animals with bovine mastitis and 15 serotypes of S. suis isolated from swine. Intramammary and intramuscular injection of Lys0859 reduced the number of bacteria in mammary tissue by 3.75 and 1.45 logs compared with the PBS group, respectively. Furthermore, 100 µg/mouse of Lys0859 administered intraperitoneally at 1 h post-infection protected 83.3% (5/6) of mice from a lethal dose of S. suis infection. CONCLUSIONS: Overall, our results enhance the understanding and development of new strategies to combat both streptococci-induced mastitis and S. suis infection.

Exploring the Benefits of Metal Ions in Phage Cocktail for the Treatment of Methicillin-Resistant Staphylococcus aureus (MRSA) Infection.

Background: Methicillin-resistant Staphylococcus aureus (MRSA) is an important zoonotic pathogen worldwide. Infections due to MRSA are associated with higher mortality rates compared with methicillin-susceptible S. aureus. Meanwhile, bacteriophages have been shown to overcome the emergence of MRSA. Methods: Phage PHB22a, PHB25a, PHB38a, and PHB40a were isolated. Here, we evaluated the ability of a phage cocktail containing phages PHB22a, PHB25a, PHB38a, and PHB40a against MRSA S-18 strain in vivo and in vitro. Phage whole-genome sequencing, host-range determination, lytic activity, and biofilm clearance experiments were performed in vitro. Galleria mellonella larvae and a mouse systemic infection model to evaluate the efficacy of phage therapy in vivo. Results: The phage cocktail exhibited enhanced antibacterial and anti-biofilm effects compared to the single phage. Phage cocktail contained with Ca2+/Zn2+ significantly reduced the number of viable bacteria (24-h or 48-h biofilm) by more than 0.81-log compared to the phage cocktail alone. Furthermore, we demonstrated that the addition of Ca2+ and Zn2+ phage cocktail could increase the survival rate of G. mellonella larvae infected with S. aureus by 10% compared with phage cocktail alone. This was further confirmed in the mouse model, which showed a 2.64-log reduction of host bacteria S-18, when Ca2+ and Zn2+ were included in the cocktail compared with the phage cocktail alone. Conclusion: Our results indicated that phage cocktail supplemented with Ca2+/Zn2+ could effectively remove bacteria in biofilms and mice tissues infected with S. aureus.

Effect of flexible linker length on the activity of fusion protein 4-coumaroyl-CoA ligase::stilbene synthase.

In order to elucidate the effect of flexible linker length on the catalytic efficiency of fusion proteins, two short flexible peptide linkers of various lengths were fused between Arabidopsis thaliana 4-coumaroyl-CoA ligase (4CL) and Polygonum cuspidatum stilbene synthase (STS) to generate fusion proteins 4CL-(GSG)n-STS (n ≤ 5) and 4CL-(GGGGS)n-STS (n ≤ 4). The fusion proteins were expressed in both Escherichia coli and Saccharomyces cerevisiae, and their bioactivities were tested in vitro and in vivo using purified proteins and engineered strains, respectively. The catalytic efficiency of the fusions decreased gradually with the increase of GSG or GGGGS repeats. In both engineered S. cerevisiae and E. coli in vivo experiments, the capacity of resveratrol production decreased gradually with increasing linker length. In silico analysis showed that the prediction of homology models of fusion proteins was consistent with the in vitro and in vivo results.