In response to the escalating antibiotic resistance in multidrug-resistant pathogens, we propose an innovative phagemid-based capsid system to generate CRISPR-Cas13a-loaded antibacterial capsids (AB-capsids) for targeted therapy against multidrug-resistant Staphylococcus aureus. Our optimized phagemid system maximizes AB-capsid yield and purity, showing a positive correlation with phagemid copy number. Notably, an 8.65-fold increase in copy number results in a 2.54-fold rise in AB-capsid generation. Phagemids carrying terL-terS-rinA-rinB (prophage-encoded packaging site genes) consistently exhibit high packaging efficiency, and the generation of AB-capsids using lysogenized hosts with terL-terS deletion resulted in comparatively lower level of wild-type phage contamination, with minimal compromise on AB-capsid yield. These generated AB-capsids selectively eliminate S. aureus strains carrying the target gene while sparing non-target strains. In conclusion, our phagemid-based capsid system stands as a promising avenue for developing sequence-specific bactericidal agents, offering a streamlined approach to combat antibiotic-resistant pathogens within the constraints of efficient production and targeted efficacy.

Staphylococcus species in food produce Staphylococcal enterotoxins (SEs) that cause Staphylococcal food poisoning (SFP). More than 20 SE types have been reported, among which Staphylococcal enterotoxin A (SEA) has been recognized as one of the most important SEs associated with SFP. However, the regulatory mechanisms underlying its production remain unclear. Previously, we identified a major SFP clone in Japan, CC81 subtype-1, which exhibits high SEA production. In this study, we attempted to identify the factors contributing to this phenomenon. Thus, we demonstrated that the attenuation of the activity of endogenous regulator, Staphylococcal accessory regulator S (SarS), and the lysogenization of a high SEA-producing phage contributed to this phenomenon in CC81 subtype-1. Furthermore, our results indicated that SarS could directly bind to the promoter upstream of the sea gene and suppress SEA expression; this low SarS repression activity was identified as one of the reasons for the high SEA production observed. Therefore, we revealed that both exogenous and endogenous factors may probably contribute to the high SEA production. Our results confirmed that SE production is a fundamental and critical factor in SFP and clarified the associated production mechanism while enhancing our understanding as to why a specific clone frequently causes SFP. IMPORTANCE: The importance of this study lies in its unveiling of a molecular regulatory mechanism associated with the most important food poisoning toxin and the evolution of Staphylococcal food poisoning (SFP)-associated clone. SFP is primarily caused by Staphylococcus aureus , with Staphylococcal enterotoxin A (SEA) being commonly involved in many cases. Thus, SEA has been recognized as a major toxin type. However, despite almost a century since its discovery, the complete mechanism of SEA production is as yet unknown. In this study, we analyzed an SEA-producing SFP clone isolated in East Asia and discovered that this strain, besides acquiring the high SEA-producing phage, exhibits remarkably high SEA production due to the low activity of SarS, an intrinsic regulatory factor. This is the first report documenting the evolution of the SFP clone through the coordinated action of exogenous mobile genetic factors and endogenous regulators on this notorious toxin.

Phage therapy has emerged as a promising alternative to conventional antimicrobial therapy for antimicrobial-resistant bacterial infections, but concerns about uncontrolled phage proliferation have limited its use. To address this issue, we established a nonproliferative phage-based DNA delivery system, called bacteria-targeting capsid particle (B-CAP), for the development of antimicrobial agents which effectively prevented phage spread while maintaining bactericidal activity. B-CAP is principally a T7 phage capsids packaged with a partial T7 phage genome, giving it the allowance to accommodate large foreign DNA up to 18 kb in length. We confirmed the efficacy of B-CAP in targeting and injecting its genome into bacteria, analogous to the wild-type phage. To demonstrate proof-of-concept and potential for developing an antimicrobial agent, we loaded colicin E1 operon onto the B-CAP system, resulting in the construction of B-CAP_ColE1, an antimicrobial agent capable of boosting colicin E1-based bacterial killing against Escherichia coli. Although no therapeutic effect was observed for B-CAP, B-CAP_ColE1 exhibited strong bactericidal activity against carbapenem-resistant E. coli both in vitro and in vivo, and significantly improved the survival of mice in an infection mouse model experiment. Finally, B-CAP_ColE1 is biologically contained or inert, reducing potential biological hazards during therapeutic use. Our results demonstrate that the B-CAP system offers a new strategy for developing nonreplicative phage-based antimicrobial agents against bacterial infections.

We have determined the complete genome sequence of a bacteriophage infecting Streptococcus equi subsp. equi , the causal agent of strangles in members of the family Equidae. This phage was isolated from wastewater and shows high similarity to C1 phage infecting other Streptococcus species.