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The bacterium Staphylococcus aureus, which colonizes healthy human skin, may cause diseases, such as atopic dermatitis (AD). Treatment for such AD cases involves antibiotic use; however, alternate treatments are preferred owing to the development of antimicrobial resistance. This study aimed to characterize the novel bacteriophage SaGU1 as a potential agent for phage therapy to treat S. aureus infections. SaGU1 that infects S. aureus strains previously isolated from the skin of patients with AD was screened from sewage samples in Gifu, Japan. Its genome was sequenced and analyzed using bioinformatics tools, and the morphology, lytic activity, stability, and host range of the phage were determined. The SaGU1 genome was 140,909 bp with an average GC content of 30.2%. The viral chromosome contained 225 putative protein-coding genes and four tRNA genes, carrying neither toxic nor antibiotic resistance genes. Electron microscopy analysis revealed that SaGU1 belongs to the Myoviridae family. Stability tests showed that SaGU1 was heat-stable under physiological and acidic conditions. Host range testing revealed that SaGU1 can infect a broad range of S. aureus clinical isolates present on the skin of AD patients, whereas it did not kill strains of Staphylococcus epidermidis, which are symbiotic resident bacteria on human skin. Hence, our data suggest that SaGU1 is a potential candidate for developing a phage therapy to treat AD caused by pathogenic S. aureus.

Atopic dermatitis is accompanied by the abnormal overgrowth of Staphylococcus aureus, a common cause of skin infections and an opportunistic pathogen. Although administration of antibiotics is effective against S. aureus, the resulting reduction in healthy microbiota and the emergence of drug-resistant bacteria are of concern. We propose that phage therapy can be an effective strategy to treat atopic dermatitis without perturbing the microbiota structure. In this study, we examined whether the S. aureus phage SaGU1 could be a tool to counteract the atopic exacerbation induced by S. aureus using an atopic mouse model. Administration of SaGU1 to the back skin of mice reduced both S. aureus counts and the disease exacerbation caused by S. aureus. Furthermore, the S. aureus-mediated exacerbation of atopic dermatitis with respect to IgE plasma concentration and histopathological findings was ameliorated by the application of SaGU1. We also found that Staphylococcus epidermidis, a typical epidermal symbiont in healthy skin, significantly attenuated the emergence of SaGU1-resistant S. aureus under co-culture with S. aureus and S. epidermidis in liquid culture infection experiments. Our results suggest that phage therapy using SaGU1 could be a promising clinical treatment for atopic dermatitis.

In response to the escalating global threat of antimicrobial resistance, our laboratory has established a phagemid packaging system for the generation of CRISPR-Cas13a-antimicrobial capsids targeting methicillin-resistant Staphylococcus aureus (MRSA). However, a significant challenge arose during the packaging process: the unintentional production of wild-type phages alongside the antimicrobial capsids. To address this issue, the phagemid packaging system was optimized by strategically incorporated silent mutations. This approach effectively minimized contamination risks without compromising packaging efficiency. The study identified the indispensable role of phage packaging genes, particularly terL-terS , in efficient phagemid packaging. Additionally, the elimination of homologous sequences between the phagemid and wild-type phage genome was crucial in preventing wild-type phage contamination. The optimized phagemid-LSAB(mosaic) demonstrated sequence-specific killing, efficiently eliminating MRSA strains carrying target antibiotic-resistant genes. While acknowledging the need for further exploration across bacterial species and in vivo validation, this refined phagemid packaging system offers a valuable advancement in the development of CRISPR-Cas13a-based antimicrobials, shedding light on potential solutions in the ongoing battle against bacterial infections.

The emergence of oxacillin-susceptible methicillin-resistant Staphylococcus aureus (OS-MRSA) strains has created new challenges for treating MRSA infections. These strains can become resistant to β-lactam antibiotics through chromosomal mutations, including those in the RNA polymerase (RNAP) genes such as rpoBC , leading to treatment failure. This study investigated the mechanisms underlying reduced β-lactam susceptibility in four rpoBC mutants of OS-MRSA. The results showed that rpoBC mutations caused RNAP transcription dysfunction, leading to an intracellular accumulation of ribonucleotides and precursors of peptidoglycan as well as wall teichoic acid. This, in turn, caused thickening of the cell wall and ultimately resulted in decreased susceptibility to β-lactam in OS-MRSA. These findings provide insights into the mechanisms of antibiotic resistance in OS-MRSA and highlight the importance of continued research in developing effective treatments to combat antibiotic resistance.

Metallo-β-lactamases (MBLs), such as those encoded by bla IMP-1 , confer resistance to carbapenem antibiotics and represent a critical challenge in treating infections caused by multidrug-resistant Pseudomonas aeruginosa . Here, we report a programmable antimicrobial strategy that restores bacterial antibiotic susceptibility through phage capsid-mediated delivery of CRISPR-Cas13a. We engineered a non-replicative phage capsid, which we called antibacterial capsid (AB-Capsid), packaged with a phagemid encoding a codon-optimized Cas13a from Leptotrichia shahii ( cas13aPA ) and a guide RNA targeting bla IMP-1 . The resulting construct, AB-Capsid_ cas13aPA _ bla IMP-1 , specifically inhibited the growth of bla IMP-1 -expressing P. aeruginosa and significantly reduced the minimum inhibitory concentration (MIC) of imipenem. No bactericidal effect was observed in the absence of the target gene or with a non-targeting AB-Capsid. Furthermore, spacer-dependent and expression-level-dependent killing activity was confirmed using inducible bla IMP-1 systems. These findings demonstrate that programmable AB-Capsids delivering Cas13a provide a gene-specific, non-replicative antimicrobial platform capable of reversing drug resistance and represent a versatile class of CRISPR-based antibiotic adjuvants.

Metallo-β-lactamases (MBLs), such as those encoded by bla , confer resistance to carbapenem antibiotics and represent a critical challenge in treating infections caused by multidrug-resistant Pseudomonas aeruginosa. Here, we report a programmable antimicrobial strategy that restores bacterial antibiotic susceptibility through phage capsid-mediated delivery of CRISPR-Cas13a. We engineered a non-replicative phage capsid, which we called antibacterial capsid (AB-Capsid), packaged with a phagemid encoding a codon-optimized Cas13a from Leptotrichia shahii (cas13aPA) and a guide RNA targeting bla . The resulting construct, AB-Capsid_cas13aPA_bla , specifically inhibited the growth of bla -expressing P. aeruginosa and significantly reduced the minimum inhibitory concentration (MIC) of imipenem. No bactericidal effect was observed in the absence of the target gene or with a non-targeting AB-Capsid. Furthermore, spacer-dependent and expression-level-dependent killing activity was confirmed using inducible bla systems. These findings demonstrate that programmable AB-Capsids delivering Cas13a provide a gene-specific, non-replicative antimicrobial platform capable of reversing drug resistance and represent a versatile class of CRISPR-based antibiotic adjuvants.

Abstract Metallo-β-lactamases (MBLs), such as those encoded by bla IMP-1, confer resistance to carbapenem antibiotics and represent a critical challenge in treating infections caused by multidrug-resistant Pseudomonas aeruginosa. Here, we report a programmable antimicrobial strategy that restores bacterial antibiotic susceptibility through phage capsid-mediated delivery of CRISPR-Cas13a. We engineered a non-replicative phage capsid, which we called antibacterial capsid (AB-Capsid), packaged with a phagemid encoding a codon-optimized Cas13a from Leptotrichia shahii (cas13aPA) and a guide RNA targeting bla IMP-1. The resulting construct, AB-Capsid_(c)as13aPA_(b)la IMP-1, specifically inhibited the growth of bla IMP-1-expressing P. aeruginosa and significantly reduced the minimum inhibitory concentration (MIC) of imipenem. No bactericidal effect was observed in the absence of the target gene or with a non-targeting AB-Capsid. Furthermore, spacer-dependent and expression-level-dependent killing activity was confirmed using inducible bla IMP-1 systems. These findings demonstrate that programmable AB-Capsids delivering Cas13a provide a gene-specific, non-replicative antimicrobial platform capable of reversing drug resistance and represent a versatile class of CRISPR-based antibiotic adjuvants.

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. Phagemid-based capsid system delivers CRISPR-Cas13a to target MRSA, showing efficient packaging, minimal wild-type phage contamination, and specific bactericidal activity, providing a promising alternative to antibiotics.

Abstract The bacterium Staphylococcus aureus, which grows on healthy human skin, may cause diseases such as atopic dermatitis (AD). Treatment for such AD cases involves antibiotic use; however, alternate treatments are preferred owing to the development of antimicrobial resistance. This study aimed to characterize the novel bacteriophage SaGU1 as a potential agent for phage therapy to treat S. aureus infections. SaGU1 that infects S. aureus strains previously isolated from the skin of patients with AD was screened from sewage samples in Gifu, Japan. Its genome was sequenced and analyzed using bioinformatics tools, and the morphology, lytic activity, stability, and host range of the phage were determined. The SaGU1 genome consisted of 140,909 bp with an average GC content of 30.2%. The viral chromosome contained putative 225 protein-coding genes and four tRNA genes, carrying neither toxic nor antibiotic resistance genes. Electron microscopy analysis revealed that SaGU1 belongs to the Myoviridae family. Stability tests showed that SaGU1 was heat-stable under physiological and acidic conditions. Host-range testing revealed that SaGU1 could infect a broad range of S. aureus clinical isolates present on the skin of patients with AD, whereas it did not kill strains of Staphylococcus epidermidis, which are symbiotic bacteria in the human skin microbiota. Our data suggest that SaGU1 is a potential candidate for developing a phage therapy to treat AD caused by pathogenic S. aureus. Competing Interest Statement The authors have declared no competing interest.