笹原 鉄平

Metallo-β-lactamases (MBLs), such as those encoded by blaIMP-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 blaIMP-1. The resulting construct, AB-Capsid_cas13aPA_blaIMP-1, specifically inhibited the growth of blaIMP-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 blaIMP-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.

Background/Objectives: Methicillin-resistant Staphylococcus aureus (MRSA) colonization in geriatric long-term care facilities (LTCFs) is a global concern. However, the transmission dynamics of MRSA among LTCF residents in Japan remain largely unknown. Methods: Whole-genome sequencing was conducted on 85 MRSA isolates obtained from 76 residents across 4 geriatric LTCFs in Japan. Single-nucleotide polymorphism (SNP) analysis was performed to identify the transmission dynamics, with a threshold of ≤15 pairwise core-genome SNP distances defining recent transmission clusters (genomic clusters). Antimicrobial susceptibility testing and investigation of antimicrobial resistance genes were also performed. Results: Among the 76 MRSA-carrying residents, 34 (44.7%) belonged to 14 genomic clusters, including strains from clinical specimens of 7 individuals. Three individuals acquired MRSA strains within the LTCFs, which were part of genomic clusters. Conversely, 14 residents who underwent testing immediately after admission carried MRSA strains within genomic clusters, suggesting transmission prior to their LTCF admission. MRSA isolates that were prevalent among LTCF residents were generally susceptible to trimethoprim-sulfamethoxazole but resistant to levofloxacin and clindamycin. Conclusions: Acquisition of MRSA genomic cluster strains among LTCF residents can occur both during and before admission to the facility. These findings underscore the need for measures that mitigate MRSA transmission inside and outside LTCFs.

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.

Leishmaniasis is caused by an obligate intracellular protozoa of the genus Leishmania. Its clinical manifestations include cutaneous, mucocutaneous, and visceral forms. Sporotrichoid cutaneous leishmaniasis (SCL) is an atypical and rare form of cutaneous leishmaniasis (CL) reported mainly in the Old World. This case report describes SCL in a Japanese man infected with Leishmania (Viannia) peruviana in Peru. His lesions occurred on both feet, with the left foot lesion being a simple CL that resolved spontaneously. However, the lesion on the right foot did not cure by itself; instead, it progressed centrally along the lymph nodes, eventually forming an SCL. Amastigotes were detected in both feet and genetically identified as L. (V.) peruviana. The lesions gradually resolved after treatment with intravenous liposomal amphotericin B. Here, we report the first case of SCL caused by L. (V.) peruviana.