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Background Mpox virus (MPXV) is a zoonotic orthopoxvirus and caused an outbreak in 2022. Although tecovirimat and brincidofovir are approved as anti-smallpox drugs, their effects in mpox patients have not been well documented. In this study, by a drug repurposing approach, we identified potential drug candidates for treating mpox and predicted their clinical impacts by mathematical modeling. Methods We screened 132 approved drugs using an MPXV infection cell system. We quantified antiviral activities of potential drug candidates by measuring intracellular viral DNA and analyzed the modes of action by time-of-addition assay and electron microscopic analysis. We further predicted the efficacy of drugs under clinical concentrations by mathematical simulation and examined combination treatment. Results Atovaquone, mefloquine, and molnupiravir exhibited anti-MPXV activity, with 50% inhibitory concentrations of 0.51–5.2 μM, which was more potent than cidofovir. Whereas mefloquine was suggested to inhibit viral entry, atovaquone and molnupiravir targeted postentry processes. Atovaquone was suggested to exert its activity through inhibiting dihydroorotate dehydrogenase. Combining atovaquone with tecovirimat enhanced the anti-MPXV effect of tecovirimat. Quantitative mathematical simulations predicted that atovaquone can promote viral clearance in patients by 7 days at clinically relevant drug concentrations. Conclusions These data suggest that atovaquone would be a potential candidate for treating mpox.

Pseudomonas aeruginosa is an opportunistic pathogen that causes nosocomial disease among immunocompromised and chronic cystic fibrosis (CF) patients. We characterized two newly isolated Pseudomonas phages, ϕPA01 and ϕPA02, with different host spectra, and examined their effect as a cocktail with antibiotics against P. aeruginosa, to indicate the possibility of combining a phage cocktail and antibiotics in treating pseudomonal infection. Phages ϕPA01 (66,220 bp) and ϕPA02 (279,095 bp) belong to the genus Pbunalikevirus and Phikzlikevirus, respectively. No virulence or lysogenic associated gene was found in their genomes, thus they are potentially safe for phage therapy. We generated respective phage-resistant strains to investigate cross-resistance between two phages. Slight cross-resistance to ϕPA02 in ϕPA01-resistant strain was observed, while ϕPA02-resistant strain remained susceptible to ϕPA01. A ϕPA01 resistant strain that was cross-resistant to ϕPA02 appeared in round 5 (R5-PA01R), revealed frameshift mutation in phosphoglucomutase (algC), which is important for the synthesis of core lipopolysaccharide (LPS). Knockout of algC was resistant to both phages. Complementation of ΔalgC restored phages' infectivity, suggesting that LPS as host receptor. Phage cocktail suppressed the growth of P. aeruginosa for longer (20 h) hour compared with single phage (8-9 h), further suggesting their potential to be used as a phage cocktail. Furthermore, application of the phage cocktail with ciprofloxacin (0.25 μg/ml) and meropenem (2 μg/ml), managed to suppress the growth of P. aeruginosa up to 96 h. Our results show the potential application of ϕPA01 and ϕPA02 as phage cocktail together with antibiotics for treatment of P. aeruginosa.

Cancer, especially the solid tumor sub-set, poses considerable challenges to modern medicine owing to the unique physiological characteristics and substantial variations in each tumor’s microenvironmental niche fingerprints. Though there are many treatment methods available to treat solid tumors, still a considerable loss of life happens, due to the limitation of treatment options and the outcomes of ineffective treatments. Cancer cells evolve with chemo- or radiation-treatment strategies and later show adaptive behavior, leading to failed treatment. These challenges demand tailored and individually apt personalized treatment methods. Bacteriophages (or phages) and phage-based theragnostic vectors are gaining attention in the field of modern cancer medicine, beyond their bactericidal ability. With the invention of the latest techniques to fine-tune phages, such as in the field of genetic engineering, synthetic assembly methods, phage display, and chemical modifications, noteworthy progress in phage vector research for safe cancer application has been realized, including use in pre-clinical studies. Herein, we discuss the distinct fingerprints of solid tumor physiology and the potential for bacteriophage vectors to exploit specific tumor features for improvised tumor theragnostic applications.

The bacteriophage (or phage for short) has been used as an antibacterial agent for over a century but was abandoned in most countries after the discovery and broad use of antibiotics. The worldwide emergence and high prevalence of antimicrobial-resistant (AMR) bacteria have led to a revival of interest in the long-forgotten antibacterial therapy with phages (phage therapy) as an alternative approach to combatting AMR bacteria. The rapid progress recently made in molecular biology and genetic engineering has accelerated the generation of phage-related products with superior therapeutic potentials against bacterial infection. Nowadays, phage-based technology has been developed for many purposes, including those beyond the framework of antibacterial treatment, such as to suppress viruses by phages, gene therapy, vaccine development, etc. Here, we highlighted the current progress in phage engineering technology and its application in modern medicine.

Staphylococcus aureus strains that are susceptible to the β-lactam antibiotic oxacillin despite carrying mecA (OS-MRSA) cause serious clinical problems globally because of their ability to easily acquire β-lactam resistance. Understanding the genetic mechanism(s) of acquisition of the resistance is therefore crucial for infection control management. For this purpose, a whole-genome sequencing-based analysis was performed using 43 clinical OS-MRSA strains and 100 mutants with reduced susceptibility to oxacillin (MICs 1.0–256 µg/mL) generated from 26 representative OS-MRSA strains. Genome comparison between the mutants and their respective parent strains identified a total of 141 mutations in 46 genes and 8 intergenic regions. Among them, the mutations are frequently found in genes related to RNA polymerase (rpoBC), purine biosynthesis (guaA, prs, hprT), (p)ppGpp synthesis (relSau), glycolysis (pykA, fbaA, fruB), protein quality control (clpXP, ftsH), and tRNA synthase (lysS, gltX), whereas no mutations existed in mec and bla operons. Whole-genome transcriptional profile of the resistant mutants demonstrated that expression of genes associated with purine biosynthesis, protein quality control, and tRNA synthesis were significantly inhibited similar to the massive transcription downregulation seen in S. aureus during the stringent response, while the levels of mecA expression and PBP2a production were varied. We conclude that a combination effect of mecA upregulation and stringent-like response may play an important role in acquisition of β-lactam resistance in OS-MRSA.

We first reported a phenomenon of cross-resistance to vancomycin (VCM) and daptomycin (DAP) in methicillin-resistant Staphylococcus aureus (MRSA) in 2006, but mechanisms underlying the cross-resistance remain incompletely understood. Here, we present a follow-up study aimed to investigate genetic determinants associated with the cross-resistance. Using 12 sets of paired DAP susceptible (DAPS) and DAP non-susceptible (DAPR) MRSA isolates from 12 patients who had DAP therapy, we (i) assessed susceptibility to DAP and VCM, (ii) compared whole-genome sequences, (iii) identified mutations associated with cross-resistance to DAP and VCM, and (iv) investigated the impact of altered gene expression and metabolic pathway relevant to the cross-resistance. We found that all 12 DAPR strains exhibiting cross-resistance to DAP and VCM carried mutations in mprF, while one DAPR strain with reduced susceptibility to only DAP carried a lacF mutation. On the other hand, among the 32 vancomycin-intermediate S. aureus (VISA) strains isolated from patients treated with VCM, five out of the 18 strains showing cross-resistance to DAP and VCM carried a mprF mutation, while 14 strains resistant to only VCM had no mprF mutation. Moreover, substitution of mprF in a DAPS strain with mutated mprF resulted in cross-resistance and vice versa. The elevated lysyl-phosphatidylglycerol (L-PG) production, increased positive bacterial surface charges and activated cell wall (CW) synthetic pathways were commonly found in both clinical isolates and laboratory-developed mutants that carry mprF mutations. We conclude that mprF mutation is responsible for the cross-resistance of MRSA to DAP and VCM, and treatment with DAP is more likely to select for mprF-mediated cross-resistance than is with VCM.

The association of Panton-Valentine leukocidin (PVL) toxin with necrotizing soft tissue infection (NSTI) caused by Staphylococcus aureus remains controversial. Here, we report the complete genome sequence of the PVL-negative S. aureus strain JMUB1273, isolated from a patient with pervasive NSTI.

The emergence of antimicrobial-resistant bacteria is an increasingly serious threat to global health, necessitating the development of innovative antimicrobials. Here we report the development of a series of CRISPR-Cas13a-based antibacterial nucleocapsids, termed CapsidCas13a(s), capable of sequence-specific killing of carbapenem-resistant Escherichia coli and methicillin-resistant Staphylococcus aureus by recognizing corresponding antimicrobial resistance genes. CapsidCas13a constructs are generated by packaging programmed CRISPR-Cas13a into a bacteriophage capsid to target antimicrobial resistance genes. Contrary to Cas9-based antimicrobials that lack bacterial killing capacity when the target genes are located on a plasmid, the CapsidCas13a(s) exhibit strong bacterial killing activities upon recognizing target genes regardless of their location. Moreover, we also demonstrate that the CapsidCas13a(s) can be applied to detect bacterial genes through gene-specific depletion of bacteria without employing nucleic acid manipulation and optical visualization devices. Our data underscore the potential of CapsidCas13a(s) as both therapeutic agents against antimicrobial-resistant bacteria and nonchemical agents for detection of bacterial genes., CRISPR technology is emerging as a potential antimicrobial against antimicrobial-resistant bacteria. Here the authors develop a bacteriophage delivered Cas13a system for killing target bacteria and detecting bacterial genes.

Emergence of antimicrobial-resistant bacteria is an increasingly serious threat to global health, necessitating the development of innovative antimicrobials. We established a series of CRISPR-Cas13a-based antibacterial nucleocapsid, termed CapsidCas13a(s), capable of sequence-specific killing of carbapenem-resistant Escherichia coli and methicillin-resistant Staphylococcus aureus through promiscuous RNA cleavage after recognizing corresponding antimicrobial resistance genes. CapsidCas13a constructs were generated by packaging CRISPR-Cas13a into a bacteriophage capsid to target antimicrobial resistance genes. Contrary to Cas9-based antimicrobials that lack bacterial killing capacity when the target genes are located on a plasmid, the CapsidCas13a(s) exhibited strong bacterial killing activities upon recognizing target genes regardless of their location. The antimicrobials’ treatment efficacy was confirmed using a Galleria mellonella larvae model. Further, we demonstrated that the CapsidCas13a(s) can assist in bacterial gene detection without employing nucleic acid amplification and optical devices.

Clustered regularly interspaced short palindromic repeats (CRISPR)-Cas13a, previously known as CRISPR-C2c2, is the most recently identified RNA-guided RNA-targeting CRISPR-Cas system and has the unique characteristics of both targeted and collateral single-stranded RNA (ssRNA) cleavage activities, which was first identified in Leptotrichia shahii CRISPR-Cas13a. Here, the complete whole genome sequences of 11 Leptotrichia strains were determined and compared with 18 publicly available Leptotrichia genomes in regard to the composition, occurrence and diversity of the CRISPR-Cas13a and other CRISPR-Cas systems. Various types of CRISPR-Cas systems, I-B, II-C, III-A, III-D, III-like and VI-A, were unevenly distributed among the Leptotrichia genomes, accounting for 10 (34.4%), 1 (2.6%), 6 (15.4%), 6 (15.4%), 3 (7.7%) and 11 (37.9%) of the 29 strains, respectively, while 8 (20.5%) strains had no CRISPR-Cas system. The Cas13a effectors were highly divergent with amino acid sequence similarities ranging from 61% to 90% to that of L. shahii and their collateral ssRNA cleavage activities, resulting in cytotoxicity to host cell, were found to be maintained. CRISPR-Cas spacers represent a sequential achievement of former intruder encounters, and the retained spacers reflect the evolutionary phylogeny or relatedness of strains. Analysis of spacer contents and numbers among Leptotrichia species showed considerable diversity with only 2 (0.5%) of 400 spacers through CRIPSR-Cas I-VI were shared by two strains. The organisation and distribution of CRISPR-Cas systems (type I-VI) encoded by all registered Leptotrichia species revealed that effector or spacer sequences of the CRISPR-Cas systems were very divergent, and the prevalence of types I, III and VI was almost equal. There was only one strain carrying type II, while none carried type IV or V. These results provide new insights into the characteristics and divergences of CRISPR-Cas systems among Leptotrichia species.

Following the emergence of antibiotic resistant bacteria such as methicillin-resistant Staphylococcus aureus (MRSA) and methicillin-resistant Staphylococcus pseudintermedius (MRSP), phage therapy has attracted significant attention as an alternative to antibiotic treatment. Bacteriophages belonging to kayvirus (previously known as Twort-like phages) have broad host range and are strictly lytic in Staphylococcus spp. Previous work revealed that kayvirus ɸSA039 has a host-recognition mechanism distinct from those of other known kayviruses: most of kayviruses use the backbone of wall teichoic acid (WTA) as their receptor; by contrast, ɸSA039 uses the β-N-acetylglucosamine (β-GlcNAc) residue in WTA. In this study, we found that ɸSA039 could switch its receptor to be able to infect S. aureus lacking the β-GlcNAc residue by acquiring a spontaneous mutation in open reading frame (ORF) 100 and ORF102. Moreover, ɸSA039 could infect S. pseudintermedius, which has a different WTA structure than S. aureus. By comparison with newly isolated S. pseudintermedius–specific phage (SP phages), we determined that glycosylation in WTA of S. pseudintermedius is essential for adsorption of SP phages, but not ɸSA039. Finally, we describe a novel strategy of S. aureus which protects the bacteria from infection of SP phages. Notably, glycosylation of ribitol phosphate (RboP) WTA by TarM or/and TarS prevents infection of S. aureus by SP phages. These findings could help to establish a new strategy for treatment of S. aureus and S. pseudintermedius infection, as well as provide valuable insights into the biology of phage–host interactions.

Bacteriophage has become an attractive alternative for the treatment of antibiotic-resistant Staphylococcus aureus. For the success of phage therapy, phage host range is an important criterion when considering a candidate phage. Most reviews of S. aureus (SA) phages have focused on their impact on host evolution, especially their contribution to the spread of virulence genes and pathogenesis factors. The potential therapeutic use of SA phages, especially detailed characterizations of host recognition mechanisms, has not been extensively reviewed so far. In this report, we provide updates on the study of SA phages, focusing on host recognition mechanisms with the recent discovery of phage receptor-binding proteins (RBPs) and the possible applications of SA phages in phage therapy.

We have previously generated strains ofStaphylococcus aureusSA003 resistant to its specific phage ϕSA012 through long-term coevolution experiment. However, the DNA mutations responsible for the phenotypic change of phage resistance are unknown. Whole-genome analysis revealed six genes that acquired unique point mutations: five missense mutations and one nonsense mutation. Moreover, one deletion, 1.779-bp, resulted in the deletion of the genes encoding glycosyltransferase, TarS, and iron-sulfure repair protein, ScdA. The deletion occurred from the second round of coculture (SA003R2) and remained through the last round. The ϕSA012 infection toward SA003R2 had decreased to 79.77±7.50% according to plating efficiency. Complementation of the phage-resistant strain by the wild-type allele showed two mutated host genes were linked to the inhibition of post-adsorption, and five genes were linked to phage adsorption of ϕSA012. Unlike ϕSA012, infection by ϕSA039, a close relative of ϕSA012, onto SA003R2 was impaired drastically. Complementation of SA003R2 by wild-typetarSrestores the infectivity of ϕSA039. Thus, we concluded that ϕSA039 requires β-GlcNAc in Wall Teichoic Acid (WTA) for its binding. In silico analysis of the ϕSA039 genome revealed that several proteins in the tail and baseplate region were different from ϕSA012; notably the partial deletion oforf96of ϕSA039, a homolog oforf99of ϕSA012.Orf100of ϕSA039, a homolog ofOrf103of ϕSA012, a previously reported receptor binding protein (RBP), had low similarity (86%) to that of ϕSA012. The difference in tail and baseplate proteins might be the factor for specificity difference between ϕSA012 and ϕSA039.