Design, synthesis, and evaluation of N1,N3-dialkyldioxonaphthoimidazoliums as antibacterial agents against methicillin-resistant Staphylococcus aureus.

Increasing antibiotic resistance of bacterial pathogens poses a serious threat to human health worldwide. Methicillin-resistant Staphylococcus aureus (MRSA) is among the most deleterious bacterial pathogens owing to its multidrug resistance, necessitating the development of new antibacterial agents against it. We previously identified a novel dioxonaphthoimidazolium agent, c5, with moderate antibacterial activity against MRSA from an anticancer clinical candidate, YM155. In this study, we aimed to design and synthesize several novel cationic amphiphilic N1,N3-dialkyldioxonaphthoimidazolium bromides with enhanced lipophilicity of the two side chains in the imidazolium scaffold and improved antibacterial activities compared to those of c5 against gram-positive bacteria in vitro and in vivo. Our new antibacterial lead, N1,N3-n-octylbenzyldioxonaphthoimidazolium bromide (11), exhibited highly potent antibacterial activities against various gram-positive bacterial strains (MICs: 0.19-0.39 µg/mL), including MRSA, methicillin-sensitive S. aureus, and Bacillus subtilis. Moreover, antibacterial mechanism of 11 against MRSA based on the generation of reactive oxygen species (ROS) was evaluated. Although compound 11 exhibited cytotoxic effects in vitro and lacked a therapeutic index against the HEK293 and HDFa mammalian cell lines, it exhibited low toxicity in the Drosophila animal model. Remarkably, 11 exhibited better in vivo antibacterial efficacy than c5 and the clinically used antibiotic, vancomycin, in SA3-infected Drosophila model. Moreover, the development of bacterial resistance to 11 was not observed after 16 consecutive passages. Therefore, rational design of antibacterial cationic amphiphiles based on ROS-generating pharmacophores with optimized lipophilicity can facilitate the identification of potent antibacterial agents against drug-resistant infections.

Diabetic Ketoacidosis Associated With Second Generation Antipsychotics: A Case Study and Review of Literature.

OBJECTIVE: Second-generation antipsychotics (SGAs) have revolutionized the treatment of psychiatric disorders, but are associated with significant metabolic risks, including diabetes and hyperglycemic crises. This review explores the complex interplay between antipsychotics, diabetes, and hyperglycemic crises, highlighting the mechanisms underlying SGA-induced diabetes. METHODS: We present the case of a patient with schizophrenia who was taking antipsychotic medication and was admitted to the emergency room due to the sudden onset of diabetic ketoacidosis (DKA) without any history of diabetes. We extensively searched databases, including Elsevier, PubMed, IEEE, SpringerLink, and Google Scholar, for papers on the effects of antipsychotic drugs on DKA from 2002 to 2021. We focused on DKA, hyperglycemia, and atypical antipsychotics, and retrieved 117 papers. After full-text review, 32 papers were included in this comprehensive review. RESULTS: DKA was significantly more frequent in patients taking SGAs. Antipsychotics can induce insulin resistance either directly or through the onset of obesity. Antipsychotics can reduce insulin secretion from pancreatic ß-cells, which is associated with absolute insulin deficiency. CONCLUSION: As the use of antipsychotics continues to increase, understanding their risks and mechanisms is crucial for clinicians to enable informed treatment decisions and prevent potentially life-threatening complications.

Autolysis of Pseudomonas aeruginosa Quorum-Sensing Mutant Is Suppressed by Staphylococcus aureus through Iron-Dependent Metabolism.

Microorganisms usually coexist as a multifaceted polymicrobial community in the natural habitats and at mucosal sites of the human body. Two opportunistic human pathogens, Pseudomonas aeruginosa and Staphylococcus aureus commonly coexist in the bacterial infections for hospitalized and/or immunocompromised patients. Here, we observed that autolysis of the P. aeruginosa quorum-sensing (QS) mutant (lasRmvfR) was suppressed by the presence of the S. aureus cells in vitro. The QS mutant still displayed killing against S. aureus cells, suggesting the link between the S. aureus-killing activity and the autolysis suppression. Independent screens of the P. aeruginosa transposon mutants defective in the S. aureus-killing and the S. aureus transposon mutants devoid of the autolysis suppression revealed the genetic link between both phenotypes, suggesting that the iron-dependent metabolism involving S. aureus exoproteins might be central to both phenotypes. The autolysis was suppressed by iron treatment as well. These results suggest that the interaction between P. aeruginosa and S. aureus might be governed by mechanisms that necessitate the QS circuitry as well as the metabolism involving the extracellular iron resources during the polymicrobial infections in the human airway.

Use of Cas9 Targeting and Red Recombination for Designer Phage Engineering.

Bacteriophages (phages) are natural antibiotics and biological nanoparticles, whose application is significantly boosted by recent advances of synthetic biology tools. Designer phages are synthetic phages created by genome engineering in a way to increase the benefits or decrease the drawbacks of natural phages. Here we report the development of a straightforward genome engineering method to efficiently obtain engineered phages in a model bacterial pathogen, Pseudomonas aeruginosa. This was achieved by eliminating the wild type phages based on the Streptococcus pyogenes Cas9 (SpCas9) and facilitating the recombinant generation based on the Red recombination system of the coliphage λ (λRed). The producer (PD) cells of P. aeruginosa strain PAO1 was created by miniTn7-based chromosomal integration of the genes for SpCas9 and λRed under an inducible promoter. To validate the efficiency of the recombinant generation, we created the fluorescent phages from a temperate phage MP29. A plasmid bearing the single guide RNA (sgRNA) gene for selectively targeting the wild type gp35 gene and the editing template for tagging the Gp35 with superfolder green fluorescent protein (sfGFP) was introduced into the PD cells by electroporation. We found that the targeting efficiency was affected by the position and number of sgRNA. The fluorescent phage particles were efficiently recovered from the culture of the PD cells expressing dual sgRNA molecules. This protocol can be used to create designer phages in P. aeruginosa for both application and research purposes.

Assessment of acetylcholinesterase activity in CD9-positive exosomes from patients with Parkinson's disease.

Introduction: Parkinson's disease (PD) is a neurodegenerative disorder characterized by dopaminergic dysfunction and associated with abnormalities in the cholinergic system. However, the relationship between PD and cholinergic dysfunction, particularly in exosomes, is not fully understood. Methods: We enrolled 37 patients with PD and 44 healthy controls (HC) to investigate acetylcholinesterase (AChE) activity in CD9-positive and L1CAM-positive exosomes. Exosomes were isolated from plasma using antibody-coupled magnetic beads, and their sizes and concentrations were assessed using transmission electron microscopy, nanoparticle tracking analysis, and western blotting. Subsequently, the AChE activity in these exosomes was analyzed in relation to various clinical parameters. Results: A significant decrease in AChE activity was observed in CD9-positive exosomes derived from patients with PD, whereas no significant differences were found in L1CAM-positive exosomes. Further analysis with a larger sample size confirmed a substantial reduction in AChE activity in CD9-positive exosomes from the PD plasma, with moderate diagnostic accuracy. The decrease in AChE activity of CD9-positive exosomes did not show an association with cognitive impairment but displayed a trend toward correlation with PD progression. Discussion: The reduction in AChE activity in CD9-positive exosomes suggests potential peripheral cholinergic dysfunction in PD, independent of the central cholinergic system. The observed alterations in AChE activity provide valuable insights into the association between cholinergic dysfunction and the pathogenesis of PD.

An outer membrane determinant for RNA phage genome entry in Pseudomonas aeruginosa.

Host range of a phage is determined at the various life cycle stages during phage infection. We reported the limited phage-receptor interaction between the RNA phage, PP7 and its host Pseudomonas aeruginosa strains: PAO1 has susceptible type IV pilus (TFP) pilin, whereas PA14 has resistant pilin. Here, we have created a PA14 derivative (PA14P) with the PAO1 pilin gene and found that other determinants than TFP pilin could limit PP7 infectivity in PA14P. Transposon mutant screens revealed that PP7 infectivity was restored in the PA14P mutants (htrB2) lacking a secondary acyltransferase in lipid A biosynthesis. The lack of this enzyme increased the RNA phage entry, which is deemed attributed to the loosened lipopolysaccharide (LPS) structure. Polymyxin B treatment also selectively increased the RNA phage entry. These results demonstrated that LPS structures could limit the entry stage of RNA phages, providing another determinant for the host range in diverse P. aeruginosa strains.

Pleiotropic effects of N-acylhomoserine lactone synthase ExpI on virulence, competition, and transmission in Pectobacterium carotovorum subsp. carotovorum Pcc21.

BACKGROUND: Pectobacterium species are necrotrophic phytopathogenic bacteria that cause soft rot disease in economically important crops. The successful infection of host plants relies on interactions among virulence factors, competition, and transmission within hosts. Pectobacteria primarily produce and secrete plant cell-wall degrading enzymes (PCWDEs) for virulence. The regulation of PCWDEs is controlled by quorum sensing (QS). Thus, the QS system is crucial for disease development in pectobacteria through PCWDEs. RESULTS: In this study, we identified a Tn-insertion mutant, M2, in the expI gene from a transposon mutant library of P. carotovorum subsp. carotovorum Pcc21 (hereafter Pcc21). The mutant exhibited reduced production and secretion of PCWDEs, impaired flagellar motility, and increased sensitivity to hydrogen peroxide, resulting in attenuated soft rot symptoms in cabbage and potato tubers. Transcriptomic analysis revealed the down-regulation of genes involved in the production and secretion in the mutant, consistent with the observed phenotype. Furthermore, the Pcc21 wild-type transiently colonized in the gut of Drosophila melanogaster within 12 h after feeding, while the mutant compromised colonization phenotype. Interestingly, Pcc21 produces a bacteriocin, carocin D, to compete with other bacteria. The mutant exhibited up-regulation of carocin D-encoding genes (caroDK) and inhibited the growth of a closely related bacterium, P. wasabiae. CONCLUSION: Our results demonstrated the significance of ExpI in the overall pathogenic lifestyle of Pcc21, including virulence, competition, and colonization in plant and insect hosts. These findings suggest that disease outcome is a result of complex interactions mediated by ExpI across multiple steps. © 2023 Society of Chemical Industry.

A TetR family regulator of an RND efflux system that directs artemisinin resistance in Vibrio cholerae.

Artemisinin (ARS) displayed bactericidal activity against Vibrio cholerae. To assess the mechanistic details of its antibacterial action, we have isolated V. cholerae mutants with enhanced ARS resistance and identified a gene (VCA0767) whose loss-of-function resulted in the ARS resistance phenotypes. This gene (atrR) encodes a TetR family transcriptional regulator, and its deletion mutant displayed the reduction in ARS-induced ROS formation and DNA damage. Transcriptomic analysis revealed that the genes encoding a resistance-nodulation-cell division (RND) efflux pump operon (vexRAB) and the outer membrane component (tolC) were highly upregulated in the artR mutant, suggesting that AtrR might act as a negative regulator of this operon and tolC. Gene deletion of vexR, vexB, or tolC abrogated the ARS resistance of the atrR mutant, and more importantly, the ectopic expression of VexAB-TolC was sufficient for the ARS resistance, indicating that the increased expression of the VexAB-TolC efflux system is necessary and sufficient for the ARS resistance of the atrR mutant. The cytoplasmic accumulation of ARS was compromised in the vexBtolC mutant, suggesting that the VexAB-TolC might be the primary efflux system exporting ARS to reduce its toxicity inside of the bacterial cells. The atrR mutant displayed resistance to erythromycin as well in a VexR-dependent manner. This result suggests that AtrR may act as a global regulator responsible for preventing intracellular accumulation of toxic chemicals by enhancing the RND efflux system.IMPORTANCEDrug efflux protein complexes or efflux pumps are considered as the major determinants of multiple antimicrobial resistance by exporting a wide range of structurally diverse antibiotics in bacterial pathogens. Despite the clinical significance of the increased expression of the efflux pumps, their substrate specificity and regulation mechanisms are poorly understood. Here, we demonstrated that VexAB-TolC, a resistance-nodulation-cell division (RND) efflux pump of V. cholerae, is responsible for the resistance to artemisinin (ARS), an antimalarial drug with bactericidal activity. Furthermore, we newly identified AtrR, a TetR family repressor, as a global regulator for VexRAB and the common outer membrane channel, TolC, where VexR functions as the pathway-specific regulator of the vexAB operon. Our findings will help improve our insight into a broad range of substrate specificity of the VexAB-TolC system and highlight the complex regulatory networks of the multiple RND efflux systems during V. cholerae pathogenesis.

Association of plasma amyloid-ß oligomerization with theta/beta ratio in older adults.

Background: Oligomeric Aß (OAß) is a promising candidate marker for Alzheimer's disease (AD) diagnosis. Electroencephalography (EEG) is a potential tool for early detection of AD. Still, whether EEG power ratios, particularly the theta/alpha ratio (TAR) and theta/beta ratio (TBR), reflect Aß burden-a primary mechanism underlying cognitive impairment and AD. This study investigated the association of TAR and TBR with amyloid burden in older adults based on MDS-OAß levels. Methods: 529 individuals (aged ≥60 years) were recruited. All participants underwent EEG (MINDD SCAN, Ybrain Inc., South Korea) and AlzOn™ test (PeopleBio Inc., Gyeonggi-do, Republic of Korea) for quantifying MDS-OAß values in the plasma. EEG variables were log-transformed to normalize the data distribution. Using the MDS-OAß cutoff value (0.78 ng/mL), all participants were classified into two groups: high MDS-OAß and low MDS-OAß. Results: Participants with high MDS-OAß levels had significantly higher TARs and TBRs than those with low MDS-OAß levels. The log-transformed TBRs in the central lobe (ß = 0.161, p = 0.0026), frontal lobe (ß = 0.145, p = 0.0044), parietal lobe (ß = 0.166, p = 0.0028), occipital lobe (ß = 0.158, p = 0.0058), and temporal lobe (beta = 0.162, p = 0.0042) were significantly and positively associated with increases in MDS-OAß levels. After adjusting for the Bonferroni correction, the TBRs in all lobe regions, except the occipital lobe, were significantly associated with increased MDS-OAß levels. Conclusion: We found a significant association of MDS-OAß with TBR in older adults. This finding indicates that an increase in amyloid burden may be associated with an increase in the low-frequency band and a decrease in the relatively high-frequency band.

Association between pseudoexfoliation and Alzheimer's disease-related brain atrophy.

BACKGROUND/AIMS: Pseudoexfoliation (PEX) syndrome is an age-related disorder characterized by the accumulation of extracellular material in the anterior eye segment. PEX pathogenesis is not fully understood, but amyloid which accumulates in the brain of patients with Alzheimer's disease (AD) is a PEX component. PEX deposition shares features with amyloid aggregation in AD, and brain atrophy is a common AD feature, with ß-amyloid accumulation among contributing factors. This study investigated whether PEX syndrome is associated with AD-related brain atrophy. METHODS: We reviewed the medical records of patients diagnosed with PEX at the Veterans Health Service Medical Center between January 2015 and August 2021. This retrospective cohort study included 48 patients with PEX and 48 healthy age- and sex-matched controls. Patients with PEX were divided into two groups: with and without glaucoma. The main outcome measure was brain atrophy, using a visual rating scale, and AD incidence. Brain atrophy was measured using the Scheltens scale for medial temporal atrophy, the posterior cortical atrophy scale for parietal atrophy, and the Pasquier scale for global cortical atrophy. RESULTS: The percentage of participants with medial temporal atrophy was 56.3% in the PEX group and 35.4% in the control group. The global cortical atrophy and parietal atrophy scores were significantly higher in the PEX group (P<0.05), whereas the PEX and PEX glaucoma groups showed no difference. Among the 96 participants, 16 and 5 participants in the PEX and control groups, respectively, were diagnosed with dementia. Patients with PEX glaucoma tended to have lower Mini-Mental State Examination scores, indicating impaired cognitive function, than those without glaucoma. CONCLUSION: PEX is associated with brain atrophy, reflecting the risk of developing AD. Patients with PEX glaucoma may present with advanced AD stages. Our results suggest that PEX may be a predictor of AD.

Double Mutations in a Patient with Early-Onset Alzheimer's Disease in Korea: An APP Val551Met and a PSEN2 His169Asn.

The etiology of early-onset Alzheimer's disease (EOAD) is associated with alterations in the production of amyloid beta (Aß) species caused by mutations in the APP, PSEN1, and PSEN2 genes. Mutations affect intra- or inter-molecular interactions and processes between the γ-secretase complex and amyloid precursor protein (APP), leading to the aberrant sequential cleavage of Aß species. A 64-year-old woman presented with progressive memory decline, mild right hippocampal atrophy, and a family history of Alzheimer's dementia (AD). Whole exome sequencing was performed to evaluate AD-related gene mutations, which were verified by Sanger sequencing. A mutation-caused structural alteration of APP was predicted using in silico prediction programs. Two AD-related mutations, in APP (rs761339914; c.G1651A; p.V551M) and PSEN2 (rs533813519; c.C505A; p.H169N), were identified. The APP Val551Met mutation in the E2 domain may influence APP homodimerization through changes in intramolecular interactions between adjacent amino acids, altering Aß production. The second mutation was PSEN2 His169Asn mutation, which was previously reported in five EOAD patients from Korea and China, with a relatively high frequency in the East Asian population. According to a previous report, the presenilin 2 protein was predicted to result in a major helical torsion by PSEN2 His169Asn mutation. Notably, the co-existence of APP Val551Met and PSEN2 His169Asn may induce a synergistic effect by both mutations. Future functional studies are needed to clarify the pathological effects of these double mutations.

A Possible Pathogenic PSEN2 Gly56Ser Mutation in a Korean Patient with Early-Onset Alzheimer's Disease.

Early-onset Alzheimer's disease (EOAD) is characterized by the presence of neurological symptoms in patients with Alzheimer's disease (AD) before 65 years of age. Mutations in pathological genes, including amyloid protein precursor (APP), presenilin-1 (PSEN1), and presenilin-2 (PSEN2), were associated with EOAD. Seventy-six mutations in PSEN2 have been found around the world, which could affect the activity of γ-secretase in amyloid beta processing. Here, a heterozygous PSEN2 point mutation from G to A nucleotide change at position 166 (codon 56; c.166G>A, Gly56Ser) was identified in a 64-year-old Korean female with AD with progressive cognitive memory impairment for the 4 years prior to the hospital visit. Hippocampal atrophy was observed from magnetic resonance imaging-based neuroimaging analyses. Temporal and parietal cortex hypometabolisms were identified using fluorodeoxyglucose positron emission tomography. This mutation was at the N-terminal portion of the presenilin 2 protein on the cytosolic side. Therefore, the serine substitution may have promoted AD pathogenesis by perturbing to the mutation region through altered phosphorylation of presenilin. In silico analysis revealed that the mutation altered protein bulkiness with increased hydrophilicity and reduced flexibility of the mutated region of the protein. Structural changes were likely caused by intramolecular interactions between serine and other residues, which may have affected APP processing. The functional study will clarify the pathogenicity of the mutation in the future.

Corrigendum: Decreased Exosomal Acetylcholinesterase Activity in the Plasma of Patients With Parkinson's Disease.

[This corrects the article DOI: 10.3389/fnagi.2021.665400.].

Decreased Exosomal Acetylcholinesterase Activity in the Plasma of Patients With Parkinson's Disease.

Exosomes, which are small extracellular vesicles produced from various cell types, contain a variety of molecular constituents, such as proteins, lipids, and RNA. Recently, exosomal biomarkers have been investigated to probe the understanding and diagnosis of neurodegenerative disorders. Previous reports have demonstrated increased exosomal α-synuclein (α-syn) in patients with Parkinson's disease (PD) in comparison to healthy controls (HC). Interestingly, the cholinergic loss was revealed in the central and peripheral nervous systems in histopathology and molecular neuroimaging. Thereby, we simultaneously examined acetylcholinesterase (AChE) with α-syn as exosomal markers. Exosomes were isolated from the plasma of 34 FP-CIT PET proven patients with PD and 29 HC. Exosomal α-syn and AChE activity were quantified andthe relationship with clinical parameters was analyzed. Remarkably, exosomal AChE activity was significantly decreased in PD compared to HC (P = 0.002). Moreover, exosomal AChE activity in PD revealed a strong negative correlation with disease severity, including H&Y (P = 0.007) and UPDRS part III (P = 0.047) scores. By contrast, no significant difference in exosomal α-syn concentration was observed between groups. These results support the occurrence of cholinergic dysfunction in PD, and they could be implicated with disease progression, especially motor deficits. Exosomal AChE activity with advanced exosome isolation techniques may be a reliable biomarker for the early diagnosis and prognosis of PD.

Cilioretinal Artery Occlusion after Endovascular Coil Embolization for Anterior Communicating Artery.

Unruptured intracranial aneurysms have a risk of rupture, so coil embolization is widely practiced as it preserves a patent artery. There are complications of coil procedures, such as patent artery occlusion and thromboembolism, which can result in retinal artery occlusion. We report onretinal artery occlusion following coil embolization of anterior communicating artery aneurysm. This is a rare case of a combination of cilioretinal and branch retinal artery occlusion, and is unusual in showing a functional recovery.

Nitrate Respiration Promotes Polymyxin B Resistance in Pseudomonas aeruginosa.

Aims: Polymyxin B (PMB) is known to require reactive oxygen species (ROS) for its bactericidal activity, but the mechanism of PMB resistance in various Pseudomonas aeruginosa strains has been poorly understood. This study examined the role of nitrate respiration (NR) of some P. aeruginosa strains in the PMB resistance. Results: We observed that the minimum inhibitory concentration (MIC) value of PMB against P. aeruginosa PA14 was eightfold reduced (from 2.0 to 0.25 µg/mL) by agitation, but not against P. aeruginosa PAO1 (from 2.0 to 1.0 µg/mL). Transcriptomic and phenotypic analyses using both strains and their NR mutants revealed that the higher NR in PAO1 than in PA14 accounted for the higher MIC value (i.e., PMB resistance) of PAO1, which was sufficient to compromise the antibacterial activity of PMB in Drosophila infections. We also confirmed the contribution of the NR to the PMB resistance is independent of the major catalase (KatA), suggesting that the NR might affect the ROS generation rather than the ROS disintegration. Furthermore, this PMB resistance was relatively common among clinical P. aeruginosa isolates and correlated with higher NR in those strains. Innovation and Conclusion: These results suggest P. aeruginosa strains could display intrinsic resistance to antibiotics in clinical settings and that NR is a crucial factor in the intrinsic antibiotic resistance, and also provide an insight into another key target for successful antibiotic treatment of P. aeruginosa infections. Antioxid. Redox Signal. 34, 442-451.

cDNA-Derived RNA Phage Assembly Reveals Critical Residues in the Maturation Protein of the Pseudomonas aeruginosa Leviphage PP7.

PP7 is a leviphage, with a single-stranded RNA genome, that infects Pseudomonas aeruginosa PAO1. A reverse genetic system for PP7 was previously created by using reverse-transcribed cDNA (PP7O) from a virion-derived RNA genome. Here, we have found that the PP7O cDNA contained 20 nucleotide differences from the PP7 genome sequence deposited in the database. We created another reverse genetic system exploiting chemically synthesized cDNA (PP7S) based on the database sequence. Unlike PP7O, which yielded infectious PP7 virions, PP7S-derived particles were incapable of plaque formation on PAO1 cells, which was restored in the PAO1 cells expressing the maturation protein (MP) from PP7O Using this reverse genetic system, we revealed two amino acid residues involved in the known roles of MP (i.e., adsorption and genome replication), fortuitously providing a lesson that the viral RNA genome sequencing needs functional verification, possibly by a reverse genetic system.IMPORTANCE The biological significance of RNA phages has been largely ignored, ironically, because few studies have focused on RNA phages. As an initial attempt to properly represent RNA phages in the phageome, we previously created, by using reverse-transcribed cDNA, a reverse genetic system for the small RNA phage PP7, which infects the opportunistic human pathogen Pseudomonas aeruginosa We report another system by using chemically synthesized cDNA based on the database genome that has 20 nucleotide differences from the previous cDNA. Investigation of those cDNA-derived phage virions revealed that two amino acids of the maturation protein are crucial for the normal phage lifecycle at different steps. Our study provides insight into the molecular basis for the RNA phage lifecycle and a lesson that the RNA genome sequencing needs to be carefully validated by cDNA-based phage assembly systems.

Reverse Genetic Systems for Pseudomonas aeruginosa Leviphages.

Reverse genetic systems for RNA viruses are the platforms to introduce mutations into the RNA genomes and thus have helped understand their life cycle and harness them for human purposes to develop vaccines and delivery systems. These systems are based on the complementary DNA (cDNA) of the RNA viruses, whose transcripts derived from bacterial RNA polymerases act not only as the primary mRNA for phage protein synthesis, but also as the template for phage RNA replicases (aka. RNA-dependent RNA polymerases). Here, we present a protocol optimized for the small RNA phages of Leviviridae (i.e., leviphages) infecting Pseudomonas aeruginosa. This protocol includes three fundamental steps: (i) Creation of a promoter-fused cDNA, (ii) generation of a clone into mini-Tn7-based vector, and (iii) introduction of the clone into non-susceptible hosts. As the representative example, we describe the reverse genetic system for PP7, which infects a set of P. aeruginosa strains such as PAO1. The cDNA was fused to the T7 promoter, which was cloned in mini-Tn7-Gm. This construct was introduced into P. aeruginosa PAK and E. coli HB101. Functional assembly of PP7 phages from the culture supernatants were assessed by plaque formation on PAO1 and the phage particles were observed under transmission microscope. We found that the host cells should be cultured at 30 °C for the maximal phage production (~1012 pfu/mL). The reverse genetic systems will provide a new insight into the life cycle of the RNA phages and help develop engineered variants with new traits for phage applications regarding selective diagnosis and efficient therapy.