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1.
Front Cell Infect Microbiol ; 14: 1296777, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38469347

RESUMO

Phage therapy is a potential approach in the biocontrol of foodborne pathogens. However, the emergence of phage resistance and the narrow host range of most phage isolates continue to limit the antimicrobial efficacy of phages. Here, we investigated the potential of the pqsA gene, encoding the anthranilate-CoA ligase enzyme, as an adjuvant for phage therapy. The knockout of the pqsA gene significantly enhanced the bactericidal effect of phages vB_Pae_QDWS and vB_Pae_S1 against Pseudomonas aeruginosa. Under phage infection pressure, the growth of the PaΔpqsA was significantly inhibited within 8 h compared to the wild-type PAO1. Furthermore, we found that altering phage adsorption is not how PaΔpqsA responds to phage infection. Although pqsA represents a promising target for enhancing phage killing, it may not be applicable to all phages, such as types vB_Pae_W3 and vB_Pae_TR. Our findings provide new material reserves for the future design of novel phage-based therapeutic strategies.


Assuntos
Bacteriófagos , Terapia por Fagos , Infecções por Pseudomonas , Fagos de Pseudomonas , Humanos , Pseudomonas aeruginosa/genética , Fagos de Pseudomonas/genética , Infecções por Pseudomonas/terapia , Mutação
2.
Methods Mol Biol ; 2793: 113-128, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38526727

RESUMO

The vast number of unknown phage-encoded ORFan genes and limited insights into the genome organization of phages illustrate the need for efficient genome engineering tools to study bacteriophage genes in their natural context. In addition, there is an application-driven desire to alter phage properties, which is hampered by time constraints for phage genome engineering in the bacterial host. We here describe an optimized CRISPR-Cas3 system in Pseudomonas for straightforward editing of the genome of virulent bacteriophages. The two-vector system combines a broad host range CRISPR-Cas3 targeting plasmid with a SEVA plasmid for homologous directed repair, which enables the creation of clean deletions, insertions, or substitutions in the phage genome within a week. After creating the two plasmids separately, a co-transformation to P. aeruginosa cells is performed. A subsequent infection with the targeted phage allows the CRISPR-Cas3 system to cut the DNA specifically and facilitate or select for homologous recombination. This system has also been successfully applied for P. aeruginosa and Pseudomonas putida genome engineering. The method is straightforward, efficient, and universal, enabling to extrapolate the system to other phage-host pairs.


Assuntos
Bacteriófagos , Fagos de Pseudomonas , Edição de Genes/métodos , Fagos de Pseudomonas/genética , Sistemas CRISPR-Cas/genética , Bacteriófagos/genética , Recombinação Homóloga
3.
Ugeskr Laeger ; 186(3)2024 01 22.
Artigo em Dinamarquês | MEDLINE | ID: mdl-38305316

RESUMO

We present a case report detailing therapeutic application of two lytic antipseudomonal bacteriophages to treat a chronic relapsing Pseudomonas aeruginosa infection of a prosthetic aortic graft. As there are currently no Danish laboratories offering phages for clinical therapy, and this case, to our knowledge represents the first applied phage therapy in Denmark, the practical and regulatory aspects of offering this treatment option in Denmark is briefly reviewed along with the clinical case.


Assuntos
Bacteriófagos , Fagos de Pseudomonas , Humanos , Pseudomonas , Prótese Vascular , Pseudomonas aeruginosa
4.
Microbiol Spectr ; 12(3): e0371923, 2024 Mar 05.
Artigo em Inglês | MEDLINE | ID: mdl-38345389

RESUMO

The ESKAPEE pathogen Pseudomonas aeruginosa is a common cause of chronic wound and cystic fibrosis lung infections, as well as acute burn and nosocomial infections. Many of these infections are recalcitrant to conventional antibiotic therapies due to both traditional antibiotic resistance mechanisms and antimicrobial tolerance. Recent successes with bacteriophage (phage) therapy to treat chronic human P. aeruginosa infections have led to a renewed interest in isolating and characterizing new P. aeruginosa phages. Here, we isolated and characterized a new lytic phage (termed PIP, pili-infecting phage) capable of infecting P. aeruginosa PA14. PIP is a tailed phage with an icosahedral head and flexible tail containing a genome that is 57,462 bp in length. Phylogenetic analysis reveals that PIP belongs to the subfamily Queuovirinae and genus Nipunavirus but is highly divergent in gene content from known Nipunaviruses. By isolating and characterizing a P. aeruginosa strain that spontaneously evolved resistance to PIP, we show that the receptor for PIP is Type IV pili. In summary, we isolated a new P. aeruginosa phage species with a unique genome, thus increasing the diversity of phages known to infect this important human pathogen.IMPORTANCEThe opportunistic pathogen Pseudomonas aeruginosa causes both acute and chronic human infections. These infections are notoriously difficult to treat due to both antibiotic resistance and antibiotic tolerance. The increasing frequency of antibiotic failure in P. aeruginosa infections has led scientists to explore other treatment options, including bacteriophage (phage) therapy. To this end, there has been a significant effort to identify new Pseudomonas phages. Here, we isolated and characterized a bacteriophage (termed PIP, pili-infecting phage) that infects P. aeruginosa PA14. Examination of the PIP genome revealed that this phage represents a new species in the subclass Queuovirinae. The isolation and characterization of spontaneous PA14 mutants that are resistant to PIP infection revealed Type IV pili as the PIP receptor. Ultimately, this study characterizes a new species of Pseudomonas phage, thus enhancing the known diversity of phages that infect this important pathogen.


Assuntos
Bacteriófagos , Infecções por Pseudomonas , Fagos de Pseudomonas , Humanos , Bacteriófagos/genética , Pseudomonas aeruginosa/genética , Filogenia , Infecções por Pseudomonas/terapia , Fagos de Pseudomonas/genética , Antibacterianos/farmacologia
5.
Sci Adv ; 10(8): eadj0341, 2024 Feb 23.
Artigo em Inglês | MEDLINE | ID: mdl-38394193

RESUMO

Prokaryotes encode multiple distinct anti-phage defense systems in their genomes. However, the impact of carrying a multitude of defense systems on phage resistance remains unclear, especially in a clinical context. Using a collection of antibiotic-resistant clinical strains of Pseudomonas aeruginosa and a broad panel of phages, we demonstrate that defense systems contribute substantially to defining phage host range and that overall phage resistance scales with the number of defense systems in the bacterial genome. We show that many individual defense systems target specific phage genera and that defense systems with complementary phage specificities co-occur in P. aeruginosa genomes likely to provide benefits in phage-diverse environments. Overall, we show that phage-resistant phenotypes of P. aeruginosa with at least 19 phage defense systems exist in the populations of clinical, antibiotic-resistant P. aeruginosa strains.


Assuntos
Bacteriófagos , Infecções por Pseudomonas , Fagos de Pseudomonas , Humanos , Bacteriófagos/genética , Pseudomonas aeruginosa , Fagos de Pseudomonas/genética , Infecções por Pseudomonas/microbiologia , Antibacterianos
6.
Sci Rep ; 14(1): 2657, 2024 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-38302552

RESUMO

Bacteriophage therapy is one potential strategy to treat antimicrobial resistant or persistent bacterial infections, and the year 2021 marked the centennial of Felix d'Hérelle's first publication on the clinical applications of phages. At the Center for Phage Biology & Therapy at Yale University, a preparatory modular approach has been established to offer safe and potent phages for single-patient investigational new drug applications while recognizing the time constraints imposed by infection(s). This study provides a practical walkthrough of the pipeline with an Autographiviridae phage targeting Pseudomonas aeruginosa (phage vB_PaeA_SB, abbreviated to ΦSB). Notably, a thorough phage characterization and the evolutionary selection pressure exerted on bacteria by phages, analogous to antibiotics, are incorporated into the pipeline.


Assuntos
Bacteriófagos , Terapia por Fagos , Infecções por Pseudomonas , Fagos de Pseudomonas , Humanos , Pseudomonas aeruginosa , Universidades , Fagos de Pseudomonas/genética , Infecções por Pseudomonas/terapia , Infecções por Pseudomonas/microbiologia
7.
Microbiol Spectr ; 12(3): e0289723, 2024 Mar 05.
Artigo em Inglês | MEDLINE | ID: mdl-38294230

RESUMO

The rise in the frequency of antibiotic resistance has made bacterial infections, specifically Pseudomonas aeruginosa, a cause for greater concern. Phage therapy is a promising solution that uses naturally isolated phages to treat bacterial infections. Ecological limitations, which stipulate a discrete host range and the inevitable evolution of resistance, may be overcome through a better understanding of phage biology and the utilization of engineered phages. In this study, we developed a synthetic biology approach to construct tailed phages that naturally target clinically relevant strains of Pseudomonas aeruginosa. As proof of concept, we successfully cloned and assembled the JG024 and DMS3 phage genomes in yeast using transformation-associated recombination cloning and rebooted these two phage genomes in two different strains of P. aeruginosa. We identified factors that affected phage reboot efficiency like the phage species or the presence of antiviral defense systems in the bacterial strain. We have successfully extended this method to two other phage species and observed that the method enables the reboot of phages that are naturally unable to infect the strain used for reboot. This research represents a critical step toward the construction of clinically relevant, engineered P. aeruginosa phages.IMPORTANCEPseudomonas aeruginosa is a bacterium responsible for severe infections and a common major complication in cystic fibrosis. The use of antibiotics to treat bacterial infections has become increasingly difficult as antibiotic resistance has become more prevalent. Phage therapy is an alternative solution that is already being used in some European countries, but its use is limited by the narrow host range due to the phage receptor specificity, the presence of antiviral defense systems in the bacterial strain, and the possible emergence of phage resistance. In this study, we demonstrate the use of a synthetic biology approach to construct and reboot clinically relevant P. aeruginosa tailed phages. This method enables a significant expansion of possibilities through the construction of engineered phages for therapy applications.


Assuntos
Infecções Bacterianas , Bacteriófagos , Infecções por Pseudomonas , Fagos de Pseudomonas , Humanos , Pseudomonas aeruginosa , Fagos de Pseudomonas/genética , Biologia Sintética , Bacteriófagos/genética , Antivirais
8.
Appl Microbiol Biotechnol ; 108(1): 6, 2024 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-38165478

RESUMO

Wound healing is a dynamic and complex process where infection prevention is essential. Chitosan, thanks to its bactericidal activity against gram-positive and gram-negative bacteria, as well as anti-inflammatory and hemostatic properties, is an excellent candidate to design dressings for difficult-to-heal wound treatment. The great advantage of this biopolymer is its capacity to be chemically modified, which allows for the production of various functional forms, depending on the needs and subsequent use. Moreover, chitosan can be an excellent polymer matrix for bacteriophage (phage) packing as a novel alternative/supportive antibacterial therapy approach. This study is focused on the preparation and characteristics of chitosan-based material in the form of a film with the addition of Pseudomonas lytic phages (KTN4, KT28, and LUZ19), which would exhibit antibacterial activity as a potential dressing that accelerates the wound healing. We investigated the method of producing a polymer based on microcrystalline chitosan (MKCh) to serve as the matrix for phage deposition. We described some important parameters such as average molar mass, swelling capacity, surface morphology, phage release profile, and antibacterial activity tested in the Pseudomonas aeruginosa bacterial model. The chitosan polysaccharide turned out to interact with phage particles immobilizing them within a material matrix. Nevertheless, with the high hydrophilicity and swelling features of the prepared material, the external solution of bacterial culture was absorbed and phages went in direct contact with bacteria causing their lysis in the polymer matrix. KEY POINTS: • A novel chitosan-based matrix with the addition of active phages was prepared • Phage interactions with the chitosan matrix were determined as electrostatic • Phages in the matrix work through direct contact with the bacterial cells.


Assuntos
Bacteriófagos , Quitosana , Fagos de Pseudomonas , Antibacterianos/farmacologia , Bactérias Gram-Negativas , Bactérias Gram-Positivas , Polímeros
9.
Microbiol Spectr ; 12(1): e0188223, 2024 Jan 11.
Artigo em Inglês | MEDLINE | ID: mdl-38014983

RESUMO

IMPORTANCE: Phage therapy is gaining traction as an alternative to antibiotics due to the rise of multi-drug-resistant (MDR) bacteria. This study assessed the pharmacokinetics and safety of PA_LZ7, a phage targeting MDR Pseudomonas aeruginosa, in mice. After intravenous administration, the phage showed an exponential decay in plasma and its concentration dropped significantly within 24 h for all dosage groups. Although there was a temporary increase in certain plasma cytokines and spleen weight at higher dosages, no significant toxicity was observed. Therefore, PA_LZ7 shows potential as an effective and safe candidate for future phage therapy against MDR P. aeruginosa infections.


Assuntos
Bacteriófagos , Infecções por Pseudomonas , Fagos de Pseudomonas , Animais , Camundongos , Fagos de Pseudomonas/genética , Antibacterianos/uso terapêutico , Antibacterianos/farmacologia , Infecções por Pseudomonas/terapia , Infecções por Pseudomonas/microbiologia , Pseudomonas aeruginosa
10.
J Virol ; 98(1): e0135923, 2024 Jan 23.
Artigo em Inglês | MEDLINE | ID: mdl-38084959

RESUMO

Phage therapy has shown great promise for the treatment of multidrug-resistant bacterial infections. However, the lack of a thorough and organized understanding of phage-body interactions has limited its clinical application. Here, we administered different purified phages (Salmonella phage SE_SZW1, Acinetobacter phage AB_SZ6, and Pseudomonas phage PA_LZ7) intravenously to healthy animals (rats and monkeys) to evaluate the phage-induced host responses and phage pharmacokinetics with different intravenous (IV) doses in healthy animals. The plasma and the organs were sampled after different IV doses to determine the phage biodistribution, phage-induced cytokines, and antibodies. The potential side effects of phages on animals were assessed. A non-compartment model revealed that the plasma phage titer gradually decreased over time following a single dose. Repeated doses resulted in a 2-3 Log10 decline of the plasma phage titer at 5 min compared to the first dose, regardless of the type of phage administered in rats. Host innate immune responses were activated including splenic enlargement following repeated doses. Phage-specific neutralization antibodies in animals receiving phages were detected. Similar results were obtained from monkeys. In conclusion, the mammalian bodies were well-tolerant to the administered phages. The animal responses to the phages and the phage biodistribution profiles could have a significant impact on the efficacy of phage therapy.IMPORTANCEPhage therapy has demonstrated potential in addressing multidrug-resistant bacterial infections. However, an insufficient understanding of phage-host interactions has impeded its broader clinical application. In our study, specific phages were administered intravenously (IV) to both rats and monkeys to elucidate phage-host interactions and evaluate phage pharmacokinetics (PK). Results revealed that with successive IV administrations, there was a decrease in plasma phage concentrations. Concurrently, these administrations elicited both innate and adaptive immune responses in the subjects. Notably, the observed immune responses and PK profiles exhibited variation contingent upon the phage type and the mammalian host. Despite these variations, the tested mammals exhibited a favorable tolerance to the IV-administered phages. This underscores the significance of comprehending these interactions for the optimization of phage therapy outcomes.


Assuntos
Infecções Bacterianas , Bacteriófagos , Terapia por Fagos , Animais , Humanos , Ratos , Infecções Bacterianas/terapia , Bacteriófagos/fisiologia , Mamíferos , Fagos de Pseudomonas , Distribuição Tecidual , Farmacorresistência Bacteriana Múltipla
11.
Methods Mol Biol ; 2734: 237-243, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38066373

RESUMO

Phage therapy can be a useful approach in a number of clinical cases associated with multidrug-resistant (MDR) bacterial infections. In this study, we describe a successful consecutive phage and antibiotic application to cure a 3-month-old girl suffering from severe bronchitis after tracheostomy. Bronchitis was associated with two bacterial agents, MDR Pseudomonas aeruginosa and a rare opportunistic pathogen Dolosigranulum pigrum. The phage cocktail "Pyobacteriophage" containing at least two different phages against isolated MDR P. aeruginosa strain was used via inhalation and nasal drops. Topical application of the phage cocktail removed most of P. aeruginosa cells and contributed to a change in the antimicrobial resistance profile of surviving P. aeruginosa cells. As a result, it became possible to choose and administer an appropriate antibiotic that was effective against both infectious agents. Complete recovery of the infant was recorded.


Assuntos
Bacteriófagos , Bronquite , Infecções por Pseudomonas , Fagos de Pseudomonas , Feminino , Humanos , Lactente , Antibacterianos/farmacologia , Antibacterianos/uso terapêutico , Pseudomonas aeruginosa , Sistema Respiratório , Infecções por Pseudomonas/tratamento farmacológico , Infecções por Pseudomonas/microbiologia
12.
Nat Commun ; 14(1): 8429, 2023 Dec 19.
Artigo em Inglês | MEDLINE | ID: mdl-38114502

RESUMO

Inoviruses are filamentous phages infecting numerous prokaryotic phyla. Inoviruses can self-assemble into mesoscale structures with liquid-crystalline order, termed tactoids, which protect bacterial cells in Pseudomonas aeruginosa biofilms from antibiotics. Here, we investigate the structural, biophysical, and protective properties of tactoids formed by the P. aeruginosa phage Pf4 and Escherichia coli phage fd. A cryo-EM structure of the capsid from fd revealed distinct biochemical properties compared to Pf4. Fd and Pf4 formed tactoids with different morphologies that arise from differing phage geometries and packing densities, which in turn gave rise to different tactoid emergent properties. Finally, we showed that tactoids formed by either phage protect rod-shaped bacteria from antibiotic treatment, and that direct association with a tactoid is required for protection, demonstrating the formation of a diffusion barrier by the tactoid. This study provides insights into how filamentous molecules protect bacteria from extraneous substances in biofilms and in host-associated infections.


Assuntos
Bacteriófagos , Inovirus , Fagos de Pseudomonas , Antibacterianos/farmacologia , Pseudomonas aeruginosa , Fagos de Pseudomonas/genética , Inovirus/genética , Bactérias , Biofilmes
13.
Antimicrob Agents Chemother ; 67(12): e0065423, 2023 12 14.
Artigo em Inglês | MEDLINE | ID: mdl-37931230

RESUMO

Antibiotic-resistant bacteria present an emerging challenge to human health. Their prevalence has been increasing across the globe due in part to the liberal use of antibiotics that has pressured them to develop resistance. Those bacteria that acquire mobile genetic elements are especially concerning because those plasmids may be shared readily with other microbes that can then also become antibiotic resistant. Serious infections have recently been related to the contamination of preservative-free eyedrops with extensively drug-resistant (XDR) isolates of Pseudomonas aeruginosa, already resulting in three deaths. These drug-resistant isolates cannot be managed with most conventional antibiotics. We sought to identify alternatives to conventional antibiotics for the lysis of these XDR isolates and identified multiple bacteriophages (viruses that attack bacteria) that killed them efficiently. We found both jumbo phages (>200 kb in genome size) and non-jumbo phages that were active against these isolates, the former killing more efficiently. Jumbo phages effectively killed the three separate XDR P. aeruginosa isolates both on solid and liquid medium. Given the ongoing nature of the XDR P. aeruginosa eyedrop outbreak, the identification of phages active against them provides physicians with several novel potential alternatives for treatment.


Assuntos
Bacteriófagos , Infecções por Pseudomonas , Fagos de Pseudomonas , Humanos , Bacteriófagos/genética , Infecções por Pseudomonas/microbiologia , Antibacterianos/farmacologia , Antibacterianos/uso terapêutico , Plasmídeos , Pseudomonas aeruginosa , Fagos de Pseudomonas/genética
14.
Viruses ; 15(11)2023 Oct 25.
Artigo em Inglês | MEDLINE | ID: mdl-38005832

RESUMO

Half a century has passed since the discovery of Pseudomonas phage phi6, the first enveloped dsRNA bacteriophage to be isolated. It remained the sole known dsRNA phage for a quarter of a century and the only recognised member of the Cystoviridae family until the year 2018. After the initial discovery of phi6, additional dsRNA phages have been isolated from globally distant locations and identified in metatranscriptomic datasets, suggesting that this virus type is more ubiquitous in nature than previously acknowledged. Most identified dsRNA phages infect Pseudomonas strains and utilise either pilus or lipopolysaccharide components of the host as the primary receptor. In addition to the receptor-mediated strictly lytic lifestyle, an alternative persistent infection strategy has been described for some dsRNA phages. To date, complete genome sequences of fourteen dsRNA phage isolates are available. Despite the high sequence diversity, similar sets of genes can typically be found in the genomes of dsRNA phages, suggesting shared evolutionary trajectories. This review provides a brief overview of the recognised members of the Cystoviridae virus family and related dsRNA phage isolates, outlines the current classification of dsRNA phages, and discusses their relationships with eukaryotic RNA viruses.


Assuntos
Bacteriófagos , Fagos de Pseudomonas , Bacteriófagos/genética , Fagos de Pseudomonas/genética , Pseudomonas , Genoma Viral
15.
Viruses ; 15(11)2023 Nov 03.
Artigo em Inglês | MEDLINE | ID: mdl-38005888

RESUMO

Therapeutic bacteriophages (phages) are primarily chosen based on their in vitro bacteriolytic activity. Although anti-phage antibodies are known to inhibit phage infection, the influence of other immune system components is less well known. An important anti-bacterial and anti-viral innate immune system that may interact with phages is the complement system, a cascade of proteases that recognizes and targets invading microorganisms. In this research, we aimed to study the effects of serum components such as complement on the infectivity of different phages targeting Pseudomonas aeruginosa. We used a fluorescence-based assay to monitor the killing of P. aeruginosa by phages of different morphotypes in the presence of human serum. Our results reveal that several myophages are inhibited by serum in a concentration-dependent way, while the activity of four podophages and one siphophage tested in this study is not affected by serum. By using specific nanobodies blocking different components of the complement cascade, we showed that activation of the classical complement pathway is a driver of phage inhibition. To determine the mechanism of inhibition, we produced bioorthogonally labeled fluorescent phages to study their binding by means of microscopy and flow cytometry. We show that phage adsorption is hampered in the presence of active complement. Our results indicate that interactions with complement may affect the in vivo activity of therapeutically administered phages. A better understanding of this phenomenon is essential to optimize the design and application of therapeutic phage cocktails.


Assuntos
Bacteriófagos , Infecções por Pseudomonas , Fagos de Pseudomonas , Humanos , Pseudomonas aeruginosa/fisiologia , Fagos de Pseudomonas/fisiologia , Bacteriólise , Infecções por Pseudomonas/terapia , Infecções por Pseudomonas/microbiologia
16.
Viruses ; 15(11)2023 Nov 05.
Artigo em Inglês | MEDLINE | ID: mdl-38005892

RESUMO

Pseudomonas aeruginosa is an opportunistic pathogen that can cause infections in humans, especially in hospital patients with compromised host defence mechanisms, including patients with cystic fibrosis. Filamentous bacteriophages represent a group of single-stranded DNA viruses infecting different bacteria, including P. aeruginosa and other human and animal pathogens; many of them can replicate when integrated into the bacterial chromosome. Filamentous bacteriophages can contribute to the virulence of P. aeruginosa and influence the course of the disease. There are just a few isolated and officially classified filamentous bacteriophages infecting P. aeruginosa, but genomic studies indicated the frequent occurrence of integrated prophages in many P. aeruginosa genomes. An analysis of sequenced genomes of P. aeruginosa isolated from upper respiratory tract (throat and nasal swabs) and sputum specimens collected from Russian patients with cystic fibrosis indicated a higher diversity of filamentous bacteriophages than first thought. A detailed analysis of predicted bacterial proteins revealed prophage regions representing the filamentous phages known to be quite distantly related to known phages. Genomic comparisons and phylogenetic studies enabled the proposal of several new taxonomic groups of filamentous bacteriophages.


Assuntos
Bacteriófagos , Fibrose Cística , Inovirus , Infecções por Pseudomonas , Fagos de Pseudomonas , Animais , Humanos , Pseudomonas aeruginosa/genética , Fagos de Pseudomonas/genética , Fibrose Cística/microbiologia , Filogenia , Inovirus/genética , Prófagos/genética , Infecções por Pseudomonas/microbiologia
17.
Sci Rep ; 13(1): 19033, 2023 11 03.
Artigo em Inglês | MEDLINE | ID: mdl-37923820

RESUMO

The present study explores the avenue of phage therapy as an alternative antimicrobial therapeutic approach to counter multidrug-resistant (MDR) Pseudomonas aeruginosa infection. Our study investigated two novel virulent phages PSPa and APPa, specific to P. aeruginosa, in which in vitro evaluations were carried out to assess the therapeutic potential of phages. Both the identified phages exhibited host specificity by showing antagonistic activity of about 96.43% (27/28) and 92.85% (26/28) towards the 28 MDR clinical isolates of P. aeruginosa. The PSPa phage was found to have linear dsDNA with a sequence length of 66,368 bp and 92 ORFs, of which 32 were encoded for known functions of the phage life cycle and the remaining 60 were hypothetical functions. The APPa phage was found to have linear dsDNA with 59,591 bp of genome length and 79 ORFs, of which 15 were found to have known phage functions and the remaining 64 were found to be hypothetical proteins. Notably, the genome of both the phages lacks genes coding for tRNA, rRNA, and tmRNA. The phylogenetic analysis revealed that PSPa and APPa share > 95% sequence similarity with previously sequenced Pseudomonas viruses of their respective families. Further, the in vivo efficacy evaluation using the zebrafish model revealed that the treatment with PSPa and APPa has remarkably improved the survival rate of bacterial-infected zebrafish, reinforcing the anti-infective potential of the isolated phages PSPa and APPa against P. aeruginosa infection.


Assuntos
Bacteriófagos , Fagos de Pseudomonas , Humanos , Animais , Pseudomonas aeruginosa/genética , Peixe-Zebra , Virulência , Filogenia , Plâncton
18.
Viruses ; 15(10)2023 10 16.
Artigo em Inglês | MEDLINE | ID: mdl-37896872

RESUMO

A nucleus-like structure composed of phage-encoded proteins and containing replicating viral DNA is formed in Pseudomonas aeruginosa cells infected by jumbo bacteriophage phiKZ. The PhiKZ genes are transcribed independently from host RNA polymerase (RNAP) by two RNAPs encoded by the phage. The virion RNAP (vRNAP) transcribes early viral genes and must be injected into the cell with phage DNA. The non-virion RNAP (nvRNAP) is composed of early gene products and transcribes late viral genes. In this work, the dynamics of phage RNAPs localization during phage phiKZ infection were studied. We provide direct evidence of PhiKZ vRNAP injection in infected cells and show that it is excluded from the phage nucleus. The nvRNAP is synthesized shortly after the onset of infection and localizes in the nucleus. We propose that spatial separation of two phage RNAPs allows coordinated expression of phage genes belonging to different temporal classes.


Assuntos
Bacteriófagos , Fagos de Pseudomonas , Bacteriófagos/genética , Proteínas Virais/metabolismo , Fagos de Pseudomonas/metabolismo , RNA Polimerases Dirigidas por DNA/metabolismo , Genes Virais
19.
Viruses ; 15(8)2023 08 12.
Artigo em Inglês | MEDLINE | ID: mdl-37632068

RESUMO

The immune system offers several mechanisms of response to harmful microbes that invade the human body. As a first line of defense, neutrophils can remove pathogens by phagocytosis, inactivate them by the release of reactive oxygen species (ROS) or immobilize them by neutrophil extracellular traps (NETs). Although recent studies have shown that bacteriophages (phages) make up a large portion of human microbiomes and are currently being explored as antibacterial therapeutics, neutrophilic responses to phages are still elusive. Here, we show that exposure of isolated human resting neutrophils to a high concentration of the Pseudomonas phage PAK_P1 led to a 2-fold increase in interleukin-8 (IL-8) secretion. Importantly, phage exposure did not induce neutrophil apoptosis or necrosis and did not further affect activation marker expression, oxidative burst, and NETs formation. Similarly, inflammatory stimuli-activated neutrophil effector responses were unaffected by phage exposure. Our work suggests that phages are unlikely to inadvertently cause excessive neutrophil responses that could damage tissues and worsen disease. Because IL-8 functions as a chemoattractant, directing immune cells to sites of infection and inflammation, phage-stimulated IL-8 production may modulate some host immune responses.


Assuntos
Bacteriófagos , Fagos de Pseudomonas , Humanos , Bacteriófago P1 , Neutrófilos , Interleucina-8
20.
Virus Res ; 335: 199184, 2023 10 02.
Artigo em Inglês | MEDLINE | ID: mdl-37532140

RESUMO

Pseudomonas aeruginosa is a clinically common conditionally pathogenic bacterium, and the abuse of antibiotics has exacerbated its drug resistance in recent years. This has resulted in extensive reports about the usage of Pseudomonas aeruginosa phage as a novel antibacterial drug. In this study, we isolated a novel phage HZ2201 with a broad lytic spectrum. The lytic rate of this phage against Pseudomonas aeruginosa reached 78.38% (29/37), including 25 multi-drug- and carbapenem-resistant Pseudomonas aeruginosa strains. Transmission electron microscopy revealed that phage HZ2201 belongs to the class Caudoviricetes. Biological characterization showed that phage HZ2201 had an latent period of 40 min, a lytic period of 20 min, and a burst size of 440 PFU/cell, with improved tolerance to temperature and pH. Considering genomic analysis, the HZ2201 genome was a circular double-stranded DNA with a size of 45,431 bp and a guanine-cytosine (G + C) content of 52.16%, and contained 3 tRNAs. 27 of the 74 open reading frames (ORFs) annotated by the Rapid Annotation using Subsystem Technology (RAST) tool could be matched to the genomes of known functions, and no genes related to virulence and antibiotic resistance were found. The phylogenetic tree suggests that phage HZ2201 is highly related to the phage ZCPS1 and PaP3, and ORF57 and ORF17 are predicted to encode a holin and an endolysin, respectively. Cell lysis by HZ2201 proceeds through the holin-endolysin system, suggesting that it is a novel phage. Additionally, we demonstrated that phage HZ2201 has a high inhibitory capacity against Pseudomonas aeruginosa biofilms. The results of our study suggest that phage HZ2201 is a novel potential antimicrobial agent for treating drug-resistant Pseudomonas aeruginosa infection.


Assuntos
Bacteriófagos , Fagos de Pseudomonas , Bacteriófagos/genética , Pseudomonas aeruginosa/genética , Filogenia , Fagos de Pseudomonas/genética , Genômica/métodos , Genoma Viral , Biofilmes
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