Genomic characterization of four novel bacteriophages infecting the clinical pathogen Klebsiella pneumoniae

Bacteriophages are an invaluable source of novel genetic diversity. Sequencing of phage genomes can reveal new proteins with potential uses as biotechnological and medical tools, and help unravel the diversity of biological mechanisms employed by phages to take over the host during viral infection. Aiming to expand the available collection of phage genomes, we have isolated, sequenced, and assembled the genome sequences of four phages that infect the clinical pathogen Klebsiella pneumoniae: vB_KpnP_FBKp16, vB_KpnP_FBKp27, vB_KpnM_FBKp34, and Jumbo phage vB_KpnM_FBKp24. The four phages show very low (0–13%) identity to genomic phage sequences deposited in the GenBank database. Three of the four phages encode tRNAs and have a GC content very dissimilar to that of the host. Importantly, the genome sequences of the phages reveal potentially novel DNA packaging mechanisms as well as distinct clades of tubulin spindle and nucleus shell proteins that some phages use to compartmentalize viral replication. Overall, this study contributes to uncovering previously unknown virus diversity, and provides novel candidates for phage therapy applications against antibiotic-resistant K. pneumoniae infections.     

噬菌体：微小却不简单

噬菌体是专一感染细菌等微生物的病毒,是地球上多样性最高和最丰富的生物体,是生物学研究中重要的模式生物,同时是抗生素耐药菌的天然抗菌剂。噬菌体研究的相关成果极大地推动了生物学各个领域的发展。     

Blood kinetics of four intraperitoneally administered therapeutic candidate bacteriophages in healthy and neutropenic mice

Due to multiple-drug resistant bacteria, phage therapy is being revisited. Although most animal experiments focus on therapeutic efficacy, the blood clearance kinetics of phages have not been well described. For further development of an efficient therapeutic strategy, information on phage blood kinetics is important. In this study, time-course concentration changes in peripheral blood of healthy and neutropenic mice were measured using four therapeutic phages (φMR11, KPP10, φEF24C, and KEP10). The results showed a two- to three-day rapid phage clearance, which fits a two-compartment model.     

Characterization of Pseudomonas aeruginosa phage KPP21 belonging to family Podoviridae genus N4‐like viruses isolated in Japan

Bacteriophages (phages) belonging to the family Podoviridae genus N4‐like viruses have been used as therapeutic agent in phage therapy against Pseudomonas aeruginosa infections. P. aeruginosa phage KPP21 was isolated in Japan, and phylogenetically investigated the phages belonging to this viral genus. Morphological and genetic analyses confirmed that phage KPP21 belongs to the family Podoviridae genus N4‐like viruses. Moreover, phylogenetic analyses based on putative DNA polymerase and major virion protein showed that P. aeruginosa phages belonging to the genus N4‐like viruses are separated into two lineages and that phage KPP21 is in the same clade as phage LUZ7.     

Prevalence, genetic conservation and transmissibility of the Chlamydia pneumoniae bacteriophage (phiCpn1)

The Chlamydia pneumoniae bacteriophage was first identified in isolate AR-39. Its relevance for chlamydial biology and pathogenicity remains unknown. In this study, a collection of 36 C. pneumoniae isolates was screened and the phage was detected in eight. As the positive isolates differed by several polymorphisms, they presumably belonged to different genetic lineages. It was investigated whether different genotypes of the phage also existed and whether they could be assigned to chlamydial genotypes as evidence of coevolution. Sequencing of >3000 bp of the 4524 bp phage genome revealed complete identity to the published sequences. Thus, it was hypothesized that the genetic conservation was related to easy transmissibility of the phage between C. pneumoniae isolates. Cocultivation of phage positive and negative isolates followed by cloning and identification of different C. pneumoniae genotypes demonstrated for the first time transmissibility of the bacteriophage from one isolate to the other. These observations indicate that the phage is capable of infecting C. pneumoniae isolates of different genetic backgrounds and suggest that all C. pneumoniae strains might be susceptible. The successful in vitro infection of C. pneumoniae with the phage provides the basis for studying its pathogenetic relevance in isolates of identical genetic background and provides a potential tool for genetic manipulation of C. pneumoniae.     

Economic evaluation of the development of a phage therapy product for the control of Salmonella in poultry

Poultry products are one of the major transmission media of Salmonella enteritidis to humans. A promising alternative to reduce the load of Salmonella in poultry are bacteriophages. Elsewhere, a mixture of six bacteriophages has been used successfully, but large-scale production would be necessary to supply potential poultry market and costs analyses have not been calculated yet. For this, a powerful tool to predict production costs is bioprocess modeling coupled with economic analyses. This work aims to model the scaled-up production of a six bacteriophages mixture based on a laboratory/pilot-scale production using Biosolve Process. For the model construction, a combination of experimental and reported data was applied, in which different production alternatives and the range of 1–100% of the Colombian poultry market (at broiler's farm and slaughterhouse) were analyzed. Results indicate that the best cost-effective process configuration/scale is to use one bioreactor (156 L) for the six bacteriophages, then a 0.45 μm filtration for removal of biomass, and a 0.22 μm filtration for sterility; this to supply the 35% of the market size for broiler farms (equivalent to 210 million chickens). This configuration gives a production cost per chicken of US$ 0.02. Additionally, a sensitivity analysis and a theoretical contrast for understanding the impact that titer and recovery have on production scale determined that titer affects the most the cost and requires optimization. The present works serves as a first, and required, approach for the development of phage therapy products that are alternatives to present-day pathogens control strategies.     

PhREEPred: Phage Resistance Emergence Prediction Web Tool to Foresee Encapsulated Bacterial Escape from Phage Cocktail Treatment

Phages, as well as phage-derived proteins, especially lysins and depolymerases, are intensively studied to become prospective alternatives or supportive antibacterials used alone or in combination. In the common phage therapy approach, the unwanted emergence of phage-resistant variants from the treated bacterial population can be postponed or reduced by the utilization of an effective phage cocktail. In this work, we present a publicly available web tool PhREEPred (Phage Resistance Emergence Prediction) (https://phartner.shinyapps.io/PhREEPred/), which will allow an informed choice of the composition of phage cocktails by predicting the outcome of phage cocktail or phage/depolymerase combination treatments against encapsulated bacterial pathogens given a mutating population that escapes single phage treatment. PhREEPred simulates solutions of our mathematical model calibrated and tested on the experimental Klebsiella pneumoniae setup and Klebsiella-specific lytic phages: K63 type-specific phage KP34 equipped with a capsule-degrading enzyme (KP34p57), capsule-independent myoviruses KP15 and KP27, and recombinant capsule depolymerase KP34p57. The model can calculate the phage-resistance emergence depending on the bacterial growth rate and initial density, the multiplicity of infection, phage latent period, its infectiveness and the cocktail composition, as well as initial depolymerase concentration and activity rate. This model reproduced the experimental results and showed that (i) the phage cocktail of parallelly infecting phages is less effective than the one composed of sequentially infecting phages; (ii) depolymerase can delay or prevent bacterial resistance by unveiling an alternative receptor for initially inactive phages. In our opinion, this customer-friendly web tool will allow for the primary design of the phage cocktail and phage-depolymerase combination effectiveness against encapsulated pathogens.     

Studies related to the head-maturation pathway of bacteriophages T4 and T2: II. Nuclear disruption,protein synthesis and particle formation with the mutant 43−·30−·46−

We describe the aberrant phage multiplication of the triple conditional lethal mutant 43^?(polymerase)· 30^?(ligase)·46^?(exonuclease) of bacteriophage T4D in which phage DNA replication is arrested but some late protein synthesis occurs (33). The nuclear disruption is indistinguishable from wild type. Forty-five empty small and empty large particles are assembled per cell when the multiplicity of infection (m.o.i.) is 100. This number corresponds closely to the 38 phage equivalents of cleaved major head protein determined biochemically. By reducing the m.o.i. the number of observable particles decreases, reaching 1–5 per cell at an m.o.i. of 5(+5). The total synthesis of phage related proteins is not significantly dependant on the m.o.i. The synthesis of late proteins is about 10% of that of wild type at high m.o.i. and decreases with the m.o.i. The different early and late proteins do not show the same relative proportions as in wild type and respond differently to an increased m.o.i. These and other results are discussed with respect to the role of phage DNA in prehead assembly and head maturation.     

Isolation of lytic bacteriophage against Vibrio harveyi

Aims: The isolation of lytic bacteriophage of Vibrio harveyi with potential for phage therapy of bacterial pathogens of phyllosoma larvae from the tropical rock lobster Panulirus ornatus. Methods and Results: Water samples from discharge channels and grow‐out ponds of a prawn farm in northeastern Australia were enriched for 24 h in a broth containing four V. harveyi strains. The bacteriophage‐enriched filtrates were spotted onto bacterial lawns demonstrating that the bacteriophage host range for the samples included strains of V. harveyi, Vibrio campbellii, Vibrio rotiferianus, Vibrio parahaemolyticus and Vibrio proteolyticus. Bacteriophage were isolated from eight enriched samples through triple plaque purification. The host range of purified phage included V. harveyi, V. campbellii, V. rotiferianus and V. parahaemolyticus. Transmission electron microscope examination revealed that six purified phage belonged to the family Siphoviridae, whilst two belonged to the family Myoviridae. The Myoviridae appeared to induce bacteriocin production in a limited number of host bacterial strains, suggesting that they were lysogenic rather than lytic. A purified Siphoviridae phage could delay the entry of a broth culture of V. harveyi strain 12 into exponential growth, but could not prevent the overall growth of the bacterial strain. Conclusions: Bacteriophage with lytic activity against V. harveyi were isolated from prawn farm samples. Purified phage of the family Siphoviridae had a clear lytic ability and no apparent transducing properties, indicating they are appropriate for phage therapy. Phage resistance is potentially a major constraint to the use of phage therapy in aquaculture as bacteria are not completely eliminated. Significance and Impact of the Study: Phage therapy is emerging as a potential antibacterial agent that can be used to control pathogenic bacteria in aquaculture systems. The development of phage therapy for aquaculture requires initial isolation and determination of the bacteriophage host range, with subsequent creation of suitable phage cocktails.     

Quantitative analysis of the bacteriophage Qβ infection cycle

In this study, the infection cycle of bacteriophage Qβ was investigated. Adsorption of bacteriophage Qβ to Escherichia coli is explained in terms of a collision reaction, the rate constant of which was estimated to be 4 × 10^− 10 ml/cells/min. In infected cells, approximately 130 molecules of β-subunit and 2 × 10⁵ molecules of coat protein were translated in 15 min. Replication of Qβ RNA proceeded in 2 steps—an exponential phase until 20 min and a non-exponential phase after 30 min. Prior to the burst of infected cells, phage RNAs and coat proteins accumulated in the cells at an average of up to 2300 molecules and 5 × 10⁵ molecules, respectively. An average of 90 infectious phage particles per infected cell was released during a single infection cycle up to 105 min.     

Phage‐selected lipopolysaccharide mutants of Pectobacterium atrosepticum exhibit different impacts on virulence

Aims: To positively select Pectobacterium atrosepticum (Pa) mutants with cell surface defects and to assess the impact of these mutations on phytopathogenesis. Methods and Results: Several phages were isolated from treated sewage effluent and were found to require bacterial lipopolysaccharide (LPS) for infection. Two strains with distinct mutations in LPS were obtained by transposon mutagenesis. Along with a third LPS mutant, these strains were characterized with respect to various virulence‐associated phenotypes, including growth rate, motility and exoenzyme production, demonstrating that LPS mutations are pleiotropic. Two of the strains were deficient in the synthesis of the O‐antigen portion of LPS, and both were less virulent than the wild type. A waaJ mutant, which has severe defects in LPS biosynthesis, was dramatically impaired in potato tuber rot assays. The infectivity of these novel phages on 32 additional strains of Pa was tested, showing that most Pa isolates were sensitive to the LPS‐dependent phages. Conclusions: Native LPS is crucial for optimal growth, survival and virulence of Pa in vivo, but simultaneously renders such strains susceptible to phage infection. Significance and Impact of the Study: This work demonstrates the power of phages to select and identify the virulence determinants on the bacterial surface, and as potential biocontrol agents for Pa infections.     

Dynamic Modulation of the Gut Microbiota and Metabolome by Bacteriophages in a Mouse Model

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Genome comparison of Pseudomonas aeruginosa large phages

Pseudomonas aeruginosa phage EL is a dsDNA phage related to the giant phiKZ-like Myoviridae. The EL genome sequence comprises 211,215 bp and has 201 predicted open reading frames (ORFs). The EL genome does not share DNA sequence homology with other viruses and micro-organisms sequenced to date. However, one-third of the predicted EL gene products (gps) shares similarity (Blast alignments of 17-55% amino acid identity) with phiKZ proteins. Comparative EL and phiKZ genomics reveals that these giant phages are an example of substantially diverged genetic mosaics. Based on the position of similar EL and phiKZ predicted gene products, five genome regions can be delineated in EL, four of which are relatively conserved between EL and phiKZ. Region IV, a 17.7 kb genome region with 28 predicted ORFs, is unique to EL. Fourteen EL ORFs have been assigned a putative function based on protein similarity. Assigned proteins are involved in DNA replication and nucleotide metabolism (NAD+-dependent DNA ligase, ribonuclease HI, helicase, thymidylate kinase), host lysis and particle structure. EL-gp146 is the first chaperonin GroEL sequence identified in a viral genome. Besides a putative transposase, EL harbours predicted mobile endonucleases related to H-N-H and LAGLIDADG homing endonucleases associated with group I intron and intein intervening sequences.     

Unlocking of the filamentous bacteriophage virion during infection is mediated by the C domain of pIII

Protein III (pIII) of filamentous phage is required for both the beginning and the end of the phage life cycle. The infection starts by binding of the N-terminal N2 and N1 domains to the primary and secondary host receptors, F pilus and TolA protein, respectively, whereas the life cycle terminates by the C-terminal domain-mediated release of the membrane-anchored virion from the cell. It has been assumed that the role of the C-terminal domain of pIII in the infection is that of a tether for the receptor-binding domains N1N2 to the main body of the virion. In a poorly understood process that follows receptor binding, the virion disassembles as its protein(s) become integrated into the host inner membrane, resulting in the phage genome entry into the bacterial cytoplasm. To begin revealing the mechanism of this process, we showed that tethering the functional N1N2 receptor-binding domain to the virion via termination-incompetent C domain abolishes infection. This infection defect cannot be complemented by in trans supply of the functional C domain. Therefore, the C domain of pIII acts in concert with the receptor-binding domains to mediate the post receptor binding events in the infection. Based on these findings, we propose a model in which binding of the N1 domain to the periplasmic portion of TolA, the secondary receptor, triggers in cis a conformational change in the C domain, and that this change opens or unlocks the pIII end of the virion, allowing the entry phase of infection to proceed. To our knowledge, this is the first virus that uses the same protein domain both for the insertion into and release from the host membrane.