Phenotypic and Genomic Comparison of Klebsiella pneumoniae Lytic Phages: vB_KpnM-VAC66 and vB_KpnM-VAC13

Klebsiella pneumoniae is a human pathogen that worsens the prognosis of many immunocompromised patients. Here, we annotated and compared the genomes of two lytic phages that infect clinical strains of K. pneumoniae (vB_KpnM-VAC13 and vB_KpnM-VAC66) and phenotypically characterized vB_KpnM-VAC66 (time of adsorption of 12 min, burst size of 31.49 ± 0.61 PFU/infected cell, and a host range of 20.8% of the tested strains). Transmission electronic microscopy showed that vB_KpnM-VAC66 belongs to the Myoviridae family. The genomic analysis of the phage vB_KpnM-VAC66 revealed that its genome encoded 289 proteins. When compared to the genome of vB_KpnM-VAC13, they showed a nucleotide similarity of 97.56%, with a 93% of query cover, and the phylogenetic study performed with other Tevenvirinae phages showed a close common ancestor. However, there were 21 coding sequences which differed. Interestingly, the main differences were that vB_KpnM-VAC66 encoded 10 more homing endonucleases than vB_KpnM-VAC13, and that the nucleotidic and amino-acid sequences of the L-shaped tail fiber protein were highly dissimilar, leading to different three-dimensional protein predictions. Both phages differed significantly in their host range. These viruses may be useful in the development of alternative therapies to antibiotics or as a co-therapy increasing its antimicrobial potential, especially when addressing multidrug resistant (MDR) pathogens.

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Isolation, phenotypic characterization and comparative genomic analysis of 2019SD1, a polyvalent enterobacteria phage

Scientific Reports ◽

10.1038/s41598-021-01419-8 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Prince Kumar ◽

Mukesh K. Meghvansi ◽

D. V. Kamboj

Keyword(s):

Sequence Data ◽

Burst Size ◽

Genomic Analysis ◽

Dna Helicase ◽

Shigella Dysenteriae ◽

Whole Genome Sequence ◽

Phenotypic Characterization ◽

Comparative Genomic ◽

Tail Fiber ◽

Parsimony Method

AbstractShigella has the remarkable capability to acquire antibiotic resistance rapidly thereby posing a significant public health challenge for the effective treatment of dysentery (Shigellosis). The phage therapy has been proven as an effective alternative strategy for controlling Shigella infections. In this study, we illustrate the isolation and detailed characterization of a polyvalent phage 2019SD1, which demonstrates lytic activity against Shigella dysenteriae, Escherichia coli, Vibrio cholerae, Enterococcus saccharolyticus and Enterococcus faecium. The newly isolated phage 2019SD1 shows adsorption time < 6 min, a latent period of 20 min and burst size of 151 PFU per bacterial cell. 2019SD1 exhibits considerable stability in a wide pH range and survives an hour at 50 °C. Under transmission electron microscope, 2019SD1 shows an icosahedral capsid (60 nm dia) and a 140 nm long tail. Further, detailed bioinformatic analyses of whole genome sequence data obtained through Oxford Nanopore platform revealed that 2019SD1 belongs to genus Hanrivervirus of subfamily Tempevirinae under the family Drexlerviridae. The concatenated protein phylogeny of 2019SD1 with the members of Drexlerviridae taking four genes (DNA Primase, ATP Dependent DNA Helicase, Large Terminase Protein, and Portal Protein) using the maximum parsimony method also suggested that 2019SD1 formed a distinct clade with the closest match of the taxa belonging to the genus Hanrivervirus. The genome analysis data indicate the occurrence of putative tail fiber proteins and DNA methylation mechanism. In addition, 2019SD1 has a well-established anti-host defence system as suggested through identification of putative anti-CRISPR and anti-restriction endonuclease systems thereby also indicating its biocontrol potential.

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Characterization and Comparative Genomic Analysis of a Novel Bacteriophage, SFP10, Simultaneously Inhibiting both Salmonella enterica and Escherichia coli O157:H7

Applied and Environmental Microbiology ◽

10.1128/aem.06231-11 ◽

2011 ◽

Vol 78 (1) ◽

pp. 58-69 ◽

Cited By ~ 84

Author(s):

Minjung Park ◽

Ju-Hoon Lee ◽

Hakdong Shin ◽

Minsik Kim ◽

Jeongjoon Choi ◽

...

Keyword(s):

Escherichia Coli ◽

Salmonella Enterica ◽

Burst Size ◽

Genomic Analysis ◽

Comparative Genomic ◽

Tail Fiber ◽

Content Type ◽

E Coli ◽

Development Of Resistance

ABSTRACTSalmonella entericaandEscherichia coliO157:H7 are major food-borne pathogens causing serious illness. Phage SFP10, which revealed effective infection of bothS. entericaandE. coliO157:H7, was isolated and characterized. SFP10 contains a 158-kb double-stranded DNA genome belonging to the Vi01 phage-like familyMyoviridae.In vitroadsorption assays showed that the adsorption constant rates to bothSalmonella entericaserovar Typhimurium andE. coliO157:H7 were 2.50 × 10−8ml/min and 1.91 × 10−8ml/min, respectively. One-step growth analysis revealed that SFP10 has a shorter latent period (25 min) and a larger burst size (>200 PFU) than ordinaryMyoviridaephages, suggesting effective host infection and lytic activity. However, differential development of resistance to SFP10 inS.Typhimurium andE. coliO157:H7 was observed; bacteriophage-insensitive mutant (BIM) frequencies of 1.19 × 10−2CFU/ml forS.Typhimurium and 4.58 × 10−5CFU/ml forE. coliO157:H7 were found, indicating that SFP10 should be active and stable for control ofE. coliO157:H7 with minimal emergence of SFP10-resistant pathogens but may not be forS.Typhimurium. Specific mutation ofrfaLinS.Typhimurium andE. coliO157:H7 revealed the O antigen as an SFP10 receptor for both bacteria. Genome sequence analysis of SFP10 and its comparative analysis with homologousSalmonellaVi01 andShigellaphiSboM-AG3 phages revealed that their tail fiber and tail spike genes share low sequence identity, implying that the genes are major host specificity determinants. This is the first report identifying specific infection and inhibition ofSalmonellaTyphimurium andE. coliO157:H7 by a single bacteriophage.

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Identification of the Lower Baseplate Protein as the Antireceptor of the Temperate Lactococcal Bacteriophages TP901-1 and Tuc2009

Journal of Bacteriology ◽

10.1128/jb.188.1.55-63.2006 ◽

2006 ◽

Vol 188 (1) ◽

pp. 55-63 ◽

Cited By ~ 51

Author(s):

Christina S. Vegge ◽

Finn K. Vogensen ◽

Stephen Mc Grath ◽

Horst Neve ◽

Douwe van Sinderen ◽

...

Keyword(s):

Host Range ◽

Electron Micrographs ◽

Promoter Region ◽

Genomic Analysis ◽

Infection Process ◽

Tail Fiber ◽

Late Promoter ◽

Gene Encoding ◽

Related Phage ◽

Tail Structure

ABSTRACT The first step in the infection process of tailed phages is recognition and binding to the host receptor. This interaction is mediated by the phage antireceptor located in the distal tail structure. The temperate Lactococcus lactis phage TP901-1 belongs to the P335 species of the Siphoviridae family, which also includes the related phage Tuc2009. The distal tail structure of TP901-1 is well characterized and contains a double-disk baseplate and a central tail fiber. The structural tail proteins of TP901-1 and Tuc2009 are highly similar, but the phages have different host ranges and must therefore encode different antireceptors. In order to identify the antireceptors of TP901-1 and Tuc2009, a chimeric phage was generated in which the gene encoding the TP901-1 lower baseplate protein (bppLTP901-1 ) was exchanged with the analogous gene (orf532009 ) of phage Tuc2009. The chimeric phage (TP901-1C) infected the Tuc2009 host strain efficiently and thus displayed an altered host range compared to TP901-1. Genomic analysis and sequencing verified that TP901-1C is a TP901-1 derivative containing the orf532009 gene in exchange for bppLTP901-1 ; however, a new sequence in the late promoter region was also discovered. Protein analysis confirmed that TP901-1C contains ORF532009 and not the lower baseplate protein BppLTP901-1, and it was concluded that BppLTP901-1 and ORF532009 constitute antireceptor proteins of TP901-1 and Tuc2009, respectively. Electron micrographs revealed altered baseplate morphology of TP901-1C compared to that of the parental phage.

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Characterization of the Three New Kayviruses and Their Lytic Activity Against Multidrug-Resistant Staphylococcus aureus

Microorganisms ◽

10.3390/microorganisms7100471 ◽

2019 ◽

Vol 7 (10) ◽

pp. 471 ◽

Cited By ~ 7

Author(s):

Natalia Łubowska ◽

Bartłomiej Grygorcewicz ◽

Katarzyna Kosznik-Kwaśnicka ◽

Agata Zauszkiewicz-Pawlak ◽

Alicja Węgrzyn ◽

...

Keyword(s):

Staphylococcus Aureus ◽

Host Range ◽

Burst Size ◽

Genomic Analysis ◽

Multidrug Resistant ◽

Biological Properties ◽

Lytic Activity ◽

Broad Host Range ◽

Transmission Electron ◽

Short Latent Period

The development of antimicrobial resistance has become a global concern. One approach to overcome the problem of drug resistance is the application of bacteriophages. This study aimed at characterizing three phages isolated from sewage, which show lytic activity against clinical isolates of multidrug-resistant Staphylococcus aureus. Morphology, genetics and biological properties, including host range, adsorption rate, latent time, phage burst size and lysis profiles, were studied in all three phages. As analyzed by transmission electron microscopy (TEM), phages vB_SauM-A, vB_SauM-C, vB_SauM-D have a myovirion morphology. One of the tested phages, vB_SauM-A, has relatively rapid adsorption (86% in 17.5 min), short latent period (25 min) and extremely large burst size (~500 plaque-forming units (PFU) per infected cell). The genomic analysis revealed that vB_SauM-A, vB_SauM-C, vB_SauM-D possess large genomes (vB_SauM-A 139,031 bp, vB_SauM-C 140,086 bp, vB_SauM-D 139,088 bp) with low G+C content (~30.4%) and are very closely related to the phage K (95–97% similarity). The isolated bacteriophages demonstrate broad host range against MDR S. aureus strains, high lytic activity corresponding to strictly virulent life cycle, suggesting their potential to treat S. aureus infections.

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Characterization of vB_Kpn_F48, a Newly Discovered Lytic Bacteriophage for Klebsiella pneumoniae of Sequence Type 101

Viruses ◽

10.3390/v10090482 ◽

2018 ◽

Vol 10 (9) ◽

pp. 482 ◽

Cited By ~ 10

Author(s):

Nagaia Ciacci ◽

Marco D’Andrea ◽

Pasquale Marmo ◽

Elisa Demattè ◽

Francesco Amisano ◽

...

Keyword(s):

Klebsiella Pneumoniae ◽

Host Range ◽

High Throughput Sequencing ◽

Burst Size ◽

Antibiotic Resistance Genes ◽

Clinical Problem ◽

Lytic Bacteriophage ◽

Transmission Electron Microscopy Analysis ◽

Narrow Host Range ◽

Physiological Characterization

Resistance to carbapenems in Enterobacteriaceae, including Klebsiella pneumoniae, represents a major clinical problem given the lack of effective alternative antibiotics. Bacteriophages could provide a valuable tool to control the dissemination of antibiotic resistant isolates, for the decolonization of colonized individuals and for treatment purposes. In this work, we have characterized a lytic bacteriophage, named vB_Kpn_F48, specific for K. pneumoniae isolates belonging to clonal group 101. Phage vB_Kpn_F48 was classified as a member of Myoviridae, order Caudovirales, on the basis of transmission electron microscopy analysis. Physiological characterization demonstrated that vB_Kpn_F48 showed a narrow host range, a short latent period, a low burst size and it is highly stable to both temperature and pH variations. High throughput sequencing and bioinformatics analysis revealed that the phage is characterized by a 171 Kb dsDNA genome that lacks genes undesirable for a therapeutic perspective such integrases, antibiotic resistance genes and toxin encoding genes. Phylogenetic analysis suggests that vB_Kpn_F48 is a T4-like bacteriophage which belongs to a novel genus within the Tevenvirinae subfamily, which we tentatively named “F48virus”. Considering the narrow host range, the genomic features and overall physiological parameters phage vB_Kpn_F48 could be a promising candidate to be used alone or in cocktails for phage therapy applications.

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Corticosteroid Catabolism by Klebsiella pneumoniae as a Possible Mechanism for Increased Pneumonia Risk

Current Pharmaceutical Biotechnology ◽

10.2174/1389201020666190313153841 ◽

2019 ◽

Vol 20 (4) ◽

pp. 309-316 ◽

Cited By ~ 1

Author(s):

Pritam Chattopadhyay ◽

Goutam Banerjee

Keyword(s):

Amino Acid ◽

Klebsiella Pneumoniae ◽

Kegg Pathway ◽

Degradation Pathway ◽

Amino Acid Sequences ◽

Biological Mechanism ◽

Clinical Strains ◽

Catabolism Pathway ◽

Steroid Catabolism ◽

Nutrition Source

Background: Several strains of Klebsiella pneumoniae are responsible for causing pneumonia in lung and thereby causing death in immune-suppressed patients. In recent year, few investigations have reported the enhancement of K. pneumoniae population in patients using corticosteroid containing inhaler. Objectives: The biological mechanism(s) behind this increased incidence has not been elucidated. Therefore, the objective of this investigating was to explore the relation between Klebsiella pneumoniae and increment in carbapenamase producing Enterobacteriaceae score (ICS). Methods: The available genomes of K. pneumoniae and the amino acid sequences of steroid catabolism pathway enzymes were taken from NCBI database and KEGG pathway tagged with UniPort database, respectively. We have used different BLAST algorithms (tBLASTn, BLASTp, psiBLAST, and delBLAST) to identify enzymes (by their amino acid sequence) involved in steroid catabolism. Results: A total of 13 enzymes (taken from different bacterial candidates) responsible for corticosteroid degradation have been identified in the genome of K. pneumoniae. Finally, 8 enzymes (K. pneumoniae specific) were detected in four clinical strains of K. pneumoniae. This investigation intimates that this ability to catabolize corticosteroids could potentially be one mechanism behind the increased pneumonia incidence. Conclusion: The presence of corticosteroid catabolism enzymes in K. pneumoniae enhances the ability to utilize corticosteroid for their own nutrition source. This is the first report to demonstrate the corticosteroid degradation pathway in clinical strains of K. pneumoniae.

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Revealing biophysical properties of KfrA-type proteins as a novel class of cytoskeletal, coiled-coil plasmid-encoded proteins

BMC Microbiology ◽

10.1186/s12866-020-02079-w ◽

2021 ◽

Vol 21 (1) ◽

Author(s):

M. Adamczyk ◽

E. Lewicka ◽

R. Szatkowska ◽

H. Nieznanska ◽

J. Ludwiczak ◽

...

Keyword(s):

Dna Binding ◽

Host Range ◽

Coiled Coil ◽

Amino Acid Sequences ◽

Broad Host Range ◽

Biophysical Properties ◽

Dna Binding Sites ◽

In Silico Studies ◽

Wide Range

Abstract Background DNA binding KfrA-type proteins of broad-host-range bacterial plasmids belonging to IncP-1 and IncU incompatibility groups are characterized by globular N-terminal head domains and long alpha-helical coiled-coil tails. They have been shown to act as transcriptional auto-regulators. Results This study was focused on two members of the growing family of KfrA-type proteins encoded by the broad-host-range plasmids, R751 of IncP-1β and RA3 of IncU groups. Comparative in vitro and in silico studies on KfrAR751 and KfrARA3 confirmed their similar biophysical properties despite low conservation of the amino acid sequences. They form a wide range of oligomeric forms in vitro and, in the presence of their cognate DNA binding sites, they polymerize into the higher order filaments visualized as “threads” by negative staining electron microscopy. The studies revealed also temperature-dependent changes in the coiled-coil segment of KfrA proteins that is involved in the stabilization of dimers required for DNA interactions. Conclusion KfrAR751 and KfrARA3 are structural homologues. We postulate that KfrA type proteins have moonlighting activity. They not only act as transcriptional auto-regulators but form cytoskeletal structures, which might facilitate plasmid DNA delivery and positioning in the cells before cell division, involving thermal energy.

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Lytic Myophage Abp53 Encodes Several Proteins Similar to Those Encoded by Host Acinetobacter baumannii and Phage phiKO2

Applied and Environmental Microbiology ◽

10.1128/aem.05116-11 ◽

2011 ◽

Vol 77 (19) ◽

pp. 6755-6762 ◽

Cited By ~ 14

Author(s):

Chia-Ni Lee ◽

Tsai-Tien Tseng ◽

Juey-Wen Lin ◽

Yung-Chieh Fu ◽

Shu-Fen Weng ◽

...

Keyword(s):

Acinetobacter Baumannii ◽

Burst Size ◽

Sputum Sample ◽

Polyacrylamide Gel Electrophoresis ◽

Multiple Drug ◽

Lytic Phage ◽

Tail Fiber ◽

Drug Resistant ◽

Content Type ◽

Multiple Drug Resistant

ABSTRACTAcinetobacter baumanniiis an important Gram-negative opportunistic pathogen causing nosocomial infections. The emergence of multiple-drug-resistantA. baumanniiisolates has increased in recent years. Directed toward phage therapy, a lytic phage ofA. baumannii, designated Abp53, was isolated from a sputum sample in this study. Abp53 has an isometric head and a contractile tail with tail fibers (belonging toMyoviridae), a latent period of about 10 min, and a burst size of approximately 150 PFU per infected cell. Abp53 could completely lyse 27% of theA. baumanniiisolates tested, which were all multiple drug resistant, but not other bacteria. Mg2+enhanced the adsorption and productivity of, and host lysis by, Abp53. Twenty Abp53 virion proteins were visualized in SDS-polyacrylamide gel electrophoresis, with a 47-kDa protein being the predicted major capsid protein. Abp53 has a double-stranded DNA genome of 95 kb. Sequence analyses of a 10-kb region revealed 8 open reading frames. Five of the encoded proteins, including 3 tail components and 2 hypothetical proteins, were similar to proteins encoded byA. baumanniistrain ACICU. ORF1176 (one of the tail components, 1,176 amino acids [aa]), which is also similar to tail protein gp21 ofKlebsiellaphage phiKO2, contained repeated domains similar to those within the ACICU_02717 protein ofA. baumanniiACICU and gp21. These findings suggest a common ancestry and horizontal gene transfer during evolution. As phages can expand the host range by domain duplication in tail fiber proteins, repeated domains in ORF1176 might have a similar significance in Abp53.

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Yersiniophage ϕR1-37 is a tailed bacteriophage having a 270 kb DNA genome with thymidine replaced by deoxyuridine

Microbiology ◽

10.1099/mic.0.28265-0 ◽

2005 ◽

Vol 151 (12) ◽

pp. 4093-4102 ◽

Cited By ~ 60

Author(s):

Saija Kiljunen ◽

Kristo Hakala ◽

Elise Pinta ◽

Suvi Huttunen ◽

Patrycja Pluta ◽

...

Keyword(s):

Yersinia Pseudotuberculosis ◽

Burst Size ◽

Outer Core ◽

International Committee ◽

Amino Acid Sequences ◽

Structural Proteins ◽

Virulence Plasmid ◽

O Antigen ◽

Serotype O ◽

Phage Receptor

Bacteriophage ϕR1-37 was isolated based on its ability to infect strain YeO3-R1, a virulence-plasmid-cured O antigen-negative derivative of Yersinia enterocolitica serotype O : 3. In this study, the phage receptor was found to be a structure in the outer core hexasaccharide of Y. enterocolitica O : 3 LPS. The phage receptor was present in the outer core of strains of many other Y. enterocolitica serotypes, but also in some Yersinia intermedia strains. Surprisingly, the receptor structure resided in the O antigen of Yersinia pseudotuberculosis O : 9. Electron microscopy demonstrated that ϕR1-37 particles have an icosahedral head of 88 nm, a short neck of 10 nm, a long contractile tail of 236 nm, and tail fibres of at least 86 nm. This implies that the phage belongs to the order Caudovirales and the family Myoviridae in the ICTV (International Committee for Taxonomy of Viruses) classification. ϕR1-37 was found to have a lytic life cycle, with eclipse and latent periods of 40 and 50 min, respectively, and a burst size of ∼80 p.f.u. per infected cell. Restriction digestions and PFGE showed that the ϕR1-37 genome was dsDNA and ∼270 kb in size. Enzymically hydrolysed DNA was subjected to HPLC-MS/MS analysis, which demonstrated that the ϕR1-37 genome is composed of DNA in which thymidine (T) is >99 % replaced by deoxyuridine (dU). The only organisms known to have similar DNA are the Bacillus subtilis-specific bacteriophages PBS1 and PBS2. N-terminal amino acid sequences of four major structural proteins did not show any similarity to (viral) protein sequences in databases, indicating that close relatives of ϕR1-37 have not yet been characterized. Genes for two of the structural proteins, p24 and p46, were identified from the partially sequenced ϕR1-37 genome.

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Characterization and Full Genome Sequence of Novel KPP-5 Lytic Phage against Klebsiella pneumoniae Responsible for Recalcitrant Infection

Biomedicines ◽

10.3390/biomedicines9040342 ◽

2021 ◽

Vol 9 (4) ◽

pp. 342

Author(s):

Ahmed R. Sofy ◽

Noha K. El-Dougdoug ◽

Ehab E. Refaey ◽

Rehab A. Dawoud ◽

Ahmed A. Hmed

Keyword(s):

Klebsiella Pneumoniae ◽

Burst Size ◽

Phylogenetic Analyses ◽

Foodborne Pathogen ◽

Gc Content ◽

Full Genome Sequence ◽

Multiple Drug ◽

Lytic Phage ◽

Trna Genes ◽

Bacteriophage Therapy

Klebsiella pneumoniae is a hazardous opportunistic pathogen that is involved in many serious human diseases and is considered to be an important foodborne pathogen found in many food types. Multidrug resistance (MDR) K. pneumoniae strains have recently spread and increased, making bacteriophage therapy an effective alternative to multiple drug-resistant pathogens. As a consequence, this research was conducted to describe the genome and basic biological characteristics of a novel phage capable of lysing MDR K. pneumoniae isolated from food samples in Egypt. The host range revealed that KPP-5 phage had potent lytic activity and was able to infect all selected MDR K. pneumoniae strains from different sources. Electron microscopy images showed that KPP-5 lytic phage was a podovirus morphology. The one-step growth curve exhibited that KPP-5 phage had a relatively short latent period of 25 min, and the burst size was about 236 PFU/infected cells. In addition, KPP-5 phage showed high stability at different temperatures and pH levels. KPP-5 phage has a linear dsDNA genome with a length of 38,245 bp with a GC content of 50.8% and 40 predicted open reading frames (ORFs). Comparative genomics and phylogenetic analyses showed that KPP-5 is most closely associated with the Teetrevirus genus in the Autographviridae family. No tRNA genes have been identified in the KPP-5 phage genome. In addition, phage-borne virulence genes or drug resistance genes were not present, suggesting that KPP-5 could be used safely as a phage biocontrol agent.

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