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

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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Isolation, Characterization, and Application in Poultry Product of a Specific Salmonella Bacteriophage S55

Journal of Food Protection ◽

10.4315/jfp-20-438 ◽

2021 ◽

Author(s):

Haojie Ge ◽

Yanping Xu ◽

Kai Zhang ◽

Shuxuan Zhang ◽

Maozhi Hu ◽

...

Keyword(s):

Prevention And Control ◽

Burst Size ◽

Gc Content ◽

Skin Surface ◽

Open Reading Frames ◽

Lytic Phage ◽

Trna Genes ◽

Host Bacterium ◽

Salmonella Pullorum ◽

And Control

Salmonellosis occurs frequently worldwide, causing serious threats to public health safety. The abuse of antibiotics is increasing the antibiotic resistance in bacteria, thereby making the prevention and control of Salmonella more difficult. A phage can help control the spread of bacteria. In this study, S55, a lytic phage, was isolated from faecal samples obtained from poultry farms using Salmonella Pullorum ( S . Pullorum) as the host bacterium. This phage belongs to Siphoviridae and has a polyhedral head and a retraction-free tail. S55 showed a strong ability to lyse Salmonella serovars, such as S . Pullorum (58/60, 96.67%) and S . Enteritidis (97/104, 93.27%). One-step growth kinetics showed that the latent period was 10 min, burst period was 80 min and burst size was 40 pfu/cell. The optimal multiplicity of infection was 0.01, and the phage was able to survive at a pH of 4–11 and temperature of 40°C–60°C for 60 min. Complete genome sequence analysis revealed that the S55 genome length is 42,781 bp (GC content, 50.28%) and it contains 58 open reading frames (ORF), including 25 ORFs with known or assumed functions, without tRNA genes. Moreover, S55 does not carry genes that encode virulence or resistance factors. At different temperatures (4°C or 25°C), S55 was found to lower the populations of S . Pullorum and S . Enteritidis on chicken skin surface. Its bacteriostatic effect at 4°C was higher than that at 25°C. In conclusion, S55 can be considered a promising biological agent for the prevention and control of Salmonella .

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Isolation and characterization of Hena1 – a novel Erwinia amylovora bacteriophage

FEMS Microbiology Letters ◽

10.1093/femsle/fnaa070 ◽

2020 ◽

Vol 367 (9) ◽

Author(s):

Natalya V Besarab ◽

Artur E Akhremchuk ◽

Maryna A Zlatohurska ◽

Liudmyla V Romaniuk ◽

Leonid N Valentovich ◽

...

Keyword(s):

Pathogenic Bacteria ◽

Fire Blight ◽

Antimicrobial Agents ◽

Erwinia Amylovora ◽

Gc Content ◽

Control Measures ◽

Effective Control ◽

Lytic Phage ◽

Trna Genes ◽

Isolation And Characterization

ABSTRACT Fire blight, caused by plant pathogenic bacterium Erwinia amylovora, is one of the most important diseases of Rosaceae plants. Due to the lack of effective control measures, fire blight infections pose a recurrent threat on agricultural production worldwide. Recently, bacterial viruses, or bacteriophages, have been proposed as environmentally friendly natural antimicrobial agents for fire blight control. Here, we isolated a novel bacteriophage Hena1 with activity against E. amylovora. Further analysis revealed that Hena1 is a narrow-host-range lytic phage belonging to Myoviridae family. Its genome consists of a linear 148,842 bp dsDNA (48.42% GC content) encoding 240 ORFs and 23 tRNA genes. Based on virion structure and genomic composition, Hena1 was classified as a new species of bacteriophage subfamily Vequintavirinae. The comprehensive analysis of Hena1 genome may provide further insights into evolution of bacteriophages infecting plant pathogenic bacteria.

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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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Complete Chloroplast Genomes of Ampelopsis humulifolia and Ampelopsis japonica: Molecular Structure, Comparative Analysis, and Phylogenetic Analysis

Plants ◽

10.3390/plants8100410 ◽

2019 ◽

Vol 8 (10) ◽

pp. 410 ◽

Cited By ~ 9

Author(s):

Xiaolei Yu ◽

Wei Tan ◽

Huanyu Zhang ◽

Han Gao ◽

Wenxiu Wang ◽

...

Keyword(s):

Phylogenetic Analysis ◽

Phylogenetic Analyses ◽

Gc Content ◽

Rrna Genes ◽

Trna Genes ◽

Protein Z ◽

Marker Selection ◽

Chloroplast Genomes ◽

Potential Marker ◽

Cp Genome

Ampelopsis humulifolia (A. humulifolia) and Ampelopsis japonica (A. japonica), which belong to the family Vitaceae, are valuably used as medicinal plants. The chloroplast (cp) genomes have been recognized as a convincing data for marker selection and phylogenetic studies. Therefore, in this study we reported the complete cp genome sequences of two Ampelopsis species. Results showed that the cp genomes of A. humulifolia and A. japonica were 161,724 and 161,430 bp in length, respectively, with 37.3% guanine-cytosine (GC) content. A total of 114 unique genes were identified in each cp genome, comprising 80 protein-coding genes, 30 tRNA genes, and 4 rRNA genes. We determined 95 and 99 small sequence repeats (SSRs) in A. humulifolia and A. japonica, respectively. The location and distribution of long repeats in the two cp genomes were identified. A highly divergent region of psbZ (Photosystem II reaction center protein Z) -trnG (tRNA-Glycine) was found and could be treated as a potential marker for Vitaceae, and then the corresponding primers were designed. Additionally, phylogenetic analysis showed that Vitis was closer to Tetrastigma than Ampelopsis. In general, this study provides valuable genetic resources for DNA barcoding marker identification and phylogenetic analyses of Ampelopsis.

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Characterization and genome analysis of novel phage vB_KpnM _Bp5 infecting Klebsiella pneumonia

10.21203/rs.2.13213/v1 ◽

2019 ◽

Author(s):

Cong Zhang ◽

Chenling Ge ◽

Xiaoye Wang ◽

Deyuan Wei ◽

Xun Li ◽

...

Keyword(s):

Klebsiella Pneumoniae ◽

Genome Analysis ◽

Bacterial Resistance ◽

Burst Size ◽

Multidrug Resistant ◽

Lytic Phage ◽

Klebsiella Pneumonia ◽

Large Outbreak ◽

Electron Microscopy Analysis ◽

Blastn Analysis

Abstract Background With the incidence of antibiotic resistance reaching crisis point, it is imperative to find alternative treatments for multidrug-resistant infections. Using phage for pathogen control might be a promising treatment option to combat bacterial resistance. Results In this study, a lytic phage, designated vB_KpnM _Bp5, was isolated from pig faecal sample in Nanning, Guangxi province of China, and classified as a member of the family Muscle virus based on electron microscopy analysis. A one-step growth curve of the phage at the optimal MOI revealed that the latent time was 40 min and the burst size was 24 PFU/cell, indicative of good lysis capacity. Whole genome sequencing showed that phage vB_KpnM _Bp5 had a small dsDNA genome of 43872 bp. BLASTn analysis showed that it shared 94.06% identity (94% genome coverage) with Klebsiella phage vB_KpnP_SU552A of complete genome idefix. RAST genome analysis showed that the phage had 50 ORFs due to its small genome size, and the number of functional proteins was consistent with other phages. To evaluate the therapeutic effect of Klebsiella pneumoniae infection in mice, the results showed that phages provided vB_KpnM _Bp5better protection. Conclusion The phage vB_KpnM _Bp5 had the characteristics of broad host spectrum, strong environmental adaptability, short incubation period, large outbreak amount, and can cure the mouse model infected by Klebsiella pneumoniae. These findings suggested that phage vB_KpnM _Bp5 could be considered a potential therapeutic or prophylactic candidate against Klebsiella pneumonia infection.

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Effectiveness of a Lytic Phage SRG1 against Vancomycin-ResistantEnterococcus faecalisin Compost and Soil

BioMed Research International ◽

10.1155/2017/9351017 ◽

2017 ◽

Vol 2017 ◽

pp. 1-8 ◽

Cited By ~ 6

Author(s):

Sidra Rahmat Ullah ◽

Saadia Andleeb ◽

Taskeen Raza ◽

Muhsin Jamal ◽

Khalid Mehmood

Keyword(s):

Soil Sample ◽

Burst Size ◽

Alternative Therapy ◽

Maximum Growth ◽

Sewage Water ◽

Infection Process ◽

Lytic Phage ◽

Lytic Bacteriophage ◽

Bacteriophage Therapy ◽

Tract Infections

Nosocomial infections caused by vancomycin-resistantEnterococcushave become a major problem. Bacteriophage therapy is proposed as a potential alternative therapy. Bacteriophages are viruses that infect bacteria and are ubiquitous in nature. Lytic bacteriophage was isolated from sewage water that infects VREF, the causative agent of endocarditis, bacteraemia, and urinary tract infections (UTIs). The phage produced clear plaques with unique clear morphology and well-defined boundaries. TEM results of phage revealed it to be108±0.2 nm long and90±0.5 nm wide. The characterization of bacteriophage revealed that infection process of phage was calcium and magnesium dependent and phage titers were highest under optimum conditions for VREF, with an optimal temperature range of 37–50°C. The maximum growth was observed at 37°C, hence having 100% viability. The latent period for phage was small with a burst size of 512 viral particles per bacterial cell. The phage was tested against various clinical strains and results proved it to be host specific. It can be used as a potential therapeutic agent for VREF infections. The phage efficiently eradicated VREF inoculated in cattle compost, poultry compost, and a soil sample which makes it a potential agent for clearing compost and soil sample.

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Efficacious antibacterial potency of novel bacteriophages against ESBL-producing Klebsiella pneumoniae isolated from burn wound infections

Iranian Journal of Microbiology ◽

10.18502/ijm.v13i5.7435 ◽

2021 ◽

Author(s):

Ladan Rahimzadeh Torabi ◽

Nafiseh Sadat Naghavi ◽

Monir Doudi ◽

Ramesh Monajemi

Keyword(s):

Klebsiella Pneumoniae ◽

Bacterial Infections ◽

Burst Size ◽

16S Rrna Gene Sequencing ◽

Morphological Characterization ◽

Host Cells ◽

Clinical Samples ◽

Rrna Gene ◽

Bacteriophage Therapy ◽

Burn Wound

Background and Objectives: Prevalence of extended spectrum β-lactamase (ESBL) leads to the development of antibi- otic resistance and mortality in burn patients. One of the alternative strategies for controlling ESBL bacterial infections is clinical trials of bacteriophage therapy. The aim of this study was to isolate and characterize specific bacteriophages against ESBL-producing Klebsiella pneumoniae in patients with burn ulcers. Materials and Methods: Clinical samples were isolated from the hospitalized patient in burn medical centers, Iran. Bio- chemical screenings and 16S rRNA gene sequencing were determined. The phages were isolated from municipal sewerage treatment plants, Isfahan, Iran. TEM and FESEM, adsorption velocity, growth curve, host range, and the viability of the phage particles as well as proteomics and enzyme digestion patterns were examined. Results: The results showed that Klebsiella pneumoniae Iaufa_lad2 (GenBank accession number: MW836954) was con- firmed as an ESBL-producing strain using combined disk method. This bacterium showed significant sensitivity to three phages including PɸBw-Kp1, PɸBw-Kp2, and PɸBw-Kp3. Morphological characterization demonstrated that the phage PɸBw-Kp3 to the Siphoviridae family (lambda-like phages) and both phages PɸBw-Kp1 and ɸBw-Kp2 to the Podoviridae family (T1-like phages). The isolated bacteriophages had a large burst size, thermal and pH viability and efficient adsorption rate to the host cells. Conclusion: In present study, the efficacy of bacteriophages against ESBL pathogenic bacterium promises a remarkable achievement for phage therapy. It seems that, these isolated bacteriophages, in the form of phage cocktails, had a strong an- tibacterial impacts and a broad-spectrum strategy against ESBL-producing Klebsiella pneumoniae isolated from burn ulcers.

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Characterisation of Bacteriophage-Encoded Depolymerases Selective for Key Klebsiella pneumoniae Capsular Exopolysaccharides

Frontiers in Cellular and Infection Microbiology ◽

10.3389/fcimb.2021.686090 ◽

2021 ◽

Vol 11 ◽

Author(s):

George Blundell-Hunter ◽

Mark C. Enright ◽

David Negus ◽

Matthew J. Dorman ◽

Gemma E. Beecham ◽

...

Keyword(s):

Klebsiella Pneumoniae ◽

Host Range ◽

Multiple Drug Resistance ◽

Sewage Water ◽

Multiple Drug ◽

Lytic Phage ◽

Broad Host Range ◽

Capsular Polysaccharides ◽

Protein Database ◽

Rolling Ball

Capsular polysaccharides enable clinically important clones of Klebsiella pneumoniae to cause severe systemic infections in susceptible hosts. Phage-encoded capsule depolymerases have the potential to provide an alternative treatment paradigm in patients when multiple drug resistance has eroded the efficacy of conventional antibiotic chemotherapy. An investigation of 164 K. pneumoniae from intensive care patients in Thailand revealed a large number of distinct K types in low abundance but four (K2, K51, K1, K10) with a frequency of at least 5%. To identify depolymerases with the capacity to degrade capsules associated with these common K-types, 62 lytic phage were isolated from Thai hospital sewage water using K1, K2 and K51 isolates as hosts; phage plaques, without exception, displayed halos indicative of the presence of capsule-degrading enzymes. Phage genomes ranged in size from 41–348 kb with between 50 and 535 predicted coding sequences (CDSs). Using a custom phage protein database we were successful in applying annotation to 30 - 70% (mean = 58%) of these CDSs. The largest genomes, of so-called jumbo phage, carried multiple tRNAs as well as CRISPR repeat and spacer sequences. One of the smaller phage genomes was found to contain a putative Cas type 1E gene, indicating a history of host DNA acquisition in these obligate lytic phage. Whole-genome sequencing (WGS) indicated that some phage displayed an extended host range due to the presence of multiple depolymerase genes; in total, 42 candidate depolymerase genes were identified with up to eight in a single genome. Seven distinct virions were selected for further investigation on the basis of host range, phage morphology and WGS. Candidate genes for K1, K2 and K51 depolymerases were expressed and purified as his6-tagged soluble protein and enzymatic activity demonstrated against K. pneumoniae capsular polysaccharides by gel electrophoresis and Anton-Paar rolling ball viscometry. Depolymerases completely removed the capsule in K-type-specific fashion from K. pneumoniae cells. We conclude that broad-host range phage carry multiple enzymes, each with the capacity to degrade a single K-type, and any future use of these enzymes as therapeutic agents will require enzyme cocktails for utility against a range of K. pneumoniae infections.

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Characterization of the complete chloroplast genomes of five Populus species from the western Sichuan plateau, southwest China: comparative and phylogenetic analyses

PeerJ ◽

10.7717/peerj.6386 ◽

2019 ◽

Vol 7 ◽

pp. e6386 ◽

Cited By ~ 6

Author(s):

Dan Zong ◽

Anpei Zhou ◽

Yao Zhang ◽

Xinlian Zou ◽

Dan Li ◽

...

Keyword(s):

Phylogenetic Analyses ◽

Molecular Genetic ◽

Gc Content ◽

Rrna Genes ◽

Trna Genes ◽

Nucleotide Substitutions ◽

Protein Coding ◽

Western Sichuan ◽

Protein Coding Genes ◽

Chloroplast Genomes

Species of the genus Populus, which is widely distributed in the northern hemisphere from subtropical to boreal forests, are among the most commercially exploited groups of forest trees. In this study, the complete chloroplast genomes of five Populus species (Populus cathayana, P. kangdingensis, P. pseudoglauca, P. schneideri, and P. xiangchengensis) were compared. The chloroplast genomes of the five Populus species are very similar. The total chloroplast genome sequence lengths for the five plastomes were 156,789, 156,523, 156,512, 156,513, and 156,465 bp, respectively. A total of 130 genes were identified in each genome, including 85 protein-coding genes, 37 tRNA genes and eight rRNA genes. Seven genes were duplicated in the protein-coding genes, whereas 11 genes were duplicated in the RNA genes. The GC content was 36.7% for all plastomes. We analyzed nucleotide substitutions, small inversions, simple sequence repeats and long repeats in the chloroplast genomes and found nine divergence hotspots (ccsA+ccsA-ndhD, ndhC-trnV, psbZ-trnfM, trnG-atpA, trnL-ndhJ, trnR-trnN, ycf4-cemA, ycf1, and trnR-trnN), which could be useful molecular genetic markers for future population genetic and phylogenetic studies. We also observed that two genes (rpoC2 and rbcL) were subject to positive selection. Phylogenetic analysis based on whole cp genomes showed that P. schneideri had a close relationship with P. kangdingensis and P. pseudoglauca, while P. xiangchengensis was a sister to P. cathayana.

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Characterization and Application of a Lytic Phage D10 against Multidrug-Resistant Salmonella

Viruses ◽

10.3390/v13081626 ◽

2021 ◽

Vol 13 (8) ◽

pp. 1626

Author(s):

Zhiwei Li ◽

Wanning Li ◽

Wenjuan Ma ◽

Yifeng Ding ◽

Yu Zhang ◽

...

Keyword(s):

Burst Size ◽

Foodborne Pathogen ◽

Food Contamination ◽

Genomic Analysis ◽

Multidrug Resistant ◽

Lytic Phage ◽

Promising Tool ◽

Short Latent Period ◽

Dynamic Growth ◽

Natural Foods

Salmonella is a widely distributed foodborne pathogen that is a serious threat to human health. The accelerated development of drug resistance and the increased demand for natural foods invoke new biocontrol agents to limit contamination by multidrug-resistant (MDR) Salmonella strains. In this study, a lytic Salmonella phage named D10 was characterized at the biological and genomic levels. D10 possesses a short latent period (10 min) and a large burst size (163 PFU/cell), as well as adequate stability under a range of pH conditions and moderate thermal tolerance. D10 effectively lysed different MDR Salmonella serovars and repressed their dynamic growth in the medium. Genomic analysis disclosed that D10 is a new member of the Siphoviridae family and lacks the genes implicated in lysogeny, pathogenicity, or antibiotic resistance. A three-ingredient phage cocktail was then developed by mixing D10 with previously identified myovirus D1-2 and podovirus Pu20. The cocktail significantly reduced the count of MDR strains in liquid eggs, regardless of the temperature applied (4 and 25 °C). These results suggest that phage D10 is a promising tool to prevent food contamination by MDR Salmonella.

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