Isolation and characterization of Hena1 – a novel Erwinia amylovora bacteriophage

2020 ◽  
Vol 367 (9) ◽  
Author(s):  
Natalya V Besarab ◽  
Artur E Akhremchuk ◽  
Maryna A Zlatohurska ◽  
Liudmyla V Romaniuk ◽  
Leonid N Valentovich ◽  
...  

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.

Biomedicines ◽  
2021 ◽  
Vol 9 (4) ◽  
pp. 342
Author(s):  
Ahmed R. Sofy ◽  
Noha K. El-Dougdoug ◽  
Ehab E. Refaey ◽  
Rehab A. Dawoud ◽  
Ahmed A. Hmed

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.


2018 ◽  
Vol 64 (7) ◽  
pp. 483-491 ◽  
Author(s):  
Urmi Bajpai ◽  
Abhishek Kumar Mehta ◽  
Kandasamy Eniyan ◽  
Avni Sinha ◽  
Ankita Ray ◽  
...  

Bacteriophages are being considered as a promising natural resource for the development of alternative strategies against mycobacterial diseases, especially in the context of the wide-spread occurrence of drug resistance among the clinical isolates of Mycobacterium tuberculosis. However, there is not much information documented on mycobacteriophages from India. Here, we report the isolation of 17 mycobacteriophages using Mycobacterium smegmatis as the bacterial host, where 9 phages also lyse M. tuberculosis H37Rv. We present detailed analysis of one of these mycobacteriophages — PDRPv. Transmission electron microscopy and polymerase chain reaction analysis (of a conserved region within the TMP gene) show PDRPv to belong to the Siphoviridae family and B1 subcluster, respectively. The genome (69 110 bp) of PDRPv is circularly permuted double-stranded DNA with ∼66% GC content and has 106 open reading frames (ORFs). On the basis of sequence similarity and conserved domains, we have assigned function to 28 ORFs and have broadly categorized them into 6 groups that are related to replication and genome maintenance, DNA packaging, virion release, structural proteins, lysogeny-related genes and endolysins. The present study reports the occurrence of novel antimycobacterial phages in India and highlights their potential to contribute to our understanding of these phages and their gene products as potential antimicrobial agents.


2019 ◽  
Vol 85 (15) ◽  
Author(s):  
Jingyu Peng ◽  
Lindsay R. Triplett ◽  
Jeffrey K. Schachterle ◽  
George W. Sundin

ABSTRACTToxin-antitoxin (TA) systems are genetic elements composed of a protein toxin and a counteracting antitoxin that is either a noncoding RNA or protein. In type I TA systems, the antitoxin is a noncoding small RNA (sRNA) that base pairs with the cognate toxin mRNA interfering with its translation. Although type I TA systems have been extensively studied inEscherichia coliand a few human or animal bacterial pathogens, they have not been characterized in plant-pathogenic bacteria. In this study, we characterized a chromosomal locus in the plant pathogenErwinia amylovoraEa1189 that is homologous to thehok-soktype I TA system previously identified in theEnterobacteriaceae-restricted plasmid R1. Phylogenetic analysis indicated that the chromosomal location of thehok-soklocus is, thus far, unique toE. amylovora. We demonstrated that ectopic overexpression ofhokis highly toxic toE. amylovoraand that the sRNAsokreversed the toxicity ofhokthroughmok, a reading frame presumably translationally coupled withhok. We also identified the region that is essential for maintenance of the main toxicity of Hok. Through ahok-sokdeletion mutant (Ea1189Δhok-sok), we determined the contribution of thehok-soklocus to cellular growth, micromorphology, and catalase activity. Combined, our findings indicate that thehok-sokTA system, besides being potentially self-toxic, provides fitness advantages toE. amylovora.IMPORTANCEBacterial toxin-antitoxin systems have received great attention because of their potential as targets for antimicrobial development and as tools for biotechnology.Erwinia amylovora, the causal agent of fire blight disease on pome fruit trees, is a major plant-pathogenic bacterium. In this study, we identified and functionally characterized a unique chromosomally encodedhok-soktoxin-antitoxin system inE. amylovorathat resembles the plasmid-encoded copies of this system in otherEnterobacteriaceae. This study of a type I toxin-antitoxin system in a plant-pathogenic bacterium provides the basis to further understand the involvement of toxin-antitoxin systems during infection by a plant-pathogenic bacterium. The new linkage between thehok-soktoxin-antitoxin system and the catalase-mediated oxidative stress response leads to additional considerations of targeting this system for antimicrobial development.


2020 ◽  
Author(s):  
Swarnali Louha ◽  
Richard J. Meinersmann ◽  
Travis C. Glenn

AbstractWe performed whole-genome multi-locus sequence typing for 2554 genes in a large and heterogenous panel of 180 Listeria monocytogenes strains having diverse geographical and temporal origins. The subtyping data was used for characterizing genetic variation and evaluating patterns of linkage disequilibrium in the pan-genome of L. monocytogenes. Our analysis revealed the presence of strong linkage disequilibrium in L. monocytogenes, with ∼99% of genes showing significant non-random associations with a large majority of other genes in the genome. Twenty-seven loci having lower levels of association with other genes were considered to be potential “hot spots” for horizontal gene transfer (i.e., recombination via conjugation, transduction, and/or transformation). The patterns of linkage disequilibrium in L. monocytogenes suggest limited exchange of foreign genetic material in the genome and can be used as a tool for identifying new recombinant strains. This can help understand processes contributing to the diversification and evolution of this pathogenic bacteria, thereby facilitating development of effective control measures.


Plant Disease ◽  
2014 ◽  
Vol 98 (1) ◽  
pp. 153-153 ◽  
Author(s):  
K. K. Bastas ◽  
F. Sahin

Fire blight, caused by Erwinia amylovora (Burr.) Winslow et al., affects plants in the Rosaceae family, which includes trees and shrubs in orchards, nurseries, and landscape plantations. During the springs and summers of 2008 and 2010, dying branches, necrotic leaves attached to shoots, and blighted twigs of meadowsweet (Spirea prunifolia) were observed at three different locations of landscape areas in Konya Province, Turkey. Disease incidence was approximately 1% on the plants during the surveys. Initial symptoms of reddish to brownish streaks on the shoots of infected plants were observed in spring. Nine representative bacterial strains were isolated from the lesions on shoots of seven meadowsweet plants on nutrient sucrose agar (NSA) medium and identified as E. amylovora on basis of biochemical, physiological (2,3) and molecular tests (1). Bacteria were gram-negative, rod shaped, aerobic, fermentative, yellow-orange on Miller and Scroth medium (2), positive for levan formation and acetoin production, did not grow at 36°C, positive for gelatin hydrolysis, and negative for esculin hydrolysis, indole, urease, catalase, oxidase, arginine dehydrolase, reduction of nitrate, acid production from lactose, and inositol. All strains were hypersensitive response-positive on tobacco (Nicotiana tabacum var. White Burley) plants. All strains were identified as E. amylovora using the species-specific primers set, A/B (1), by PCR assay, and by fatty acid methyl ester (FAME) profiles determined by Sherlock Microbial Identification System software (TSBA 6 v. 6.00; Microbial ID, Newark, DE) with similarity indices ranging from of 79 to 99%. Pathogenicity was tested by injecting of petioles and actively growing three shoot tips of 2-year-old S. prunifolia seedlings cv. number 29 using a 0.46 mm-diameter hypodermic needle with bacterial suspensions containing 108 CFU mL–1 in sterile distilled water (SDW) Plants were inoculated with each of the nine bacterial strains and two references strains, Ea29 and NCPPB 2791 (Selcuk University, Department of Plant Protection, Konya, Turkey). Symptoms resembling those associated with natural infection appeared on the inoculated plants 7 days after inoculation. Plants inoculated with SDW served as a negative control treatment, and no symptoms were observed on these plants. All tests were repeated three times with the same results. Bacterial re-isolations were attempted from the control plants as well as shoots and leaves inoculated with the two reference strains and the nine bacteria identified as E. amylovora. Bacteria isolated from inoculated plants were identified as E. amylovora using the biochemical, physiological, and molecular tests described above, but this bacterium was not isolated from the control plants. Phytosanitary measures must be taken to avoid spread of the pathogen to ornamentals in new landscape areas in Turkey. This report is important because infected Spirea spp. can be a potential inoculum source for other rosaceous ornamentals. To our knowledge, this is the first report of the occurrence of fire blight on meadowsweet in Turkey. References: (1) S. Bereswill et al. Appl. Environ. Microbiol. 58:3522, 1992. (2) A. L. Jones and K. Geider. Laboratory Guide for Identification of Plant Pathogenic Bacteria, pp. 40-55. American Phytopathological Society, St. Paul, MN, 2001. (3) R. A. Lelliott and D. E. Stead. Methods for Diagnosis of Bacterial Diseases of Plants (Methods in Plant Pathology). Oxford, UK, 1987.


Author(s):  
Haojie Ge ◽  
Yanping Xu ◽  
Kai Zhang ◽  
Shuxuan Zhang ◽  
Maozhi Hu ◽  
...  

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 .


2018 ◽  
Vol 63 (3) ◽  
pp. 586-594 ◽  
Author(s):  
Mehul M. Jadav ◽  
Niranjan Kumar ◽  
Bhupamani Das ◽  
Jaesh B. Solanki

Abstract Morphological and molecular identification can pave the way to design the most effective control measures against the Paramphistomum epiclitum in small ruminants. Morphology of the flukes had described the features of Paramphistomum genus. Body was conical with concave ventral and convex dorsal surface, tegumental spines all around the body in the immature stage, terminal funnel shape oral sucker, sub-terminal acetabulum, blind caeca with a serpentine course touching the anterior level of the acetabulum. Vitelline glands were at the lateral margins of the body extended from the pharynx to the posterior sucker. Testes were lobed and tandem, wavy post-testicular uterus and genital pore behind intestinal bifurcation. Sequence analyses of internal transcribed spacer (ITS)-2+ (PCR products of approximately 500 bp) of 10 flukes yielded 2 genotypes, Navsari isolate 1 and 2. In BLAST analysis, ITS-2+ genotypes were 97.3–99% similar with published sequences (KF564870, JF834888, KF642983 and JX678254) of P. epiclitum of Paramphistomatidae. Two genotypes depicted 4 single nucleotide polymorphisms (NPs) in the form of transitions (C-T at 10 and 18; G-A at 255; A-G at 367 locus), 1 triple NPs (CGT-GAA between 21–23 loci) and missing A base at codon 40 in the genotype 1. Average AT and GC content was 49.61% and 50.38%, respectively. Trees topology inferred by Neighbor Joining and Maximum Likelihood methods of ITS2+ of trematodes were similar, with small difference of bootstrap values. Navsari genotypes formed a tight cluster with the P. epiclitum, originated from different location with high bootstrap value and 0.004–0.011 estimated evolutionary divergence.


Author(s):  
Alphonce B. Chandika ◽  
Reuben S. Mkala ◽  
Bushi Lugoba ◽  
Benjamin C. Kipilipili ◽  
Witness Saitot ◽  
...  

Background: Hospital acquired infection pose a great challenge in provision of healthcare services to many settings  particulary in developing countries where there is limited availability of resources. The roles played by exposed surfaces in spreading of potential bacterial pathogens within the hospital environment have certainly contributed to the increased burden of bacterial infectious diseases such as morbidness, death as well as cost imolecation in healthcare. Our study aimed to determine common bacteria isolated on exposed surfaces and the antimicrobial sensitivity patterns. Methods: A sum of 516 specimens gathered and enrolled for study at Benjamin Mkapa Hopsital (BMH).  The swab specimens were continuously gathered (collected) from different exposed objects in hospital environmental surfaces. All microbiological procedures or tests were performed using appropriate standard operating procedure. The obtained data were analysed using SPSS version 16.0. Results: Among 516 samples, 317 (61.4%) were positive with variety of bacterial isolates from different sites. Out of 317 positive cultures, 120 (37.9%) Staphylococcus aureus, 97 (30.6%) E. coli, 28 (8.8%) Enterobacter aerogenes, 25 (7.9%) Pseudomonas aeruginosa, 23 (7.3%) other Citrobacter species, 20 (6.3%) Citrobacter freundii, 3 (0.9%) Morganella morganii and 1 (0.3%) Serratia mercesens were isolated from different source of exposed surfaces at BMH. Serratia mercesens were highly (100%) resistant to most of the antimicrobial agents including erythromycin, cefotaxime, ciprofloxacin, gentamicin, amikacin, penicillin,ceftriaxone, ceftazidime, levofloxacin, ampicillin, azithromycin and cotrimoxazoleTrimethoprim/sulfamethoxazole. Conclusion: High levels of potentially pathogenic bacteria were isolated in swab specimens from a wide range of exposed surfaces at BMH. Variable pattern of antiobiotic resistance were observed among bacterial isolates with alarming levels demonstrated by isolates of Serratia mercesens which is amongst the common causes of surgical site infections. This findings call for improved actions for infection prevention and control measures at BMH.


2021 ◽  
Vol 78 (4) ◽  
pp. 1267-1276
Author(s):  
Yuzuki Shimamori ◽  
Ajeng K. Pramono ◽  
Tomoe Kitao ◽  
Tohru Suzuki ◽  
Shin-ichi Aizawa ◽  
...  

AbstractThe bacterium Staphylococcus aureus, which colonizes healthy human skin, may cause diseases, such as atopic dermatitis (AD). Treatment for such AD cases involves antibiotic use; however, alternate treatments are preferred owing to the development of antimicrobial resistance. This study aimed to characterize the novel bacteriophage SaGU1 as a potential agent for phage therapy to treat S. aureus infections. SaGU1 that infects S. aureus strains previously isolated from the skin of patients with AD was screened from sewage samples in Gifu, Japan. Its genome was sequenced and analyzed using bioinformatics tools, and the morphology, lytic activity, stability, and host range of the phage were determined. The SaGU1 genome was 140,909 bp with an average GC content of 30.2%. The viral chromosome contained 225 putative protein-coding genes and four tRNA genes, carrying neither toxic nor antibiotic resistance genes. Electron microscopy analysis revealed that SaGU1 belongs to the Myoviridae family. Stability tests showed that SaGU1 was heat-stable under physiological and acidic conditions. Host range testing revealed that SaGU1 can infect a broad range of S. aureus clinical isolates present on the skin of AD patients, whereas it did not kill strains of Staphylococcus epidermidis, which are symbiotic resident bacteria on human skin. Hence, our data suggest that SaGU1 is a potential candidate for developing a phage therapy to treat AD caused by pathogenic S. aureus.


2009 ◽  
Vol 75 (7) ◽  
pp. 2139-2147 ◽  
Author(s):  
Susan M. Lehman ◽  
Andrew M. Kropinski ◽  
Alan J. Castle ◽  
Antonet M. Svircev

ABSTRACT The first complete genome sequence for a myoviridal bacteriophage, ΦEa21-4, infecting Erwinia amylovora, Erwinia pyrifoliae, and Pantoea agglomerans strains has been determined. The unique sequence of this terminally redundant, circularly permuted genome is 84,576 bp. The ΦEa21-4 genome has a GC content of 43.8% and contains 117 putative protein-coding genes and 26 tRNA genes. ΦEa21-4 is the first phage in which a precisely conserved rho-independent terminator has been found dispersed throughout the genome, with 24 copies in all. Also notable in the ΦEa21-4 genome are the presence of tRNAs with six- and nine-base anticodon loops, the absence of a small packaging terminase subunit, and the presence of nadV, a principle component of the NAD+ salvage pathway, which has been found in only a few phage genomes to date. ΦEa21-4 is the first reported Felix O1-like phage genome; 56% of the predicted ΦEa21-4 proteins share homology with those of the Salmonella phage. Apart from this similarity to Felix O1, the ΦEa21-4 genome appears to be substantially different, both globally and locally, from previously reported sequences. A total of 43 of the 117 genes are unique to ΦEa21-4, and 32 of the Felix O1-like genes do not appear in any phage genome sequences other than ΦEa21-4 and Felix O1. N-terminal sequencing and matrix-assisted laser desorption ionization-time of flight analysis resulted in the identification of five ΦEa21-4 genes coding for virion structural proteins, including the major capsid protein.


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