scholarly journals Comparison of Enterococcus faecalis Biofilm Removal Efficiency among Bacteriophage PBEF129, Its Endolysin, and Cefotaxime

Viruses ◽  
2021 ◽  
Vol 13 (3) ◽  
pp. 426
Author(s):  
Hyun Keun Oh ◽  
Yoon Jung Hwang ◽  
Hye-Won Hong ◽  
Heejoon Myung
Keyword(s):  
Enterococcus Faecalis ◽  
Gc Content ◽  
Bacterial Biofilm ◽  
Open Reading Frames ◽  
Base Pairs ◽  
Double Stranded Dna ◽  
Biofilm Removal ◽  
Query Coverage ◽  
Encoding Genes

Enterococcus faecalis is a Gram-positive pathogen which colonizes human intestinal surfaces, forming biofilms, and demonstrates a high resistance to many antibiotics. Especially, antibiotics are less effective for eradicating biofilms and better alternatives are needed. In this study, we have isolated and characterized a bacteriophage, PBEF129, infecting E. faecalis. PBEF129 infected a variety of strains of E. faecalis, including those exhibiting antibiotic resistance. Its genome is a linear double-stranded DNA, 144,230 base pairs in length. Its GC content is 35.9%. The closest genomic DNA sequence was found in Enterococcus phage vB_EfaM_Ef2.3, with a sequence identity of 99.06% over 95% query coverage. Furthermore, 75 open reading frames (ORFs) were functionally annotated and five tRNA-encoding genes were found. ORF 6 was annotated as a phage endolysin having an L-acetylmuramoyl-l-alanine amidase activity. We purified the enzyme as a recombinant protein and confirmed its enzymatic activity. The endolysin’s host range was observed to be wider than its parent phage PBEF129. When applied to bacterial biofilm on the surface of in vitro cultured human intestinal cells, it demonstrated a removal efficacy of the same degree as cefotaxime, but much lower than its parent bacteriophage.

scholarly journals Isolation and characterization of bacteriophages from India, with lytic activity againstMycobacterium tuberculosis

2018 ◽  
Vol 64 (7) ◽  
pp. 483-491 ◽  
Author(s):  
Urmi Bajpai ◽  
Abhishek Kumar Mehta ◽  
Kandasamy Eniyan ◽  
Avni Sinha ◽  
Ankita Ray ◽  
...  
Keyword(s):  
Antimicrobial Agents ◽  
Sequence Similarity ◽  
Gc Content ◽  
Polymerase Chain Reaction Analysis ◽  
Open Reading Frames ◽  
Genome Maintenance ◽  
Isolation And Characterization ◽  
Virion Release ◽  
Double Stranded Dna ◽  
Transmission Electron

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.

scholarly journals Genome Analysis of a Novel Clade b Betabaculovirus Isolated from the Legume Pest Matsumuraeses phaseoli (Lepidoptera: Tortricidae)

Viruses ◽  
2020 ◽  
Vol 12 (10) ◽  
pp. 1068
Author(s):  
Ruihao Shu ◽  
Qian Meng ◽  
Lin Miao ◽  
Hongbin Liang ◽  
Jun Chen ◽  
...  
Keyword(s):  
Northeast Asia ◽  
Gc Content ◽  
Open Reading Frames ◽  
Chinese Medicinal Herb ◽  
Double Stranded Dna ◽  
Clade B ◽  
Potential Biocontrol Agent ◽  
Conserved Genes ◽  
Inhibitors Of Apoptosis Proteins ◽  
Lepidopteran Pest

Matsumuraeses phaseoli is a Lepidopteran pest that primarily feeds on numerous species of cultivated legumes, such as Glycine and Phaseolus. It is widely distributed in northeast Asia. A novel granulovirus, designated as Matsumuraeses phaseoli granulovirus (MaphGV), was isolated from pathogenic M. phaseoli larvae that dwell in rolled leaves of Astragalus membranaceus, a Chinese medicinal herb. In this study, using next-generation sequencing, we report the complete genome of MaphGV. MaphGV genome comprises a double-stranded DNA of 116,875 bp, with 37.18% GC content. It has 128 hypothetical open reading frames (ORFs). Among them, 38 are baculovirus core genes, 18 are lepidopteran baculovirus conserved genes, and 5 are unique to Baculoviridae. MaphGV has one baculovirus repeat ORF (bro) and three inhibitors of apoptosis proteins (iap), including a newfound iap-6. We found two atypical baculoviral homologous regions (hrs) and four direct repeats (drs) in the MaphGV genome. Based on phylogenetic analysis, MaphGV belongs to Clade b of Betabaculovirus and is closely related to Cydia pomonellagranulovirus (CpGV) and Cryptophlebia leucotretagranulovirus (CrleGV). This novel baculovirus discovery and sequencing are invaluable in understanding the evolution of baculovirus and MaphGV may be a potential biocontrol agent against the bean ravaging pest.

scholarly journals An in vivo assay for the reverse transcriptase of human retrotransposon L1 in Saccharomyces cerevisiae.

1994 ◽  
Vol 14 (7) ◽  
pp. 4485-4492 ◽  
Author(s):  
B A Dombroski ◽  
Q Feng ◽  
S L Mathias ◽  
D M Sassaman ◽  
A F Scott ◽  
...  
Keyword(s):  
Reverse Transcriptase ◽  
Consensus Sequence ◽  
Unknown Origin ◽  
Open Reading Frames ◽  
Long Terminal Repeats ◽  
Base Pairs ◽  
In Vivo Assay ◽  
Reading Frames

L1 elements constitute a highly repetitive human DNA family (50,000 to 100,000 copies) lacking long terminal repeats and ending in a poly(A) tail. Some L1 elements are capable of retrotransposition in the human genome (Kazazian, H. H., Jr., C. Wong, H. Youssoufian, A. F. Scott, D. G. Phillips, and S.E. Antonarakis, Nature (London) 332:164-166, 1988). Although most are 5' truncated, a consensus sequence of complete L1 elements is 6 kb long and contains two open reading frames (ORFs) (Scott, A. F., B. J. Schmeckpeper, M. Abdelrazik, C. T. Comey, B. O'Hara, J. P. Rossiter, T. Cooley, P. Health, K. D. Smith, and L. Margolet, Genomics 1:113-125, 1987). The protein encoded by ORF2 has reverse transcriptase (RT) activity in vitro (Mathias, S. L., A. F. Scott, H. H. Kazazian, Jr., J. D. Boeke, and A. Gabriel, Science 254:1808-1810, 1991). Because L1 elements are so numerous, efficient methods for identifying active copies are required. We have developed a simple in vivo assay for the activity of L1 RT based on the system developed by Derr et al. (Derr, L. K., J. N. Strathern, and D. J. Garfinkel, Cell 67:355-364, 1991) for yeast HIS3 pseudogene formation. L1 ORF2 displays an in vivo RT activity similar to that of yeast Ty1 RT in this system and generates pseudogenes with unusual structures. Like the HIS3 pseudogenes whose formation depends on Ty1 RT, the HIS3 pseudogenes generated by L1 RT are joined to Ty1 sequences and often are part of complex arrays of Ty1 elements, multiple HIS3 pseudogenes, and hybrid Ty1/L1 elements. These pseudogenes differ from those previously described in that there are base pairs of unknown origin inserted at several of the junctions. In two of three HIS3 pseudogenes studied, the L1 RT appears to have jumped from the 5' end of a Ty1/L1 transcript to the poly(A) tract of the HIS3 RNA.

scholarly journals In vitro estimation of the infectious potential of the enterococcal strain by an analysis of monocytes’ response to the formed biofilm

2018 ◽  
Vol 72 ◽  
pp. 290-294
Author(s):  
Tomasz Jarzembowski ◽  
Agnieszka Daca ◽  
Jacek M. Witkowski ◽  
Ewa Bryl ◽  
Bolesław Rutkowski ◽  
...  
Keyword(s):  
Enterococcus Faecalis ◽  
Morphological Changes ◽  
Blood Stream ◽  
Bacterial Biofilm ◽  
Commensal Bacteria ◽  
Adhesive Properties ◽  
Enterococcal Strain ◽  
Potential Pathogenicity ◽  
Infectious Potential

The aim of our study was to assess the possibility of predicting Enterococcus faecalis pathogenicity based on morphological changes of monocytes interacting with of bacteria and their adherence to biofilm. Changes in the size and granularity of monocytes, as well as their adherence to biofilm were assessed using FACScan flow cytometer after 1h co-incubation of monocytes and enterococcal biofilm in 37°C. The obtained results were validated with respect to monocytes incubated without bacteria. The most prominent changes in size of monocytes were observed in the case of commensal bacteria. The interaction with bacteria isolated from the blood stream and urine caused comparable changes in the size of THP-1 cells and were smaller than the changes of cells incubated with commensal bacteria. Changes in THP-1 granularity were comparable regardless of the source of bacteria forming biofilm. The parameter which differed the most was connected to the adherence index relating to the monocytes remaining on the surface of biofilm after 1h of incubation. Taking into consideration the obtained results, we conclude that changes in the morphology of monocytes can be treated as a tool assessing the potential pathogenicity of the bacteria. What is more, the adhesive properties towards bacterial biofilm alone might be considered as a tool allowing for the assessment of the pathogenicity of the isolated strain bypassing time-consuming techniques.

scholarly journals Isolation and Characterization of a Bacteriophage Infecting Pseudomonas Aeruginosa and Its Application as a Potential Decontaminating Agent

2021 ◽  
Author(s):  
Sonika Sharma ◽  
Sibnarayan Datta ◽  
Soumya Chatterjee ◽  
Moumita Dutta ◽  
Jhuma Samanta ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Burst Size ◽  
Gc Content ◽  
Alginate Beads ◽  
Open Reading Frames ◽  
Isolation And Characterization ◽  
The Family ◽  
Infected Cells

Abstract To treat antibiotic resistance bacteria, bacteriophage (also called 'phage') application has recently drawn considerable attention from researchers globally. Bacteria like Pseudomonas aeruginosa are known to be associated with nosocomial infections especially in patients with compromised immune systems. In the present work, phage against P. aeruginosa (named 'DRLP1') was isolated from wastewater, enriched and characterized. Morphologically DRLP1 belongs to the family Myoviridae with a high lytic ability. DRLP1 has a burst size of approximately 100 PFU/infected cells, a rapid adsorption time when supplemented with MgCl2, and has viability in a wide temperature range and pH. Genomic sequencing and bioinformatics analysis showed that the phage genome is linear double-stranded, 66,243 bp in length and have a GC content of 54.9%. the genome encodes 93 phage related ORFs open reading frames (ORFs). Phage stability in lyophilized state, adsorption study on sodium alginate beads, and in-vitro pathogen reduction assays were also investigated. Study carried out with artificially contaminated fomites suggests that this phage has the potential for application as a biological decontaminant agent against P. aeruginosa in different conditions.

scholarly journals Hoogsteen base pairs increase the susceptibility of double-stranded DNA to cytotoxic damage

2020 ◽  
Vol 295 (47) ◽  
pp. 15933-15947
Author(s):  
Yu Xu ◽  
Akanksha Manghrani ◽  
Bei Liu ◽  
Honglue Shi ◽  
Uyen Pham ◽  
...  
Keyword(s):  
Dynamic Equilibrium ◽  
Alkylating Agents ◽  
Dimethyl Sulfate ◽  
Dot Blot ◽  
Base Pairs ◽  
Double Stranded Dna ◽  
Genome Wide ◽  
Damage Susceptibility

As the Watson–Crick faces of nucleobases are protected in dsDNA, it is commonly assumed that deleterious alkylation damage to the Watson–Crick faces of nucleobases predominantly occurs when DNA becomes single-stranded during replication and transcription. However, damage to the Watson–Crick faces of nucleobases has been reported in dsDNA in vitro through mechanisms that are not understood. In addition, the extent of protection from methylation damage conferred by dsDNA relative to ssDNA has not been quantified. Watson–Crick base pairs in dsDNA exist in dynamic equilibrium with Hoogsteen base pairs that expose the Watson–Crick faces of purine nucleobases to solvent. Whether this can influence the damage susceptibility of dsDNA remains unknown. Using dot-blot and primer extension assays, we measured the susceptibility of adenine-N1 to methylation by dimethyl sulfate (DMS) when in an A-T Watson–Crick versus Hoogsteen conformation. Relative to unpaired adenines in a bulge, Watson–Crick A-T base pairs in dsDNA only conferred ∼130-fold protection against adenine-N1 methylation, and this protection was reduced to ∼40-fold for A(syn)-T Hoogsteen base pairs embedded in a DNA-drug complex. Our results indicate that Watson–Crick faces of nucleobases are accessible to alkylating agents in canonical dsDNA and that Hoogsteen base pairs increase this accessibility. Given the higher abundance of dsDNA relative to ssDNA, these results suggest that dsDNA could be a substantial source of cytotoxic damage. The work establishes DMS probing as a method for characterizing A(syn)-T Hoogsteen base pairs in vitro and also lays the foundation for a sequencing approach to map A(syn)-T Hoogsteen and unpaired adenines genome-wide in vivo.

scholarly journals Three Novel Virophage Genomes Discovered from Yellowstone Lake Metagenomes

2014 ◽  
Vol 89 (2) ◽  
pp. 1278-1285 ◽  
Author(s):  
Jinglie Zhou ◽  
Dawei Sun ◽  
Alyson Childers ◽  
Timothy R. McDermott ◽  
Yongjie Wang ◽  
...  
Keyword(s):  
Phylogenetic Analyses ◽  
Evolutionary Relationship ◽  
Gc Content ◽  
Open Reading Frames ◽  
Lake Ecosystem ◽  
Photic Zone ◽  
Yellowstone Lake ◽  
Dna Viruses ◽  
Double Stranded Dna ◽  
Unique Distribution

ABSTRACTVirophages are a unique group of circular double-stranded DNA viruses that are considered parasites of giant DNA viruses, which in turn are known to infect eukaryotic hosts. In this study, the genomes of three novel Yellowstone Lake virophages (YSLVs)—YSLV5, YSLV6, and YSLV7—were identified from Yellowstone Lake through metagenomic analyses. The relative abundance of these three novel virophages and previously identified Yellowstone Lake virophages YSLV1 to -4 were determined in different locations of the lake, revealing that most of the sampled locations in the lake, including both mesophilic and thermophilic habitats, had multiple virophage genotypes. This likely reflects the diverse habitats or diversity of the eukaryotic hosts and their associated giant viruses that serve as putative hosts for these virophages. YSLV5 has a 29,767-bp genome with 32 predicted open reading frames (ORFs), YSLV6 has a 24,837-bp genome with 29 predicted ORFs, and YSLV7 has a 23,193-bp genome with 26 predicted ORFs. Based on multilocus phylogenetic analysis, YSLV6 shows a close evolutionary relationship with YSLV1 to -4, whereas YSLV5 and YSLV7 are distantly related to the others, and YSLV7 represents the fourth novel virophage lineage. In addition, the genome of YSLV5 has a G+C content of 51.1% that is much higher than all other known virophages, indicating a unique host range for YSLV5. These results suggest that virophages are abundant and have diverse genotypes that likely mirror diverse giant viral and eukaryotic hosts within the Yellowstone Lake ecosystem.IMPORTANCEThis study discovered novel virophages present within the Yellowstone Lake ecosystem using a conserved major capsid protein as a phylogenetic anchor for assembly of sequence reads from Yellowstone Lake metagenomic samples. The three novel virophage genomes (YSLV5 to -7) were completed by identifying specific environmental samples containing these respective virophages, and closing gaps by targeted PCR and sequencing. Most of the YSLV genotypes were associated primarily with photic-zone and nonhydrothermal samples; however, YSLV5 had a unique distribution with an occurrence in vent samples similar to that in photic-zone samples and with a higher GC content that suggests a distinct host and habitat compared to other YSLVs. In addition, genome content and phylogenetic analyses indicate that YSLV5 and YSLV7 are distinct from known virophages and that additional as-yet-uncharacterized virophages are likely present within the Yellowstone Lake ecosystem.

In vitro excision of adeno-associated virus DNA from recombinant plasmids: isolation of an enzyme fraction from HeLa cells that cleaves DNA at poly(G) sequences

1988 ◽  
Vol 8 (6) ◽  
pp. 2513-2522
Author(s):  
J Gottlieb ◽  
N Muzyczka
Keyword(s):  
Dna Sequences ◽  
Base Pairs ◽  
Adeno Associated Virus ◽  
Recombinant Plasmids ◽  
Culture Cells ◽  
Double Stranded Dna ◽  
Enzyme Fraction ◽  
Nuclear Extracts

When circular recombinant plasmids containing adeno-associated virus (AAV) DNA sequences are transfected into human cells, the AAV provirus is rescued. Using these circular AAV plasmids as substrates, we isolated an enzyme fraction from HeLa cell nuclear extracts that excises intact AAV DNA in vitro from vector DNA and produces linear DNA products. The recognition signal for the enzyme is a polypurine-polypyrimidine sequence which is at least 9 residues long and rich in G.C base pairs. Such sequences are present in AAV recombinant plasmids as part of the first 15 base pairs of the AAV terminal repeat and in some cases as the result of cloning the AAV genome by G.C tailing. The isolated enzyme fraction does not have significant endonucleolytic activity on single-stranded or double-stranded DNA. Plasmid DNA that is transfected into tissue culture cells is cleaved in vivo to produce a pattern of DNA fragments similar to that seen with purified enzyme in vitro. The activity has been called endo R for rescue, and its behavior suggests that it may have a role in recombination of cellular chromosomes.

scholarly journals Characterization of Lactococcus lactis Phage 949 and Comparison with Other Lactococcal Phages

2010 ◽  
Vol 76 (20) ◽  
pp. 6843-6852 ◽  
Author(s):  
Julie E. Samson ◽  
Sylvain Moineau
Keyword(s):  
Lactococcus Lactis ◽  
Defense Mechanisms ◽  
Gc Content ◽  
Tail Length ◽  
Open Reading Frames ◽  
Phage Group ◽  
Double Stranded Dna ◽  
Group I ◽  
Sds Page ◽  
Genetic Clusters

ABSTRACT The virulent Lactococcus lactis phage 949 was isolated in 1975 from cheese whey in New Zealand. This phage is a member of the Siphoviridae family and of a rare lactococcal phage group that bears its name (949 group). It has an icosahedral capsid (79-nm diameter) and a very long noncontractile tail (length, 500 nm; width, 12 nm). It infected 7 of 59 tested L. lactis strains, a somewhat expanded host range for a rare lactococcal phage. The abortive phage infection defense mechanisms AbiQ and AbiT strongly inhibited the multiplication of phage 949, but AbiK and AbiV did not. Its double-stranded DNA (dsDNA) genome of 114,768 bp is, to date, the largest among lactococcal phages. Its GC content was calculated at 32.7%, which is the lowest reported for a lactococcal phage. Its 154 open reading frames (ORFs) share limited identity with database sequences. In addition, terminal redundancy was observed as well as the presence of six tRNAs, one group I intron, and putative recombinases. SDS-PAGE coupled with mass spectrometry identified 13 structural proteins. The genomes of the members of the 10 currently known L. lactis phage groups were used to construct a proteomic tree. Each L. lactis phage group separated into distinct genetic clusters, validating the current classification scheme. Of note, members of the polythetic P335 groups were clearly separated into subgroups.

scholarly journals Once-versus thrice-daily netilmicin combined with amoxicillin, penicillin, or vancomycin against Enterococcus faecalis in a pharmacodynamic in vitro model.

1996 ◽  
Vol 40 (10) ◽  
pp. 2258-2261 ◽  
Author(s):  
S Schwank ◽  
J Blaser
Keyword(s):  
Enterococcus Faecalis ◽  
Half Life ◽  
In Vitro Model ◽  
Bacterial Biofilm ◽  
In Vivo Studies ◽  
Bacterial Killing ◽  
Once Daily ◽  
Vitro Model

Several in vitro and in vivo studies as well as clinical trials have demonstrated that once-daily aminoglycoside regimens are as effective as or more effective than multiple daily dosings. However, the most favorable aminoglycoside dosing regimen for treating enterococcal endocarditis remains controversial. The same total dose of netilmicin was administered as once-daily (24-micrograms/ml peaks) and thrice-daily (8 micrograms/ml) regimens in a pharmacodynamic in vitro model simulating exposure of Enterococcus faecalis to human serum kinetics. Netilmicin was administered in combination with continuous infusions of amoxicillin, vancomycin, or penicillin against a bacterial biofilm adhering to glass beads. No significant differences in bacterial killing were found after 24 or 48 h between the once- and thrice-daily regimens. Additional experiments considering animal kinetics (half-life of netilmicin, 20 min) instead of human kinetics (half-life, 2.5 h) in the pharmacodynamic model also revealed similar results. The addition of netilmicin synergistically increased the activity of vancomycin (P < 0.05). In contrast, amoxicillin alone was as effective as the combination with netilmicin. Thus, it could not be established in this model that once-daily dosing of aminoglycosides is contraindicated for treating infections caused by E. faecalis.

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