Genetic modifications to temperate Enterococcus faecalis phage ϕEf11 that abolish the establishment of lysogeny and sensitivity to repressor, and increase host range and productivity of lytic infection

Zhang, H.; Fouts, Derrick E.; DePew, Jessica; Stevens, R. H.

doi:10.1099/mic.0.067116-0

Cited by 58 publications

(45 citation statements)

References 67 publications

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“…E. faecalis V583 (vancomycin resistant) was originally isolated by Sahm et al (Sahm, Kissinger, Gilmore, Murray, Mulder, Solliday & Clarke,1989). Both strains were grown in brain heart infusion (BHI) broth, and transformed with plasmid pMSP3535, cat , nis R nis K, using procedures previously described (Zhang et al, 2013). JH2-2[ϕEf11(Δ61-1, ϕFL1C40-44)] is a lysogenic E. faecalis strain harboring prophage ϕEf11(Δ61-1, ϕFL1C40-44), that was prepared previously (Zhang et al, 2013).…”

Section: Methodsmentioning

confidence: 99%

“…Both strains were grown in brain heart infusion (BHI) broth, and transformed with plasmid pMSP3535, cat , nis R nis K, using procedures previously described (Zhang et al, 2013). JH2-2[ϕEf11(Δ61-1, ϕFL1C40-44)] is a lysogenic E. faecalis strain harboring prophage ϕEf11(Δ61-1, ϕFL1C40-44), that was prepared previously (Zhang et al, 2013). E. faecalis JH2-2(pMSP3535, cat , nis R/K) was prepared by transforming strain JH2-2 with plasmid (pMSP3535, cat , nis R/K), and selecting transformants on chloramphenicol-containing media.…”

Section: Methodsmentioning

confidence: 99%

“…A recombinant plasmid containing a 1kb DNA fragment upstream of ϕEF11 ORF36, a nisin promoter (P nisA ), plus an erythromycin selection marker ( erm ), and a 1kb fragment downstream of phage ϕEf11 P cro (the wild type promoter controlling lytic cycle functions in phage ϕEf11) was constructed essentially as described previously (Zhang et al, 2013). This plasmid was electroporated into a competent strain of E. faecalis JH2-2[ϕEf11(Δ61-1, ϕFL1C40-44], harboring the spontaneous recombinant prophage ϕEf11(Δ61-1,ϕFL1C40-44) (Zhang et al, 2013).…”

Section: Methodsmentioning

confidence: 99%

“…This plasmid was electroporated into a competent strain of E. faecalis JH2-2[ϕEf11(Δ61-1, ϕFL1C40-44], harboring the spontaneous recombinant prophage ϕEf11(Δ61-1,ϕFL1C40-44) (Zhang et al, 2013). Following homologous recombination, transformant JH2-2[ϕEf11(Δ61-1, ϕFL1C40-44, Δ36, erm , ΔP cro , P nisA )] clones were selected on erythromycin-containing BHI agar plates.…”

Section: Methodsmentioning

confidence: 99%

“…In this regard, we previously isolated a bacteriophage, ØEf11, that we induced from a root canal isolate of E. faecalis (Stevens, Porras, & Delisle, 2009). Subsequently, we sequenced and annotated the genome of this phage (Stevens, Ektefaie, & Fouts, 2011) and then genetically engineered it (Zhang, Fouts, DePew, & Stevens, 2013) to modify its properties in order to render it useful as a therapeutic agent in phage therapy. Several iterations of genetic constructs of phage ϕEf11 have been produced in the course of our genetic engineering of the phage.…”

Section: Introductionmentioning

confidence: 99%

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Effect of a genetically engineered bacteriophage on Enterococcus faecalis biofilms

Tinoco

Buttaro

Zhang

et al. 2016

Archives of Oral Biology

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Objective-Enterococcus faecalis is a Gram-positive, facultative anaerobic bacterium that is associated with failed endodontic cases and nosocomial infections. E. faecalis can form biofilms, penetrate dentinal tubules and survive in root canals with scarce nutritional supplies. These properties can make E. faecalis resistant to conventional endodontic disinfection therapy. Furthermore, treatment may be complicated by the fact that many E. faecalis strains are resistant to antibiotics. A potential alternative to antibiotic therapy is phage therapy. ϕEf11 is a temperate phage that infects strains of E. faecalis. It was previously sequenced and genetically engineered to modify its properties in order to render it useful as a therapeutic agent in phage therapy. In the current study, we have further genetically modified the phage to create phage ϕEf11/ ϕFL1C(Δ36) PnisA . The aim of this study was to evaluate the efficacy of bacteriophage ϕEf11/ ϕFL1C(Δ36) PnisA , to disrupt biofilms of two Enterococcus faecalis strains: JH2-2 (vancomycinsensitive) and V583 (vancomycin-resistant).Methods-24 hour static biofilms of E. faecalis strains JH2-2(pMSP3535 nisR/K) and V583 (pMSP3535nisR/K), formed on cover slips, were inoculated with bacteriophage ϕEf11/ ϕFL1C(Δ36) PnisA . After 24 and 48 hours incubation, the bacterial biomass was imaged by confocal microscopy and viable cells were quantified by colony forming unit measurement. Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.Competing interests: None declared. Ethical approval: Not required. HHS Public Access Author Manuscript Author ManuscriptAuthor Manuscript Author ManuscriptResults-The results showed a 10-100-fold decrease in viable cells (CFU/biofilm) after phage treatment, which was consistent with comparisons of treated and untreated biofilm images visualized as max projections of the Z-series.Conclusion-The biomass of both vancomycin-sensitive and vancomycin-resistant E. faecalis biofilms is markedly reduced following infection by bacteriophage ϕEf11/ϕFL1C(Δ36) PnisA .

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%