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Fluoromycobacteriophages for rapid, specific, and sensitive antibiotic susceptibility testing of Mycobacterium tuberculosis.

Piuri M, Jacobs WR, Hatfull GF - PLoS ONE (2009)

Bottom Line: We describe here a virus-based assay in which fluoromycobacteriophages are used to deliver a GFP or ZsYellow fluorescent marker gene to M. tuberculosis, which can then be monitored by fluorescent detection approaches including fluorescent microscopy and flow cytometry.Pre-clinical evaluations show that addition of either Rifampicin or Streptomycin at the time of phage addition obliterates fluorescence in susceptible cells but not in isogenic resistant bacteria enabling drug sensitivity determination in less than 24 hours.Fluorescence withstands fixation by paraformaldehyde providing enhanced biosafety for testing MDR-TB and XDR-TB infections.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, University of Pittsburgh, Pittsburgh, Pennsylvania, United States of America.

ABSTRACT
Rapid antibiotic susceptibility testing of Mycobacterium tuberculosis is of paramount importance as multiple- and extensively-drug resistant strains of M. tuberculosis emerge and spread. We describe here a virus-based assay in which fluoromycobacteriophages are used to deliver a GFP or ZsYellow fluorescent marker gene to M. tuberculosis, which can then be monitored by fluorescent detection approaches including fluorescent microscopy and flow cytometry. Pre-clinical evaluations show that addition of either Rifampicin or Streptomycin at the time of phage addition obliterates fluorescence in susceptible cells but not in isogenic resistant bacteria enabling drug sensitivity determination in less than 24 hours. Detection requires no substrate addition, fewer than 100 cells can be identified, and resistant bacteria can be detected within mixed populations. Fluorescence withstands fixation by paraformaldehyde providing enhanced biosafety for testing MDR-TB and XDR-TB infections.

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Related in: MedlinePlus

Fluoromycobacteriophages construction.Schematic representation of phAE87::hsp60-EGFP construction. Shuttle phasmid phAE87 is a conditionally replicating derivative of phage TM4 in which the cosmid moiety is flanked by Pac I restriction sites. A plasmid derivative of pYUB854 containing the EGFP gene (pMP14) was used to replace the cosmid in phAE87 followed by lambda packaging and recovery in E. coli.
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pone-0004870-g001: Fluoromycobacteriophages construction.Schematic representation of phAE87::hsp60-EGFP construction. Shuttle phasmid phAE87 is a conditionally replicating derivative of phage TM4 in which the cosmid moiety is flanked by Pac I restriction sites. A plasmid derivative of pYUB854 containing the EGFP gene (pMP14) was used to replace the cosmid in phAE87 followed by lambda packaging and recovery in E. coli.

Mentions: To construct fluorophage derivatives of TM4, gfp or ZsYellow genes were fused to the constitutive M. bovis BCG Hsp60 promoter in plasmid derivatives of pYUB854 [27] and transferred into the phAE87 shuttle phasmid to generate phAE87::hsp60-EGFP and phAE87::hsp60-ZsYellow respectively (Figure 1). DNA was extracted from the E. coli-propagated phasmids and their structures verified by restriction analysis. High titer stocks (>1010pfu/ml) were prepared from individual plaques following transfection of M. smegmatis, and used in subsequent assays.


Fluoromycobacteriophages for rapid, specific, and sensitive antibiotic susceptibility testing of Mycobacterium tuberculosis.

Piuri M, Jacobs WR, Hatfull GF - PLoS ONE (2009)

Fluoromycobacteriophages construction.Schematic representation of phAE87::hsp60-EGFP construction. Shuttle phasmid phAE87 is a conditionally replicating derivative of phage TM4 in which the cosmid moiety is flanked by Pac I restriction sites. A plasmid derivative of pYUB854 containing the EGFP gene (pMP14) was used to replace the cosmid in phAE87 followed by lambda packaging and recovery in E. coli.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2654538&req=5

pone-0004870-g001: Fluoromycobacteriophages construction.Schematic representation of phAE87::hsp60-EGFP construction. Shuttle phasmid phAE87 is a conditionally replicating derivative of phage TM4 in which the cosmid moiety is flanked by Pac I restriction sites. A plasmid derivative of pYUB854 containing the EGFP gene (pMP14) was used to replace the cosmid in phAE87 followed by lambda packaging and recovery in E. coli.
Mentions: To construct fluorophage derivatives of TM4, gfp or ZsYellow genes were fused to the constitutive M. bovis BCG Hsp60 promoter in plasmid derivatives of pYUB854 [27] and transferred into the phAE87 shuttle phasmid to generate phAE87::hsp60-EGFP and phAE87::hsp60-ZsYellow respectively (Figure 1). DNA was extracted from the E. coli-propagated phasmids and their structures verified by restriction analysis. High titer stocks (>1010pfu/ml) were prepared from individual plaques following transfection of M. smegmatis, and used in subsequent assays.

Bottom Line: We describe here a virus-based assay in which fluoromycobacteriophages are used to deliver a GFP or ZsYellow fluorescent marker gene to M. tuberculosis, which can then be monitored by fluorescent detection approaches including fluorescent microscopy and flow cytometry.Pre-clinical evaluations show that addition of either Rifampicin or Streptomycin at the time of phage addition obliterates fluorescence in susceptible cells but not in isogenic resistant bacteria enabling drug sensitivity determination in less than 24 hours.Fluorescence withstands fixation by paraformaldehyde providing enhanced biosafety for testing MDR-TB and XDR-TB infections.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, University of Pittsburgh, Pittsburgh, Pennsylvania, United States of America.

ABSTRACT
Rapid antibiotic susceptibility testing of Mycobacterium tuberculosis is of paramount importance as multiple- and extensively-drug resistant strains of M. tuberculosis emerge and spread. We describe here a virus-based assay in which fluoromycobacteriophages are used to deliver a GFP or ZsYellow fluorescent marker gene to M. tuberculosis, which can then be monitored by fluorescent detection approaches including fluorescent microscopy and flow cytometry. Pre-clinical evaluations show that addition of either Rifampicin or Streptomycin at the time of phage addition obliterates fluorescence in susceptible cells but not in isogenic resistant bacteria enabling drug sensitivity determination in less than 24 hours. Detection requires no substrate addition, fewer than 100 cells can be identified, and resistant bacteria can be detected within mixed populations. Fluorescence withstands fixation by paraformaldehyde providing enhanced biosafety for testing MDR-TB and XDR-TB infections.

Show MeSH
Related in: MedlinePlus