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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

Paraformaldhyde fixation of fluoromycobacteriophage-infected mycobacteria.M. smegmatis mc2155 cells were infected with phAE87::hsp60-EGFP and fluorescence detected in A, live cells, B, fixed cells and C, fixed cells after 2 weeks at 4°C. Top, fluorescence micrograph images; bottom, merge of fluorescence and phase contrast images. Scale bar, 10 µm.
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pone-0004870-g005: Paraformaldhyde fixation of fluoromycobacteriophage-infected mycobacteria.M. smegmatis mc2155 cells were infected with phAE87::hsp60-EGFP and fluorescence detected in A, live cells, B, fixed cells and C, fixed cells after 2 weeks at 4°C. Top, fluorescence micrograph images; bottom, merge of fluorescence and phase contrast images. Scale bar, 10 µm.

Mentions: A potential advantage of using GFP as a reporter is that it is known to withstand fixation when expressed in eukaryotic cells [28]. Paraformaldehyde fixation of fluoromycobacteriophage-infected mycobacteria has two key potential advantages. First, it should confer significant biosafety advantages especially when analyzing notably hazardous strains such as XDR-TB, simplifying sample handling procedures such as microscopy and flow cytometry. Secondly, by preserving fluorescence for extended periods of time, samples could be readily stored or transported for off-site analysis. We evaluated this using M. smegmatis, adding 2% paraformaldehyde – which effectively kills mycobacteria [29] – after phage incubation. Samples were stored at 4°C for varying periods of time and examined by microscopy. As shown in Figure 5, fluorescence was maintained following fixation and showed only modest decay during a two-week incubation. Incubation for a shorter period (48 hours) at room temperature showed no reduction of fluorescence (data not shown).


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

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

Paraformaldhyde fixation of fluoromycobacteriophage-infected mycobacteria.M. smegmatis mc2155 cells were infected with phAE87::hsp60-EGFP and fluorescence detected in A, live cells, B, fixed cells and C, fixed cells after 2 weeks at 4°C. Top, fluorescence micrograph images; bottom, merge of fluorescence and phase contrast images. Scale bar, 10 µm.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2654538&req=5

pone-0004870-g005: Paraformaldhyde fixation of fluoromycobacteriophage-infected mycobacteria.M. smegmatis mc2155 cells were infected with phAE87::hsp60-EGFP and fluorescence detected in A, live cells, B, fixed cells and C, fixed cells after 2 weeks at 4°C. Top, fluorescence micrograph images; bottom, merge of fluorescence and phase contrast images. Scale bar, 10 µm.
Mentions: A potential advantage of using GFP as a reporter is that it is known to withstand fixation when expressed in eukaryotic cells [28]. Paraformaldehyde fixation of fluoromycobacteriophage-infected mycobacteria has two key potential advantages. First, it should confer significant biosafety advantages especially when analyzing notably hazardous strains such as XDR-TB, simplifying sample handling procedures such as microscopy and flow cytometry. Secondly, by preserving fluorescence for extended periods of time, samples could be readily stored or transported for off-site analysis. We evaluated this using M. smegmatis, adding 2% paraformaldehyde – which effectively kills mycobacteria [29] – after phage incubation. Samples were stored at 4°C for varying periods of time and examined by microscopy. As shown in Figure 5, fluorescence was maintained following fixation and showed only modest decay during a two-week incubation. Incubation for a shorter period (48 hours) at room temperature showed no reduction of fluorescence (data not shown).

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