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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

Sensitivity of Fluoromycobacteriophages.Varying numbers of M. smegmatis mc2155 cells were infected with pHAE87::hsp60-EGFP at a moi of 100. After infection at 37°C for 4 hrs, cells were concentrated by filtration and visualize using by fluorescence microscopy. A, 106; B,105; C, 104; D, 103 and E, 102 cells. Scale bar, 20 µm.
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pone-0004870-g004: Sensitivity of Fluoromycobacteriophages.Varying numbers of M. smegmatis mc2155 cells were infected with pHAE87::hsp60-EGFP at a moi of 100. After infection at 37°C for 4 hrs, cells were concentrated by filtration and visualize using by fluorescence microscopy. A, 106; B,105; C, 104; D, 103 and E, 102 cells. Scale bar, 20 µm.

Mentions: Since a high proportion of cells fluoresce following infection, we reasoned that we should be able to detect relatively small numbers of M. smegmatis bacteria. To evaluate this we made dilutions of an M. smegmatis culture and infected 200 µl aliquots each with phAE87::hsp60-EGFP at an moi of 100. In order to recover all of the bacteria present in the sample, infected cells were collected on a 0.45 µm filter (GH polypro, PALL Life Sciences) and examined directly by fluorescence microscopy (Figure 4). When as few as 100 cells were present in the sample, we could readily recover and identify them by examination of just a few microscopic fields (at 400-fold magnification) (Figure 4E). This experiment shows that infection is sufficiently efficient even when cells are dilute, and that because microscopic detection enables identification of individual infected cells the overall sensitivity of the assay is restricted primarily by the efficiency with which cells can be recovered for microscopy. We note that the opacity of these filters eliminates the ability to determine total bacterial counts using bright field optics.


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

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

Sensitivity of Fluoromycobacteriophages.Varying numbers of M. smegmatis mc2155 cells were infected with pHAE87::hsp60-EGFP at a moi of 100. After infection at 37°C for 4 hrs, cells were concentrated by filtration and visualize using by fluorescence microscopy. A, 106; B,105; C, 104; D, 103 and E, 102 cells. Scale bar, 20 µm.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0004870-g004: Sensitivity of Fluoromycobacteriophages.Varying numbers of M. smegmatis mc2155 cells were infected with pHAE87::hsp60-EGFP at a moi of 100. After infection at 37°C for 4 hrs, cells were concentrated by filtration and visualize using by fluorescence microscopy. A, 106; B,105; C, 104; D, 103 and E, 102 cells. Scale bar, 20 µm.
Mentions: Since a high proportion of cells fluoresce following infection, we reasoned that we should be able to detect relatively small numbers of M. smegmatis bacteria. To evaluate this we made dilutions of an M. smegmatis culture and infected 200 µl aliquots each with phAE87::hsp60-EGFP at an moi of 100. In order to recover all of the bacteria present in the sample, infected cells were collected on a 0.45 µm filter (GH polypro, PALL Life Sciences) and examined directly by fluorescence microscopy (Figure 4). When as few as 100 cells were present in the sample, we could readily recover and identify them by examination of just a few microscopic fields (at 400-fold magnification) (Figure 4E). This experiment shows that infection is sufficiently efficient even when cells are dilute, and that because microscopic detection enables identification of individual infected cells the overall sensitivity of the assay is restricted primarily by the efficiency with which cells can be recovered for microscopy. We note that the opacity of these filters eliminates the ability to determine total bacterial counts using bright field optics.

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