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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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Detection of M. tuberculosis mc26230 RifR cells in a mixed population using flow cytometry.M. tuberculosis mc26230 wt was mixed with the isogenic rifR strain at different proportions. A total of 107 mixed cells were infected with phAE87::hsp60-EGFP in the presence of Rifampicin; cells were fixed and analyze by flow cytometry. (A) 100% wt: 0% RifR (B) 50% wt: 50% RifR; (C) 75% wt : 25% RifR ; (D) 90% wt :10% RifR; (E) 99% wt :1% RifR . Left panels, the intensity of fluorescence is plotted against light side scatter; right panels, the level of fluorescence is plotted against the number of events counted.
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pone-0004870-g010: Detection of M. tuberculosis mc26230 RifR cells in a mixed population using flow cytometry.M. tuberculosis mc26230 wt was mixed with the isogenic rifR strain at different proportions. A total of 107 mixed cells were infected with phAE87::hsp60-EGFP in the presence of Rifampicin; cells were fixed and analyze by flow cytometry. (A) 100% wt: 0% RifR (B) 50% wt: 50% RifR; (C) 75% wt : 25% RifR ; (D) 90% wt :10% RifR; (E) 99% wt :1% RifR . Left panels, the intensity of fluorescence is plotted against light side scatter; right panels, the level of fluorescence is plotted against the number of events counted.

Mentions: Because sputum samples may not be phenotypically homogenous cultures, we evaluated the ability of fluoromycobacteriophages to detect drug resistant strains within mixed cultures. Rifampicin-resistant and rifampicin-sensitive cells were mixed at varying ratios, and tested with phAE87::hsp60-EGFP in the presence of rifampicin (Figure 9). To optimize recovery of bacteria present in the sample, cells were fixed and recovered on a 0.45 µM filter after phage infection as described above. In samples containing a total of approximately 106 cells in a 200 µl aliquot, rifampicin-resistant cells could be readily detected when they constituted as few at 1% of the total population using a 400× or 1000× fold magnification (Figure 9E). A similar experiment was performed using flow cytometry and similar results were observed with detection of a very small population (1%) of rifampicin resistant cells when a mixed culture of sensitive and resistant cells was infected in the presence of rifampicin (Figure 10). We note that the number of drug-resistant cells rather than the total number of bacteria determines the sensitivity of detection in such mixed cultures.


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

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

Detection of M. tuberculosis mc26230 RifR cells in a mixed population using flow cytometry.M. tuberculosis mc26230 wt was mixed with the isogenic rifR strain at different proportions. A total of 107 mixed cells were infected with phAE87::hsp60-EGFP in the presence of Rifampicin; cells were fixed and analyze by flow cytometry. (A) 100% wt: 0% RifR (B) 50% wt: 50% RifR; (C) 75% wt : 25% RifR ; (D) 90% wt :10% RifR; (E) 99% wt :1% RifR . Left panels, the intensity of fluorescence is plotted against light side scatter; right panels, the level of fluorescence is plotted against the number of events counted.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0004870-g010: Detection of M. tuberculosis mc26230 RifR cells in a mixed population using flow cytometry.M. tuberculosis mc26230 wt was mixed with the isogenic rifR strain at different proportions. A total of 107 mixed cells were infected with phAE87::hsp60-EGFP in the presence of Rifampicin; cells were fixed and analyze by flow cytometry. (A) 100% wt: 0% RifR (B) 50% wt: 50% RifR; (C) 75% wt : 25% RifR ; (D) 90% wt :10% RifR; (E) 99% wt :1% RifR . Left panels, the intensity of fluorescence is plotted against light side scatter; right panels, the level of fluorescence is plotted against the number of events counted.
Mentions: Because sputum samples may not be phenotypically homogenous cultures, we evaluated the ability of fluoromycobacteriophages to detect drug resistant strains within mixed cultures. Rifampicin-resistant and rifampicin-sensitive cells were mixed at varying ratios, and tested with phAE87::hsp60-EGFP in the presence of rifampicin (Figure 9). To optimize recovery of bacteria present in the sample, cells were fixed and recovered on a 0.45 µM filter after phage infection as described above. In samples containing a total of approximately 106 cells in a 200 µl aliquot, rifampicin-resistant cells could be readily detected when they constituted as few at 1% of the total population using a 400× or 1000× fold magnification (Figure 9E). A similar experiment was performed using flow cytometry and similar results were observed with detection of a very small population (1%) of rifampicin resistant cells when a mixed culture of sensitive and resistant cells was infected in the presence of rifampicin (Figure 10). We note that the number of drug-resistant cells rather than the total number of bacteria determines the sensitivity of detection in such mixed cultures.

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