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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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Fluoromycobacteriophage M. tuberculosis susceptibility testing using flow cytometry.A. M. tuberculosis mc26230 strains grown in the absence of Tween detergent were infected with phAE87::hsp60-EGFP with or without antibiotic treatment, fixed with paraformaldehyde, and analyzed by flow cytometry. In panels a, mock-infected control cells and b, phage infected cells, the intensity of fluorescence is plotted against light side scatter. The signal observed in mock-infected cells corresponds to autofluorescence of M. tuberculosis and the shift towards greater fluorescence in phage-infected cells shows that a high proportion of cells are infected and expressed GFP. In panels c–i the level of fluorescence is plotted against the number of events counted. In each experiment mock-infected cells are shown in red, phage infected cells in green, and antibiotic-treated phage infected cells in blue. When treated with rifampicin (Rif) or streptomycin (Str) antibiotics were added simultaneously with phage (φ), incubated for 16 hours, fixed and analyzed; for INH treatment cells were pre-incubated with antibiotic for 48 hours, incubated with phage for 16 hours, fixed, and analyzed. B. M. tuberculosis mc26230 strains were grown in the presence of Tween, washed, preincubated with isoniazid for 24 hours, infected with phAE87::hsp60-EGFP, fixed, and analyzed by flow cytometry. Panels a–e correspond to panels a–c, f, and i of Figure 8A respectively.
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pone-0004870-g008: Fluoromycobacteriophage M. tuberculosis susceptibility testing using flow cytometry.A. M. tuberculosis mc26230 strains grown in the absence of Tween detergent were infected with phAE87::hsp60-EGFP with or without antibiotic treatment, fixed with paraformaldehyde, and analyzed by flow cytometry. In panels a, mock-infected control cells and b, phage infected cells, the intensity of fluorescence is plotted against light side scatter. The signal observed in mock-infected cells corresponds to autofluorescence of M. tuberculosis and the shift towards greater fluorescence in phage-infected cells shows that a high proportion of cells are infected and expressed GFP. In panels c–i the level of fluorescence is plotted against the number of events counted. In each experiment mock-infected cells are shown in red, phage infected cells in green, and antibiotic-treated phage infected cells in blue. When treated with rifampicin (Rif) or streptomycin (Str) antibiotics were added simultaneously with phage (φ), incubated for 16 hours, fixed and analyzed; for INH treatment cells were pre-incubated with antibiotic for 48 hours, incubated with phage for 16 hours, fixed, and analyzed. B. M. tuberculosis mc26230 strains were grown in the presence of Tween, washed, preincubated with isoniazid for 24 hours, infected with phAE87::hsp60-EGFP, fixed, and analyzed by flow cytometry. Panels a–e correspond to panels a–c, f, and i of Figure 8A respectively.

Mentions: Flow cytometry offers an alternative approach to the detection of fluorescence following fluoromycobacteriophage infection. While the instrumentation required might seem prohibitively costly for routine use in many areas of the world where the incidence of tuberculosis is high, if the HIV infection rates are also high, flow cytometry may have been previously established for CD4 count determination; the province of KwaZulu-Natal in South Africa is one example of this. To evaluate this approach, M. tuberculosis cells infected with phAE87::hsp60-EGFP cells were fixed, and analyzed by flow cytometry to measure side light scatter as well as fluorescence (Figure 8A). Although M. tuberculosis has significant autofluorescence as seen in the mock-infected cells (Figure 8A panels a and c), fluoromycobacteriophage-infected cells can be readily distinguished (Figure 8A panels b and c) and a very high proportion of cells fluoresced following fluoromycobacteriophage infection (Figure 8).


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

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

Fluoromycobacteriophage M. tuberculosis susceptibility testing using flow cytometry.A. M. tuberculosis mc26230 strains grown in the absence of Tween detergent were infected with phAE87::hsp60-EGFP with or without antibiotic treatment, fixed with paraformaldehyde, and analyzed by flow cytometry. In panels a, mock-infected control cells and b, phage infected cells, the intensity of fluorescence is plotted against light side scatter. The signal observed in mock-infected cells corresponds to autofluorescence of M. tuberculosis and the shift towards greater fluorescence in phage-infected cells shows that a high proportion of cells are infected and expressed GFP. In panels c–i the level of fluorescence is plotted against the number of events counted. In each experiment mock-infected cells are shown in red, phage infected cells in green, and antibiotic-treated phage infected cells in blue. When treated with rifampicin (Rif) or streptomycin (Str) antibiotics were added simultaneously with phage (φ), incubated for 16 hours, fixed and analyzed; for INH treatment cells were pre-incubated with antibiotic for 48 hours, incubated with phage for 16 hours, fixed, and analyzed. B. M. tuberculosis mc26230 strains were grown in the presence of Tween, washed, preincubated with isoniazid for 24 hours, infected with phAE87::hsp60-EGFP, fixed, and analyzed by flow cytometry. Panels a–e correspond to panels a–c, f, and i of Figure 8A respectively.
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pone-0004870-g008: Fluoromycobacteriophage M. tuberculosis susceptibility testing using flow cytometry.A. M. tuberculosis mc26230 strains grown in the absence of Tween detergent were infected with phAE87::hsp60-EGFP with or without antibiotic treatment, fixed with paraformaldehyde, and analyzed by flow cytometry. In panels a, mock-infected control cells and b, phage infected cells, the intensity of fluorescence is plotted against light side scatter. The signal observed in mock-infected cells corresponds to autofluorescence of M. tuberculosis and the shift towards greater fluorescence in phage-infected cells shows that a high proportion of cells are infected and expressed GFP. In panels c–i the level of fluorescence is plotted against the number of events counted. In each experiment mock-infected cells are shown in red, phage infected cells in green, and antibiotic-treated phage infected cells in blue. When treated with rifampicin (Rif) or streptomycin (Str) antibiotics were added simultaneously with phage (φ), incubated for 16 hours, fixed and analyzed; for INH treatment cells were pre-incubated with antibiotic for 48 hours, incubated with phage for 16 hours, fixed, and analyzed. B. M. tuberculosis mc26230 strains were grown in the presence of Tween, washed, preincubated with isoniazid for 24 hours, infected with phAE87::hsp60-EGFP, fixed, and analyzed by flow cytometry. Panels a–e correspond to panels a–c, f, and i of Figure 8A respectively.
Mentions: Flow cytometry offers an alternative approach to the detection of fluorescence following fluoromycobacteriophage infection. While the instrumentation required might seem prohibitively costly for routine use in many areas of the world where the incidence of tuberculosis is high, if the HIV infection rates are also high, flow cytometry may have been previously established for CD4 count determination; the province of KwaZulu-Natal in South Africa is one example of this. To evaluate this approach, M. tuberculosis cells infected with phAE87::hsp60-EGFP cells were fixed, and analyzed by flow cytometry to measure side light scatter as well as fluorescence (Figure 8A). Although M. tuberculosis has significant autofluorescence as seen in the mock-infected cells (Figure 8A panels a and c), fluoromycobacteriophage-infected cells can be readily distinguished (Figure 8A panels b and c) and a very high proportion of cells fluoresced following fluoromycobacteriophage infection (Figure 8).

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