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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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Antibiotic susceptibility testing of M. tuberculosis using fluoromycobacteriophages.M. tuberculosis mc26230 wt and antibiotic resistant strains were infected with phAE87::hsp60-EGFP with or without antibiotic addition as indicated. A. Cultures were grown in the absence of Tween, washed, and tested. In panels a–d antibiotics and phage were added simultaneously, incubated for 16 hours, and examined by microscopy. In panels e–f INH was added first for 48 hours followed by phage addition, incubation for a further 16 hours, and examined by microscopy. Scale bar, 10 µm. B. Cultures were grown in the presence of Tween, centrifuged and washed twice, and then incubated with antibiotic (as indicated) for 24 hours. Phage were then added, cultures incubated for 16 hour, and examined by microscopy. Scale bar, 20 µm.
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pone-0004870-g007: Antibiotic susceptibility testing of M. tuberculosis using fluoromycobacteriophages.M. tuberculosis mc26230 wt and antibiotic resistant strains were infected with phAE87::hsp60-EGFP with or without antibiotic addition as indicated. A. Cultures were grown in the absence of Tween, washed, and tested. In panels a–d antibiotics and phage were added simultaneously, incubated for 16 hours, and examined by microscopy. In panels e–f INH was added first for 48 hours followed by phage addition, incubation for a further 16 hours, and examined by microscopy. Scale bar, 10 µm. B. Cultures were grown in the presence of Tween, centrifuged and washed twice, and then incubated with antibiotic (as indicated) for 24 hours. Phage were then added, cultures incubated for 16 hour, and examined by microscopy. Scale bar, 20 µm.

Mentions: To confirm that gfp expression following fluoromycobacteriophage infection is responsive to antibiotics, an M. tuberculosis mc26230 culture was grown (in the absence of Tween), spun and washed, and phage added with either rifampicin or streptomycin simultaneously (Figure 7A panels a and c), incubated for 16 hours and examined by microscopy; in both experiments fluorescence was eliminated by antibiotic addition. Isogenic rifampicin-resistant and streptomycin-resistant derivatives of M. tuberculosis mc26230 were constructed by recombineering with ssDNA substrates [32] – introducing a S456L substitution in rpoB (Rv0667) and a K43R substitution in rpsL (Rv0682), respectively – and tested in a similar assay (Figure 7A panels b and d). Antibiotic resistant strains fluoresced well in the presence of antibiotic (Figure 7A panels b and d) demonstrating that fluoromycobacteriophages can provide a report of resistance/susceptible profiles of M. tuberculosis to rifampicin and streptomycin in approximately 16 hours. Because isoniazid (INH) is an inhibitor of mycobacterial cell wall synthesis rather then gene expression, cultures were pre-incubated with INH for 48 hours prior to addition of fluoromycobacteriophage; however, fluorescence was unaffected even in INH-sensitive bacteria (Figure 7A, panel e). Although a variety of pre-incubation times and drug concentrations have been examined using cells grown in these conditions, no significant influence of INH on fluorescence was observed (data not shown).


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

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

Antibiotic susceptibility testing of M. tuberculosis using fluoromycobacteriophages.M. tuberculosis mc26230 wt and antibiotic resistant strains were infected with phAE87::hsp60-EGFP with or without antibiotic addition as indicated. A. Cultures were grown in the absence of Tween, washed, and tested. In panels a–d antibiotics and phage were added simultaneously, incubated for 16 hours, and examined by microscopy. In panels e–f INH was added first for 48 hours followed by phage addition, incubation for a further 16 hours, and examined by microscopy. Scale bar, 10 µm. B. Cultures were grown in the presence of Tween, centrifuged and washed twice, and then incubated with antibiotic (as indicated) for 24 hours. Phage were then added, cultures incubated for 16 hour, and examined by microscopy. Scale bar, 20 µm.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0004870-g007: Antibiotic susceptibility testing of M. tuberculosis using fluoromycobacteriophages.M. tuberculosis mc26230 wt and antibiotic resistant strains were infected with phAE87::hsp60-EGFP with or without antibiotic addition as indicated. A. Cultures were grown in the absence of Tween, washed, and tested. In panels a–d antibiotics and phage were added simultaneously, incubated for 16 hours, and examined by microscopy. In panels e–f INH was added first for 48 hours followed by phage addition, incubation for a further 16 hours, and examined by microscopy. Scale bar, 10 µm. B. Cultures were grown in the presence of Tween, centrifuged and washed twice, and then incubated with antibiotic (as indicated) for 24 hours. Phage were then added, cultures incubated for 16 hour, and examined by microscopy. Scale bar, 20 µm.
Mentions: To confirm that gfp expression following fluoromycobacteriophage infection is responsive to antibiotics, an M. tuberculosis mc26230 culture was grown (in the absence of Tween), spun and washed, and phage added with either rifampicin or streptomycin simultaneously (Figure 7A panels a and c), incubated for 16 hours and examined by microscopy; in both experiments fluorescence was eliminated by antibiotic addition. Isogenic rifampicin-resistant and streptomycin-resistant derivatives of M. tuberculosis mc26230 were constructed by recombineering with ssDNA substrates [32] – introducing a S456L substitution in rpoB (Rv0667) and a K43R substitution in rpsL (Rv0682), respectively – and tested in a similar assay (Figure 7A panels b and d). Antibiotic resistant strains fluoresced well in the presence of antibiotic (Figure 7A panels b and d) demonstrating that fluoromycobacteriophages can provide a report of resistance/susceptible profiles of M. tuberculosis to rifampicin and streptomycin in approximately 16 hours. Because isoniazid (INH) is an inhibitor of mycobacterial cell wall synthesis rather then gene expression, cultures were pre-incubated with INH for 48 hours prior to addition of fluoromycobacteriophage; however, fluorescence was unaffected even in INH-sensitive bacteria (Figure 7A, panel e). Although a variety of pre-incubation times and drug concentrations have been examined using cells grown in these conditions, no significant influence of INH on fluorescence was observed (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