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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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Specificity and efficiency of fluoromycobacteriophage infections.A. A mixture of E. coli cells expressing DsRed-Express and M. smegmatis were infected with (a) phAE87::hsp60-EGFP or (b) mock infected. From left to right: fluorescence micrograph images using a ZsGreen1 filter (green); fluorescence micrograph images using a HQ:R NX filter (red), merged fluorescence images; phase contrast images. Red cells: E. coli; green cells: M. smegmatis mc2155 infected with pHAE87::hsp60-EGFP. Scale bar, 10 µm. B. M. smegmatis mc2155 cells were infected with phAE87::hsp60-EGFP at different multiplicities of infection (moi). Green cells were counted in 15 individual fields and the percentage of fluorescent cells compared to the total number of cells in phase contrast images determined.
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pone-0004870-g003: Specificity and efficiency of fluoromycobacteriophage infections.A. A mixture of E. coli cells expressing DsRed-Express and M. smegmatis were infected with (a) phAE87::hsp60-EGFP or (b) mock infected. From left to right: fluorescence micrograph images using a ZsGreen1 filter (green); fluorescence micrograph images using a HQ:R NX filter (red), merged fluorescence images; phase contrast images. Red cells: E. coli; green cells: M. smegmatis mc2155 infected with pHAE87::hsp60-EGFP. Scale bar, 10 µm. B. M. smegmatis mc2155 cells were infected with phAE87::hsp60-EGFP at different multiplicities of infection (moi). Green cells were counted in 15 individual fields and the percentage of fluorescent cells compared to the total number of cells in phase contrast images determined.

Mentions: To evaluate the ability of phAE87::hsp60-EGFP and phAE87::hsp60-ZsYellow to confer fluorescence following infection, a mid-logarithmic culture of M. smegmatis mc2155 was infected at a multiplicity of infection (moi) of approximately 100, incubated for 4 hours at 37°C, spun and washed, and examined by fluorescence microscopy (Figure 2). Little or no fluorescence was detected from uninfected cells, and individual cells could be easily detected following infection with either phage (Figure 2). To confirm that fluorescence was specific to infection of M. smegmatis, fluoromycobacteriophages were used to infect a mixed culture containing approximately equivalent amounts of M. smegmatis and an Escherichia coli strain expressing the fluorescent reporter gene DsRed (Figure 3). Green fluorescence was readily observed only in infected M. smegmatis cells and in each case was distinct and non-overlapping with the red fluorescence from E. coli (Figure 3A panels a and b,).


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

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

Specificity and efficiency of fluoromycobacteriophage infections.A. A mixture of E. coli cells expressing DsRed-Express and M. smegmatis were infected with (a) phAE87::hsp60-EGFP or (b) mock infected. From left to right: fluorescence micrograph images using a ZsGreen1 filter (green); fluorescence micrograph images using a HQ:R NX filter (red), merged fluorescence images; phase contrast images. Red cells: E. coli; green cells: M. smegmatis mc2155 infected with pHAE87::hsp60-EGFP. Scale bar, 10 µm. B. M. smegmatis mc2155 cells were infected with phAE87::hsp60-EGFP at different multiplicities of infection (moi). Green cells were counted in 15 individual fields and the percentage of fluorescent cells compared to the total number of cells in phase contrast images determined.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2654538&req=5

pone-0004870-g003: Specificity and efficiency of fluoromycobacteriophage infections.A. A mixture of E. coli cells expressing DsRed-Express and M. smegmatis were infected with (a) phAE87::hsp60-EGFP or (b) mock infected. From left to right: fluorescence micrograph images using a ZsGreen1 filter (green); fluorescence micrograph images using a HQ:R NX filter (red), merged fluorescence images; phase contrast images. Red cells: E. coli; green cells: M. smegmatis mc2155 infected with pHAE87::hsp60-EGFP. Scale bar, 10 µm. B. M. smegmatis mc2155 cells were infected with phAE87::hsp60-EGFP at different multiplicities of infection (moi). Green cells were counted in 15 individual fields and the percentage of fluorescent cells compared to the total number of cells in phase contrast images determined.
Mentions: To evaluate the ability of phAE87::hsp60-EGFP and phAE87::hsp60-ZsYellow to confer fluorescence following infection, a mid-logarithmic culture of M. smegmatis mc2155 was infected at a multiplicity of infection (moi) of approximately 100, incubated for 4 hours at 37°C, spun and washed, and examined by fluorescence microscopy (Figure 2). Little or no fluorescence was detected from uninfected cells, and individual cells could be easily detected following infection with either phage (Figure 2). To confirm that fluorescence was specific to infection of M. smegmatis, fluoromycobacteriophages were used to infect a mixed culture containing approximately equivalent amounts of M. smegmatis and an Escherichia coli strain expressing the fluorescent reporter gene DsRed (Figure 3). Green fluorescence was readily observed only in infected M. smegmatis cells and in each case was distinct and non-overlapping with the red fluorescence from E. coli (Figure 3A panels a and b,).

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