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. 2012 Mar 13;3(2):e00312-11.
doi: 10.1128/mBio.00312-11. Print 2012.

Generation of a Novel Nucleic Acid-Based Reporter System to Detect Phenotypic Susceptibility to Antibiotics in Mycobacterium Tuberculosis

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Free PMC article

Generation of a Novel Nucleic Acid-Based Reporter System to Detect Phenotypic Susceptibility to Antibiotics in Mycobacterium Tuberculosis

Matthew C Mulvey et al. mBio. .
Free PMC article

Abstract

We designed, constructed, and evaluated a prototype novel reporter system comprised of two functional cassettes: (i) the SP6 RNA polymerase gene under transcriptional control of a promoter active in mycobacteria and (ii) the consensus SP6 polymerase promoter that directs expression of an otherwise unexpressed sequence. We incorporated the reporter system into a mycobacteriophage for delivery into viable Mycobacterium tuberculosis, and introduction led to synthesis of an SP6 polymerase-dependent surrogate marker RNA that we detected by reverse transcriptase PCR (RT-PCR). The reporter confirmed the susceptibility profile of both drug-susceptible and drug-resistant M. tuberculosis strains exposed to first-line antitubercular drugs and required as little as 16 h of exposure to antibacterial agents targeting bacterial metabolic processes to accurately read the reaction. The reporter system translated the bacterial phenotype into a language interpretable by rapid and sensitive nucleic acid detection. As a phenotypic assay that works only on viable M. tuberculosis, it could be used to rapidly assess resistance to any drug, including drugs for which the mechanism of resistance is unknown or which result from many potential known (and unknown) genetic alterations.

Importance: The ability to detect antibiotic resistance of slow-growing bacteria (i.e., Mycobacterium tuberculosis) is hampered by two factors, the time to detection (weeks to months) and the resistance mechanism (unknown for many drugs), delaying the appropriate treatment of patients with drug-resistant or multidrug-resistant tuberculosis (TB). The novel technique described in this article uses a unique surrogate nucleic acid marker produced by phage that infects M. tuberculosis to record phenotypic antibiotic susceptibility in less than a day.

Figures

FIG 1
FIG 1
Design and characterization of phSP6-ProPol. (A) The SGM was comprised of 2 sections contained within a XbaI-NotI restriction fragment: the SP6 RNA polymerase gene (SP6 RNA Pol) under transcriptional control of the mycobacteriophage L5 Pleft promoter (open star) and the consensus SP6 promoter fused to the SML-encoding sequence (filled star). The SP6-SML section is flanked by 2 transcription terminators: the upstream terminator (filled circle) is E. coli rrnBT2 and precludes basal transcription through the SML-encoding sequence by host RNA polymerase; the downstream terminator (open circle) is the SP6 RNA polymerase terminator from the region downstream of the SP6 phage major capsid subunit described by Dobbins et al. (22). After expression of SP6 RNA polymerase from Pleft, the SML-encoding sequence downstream of the SP6 promoter was transcribed by SP6 RNA polymerase. SP6-dependent transcription of the SML-encoding sequence constituted generation of the SML. SML RNA could then be amplified and detected using primers that bind the SML. (B) The TM4 genome is depicted by the solid black line at the top of the figure. Expression of phage genes occurs on one strand of the genome, and the direction of transcription is indicated by the dashed arrow above the phage genome. Transgenic functions inserted into TM4 are contained on a NotI fragment, which is indicated and expanded. phAE142 encodes an ampicillin resistance cassette (Ampr) and an origin of replication (oriE) for maintenance and selection of the phasmid in E. coli. phAE142 also encodes the luciferase open reading frame fused to Pleft. phSP6-ProPol was derived from phAE142 and replaced the luciferase-encoding XbaI-NotI fragment with the XbaI-NotI SGM. In addition, phSP6-ProPol contained a kanamycin resistance cassette (Kmr) in place of phAE142 Ampr. Pleft transcription occurred on the strand opposite the endogenous phage functions in both phAE142 and phSP6-ProPol. The binding sites and orientation of oligonucleotide primers Ul53-UpSt-113348 and Ul53-DnSt-112112 (UpSt and DnSt, respectively) used for detection of SML generation and those used to characterize transgene structure in phSP6-ProPol are indicated. (C) Phage eluted from primary (1°) plaques originating with transformation of 2 independent phSP6-ProPol-Kan phasmid DNA clones (#4 and #5), as well as phAE142 phasmid DNA, into mc2 4502 were added to a PCR with the primers PLLF and TM4-50133.52. Phasmid DNA with and without the addition of MP buffer was included as controls. PLLF and TM4-50133.52 were predicted to generate a 667-bp product using phSP6-ProPol as a template, compared to a 181-bp product when phAE142 was the substrate. Products were separated on a 2% agarose gel and visualized by ethidium bromide staining. Locations of DNA size markers are indicated. (D) Phage eluted from the 1° plaques in panel C were amplified using primers PLLU and PLLD, which were predicted to mediate amplification of 2,854-bp and 1,882-bp products in phSP6-ProPol and phAE142, respectively. Products were then separated on a 1% agarose gel and visualized by ethidium bromide staining. The locations of DNA size markers are indicated.
FIG 2
FIG 2
SML RNA present in phSP6-ProPol stocks. A crude phSP6-ProPol preparation (108 PFU) was either left untreated or treated with MRI to a final concentration of 1 U/μl. Either 5 ng (++) or 1 ng (+) RNase A was then added, and the mixture was incubated for 30 min at 37°C. After purification of RNA and digestion with DNase I, reverse transcription was carried out using the DnSt primer. After reverse transcription but prior to PCR, cDNA from each sample was diluted 1:10 to estimate a 10-fold signal reduction. The primers DnSt and UpSt were then used to amplify a 150-bp section of SML cDNA using PCR. Products were separated on a 2% agarose gel and visualized by ethidium bromide staining. The locations of DNA size markers are indicated.
FIG 3
FIG 3
SML detection occurs at 4 h postinfection (p.i.). H37Rv was infected with phSP6-ProPol. RNase A was added to phSP6-ProPol prior to initiation of infection, and MRI was added at 0.5 h p.i. At 0.5, 3, and 4 h p.i., RNA was purified, digested with DNase I, and amplified using RT-PCR. Products were then separated on a 2% agarose gel and visualized by ethidium bromide staining. The locations of DNA size markers are indicated.
FIG 4
FIG 4
SML generation observed in phSP6-ProPol-infected cells. H37Rv cells were either mock infected or infected with phSP6-ProPol. RNA was purified at 4 h p.i., and SML generation was detected using RT-PCR. Products were then separated on a 2% agarose gel and visualized by ethidium bromide staining. The locations of DNA size markers are indicated.
FIG 5
FIG 5
Detection of transcription by a cryptic promoter upstream of the SP6 promoter. (A) SP6 promoter-SML transcription unit and the locations of primers UpSt and DnSt, used to amplify SML RNA, are indicated. The primer SP6-Dep-UpSt overlaps the SP6 promoter and terminates one nucleotide 5′ to the transcription initiation site for SP6 polymerase. (B) RNA purified at 4 h p.i. from H37Rv M. tuberculosis infected with phSP6-ProPol was digested with DNase I prior to reverse transcription with the DnSt primer. A portion of cDNA was then left undiluted or diluted 1:10 and was PCR amplified using the primers DnSt and UpSt or DnSt and SP6-Dep-UpSt. The targeting plasmid pSP6-ProPol-Kan was included in separate amplification reactions as a control for successful PCR using each primer set. Products were then separated on a 2% agarose gel and visualized by ethidium bromide staining. The locations of DNA size markers are indicated.
FIG 6
FIG 6
phSP6-ProPol determined H37Rv susceptibility to first-line antitubercular antibiotics with a high signal-to-noise ratio. H37Rv was either left untreated or treated with rifampin, isoniazid, streptomycin, or ethambutol for 16 h or 40 h. Bacteria were then infected with phSP6-ProPol for 4 h. RNA was isolated, purified, and amplified using RT-PCR. The resultant product was separated by 2% agarose gel electrophoresis and was stained with ethidium bromide. Dilutions (1:10 and 1:100) of cDNA from the untreated controls were made prior to PCR amplification to provide a semiquantitative estimation of the effect of each drug on SML synthesis during infection of drug-susceptible M. tuberculosis. The locations and molecular weights of DNA size markers in lane M and the location of the SML signal are indicated.
FIG 7
FIG 7
phSP6-ProPol can detect M. tuberculosis drug resistance. RIFr M. tuberculosis and EMBr M. tuberculosis were either left untreated or treated with rifampin or ethambutol for 16 h. Bacteria were then infected with phSP6-ProPol for 4 h. RNA from each sample was then purified and amplified using RT-PCR. The resultant product was separated by 2% agarose gel electrophoresis and visualized by staining with ethidium bromide. A 1:10 dilution of cDNA derived from the untreated and ethambutol-treated phSP6-ProPol-infected RIFr strain was made prior to PCR amplification to provide a semiquantitative determination of the reduction of SML synthesis by ethambutol in this strain. The locations of DNA size markers are indicated.

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