All manipulation of live M. tuberculosis was performed under biohazard category 3 safety conditions using a microbiological safety cabinet in accordance with local regulations. One hundred and forty-nine cultures of M. tuberculosis isolated from 129 patients were selected for testing at the Mycobacteriology Laboratory of the Joint Clinical Research Centre (JCRC) in Kampala, Uganda. Strains for the study were selected from stored cultures previously isolated from subjects enrolled into several IRB-approved studies at the Tuberculosis Research Unit in Uganda. Prior to testing all isolates were freshly sub-cultured on Middlebrook 7H10 agar or Lowenstein-Jensen (LJ) medium. M. tuberculosis H37Rv was used as a susceptible control strain and M. tuberculosis TMC 331 as a resistant control strain. All strains were subjected to testing for susceptibility to rifampicin by the phage assay and a traditional phenotypic test. Statistical analysis of results was performed using STATA 9.0 (Texas, USA)
Phage assay
The details of the assay and production of phage D29 have been described previously [8]. Indicator plates for detection of progeny phage were prepared by adding 10% v/v of a stationary phase culture of M. smegmatis mc2155 to 1.5% agar in Luria-Bertani broth (Difco, Becton Dickinson, Sparks, USA.). Rifampicin was prepared from a stock solution of 20 mg/ml in dimethylformamide (Sigma-Aldrich, Poole, UK). 75 μl of drug at 4, 8 and 20 μg/ml were placed to the wells of a sterile 96-well plate (Greiner Labortechnik, Stonehouse, UK) to give a final working concentration of 2, 4 and 10 μg/ml. Aliquots containing no drug were also plated.
Bacterial suspensions of isolates under test were prepared from growth on solid medium in 2 ml of Luria-Bertani broth supplemented with 1 mM calcium chloride (assay broth). Samples were vortexed in the presence of glass beads to disaggregate clumps and left to stand for at least 5 min to allow aerosols to settle. Aliquots of 75 μl were placed in each well of the microwell plate containing drug solutions. The plate was then covered, sealed in a plastic bag and incubated at 37°C for 24 hours. D29 phage suspension was diluted in the assay broth and an aliquot of 50 μl added to each well, giving a final concentration of 2.5 × 107 phage/ml. The plate was resealed and incubated at 37°C for 90 min. Aliquots of 100 μl of freshly prepared solution of 30 mM ferrous ammonium sulphate (Sigma-Aldrich, Poole, UK) were added to each well and mixed by pipetting. Aliquots of 10 μl from each well were then spotted onto the surface of the M. smegmatis indicator plate. After absorption of drops in the agar medium the plates were sealed in plastic bags and incubated overnight at 37°C. The number of plaque forming units (pfu) was recorded on the drug-containing assay and compared with the number of pfu on the drug-free control. An isolate was recorded as susceptible when no pfu were observed from drug containing samples and as resistant when pfu were observed for the assay corresponding to each drug concentration. The assay and interpretation of results was performed blinded, without prior knowledge of the outcome of other susceptibility tests.
Phenotypic susceptibility testing
Drug susceptibility testing was performed using the BACTEC 460 system (Becton Dickinson, Sparks, Maryland, USA) following the manufacturer's recommendations [9]. One standard drug concentration (2 μg/ml) was reconstituted from lyophilized drugs supplied by the manufacturer. The test inoculates were standardized prior to the addition of 0.1 ml volumes to the vials containing drug, and to a no drug control. Samples were incubated at 37°C and daily readings were taken and interpretation performed after comparing the changes in the growth indices of the inoculated control with that of the test drug.
Nucleotide sequencing
Strains found resistant by either by the BACTEC or the Phage assay were investigated for mutations in a region of the rpoB gene. Sequencing was performed on in the Genome Research Centre at the London School of Hygiene & Tropical Medicine. DNA was extracted from cells grown on Middlebrook 7H11 agar using the CTAB (hexadecyl trimethyl ammonium bromide)-NaCl method described by Van Embden and collaborators [10]. A 255-bp fragment of the rpoB gene including the 81-bp core region was amplified by PCR using primers RP4T (5'-GAG GCG ATC ACA CCG CAG ACG T-3') and RP8T (5'-GAT GTT GGG CCC CTC AGG GGT T-3') [11] and a two-step amplification programme (3 min at 95°C followed by 30 cycles of [95°C for 30s; 65°C for 45s and 72°C for 1 min]). PCR products were purified through QIAquick PCR Purification columns (Qiagen, Crawley, UK). Sequencing of both forward and reverse strands of the amplicons was performed using the amplification primers (RP4T and RP8T) and the BigDye Terminator Cycle Sequencing kit v.3.1. (Applied Biosystems, Foster City, CA, USA). The sequences obtained were compared to wild type M. tuberculosis H37Rv RNA-polymerase beta subunit (rpoB) gene (partial cds U12205) using DNAStar MegAlign programme (DNAStar, Wisconsin, USA).
Minimal inhibitory concentration
Minimal inhibitory concentrations (MIC) were determined using the assay described by Abate et al [12]. This assay is based on detection of bacterial growth by a redox dye 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) (Sigma-Aldrich, Poole, UK). Serial two-fold dilutions of rifampicin were prepared in the wells of a sterile flat bottom 96-well plate (Greiner Labortechnik, Stonehouse, UK) to achieve concentrations ranging from 0.4 – 100 μg/ml in 100 μl Middlebrook 7H9 broth supplemented with OADC (Becton Dickinson, Sparks, USA). The inoculum was prepared from fresh Lowenstein Jensen cultures in Middlebrook broth and 100 μl of bacterial suspension added to each well. A growth control well containing no drug was included for each isolate. After seven days of incubation at 37°C, 10 μl of the MTT solution (5 mg/ml) was added to each well and the plate was re-incubated overnight. Aliquots of 50 μl of formazan solubilising buffer [1:1 (v/v) 20% SDS: 50% N,N-dimethylformamide (Sigma, Poole, UK)] were then added to the wells and the plate re-incubated for three hours. A change in colour from yellow to violet indicated growth of bacteria and the MIC was interpreted as the lowest concentration that inhibited bacterial growth.
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