the isoniazid - monoresistant and multidrug - resistant tuberculosis ( mdr - tb ) isolates were obtained from the patients ' source hospital or mru . all isolates were identified as belonging to the same strain of m. tuberculosis ( rapet or is6110 typing ) , and all drug - susceptibility testing was carried out at mru according to standard procedures . the wildtype control isolate used for these studies was m. tuberculosis , h37rv ( atcc , 9360 national collection of type culture , london , uk ) . three isolates , 018 , 483 , and 915 , were from patients in whom mdr - tb developed as a result of poor compliance with therapy , whereas isolates 604 , t7 , and 371 were from patients who contracted primary mdr - tb . dna was prepared from the 6 isolates of m. tuberculosis by emulsifying 23 colonies in 400 l tris - edta buffer and heating the suspension in a water bath for 40 min at 80c . polymerase chain reaction ( pcr ) was performed on the extracted dna with 6 sets of primers designed to amplify 6 overlapping fragments of the rpob gene from the 6 m. tuberculosis isolates ( table ) . ten microliters of dna was added to the pcr mix containing 81.4 l pcr - quality water , 10 l potassium chloride buffer ( bioline ltd . , london , uk ) , 0.4 l of each primer ( 100 mmol ) ( sigma - genosys ltd . , haverhill , uk ) , 3 l deoxynucleoside triphosphates ( 5 mmol ) , and 1 l taq ( bioline ) . the amplification was performed on a techgene thermal cycler ( techne , princeton , nj , usa ) . pcr products were separated by gel electrophoresis on a 1.5% agarose gel , and dna bands were stained with ethidium bromide . primers and excess nucleotides were removed from the amplified dna with a pcr clean - up kit ( qiagen , inc . , the amount of dna in the cleaned - up product was quantified by comparing the intensity of the band to bands of known intensity in a hyperladder marker ( bioline ) . * denotes a primer position in the sequence before the start (  ) or after the end ( + ) of the rpob gene sequence . forward and reverse cycle sequencing reactions were performed with the big dye terminator cycle sequencing ready reaction dna sequencing kit ( applied biosystems , inc . , foster city , ca , usa ) . briefly , 40 ng of cleaned - up dna was added to 10.8 l pcr - quality water , 3 l buffer , 3.2 l of forward or reverse 1 mmol primer , and 1 l of cycle sequencing ready reaction mix . the labeled dna was precipitated by adding 14.5 l pcr - quality water , 62.5 l 95% ethanol , and 3 l sodium acetate solution ( 2.3 mol / l ) and centrifuging ( 13,000  g , 15 min , 4c ) . the supernatant was removed with a fine - tipped pipette , and the pellet was cleaned with 200 l 70% ethanol and then recentrifuged ( 13,000  g , 15 min , 4c ) . again the supernatant was removed , and the pellet was dried at 37c for 30 min . four microliters of formamide and 1 l of dextran loading buffer were added to each pellet , and 1.5 l of sample was added to each well of the sequencing gel . the 6 fragments of the rpob gene from each of the 6 isolates were then sequenced with an abi 377 applied biosystems sequencer . the sequences obtained from each isolate were joined together to form a continuous whole gene sequence , aligned with clustalw ( http://www.ebi.ac.uk/clustalw/ ) and compared to the wildtype to identify base - pair mismatches . in addition , all 6 isolates were tested with a line probe resistance  determining hybridization assay , inno - lipa ( innogenetics belgium ) , as described in the manufacturer 's instructions . briefly , an 81-bp region of the rpob gene was amplified with biotinylated primers , which yielded a biotinylated target sequence , and hybridized with specific oligonucleotide probes immobilized on a parallel strip . inno - lipa consists of 10 oligonucleotide probes ( 1923 bases in length ) , encompassing the 81-bp region ( rrdr ) of the rpob gene . one is specific for m. tuberculosis complex , whereas the other 5 partially overlapping wildtype probes ( s1s5 ) cover the region from positions 507 to 534 of the rpob gene . these s - probes hybridize to the wildtype ( rifampicin - sensitive ) dna sequence . failure of any of these s - probes to hybridize indicates that a mutation has occurred . four other probes ( r2 , r4a , r4b , and r5 ) are specific for amplicons carrying the most common rpob mutation that confers rifampicin resistance . the results of the inno - lipa assay showed 5 of the 6 isolates , which were phenotypically rifampicin - resistant , were negative . the target dna hybridized to all 5 wildtype s - probes and none of the r - probes , which demonstrated that the assay failed to detect rifampicin resistance in these 5 isolates . failure to detect rare mutations within the rrdr may be caused by nonspecific hybridization of the wildtype s - probes because of slight fluctuations in temperature during the hybridization process . isolate 483 showed a weak dna hybridization reaction with the s4 probe , indicating that a mutation was present on codon 451 ( codon 526 in escherichia coli ) . analysis of the sequence data identified specific mutations in rpob in all 6 strains studied ( figure 1 ) . three had mutations within the rrdr , and of these , 2 were c - to - g mutations at codon 456 , inducing a serine to tryptophan amino acid conversion ( s456w ) . the third was an a - to - g mutation at codon 451 , resulting in a change from histidine to arginine ( h451r ) ( figure 1 ) . no other mutations were identified in the dna sequences outside the rrdr in these 3 isolates . the 3 other isolates had the mutation g to t at codon 176 , outside the rrdr , which caused a change from valine to phenylalanine ( v176f ) . neither rrdr mutations nor any other mutations were found in the rpob gene sequences of these 3 isolates . wt , wildtype ; t7 , 018 , 604  v176f ( g to t , highlighted in blue ) ; 483 , h526r ( a to g , highlighted in yellow ) ; 915 , 317 , s531w ( c to g , highlighted in pink ) . of the mutations found within the rrdr , h526r occurs in < 4% , and s531w occurs in 1.4% of all rifampicin - resistant isolates , respectively ( 7 ) . mutations outside the rrdr account for < 4% of rifampicin resistance , and few have been described ( 79 ) . in a previous study ( 7 ) , v176f was found in 5 of 18 isolates with no mutations in the rrdr from asia ( 9,10 ) and africa ( 11,12 ) . to our knowledge , this is the first time this type of mutation has been detected in strains isolated in the united kingdom . three different mutations in the rpob gene , v176f , h526r and s531w , were detected in the isolates associated with the isoniazid - resistant outbreak . given that all the mutations found in this study are rare and that mdr - tb developed during treatment in 3 patients ( 018 , 483 , and 915 ) ( figure 2 ) , the rifampicin - resistance mutations observed in these 3 patients likely occurred on 3 independent occasions , as a result of poor compliance with therapy . two of these patients , 018 and 915 , were contacts of the 3 patients who subsequently had primary mdr - tb ( patients t7 and 604 [ v176f ] and 317 [ s531w ] ) ( figure 2 ) . suggested epidemiologic relationship between 6 cases of multidrug - resistant tuberculosis ( mdr - tb ) . resistance to rifampicin developed in patients 018 , 483 , and 915 while on therapy , whereas patients t7 , 604 , and 317 contracted primary mdr - tb . the type of mutation present in each patient 's strain of mdr - tb is highlighted in bold . our study highlights problems associated with using a line probe hybridization assay to detect rifampicin resistance in m. tuberculosis . the inno - lipa assay can not detect mutations outside the rrdr , and failure to detect rare mutations within the rrdr may be caused by nonspecific hybridization of the wildtype s - probes because of slight fluctuations in temperature during the hybridization process . in this study , all the rare mutations , inside and outside the rrdr , were detected by sequencing the entire rpob gene . the inability to identify mdr - tb isolates results in treatment failure and increased risk for transmission of resistant disease in the community . we recommend that , when the index of suspicion for mdr - tb is high and the line probe assays fail to detect mutations conferring rifampicin resistance , the entire rpob gene should be sequenced to prevent unnecessary delay in diagnosing mdr - tb .
mycobacterium tuberculosis isolates cultured from 6 patients associated with an isoniazid - resistant m. tuberculosis outbreak acquired rifampicin resistance . 
 the rpob gene sequence showed that resistance was associated with rare mutations in each isolate . 
 three isolates had a mutation outside the rifampicin resistance  determining region .