Frequency of Mutations in the Rpob Gene of Multidrug-resistant Mycobacterium Tuberculosis Clinical Isolates from Sudan

This is an open-access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Multidrug-resistant (MDR) strains of Mycobacterium tuberculosis represent an obstacle in tuberculosis control and stress the need for rapid and reliable diagnostic tools for drug susceptibility testing in clinical isolates [1]. Mutations in the 81-bp region of the rpoB gene are responsible for resistance to rifampicin, an essential drug for the therapy of tuberculosis (TB) [2]. Mycobacteria laboratory facilities in Sudan exist only in Khartoum state whereas TB patients from other states are referred to Khartoum state for diagnosis and treatment. The MDR-TB prevalence in Sudan was found in 5% of new cases and 24% of previously treated patients [3]. Recent development of the Xpert MTB/RIF test [4] and of the line probe assay [5] which span 81-bp fragment of the RNA polymerase beta subunit (rpoB) gene have allowed for the rapid detection of resistance to rifampicin (RIF). Knowledge on the geographical distribution of the rpoB resistant alleles is essential for the development of diagnostic tools. Although sequence analysis of the rpoB gene has been shown to be effective for detecting RIF-resistance alleles in over 90% of RIF-resistant isolates from different geographical regions [6,7], there is only limited information from Sudan [8]. In order to find Sudan-specific mutations, that could potentially be used for the optimization of the current detection tools of RIF resistance alleles, we determined in this study the mutation profile occurring within an 81-bp fragment of the rpoB gene, in a collection of MDR clinical isolates of Mycobacterium tuberculosis isolated from Khartoum, Sudan. Forty-nine clinical isolates consisting of RIF-resistant M. tuberculosis were isolated from sputum of TB patients during the period between 2007 and 2009, into the Khartoum state, Sudan. The drug susceptibility testing was performed using standard proportional method and the drugs tested were rifampicin, isoniazid (INH), ethambutol (EMB), and streptomycin (SM) [9]. Genomic DNA was extracted using the boiling method. An 81-bp region of the rpoB gene was amplified by polymerase chain reaction (PCR) [10] and DNA sequencing was performed by Macrogen Inc. (Seoul, South Korea) on a ABI3730XL DNA sequencer (Applied Biosystems, Foster City, USA). Water was used as a negative control to replace DNA, to rule out carry-over contamination of the amplicon throughout the steps of the protocol. We report here the determination of the …


Multidrug-resistant
(MDR) strains of Mycobacterium tuberculosis represent an obstacle in tuberculosis control and stress the need for rapid and reliable diagnostic tools for drug susceptibility testing in clinical isolates [1].Mutations in the 81-bp region of the rpoB gene are responsible for resistance to rifampicin, an essential drug for the therapy of tuberculosis (TB) [2].Mycobacteria laboratory facilities in Sudan exist only in Khartoum state whereas TB patients from other states are referred to Khartoum state for diagnosis and treatment.The MDR-TB prevalence in Sudan was found in 5% of new cases and 24% of previously treated patients [3].Recent development of the Xpert MTB/RIF test [4] and of the line probe assay [5] which span 81-bp fragment of the RNA polymerase beta subunit (rpoB) gene have allowed for the rapid detection of resistance to rifampicin (RIF).Knowledge on the geographical distribution of the rpoB resistant alleles is essential for the development of diagnostic tools.Although sequence analysis of the rpoB gene has been shown to be effective for detecting RIF-resistance alleles in over 90% of RIF-resistant isolates from different geographical regions [6,7], there is only limited information from Sudan [8].In order to find Sudanspecific mutations, that could potentially be used for the optimization of the current detection tools of RIF resistance alleles, we determined in this study the mutation profile occurring within an 81-bp fragment of the rpoB gene, in a collection of MDR clinical isolates of Mycobacterium tuberculosis isolated from Khartoum, Sudan.
Forty-nine clinical isolates consisting of RIFresistant M. tuberculosis were isolated from sputum of TB patients during the period between 2007 and 2009, into the Khartoum state, Sudan.The drug susceptibility testing was performed using standard proportional method and the drugs tested were rifampicin, isoniazid (INH), ethambutol (EMB), and streptomycin (SM) [9].
Genomic DNA was extracted using the boiling method.An 81-bp region of the rpoB gene was amplified by polymerase chain reaction (PCR) [10] and DNA sequencing was performed by Macrogen Inc. (Seoul, South Korea) on a ABI3730XL DNA sequencer (Applied Biosystems, Foster City, USA).Water was used as a negative control to replace DNA, to rule out carry-over contamination of the amplicon throughout the steps of the protocol.
We report here the determination of the rpoB gene mutation profile in MDR M. tuberculosis clinical isolates from the Khartoum state and the comparison with mutation profiles from different countries.The resistance profile and mutations of rpoB are listed in Table 1.The mutations observed in this study were at codon 531 (64.1%), 526 (17.9%), 516 (7.7%), 511 (2.6%) and two isolates (5.1%) had insertion between codon 514-515.All genetic alterations were caused by single base substitutions, and the most frequent mutation was observed at codon 531 (Ser ⇒ Leu 61.5%).Missense mutations with different frequencies were observed at codon 526 (His ⇒ tyr 12.8%), (His ⇒ leu 5.1%) and (His ⇒ cys 2.5%).Other mutations were at codon 531 (ser ⇒ Trp 2.5%), 516 (asp ⇒ val 7.6%) and two isolates had insertion of Phe.Allele10 contained novel mutation at codon 511 (CTG leu to CCC pro) that has never been previously reported.The sensitivity of rpoB mutations within the 81-bp region for predicting the RIF-resistance of M. tuberculosis is 80%.The new allele sequence has been deposited in GenBank under the accession number KF877732.
A comparison of mutation frequency at codon 531, 526, 516 and 511 was similar to mutation frequency reported from Taiwan, India, Italy and China [11,12,13,14].Mutations within the 81-bp hot-spot region (codons 507 to 533) account for more than 90% of RIF-resistant M. tuberculosis strains [6,7], but still there was a proportion of strains phenotypically RIFresistant but lacking mutation within the hot-spot region.Yue et al., 2003 [14] have reported that 10% of phenotypically RIF-resistant M. tuberculosis isolates did not show any mutations at the 81-bp region.According to Adikaram et al., 2012 [15], 41.9% of RIF-resistant M. tuberculosis isolates contained mutation outside the hot-spot region.In the current study, 20% of our isolates contained no mutations within the 81-bp region.This disagreement between phenotypic resistant and genotypic sensitive isolates can be explained by occurrence of mutation elsewhere outside 81-bp region as shown by Adikaram et al., 2012 [15], or may be due to other mechanism.The 80% sensitivity obtained herein for rpoB mutations within the 81-bp region for predicting the RIFresistance of M. tuberculosis was lower than the sensitivity reported from other countries which was > 90 %.
We conclude that the discovery of one new mutation and isolates lacking mutations mentioned herein emphasizes the need for mutations surveillance prior to the implementation of commercial molecular tools [4], and optimization of these techniques to cover different alleles of rpoB gene in Sudan.In addition, the detection of RIF-resistance by molecular technique [4] must always be confirmed by phenotypic methods in case of absence of mutations.Study limitations include the relatively short region of rpoB sequenced and the small sample size.Despite these limitations, this work characterizes the rpoB mutations in MDR TB in Sudan.In addition, our finding is valuable for the design of screening diagnostic tests for rapid detection of mutations associated with rifampicin resistance in Sudanese clinical isolates.

Table 1 .
Frequency of rpoB mutation and antibiotics profile in MDR M. tuberculosis isolates