Early detection of drug resistance constitutes one of the priorities of TB control programs. It allows initiation of the appropriate treatment in patients and avoids dissemination of resistant strains in the community. In the context of a poor resource country, detection of drug resistance is performed in the reference laboratory by so-called ‘conventional methods’ based on detection of growth of M. tuberculosis in the presence of the respective antibiotics. Depending on the method, this process requires at least 10 days to 8 weeks before drug sensitivity results are available. During this time the infected patient may be treated incorrectly which may have serious health implications in particular in patients with HIV-TB coinfection. The disclosure of the genetic basis of resistance to anti-tuberculous agents has enabled development of new molecular tests to detect mutations associated with reduced susceptibility to antituberculous drugs [9, 10]. In order to detect and validate the drug resistance associated mutations, DNA sequencing is the most accurate among the molecular techniques. We used PCR fragment sequencing since molecular mechanisms explaining resistance to anti-tuberculous agents are not fully understood . It presents the advantage, over methods that use DNA probes, to detect unknown mutations. Recently the GeneXpert has been endorsed by the WHO for point of care testing . Drug sensitivity testing with this method is based on the detection of mutations in the core region of the rpoB gene, thus only RIF-resistance or MDR would be detected.
In this study, we set out to investigate the association of phenotypic resistance with genetic mutations in drug resistance TB isolates in Cameroon. The majority of the isolates in this study were from the Jamot Hospital (Central Region of Cameroon), the reference hospital for diagnostic and treatment of pulmonary diseases throughout the country. Therefore, the data obtained in this study can be considered to be representative of the make-up of resistance conferring mutations present in M. tuberculosis strains in this region.
A 158-bp fragment of the rpoB gene from codon 507 to 533 was amplified and sequenced to detect mutations in RIFR strains. Of the 7 phentotypically RIFR strains, mutations were found in the rifampicin resistant determining region (RRDR) for all the 7 isolates. These alterations affected the codons Ser531Thr (71.4%), His526Asp (14.3%) and Asp516Val (14.3%). The rpoB codons 531, 526, and 516 are the most frequently mutated codons worldwide, although variations in the relative frequencies of mutations in these codons have been described for M. tuberculosis isolates from different geographic locations. The most common site of nucleotide substitutions in RIFR isolates was codon 531. This finding was similar to those reported in Russia , the US , Tunisia  Ghana  and Germany . The codon 531 mutation was also reported as the most frequent (68%) in M. tuberculosis isolates of the LAM family in Cameroon . For codons 526 and 516 involved in RIFR, mutations in our strains occurred at equal frequencies than in strains from other geographical regions [31–33]. It has been shown that various substitutions in the same codon can lead to different level of resistance. Mutations at codon 516 of the rpoB gene can confer either low or high level resistance depending on the codon change . It has been reported that substitution of aspartate by tyrosine in codon 516 induced RIF-resistance of M. tuberculosis with minimum inhibitory concentration (MIC) between 15 μg/ml and 25 μg/ml in BACTEC 460-TB system . In our study, RIF susceptibility was evaluated in Lowenstein Jensen at a concentration of 50 μg/ml. This might explain why strains harbouring this mutation in our study were phenotypically RIF-susceptible. Among the 7 isolates which were altered genetically, 6 were MDR strains and one a RIF-SM-resistant isolate. Thus, rpoB could be an indicator of multidrug resistance among M. tuberculosis strains. This observation was previously reported among Cameroonian M. tuberculosis isolates .
It has been previously shown that about 10–15.9% of RIF -resistant isolates do not have mutations in the RRDR . More than 90% of RIF -resistant strains from other regions had mutations located in the 81-bp core region [35–38]. This indicated a possible occurrence of alteration outside the core region of 81 bp of the examined rpoB. Among other explanations, several additional genes might be involved in RIF-resistance such as rpoA, rpoC or rpoD. The natural resistance to RIF in some M. avium and M. intracellular strains is known to be a result of efficient cell wall permeability and exclusion barrier, suggesting that these elements could also play an important role in M. tuberculosis. However, in our study, all the isolates harboured mutations in the RRDR core region.
Common genes known to be involved in INH-resistance are katG, inhA, ahpC, oxyR. Several investigators have shown that INH-resistance in M. tuberculosis isolates arise principally from a katG gene alteration [40–42] that corresponds essentially to point mutations in codon 315 (point mutations in two bases 944 and 945). In this study, 18 (40.0%) INH -resistant isolates were genetically altered in the katG codon 315. Others studies have reported 95% of all INH-resistant isolates with mutations in codon 315 . Out of the 6 MDR strains identified in this study, 5 displayed a high level resistance to isoniazid with a katG alteration and the remaining one displayed a low level INH-resistance with -32G → A mutation in oxyR-ahpC intergenic region. Therefore, it will be useful to combine katG315 and -15 point mutation inhA promoter region with rpoB in molecular assays looking at drug resistance. Since some of the INHR strains in this study had no mutation in katG315 and -15 inhA promoter region, it is likely that mutations in other genes, such as the inhA locus, contribute to resistance. Previous studies have shown that mutations in the upstream region of the inhA locus result in increased levels of InhA (NADH-dependent enoyl-acyl carrier protein reductase) expression, thereby elevating the drug target levels and producing INH -resistance via a titration mechanism . We assessed for mutations in the inhA regulatory region of all the 44 INHR
M. tuberculosis strains and found a substitution at position 15 upstream of the start codon in 13 (28.9%) isolates. The frequency of the occurrence of specific INH-resistance conferring mutations varied between geographical regions in the world . A study in Equatorial Guinea reported the absence of mutation in the katG gene of M. tuberculosis INH -resistant isolates . The unique katG mutation observed in this study was the substitution of Serine to Threonine at codon 315. High proportion of Ser315katG mutations has been reported in Russia (76.9%) , in Morocco (68.6%) , in isolates of the LAM family in Cameroon  and also in Korea (49.1%) . In INHR strains, neither insertions nor complete deletions of katG were found, which is evidence of the rare occurrence of these mutations in clinical isolates, although they were reported previously by other authors [47, 48]. Fourteen (31.8%) INHR isolates did not show mutations at the four loci analyzed. This discrepancy between the phenotypic results and the genotypic drug resistance tests could be attributed to the presence of other mutations located either outside the selected target region or the selected genes. Several others studies have reported that mutations in inhA or its promoter region are usually associated with low-level resistance of INH. Moreover, INH-resistant isolates with inhA mutations can have additional mutations in katG, conferring higher levels of INH-resistance .
All mutations found in fabG1-inhA promoter region were not associated with phenotypic resistance. The substitution of G to C at position -47 first described by Homolka and al.  in an INH-resistant strain has been found in both susceptible (24/44; 54.5%) and resistant isolates (5/44; 11.4%). Thus, this mutation seems to not correlate with INH-resistance. The mutation -102 C → T not yet described is also not relevant to INH-resistance since it was found only in susceptible isolates.
The analysis of SM-resistance mechanism revealed that none of the SM-resistant strains carried a mutation in the rrs gene although those mutations have been described as main resistance mechanism that confer high level SM -resistance . Clinical isolates showing no mutations in rpsL or rrs gene have been reported in the literature . A previous investigation from Cameroon encountered rpsL or rrs mutations in SM-resistant isolates from the Central Region of Cameroon . In contrast in the current investigation only rpsL mutations were associated with SM-resistance. This indicates that further studies are necessary to delineate the molecular markers for SM-resistance. Mutations in the rpsL locus have been hypothesized to be an alternative mechanism of SM-resistance like mutations in the gidB or efflux pumps . Overall, we detected gidB mutations in 18.5% of SM-resistant isolates and 6% of SM susceptible isolates. Although the encountered mutations in resistant samples were not observed in susceptible isolates, their association with SM-resistance needs to be confirmed.
Three contiguous genes encoding arabinosyl transferases and designated embC, embA, and embB were analyzed in the present study. These 3 genes have been identified in M. tuberculosis. Previous studies based on limited sequencing region containing the embCAB genes have identified mutations that result in replacement of amino acid residues and are found only in EMB-resistant organisms cultured from humans . In this study, the embB analysis gene identified 1 of 2 resistant isolates with EMB-resistance-associated nucleotide substitutions in codon 306ATG → GTG that result in amino acid replacement (Met → Val). This is in accordance with others studies analyzing EMB-resistant clinical isolates of M. tuberculosis that identified embB amino acid-conferring mutations in approximately 50 to 70% isolates with resistance-associated polymorphisms . Certain variations affecting embA (330CTG → TTG) and embC (-20A → C and -230 A → C) appeared to be not associated with drug resistance. Given the low number of EMB-resistant isolates in our investigation further studies are needed to confirm these findings.