The results of this study demonstrate the utility of detection of mutations associated with drug resistance to rapidly and accurately determine the drug susceptibility of M. tuberculosis complex isolates. This study also demonstrates the advantages of DNA sequencing over methods that use DNA probes to detect deviation from the wild-type sequence or a limited set of specific mutations, since several specific mutations identified in this study were not associated with drug resistance. Each resistance-associated locus examined has its own unique properties which must be taken into consideration when the molecular drug sequencing results are interpreted.
The molecular mechanism of resistance to RIF is the most completely understood of the resistance mechanisms of all the drugs used in the treatment of tuberculosis, and it has been well established that mutations within the RRDR of rpoB
occur in 95% or more of RIF-resistant isolates. Within the RRDR, three specific mutations predominate (Ser531Leu, His526Tyr, and Asp516Val), presumably because these mutations minimally impact the fitness of the bacilli (16
). In this study, 140 of the 174 (80.5%) RIF-resistant isolates harbored one of these three rpoB
mutations. However, many other RRDR mutations have been described, including 25 within our study (see Table S3 in the supplemental material). Since these other mutations are far less frequently encountered, their association with RIF resistance has not been well established. Some of these rpoB
mutations, including Leu511Pro, Asp516Tyr, His526Leu/Ser, and Leu533Pro, are associated with discordant susceptibility test results (37
). In a previous study, 11 isolates harboring these mutations were classified RIF susceptible by either the Bactec 460 TB or Bactec 960 MGIT system, while only 1 was susceptible by the proportion method on Löwenstein-Jensen medium and only 2 were susceptible on Middlebrook 7H10 agar (37
). The authors proposed that these mutations could confer low-level but clinically relevant RIF resistance (37
). Others have considered isolates harboring the Leu533Pro substitution to be susceptible (17
). In this study, the His526Asn, His526Leu, and Leu533Pro mutations were identified in eight susceptible and eight resistant isolates. Four of the eight resistant isolates contained other resistance-associated rpoB
mutations. These data weakly support the association of these mutations with low-level resistance. However, further studies are required to definitively characterize their role in RIF resistance. The relative rarity of these mutations may be partially attributable to selection bias, as most studies have examined primarily or exclusively RIF-resistant isolates. With increased use of genotypic methods to diagnose RIF resistance, these mutations may be more frequently observed, and subsequently, their clinical and epidemiological significance may be better understood. The specificity of DNA sequencing as a diagnostic tool will increase, should future research establish that certain RRDR mutations do not impart a clinically relevant level of RIF resistance. The possibility that such mutations exist has important ramifications for methods which detect the presence of any mutation and not the mutation of interest.
The mode of action of isoniazid, though extensively investigated, remains incompletely understood. Several different loci are known to be involved in resistance, especially katG
. Mutations in codon 315 of katG
and the inhA
promoter are proven mechanisms of INH resistance. None of the INH-susceptible isolates in this study had a mutation in either katG
codon 315 or the inhA
promoter, making the test 100% specific. Nineteen of the INH-resistant isolates were wild type at both these locations and also in the region of the inhA
structural gene where resistance-associated mutations have been reported (data not shown) (3
). Although mutations in other loci, such as ndh
, may account for the INH-resistant phenotype in some of these isolates, we chose not to sequence these loci because there are very limited data on their role in INH resistance.
Until recently, the significance of mutations within the M. tuberculosis embB
gene, especially those at codon 306, had been controversial because such mutations had been reported in both EMB-susceptible and -resistant isolates (11
). Allelic exchange experiments have now convincingly shown that mutations at embB
codons 306, 406, and 497 confer EMB resistance (27
). In those earlier reports of EMB-susceptible isolates harboring embB
mutations, the drug susceptibility results were presumably inaccurate. Conventional culture-based methods of EMB susceptibility testing are notoriously problematic (14
). The allelic exchange studies demonstrated that the mutants with Met306Val and Met306Leu mutations had EMB MICs well above the critical concentration used for drug susceptibility testing, and indeed, we found that all of those isolates with only a Met306Val (n
= 53) or Met306Leu (n
= 3) substitution were EMB resistant. In contrast, MICs for the mutants with the Met306Ile mutation were only modestly higher than the critical concentration, a confounding circumstance that could result in false-susceptible EMB testing results. This expectation is supported by our finding that 5 of the 29 isolates possessing only a Met306Ile substitution were EMB susceptible.
The third most frequent embB
substitution in this study was Glu378Ala. This was the only embB
mutation in 22 isolates, of which 16 were EMB susceptible. This substitution was previously reported in a single isolate (26
) and in combination with an Asp299Glu substitution in two isolates (31
), and in both reports, the isolates were characterized to be EMB resistant. The probable explanation for the paucity of reports of this mutation is that prior studies have examined primarily EMB-resistant strains. In this study, we examined a large number of EMB-susceptible and pansusceptible isolates to more accurately assess the drug susceptibility phenotype of the mutations identified. Of the 22 isolates, 18 were determined to belong to the IndoOceanic lineage by spoligotyping. Therefore, our data indicate that the mutation resulting in the Glu378Ala substitution is a naturally occurring polymorphism and possible lineage marker that does not confer EMB resistance. Functional genetic analysis of this mutation is needed to definitively validate this conclusion.
We identified 20 different embB mutations or combination of mutations, including the previously discussed Glu378Ala mutation. This diversity of mutations gives DNA sequencing a definite advantage over hybridization-based methods, particularly given that a putative polymorphism occurs within the ERDR. Although further functional genetic data are needed to better define the phenotype of additional embB mutations, complementing inherently problematic culture-based DST with DNA sequencing would substantially improve diagnostic accuracy.
The presence of a mutation within pncA
has been shown to correlate well with PZA resistance (22
), and a diverse and widely distributed array of pncA
mutations has been reported (29
). We also found a diverse group of pncA
mutations that correlated well with PZA resistance. Conventional DST of PZA is challenging and problematic due to the poor growth of M. tuberculosis
under the acidic conditions (pH 5.5 to 6.0) required for optimal drug activity (13
). The good correlation between the presence of a pncA
mutation and PZA resistance makes DNA sequencing of this gene a very useful adjunctive diagnostic test. Unfortunately, the tremendous diversity of reported pncA
mutations and the lack of any predominant mutations represent a substantial limitation. Without strong statistical evidence linking specific mutations with PZA resistance, further experimental evidence is needed to determine which pncA
mutations confer resistance. Mindful of these limitations, the use of pncA
sequencing in conjunction with phenotypic PZA testing could aid in determining PZA susceptibility. Additional studies to characterize the phenotype of specific pncA
mutations through the determination of MIC values or functional genetics would greatly enhance the diagnostic value of pncA
The FQs are known to be effective against M. tuberculosis
by targeting and inhibiting the essential bacterial enzyme DNA gyrase (44
). It has been well established that mutations within gyrA
, which encodes subunit A of the DNA gyrase, are often associated with resistance (33
). The most common resistance-associated mutations within gyrA
are found within the QRDR at codons 94 and 90, as was the case with our study (35
). In addition to the known mutations, we discovered the novel Asp89Asn and Asp89Gly substitutions. Because these mutations have not been previously reported, their association with resistance is unknown. Lastly, 18 FQ-resistant isolates did not harbor any mutations within the gyrA
locus. These isolates could be explained by mutations within the gyrB
locus (unexamined in this study) or by unknown mechanisms of FQ resistance.
The FQ-susceptible isolates harbored primarily wild-type gyrA
loci, with few exceptions. One isolate contained the resistance-associated Ala90Val mutation, and repeat sequencing confirmed the discordant result. However, repeat DST was unable to be completed due to the lack of subsequent growth from the original isolate, leaving the possibility of irreproducible phenotypic results. In addition, four FQ-susceptible isolates harbored the Thr80Ala mutation, which has been reported as providing a strain with increased susceptibility, and are therefore not discordant (2
Because resistance to one of the second-line injectable drugs is part of the definition of XDR TB, the establishment of rapid assays for detecting resistance to these drugs is of particularly high importance. All of these drugs are known to target the bacterial ribosome, and mutations within the 16S rRNA, encoded by rrs
, are well established as being associated with second-line drug resistance. Most of the resistance-associated mutations within rrs
are found within a 516-bp region of the gene. In this study, rrs
mutations associated with AMK-resistant isolates included a large number of expected A1401G mutations as well as a resistance-associated G1484T mutation. Both of these mutations have been previously described as playing a central role in AMK resistance (18
). In addition to the established mutations, we also discovered a previously unreported T1322C and A1401G combination of mutations in an isolate resistant to AMK, KAN, and CAP. Since the T1322C mutation has not been previously identified, its role in aminoglycoside resistance is unclear, both as a lone mutation and in combination with the A1401G substitution. However, its presence in an isolate that is 100% resistant to both KAN and AMK suggests that it is associated with aminoglycoside resistance. We did not detect any resistance-associated mutations in the vast majority of AMK-susceptible isolates; however, one contained the C1402T mutation for AMK susceptibility and two harbored novel A1196G SNPs (18
). Because the substitution was found only in susceptible isolates, it is unlikely that it is associated with AMK resistance.
Alterations within the rrs
locus also play a significant role in KAN resistance, with the 65 detected RRDR mutations accounting for 60% of substitutions within KAN-resistant isolates (1
). The detected A1401G, C1402T, and G1484T mutations have all been well established as resistance-associated substitutions, leaving the novel A1401G and T1322C mutation combination to be the only allele with unknown implications (18
). Two study isolates that were both AMK and KAN susceptible harbored previously unknown A1196G mutations, suggesting that these substitutions do not confer resistance.
In addition to rrs
, mutations within the promoter region of eis
, which encodes an aminoglycoside acetyltransferase, are associated with low-level KAN resistance (45
). In our survey, we identified 38 eis
mutations, including 3 previously unreported substitutions. The most common previously reported resistance-associated eis
mutations included the G(−10)A, C(−14)T, and G(−37)T polymorphisms (45
). Our study supports these findings, as they were also the most prominent in our KAN-resistant isolates (see Table S9 in the supplemental material). Also in agreement with the previous study was our finding of the C(−12)T mutation in a KAN-resistant isolate and three KAN-susceptible isolates (see Table S9 in the supplemental material). These data support the proposed hypothesis that this mutation may result in an MIC value close to the recommended critical concentration for testing KAN susceptibility (45
). As a result, the C(−12)T mutation is considered a polymorphism with little predictive value for KAN resistance. It is also of note that we identified one G(−10)A mutation in a KAN-susceptible isolate from the 2008 WHO proficiency testing panel. Because WHO isolates were not subjected to repeat testing, this result remains unconfirmed and may be explained by undetected low-level KAN resistance. Lastly, our detection of the Ser48Ser and Met100Thr mutation combination within the open reading frame of eis
is novel, and the potential effects of open reading frame mutations on KAN resistance are unknown. However, eis
-mediated resistance is due to promoter mutations that alter eis
expression levels, and the impact of open reading frame mutations on KAN resistance would potentially require a separate mechanism.
Although the combination of mutations within rrs and eis resulted in a relatively high specificity (86.5%), 15 KAN-resistant isolates did not harbor mutations in either gene. Two of these 15 isolates were determined to be KAN susceptible upon repeat drug susceptibility testing, suggesting that they may display a level of resistance close to the current testing concentration. Two of the wild-type isolates were duplicates of each other from the 2008 WHO panel, thereby representing one isolate whose DST result was not confirmed with repeat testing. In addition, two isolates were susceptible to 5 μg/ml KAN with subsequent repeat testing and were therefore no longer considered discordant. The nine remaining isolates were confirmed to be discordant by repeat testing and exhibited a range of resistance levels, suggesting that there remains at least one unknown mechanism for KAN resistance (data not shown).
Similar to KAN, resistance to the macrocyclic polypeptide CAP is associated with mutations within two separate loci: rrs
). Polymorphisms within the rrs
locus affect CAP binding to the ribosome, while those within tlyA
prevent necessary methylation of rRNA, rendering their ribosomes CAP resistant (19
). Common rrs
mutations associated with CAP resistance include the prominent A1401G mutation and the C1402T and G1484T mutations. Our study detected all of these polymorphisms as well as two novel A1196G mutations, whose role in aminoglycoside resistance is currently unknown. Previous studies have often demonstrated a strong correlation between the A1401G mutation and CAP resistance (10
). However, other reports have found the same mutation in isolates determined to be CAP susceptible (5
). In this study, we detected a large number of A1401G mutations in both CAP-resistant and CAP-susceptible isolates (see Table S8 in the supplemental material). The mutations were verified by repeat DNA sequencing; however, the drug susceptibility testing result was found to vary widely upon repeat testing. In fact, select isolates were retested up to five times, with different results obtained for each testing cycle (data not shown). These data suggest that the CAP MIC value for isolates harboring the A1401G mutation may be very close to the current recommended critical concentration for testing CAP susceptibility on 7H10 agar. Previous studies may not have detected this variation due to relatively smaller study sizes, limited numbers of susceptible isolates, or variation in CAP drug susceptibility testing methods.
Mutations within tlyA
are uncommon, possibly due to limited and local usage of CAP to treat M. tuberculosis
). Polymorphisms within tlyA
in our study were less common than those within rrs
, with the total number accounting for 13% of study mutations associated with CAP resistance. The addition of tlyA
mutation data to rrs
mutation data in our study increased our sensitivity and specificity for CAP resistance from 55.1% and 88.6% to 60.9% and 87.3%, suggesting that tlyA
currently plays a minor role in the detection of CAP resistance and could be used sparingly as a resistance marker, as long as usage of the drug remains low.
Cross-resistance between the second-line injectables has been well documented and is further supported by our study data (5
). As expected, the ribosomal rrs
mutation A1401G was the most common polymorphism associated with cross-resistance between KAN, AMK, and CAP, as well as KAN and AMK (see Tables S8 and S11 in the supplemental material). One isolate, resistant to both KAN and AMK, harbored an A14041G substitution in combination with a T1322C mutation (see Tables S8 and S11 in the supplemental material). Because the latter polymorphism has not been previously described and resistance to both drugs can be explained by the rrs
substitution, association of this novel mutation with resistance to either drug is unclear. It is worth noting that none of the isolates were resistant only to AMK, suggesting that detection of AMK resistance may be a greater indication of second-line cross-resistance. Lastly, although there was a high level of agreement between our genotypic and phenotypic data for injectable cross-resistance, some isolates were wild type for all resistance-associated alleles (see Table S11 in the supplemental material). This result suggests that there is still at least one mechanism of second-line injectable drug cross-resistance that has yet to be discovered.
There is a current need for the development of rapid molecular tests that detect mutations associated with drug resistance in strains of M. tuberculosis. Conventional culture-based techniques are technically demanding and have long turnaround times. Molecular techniques, such as the Sanger-based DNA sequencing method presented here, provide specific and prompt strain resistance data that aid in the development of appropriate antituberculosis treatment regimens. The development of genetic assays requires the verification of molecular data with respect to conventional drug susceptibility data. Here we completed a comprehensive survey of nine loci known to harbor mutations associated with resistance to both first-line and second-line anti-TB drugs. The resultant genotypic data set was compared with drug susceptibility data to determine sensitivity and specificity values for each locus (). These values served to verify the new CDC MDDR service and have the potential to aid in the development of other, in-house molecular assays. Developed genetic tests will inevitably produce more rapid results for drug-resistant isolates, which will lead to faster identification of MDR and XDR strains, more tailored treatment regimens, and a reduction in the transmission of TB.