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 Table of Contents  
ARTICLE
Year : 2016  |  Volume : 5  |  Issue : 1  |  Page : 1-6

Correlating rrs and eis promoter mutations in clinical isolates of Mycobacterium tuberculosis with phenotypic susceptibility levels to the second-line injectables


1 Microbiology Section, Department of Laboratory Medicine, P. D. Hinduja Hospital & Medical Research Centre, Mumbai, India
2 Pulmonology Section, Department of Medicine, P. D. Hinduja Hospital & Medical Research Centre, Mumbai, India
3 Department of Medicine, University of California, San Diego, San Diego, CA, United States

Date of Web Publication8-Feb-2017

Correspondence Address:
Camilla Rodrigues
Microbiology Section, Department of Laboratory Medicine, P. D. Hinduja Hospital & Medical Research Centre, Mumbai
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.1016/j.ijmyco.2015.09.001

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  Abstract 

Objective/background: The in vitro drug-susceptibility testing of Mycobacterium tuberculosis reports isolates as resistant or susceptible on the basis of single critical concentrations. It is evident that drug resistance in M. tuberculosis is quite heterogeneous, and involves low level, moderate level, and high level of drug-resistant phenotypes. Thus, the aim of our study was to correlate rrs (X52917) and eis (AF144099) promoter mutations, found in M. tuberculosis isolates, with corresponding minimum inhibitory concentrations of amikacin, kanamycin, and capreomycin.
Methods: Ninety M. tuberculosis clinical isolates were analyzed in this study. The minimum inhibitory concentrations were determined by MGIT 960 for 59 isolates with resistance-associated mutations in the rrs and eis promoter gene regions, and 31 isolates with wild-type sequences, as determined by the GenoType MTBDRsl (version 1) assay.
Results: The rrs A1401G mutation was identified in 48 isolates resistant to the second-line injectables. The eis promoter mutations C-14T (n=3), G-10C (n=3), G-10A (n=3), and C-12T (n=2) were found within 11 isolates with various resistance profiles to the second-line injectables. Thirty-one isolates had wild-type sequences for the rrs and eis promoter gene regions of interest, one of which was amikacin, kanamycin, and capreomycin resistant. The isolates with the rrs A1401G mutation had amikacin, kanamycin, and capreomycin minimum inhibitory concentrations of >40 mg/L, >20 mg/L, and 5–15 mg/L, respectively. The isolates with eis promoter mutations had amikacin, kanamycin, and capreomycin minimum inhibitory concentrations of 0.25–1.0 mg/L, 0.625–10 mg/L, and 0.625–2.5 mg/L, respectively.
Conclusion: This study provides a preliminary basis for the prediction of phenotypic-resistance levels to the second-line injectables based upon the presence of genetic mutations associated with amikacin, kanamycin, and capreomycin resistance. The results suggest that isolates with eis promoter mutations have consistently lower resistance levels to amikacin, kanamycin, and capreomycin than isolates with the rrs A1401G mutation.

Keywords: Drug-susceptibility testing, Extremely drug-resistant tuberculosis, Level of resistance, Mutations


How to cite this article:
Kambli P, Ajbani K, Nikam C, Sadani M, Shetty A, Udwadia Z, Georghiou SB, Rodwell TC, Catanzaro A, Rodrigues C. Correlating rrs and eis promoter mutations in clinical isolates of Mycobacterium tuberculosis with phenotypic susceptibility levels to the second-line injectables. Int J Mycobacteriol 2016;5:1-6

How to cite this URL:
Kambli P, Ajbani K, Nikam C, Sadani M, Shetty A, Udwadia Z, Georghiou SB, Rodwell TC, Catanzaro A, Rodrigues C. Correlating rrs and eis promoter mutations in clinical isolates of Mycobacterium tuberculosis with phenotypic susceptibility levels to the second-line injectables. Int J Mycobacteriol [serial online] 2016 [cited 2020 Jun 1];5:1-6. Available from: http://www.ijmyco.org/text.asp?2016/5/1/1/199734


  Introduction Top


Drug-resistant tuberculosis (TB) poses a major threat to TB control efforts worldwide. There is an increasing global incidence of multidrug-resistant TB (MDR-TB) and extremely drug-resistant TB (XDR-TB), characterized by resistance to isoniazid, rifampin, a fluoroquinolone, and at least one of three second-line injectable (SLI) drugs: amikacin (AMK), kanamycin (KAN), and/or capreomycin (CAP). The effective treatment of MDR-TB, as well as the prevention of XDR-TB, is reliant upon the appropriate use of these SLIs. In order to establish appropriate treatment regimens, and to prevent the amplification of SLI resistance, it is critical that MDR-TB patients have full drug-susceptibility profiles established for their particular Mycobacterium tuberculosis infections prior to treatment [1].

The conventional diagnosis of drug-resistant M. tuberculosis relies upon the slow growth of mycobacterium cultures, and can take anywhere from several weeks to months to generate results [2]. While methods for the conventional diagnosis of MDR-TB are well established and generally effective, the culture-based detection of SLI resistance is more complicated and results are less reliable [3]. As such, rapid molecular diagnostic tests, detecting genetic mutations associated with drug resistance, are essential to the future diagnosis and management of M/XDR-TB.

A high level of cross-resistance has been observed between the aminoglycosides AMK and KAN [4],[5]. CAP, a cyclic polypeptide, can be used as a substitute, although it is structurally dissimilar to the aminoglycosides [4],[6]. For patients with MDR-TB, if CAP resistance additionally occurs, an increased incidence of treatment failure and patient mortality is observed [7]. Furthermore, studies conducted in clinical isolates and laboratory-generated M. tuberculosis mutants have noted the additional occurrence of cross-resistance between AMK/KAN and CAP, limiting the treatment options for these infections [4],[5],[8]. SLI resistance is mainly conveyed by mutations in the rrs and eis promoter gene regions of M. tuberculosis isolates. AMK/KAN and CAP are known to effect protein synthesis in M. tuberculosis, and resistance to these compounds is primarily conveyed by changes in the 16S ribosomal RNA (encoded by the rrs gene) [4],[5],[9]. A high-level AMK/KAN resistance and a lower-level CAP resistance can be caused by mutation in rrs A1401G [4]. The transfer of an acetyl group from acetyl coenzyme A to an amine group of aminoglycoside is catalyzed by eis (aminoglycoside acetyltransferase); a multiacetylation capability at the 2’-, 3-, or 6’ positions of aminoglycoside antibiotics is shown by the eis of M. tuberculosis [10]. Although these resistance mechanisms have been well defined, it is critical to additionally correlate specific genetic mutations with levels of SLI resistance in order to guide M/XDR-TB treatment regimens and improve patient outcomes. Herein, we establish the minimum inhibitory concentrations (MICs) for 90 clinical M. tuberculosis isolates with specific rrs and eis promoter mutations.


  Materials and methods Top


Setting and ethical approval

A written consent was waived for all participants, as the study was carried out on 90 archived isolates for which pyrosequencing (PSQ), GenoType MTBDRsl assay (version 1) and MGIT 960 drug-susceptibility testing (DST) (utilizing the World Health Organization-approved critical concentrations), was performed previously. Sample collection, MGIT culture, GenoType MTBDRsl assay, and PSQ were performed at the Mycobacteriology Laboratory of the P. D. Hinduja Hospital & Medical Research Centre, a tertiary-care hospital in Mumbai, India with a referral bias toward TB treatment nonresponders. This study was approved by the Institutional Review Board of P. D. Hinduja Hospital & Medical Research Centre.

Phenotypic MGIT DST

We performed a standardized DST to KAN using the BACTEC MGIT 960 System (Becton, Dickinson and Company Diagnostic Systems, Sparks, MD, USA) with the EpiCenter software. The manufacturer's protocol was strictly followed for preparation of the inocula, inoculation, and incubation. For KAN, a break-point concentration of 2.5 mg/L was utilized based upon previous findings [11]. The critical concentrations of AMK and CAP were 1.0 mg/L and 2.5 mg/L, respectively. The stock solutions of AMK, KAN, and CAP (Sigma–Aldrich, St. Louis, Missouri, USA) were prepared by dissolving the compounds in distilled water. The drugs were filtered, further diluted with distilled water, and stored at −80°C for up to 6 months. A genotypically confirmed resistant strain and a pan-susceptible strain, H37Rv, were tested weekly by the MGIT 960 DST as a quality control.

Quantitative DST

Six concentrations of KAN and seven concentrations of AMK and CAP were used to establish the MICs for the M. tuberculosis isolates included in this study. Two concentrations below the critical concentration (0.25 mg/L and 0.5 mg/L) and three concentrations above the critical concentration (4.0 mg/L, 20.0 mg/L, and 40.0 mg/L) were used to establish the AMK MICs; two concentrations below the critical concentration (0.625 mg/L and 1.25 mg/L) and three concentrations above the critical concentration (5.0 mg/L, 10.0 mg/L, and 20.0 mg/L) were used for KAN; and two concentrations below the critical concentration (0.625 mg/L and 1.25 mg/L) and four concentrations above the critical concentration (5.0 mg/L, 10.0 mg/L, 15.0 mg/L, and 30.0 mg/L) were used for CAP.

GenoType MTBDRsl assay

The procedure for the GenoType MTBDRsl assay was as follows:

  1. DNA extraction: DNA was extracted from all decontaminated patient samples using a GenoLyse kit (Hain Lifescience, Nehren, Germany).
  2. Polymerase chain reaction (PCR): Multiplex PCR amplification was conducted for the rrs gene of interest utilizing biotinylated primers. PCR was performed with the following cycling conditions: initial denaturation 95°C/15 min; (denaturation 95°C/30s, annealing 58°C/2 min [10 cycles]); (denaturation 95°C/25s, annealing 53°C/40s, extension 70°C/40s [30 cycles]); and final extension 70°C/8 min.
  3. Hybridization: Reverse hybridization/GenoType MTBDRsl assay was performed as per manufacturer's instructions (Hain Lifescience) [12].
  4. Analysis: The GenoType MTBDRsl assay evaluates the rrs gene for mutations at positions 1401/1402 and 1484. The absence of the test wild-type (WT) marker, WT1, and the presence of the mutation (MUT) probe, MUT1, correspond to the A1401G mutation, while the absence of the test WT2 marker and the presence of the probe MUT2 correspond to the G1484T mutation.


PSQ

A total of 20 M. tuberculosis isolates were additionally rrs sequence confirmed and evaluated for the presence of the eis promoter mutations by PSQ. Reagents from the HotStarTaq kit and deoxynucleotide-triphosphate mixtures (Qiagen, Valencia, CA, USA) were used in the PCR master mix. PSQ was performed as follows:

  1. DNA extraction: The PSQ assay utilized crude DNA, extracted by a simple heating procedure (95°C, 25 min).
  2. PCR solutions: Each PCR reaction contained 2.5 μL of extracted isolate DNA and 22.5 μL of the PCR master mix (1× PCR buffer, 2.5 mM MgCl2, 0.96 mM deoxynucleotide-triphosphate mixture, 1× Q-Solution, 0.5 μM rrs or eis promoter primers, and 1 U of HotStarTaq).
  3. PCR steps: The initial activation was done with HotstarTaq polymerase at 95 °C for 15 min, followed by 50 cycles of amplification (94 °C for 15s, 60 °C for 30s, and 72 °C for 20s), and a final extension at 72 °C for 5 min.
  4. PSQ reaction: PSQ was conducted for the −6 to −47 regions of the eis promoter, and the 1401/1402 region of the rrs gene. PSQ was performed with PyroMark Q96 reagents, utilizing the sequence-analysis mode of the PyroMark Q96 ID system (Qiagen) [13].



  Results Top


Phenotypic DST and MIC results

Sixty phenotypically XDR-TB strains were included in this study. Eleven (found to have eis promoter mutations) were resistant to KAN at the critical concentration, but sensitive to AMK and CAP. Forty-eight isolates (determined to have the rrs A1401G mutation) were found to be resistant to KAN, AMK, and CAP. One isolate (with WT sequences for both the rrs and eis promoter) was also resistant to KAN, AMK, and CAP at the critical concentration. Thirty isolates were pan susceptible to all drugs tested. The MICs of AMK, KAN, and CAP for all isolates are shown in [Table 1].
Table 1: Mutations found within Mycobacterium tuberculosis clinical isolates and their associated minimum inhibitory concentrations against the second-line injectables.

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Genotypic results

The agreement between the GenoType MTBDRsl assay results (based solely upon the presence of resistance-associated rrs mutations) and the phenotypic DST was 97% for AMK, 96% for KAN, and 86% for CAP. The sequencing and DST results for the 20 isolates subjected to PSQ are summarized in [Table 2]. One isolate, found to be phenotypically resistant to the three SLIs, had rrs and eis promoter WT sequences by both the GenoType MTBDRsl and PSQ assays.
Table 2: Pyrosequencing results for 20 Mycobacterium tuberculosis clinical isolates with wild-type MTBDRsl assay results.

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Correlation of KAN MICs with resistance-associated mutations

The isolates harboring the rrs A1401G mutation had AMK, KAN, and CAP MICs of >40 mg/L, >20 mg/L, and 5–15 mg/L, respectively, whereas the isolates with eis promoter mutations were found to have AMK, KAN, and CAP MICs ranging from 0.25 mg/L to 1.0 mg/L, 5.0 mg/L to 10 mg/L, and 0.625 mg/L to 2.5 mg/L, respectively ([Table 1]). As such, the rrs A1401G mutation correlated with high-level AMK and KAN resistance, and moderate-level CAP resistance. The eis promoter mutations correlated with low-level AMK and CAP resistance, and moderate-level KAN resistance.


  Discussion Top


This study correlates specific rrs and eis promoter mutations identified in M. tuberculosis clinical isolates with AMK, KAN, and CAP phenotypic-resistance levels. Overall, the MIC ranges for M. tuberculosis isolates with WT rrs and eis gene sequences were different from the MIC ranges for the isolates harboring resistance-associated mutations in these gene regions. The rrs and eis promoter mutations associated with SLI resistance were found in 59 of the 90 M. tuberculosis isolates evaluated in this study. These two genes, alone, appeared to be highly sensitive markers for SLI resistance, as only one of the 60 SLI-resistant isolates evaluated in the study was determined to have WT sequences for the gene regions evaluated. This isolate may have had SLI resistance-associated mutations in other genes, such as tlyA and gidB, that were not assessed in this study. Overall, our results provide a preliminary basis for the prediction of SLI phenotypic-resistance levels based upon the presence or absence of specific resistance-associated mutations in both the rrs and eis promoter gene regions of M. tuberculosis clinical isolates.

The peak serum concentrations for KAN, AMK, and CAP have been previously reported at 22.5 mg/L, 20–40 mg/L, and 10–30 mg/L, respectively [14],[15]. Taking these concentrations into account, M. tuberculosis resistance levels to each drug may be categorized as low, moderate, or high. As noted in [Table 1], the rrs A1401G mutation appears to consistently correspond to a high level of AMK and KAN resistance (MICs >40 mg/L, >20 mg/L). The KAN MICs for isolates with the A1401G mutation have been previously documented to be as high as >80 mg/L, and clinical studies have confirmed that this particular rrs mutation confers KAN resistance even at these high concentrations, with additional cross-resistance to AMK with MICs of >64 mg/L [5] and, to a lesser extent, CAP with MICs of 5–10 mg/L [16]. Conversely, isolates with eis promoter mutations have been documented to be resistant to KAN, but susceptible to AMK and CAP at their respective break points [4],[16],[17],[18]. Our study identified 11 isolates with eis promoter mutations. These isolates had low- to moderate-level KAN resistance, and a low level of resistance to AMK and CAP. These eis promoter mutations, C-14T, G-10C, G-10A, and C-12T, have been shown to result in a significant increase in eis transcript levels, as well as in a corresponding increase in the expression of an enzyme that acetylates KAN, inactivating the drug [18],[19].

Importantly, although the Eis enzyme was found to have some cross-reactivity with AMK, the activity was limited, and eis mutants are found to be largely susceptible to this other aminoglycoside, as reflected by current testing and treatment guidelines [17],[18]. As Eis utilizes KAN as a substrate up to three times more efficiently than AMK, the noted lack of cross-resistance is unsurprising. This preferential substrate utilization by Eis is likely a result of aminoglycoside structural differences; AMK sterically delays acetylation, as it contains an l-hydroxyaminobuteroyl amide group substitution in the N1 position of the deoxystreptamine ring [18]. It is notable that, even though rrs mutations confer cross-resistance to KAN and AMK, KAN-resistant isolates with eis promoter mutations may still be sensitive to AMK, and thus, both KAN and AMK should be considered individually when determining phenotypic drug-susceptibility profiles [17]. Indeed, the eis promoter mutations were found only within AMK- and CAP-susceptible isolates in our study, suggesting that these drugs could potentially be used to treat strains housing eis promoter mutations.

Our observation that different eis promoter mutations correspond to varying levels of phenotypic resistance to KAN, AMK, and CAP is also notable. The eis promoter mutations C-14T and G-10A corresponded to low- or moderate-level resistance to KAN, with KAN MICs generally ranging from 2.5 μg/mL to 10 μg/mL, and MICs corresponding with low-level resistance to AMK and CAP (0.25–1.0 mg/L and 0.625–2.5 mg/L), comparable with values reported by previous studies [16],[17],[18]. The association of the C-12T mutation with KAN resistance, however, has been a point of contention. In one study, C-12T mutants were shown to have slightly increased KAN MICs, although they retained sensitivity to KAN and AMK [18]. Another study found isolates with the C-12T mutation to have KAN resistance at an MIC of 5.0 mg/L, although the CAP MICs were <2.5 mg/L [16]. Overall, all studies found aminoglycoside MICs to be lower for eis promoter mutants compared to rrs mutants [10],[16],[18]. This supports our characterization of eis promoter mutants as “low- to moderate-level” KAN resistant with MICs ranging from 0.625 mg/L to 10 mg/L, “low-level” AMK resistant with MICs from 0.25 mg/L to 1.0 mg/L, and “low-level” CAP resistant with MICs from 0.625 mg/L to 2.5 mg/L. However, the overlap seen for KAN MIC ranges for the various mutants (i.e., MICs for -14T mutants were not very different than -10A or -12T mutants) suggests the need for further studies to clearly define associated phenotypic-resistance levels.

The sequencing of additional gene regions not evaluated in this study, such as gidB and tlyA, might additionally contribute to our understanding of the genetic basis of the observed SLI phenotypic-resistance levels of M. tuberculosis clinical isolates. These initial results, however, confirm that specific mutations in the rrs and eis promoter are promising markers for improved diagnostic assay sensitivity and specificity for the detection of injectable-drug resistance. Furthermore, our findings suggest that each drug should be considered independently when considering the results of molecular diagnostic assays, in order to determine the most effective treatment regimens for M/XDR-TB cases.


  Conclusion Top


These results suggest that the various mutations associated with SLI resistance may be correlated with specific SLI phenotypic-resistance levels. The rrs canonical mutation, A1401G, was found to confer a high level of AMK and KAN resistance to M. tuberculosis isolates, but a moderate level of CAP resistance. The eis promoter mutations were generally determined to confer lower levels of AMK and CAP resistance, and low to moderate levels of KAN resistance. Thus, together, the rrs and eis promoter mutations appear to have utility as molecular markers for aminoglycoside-resistance levels, directing optimal treatment regimens for M/XDR-TB cases.

Conflicts of interest

The authors declared no conflicts of interest.

Acknowledgments

The authors are thankful to the National Health and Education Society, P. D. Hinduja Hospital & Medical Research Centre. The work was supported by the National Health and Education Society of the P. D. Hinduja Hospital & Medical Research Centre. Antonino Catanzaro, Timothy C. Rodwell and Sophia B. Georghiou were funded by the National Institute of Allergy and Infectious Diseases grants U01-AI082229 and R01-A111435-04.



 
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    Tables

  [Table 1], [Table 2]


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14 Impact of gyrB and eis Mutations in Improving Detection of Second-Line-Drug Resistance among Mycobacterium tuberculosis Isolates from Georgia
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Antimicrobial Agents and Chemotherapy. 2017; 61(9)
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15 Amikacin: Uses, Resistance, and Prospects for Inhibition
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16 Antimicrobial resistance in Mycobacterium tuberculosis: mechanistic and evolutionary perspectives
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17 Evaluation of the GenoType MTBDR sl Version 2.0 Assay for Second-Line Drug Resistance Detection of Mycobacterium tuberculosis Isolates in South Africa
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18 Increased Tuberculosis Patient Mortality Associated with Mycobacterium tuberculosis Mutations Conferring Resistance to Second-Line Antituberculous Drugs
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