Mutations in the rrs A1401G Gene and Phenotypic Resistance to Amikacin and Capreomycin in Mycobacterium tuberculosis

Abstract

The aminoglycosides amikacin (AMK)/kanamycin (KAN) and the cyclic polypeptide capreomycin (CAP) are important injectable drugs in the treatment of multidrug-resistant tuberculosis. Cross-resistance among these drug classes occurs and information on the minimum inhibitory concentrations (MICs), above the normal wild-type distribution, may be useful in identifying isolates that are still accessible to drug treatment. Isolates from the Eastern Cape Province of South Africa were subjected to DNA sequencing of the rrs (1400–1500 region) and tlyA genes. Sequencing data were compared with (i) conventional susceptibility testing at standard critical concentrations (CCs) on Middlebrook 7H11 agar and (ii) MGIT 960-based MIC determinations to assess the presence of AMK- and CAP-resistant mutants. Isolates with an rrs A1401G mutation showed high-level resistance to AMK (>20 mg/L) and decreased phenotypic susceptibility to CAP (MICs 10–15 mg/L). The MICs of CAP were below the bioavailability of the drug, which suggests that it may still be effective against multi- or extensively drug resistant tuberculosis [M(X)DR-TB]. Agar-based CC testing was found to be unreliable for resistance recognition of CAP in particular.

Introduction

Therapeutic options in the treatment of pulmonary tuberculosis (TB) are limited and this is a major concern in view of the increasing incidence of drug-resistant Mycobacterium tuberculosis. First-line drugs, especially isoniazid (INH) and rifampicin (RMP), are the most effective and best tolerated amongst the antituberculosis drugs.^3,23 Resistance to at least INH and RMP indicates that multidrug-resistant tuberculosis (MDR-TB) has emerged.³ Treatment of MDR-TB requires prolonged administration of expensive and less-potent second-line drugs, which are often poorly tolerated.^3,23,24 MDR-TB patients are thus at an increased risk of acquiring additional resistance, which will eventually give rise to extensively drug resistant (XDR)-TB.⁶ XDR-TB has independently emerged worldwide and is defined as MDR-TB that has acquired additional resistance to any fluoroquinolone (FQN) such as ofloxacin (OFX) and also at least one of the three injectable second-line drugs: amikacin (AMK) kanamycin (KAN), or capreomycin (CAP).^3,6

The standard MDR-TB treatment regimen used in South Africa is based on the recommended guidelines of the World Health Organization (WHO) and includes the following drugs: KAN or AMK (injectable), OFX, terizidone, ethionamide, and pyrazinamide.^23,24 The FQNs and the injectables (aminoglycosides and polypeptides) are the only bactericidal agents among the second-line drugs.^8,24 These drugs are therefore essential in the treatment of MDR-TB, because they have an equal role to play as INH and RMP among the first-line drugs.^14,24 AMK and KAN are aminoglycosides that have a high level of cross-resistance between them.^1,10,12 The cyclic polypeptide CAP is structurally unrelated to the aminoglycosides and thus is a potential candidate to replace AMK or KAN if resistance to either of them is suspected.^2,9,24 Evidence that CAP is bactericidal against nonreplicating M. tuberculosis has renewed interest in this drug, despite its limitations because of renal and auditory toxicities.⁸ It has been also demonstrated that the risk of treatment failure and mortality increase when CAP resistance emerges among MDR-TB cases.¹⁴ However, cross resistance in M. tuberculosis between AMK/KAN and CAP has been observed in both clinical isolates and laboratory-generated mutants.^4,10,12,22 AMK/KAN and CAP primarily affect protein synthesis in M. tuberculosis and resistance to these drugs is associated with changes in the 16S rRNA (rrs).^{1,9,12,13,16,22} The rrs mutation A1401G causes high-level AMK/KAN and low-level CAP resistance. C1402T is associated with CAP resistance (also viomycin) and low-level KAN resistance. G1484T is linked to high-level AMK/KAN and CAP resistance (including viomycin).^{4,10,12,13,19} Various single-nucleotide polymorphisms (SNPs) in the tlyA gene have been also associated with CAP resistance.¹⁷ The tlyA gene encodes a putative rRNA methyltransferase and mutations that inactivate this gene in M. tuberculosis result in CAP and viomycin resistance.^9,13

The Eastern Cape Province is a region in South Africa where the incidence of drug-resistant TB is high. The purpose of this study was to investigate a subset of M(X)DR-TB clinical isolates of M. tuberculosis from this region to (i) correlate the phenotypic susceptibility levels of AMK and CAP with the molecular mechanisms that cause drug resistance and (ii) consider possible applications of our findings in clinical studies, particularly in regions with a high incidence of M(X)DR-TB.

Materials and Methods

Clinical isolates

Between June 2008 and November 2009, 310 M(X)DR TB isolates of M. tuberculosis, collected by the National Health Laboratory Services (NHLS) in the Eastern Cape of South Africa, were sent to Stellenbosch University for genotyping. The isolates were initially cultured by the NHLS in Port Elizabeth and subjected to routine drug susceptibility testing (DST) of first- and second-line drugs. AMK was tested at a critical concentration (CC) of 4.0 mg/L⁷ and CAP at 10 mg/L²⁴ on Middlebrook 7H11 agar. A subset of 50 M(X)DR-TB isolates was selected from the above group with each isolate representing a separate patient. The cohort included strains that were susceptible to both AMK and CAP, resistant to AMK, and resistant to both drugs (Table 1). The test isolates were subdivided according to their drug susceptibility patterns into three groups: MDR (19 of these showed additional resistance to one or more of the alternative antituberculosis drugs, excluding the FQNs and the injectable drugs), pre-XDR (MDR with additional resistance to either an FQN or an injectable),²² and XDR.

Table 1.

Mutations and Susceptibility Profiles of Amikacin and Capreomycin in 50 Mycobacterium tuberculosis Isolates

	Routine DST profile (7H11 agar) ^a		QDST MGIT 960^b MIC in mg/L^c		Gene mutations ^d
Resistance classification No. of clinical isolates	AMK	CAP	AMK	CAP	rrs gene	tlyA gene
XDR
6	R	S	>20 (R)
7	R	R	>20 (R)		A1401G	None
2	R	R	>20 (R)
Pre-XDR
6	R	S	>20 (R)
1	R	S	>20 (R)
7	R	R	>20 (R)		A1401G	None
1	R	R	>20 (R)
MDR
5	S	S	>20 (R)		A1401G	None
15	S	S	<1.0 (S)		None	None
Control
H37Rv (ATCC 27294)	S	S	<1.0 (S)	≤2.5 (S)	Not done	Not done

Critical concentrations on 7H11 agar: AMK, 4.0 mg/L; CAP, 10 mg/L.

QDST, quantitative drug susceptibility testing.

Critical concentrations in MGIT 960 medium: AMK, 1.0 mg/L; CAP, 2.5 mg/L.

The entire tlyA gene and region 1400–1500 of the rrs gene were sequenced.

DST, drug susceptibility testing; QDST, quantitative DST; MIC, minimum inhibitory concentration; AMK, amikacin; CAP, capreomycin; S, susceptible; R, resistant.

Each of the 50 isolates was retested in our laboratory by semiquantitative DST (QDST) to assess the level of AMK and CAP resistance. The automated BACTEC MGIT 960 instrument (BD Bioscience) equipped with the TBeXiST application and EpiCentre™ V5.69A software (BD Bioscience) was used for QDST as previously described.²⁰ AMK was tested at 1.0, 4.0, and 20.0 mg/L and CAP at 2.5, 5.0, 10.0, 15.0, and 25.0 mg/L. M. tuberculosis strain H37Rv (ATCC 27294) was included as a control and subjected to all the relevant drug concentrations. Strains with minimum inhibitory concentrations (MICs) of ≥1.0 mg/L for AMK and ≥2.5 mg/L for CAP were considered as resistant based on the CCs suggested by the WHO.²⁴ Ethical approval for the study (N09/11296) was given by the Faculty of Health Science at Stellenbosch University.

Polymerase chain reaction amplification and DNA sequencing

Mutations in the rrs and tlyA genes were identified by polymerase chain reaction (PCR) amplification with primers that were designed in-house using Primer3 (v. 0.40).¹⁵ The 1400-bp region of the rrs gene (position 1339–1528) was amplified with the following primers: rrs290_F 5′-TGCTAC AATGGCCGGTACAA-3′ and rrs290_R 5′-CTTCCGGTACG GCTACCTTG-3′.

Amplification of the entire tlyA gene (including 31 and 33 bases upstream and downstream of the gene, respectively) was done with the following primers: tlyA_F 5′-CTGGAG TCGGCGGAGAAG-3′ and tlyA_R 5′-GGACGACCAGCAG AACACTG-3′.

Briefly, 200 μl of a primary MGIT subculture was heat inactivated by incubating at 100°C for 30 min to generate a crude DNA lysate. Each reaction mixture used for PCR amplification contained 2 μl crude DNA template, 5 μl Q-Buffer, 2.5 μl of 10 × Buffer, 2 μl of 25 mM MgCl₂, 4 μl of 10 mM dNTPs, 1 μl of the forward and reverse primers, and 0.125 μl HotStarTaq DNA polymerase (Qiagen), and double-distilled H₂O was then added to make a final volume of 25 μl. Amplification was initiated by incubation at 95°C for 15 min, followed by 35–45 cycles at 94°C for 45 sec, 62°C for 45 sec, and 72°C for 45 sec. After the last cycle, the samples were incubated at 72°C for 10 min. The preparation of the PCR reaction mixes, the addition of the DNA, and the PCR amplification were conducted in separated rooms to minimize laboratory cross-contamination. Negative controls (H₂O) were included to detect reagent contamination. Amplification was confirmed by electrophoretic fractionation in 1% agarose containing Tris-borate (TBE)-EDTA (pH 8.3). Amplification products were sequenced using the ABI3130XL genetic analyzer (Applied Biosystems) and the resulting chromatograms were analyzed using Chromas software.

Antimicrobial agents

AMK (disulfate salt) and CAP (capreomycin sulfate) were purchased from Sigma Aldrich. Stock solutions of the drugs were prepared in distilled water. The drugs were then filter-sterilized and stored at −80°C for up to 6 months.

Results

DNA sequencing of the rrs gene detected the A1401G mutation in 181 of the 310 isolates (58%). Routine DST on Middlebrook 7H11 agar at standard AMK and CAP CCs^7,24 indicated that 89.5% (162/181) of the isolates with an rrs A1401G mutation were resistant to AMK and 13% (21/162) of these had resistance to both AMK and CAP. Of the remaining 129 isolates that lacked an rrs A1401G mutation, 6% (8/129) were resistant to AMK and one of these (1/8) was resistant to both AMK and CAP. Of the 310 isolates, 50 were selected to represent a variety of drug-resistant patterns for further analysis (Table 1). This selection was based on data initially obtained from routine DST on Middlebrook 7H11 agar plates. The level of AMK and CAP resistance was subsequently quantified by QDST in MGIT 960 and these findings were then compared with those obtained from CC testing. A comparison of the phenotypic and genotypic drug resistance is summarized in Table 1. According to routine DST on 7H11 agar, 20 of the 50 M(X)DR isolates tested were susceptible to both AMK (CC of 4.0 mg/L)⁷ and CAP (CC of 10.0 mg/L).²⁴ However, QDST by MGIT 960 at CCs of 1.0 mg/L for AMK and 2.5 mg/L for CAP showed that five of the former isolates (5/20) were resistant to both drugs. The latter five isolates also displayed an rrs A1401G mutation, which is expected to mediate cross-resistance between AMK and CAP.^4,10,12,22 Both phenotypic methods found the other 15 isolates (15/20), which lacked mutations in the genes investigated to be truly susceptible to AMK and CAP. The remaining 30/50 strains also tested resistant to both AMK and CAP by MGIT 960 QDST. On the other hand, routine DST only found 17 of the 30 strains resistant to both AMK and CAP, and 13 were resistant to AMK only. The MICs of AMK and CAP in the resistant strains by MGIT 960 were >20 mg/L and 10–15 mg/L, respectively. These results correlate with recently published data.⁴ No mutations were detected in the 1400–1500 region of the rrs gene in any of the 15 AMK-CAP–susceptible isolates, and all 50 isolates had wild-type tlyA genes. The agreement between the two phenotypic methods to detect isolates susceptible to both AMK and CAP was 15/20 (75%), to indicate AMK resistance was 30/35 (85.7%), and to predict the presence of both AMK and CAP resistance was 17/35 (48.6%).

Discussion

In this study, a complete correlation between the MGIT 960 QDST results and the presence of a nucleotide substitution at position 1401(A→G) in the rrs gene of all 35 AMK-resistant strains was observed. Our findings are therefore in accordance with existing data, which clearly demonstrate that a mutation at rrs position 1401 not only confers high-level AMK/KAN resistance in M. tuberculosis, but also decreases susceptibility to CAP.^4,5,10,22 In contrast, agar-based CC testing was compromised by significant discordance between phenotypic and genetic resistance testing, particularly for CAP. Methodological differences may account for the discrepancies between CC testing on 7H11 plates and QDST in MGIT 960, especially where borderline results were anticipated. Borderline resistance is associated with increased MICs that are only moderately higher than the CC of a given drug. An MIC range of 4–32 mg/L on 7H10 agar has been recently demonstrated for CAP against M. tuberculosis isolates that contain the rrs (A1401G) SNP.⁴ These MIC values were scattered around the CC (10 mg/L) that is currently used in 7H10 agar for routine DST of CAP.^4,24 Clustering of MICs around the CC reflects borderline resistance, which may lead to false-susceptibility results and variability in reporting methods. It is also evident from related studies that the current CC for CAP in 7H10 agar is probably too high and 4 mg/L has been suggested to match the 2.5 mg/L for MGIT 960.^4,11,24 Disparity in CCs and borderline resistance to CAP could have contributed to the discrepancies between the two methods of susceptibility testing that we observed in this study. However, an SNP at position 1401 in the rrs gene confers high-level resistance to AMK^4,10 and these reasons do not justify the conflicting results obtained with this drug.

The results of the test isolates from patients in the Eastern Cape reflect a history of AMK/KAN usage prior to CAP treatment. It is likely that decreased susceptibility to CAP occurred even before it was added to the treatment regimen. The preselection of low-level CAP resistance as a result of AMK/KAN therapy may imply that the drug has no further clinical relevance in the treatment of XDR patients. However, conventional DST is based on a single CC, which does not necessarily reflect clinical resistance.²⁰ Peak serum concentration levels of 20–47 mg/L for CAP are achieved between 1 and 2 hr after a single daily dosage of 15–20 mg/kg by intramuscular injection.² In this study, the MICs (10–15 mg/L) of the CAP-resistant isolates were above the CC of 2.5 mg/L as recommended by WHO,²⁴ but substantially below the achievable peak serum levels.² Protein binding of CAP is relatively low at 20% and the concentration of free drug at the point of infection may be sufficient for an adequate therapeutic effect in patients infected with low-level CAP-resistant mutants.² Therapeutic options for M(X)DR-TB are severely restricted and the omission of CAP based on the current DST criteria needs to be considered with caution. QDST provided valuable information on the degree of CAP resistance, which could be useful in classifying clinical isolates as susceptible (wt), intermediate, or resistant.¹⁸ We associate intermediate resistance to CAP with a distinct MIC cluster between susceptible and definite resistant strains. The results obtained in this study demonstrate that the rrs A1401G mutation confers an intermediate level of resistance to CAP in the isolates from the Eastern Cape. However, the therapeutic effect of CAP against such strains is currently uncertain. It is therefore recommended that a clear correlation between multiple breakpoints above the standard CC for CAP and patient outcome is established through well-designed clinical studies.

Conclusions

This investigation supports existing data that the A1401G mutation in the rrs gene mediates resistance to AMK/KAN and decreased susceptibility to CAP.^4,10 The susceptibility results obtained in this study with MGIT 960 for AMK and CAP were in exact concordance with the nucleic acid sequence data, whereas significant differences were found with 7H11 agar. In addition, the MGIT 960 results were based on multiple MIC concentrations as opposed to a single CC used for 7H11. In view of these reasons, we consider the MGIT results superior to those obtained by 7H11 agar. Further, the automated MGIT 960 system is technically less demanding and has a much shorter turnaround time than the standard 7H11 agar method. Given the unreliability of routine agar-based CC testing, we favor the view that MGIT 960-based testing should become the standard for second-line DST,²¹ as it is suitable for both CC testing and QDST when equipped with TBeXiST and EpiCentre software.²⁰

Our findings recommend the use of this technology together with pharmacokinetic and pharmacodynamic parameters for the management of M(X)DR-TB in the Eastern Cape, where nearly 60% of MDR cases harbor the rrs A1401G mutation.

Footnotes

Acknowledgments

The authors thank Marianna de Kock and Claudia Spies for excellent technical assistance and Dr. Alpa Somaiya from the South African MRC for manuscript review. The National Center for Mycobacteria (ECB) is in part supported by the Bundesamt für Gesundheit (BAG), Bern, Switzerland.

Disclosure Statement

The authors declare that they have no competing interests.

References

Alangadan

G.J.

, Kreiswirth

B.N.

, Aouad

, Khetarpal

, Igno

F.R.

, Moghazeh

S.L.

, Manavathu

E.K.

, Lerner

S.A.

1998. Mechanisms of resistance to amikacin and kanamycin in Mycobacterium tuberculosis. Antimicrob. Agents. Chemother., 42:1295–1297.

Anonymous. 2008. Capreomycin. Tuberculosis, 88:89–91.

Chan

E.D.

, Iseman

M.D.

2008. Multidrug-resistant and extensively drug-resistant tuberculosis: a review. Curr. Opin. Infect. Dis., 21:587–595.

Engström

, Perskvist

, Werngren

, Hoffner

S.V.

, Juréen

2011. Comparison of clinical isolates and in vitro selected mutants reveals that tlyA is not a sensitive genetic marker for capreomycin resistance in Mycobacterium tuberculosis. J. Antimicrob. Chemother., 66:1247–1254.

Feuerriegel

, Cox

H.S.

, Zarkua

, Karimovich

H.A.

, Braker

, Rüsch-Gerdes

, Stefan

Niemann

. 2009. Sequence analyses of just four genes to detect extensively drug-resistant Mycobacterium tuberculosis strains in multidrug-resistant tuberculosis patients undergoing treatment. Antimicrob. Agents Chemother., 53:3353–3356.

Gandhi

N.L.

, Moll

, Sturm

A.W.

, Pawinski

, Govender

, Lalloo

, Zeller

, Andrews

, Friedland

2006. Extensively drug-resistant tuberculosis as a cause of death in patients co-infected with tuberculosis and HIV in a rural area of South Africa. Lancet, 368:1575–1580.

Heifets

, Desmond

2005. Clinical mycobacteriology (tuberculosis) laboratory: services and methods. Cole

S.T.

, Eisenach

K.D.

, Murray

D.N.

, Jacobs

W.J.

Jr. Tuberculosis and the Tubercle Bacillus. American Society for Microbiology. Washington, DC, 49–70.

Heifets

, Simon

, Pham

2005. Capreomycin is active against non-replicating M. tuberculosis. Ann. Clin. Microbiol. Antimicrob., 4:6.

Johansen

, Maus

C.E.

, Plikaytis

B.B.

, Douthwaite

2006. Capreomycin binds across the ribosome subunit interface using tlyA-encoded 2’-O-methylations in 16S and 23S rRNAs. Mol. Cell, 23:173–182.

10.

Jugheli

, Bzekalava

, de Rijk

, Fisette

, Portaels

, Rigouts

2009. High level of cross-resistance between kanamycin, amikacin, and capreomycin among Mycobacterium tuberculosis isolates from Georgia and a close relation with mutations in the rrs gene. Antimicrob. Agents Chemother., 53:5064–5068.

11.

Juréen

, Ängeby

, Sturegård

, Chryssanthou

, Giske

C.G.

, Werngren

, Nordvall

, Johansson

, Kahlmeter

, Hoffner

, Schön

2010. Wild-type MIC distributions for aminoglycoside and cyclic polypeptide antibiotics used for treatment of Mycobacterium tuberculosis infections. J. Clin. Microbiol., 48:1853–1858.

12.

Maus

C.E.

, Plikaytis

B.B.

, Shinnick

T.M.

2005. Molecular analysis of cross-resistance to capreomycin, kanamycin, amikacin, and viomycin in Mycobacterium tuberculosis. Antimicrob. Agents Chemother., 49:3192–3197.

13.

Maus

C.E.

, Plikaytis

B.B.

, Shinnick

T.M.

2005. Mutation of tlyA confers capreomycin resistance in Mycobacterium tuberculosis. Antimicrob. Agents Chemother., 49:571–577.

14.

Migliori

G.B.

, Lange

, Centis

, Sotgiu

, Mütterlein

, Hoffmann

, Kliiman

, De Iaco

, Lauria

F.N.

, Richardson

M.D.

, Spanevello

, Cirillo

D.M.

TBNET Study Group. 2008. Resistance to second-line injectables and treatment outcomes in multidrug-resistant and extensively drug-resistant tuberculosis cases. Eur. Respir. J., 31:1155–1159.

15.

Rozen

, Skaletsky

H.J.

2009. Primer3 on the WWW for general users and for biologist programmers. (v. 0.40) pick primers from a DNA sequence. http://frodo.wi.mit.edu/primer3/(Online.)

16.

Sander

, Prammananan

, Böttger

E.C.

1996. Introducing mutations into a chromosomal rRNA gene using a genetically modified eubacterial host with a single rRNA operon. Mol. Microbiol., 22:841–848.

17.

Sandgren

, Strong

, Muthukrishnan

, Weiner

B.K.

, Church

G.M.

, Murray

M.B.

2009. Tuberculosis drug resistance mutation database. PloS Med., 6:e2.

18.

Schön

, Juréen

, Giske

C.G.

, Chryssanthou

, Sturegård

, Werngren

, Kahlmeter

, Hoffner

S.E.

, Ängeby

K.A.

2009. Evaluation of wild-type MIC distributions as a tool for determination of clinical breakpoints for Mycobacterium tuberculosis. J. Antimicrob. Chemother., 64:786–793.

19.

Shcherbakov

, Akbergenov

, Matt

, Sander

, Anderson

D.I.

, Böttger

E.C.

2010. Directed mutagenesis of Mycobacterium smegmatis 16S rRNA to reconstruct the in vivo evolution of aminoglycoside resistance in Mycobacterium tuberculosis. Mol. Microbiol., 77:830–840.

20.

Springer

, Lucke

, Calligaris-Maibach

, Ritter

, Böttger

E.C.

2009. Quantitative drug susceptibility testing of Mycobacterium tuberculosis by use of MGIT 960 and EpiCenter Instrumentation. J. Clin. Microbiol., 47:1773–1780.

21.

Van Ingen

, Simons

, de Zwaan

, van der Laan

, Kampst-van Agterveld

, Boeree

M.J.

2010. Comparative study on genotypic and phenotypic second-line drug resistant testing of Mycobacterium tuberculosis complex isolates. J. Clin. Microbiol., 48:2749–2753.

22.

Via

L.E.

, Cho

S.-N.

, Hwang

, Bang

, Park

S.K.

, Kang

H.S.

et al. 2010. Polymorphisms associated with resistance and cross-resistance to aminoglycosides and capreomycin in Mycobacterium tuberculosis isolates from South Korean patients with drug-resistant tuberculosis. J. Clin. Microbiol., 48:402–411.

23.

World Health Organization (WHO). 2008. Treatment strategies for MDR-TB. Guidelines for the Programmatic Managament of Drug-Resistant Tuberculosis (WHO):50–74. (WHO/HTM/TB/2008.402) WHO: Geneva, Switzerland.

24.

World Health Organization (WHO). 2008. Policy Guidance on Drug-Susceptibility Testing (DST) of Second-Line Antituberculosis Drugs. Report (WHO/HTM/TB/2008.392):1–20. WHO: Geneva, Switzerland.