A First Insight into the katG and rpoB Gene Mutations of Multidrug-Resistant Mycobacterium tuberculosis Strains from Ecuador

Abstract

The aim of this study was to characterize the most frequent mutations associated with rifampicin (RIF) and isoniazid (INH) resistance of Mycobacterium tuberculosis isolates from Ecuador. Sequence analysis of 40 strains, resistant for the tuberculosis drugs INH, RIF, or for both showed that of the 31 strains with resistance to INH, 20 strains (64.5%) carried a mutation in the katG gene (codon 315). Eight INH-resistant strains carried a mutation in the katG gene at codon 463. This katG463 mutation, considered a phylogenetic marker, was exclusively found in INH-resistant strains and not in 121 INH-susceptible strains. Of the 35 strains resistant to RIF, 33 (93.9%) had mutations in the hot spot region of the rpoB gene, predominantly in codons 531, 516, and 526. Our results show that sequence-based detection for drug resistance of the katG will identify, respectively, 64.5% or, considering katG463 as a marker, 90.3% of the INH-resistant strains. Sequencing of the hot spot region of the rpoB gene will detect 94.3% of the RIF drug-resistant isolates in Ecuador. This is appropriate for fast screening for drug resistance with the GeneXpert MTB/RIF assay or by direct sequencing of a part of the genes katG and rpoB of PCR products obtained from DNA isolation from primary cultures.

Introduction

The emergence of drug-resistant and multidrug-resistant (MDR) strains has complicated the treatment of tuberculosis (TB). Early detection of these strains, preferably before starting treatment, is essential for the implementation of an effective treatment regimen and hence interrupting the transmission of these strains, avoiding further spread of drug-resistant TB. Initiation of an adequate treatment in case of drug resistance is often being hampered because of the delayed reporting of the resistance profile of the TB isolate, which in general comes weeks or months after the beginning the treatment program. The use of the GeneXpert Mycobacterium tuberculosis (MTB)/rifampicin (RIF) assay has overcome this problem for RIF resistance, but the resistance testing for isoniazid (INH) still needs in the majority of the cases a mycobacterial culture and classical drug resistance tests.

According to the World Health Organization, Ecuador has a relatively high prevalence rate of MDR-TB of 7.3% (5.4–9.2) in newly diagnosed cases and 28% (25–31) in previously treated TB patients.¹

Concerning the genetic mutations that lead to drug resistance for the drugs INH and RIF, the resistance to INH is in general mediated by a mutation in the katG gene at codon 315 resulting in partial loss of catalase-peroxidase activity.^2–4 Mutations responsible for RIF resistance are primarily located in a “hot spot” 81 bp region of the rpoB gene, called rifampicin resistance-determining region (RRDR). The gene rpoB codes for the beta subunit of the RNA polymerase and mutations are predominantly at codons 531, 526, and 516.^2,5,6

In this article we focus on the genetic mutations converting MTB strains in Ecuador from susceptibility to resistance for the drugs INH and RIF. We determine if sequencing of the core genes for drug resistance for these antibiotics constitutes a rapid alternative to conventional drug susceptibility testing in Ecuador. In Ecuador there is no information on specific antibiotic resistance gene mutations of MTB.¹

Materials and Methods

MTB clinical isolates

A total of 152 MTB strains, 47 isolated from new patients and 105 from previously treated patients, were used in this study. The strains were isolated in the years 2006–2012 and came from 15 of the 24 provinces in Ecuador. All strains were isolated in the National Reference Laboratory for Mycobacteria at the Instituto Nacional de Salud Pública e Investigación “Leopoldo Izquieta Pérez” in Guayaquil (Ecuador) from sputum samples using Löwenstein–Jensen (L–J) medium. Drug susceptibility testing for first-line drugs INH and RIF was performed with the proportion method of Canetti et al.⁷ on L–J medium with a minimal inhibitory concentrations of 40 μg/mL for RIF and 0.2 μg/mL for INH. MTB H37Rv strain was used as a positive control for RIF and INH susceptibility.

DNA Isolation, PCR, and sequencing for the detection of mutations in the katG and rpoB genes

DNA was isolated from heat-inactivated lysates of the MTB strains, harvested from L–J slants and following the supplier's protocol, using spin columns and the DNeasy Blood & Tissue Kit (Qiagen). For the katG gene an amplicon of 620 bp (codons 300–505) was amplified using the forward and reverse primers 5′-AGCTCGTATGGCACCGGAAC-3′ and 5′-TTGACCTCCCACCCGACTTG-3′. For rpoB sequencing, an amplicon of 210 bp (codons 493–563), which encloses the RRDR region, was generated using the forward and reverse primers 5′-CGTGGAGGCGATCACACCGCAGACGT-3′ and 5′-AGTGCGACGGGTGCACGTGCGGGACCT-3′. The PCR products were isolated using a PCR fragment purification kit (Qiagen) and sequenced using an ABI 310 Genetic Analyzer (Applied Biosystems, CA). The sequence data were analyzed using CHROMAS, EDITSEQ, and BIOEDIT ALIGN software with the MTB H37Rv katG and rpoB sequences as the reference sequence.

Results

Drug resistance testing with the proportion method of the 152 strains for INH and RIF showed that 7 strains were resistant to INH only, 11 strains were resistant to RIF only, and 24 were resistant to both RIF and INH (MDR strains). Of these 42 strains, 5 were from the 47 newly diagnosed patients and 37 from the 105 previously treated patients supporting the hypothesis of high prevalence of drug resistance in Ecuador: in this study 10.6% from newly diagnosed patients and 35.2% from the previously treated patients.

Sequencing of the katG gene of the 31 INH-resistant strains showed that 64.5% (20/31) carried a single mutation in katG at codon 315 (Table 1). In 3 of the 31 strains (9.7%) no mutation was detected in the amplified gene fragment, and 8 INH resistance strains (25.8%) had a single mutation at codon 463 (CGG-CTG) (Table 1). Although this mutation is sometimes considered as a mutation associated with INH resistance,^8,9 katG463 is also considered a phylogenetic marker^10–12 for the early subdivision of MTB into three principal genetic lineages. Sequencing of the 121 strains sensible to INH did not show any mutation in the amplified fragment of katG.

Table 1.

Identification of the katG Mutations for Isoniazid-Resistant Mycobacterium tuberculosis Strains Using Sequencing

Altered codon	Nucleotide change	Amino acid change	No. strains with mutations
315	AGC-AAC	ser-thr	20
463	CGG-CTG	arg-leu	8

For three INH-resistant strains and 121 INH-susceptible strains no mutations in the katG gene were detected.

INH, isoniazid.

Among the RIF-resistant strains, 30.3% (10/33) carried a mutation in rpoB at codon 531. The second most frequent mutation was found at codon 516 in 28.6% (10/35) of the strains. In addition, 25.7% (9/35) had a mutation in codon 526, and two strains each had mutations in, respectively, codons 512 and 533. No novel mutations or deletions across the 81 bp RRDR region were found. For 2 of the 35 RIF-resistant strains (5.7%) no mutations were found in the amplified region of the rpoB gene (Table 2). To determine the presence of silent mutations in the rpoB gene, the 110 strains susceptible to RIF were sequenced. Only wild-type rpoB sequences were found and no silent or missense mutations were detected.

Table 2.

Identification of the rpoB Mutations for Rifampicin-Resistant Mycobacterium tuberculosis Strains Using Sequencing

Altered codon	Nucleotide change(s)	Amino acid change(s)	No. of strains with mutations
512	AGC-ATG	ser-met	2
516	GAC-GTC	asp-val	10
526	CAC-GAC, CAC-TAC	his-asp, his-tyr	9,2
531	TCG-TTG	ser-leu	10
533	CTG-CCG	leu-pro	2

For two RIF-resistant strains and 110 RIF-susceptible strains no mutations in the rpoB gene were detected.

RIF, rifampicin.

Discussion

This study is the first for Ecuador showing the distribution of mutations in the genes katG and rpoB of INH and RIF resistance strains. All the mutations we found in these genes have been described earlier.¹³

For detection of INH resistance by sequencing of the katG gene in Ecuador, the katG315 mutation would detect 64.5% of the INH-resistant strains. In addition, globally ∼64% of the INH resistance has been associated with the katG315 mutation. We did not assess the second most frequently observed mutation, inhA-15, reported worldwide among 19% of phenotypically resistant isolates.¹¹

Regarding the mutation in codon 463 of katG in eight INH-resistant strains in this study, we always found that this mutation is associated with INH resistance. In other studies this mutation was also found in INH-susceptible strains.^10–12 However, in our study 8 INH resistance isolates carried only this mutation in the katG gene, whereas none of the 121 INH-susceptible isolates had the mutation. Considering this mutation in Ecuador as a marker for INH resistance, the protocol described in this study for sequencing a katG gene region for PCR products from DNA isolations from MTB primary cultures would detect 90.3% (28/31 strains) of the INH-resistant strains.

For detection of RIF resistance, sequence analyses of the RRDR of the rpoB gene had a sensitivity of 94.3%. This guarantees a high sensitivity for RIF testing with the GeneXpert MTB/RIF assay in our setting and its use should be encouraged. The GeneXpert assay cannot differentiate silent mutations in the rpoB gene causing misinterpretation of RIF susceptibility.^14–17 However, no silent mutations were found in the rpoB gene of 110 susceptible strains guaranteeing a high specificity of this molecular assay.

We conclude that screening for rpoB and katG gene mutations is useful for early detection of MDR-TB. Although MTB primary culture on L–J medium is the golden standard for TB diagnosis for public health authorities in Ecuador, it would be interesting to improve MDR-TB screening by implementing the method described in this study, directly from sputum samples. Nevertheless, it is important to note that 9.7% of isolates resistant to INH and 5.7% of isolates resistant to RIF cannot be detected with sequence analysis. This means that phenotypic testing of the strains will always be necessary to follow-up on the results of genetic testing. We are also aware of the possibility that a part of the strain collection we used in this study was the result of the clonal spread of a specific strain. This would mean that the mutation frequency found in this study is not at random and some mutations could be overrepresented. However, we almost exclude this possibility for the following reasons: the strains came from most of the provinces of Ecuador and were isolated in a period of 6 years. In addition, genotyping of all the strains used in this study is in progress. All 152 strains will be analyzed with 24-loci MIRU-VNTR typing and lineages will be assigned by comparing their MIRU-VNTR patterns with those in the MIRU-VNTR plus platform (www.miru-vntrplus.org).

Transparency Declarations

This study used only culture isolates from Instituto Nacional de Salud Pública e Investigación Leopoldo Izquieta Pérez (INSPI) without involving direct collection of clinical samples.

Footnotes

Acknowledgments

This study was supported by internal funding from the Instituto Nacional de Salud Pública e Investigación Leopoldo Izquieta Pérez (INSPI). G.F.S. was recipient of a fellowship from SENESCYT. G.F.S. conceived and designed the experiments. G.F.S. and M.L.B. performed the experiments. G.F.S., D.G.-C., J.H.D.W., and M.A.G.B. analyzed the data, wrote the article, read, and approved the final version. The authors of this article thank Instituto de Salud Pública de Chile for support with DNA sequencing.

Disclosure Statement

No competing financial interests exist.

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