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J Clin Microbiol. Jul 2011; 49(7): 2625–2630.
PMCID: PMC3147840

Streptomycin Resistance and Lineage-Specific Polymorphisms in Mycobacterium tuberculosis gidB Gene [down-pointing small open triangle]

Abstract

Mutations related to streptomycin resistance in the rpsL and rrs genes are well known and can explain about 70% of this phenotypic resistance. Recently, the gidB gene was found to be associated with low-level streptomycin resistance in Mycobacterium tuberculosis. Mutations in gidB have been reported with high frequency, and this gene appears to be very polymorphic, with frameshift and point mutations occurring in streptomycin-susceptible and streptomycin-resistant strains. In this study, mutations in gidB appeared in 27% of streptomycin-resistant strains that contained no mutations in the rpsL or rrs genes, and they were associated with low-level streptomycin resistance. However, the association of certain mutations in gidB with streptomycin resistance needs to be further investigated, as we also found mutations in gidB in streptomycin-susceptible strains. This occurred only when the strain was resistant to rifampin and isoniazid. Two specific mutations appeared very frequently in this and other studies of streptomycin-susceptible and -resistant strains; these mutations were not considered related to streptomycin resistance, but as a polymorphism. We stratified the strains according to the different phylogenetic lineages and showed that the gidB16 polymorphism (16G allele) was exclusively present in the Latin American-Mediterranean (LAM) genotype, while the gidB92 polymorphism (92C allele) was associated with the Beijing lineage in another population. In the sample studied, the two characterized single-nucleotide polymorphisms could distinguish LAM and Beijing lineages from the other lineages.

INTRODUCTION

Acquired drug resistance in Mycobacterium tuberculosis arises from spontaneous chromosomal mutations. Clinical drug-resistant tuberculosis (TB) occurs when these genetic alterations are selected for during disease treatment. This can occur via erratic drug supply, suboptimal physician prescription, and poor patient adherence (34).

The aminoglycoside antibiotic streptomycin (STR) was the first antibiotic used to control TB. It interacts directly with the small ribosomal subunit 16S rRNA and interferes with translational proofreading, thereby leading to inhibition of protein synthesis (14, 33). Mutations associated with high-level STR resistance in M. tuberculosis have been identified in the genes encoding ribosomal protein S12 (rpsL) and 16S rRNA (rrs) (9). However, mutations in these two genes explain only about 70% of the cases of STR resistance in clinical isolates (22, 26), implying that there must be other loci involved or another mechanism for STR resistance.

Recently, it was shown that mutations in the gene encoding a 7-methylguanosine (m7G) methyltransferase (gidB) specific for the 16S rRNA resulted in low-level STR resistance (20). However, in clinical isolates of M. tuberculosis, mutations in gidB have been observed in strains that are both susceptible and resistant to STR (20, 25).

Genotyping of M. tuberculosis strains is useful to answer evolutionary questions and for surveying its transmission dynamics in epidemiological studies (2). In addition, molecular typing improves our understanding of the basic biology of bacterial pathogens, including differences in virulence and transmissibility and the variable effectiveness of vaccines (7). Spoligotyping is a PCR-based genotyping technique that exploits the variability of the direct repeat (DR) region in M. tuberculosis (16). Since 1999, genetic diversity databases have been organized for the M. tuberculosis complex DR locus as an attempt to analyze population structure and to assess the complexity of global TB transmission underlying the spatial and temporal evolution of the TB genetic landscape (3). The accumulated data demonstrate that a few major lineages of conserved spoligotypes are well distributed throughout the world, whereas others are specific for certain geographic regions (3, 24).

Single-nucleotide polymorphisms (SNPs) are the most robust and appropriate phylogenetically informative markers (7). Therefore, in recent years, some authors have proposed SNPs in phylogenetic studies (2, 8, 12). Some SNPs have been linked to specific M. tuberculosis phylogenetic lineages, such as an SNP in the mgtC gene (R182H) that can differentiate between Haarlem and non-Haarlem lineages (1), the variation in Rv2629 (N64A), which is found exclusively in lineages from Beijing (5, 13), and an SNP in fadD28 codon 507, which acts as a specific marker for the East Asia lineage (6).

In the present study, we investigated mutations in the gidB gene in clinical isolates representative of the genetic diversity of M. tuberculosis to explore its possible involvement in STR resistance in strains from southern Brazil. Furthermore, we stratified the strains according to the different phylogenetic lineages and showed that the gidB16 polymorphism (16G allele) was exclusively present in the Latin American-Mediterranean (LAM) genotype and that the gidB92 polymorphism (92C allele) was associated with the Beijing lineage in another population.

MATERIALS AND METHODS

Strains.

One hundred six M. tuberculosis strains originating from the south of Brazil were isolated between 2005 and 2006 at LACEN-RS (Central Laboratory of Rio Grande do Sul State, Brazil), and 22 M. tuberculosis strains of Beijing genotype were acquired from the collection at the Institute of Tropical Medicine (ITM) in Belgium.

MIC determinations.

The resazurin microtiter assay (REMA) was used for MIC determinations (21). Briefly, 96-well plates were filled with 7H9-oleic acid-albumin-dextrose-catalase medium. Serial 1:2 dilutions of STR (125 μg/ml to 0.12 μg/ml), rifampin (RIF; 16 μg/ml to 0.03 μg/ml), and isoniazid (INH; 12.8 μg/ml to 0.01 μg/ml) were performed in each column. The breakpoint used to determine STR resistance was an MIC of ≥1 μg/ml; INH and RIF resistance was considered as an MIC of ≥0.25 μg/ml (19).

DNA extraction and sequencing.

DNA was isolated from mycobacterial cultures by a lysozyme/proteinase K cetyltrimethylammonium bromide procedure (28). A 306-bp fragment of the M. tuberculosis rpsL gene (GenBank accession number L08011) and the 530 region (238 bp) and 912 region (238 bp) of the rrs gene (GenBank accession number X52917) were amplified as described by Tracevska et al. (27), and a 675-bp fragment of the gene gidB (GenBank accession number AAK48404) was amplified as described by Spies et al. (25). Amplification products were sequenced using the ABI Prism 3100 DNA sequencer (Applied Biosystems) and MegaBACE 1000 DNA analysis system (GE Healthcare Life Sciences). Nucleotide sequences were analyzed using the programs PREGAP and GAP4 of the STADEN software package ver. 10.0. Nucleotide sequences with Phred values of >20 were considered for analysis.

Genotyping.

Spoligotyping was performed using a commercial kit (Isogen Biosciences B.V., Netherlands) according to the manufacturer's instructions. Spoligopatterns obtained were entered in a binary format as Excel spreadsheets (Microsoft, Redmond, WA) and compared to information in the SITVIT database (http//www.pasteur-guadaloupe.fr:0881/SITVITDemo/) (3).

LAM-specific PCR.

The strains were further characterized by a LAM-specific PCR to classify strains that could not be assigned to an internationally recognized genotype lineage and to confirm the spoligotyping result. In this assay, PCR primers identified the presence of an IS6110 insertion (position 932204 according to the whole H37Rv genome sequence), which is unique to all members of the LAM lineage (17). PCR amplification products were electrophoretically fractionated in a 3.0% agarose gel at 85 V for 2 h. The presence of a LAM strain was represented by a band of 205 bp, while a non-LAM strain was represented by a band of 141 bp (17).

RESULTS

Mutations in the rpsL, rrs, and gidB genes and STR resistance.

To analyze if mutations in the gidB gene were related to STR resistance, we compared the mutations present in resistant and susceptible strains with sequencing data from rpsL and rrs, genes known to be related to STR resistance.

Among the 40 STR-resistant strains, 10 highly STR-resistant strains (MIC ≥ 125 μg/ml) presented only the mutation in codon 43 (AAG→AGG; K43R) of the rpsL gene. One strain highly resistant to STR presented a mutation in codon 88 (AAG→AGG; K88Q) of the rpsL gene and a mutation in codon 183 of the gidB gene (GCG→ACG; A183T), while another strain highly resistant to STR contained a silent mutation in codon 81 of the rpsL gene (CTG→TTG; L81L), a frameshift mutation in codon 117 of the gidB gene (nucleotide 350 G insertion) and a mutation in position 905 of the rrs gene (Table 1).

Table 1.
Mutations in rpsL, rrs, and gidB genes in resistant strains

Three resistant strains presented a single C→T mutation in position 513 of the rrs gene, and one strain contained a C→G mutation in position 904. The C→T mutation in position 491 of the rrs gene appeared in four low-level resistant strains: three without mutations in the other studied genes and one with a mutation in codon 200 of the gidB gene (GCG→GAG; A200E) (Table 1). This mutation in position 491 of the rrs gene is also present in two STR-susceptible strains, one of which also contains a mutation in codon 115 (GTG→GGG; V155G) of the gidB gene (Table 2).

Table 2.
Mutations in rrs and gidB genes in STR-susceptible strains

Ten strains with low-level STR resistance presented different mutations in the gidB gene (TGG→TCG [W45S], CCG→CTG [P84L], CAT→TAT [H48Y], GGT→CGT [G30R], TGC→TGA [C52stop], 51 AAC→ACC [N51T], 39 frameshift; TTG→TTT [L79F], CTA→CCA [L49P], 164 GGC→TGC [G164C]), and one with intermediate-level resistance presented the mutation GGG→GAG (G117E) (Table 1). Nine STR-resistant strains presented no mutations in the fragments of the genes studied (STR MICs from 8 to 2 μg/ml).

Of the 66 STR-susceptible strains, 60 did not present any mutation in the DNA fragments of the studied genes. Six strains presented mutations, five of which contained mutations in the gidB gene (34 frameshift; CCG→CTG [P84L], CCG→CGG [P93R], TCT→TTT [S100F], GTG→GGG [V115G]). Additionally, all five of the strains containing gidB mutations were RIF and INH resistant (Table 2). No susceptible strain had mutations in the rpsL gene.

Highly frequent gidB16 polymorphism and LAM lineage.

Apart from the several gidB mutations that appeared in single strains (Tables 1 and and2),2), one particular mutation, gidB16 (CTT→CGT; L16R), appeared frequently (n = 63; 59%) in this study population and was identified in 24 resistant and 39 susceptible strains. Therefore, this mutation was not considered a mutation related to STR resistance but as a polymorphism and was not included in Tables 1 and and22.

The presence of this polymorphism was compared according to the distribution of spoligotype lineages, and we observed a very strong association between the 16G allele and the LAM lineage (Table 3). To confirm this, all strains were analyzed using a LAM-specific PCR test.

Table 3.
Spoligotyping of the strains and gidB16 polymorphism

Of the 54 strains for which the spoligopattern was determined to be LAM, 53 presented the 16G allele and were confirmed by the LAM-specific PCR test. One strain that belongs to the LAM3 and S/convergent sublineage had a 16T allele in the gidB gene and was not considered to belong to the LAM lineage by the LAM-specific PCR (Table 3).

All strains belonging to the Haarlem, T, S, and X lineages were considered non-LAM by the LAM-specific PCR test and contained the 16T allele in gidB gene (Table 3).

From the 10 strains belonging to the unknown lineage, 6 were classified as LAM by the LAM-specific PCR test, and all 6 also presented the 16G allele. Nine strains presented no shared international type (SIT), and four of them also had the same gidB16 polymorphism (16G allele) and were identified as LAM by the LAM-specific PCR test (Table 3).

Polymorphism in gidB92 and Beijing family.

Interestingly, previous reports identified a different gidB mutation that appears frequently within a population. According to Via et al. (29), the majority of the isolates studied (85%) contained an A-C SNP at nucleotide 276 (GAA→GAC; E92D). According to Okamoto et al. (20), the majority of the clinical isolates studied (70%) had this same polymorphism, E92D, and they did not consider this alteration to be related to STR resistance.

This polymorphism was not found in our Brazilian sample, and because there is a different distribution of lineages around the world, we decided to test the possible relationship of this polymorphism with the Beijing genotype. We sequenced the gidB gene in 22 Beijing strains to better understand this correlation and found that all of these strains had the 92C allele in the gidB gene (GAA→GAC; E92D) as well as the 16T allele. Consequently, they were classified as non-LAM strains and confirmed the relationship of the gidB92 polymorphism with the Beijing genotype.

DISCUSSION

Genetic resistance to antituberculosis drugs is due to spontaneous chromosomal mutations, because no mobile genetic elements, such as plasmids or transposons, have been characterized in M. tuberculosis. Therefore, it is of critical importance to determine the mutations related to STR resistance.

Mutations associated with STR resistance in rpsL and rrs are well known. For example, the mutation in codon 43 of rpsL (found in 10 strains in this study) is the most frequent mutation associated with high-level STR resistance (4, 10, 26), while the mutation at position 491 of rrs (found in 4 strains in this study) is not related to STR resistance (30, 31). However, mutations in the gidB gene of M. tuberculosis possibly related to STR resistance have been described in other studies (20, 25, 29).

In this study, we analyzed 106 M. tuberculosis clinical isolates from Brazil and found that 18% (19/106) had mutations in gidB (without the L16R polymorphism). Only three resistant strains also had mutations in other genes associated with STR resistance. Eleven of 40 STR-resistant strains (27%) had mutations only in gidB; 10 had a low level of STR resistance and 1 had intermediate resistance. According to other reports (22, 26), approximately 30% of STR-resistant strains lack mutations in rpsL or rrs. In our study, gidB mutations may explain the 27% of strains that were resistant to STR without mutations in rpsL or rrs. However, nine strains (22%) considered STR resistant had no mutations in rpsL, rrs or gidB.

We found five STR-susceptible strains with mutations in gidB that occurred only in RIF- and INH-resistant strains. No gidB mutation was observed in fully susceptible strains. Okamoto et al. (20) came to the same conclusion: gidB mutations in STR-susceptible strains occurred in 15/51 strains resistant to INH and RIF but in only 1 (1/24) fully susceptible strain.

Mutations in gidB have been reported frequently in other studies. Okamoto et al. (20) reported gidB mutations in 33% of STR-resistant strains. Spies et al. (25) found that 73% of isolates presented nucleotide mutations, and 5% with low-level STR resistance had mutations only in gidB. In the study by Via et al. (29), 15 different polymorphisms in gidB from 21 isolates were described, including drug-susceptible and -resistant strains.

In addition to being frequently mutated, gidB seems to be very polymorphic. Apparently, mutations are not present at conserved sites, with the exception of the gidB16 and gidB92 polymorphisms, which are not associated with STR resistance. Taking into account all published studies (20, 25, 29) and this study, a total of 414 M. tuberculosis strains have been sequenced for the gidB gene, comprising strains from Japan (132), South Korea (97), and Brazil (185).

The frameshift mutations at codons 14, 36, and 64 each appeared in one susceptible strain. The frameshift mutations at codons 34, 39, 117, and 118 appeared in several strains each, both in STR-resistant and -susceptible strains. The frameshift mutation at codon 40 appeared only in one resistant strain which contained no additional mutations in the other STR resistance-associated genes.

Point mutations (substitutions) are more variable than the frameshift mutations and did not occur at conserved sites. Fifty-five different point mutations have been found in the gidB gene. Forty-three of them occurred only once: 21 in susceptible strains (L35R, W45R, V65A, V66M, L74S, V74A, V77A, R83L, P84R, P84C, P93R, P93Q, S100F, V115G, R116P, R118C, D132V, R137G, V139M, A183E, and V188M), 10 in resistant strains with mutations in rpsL and/or rrs (E40stop, R47Q, I55S, E57stop, R96R, E103stop, L128S, K147T, A183T, and V188G), and 12 in resistant strains with no mutations in the rpsL and/or rrs genes (G30R, W45C/V110V/W148R, W45S, H48Y, L49P, N51T, C52stop, D67H, P75S/V110V/A141A, P75A, G117E, and G164C).

While investigating the evolution of drug resistance in the KwaZulu-Natal family of M. tuberculosis, Ioerger et al. (15) reported a distinct gidB mutation, a 130-bp deletion (spanning amino acids 50 to 93 encompassing the SAM-binding site) that causes a frameshift in the C-terminal remainder, which they presumed to completely abrogate the function of the protein. This deletion was found in both drug-resistant (multidrug resistant and extremely drug resistant) and in one STR-susceptible strain.

It will be necessary to sequence more clinical isolates of M. tuberculosis to better understand the role of gidB gene mutations in STR resistance, thus allowing a more accurate identification of mutations relevant for STR resistance. Nevertheless, gidB gene mutations appearing in STR-resistant strains with no additional mutations in other genes seem to be associated with low-level resistance to STR. Low-level antibiotic resistance is frequently required as an initial step for the emergence of high-level resistance. If the bacterial population is not killed effectively by a given antibiotic, the cells will remain under stress, and this may increase the mutation rate (18).

Okamoto et al. (20) found that 70% (93/132) of clinical isolates had the amino acid substitution E92D in gidB. We previously found the L16R substitution in 49% (39/79) of clinical isolates (25). Via et al. (29) found 85% (83/97) of isolates contained a point mutation in E92D of gidB.

Taking all this into account, we can report that polymorphism gidB92 (GAA→GAC; E92D) was found more frequently among strains from Asia and polymorphism gidB16 (CTT→CGT; L16R) was very frequent among strains from Brazil. According to Brudey et al. (3), Beijing and Beijing-like strains represent about 50% of the strains from Far East Asia (such as Japan and South Korea), and the LAM lineage corresponds only to 5% of the strains. In South America, about 50% of the strains belong to the LAM lineage, and the Beijing genotype is rarely observed. In Brazil, only 0.8% of the strains have been reported as Beijing genotype (23).

We have successfully characterized two SNPs that can distinguish the LAM and Beijing lineages. All strains belonging to the LAM lineage by LAM-specific PCR had the SNPs in codon 16 (G allele) and codon 92 (A allele) of gidB, indicating their belonging to the LAM and non-Beijing lineages. Similarly, all Beijing strains characterized by spoligotyping had the SNPs in codon 16 (T allele) and codon 92 (C allele) in gidB; thus, they were classified as non-LAM Beijing strains. However, since we have analyzed only samples from Brazil and Asian countries, it will be important to extend these studies to samples from other geographic regions.

One strain gave discordant results; it was classified as LAM3 and S/convergent sublineage by spoligotyping but as non-LAM by the LAM-specific PCR and by the gidB16 polymorphism. Spoligotypes evolve through successive loss of spacer DNA sequences (16), and since lineages are defined by spacer patterns, the independent loss of similar spacers sets can lead to the convergent evolution of spoligotypes (32). This is problematic, because not all strains may truly represent their designed spoligotype lineage (11). This may explain the discrepant result found in this study, indicating that a convergence of spoligotypes may have occurred. The same has been reported from a study by Gibson et al. (11), where strains classified as LAM by spoligotyping were considered non-LAM by LAM-specific PCR and SNP analysis.

When some spoligotyping patterns could not be assigned to an internationally recognized genotype lineage, we were able to differentiate these strains as LAM or non-LAM by the gidB16 polymorphism and LAM-specific PCR, indicating that spoligotyping alone is not always the best method to differentiate LAM strains.

In conclusion, we have described two SNPs in the gidB gene that allowed us to distinguish LAM and Beijing lineages from the other lineages for M. tuberculosis. The previously reported association of certain mutations in gidB with resistance to STR needs to be further investigated.

ACKNOWLEDGMENTS

This work was supported by CNPq and the Centro de Desenvolvimento Científico e Tecnológico, Fundação Estadual de Produção e Pesquisa em Saúde. F.S.S. is a recipient of a CNPq fellowship.

Footnotes

[down-pointing small open triangle]Published ahead of print on 18 May 2011.

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