Abstract
Streptomycin (STR) is a component of first-line drugs used to treat multidrug-resistant tuberculosis. The purpose of this study was to investigate the proportion and type of mutations in Mycobacterium tuberculosis isolates resistant to STR and their relationship with the STR-resistant phenotype and with the epidemiological molecular model of the isolates. A total of 302 clinical isolates, including 215 STR-resistant and 87 STR-susceptible isolates, were characterized using the proportion method with Lowenstein-Jensen medium. The genes rpsL, rrs and gidB were screened for mutations using DNA sequencing methodology. All strains were genotyped using the spoligotyping technique. Mutations in rpsL and in rrs were observed in 63.3% and 15.8% of the STR-resistance isolates, respectively. The most prevalent mutations were the Lys43Arg substitution in the rpsL gene and the A514C change in the rrs gene. Ten novel mutations were identified in gidB. These novel mutations might be new potential markers for predicting STR-resistance in clinical Mycobacterium tuberculosis isolates. Mutations in rpsL, rrs, and gidB had a sensitivity of 84.2% and a specificity of 77.0% for the detection of STR-resistance isolates. The Beijing lineage strains were associated with the rpsL mutation Lys43Arg (P = 0.051), as well as the dual gidB mutations Glu92Asp and Ala205Ala (P < 0.001). Our study suggested that rpsL and rrs can act as useful genetic markers for predicting STR-resistance, and gidB polymorphisms play an important role in STR-resistant clinical Mycobacterium tuberculosis isolates from Hebei, China.
Keywords: Mycobacterium tuberculosis, streptomycin, gene mutation, Spoligotyping
Introduction
Tuberculosis (TB) is a devasting infectious disease globally caused by Mycobacterium tuberculosis. According to the recent WHO report, about 107,000 newly infected TB cases are estimated to emerge, and 28,000 cases with TB die in China [1]. The spread of TB, especially drug-resistant TB (DR-TB) and multidrug-resistant TB (MDR-TB, defined as resistance to rifampicin and isoniazid), produces a real challenge for the prevention and treatment of this fatal disease. Streptomycin (STR) was the first aminocyclitol glycoside antibiotic for the treatment of TB in 1940s. It is still included in the first-line TB treatment prescription (isoniazid, rifampicin, ethambutol and STR) for retreated patients. However, the monotherapy regimen, due to its resistance and toxicity, is gradually replaced by a short course of MDR-TB regimens [2,3].
STR targets the 16S rRNA of the 30S ribosomal subunit, interfering with the protein synthesis with subsequent bacteria death [4]. Although the molecular mechanisms of STR-resistance have not been fully elucidated, several studies have linked them to distinct mutations in various genetic loci of the Mycobacterium tuberculosis genome. Mutations in three genes, namely rpsL, rrs, and gidB, coding for the S12 ribosomal protein, 16S rRNA and 16S rRNA specific methyltransferase, respectively, are thought to be involved in conferring resistance to streptomycin (STR) in clinical Mycobacterium tuberculosis isolates [5,6]. The majority of the mutations at rpsL and rrs are responsible for high-level or intermediate-level STR-resistance [7]. Approximately, 70% of the mutations of STR- resistance isolates were detected in codon 43 and 88 of rpsL and in the 530 loop and the 912 region of rrs [8,9]. Mutations in the gidB gene commonly confer a low-level of STR resistance [10].
In this study, we identified 302 DR strains in sputum cultures using conventional drug sensitivity tests. All isolates were sequenced for STR-resistance associated-mutations in rpsL, rrs and gidB. To the best of our knowledge, this is the first report of a description of the mutations in three typical genetic loci related with STR-resistance in clinical isolates from Hebei Province. In addition, the aim of this study was also to explore the relationship between the polymorphisms of STR-resistant associated-genes and the spoligotyping patterns in STR-resistant clinical Mycobacterium tuberculosis isolates from Hebei Province, China.
Materials and methods
Mycobacterium tuberculosis strains
From January to December 2014, a total of 1,017 Mycobacterium tuberculosis strains were isolated from individual patients with pulmonary TB across Hebei Province (Shijiazhuang, Xingtai, Handan, Hengshui, Baoding, Tangshan Cangzhou and Qinhuangdao). Among the 1,017 Mycobacterium tuberculosis strains, 302 DR strains were successfully revived in Lowenstein-Jensen (L-J) medium after 3-4 weeks.
Drug sensitivity test
A drug sensitivity test (DST) against the first-line drugs was performed using the traditional proportion method on an L-J medium incubated for 4 weeks, with the following cut-off concentrations: isoniazid (INH), 0.2 μg/ml; rifampicin (RFP), 40 μg/ml; ethambutol (EMB), 2 μg/ml; and STR, 4 μg/ml. Resistance was defined as the growth ≥ 1% of bacterial colonies on the drug-containing medium compared with the drug-free control culture. H37Rv was used as a growth control for each DST.
DNA extraction, PCR amplification and sequencing
Genomic DNA was extracted from Mycobacterium tuberculosis cultures on a slant of L-J medium. One loopful of the bacillus was suspended in a fast lysis buffer (DNA bacteria kit, Qiagen, Germany) boiling for 10 minutes. After centrifugation for 5 minutes at 13,000 × g, the supernatant was transferred to a fresh tube and stored at -20°C for further use. PCR amplification and sequencing of rpsL, rrs and gidB was performed using specific primers either based on previously reported studies or newly designed methods in the present study (Table 1). Each 20 μL PCR mixture contained 10 μL 2 × Taq Master Mix (CWBOIO, Beijing, China), 1 μL of the forward and reverse 10 μM primers, 1 μL genomic DNA and final 7 μL distilled H2O complement. The reaction conditions consisted of a denaturation step of 5 min at 94°C , followed by 35 cycles of 30 s at 94°C, 30 s at 60°C, 30 s at 72°C, and a completely final extension at 72°C for 10 min. All PCR products were sent to TsingKe Biological Technology Company (TsingKe, Beijing, China) for sequencing. All the sequence data were aligned and analyzed with the reference Mycobacterium tuberculosis H37Rv strain (NC_000962.3) performed by BioEdit version 7.2.5 software.
Table 1.
Primers used for PCR and sequencing in this study
| Locus | Sequence (5’-3’) | Product size (bp) | Reference |
|---|---|---|---|
| rpsL-F | GGCGGCTTACGCTTGATG | 608 | This study |
| rpsL-R | TCCGTAGACCGGGTCGTTG | ||
| rrs-F | CGGCCACACTGGGACTGA | 724 | This study |
| rrs-R | AGGCCACAAGGGAACGCCT | ||
| gidB-F | GTCCCTCCACTCGCCATC | 675 | [11] |
| gidB-R | GCGGAGTGCGTAATGTCTC |
Resolution of discrepant results
When discrepant results occurred between DST and DNA sequencing, repeat testing was performed using both methods. If the repeated result conflicted with the original data, a third round of testing was conducted, with the two concordant results accepted as the final value.
Spoligotyping
All Mycobacterium tuberculosis isolates were subjected to spoligotyping as previously described [12]. In brief, this method used the primers DRa (5’-GGTTTTGGGTCTGACGAC-3’) and DRb (5’-CCGAGAGGGGACGGAAAC-3’) to amplify the direct region of Mycobacterium tuberculosis, and the PCR products were hybridized to a hybrid membrane immobilized with 43 spacer oligonucleotide probes. The identification results were entered in an excel sheet and matched with the database SpolDB4.0 (http://www.pasteur-uadeloupe.fr:8081/SITVITDemo/index.jsp).
Statistical analysis
All analyses were carried out using the SPSS version 22.0 (SPSS Inc. Chicago, IL, USA) statistical software package. A comparison of the differences between variables (DST patterns, individual mutations, Spoligotyping lineages) using the χ2 test or Fisher’s exact test. The differences were considered statistically significant when the P-value < 0.05.
Results
DST profiles
Of the 302 resistant bacterial strains, 33 isolates (33/302, 10.9%) were resistant to INH alone and 16 isolates (16/302, 5.3%) were resistant to RFP alone, and 5 isolates (5/302, 1.7%) were resistant to EMB alone. Furthermore, 44.7% of the isolates (135/302) tested were MDR, with 17.2% (52/302) being resistant to all four first-line agents. A total of 71.2% of the isolates (215/302) showed some resistance to STR, while 28.8% isolates (87/302) were susceptible to STR (Table 2).
Table 2.
Drug susceptibility characteristics in different spoligotyping families obtained from 302 clinical Mycobacterium tuberculosis isolates
| Characteristic | No. (%) of different genotyping isolates | ||||||
|---|---|---|---|---|---|---|---|
|
| |||||||
| Beijing | T | H | LAM | MANU | U | New | |
| Mono drug-resistance | |||||||
| STR | 39 (10.5) | 3 (13.6) | 2 (50.0) | 0 (0.0) | 1 (50.0) | 0 (0.0) | 2 (100.0) |
| INH | 30 (8.0) | 2 (9.1) | 1 (25.0) | 0 (0.0) | 0 (0.0) | 0 (0.0) | 0 (0.0) |
| RFP | 16 (4.3) | 0 (0.0) | 0 (0.0) | 0 (0.0) | 0 (0.0) | 0 (0.0) | 0 (0.0) |
| EMB | 3 (0.8) | 2 (9.1) | 0 (0.0) | 0 (0.0) | 0 (0.0) | 0 (0.0) | 0 (0.0) |
| Poly drug-resistance | |||||||
| STR+INH | 29 (7.8) | 1 (4.5) | 0 (0.0) | 0 (0.0) | 0 (0.0) | 0 (0.0) | 0 (0.0) |
| STR+RFP | 13 (3.5) | 2 (9.1) | 0 (0.0) | 0 (0.0) | 0 (0.0) | 0 (0.0) | 0 (0.0) |
| STR+EMB | 6 (1.6) | 0 (0.0) | 0 (0.0) | 0 (0.0) | 0 (0.0) | 0 (0.0) | 0 (0.0) |
| INH+EMB | 0 (0.0) | 0 (0.0) | 0 (0.0) | 0 (0.0) | 0 (0.0) | 0 (0.0) | 0 (0.0) |
| RFP+EMB | 6 (1.6) | 0 (0.0) | 0 (0.0) | 0 (0.0) | 0 (0.0) | 0 (0.0) | 0 (0.0) |
| STR+RFP+EMB | 6 (1.6) | 1 (4.5) | 0 (0.0) | 0 (0.0) | 0 (0.0) | 0 (0.0) | 0 (0.0) |
| STR+INH+EMB | 2 (0.5) | 0 (0.0) | 0 (0.0) | 0 (0.0) | 0 (0.0) | 0 (0.0) | 0 (0.0) |
| Overall multi-drug resistance | |||||||
| INH+RFP | 21 (5.6) | 1 (4.5) | 0 (0.0) | 0 (0.0) | 0 (0.0) | 0 (0.0) | 0 (0.0) |
| INH+RFP+STR | 52 (13.9) | 2 (9.1) | 1 (25.0) | 0 (0.0) | 0 (0.0) | 1 (100.0) | 0 (0.0) |
| INH+RFP+EMB | 5 (1.3) | 0 (0.0) | 0 (0.0) | 0 (0.0) | 0 (0.0) | 0 (0.0) | 0 (0.0) |
| INH+RFP+STR+EMB | 47 (12.6) | 3 (13.6) | 0 (0.0) | 1 (100.0) | 1 (50.0) | 0 (0.0) | 0 (0.0) |
| Total | 269 | 17 | 4 | 1 | 2 | 1 | 2 |
Abbreviations: STR, streptomycin; INH, isoniazid; RFP: rifampicin; EMB, ethambutol.
rpsL
Six types of point mutations were identified in the rpsL gene of 136 (136/215, 63.3%) STR-resistance isolates (Table 3). The mutated position at 128 (A-G) (115/215, 53.5%) was the most frequent site, while 263 (A-G) (17/215, 7.9%) was the next most common mutation. Of the remaining four mutation types, 128 (A-C), 128 (A-T), 129 (G-T), and 262 (A-C) were found in one isolate, respectively.
Table 3.
Distribution of mutations in rpsL and rrs conferring the spoligotype of 302 clinical Mycobacterium tuberculosis isolates
| Locus | Mutation patterns | No. of isolates | No. of genotype isolates | ||||
|---|---|---|---|---|---|---|---|
|
|
|
|
|||||
| DNA | Protein | STR-S | STR-R | MDR-TB | Beijing | Non-Beijing | |
| rpsL | AAG128AGG | Lys43Arg | 4 | 115 | 0 | 115 | 4 |
| AAG128ACG | Lys43Thr | 0 | 1 | 66 | 1 | 0 | |
| AAG128ATG | Lys43Met | 1 | 1 | 1 | 2 | 0 | |
| AAG129AAT | Lys43Asn | 0 | 1 | 0 | 1 | 0 | |
| AAG262CAG | Lys88Gln | 1 | 1 | 1 | 2 | 0 | |
| AAG263AGG | Lys88Arg | 2 | 17 | 7 | 19 | 0 | |
| rrs | A514T | 2 | 1 | 1 | 2 | 1 | |
| A514C | 3 | 26 | 12 | 29 | 0 | ||
| C517T | 2 | 5 | 2 | 6 | 1 | ||
| G848T | 0 | 1 | 1 | 1 | 0 | ||
| A906G | 0 | 1 | 0 | 1 | 0 | ||
| Total | 15 | 170 | 91 | 179 | 6 | ||
Abbreviations: STR, streptomycin; S, sensitive; R, resistant.
rrs
For rrs, nucleotide changes were observed in 34 (34/215, 15.8%) STR-resistance isolates (Table 3). Base substitutions at nucleotide positions 514 (A-T/C) and 517 (C-T) of rrs 530 loop were observed in 27 (27/215, 12.6%) isolates and 5 (5/215, 2.3%) isolates, respectively. The mutations of the 912 region occurred at nucleotide position 848 (G-T) in one isolate and 906 (A-G) in another isolate. Mutations in rrs and rpsL were not co-occurring in both STR-resistant and STR-susceptible isolates.
gidB
In gidB, 19 different types of mutations were identified in both the STR-resistant and STR- susceptible strains (Table 4). Among them, the changes were present in other studies such as codons 16 (Leu-Arg), 20 (Arg-Glu), 79 (Leu-Ser), 86 (Leu-Phe), 163 (Lys-Glu), and 178 (Met-Ile), et al. Apart from those canonical mutations in gidB, mutation at codons 74 (Leu-Ser), 77 (Val-Ala), 85 (Asp-His), 91 (Leu-Arg), 166 (Arg-Trp), 174 (His-Gln) were newly identified in 10 (10/215, 4.7%) isolates. A new silent mutation, codon 7 (Ala-Ala), was observed in only one STR-resistant isolate. The frameshifts included eight isolates in which only a single nucleotide deletion or insertion was detected. We also found that mutations at codons 92 (Glu-Asp) and 205 (Ala-Ala) were identified in 197 (197/302, 65.2%) STR-resistance isolates and 83 (27.45%, 83/302) STR-susceptible isolates. Nine strains were correlated with STR resistance, since they co-occurred with mutations in the rpsL or rrs loci.
Table 4.
Spoligotyping lineage conferring in gidB mutations among 302 clinical Mycobacterium tuberculosis isolates
| Codon | DNA/Protein changes | STRR | STRS | Spoligotyping lineages | Spoligotype description octonary | No. of isolates | Discovery |
|---|---|---|---|---|---|---|---|
| 7 | GCG-GCA/Silent | 1 | 0 | Beijing | 000000000003771 | 1 | Novel |
| 74 | TTG-TCG/Leu-Ser | 1 | 0 | Beijing | 000000000003731 | 1 | Novel |
| TTG-TTC/Leu-Phe | 1 | 0 | Beijing | 000000000003771 | 1 | Novel | |
| 77 | GTG-GCG/Val-Ala | 0 | 1 | Beijing | 000000000003771 | 1 | Novel |
| 85 | GAC-CAC/Asp-His | 0 | 1 | H3 | 777777777720771 | 1 | Novel |
| 91 | CTA-CGA/Leu-Arg | 1 | 0 | T3 | 577737777760771 | 1 | Novel |
| 166 | CGG-TGG/Arg-Trp | 0 | 1 | Beijing | 000000000003771 | 1 | Novel |
| 174 | CAC-CAG/His-Gln | 1 | 1 | Beijing | 000000000003771 | 2 | Novel |
| 194 | 582A insertion/Frameshift | 1 | 0 | T2 | 777777777760731 | 1 | Novel |
| 199 | 597C deletion/Frameshift | 0 | 1 | Beijing | 000000000003771 | 1 | Novel |
| 16 | CTT-CGT/Leu-Arg | 1 | 0 | LAM1 | 407777607760771 | 1 | Reported |
| 20 | CGG-GGG/Arg-Glu | 1 | 0 | Beijing | 000000000003771 | 1 | Reported |
| 34 | 102G deletion/frameshift | 1 | 0 | Beijing | 000000000003771 | 1 | Reported |
| 39 | 115C deletion/frameshift | 1 | 0 | T1 | 777777777760771 | 1 | Reported |
| 79 | TTG-TCG/Leu-Ser | 1 | 0 | T2 | 777777777760731 | 1 | Reported |
| 86 | CTC-TTC/Leu-Phe | 2 | 1 | Beijing | 000000000003771 | 3 | Reported |
| 117 | 351G deletion/Frameshift | 2 | 0 | Beijing | 000000000003771 | 2 | Reported |
| 118 | 352G insertion/Frameshift | 1 | 0 | Beijing | 000000000003771 | 1 | Reported |
| 352G insertion/Frameshift | 1 | 0 | New | 037777777740731 | 1 | Reported | |
| 163 | AAA-GAA/Lys-Glu | 1 | 0 | H3 | 777777677720771 | 1 | Reported |
| 178 | ATG-ATA/Met-Ile | 2 | 0 | Beijing | 000000000003771 | 2 | Reported |
| 92, 205 | GAA-GAC/Glu-Asp, GCA-GCG/Silent | 193 | 81 | Beijing | 000000000003771 | 274 | Reported |
| GAA-GAC/Glu-Asp, GCA-GCG/Silent | 2 | 1 | T1 | 777777777760771 | 3 | Reported | |
| GAA-GAC/Glu-Asp, GCA-GCG/Silent | 1 | 1 | T2 | 777777777760731 | 2 | Reported | |
| GAA-GAC/Glu-Asp, GCA-GCG/Silent | 1 | 0 | MANU2 | 777777777763771 | 1 | Reported |
Abbreviations: R, resistance; S, sensitive.
Evaluation of rpsL, rrs, and gidB by DNA sequencing
Based on the phenotypic data, the sensitivity and specificity of the rpsL, rrs, and gidB mutations for predicting STR-resistance were 63.3% and 90.8%, 15.8% and 92.0%, and 9.3% and 93.1%, respectively. The detection of STR-resistance with mutations in the rpsL and rrs had a sensitivity of 79.1% and a specificity of 82.8%, respectively. DNA sequencing of the rpsL, rrs and gidB was 84.2% sensitive and 77.0% specific for the detection of STR-resistance isolates (Table 5).
Table 5.
Evaluation of the DNA sequencing analysis of rpsL, rrs and gidB and phenotypic drug susceptibility testing
| STR phenotypic results | No. of isolates with the indicated result by DNA sequencing | |||||||||
|---|---|---|---|---|---|---|---|---|---|---|
|
| ||||||||||
| rpsL | rrs | gidB | rpsL, rrs | rpsL, rrs and gidB | ||||||
|
|
|
|
|
|
||||||
| WT | MT | WT | MT | WT | MT | WT | MT | WT | MT | |
| R | 79 | 136 | 181 | 34 | 195 | 20 | 45 | 170 | 34 | 181 |
| S | 79 | 8 | 80 | 7 | 81 | 6 | 72 | 15 | 67 | 20 |
| Sensitivity (%) | 63.3 | 15.8 | 9.3 | 79.1 | 84.2 | |||||
| Specificity (%) | 90.8 | 92.0 | 93.1 | 82.8 | 77.0 | |||||
Abbreviations: STR, streptomycin; WT, wild type; MT, mutant type; R, resistance; S, sensitive.
Spoligotyping
For these 302 clinical Mycobacterium tuberculosis isolates, the Beijing lineage (275/302, 91.1%) was the majority clade, followed by T (17/302, 5.6%), H (4/302, 1.3%), MANU (2/302, 0.6%), New (2/302, 0.6%), U (1/302, 0.3%), and LAM (1/302, 0.3%) (Table 2). The statistical analysis showed that the Beijing lineage existed in 194 (194/215, 90.2%) STR-resistant isolates and 81 (81/87, 93.1%) STR-susceptible isolates, revealing no significant association with STR-resistance (P = 0.051). No associations were observed between any mutations and the Beijing lineage, but rpsL Lys43Arg was predominant among the Beijing lineage isolates (115/275, 41.8%) (P = 0.007), but their presence was significantly lower among non-Beijing lineage isolates. Also, a majority of dual-mutation Glu92Asp (99.6%, 274/275) and Ala205Ala (99.6%, 274/275) was considered to belong to the Beijing lineage (P < 0.001).
Discussion
Hebei lies in the large Beijing-Tianjin region that plays a crucial role in the economy of northern China. In this population mobility, special location and environmental conditions contribute to the spread of DR-TB. Pathogenic monitoring and regular surveillance of drug-resistance based on conventional DST is important in understanding the trend of drug resistance to TB drugs in Hebei Province. Among the 306 DR strains with first-line drug resistance, 71.2% were resistant to STR, and 44.7% of strains had MDR. The drug resistance rate to STR was similar to Kuwait, North Korea, Thailand, and Iran [13-16]. The high prevalence rate of our research revealed that STR susceptibility testing is necessary before establishing appropriate anti-TB therapeutic schemes.
Previous studies have indicated that most STR-resistance Mycobacterium tuberculosis isolates can be determined by mutations in rpsL, rrs and gidB [10,17,18]. In a meta-analytical study of 1372 STR-resistant strains done by sequencing the rpsL gene, an estimated 64.0% frequency was found, which is equivalent to our finding of 63.3% frequency [19]. Nevertheless, a mutation at codon 43 (Lys-Arg) in the rpsL gene was shown to have a prevalence of 53.5%. A high detection rate of rpsL 43 mutations has been reported from Thailand, Iran, and Singapore [15,16,20]. Codon 43 in rpsL has been generally reported in both STR-resistance and MDR isolates and has thus been concluded to be a genetic marker of STR resistance. Another interesting finding, the uniqueness of mutation Lys43Arg in the rpsL gene was overwhelmingly found in Beijing lineage isolates, so it can be speculated that the rpsL mutation Lys43Arg in the broad transmission of the Beijing lineage may be due to the evolutionary advantage under STR pressure, which was in accordance with the preceding ones [21,22].
After mutations in rpsL, mutations in the rrs gene, including the 530 loop and the 912 region, showed the second most common mechanism of drug resistance to STR. In this study, mutations in rrs were found in 15.8% of the STR-resistance isolates, within the range of 3.5%-44% [23-25]. Substitutions at nucleotide positions 514 (A-T/C) and 517 (C-T) of the rrs 530 loop were observed in 12.6% of the isolates and 2.3% of the isolates, respectively. It should be noted that the uniqueness of the substitutions has been frequently reported [26,27]. One isolate was found at a nucleotide change at position 906 (A-G) in our study, and this position could be responsible for the STR-resistance. This result is supported by Sun et al. [28]. Our study also demonstrated that no rrs mutations occurred in conjunction with mutations in rpsL, because of their incompatibility.
Recently, several studies implied that gidB mutations conferring low-level resistance which functions for the methylation of G527 in 530 loop of 16S rRNA [10,17,29]. A total of 19 types of mutations were detected in both STR-susceptible and STR-resistant strains. This highly polymorphic nature of gidB gene was also observed in earlier studies [29,30]. Codons 16 (Leu-Arg), 20 (Arg-Glu), 79 (Leu-Ser), 86 (Leu-Phe), 163 (Lys-Glu), 78 (Met-Ile), and so on, have been repeatedly reported on before [15,16,21,23,28,31]. In our study, seven types of mutations, including codons 7 (Ala-Ala), 74 (Leu-Ser), 77 (Val-Ala), 85 (Asp-His), 91 (Leu-Arg), 166 (Arg-Trp) and 174 (His-Gln), were newly identified in both STR-resistance and STR-susceptible isolates. Further work is required to determine the role of these mutations in the development of STR resistance. Eight isolates had a single-base insertion or deletion, i.e. codons 102, 115, 351, 352, 582, and 597, causing a frameshift mutation. The higher risk of frameshift mutations we observed in STR-resistance suggests that the potential low-level STR-resistance may be attributed to frameshift mutations. Interestingly, a type of double substitution mutation in gidB was identified in the Beijing lineage. A high proportion of dual-mutation Glu92Asp and Ala205Ala (99.6%, 274/275) was observed in strains with the Beijing lineage, showing a significant association between dual-mutation Glu92Asp and Ala205Ala and Beijing lineage (P < 0.001). Consequently, the occurrence of dual-mutations at codon 92 and codon 205 was used as a specific marker for predicting the Beijing lineage phylogenetic strains.
In our study, we performed a DNA sequencing technique to evaluate the accuracy of rpsL, rrs, and gidB for the rapid detection of STR-resistance. Detection of STR-resistance with mutations in the rpsL and rrs exhibited a sensitivity of 79.1%, which is similar to the level found in previous reports from Japan (77.8%) [32], but higher than the level reported from Mexico (48.0%) [33]. The combination of mutations in rpsL, rrs, and gidB could detect 84.2% STR-resistance. This was in accordance with previous studies [24,28]. Using a combination of three loci, the DNA sequencing assay is able to achieve the best evaluation for predicting STR-resistance. The specificity for the detection of STR-resistance by the three loci analysis was 77.0%, which was lower than the value obtained in other studies [24]. Regional variation may explain this discrepancy.
Conclusions
To conclude, we explored the types of mutation in rpsL, rrs, and gidB genetic loci reported to be associated with STR-resistance in 302 clinical Mycobacterium tuberculosis isolates circulating in Hebei. Here, the mutation frequencies of rpsL and rrs were 63.3% and 58.0%, respectively. For gidB, we discovered ten different types of mutations not previously reported, which may serve as new potential bio-markers of STR-resistance in Mycobacterium tuberculosis. Also, we demonstrated that Lys43Arg was closely associated with the Beijing lineage. Moreover, gidB polymorphisms at codons Glu92Asp or Ala205Ala were found to be signatures for the Beijing lineage rather than STR-resistance markers. Furthermore, the absence of mutations in any rpsL, rrs, and gidB genes investigated in 34 STR-resistant strains from this study implies that other mechanisms may be involved, such as the overexpression of drug efflux or drug target alteration [7]. Further studies are required to confirm this speculation. Knowledge of the frequency and distribution of STR-resistance conferring mutations guides the advancement of rapid methods for detecting STR-resistance and the development of lineage-specific markers for the epidemiological survey of TB in clinically and geographically diverse settings.
Acknowledgements
This work was supported by the National Key Program of Mega Infectious Disease of the 12th Five Year Plan (Grant no. 2015ZX10004801) and the Scientific Research Fund Project of Health Department from Hebei Province (Grant no. 20130650). We gratefully thank the staffs from the laboratory of Shijiazhuang Fifth Hospital for supplying clinical profiles, strains and DNA samples. We also thank the staffs of the Chinese Centre for Disease Control and Prevention for their excellent technical assistance.
Disclosure of conflict of interest
None.
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