Objective: Increasing reports of resistance to newer antituberculosis drugs have prompted the search for other alternative drugs. Streptomycin (STR) could be used for the treatment of drug-resistant tuberculosis if susceptibility of Mycobacterium tuberculosis isolate to STR could be accurately detected. We performed phenotypic and genotypic drug susceptibility testing (DST) of 118 M. tuberculosis isolates for STR. Materials and Methods: Fifty pansusceptible and 68 multidrug-resistant M. tuberculosis (MDR-TB) isolates were used. Phenotypic DST for STR, rifampicin, isoniazid, and ethambutol was performed by mycobacteria growth indicator tube 960 System. Genotypic DST was done by GenoTypeMTBDRplus assay for rifampicin and isoniazid and by PCR-sequencing of rpsL, rrs, and gidB genes for STR. MDR-TB isolates were genotyped by spoligotyping. Results: Phenotypic DST identified 50 isolates susceptible to all four drugs (pansusceptible). Sixty-one of 68 MDR-TB isolates were resistant to STR. Genotypic testing for rifampicin and isoniazid yielded expected results. Fifty pansusceptible and 7 STR-susceptible MDR-TB isolates contained no mutation in rpsL or rrs, while 47, 2, and 1 STR-resistant isolate contained rpsL, rrs, and rpsL + rrs mutations, respectively. Of the remaining 11 STR-resistant MDR-TB, 9 isolates contained deletion frame-shift/nonsynonymous mutations in gidB. Surprisingly, 13 pansusceptible isolates also contained deletion frame-shift/nonsense/nonsynonymous mutations in gidB. Also, 30 of 68 MDR-TB but only 2 of 50 pansusceptible isolates belonged to the Beijing genotype. Conclusions: Our data show that, like rifampicin, ethambutol, and pyrazinamide, STR also exhibits discordant phenotypic and genotypic DST results for some M. tuberculosis isolates. Hence, STR should be included in therapy regimens only if both phenotypic and genotypic resistance testing indicate susceptibility to avoid amplification of resistance and drug toxicity.

Journal Section:
Original Paper
Keywords:
Mycobacterium tuberculosis, Streptomycin, Phenotypic susceptibility testing, Genotypic susceptibility testing, rpsL, rrs, gidB, Mutations

Highlights of the Study

  • Phenotypic drug susceptibility testing for first-line drugs, rifampicin, pyrazinamide and ethambutol by mycobacteria growth indicator tube 960 system is imperfect.

  • Discordant results for phenotypic and genotypic drug susceptibility testing of streptomycin (STR) are also seen among M. tuberculosis isolates with mutations in gidB alone.

  • Both phenotypic and genotypic drug susceptibility testing should guide inclusion of STR in MDR-TB regimens to avoid amplification of resistance and toxic side effects.

Tuberculosis (TB), caused primarily by the obligate human pathogen, Mycobacterium tuberculosis, is still a major cause of morbidity and mortality worldwide. Although TB disease cases and deaths were declining in the last decade, they increased worldwide during 2020–2021 due to COVID-19 [1]. Nearly, 10.6 million people developed active TB disease (an increase of 4.5% from 10.1 million in 2020) and 1.6 million people died in 2021 (an increase of 6.6% from 1.5 million in 2020) [1]. One of the major factors contributing to the worldwide problem of TB is the increasing rate of resistance of M. tuberculosis to anti-TB drugs [2]. The global burden of rifampicin (RIF)-resistant (RR)-TB and multidrug-resistant TB (TB resistant to RIF and isoniazid [INH]; the two main first-line drugs) also increased between 2020 and 2021 and nearly 450,000 cases of RR-TB were estimated to have occurred in 2021 [1]. Nearly, 6.4% of MDR-TB cases were estimated to have pre-extensively drug-resistant (pre-XDR)-TB (i.e., MDR-TB additionally resistant to a fluoroquinolone) or XDR-TB (MDR-TB strains additionally resistant to a fluoroquinolone and bedaquiline or linezolid) [1].

Recognition of genotypic resistance determinants in M. tuberculosis isolates from patients without prior exposure to new/repurposed drugs, bedaquiline or clofazimine and rapid development of resistance to bedaquiline, linezolid, delamanid, clofazimine, and pretomanid in patients exposed to these drugs has prompted the search for other alternative drugs [2, 3]. Although streptomycin (STR) is no longer used as a first-line drug due to relatively higher rates of resistance among M. tuberculosis isolates and the availability of other active drugs, it could still be used as an alternative drug for the treatment of some MDR-TB patients [4, 5]. According to WHO updated treatment recommendations for longer regimens for RR/MDR-TB, STR may be used only as a substitute for amikacin in the following specific situations: when amikacin is not available, when there is confirmed resistance to amikacin but confirmed susceptibility to STR, and when an all-oral regimen cannot be constituted [2, 5]. Susceptibility to STR in MDR-TB strains susceptible to fluoroquinolones has been shown to improve median survival and higher treatment success rate among TB patients [2, 5].

STR binds to S12 ribosomal protein (rpsL) and 16S rRNA (rrs) and disrupts protein translation [2, 4]. Consequently, resistance to STR in M. tuberculosis isolates is mainly due to mutations at codon 43 and 88 in rpsL (high-level of resistance) and 500 and 900 regions of rrs (intermediate-level of resistance) [2, 6‒9]. Mutations in gidB encoding 7-methylguanosine methyltransferase which methylates g527 nucleotide of rrs or changes in efflux pumps also confer resistance to STR [2, 6‒12]. However, gidB mutations confer only low-level of STR resistance [2, 6‒10, 12]. Previous studies have shown that phenotypic drug susceptibility testing (DST) for first-line drugs, RIF and ethambutol (EMB) by mycobacteria growth indicator tube (MGIT) 960 system is imperfect as M. tuberculosis isolates with specific mutations conferring-borderline resistance are missed [13, 14]. Phenotypic DST for pyrazinamide (PZA), another first-line drug, is also problematic due to the requirement for acidic pH [14]. Recent studies have shown that low-level STR resistance due to gidB mutations is also often missed by phenotypic DST methods [6‒10, 12]. However, the extent of the discrepancy between phenotypic and genotypic resistance testing for STR is not fully defined as only few studies have simultaneously used STR-susceptible, and more importantly, pansusceptible M. tuberculosis (phenotypically susceptible to STR, RIF, INH, and EMB [SIRE]) isolates [8, 9, 12]. This information is critical if STR is to be used as an alternative drug for the treatment of RR/MDR-TB. We have previously described mutations in rpsL and rrs genes among some MDR-TB strains [15, 16]. This study detected resistance-conferring mutations in rpsL, rrs, and gidB genes among pansusceptible and STR-resistant and STR-susceptible MDR-TB isolates and correlated the findings with results of phenotypic DST.

Clinical M. tuberculosis Isolates

A total of 118 M. tuberculosis isolates were used. These included 68 MDR-TB strains and 50 pansusceptible M. tuberculosis isolates cultured from 85 pulmonary and 33 extrapulmonary samples obtained from 118 adult newly diagnosed TB patients, before initiation of treatment with anti-TB drugs, during 2005–2021. All specimens were collected after obtaining verbal consent as part of routine care for suspected TB patients visiting TB clinic (Kuwait National Tuberculosis Control Center, KNTCC) and were processed anonymously. Nationwide, nearly 800 culture-confirmed cases of active TB are diagnosed at KNTCC every year [17, 18]. Nearly, 80% of all active disease cases and nearly 90% of MDR-TB cases in Kuwait occur among expatriate patients mainly originating from TB endemic countries of south/southeast Asia and Africa [17, 18]. The clinical specimens were processed by using MGIT 960 system according to the manufacturer’s instructions (Becton Dickinson, Sparks, MD, USA), the presence of M. tuberculosis in growth-positive cultures was detected by Ziehl-Neelsen staining and by an in-house PCR, as described previously [19].

Phenotypic Drug Susceptibility Testing

M. tuberculosis cultures were subjected to phenotypic DST against STR (1 µg/mL), INH (0.1 µg/mL), EMB (5 µg/mL), and RIF (1 µg/mL) by using the MGIT 960 system and SIRE drug kit, according to the manufacturer’s recommendations and as described previously [19]. The M. tuberculosis isolates phenotypically susceptible to all four (SIRE) drugs were classified as pansusceptible isolates while isolates resistant to RIF and INH with/without additional resistance to other (STR and/or EMB) drugs were categorized as MDR-TB strains.

Molecular Detection of Resistance to RIF and INH

For genotypic characterization, the MGIT 960 system-positive cultures were used for the extraction of DNA by the rapid Chelex-100-based method, as described previously [20]. All isolates were tested by the GenoTypeMTBDRplus assay for detection of genotypic resistance to RIF and INH, as described previously [20]. PCR-sequencing of rpoB, katG, and inhA was also done for M. tuberculosis isolates with wild-type probe patterns and for isolates yielding resistance genotype by lack of hybridization with a wild-type probe only, as described previously [20].

Molecular Detection of Resistance to STR

Genotypic resistance testing for STR was performed by detecting resistance-associated mutations in rpsL, rrs (500 and 900 regions), and gidB genes. Gene-specific primers for PCR amplification and sequencing of target DNA are listed in online supplementary Table S1 (for all online suppl. material, see https://doi.org/10.1159/000538584). PCR amplification was performed, the amplicons were purified by using PCR product purification kit (Qiagen, Hilden, Germany) according to the kit instructions and bidirectional sequencing with gene-specific primers (online suppl. Table S1) was done, as described previously [16, 20].

Spoligotyping

The MDR-TB isolates were genotyped by spoligotyping method using SPOLIGO TB kit (Mapmygenome Co., Hyderabad, India), and the results were interpreted by following the instructions supplied with the kit. Phylogenetic lineages (spoligotypes) were assigned according to SITVIT database and MIRU-VNTRplus software, as described previously [16]. The gidB gene also contains lineage-specific polymorphisms. These include a276c + a615g (E92D + A205A) specific for lineage 2 (Beijing genotype) and g206a (G69D) for lineage 4 (Euro-American lineage) [6‒9, 12, 21‒23].

Statistical Analyses

The data were analyzed by using two-tailed Fisher’s exact test and probability values <0.05 were considered as significant. Statistical analyses were performed by using WinPepi software ver. 11.65 (PEPI for Windows, Microsoft Inc., Redmond, WA, USA). GraphPad Software (GraphPad, La Jolla, CA, USA) was also used and a Kappa coefficient (κ) value of 0–0.2, 0.21–0.4, 0.4–0.6, 0.61–0.8, and 0.81–1.0 indicated poor, fair, moderate, substantial, and almost perfect agreement, respectively.

Demographic Data of TB Patients and Clinical Specimens for M. tuberculosis Isolates

The demographic data of TB patients yielding STR-resistant MDR-TB (n = 61), STR-susceptible MDR-TB (n = 7), and pansusceptible M. tuberculosis (n = 50) isolates are shown in online supplementary Table S2, and the details of clinical specimens yielding the same three categories of isolates are shown in online supplementary Table S3. The isolates were cultured from newly diagnosed TB patients before initiation of treatment with anti-TB drugs. The presence of acid-fast bacilli in all 118 growth-positive MGIT 960 System tubes was confirmed by Ziehl-Neelsen staining. All 118 samples were also positive for M. tuberculosis complex DNA by PCR.

Results of Phenotypic DST

The phenotypic DST results against SIRE anti-TB drugs showed that 50 drug-susceptible M. tuberculosis isolates were susceptible to all four SIRE drugs tested (pansusceptible strains). Among 68 multidrug-resistant isolates, 5 isolates were resistant to RIF and INH only, 2 isolates were resistant to RIF, INH, and EMB, 29 isolates were resistant to STR, RIF, and INH, and 32 isolates were resistant to all four (SIRE) drugs. Thus, 61 of 68 MDR-TB strains were additionally resistant to STR.

Mutations Conferring Resistance to RIF and INH

The combined results of GenoTypeMTBDRplus assay and PCR-sequencing of rpoB, katG, and inhA confirmed simultaneous genotypic resistance to RIF and INH in all 68 MDR-TB strains. The rpoB mutations detected among MDR-TB isolates included S450L (n = 48), H445Y (n = 5), D435V (n = 3), S450W (n = 3), M434I + D435Y (n = 2) and one isolate each with V172F, Q432E, Q432P, H445R, H445N, H445L, and D435V + H445Q mutations. The INH-resistance-associated mutations detected among MDR-TB isolates included katG S315T (n = 61), inhA −15 c/t (n = 5), and katG S315T + inhA −15 c/t (n = 2). All 50 pansusceptible M. tuberculosis isolates yielded wild-type sequence patterns for rpoB, katG, and inhA, as expected.

Mutations Conferring Resistance to STR in rpsL, rrs, and gidB

Of 61 STR-resistant MDR-TB isolates, 36, 6, 4, and 1 isolate contained K43R, K88R, K88T, and K88M mutations, respectively, in rpsL only (Table 1). Two other isolates contained c517t mutation in rrs while 1 isolate contained two mutations (K88T mutation in rpsL and c517t mutation in rrs) (Table 1). The remaining 11 isolates contained wild-type sequence (no mutation) in rpsL and rrs (Table 1). The presence of K43R mutation was significantly associated with Beijing genotype (lineage 2) isolates (p = 0.001).

Table 1.

Phenotypic resistance patterns and mutations detected in rpsL, rrs, and gidB genes in 118 M. tuberculosis isolates

Phynotypic resistance patternMutation detected inRole of gidB mutation in STR resistanceM. tuberculosis isolates, n
rpsLrrsgidB
DNAproteinDNADNAprotein
STRr MDR-TB (n = 61) a128g K43R NM NM NM N. A. 1 
 a128g K43R NM a276c + a615g E92Da + A205Aa No 14 
 a128g K43R NM g330t + a615g V110V + A205A No 2 
 a263g K88R NM g206a G69Db No 1 
 a128g K43R NM N. D. N. D. N. A. 19 
 a263g K88R NM N. D. N. D. N. A. 5 
 a263c K88T NM N. D. N. D. N. A. 4 
 a263t K88M NM N. D. N. D. N. A. 1 
 a263g K88T c517t N. D. N. D. N. A. 1 
 NM NM c517t NM NM N. A. 1 
 NM NM c517t a615g A205A No 1 
 NM NM NM del g350 + a615g G117 fsc + A205A Yes 2 
 NM NM NM del t454c L152 fsc Yes 1 
 NM NM NM a1gc + c276tc M1Mc + P93Sc Yes 1 
 NM NM NM g217a G73Rc Likely 1 
 NM NM NM c301t + g330t + a615g L101F + V110V + A205A Likely 1 
 NM NM NM g91ac V31Mc Likely 1 
 NM NM NM g206a G69Db No 1 
 NM NM NM a615g A205A No 2 
 NM NM NM g206a + g330t + a615g G69Db + V110V + A205A No 1 
STRs MDR-TB (n = 7) NM NM NM g206a G69Db No 2 
 NM NM NM g330t + a615g V110V + A205A No 2 
 NM NM NM c270g L90L No 1 
 NM NM NM a276c + a615g E92Da + A205Aa No 1 
 NM NM  t104g + a276c L35Rc + V110V Likely 1 
Pansusceptible (n = 50) NM NM NM NM NM N. A. 7 
 NM NM NM a615g A205A No 8 
 NM NM NM g330t + a615g V110V + A205A No 18 
 NM NM NM g330t + g423a + a615g V110V + A141A + A205A No 2 
 NM NM NM a276c + a615g E92Da + A205Aa No 2 
 NM NM NM g192a + g330t + a615g R64R + V110V + A205A No 1 
 NM NM NM g330t + c378t + a615g V110V + D126D + A205A No 1 
 NM NM NM del c112 P38 fs Yes 1 
 NM NM NM del c112 + g330t + g423a + a615g P38 fs + V110V + A141A + A205A Yes 1 
 NM NM NM del g385 G129 fsc Yes 1 
 NM NM NM c66a + g330t + a615g Y22c, d + V110V + A205A Yes 1 
 NM NM NM c277t P93S Likely 1 
 NM NM NM g91a V31Mc Likely 1 
 NM NM NM a95t + g330t + a615g E32Vc + V110V + A205A Likely 1 
 NM NM NM g218a + g330t + a615g G73E + V110V + A205A Likely 1 
 NM NM NM g330t + g355a + a615g V110V + A119Tc + A205A Likely 2 
 NM NM NM g330t + g288t + a615g V110V + L196Fc + A205A Likely 1 
Phynotypic resistance patternMutation detected inRole of gidB mutation in STR resistanceM. tuberculosis isolates, n
rpsLrrsgidB
DNAproteinDNADNAprotein
STRr MDR-TB (n = 61) a128g K43R NM NM NM N. A. 1 
 a128g K43R NM a276c + a615g E92Da + A205Aa No 14 
 a128g K43R NM g330t + a615g V110V + A205A No 2 
 a263g K88R NM g206a G69Db No 1 
 a128g K43R NM N. D. N. D. N. A. 19 
 a263g K88R NM N. D. N. D. N. A. 5 
 a263c K88T NM N. D. N. D. N. A. 4 
 a263t K88M NM N. D. N. D. N. A. 1 
 a263g K88T c517t N. D. N. D. N. A. 1 
 NM NM c517t NM NM N. A. 1 
 NM NM c517t a615g A205A No 1 
 NM NM NM del g350 + a615g G117 fsc + A205A Yes 2 
 NM NM NM del t454c L152 fsc Yes 1 
 NM NM NM a1gc + c276tc M1Mc + P93Sc Yes 1 
 NM NM NM g217a G73Rc Likely 1 
 NM NM NM c301t + g330t + a615g L101F + V110V + A205A Likely 1 
 NM NM NM g91ac V31Mc Likely 1 
 NM NM NM g206a G69Db No 1 
 NM NM NM a615g A205A No 2 
 NM NM NM g206a + g330t + a615g G69Db + V110V + A205A No 1 
STRs MDR-TB (n = 7) NM NM NM g206a G69Db No 2 
 NM NM NM g330t + a615g V110V + A205A No 2 
 NM NM NM c270g L90L No 1 
 NM NM NM a276c + a615g E92Da + A205Aa No 1 
 NM NM  t104g + a276c L35Rc + V110V Likely 1 
Pansusceptible (n = 50) NM NM NM NM NM N. A. 7 
 NM NM NM a615g A205A No 8 
 NM NM NM g330t + a615g V110V + A205A No 18 
 NM NM NM g330t + g423a + a615g V110V + A141A + A205A No 2 
 NM NM NM a276c + a615g E92Da + A205Aa No 2 
 NM NM NM g192a + g330t + a615g R64R + V110V + A205A No 1 
 NM NM NM g330t + c378t + a615g V110V + D126D + A205A No 1 
 NM NM NM del c112 P38 fs Yes 1 
 NM NM NM del c112 + g330t + g423a + a615g P38 fs + V110V + A141A + A205A Yes 1 
 NM NM NM del g385 G129 fsc Yes 1 
 NM NM NM c66a + g330t + a615g Y22c, d + V110V + A205A Yes 1 
 NM NM NM c277t P93S Likely 1 
 NM NM NM g91a V31Mc Likely 1 
 NM NM NM a95t + g330t + a615g E32Vc + V110V + A205A Likely 1 
 NM NM NM g218a + g330t + a615g G73E + V110V + A205A Likely 1 
 NM NM NM g330t + g355a + a615g V110V + A119Tc + A205A Likely 2 
 NM NM NM g330t + g288t + a615g V110V + L196Fc + A205A Likely 1 

STR, streptomycin; MDR-TB, multidrug-resistant M. tuberculosis; STRr MDR-TB, STR-resistant MDR-TB; STRs MDR-TB; STR-susceptible MDR-TB; NM, no mutation; N. A., not applicable; N. D., not done; fs, frame-shift mutation.

aGenetic polymorphisms specific for the Beijing genotype (lineage 2).

bGenetic polymorphism specific for Euro-American lineage (lineage 4).

cNovel mutations detected in this study.

dNonsense (termination) mutation.

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Initially, the gidB gene was sequenced from 18 of 48 STR-resistant MDR-TB isolates with an rpsL mutation. Consistent with several previous reports [7, 9, 12, 21‒24], 17 isolates with K43R and 1 isolate with K88R mutation either contained wild-type sequence or lineage-specific polymorphisms or synonymous mutations only in gidB (Table 1). Consequently, PCR-sequencing of gidB was not done for the remaining 30 STR-resistant MDR-TB isolates containing K43R or K88R/T/M mutations. Two other isolates with c517t rrs mutation also either contained wild-type sequence or a synonymous (A205A) mutation only in gidB (Table 1). Of the remaining 11 STR-resistant MDR-TB isolates, 4 isolates contained synonymous and/or lineage-specific polymorphisms while 7 isolates contained deletion frame-shift (3 isolates) or other nonsynonymous mutations (4 isolates) in gidB (Table 1). Considering 3 deletion frame-shift mutations and 4 nonsynonymous mutations in gidB as indicative of STR resistance, 61 and 57 MDR-TB isolates were detected as STR-resistant by phenotypic and combined genotypic (rpsL, rrs, and gidB) resistance testing (p = 0.696), respectively. No rpsL or rrs mutation was detected in 7 STR-susceptible MDR-TB isolates; however, synonymous mutations or lineage-specific polymorphisms were found in 6 isolates while 1 isolate contained a nonsynonymous mutation (L35R) plus a synonymous (V110V) mutation in gidB (Table 1).

No rpsL or rrs mutation was detected in 50 pansusceptible M. tuberculosis isolates, as expected. However, gidB results were different. Seven isolates contained no mutation and another 32 isolates either contained lineage-specific polymorphisms or synonymous mutation(s) in gidB (Table 1). The remaining isolates (n = 11) contained various other mutations. These included deletion frame-shift mutations in 3 isolates, a nonsense mutation in 1 isolate and a nonsynonymous mutation in 7 isolates either alone or in combination with other synonymous mutation(s). The deletion frame-shift mutations included del 112c (C38 frame-shift) mutation in 1 isolate, del 112c + g330t + g423a + a615g (C38 frame shift + V110V + A141A + A205A) mutations in 1 isolate and del g385 (G129 frame-shift) mutation in 1 isolate (Table 1). The isolate with a nonsense mutation at codon 22 also contained two synonymous mutations (c66a + g330t + a615g [Y22* resulting in premature termination + V110V + A205A]) (Table 1). Six different nonsynonymous (V31M, E32V, G73E, P93S, A119T, and L196f) mutations with/without other synonymous mutation(s) were detected in 7 isolates (Table 1). Another interesting observation of our study was that 30 of 68 (44%) MDR-TB but only 2 of 50 (4%) pansusceptible isolates belonged to the Beijing genotype and this difference was statistically significant (p = 0.001). There was no correlation between a specific mutation with the gender or country of origin of TB patient or clinical specimen used as the number of isolates with a particular kind of gidB mutation were very small (1 or 2 isolates only in each case).

Overall, 45 and 57 isolates were detected as STR-susceptible and STR-resistant by both phenotypic and genotypic resistance testing, respectively, while 16 isolates yielded discrepant (12 isolates as STR-susceptible by phenotypic testing but STR-resistant by genotypic testing and 4 isolates as STR-resistant by phenotypic testing but STR-susceptible by genotypic testing) results (Kappa = 0.746, substantial agreement; 95% CI: 0.631–0.862). Furthermore, the presence of frame-shift, nonsense, or well-defined nonsynonymous mutations which will abrogate GidB protein function, likely contributed toward STR resistance in 7 of 11 (63.6%) phenotypically STR-resistant isolates but only among 12 of 57 (21%) phenotypically STR-susceptible isolates among 78 M. tuberculosis isolates lacking a mutation in rpsL or rrs gene and the difference was statistically significant (p = 0.008) (Table 2).

Table 2.

Contribution of gidB mutations among 78 M. tuberculosis isolates lacking an rpsL or rrs mutation and phenotypically detected as STR resistant or STR susceptible

No. of M. tuberculosis isolates detected asContribution of gidB mutation in STR resistance
STR susceptibleSTR resistant
4 4 Yes 
8 3 Likely 
45 4 None 
No. of M. tuberculosis isolates detected asContribution of gidB mutation in STR resistance
STR susceptibleSTR resistant
4 4 Yes 
8 3 Likely 
45 4 None 

STR, streptomycin.

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The results presented in this study showed that 48 of 61 (78.6%) STR-resistant MDR-TB isolates contained rpsL mutations with 34 of 61 (55.7%) isolates containing K43R mutation alone. The frequency of rpsL K43R mutation which confers high-level STR resistance is usually high in areas where the Beijing genotype (lineage 2) strains predominate [8, 9, 21‒23]. The frequency of rpsL K43R mutation in MDR-TB isolates from Kuwait was also significantly higher among Beijing genotype strains. Previous studies have also shown that M. tuberculosis isolates with rpsL mutations either contain wild-type sequence or synonymous mutations/lineage-specific polymorphisms in gidB [7, 9, 12, 21‒24]. This was also observed in Kuwait as all 18 STR-resistant MDR-TB isolates with an rpsL mutation for which gidB was sequenced either contained wild-type sequence or only lineage-specific polymorphisms. Furthermore, 30 of 68 (44%) MDR-TB isolates but only 2 of 50 (4%) pansusceptible isolates belonged to the Beijing genotype. The data are consistent with observations showing that Beijing genotype is strongly associated with multidrug-resistant phenotype [25].

Of 11 STR-resistant MDR-TB isolates with no rpsL/rrs mutation, deletion of a single nucleotide resulting in frame-shift of gidB was seen in 3 isolates. Frame-shift mutations, which abolish GidB function, have been reported frequently among clinical M. tuberculosis isolates lacking rpsL/rrs mutations and exhibit low-level STR resistance [6‒9, 12]. One isolate exhibited initiation codon (aug to gug) change, likely to reduce translation efficiency of gidB mRNA, together with P93S mutation. Other mutations at codon P93 (P93L or P93Q) have been described in some M. tuberculosis isolates exhibiting low-level STR resistance [9, 10, 26]. Among other nonsynonymous mutations with/without other synonymous mutation(s) detected in this study, G73R and L101F likely conferred STR resistance as these mutations have also been detected previously in some STR-resistant isolates lacking rpsL/rrs mutations [6, 22, 26, 27]. The role of V31M mutation in STR resistance in 1 isolate remains unknown as, to the best of our knowledge, this mutation has not been described previously. However, considering that GidB, a relatively small protein, is highly vulnerable to mutations as its active site consists of 16 amino acid residues spread over its entire length, many nonsynonymous mutations cause low-level STR resistance due to disruption of binding-site structure [26]. Biocomputational analyses and molecular dynamics simulations coupled with binding-free energy calculations have also shown that the active site topography is affected by several nonsynonymous mutations such as S70R, G76C, V89M, L101F, T146M, S149R, V171I, and R187M that are spread over nearly the entire length of this small protein [11, 27, 28]. Thus, V31M may also confer low-level STR resistance.

The G69D mutation in 2 STR-resistant MDR-TB isolates with/without other synonymous mutations likely represents a polymorphism connected with Euro-American lineage as it was also detected in another STR-resistant MDR-TB isolate containing K88R rpsL mutation and 2 STR-susceptible MDR-TB isolates. Only one previous study has reported G69D mutation in 1 STR-resistant MDR-TB isolate carrying rpsL K43R mutation which also belonged to Euro-American lineage [23]. The remaining 2 STR-resistant isolates only contained a synonymous (A205A) mutation in gidB. Taken together, 4 of 61 STR-resistant MDR-TB isolates did not contain a resistance-conferring mutation in rpsL/rrs/gidB. Other studies have also reported a minority of STR-resistant M. tuberculosis isolates with none or containing only synonymous mutations in rpsL/rrs/gidB [7, 9, 10, 21‒23], and the molecular basis of resistance in these isolates may involve other mechanisms such as efflux pumps [2, 4].

Among 7 STR-susceptible MDR-TB isolates, only 1 isolate contained nonsynonymous mutation L35R together with a synonymous (A205A) mutation not linked with a particular lineage. As explained above, L35R is also likely to be associated with STR resistance but was missed by the faulty phenotypic testing. The L35R mutation in gidB has also previously been detected in 1 STR-susceptible isolate [7].

The gidB results for 50 pansusceptible M. tuberculosis isolates were interesting. Although 39 isolates either contained wild-type sequence or lineage-specific polymorphisms or synonymous mutations in gidB, 11 isolates contained various other mutations. The 3 isolates with deletion frame-shift and 1 isolate with a nonsense mutation will lack a functional GidB and so are likely to exhibit low-level STR resistance, as has been shown for STR-resistant isolates in several previous studies [6‒10, 12]. Furthermore, V31M, P93S, and mutation at codon G73 (G73R) were also detected in STR-resistant MDR-TB isolates from Kuwait. Mutations at codons G73 and P93 causing low-level STR resistance have been described in other studies [6, 7, 9, 10, 12, 26]. The remaining nonsynonymous mutations E32V, A119T (detected in 2 isolates), and L196F have not been described previously in gidB. However, it is probable that these nonsynonymous mutations also likely disrupt the highly vulnerable binding-site structure of this relatively small protein [26] and thus confer low-level STR resistance which is missed by the faulty phenotypic DST.

The detection of 4 of 50 (8%) pansusceptible isolates carrying deletion frame-shift/nonsense gidB mutations which clearly abrogate GidB function and so confer low-level resistance to STR is rather surprising but can be explained as follows. Previous studies have shown that gidB is a hot-spot region for mutations on M. tuberculosis genome, and these mutations have little effect on fitness [29, 30]. One study has also recently shown that specific gidB mutations are transmitted at cluster level among cohorts of TB patients involved in outbreaks allowing evolution of low-to-high level STR resistance without significantly affecting fitness [31]. Furthermore, GidB, a small protein, has a substrate-binding site comprising 16 interacting amino acids and so many nonsynonymous mutations abrogate its function [26]. This is also supported by the relatively higher frequency of gidB mutations in STR-susceptible drug-resistant strains, particularly MDR-TB strains [7, 10, 21]. Since only few studies have analyzed pansusceptible M. tuberculosis isolates for gidB [8, 9, 12], gidB mutations have not been reported frequently among pansusceptible isolates. Another possible reason for the high frequency of gidB mutations among pansusceptible isolates detected in our study is the ethnic origin of the TB patients from Kuwait. All 11 pansusceptible isolates with gidB mutations were cultured from TB patients originating from India (n = 6), Bangladesh (n = 3), Nepal (n = 1), and Indonesia (n = 1) where TB, drug-resistant TB and MDR-TB are endemic [1] and resistance rates to anti-TB drugs, particularly STR, are high [32‒34]. Furthermore, none of the previous studies from these four countries on STR resistance have included pansusceptible/STR-susceptible M. tuberculosis isolates for mutations in gidB [11, 30].

A pertinent question arising out of the results described in this paper and other recent studies is the relevance of gidB mutations conferring low-level resistance on therapy of TB patients with STR. Accurate DST of M. tuberculosis isolates to anti-TB drugs is crucial for effective management of drug-resistant TB [1, 4, 5]. Discrepant DST results by phenotypic and genotypic methods have been reported recently for M. tuberculosis isolates with borderline resistance (minimum inhibitory concentration, MIC values close to critical concentration of the drug) for three first-line (RIF, EMB, and PZA) and some second-line drugs [13‒15, 35, 36]. The clinical impact of borderline resistance on patient outcome remains unknown for individual drugs in combination therapy except for RIF [14, 35, 36]. Recent studies have shown that treatment failure/relapse rates are same among TB patients infected with isolates with high-level RIF resistance due to well-known rpoB mutations or borderline resistance due to some specific (disputed) rpoB mutations [35, 36]. PZA is also not recommended in treatment regimens if genotypic DST results indicate resistance [37]. These observations strongly suggest that STR should also be included in treatment regimens for RR-/MDR-TB only if both phenotypic and genotypic resistance testing indicate susceptibility and thus usefulness of the drug to avoid amplification of resistance and toxic side effects [14, 31].

Our study has a few limitations. The MIC values for STR among M. tuberculosis isolates were not determined. The gidB gene was not sequenced from all isolates carrying an rpsL mutation. The clinical details, treatment given, and outcome were not available for TB patients as most of the patients were expatriate subjects who were sent back to their respective countries after initial treatment to achieve sputum smear-negative status was fulfilled.

Phenotypic DST of M. tuberculosis isolates against three first-line anti-TB drugs is imperfect. Discordance among phenotypic and genotypic resistance testing for RIF has been noted in isolates with specific rpoB mutations which cause only borderline resistance. However, treatment failure rates among isolates with high-level or borderline resistance for RIF are nearly same. Previous studies have shown that deletion frame-shift, nonsense, or well-defined nonsynonymous mutations in gidB also confer low-level of resistance to STR. The presence of such mutations in some pansusceptible M. tuberculosis isolates in Kuwait suggests that low-level STR resistance in these isolates was missed by the faulty phenotypic testing by MGIT 960 system. As a few phenotypically STR-resistant M. tuberculosis isolates also lacked a mutation in rpsL, rrs, or gidB, both phenotypic and genotypic resistance testing should guide inclusion of STR in therapy regimens to avoid amplification of resistance and toxic side effects.

The study was approved by the Ethical Committee of the Health Sciences Center, Kuwait University (Approval No. VDR/EC/3451).

The authors have no conflicts of interest to declare.

This study was funded by the Department of Microbiology, College of Medicine, and Research Core Facility grant SRUL02/13, Kuwait University.

Noura M. Al-Mutairi, Suhail Ahmad, and Eiman Mokaddas designed the study. Noura M. Al-Mutairi performed the experiments and collected the data. Noura M. Al-Mutairi and Suhail Ahmad analyzed the data and wrote the first draft of the manuscript. All the authors edited and approved the final version of the manuscript.

The DNA sequencing data for rpsL, rrs, and gidB reported in this study have been submitted to GenBank under Accession No. OR633395-OR633431 and European Nucleotide Archive (ENA) under the accession numbers LR782538 and LR782098 to LR782121 and are freely available. All other data are available in the manuscript and the information will be provided by the corresponding author upon reasonable request.

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