rpsL Mutations Research Articles

Abattoir survey of bovine tuberculosis in tanta, centre of the Nile delta, with in silico analysis of gene mutations and protein-protein interactions of the involved mycobacteria.

Bovine tuberculosis is a transboundary disease of high economic and public health burden worldwide. In this study, post-mortem examination of 750 cattle and buffalo in Tanta abattoir, Centre of the Nile Delta, revealed visible TB in 4% of animals and a true prevalence of 6.85% (95% CI: 5.3%-8.9%). Mycobacterial culture, histopathology and RT-PCR targeting all members of M.tuberculosis complex were performed, upon which 85%, 80% and 100% of each tested lesions were confirmed as TB, respectively. Mpb70-targeting PCR was conducted on ten RT-PCR positive samples for sequencing and identified nine Mycobacterium (M.) bovis strains and, interestingly, one M. tuberculosis (Mtb) strain from a buffalo. Bioinformatics tools were used for prediction of mutations, nucleotide polymorphisms, lineages, drug resistance and protein-protein interactions (PPI) of the sequenced strains. The Mtb strain was resistant to rifampicin, isoniazid and streptomycin, and to the best of our knowledge, this is the first report of multidrug resistant (MDR)-Mtb originating from buffaloes. Seven M.bovis strains were resistant to ethambutol and ethionamide. Such resistances were associated with KatG, rpoB, rpsL, embB and ethA genes mutations. Other mutations and nucleotide polymorphisms were also predicted, some are reported for the first time and require experimental work for validation. PPI revealed more interactions than what would be expected for a random set of proteins of similar size and had dense interactions between nodes that are biologically connected, as a group. Two M.bovis strains belonged to BOV AFRI lineage (Spoligotypes BOV 1; BOV 2) and eight strains belonged to East-Asian (Beijing) lineage. In conclusion, visible TB was prevalent in the study area, RT-PCR is the best to confirm the disease, MDR-Mtb is associated with buffalo TB, and mycobacteria of different lineages carry many resistance genes to chemotherapeutic agents used in treatment of human TB constituting a major public health risk.

Drug-sensitivity test and analysis of drug-resistant mutations in Mycobacterium tuberculosis isolates from Kashgar, China

Introduction In recent years, drug-resistant Mycobacterium tuberculosis strains have gradually become widespread. Most drug resistance is related to specific mutations. We investigated M. tuberculosis drug resistance in the Kashgar area, China. Methods The drug-susceptibility test was conducted to clinical isolates of M. tuberculosis. Genomic-sequencing technology was used for the drug-resistant strains and the significance of DNA sequencing as a rapid aid for drug-resistance detection and the diagnosis method was evaluated. Results The resistance rates of clinical isolates to rifampicin (RFP), isoniazid (INH), streptomycin (SM), ethambutol (EMB), and ofloxacin (OFX) were, respectively, 4.4%, 12.3%, 8.8%, 2.6%, and 3.5%. The single- and multi-drug resistance rates were, respectively, 80.0% and 20.0%. The resistance genes RopB, katG, InhA, RpsL, rrs, gyrA, and embB displayed codon mutations, while InhA was mutated in its promoter region. Kappa scores, evaluating the consistency between DNA sequencing and the resistance ratio methods for the detection of isolates’ resistance to RFP, INH, SM, OFX, and EMB, were 1, 0.955, 0.721, 0.796, and 1, respectively. Conclusion The resistance rate of INH and SM is relatively high in the Kashgar area. Detection of mutations in RopB, katG, InhA, RpsL, rrs, gyrA, and embB by DNA sequencing can predict drug resistance of M. tuberculosis strains with high sensitivity and specificity, and can be used for diagnosis.

Molecular analysis of streptomycin-resistance associating genes in Mycobacterium tuberculosis isolates from Nepal

Mutation in rpsL (encoding ribosomal protein S12), rrs (encoding 16S ribosomal RNA) and gidB (encoding 7-methylguanosine methyltransferase) are associated with resistance to streptomycin (STR), which is used for the treatment of multi-drug resistant tuberculosis (MDR-TB) in Nepal. The aim of our study is to analyze the correlation between mutations in the target genes and STR-resistance in 197 Mycobacterium tuberculosis (MTB) isolates from Nepal. Mutations in rpsL was harbored by 65.9% of isolates, in which the most common mutation in rpsL is caused by K43R (58.8%) and were significantly associated with Beijing genotype (P < 0.001). About 13.2% of isolates harbored mutations in two highly mutable regions of rrs, the 530 loop and the 912 region. About 13.2% of gidB mutants do not show any mutation in rpsL and rrs, which might suggest the role of gidB mutations in STR-resistance in MTB. In addition, 5.6% of isolates do not show any mutations in three genes examined, suggesting the involvement of other mechanism in STR-resistance in MTB. Our findings can be implemented for the establishment of molecular STR-susceptibility testing, in which tuberculosis can be treated with appropriate drugs and can improve control strategies for DR-TB.

Characterization and direct molecular discrimination of rpsL mutations leading to high streptomycin resistance in Erwinia amylovora

Erwinia amylovora can rapidly become highly resistant to applications of the antibiotic streptomycin through a single nucleotide mutation in the rpsL gene, which causes an amino acid substitution that prevents inhibitory binding of streptomycin while preserving the functionality of the ribosome. Several viable mutations are described in the literature, but screenings in the orchards consistently revealed that the one leading to the substitution of lysin by arginine at position 43 (K43R) is by far the most abundant. In this work, we tested the rate of emergence of the various spontaneous mutants, their tolerance to different antibiotic concentrations and their general fitness in competition experiments. Furthermore, we developed a molecular assay based on the SNaPshot™ Multiplex Kit from Thermo Fisher Scientific that allows swift determination of rpsL mutations using a single-base primer extension approach directly from colonies on plate. Or results indicate that the prevalence of variant K43R is largely due to fact that it retains the highest environmental fitness even in absence of antibiotic pressure. Sequence analysis of two resistant colonies that did not respond as expected in the SNaPshot™ assay revealed the existence of two yet undescribed rpsL mutations resulting in conditional-lethal streptomycin-dependent (SmD) phenotypes, which are unable to grow in absence of the antibiotic.

Phenotypic and Genotypic Characterization of Streptomycin-Resistant Multidrug-Resistant Mycobacterium tuberculosis Clinical Isolates in Southern China.

Streptomycin (STR) is the first antibiotic used in the treatment of tuberculosis (TB) and the earliest antituberculosis drug with acquired resistance developed by Mycobacterium tuberculosis. The high prevalence of such resistance in many parts of the world limits its use for treating multidrug-resistant (MDR) TB. The aims of this study are to characterize of mutations in rpsL, rrs, and gidB genes in MDR M. tuberculosis isolates originating from southern China and to investigate possible relationship between mutations and strain genotypes for precise diagnosis and treatment. Sequences of rpsL, rrs, and gidB genes and the resistance profiles were analyzed for 218 MDR M. tuberculosis isolates. Our study showed that 68.35% of MDR M. tuberculosis isolates were resistant to STR and 89.91% of STR-resistant (STRR) isolates were Beijing lineage strains. Mutations were observed in STRR MDR M. tuberculosis isolates at the following rates: 72.48% in rpsL, 36.91% in rrs, and 15.44% in gidB. Compared with the phenotypic data, the combination of mutations in rpsL, rrs, and gidB has sensitivity and specificity of 96.64% and 100.00%, respectively. The most common mutations in STRR isolates were rpsL128,263 and rrs514,1401, of which rpsL128 showed association with Beijing lineage (p < 0.001). It is noteworthy that a1401g mutation was present in rrs, while MDR M. tuberculosis isolates were resistant to both STR and amikacin. Twenty two novel mutations were found in STRR isolates. These findings could be helpful to develop rapid molecular diagnostic methods and understand STR resistance in China for developing TB precision medicine and disturbance of drug-resistant TB transmission.

Bacillus Calmette–Guérin strains with defined resistance mutations: a new tool for tuberculosis laboratory quality control

ObjectiveLaboratory quality control (QC) is essential to assess the reliability of tuberculosis diagnostic testing. To provide safe QC reagents for the detection of drug-resistant Mycobacterium tuberculosis, we generated antibiotic-resistant mycobacterial strains of attenuated virulence (M. bovis bacillus Calmette–Guérin (BCG)). MethodsSeven mono-resistant BCG strains were developed by introducing resistance-conferring mutations into wild-type BCG strains. Mutations were confirmed by dideoxynucleotide sequencing. Phenotypic resistance was quantified by microbroth dilution to determine the MIC90. The capacity of two commercial tests (GeneXpert TB/RIF and Genotype MTBDRplus) to detect resistance-conferring mutations was evaluated independently. ResultsOur panel included BCG strains with mutations in rpoB (S450L, I491F), katG (deletion at AA428), gyrA (D94G), rpsL (K43R) and Rv0678c (S63R). These mutations translated respectively into phenotypic resistance to rifampin (MIC ≥8 mg/L), isoniazid (MIC ≥8 mg/L), moxifloxacin (MIC 4 mg/L) and streptomycin (MIC ≥8 mg/L); the Rv0678c mutant showed decreased susceptibility to both clofazimine (MIC 4 mg/L) and bedaqualine (MIC 1 mg/L). GeneXpert (Cepheid) and Genotype MTBDRplus (Hain Lifesciences) both called the rpoB S450L strain rifampin-resistant and the I491F mutant rifampin-susceptible, as expected based on single nucleotide polymorphism positions. Likewise, MTBDRplus called the novel katG deletion mutant isoniazid susceptible despite phenotypic resistance. ConclusionBCG strains engineered to be mono-resistant to anti-tuberculosis drugs can be used as safe QC reagents for tuberculosis diagnostics and drug susceptibility testing.

Effect of "ribosome engineering" on the transcription level and production of S. albus indigenous secondary metabolites.

Significant resources are invested into efforts to improve the production yields of natural products from Actinobacteria, a well-recognized source of leads for several industries, most notably pharmaceutical one. Introduction of changes into genes for ribosomal protein S12 (rpsL) and/or 16S rRNA methylation (rsmG) is one of traditional approaches (referred to as ribosomal engineering) towards actinobacterial strain improvement. Yet, true potential of ribosome engineering remains unknown as it is currently coupled to empirical selection for aminoglycoside-resistance; rpsL mutations without such phenotypic expression could not be isolated. Here, we report a systematic and rational ribosome engineering approach to study the effect of a range of rpsL mutations on the production level of different biosynthetic gene clusters (BGC). The severe effect of diverse rpsL mutations together with deletion of rsmG engineered in Streptomyces albus has been revealed on the transcription level of several indigenous BGCs. The aforementioned mutations strongly impacted the transcription of indigenous BGCs, possibly because they alter the transcription of BGC-situated and global regulatory genes. The rsmG deletion with certain rpsL mutations can have a synergistic effect on the transcription level of indigenous BGCs. Our work thus provides the first streptomycete platform for rational engineering and study of virtually any nonlethal rpsL mutation. The tremendous effect of ribosome engineering on the transcription profile of the strains was reported for the first time. A library of described S. albus rpsL*/ΔrsmG strains represents a useful tool for overproducing known secondary metabolites and activating silent biosynthetic gene clusters in Actinobacteria.

Deciphering extracellular antibiotic resistance genes (eARGs) in activated sludge by metagenome

Antibiotic resistance genes (ARGs) including extracellular ARGs (eARGs) and intracellular ARGs (iARGs), are recognized as emerging environmental contaminants. Despite extensive efforts to profile ARGs in their “hotspots” wastewater treatment plants (WWTPs), the contribution of eARGs to antibiotic resistance spread remains unclear. Here, we applied metagenomic sequencing to investigate the distribution, mobility and microbial hosts of eARGs in activated sludge from five WWTPs. The total relative abundance eARGs ranged from 9.5 × 10−6 to 1.3 × 10−4, mainly encompassing elfamycin, dual drug, and aminoglycoside resistance genes. Multiple eARGs (e.g., EF-Tu, ropB, and rpsL mutants) were shared among the five WWTPs, and some clustered in the same genetic element (e.g., EF-Tu-rpsJ). eARGs were found to frequently co-localize with the eMGEs (e.g., sul1-3′CS-TnAs3, sul2-intI1-ISVsa3, and tetX-p63039), which may facilitate the mobilization of eARGs. Most eARGs likely originated from the genera Mycobacterium (6.7%), Nitrosomonas (5.3%), Steroidobacter (5.3%), Nitrospira (5.2%) and Pseudomonas (5.1%). No significant difference in the diversity, abundance, and mobility was observed between eARGs and iARGs. The host composition of eARGs and iARGs from municipal WWTPs are consistently dominated by Nitrosomonas, Steroidobacter, Nitrospira and Pseudomonas, while some differentially enriched genera (especially Nitrospira) in the hosts of iARGs compared with those of eARGs from the swine WWTP were identified. Our findings corroborate the mobile eARGs reservoir in WWTPs, thereby laying foundation for mitigating widespread antibiotic resistance.

Ant(6)-I Genes Encoding Aminoglycoside O-Nucleotidyltransferases Are Widely Spread Among Streptomycin Resistant Strains of Campylobacter jejuni and Campylobacter coli.

Thermotolerant Campylobacter species C. jejuni and C. coli are actually recognized as the major bacterial agent responsible for food-transmitted gastroenteritis. The most effective antimicrobials against Campylobacter are macrolides and some, but not all aminoglycosides. Among these, susceptibility to streptomycin is reduced by mutations in the ribosomal RPSL protein or by expression of ANT(6)-I aminoglycoside O-nucleotidyltransferases. The presence of streptomycin resistance genes was evaluated among streptomycin-resistant Campylobacter isolated from humans and animals by using PCR with degenerated primers devised to distinguish ant(6)-Ia, ant(6)-Ib and other ant-like genes. Genes encoding ANT(6)-I enzymes were found in all possible combinations with a major fraction of the isolates carrying a previously described ant-like gene, distantly related and belonging to the new ant(6)-I sub-family ant(6)-Ie. Among Campylobacter isolates, ant(6)-Ie was uniquely found functional in C. coli, as shown by gene transfer and phenotype expression in Escherichia coli, unlike detected coding sequences in C. jejuni that were truncated by an internal frame shift associated to RPSL mutations in streptomycin resistant strains. The genetic relationships of C. coli isolates with ANT(6)-Ie revealed one cluster of strains presented in bovine and humans, suggesting a circulation pathway of Campylobacter strains by consuming contaminated calf meat by bacteria expressing this streptomycin resistance element.

Characterization of mutations conferring streptomycin resistance to multidrug-resistant Mycobacterium tuberculosis isolates from Myanmar

Numerous studies report that mutations of rpsL (encoding the S12 protein), rrs (encoding 16S rRNA) and gidB (encoding rRNA methyltransferase) are responsible for conferring resistance to streptomycin (STR), which is usually used in both multidrug-resistant tuberculosis (MDR-TB) treatments and re-treatments in Myanmar. The aim of this study was to explore the variation and frequency of mutations in rpsL, rrs and gidB in 141 STR-resistant MDR-TB isolates from Myanmar. Most isolates belonged to the Beijing genotype (105, 74.5%). Moreover, mutations in rpsL were identified in 69.5% (98/141) of the STR-resistant isolates, where the most prevalent (92.0%, 90/98) and significantly associated mutation with the Beijing genotype (P < 0.001) was Lys43Arg. Fifteen different mutations in gidB were found in 16.3% (23/141) of the isolates, and most of them were novel mutations. Moreover, based on our results, we suggest A276C nucleotide substitution in gidB as a phylogenetic marker for the Beijing family in Myanmar. Sequence analysis of rpsL, rrs and gidB with a sensitivity of 83.7% satisfactorily predicted STR resistance in Myanmar isolates. However, in 16.3% (23/141) of the isolates, none of the examined genes showed mutation. Hence, further studies are strongly recommended to elucidate other possible resistance mechanisms. The present findings may be useful in developing molecular STR susceptibility assays, which in turn could contribute to develop TB treatments and control strategies in Myanmar.

Evolution of high-level resistance during low-level antibiotic exposure

It has become increasingly clear that low levels of antibiotics present in many environments can select for resistant bacteria, yet the evolutionary pathways for resistance development during exposure to low amounts of antibiotics remain poorly defined. Here we show that Salmonella enterica exposed to sub-MIC levels of streptomycin evolved high-level resistance via novel mechanisms that are different from those observed during lethal selections. During lethal selection only rpsL mutations are found, whereas at sub-MIC selection resistance is generated by several small-effect resistance mutations that combined confer high-level resistance via three different mechanisms: (i) alteration of the ribosomal RNA target (gidB mutations), (ii) reduction in aminoglycoside uptake (cyoB, nuoG, and trkH mutations), and (iii) induction of the aminoglycoside-modifying enzyme AadA (znuA mutations). These results demonstrate how the strength of the selective pressure influences evolutionary trajectories and that even weak selective pressures can cause evolution of high-level resistance.

Interaction between rpsL and gyrA mutations affects the fitness and dual resistance of Mycobacterium tuberculosis clinical isolates against streptomycin and fluoroquinolones.

BackgroundThe interaction between different drug-resistant mutations is important to the development of drug resistance and its evolution. In this study, we aimed to reveal the potential relationships between mutations conferring resistance to two important antituberculosis drugs streptomycin (STR) and fluoroquinolones (FLQ).Materials and methodsWe used an in vitro competitive fitness assay to reveal the interactions between different mutations of rpsL and gyrA in drug-resistant Mycobacterium smegmatis, followed by the analysis of the frequency of rpsL and gyrA mutation combinations in 213 STR–FLQ dual-resistant clinical Mycobacterium tuberculosis isolates from Sichuan region, which was also investigated by the whole genome data from 3,056 global clinical M. tuberculosis isolates.ResultsThe strains with K43R and K88R mutation in rpsL showed no difference in relative fitness compared with their susceptible ancestor, while K43N, K43M, K43T, and K88E exhibited a significantly lower relative fitness (P<0.05). For the FLQ-resistant mutants, all mutation types showed no difference in their relative fitness. Among STR–FLQ dual-resistant M. smegmatis strains, a lower fitness was detected in those with K43N/M/T and K88E instead of K43R and K88R mutations in rpsL. Among M. tuberculosis isolates harboring rpsL and gyrA dual mutations, the most two frequent combinatorial mutation types were K43R/D94G (n=37) and K43R/A90V (n=24), with the former being the most frequent one by both in vitro tests and clinical survey.ConclusionOur results suggest that the interaction between rpsL and gyrA mutations affects the fitness cost in STR–FLQ dual-resistant M. smegmatis and also the predilection of mutation combinations in clinical M. tuberculosis isolates.

Antibiotic resistance mutations induced in growing cells of Bacillus-related thermophiles.

Stress-induced mutagenesis can assist pathogens in generating drug-resistant cells during antibiotic therapy; however, if and how antibiotics induce mutagenesis in microbes remains poorly understood. A non-pathogenic thermophile, Geobacillus kaustophilus HTA426, efficiently produces derivative cells resistant to rifampicin and streptomycin via rpoB and rpsL mutations, respectively. Here, we examined this phenomenon to suggest a novel mutagenic mode induced by antibiotics. Fluctuation analysis indicated that mutations occurred via spontaneous mutations during culture. However, mutations were much more frequent in growing cells than stationary cells, and mutation sites were varied through cell growth. These observations suggested that growing cells induced mutagenesis in response to antibiotics. An in-frame deletion of mfd, which governs transcription-coupled repair to correct DNA lesions on the transcribed strand, caused mutations that were comparable between growing and stationary cells; therefore, the mutagenic mechanism was attributable to DNA repair defects where growing cells depressed mfd function. Mutations occurred more frequently at optimal growth temperatures for G. kaustophilus than at a higher growth temperature, suggesting that the mutagenesis relies on active cellular activities rather than high temperature-associated DNA damage. In addition, the mutagenesis may involve a mutagenic factor targeting these sites, in addition to mfd depression, because rpoB and rpsL mutations were dominant at thymine and guanine sites on the transcribed strand. A similar mutagenic profile was observed for other Geobacillus and thermophilic Bacillus species. This suggests that Bacillus-related thermophiles commonly induce mutagenesis in response to rifampicin and streptomycin to produce resistant cells.

Molecular Screening Versus Phenotypic Susceptibility Testing of Multidrug-Resistant Mycobacterium tuberculosis Isolates for Streptomycin and Ethambutol.

Proper management of multidrug-resistant tuberculosis (MDR-TB) requires accurate drug susceptibility testing (DST) of Mycobacterium tuberculosis isolates to other (ethambutol [EMB], pyrazinamide, and streptomycin [SM]) first-line drugs. This study compared the performance of Mycobacterium Growth Indicator Tube (MGIT) 960 system for DST of MDR-TB isolates with polymerase chain reaction (PCR) sequencing of embB, rpsL, and rrs genes for detecting resistance to EMB and SM. MDR-TB strains (n = 60) and 25 pansusceptible M. tuberculosis isolates collected during 2011-2016 were tested. Phenotypic DST was performed by MGIT 960 system by using SIRE drug kit. EMB and SM resistance-conferring mutations in embB and rpsL+rrs genes, respectively, were detected by PCR sequencing. No mutations were detected in pansusceptible isolates. Among 60 MDR-TB strains, 35 of 40 SM-resistant and none of 20 SM-susceptible isolates contained rpsL and/or rrs mutations (κ = 0.82, very good agreement). However, all 18 EMB-resistant MDR-TB strains and 33 of 42 EMB-susceptible MDR-TB strains contained an embB mutation (κ = 0.14, poor agreement). Thus, 40 of 60 (67%) and 35 of 60 (58%) isolates were resistant to SM (p = 0.451), while 18 of 60 (30%) and 51 of 60 (85%) isolates were resistant to EMB (p = 0.000) by MGIT 960 system and PCR sequencing, respectively. MGIT 960 system showed acceptable performance for DST for SM; however, it performed poorly for EMB as many MDR-TB strains with embB mutations, which confer low-level resistance to EMB, were detected as EMB susceptible. Molecular screening for resistance-conferring mutations in embB gene is thus superior to MGIT 960 system when accurate EMB susceptibility results are needed for proper management of MDR-TB patients.

Frequency of rrs and rpsL mutations in streptomycin-resistant Mycobacterium tuberculosis isolates from Iranian patients.

Streptomycin (SM) is one of the most effective drugs for the treatment of multidrug-resistant (MDR) tuberculosis. However, resistance to SM is increasingly reported, mainly due to mutations in the rpsL and rrs genes. This study was designed with the aim of determining the nature of SM resistance and the type and frequency of rpsL and rrs mutations among SM-resistant Mycobacterium tuberculosis (MTB) isolates from Iran. A total of 100 clinical monoresistant and MDR MTB isolates were subjected to drug susceptibility testing (DST) for SM. SM-resistant isolates were genotyped by MIRU-VNTR typing. Fragments of the rpsL and rrs genes were amplified to investigate the most common mutations, with subsequent sequence analysis. By DST, 32 isolates (32%) were identified as SM-resistant, of which 50% (16/32) were MDR. By MIRU-VNTR typing, the SM-resistant isolates were classified into 20 different MIRU types and 8 clusters, with Beijing (22%) being the most prevalent genotype. Mutations in the rrs and rpsL genes were identified in 14 (44%) and 10 (31%) of the 32 SM-resistant isolates, respectively. The most common mutations were at rpsL nucleotide 128 (AAG→AGG, Lys43Arg), found in 7 SM-resistant isolates (22%) and nucleotide 263 (A→G, Lys88Arg) in 3 SM-resistant isolates (9%). The results suggest an association between rpsL mutation and SM-resistant strains of Beijing genotype. The existence of SM resistance in 25% of isolates without mutation in rrs and rpsL suggests the occurrence of further mechanisms associated with SM resistance in these isolates.

Mutations in Streptomycin Resistance Genes and Their Relationship to Streptomycin Resistance and Lineage of Mycobacterium tuberculosis Thai Isolates.

BackgroundStreptomycin (SM) is recommended by the World Health Organization (WHO) as a part of standard regimens for retreating multidrug-resistant tuberculosis (MDR-TB) cases. The incidence of MDR-TB in retreatment cases was 19% in Thailand. To date, information on SM resistance (SMR) gene mutations correlated to the SMR of Mycobacterium tuberculosis Thai isolates is limited. In this study, the mutations in rpsL, rrs, gidB, and whiB7 were investigated and their association to SMR and the lineage of M. tuberculosis were explored.MethodsThe lineages of 287 M. tuberculosis collected from 2007 to 2011 were identified by spoligotyping. Drug susceptibility profiles were evaluated by the absolute concentration method. Mutations in SMR genes of 46 SM-resistant and 55 SM-susceptible isolates were examined by DNA sequencing.ResultsThree rpsL (Lys43Arg, Lys88Arg, and Lys88Thr) and two gidB (Trp45Ter and Gly69Asp) mutations were present exclusively in the SM resistant M. tuberculosis. Lys43Arg rpsL was the most predominant SMR mutations (69.6%) and prevailed among Beijing isolates (p<0.001). No SMR-related mutation in was found rrs. The combination of rpsL and gidB mutations provided 76.1% sensitivity for detecting SMR in M. tuberculosis Thai isolates. whiB7 was not responsible for SMR in SM resistant isolates lacking rpsL and rrs mutations. The significance of the three gidB mutations, 276A>C, 615A>G, and 330G>T, as lineage signatures for Beijing and EAI were underscored. This study identified 423G>A gidB as a novel sub-lineage marker for EAI6-BGD1.ConclusionOur study suggested that the majority of SMR in M. tuberculosis Thai isolates were responsible by rpsL and gidB polymorphisms constantly providing the novel lineage specific makers.

Genetic Patterns of rpsL and rrs Genes in Clinical Isolates of Mycobacterium tuberculosis, Isfahan‚ Iran

Drug-resistant tuberculosis is considered a major universal problem. Based on knowledge on certain mutations occurring in Mycobacterium tuberculosis genome, drug resistance could be detected timely. The goal of this study was to determine the prevalence of the most common mutations likely to result from resistance to streptomycin in M. tuberculosis isolates, as well as genetic patterns of rpsL and rrs genes, in the province of Isfahan, Iran. Clinical specimens were collected from individuals suspected of tuberculosis who referred to the Tuberculosis Center of Isfahan among whom 205 isolates were diagnosed with M. tuberculosis by conventional methods. The minimum inhibitory concentration of streptomycin in these isolates was determined with proportion method using Lowenstein- Jensen medium from which 10 isolates were recognized with streptomycin-resistant tuberculosis. The nucleotide sequence of rpsL and 530 loop of rrs genes were analyzed in all streptomycin-resistant isolates, in addition to five randomly selected streptomycin-susceptible isolates. Six (6/10, 60%) streptomycin-resistant isolates represented a mutation in either rpsL gene and/or rrs530 loop. Four (40%) isolates showed rpsL mutations (codons 43 and 88), and two (20%) of them alterations in rrs gene (A514C and C517T). However, no mutation was found in streptomycin-susceptible isolates in either of the genes. The study could successfully highlight the positive effects of rpsL and rrs mutations as molecular markers of streptomycin resistance in M. tuberculosis strains. Diversity and presence or absence of mutations suggested possible circulation of a variety of strains and the role of additional mechanisms contributing to strstreptomycin resistance in various regions.

Mutations in rrs, rpsL and gidB in streptomycin-resistant Mycobacterium tuberculosis isolates from Thailand.

The objectives of this study were to characterise mutations in rrs, rpsL and gidB genes in Mycobacterium tuberculosis isolates from Thailand and to examine possible associations between mutations and strain genotypes. In total, 110 streptomycin (STR)-resistant M. tuberculosis isolates and 51 STR-susceptible isolates obtained from a sample collection in Thailand during 1999-2011 were sequenced for mutation analysis in rrs, rpsL and gidB. Genotypes of the isolates were identified using spoligotyping and large sequence polymorphisms. Mutations at codons 43 and 88 in rpsL represented 63.6% of the STR-resistant isolates and were mostly associated with Beijing strains. Mutations in rrs existed in 17.3% of the STR-resistant isolates; only 8.2% harboured resistance-associated mutations. Twenty-five different mutations were found in gidB, twelve of which are new. Eight gidB mutations were likely to contribute to STR resistance in ca. 14% of the resistant isolates; about one-half of the isolates also had a mutation in rrs or rpsL. Nearly all of the double mutants belonged to Beijing strains, whereas isolates carrying only STR-associated gidB mutation were non-Beijing strains. Three different alleles in gidB were also found, each specific to Beijing, East-African Indian and Euro-American lineages, respectively. Most of the STR-resistant isolates (80.9%) carried putative resistance-associated mutations in the analysed genes. Beijing strains were related not only to single resistance-associated mutations in rpsL or rrs but usually harboured a second mutation in gidB. Strains harbouring resistance-associated gidB mutations without rrs or rpsL mutations were more associated with non-Beijing isolates. Certain gidB mutations were also potential lineage markers.

Characterization of mutations in streptomycin-resistant Mycobacterium tuberculosis isolates in Sichuan, China and the association between Beijing-lineage and dual-mutation in gidB

Mutations in rpsL, rrs, and gidB are well linked to streptomycin (STR) resistance, some of which are suggested to be potentially associated with Mycobacterium tuberculosis genotypic lineages in certain geographic regions. In this study, we aimed to investigate the mutation characteristics of streptomycin resistance and the relationship between the polymorphism of drug-resistant genes and the lineage of M. tuberculosis isolates in Sichuan, China. A total of 227 M. tuberculosis clinical isolates, including 180 STR-resistant and 47 pan-susceptible isolates, were analyzed for presence of mutations in the rpsL, rrs and gidB loci. Mutation K43R in rpsL was strongly associated with high-level streptomycin resistance (P < 0.01), while mutations in rrs and gidB potentially contributed to low-level resistance (P < 0.05). No general association was exhibited between STR resistance and Beijing genotype, however, in STR-resistant strains, Beijing genotype was significantly correlated with high-level STR resistance, as well as the rpsL mutation K43R (P < 0.01), indicating that Beijing genotype has an evolutionary advantage under streptomycin pressure. Notably, in all isolates of Beijing genotype, a dual mutation E92D (a276c) and A205A (a615g) in gidB was detected, suggesting a highly significant association between this dual mutation and Beijing genotype.

Identification of mutations conferring streptomycin resistance in multidrug-resistant tuberculosis of China

We investigated the spectrum and frequency of mutations in rpsL, rrs, and gidB among 140 multidrug-resistant tuberculosis (MDR-TB) clinical isolates from China. The association between mutations and different genotypes was also analyzed. Our data revealed that 65.7% of MDR-TB were resistant to streptomycin (STR), and 90.2% of STR-resistant isolates were Beijing strains. STR resistance was correlated with Beijing family (P=0.00). Compared with phenotypic data, detection of mutations for the combination of these 3 genes exhibited 94.6% sensitivity, 91.7% specificity, and 93.6% accuracy. The most common mutations in STR-resistant isolates were rpsL128, 262, and rrs514, of which rpsL128 showed association with Beijing lineage (P=0.00). A combination of these 3 mutations can serve as the reliable predictors for STR resistance, showing the sensitivity, specificity, and accuracy of 85.9%, 97.9%, and 90.0%, respectively. Furthermore, gidBA276C, not A615G, was Beijing lineage specific. These findings are useful to develop rapid molecular diagnostic methods for STR resistance in China.