Granzyme B Disrupts Central Metabolism and Protein Synthesis in Bacteria to Promote an Immune Cell Death Program.

Human cytotoxic lymphocytes kill intracellular microbes. The cytotoxic granule granzyme proteases released by cytotoxic lymphocytes trigger oxidative bacterial death by disrupting electron transport, generating superoxide anion and inactivating bacterial oxidative defenses. However, they also cause non-oxidative cell death because anaerobic bacteria are also killed. Here, we use differential proteomics to identify granzyme B substrates in three unrelated bacteria: Escherichia coli, Listeria monocytogenes, and Mycobacteria tuberculosis. Granzyme B cleaves a highly conserved set of proteins in all three bacteria, which function in vital biosynthetic and metabolic pathways that are critical for bacterial survival under diverse environmental conditions. Key proteins required for protein synthesis, folding, and degradation are also substrates, including multiple aminoacyl tRNA synthetases, ribosomal proteins, protein chaperones, and the Clp system. Because killer cells use a multipronged strategy to target vital pathways, bacteria may not easily become resistant to killer cell attack.

Bactericidal Activity of Liposomal Form of Lytic Mycobacteriophage D29 in Cell Models of Tuberculosis Infection In Vitro.

The use of lytic mycobacteriophages to treat tuberculosis under conditions of acquired resistance to anti-tuberculosis drugs is one of the most practical ways to improve the effectiveness of therapy and reduce the spread of this disease. We studied the efficacy of antimycobacterial action of mycobacteriophage D29 encapsulated into 400-nm liposomes in cell models of tuberculosis infection in vitro. The antimycobacterial action of lytic mycobacteriophage D29 used in free or liposome-encapsulated forms was demonstrated on cell models of intracellularly infected RAW264.7 macrophages and tuberculous granuloma formed by human blood mononuclear cells. The experiments demonstrated pronounced advantage of liposomal form of mycobacteriophage according to the criteria of their penetration into macrophages and lysis of Mycobacterium tuberculosis in culture.

Location of Pulmonary Mycobacteria Tuberculosis and Effectiveness of Various Dextrazide Compositions in Treatment of Mice with BCG-Induced Granulomatosis.

The study examined effectiveness of liposomal form of dextrazide (inhaled or intraperitoneal), free dextrazide (intraperitoneal), and isoniazid (intraperitoneal) in the treatment of BALB/c mice with BCG-induced granulomatosis. The mice were infected with mycobacteria tuberculosis 3 months prior to onset of treatment. The preparations under examinations were administered twice a week over 2 months. The decrease of the number and size of macrophagal granulomas in mice BCG-induced granulomatosis during treatment was determined by the number of living mycobacteria tuberculosis in these granulomas. The most effective treatment was achieved with liposomal form of dextrazide (a conjugate of oxidized dextran with isonicotinic acid hydrazide). Macrophages with captured mycobacteria tuberculosis, dextrazide, and dextrazide-loaded liposomes can be incorporated into granulomas. The antimycobacterial effect of dextrazide is an important factor preventing the destructive processes in granulomas and organs via a decrease in the prodestructive potential of lysosomes in macrophages realized after their migration from granulomas.

Imaging flow cytometry analysis of intracellular pathogens.

Imaging flow cytometry has been applied to address questions in infection biology, in particular, infections induced by intracellular pathogens. This methodology, which utilizes specialized analytic software makes it possible to analyze hundreds of quantified features for hundreds of thousands of individual cellular or subcellular events in a single experiment. Imaging flow cytometry analysis of host cell-pathogen interaction can thus quantitatively addresses a variety of biological questions related to intracellular infection, including cell counting, internalization score, and subcellular patterns of co-localization. Here, we provide an overview of recent achievements in the use of fluorescently labeled prokaryotic or eukaryotic pathogens in human cellular infections in analysis of host-pathogen interactions. Specifically, we give examples of Imagestream-based analysis of cell lines infected with Toxoplasma gondii or Mycobacterium tuberculosis. Furthermore, we illustrate the capabilities of imaging flow cytometry using a combination of standard IDEAS™ software and the more recently developed Feature Finder algorithm, which is capable of identifying statistically significant differences between researcher-defined image galleries. We argue that the combination of imaging flow cytometry with these software platforms provides a powerful new approach to understanding host control of intracellular pathogens.

[The informative value of various methods for identifying acid-fast bacilli in relation to the degree of tuberculosis process activity]. / Informativnost' razlichnykh metodov identifikatsii kislotoustoichivykh mikobakterii v zavisimosti ot stepeni aktivnosti tuberkuleznogo protsessa.

Carbol fuchsin (Ziehl-Neelsen) staining is a major method used since the late nineteenth century to detect Mycobacterium tuberculosis in the tissues. At the same time, the number of mycobacteria does not frequently show a clinical and morphological correspondence to the activity of the infectious process. OBJECTIVE: To reveal the morphological characteristics of Mycobacterium tuberculosis in patients with long-lasting tuberculosis. MATERIAL AND METHODS: Twenty-four autopsies of verified fibrous-cavernous tuberculosis were examined. Paraffin sections were stained with hematoxylin and eosin, carbol fuchsin (Ziehl-Neelsen), or auramine-rhodamine (followed by luminescence microscopic examination); an immunohistochemical (IHC) study with serum to PAV (Protein antigen B) was conducted. RESULTS AND DISCUSSION: The changes characteristic of progressive tuberculosis were revealed in all the cases. The Ziehl-Neelsen staining revealed isolated clusters of acid-fast bacilli (a total of 1,000 in less than 10 fields of view). When stained with auramine-rhodamine, the number of found mycobacteria proved to be significantly larger (a total of 1,000 to 10,000 in 10-50 fields of view) and that was greatest in the IHC study (a total of over 10,000 in more than 50 fields of view). At the same type, all types of studies revealed that the localization of mycobacteria was exclusively extracellular. The Ziehl-Neelsen staining indicated that the proportion of typical bacilli was much higher (85-95%; mean 88.13±2.14) than that (50-85%; mean 64.38±4.24%) identified when stained with auramine-rhodamine and even more significantly more than that (50-70%; mean 57.29±2.78%) detected during the IHC study. The indicators were equally different for the atypical morphological forms of mycobacteria identified in minimal quantities by the Ziehl-Nielsen staining and in large quantities by fluorescence and IHC studies. CONCLUSION: There is evidence for the ability of mycobacteria to have morphological polymorphism and for the need to clarify the pathogenesis of tuberculosis.

Mycobacterial PPE36 Modulates Host Inflammation by Promoting E3 Ligase Smurf1-Mediated MyD88 Degradation.

Mycobacterium tuberculosis (Mtb) PPE36, a cell-wall-associated protein, is highly specific and conserved for the Mtb complex group. Although PPE36 has been proven essential for iron utilization, little is known about it in regulating host immune responses. Here we exhibited that PPE36 was preferentially enriched in Mtb virulent strains and could efficiently inhibit host inflammatory responses and increase bacterial loads in infected macrophages and mice. In exploring the underlying mechanisms, we found that PPE36 could robustly inhibit the activation of inflammatory NF-κB and MAPK (Erk, p38, and Jnk) pathways by promoting E3 ligase Smurf1-mediated ubiquitination and proteasomal degradation of MyD88 protein. Our research revealed a previously unknown function of PPE36 on modulating host immune responses and provided some clues to the development of novel tuberculosis treatment strategies based on immune regulation.

In Vitro and In Vivo Antigen Presentation and Diagnosis Development of Recombinant Overlapping Peptides Corresponding to Mtb ESAT-6/CFP-10.

Cellular immunity in Mycobacteria tuberculosis (Mtb) infection is important for the pathogenesis and final clearance of intracellular Mtb infection. In addition, it is valuable for the diagnosis of tuberculosis. In this pioneering work, we tested in vitro and in vivo antigen presentation and diagnostic application of a recombinant overlapping peptide-protein derived from two Mtb RD1 antigens ESAT-6 and CFP-10 (ROP-TB). The overlapping peptide sequence of ROP-TB is cleaved by the cathepsin S enzyme and covers the entire length of the two proteins. ROP-TB can be expressed and purified from E. coli. Once taken in by antigen-presenting cells, ROP-TB can be cleaved into a peptide pool by cathepsin S within the cells. We found that in dendritic cells, ROP-TB can be processed in 6 hours of co-culture, while the ESAT-6/CFP-10 fusion protein remained in the endosomal compartment. In Mtb-infected mice, ROP-TB stimulated stronger specific T cell responses than pooled synthetic peptides derived from ESAT-6 and CFP-10. With regard to the presentation of in vivo antigens, in a guinea pig model infected with Mtb, ROP-TB induced delayed type hypersensitivity (DTH) responses comparable to those of the tuberculin purified protein derivative (PPD) and ESAT-6/CFP-10 fusion protein. In Mycobacterium bovis (Bovine TB)-infected cattle, ROP-TB elicited DTH responses. Finally, in Mtb infected patients, ROP-TB stimulated cellular immune responses in majority of patients (16/18) of different HLA phenotypes while a single peptide derived from the same proteins did not elicit the immune responses in all patients. In summary, in vitro and in vivo data suggest that ROP-TB stimulates a strong cellular immune response irrespective of HLA phenotypes and is therefore suitable for use in vitro and in vivo diagnostics.

Primary lymphatic tuberculosis in children - Literature overview and case report.

Tuberculosis bacilli can enter the human body through the digestive system, the skin, and the mucous membranes, although they mainly enter through the respiratory tract. TB bacilli can enter the bloodstream and attack other organs including the lymphatic system. The TB bacillus can cause miliary tuberculosis once they have entered the bloodstream and infiltrated the lymphatic system, which can then manifest as large lymph nodes in the hilum, mediastinum, and lung. Complicated primary TB infection occurs when enlarged lymph nodes compress the airways, causing a partial or complete obstruction that can lead to air retention or atelectasis. More serious complications can occur if the lymph nodes fill with pus and burst, as this can lead to TB spreading through the airways via a humoral mechanism. Making a differential diagnosis of hilar and mediastinal lymphadenopathy is often difficult because diagnostic interventions in this area are problematic. We report on a clinical case of a child with primary TB of the lymphatic system. The patient presented with mediastinal lymphadenopathy and miliary lesions in the lung, which was confirmed by a transthoracic biopsy performed under CT guidance. It is hoped that this report can provide doctors with a more comprehensive approach when diagnosing this disease.

Primary mediastinal lymph node tuberculosis diagnosed using endobronchial ultrasound-guided transbronchial needle aspiration: Literature review and case report.

Tuberculosis bacilli can enter the human body through the respiratory system, digestive system, or skin and mucous membranes, with the respiratory tract representing the primary point of entry. Once inside the body, tuberculosis bacilli can enter the bloodstream and attack other organs, including the lymphatic system. One manifestation associated with lymphatic tuberculosis infiltration is the presence of large hilar and mediastinal lymph nodes, which are common in children and classified as primary tuberculosis. A diagnosis of primary tuberculosis in adults is often overlooked by doctors due to its low frequency. When a single enlarged mediastinal lymph node is observed, most doctors suspect other causes, especially malignancy. Determining the correct diagnosis for enlarged hilar and mediastinal lymph nodes can be difficult because diagnostic interventions in this area are challenging to perform. We report a clinical case of primary lymph node tuberculosis in an adult, confirmed by endobronchial ultrasound-guided transbronchial needle aspiration. We aim to provide doctors with a more comprehensive approach for diagnosing this disease.

GSK-3α/ß Activity Negatively Regulates MMP-1/9 Expression to Suppress Mycobacterium tuberculosis Infection.

Tuberculosis (TB) caused by Mycobacterium tuberculosis (Mtb) infection is the deadliest infectious disease and a global health problem. Macrophages (Mφs) and neutrophils that can phagocytose Mtb represent the first line of immune response to infection. Glycogen synthase kinase-3α/ß (GSK-3α/ß) represents a regulatory switch in host immune responses. However, the efficacy and molecular mechanisms of how GSK-3α/ß interacts with Mtb infection in Mφs remain undefined. Here, we demonstrated that Mtb infection downregulated GSK-3α/ß activity and promoted matrix metalloproteinase-1 (MMP-1) and MMP-9 expressions in Mφs derived from acute monocytic human leukemia THP-1 cells (THP-1-Mφs). We confirmed the upregulation of MMP-9 expression in tissues of TB patients compared with patients of chronic inflammation (CI). In THP-1-Mφs and C57BL/6 mice, GSK-3α/ß inhibitor SB216763 significantly increased MMP-1/9 production and facilitated Mtb load, while MMP inhibitors blocked MMP-1/9 expression and Mtb infection. Consistently, GSK-3α/ß silencing significantly increased MMP-1/9 expression and Mtb infection, while overexpression of GSK-3α/ß and constitutive activated GSK-3α/ß mutants significantly reduced MMP-1/9 expression and Mtb infection in THP-1-Mφs. MMP-1/9 silencing reduced Mtb infection, while overexpression of MMP-1/9 promoted Mtb infection in THP-1-Mφs. We further found that GSK-3α/ß inhibition increased Mtb infection and MMP-1/9 expression was blocked by ERK1/2 inhibitor. Additionally, we showed that protein kinase C-δ (PKC-δ) and mammalian target of rapamycin (mTOR) reduced GSK-3α/ß activity and promoted MMP-1/9 production in Mtb-infected THP-1-Mφs. In conclusion, this study suggests that PKC-δ-mTOR axis suppresses GSK-3α/ß activation with acceleration of MMP-1/9 expression through phospho-ERK1/2. These results reveal a novel immune escape mechanism of Mtb and a novel crosstalk between these critical signaling pathways in anti-TB immunity.

IMB-XMA0038, a new inhibitor targeting aspartate-semialdehyde dehydrogenase of Mycobacterium tuberculosis.

The emergence of drug-resistant tuberculosis (TB) constitutes a major challenge to TB control programmes. There is an urgent need to develop effective anti-TB drugs with novel mechanisms of action. Aspartate-semialdehyde dehydrogenase (ASADH) is the second enzyme in the aspartate metabolic pathway. The absence of the pathway in humans and the absolute requirement of aspartate in bacteria make ASADH a highly attractive drug target. In this study, we used ASADH coupled with Escherichia coli type III aspartate kinase (LysC) to establish a high-throughput screening method to find new anti-TB inhibitors. IMB-XMA0038 was identified as an inhibitor of MtASADH with an IC50 value of 0.59 µg/mL through screening. The interaction between IMB-XMA0038 and MtASADH was confirmed by surface plasmon resonance (SPR) assay and molecular docking analysis. Furthermore, IMB-XMA0038 was found to inhibit various drug-resistant MTB strains potently with minimal inhibitory concentrations (MICs) of 0.25-0.5 µg/mL. The conditional mutant strain MTB::asadh cultured with different concentrations of inducer (10-5 or 10-1 µg/mL pristinamycin) resulted in a maximal 16 times difference in MICs. At the same time, IMB-XMA0038 showed low cytotoxicity in vitro and vivo. In mouse model, it encouragingly declined the MTB colony forming units (CFU) in lung by 1.67 log10 dosed at 25 mg/kg for 15 days. In conclusion, our data demonstrate that IMB-XMA0038 is a promising lead compound against drug-resistant tuberculosis.

Crystal Structure of Mycobacterium tuberculosis Elongation Factor G1.

Mycobacterium tuberculosis (Mtb) caused an estimated 10 million cases of tuberculosis and 1.2 million deaths in 2019 globally. The increasing emergence of multidrug-resistant and extensively drug-resistant Mtb is becoming a public health threat worldwide and makes the identification of anti-Mtb drug targets urgent. Elongation factor G (EF-G) is involved in tRNA translocation on ribosomes during protein translation. Therefore, EF-G is a major focus of structural analysis and a valuable drug target of antibiotics. However, the crystal structure of Mtb EF-G1 is not yet available, and this has limited the design of inhibitors. Here, we report the crystal structure of Mtb EF-G1 in complex with GDP. The unique crystal form of the Mtb EF-G1-GDP complex provides an excellent platform for fragment-based screening using a crystallographic approach. Our findings provide a structure-based explanation for GDP recognition, and facilitate the identification of EF-G1 inhibitors with potential interest in the context of drug discovery.

Robust barcoding and identification of Mycobacterium tuberculosis lineages for epidemiological and clinical studies.

BACKGROUND: Tuberculosis, caused by bacteria in the Mycobacterium tuberculosis complex (MTBC), is a major global public health burden. Strain-specific genomic diversity in the known lineages of MTBC is an important factor in pathogenesis that may affect virulence, transmissibility, host response and emergence of drug resistance. Fast and accurate tracking of MTBC strains is therefore crucial for infection control, and our previous work developed a 62-single nucleotide polymorphism (SNP) barcode to inform on the phylogenetic identity of 7 human lineages and 64 sub-lineages. METHODS: To update this barcode, we analysed whole genome sequencing data from 35,298 MTBC isolates (~ 1 million SNPs) covering 9 main lineages and 3 similar animal-related species (M. tuberculosis var. bovis, M. tuberculosis var. caprae and M. tuberculosis var. orygis). The data was partitioned into training (N = 17,903, 50.7%) and test (N = 17,395, 49.3%) sets and were analysed using an integrated phylogenetic tree and population differentiation (FST) statistical approach. RESULTS: By constructing a phylogenetic tree on the training MTBC isolates, we characterised 90 lineages or sub-lineages or species, of which 30 are new, and identified 421 robust barcoding mutations, of which a minimal set of 90 was selected that included 20 markers from the 62-SNP barcode. The barcoding SNPs (90 and 421) discriminated perfectly the 86 MTBC isolate (sub-)lineages in the test set and could accurately reconstruct the clades across the combined 35k samples. CONCLUSIONS: The validated 90 SNPs can be used for the rapid diagnosis and tracking of MTBC strains to assist public health surveillance and control. To facilitate this, the SNP markers have now been incorporated into the TB-Profiler informatics platform ( https://github.com/jodyphelan/TBProfiler ).

Endeavors towards transformation of M. tuberculosis thymidylate kinase (MtbTMPK) inhibitors into potential antimycobacterial agents.

As the last enzyme in nucleotide synthesis as precursors for DNA replication, thymidylate kinase of M. tuberculosis (MtbTMPK) attracts significant interest as a target in the discovery of new anti-tuberculosis agents. Earlier, we discovered potent MtbTMPK inhibitors, but these generally suffered from poor antimycobacterial activity, which we hypothesize is due to poor bacterial uptake. To address this, we herein describe our efforts to equip previously reported MtbTMPK inhibitors with targeting moieties to increase the whole cell activity of the hybrid analogues. Introduction of a simplified Fe-chelating siderophore motif gave rise to analogue 17 that combined favorable enzyme inhibitory activity with significant activity against M. tuberculosis (MIC of 12.5 µM). Conjugation of MtbTMPK inhibitors with an imidazo[1,2-a]pyridine or 3,5-dinitrobenzamide scaffold afforded analogues 26, 27 and 28, with moderate MtbTMPK enzyme inhibitory potency, but sub-micromolar activity against mycobacteria without significant cytotoxicity. These results indicate that conjugation with structural motifs known to favor mycobacterial uptake may be a valid approach for discovering new antimycobacterial agents.

Synthesis and Anti-mycobacterium Study on Halo-substituted 2-aryl oxyacetohydrazones.

BACKGROUND: The treatment of multiple-drug-resistant tuberculosis (MDR-TB) with currently available marketed drugs remains a global health concern. The cases of resistant tuberculosis patients are increasing day by day. OBJECTIVE: The objective of this study is to highlight the need of developing shorter, simpler and tolerable drug regimens. METHODS: In the present study, we synthesized various halo-substituted 2-aryloxyacetohydrazones via a series of reactions from halo-substituted phenols. All the compounds were characterized by using various spectroscopic methods, such as NMR, FT-IR, UV spectroscopy, etc. Results: All the synthesized hydrazones showed theoretically good interactions with enzyme enoyl reductase (pdb id: 4tzk). All the synthesized compounds (5a-5o) showed moderate to good activity (3.125-100 µg/mL) against Mycobacteria tuberculosis, H37RV strain. CONCLUSION: Our results would pave a new way for the development of more effective Anti-TB agents in the future.

Tuberculous spondylitis after percutaneous vertebroplasty: A case series of 9 cases.

BACKGROUND: Percutaneous vertebroplasty has become the treatment of choice for compression fractures. Although the incidence is low, infection after vertebroplasty is a serious complication. The pathogens most often responsible for infection are bacteria. Meanwhile, mycobacterium tuberculosis-induced infection is extremely rare. In this study, we reported our treatment experience with 9 cases of tuberculous spondylitis after vertebroplasty. METHODS: Between January 2001 and December 2015, 5749 patients underwent vertebroplasty or kyphoplasty in our department. Nine cases developed tuberculous spondylitis after vertebroplasty (0.16%). Data on clinical history, laboratory examinations, image, treatment and outcomes were examined. RESULTS: One male and 8 female patients with a mean age of 75.1 years developed tuberculous spondylitis after vertebroplasty. 5 patients had a history of pulmonary tuberculosis (TB). Revision surgeries were performed from 5 days to 1124 days after vertebroplasty. Seven patients underwent anterior debridement and fusion with or without posterior instrumentation, and 2 cases received posterior decompression and instrumentation only. After operation, the diagnosis of tuberculous spondylitis was confirmed by TB polymerase chain reaction (TB-PCR) or mycobacteria culture. Mean follow-up period after revision surgery was 36.8 months. At the end of follow-up, 1 patient with paraplegia had passed away, 2 needed a wheel chair, 4 required a walker and 2 were able to walk unassisted. CONCLUSIONS: Vertebroplasty is a minimally invasive procedure but still retains some possibility of complications, including TB infection. Patients with a history of pulmonary TB or any elevation of infection parameters should be reviewed carefully to avoid infective complications.

The role of FOXO3 polymorphisms in susceptibility to tuberculosis in a Chinese population.

BACKGROUND: Tuberculosis (TB) is a significant worldwide health problem, and is caused by Mycobacteria tuberculosis. Recent studies have suggested that FOXO3 plays vital roles in the risk of immune-related infectious diseases such as TB. METHODS AND RESULTS: The present study aimed to evaluate FOXO3 genetic variants and TB risk. We recruited 510 TB patients and 508 healthy controls in this study. All subjects were genotyped with the Agena MassARRAY platform. Odds ratios (ORs) and 95% confidence intervals (CIs) were calculated using logistic regression adjusted for age and gender. Our result revealed that rs3800229 T/G and rs4946935 G/A genotypes significantly increased the risk of TB (OR = 1.34, 95% CI = 1.04-1.74, p = 0.026; OR = 1.34, 95% CI = 1.03-1.73, p = 0.029, respectively). In stratified analysis according to gender and age, we observed that rs3800229 T/G and rs4946935 G/A genotypes were associated with an increase the risk of TB among males and age ≤41 years, respectively (OR = 1.47, 95% CI = 1.06-2.04, p = 0.022 and OR = 1.45, 95% CI = 1.05-2.02, p = 0.025). CONCLUSIONS: Our study showed that rs3800229 and rs4946935 in FOXO3 were associated with a risk of TB in the Chinese population.

The Inhibitory Effect of GlmU Acetyltransferase Inhibitor TPSA on Mycobacterium tuberculosis May Be Affected Due to Its Methylation by Methyltransferase Rv0560c.

Mycobacterium tuberculosis bifunctional enzyme GlmU is a novel target for anti-TB drugs and is involved in glycosyl donor UDP-N-acetylglucosamine biosynthesis. Here, we found that TPSA (2-[5-(2-{[4-(2-thienyl)-2-pyrimidinyl]sulfanyl}acetyl)-2-thienyl]acetic acid) was a novel inhibitor for GlmU acetyltransferase activity (IC50: 5.3 µM). The interaction sites of GlmU and TPSA by molecular docking were confirmed by site-directed mutagenesis. TPSA showed an inhibitory effect on Mtb H37Ra growth and intracellular H37Ra in macrophage cells (MIC: 66.5 µM). To investigate why TPSA at a higher concentration (66.5 µM) was able to inhibit H37Ra growth, proteome and transcriptome of H37Ra treated with TPSA were analyzed. The expression of two methyltransferases MRA_0565 (Rv0558) and MRA_0567 (Rv0560c) were markedly increased. TPSA was pre-incubated with purified Rv0558 and Rv0560c in the presence of S-adenosylmethionine (methyl donor) respectively, resulting in its decreased inhibitory effect of GlmU on acetyltransferase activity. The inhibition of TPSA on growth of H37Ra with overexpressed Rv0558 and Rv0560c was reduced. These implied that methyltransferases could modify TPSA. The methylation of TPSA catalyzed by Rv0560c was subsequently confirmed by LC-MS. Therefore, TPSA as a GlmU acetyltransferase activity inhibitor may offer a structural basis for new anti-tuberculosis drugs. TPSA needs to be modified further by some groups to prevent its methylation by methyltransferases.

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.