Increased expression of Mycobacterium tuberculosis Rv3737 gene associated with low-level amikacin resistance.

INTRODUCTION: As an infectious disease, tuberculosis (TB) poses a serious threat to public health. Although amikacin (AMK) is an important antibiotic for the treatment of drug-resistant TB, its resistance mechanisms are not fully understood. METHODS: To investigate the role of Rv3737 gene on AMK drug susceptibility, a Mycobacterium tuberculosis (M.tb) Rv3737 knockout strain (H37Rv△Rv3737) and a Mycobacterium smegmatis (M.sm) Rv3737 overexpressing strain (Msm/pMV261-Rv3737) were used to detect their minimal inhibitory concentrations (MICs) in this study. RESULTS: The AMK MICs of Rv3737 knockout and overexpressing strains were 4-fold lower and 2-fold higher than those of the wild-type and empty plasmid strains, respectively. The results of clinical isolates showed that no Rv3737 gene mutation was found to be associated with AMK susceptibility, while the rrs A1401G mutation remained the main mechanism of high level of AMK resistance (MIC>32 µg/ml). There was a positive correlation between Rv3737 mRNA expression level and AMK MIC. In the isolates with low-level AMK resistance (MIC = 4 µg/ml) without rrs A1401G mutation, the expression level of Rv3737 gene was significantly higher than those of susceptible isolates. CONCLUSIONS: In this study, the Rv3737 gene was reported for the first time for its effect on AMK susceptibility in M.tb. Although the rrs A1401G mutation remains the main reason of high-level AMK resistance, high expression of the Rv3737 gene was associated with low-level AMK resistance in clinical isolates.

Discovery and characterization of genes conferring natural resistance to the antituberculosis antibiotic capreomycin.

Metagenomic-based studies have predicted an extraordinary number of potential antibiotic-resistance genes (ARGs). These ARGs are hidden in various environmental bacteria and may become a latent crisis for antibiotic therapy via horizontal gene transfer. In this study, we focus on a resistance gene cph, which encodes a phosphotransferase (Cph) that confers resistance to the antituberculosis drug capreomycin (CMN). Sequence Similarity Network (SSN) analysis classified 353 Cph homologues into five major clusters, where the proteins in cluster I were found in a broad range of actinobacteria. We examine the function and antibiotics targeted by three putative resistance proteins in cluster I via biochemical and protein structural analysis. Our findings reveal that these three proteins in cluster I confer resistance to CMN, highlighting an important aspect of CMN resistance within this gene family. This study contributes towards understanding the sequence-structure-function relationships of the phosphorylation resistance genes that confer resistance to CMN.

Molecular Characterization of Resistance to Second-Line Anti-Mycobacterial Drugs among Clinical Isolates of Multidrug-Resistant Mycobacterium tuberculosis.

BACKGROUND: The emergence of multidrug resistance and extensively drug-resistant tuberculosis is a serious public health crisis. Using rapid and inexpensive molecular methods such as HRM assay in the detection of second-line drugs resistance in M. tuberculosis would be helpful in the treatment and control of XDR tuberculosis cases. METHODS: MDR-TB isolates were collected from Iranian tuberculosis laboratories. Drug susceptibility test performed via the indirect proportion method utilizing LJ Medium. Susceptibility to ciprofloxacin, ofloxacin, amikacin, kanamycin, and capreomycin, as second-line anti-tuberculosis agents were assessed. Single point mutations in gyrA, rrs and eis genes were detected via HRM assay and DNA sequencing. RESULTS: A DST test was performed for 56 MDR isolates and at least 27 (48.2%) isolates were resistant to CIP or OFL. Also, 14 (25%), 12 (21.4%), and 15 (26.7%) isolates were resistant to capreomycin, amikacin, and kanamycin, respectively. D94G, A90V, and G88C mutations were the most frequent mutations in gyrA gene. Also, A1401G mutation was detected more than the other mutations in rrs gene. CONCLUSIONS: The frequency of CIP/OFL and AMK/CAP/KAN-resistant TB is considerable among Iranian tuberculosis cases. HRM assay is a rapid and inexpensive test and can detect important mutation-based drug resistance in MDR-TB and XDR-TB isolates.

GenoType MTBDRsl for detection of second-line drugs and ethambutol resistance in multidrug-resistant Mycobacterium tuberculosis isolates at a high-throughput laboratory.

We assessed the performance of MTBDRsl for detection of resistance to fluoroquinolones, aminoglycosides/cyclic peptides, and ethambutol compared to BACTEC MGIT 960 by subjecting simultaneously to both tests 385 phenotypically multidrug-resistant-Mycobacterium tuberculosis isolates from Sao Paulo, Brazil. Discordances were resolved by Sanger sequencing. MTBDRsl correctly detected 99.7% of the multidrug-resistant isolates, 87.8% of the pre-XDR, and 73.9% of the XDR. The assay showed sensitivity of 86.4%, 100%, 85.2% and 76.4% for fluoroquinolones, amikacin/kanamycin, capreomycin and ethambutol, respectively. Specificity was 100% for fluoroquinolones and aminoglycosides/cyclic peptides, and 93.6% for ethambutol. Most fluoroquinolone-discordances were due to mutations in genome regions not targeted by the MTBDRsl v. 1.0: gyrA_H70R and gyrB_R446C, D461N, D449V, and N488D. Capreomycin-resistant isolates with wild-type rrs results on MTBDRsl presented tlyA mutations. MTBDRsl presented good performance for detecting resistance to second-line drugs and ethambutol in clinical isolates. In our setting, multidrug-resistant. isolates presented mutations not targeted by the molecular assay.

Case report: A 9-year systematic treatment failure of a pulmonary tuberculosis patient.

Objective: To explore the reasons of failure in a case of pulmonary tuberculosis (PTB) after 9 years systematic treatment. Methods: We extracted the patients' treatment history, drug susceptibility testing (DST), Computed tomography (CT) images, and sequenced the isolated strains by whole gene sequencing (WGS). Results: Although most results of the phenotypical DSTs were consistent with the genotype DST, the occurrence of gene resistance to amikacin (AMK), capreomycin (CAP), moxifloxacin (MFX) was earlier than the phenotypical DST. Based on the continuously reversed results of phenotypical DSTs, CT images in different stages and WGS, it can be confirmed that the patient was infected with two different strains of Mycobacterium tuberculosis (M.TB). Moreover, severe cavities may be another factor leading to treatment failure. Conclusion: Given the suggestive effect of genotype DST is earlier than the phenotypical DST, so genotype DST can play a better guiding role in patients with MDR-TB. Additionally, for patients who have not been cured for a long time, medication should be more cautious and the role of WGS in drug resistance surveillance should be fully utilized.

Evaluation of Susceptibility of the Human Pathogen Helicobacter pylori to the Antibiotic Capreomycin.

Helicobacter pylori infection causes gastritis, peptic ulcer disease, mucosa-associated lymphoid tissue lymphoma, and gastric cancer and can also promote thrombosis. It is estimated that approximately 4.5 billion individuals are infected, thus rendering H. pylori the most prevalent microbial pathogen. Currently established regimes for antibiotic treatment are massively challenged by increasing drug resistance and the development of novel antimicrobial therapies is urgently required. The antibiotic capreomycin is clinically used against multiple drug-resistant strains of Mycobacterium tuberculosis. It targets the complex between TlyA, a hemolysin- and RNA-binding protein, and the bacterial rRNA. In this study we have explored the possible antibacterial effects of capreomycin against several strains of H. pylori and found only moderate activity which was comparable to metronidazole-resistant strains. Molecular docking of capreomycin to TlyA proteins from H. pylori and M. tuberculosis identified several residues within TlyA which interact with the drug; however, binding affinities of H. pylori- TlyA for capreomycin appear to be higher than those of Mycobacterium- TlyA. The data suggest that capreomycin may warrant further investigations into its potential use as antibiotic against H. pylori.

[Exploratory study on detection of drug resistance of Mycobacterium tuberculosis in sputum specimens by next-generation sequencing].

Objective: To compare the diagnostic performance of next-generation sequencing (NGS) detection methods in sputum samples and Mycobacterium tuberculosis strains, in order to explore the feasibility of the NGS method to detect drug resistance in sputum specimens. Methods: In this retrospective study, the sputum specimens and corresponding clinical isolates of 50 pulmonary tuberculosis patients admitted to Beijing Chest Hospital from January 2017 to December 2017 were collected. The gene mutations of katG, inhA, rpoB, embA, embB, rpsL, rrs, gyrA, gyrB and tlyA in sputum specimens and corresponding clinical isolates were detected by NGS method. The phenotypic drug susceptibility test (DST) of the strains was carried out by the proportion method. Using DST results as a reference, the sensitivity, specificity, positive predictive value and negative predictive value of the NGS method for clinical strains and sputum specimens, as well as the consistency statistic (Kappa) with phenotype DST were calculated respectively. The Chi-square test was used to compare the accuracy of the NGS testing in sputum samples and strain samples. Results: The results showed that rpoB(63.83%, 30/47) and rrs(57.45%, 27/47) were the most common mutated genes, followed by katG(46.81%, 22/47), rpsL(29.79%, 14/47), gyrA(27.66%, 13/47), embB(21.28%, 10/47), tlyA(12.77%, 6/47), gyrB(8.51%, 4/47), and inhA promoter(19.15%, 9/47), embA promoter region (12.77%, 6/47) mutation. when the NGS method was compared with the resistance phenotype of isoniazid, rifampicin, ethambutol, second-line injectable drugs (streptomycin, capreomycin, kanamycin, amikacin), levofloxacin, the sensitivity were 85.71%, 91.67%, 77.78%, 81.82%, 100.00%, 87.50%, 100.00%, 69.23%, and the specificity were 100.00%, 94.12, 87.50%, 89.47%, 97.06%, 96.97%, 94.29%, 89.29% in sputum samples, while in strain samples, the sensitivity were 92.86%, 100.00%, 81.82%, 86.96%, 88.89%, 80.00%, 100.00%, 85.71%. The specificity were 100.00%, 92.86%, 87.10%, 94.74%, 100.00%, 100.00%, 97.14%, 92.86%. Compared with the phenotypic drug susceptibility results, the NGS method has better detection performance for isoniazid, rifampicin, capreomycin, kanamycin, and amikacin in sputum specimens (Kappa≥0.75); while among the strains, the NGS method had a good detection performance for isoniazid, rifampicin, streptomycin, capreomycin, kanamycin, amikacin and levofloxacin (Kappa≥0.75). With the accuracy of the NGS method for detecting strains as a reference, there was no statistically significant difference in the accuracy of all drug resistance detected between strains and sputum specimens. Conclusions: This study showed that the NGS technology was effective in predicting the resistance of isoniazid, rifampicin, and second-line injectable drugs (capreomycin, kanamycin and amikacin) by detecting sputum samples and strain genotypes, suggesting the feasibility and potential of direct detection of sputum samples by the NGS method as an early detection method for drug resistance.

Distribution of Common and Rare Genetic Markers of Second-Line-Injectable-Drug Resistance in Mycobacterium tuberculosis Revealed by a Genome-Wide Association Study.

Point mutations in the rrs gene and the eis promoter are known to confer resistance to the second-line injectable drugs (SLIDs) amikacin (AMK), capreomycin (CAP), and kanamycin (KAN). While mutations in these canonical genes confer the majority of SLID resistance, alternative mechanisms of resistance are not uncommon and threaten effective treatment decisions when using conventional molecular diagnostics. In total, 1,184 clinical Mycobacterium tuberculosis isolates from 7 countries were studied for genomic markers associated with phenotypic resistance. The markers rrs:A1401G and rrs:G1484T were associated with resistance to all three SLIDs, and three known markers in the eis promoter (eis:G-10A, eis:C-12T, and eis:C-14T) were similarly associated with kanamycin resistance (KAN-R). Among 325, 324, and 270 AMK-R, CAP-R, and KAN-R isolates, 274 (84.3%), 250 (77.2%), and 249 (92.3%) harbored canonical mutations, respectively. Thirteen isolates harbored more than one canonical mutation. Canonical mutations did not account for 103 of the phenotypically resistant isolates. A genome-wide association study identified three genes and promoters with mutations that, on aggregate, were associated with unexplained resistance to at least one SLID. Our analysis associated whiB7 5'-untranslated-region mutations with KAN resistance, supporting clinical relevance for this previously demonstrated mechanism of KAN resistance. We also provide evidence for the novel association of CAP resistance with the promoter of the Rv2680-Rv2681 operon, which encodes an exoribonuclease that may influence the binding of CAP to the ribosome. Aggregating mutations by gene can provide additional insight and therefore is recommended for identifying rare mechanisms of resistance when individual mutations carry insufficient statistical power.

Rv1258c acts as a drug efflux pump and growth controlling factor in Mycobacterium tuberculosis.

The possible role of efflux pump as a survival mechanism in Mycobacterium tuberculosis (M. tb) is gaining an increasing attention. Previously, Rv1258c (Tap) and its certain mutations confer the clinically relevant drug resistance. In this study, we found new mutations of Rv1258c in G195C, T297P and I328T. Effect of modulating T297P and I328T on the drug resistance by knockout and complement in M. tb H37Rv showed that M. tb ΔRv1258c showed a slightly lower MIC for rifampin, ethambutol, ofloxacin, amikacin, capreomycin and streptomycin than M. tb H37Rv WT and the complement. Rv1258c T297P and Rv1258c I328T showed an increased drug resistance to ethambutol and capreomycin than the complement of Rv1258c WT. Most importantly, M. tb ΔRv1258c exhibited a slow growth in the normal culture medium. TMT-based quantitative proteomics analysis of M. tb ΔRv1258c and WT showed that the knockout of Rv1258c greatly down-regulated the expression of the ribosome system and one of the special five type VII secretion systems, ESX-3, which impaired the bacterial growth. These results indicate that the newly found T297P and I328T mutations of Rv1258c contributed to an increased resistance to ethambutol and capreomycin, and Rv1258c as growth controlling factor influencing the growth of M. tb.

Dual-Mechanism Confers Self-Resistance to the Antituberculosis Antibiotic Capreomycin.

Capreomycin (CMN) is an important second-line antituberculosis antibiotic isolated from Saccharothrix mutabilis subspecies capreolus. The gene cluster for CMN biosynthesis has been identified and sequenced, wherein the cph gene was annotated as a phosphotransferase likely engaging in self-resistance. Previous studies reported that Cph inactivates two CMNs, CMN IA and IIA, by phosphorylation. We, herein, report that (1) Escherichia coli harboring the cph gene becomes resistant to both CMN IIA and IIB, (2) phylogenetic analysis regroups Cph to a new clade in the phosphotransferase protein family, (3) Cph shares a three-dimensional structure akin to the aminoglycoside phosphotransferases with a high binding affinity (KD) to both CMN IIA and IIB at micromolar levels, and (4) Cph utilizes either ATP or GTP as a phosphate group donor transferring its γ-phosphate to the hydroxyl group of CMN IIA. Until now, Cph and Vph (viomycin phosphotransferase) are the only two known enzymes inactivating peptide-based antibiotics through phosphorylation. Our biochemical characterization and structural determination conclude that Cph confers the gene-carrying species resistance to CMN by means of either chemical modification or physical sequestration, a naturally manifested belt and braces strategy. These findings add a new chapter into the self-resistance of bioactive natural products, which is often overlooked while designing new bioactive molecules.

Insights into the mutations leading to capreomycin resistance in S-adenosyl-L-methionine binding motif in TlyA from Mycobacterium tuberculosis.

Capreomycin is a second line antibiotic used for the treatment of drug resistant Tuberculosis (TB), primary reason of death from a solo infectious organism, Mycobacterium tuberculosis (M.tb). Capreomycin targets the ribosome of bacteria and is known to bind at the interface where the large and small ribosomal subunits interact in M.tb using an S-Adenosyl Methionine (SAM) dependent methyltransferase, TlyA (Rv1794). Besides the methyltransferase activity, TlyA has also been found to show substantial haemolytic activity. The dual activity of TlyA highlights its crucial role in pathogenesis and virulence of M.tb. In the present study, docking and molecular dynamics (MD) simulations were carried out to explore the impact of mutations in a conserved SAM binding motif, 90GASTG94, on the affinity of TlyA enzyme for SAM. Two already reported mutations, A91E and S92L, and the remaining wild type residues, Gly90, Thr93, Gly94 mutated to alanine were taken into consideration resulting in a total of six systems, wild type + SAM, G90A + SAM, A91E + SAM, S92L + SAM, T93A + SAM and G94A + SAM that were subjected to 100 ns MD simulations. Docking scores and MD simulations analyses revealed that in contrast to wild type, mutants reduced the affinity of SAM for TlyA with most prominent effect observed in case of alanine mutants. Mutations also led to the loss of hydrogen bond and hydrophobic interactions and large-scale movement of atoms evident from the principal component analyses indicating their destabilizing impact on TlyA. The present study gives insights into influence of mutations on binding of SAM to TlyA in M.tb and promoting capreomycin resistance.Communicated by Ramaswamy H. Sarma.

Detection of isoniazid, fluoroquinolone, ethionamide, amikacin, kanamycin, and capreomycin resistance by the Xpert MTB/XDR assay: a cross-sectional multicentre diagnostic accuracy study.

BACKGROUND: The WHO End TB Strategy requires drug susceptibility testing and treatment of all people with tuberculosis, but second-line diagnostic testing with line-probe assays needs to be done in experienced laboratories with advanced infrastructure. Fewer than half of people with drug-resistant tuberculosis receive appropriate treatment. We assessed the diagnostic accuracy of the rapid Xpert MTB/XDR automated molecular assay (Cepheid, Sunnyvale, CA, USA) to overcome these limitations. METHODS: We did a prospective study involving individuals presenting with pulmonary tuberculosis symptoms and at least one risk factor for drug resistance in four sites in India (New Delhi and Mumbai), Moldova, and South Africa between July 31, 2019, and March 21, 2020. The Xpert MTB/XDR assay was used as a reflex test to detect resistance to isoniazid, fluoroquinolones, ethionamide, amikacin, kanamycin, and capreomycin in adults with positive results for Mycobacterium tuberculosis complex on Xpert MTB/RIF or Ultra (Cepheid). Diagnostic performance was assessed against a composite reference standard of phenotypic drug-susceptibility testing and whole-genome sequencing. This study is registered with ClinicalTrials.gov, number NCT03728725. FINDINGS: Of 710 participants, 611 (86%) had results from both Xpert MTB/XDR and the reference standard for any drug and were included in analysis. Sensitivity for Xpert MTB/XDR detection of resistance was 94% (460 of 488, 95% CI 92-96) for isoniazid, 94% (222 of 235, 90-96%) for fluoroquinolones, 54% (178 of 328, 50-61) for ethionamide, 73% (60 of 82, 62-81) for amikacin, 86% (181 of 210, 81-91) for kanamycin, and 61% (53 of 87, 49-70) for capreomycin. Specificity was 98-100% for all drugs. Performance was equivalent to that of line-probe assays. The non-determinate rate of Xpert MTB/XDR (ie, invalid M tuberculosis complex detection) was 2·96%. INTERPRETATION: The Xpert MTB/XDR assay showed high diagnostic accuracy and met WHO's minimum target product profile criteria for a next-generation drug susceptibility test. The assay has the potential to diagnose drug-resistant tuberculosis rapidly and accurately and enable optimum treatment. FUNDING: German Federal Ministry of Education and Research through KfW, Dutch Ministry of Foreign Affairs, and Australian Department of Foreign Affairs and Trade.

Whole genome sequencing of clinical samples reveals extensively drug resistant tuberculosis (XDR TB) strains from the Beijing lineage in Nigeria, West Africa.

Multi-drug (MDR) and extensively drug-resistant (XDR) tuberculosis (TB) continues to be a global public health problem especially in high TB burden countries like Nigeria. Many of these cases are undetected and go on to infect high risk individuals. Clinical samples from positive rifampicin resistant Xpert®MTB/Rif assay were subjected to direct whole genome sequencing and bioinformatics analysis to identify the full antibiotics resistance and lineage profile. We report two (2) XDR TB samples also belonging to the East-Asian/Beijing family of lineage 2 Mycobacterium tuberculosis complex from clinical samples in Nigeria. Our findings further reveal the presence of mutations that confer resistance to first-line drugs (rifampicin, isoniazid, ethambutol and pyrazanimide), second-line injectables (capreomycin, streptomycin, kanamycin and/or amikacin) and at least one of the fluoroquinolones (ofloxacin, moxifloxacin, levofloxacin and/or ciprofloxacin) in both samples. The genomic sequence data from this study not only provide the first evidence of XDR TB in Nigeria and West Africa, but also emphasize the importance of WGS in accurately detecting MDR and XDR TB, to ensure adequate and proper management treatment regimens for affected individuals. This will greatly aid in preventing the spread of drug resistance TB in high burden countries.

Aminoglycosides and Capreomycin in the Treatment of Multidrug-resistant Tuberculosis: Individual Patient Data Meta-analysis of 12 030 Patients From 25 Countries, 2009-2016.

BACKGROUND: As new drugs are developed for multidrug-resistant tuberculosis (MDR-TB), the role of currently used drugs must be reevaluated. METHODS: We combined individual-level data on patients with pulmonary MDR-TB published during 2009-2016 from 25 countries. We compared patients receiving each of the injectable drugs and those receiving no injectable drugs. Analyses were based on patients whose isolates were susceptible to the drug they received. Using random-effects logistic regression with propensity score matching, we estimated the effect of each agent in terms of standardized treatment outcomes. RESULTS: More patients received kanamycin (n = 4330) and capreomycin (n = 2401) than amikacin (n = 2275) or streptomycin (n = 1554), opposite to their apparent effectiveness. Compared with kanamycin, amikacin was associated with 6 more cures per 100 patients (95% confidence interval [CI], 4-8), while streptomycin was associated with 7 (95% CI, 5-8) more cures and 5 (95% CI, 4-7) fewer deaths per 100 patients. Compared with capreomycin, amikacin was associated with 9 (95% CI, 6-11) more cures and 5 (95% CI, 2-8) fewer deaths per 100 patients, while streptomycin was associated with 10 (95% CI, 8-13) more cures and 10 (95% CI, 7-12) fewer deaths per 100 patients treated. In contrast to amikacin and streptomycin, patients treated with kanamycin or capreomycin did not fare better than patients treated with no injectable drugs. CONCLUSIONS: When aminoglycosides are used to treat MDR-TB and drug susceptibility test results support their use, streptomycin and amikacin, not kanamycin or capreomycin, are the drugs of choice.

Capreomycin resistance prediction in two species of Mycobacterium using a stacked ensemble method.

AIMS: Predicting bacterial resistance provides valuable information that can assist in clinical decisions. With recent advances in whole genome sequencing technology, the detection of antibiotic resistance (AR) proteins directly from genomic data is becoming feasible. AR genes/proteins can be identified using best-hit methods that work by comparing candidate sequences with known AR genes in public databases. However, these approaches may fail to detect resistance genes with sequences that differ significantly from known sequences. Our goal is to develop a machine learning technique to accurately predict capreomycin resistance in Mycobacteria with low false discovery rates. METHODS AND RESULTS: We present a stacked ensemble learning model as an alternative to traditional DNA sequence alignment-based methods using optimal features generated from the physicochemical, evolutionary and secondary structure properties of protein sequences. We train logistic regression, C5.0 and support vector machine (SVM) algorithms as our base classifiers, and our stacked ensemble predictors combine the results from the base classifiers to achieve higher accuracy. Compared with our most accurate base classifier (SVM), our most accurate stacked ensemble predictor increases training accuracy by 2·43%. Our stacked ensemble predictors achieve test accuracy up to 81·25%. CONCLUSIONS: We developed a stacked ensemble model to predict capreomycin resistance for Mycobacteria with an accuracy >80% using protein sequences with sequence similarity ranging between 10% and 70%. This performance cannot be achieved with best-hit methods due to differences in sequence similarity. SIGNIFICANCE AND IMPACT OF THE STUDY: Today an estimated one-half million cases of multidrug-resistant (MDR) and extensively drug-resistant (XDR) tuberculosis (TB) occur annually worldwide at a great cost. Because capreomycin is a second-line drug used to treat drug-resistant TB, the ability to use a machine learning approach to classify capreomycin-resistant TB in a timely manner is crucial for the successful treatment of MDR or XDR TB.

Assessing capreomycin resistance on tlyA deficient and point mutation (G695A) Mycobacterium tuberculosis strains using multi-omics analysis.

Capreomycin (CAP), a cyclic peptide antibiotic, is considered to be an ideal second-line drug for tuberculosis (TB). However, in the past few years, the emergence of more CAP-resistant (CAPr) TB patients has limited its use. Although it has been reported that CAP resistance to Mycobacterium tuberculosis (Mtb) is associated with rrs or tlyA mutation, the exact mechanism of CAPr Mtb strains, especially the mechanism associated with tlyA deficient or mutation, is not fully understood. Herein, we utilized a multi-omics (genome, proteome, and metabolome) approach to assess CAP resistance on tlyA deficient CAPr Mtb strains (CAPr1) and tlyA point mutation CAPr Mtb strains (CAPr2) that we established for the first time in vitro to investigate the CAP-resistant mechanism. Our results showed that the CAPr1 strains (> 40 µg/ml) was more resistant to CAP than the CAPr2 strains (G695A, 10 µg/ml). Furthermore, multi-omics analysis indicated that the CAPr1 strains exhibited greater drug tolerance than the CAPr2 strains may be associated with the weakening of S-adenosyl-L-methionine-dependent methyltransferase (AdoMet-MT) activity and abnormal membrane lipid metabolism such as suppression of fatty acid metabolism, promotion of glycolipid phospholipid and glycerolipid metabolism. As a result, these studies reveal a new mechanism for CAP resistance to tlyA deficient or mutation Mtb strains, and may be helpful in developing new therapeutic approaches to prevent Mtb resistance to CAP.

Impact of bedaquiline and capreomycin on the gene expression patterns of multidrug-resistant Mycobacterium tuberculosis H37Rv strain and understanding the molecular mechanism of antibiotic resistance.

The emergence of multidrug resistance (MDR), extensively drug-resistant, and total drug-resistant Mycobacterium tuberculosis (Mtb) strains have hampered the treatment of tuberculosis (TB). Capreomycin and Bedaquiline are currently used for MDR-TB treatment. To understand the impact of these antibiotics on Mtb genes, we have curated the gene expression data where the Mtb cultures were exposed to the Bedaquiline and Capreomycin. Based on the P value cut off (<0.05) and logFC (<-0.5 and >+0.5) values, we have selected the top differentially expressed genes during the antibiotic exposures. We have observed that the top differentially expressed Mtb genes were related to universal stress genes, two-component regulatory systems, and drug efflux pumps. We have curated the Mtb gene datasets and carried out the functional over-representation analysis using the individual gene expression values. We further, constructed the gene interaction networks of antibiotic resistance genes and virulence genes of Mtb to understand the impact of the antibiotics at the molecular level and thus to understand the antimicrobial resistance and virulence patterns. Our study elucidates the impact of antibiotics on the Mtb genes at the molecular level and the positively enriched pathways, operons, and regulons data are helpful in understanding the resistance patterns in Mtb. The upregulated genes during the exposure of Bedaquiline and Capreomycin can be considered as potent drug targets for the development of new anti-TB drugs.

Prevalence and detection of drug resistant mutations in Mycobacterium tuberculosis among drug naïve patients in Nairobi, Kenya.

BACKGROUND: Tuberculosis (TB), an ancient scourge of humanity known for several thousands of years, is still a significant public health challenge in many countries today even though some progress has been made in recent years in controlling the disease. The study's aim was to determine the prevalence of mutations responsible for drug resistance in Mycobacterium tuberculosis among patients visiting selected health centers in Nairobi, Kenya. METHODS: The cross-sectional study involved 132 TB positive patients visiting Mbagathi and Chandaria hospitals between September 2015 and August 2016. Sputum samples were collected from the participants and handled in a biosafety level 3 laboratory at the Kenya Medical Research Institute (KEMRI). Samples were decontaminated using N-Acetyl-L-Cysteine (NALC) - Sodium Hydroxide (NALC-NaOH), stained using Zeihl-Neelsen (ZN), and cultured in Mycobacterium Growth Indicator Tube (MGIT). DNA extracted from cultured isolates using Genolyse™ technique was subjected to Multiplex PCR amplification and reverse hybridization for detection of drug resistance mutations on rpoB, katG, inhA, gyrA, gyrB, rrs and eis genes using Hain Genotype MTBDRplus and MTBDRsl. RESULTS: All 132 (100%) patients included in the study were culture positive for M. tuberculosis. Among them, 72 (54%) were male while the remaining 60 (46%) were female. The mean age of the patients was 26.4 ± 19.4 (SD) with a range of 18 to 60 years. Overall, the prevalence of the resistance to first and second-line TB drugs was 1.5% (2/132). Resistance to isoniazid (INH) was observed in 1 of 132 patients (0.8%), as was multi-drug resistant tuberculosis (MDR-TB), also at 0.8%. No resistance to fluoroquinolones (FQ) or kanamycin (KAN) was observed. The INH resistant strain had the katG mutations S315 T, while mutations detected for the MDR-TB were katG S513 T for INH, rpoB S531 L for rifampicin (RIF) and rrs G1484 T for cross-resistance to aminoglycosides/capreomycin (AG/CP). CONCLUSIONS: Molecular analysis confirms transmission of the drug-resistant M. tuberculosis strains. The data suggested that there is homogeneity when it comes to the type of drug resistance and mutation that occurs in the region. This calls for intensified drug resistance surveillance and drug adherence among patients infected with TB.

Isolation of Mycobacterium arupense from pleural effusion: culprit or not?

BACKGROUND: Mycobacterium arupense, first identified in 2006, is a slow-growing nontuberculous mycobacterium (NTM) and an emerging cause of tenosynovitis, potentially associated with immunosuppression. However, unlike the diagnostic value of its isolation from osteoarticular specimens, the significance of detecting M. arupense in respiratory specimens is not yet clear. CASE PRESENTATION: To our knowledge, we, for the first time, described the identification of M. arupense from the pleural effusion of an immunocompetent patient, who presented with fever and chylothorax. The symptoms resolved with doxycycline treatment for 45 days and a low-fat, high-protein diet. Follow-up at 14 months showed no relapse. CONCLUSIONS: Because the patient fully recovered without combined anti-NTM treatment, we did not consider M. arupense the etiological cause in this case. This indicates that M. arupense detected in pleural effusion is not necessarily a causative agent and careful interpretation is needed in terms of its clinical relevance.

Capreomycin inhibits the initiation of amyloid fibrillation and suppresses amyloid induced cell toxicity.

Protein aggregation and amyloid fibrillation are responsible for several serious pathological conditions (like type II diabetes, Alzheimer's and Parkinson's diseases etc.) and protein drugs ineffectiveness. Therefore, a molecule that can inhibit the amyloid fibrillation and potentially clear amyloid fibrils is of great therapeutic value. In this manuscript, we investigated the antiamyloidogenic, fibril disaggregating, as well as cell protective effect of an anti-tuberculosis drug, Capreomycin (CN). Aggregation kinetics data, as monitored by ThT fluorescence, inferred that CN retards the insulin amyloid fibrillation by primarily targeting the fibril elongation step with little effect on lag time. Increasing the dose of CN boosted its inhibitory potency. Strikingly, CN arrested the growth of fibrils when added during the elongation phase, and disaggregated mature insulin fibrils. Our Circular Dichroism (CD) results showed that, although CN is not able to maintain the alpha helical structure of protein during fibrillation, reduces the formation of beta sheet rich structure. Furthermore, Dynamic Light Scattering (DLS) and Transmission Electronic Microscopy (TEM) analysis confirmed that CN treated samples exhibited different size distribution and morphology, respectively. In addition, molecular docking results revealed that CN interacts with insulin through hydrophobic interactions as well as hydrogen bonding, and the Hemolytic assay confirmed the non-hemolytic activity of CN on human RBCs. For future research, this study may assist in the rational designing of molecules against amyloid formation.