Genotypic and phenotypic drug resistance patterns of Mycobacterium tuberculosis isolated from presumptive pulmonary tuberculosis patients in Ethiopia: A multicenter study.

BACKGROUND: The emergence of drug-resistant tuberculosis (DR-TB) has been a major obstacle to global tuberculosis control programs, especially in developing countries, including Ethiopia. This study investigated drug resistance patterns and associated mutations of Mycobacterium tuberculosis Complex (MTBC) isolates from the Amhara, Gambella, and Benishangul-Gumuz regions of Ethiopia. METHODS: A cross-sectional study was conducted using 128 MTBC isolates obtained from patients with presumptive tuberculosis (TB). Phenotypic (BACTEC MGIT 960) and genotypic (MTBDRplus and MTBDRsl assays) methods were used for drug susceptibility testing. Data were entered into Epi-info and analyzed using SPSS version 25. Frequencies and proportions were determined to describe drug resistance levels and associated mutations. RESULTS: Of the 127 isolates recovered, 100 (78.7%) were susceptible to four first-line anti-TB drugs. Any drug resistance, polydrug resistance, and multi-drug resistance (MDR) were detected in 21.3% (27), 15.7% (20), and 15% (19) of the isolates, respectively, by phenotypic and/or genotypic methods. Mono-resistance was observed for Isoniazid (INH) (2, 1.6%) and Streptomycin (STR) (2, 1.6%). There were two genotypically discordant RIF-resistant cases and one INH-resistant case. One case of pre-extensively drug-resistant TB (pre-XDR-TB) and one case of extensively drug-resistant TB (XDR-TB) were identified. The most frequent gene mutations associated with INH and rifampicin (RIF) resistance were observed in the katG MUT1 (S315T1) (20, 76.9%) and rpoB (S531L) (10, 52.6%) genes, respectively. Two MDR-TB isolates were resistant to second-line drugs; one had a mutation in the gyrA MUT1 gene, and the other had missing gyrA WT1, gyrA WT3, and rrs WT1 genes without any mutation. CONCLUSIONS: The detection of a significant proportion of DR-TB cases in this study suggests that DR-TB is a major public health problem in Ethiopia. Thus, we recommend the early detection and treatment of DR-TB and universal full first-line drug-susceptibility testing in routine system.

A dose-response model for statistical analysis of chemical genetic interactions in CRISPRi screens.

An important application of CRISPR interference (CRISPRi) technology is for identifying chemical-genetic interactions (CGIs). Discovery of genes that interact with exposure to antibiotics can yield insights to drug targets and mechanisms of action or resistance. The objective is to identify CRISPRi mutants whose relative abundance is suppressed (or enriched) in the presence of a drug when the target protein is depleted, reflecting synergistic behavior. Different sgRNAs for a given target can induce a wide range of protein depletion and differential effects on growth rate. The effect of sgRNA strength can be partially predicted based on sequence features. However, the actual growth phenotype depends on the sensitivity of cells to depletion of the target protein. For essential genes, sgRNA efficiency can be empirically measured by quantifying effects on growth rate. We observe that the most efficient sgRNAs are not always optimal for detecting synergies with drugs. sgRNA efficiency interacts in a non-linear way with drug sensitivity, producing an effect where the concentration-dependence is maximized for sgRNAs of intermediate strength (and less so for sgRNAs that induce too much or too little target depletion). To capture this interaction, we propose a novel statistical method called CRISPRi-DR (for Dose-Response model) that incorporates both sgRNA efficiencies and drug concentrations in a modified dose-response equation. We use CRISPRi-DR to re-analyze data from a recent CGI experiment in Mycobacterium tuberculosis to identify genes that interact with antibiotics. This approach can be generalized to non-CGI datasets, which we show via an CRISPRi dataset for E. coli growth on different carbon sources. The performance is competitive with the best of several related analytical methods. However, for noisier datasets, some of these methods generate far more significant interactions, likely including many false positives, whereas CRISPRi-DR maintains higher precision, which we observed in both empirical and simulated data.

The Structural and Molecular Mechanisms of Mycobacterium tuberculosis Translational Elongation Factor Proteins.

Targeting translation factor proteins holds promise for developing innovative anti-tuberculosis drugs. During protein translation, many factors cause ribosomes to stall at messenger RNA (mRNA). To maintain protein homeostasis, bacteria have evolved various ribosome rescue mechanisms, including the predominant trans-translation process, to release stalled ribosomes and remove aberrant mRNAs. The rescue systems require the participation of translation elongation factor proteins (EFs) and are essential for bacterial physiology and reproduction. However, they disappear during eukaryotic evolution, which makes the essential proteins and translation elongation factors promising antimicrobial drug targets. Here, we review the structural and molecular mechanisms of the translation elongation factors EF-Tu, EF-Ts, and EF-G, which play essential roles in the normal translation and ribosome rescue mechanisms of Mycobacterium tuberculosis (Mtb). We also briefly describe the structure-based, computer-assisted study of anti-tuberculosis drugs.

Targeting Mycobacterium tuberculosis pH-driven adaptation.

Mycobacterium tuberculosis (Mtb) senses and adapts to host environmental cues as part of its pathogenesis. One important cue sensed by Mtb is the acidic pH of its host niche - the macrophage. Acidic pH induces widespread transcriptional and metabolic remodelling in Mtb. These adaptations to acidic pH can lead Mtb to slow its growth and promote pathogenesis and antibiotic tolerance. Mutants defective in pH-dependent adaptations exhibit reduced virulence in macrophages and animal infection models, suggesting that chemically targeting these pH-dependent pathways may have therapeutic potential. In this review, we discuss mechanisms by which Mtb regulates its growth and metabolism at acidic pH. Additionally, we consider the therapeutic potential of disrupting pH-driven adaptations in Mtb and review the growing class of compounds that exhibit pH-dependent activity or target pathways important for adaptation to acidic pH.

Prevalence and genetic basis of Mycobacterium tuberculosis resistance to pretomanid in China.

BACKGROUND: Pretomanid is a key component of new regimens for the treatment of drug-resistant tuberculosis (TB) which are being rolled out globally. However, there is limited information on the prevalence of pre-existing resistance to the drug. METHODS: To investigate pretomanid resistance rates in China and its underlying genetic basis, as well as to generate additional minimum inhibitory concentration (MIC) data for epidemiological cutoff (ECOFF)/breakpoint setting, we performed MIC determinations in the Mycobacterial Growth Indicator Tube™ (MGIT) system, followed by WGS analysis, on 475 Mycobacterium tuberculosis (MTB) isolated from Chinese TB patients between 2013 and 2020. RESULTS: We observed a pretomanid MIC distribution with a 99% ECOFF equal to 0.5 mg/L. Of the 15 isolates with MIC values > 0.5 mg/L, one (MIC = 1 mg/L) was identified as MTB lineage 1 (L1), a genotype previously reported to be intrinsically less susceptible to pretomanid, two were borderline resistant (MIC = 2-4 mg/L) and the remaining 12 isolates were highly resistant (MIC ≥ 16 mg/L) to the drug. Five resistant isolates did not harbor mutations in the known pretomanid resistant genes. CONCLUSIONS: Our results further support a breakpoint of 0.5 mg/L for a non-L1 MTB population, which is characteristic of China. Further, our data point to an unexpected high (14/475, 3%) pre-existing pretomanid resistance rate in the country, as well as to the existence of yet-to-be-discovered pretomanid resistance genes.

Drug-resistant Mycobacterium tuberculosis among Nepalese patients at a tuberculosis referral center.

BACKGROUND: Multidrug-resistant tuberculosis (MDR-TB), characterized by isoniazid and rifampicin resistance, is caused by chromosomal mutations that restrict treatment options and complicate tuberculosis management. This study sought to investigate the prevalence of pre-extensively drug-resistant (pre-XDR) and extensively drug-resistant (XDR) tuberculosis, as well as mutation pattern, in Nepalese patients with MDR/rifampicin-resistant (RR)-TB strains. METHODS: A cross-sectional study was conducted on MDR/RR-TB patients at the German Nepal Tuberculosis Project from June 2017 to June 2018. The MTBDRsl line probe assay identified pre-XDR-TB and XDR-TB. Pre-XDR-TB included MDR/RR-TB with resistance to any fluoroquinolone (FLQ), while XDR-TB included MDR/RR-TB with resistance to any FLQ and at least one additional group A drug. Mutation status was determined by comparing bands on reaction zones [gyrA and gyrB for FLQ resistance, rrs for SILD resistance, and eis for low-level kanamycin resistance, according to the GenoType MTBDRsl VER 2.0, Hain Lifescience GmbH, Nehren, Germany definition of pre-XDR and XDR] to the evaluation sheet. SPSS version 17.0 was used for data analysis. RESULTS: Out of a total of 171 patients with MDR/RR-TB, 160 had (93.57%) had MTBC, of whom 57 (35.63%) had pre-XDR-TB and 10 (6.25%) had XDR-TB. Among the pre-XDR-TB strains, 56 (98.25%) were FLQ resistant, while 1 (1.75%) was SLID resistant. The most frequent mutations were found at codons MUT3C (57.14%, 32/56) and MUT1 (23.21%, 13/56) of the gyrA gene. One patient had SLID resistant genotype at the MUT1 codon of the rrs gene (100%, 1/1). XDR-TB mutation bands were mostly detected on MUT1 (30%, 3/10) of the gyrA and rrs, MUT3C (30%, 3/10) of the gyrA, and MUT1 (30%, 3/10) of the rrs. CONCLUSIONS: Pre-XDR-TB had a significantly higher likelihood than XDR-TB, with different specific mutation bands present in gyrA and rrs genes.

Mycobacterium tuberculosis PhoP integrates stress response to intracellular survival by regulating cAMP level.

Survival of Mycobacterium tuberculosis within the host macrophages requires the bacterial virulence regulator PhoP, but the underlying reason remains unknown. 3',5'-Cyclic adenosine monophosphate (cAMP) is one of the most widely used second messengers, which impacts a wide range of cellular responses in microbial pathogens including M. tuberculosis. Herein, we hypothesized that intra-bacterial cAMP level could be controlled by PhoP since this major regulator plays a key role in bacterial responses against numerous stress conditions. A transcriptomic analysis reveals that PhoP functions as a repressor of cAMP-specific phosphodiesterase (PDE) Rv0805, which hydrolyzes cAMP. In keeping with these results, we find specific recruitment of the regulator within the promoter region of rv0805 PDE, and absence of phoP or ectopic expression of rv0805 independently accounts for elevated PDE synthesis, leading to the depletion of intra-bacterial cAMP level. Thus, genetic manipulation to inactivate PhoP-rv0805-cAMP pathway decreases cAMP level, stress tolerance, and intracellular survival of the bacillus.

The Role of Efflux Pumps transporter in Multi-drug Resistant Tuberculosis: Mycobacterial memberane protein(MmpL5).

BACKGROUND: The overexpression of efflux pumps (Eps) was reported to contribute to multidrug resistant tuberculosis (MDR-TB). Increases in Eps that expel structurally unrelated drugs contribute to reduced susceptibility by decreasing the intracellular concentration of antibiotics. In the present study, an association of mycobacterial membrane protein (MmpS5-MmpL5) Ep and its gene regulator (Rv0678) was investigated in MDR-tuberculosis isolates. METHODS: MTB strains were isolated from patients at two different intervals, i.e., once when they had persistent symptoms despite 3-15 ≥ months of treatment and once when they had started new combination therapy ≥2-3 months. Sputum specimens were subjected to Xpert MTB/rifampicin test and then further susceptibility testing using proportional method and multiplex polymerase chain reaction (PCR) were performed on them. The isolates were characterized using both 16S-23S RNA and hsp65 genes spacer (PCR-restriction fragment length polymorphism). Whole-genome sequencing (WGS) was investigated on two isolates from culture-positive specimen per patient. The protein structure was simulated using the SWISS-MODEL. The input format used for this web server was FASTA (amino acid sequence). Protein structure was also analysis using Ramachandran plot. RESULTS: WGS documented deletion, insertion, and substitution in transmembrane transport protein MmpL5 (Rv0676) of Eps. Majority of the studied isolates (n = 12; 92.3%) showed a unique deletion mutation at three positions: (a) from amino acid number 771 (isoleucine) to 776 (valine), (b) from amino acid number 785 (valine) to 793 (histidine), and (c) from amino acid number 798 (leucine) to 806 (glycine)." One isolate (7.6%) had no deletion mutation. In all isolates (n = 13; 100%), a large insertion mutation consisting of 94 amino acid was observed "from amino acid number 846 (isoleucine) to amino acid number 939 (leucine)". Thirty-eight substitutions in Rv0676 were detected, of which 92.3% were identical in the studied isolates. WGS of mycobacterial membrane proteins (MmpS5; Rv0677) and its gene regulator (Rv0678) documented no deletion, insertion, and substitution. No differences were observed between MmpS5-MmpL5 and its gene regulator in isolates that were collected at different intervals. CONCLUSIONS: Significant genetic mutation like insertion, deletion, and substitution within transmembrane transport protein MmpL5 (Rv0676) can change the functional balance of Eps and cause a reduction in drug susceptibility. This is the first report documenting a unique amino acid mutation (insertion and deletion ≥4-94) in Rv0676 among drug-resistant MTB. We suggest the changes in Mmpl5 (Rv0676) might occurred due to in-vivo sub-therapeutic drug stress within the host cell. Changes in MmpL5 are stable and detected through subsequent culture-positive specimens.

Diagnostic Performance of STANDARD™ M10 Multidrug-resistant Tuberculosis Assay for Detection of Mycobacterium tuberculosis and Rifampicin and Isoniazid Resistance in Zimbabwe.

BACKGROUND: Although Zimbabwe has transitioned out of the 30 high-burden countries, it still remained in the 30 high multidrug-resistant (MDR)/rifampicin-resistant tuberculosis (TB) burden. Rapid detection of rifampicin (RIF) and isoniazid (INH) is essential for the diagnosis of MDR-TB. The World Health Organization has recommended the use of molecular WHO-recommended rapid diagnostic (mWRD) for TB and DR-TB. STANDARD™ M10 MDR-TB assay is a new molecular rapid diagnostic assay developed by SD Biosensor for the detection of Mycobacterium tuberculosis (MTB) and RIF and INF resistance. This study aims to determine the diagnostic accuracy of STANDARD™ M10 MDR-TB assay. METHODS: The study was conducted on 214 samples with different MTB and RIF and INH resistance status. The STANDARD™ M10 MDR-TB assay was performed according to the manufacturer's instructions. Xpert MTB/RIF Ultra, MGIT culture, and phenotypic drug susceptibility testing are used as comparative methods. RESULTS: The sensitivity and specificity of STANDARD™ M10 MDR-TB assay for the detection of MTB are 99% and 97.9%, respectively. The sensitivity and specificity of the assay for detection of MDR-TB were 97.8% and 100%, respectively. CONCLUSION: The STANDARD™ M10 MDR-TB assay demonstrated high diagnostic accuracy in the detection of MTB and RIF and INH resistance. This molecular assay can also be used as an alternative to other mWRD assays.

Correlation of Cyclic Threshold Values Generated by GeneXpert Ultra MTB/RIF and Fluorescence Microscopy to Predict Mycobacterial Burden in Suspected Cases of Pulmonary Tuberculosis.

BACKGROUND: Smear microscopy for acid-fast bacilli visualization is important to assess the infectivity rate in patients with pulmonary tuberculosis (PTB), but it has limited sensitivity; hence, it is important to find an alternative strategy. The aim of our study was to compare the fluorescence microscopy grading by Auramine O phenol staining technique of respiratory samples with the cyclic threshold (Ct) values of GeneXpert Ultra (Mycobacterium tuberculosis/rifampicin [MTB/RIF]) and assess the diagnostic efficacy of GeneXpert Ultra (MTB/RIF) compared to microscopy in suspected cases of PTB. METHODS: The study was conducted in the Mycobacteriology Laboratory, Department of Microbiology, in Kasturba Hospital, Manipal. The study was a prospective, single-centered, cross-sectional study. Four hundred and fifty-two respiratory samples were included in the study. An optimal Ct cutoff value for ruling smear-positivity and smear-negativity and the mean Ct cutoff value were calculated. Clinical and radiological data from the requisition forms were assessed. IBM SPSS statistics software version 22 was used. The correlation between GeneXpert Ultra (MTB/RIF) Ct values and smear status was calculated by polychoric correlation. The extended McNemar's test was used to find the association between the variables. RESULTS: GeneXpert Ultra (MTB/RIF) yielded a higher positivity rate of 22.2% compared to smear microscopy 17.2%. Ct value and smear grading yielded a positive correlation (P = 0.8681; P < 0.05). GeneXpert Ultra (MTB/RIF) yielded nontuberculous mycobacteria in five undetected cases and speciated as Mycobacterium abscessus complex. CONCLUSIONS: Our study confirms the GeneXpert Ultra (MTB/RIF) Ct value levels as a predictor of smear positivity.

Rapid Detection of M. tuberculosis and Its Resistance to Rifampicin and Isoniazid with the mfloDx™ MDR-TB test.

BACKGROUND: Rapid detection of tuberculosis (TB) and its resistance are essential for the prompt initiation of correct drug therapy and for stopping the spread of drug-resistant TB. There is an urgent need for increased use of rapid diagnostic tests to control the threat of increased TB and multidrug-resistant TB (MDR-TB). METHODS: EMPE Diagnostics has developed a multiplex molecular diagnostic platform called mfloDx™ by combining nucleotide-specific padlock probe-dependent rolling circle amplification with sensitive lateral flow biosensors, providing visual signals, similar to a COVID-19 test. The first test kit of this platform, mfloDx™ MDR-TB can identify Mycobacterium tuberculosis (MTB) complex and its clinically significant mutations in the rpoB and katG genes and in the inhA promotor contributing resistance to rifampicin (RIF) and isoniazid (INH), causing MDR-TB. RESULTS: We have evaluated the performance of the mfloDx™ MDR-TB test on 210 sputum samples (110 from suspected TB cases and 100 from TB-negative controls) received from a tertiary care center in India. The clinical sensitivity for detecting MTB compared to acid-fast microscopy and mycobacteria growth indicator tube (MGIT) cultures was 86.4% and 84.9%, respectively. All the 100 control samples were negative indicating excellent specificity. In smear-positive sputum samples, the mfloDx™ MDR-TB test showed a sensitivity of 92.5% and 86.4% against MGIT culture and Xpert MTB/RIF, respectively. The clinical sensitivity for the detection of RIF and INH resistance in comparison with MGIT drug susceptibility testing was 100% and 84.6%, respectively, while the clinical specificity was 100%. CONCLUSION: From the above evaluation, we find mfloDx™ MDR-TB to be a rapid and efficient test to detect TB and its multidrug resistance in 3 h at a low cost making it suitable for resource-limited laboratories.

Spinal Tuberculosis: An Exhaustive Diagnosis.

BACKGROUND: The most common organ affected due to tuberculosis (TB) is the lungs. Extrapulmonary TB is less common. Musculoskeletal organs are affected in around 8% of all tubercular patients, of which the spine is affected in almost half of the patients. The criteria for diagnosing spinal TB are quite difficult and we use an array of investigations for the same. METHODS: A retrospective study was carried out in the Neurosurgery and Microbiology Department at IMS and SUM Hospital between January 2021 and November 2023, and data were collected and tabulated in an Excel sheet. One hundred patients with spinal TB were evaluated, and their age, sex, samples sent, diagnostic investigation, duration of diagnosis from hospital admission, histopathology results, and surgical intervention (done or not) were recorded. RESULTS: The best investigation done to diagnose spinal TB was imaging and surgical/computed tomography (CT)-guided biopsy. The earliest result to diagnose spinal TB was histopathology. The yield of positivity in pus culture, smear microscopy, and true nucleic acid amplification test (NAAT) was found to be low even though sensitivity was on the higher side. CONCLUSION: Even though we have an array of investigations for diagnosing spinal TB, the best and the earliest diagnosing test was imaging plus CT-guided biopsy. The confirmation is made in the biopsy. Finding acid-fast bacteria (AFB) and NAAT tests are additional beneficial tests to supplement the diagnosis. Hence, we can conclude that sending for tests like AFB in pus, NAAT, and GeneXpert is a wastage of biological samples and delays in diagnosis.

Prevalence, drug resistance, and genotypic diversity of the RDRio subfamily of Mycobacterium tuberculosis in Ecuador: a retrospective analysis for years 2012-2016.

Introduction: A major sublineage within the Mycobacterium tuberculosis (MTB) LAM family characterized by a new in-frame fusion gene Rv3346c/55c was discovered in Rio de Janeiro (Brazil) in 2007, called RDRio, associated to drug resistance. The few studies about prevalence of MTB RDRio strains in Latin America reported values ranging from 3% in Chile to 69.8% in Venezuela, although no information is available for countries like Ecuador. Methods: A total of 814 MTB isolates from years 2012 to 2016 were screened by multiplex PCR for RDRio identification, followed by 24-loci MIRU-VNTR and spoligotyping. Results: A total number of 17 MTB RDRio strains were identified, representing an overall prevalence of 2.09% among MTB strains in Ecuador. While 10.9% of the MTB isolates included in the study were multidrug resistance (MDR), 29.4% (5/17) of the RDRio strains were MDR. Discussion: This is the first report of the prevalence of MTB RDRio in Ecuador, where a strong association with MDR was found, but also a very low prevalence compared to other countries in Latin America. It is important to improve molecular epidemiology tools as a part of MTB surveillance programs in Latin America to track the transmission of potentially dangerous MTB stains associated to MDR TB like MTB RDRio.

Population structure and spatial distribution of Mycobacterium tuberculosis in Ethiopia.

Ethiopia is one of the countries with a high tuberculosis (TB) burden, yet little is known about the spatial distribution of Mycobacterium tuberculosis (Mtb) lineages. This study identifies the spoligotyping of 1735 archived Mtb isolates from the National Drug Resistance Survey, collected between November 2011 and June 2013, to investigate Mtb population structure and spatial distribution. Spoligotype International Types (SITs) and lineages were retrieved from online databases. The distribution of lineages was evaluated using Fisher's exact test and logistic regression models. The Global Moran's Index and Getis-Ord Gi statistic were utilized to identify hotspot areas. Our results showed that spoligotypes could be interpreted and led to 4 lineages and 283 spoligotype patterns in 91% of the isolates, including 4% of those with multidrug/rifampicin resistance (MDR/RR) TB. The identified Mtb lineages were lineage 1 (1.8%), lineage 3 (25.9%), lineage 4 (70.6%) and lineage 7 (1.6%). The proportion of lineages 3 and 4 varied by regions, with lineage 3 being significantly greater than lineage 4 in reports from Gambella (AOR = 4.37, P < 0.001) and Tigray (AOR = 3.44, P = 0.001) and lineage 4 being significantly higher in Southern Nations Nationalities and Peoples Region (AOR = 1.97, P = 0.026) than lineage 3. Hotspots for lineage 1 were located in eastern Ethiopia, while a lineage 7 hotspot was identified in northern and western Ethiopia. The five prevalent spoligotypes, which were SIT149, SIT53, SIT25, SIT37 and SIT26 account for 42.8% of all isolates under investigation, while SIT149, SIT53 and SIT21 account for 52-57.8% of drug-resistant TB cases. TB and drug resistant TB are mainly caused by lineages 3 and 4, and significant proportions of the prevalent spoligotypes also influence drug-resistant TB and the total TB burden. Regional variations in lineages may result from both local and cross-border spread.

Immunohistochemistry and real-time Polymerase Chain Reaction: importance in the diagnosis of intestinal tuberculosis in a Peruvian population.

INTRODUCTION: The diagnosis of intestinal tuberculosis is challenging even nowadays. This study aims to report the positivity rates of new diagnostic methods such as immunohistochemistry and Real-Time Polymerase Chain Reaction in patients with intestinal tuberculosis, as well as describe the pathological and endoscopic features of intestinal tuberculosis in our population. METHODS: This was a retrospective observational study conducted in patients diagnosed with intestinal tuberculosis, between 2010 to 2023 from the Hospital Nacional Daniel Alcides Carrion and a Private Pathology Center, both located in Peru. Clinical data was obtained, histologic features were independently re-evaluated by three pathologists; and immunohistochemistry and real-time Polymerase Chain Reaction evaluation were performed. The 33 patients with intestinal tuberculosis who fulfilled the inclusion criteria were recruited. RESULTS: Immunohistochemistry was positive in 90.9% of cases, while real-time Polymerase Chain Reaction was positive in 38.7%. The ileocecal region was the most affected area (33.3%), and the most frequent endoscopic appearance was an ulcer (63.6%). Most of the granulomas were composed solely of epithelioid histiocytes (75.8%). Crypt architectural disarray was the second most frequent histologic finding (78.8%) after granulomas, but most of them were mild. CONCLUSION: Since immunohistochemistry does not require an intact cell wall, it demonstrates higher sensitivity compared to Ziehl-Neelsen staining. Therefore, it could be helpful for the diagnosis of paucibacillary tuberculosis.

Identification of novel single nucleotide variants in the drug resistance mechanism of Mycobacterium tuberculosis isolates by whole-genome analysis.

BACKGROUND: Tuberculosis (TB) represents a major global health challenge. Drug resistance in Mycobacterium tuberculosis (MTB) poses a substantial obstacle to effective TB treatment. Identifying genomic mutations in MTB isolates holds promise for unraveling the underlying mechanisms of drug resistance in this bacterium. METHODS: In this study, we investigated the roles of single nucleotide variants (SNVs) in MTB isolates resistant to four antibiotics (moxifloxacin, ofloxacin, amikacin, and capreomycin) through whole-genome analysis. We identified the drug-resistance-associated SNVs by comparing the genomes of MTB isolates with reference genomes using the MuMmer4 tool. RESULTS: We observed a strikingly high proportion (94.2%) of MTB isolates resistant to ofloxacin, underscoring the current prevalence of drug resistance in MTB. An average of 3529 SNVs were detected in a single ofloxacin-resistant isolate, indicating a mutation rate of approximately 0.08% under the selective pressure of ofloxacin exposure. We identified a set of 60 SNVs associated with extensively drug-resistant tuberculosis (XDR-TB), among which 42 SNVs were non-synonymous mutations located in the coding regions of nine key genes (ctpI, desA3, mce1R, moeB1, ndhA, PE_PGRS4, PPE18, rpsA, secF). Protein structure modeling revealed that SNVs of three genes (PE_PGRS4, desA3, secF) are close to the critical catalytic active sites in the three-dimensional structure of the coding proteins. CONCLUSION: This comprehensive study elucidates novel resistance mechanisms in MTB against antibiotics, paving the way for future design and development of anti-tuberculosis drugs.

Mycobacterial biotin synthases require an auxiliary protein to convert dethiobiotin into biotin.

Lipid biosynthesis in the pathogen Mycobacterium tuberculosis depends on biotin for posttranslational modification of key enzymes. However, the mycobacterial biotin synthetic pathway is not fully understood. Here, we show that rv1590, a gene of previously unknown function, is required by M. tuberculosis to synthesize biotin. Chemical-generic interaction experiments mapped the function of rv1590 to the conversion of dethiobiotin to biotin, which is catalyzed by biotin synthases (BioB). Biochemical studies confirmed that in contrast to BioB of Escherichia coli, BioB of M. tuberculosis requires Rv1590 (which we named "biotin synthase auxiliary protein" or BsaP), for activity. We found homologs of bsaP associated with bioB in many actinobacterial genomes, and confirmed that BioB of Mycobacterium smegmatis also requires BsaP. Structural comparisons of BsaP-associated biotin synthases with BsaP-independent biotin synthases suggest that the need for BsaP is determined by the [2Fe-2S] cluster that inserts sulfur into dethiobiotin. Our findings open new opportunities to seek BioB inhibitors to treat infections with M. tuberculosis and other pathogens.

Targeted protein degradation in mycobacteria uncovers antibacterial effects and potentiates antibiotic efficacy.

Proteolysis-targeting chimeras (PROTACs) represent a new therapeutic modality involving selectively directing disease-causing proteins for degradation through proteolytic systems. Our ability to exploit targeted protein degradation (TPD) for antibiotic development remains nascent due to our limited understanding of which bacterial proteins are amenable to a TPD strategy. Here, we use a genetic system to model chemically-induced proximity and degradation to screen essential proteins in Mycobacterium smegmatis (Msm), a model for the human pathogen M. tuberculosis (Mtb). By integrating experimental screening of 72 protein candidates and machine learning, we find that drug-induced proximity to the bacterial ClpC1P1P2 proteolytic complex leads to the degradation of many endogenous proteins, especially those with disordered termini. Additionally, TPD of essential Msm proteins inhibits bacterial growth and potentiates the effects of existing antimicrobial compounds. Together, our results provide biological principles to select and evaluate attractive targets for future Mtb PROTAC development, as both standalone antibiotics and potentiators of existing antibiotic efficacy.

Decreased mRNA expression of NR1H3 and ABCA1 in pulmonary tuberculosis patients from population of Punjab, India.

BACKGROUND: Mycobacterium tuberculosis (MTB) is the causative organism of tuberculosis. Cholesterol is a crucial carbon source required for the survival of MTB in host cells. Transcription factor NR1H3 along with its important target genes ABCA1 and ApoE play important role in removal of extra cholesterol from cells. Changes in the gene expression of NR1H3, ABCA1 and ApoE can affect cholesterol homeostasis and thus the survival of MTB in host cells.Therefore, the present study was designed to analyze the mRNA expression of NR1H3, ABCA1 and ApoE in pulmonary TB (PTB) patients from the population of Punjab, India. METHODS AND RESULTS: In this study, mRNA expression of the transcription factor NR1H3 and its target genes ABCA1 and ApoE was analyzed in 89 subjects, including 41 PTB patients and 48 healthy controls (HCs) by real-time quantitative PCR. It was found that the mRNA expression of both NR1H3 and ABCA1 genes was significantly lower in TB patients than in HCs (p < 0.001). Even after sex-wise stratification of the subjects, mRNA expression of NR1H3 and ABCA1 was found to be down-regulated in both male and female TB patients. No significant difference was observed in expression of ApoE (p = 0.98). CONCLUSIONS: The present study found that the mRNA expression of NR1H3 and ABCA1 is down-regulated in TB patients from Punjab state of India.

Exploring optimal drug targets through subtractive proteomics analysis and pangenomic insights for tailored drug design in tuberculosis.

Tuberculosis (TB), caused by Mycobacterium tuberculosis, ranks among the top causes of global human mortality, as reported by the World Health Organization's 2022 TB report. The prevalence of M. tuberculosis strains that are multiple and extensive-drug resistant represents a significant barrier to TB eradication. Fortunately, having many completely sequenced M. tuberculosis genomes available has made it possible to investigate the species pangenome, conduct a pan-phylogenetic investigation, and find potential new drug targets. The 442 complete genome dataset was used to estimate the pangenome of M. tuberculosis. This study involved phylogenomic classification and in-depth analyses. Sequential filters were applied to the conserved core genome containing 2754 proteins. These filters assessed non-human homology, virulence, essentiality, physiochemical properties, and pathway analysis. Through these intensive filtering approaches, promising broad-spectrum therapeutic targets were identified. These targets were docked with FDA-approved compounds readily available on the ZINC database. Selected highly ranked ligands with inhibitory potential include dihydroergotamine and abiraterone acetate. The effectiveness of the ligands has been supported by molecular dynamics simulation of the ligand-protein complexes, instilling optimism that the identified lead compounds may serve as a robust basis for the development of safe and efficient drugs for TB treatment, subject to further lead optimization and subsequent experimental validation.