Mycobacterium Tuberculosis Complex Strains Research Articles

Detection of multidrug-resistance in Mycobacterium tuberculosis by phenotype- and molecular-based assays

BackgroundThe whole-genome sequencing (WGS) is becoming an increasingly effective tool for rapid and accurate detection of drug resistance in Mycobacterium tuberculosis complex (MTBC). This approach, however, has still been poorly evaluated on strains from Central and Eastern European countries. The purpose of this study was to assess the performance of WGS against conventional drug susceptibility testing (DST) for the detection of multi-drug resistant (MDR) phenotypes among MTBC clinical strains from Poland and Lithuania.MethodsThe study included 208 MTBC strains (130 MDR; 78 drug susceptible), recovered from as many tuberculosis patients in Lithuania and Poland between 2018 and 2021. Resistance to rifampicin (RIF) and isoniazid (INH) was assessed by Critical Concentration (CC) and Minimum Inhibitory Concentration (MIC) DST as well as molecular-based techniques, including line-probe assay (LPA) and WGS. The analysis of WGS results was performed using bioinformatic pipeline- and software-based tools.ResultsThe results obtained with the CC DST were more congruent with those by LPA compared to pipeline-based WGS. Software-based tools showed excellent concordance with pipeline-based analysis in prediction of RIF/INH resistance. The RIF-resistant strains demonstrated a relatively homogenous MIC distribution with the mode at the highest tested MIC value. The most frequent RIF-resistance conferring mutation was rpoB S450L. The mode MIC for INH was two-fold higher among double katG and inhA mutants than among single katG mutants. The overall rate of discordant results between all methods was calculated at 5.3%. Three strains had discordant results by both genotypic methods (LPA and pipeline-based WGS), one strain by LPA only, three strains by MIC DST, two strains by both MIC DST and pipeline-based WGS, and the remaining two strains showed discordant results with all three methods, compared to CC DST.ConclusionsConsidering MIC DST results, current CCs of the first-line anti-TB drugs might be inappropriately high and may need to be revised. Both molecular methods demonstrated 100% specificity, while pipeline-based WGS had slightly lower sensitivity for RIF and INH than LPA, compared to CC DST.

Antimicrobial resistance profile and prevalence of Mycobacterium tuberculosis complex in Western Iran using spoligotyping method

Tuberculosis (TB) is a chronic infectious disease with multiple manifestations and gradual progression that remains a major health problem and a leading cause of death worldwide. In recent years, a number of DNA fingerprinting techniques have been developed to identify strains of the Mycobacterium tuberculosis (MTB) complex. Spoligotyping is one of the first PCR-based genotyping methods. Information on the number and identification of common strains among MTB complex samples in clinical samples from Kermanshah city is needed to develop more effective therapeutic strategies.This is a descriptive cross-sectional study of 41 sample patients with TB referred to Kermanshah Tuberculosis Centre between December 2021 and June 2022, including sputum, aspiration, urine, etc. First, the susceptibility of the developed bacteria to culture media was compared with that of isoniazid using the proportional method, and rifampin was determined according to the standard protocol. Demographic data of patients referred to the Centre for the Control of Lung Diseases were also recorded.In the next step, spoligotyping was carried out using the standard method and each strain pattern was recorded as an OCTAL code and compared with the information available at the World Bank on spoligotyping and its strains. Forty-one patients with pulmonary TB were tested using spoligotyping. Four MTB strains were identified, including H4, CAS, T1 and H1.The H4 strain also had the highest frequency with 16 samples (39%) among the MTB complex strains isolated using spoligotyping.The highest frequency of strains isolated using spoligotyping was associated with the H4 strain. It can be concluded that spoligotyping is very cost effective, simple, repeatable and highly sensitive.

Metagenomic and paleopathological analyses of a historic documented collection explore ancient dental calculus as a diagnostic tool

Dental calculus is a microbial biofilm that contains biomolecules from oral commensals and pathogens, including those potentially related to cause of death (CoD). To assess the utility of calculus as a diagnostically informative substrate, in conjunction with paleopathological analysis, calculus samples from 39 individuals in the Smithsonian Institution’s Robert J. Terry Collection with CoDs of either syphilis or tuberculosis were assessed via shotgun metagenomic sequencing for the presence of Treponema pallidum subsp. pallidum and Mycobacterium tuberculosis complex (MTBC) DNA. Paleopathological analysis revealed that frequencies of skeletal lesions associated with these diseases were partially inconsistent with diagnostic criteria. Although recovery of T. p. pallidum DNA from individuals with a syphilis CoD was elusive, MTBC DNA was identified in at least one individual with a tuberculosis CoD. The authenticity of MTBC DNA was confirmed using targeted quantitative PCR assays, MTBC genome enrichment, and in silico bioinformatic analyses; however, the lineage of the MTBC strain present could not be determined. Overall, our study highlights the utility of dental calculus for molecular detection of tuberculosis in the archaeological record and underscores the effect of museum preparation techniques and extensive handling on pathogen DNA preservation in skeletal collections.

Sustained transmission over two decades of a previously unrecognised MPT64 negative Mycobacterium tuberculosis strain in Queensland, Australia: a whole genome sequencing study

MPT64 is a key protein used for Mycobacterium tuberculosis (MTB) complex strain identification. We describe protracted transmission of an MPT64 negative MTB strain in Queensland, Australia, and explore genomic factors related to its successful spread. All MPT64 negative strains identified between 2002 and 2022 by the Queensland Mycobacteria Reference Laboratory, and an additional 2 isolates from New South Wales (NSW), were whole genome sequenced. Bayesian modelling and phylogeographical analyses were used to assess their evolutionary history and transmission dynamics. Protein structural modelling to understand the putative functional effects of the mutated gene coding for MPT64 protein was performed. Forty-three MPT64 negative isolates were sequenced, belonging to a single MTB cluster of Lineage 4.1.1.1 strains. Combined with a UK dataset of the same lineage, molecular dating estimated 1990 (95% HPD 1987-1993) as the likely time of strain introduction into Australia. Although the strain has spread over a wide geographic area and new cases linked to the cluster continue to arise, phylodynamic analysis suggest the outbreak peaked around 2003. All MPT64 negative strains had a frame shift mutation (delAT, p.Val216fs) within the MPT64 gene, which confers two major structural rearrangements at the C-terminus of the protein. This study uncovered the origins of an MPT64 negative MTB outbreak in Australia, providing a richer understanding of its biology and transmission dynamics, as well as guidance for clinical diagnosis and public health action. The potential spread of MPT64 negative strains undermines the diagnostic utility of the MPT64 immunochromatographic test. This study was funded from an operational budget provided to the Queensland Mycobacterium Reference Laboratory by Pathology Queensland, Queensland Department of Health.

Analysis of Drug Resistance Characteristics of Mycobacterium Tuberculosis in Anhui Province, 2016-2022.

To preliminarily assess the prevalence and control effect of tuberculosis and drug-resistant tuberculosis (TB) in Anhui province, and analyze the trends in the changing drug resistance spectrum of Mycobacterium tuberculosis (Mtb) isolated in Anhui province from 2016 to 2022. From 2016 to 2022, a total of 2336 culture-positive tuberculosis strains were collected from four drug resistance monitoring sites. Patient demographic information was collected and drug susceptibility testing was conducted. Among the 2336 Mycobacterium tuberculosis complex strains, 1788 (76.54%) were from male patients and 548 (23.46%) were from female patients. The majority were of Han ethnicity, from rural areas, and employed in agriculture, with 12.54% (285/2273) having diabetes. A total of 1893 (81.04%) strains were sensitive to all six anti-TB drugs tested, and 443 (18.96%) strains were resistant to at least one or more anti-TB drugs. The drug resistance rate for patients undergoing initial treatment was 16.80% (348/2071), and 35.85% (95/265) for those receiving retreatment. Among the six anti-TB drugs, the resistance rates from highest to lowest were: INH (10.55%, 236/2336), SM (8.18%, 183/2336), OFX (6.53%, 146/2336), RFP (5.95%, 133/2336), EMB (2.37%, 53/2336), KM (1.97%, 44/2336). Significant differences were observed in MDR strains across different ages, types, with or without diabetes, and geographical sources (χ2=14.895,76.534,6.032,5.109, all P<0.05). The tuberculosis prevention and control measures have controlled the drug resistance rate of Mycobacterium tuberculosis to a certain extent. However, there are still statistical differences in drug resistance rates among TB patients with different categories, age groups, regions, and diabetic diseases. Early detection and prompt treatment of patients with drug-resistant TB remain critical to controlling the spread of this disease.

Opinion review of drug resistant tuberculosis in West Africa: tackling the challenges for effective control.

Drug-resistant (DR) tuberculosis (TB) is a major public health concern globally, complicating TB control and management efforts. West Africa has historically faced difficulty in combating DR-TB due to limited diagnostic skills, insufficient access to excellent healthcare, and ineffective healthcare systems. This has aided in the emergence and dissemination of DR Mycobacterium tuberculosis complex (MTBC) strains in the region. In the past, DR-TB patients faced insufficient resources, fragmented efforts, and suboptimal treatment outcomes. However, current efforts to combat DR-TB in the region are promising. These efforts include strengthening diagnostic capacities, improving access to quality healthcare services, and implementing evidence-based treatment regimens for DR-TB. Additionally, many West African National TB control programs are collaborating with international partners to scale up laboratory infrastructure, enhance surveillance systems, and promote infection control measures. Moreso, novel TB drugs and regimens, such as bedaquiline and delamanid, are being introduced to improve treatment outcomes for DR-TB cases. Despite these obstacles, there is optimism for the future of DR-TB control in West Africa. Investments are being made to improve healthcare systems, expand laboratory capacity, and support TB research and innovation. West African institutions are now supporting knowledge sharing, capacity building, and resource mobilization through collaborative initiatives such as the West African Network for TB, AIDS, and Malaria (WANETAM), the West African Health Organization (WAHO), and other regional or global partners. These efforts hold promise for improved diagnostics, optimized treatment regimens, and provide better patient outcomes in the future where drug-resistant TB in WA can be effectively controlled, reducing the burden of the disease, and improving the health outcomes of affected individuals.

Resistance Rates of Mycobacterium tuberculosis Complex Isolates to Primary Anti-tuberculosis Drugs: A 5-Year Retrospective Study

Background/Aim: Tuberculosis remains a major global health problem with a high morbidity and mortality rate, approximately a quarter of the population is infected with tuberculosis. Drug susceptibility testing is an essential tool for identifying and managing drug-resistant tuberculosis. This study was conducted to evaluate the drug susceptibility pattern of Mycobacterium tuberculosis complex strains isolated from a university hospital. Methods: A total of 10900 samples sent to the microbiology laboratory with the suspicion of tuberculosis clinically between January 2018 and January 2022 were analyzed retrospectively. The automated BACTEC MGIT 960 (Becton Dickinson, USA) was used for sample culture and susceptibility testing. The obtained data were statistically analyzed with the Statistical Package for Social Sciences (SPSS version 20). Results: Out of the 154 isolated positive samples, males and females constituted equal parts of the study population (50%). The majority of tuberculosis cases were in the age group 56–75 years (42.2%), Pulmonary TB was detected in (90.3%) of the patients, while extrapulmonary TB cases were observed in (9.7%). As a result of susceptibility studies on positive samples, isoniazid resistance was 5.2%; streptomycin resistance 1.3%; ethambutol resistance was detected at a rate of 0.6%, while no rifampicin resistant sample was found. Both streptomycin and isoniazid resistance were seen together in 1.3% of the samples. Conclusion: A similar resistance pattern of the first-line antituberculosis drugs was observed in other studies conducted in different provinces of Turkey. The absence of multi-drug resistant and extensively drug-resistant tuberculosis in our study indicates that the tuberculosis surveillance program implemented in our region was successful.

Bacterial diversity dominates variable macrophage responses of tuberculosis patients in Tanzania

The Mycobacterium tuberculosis complex (MTBC) comprises nine human-adapted lineages that differ in their geographical distribution. Local adaptation of specific MTBC genotypes to the respective human host population has been invoked in this context. We aimed to assess if bacterial genetics governs MTBC pathogenesis or if local co-adaptation translates into differential susceptibility of human macrophages to infection by different MTBC genotypes. We generated macrophages from cryopreserved blood mononuclear cells of Tanzanian tuberculosis patients, from which the infecting MTBC strains had previously been phylogenetically characterized. We infected these macrophages ex vivo with a phylogenetically similar MTBC strain (“matched infection”) or with strains representative of other MTBC lineages (“mismatched infection”). We found that L1 infections resulted in a significantly lower bacterial burden and that the intra-cellular replication rate of L2 strains was significantly higher compared the other MTBC lineages, irrespective of the MTBC lineage originally infecting the patients. Moreover, L4-infected macrophages released significantly greater amounts of TNF-α, IL-6, IL-10, MIP-1β, and IL-1β compared to macrophages infected by all other strains. While our results revealed no measurable effect of local adaptation, they further highlight the strong impact of MTBC phylogenetic diversity on the variable outcome of the host–pathogen interaction in human tuberculosis.

Genotypic Characterization of Mycobacterium Tuberculosis Strains Resistant to Rifampicin, Isoniazid and Second-Line Antibiotics in Chad

Monitoring drug resistance and identifying the genetic basis of resistance to first- and second-line anti-tuberculosis (anti-TB) drugs provides important information to optimize patient care. The present study was an observational, cross-sectional and analytical study aimed at determining the prevalence of resistance genes to rifampicin, isoniazid and resistant and multi-resistant genes with mutations associated with second-line anti-tuberculosis drugs (Levofloxacin Amikacin, Kanamycin and Capreomycin) among strains of Mycobacterium tuberculosis in nine hospitals in four provinces of Chad with three new molecular techniques. The TB-LAMP molecular technique made it possible to detect 264 (25.14%) strains of Mycobacterium tuberculosis complex among 1050 people referred for tuberculosis research, including 37 cases in relapse and 237 new cases under treatment or not without clinical improvement. The average age of the patients was 34.15 years with the extremes ranging from 12 to 77 years. The sex ratio was 3.33 in favor of men. The GeneXpert made it possible to confirm the 264 positive cases of Mycobacterium tuberculosis (MTB+) of which 76% of men and 24% of women had the MTB+ profile sensitive to rifampicin, and 80% of men and 20% of women harbored the Mycobacterium complex. rifampicin-resistant tuberculosis. The determination of resistance genes including 13% (rpoB) to rifampicin, 4% (inhA) to isoniazid and 8% (rpoB/KatG and/or KatG/inhA) multi-resistant (MDR) genes was carried out using the Line Probe Assay technique. This technique also made it possible to determine resistance genes with associated mutations including 13.6% (gyrA and gyrB), 7.6% (eis), 6.1% (rrs and eis) and 10.6% being mutations affecting the different alleles on the chromosomes of the Mycobacterium tuberculosis complex. This study highlighted the emergence of resistance to rifampicin and isoniazid as well as resistance with mutations associated with second-line anti-tuberculosis drugs. It raises the need to implement an effective surveillance system to detect the resistance of Mycobacterium tuberculosis to anti-tuberculosis drugs in Chad and even in Central Africa.

Mycobacterium tuberculosis complex in domestic goats in Southern Spain

Tuberculosis (TB) is a zoonotic infectious disease caused by bacteria belonging to the Mycobacterium tuberculosis complex (MTC), which can affect a wide variety of domestic and wild animal species. Although the role of goats as a reservoir of MTC bacteria has been evidenced, information about the circulation of MTC strains in this species is still very scarce. The aim of the present study was to determine the seroprevalence, spatial distribution, risk factors and MTC spoligotypes circulating in goats from Andalusia (Southern Spain), the Spanish region with the largest goat census and a hotspot area of TB in both cattle and wild ungulates. A total of 2155 serum samples from 80 goat flocks were analyzed by an in-house ELISA using the P22 protein complex as a coating antigen. Antibodies against MTC were detected in 473 goats (21.9%, 95% CI: 20.2–23.7) and the true seroprevalence was 22.3% (95% CI: 20.6–24.1). Seropositivity was found in 72 (90.0%) of the 80 flocks analyzed. The generalized estimating equation model showed that the management system (higher seroprevalence on intensive and semi-intensive farms), and the presence of hospital pens inside the regular stables, were risk factors potentially associated with MTC exposure in goats in Southern Spain. The spatial analysis identified a significant spatial cluster (p < 0.001) in Eastern Andalusia. A total of 16 different MTC spoligotypes, including five of M. caprae and eleven of M. bovis, were identified in goats between 2015 and 2022 in the study area, with SB0157 as the most frequently isolated. The results obtained indicate widespread and non-homogeneous spatial distribution of MTC in goat herds from Southern Spain. The high individual and herd-level seroprevalence values found suggest that goats could play a significant role in the maintenance and transmission of MTC in the study area. Our results highlight the importance of implementing control measures in this species.

Dependency on host vitamin B12 has shaped Mycobacterium tuberculosis Complex evolution

Human and animal tuberculosis is caused by the Mycobacterium tuberculosis Complex (MTBC), which has evolved a genomic decay of cobalamin (vitamin B12) biosynthetic genes. Accordingly, and in sharp contrast to environmental, opportunistic and ancestor mycobacteria; we demonstrate that M. tuberculosis (Mtb), M. africanum, and animal-adapted lineages, lack endogenous production of cobalamin, yet they retain the capacity for exogenous uptake. A B12 anemic model in immunocompromised and immunocompetent mice, demonstrates improved survival, and lower bacteria in organs, in B12 anemic animals infected with Mtb relative to non-anemic controls. Conversely, no differences were observed between mice groups infected with M. canettii, an ancestor mycobacterium which retains cobalamin biosynthesis. Interrogation of the B12 transcriptome in three MTBC strains defined L-methionine synthesis by metE and metH genes as a key phenotype. Expression of metE is repressed by a cobalamin riboswitch, while MetH requires the cobalamin cofactor. Thus, deletion of metE predominantly attenuates Mtb in anemic mice; although inactivation of metH exclusively causes attenuation in non-anemic controls. Here, we show how sub-physiological levels of B12 in the host antagonizes Mtb virulence, and describe a yet unknown mechanism of host-pathogen cross-talk with implications for B12 anemic populations.

Genetic diversity of Mycobacterium tuberculosis strains isolated from spiritual holy water site attendees in Northwest Ethiopia. A cross-sectional study

BackgroundThe genetic diversity of Mycobacterium tuberculosis complex (MTBC) strains was characterized among isolates from individuals with pulmonary tuberculosis (PTB) symptoms attended holy water sites (HWSs) in the Amhara region, Ethiopia. MethodsA cross-sectional study was done from June 2019 to March 2020 to describe the genetic diversity and drug-resistance profiles of MTBC isolates. Sputum specimens were collected and cultured in the Löwenstein-Jensen culture medium. Line Probe Assay, MTBDRplus VER 2.0, and MTBDRsl VER 2.0 were used to detect first-and second-line anti-TB drug-resistance patterns. A spoligotyping technique was utilized to characterize the genetic diversity. Statistical analysis was performed using STATA 15. ResultsOf 560 PTB-symptomatic participants, 122 (21.8%) were culture-positive cases. Spoligotyping of 116 isolates revealed diverse MTBC sublineages, with four major lineages: Euro-American (EA) (Lineage 4), East-African-Indian (EAI) (Lineage 3), Ethiopian (ETH) (Lineage 7), East Asian (EA) (Lineage 2). The majority (96.6%) of the isolates were EA (lineage 4) and EAI, with proportions of 54.3% and 42.2%, respectively. A total of 31 spoligotype patterns were identified, 26 of which were documented in the SITVIT2 database. Of these, there were 15 unique spoligotypes, while eleven were grouped with 2-17 isolates. SIT149/T3-ETH (n = 17), SIT26/CAS1-DELHI (n = 16), SIT25/CAS1-DELHI (n = 12), and SIT52/T2 (n = 11) spoligotypes were predominant. A rare spoligotype pattern: SIT41/Turkey and SIT1/Beijing, has also been identified in North Shewa. The overall clustering rate of sub-lineages with known SIT was 76.4%.Of the 122 culture-positive isolates tested, 16.4% were resistant to rifampicin (RIF) and/or isoniazid (INH). Multidrug-resistant TB (MDR-TB) was detected in 12.3% of isolates, five of which were fluoroquinolones (FLQs) resistant. SIT149/T3-ETH and SIT21/CAS1-KILI sublineages showed a higher proportion of drug resistance. ConclusionsDiverse MTBC spoligotypes were identified, with the T and CAS families and EA (lineage 4) predominating. A high prevalence of drug-resistant TB, with SIT149/T3-ETH and CAS1-KILI sublineages comprising a greater share, was observed. A study with large sample size and a sequencing method with stronger discriminatory power is warranted to understand better the genetic diversity of circulating MTBC in this cohort of study, which would help to adopt targeted interventions.

A first insight into tuberculosis transmission at the border of Ecuador and Colombia: a retrospective study of the population structure of Mycobacterium tuberculosis in Esmeraldas province.

Tuberculosis (TB) is a major public health concern in Ecuador and Colombia, considering that both countries are high-burden TB settings. Molecular epidemiology is crucial to understand the transmission dynamics of Mycobacterium tuberculosis complex (MTBC) and to identify active transmission clusters of regional importance. We studied the potential transmission of TB between Colombia and Ecuador through the analysis of the population structure of MTBC lineages circulating in the Ecuadorian province of Esmeraldas at the border with Colombia. A total of 105 MTBC strains were characterized by 24-loci MIRU-VNTR and spoligotyping. MTBC lineage 4 is only present in Esmeraldas; no MTBC strains belonging to Lineage 2-sublineage Beijing were found despite its presence in other provinces of Ecuador and, in Colombia. Genotyping results revealed a high degree of diversity for MTBC in Esmeraldas: Neither active transmission clusters within this province nor including MTBC strains from Colombia or other provinces of Ecuador were found. Our data suggest that tuberculosis dynamics in this rural and isolated area may be not related to highly transmitted strains but could be influenced by other health determinants that favor TB relapse such as poverty and poor health system access. Further studies including a larger number of MTBC strains from Esmeraldas are necessary to test this hypothesis.

Genetic diversities of Mycobacterium tuberculosis complex species in Western Kenya.

Tuberculosis (TB) remains a high-burden infectious disease worldwide. Mycobacterium tuberculosis complex (MTBC) is the aetiological agent of TB. The TB burden is significantly linked to the development of drug-resistant strains. Thus, there is an urgent need for close surveillance of MTBC circulating in a given region, such as Western Kenya, for treatment of TB. To determine the proportion of MTBC species, strains and genetic diversities in circulation in HIV/AIDS-prevalent regions, and Western Kenya in particular. The clinical MTBC isolates were collected from Moi Teaching and Referral Hospital (MTRH) at Eldoret-Kenya during 2013-14. All clinical MTBC isolates were confirmed by the gold standard method (Löwenstein-Jensen medium culture) before inclusion in the investigation. Twelve-loci mycobacterium interspersed repetitive unit - variable-number tandem repeats (MIRU-VNTR) genotyping was performed to determine the circulating species/strains of MTBC using the www.miru-vntrplus.org web platform. Allelic diversity was calculated using the Hunter-Gaston diversity index (HGDI). The species M. tuberculosis, Mycobacterium bovis, Mycobacterium africanum, Mycobacterium pinnipedii, Mycobacterium microti, Mycobacterium caprae and Mycobacterium canetti were identified in the MTBC population. These strains were found in the Beijing, Latin American Mediterranean, Uganda 1/2, East African Indian, Ilama, West African 1/2, Harlem, URAL, Ghana, Seal, Cameroon and Vole etc. regions of Western Kenya. Notably, some isolates had unknown (new/unassigned) species. The strains were grouped into nine clusters with a clustering rate of 31.18 % and a high allelic diversity index of 0.53 was observed. The present findings suggest that there is an urgent need for more awareness among healthcare professionals and stakeholders concerning the existence of foreign MTBC species/strains in Kenya. Furthermore, 12-loci MIRU-VNTR may not be suitable for the surveillance of MTBC strains in circulation in Kenya. Thus, high-resolution techniques such as whole-genome sequencing need to be adopted to resolve the genetic diversity and establish evolutionary trends for future and archived samples. This knowledge will be crucial in restraining TB, providing insights into new drug development, and developing prevention, control and treatment strategies for TB.

Molecular determinants of multidrug-resistant tuberculosis in Sierra Leone.

A substantial proportion of MDR-TB strains in Sierra Leone were resistant against all first line drugs; however this makes the all-oral-six-month BPaLM regimen or other 6-9 months all oral regimens still viable, mainly because there was no FQ resistance.Resistance to BDQ was detected, as well as RR, due to mutations outside of the hotspot region. While the prevalence of those resistances was low, it is still cause for concern and needs to be closely monitored.

Prison as a driver of recent transmissions of multidrug-resistant tuberculosis in Callao, Peru: a cross-sectional study

We sought to identify resistance patterns and key drivers of recent multidrug-resistant tuberculosis (MDR-TB) transmission in a TB-prevalent area in Peru. Cross-sectional study including MDR Mycobacterium tuberculosis complex (Mtbc) strains identified in Callao-Peru between April 2017 and February 2019. Mtbc DNA was extracted for whole genome sequencing which was used for phylogenetic inference, clustering, and resistance mutation analyses. Clusters indicative of recent transmission were defined based on a strain-to-strain distance of ≤5 (D5) single nucleotide polymorphisms (SNPs). Epidemiologic factors linked to MDR-TB clustering were analyzed using Poisson regression. 171 unique MDR-Mtbc strains were included; 22 (13%) had additional fluoroquinolone resistance and were classified as pre-XDR. Six strains (3.5%) harboured bedaquiline (BDQ) resistance mutations and were classified as MDR+BDQ. 158 (92%) Mtbc strains belonged to lineage 4 and 13 (8%) to lineage 2. Using a cluster threshold of ≤5 SNPs, 98 (57%) strains were grouped in one of the 17 D5 clusters indicative of recent transmission, ranging in size from 2 to the largest cluster formed by 53 4.3.3 strains (group_1). Lineage 4.3.3 strains showed the overall highest cluster rate (43%). In multivariate analyses, current or previous imprisonment was independently associated with being part of any MDR-TB transmission clusters (adjusted prevalence ratio [aPR], 1.45; 95% CI, 1.09-1.92). Pre-XDR-TB emerged in more than 10% of the MDR-TB strains investigated. Transmission of 4.3.3 Mtbc strains especially of the dominant group_1 clone is a major driver of the MDR-TB epidemic in Callao. Current or previous imprisonment was linked to recent MDR-TB transmissions, indicating an important role of prisons in driving the MDR-TB epidemic. This work was supported in part by the ERANet-LAC Network of the European Union, Latin America and the Caribbean Countries on Joint Innovation and Research Activities, and FONDECYT. Additional support was received from Leibniz Science Campus Evolutionary Medicine of the Lung, the Deutsche Forschungsgemeinschaft (German Research Foundation, under Germany's Excellence Strategy-EXC 2167 Precision Medicine in Inflammation), and the Research Training Group 2501 TransEvo.

Genomic Characterization of IS6110 Insertions in Mycobacterium orygis.

Mycobacterium orygis, a subspecies of the Mycobacterium tuberculosis complex (MTBC), has emerged as a significant concern in the context of One Health, with implications for zoonosis or zooanthroponosis or both. MTBC strains are characterized by the unique insertion element IS6110, which is widely used as a diagnostic marker. IS6110 transposition drives genetic modifications in MTBC, imparting genome plasticity and profound biological consequences. While IS6110 insertions are customarily found in the MTBC genomes, the evolutionary trajectory of strains seems to correlate with the number of IS6110 copies, indicating enhanced adaptability with increasing copy numbers. Here, we present a comprehensive analysis of IS6110 insertions in the M. orygis genome, utilizing ISMapper, and elucidate their genetic consequences in promoting successful host adaptation. Our study encompasses a panel of 67 paired-end reads, comprising 11 isolates from our laboratory and 56 sequences downloaded from public databases. Among these sequences, 91% exhibited high-copy, 4.5% low-copy, and 4.5% lacked IS6110 insertions. We identified 255 insertion loci, including 141 intragenic and 114 intergenic insertions. Most of these loci were either unique or shared among a limited number of isolates, potentially influencing strain behavior. Furthermore, we conducted gene ontology and pathway analysis, using eggNOG-mapper 5.0, on the protein sequences disrupted by IS6110 insertions, revealing 63 genes involved in diverse functions of Gene Ontology and 45 genes participating in various KEGG pathways. Our findings offer novel insights into IS6110 insertions, their preferential insertion regions, and their impact on metabolic processes and pathways, providing valuable knowledge on the genetic changes underpinning IS6110 transposition in M. orygis.

Diagnostic performance of two commercial MPT64 assays for the identification of Mycobacterium tuberculosis complex from liquid culture

BackgroundTuberculosis (TB) is a chronic infectious disease caused by bacteria of the Mycobacterium tuberculosis complex (MTBC). Early diagnosis of TB is the most essential component to control TB. In this study, we evaluated the diagnostic performance of SD Bioline TB Ag MPT64 (SD Bioline) and BD MGIT TBc Identification (BD TBc ID) kits for the detection of MTBC from MGIT (Mycobacteria Growth Indicator Tube) 960 culture tube, positive for acid-fast bacilli (AFB). MethodsA total of 198 AFB positive MGIT cultures [MTBC, 150 and non-tuberculous mycobacteria (NTM), 48] were collected during the study period (April 2021 to March 2023) and subjected to SD Bioline and BD TBc ID assays, respectively. All the 150 MTBC positive cultures were confirmed by multiplex PCR targeting MPB64, protein B and IS6110 genes, and the 48 NTM cultures were speciated by using line probe assays technique- Genotype Mycobacterium CM assay. ResultsSD Bioline and BD TBc ID kits detected all 150 MTBC strains correctly from MGIT positive cultures and no false negative was observed. However, there was one false positive for M. abscessus by SD Bioline and two false positive results for M. abscessus and M. intracellulare each by BD TBc ID. Overall, the sensitivity of both SD Bioline and BD TBc ID tests was 100 % and specificity was 97.9 % and 95.8 %, respectively. ConclusionOur results highlighted that SD Bioline and BD TBc ID assays played a promising role in the earlier identification of MTBC present in liquid culture and may act as a potential alternative to biochemical and expensive molecular techniques.

Mycobacterium tuberculosis complex molecular networks and their regulation: Implications of strain heterogeneity on epigenetic diversity and transcriptome regulation

Tuberculosis has been a public health crisis since the 1900, which has caused the highest mortalities due to a single bacterial infection worldwide, that was recently further complicated by the Coronavirus disease 2019 pandemic. The causative agent of Tuberculosis, Mycobacterium tuberculosis, belongs to a genetically well-characterized family of strains known as the Mycobacterium tuberculosis complex, which has complicated progress made towards eradicating Tuberculosis due to pathogen-specific phenotypic differences in the members of this complex. Mycobacterium tuberculosis complex strains are genetically diverse human- and animal-adapted pathogens belonging to 7 lineages (Indo-Oceanic, East-Asian, East-African Indian, Euro-American, M. africanum West Africa 1, M. africanum West Africa 2 and Ethopia), respectively and the recently identified Lineage 8 and M. africanum Lineage 9. Genomic studies have revealed that Mycobacterium tuberculosis complex members are ∼99 % similar, however, due to selective pressure and adaptation to human host, they are prone to mutations that have resulted in development of drug resistance and phenotypic heterogeneity that impact strain virulence. Furthermore, members of the Mycobacterium tuberculosis complex have preferred geographic locations and possess unique phenotypic characteristics that is linked to their pathogenicity. Due to the recent advances in development next generation sequencing platforms, several studies have revealed epigenetic changes in genomic regions combined with “unique” gene regulatory mechanisms through non-coding RNAs that are responsible for strain-specific behaviour on in vitro and in vivo infection models. The current review provides up to date epigenetic patterns, gene regulation through non-coding RNAs, together with implications of these mechanisms in down-stream proteome and metabolome, which may be responsible for “unique” responses to infection by members of the Mycobacterium tuberculosis complex. Understanding lineage-specific molecular mechanisms during infection may provide novel drug targets and disease control measures towards World Health organization END-TB strategy.

Molecular identification of multiple drug resistance (MDR) strain of Mycobacterium tuberculosis.

Isoniazid and rifampin are the first -line drugs against Mycobacterium tuberculosis. Resistance to these important drugs is a serious threat to human public health. Therefore, this study aimed at molecular detection of resistance to these valuable drugs. In this descriptive cross-sectional study, 111 non - duplicated clinical samples including sputum and Bronchoalveolar lavage (BAL) samples were collected from patients referred to the Ardabil Health Center between 2017 and 2020. The samples were first examined by microscopic method, then their DNA was extracted using the boiling method. Specific primers and MAS-PCR method were employed for the detection resistance to isoniazid and rifampin drugs and identification of MDR strain. of 111 specimens, 15.3% belonged to NTM. In total, the resistance rate to isoniazid and rifampin was 17% and 27% respectively while the resistance rate to isoniazid and rifampin among NTM was 61.54% and 38.46%. In our study, the prevalence of resistance to isoniazid and rifampin among Mycobacterium tuberculosis complex(MTC) and non-tuberculous mycobacteria(NTM) was investigated using the MAS-PCR method. This work highlighted the high anti- tuberculosis resistance rate among NTM compared to MTC strains.