Genetic Diversity of Legionella pneumophila Isolates from Artificial Water Sources in Brazil.

Legionella pneumophila (Lp) is a Gram-negative bacterium found in natural and artificial aquatic environments and inhalation of contaminated aerosols can cause severe pneumonia known as Legionnaires' Disease (LD). In Brazil there is hardly any information about this pathogen, so we studied the genetic variation of forty Legionella spp. isolates obtained from hotels, malls, laboratories, retail centers, and companies after culturing in BCYE medium. These isolates were collected from various sources in nine Brazilian states. Molecular identification of the samples was carried out using Sequence-Based Typing (SBT), which consists of sequencing and analysis of seven genes (flaA, pilE, asd, mip, mompS, proA, and neuA) to define a Sequence Type (ST). Eleven STs were identified among 34/40 isolates, of which eight have been previously described (ST1, ST80, ST152, ST242, ST664, ST1185, ST1464, ST1642) and three were new STs (ST2960, ST2962, and ST2963), the former identified in five different cooling towers in the city of São Paulo. The ST1 that is widely distributed in many countries was also the most prevalent in this study. In addition, other STs that we observed have also been associated with legionellosis in other countries, reinforcing the potential of these isolates to cause LD in Brazil. Unfortunately, no human isolates could be characterized until presently, but our observations strongly suggest the need of surveillance implementation system and control measures of Legionella spp. in Brazil, including the use of more sensitive genotyping procedures besides ST.

Description of new species of Mycobacterium terrae complex isolated from sewage at the São Paulo zoological park foundation in Brazil.

Five mycobacterial isolates from sewage were classified as members of the genus Mycobacterium but presented inconclusive species assignments. Thus, the isolates (MYC017, MYC098, MYC101, MYC123 and MYC340) were analyzed by phenotypical, biochemical, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) and genomic features to clarify their taxonomic position. Phenotypic analysis and biochemical tests did not distinguish these isolates from other non-pigmented mycobacteria. In contrast, MALDI-TOF MS analysis showed that isolates were not related to any previously described Mycobacterium species. Comparative genomic analysis showed values of ANI and dDDH between 81.59-85.56% and 24.4-28.8%, respectively, when compared to the genomes of species of this genus. In addition, two (MYC101 and MYC123) presented indistinguishable protein spectra from each other and values of ANI = 98.57% and dDDH = 97.3%, therefore being considered as belonging to the same species. Phylogenetic analysis grouped the five isolates within the Mycobacterium terrae complex (MTC) but in a specific subclade and separated from the species already described and supported by 100% bootstrap value, confirming that they are part of this complex but different from earlier described species. According to these data, we propose the description of four new species belonging to the Mycobacterium genus: (i) Mycobacterium defluvii sp. nov. strain MYC017T (= ATCC TSD-296T = JCM 35364T), (ii) Mycobacterium crassicus sp. nov. strain MYC098T (= ATCC TSD-297T = JCM 35365T), (iii) Mycobacterium zoologicum sp. nov. strain MYC101T (= ATCC TSD-298T = JCM 35366T) and MYC123 (= ATCC BAA-3216 = JCM 35367); and (iv) Mycobacterium nativiensis sp. nov. strain MYC340T (= ATCC TSD-299T = JCM 35368T).

Human N-acetyltransferase 2 (NAT2) gene variability in Brazilian populations from different geographical areas.

Introduction: Several polymorphisms altering the NAT2 activity have already been identified. The geographical distribution of NAT2 variants has been extensively studied and has been demonstrated to vary significantly among different ethnic population. Here, we describe the genetic variability of human N-acetyltransferase 2 (NAT2) gene and the predominant genotype-deduced acetylation profiles of Brazilians. Methods: A total of 964 individuals, from five geographical different regions, were genotyped for NAT2 by sequencing the entire coding exon. Results: Twenty-three previously described NAT2 single nucleotide polymorphisms (SNPs) were identified, including the seven most common ones globally (c.191G>A, c.282C>T, c.341T>C, c.481C>T, c.590G>A, c.803A>G and c.857G>A). The main allelic groups were NAT2*5 (36%) and NAT2*6 (18.2%), followed to the reference allele NAT2*4 (20.4%). Combined into genotypes, the most prevalent allelic groups were NAT2*5/*5 (14.6%), NAT2*5/*6 (11.9%) and NAT2*6/*6 (6.2%). The genotype deduced NAT2 slow acetylation phenotype was predominant but showed significant variability between geographical regions. The prevalence of slow acetylation phenotype was higher in the Northeast, North and Midwest (51.3%, 45.5% and 41.5%, respectively) of the country. In the Southeast, the intermediate acetylation phenotype was the most prevalent (40.3%) and, in the South, the prevalence of rapid acetylation phenotype was significantly higher (36.7%), when compared to other Brazilian states (p < 0.0001). Comparison of the predicted acetylation profile among regions showed homogeneity among the North and Northeast but was significantly different when compared to the Southeast (p = 0.0396). The Southern region was significantly different from all other regions (p < 0.0001). Discussion: This study contributes not only to current knowledge of the NAT2 population genetic diversity in different geographical regions of Brazil, but also to the reconstruction of a more accurate phenotypic picture of NAT2 acetylator profiles in those regions.

Hi-plex deep amplicon sequencing for identification, high-resolution genotyping and multidrug resistance prediction of Mycobacterium leprae directly from patient biopsies by using Deeplex Myc-Lep.

BACKGROUND: Expansion of antimicrobial resistance monitoring and epidemiological surveillance are key components of the WHO strategy towards zero leprosy. The inability to grow Mycobacterium leprae in vitro precludes routine phenotypic drug susceptibility testing, and only limited molecular tests are available. We evaluated a culture-free targeted deep sequencing assay, for mycobacterial identification, genotyping based on 18 canonical SNPs and 11 core variable-number tandem-repeat (VNTR) markers, and detection of rifampicin, dapsone and fluoroquinolone resistance-associated mutations in rpoB/ctpC/ctpI, folP1, gyrA/gyrB, respectively, and hypermutation-associated mutations in nth. METHODS: The limit of detection (LOD) was determined using DNA of M. leprae reference strains and from 246 skin biopsies and 74 slit skin smears of leprosy patients, with genome copies quantified by RLEP qPCR. Sequencing results were evaluated versus whole genome sequencing (WGS) data of 14 strains, and versus VNTR-fragment length analysis (FLA) results of 89 clinical specimens. FINDINGS: The LOD for sequencing success ranged between 80 and 3000 genome copies, depending on the sample type. The LOD for minority variants was 10%. All SNPs detected in targets by WGS were identified except in a clinical sample where WGS revealed two dapsone resistance-conferring mutations instead of one by Deeplex Myc-Lep, due to partial duplication of the sulfamide-binding domain in folP1. SNPs detected uniquely by Deeplex Myc-Lep were missed by WGS due to insufficient coverage. Concordance with VNTR-FLA results was 99.4% (926/932 alleles). INTERPRETATION: Deeplex Myc-Lep may help improve the diagnosis and surveillance of leprosy. Gene domain duplication is an original putative drug resistance-related genetic adaptation in M. leprae. FUNDING: EDCTP2 programme supported by the European Union (grant number RIA2017NIM-1847 -PEOPLE). EDCTP, R2Stop: Effect:Hope, The Mission To End Leprosy, the Flemish Fonds Wetenschappelijk Onderzoek.

Genetic diversity of Mycobacterium leprae in the state of São Paulo, an area of low-leprosy incidence in Brazil.

BACKGROUND: Brazil has the second largest number of leprosy cases worldwide, and the state of São Paulo has been considered non-endemic since 2006. METHODS: We analyzed 16 variable number tandem repeats loci and three single nucleotide polymorphisms loci of Mycobacterium leprae (M. leprae) in 125 clinical isolates from patients in different municipalities in the state. RESULTS: The clustering pattern of M. leprae indicated that the transmission of leprosy persisted in the state and included scenarios of intra-extra-familial transmission in areas with low endemicity. CONCLUSIONS: A significantly active circulation of M. leprae was observed. Therefore, surveillance and control measures must be implemented.

Genomic Diversity of the Rarely Observed Genotype of the Mycobacterium tuberculosis Central Asian (CAS) Lineage 3 from North Brazil.

Mycobacterium tuberculosis (Mtb) Central Asian Strain (CAS) Lineage 3 (L3) genotype is predominantly found in East-Africa, Central-Asia, Western-Asia, and South-Asia; however, a new spoligotyping CAS/SIT2545 was found in northern regions of Brazil. We aimed to characterize and describe the genetic diversity and perform a phylogenetic assessment of this novel genotype. We performed 24-MIRU-VNTR loci and Whole-genome sequencing (WGS) of six Brazilian isolates previously spoligotyped. The libraries were prepared using a Nextera-XT kit and sequenced in a NextSeq 550 Illumina instrument. We performed lineage assignment and genomic characterization. From publicly available genomes of Mtb L3 and other lineages, we created a robust dataset to run the MTBSeq pipeline and perform a phylogenetic analysis. MIRU-VNTR and WGS confirmed CAS/SIT2545 belongs to L3. Out of 1691 genomes, 1350 (79.83%) passed in quality control (genomic coverage > 95%). Strain 431 differed in 52 single nucleotide variants (SNV), confirming it does not belong to the same transmission chain. The eight genomes from a global dataset clustered closer to Brazilian strains differed in >52 SNVs. We hypothesized L3 and L1 were introduced in Brazilian Northern in the same historical event; however, there is a need for additional studies exploring the genetic diversity of Mtb Brazilian Northern.

Genetic diversity of Mycobacterium leprae in the state of São Paulo, an area of low-leprosy incidence in Brazil

ABSTRACT Background: Brazil has the second largest number of leprosy cases worldwide, and the state of São Paulo has been considered non-endemic since 2006. Methods: We analyzed 16 variable number tandem repeats loci and three single nucleotide polymorphisms loci of Mycobacterium leprae (M. leprae) in 125 clinical isolates from patients in different municipalities in the state. Results: The clustering pattern of M. leprae indicated that the transmission of leprosy persisted in the state and included scenarios of intra-extra-familial transmission in areas with low endemicity. Conclusions: A significantly active circulation of M. leprae was observed. Therefore, surveillance and control measures must be implemented.

Genomic analysis of Mycobacterium tuberculosis variant bovis strains isolated from bovine in the state of Mato Grosso, Brazil.

The species Mycobacterium tuberculosis variant bovis (M. tuberculosis var. bovis) is associated with tuberculosis, mainly in cattle and buffaloes. This pathogen has the potential to infect other mammals, including humans. Tuberculosis caused by M. tuberculosis var. bovis is a zoonosis clinically identical to tuberculosis caused by Mycobacterium tuberculosis, and the recommended treatment in humans results in the use of antibiotics. In this study, we used the whole genome sequencing (WGS) methodology Illumina NovaSeq 6000 System platform to characterize the genome of M. tuberculosis var. bovis in cattle circulating in Mato Grosso, identify mutations related to drug resistance genes, compare with other strains of M. tuberculosis var. bovis brazilian and assess potential drug resistance. Four isolates of M. tuberculosis var. bovis of cattle origin representing the main livestock circuits, which had been more prevalent in previous studies in the state of Mato Grosso, were selected for the genomic study. The genome sizes of the sequenced strains ranged from 4,306,423 to 4,332,964 bp, and the GC content was 65.6%. The four strains from Mato Grosso presented resistance genes to pncA (pyrazinamide), characterized as drug-resistant strains. In addition to verifying several point mutations in the pncA, rpsA, rpsL, gid, rpoB, katG, gyrB, gyrA, tlyA, embA, embB, embC, fgd, fbiB, and fbiC genes, these genes were similar to antibiotic resistance in more than 92% of the Brazilian strains. Therefore, our results indicated a high genetic diversity between our isolates and other M. tuberculosis var. bovis isolated in Brazil. Thus, multiple transmission routes of this pathogen may be present in the production chain. So, to achieve a bovine tuberculosis-free health status, the use of the WGS as a control and monitoring tool will be crucial to determine these transmission routes.

Heterogeneous persistence of Mycobacterium leprae in oral and nasal mucosa of multibacillary patients during multidrug therapy.

BACKGROUND: Leprosy is curable by multidrug therapy (MDT) treatment regimen ranging from six to 12 months. The variable levels of tolerance and adherence among patients can, however, result in treatment failure and the emergence of drug-resistant strains. OBJECTIVES: Describe the impact of MDT over Mycobacterium leprae viability in patient's oral and nasal mucosa along treatment. METHODS: Mycobacterium leprae viability was monitored by quantitative polymerase chain reaction (qPCR) quantification of 16S rRNA in lateral and contralateral scrapings of oral and nasal mucosa of 10 multibacillary patients along the initial five months of treatment. FINDINGS: The results demonstrated high heterogenicity of M. leprae viability among patients and between nasal and oral samples. Of six patients who presented good adherence and tolerance to the treatment, only four displayed absence of M. leprae viability in both samples three months after the first MDT dose, while for the other two, the absence of M. leprae viability in the oral and nasal cavities was only detected five months after the first dose. MAIN CONCLUSIONS: We concluded that qPCR of 16S rRNA for the determination of M. leprae viability in nasal and oral scraping samples could represent an interesting approach to monitor treatment efficacy.

Investigating drug resistance of Mycobacterium leprae in the Comoros: an observational deep-sequencing study.

BACKGROUND: Despite strong leprosy control measures, including effective treatment, leprosy persists in the Comoros. As of May, 2022, no resistance to anti-leprosy drugs had been reported, but there are no nationally representative data. Post-exposure prophylaxis (PEP) with rifampicin is offered to contacts of patients with leprosy. We aimed to conduct a countrywide drug resistance survey and investigate whether PEP led to the emergence of drug resistance in patients with leprosy. METHODS: In this observational, deep-sequencing analysis we assessed Mycobacterium leprae genomes from skin biopsies of patients in Anjouan and Mohéli, Comoros, collected as part of the ComLep (NCT03526718) and PEOPLE (NCT03662022) studies. Skin biopsies that had sufficient M leprae DNA (>2000 bacilli in 2 µl of DNA extract) were assessed for the presence of seven drug resistance-associated genes (ie, rpoB, ctpC, ctpI, folP1, gyrA, gyrB, and nth) using Deeplex Myc-Lep (targeted next generation deep sequencing), with a limit of detection of 10% for minority M leprae bacterial populations bearing a polymorphism in these genes. All newly registered patients with leprosy for whom written informed consent was obtained were eligible for inclusion in the survey. Patients younger than 2 years or with a single lesion on the face did not have biopsies taken. The primary outcome of our study was the proportion of patients with leprosy (ie, new cases, patients with relapses or reinfections, patients who received single (double) dose rifampicin-PEP, or patients who lived in villages where PEP was distributed) who were infected with M leprae with a drug-resistant mutation for rifampicin, fluoroquinolone, or dapsone in the Comoros. FINDINGS: Between July 1, 2017, and Dec 31, 2020, 1199 patients with leprosy were identified on the basis of clinical criteria, of whom 1030 provided a skin biopsy. Of these 1030 patients, 755 (73·3%) tested positive for the M leprae-specific repetitive element-quantitative PCR (qPCR) assay. Of these 755 patients, 260 (34·4%) were eligible to be analysed using Deeplex Myc-Lep. 251 (96·5%) were newly diagnosed with leprosy, whereas nine (3·4%) patients had previously received multidrug therapy. 45 (17·3%) patients resided in villages where PEP had been administered in 2015 or 2019, two (4·4%) of whom received PEP. All seven drug resistance-associated targets were successfully sequenced in 216 samples, 39 samples had incomplete results, and five had no results. No mutations were detected in any of the seven drug resistance-related genes for any patient with successfully sequenced results. INTERPRETATION: This drug resistance survey provides evidence to show that M leprae is fully susceptible to rifampicin, fluoroquinolones, and dapsone in the Comoros. Our results also show, for the first time, the applicability of targeted sequencing directly on skin biopsies from patients with either paucibacillary or multibacillary leprosy. These data suggest that PEP had not selected rifampicin-resistant strains, although further support for this finding should be confirmed with a larger sample size. FUNDING: Effect:Hope, The Mission To End Leprosy, the Fonds Wetenschappelijk Onderzoek, the EU.

Tuberculosis drug resistance profiling based on machine learning: A literature review.

Tuberculosis (TB), caused by Mycobacterium tuberculosis (MTB), is one of the top 10 causes of death worldwide. Drug-resistant tuberculosis (DR-TB) poses a major threat to the World Health Organization's "End TB" strategy which has defined its target as the year 2035. In 2019, there were close to 0.5 million cases of DRTB, of which 78% were resistant to multiple TB drugs. The traditional culture-based drug susceptibility test (DST - the current gold standard) often takes multiple weeks and the necessary laboratory facilities are not readily available in low-income countries. Whole genome sequencing (WGS) technology is rapidly becoming an important tool in clinical and research applications including transmission detection or prediction of DR-TB. For the latter, many tools have recently been developed using curated database(s) of known resistance conferring mutations. However, documenting all the mutations and their effect is a time-taking and a continuous process and therefore Machine Learning (ML) techniques can be useful for predicting the presence of DR-TB based on WGS data. This can pave the way to an earlier detection of drug resistance and consequently more efficient treatment when compared to the traditional DST.

Tuberculosis drug resistance profiling based on machine learning: A literature review

Abstract Tuberculosis (TB), caused by Mycobacterium tuberculosis (MTB), is one of the top 10 causes of death worldwide. Drug-resistant tuberculosis (DR-TB) poses a major threat to the World Health Organization's "End TB" strategy which has defined its target as the year 2035. In 2019, there were close to 0.5 million cases of DRTB, of which 78% were resistant to multiple TB drugs. The traditional culture-based drug susceptibility test (DST - the current gold standard) often takes multiple weeks and the necessary laboratory facilities are not readily available in low-income countries. Whole genome sequencing (WGS) technology is rapidly becoming an important tool in clinical and research applications including transmission detection or prediction of DR-TB. For the latter, many tools have recently been developed using curated database(s) of known resistance conferring mutations. However, documenting all the mutations and their effect is a time-taking and a continuous process and therefore Machine Learning (ML) techniques can be useful for predicting the presence of DR-TB based on WGS data. This can pave the way to an earlier detection of drug resistance and consequently more efficient treatment when compared to the traditional DST.

Heterogeneous persistence of Mycobacterium leprae in oral and nasal mucosa of multibacillary patients during multidrug therapy

BACKGROUND Leprosy is curable by multidrug therapy (MDT) treatment regimen ranging from six to 12 months. The variable levels of tolerance and adherence among patients can, however, result in treatment failure and the emergence of drug-resistant strains. OBJECTIVES Describe the impact of MDT over Mycobacterium leprae viability in patient's oral and nasal mucosa along treatment. METHODS Mycobacterium leprae viability was monitored by quantitative polymerase chain reaction (qPCR) quantification of 16S rRNA in lateral and contralateral scrapings of oral and nasal mucosa of 10 multibacillary patients along the initial five months of treatment. FINDINGS The results demonstrated high heterogenicity of M. leprae viability among patients and between nasal and oral samples. Of six patients who presented good adherence and tolerance to the treatment, only four displayed absence of M. leprae viability in both samples three months after the first MDT dose, while for the other two, the absence of M. leprae viability in the oral and nasal cavities was only detected five months after the first dose. MAIN CONCLUSIONS We concluded that qPCR of 16S rRNA for the determination of M. leprae viability in nasal and oral scraping samples could represent an interesting approach to monitor treatment efficacy.

Practical approach to detection and surveillance of emerging highly resistant Mycobacterium tuberculosis Beijing 1071-32-cluster.

Ancient sublineage of the Mycobacterium tuberculosis Beijing genotype is endemic and prevalent in East Asia and rare in other world regions. While these strains are mainly drug susceptible, we recently identified a novel clonal group Beijing 1071-32 within this sublineage emerging in Siberia, Russia and present in other Russian regions. This cluster included only multi/extensive drug resistant (MDR/XDR) isolates. Based on the phylogenetic analysis of the available WGS data, we identified three synonymous SNPs in the genes Rv0144, Rv0373c, and Rv0334 that were specific for the Beijing 1071-32-cluster and developed a real-time PCR assay for their detection. Analysis of the 2375 genetically diverse M. tuberculosis isolates collected between 1996 and 2020 in different locations (European and Asian parts of Russia, former Soviet Union countries, Albania, Greece, China, Vietnam, Japan and Brazil), confirmed 100% specificity and sensitivity of this real-time PCR assay. Moreover, the epidemiological importance of this strain and the newly developed screening assay is further stressed by the fact that all identified Beijing 1071-32 isolates were found to exhibit MDR genotypic profiles with concomitant resistance to additional first-line drugs due to a characteristic signature of six mutations in rpoB450, rpoC485, katG315, katG335, rpsL43 and embB497. In conclusion, this study provides a set of three concordant SNPs for the detection and screening of Beijing 1071-32 isolates along with a validated real-time PCR assay easily deployable across multiple settings for the epidemiological tracking of this significant MDR cluster.

Genotypic Characterization of Mycobacterium bovis Isolates From Dairy Cattle Diagnosed With Clinical Tuberculosis.

Molecular diagnosis of bovine tuberculosis plays an essential role in the epidemiological knowledge of the disease. Bovine tuberculosis caused by Mycobacterium bovis represents a risk to human health. This study aimed to perform the genotypic characterization of M. bovis isolated from bovines diagnosed as tuberculosis from dairy herds in the state of Pernambuco, Brazil. Granulomas from 30 bovines were sent for microbiological culture, and colonies compatible with Mycobacterium spp. were obtained in at least one culture from 17/30 granulomas. All isolates were confirmed to be M. bovis by spoligotyping and 24loci MIRU-VNTR typing. While spoligotyping characterized the isolates as SB0121, SB0295, SB0852, SB0120, and an unclassified genotype, 24loci MIRU-VNTR rendered two clusters of two isolates each and 13 unique profiles. Loci ETR-A showed higher discriminatory power, and loci (ETR-B, ETR-C, MIRU16, MIRU27, and QUB26) showed moderate allelic diversity. This is the first study on the genetic variability of the infectious agent cause of bovine TB in Pernambuco and demonstrates variability of strains in the state. Thus, it corroborates the importance of this microorganism as agent of bovine tuberculosis and its zoonotic potential, this epidemiological tool being a determinant in the rigor of the sanitary practices of disease control in dairy herds.

A Murine Model of Mycobacterium kansasii Infection Reproducing Necrotic Lung Pathology Reveals Considerable Heterogeneity in Virulence of Clinical Isolates.

Among non-tuberculous mycobacteria, Mycobacterium kansasii is one of the most pathogenic, able to cause pulmonary disease indistinguishable from tuberculosis in immunocompetent susceptible adults. The lack of animal models that reproduce human-like lung disease, associated with the necrotic lung pathology, impairs studies of M. kansasii virulence and pathogenicity. In this study, we examined the ability of the C57BL/6 mice, intratracheally infected with highly virulent M. kansasii strains, to produce a chronic infection and necrotic lung pathology. As a first approach, we evaluated ten M. kansasii strains isolated from Brazilian patients with pulmonary disease and the reference strain M. kansasii ATCC 12478 for virulence-associated features in macrophages infected in vitro; five of these strains differing in virulence were selected for in vivo analysis. Highly virulent isolates induced progressive lung disease in mice, forming large encapsulated caseous granulomas in later stages (120-150 days post-infection), while the low-virulent strain was cleared from the lungs by day 40. Two strains demonstrated increased virulence, causing premature death in the infected animals. These data demonstrate that C57BL/6 mice are an excellent candidate to investigate the virulence of M. kansasii isolates. We observed considerable heterogeneity in the virulence profile of these strains, in which the presence of highly virulent strains allowed us to establish a clinically relevant animal model. Comparing public genomic data between Brazilian isolates and isolates from other geographic regions worldwide demonstrated that at least some of the highly pathogenic strains isolated in Brazil display remarkable genomic similarities with the ATCC strain 12478 isolated in the United States 70 years ago (less than 100 SNPs of difference), as well as with some recent European clinical isolates. These data suggest that few pathogenic clones have been widely spread within M. kansasii population around the world.

Whole-Genome Sequences of Mycobacterium abscessus subsp. massiliense Isolates from Brazil.

The Mycobacterium abscessus complex comprises multidrug-resistant, opportunistic, and rapidly growing pathogens responsible for severe infections. Here, we report the genome composition of four Mycobacterium abscessus subsp. massiliense isolates from three sources: two from the lung of a cystic fibrosis patient, one from a mammary cyst, and one from a gutter system.

Multispacer Sequence Typing for Mycobacterium bovis Genotyping.

The molecular typing of Mycobacterium bovis, which causes bovine tuberculosis, can be accomplished by combining different polymorphic markers, contributing to its epidemiological investigation. Multispacer sequence typing (MST) is a sequencing-based method that employs intergenic regions susceptible to higher mutation rates given the low selection pressure. It has been applied to M. tuberculosis, but not to M. bovis. The aim of this study was to evaluate a MST for M. bovis. A total of 58 strains isolated from tissues with lesions suggestive of bovine tuberculosis, coming from cattle herds in six Brazilian states and four standard samples of M. bovis were typified employing the MST technique. Fourteen intergenic regions were used, and four types of genetic events were reported: single nucleotide mutation (SNP), insertion, deletion, and tandem repeat (TR). Seven loci were chosen for typing. Twenty-eight type sequences (ST) were identified, indicating type sequences (ST) were identified, indicating a 92.9% HGDI (Hunter Gaston Discriminatory Index). The data were used to analyze the evolutionary patterns of these isolates and correlate them to phylogeographic lineages based on the formation of clonal complexes generated from eBURST software. Later, we associated the MST with spoligotyping technique, currently considered the gold standard for classification of M. bovis. The results support the MST as an alternative method for genotyping of M. bovis. The method has the advantage of sequencing and the availability of sequences analyzed in public databases, which can be used by professionals around the world as a tool for further analysis. This was the first study to identify the variability of isolates of M. bovis by the MST method.

Local adaptation in populations of Mycobacterium tuberculosis endemic to the Indian Ocean Rim.

Background: Lineage 1 (L1) and 3 (L3) are two lineages of the Mycobacterium tuberculosis complex (MTBC) causing tuberculosis (TB) in humans. L1 and L3 are prevalent around the rim of the Indian Ocean, the region that accounts for most of the world's new TB cases. Despite their relevance for this region, L1 and L3 remain understudied. Methods: We analyzed 2,938 L1 and 2,030 L3 whole genome sequences originating from 69 countries. We reconstructed the evolutionary history of these two lineages and identified genes under positive selection. Results: We found a strongly asymmetric pattern of migration from South Asia toward neighboring regions, highlighting the historical role of South Asia in the dispersion of L1 and L3. Moreover, we found that several genes were under positive selection, including genes involved in virulence and resistance to antibiotics. For L1 we identified signatures of local adaptation at the esxH locus, a gene coding for a secreted effector that targets the human endosomal sorting complex, and is included in several vaccine candidates. Conclusions: Our study highlights the importance of genetic diversity in the MTBC, and sheds new light on two of the most important MTBC lineages affecting humans.

Molecular epidemiology of Mycobacterium tuberculosis in Brazil before the whole genome sequencing era: a literature review.

Molecular-typing can help in unraveling epidemiological scenarios and improvement for disease control strategies. A literature review of Mycobacterium tuberculosis transmission in Brazil through genotyping on 56 studies published from 1996-2019 was performed. The clustering rate for mycobacterial interspersed repetitive units - variable tandem repeats (MIRU-VNTR) of 1,613 isolates were: 73%, 33% and 28% based on 12, 15 and 24-loci, respectively; while for RFLP-IS6110 were: 84% among prison population in Rio de Janeiro, 69% among multidrug-resistant isolates in Rio Grande do Sul, and 56.2% in general population in São Paulo. These findings could improve tuberculosis (TB) surveillance and set up a solid basis to build a database of Mycobacterium genomes.