Whole Genome Sequencing Analysis Revealed High Genetic Diversity and Drug-Resistant Characteristics of Mycobacterium bovis From Chinese Farms.

To the Editor: Documentation of possible tuberculosis (TB) in goats in Nigeria was reported by Ojo (1) on the basis of gross lesions without culture confirmation. Livestock owners in Nigeria normally graze cattle and goats together, and this practice poses a high risk for transmission of bovine TB among these animals (1). This practice is especially a threat to goats in Nigeria because several reports have described bovine TB in cattle in Nigeria (2–5). However, reports of diagnosis of TB in goats in Nigeria are lacking. Molecular epidemiologic techniques such as deletion typing and spoligotyping have been used to characterize members of the Mycobacterium tuberculosis complex (MTC) and to provide information on transmission of mycobacterial diseases between animals and humans (6). However, because of limited resources and lack of expertise, these techniques are not commonly used in most developing nations such as Nigeria, where TB is endemic (3). Because slaughterhouses provide excellent opportunities for detecting diseases of economic and public health importance, we investigated the presence of mycobacteria in slaughtered goats with lesions suggestive of TB. The investigation was conducted at the Bodija Municipal Abattoir in Ibadan in southwestern Nigeria over a period of 6 months. Slaughtered goats were obtained from local herds and herds in northern Nigeria. A total of 10,389 male and female goats of 2 breeds (West African Dwarf and Red Sokoto) and 1–2 years of age were slaughtered; 1,387 were inspected for gross lesions of TB. Of 1,387 animals screened, 62 (4.47%) had lesions suggestive of TB in the liver, lungs, and mesenteric lymph nodes. Five (0.36%) goats were confirmed positive by culture as described (2). Deletion typing (6) with the RD9 deletion was used to distinguish M. tuberculosis from other members of the MTC. Those isolates with a deletion in this region were further investigated with primers specific for RD4. This reaction distinguishes between M. bovis, M. caprae, and other members of the MTC. Spoligotyping was performed as described (7) to type an M. tuberculosis isolate from a goat after identification of this bacterium by deletion typing. We isolated 4 strains of M. bovis and 1 strain of M. tuberculosis from the goats (Table). Spoligotyping identified the M. tuberculosis isolate as belonging to the East African Indian (EAI)–5 family in the SpolDB4 database. All M. bovis isolates were M. bovis bovis, not M. bovis caprae, according to their deletion typing profile (6). One M. bovis isolate was obtained from a male goat; the 3 remaining M. bovis isolates and the M. tuberculosis isolate were obtained from female goats. Table Results of deletion typing for Mycobacterium tuberculosis and M. bovis in goats, Nigeria* Epidemiologic inferences can be made from the results of our study. First, M. bovis, which is naturally found in cattle, was isolated from 4 slaughtered goats. Although M. bovis caprae was the M. bovis variant most frequently isolated from goats in some areas (8), in our study, only M. bovis bovis was isolated. This finding is consistent with results reported by Crawshaw et al. (9), and suggests transmission from cattle, rather than transmission from the goat reservoir. Second, because the infected goats were adult females, infection may be transmitted to their offspring. Third, M. tuberculosis was isolated from a goat. Its presence in this goat may have been caused by direct transmission from humans because this bacterium may be a natural pathogen of humans. Transmission caused by close cohabitation of goats and humans with advanced TB may occur, given the endemic nature of TB in humans in Nigeria (10). TB cases caused by EAI strains have been found in humans in southwestern Nigeria (4; S.I. Cadmus et al., unpub. data), a finding that supports zoonotic transmission of this organism from humans to goats. However, different lineages of M. tuberculosis may vary in host range, and EAI genotype strains may be adapted to human and animal hosts. Conversely, human-to-animal transmission of M. tuberculosis has been reported in Nigeria relative to infection in cattle (3,4). Thus, confirmation of TB in goats supports the possibility of risk for TB transmission between humans and animals in Nigeria. This study should be interpreted in the context of its limitations. Because the sources of the animals were unknown, we could not determine whether the organisms were imported from a neighboring country (3). In addition, we lacked information on the breed and condition of the animals. However, we have identified M. tuberculosis and TB in goats in Nigeria. Additional studies of other slaughterhouses in Nigeria are needed to confirm our results.

This work is an approach to the molecular epidemiology of Mycobacterium tuberculosis complex (MTBC) bovine infections in Tunisia. A total of 35 MTBC isolates from both lateral retropharyngeal lymph node samples of cattle slaughtered in different Tunisian regions were genotyped by spoligotyping and variable number tandem repeat typing (VNTR)-typing. Spoligotyping allowed to identify two profiles not previously registered, namely SB2024, a Mycobacterium caprae isolate from Nabeul Region (North East Tunisia), the first description of this species in the country, and SB2025 (Mycobacterium bovis) from Sfax Region (Southern Tunisia). A second M. caprae isolate with a spoligotyping profile previously described in Europe mainland, SB0418, was also isolated from a bovine of Sfax region. Both isolates suggest the possibility of a widespread distribution of this species in the country. The predominant spoligotype was SB0120, present in all Tunisian regions selected for the study but Nabeul. Molecular typing also allowed to describe a mixed infection caused by two different M. bovis isolates (SB0120 and SB0848) in the same animal. VNTR typing was highly discriminant by testing a panel of six loci. Loci QUB3232 and QUB11b were the most discriminant, whereas ETR-D and QUB11a had the lower diversity index. The value of allelic diversity can significantly vary among countries; thus, it is important to standardize a panel of loci for future inter-laboratory comparisons. Although VNTR typing proved to be useful for an efficient discrimination among MTBC isolates, especially in combination with spoligotyping, further studies are needed in order to assess the genetic diversity of the MTBC in Tunisia.

BackgroundWe aimed to investigate the molecular epidemiology of Mycobacterium tuberculosis complex (MTBC) isolates in the province of Palermo, Sicily, Italy, by characterizing 183 isolates identified in the years 2004-2012. A comparison with 104 MTBC strains identified in the same geographic area in the years 1994-2000 was also carried out.MethodsOne hundred eighty-three MTBC isolates identified in Palermo, Italy, in the years 2004-2012 were analyzed by spoligotyping and the 24 mycobacterial interspersed repetitive unit (MIRU)-variable-number tandem-repeat (VNTR) method typing. Susceptibility testing to streptomycin, isoniazid, rifampin and ethambutol was also performed. Furthermore, the spoligotyping dataset obtained from 104 MTBC isolates identified from 1994 to 2000 was reanalyzed. Distribution into lineages and clustering of isolates in the two periods was compared.ResultsOne hundred seventy-seven out of the 183 isolates of MTBC submitted to molecular typing were fully characterized. Of these, 108 were from Italian-born and 69 from foreign-born individuals. Eleven different lineages and 35 families-subfamilies were identified with the most represented lineages being Haarlem (26.5%), T (19.2%), LAM (13.6%) and S (8.5%). Except for the Haarlem lineage, where isolates from foreign-born patients were overrepresented, the distribution of isolates in the families belonging to the Euro-American clone reflected the proportions of the two subpopulations. A total of 27 (15.2%) strains were clustered and three clusters were mixed. Approximately 25% of the 183 MTBC isolates under study proved to be resistant to at least one antiTB drug, with only three isolates categorized as multidrug resistant (MDR). When MTBC isolates identified in the years 1994-2000 were reanalyzed, lineages T (30.8%), LAM (29.8%), Haarlem (16.3%) and S (13.5%) proved to be predominant. No MTBC isolates belonging to CAM, U, CAS, Turkish and Ural lineages were identified.ConclusionsA wide heterogeneity was detected among the MTBC strains isolated in the years 2004-2012. Six lineages were not present among the isolates of the period 1994-2000. Comparison between distribution of lineages in the two consecutive periods depicts rapid and deep changes in the TB epidemiology in Palermo, Italy. An universal and continued laboratory-based surveillance of TB in Sicily is required.Electronic supplementary materialThe online version of this article (doi:10.1186/s12879-014-0602-4) contains supplementary material, which is available to authorized users.

Dealing With Threat of Drug-Resistant Tuberculosis: Background Information for Interpreting the Andrew Speaker and Related Cases

IntroductionDespite the moderate incidence of tuberculosis (TB) in many parts of Iran, Golestan province had a permanently higher TB incidence rate than the national average. Moreover, Golestan province receives immigrants, mainly from TB-endemic areas of Iran and neighbor countries. Here, we aimed to characterize the circulating Mycobacterium tuberculosis complex (MTBC) isolates in terms of the spoligotype and drug resistance patterns, across Golestan province.Materials and MethodsA set of 166 MTBC isolates was collected during July 2014 to July 2015 and subjected to drug susceptibility testing for first- and second-line anti-TB drugs and spoligotyping.ResultsOf 166 MTBC isolates, 139 (83.7%) isolates were assigned to 28 spoligotype international types (SITs). The most frequent SITs were SIT127/Ural-2 (n=25, 15.1%), followed by SIT1/Beijing (n=21, 12.7%) and SIT3427/Ural-2 (n=18, 10.8%). The set of 18 isolates (10.8%) showed resistance to at least one drug, which mainly belonged to SIT1/Beijing (n=7, 38.9%), orphan patterns (n=4, 22.2%) and SIT357/CAS1-Delhi (n=3, 16.7%). In addition, four isolates (2.4%) were resistant to pyrazinamide. The analysis of mutation corresponded to resistance to rifampin and isoniazid showed that two isolates had Ser531Leu substitution in rpoB, four isolates had Ser315Thr substitution in katG and one isolate had [C(−15)T] in inhA locus.ConclusionHigh diversity in spoligotypes of the MTBC isolates and lack of dominant genotype might be due to residence of immigrants in this region and consequent reactivation of latent infection. In addition, due to the presence of extensively drug-resistant (XDR) isolates in Golestan province, it is important to conduct future studies to determine transmission pattern of drug-resistant isolates in this region.

BackgroundDrug resistance surveillance is crucial for tuberculosis (TB) control. Therefore, our goal was to determine the prevalence of second-line anti-TB drug resistance among diverse primary drug-resistant Mycobacterium tuberculosis complex (MTBC) isolates in Ghana.Materials and methodsOne hundred and seventeen MTBC isolates with varying first-line drug resistance were analyzed. Additional resistance to second-line anti-TB drugs (streptomycin [STR], amikacin [AMK] and moxifloxacin [MOX]) was profiled using the Etest and GenoType MTBDRsl version 2.0. Genes associated with resistance to AMK and MOX (gyrA, gyrB, eis, rrs, tap, whiB7 and tlyA) were then analyzed for mutation.ResultsThirty-seven (31.9%) isolates had minimum inhibitory concentration (MIC) values ≥2 µg/mL against STR while 12 (10.3%) isolates had MIC values ≥1 µg/mL for AMK. Only one multidrug-resistant (MDR) isolate (Isolate ID: TB/Nm 919) had an MIC value of ≥0.125 µg/mL for MOX (MIC = 3 µg/mL). This isolate also had the highest MIC value for AMK (MIC = 16 µg/mL) and was confirmed as resistant to AMK and MOX by the line probe assay GenoType MTBDRsl version 2.0. Mutations associated with the resistance were: gyrA (G88C) and rrs (A514C and A1401G).ConclusionOur findings suggest the need to include routine second-line anti-TB drug susceptibility testing of MDR/rifampicin-resistant isolates in our diagnostic algorithm.

Objective To investigate the correlation between the mutation of pncA gene and the susceptibility to pyrazinamide (PZA) in Mycobacterium tuberculosis complex (MTBC) strains and to analyze the mutation of panD and rpsA genes in wild type isolates without pncA gene mutation. Methods The susceptibilities of 108 MTBC strains to first-line drugs including PZA were detected by using the MGIT 960 TB system. PCR was performed to amplify the 16S rDNA and pncA, panD and rpsA genes. The PCR products were analyzed by DNA sequencing analysis. Results Among the 78 multidrug-resistant MTBC strains, 47 isolates (60%) were resistant to PZA. Four out of 30 (13%) strains that were sensitive to ethambutol, isoniazid, rifampicin and streptomycin (EIRS) were resistant to PZA. The drug-resistant MTBC strains showed higher resistance rate to PZA than that of the EIRS sensitive strains. There were 49 (96%) PZA-resistant isolates and 4 (7%) PZA-sensitive isolates occurred pncA gene mutation. Most of the pncA gene mutations in the genomes of PZA-resistant strains were base substitution mutation, especially the His57Asp substitution. The pncA gene mutations centralized in the regions of 160-169, 203-289, 309-396 and 413-467. Seven novel mutation sites of pncA gene were observed including T175C, C188A, G insertion at 68, AGC insertion at 235, C insertion at 339, CC insertion at 392 and GT deletion at 395. The mutation sites founded in the genomes of PZA-sensitive strains were different from those of the PZA-resistant strains. No mutation of the pncA gene and the upstream regulatory sequence was found in two PZA-resistant strains, NJ44 and NJ108. The sequence analysis of panD and rpsA gene showed that the NJ108 strain had panD gene mutation at G419A, but no mutation was detected in the NJ44 strain. Conclusion The multidrug-resistant MTBC strains showed higher resistance rate to PZA. The pncA gene mutation was common in PZA-resistant MTB strains and the panD gene mutation was also worthy of attention. Key words: Mycobacterium tuberculosis; Pyrazinamide; Drug resistance; Molecular characteristic

Tuberculosis caused by Mycobacterium tuberculosis complex is a significant global health burden, with drug-resistant TB, especially multidrug-resistant TB, causing severe challenges to treatment. In Ethiopia, a high TB-burden country, drug resistance has continued spreading. However, some studies indicate genetic diversity, transmission dynamics, and resistance-conferring mutations by using targeted amplification, there are limited reports of whole genome sequencing analysis to uncover the antimicrobial resistance and virulent genes. Based on that, the objective of this project was to identify antimicrobial resistance regions and characterize virulence factors in M. tuberculosis isolates through in silico whole-genome sequence analysis. A FASTQ file of 45 M. tuberculosis isolates whole genome sequence was downloaded from the SAR database. Following quality control using FASTQC coupled with MultiQC and trimming with Trimmomatic, de novo assembly was conducted using SPAdes. The Burrows-Wheeler Aligner was used for mapping against the M. tuberculosis H37Rv reference genome, followed by variant calling with FreeBayes. In silico spoligotyping was performed using SpoTyping, and drug resistance mutations were identified with TB-Profiler and validated using Mykrobe. Virulence factors were detected through ABRicate and the Virulence Factor Database. STRING was used to network the virulent genes. All statistical analyses were performed using R software. This study revealed the most prevalent TB-lineage in the Amhara region was L4 (58.53%), followed by L3 (34.15%), and L1 (4.88%), and in silico spoligotyping classified 90.24% of the isolates into 12 shared types, with SIT 149 (41.46%) and SIT 21 (14.63%) as the most frequent spoligotypes. Seven major genotypic families were identified, with T3-ETH being the dominant family (48.78%). Drug resistance analysis revealed that 38 isolates (92.7%) were multidrug-resistant, and 1 (2.4%) was pre-extensively drug-resistant. Lineage 4 (59%) and its sub-lineage 4.2.2 (51.3%) show the highest resistance. The most frequent mutations to rifampicin, isoniazid, pyrazinamide, ethambutol, streptomycin, ethionamide, fluoroquinolone, and 2nd-line injectable drugs occurred at rpoB Ser450Leu, katG Ser315Thr, pncA c.-11A > G, embB Gly406Ala, rpsL Lys43Arg, Lys88Thr, ethA Met1, gyrA Ala90Val, Asp94Asn, and rrs 1401A > G, respectively. Additionally, a mutation at the mmpR5 gene for bedaquiline and clofazimine resistance occurred in one isolate. A total of 67 virulence genes were identified and 63 of them occurred in all isolates. The high prevalence of MDR-TB and the detection of resistance to both first- and second-line drugs in this study underscore the urgent need for enhanced TB control measures in the Amhara region.

BackgroundDrug resistance in tuberculosis poses challenges to both the control and prevention of the disease. The extent of resistance is not well known in developing countries, including Ethiopia. This study was conducted to determine the drug resistance patterns and mutation characteristics of Mycobacterium tuberculosis among extra pulmonary tuberculosis patients in selected health facilities in Addis Ababa.Material and MethodsA cross-sectional study was conducted from February 2022 to August 2022 in selected hospitals in Addis Ababa. Socio-demographic and clinical data were collected using structured questionnaire. Mycobacterium tuberculosis complex (MTBC) isolates were tested for phenotypic drug susceptibility patterns using the Mycobacterium growth indicator tube (MGIT) method for first-line drugs and mutation characteristics using the Line Probe Assay (LPA) method. The data were analyzed using: SPSS version 23, and a P-value ≤ 0.05 was considered statistically significant.ResultsFrom a total of 308 patient samples from presumptive extra pulmonary patients, 44 (14.3%) were positive for MTBC. Any drug resistance was discovered in 25% of 44 MTBC isolates evaluated for five first-line drugs phenotypically, with isoniazid (INH) and pyrazinamide (PZA) resistance accounting for a greater proportion with 13.6% and 11.4% of the isolates, respectively. Two (4.5%) of the isolates were MDR-TB. Out of 44 isolates tested using the Geno Type MTBDRplus assay, 5 (11.4%) showed mutations at katG and 2 (4.5%) showed mutations in the rpoB genes.ConclusionBoth the phenotypic and genotypic drug susceptibility test results showed a high proportion of INH resistance. All INH resistance-conferring mutations were identified from katG gene. The overall prevalence of MDR-TB was also high. For early case detection and treatment, expanding diagnostic capacity for first-line DST is a vital step to limit further spread of drug resistant TB strains in the study area.

Molecular epidemiology and diagnosis of "Mycobacterium bovis" infections in African cattle

Bovine tuberculosis is a chronic disease, caused by M. bovis, mycobacteria which belongs to the mycobacterium tuberculosis complex; it is notable for having one of the broadest spectrum of hosts. The preferred host of M. bovis is cattle, but it has the ability to infect humans and a wide range of domestic animals.
\nIn Morocco, cattle production is one of the most important components of the agricultural economy; a sector which contributes heavily to the development of the Moroccan economy. The development of this sector is faced by many problems, like poor infrastructure, lack of services and climate change, in addition to infectious diseases like bovine tuberculosis
\nBovine tuberculosis is a zoonosis which affects the livestock industry, the public health sector and wildlife reservoirs. BTB has also effects like international trade restrictions for countries where BTB is endemic. Tourism and other areas of public and private interest could also be affected indirectly by BTB infection. 
\nThe respiratory route is considered to be the primary mode of infection between cattle. In addition, M. bovis is largely transmitted to humans through consumption of unpasteurized milk, but there is also the possibility of inhalation of aerosols due to contact with cattle.
\nBovine tuberculosis in endemic in Morocco, the prevalence in Moroccan cattle is estimated at 18% (95% CI: 16.5%-20.3%), and 33% (95% CI: 31%-35%) at the individual and the herd level respectively, but the human burden needs further clarification.
\nA prevalence study have been conducted in Sidi Kacem province in Morocco in 2012, 1201 cattle were screened using single comparative intradermal tuberculin skin test, the apparent prevalence was 20.4% and 57.7% in the individual and herd level respectively. The individual prevalence found in the present study is in line with the last national survey conducted in 2004 in collaboration with the FAO in Morocco. Consequently, Morocco is in an endemic stable state, similarly to other African countries.
\nThe livestock production sector in Morocco is continuously growing, due to the ambitious “plan Maroc Vert” launched in 2008, and also to the increasing demand of animal protein in Morocco. Consequently, livestock production system in Morocco is moving to intensified and irrigation rearing systems. Those factors in Sidi Kacem have been shown to be associated with higher risk of BTB compared to the extensive livestock system.
\nIn order to investigate BTB molecular epidemiology in Morocco. Bovine tuberculosis samples were collected from two slaughterhouses in Morocco, Rabat and El Jadida, 8658 animals were examined, 3.7% of them showed gross visible lesions suggesting bovine tuberculosis. However this prevalence reflects the prevalence in young bulls and old cows rather than the prevalence in the whole cattle population. 
\nMolecular characterization of the samples collected from the previously reported slaughterhouses has shown grown cultures in 225 isolates, 63.6% (n=143) have been confirmed to be M. bovis (absence of the RD4). 
\nFrom 134 samples analyzed using spoligotyping, 43 different spoligotypes were found; ten of them were new patterns (23%), they were submitted to the M.bovis database and they were given new reference numbers. The most prevalent spoligotypes were SB0121, SB0265, and SB0120, which were already reported in many other countries, mainly in Algeria, Spain, Tunisia, and also in the United States and Argentina. 
\nSpoligotypes of African 1 and African 2 clonal complexes were not reported among the characterized isolates. Considering the localization of Af 1 and Af 2 in West Central Africa and East Africa respectively, we could consider Sahara as a potential efficient barrier preventing the introduction of BTB to Morocco from West Central and East Africa.
\nMore molecular characterization is needed to investigate the strains circulating in the south and the north of Morocco. In order to investigate more deeply transmission dynamics of BTB in Africa, an overall study using whole genome sequencing and including several African countries is needed.
\nThe present thesis presents the first cattle to cattle and cattle to human compartmental deterministic mathematic model. Bovine tuberculosis reproductive number was consequently calculated, it was found to be equal to 1.375, in the range of both low and high risk areas.
\nThe sensitivity analysis of the model showed that the birth rate and the sensitivity of the single comparative intradermal tuberculin skin test are the most sensitive parameters of the model for the total cost and the time to elimination respectively. High birth rate values lead to an increased cattle population yielding higher costs for elimination. In the other hand, low test sensitivity cases low detection of infected animals and therefore less culling which leads to a longer time to elimination.
\nSimulation of test and slaughter interventions led to a decline of BTB prevalence depending on the proportion of testing (p). Using a severe cut off (2mm) for the SICTT, the time of freedom from BTB ranged from 75 years for p=20% to 25 years for p=50%. The cumulated cost was largely stable ranging from 1.47*10^9 (p=100%, time to disease freedom of 12 years) to 1.87*10^9 (p=20%, time to disease freedom of 12 years).
\nDeterministic and matrix models were used to develop a demographic model of Moroccan cattle population based on real data. The cost of bovine tuberculosis was consequently calculated using the established model. 
\nThe productivity losses triggered by BTB (5%) were estimated for 18 years, applying Leslie matrix with and without BTB. Cattle Moroccan population was compared with and without the disease, and the loss in term of animal numbers was then calculated. Considering the productivity loss, the asset value of the living animals lost due to BTB in year 18 is 98 Million Euro.
\nThe present thesis informs Moroccan stakeholders involved in bovine tuberculosis regarding the updated prevalence in Sidi Kacem Area, molecular epidemiology of BTB among slaughtered cattle, the time frame, and range of cost and levels of intervention, in addition to the cost of BTB considering productivity losses. 
\nFurther research is needed in Morocco, in one hand, investigations of the molecular epidemiology of BTB in the north and the south of the country will give more insight about the dynamics of BTB in Morocco, a broader investigation using whole genome sequencing including several African countries could be even more efficient. In the other hand a herd based transmission model will provide a more realistic cost estimation of BTB intervention in Morocco.
\nElimination of bovine tuberculosis is a costly and long process, the achievement of BTB control of Morocco will need the commitment of the different stakeholders involved. In addition, public-private collaborations could be helpful in order to achieve a sustainable control intervention of BTB in Morocco.
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Mycobacterium bovis is the cause of bovine tuberculosis, and it can also cause disease in humans, with symptoms similar to those caused by M. tuberculosis. However, our understanding of its genomic diversity, biogeography, and drug resistance remains incomplete. We performed a comparative and phylogenetic analysis of 3228 M. bovis genomes from 24 countries. Following drug susceptibility testing, we applied a bacterial genome-wide association study to capture associations between genomic variation and drug resistance in 74 newly isolated strains from China. The data show that the cattle-adapted M. bovis were divided into six lineages with a strong phylogeographical population structure. Lineages 1 and 6 are the most widespread globally, while others show a strong geographical restriction. Note that 17.39% of M. bovis isolates were resistant to at least one drug in China. Furthermore, we identify genomic variations associated with an increased risk of resistance acquisition. This study furthers our knowledge of M. bovis diversity, biogeography, and drug resistance and will facilitate more deeply informed genomic tracking and surveillance to minimize its threat to human health, as a cause of zoonotic tuberculosis.

Drug-resistance in Mycobacterium tuberculosis complex remains a major health burden in human history and still is a major leading cause of death in developing countries including Ethiopia. Early detection of all forms of drug-resistant Tuberculosis(TB) is a key factor to reduce and contain the spread of these resistant strains. A health facility-based cross-sectional study was employed, based on demographic, clinical, and laboratory data collected from 204 patients with bacteriological confirmed TB. Sputum samples were analyzed using conventional TB culture and identification test followed by molecular species identification, and then phenotypic drug susceptibility tests. Data were entered using an excel spreadsheet and exported to SPSS version 20 for analysis. Descriptive analysis; frequencies, and proportions were computed. Among the 204 sputum samples inoculated in culture media, Mycobacterium species were recovered from 165 specimens, with 160 Mycobacterium tuberculosis complex and five Non- Tuberculosis Mycobacterium(NTM) species. All Mycobacterium tuberculosis complex was found to be M. tuberculosis. Of the five NTM species, 2 M.fortuitum, 2 M.intracellulare, and 1 M.gordonae were identified. Among 160 species of M. tuberculosis isolates, 110(68.8%) were resistant to any of the anti-TB drugs. The resistance pattern was; INH (109, 68.1%), RIF (99, 61.9%), STM (73,45.6%), and EMB (32,20.0%). Mono-resistance was found for INH (7,4.3%) and STM (1,0.6%). Ninety-nine (61.9%) isolates become MDR, while resistance to any of the second-line anti-TB drugs was detected in 9 (5.6%) strains, with 8(5%) Pre-XDR and one (0.6%) XDR cases. Our findings highlight high frequencies of drug resistance to first and second-line anti-TB drugs.Determining the drug-resistance pattern of MTB is important for programmatic management of drug-resistant TB in Ethiopia. The circulating Pre-XDR and XDR case identified in the current study is alarming to the tuberculosis control program in the country.

ObjectiveMultidrug-resistant tuberculosis (MDR-TB) remains a major public health challenge in China. Hainan, China’s largest tropical island, possesses distinct socio-geographical features. However, the drug resistance patterns and molecular epidemiology of MDR-TB in this region have not been fully elucidated. This study aimed to assess the correlation between drug resistance genotypes and phenotypic resistance levels in multidrug-resistant Mycobacterium tuberculosis (MDR-MTB) strains collected from Hainan Island, using whole-genome sequencing (WGS) and phenotypic drug susceptibility testing (DST).MethodsMDR-MTB strains isolated from patients on Hainan Island (2019–2021) were analyzed. Minimum inhibitory concentrations (MIC) for 15 anti-TB drugs were determined by broth microdilution (BMD). Whole-genome sequencing (WGS) was performed using Illumina NovaSeq 6000. Genotypic resistance was predicted via TB-Profiler, and correlations between resistance mutations and MIC levels were assessed.ResultsA total of 209 MDR-MTB strains were analyzed. Strains of lineage 2.2 exhibited significantly higher resistance to ethambutol (EMB) compared to non-lineage 2 strains (P < 0.05). The sensitivity of WGS in predicting resistance to first-line drugs isoniazid (INH), rifampicin (RIF), EMB, and pyrazinamide (PZA) was 94.7%, 99.0%, 96.5%, and 80.8%, respectively. However, specificity for EMB and PZA was lower at 60.2% and 79.4%. WGS also demonstrated high sensitivity and specificity (> 95%) for second-line injectable aminoglycosides (amikacin [AMK], capreomycin [CPM], and kanamycin [KM]), but sensitivity for other second-line drugs except for fluoroquinolone drug moxifloxacin (MOX, 94.4%) was below 80.0%. Notably, mutations in katG_S315T, rpoB_S450L, and gyrA_D94G were strongly associated with high-level resistance, while mutations in fabG1, ahpC, embA promoters, and gyrA at codon 90 were linked to low-level resistance.ConclusionsThis study quantitatively demonstrates the relationship between specific drug resistance genotypes and resistance levels. It is the first to characterize the regional resistance spectrum of MDR-MTB strains on Hainan Island. These findings offer a novel foundation for MIC-based dose adjustment and the optimization of treatment strategies in this region. Trial registrationMR-46–23-020530. Date of registration:2023–07-03.Supplementary InformationThe online version contains supplementary material available at 10.1186/s12879-025-11312-8.

ABSTRACTBackground: In this study, we aimed to describe the mutations associated with first-line drug resistance in Mycobacterium tuberculosis complex (MTBC) isolates from São Paulo, Brazil, between 2019 and 2021. Methods: Mutations in the coding regions of rpoB and katG genes and in the promoter region of the inhA gene in MTBC clinical isolates were detected using the GenoType MTBDRplus assay (LPA). All mutations inferred by LPA were sequenced. Results: Of the 13,489 MTBC isolates with valid LPA results, 657 (4.9%) harbored mutations. The overall prevalence rates of rifampicin-resistant (RIF-R) tuberculosis (TB), isoniazid-resistant (INH-R) TB, and multidrug-resistant (MDR) TB were 1.5, 2.0, and 1.2%, respectively. A significant proportion of RIF-R isolates presented inferred rpoB mutations (89.1%), most of which were the borderline H445N mutation. The inhA promoter C-15T mutation was predominant among the INH-R isolates (52.8%). Most MDR isolates presented rpoB S450L + katG S315T1 mutations. Gene sequencing identified mutations not included in the catalogue of mutations published by the World Health Organization. Phenotypic drug susceptibility testing on isolates with inferred rpoB mutations revealed that the 0.5 µg/mL critical concentration of RIF failed to detect most borderline mutations when using the BACTEC MGIT 960 system. Conclusions: These findings emphasize the need for continuous surveillance and the integration of molecular and phenotypic methods to ensure an accurate detection and management of drug-resistant TB in high-burden settings.