Phylogenetically Informative Mutations in Drug Resistance Genes of Human-Infecting Mycobacterium bovis

Abstract

The diagnosis of drug-resistant tuberculosis (TB) by molecular testing of Mycobacterium tuberculosis drug resistance genes is becoming increasingly common clinically. However, M. bovis, as an uncommon pathogen of human TB, may interfere with the test results. A comprehensive understanding of phylogenetically informative mutations in the drug resistance genes of M. bovis is required to distinguish true resistance-conferring mutations. We analyzed 53 drug resistance genes in 165 M. bovis isolated from humans using whole-genome sequencing data and found that 98.2% (162/165) of isolates have pyrazinamide intrinsic genotypic resistance, owing to the H57D mutation in the pncA gene. 12.1% (20/165) of M. bovis isolates were resistant to drugs other than pyrazinamide. Furthermore, we discovered 18 phylogenetically informative mutations that differed between M. bovis and the major lineages 1–4 of M. tuberculosis. Additionally, we reported false-positive ethambutol resistance caused by M. bovis infection due to the phylogenetically informative mutation embB E378A. This study is crucial for gaining insights into the genetic characterization and drug resistance of M. bovis prevalent in humans, as well as contributing to the development of more accurate molecular diagnostic methods and detection tools for drug resistance.

This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

1. Introduction

Tuberculosis (TB) remains a global health challenge, with the emergence of drug-resistant isolates leading to unfavorable clinical outcomes. The etiological agents of TB are a group of closely related (> 99% nucleotide sequence identity) bacteria known as the Mycobacterium tuberculosis complex (MTBC) [1]. The MTBC achieves genetic diversity through genetic mutations, recombination, and natural selection, enabling its widespread global propagation [2]. Genetic mutations within the MTBC primarily drive drug resistance, impacting drug sensitivity testing (DST) and clinical drug use [3]. Certain strains of MTBC exhibit intrinsic resistance to specific drugs due to genetic alterations [4–6]. Owing to the strictly clonal population structure of MTBC and the absence of horizontal gene transfer, these mutations are often unique and phylogenetically informative, serving as markers for specific subgroups within the global MTBC diversity [6]. For instance, the phylogenetic marker tlyA N236K of a cluster of M. tuberculosis lineage 4.6.2 leads to intrinsic capreomycin (CPM) resistance [5]. H57D in the pncA gene of M. bovis results in intrinsic resistance to pyrazinamide (PZA) [4].

Mycobacterium bovis, a member of the MTBC with a broad range of host species as well as the etiological agent of bovine TB, can be transmitted to humans through the consumption of contaminated dairy products or meat [7–9]. Compared with M. tuberculosis, TB caused by M. bovis is relatively less common in humans [7, 9]. However, due to the similarities in symptoms and clinical manifestations with M. tuberculosis infection, the lack of immediate diagnostic tools to accurately differentiate between the two, and the limited systematic monitoring of M. bovis as the cause of TB (especially in underdeveloped countries), the incidence of human M. bovis infection is underestimated, and it constitutes a potential serious public health threat [10].

Drug-resistant TB threatens global public health security, and although most human cases of drug-resistant TB are caused by M. tuberculosis, infections of humans with drug-resistant M. bovis have been reported [11–14]. Due to its broad host range, drug-resistant M. bovis has been identified in cattle, sheep, dogs, and other domesticated animals [14–17]. Whole-genome sequencing (WGS) can rapidly and accurately obtain genomic information about isolates, reveal the genetic variation of drug-resistant M. bovis, and play an important role in tracing the transmission pathway of M. bovis [15, 18, 19]. WGS can provide an important tool and data support for studying the transmission, genetic diversity, and evolution of drug-resistant M. bovis among hosts of different species.

In this study, we utilized the WGS data of 165 M. bovis isolated from humans to examine 53 genes associated with resistance against 19 drugs. These genes were selected based on their documented roles in drug resistance [3, 6]. Resistance-associated genes with no observed mutations were excluded from the analysis. This analysis aims to enhance our understanding of the genetic traits and drug resistance of M. bovis in human infections and to aid in the development of more precise diagnostic methods and detection tools.

2. Materials and Methods

2.1. Strain Collection

We analyzed the genomes of 165 M. bovis isolated from humans, of which 160 were downloaded from SRA, NCBI (http://www.ncbi.nlm.nih.gov/sra/, downloaded on 27 November 2023). Among these, 68 genomes were sourced from Zwyer et al. [20] who collected livestock-associated MTBC genomes and defined phylogenetic lineages. This dataset was supplemented with 17 genomes from the TB clinic of the Tijuana General Hospital in Baja, California, USA, and 75 genomes from publicly available WGS datasets [21] (Supporting Information S1). By analyzing the presence or absence of the RD1 and RD4 genomic regions, we confirmed that all genomes were M. bovis rather than BCG or other MTBC species, as described previously [22]. In addition, five M. bovis genomes were obtained from the Chinese Center for Disease Control and Prevention, specifically four from Xinjiang and one from Jiangsu. One of the isolates from Xinjiang was determined to be ethambutol (EMB) resistant by a high-resolution melting (HRM) assay [23]. Furthermore, we included 117 M. tuberculosis isolates from lineages 1–4 in our genomic analysis to assess the phylogenetic informativeness of the identified mutations (Supporting Information S1).

2.2. Whole-Genome Sequence Analysis

The FASTQ files containing the raw sequencing data for all the isolate genomes examined can be obtained from SRA, NCBI, and additional information can be found in Supporting Information S1. Fastp v0.23.4 was used to filter out low-quality bases (Phred score < 20) and residual Illumina adapter contaminations in the FASTQ files [24]. Clean reads from each M. bovis genome were analyzed with the Snippy 4.6.0 pipeline [25]. First, reads were mapped to the M. tuberculosis H37Rv genome (GenBank ID: NC_000962.3) with BWA v0.7.17 [26]. The variant calling was done by Freebayes v1.3.6, and the vcf files were further annotated using SnpEff v5.0 [27, 28]. A comparative circular genome visualization was mapped using Proksee [29].

2.3. Phylogenetic Analysis

Single-nucleotide polymorphisms (SNPs) located in PE/PPE gene families were excluded for the phylogenetic reconstruction due to the difficulties in reliably aligning sequences to the high GC repetitive regions [30, 31, and 32]. We then estimated a maximum likelihood tree using IQ-TREE v2.2.0 [33]. The best-fitting substitution model was selected automatically using the ModelFinder program implemented in IQ-TREE [34]. The phylogenetic analysis was performed with 1000 ultrafast bootstrap replicates. The TVM + F, determined as the best-fit model of nucleotide substitution based on the BIC values reported by the ModelFinder program, was employed [34].

2.4. Genotypic Drug Resistance Analysis

For each genome, genotypic drug resistance was predicted using TB-Profiler v4.4.0 [35], covering rifampicin (RIF), isoniazid (INH), EMB, PZA, streptomycin (STM), fluoroquinolones (FQ, including moxifloxacin, ofloxacin, levofloxacin, and ciprofloxacin), amikacin (AMK), CPM, kanamycin (KAN), cycloserine (CYC), ethionamide (ETH), clofazimine (CFZ), para-aminosalicylic acid (PAS), delamanid (DLM), bedaquiline (BDQ), and linezolid (LZD).

3. Results

3.1. Phylogeny and Genotypic Drug Resistance

Our collection featured 165 virulent M. bovis genomes isolated from humans, originating from Algeria (n = 4), Cameroon (n = 2), Germany (n = 6), Ghana (n = 3), Italy (n = 2), Malawi (n = 3), Mexico (n = 17), the Netherlands (n = 27), New Zealand (n = 1), Russia (n = 1), Tunisia (n = 1), Turkey (n = 68), the United Kingdom (n = 11), and the United States of America (n = 14), as well as five newly sequenced genomes from China. To better understand the genetic structures of these M. bovis genomes, a maximum likelihood tree was constructed based on 3297 SNPs. We used TB-Profiler v4.4.0 to predict the genotypic drug resistance of each M. bovis isolate [35]. Overall, 98.2% (162/165) of isolates were resistant to PZA, owing to the H57D mutation in the pncA gene. 12.1% (20/165) of M. bovis isolates were resistant to drugs other than PZA. Among the 68 isolates in Turkey, in addition to intrinsic resistance to PZA, eight isolates were resistant to INH due to the P2S mutation in the ahpC gene, and another eight isolates were resistant to FQ due to the D94G mutation in the gyrA gene. An isolate originating from Algeria was resistant to STM due to the rrs 888G> A mutation, while two isolates from the Netherlands and the USA were resistant to STM due to the rpsL K43R mutation. It is worth noting that this isolate in the USA was also resistant to aminoglycosides (KAN, CPM, and AMK) simultaneously due to the rrs 1401A> G mutation. Moreover, a M. bovis isolated from Ghana was resistant to RIF due to the rpoB Q432R mutation (Figure 1).

[figure(s) omitted; refer to PDF]

3.2. Phylogenetically Informative Mutations

The pncA H57D mutation confers intrinsic PZA resistance to M. bovis and serves as a phylogenetically informative mutation [3, 4, 36]. Additionally, we analyzed the mutation frequencies of 53 drug-resistant genes to reveal the phylogenetic polymorphisms that are specific to M. bovis but are not associated with drug resistance (Figure 2 and Supporting Information S2). Among the 294 mutations that occurred in these 53 genes, 43.5% (128/294) were low-frequency variations (occurred in only one isolate), compared with 44.9% of SNVs occurring in one isolate of M. bovis isolated from cattle, suggesting that M. bovis exhibits similar genetic diversity across hosts [15]. Furthermore, we discovered 40 variations with mutation frequencies exceeding 90% (Supporting Information S2). To identify phylogenetically informative markers specific to M. bovis, we included 117 genomes from the major lineages 1–4 of M. tuberculosis to analyze whether the 40 mutations are also present in these lineages. We found that 18 of these mutations were unique to M. bovis and absent in M. tuberculosis lineages 1–4 (Table 1). Among them, we identified four mutations that had not previously been recognized as phylogenetically informative markers for M. bovis: clpC1 L768L, aftB I327V, ndh-132delG, and ald 266delA [6]. The remaining 22 mutations were also present in some M. tuberculosis lineages.

[figure(s) omitted; refer to PDF]

Table 1

Phylogenetically informative markers distinguishing M. bovis from major M. tuberculosis lineages 1–4.

Position	Gene name	M. tuberculosis H37Rv	M. bovis AF2122/97	Variant type	Change
5752	gyrB	Rv0005	Mb0005	synonymous_variant	V171V (gtg/gtA)
6406	gyrB	Rv0006	Mb0005	synonymous_variant	N389N (aac/aaT)
6446	gyrB	Rv0005	Mb0005	missense_variant	A403S (gcg/Tcg)
8285	gyrA	Rv0006	Mb0006	synonymous_variant	I328I (atc/atT)
1302899	fbiC_upstream	—	—	upstream_gene_variant	c.-32A > G
1834859	rpsA	Rv1630	Mb1656	missense_variant	A440T (gcg/Acg)
2103173	ndh_upstream	—	—	upstream_gene_variant	c.-132delG
2155503	katG	Rv1908c	Mb1943c	synonymous_variant	T203T (acc/acT)
2156025	katG	Rv1908c	Mb1943c	synonymous_variant	P29P (ccc/ccA)
3087084	ald	Rv2780	Mb2802, Mb2803	frameshift_variant	c.266delA p.Gln89fs
3448783	Rv3083	Rv3083	Mb3110	missense_variant	V94I (gtc/Atc)
4038403	clpC1	Rv3596c	Mb3627c	synonymous_variant	L768L (ttg/Ctg)
4242970	embC	Rv3794	Mb3822	upstream_gene_variant	T1036T (acc/acT)
4244220	embA	Rv3794	Mb3822	synonymous_variant	L330L (ctg/Ttg)
4246551	embB	Rv3795	Mb3824	missense_variant	N13S (aat/aGt)
4246864	embB	Rv3795	Mb3824	synonymous_variant	V117V (gtc/gtT)
4267858	aftB	Rv3805c	Mb3835c	missense_variant	I327V (att/Gtt)
4269351	ubiA	Rv3806c	Mb3836c	synonymous_variant	A161A (gcc/gcT)

Note: Italic value highlights gene names and locus tags.

3.3. Impact of Phylogenetic Polymorphisms on DSTs

The HRM assay is a rapid and simple PCR-based method for detecting SNPs by measuring fluorescence changes in the melting temperature of amplified products [37]. This assay is commonly used for screening drug resistance in M. tuberculosis [23, 38]. However, ignoring phylogenetic polymorphisms may lead to false-positive reports of drug resistance [39]. In one case from Xinjiang described in this study, the hospital used a HRM assay to test EMB drug sensitivity in an isolate before confirming it as M. bovis. This assay covers the detection of mutations in six codons (306, 368, 378, 380, 406, and 497) located on the embB gene [23]. The determination of EMB resistance was made by comparing the melting temperature differences in the melting curves between the tested samples and the positive control. Given the determination of EMB resistance, a customized treatment regimen consisting of INH, RIF, PZA, and levofloxacin (LVX) was implemented, extending the treatment duration to 9 months. However, after WGS, we found that this isolate belongs to M. bovis. Through whole-genome BLAST comparisons with the reference strains M. bovis AF2122/97 and M. tuberculosis H37Rv, we identified an H57D mutation in the pncA gene and an E378A mutation in the embB gene (Figure 3). These two mutations were present in both the isolate and the M. bovis AF2122/97 reference strain. The H57D mutation in the pncA gene is associated with the inherent PZA resistance of M. bovis, while the E378A mutation in the embB gene is unrelated to EMB resistance [3, 40].

[figure(s) omitted; refer to PDF]

Despite emerging from a common progenitor, the MTBC is divided into different phylogenetic lineages. After the loss of the M. tuberculosis-specific deletion region 1 (TbD1), M. tuberculosis diverged into lineages 2, 3, and 4, known as modern MTBC. In contrast, strains with an intact TbD1 region are referred to as “ancestral” strains [41, 42]. The embB E378A mutation is not a marker for EMB resistance but a phylogenetically informative marker of the MTBC [40]. At this codon, A represents the ancestral amino acid, whereas E is present in modern MTBC (Figure 4). Therefore, when the tested isolate belongs to M. tuberculosis lineages L1, L5, L6, L7, L8, and L9 or animal-adapted members of the MTBC, using this assay kit can lead to erroneous reports of EMB resistance, impacting drug treatment regimen selection. In this case, due to the false-positive report for EMB resistance, the treatment regimen for the patient excluded EMB and instead used the second-line anti-TB drug LVX, with the treatment duration extended from 6 to 9 months. Following our feedback, the assay used in this case has removed the detection of this mutation. This case highlights the importance of studying phylogenetically informative mutations related to drug resistance genes in M. bovis. Ignoring phylogenetic diversity in molecular detection can lead to false-positive results, thereby affecting clinical treatment decisions.

[figure(s) omitted; refer to PDF]

4. Discussion

While some countries have made progress in eradicating bovine TB, the disease continues to threaten people who depend on livestock for their livelihoods, particularly in underdeveloped and rural areas. The genome sequence of M. bovis is over 99.95% identical to that of M. tuberculosis, and many pathogen-associated molecular patterns are identical between the two, allowing recognition by various pattern recognition receptors on macrophages [43, 44]. M. bovis can be transmitted to humans through direct contact with infected animals or by consuming animal products such as unpasteurized milk [8]. Though rare, human-to-human transmission of M. bovis is also possible [45–47]. As a result, the risk of M. bovis infection cannot be entirely eliminated, even in individuals with no prior exposure to farm animals or consumption of pasteurized milk [21, 45, 47]. According to the WHO statistics, M. bovis causes ~140,000 new cases of TB and 11,400 deaths in humans each year [48].

Infection with M. bovis in humans can result in various symptoms of TB, including cough, poor appetite, fatigue, weight loss, and night sweats [49]. Given that 98.2% (162/165) of M. bovis isolated from humans are inherently genotypically resistant to PZA, it is critical to accurately identify M. bovis as the cause of human TB before initiating treatment. Although PZA is commonly used in first-line anti-TB treatment, the treatment regimen for M. bovis infection is usually adjusted to a combination of RIF, INH, and EMB, with a duration of 9 months [50]. However, due to limitations in PZA resistance testing, cases of M. bovis infection may lead to ineffective use of PZA if not accurately diagnosed.

The prevalence of drug-resistant M. bovis varies widely between regions. A study showed that of 167 human M. bovis infection cases in San Diego, 7% were resistant to INH and 1% to RIF [51]. Another study based on 15 years of laboratory surveillance in Mexico found a high rate of resistance to first-line anti-TB drugs in M. bovis isolates from humans, of which 16.1% were resistant to INH, 9.5% to RIF, 0.7% to EMB, 11.8% to STM, and 7.6% were multidrug-resistant [52]. About 70% of isolates were resistant to first- and second-line anti-TB drugs in Peshawar, Pakistan [53]. Our data support the prevalence of drug-resistant M. bovis in humans and account for 12.1% of human M. bovis infections. The heterogeneity of drug-resistant M. bovis prevalence in different regions reflects the genetic diversity and adaptability of M. bovis. Ongoing surveillance and testing efforts are key to the timely detection of drug-resistant strains and the adoption of isolation and treatment measures.

Moreover, if the M. bovis infection cannot be accurately identified, as illustrated in our case, it can result in false-positive DST, posing a hidden challenge in accurately diagnosing TB and potentially leading to the inability to use the most appropriate treatment scheme. We are confronted with the reality that drug-resistant M. bovis is spreading in humans, presenting significant challenges to global public health and clinical treatment [13]. If left unaddressed, this drug resistance could lead to a myriad of adverse outcomes, including reduced cure rates, the need for prolonged treatment, increased overall costs, and potentially treatment failure or even death [54, 55]. Given the gravity of this situation, it is imperative to intensify our efforts in surveillance, prevention, and control measures for zoonotic TB [9]. Early identification of cases of this specific strain of TB is crucial, especially in patients with a history of contact with farm animals or occupational risks. Timely detection and diagnosis of TB cases are essential for identifying the involved strains and implementing appropriate safety measures to prevent further spread [56, 9]. Furthermore, a comprehensive understanding of the epidemiology of drug-resistant M. bovis strains is vital for interrupting the transmission chain. This underscores the need for increased emphasis on epidemiological research to gain insights into the disease’s spread and its evolution over time. Monitoring resistance genes and utilizing this information to develop effective strategies for combating zoonotic TB is also essential.

Besides the pncA H57D mutation being identified as a phylogenetic marker for M. bovis and conferring intrinsic resistance to PZA, we also analyzed mutations in 53 genes associated with drug resistance [3, 6]. We discovered 18 phylogenetically informative mutations that can be used to distinguish M. bovis from the major lineages 1–4 of M. tuberculosis. By recognizing specific phylogenetically informative mutations, we can track the origin and transmission pathways of M. bovis more accurately. Additionally, understanding mutations that do not affect drug resistance but have important phylogenetic implications helps us better comprehend the population structure and genetic diversity of M. bovis. The most important aspect is to utilize the information obtained from systematic phylogenetic diversity to develop more precise and efficient molecular diagnostic methods, enabling accurate and rapid detection of drug-resistant M. bovis, guiding clinical treatment choices, and ultimately achieving effective therapeutic outcomes.

In conclusion, this study revealed the threat of M. bovis to human health and the prevalence of drug-resistant M. bovis in humans. The association between drug resistance in M. bovis and its phylogenetic diversity provides important evidence for accurately tracking the transmission pathways of M. bovis and developing precise and efficient molecular diagnostic methods.

Ethics Statement

All authors confirm the ethical policies of the journal. Ethical approval was not required for this study, as the isolates were obtained from routine surveillance and treatment programs. Specifically, the five isolates from China were collected as part of ongoing public health surveillance efforts conducted by the Chinese Center for Disease Control and Prevention.

Author Contributions

Yuhui Dong and Xichao Ou designed and coordinated the study. Yanlin Zhao and Xiangmei Zhou supervised the study. Bing Zhao collected samples. Yuanzhi Wang, Yiduo Liu, Ziyi Liu, Haoran Wang, Xin Ge, and Yue Nan provided critical revisions to the manuscript. All authors have read and approved the final manuscript. Yuhui Dong and Xichao Ou contributed equally to this study.

References

[1] S. Gagneux, "Ecology and Evolution of Mycobacterium tuberculosis," Nature Reviews Microbiology, vol. 16 no. 4, pp. 202-213, DOI: 10.1038/nrmicro.2018.8, 2018.

[2] A. Namouchi, X. Didelot, U. Schöck, B. Gicquel, E. P. C. Rocha, "After the Bottleneck: Genome-Wide Diversification of the Mycobacterium tuberculosis Complex by Mutation, Recombination, and Natural Selection," Genome Research, vol. 22 no. 4, pp. 721-734, DOI: 10.1101/gr.129544.111, 2012.

[3] T. M. Walker, P. Miotto, C. U. Koser, "The 2021 WHO Catalogue of Mycobacterium tuberculosis Complex Mutations Associated with Drug Resistance: A Genotypic Analysis," Lancet Microbe, vol. 3, pp. e265-e273, DOI: 10.1016/S2666-5247(21)00301-3, 2022.

[4] C. Loiseau, D. Brites, I. Moser, "Revised Interpretation of the Hain Lifescience GenoType MTBC To Differentiate Mycobacterium Canettii and Members of the Mycobacterium tuberculosis Complex," Antimicrob Agents Chemother, vol. 63,DOI: 10.1128/AAC.00159-19, 2019.

[5] T. M. Walker, M. Merker, A. M. Knoblauch, "A Cluster of Multidrug-Resistant Mycobacterium tuberculosis among Patients Arriving in Europe from the Horn of Africa: A Molecular Epidemiological Study," Lancet Infectious Diseases, vol. 18 no. 4, pp. 431-440, DOI: 10.1016/S1473-3099(18)30004-5, 2018.

[6] M. Merker, T. A. Kohl, I. Barilar, "Phylogenetically Informative Mutations in Genes Implicated in Antibiotic Resistance in Mycobacterium tuberculosis Complex," Genome Medicine, vol. 12 no. 27,DOI: 10.1186/s13073-020-00726-5, 2020.

[7] A. S. Dean, S. Forcella, F. Olea-Popelka, "A Roadmap for Zoonotic Tuberculosis: A One Health Approach to Ending Tuberculosis," Lancet Infectious Diseases, vol. 18 no. 2, pp. 137-138, DOI: 10.1016/S1473-3099(18)30013-6, 2018.

[8] F. Olea-Popelka, A. Muwonge, A. Perera, "Zoonotic Tuberculosis in Human Beings Caused by Mycobacterium bovis— A Call for Action," The Lancet Infectious Diseases, vol. 17 no. 1, pp. e21-e25, DOI: 10.1016/S1473-3099(16)30139-6, 2017.

[9] A. Zumla, D. Yeboah-Manu, A. L. Michel, "Zoonotic Tuberculosis—A Call for an Open One Health Debate," The Lancet Infectious Diseases, vol. 20 no. 6, pp. 642-644, DOI: 10.1016/S1473-3099(20)30166-3, 2020.

[10] J. Sawyer, S. Rhodes, G. J. Jones, P. J. Hogarth, H. M. Vordermeier, "Mycobacterium bovis and Its Impact on Human and Animal Tuberculosis," Journal of Medical Microbiology, vol. 72 no. 11,DOI: 10.1099/jmm.0.001769, 2023.

[11] A. Rivero, M. Marquez, J. Santos, "High Rate of Tuberculosis Reinfection during a Nosocomial Outbreak of Multidrug-Resistant Tuberculosis Caused by Mycobacterium bovis Strain B," Clinical Infectious Diseases, vol. 32 no. 1, pp. 159-161, DOI: 10.1086/317547, 2001.

[12] J. Blazquez, L. E. E. de Los Monteros, S. Samper, "Genetic Characterization of Multidrug-Resistant Mycobacterium bovis Strains from a Hospital Outbreak Involving Human Immunodeficiency Virus-Positive Patients," Journal of Clinical Microbiology, vol. 35 no. 6, pp. 1390-1393, DOI: 10.1128/jcm.35.6.1390-1393.1997, 1997.

[13] C. A. Vazquez-Chacon, A. Martinez-Guarneros, D. Couvin, "Human Multidrug-Resistant Mycobacterium bovis Infection in Mexico," Tuberculosis, vol. 95 no. 6, pp. 802-809, DOI: 10.1016/j.tube.2015.07.010, 2015.

[14] B. P. de Val, B. Romero, M. T. Tortola, "Polyresistant Mycobacterium bovis Infection in Human and Sympatric Sheep, Spain, 2017–2018," Emerging Infectious Diseases, vol. 27 no. 4, pp. 1241-1243, DOI: 10.3201/eid2704.204467, 2021.

[15] Y. Dong, Y. Feng, X. Ou, "Genomic Analysis of Diversity, Biogeography, and Drug Resistance in Mycobacterium bovis," Transboundary and Emerging Diseases, vol. 69 no. 5,DOI: 10.1111/tbed.14628, 2022.

[16] H.-S. Cho, U.-S. Choi, Y. Oh, "Isolation of Multidrug-Resistant (MDR) Mycobacterium bovis from a Dog in Korea," Journal of Veterinary Medical Science, vol. 84 no. 10, pp. 1358-1362, DOI: 10.1292/jvms.21-0347, 2022.

[17] M. Borham, A. Oreiby, A. El-Gedawy, "Review on Bovine Tuberculosis: An Emerging Disease Associated with Multidrug-Resistant Mycobacterium Species," Pathogens, vol. 11 no. 7,DOI: 10.3390/pathogens11070715, 2022.

[18] A. C. Pereira, D. Pinto, M. V. Cunha, "First Time Whole Genome Sequencing of Mycobacterium bovis from the Environment Supports Transmission at the Animal-Environment Interface," Journal of Hazardous Materials, vol. 472,DOI: 10.1016/j.jhazmat.2024.134473, 2024.

[19] C. M. O’Connor, M. Abid, A. L. Walsh, "Cat-to-Human Transmission of Mycobacterium bovis , United Kingdom," Emerging Infectious Diseases, vol. 25 no. 12, pp. 2284-2286, DOI: 10.3201/eid2512.190012, 2019.

[20] M. Zwyer, C. Cavusoglu, G. Ghielmetti, "A New Nomenclature for the Livestock-Associated Mycobacterium tuberculosis Complex Based on Phylogenomics," Open Research Europe, vol. 1,DOI: 10.12688/openreseurope.14029.2, 2021.

[21] S. E. Sandoval-Azuara, R. Muñiz-Salazar, R. Perea-Jacobo, "Whole Genome Sequencing of Mycobacterium bovis to Obtain Molecular Fingerprints in Human and Cattle Isolates from Baja California, Mexico," International Journal of Infectious Diseases, vol. 63, pp. 48-56, DOI: 10.1016/j.ijid.2017.07.012, 2017.

[22] K. Faksri, E. Xia, J. H. Tan, Y.-Y. Teo, R. T.-H. Ong, "In Silico Region of Difference (RD) Analysis of Mycobacterium tuberculosis Complex from Sequence Reads Using RD-Analyzer," BMC Genomics, vol. 17 no. 1,DOI: 10.1186/s12864-016-3213-1, 2016.

[23] J. Wang, W. Zhao, R. Liu, "Rapid Detection of Ethambutol-Resistant Mycobacterium tuberculosis from Sputum by High-Resolution Melting Analysis in Beijing, China," Infection and Drug Resistance, vol. 13, pp. 3707-3713, DOI: 10.2147/IDR.S270542, 2020.

[24] S. Chen, "Ultrafast One-Pass FASTQ Data Preprocessing, Quality Control, and Deduplication Using Fastp," iMeta, vol. 2 no. 2,DOI: 10.1002/imt2.107, 2023.

[25] T. Seemann, "Snippy: Fast Bacterial Variant Calling from NGS Reads," 2015. https://github.com/tseemann/snippy

[26] M. Vasimuddin, S. Misra, H. Li, S. Aluru, "Efficient Architecture-Aware Acceleration of BWA-MEM for Multicore Systems," 2019 IEEE 33rd International Parallel and Distributed Processing Symposium (IPDPS), pp. 314-324, DOI: 10.1109/IPDPS.2019.00041, .

[27] E. Garrison, G. Marth, "Haplotype-Based Variant Detection from Short-Read Sequencing," 2012. arXiv: 1207.3907

[28] P. Cingolani, A. Platts, L. le Wang, "A Program for Annotating and Predicting the Effects of Single Nucleotide Polymorphisms, SnpEff: SNPs in the Genome of Drosophila melanogaster Strain w1118; ISO-2; ISO-3," Fly, vol. 6 no. 2, pp. 80-92, DOI: 10.4161/fly.19695, 2014.

[29] J. R. Grant, E. Enns, E. Marinier, "Proksee: In-Depth Characterization and Visualization of Bacterial Genomes," Nucleic Acids Research, vol. 51 no. W1, pp. W484-W492, DOI: 10.1093/nar/gkad326, 2023.

[30] L. S. Ates, "New Insights into the Mycobacterial PE and PPE Proteins Provide a Framework for Future Research," Molecular Microbiology, vol. 113 no. 1,DOI: 10.1111/mmi.14409, 2020.

[31] C. J. Meehan, G. A. Goig, T. A. Kohl, "Whole Genome Sequencing of Mycobacterium tuberculosis : Current Standards and Open Issues," Nature Reviews Microbiology, vol. 17 no. 9, pp. 533-545, DOI: 10.1038/s41579-019-0214-5, 2019.

[32] P. J. Gomez-Gonzalez, A. D. Grabowska, L. D. Tientcheu, "Functional Genetic Variation in pe / ppe Genes Contributes to Diversity in Mycobacterium tuberculosis Lineages and Potential Interactions with the Human Host," Frontiers in Microbiology, vol. 14,DOI: 10.3389/fmicb.2023.1244319, 2023.

[33] B. Q. Minh, H. A. Schmidt, O. Chernomor, "IQ-TREE 2: New Models and Efficient Methods for Phylogenetic Inference in the Genomic Era," Molecular Biology and Evolution, vol. 37 no. 5, pp. 1530-1534, DOI: 10.1093/molbev/msaa015, 2020.

[34] S. Kalyaanamoorthy, B. Q. Minh, T. K. F. Wong, A. von Haeseler, L. S. Jermiin, "ModelFinder: Fast Model Selection for Accurate Phylogenetic Estimates," Nature Methods, vol. 14 no. 6, pp. 587-589, DOI: 10.1038/nmeth.4285, 2017.

[35] J. E. Phelan, D. M. O’Sullivan, D. Machado, "Integrating Informatics Tools and Portable Sequencing Technology for Rapid Detection of Resistance to Anti-Tuberculous Drugs," Genome Medicine, vol. 11 no. 1,DOI: 10.1186/s13073-019-0650-x, 2019.

[36] C. Loiseau, F. Menardo, A. Aseffa, "An African Origin for Mycobacterium bovis," Evolution, Medicine, and Public Health, vol. 2020 no. 1, pp. 49-59, DOI: 10.1093/emph/eoaa005, 2020.

[37] M. Liew, R. Pryor, R. Palais, "Genotyping of Single-Nucleotide Polymorphisms by High-Resolution Melting of Small Amplicons," Clinical Chemistry, vol. 50 no. 7, pp. 1156-1164, DOI: 10.1373/clinchem.2004.032136, 2004.

[38] X. Chen, F. Kong, Q. Wang, C. Li, J. Zhang, G. L. Gilbert, "Rapid Detection of Isoniazid, Rifampin, and Ofloxacin Resistance in Mycobacterium tuberculosis Clinical Isolates Using High-Resolution Melting Analysis," Journal of Clinical Microbiology, vol. 49 no. 10, pp. 3450-3457, DOI: 10.1128/JCM.01068-11, 2011.

[39] C. U. Köser, S. Feuerriegel, D. K. Summers, J. A. C. Archer, S. Niemann, "Importance of the Genetic Diversity Within the Mycobacterium tuberculosis Complex for the Development of Novel Antibiotics and Diagnostic Tests of Drug Resistance," Antimicrobial Agents and Chemotherapy, vol. 56 no. 12, pp. 6080-6087, DOI: 10.1128/AAC.01641-12, 2012.

[40] C. U. Koser, J. M. Bryant, I. Comas, "Comment on: Characterization of the embB Gene in Mycobacterium tuberculosis Isolates from Barcelona and Rapid Detection of Main Mutations Related to Ethambutol Resistance Using a Low-Density DNA Array," Journal of Antimicrobial Chemotherapy, vol. 69 no. 8, pp. 2298-2299, DOI: 10.1093/jac/dku101, 2014.

[41] D. Bottai, W. Frigui, F. Sayes, "TbD1 Deletion as a Driver of the Evolutionary Success of Modern Epidemic Mycobacterium tuberculosis Lineages," Nature Communications, vol. 11 no. 1,DOI: 10.1038/s41467-020-14508-5, 2020.

[42] R. Brosch, S. V. Gordon, M. Marmiesse, "A New Evolutionary Scenario for the Mycobacterium tuberculosis Complex," Proceedings of the National Academy of Sciences, vol. 99 no. 6, pp. 3684-3689, DOI: 10.1073/pnas.052548299, 2002.

[43] T. Garnier, K. Eiglmeier, J.-C. Camus, "The Complete Genome Sequence of Mycobacterium bovis," Proceedings of the National Academy of Sciences of the United States of America, vol. 100 no. 13, pp. 7877-7882, DOI: 10.1073/pnas.1130426100, 2003.

[44] P. Li, R. Wang, W. Dong, "Comparative Proteomics Analysis of Human Macrophages Infected with Virulent Mycobacterium bovis," Frontiers in Cellular and Infection Microbiology, vol. 7,DOI: 10.3389/fcimb.2017.00065, 2017.

[45] J. T. Evans, E. G. Smith, A. Banerjee, "Cluster of Human Tuberculosis Caused by Mycobacterium bovis : Evidence for Person-to-Person Transmission in the UK," The Lancet, vol. 369 no. 9569, pp. 1270-1276, DOI: 10.1016/S0140-6736(07)60598-4, 2007.

[46] I. Etchechoury, G. E. Valencia, N. Morcillo, "Molecular Typing of Mycobacterium bovis Isolates in Argentina: First Description of a Person-to-Person Transmission Case," Zoonoses and Public Health, vol. 57 no. 6, pp. 375-381, DOI: 10.1111/j.1863-2378.2009.01233.x, 2010.

[47] S. Sunder, P. Lanotte, S. Godreuil, C. Martin, M. L. Boschiroli, J. M. Besnier, "Human-to-Human Transmission of Tuberculosis Caused by Mycobacterium bovis in Immunocompetent Patients," Journal of Clinical Microbiology, vol. 47 no. 4, pp. 1249-1251, DOI: 10.1128/JCM.02042-08, 2009.

[48] World Health Organization, "Global tuberculosis report 2020," 2020. Retrieved from https://www.who.int/publications/i/item/9789240013131

[49] J. R. Yates, P. J. Lillie, E. Helbren, A. D. Samson, "Mycobacterium bovis Infection Mimicking Pancreatic Cancer," Lancet Infectious Diseases, vol. 1099,DOI: 10.1016/S1473-3099(19)30560-2, 2020.

[50] C. Allix-Beguec, M. Fauville-Dufaux, K. Stoffels, "Importance of Identifying Mycobacterium bovis as a Causative Agent of Human Tuberculosis," European Respiratory Journal, vol. 35 no. 3, pp. 692-694, DOI: 10.1183/09031936.00137309, 2010.

[51] P. A. LoBue, K. S. Moser, "Treatment of Mycobacterium bovis Infected Tuberculosis Patients: San Diego County, California, United States, 1994–2003," International Journal of Tuberculosis and Lung Disease, vol. 9, pp. 333-338, 2005.

[52] M. Bobadilla-del Valle, P. Torres-González, M. E. Cervera-Hernández, "Trends of Mycobacterium bovis Isolation and First-Line Anti-Tuberculosis Drug Susceptibility Profile: A Fifteen-Year Laboratory-Based Surveillance," PLOS Neglected Tropical Diseases, vol. 9 no. 9,DOI: 10.1371/journal.pntd.0004124, 2015.

[53] I. Khattak, M. H. Mushtaq, S. Ayaz, "Incidence and Drug Resistance of Zoonotic Mycobacterium bovis Infection in Peshawar, Pakistan," Yeast Membrane Transport, vol. 1057, pp. 111-126, DOI: 10.1007/978-3-319-79017-6, 2018.

[54] Q. Liu, J. Zhu, C. L. Dulberger, "Tuberculosis Treatment Failure Associated with Evolution of Antibiotic Resilience," Science, vol. 378 no. 6624, pp. 1111-1118, DOI: 10.1126/science.abq2787, 2022.

[55] S. Bobba, S. A. Khader, "Rifampicin Drug Resistance and Host Immunity in Tuberculosis: More than Meets the Eye," Trends in Immunology, vol. 44 no. 9, pp. 712-723, DOI: 10.1016/j.it.2023.07.003, 2023.

[56] Z.-P. Li, W.-H. Mao, F. Huang, "Access to Quality Diagnosis and Rational Treatment for Tuberculosis: Real-World Evidence from China—Gates Tuberculosis Control Project Phase III," Infectious Diseases of Poverty, vol. 10 no. 1,DOI: 10.1186/s40249-021-00875-8, 2021.