International Journal of Molecular Sciences

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16 pages, 3191 KiB

Open AccessArticle

Biochip-Based Identification of Mycobacterial Species in Russia

by Danila Zimenkov, Vyacheslav Zhuravlev, Anastasia Ushtanit, Marina Filippova, Uliana Semenova, Natalia Solovieva, Maria Sviridenko, Anastasia Khakhalina, Svetlana Safonova, Marina Makarova, Elizaveta Gordeeva, Elena Guselnikova, Yakov Schwartz, Natalia Stavitskaya and Peter Yablonsky

Int. J. Mol. Sci. 2024, 25(23), 13200; https://doi.org/10.3390/ijms252313200 - 8 Dec 2024

Viewed by 618

Abstract

Infections caused by nontuberculous mycobacteria (NTM) are rising globally throughout the world. The number of species isolated from clinical samples is steadily growing, which demands the implementation of a robust diagnostic method with wide specificity. This study was carried out in in 2022–2024 [...] Read more.

Infections caused by nontuberculous mycobacteria (NTM) are rising globally throughout the world. The number of species isolated from clinical samples is steadily growing, which demands the implementation of a robust diagnostic method with wide specificity. This study was carried out in in 2022–2024 in three clinical antituberculosis centers in the biggest cities of Russia: Moscow, Saint Petersburg, and Novosibirsk. We developed the DNA hybridization assay ‘Myco-biochip’ that allows the identification of 79 mycobacterial species and analyzed 3119 samples from 2221 patients. Sixty-eight mycobacterial species were identified in clinics, including the three novel species phylogenetically related to M. duvalii, M. lentiflavum, and M. talmoniae. The identification of a close relative of M. talmoniae adds to the existence of separate clade between M. terrae, M. triviale complexes and other slow-growing Mycobacteria, which supports the thesis against the splitting of Mycobacteria into five separate genera. Adding to the list of potentially pathogenic species, we identified M. adipatum and M. terramassiliense, which were previously described as natural habitats. The diversity of acid-fast bacilli identified in TB-suspected persons was not limited to the Mycobacteria genus and also includes species from genera Nocardia, Gordonia, Corynebacterium, Tsukamurella, and Rhodococcus of the order Mycobacteriales. The revealed bacterial diversity in patients with suspected NTM-diseases requires the implementation of relevant species identification assays as the first step in the laboratory diagnostic pipeline. Full article

(This article belongs to the Special Issue Advances in Molecular Biology on Mycobacteria)

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15 pages, 5901 KiB

Open AccessArticle

Binding of MAP3773c Protein of Mycobacterium avium subsp. paratuberculosis in the Mouse Ferroportin1 Coding Region

by Dulce Liliana Dueñas Mena, José A. Gutiérrez-Pabello, Kaina Quintero Chávez, Mirna Del Carmen Brito-Perea, Dania Melissa Díaz Padilla, Omar Cortez Hernández, José Román Chávez Mendez, Jocelyn Marcela Alcalá Zacarias, Giselle Berenice Vela Sancho and Bertha Landeros Sánchez

Int. J. Mol. Sci. 2024, 25(23), 12687; https://doi.org/10.3390/ijms252312687 - 26 Nov 2024

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Abstract

Mycobacterium avium subsp. paratuberculosis (MAP) is known to cause paratuberculosis. One notable protein, MAP3773c, plays a critical role in iron metabolism as a transcription factor. This study aims to investigate the binding affinity of MAP3773c to the chromatin of the Ferroportin1 (FPN1) gene [...] Read more.

Mycobacterium avium subsp. paratuberculosis (MAP) is known to cause paratuberculosis. One notable protein, MAP3773c, plays a critical role in iron metabolism as a transcription factor. This study aims to investigate the binding affinity of MAP3773c to the chromatin of the Ferroportin1 (FPN1) gene in murine macrophage J774 A.1. We conducted a sequence alignment to identify potential interaction sites for MAP3773c. Following this, we used in silico analysis to predict binding interactions, complemented by electrophoretic mobility shift assay (EMSA) to confirm in vitro binding of MAP3773c. The map3773c gene was cloned into the pcDNA3.1 vector, with subsequent expression analysis carried out via Western blotting and real-time PCR. Chromatin immunoprecipitation (CHiP) assays were performed on transfected macrophages to confirm binding in the native chromatin context. Our in silico and in vitro analysis indicated that MAP3773c interacts with two binding motifs within the FPN1 coding region. The ChiP results provided additional validation, demonstrating the binding of MAP3773c to the FPN1 chromatin through successful amplification of the associated chromatin fragment via PCR. Our study demonstrated that MAP3773c binds to FPN1 and provides insight into the role of MAP3773c and its effect on host iron transport. Full article

(This article belongs to the Special Issue Advances in Molecular Biology on Mycobacteria)

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19 pages, 340 KiB

Open AccessArticle

The Intriguing Pattern of Nontuberculous Mycobacteria in Bulgaria and Description of Mycobacterium bulgaricum sp. nov.

by Danila Zimenkov, Yuliana Atanasova, Anastasia Ushtanit, Stanislava Yordanova, Ana Baykova, Marina Filippova, Uliana Semenova, Igor Mokrousov and Elizabeta Bachiyska

Int. J. Mol. Sci. 2024, 25(19), 10434; https://doi.org/10.3390/ijms251910434 - 27 Sep 2024

Cited by 1 | Viewed by 886

Abstract

We investigated the rise of nontuberculous mycobacteria (NTM) infections in Bulgaria, focusing on species identification and distribution from 2018 to 2022. Utilizing advanced diagnostic tools, including the Hain Mycobacterium CM/AS method, Myco-biochip assay, and whole-genome sequencing, the study identifies and characterizes a diverse [...] Read more.

We investigated the rise of nontuberculous mycobacteria (NTM) infections in Bulgaria, focusing on species identification and distribution from 2018 to 2022. Utilizing advanced diagnostic tools, including the Hain Mycobacterium CM/AS method, Myco-biochip assay, and whole-genome sequencing, the study identifies and characterizes a diverse range of Mycobacterium species from clinical samples. While M. avium, M. gordonae, M. fortuitum, and M. chelonae were dominating, a number of rare species were also found. They include such species as M. marseillense and M. celatum. Moreover, the noticeable prevalence of M. terrae complex species missed by conventional testing was observed. We identified a rare species, highly homologous to previously described strains from Japan; based on genome–genome distance data, we propose its reannotation as a new species. Further, a novel species was identified, which is significantly distinct from its closest neighbor, M. iranicum, with ANI = 87.18%. Based on the SeqCode procedure, we propose to name this new species Mycobacterium bulgaricum sp. nov. Dynamic changes in NTM species prevalence in Bulgaria observed from 2011 to 2022 highlight the emergence of new species and variations tied to environmental and demographic factors. This underscores the importance of accurate species identification and genotyping for understanding NTM epidemiology, informing public health strategies, and enhancing diagnostic accuracy and treatment protocols. Full article

(This article belongs to the Special Issue Advances in Molecular Biology on Mycobacteria)

17 pages, 4260 KiB

Open AccessArticle

Rescue of Mycobacterium bovis DNA Obtained from Cultured Samples during Official Surveillance of Animal TB: Key Steps for Robust Whole Genome Sequence Data Generation

by Daniela Pinto, Gonçalo Themudo, André C. Pereira, Ana Botelho and Mónica V. Cunha

Int. J. Mol. Sci. 2024, 25(7), 3869; https://doi.org/10.3390/ijms25073869 - 30 Mar 2024

Cited by 1 | Viewed by 1439

Abstract

Epidemiological surveillance of animal tuberculosis (TB) based on whole genome sequencing (WGS) of Mycobacterium bovis has recently gained track due to its high resolution to identify infection sources, characterize the pathogen population structure, and facilitate contact tracing. However, the workflow from bacterial isolation [...] Read more.

Epidemiological surveillance of animal tuberculosis (TB) based on whole genome sequencing (WGS) of Mycobacterium bovis has recently gained track due to its high resolution to identify infection sources, characterize the pathogen population structure, and facilitate contact tracing. However, the workflow from bacterial isolation to sequence data analysis has several technical challenges that may severely impact the power to understand the epidemiological scenario and inform outbreak response. While trying to use archived DNA from cultured samples obtained during routine official surveillance of animal TB in Portugal, we struggled against three major challenges: the low amount of M. bovis DNA obtained from routinely processed animal samples; the lack of purity of M. bovis DNA, i.e., high levels of contamination with DNA from other organisms; and the co-occurrence of more than one M. bovis strain per sample (within-host mixed infection). The loss of isolated genomes generates missed links in transmission chain reconstruction, hampering the biological and epidemiological interpretation of data as a whole. Upon identification of these challenges, we implemented an integrated solution framework based on whole genome amplification and a dedicated computational pipeline to minimize their effects and recover as many genomes as possible. With the approaches described herein, we were able to recover 62 out of 100 samples that would have otherwise been lost. Based on these results, we discuss adjustments that should be made in official and research laboratories to facilitate the sequential implementation of bacteriological culture, PCR, downstream genomics, and computational-based methods. All of this in a time frame supporting data-driven intervention. Full article

(This article belongs to the Special Issue Advances in Molecular Biology on Mycobacteria)

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21 pages, 7130 KiB

Open AccessArticle

Differential Immunogenicity and Lung Disease-Inducing Potential of Mycobacterium immunogenum Genotypes and Impact of Co-Exposure with Pseudomonas: Optimizing a Mouse Model of Chronic Hypersensitivity Pneumonitis

by Elisabet Johansson and Jagjit S. Yadav

Int. J. Mol. Sci. 2024, 25(4), 2058; https://doi.org/10.3390/ijms25042058 - 8 Feb 2024

Cited by 1 | Viewed by 1137

Abstract

Mycobacterium immunogenum (MI) colonizing metalworking fluids (MWFs) has been associated with chronic hypersensitivity pneumonitis (HP) in machinists. However, it is etiologically unclear why only certain mycobacteria-contaminated fluids induce this interstitial lung disease. We hypothesized that this may be due to differential immunogenicity and [...] Read more.

Mycobacterium immunogenum (MI) colonizing metalworking fluids (MWFs) has been associated with chronic hypersensitivity pneumonitis (HP) in machinists. However, it is etiologically unclear why only certain mycobacteria-contaminated fluids induce this interstitial lung disease. We hypothesized that this may be due to differential immunogenicity and the HP-inducing potential of MI strains/genotypes as well as the confounding effect of co-inhaled endotoxin-producers. To test this hypothesis, we optimized a chronic HP mouse model in terms of MI antigen dose, timepoint of sacrifice, and form of antigen (cell lysates vs. live cells) and compared six different field-isolated MI strains. Overall, MJY10 was identified as the most immunogenic and MJY4 (or MJY13) as the least immunogenic genotype based on lung pathoimmunological changes as well as Th1 cellular response (IFN-γ release). Infection with MI live cells induced a more severe phenotype than MI cell lysate. Co-exposure with Pseudomonas fluorescens caused a greater degree of lung innate immune response and granuloma formation but a diminished adaptive (Th1) immune response (IFN-γ) in the lung and spleen. In summary, this study led to the first demonstration of differential immunogenicity and the disease-inducing potential of field strains of MI and an interfering effect of the co-contaminating Pseudomonas. The improved chronic MI-HP mouse model and the identified polar pair of MI strains will facilitate future diagnostic and therapeutic research on this poorly understood environmental lung disease. Full article

(This article belongs to the Special Issue Advances in Molecular Biology on Mycobacteria)

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13 pages, 7025 KiB

Open AccessArticle

Epinephrine Stimulates Mycobacterium tuberculosis Growth and Biofilm Formation

by Yingying Lei, Khaista Rahman, Xiaojian Cao, Bing Yang, Wei Zhou, Aikebaier Reheman, Luxia Cai, Yifan Wang, Rohit Tyagi, Zhe Wang, Xi Chen and Gang Cao

Int. J. Mol. Sci. 2023, 24(24), 17370; https://doi.org/10.3390/ijms242417370 - 12 Dec 2023

Cited by 2 | Viewed by 1856

Abstract

The human stress hormones catecholamines play a critical role in communication between human microbiota and their hosts and influence the outcomes of bacterial infections. However, it is unclear how M. tuberculosis senses and responds to certain types of human stress hormones. In this [...] Read more.

The human stress hormones catecholamines play a critical role in communication between human microbiota and their hosts and influence the outcomes of bacterial infections. However, it is unclear how M. tuberculosis senses and responds to certain types of human stress hormones. In this study, we screened several human catecholamine stress hormones (epinephrine, norepinephrine, and dopamine) for their effects on Mycobacterium growth. Our results showed that epinephrine significantly stimulated the growth of M. tuberculosis in the serum-based medium as well as macrophages. In silico analysis and molecular docking suggested that the extra-cytoplasmic domain of the MprB might be the putative adrenergic sensor. Furthermore, we showed that epinephrine significantly enhances M. tuberculosis biofilm formation, which has distinct texture composition, antibiotic resistance, and stress tolerance. Together, our data revealed the effect and mechanism of epinephrine on the growth and biofilm formation of M. tuberculosis, which contributes to the understanding of the environmental perception and antibiotic resistance of M. tuberculosis and provides important clues for the understanding of bacterial pathogenesis and the development of novel antibacterial therapeutics. Full article

(This article belongs to the Special Issue Advances in Molecular Biology on Mycobacteria)

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16 pages, 3410 KiB

Open AccessArticle

Genome-Scale Characterization of Mycobacterium abscessus Complex Isolates from Portugal

by Sofia Carneiro, Miguel Pinto, Sónia Silva, Andrea Santos, Irene Rodrigues, Daniela Santos, Sílvia Duarte, Luís Vieira, João Paulo Gomes and Rita Macedo

Int. J. Mol. Sci. 2023, 24(20), 15402; https://doi.org/10.3390/ijms242015402 - 20 Oct 2023

Cited by 2 | Viewed by 1444

Abstract

The Mycobacterium abscessus complex (MABC) is an emerging, difficult to treat, multidrug-resistant nontuberculous mycobacteria responsible for a wide spectrum of infections and associated with an increasing number of cases worldwide. Dominant circulating clones (DCCs) of MABC have been genetically identified as groups of [...] Read more.

The Mycobacterium abscessus complex (MABC) is an emerging, difficult to treat, multidrug-resistant nontuberculous mycobacteria responsible for a wide spectrum of infections and associated with an increasing number of cases worldwide. Dominant circulating clones (DCCs) of MABC have been genetically identified as groups of strains associated with higher prevalence, higher levels of antimicrobial resistance, and worse clinical outcomes. To date, little is known about the genomic characteristics of MABC species circulating in Portugal. Here, we examined the genetic diversity and antimicrobial resistance profiles of 30 MABC strains isolated between 2014 and 2022 in Portugal. The genetic diversity of circulating MABC strains was assessed through a gene-by-gene approach (wgMLST), allowing their subspecies differentiation and the classification of isolates into DCCs. Antimicrobial resistance profiles were defined using phenotypic, molecular, and genomic approaches. The majority of isolates were resistant to at least two antimicrobials, although a poor correlation between phenotype and genotype data was observed. Portuguese genomes were highly diverse, and data suggest the existence of MABC lineages with potential international circulation or cross-border transmission. This study highlights the genetic diversity and antimicrobial resistance profile of circulating MABC isolates in Portugal while representing the first step towards the implementation of a genomic-based surveillance system for MABC at the Portuguese NIH. Full article

(This article belongs to the Special Issue Advances in Molecular Biology on Mycobacteria)

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18 pages, 3070 KiB

Open AccessArticle

Dynamic Transcriptional Landscape of Mycobacterium smegmatis under Cold Stress

by Artem S. Grigorov, Yulia V. Skvortsova, Oksana S. Bychenko, Leonid V. Aseev, Ludmila S. Koledinskaya, Irina V. Boni and Tatyana L. Azhikina

Int. J. Mol. Sci. 2023, 24(16), 12706; https://doi.org/10.3390/ijms241612706 - 11 Aug 2023

Viewed by 1644

Abstract

Bacterial adaptation to cold stress requires wide transcriptional reprogramming. However, the knowledge of molecular mechanisms underlying the cold stress response of mycobacteria is limited. We conducted comparative transcriptomic analysis of Mycobacterium smegmatis subjected to cold shock. The growth of M. smegmatis cultivated at [...] Read more.

Bacterial adaptation to cold stress requires wide transcriptional reprogramming. However, the knowledge of molecular mechanisms underlying the cold stress response of mycobacteria is limited. We conducted comparative transcriptomic analysis of Mycobacterium smegmatis subjected to cold shock. The growth of M. smegmatis cultivated at 37 °C was arrested just after exposure to cold (acclimation phase) but later (by 24 h) was resumed at a much slower rate (adaptation phase). Transcriptomic analyses revealed distinct gene expression patterns corresponding to the two phases. During the acclimation phase, differential expression was observed for genes associated with cell wall remodeling, starvation response, and osmotic pressure stress, in parallel with global changes in the expression of transcription factors and the downregulation of ribosomal genes, suggesting an energy-saving strategy to support survival. At the adaptation phase, the expression profiles were recovered, indicating restoration of the processes repressed earlier. Comparison of transcriptional responses in M. smegmatis with those in other bacteria revealed unique adaptation strategies developed by mycobacteria. Our findings shed light on the molecular mechanisms underlying M. smegmatis survival under cold stress. Further research should clarify whether the discovered transcriptional mechanisms exist in other mycobacterial species, including pathogenic Mycobacterium tuberculosis, which could be important for transmission control. Full article

(This article belongs to the Special Issue Advances in Molecular Biology on Mycobacteria)

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