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PCR Development for Analysis of Some Type II Toxin–Antitoxin Systems, relJK, mazEF3, and vapBC3 Genes, in Mycobacterium tuberculosis and Mycobacterium bovis

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Abstract

Toxin–Antitoxin (TA) systems are some small genetic modules in bacteria that play significant roles in resistance and tolerance development to antibiotics. Whole genome sequencing (WGS) is an effective method to analyze TA systems in pathogenic Mycobacteria. However, this study aimed to use a simple and inexpensive PCR-Sequencing approach to investigate the type II TA system. Using data from the WGS of Mycobacterium tuberculosis (M. tuberculosis) strain H37Rv and Mycobacterium bovis (M. bovis) strain BCG, primers specific to the relJK, mazEF3, and vapBC3 gene families were designed by Primer3 software. Following that, a total of 90 isolates were examined using the newly developed PCR assay, consisting of 64 M. tuberculosis and 26 M. bovis isolates, encompassing both 45 rifampin-sensitive and 45 rifampin-resistant strains. Finally, 28 isolates (including 14 rifampin-resistant isolates) were sent for sequencing, and their sequences were aligned and compared to the mentioned reference sequences. The amplicons size of mazEF3, relJK, and vapBC3 genes were 825, 875, and 934 bp, respectively. Furthermore, all tested isolates showed the specific amplicons for these TA families. To evaluate the specificity of the primers, PCR was performed on S. aureus and E.coli isolates. None of the examined samples had the desired amplicons. Therefore, the primers had acceptable specificity. The results indicated that the developed PCR-Sequencing approach can be used to effectively investigate certain types of TA systems. Considering high costs of WGS and difficulty in interpreting its results, such a simple and inexpensive method is beneficial in the evaluation of TA systems in Mycobacteria.

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Acknowledgements

We would like to express our gratitude to the staff of the Department of Pathobiology at Bu-Ali Sina University and the Microbiology Department of Golestan University of Medical Sciences, both in Iran, for their kind cooperation. Additionally, we extend our sincere thanks to the Razi Vaccine and Serum Institute in Iran for providing us with the Mycobacterium bovis isolates.

Funding

The work was supported by Bu-Ali Sina University (Grant Number: 1677), Hamedan, Iran.

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Authors and Affiliations

  1. Department of Pathobiology, Faculty of Veterinary Medicine, Bu-Ali Sina University, Hamedan, Iran

    Maryam Shafipour, Abdolmajid Mohammadzadeh & Pezhman Mahmoodi

  2. Infectious Diseases Research Center, Golestan University of Medical Sciences, Gorgan, Iran

    Ezzat Allah Ghaemi

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  1. Maryam Shafipour
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  2. Abdolmajid Mohammadzadeh
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Contributions

(i) The conception or design of the study: A.M., E.A.G., P.M. (ii) the acquisition: M.S. analysis: M.S., P.M., E.A.G. interpretation of the data: M.S., A.M., P.M.; and (iii) writing of the manuscript: M.S., A.M., and P.M. All authors approved the final version.

Corresponding author

Correspondence to Abdolmajid Mohammadzadeh.

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The authors have no conflict of interest to declare for this study.

Ethical Approval

This study was approved by the Ethical Committee of the Golestan University of Medical Sciences, Iran (Ethical code: IR.GOUMS.REC.1401.224).

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Shafipour, M., Mohammadzadeh, A., Ghaemi, E.A. et al. PCR Development for Analysis of Some Type II Toxin–Antitoxin Systems, relJK, mazEF3, and vapBC3 Genes, in Mycobacterium tuberculosis and Mycobacterium bovis. Curr Microbiol 81, 90 (2024). https://doi.org/10.1007/s00284-023-03599-0

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