DEVELOPMENT OF A NOVEL MULTIPLEX PCR ASSAY FOR THE DETECTION OF MOBILE CARBAPENEM, TETRACYCLINE RESISTANCE GENES,  AND CLASS 1 INTEGRON IN ENVIRONMENTAL AND CLINICAL SPECIMENS

Bùi Đức Thịnh; Lê Thị Thu Hằng; Nguyễn Quang Huy

doi:10.34238/tnu-jst.12494

DEVELOPMENT OF A NOVEL MULTIPLEX PCR ASSAY FOR THE DETECTION OF MOBILE CARBAPENEM, TETRACYCLINE RESISTANCE GENES, AND CLASS 1 INTEGRON IN ENVIRONMENTAL AND CLINICAL SPECIMENS

About this article

Received: 06/04/25 Revised: 21/05/25 Published: 22/05/25

Authors

1. Bui Duc Thinh, University of Science and Technology of Hanoi - Vietnam Academy of Science and Technology
2. Le Thi Thu Hang, University of Science and Technology of Hanoi - Vietnam Academy of Science and Technology
3. Nguyen Quang Huy , University of Science and Technology of Hanoi - Vietnam Academy of Science and Technology

Abstract

The rapid spread of antibiotic-resistant bacteria and antimicrobial resistance genes threatens the global health. Polymerase chain reaction based assays offer rapid detection of antibiotic-resistant bacteria and resistance determinants. This study develops a novel multiplex polymerase chain reaction assay for the simultaneous detection of genes: tetM (tetracycline resistance), blaNDM and blaKPC (carbapenem resistance), and intI1 (class 1 integron-integrase gene). The assay demonstrated 100% sensitivity and specificity for the detection of these target genes, both individually and in mixtures. The assay’s limit of detection was established at 10¹ copies/mL for blaKPC and blaNDM, and at 10⁴ copies/mL for tetM and intI1. Furthermore, the multiplex polymerase chain reaction assay was successfully applied to the direct screening of target genes in environmental and clinical samples. In conclusion, the multiplex polymerase chain reaction assay developed in this study represents a valuable tool for the surveillance of tetM, blaNDM, blaKPC, and intI1 genes, and for the detection of antibiotic-resistant bacteria.

Keywords

Multiplex PCR; Intergon class 1; Carbapenem resistance; Tetracycline resistance; Multidrug resistance

Full Text:

PDF

References

[1] World Health Organization, “Global antimicrobial resistance and use surveillance system (‎GLASS)‎ report: 2022,” 2022. [Online]. Available: https://www.who.int/publications/i/item/9789240062702. [Accessed April 5, 2025].

[2] World Health Organization, "WHO publishes list of 12 antibiotic-resistant priority pathogens," 2017. [Online]. Available: https://www.rapidmicrobiology.com/news/who-publishes-list-of-12-antibiotic- resistant-priority-pathogens. [Accessed April 5, 2025].

[3] World Health Organization, “WHO bacterial priority pathogens list, 2024: Bacterial pathogens of public health importance to guide research, development and strategies to prevent and control antimicrobial resistance,” 2024. [Online]. Available: https://www.who.int/publications/i/item/9789240093461. [Accessed April 5, 2025].

[4] C. Perez-Gonzalez et al., "Rapid detection of antibiotic resistance: current status and future perspectives," Critical Reviews in Microbiology, vol. 46, no. 5, pp. 585–607, 2020, doi: 10.1080/1040841X.2020.1783448.

[5] M. A. Fischbach and C. T. Walsh, "Antibiotics for emerging pathogens," Science, vol. 353, no. 6295, pp. 126-129, Jul. 2016, doi: 10.1126/science.aaf9572.

[6] S. S. Jean, D. Harnod, and P. R. Hsueh, “Global Threat of Carbapenem-Resistant Gram-Negative Bacteria,” Front Cell Infect Microbiol, vol. 12, Mar 15, 2022, Art. no. 823684, doi: 10.3389/fcimb.2022.823684.

[7] F. Y. Ramírez-Castillo, A. L. Guerrero-Barrera, and F. J. Avelar-González, “An overview of carbapenem-resistant organisms from food-producing animals, seafood, aquaculture, companion animals, and wildlife,” Front Vet Sci., vol. 10, Jun 15, 2023, Art. no. 1158588, doi: 10.3389/fvets.2023.1158588.

[8] P. Nordmann and L. Poirel, “Epidemiology and Diagnostics of Carbapenem Resistance in Gram-negative Bacteria,” Clinical Infectious Diseases, vol, 69, no. 7, pp. S521–S528, December 2019, doi: 10.1093/cid/ciz824.

[9] A. D. Cerbo, F. Pezzuto, G. Guidetti, S. Canello, and L. Corsi, “Tetracyclines: Insights and Updates of their Use in Human and Animal Pathology and their Potential Toxicity,” The Open Biochemistry Journal, vol. 13, pp. 1-12, 2019, doi: 10.2174/1874091X01913010001.

[10] X. X. Zhang et al., "Occurrence and distribution of tetracycline antibiotics and resistance genes in various environmental matrices: A review," Science of the Total Environment, vol. 521-522, pp. 587–598, Aug. 2015, doi: 10.1016/j.scitotenv.2015.03.082.

[11] J. H. Jeon, K.-M. Jang, J. H. Lee, L.-W. Kang, and S. H. Lee, “Transmission of antibiotic resistance genes through mobile genetic elements in Acinetobacter baumannii and gene-transfer prevention,” Science of The Total Environment, vol. 857, 2023, Art. no. 159497, doi: 10.1016/j.scitotenv.2022.159497.

[12] H. S. Tran et al., "Occurrence of Multidrug-Resistant Enterobacteriaceae and Antibiotic-Resistant Genes in the Anthropogenic Impacted Bay of Nha Trang, Vietnam," Regional Studies in Marine Science, vol. 83, 2025, Art. no. 104084, doi: 10.1016/j.rsma.2025.104084.

[13] ECDC, Carbapenem- and colistin-resistant Enterobacteriaceae: Laboratory manual,” Stockholm: European Centre for Disease Prevention and Control (ECDC), 2023. [Online]. Available: https://www.ecdc.europa.eu/sites/default/files/documents/carbapenem-colistin-resistant- enterobacteriaceae-laboratory-manual.pdf. [Accessed April 5, 2025].

[14] ECDC, “Antimicrobial resistance surveillance in Europe 2022 - Annual report of the European Antimicrobial Resistance Surveillance Network (EARS-Net),” Stockholm: European Centre for Disease Prevention and Control (ECDC), 2024. [Online]. Available: https://www.ecdc.europa.eu/sites/default/ files/documents/EURGen-Net-manual.pdf. [Accessed April 5, 2025].

[15] A. M. Queenan and G. A. Bushman, "Antimicrobial Resistance in Bacteria from Companion Animals," Animal Health Research Reviews, vol. 14, no. 2, pp. 191-217, Dec. 2013, doi: 10.1017/S146625231300027X.

[16] G. Gaggero et al., "Characterization of Vibrio parahaemolyticus strains isolated in Chile in 2005 and in 2007," Journal of Infection in Developing Countries, vol. 5, no. 10, pp. 718-725, Nov. 2011.

[17] L. T. Thuy et al., "Initial establishment of a multiplex real-time PCR assay for simultaneous detection of common colistin and carbapenemase genes," Vietnam Journal of Science and Technology - Vietnam Academy of Science and Technology, vol. 62, no. 3, pp. 521–529, 2024, doi: 10.15625/2525- 2518/19130.

[18] J. Davies and D. I. Davies, "Origins and evolution of antibiotic resistance," Microbiology and Molecular Biology Reviews, vol. 74, no. 3, pp. 417-433, Sep. 2010, doi: 10.1128/MMBR.00016-10.

[19] M. A. Webber et al., "Surveillance of antibiotic resistance in bacteria from companion animals in the UK," Journal of Antimicrobial Chemotherapy, vol. 72, no. 10, pp. 2897-2905, Oct. 2017, doi: 10.1093/jac/dkx231.

[20] L. T. Thuy et al., "Initial establishment of a multiplex real-time PCR assay for simultaneous detection of common colistin and carbapenemase genes," Vietnam Journal of Science and Technology - Vietnam Academy of Science and Technology, vol. 62, no. 3, pp. 521–529, 2024, doi: 10.15625/2525- 2518/19130.

[21] T. Ceccarelli et al., "Occurrence of integrons and resistance genes in Escherichia coli isolates from intensive pig farms in Italy," FEMS Microbiology Ecology, vol. 62, no. 3, pp. 319-326, Dec. 2007, doi: 10.1111/j.1574-6941.2007.00391.x.

[22] S. Aminov, "The Role of Metals in Antibiotic Resistance and the Use of Metals as Antimicrobial Agents," Journal of Microbiology and Biotechnology, vol. 29, no. 2, pp. 149-157, Feb. 2019, doi: 10.4014/jmb.1810.10030.

DOI: https://doi.org/10.34238/tnu-jst.12494

Refbacks

There are currently no refbacks.



Remember me