The Use of Gel Electrophoresis to Separate Multiplex Polymerase Chain Reaction Amplicons Allows for the Easy Identification and Assessment of the Spread of Toxigenic Strains

Overview

Journal Gels

Date 2024 Dec 27

PMID 39727576

Authors

Tomasz Bogiel

Patrycja Kwiecinska

Robert Gorniak

Piotr Kanarek

Agnieszka Mikucka

Affiliations

Soon will be listed here.

Abstract

is a common etiological factor of hospital infections, which, in extreme cases, can lead to the death of patients. Most strains belonging to this bacterium species synthesize very dangerous toxins: toxin A (TcdA) and B (TcdB) and binary toxin (CDT). The aim of this study was to assess the suitability of agarose gel electrophoresis separation of multiplex PCR amplicons to investigate the toxinogenic potential of strains. Additionally, the frequency of toxin genes and the genotypes of toxin-producing strains were determined. Ninety-nine strains were used in the detection of the presence of genes encoding all of these toxins using the multiplex PCR method. In 85 (85.9%) strains, the presence of genes encoding enterotoxin A was detected. In turn, in 66 (66.7%) isolates, the gene encoding toxin B () was present. The lowest number of strains tested was positive for genes encoding a binary toxin. Only 31 (31.3%) strains possessed the gene and 22 (22.2%) contained both genes for the binary toxin subunits (the and genes). A relatively large number of the strains tested had genes encoding toxins, whose presence may result in a severe course of disease. Therefore, the accurate diagnosis of patients, including the detection of all known toxin genes, is very important. The multiplex PCR method allows for the quick and accurate determination of whether the tested strains of this bacterium contain toxin genes. Agarose gel electrophoresis is a useful tool for visualizing amplification products, allowing one to confirm the presence of specific toxin genes as well as investigate their dissemination for epidemiological purposes.

References

Bustin S, Benes V, Garson J, Hellemans J, Huggett J, Kubista M . The MIQE guidelines: minimum information for publication of quantitative real-time PCR experiments. Clin Chem. 2009; 55(4):611-22. DOI: 10.1373/clinchem.2008.112797. View

Pradhan R, Ngo P, Martinez-Sanchez L, Neurath M, Lopez-Posadas R . Rho GTPases as Key Molecular Players within Intestinal Mucosa and GI Diseases. Cells. 2021; 10(1). PMC: 7823293. DOI: 10.3390/cells10010066. View

Gnocchi M, Gagliardi M, Gismondi P, Gaiani F, De Angelis G, Esposito S . Updated Management Guidelines for in Paediatrics. Pathogens. 2020; 9(4). PMC: 7238231. DOI: 10.3390/pathogens9040291. View

Ruiz-Villalba A, Ruijter J, van den Hoff M . Use and Misuse of C in qPCR Data Analysis and Reporting. Life (Basel). 2021; 11(6). PMC: 8229287. DOI: 10.3390/life11060496. View

Alam M, Markantonis J, Fallon J . Host Immune Responses to Infection and Potential Novel Therapeutic Approaches. Trop Med Infect Dis. 2023; 8(12). PMC: 10747268. DOI: 10.3390/tropicalmed8120506. View

Waker E, Ambrozkiewicz F, Kulecka M, Paziewska A, Skubisz K, Cybula P . High Prevalence of Genetically Related Strains at a Single Hemato-Oncology Ward Over 10 Years. Front Microbiol. 2020; 11:1618. PMC: 7384382. DOI: 10.3389/fmicb.2020.01618. View

Gerding D, Johnson S, Rupnik M, Aktories K . Clostridium difficile binary toxin CDT: mechanism, epidemiology, and potential clinical importance. Gut Microbes. 2013; 5(1):15-27. PMC: 4049931. DOI: 10.4161/gmic.26854. View

Martinez-Melendez A, Cruz-Lopez F, Morfin-Otero R, Maldonado-Garza H, Garza-Gonzalez E . An Update on Binary Toxin. Toxins (Basel). 2022; 14(5). PMC: 9146464. DOI: 10.3390/toxins14050305. View

Alam M, Madan R . Toxins: Host Cell Interactions and Their Role in Disease Pathogenesis. Toxins (Basel). 2024; 16(6). PMC: 11209539. DOI: 10.3390/toxins16060241. View

10.

Deniz U, Ulger N, Aksu B, Karavus M, Soyletir G . [Investigation of toxin genes of Clostridium difficile strains isolated from hospitalized patients with diarrhoea at Marmara University Hospital]. Mikrobiyol Bul. 2011; 45(1):1-10. View

11.

Aptekorz M, Sacha K, Gofron Z, Kabala M, Harmanus C, Kuijper E . Antibiotic Resistance Profile of RT 027/176 Versus Other Isolates in Silesia, Southern Poland. Pathogens. 2022; 11(8). PMC: 9416131. DOI: 10.3390/pathogens11080949. View

12.

Pituch H, van Leeuwen W, Maquelin K, Wultanska D, Obuch-Woszczatynski P, Nurzynska G . Toxin profiles and resistances to macrolides and newer fluoroquinolones as epidemicity determinants of clinical isolates of Clostridium difficile from Warsaw, Poland. J Clin Microbiol. 2007; 45(5):1607-10. PMC: 1865912. DOI: 10.1128/JCM.00306-07. View

13.

Mitchell M, Nguyen S, Macori G, Bolton D, McMullan G, Drudy D . as a Potential Pathogen of Importance to One Health: A Review. Foodborne Pathog Dis. 2022; 19(12):806-816. DOI: 10.1089/fpd.2022.0037. View

14.

Hulme J . Emerging Diagnostics in Infection. Int J Mol Sci. 2024; 25(16). PMC: 11354687. DOI: 10.3390/ijms25168672. View

15.

Wittwer C, Herrmann M, Gundry C, Elenitoba-Johnson K . Real-time multiplex PCR assays. Methods. 2002; 25(4):430-42. DOI: 10.1006/meth.2001.1265. View

16.

Bogiel T, Dura A, Wozniak M, Mikucka A, Kanarek P . Usefulness of Capillary Gel Electrophoresis-Based PCR for Detection of Strains with Hypervirulent Ribotypes. Gels. 2024; 10(5). PMC: 11121280. DOI: 10.3390/gels10050343. View

17.

Azimirad M, Naderi Noukabadi F, Lahmi F, Yadegar A . Prevalence of binary-toxin genes ( and ) among clinical strains of isolated from diarrheal patients in Iran. Gastroenterol Hepatol Bed Bench. 2019; 11(Suppl 1):59-65. PMC: 6347996. View

18.

Tokimatsu I, Shigemura K, Osawa K, Kinugawa S, Kitagawa K, Nakanishi N . Molecular epidemiologic study of Clostridium difficile infections in university hospitals: Results of a nationwide study in Japan. J Infect Chemother. 2018; 24(8):641-647. DOI: 10.1016/j.jiac.2018.03.015. View

19.

Mendo-Lopez R, Alonso C, Villafuerte-Galvez J . Best Practices in the Management of Infection in Developing Nations. Trop Med Infect Dis. 2024; 9(8). PMC: 11359346. DOI: 10.3390/tropicalmed9080185. View

20.

Suleiman M, Tang P, Imam O, Morales P, Altrmanini D, Roberts J . Infections in Children: What Is the Optimal Laboratory Diagnostic Method?. Microorganisms. 2024; 12(9). PMC: 11434531. DOI: 10.3390/microorganisms12091785. View