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

Abstract

Clostridioides difficile 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 C. difficile strains. Additionally, the frequency of C. difficile toxin genes and the genotypes of toxin-producing strains were determined. Ninety-nine C. difficile 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 tcdA genes encoding enterotoxin A was detected. In turn, in 66 (66.7%) isolates, the gene encoding toxin B (tcdB) was present. The lowest number of strains tested was positive for genes encoding a binary toxin. Only 31 (31.3%) strains possessed the cdtB gene and 22 (22.2%) contained both genes for the binary toxin subunits (the cdtB and cdtA 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 C. difficile 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 C. difficile toxin genes as well as investigate their dissemination for epidemiological purposes.

Keywords: Clostridioides difficile; Clostridioides difficile toxinotypes; agarose gel electrophoresis; antibiotic-associated diarrhea; binary toxin; multiplex PCR; toxin A; toxin B; toxinogenic potential.

Figure 1

The diversity and number of the detected toxinogenotypes among Clostridioides difficile strains (n = 99), where cdtA+—presence of the gene encoding the A subunit of the binary toxin, cdtA−—no gene present, cdtB+—presence of the gene encoding the B subunit of the binary toxin, cdtB−—no gene present, gluD+—presence of the gene encoding glutamate dehydrogenase, gluD−—no gene present, tcdA+—presence of the gene encoding toxin A, tcdA−—no gene present, tcdB+—presence of the gene encoding toxin B, tcdB−—no gene present.

Figure 2

Example of agarose gel electrophoretic separation of the amplification products using the multiplex PCR technique for the tcdA (629 bp), tcdB (410 bp), cdtB (262 bp), cdtA (221 bp), and gluD (158 bp) genes, where cdtA—binary toxin subunit A gene; cdtB—binary toxin subunit B gene; gluD—glutamate dehydrogenase gene; tcdA—toxin A gene; tcdB—toxin B gene; M—DNA size marker 100–1000 base pairs; the lines labelled 40–46, 117—numbers of the tested strains; 42, 46, and K (+)—positive controls of the amplification reaction for all the investigated genes, and the remaining samples with the gluD, tcdA, and tcdB genes only; K (−)—negative control of the amplification reaction.