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Molecular Detection of Toxigenic Clostridioides difficile among Diarrheic Dogs and Cats: A Mounting Public Health Concern.

ABSTRACT: Nowadays, pet animals are known to be asymptomatic carriers of Clostridioidesdifficile. This study was conducted to investigate the burden of toxigenic C. difficile among diarrheic dogs and cats using direct PCR on fecal samples to reveal better insights about the epidemiology of such toxigenic strains referring to its public health significance. For this purpose, fecal samples were obtained from 58 dogs and 42 cats experiencing diarrhea. Following DNA extraction, the extracted DNA was examined for the occurrence of C. difficile as well as toxigenic strains through the detection of C. difficile 16S rRNA and toxin encoding genes (tcdA, tcdB, cdtA and cdtB) using PCR. Moreover, partial DNA sequencing of toxigenic strains retrieved from dog and cat was carried out. Of 100 examined diarrheic animals, 90 (90%) were C. difficile positive, including 93.1% and 85.7% of dogs and cats, respectively. In addition, toxigenic strains were detected in 13 animals, giving an overall prevalence 13% with the following prevalence rates among dogs and cats 12.1% and 14.3%, respectively. Furthermore, the phylogenetic analysis of the obtained sequence revealed high genetic relatedness of tcdA sequence obtained from a cat to strains of human diarrheic cases to point out the public health threat of such sequence. In conclusion, the direct detection of toxigenic C. difficile using PCR among dogs and cats highlights the potential role of household pets as a source for such strains to human contacts.

SUBMITTER: Samir A

PROVIDER: S-EPMC8224758 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:This study developed a new single-tube multiplex real-time PCR method for detecting toxigenic C. difficile directly from fecal samples using tcdA, tcdB, cdtB, and internal gene tpi as targets, which could be performed on kinds of polymerase chain reaction device including point-of-care testing (POCT), with improved detection efficiency. The specificity, sensitivity, and repeatability of each gene was evaluated using 69 C. difficile isolates and 74 fecal samples. Results were compared with established PCR, qPCR, and ELISA methods. Interspecies specificity was 100% based on six common intestinal pathogens (Escherichia coli, Enterococcus Faecium, Enterococcus faecalis, Clostridium perfringens, Bacteroides fragilis, Clostridium botulinum). The lower detection limit (LDL) for tcdA, tcdB, and cdtB with pure C. difficile DNA was 101,100, and 100 copies/μL, respectively, the coefficients of variation among different experimental batches and within each experimental batch were both less than 3%, which shows that this method has strong repeatability. And the LDL of fecal DNA was 5 × 100, 5 × 103, and 5 × 102 colony-forming units (CFU)/g, respectively. In addition, the efficiency for detection of tcdA was compared with established PCR and real-time PCR methods, demonstrating high consistency (98.4%) and similar sensitivity. ELISA was used to confirm inconsistent results, which were identical with our method. The sensitivity and specificity for detecting toxigenic C. difficile in fecal samples were 96.49% and 94.12% compared with the toxigenic culture (TC). This method effectively identified the toxigenic and non-toxigenic strains with high specificity, sensitivity, and repeatability, and could reduce the false positive rate of tcdA, and accurately identify the typical Asian strain RT017, making it potentially contribute to the surveillance of CDI in China.Supplementary informationThe online version contains supplementary material available at 10.1007/s13205-022-03434-6.

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Molecular Detection of Toxigenic Clostridioides difficile among Diarrheic Dogs and Cats: A Mounting Public Health Concern.

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