Detection of CDT toxin genes in Campylobacter spp.
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Hamid Mahmoodipour,1,*
1. Department of Nursing and Midwifery, Behbahan Branch, Islamic Azad University, Behbahan, Iran
- Introduction: Campylobacter subspecies represent a highly frequent cause of foodborne gastrointestinal (GI) illnesses in humans worldwide. The incidence and prevalence of campylobacteriosis due to infection with Campylobacter jejuni and campylobacter coli has increased over the past decades, both in developed and developing countries. The clinical outcome of C. jejuni and C.coli infection ranges from mild to severe diarrheal disease, and some postinfection sequelae, including Guillain–Barré syndrome, reactive arthritis, and others.
Several genes have been linked to Campylobacter virulence, but the most important are cytolethal distending toxin (cdt), which disrupts mucosal ¬barriers by causing host cell death.
Cytolethal distending toxin (cdt) in Campylobacter spp. is among the significant virulence factors of these bacteria in the intestine.
- Methods: Campylobacter detection in the present study was pre-treatment- Kapandis Baseri (prêt-KB) technique and the medium was blood and antibiotic free Kapandis Baseri (KB) medium (HiMedia, Mumbai, India). To perform the method faecal samples were added (10% (w/v)) in sterile phosphate-buffered saline (0.1 mol l-1, pH 7) (Merck, Germany) to obtained 10% suspension. The suspension was centrifuged at 8500 rpm for 10 min, then it was placed at room temperature. Afterward 10–15 min, 0.1 ml supernatant from the tube was plated on the KB medium (HiMedia, Mumbai, India). The plates were incubated at 37°C for 48 h in microaerophilic conditions and tested daily for 5 days.
All suspected colonies grew on the KB medium confirmed by typical morphology, darting motility, gram staining, oxidase, and catalase tests. The isolates with presumptive Campylobacter character were subjected to standard Campylobacter phenotypic identification tests recommended by Atabay and Corry . These tests included H2S by lead acetate strip, nitrate reduction, growth in 1% glycine and 3.5% NaCl, growth at temperatures 25°C, 37°C, and 42°C, hippurate hydrolysis, indoxyl acetate hydrolysis, urease production, and resistance to Nalidixic acid (30 μg) and Cephalothin (30 μg). Additional tests for identification of Campylobacters were alkaline phosphatase production, oxidative-fermentative test (OF Test), and glucose fermentation. All items used in the phenotypic identification tests were purchased from Parsalab (Tehran, Iran). At the end, the PCR method was carried out in order to confirm the phenotyping results and detection of cdtA, cdtB and cdtC genes.
The PCR assay was done for authentication of Campylobacter and detection of cdtA, cdtB and cdtC genes. DNA was extracted from suspected colony using phenol chloroform method. The PCR was performed in 25 ml of the reaction mixture with a final concentration of 1×PCR reaction mix, 10pg–1μg concentration of template deoxynucleotide triphosphate (DNA), 0.1 –1 µmol L-1 concentrations of each primer (Macrogen, Inc, Seoul, Korea), 3.2 m mol L-1 MgCl2 solution, and 1.25–2.5 U/50 μl of GoTaq DNA polymerase. All items used in the PCR were purchased from Yekta Tajhiz Azma (Tehran, Iran) and the experiment was performed by thermal cycler (Bio-Red, Singapore). A 100-bp DNA ladder (Yekta Tajhiz Azma, Tehran, Iran) was used as a DNA molecular ladder. PCR products were electrophorized using 1% agarose gel (Yekta Tajhiz Azma, Tehran, Iran) at 80 V for 60 minutes. In addition detection of cdtA, cdtB and cdtC genes from each DNA extract was carried out and all amplified DNAs were visualized with UV transilluminator (Heidolph, Germany).
- Results: Sampling from the poultry faeces was conducted between May 2020 and September 2020 in behbahan city, khuzestan province, iran. The results obtained indicated that 27 strains of Campylobacter spp. were isolated. Among the 27 Campylobacter strains, including 22 Campylobacter jejuni and 5 Campylobacter coli, the prevalence of cdtA, cdtB, cdtC, virulence genes were 40.74% (11/27), 85.19% (23/27), 77.77% (21/27), respectively
- Conclusion: The research works on the virulence characteristics of potentially pathogenic bacteria in domestic animals and in foods containing animal origin are essential for the safety of the user. Although for assessing campylobacteriosis risk, heterogenic identification and virulence of genes of Campylobacter species isolated from the faeces samples of poultry are needed, so far it has not been done in the Khuzestan province.
The results of our study showed that over 80% of the Campylobacter isolates have genes interfering in toxin production (cdtB), which signifies that most of the Campylobacter isolates have powerful pathogenic .
- Keywords: Detection, CDT toxin genes, Campylobacter spp.
