Investigation of racR gene in thermophilic campylobacter Spp.
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Hamid Mahmoodipour,1,*
1. Department of Nursing and Midwifery, Behbahan Branch, Islamic Azad University, Behbahan, Iran
- Introduction: Campylobacter is one of the most common causative agents of bacterial food-borne gastroenteritis in humans worldwide. Thermophilic Campylobacter species like C.jejuni grow between 37°C and 42°C, (optimally at 41.5°C ) and are incapable to grow below 30°C. The RacR-RacS signal transduction system have a key function in altering gene expression at 42°C.
The racR gene (reduced ability to colonize), a member of the RacR-RacS system, codes for a regulatory protein that has an effect on the growth of Campylobacter in a temperature-dependent way and on the colonization of the intestinal tract of chickens.
- Methods: In all, 136 faecal samples were collected from poultry. The fecal samples were collected using sterile sticks and polyethylene bags (Isun Medical, Tehran, Iran) and transferred to the laboratory of Islamic Azad University, Behbahan branch within one hour of sampling. The samples were subjected for detection of Campylobacter immediately upon arrive to the laboratory.
Campylobacter detection in the present study was pre-treatment- apandis 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 racR gene.
The PCR assay was done for authentication of Campylobacter and detection of racR gene. 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 racR gene from each DNA extract was carried out and all amplified DNAs were visualized with UV transilluminator (Heidolph, Germany).
- Results: Among 27 Campylobacter strains, including 23 C.jejuni and 4 C.coli, prevalence of racR gene in poultry was 88.89% (24/27) .
- Conclusion: The results obtained from present study achieved during March 2020 and May 2020 from 136 samples were collected of which 27 strain of Campylobacter spp. isolated. Of the 27 strains, 23 C.jejuni and 4 C.coli were isolated. This is evidently could concluded that there exist C.jejuni and C.coli in Poultry in behbahan city. The racR gene of Campylobacter spp. Has been reported to be important in the ability to colonize chickens and support optimal growth rates at 42°C, suggesting that the racR gene regulates genes important for in vivo colonization in a temperature-dependent manner. In our research, the prevalence rate of racR gene was 88.89%. The results, similar to our research, have been reported where in the prevalence rate of racR gene is high .
- Keywords: racR gene, Thermophilic Campylobacters
