Prevalence and Distribution of Arcobacter Butzelri Virulence
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www.journals.rifst.ac.ir Journal of Research and Innovation in Food Science and Technology 9 (2021) 4, 423-432 Original Paper DOI: 10.22101/JRIFST.2021.260335.1207 Prevalence and Distribution of Arcobacter Butzelri Virulence Genes in Poultry Slaughterhouse Effluent Samples in Tonekabon Sara Fallahchay¹, Nima Bahador²*, Masood Ghane³ 1- PhD. Student, Department of Microbiology, College of Sciences, Shiraz Branch, Islamic Azad University, Shiraz, Iran 2- Associate Professor, Department of Microbiology, College of Sciences, Shiraz Branch, Islamic Azad University, Shiraz, Iran * Corresponding author ([email protected]) 3- Associate Professor, Department of Microbiology, Tonekabon Branch, Islamic Azad University, Tonekabon, Iran Abstract Received: 2020.12.08 Arcobacter butzelri is known as the cause of enteritis, abdominal Accepted: 2021.02.08 cramps, bacteremia, and appendicitis in humans, it is also the cause of enteritis and abdominal pain in animals. It has been Keywords introduced as the most dangerous species for human health by the Arcobacter butzleri International Committee on Food Microbiology and recently as Virulence gene an important zoonotic pathogen. The aim of this study was Polymerase chain reaction isolatation and characterization of Arcobacter butzelri based on pathogenic genes (tlyA, ciaB, mviN) in different seasons. Therefore, 238 samples were collected from the effluent of poultry slaughterhouse in Tonekabon, Iran. The suspected colonies were identified using biochemical test and then confirmed using the Polymerase chain reaction (PCR) technique. Out of 42 isolated of A. butzleri, the virulence genes were detected in 15 isolates. The results showed that ciaB gene was present in 13 strains with a frequency of 30.9%, mviN in 11 samples with a frequency of 26.2%, and tlyA gene in 9 samples with a frequency of 21.4%. The results also revealed that the highest frequency of A. butzelri was 8.4% in spring and the lowest rate was 1.2% in winter. 1 Introduction butzelri is the most common species which Arcobacter genus is a member of is usually isolated from a variety of Campylobacter family and belongs to the environments (Figueras et al., 2014; class of Epsilon proteobacteria (Ellis, Neill, Jyothsna, Rahul, Ramaprasad, Sasikala, & O'brien, Ferguson, & Hanna, 1977). Ramana, 2013; Levican, Collado, Yustes, Arcobacteria can cause infertility, Aguilar, & Figueras, 2014) Most studies on miscarriage, and digestive disorders in the prevalence of Arcobacter in foods are animals, and bacterial gastroenteritis, related to the poultry that have the highest endocarditis, and peritonitis in humans prevalence, followed by pork and raw milk (Badilla-Ramirez, Fallas-Padilla, products (Collado, Guarro, & Figueras, Fernandez-Jaramillo, & Arias-Echandi, 2009; Figueras et al., 2014) The genus 2016). They are widely found in water and Arcobacter has become very important in the sources of animal foods. Arcobacter recent years because its members are Research and Innovation in Food Science and Technology, 9 (2021) 4, 424 considered as a potential zoonotic agent. Rojas, Ham, Deng, Doyle, & Collmer, The global prevalence of Arcobacter 2002). IrgA gene encodes an outer infection is unclear since the usual membrane protein and the periplasmic diagnostic method is not applicable to this enzyme of the iroE gene, both of which bacterium. There is no doubt that the regulate iron (Mey et al., 2002; Rashid, Arcobacter species can cause disease in Tarr, & Moseley, 2006; Zhu, Valdebenito, humans but more clinical observations are Winkelmann, & Hantke, 2005). Although essential to determine their pathogenicity it remains unclear whether these virulence in human (Levican, Alkeskas, Günter, genes have similar functions to their Forsythe, & Figueras, 2013). Preliminary homologs in different pathogenic bacteria, studies on A. butzelri have shown that showed little or no invasion of cells by these bacteria have clinical and microbial Arcobacter strains that lack virulence characteristics similar to Campylobacter genes. Therefore, the detection and jejuni strain but they are more associated quantification of virulence genes of with severe and persistent diarrhea and less Arcobacter may elucidate its role in associated with C. jejuni (Vandenberg et waterborne infections (Ghaju Shrestha et al., 2004; Wesley & Miller, 2010). In the al., 2019). Arcobacter can be considered as recent decade, sequencing of the RM4018 a relatively resistant bacteria which can genome of A. butzelri was an important survive in adverse conditions and affect the step in the development of research on pathogenicity process in human (Ferreira et Arcobacter, in which 10 pathogenic genes al., 2016). Thus, the aim of this work was were identified (Douidah et al., 2012; to isolate and identify this bacteria from Ferreira, Queiroz, Oleastro, & Domingues, wastewater of poultry slaughterhouse in 2016; Miller et al., 2007). The Tonekabon to examine the presence of pathogenicity of an organism is genes. characterized according to the presence of virulence genes (Ghaju Shrestha, Tanaka, Materials and methods Sherchand, & Haramoto, 2019). The cadF Sample Collection and screening of the and cj1349 genes encode outer membrane isolates proteins, which promote bacteria to cell 238 samples were collected from contact by adherence to fibronectin (Dasti, wastewater of poultry slaughterhouse in Tareen, Lugert, Zautner, & Groß, 2010). Tonekabon located in the geographical CiaB gene promotes host cell invasion position of x=0494258 and y=4069580 through the secretion system (Tabatabaei, (N=36.752036, E=50.929654) in different Aski, Shayegh, & Khoshbakht, 2014). seasons. To isolate A. butzelri, 50 mL of MviN gene is a vital protein required for the samples were transferred to sterile peptidoglycan biosynthesis (Inoue et al., screw cap tubes and transferred to the 2008). PldA encodes the outer membrane microbiology laboratory. Then they were phospholipase A (Grant, Belandia, Dekker, cultivated onto the Preston medium Broth Richardson, & Park, 1997). TlyA gene enriched with supplements (Preston encodes hemolysin which plays an supplement, biomark) and incubated at 25 important role of C. jejuni in adhesion to °C for 24 h. After enrichment, the Caco-2 cells (Slater, Bailey, Tett, & membrane filtration technique was applied Turner, 2008). The hecA gene encodes a using 0.45 μm pore size nitrocellulose protein belonging to a group of adhesives membrane filters. Then the samples (Filamentous haemagglutinin family). The transferred to Campylobacter Selective hecB encodes a related haemolysin Agar enriched with defibrillated sheep activation protein (Miller et al., 2007; blood and Campylobacter supplement, and Fallahchay et al. Prevalence and Distribution of Arcobacter Butzelri Virulence Genes … 425 the plates were incubated at 25 °C for 48 to butzelri with a frequency of 17.6%. In this 72 h. Finally, the suspected colonies were regard, the highest rate of isolation of A. identified using biochemical tests (Atabay butzelri was 8.4% in spring and the lowest & Corry, 1997). rate was 1.2% in winter. In spring, 20 strains with a frequency of 8.4% in Identification of Arcobacter butzelri and its summer 8 strains with a frequency of 3.3% pathogenic genes in fall, 11 strains with a frequency of 4.6%, All isolates were identified using and in winter, 3 strains of Arcobacter with polymerase chain reaction (PCR) a frequency of 1.2% were isolated from the techniques. For this purpose, DNA was slaughterhouse wastewater. extracted using Gene all kit (GeneAll Biotechnology Co., LTD.Korea, Cat.No. Identification of A. butzelri 106-152). The 16S r-RNA gene and The results of PCR and sequencing of 16S virulence genes of mviN, ciaB, and tlyA of r-RNA specific genes confirmed the A. butzelri were amplified using their phenotypic results obtained in 42 isolated special primers. Moreover, all PCRs were strains (Fig. 1). prepared with a final volume of 20 µL containing 10 µl of Mastermix (Hot Start Taq Polymerase, dNTP, buffer, Mgcl), 1 µL of forward primer, 1 µl of reverse primer, 3 µl of distilled water, and 5 µl of DNA To begin the process of polymerization of the 16S r-RNA gene, the thermocycler was set at 94 °C for 4 min for initial denaturation. Afterward, 25 cycles of PCR were performed including 94, 56, and 72 °C for 60s. The final extension was Fig. 1. PCR products on agarose gel (1.5%), Lane carried out at 72 °C for 7 min. In addition, 1: negative control, Lane 2: positive control, Lanes the cycling condition for mviN and ciaB 3-17: samples of isolated A. butzleri. M: Marker genes were placed in a thermocycler (1.5-k bp) device at 94 °C for 3 min for initial denaturation. In the next stage, 35 PCR Evaluation of the presence of A. butzelri cycles were performed including 94, 56, pathogenic genes and 72 °C for 45 s. The final extension was The presence of pathogenic genes of tlyA, carried out at 72 °C for 10 min. To carry ciaB, mviN, in A. butzelri was evaluated out the polymerization process of the tlyA using polymerase chain reaction. gene, the thermocycler device was set on According to the results obtained from this 94 °C for 3 min for initial denaturation. study indicated that 13 strains showed the Then, 35 PCR cycles were performed presence of ciaB gene (frequency of including 94, 55, and 72 °C for 45 s. 30.9%) (Fig. 2), 11 strains, mviN gene Finally, the final extension was carried out (frequency of 26.2%) (Fig. 3), and 9 strains at 72 °C for 10 min (Douidah et al., 2012; showed tlyA gene (frequency of 21.4%) Tabatabaei et al., 2014). (Fig. 4). The results also revealed that out of 15 strains of A. butzelri, 9 strains with 60% frequency had all 3 pathogenic genes Results and discussion Isolation of A. butzelri from wastewater of a (Table 1). poultry slaughterhouse in Tonekabon Out of 238 collected samples in 4 different seasons, 42 samples were positive for A. Research and Innovation in Food Science and Technology, 9 (2021) 4, 426 Fig. 4. PCR products on agarose gel (1.5%), Lane Fig.