Virulence gene profiles of Arcobacter species isolated from animals, foods of animal origin, and humans in Andhra Pradesh, India

Aim:: This study aimed to detect putative virulence genes in Arcobacter species of animal and human origin. Materials and Methods:: A total of 41 Arcobacter isolates (16 Arcobacter butzleri, 13 Arcobacter cryaerophilus, and 12 Arcobacter skirrowii) isolated from diverse sources such as fecal swabs of livestock (21), raw foods of animal origin (13), and human stool samples (7) were subjected to a set of six uniplex polymerase chain reaction assays targeting Arcobacter putative virulence genes (ciaB, pldA, tlyA, mviN, cadF, and cj1349). Results:: All the six virulence genes were detected among all the 16 A. butzleri isolates. Among the 13 A. cryaerophilus isolates, cadF, ciaB, cj1349, mviN, pldA, and tlyA genes were detected in 61.5, 84.6, 76.9, 76.9, 61.5, and 61.5% of isolates, respectively. Among the 12 A. skirrowii isolates, cadF, ciaB, cj1349, mviN, pldA, and tlyA genes were detected in 50.0, 91.6, 83.3, 66.6, 50, and 50% of isolates, respectively. Conclusion:: Putative virulence genes were detected in majority of the Arcobacter isolates examined. The results signify the potential of Arcobacter species as an emerging foodborne pathogen.


Introduction
The genus Arcobacter encompasses a group of Gram-negative, fastidious, nonspore forming, motile, spirally curved rods of the family Campylobacteraceae [1]. Arcobacter species differ from Campylobacter species in their aerotolerance and growth at 15°C [2]. In contrast with the acceptance of Escherichia coli, Salmonella, and Campylobacter species as the main foodborne pathogens, the reports dealing with the association of Arcobacter species are limited. Over the past few years, studies regarding the isolation of Arcobacter species from animals, raw foods of animal origin, and diarrheic humans signify the potential food safety concern associated with them [3][4][5]. Among several Arcobacter species, Arcobacter butzleri, Arcobacter cryaerophilus, and Arcobacter skirrowii are reported to have veterinary and public health significance [2]. Furthermore, the presence of virulence genes and its cytopathogenic effect on in vitro cell lines resulted in the categorization of A. butzleri as a "serious hazard" to human health by the International Commission on Microbiological Specifications for Foods [6].
Putative virulence determinants such as ciaB (encodes Campylobacter jejuni invasion antigen B that contributes to host cell invasion), mviN (encodes virulence factor, inner membrane protein required for peptidoglycan biosynthesis), pldA (encodes outer membrane phospholipase A associated with lysis of erythrocytes), tlyA (the hemolysin gene), and cadF and cj1349 (encodes fibronectin-binding proteins which promote the binding of bacteria to intestinal cells) were reported to be present in A. butzleri (RM4018) genome [7]. In addition, cadF (Campylobacter adhesion to fibronectin) protein also induces the internalization of bacterial cells by the activation of GTPases [7]. Despite increasing reports of the association of Arcobacter species with livestock and human diseases, the mechanisms of pathogenicity of Arcobacter species are still poorly understood [8,9].
Perusal of the available literature revealed a lack of information on virulence gene profiles of Arcobacter species of animal and human origin in India. The present study was carried out to characterize virulence gene profiles of Arcobacter species Available at www.veterinaryworld.org/Vol.10/June-2017/23.pdf isolated from animals, foods of animal origin, and human sources in Andhra Pradesh, India.

Ethical approval
This work does not require ethical approval as we have collected fecal swabs after defecation.

Reference strain
The reference strain A. butzleri (ATCC 49616) used in the present study was obtained from the Division of Veterinary Public Health, Indian Veterinary Research Institute, Izatnagar.

Detection of putative virulence genes by PCR
All the 41 Arcobacter isolates were subjected to a set of six uniplex PCR assays for the detection of six putative virulence genes (ciaB, pldA, tlyA, mviN, cadF, and cj1349) [13]. Primer sequences, expected amplicon sizes, and virulence gene details are listed in Table-1. A preliminary gradient PCR was conducted with DNA of A. butzleri (ATCC 49616) which carried all the six putative virulence genes to assess the correct annealing temperature. With annealing temperatures lower than 56.0°C (for ciaB, cj1349, and mviN genes) and 58.0°C (for cadF, pldA, and tlyA genes), lower molecular weight products were detected in addition to expected amplicons. Thus, annealing temperatures of 56.0°C (for amplification of ciaB, cj1349, and mviN genes) and 58.0°C (for cadF, pldA, and tlyA genes) were optimized for future PCR reactions. All the six PCR assays were carried out in Eppendorf thermal cycler (USA) with a heated lid.

Results and Discussion
Putative virulence genes were detected in majority of screened Arcobacter isolates. virulence genes. None of the 13 A. cryaerophilus and 12 A. skirrowii isolates possessed all the six virulence genes. The present results were in accordance with the previous studies from Berlin [9] and Belgium [13] where 100% prevalence of these putative virulence genes in A. butzleri strains was reported.
In a study from Belgium [13], three Arcobacter type strains (i.e., A. butzleri LMG 10828, A. cryaerophilus LMG 10210, and A. skirrowii LMG 6621) were reported to carry nine putative Campylobacter virulence genes (cadF, ciaB, cj1349, hecA, hecB, irgA, mviN, pldA, and tlyA). In addition, no significant difference was noticed in cadF gene detected among different strains of Arcobacter isolated from humans, cattle, horses, sheep, dogs, and chicken [13]. However, the role of these putative virulence determinants or Campylobacter homologs in the pathogenicity Arcobacter species was still contradictory [9,16]. Arcobacter species were reported to have ability to adhere and invade the intestinal epithelial cells of host and induce inflammatory responses [17]. Arcobacter infection was reported to be associated with leak flux type of watery diarrhea resulting from epithelial barrier dysfunction [18], and adhesion, invasion and toxin production could be the mechanisms of Arcobacter pathogenicity [9,19].

Conclusion
Food safety needs a thorough investigation of virulence properties of potentially emerging pathogenic bacteria in animals and foods of animal origin. The presence of Arcobacter strains carrying virulence markers in animals, humans and foods of animal origin have not yet been a subject of investigation in India. The present study reporting the presence of six virulence-associated genes of three emerging Arcobacter species isolated from animal and human sources adds to the significance of Arcobacter as an emerging foodborne pathogen with zoonotic potential. Further studies should focus on examining the interactions of Arcobacter species virulence factors with eukaryotic cells.

Authors' Contributions
MSS is the student worked for M.V.Sc thesis. SRT as a major guide and BKC and VSK as minor guides designed and supervised the research work. NMS collected the samples and executed the isolation. MSS was involved in the molecular characterization. The manuscript was drafted and revised by MSS and NMS under the guidance of SRT, BKC, and VSK. All authors read and approved the final manuscript.