Molecular Detection of Arcobacter in Human Stool Samples Using Housekeeping Genes

Background: Arcobacter is one of the most common bacteria in humans and livestock, leading to gastroenteritis in humans as well as genital and enteric diseases in animals. This bacterium is known to be the main cause of diarrhea. In molecular studies, the 16SrRNA gene was primarily used as the standard gene for the determination of the Arcobacter. The purpose of this study was to investigate the molecular detection of Arcobacter using glyA, atpA, and gyrA genes compared to16SrRNA. Methods: In this study, 61 samples of Arcobacter DNA isolated from fecal specimens of patients and healthy individuals in the sample bank were used. In order to detect Arcobacter, the intended primers for 16SrRNA as well as glyA, atpA, and gyrA genes were used for polymerase chain reaction (PCR). The products obtained from the PCR were sequenced. Results: The results of the proliferation reactions indicated the accuracy of the intended primers and the associated molecular experiments. Our results showed that 65.57% of the cases were detected to be positive for Arcobacter among 61 samples using the glyA gene. This percentage was higher compared to 16SrRNA (42.62%), gyrA (42.62%), and atpA (24.59%). The analysis was statistically significant. Conclusions: Given the presence of repetitive sequences in the 16SrRNA in most bacteria, the interpretation of the results is likely to be difficult for researchers. The results of this study showed more sensitivity and accurate diagnosis of Arcobacter using the glyA gene than other studied genes. In diagnostic studies of Arcobacter, the glyA gene is proposed as an alternative to the 16SrRNA.

on accurate molecular detection of Arcobacter spp. using the glyA, atpA, and gyrA genes simultaneously.
By targeting 16SrRNA and 23SrRNA genes, one-step PCR can be used to detect A. butzleri, A. cryaerophilus, and A. skirrowi at the same time (21). Other molecular methods have been proposed, including real-time PCR, DGGE, and AFLP (22). 16SrRNA gene is significantly conserved within the species of the genus so that it can be used as a golden standard for bacterial diagnosis. Although 16SrRNA gene is an indicator gene and the design of the primers is easy to amplify, it has many variables for the differentiation of microbial taxa. Almost all the studies in this field have been done on the molecular determination of Arcobacter using this gene. However, there are also some disadvantages. The 16SrRNA nucleotide sequence has multiple copies and low molecular resolution and cannot be easily interpreted in a research framework (23,24). In this way, the use of other housekeeping genes such as gyrA (encoding DNA gyrase submit A), glyA (encoding serine transhydroxymethylase), and atpA (encoding the submission of F1 ATPase) may offer different potential benefits for the molecular detection of Arcobacter (25).
The objective of this study is to identify Arcobacter in human stool samples using other housekeeping genes, including gyrA, glyA, and atpA, in order to develop target genes in addition to the 16SrRNA gene for molecular detection.

Collection of Samples
In this study, we selected 61 genomic DNA samples extracted from human fecal specimens available from the DNA Bank of Infectious Diseases Research Center (Arak University of Medical Sciences). These DNA samples were extracted from stool samples and were enriched with special media (arco broth) containing antibiotics incubated at 28°C for 48 hours. Then, they were inoculated on Brucella agar medium followed by passive filtration of the broth through a 0.45 μm membrane filter placed on the blood agar medium (26). Among these DNA samples, 29 samples were from healthy people who were exposed to poultry meat, and 32 samples were from individuals with diarrhea.

Genus-Specific PCR
Genus-specific PCR has been used to detect Arcobacter at the level of the genus. Primers for gyrA, glyA, and atpA genes have been designed using specialized programs such as Primer Blast, Mega 4.0, Oligo 6.0, and Primer3. The sequences of 16SrRNA-specific primers were provided by Gonzalez et al (15). The specifications of the used primers are shown in Table 1. The PCR reaction mix for each gene included 1.5 μL of extracted DNA (20-50 μg), 0.7 μL of each primer (Copenhagen, Denmark), 7.5 μL of 2x super master-mix (YTA, Iran), and 4.6 μL of DDW in the final volume of 15 μL. The amplification was performed with initial denaturation at 94°C (5 minutes) followed by 28 cycles of denaturation at 94°C (1 minute), annealing at specific temperatures (55 seconds), and extension at 72°C (55 seconds) ( Table 1). Moreover, the final extension was carried out at 72°C (8 minutes). DNA extracted from the Arcobacter colonies and the water (no template) were considered positive and negative, respectively.

Gel Electrophoresis
Gel electrophoresis was used to evaluate the PCR results of each amplifier. The products of PCR reactions were loaded on a 1.3% agarose gel (Genefanavaran, Iran). The results were analyzed using the gel doc system (Quantum ST4, Germany).

Sequencing
PCR products were sequenced on an ABI automatic sequencer (Applied Biosystem Inc., CA, USA) using Macrogen (South Korea) facilities for confirmation of the amplification reaction.

Statistical Analysis
Statistical analyses were carried out using related software packages such as Excel 2007 and MedCalc 18.11. Figure 1 shows the PCR products electrophoresed on 1.3% agarose gel.

Evaluation of PCR Products for Electrophoresis
The sequencing results were analyzed using related software packages (Mega4 and Chromas). The amplicons   Table 2.

Discussion
The increased isolation of Arcobacter from clinical samples and healthy people has increased its importance in general health (27). Due to the existence of Arcobacter in food products containing animal resources, vegetables, and water, this bacterium is introduced as a food-borne pathogen (28). In addition, Arcobacter can cause gastroenteritis in human and genital diseases in animals, which can confirm the need to detect Arcobacter (29). Because molecular detection methods are faster and more accurate than cultural ones, we used molecular methods to identify Arcobacter in fecal samples of healthy individuals and patients. In previous studies, the 16SrRNA gene is mostly used for the diagnosis of the Arcobacter gene. Due to the existence of different bacterial genomes in stool samples as well as repeated nucleotide sequences in the 16SrRNA gene of some bacteria, the detection of Arcobacter probably cannot be accurate (25). Therefore, in this study, we evaluated the molecular detection of Arcobacter using housekeeping genes such as gyrA, glyA, and atpA genes compared to 16SrRNA genes. Based on the suggestions of other studies, these genes have been selected. To date, no study has been conducted in our study environment reporting similar prevalence rates for Arcobacter using these genes. To the best of our knowledge, no previous study has used four housekeeping genes (gyrA, glyA, atpA, and 16SrRNA) simultaneously in the stool samples to differentiate    (32). Therefore, in the genes used in our study, glyA was also selected. Abdelbaqi et al studied the development of real-time PCR for investigating the quinolone resistance-determining regions in the gyrA gene of Arcobacter spp. in France (33). According to their study, the nucleotide sequences of the gyrA genes of A. butzleri, A. cryaerophilus, A. cibarius, and A. skirrowi were determined. Phylogenetic analysis of gyrA sequences provides results similar to phylogenetic analysis of the 16SrRNA gene sequence and allows for differentiation between A. butzleri species (33). In our study, in addition to the 16SrRNA gene, we used other in-house genes ( gyrA, glyA, and atpA) to detect Arcobacter at the molecular level. As shown in Table 2, 19 (31.14%), 9 (14.75%), 9 (14.75%), 9 (14.75%) of healthy people, who were exposed to poultry meat, and 21 (34.42%), 17 (27.86%), 17 (27.86%), 6 (9.83%) of patients with diarrhea were detected positive using proliferation of glyA, 16SrRNA, gyrA, and atpA genes, respectively, among a total of 61 samples. Recently, it has been shown that Arcobacter can be better identified by glyA gene than other genes in both groups. Although this bacterium has been detected more frequently in the patient group than in healthy people, glyA may be useful for the identification of Arcobacter in both groups (P=0.01). In addition, according to statistical data, the sensitivity of the use of the glyA gene to detect the Arcobacter is higher compared to the 16SrRNA gene. As shown in Table 3, the difference in the prevalence between groups was statistically significant (P < 0.05). Therefore, the proliferation of glyA gene by designing correct primers, which can be attached to genomes of different species of the Arcobacter, may be more useful than other studied genes for the detection of Arcobacter. In this study, due to limited financial resources, we did not examine all the housekeeping genes. We used only 4 genes for screening, and in future studies, other housekeeping genes can also be used. Despite the above discussion, it is recommended that this study should be carried out with a larger sample size and that the bacterial load of Arcobacter be studied in fecal samples of both healthy people and patients.

Conclusions
To date, Arcobacter has not been detected using gyrA, atpA, and glyA genes in clinical samples. The results of this study have shown that the glyA gene is more acceptable than other used housekeeping genes for molecular detection of this bacteria. Proliferation of the glyA gene may be considered as an alternative to the 16SrRNA gene to detect Arcobacter genus.

Ethical Approval
This study was derivate from research project of Arak University of medical science with IR.ARAKMU.REC.1397.228 ethical code.