Development and evaluation of multiplex PCR assays for rapid detection of virulence-associated genes in Arcobacter species

    Development and Evaluation of Multiplex PCR Assays for Rapid Detection of Virulence-associated Genes in Arcobacter Species Jenni Whiteduck-L´eveill´ee, Michel Cloutier, Edward Topp, David R. Lapen, Guylaine Talbot, Richard Villemur, Izhar U.H. Khan PII: DOI: Reference:

S0167-7012(15)30138-X doi: 10.1016/j.mimet.2015.12.017 MIMET 4814

To appear in:

Journal of Microbiological Methods

Received date: Revised date: Accepted date:

6 October 2015 17 December 2015 31 December 2015

Please cite this article as: Whiteduck-L´eveill´ee, Jenni, Cloutier, Michel, Topp, Edward, Lapen, David R., Talbot, Guylaine, Villemur, Richard, Khan, Izhar U.H., Development and Evaluation of Multiplex PCR Assays for Rapid Detection of Virulenceassociated Genes in Arcobacter Species, Journal of Microbiological Methods (2016), doi: 10.1016/j.mimet.2015.12.017

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ACCEPTED MANUSCRIPT REVISED Development and Evaluation of Multiplex PCR Assays for Rapid Detection of Virulence-associated Genes in Arcobacter Species

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Jenni Whiteduck-Léveillée1, Michel Cloutier1, Edward Topp2, David R. Lapen1, Guylaine Talbot3, Richard Villemur4, Izhar U.H. Khan1*

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Ottawa Research and Development Centre (ORDC), Agriculture and Agri-Food Canada, Ottawa, ON, Canada. 2

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London Research and Development Centre (LRDC), Agriculture and Agri-Food Canada, London, ON, Canada. 3

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Sherbrooke Research and Development Centre (SRDC), Agriculture and Agri-Food Canada, Sherbrooke, QC, Canada. INRS-Institute Armand-Frappier Research Centre, Laval, QC, Canada.

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Arcobacter spp.

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Running Title: Multiplex PCR assays for the detection of virulence-associated genes in

Keywords: Multiplex PCR assays, Virulence-associated genes, Arcobacter butzleri, A. cryaerophilus, A. skirrowii, Fecal matter

*Corresponding author: Mailing address: Eastern Cereal and Oilseed Research Centre (ECORC), Agriculture and Agri-Food Canada, 960 Carling Ave., Ottawa, K1A 0C6, Ontario, Canada. Tel.: +613-759-7702; Fax: +613-759-1924; E- mail: [email protected]

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ACCEPTED MANUSCRIPT Abstract

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As the pathogenicity of Arcobacter species might be associated with various virulence factors, this study was aimed to develop and optimize three single-tube multiplex PCR (mPCR) assays

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that can efficiently detect multiple virulence-associated genes (VAGs) in Arcobacter spp.

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including the A. butzleri, A. cryaerophilus and A. skirrowii, respectively. The recognized target

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virulence factors used in the study were fibronectin binding protein (cj1349), filamentous hemagglutinin (hecA), hemolysin activation protein (hecB), hemolysin (tlyA), integral

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membrane protein virulence factor (mviN), invasin (ciaB), outer membrane protein (irgA) and phospholipase (pldA). Identical results were obtained between singleplex PCR and mPCR assays

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and no cross- and/ or non-specific amplification products were obtained when tested against

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other closely related bacterial species. The sensitivities of these three mPCR assays were ranging

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from 1 ng µL-1 to 100 ng µL-1 DNA. The developed assays with combinations of duplex or triplex PCR primer pairs of VAGs were further evaluated and validated by applying them to isolates of the A. butzleri, A. cryaerophilus and A. skirrowii recovered from fecal samples of human and animal origins. The findings revealed that the distribution of the ciaB (90%), mviN (70%), tlyA (50%) and pldA (45%) genes among these target species were significantly higher than the hecA (16%), hecB (10%) and each of irgA and cj1349 (6%) genes, respectively. The newly developed mPCR assays can be used as rapid technique and useful markers for the detection, prevalence and profiling of VAGs in the Arcobacter spp. Moreover, these assays can easily be performed with a high throughput to give a presumptive identification of the causal pathogen in epidemiological investigation of human infections.

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ACCEPTED MANUSCRIPT 1.

Introduction Arcobacters are Gram-negative, rod-shaped aerotolerant bacteria (Vandamme et al., 1991)

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isolated from a wide variety of ecological niches ranging from animal and human feces to food

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products (Fera et al., 2004; Figueras et al., 2011; Gonzalez and Ferru´s, 2010; Ho et al., 2006).

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Among twenty-one well-defined Arcobacter species, several species have been associated with diseases in humans and animals such as gastroenteritis, mastitis, bacteremia, reproductive

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disorders in livestock, and abortion (Ho et al., 2006). In recent years, the A. butzleri, A. cryaerophilus, A. cibarius and A. skirrowii have become increasingly recognized as emergent

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pathogens and known as potential zoonotic agents (Collado et al., 2011; Collado and Figueras,

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2011; Ho et al., 2006; Houf and Stephan 2007).

Housekeeping genes such as those encoding 16S rRNA, 23S rRNA and gyrase (gyrA and

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gyrB) can be used to detect arcobacters in DNA extracted from environmental matrices; but these genes do not reveal the pathogenic potential of Arcobacter spp. that may be present (Douidah et al., 2010; Harmon and Wesley, 1996). The pathogenicity of Arcobacter that can potentially cause health risks for humans and animals would require information on associated virulence factors. However, current knowledge of key mechanisms (e.g., adhesion, invasion, and cytotoxicity capacity) and potential virulence factors of Arcobacter is still limited (Douidah et al., 2012; Karadas et al., 2013; Levican et al., 2013). The complete genome sequence of the Arcobacter butzleri ATCC 49616 reference strain has revealed the presence of nine putative virulence-associated genes (VAGs). The key VAGs that have been identified to date include: i) ciaB, an invasive antigen that contributes to host cell invasion through a secretion system; ii) the virulence factor mviN which is an essential protein required for peptidoglycan biosynthesis; iii)

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ACCEPTED MANUSCRIPT two genes encoding for fibronectin binding proteins the cadF and cj1349 code the fibronectin binding proteins which promote the binding of bacteria to intestinal cells, whereas the cadF

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protein also induces the internalization of bacterial cells by the activation of GTPases; iv) the

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phospholipase gene pldA that encodes the outer membrane phospholipase A that is associated with lysis of erythrocytes; v) the hemolysin gene tlyA which is the gene encoding for an iron-

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regulated outer membrane protein irgA that encodes an outer membrane receptor for

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enterobactin, and the gene encoding hemolysin activation proteins hecB and hecA, a member of the filamentous hemagglutinin (FHA), that participate in attachment and aggregation and is also

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implicated in epidermal cell killing (Miller et al., 2007). Other studies have previously evaluated the occurrence and potential role of VAGs in the pathogenesis of human and animal infections

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by the Arcobacter spp. (Douidah et al., 2012; Tabatabaei et al., 2014).

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Molecular identification of pathogenic vs. non-pathogenic Arcobacter strains can potentially be differentiated by detection of their virulence markers and gene expression analysis. DNAbased methods, such as PCR, are more practical, rapid, specific and sensitive than conventional phenotypic assays and have been used to detect one or more virulence genes (Kingombe et al., 1999; Thoerner et al., 2003). Moreover, a multiplex PCR approach, can further improve the efficiency of a PCR assay to Arcobacter spp., overcoming a challenge observed using only single gene target PCR formats by this method which is more rapid and economical in terms of time and labor. Information on important VAG markers, such as mviN, tlyA and hecB genes, can be obtained in one step. Thus, multiplex PCR-based approaches offer the potential to be more rapid than other methods of detection which has also certain advantages for assessing the pathogenic potential of strains in environmental samples, where virulence determinant is important in

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ACCEPTED MANUSCRIPT assessing the risk of human illness. It also presents advantages in terms of speed of execution, lower cost and high throughput screening of pathogenic organisms. Regarding the detection of

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several genes in a single-tube PCR reaction, many multiplex PCR assays have been developed

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and are widely used as diagnostic tools for various pathogens such as E. coli, Staphylococcus, Clostridium difficile and Pasteurella multocida (Andrade et al., 2012; Antikainen et al., 2009;

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Franck et al., 1998; Furian et al., 2013; McClure et al., 2006). Current conventional single-target

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PCR assays require detection of each target VAG individually in the Arcobacter spp. (Douidah et al., 2012; Tabatabaei et al., 2014). These VAGs have not been used as a multiplex PCR assay

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by combining duplex or triplex set of VAG-specific oligonucleotide primer pairs that can specifically differentiate multiple target genes in a single-tube PCR amplification reaction.

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Therefore, this study sought to develop and evaluate an accurate, simple and rapid three multiplex PCR-based (mPCR) assays relative to the previously validated singleplex PCR assays

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that can simultaneously detect two or three major VAGs in a single-tube PCR assay. Specificity of the assays was evaluated using 145 Arcobacter isolates in order to provide an efficient tool for the screening of these genes in the Arcobacter spp.

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Materials and Methods 2.1.

Reference strain culture conditions and isolation of Arcobacter fecal cultures:

Sixteen strains of human and animal-associated Arcobacter spp. and 32 other bacterial reference species originally isolated from clinical and environmental sources obtained from the American Type Culture Collection (ATCC), National Collection of Type Cultures (NCTC) and Laboratory for Microbiology Gent (LMG) were used as positive and negative controls for this study to test

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ACCEPTED MANUSCRIPT the specificity of the multiplex PCR (mPCR) assays (Table 1). The reference strains were grown

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on selective growth media according to specified culture conditions.

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In addition, 145 Arcobacter culture isolates including the A. butzleri, A. cryaerophilus and A. skirrowii isolated from various human and animal fecal sources were tested for

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evaluation, validation and sensitivity of the developed mPCR assays. The cultures were isolated

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by diluting 1 g of fecal sample in 9 ml of peptone water (PW) using a ten-fold serial dilution approach. The suspension (100 µl) was directly plated on Arcobacter selective isolation agar

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(ASIA) (Oxoid, Nepean, ON) containing antibiotic (fluorouracil, amphotericin-B, cefoperazone, novobiocin and trimethoprim) supplements. The plates were incubated at 30°C under

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microaerophilic conditions (85% N2, 10% CO2 and 5% O2) for 3 to 6 days as described by Whiteduck-Léveillée et al. (2015). The putative Arcobacter cultures selected based on their

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growth characteristics and colony morphology were further purified on modified Agarised Arcobacter Medium (m-AAM) (Oxoid) containing selective antibiotic (cefoperazone, amphotericin-B and teicoplanin) supplements, and the plates were incubated according to the conditions mentioned above. The purified putative culture isolates were confirmed by colony morphology, Gram staining reaction and genus- and species-specific PCR assays (Douidah et al., 2010; Harmon & Wesley, 1996) using the DNA extracted by the method as described in the following section.

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Nucleic acid extraction: The extraction of genomic DNA from reference strains

and Arcobacter culture isolates recovered from fecal samples was performed by re-suspending a purified single colony in a sterile 1.5 mL microfuge tube containing 100 μL TE buffer (10 mM Tris-HCl and 1 mM EDTA, pH 8.0). The cells were gently mixed and boiled for 10 min as per 6

ACCEPTED MANUSCRIPT Khan et al. (2013). The tube was then centrifuged at high speed for 1 min and the supernatant containing purified DNA was further examined and quantified by agarose gel electrophoresis and

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an ND-1000 spectrophotometer using low range quantitative DNA marker (Fisher Scientific,

Development and optimization of mPCR amplification reactions: Three

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2.3.

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Ottawa, ON), respectively. The DNA extract was stored at -20°C for further PCR analyses.

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mPCR-based assays consisting of a combination of two or three VAGs were developed and optimized using oligonucleotide primers previously designed by Douidah et al. (2012). The

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duplex or triplex PCR primer pairs that have a similar thermal melting temperature (Tm) were selected in order to have minimal base pairing interactions with other primers in the reaction, and

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to yield products that differed from each other by approx. 60-100 bp, which could be resolved by agarose gel electrophoresis (Table 2A-C). The specificity of each PCR protocol was initially

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confirmed by setting up singleplex and mPCR amplification reactions, for all PCR reactions, with the A. butzleri ATCC 49616, Helicobacter pylori NCTC 11637 and Campylobacter jejuni ATCC 33291 reference strains used as positive and negative controls, respectively. Each mPCR amplification assay was performed in 25 μL of reaction mixtures containing 1 U of Ex-Taq DNA polymerase and the compatible PCR reagents including 1x buffer with MgCl2, 200 µM each of the dNTPs (Fisher Scientific, Nepean, ON), 0.1 µM of the each set of forward and reverse primer pair and 1 μL (1 to 100 ng μL-1) of extracted DNA template. The volume was adjusted with sterile distilled water to obtain 25 µL. The PCR reaction was performed in a Mastercycler Gradient PCR system (Eppendorf, Hauppauge, NY). In this study, three tubes for the simultaneous detection of eight target virulence genes using three sets of PCR primers (one set containing duplex and other two sets containing triplex PCR primer pairs each; Table 2A-C)

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ACCEPTED MANUSCRIPT using the same PCR reaction conditions. The PCR program consisted of an initial template denaturation step at 95 ºC for 4 min followed by 30 cycles of amplification (denaturation at 95

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ºC for 30 s, annealing at 56 ºC for 45 s and extension at 72 ºC for 45 s) ending with a 5 min final

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extension at 72 ºC. Due to expected small amplicon fragment sizes, amplified PCR products were analysed by gel electrophoresis on 2% agarose gel at 100 V for at least 90 min using 100 bp

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DNA size marker (Life Technologies, Grand Island, NY). The gels were stained in ethidium

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bromide (0.5µg mL-1), visualized on an ultraviolet (UV) transilluminator and photographed using

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an Alpha Imager (Fisher Scientific) gel documentation system.

The positive PCR amplicons of each target VAG were further sequenced in order to

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determine specificity of VAG present in the Arcobacter reference strains. Amplified PCR

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products were purified using the Qiaquick PCR purification kit (Qiagen, Toronto, ON, Canada)

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and electrophoresed on a 1.5% agarose gel to check for purity and band intensity. Sequencing reactions were performed as previously described (Whiteduck-Léveillée et al., 2015) using BigDye terminator chemistry and the amplified products were sequenced on an ABI 3130xL Genetic analyzer instrument (Applied Biosystems, Burlington, ON, Canada) according to the manufacturer’s recommendation. The sequence data were analyzed using a BLAST search against the global database to identify and confirm VAG in Arcobacter spp.

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Sensitivity and specificity of mPCR assays: The sensitivity of the mPCR assays

was tested using the 10-fold serial dilution (ranging from 100 ng µL-1 to 1 ng µL-1) of the DNA of A. butzleri ATCC 49616, A. cryaerophilus NCTC 11885 and A. skirrowii LMG 6621 prepared in 1xTE buffer. Each experiment consisted of triplicate tests with two replicates (each experiment with two replicates). Moreover, the specificity of each mPCR assay was determined 8

ACCEPTED MANUSCRIPT by examining the ability of the test to detect and distinguish the other Arcobacter spp. reference strains for the presence of eight virulence genes. In addition, 32 other bacterial species were used

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to evaluate the specificity of the newly developed mPCR assays (Table 1).

Evaluation of mPCR assays for detection of VAGs in field isolates: In parallel,

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the 145 fecal samples including 100 identified as the A. butzleri, 42 A. cryaerophilus and 3 A.

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skirrowii strains, from various human and animal fecal sources, were used for this study. The isolates were previously tested using standard conventional microbiology procedures and further

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confirmed by genus- and species-specific PCR assays as described above in order to not only confirm the newly developed mPCR assays, but also evaluate their performance. All DNA from

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the field samples were processed simultaneously by singleplex and mPCR assays to determine

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the comparative efficiency of the two methods.

Results 3.1.

Optimization of three multiplex PCR (mPCR) protocols: Initially, the reaction

conditions for three mPCR assays were optimized to ensure that all target gene sequences could be satisfactorily amplified, rendering amplicons easily visible on an agarose gel. First, to standardize the conditions of the assay each one of the eight genes (ciaB, cj1349, pldA, irgA, hecA, tlyA, mviN and hecB) was amplified alone, testing two annealing temperatures and primer concentrations using the A. butzleri ATCC 49616, Helicobacter pylori NCTC 11637 and Campylobacter jejuni ATCC 33291 reference strains as positive and negative controls (Fig. 1). After optimizing the primer concentration (0.1 µM) and annealing temperature (56°C) for each VAG, three mPCR assays were developed by using a combination of duplex (ciaB and cj1349),

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ACCEPTED MANUSCRIPT and triplex [(tlyA, mviN and hecB) and (pldA, irgA and hecA)] Arcobacter-specific VAG primer pairs. In each mPCR assay, the PCR amplification protocol and primer pairs used in the assay

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did not interfere with each other and generated expected amplification products of 284 and 659

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bp for the ciaB and cj1349; 230, 294 and 528 bp for the tlyA, mviN, hecB; 293, 437 and 537 bp for the pldA, irgA and hecA VAGs, respectively (Fig. 2A-C). All eight VAGs were detected in

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the A. butzleri reference strain with good efficiency in terms of intensity and expected size of

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amplified product of each VAG in each mPCR assay and the reaction conditions resulted in strong reproducible PCR amplicons of the predicted sizes. The eight PCR products could still be

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seen in the sample that contained a 103-fold-diluted (1 ng μL-1) DNA concentration. The PCR products of each mPCR assay had sizes different enough to be easily identified and resolve all

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the bands on 2% agarose gels (Fig. 1). Clear PCR products were obtained for duplex and triplex genes from A. butzleri reference strain DNA, indicating a lack of interference between any of the

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primer pairs or amplicons (Fig. 2A-C). Of the 15 other Arcobacter reference species tested, however, the A. cryaerophilus and A. skirrowii only showed positive amplification reactions for the ciaB, mviN and hecA; the A. lanthieri, A. molluscurum, A. trophiarum, A. venerupis and A. cibarius for ciaB; A. ellisii and the A. defluvii for the ciaB and mviN; and the A. bivalviorum for the mviN VAGs. On the other hand, none of the VAGs were detected in the A. nitrofigilis, A. marinus, A. mytili, A. halophilus and A. thereius reference strains (Fig. 2A-C). The specificity of the developed mPCR protocols was also tested on DNA templates prepared from the 32 different bacterial reference strains (Table 1). The VAGs were not detected in any of the non-target reference strains, indicating specificity of the targets for Arcobacter spp.

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ACCEPTED MANUSCRIPT The specificity of the assays were further confirmed by sequencing where all Arcobacter reference strains positive for target VAGs were confirmed with available sequence data analysis

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and showed sequence homology with the Arcobacter spp.

Evaluation of mPCR assays for detection of VAGs in Arcobacter cultures

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isolated from fecal samples: Considering the specific amplification ability of eight VAGs in the

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three developed mPCR assays, for validation purpose, this approach was further used for the detection and identification of VAGs in the A. butzleri (n= 100), A. cryaerophilus (n= 42) and A.

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skirrowii (n= 3) strains isolated from various fecal sources (Fig. 3). Of the total 145 isolates, overall, the ciaB (90%; n= 130) and mviN (77%; n= 112) were detected more commonly

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compared to the tlyA (50%; n= 73) and pldA (45%; n= 65) VAGs, respectively. However, the cj1349 (16%; n= 23) and hecB (10%; n= 15) were detected at a high relative frequency than each

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of the irgA and hecA (6%; n= 9) VAGs (Table 3). On the other hand, none of the eight VAGs were detected in 11 (8%) of the Arcobacter isolates tested in this study.

Analysis of the A. butzleri (n= 100) tested strains indicated that the ciaB (90%) and mviN (82%) were detected more frequently than the tlyA (67%), pldA (58%), cj1349 (20%), hecB (14%) and each of the irgA and hecA (9%) VAGs. Similar rate of frequency, in the A. cryaerophilus (n= 42) strains, was detected for the ciaB (90%) and mviN (69%) with a relatively low frequency of each of the tlyA and pldA (17%), cj1349 (7%) and hecB (2%) VAGs, respectively. However, none of the A. cryaerophilus strains were positive for the irgA and hecA VAGs. On the other hand, of eight VAGs, only the ciaB (100%) and mviN (67%) VAGs were detected in three strains of the A. skirrowii (Table 3). The results showed that multiple VAGs

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ACCEPTED MANUSCRIPT were present in the A. butzleri and A. cryaerophilus strains; however, none of the A. butzleri strains were positive for all eight VAGs. All the Arcobacter strains tested were previously

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screened for the presence of individual VAGs genes detected by using single PCR assay. There

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was a total correlation between the previous and current results for these genes obtained by the

Discussion

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single and mPCR assays described in this study.

Several studies have investigated adhesion capacity, invasiveness, and cytotoxicity of

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Arcobacter species. However, pathogenicity and virulence mechanisms, dose response, and toxin production in the development of disease are not fully understood (Ferreira et al., 2015).

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Therefore, Arcobacter spp. is considered a low public health risk due to the fact that the number of outbreaks and incidence are unknown and clinical samples are not routinely tested for

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Arcobacter spp. (Dhama et al., 2013). Nonetheless, recent studies demonstrated the presence of virulence-associated genes (VAGs) in multiple Arcobacter spp. (Collado et al., 2011; Douidah et al., 2012; Ho et al., 2006; Levican et al., 2013) making it perhaps a more important pathogen in the context of public health risk than initially believed. Nine putative Arcobacter virulence genes share similarities (ranging from 51% to 76%) with virulence genes of other bacteria (Miller et al., 2007). Hence, Arcobacter VAG-specific PCR assays were developed as an analytical tool for the detection of VAGs in Arcobacter spp. (Douidah et al., 2012), but these assays were designed to detect only a single gene at a time therefore requiring several such single PCRs for the detection of multiple genes which is labour intensive and time consuming. In comparison to the singleplex PCR assays, mPCR is a powerful tool in microbiology and has been widely applied to detect bacteria and genes of interest (Kong et al., 2002; Lehmann et al., 2008). The mPCR assay

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ACCEPTED MANUSCRIPT is rapid and simple for sample preparation. Moreover, mPCR has the potential to produce considerable savings of time and effort within the laboratory without compromising test utility.

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The mPCR assay has been successfully applied in many research areas including gene deletion

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analysis (Sbiti et al., 2002), mutation and polymorphism analysis (Cheng et al., 2004; FerrãoBeck et al., 2006), quantitative analysis (Abdeldaim et al. 2010; Weltia et al., 2003) and RNA

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detection (Gröndahl et al., Wang et al., 1999).

Considering the importance of mPCR assay, it was needed to develop more reliable and

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rapid identification system for Arcobacter spp. to detect multiple VAGs in a single-tube PCR assay. Therefore, this study describes the development and evaluation of three novel mPCR

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assays for rapid detection of eight major VAGs of Arcobacter spp. The VAGs included in the three mPCR assays, in this study, were selected because they are well characterized and have

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been shown to be prevalent in the Arcobacter butzleri (Douidah et al., 2012; Ferreira et al., 2014; Karadas et al., 2013). Specific and sensitive amplification of target gene sequences by mPCR are dependent on a number of key parameters such as primer and DNA template concentration, annealing temperature, extension time and the amount and quality of Taq polymerase used (Henegariu et al., 1997). Similarly, multiple aspects, in this study, were assessed to determine which component can offer maximum amplicon quality in terms of specific amplicon size and reaction intensity for rapid detection of the genes. Initially, different and equimolar primer concentrations (ranging from 0.1 to 0.4 µM) for each mPCR assay were used in order to assess the quality of amplified product and reproducibility of the assay for each target VAG. There was an even amplification results, with the same concentrations of 0.1 µM for each primer pair used in each mPCR assay, were observed. Secondly, the two annealing temperatures (55°C and 56°C)

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ACCEPTED MANUSCRIPT described by Douidah et al. (2012) along with other parameters were separately evaluated for all target VAGs, and the amplicon quality was compared to determine if only one annealing

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temperature can be used for all three mPCR assays. Our developed mPCR assays compared

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favourably with individual PCR assays in which single target genes were detected at 56°C with the same PCR band intensity following the optimized mPCR protocols. The results also

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demonstrated that the amplification of the two or three sets of primers was efficient for

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producing 8 clear bands in three mPCR assays. Furthermore, each mPCR assay was applied to 15 other Arcobacter reference species to detect occurrence of VAGs where it was observed that

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none of the Arcobacter spp., except the A. butzleri, showed positive amplification for all VAGs (Douidah et al., 2012). Although, the results are similar to the previously reported findings, but

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to our knowledge, this is the first study where a total of 13 strains of Arcobacter reference spp., other than A. butzleri, A. cryaerophilus and A. skirrowii, were tested for the detection and

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occurrence of VAGs using newly developed mPCR assays. In contrast to the A. butzleri, no other Arcobacter reference spp. showed positive amplification to all eight VAGs indicates that there might be heterogeneity in the genomes of other species that may lead to detect VAGs with less accuracy. It is also possible that the genus-specific VAGs primers developed by Douidah et al. (2012) are not specific to all Arcobacter spp. and may not amplify VAGs present in the other Arcobacter spp. (e.g., A. nitrofigilis, A. marinus, A. mytili, A. halophilus and A. thereius reference strains did not show amplification reactions for any VAGs tested). Therefore, there is a need to investigate and develop other genus- or species-specific VAGs-based primers and establish optimal detection protocol that can be used to accurately detect VAGs in all Arcobacter spp.

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ACCEPTED MANUSCRIPT The developed mPCR assays were further evaluated and validated using 145 cultures of the A. butzleri, A. cryaerophilus and A. skirrowii strains isolated from various fecal samples. In

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comparison to the three VAGs detected in the A. cryaerophilus and A. skirrowii reference strains,

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fecal isolates showed occurrence of six VAGs (ciaB, cj1349, tlyA, mviN, hecB and pldA) in the A. cryaerophilus and two (ciaB and mviN) in the A. skirrowii strains at various frequency similar

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to the results reported by (Douidah et al., 2012; Ferreira et al., 2014; Tabatabaei et al., 2014).

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Similarly, our results for validation of the developed assays for the detection and occurrence of eight VAGs in the A. butzleri are in agreement with other findings (Douidah et al., 2012; Ferreira

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et al., 2014; Karadas et al., 2013; Tabatabaei et al., 2014; Zacharow et al., 2015) where each VAG was detected at a variable frequency. This validation of mPCR assays indicated that each

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assay was capable of simultaneously detecting and differentiating multiple VAGs in the Arcobacter spp. In addition, these assays can be applied to characterize pathogenic isolates that

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might play a pivotal role in the epidemiological investigation of disease, generating the information necessary for identifying, tracking, and intervening against disease outbreaks.

Another potential advantage of the three mPCR assays developed in this study is that they have a significant level of sensitivity. Each mPCR assay, in this study, showed that a least detection limit of 1 ng μL-1 of DNA concentration can simultaneously detect all target VAGs in different strains of the Arcobacter spp. Although the infectious dose varies among pathogen types, it is generally believed that most bacterial pathogens are able to cause infection when more than 103 CFU mL-1 are ingested (US EPA, 1992). Thus, the detection sensitivity of the mPCR assays described in this study is within the range of infectious dose of most pathogens.

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ACCEPTED MANUSCRIPT This study demonstrates the interesting potential for use of mPCR techniques to rapidly (<4 h) identify the presence of eight Arcobacter VAGs. Moreover, all three mPCR assays with

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the same PCR protocol specific to each VAG, with minimized risk of cross-amplification with

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other primer pairs, are potentially adaptable as a rapid and high-throughput screening tool for the Arcobacter spp. The detection time could be further shortened by using real-time PCR format

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which would allow early detection of the amplicons as well as confirmation of each target VAG

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amplicon based on melting-curve analysis. Since the study results show that many Arcobacter strains isolated from fecal origin have multiple VAGs and thus have the potential to be

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pathogenic; therefore, additional evaluation including gene expression analysis is needed in order to completely determine the applicability of this approach to virulence factor determination and

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Acknowledgements

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that role in assessing the Arcobacter influences on human and animal health.

This study was funded by Agriculture and Agri-Food Canada (AAFC) (project # 1800). We also thank the field crew and coop students including Kerri Whiteduck-Léveillée, Cynthia A. Kaneza, Mark Sunohara and Nathalie Gagnon who have assisted with fecal sample collection and laboratory analysis.

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ACCEPTED MANUSCRIPT References: 1. Abdeldaim, G.M.K., Strålin, K., Korsgaard, J., Blomberg, J., Welinder-Olsson, C., Herrmann,

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B. 2010. Multiplex quantitative PCR for detection of lower respiratory tract infection and

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meningitis caused by Streptococcus pneumoniae, Haemophilus influenzae and Neisseria

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meningitides. BMC Microbiol. 10, 310.

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ACCEPTED MANUSCRIPT 32. Tabatabaei, M., Askia, H.S. Shayegh, H., Khoshbakht, R. 2014. Occurrence of six virulenceassociated genes in Arcobacter species isolated from various sources in Shiraz, Southern Iran.

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ACCEPTED MANUSCRIPT 39. Zacharow, I., Bystroń, J., Wałecka-Zacharska, E., Podkowik, M., Bania, J. 2015. Genetic diversity and incidence of virulence-associated genes of Arcobacter butzleri and Arcobacter

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http://dx.doi.org/10.1155/2015/956507

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ACCEPTED MANUSCRIPT Figure legends: Figure 1: Typical PCR amplicons of the Arcobacter butzleri ATCC 49616 strain obtained from

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eight individual Arcobacter-specific VAGs (ciaB, cj1349, tlyA, mviN, hecB, pldA, irgA and

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hecA) with expected sizes of 284, 659, 230, 294, 528, 293, 437 and 537 bp (lanes 1-8). Lanes 9-

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10: Helicobacter pylori NCTC 11637 and Campylobacter jejuni served as a negative control;

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Lane M: 100 bp DNA ladder size marker (GeneRuler; Life Technologies).

Figure 2: Typical PCR amplicons obtained for each multiplex PCR assay developed for the

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detection of VAGs in Arcobacter reference spp.: Panel A: ciaB (284 bp) and cj1349 (659bp); Panel B: tlyA (230 bp), mviN (294 bp) and hecB (528 bp); Panel C: pldA (293 bp), irgA (437

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bp) and hecA (537 bp). Lanes: (1) A. butzleri, (2) A. cryaerophilus, (3) A. skirrowii, (4) A. lanthieri, (5) A. bivalviorum, (6) A. nitrofigilis, (7) A. molluscorum, (8) A. ellisii, (9) A.

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trophiarum, (10) A. marinus, (11) A. mytili, (12) A. venerupis, (13) A. defluvii, (14) A. halophilus, (15) A. thereius and (16) A. cibarius. Lane 17: negative control with no template DNA; Lane M: 100 bp DNA ladder size marker.

Figure 3: Evaluation and confirmation of optimized multiplex PCR assays using oligonucleotide primers and PCR protocol specific for Arcobacter VAGs applied to the A. butzleri (Lanes 6, 1112), A. cryaerophilus (Lanes 7 and 10), and A. skirrowii (Lane 8) culture isolates recovered from different fecal sources. A. butzleri reference strain (Lanes 2-4) served as control positive for each multiplex PCR assay; Lanes 1, 5 and 9: negative control with no template DNA; Lanes M: 100 bp DNA ladder size marker.

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Figure 2

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ACCEPTED MANUSCRIPT Table 1: List of reference strains of Arcobacter and other bacterial species used in this study

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A. mytilii A. venerupis A. defluvii A. cibarius A. halophilus A. thereius A. skirrowii A. lanthieri A. cryaerophilus Aeromonas bv. veronii A. encheleia

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13. 14. A. 15. 16. 17. 18.

Source Human Diarrheic Stool Shellfish Roots Mussels and Oysters Mussels Feces of Fattening Pigs Mix seawater, Starfish and Seaweeds Mussels Shellfish Sewage Broiler Carcasses Hypersaline Lagoon

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Species Arcobacter butzleri A. bivalviorum A. nitrofigilis A. molluscorum A. ellisii A. trophiarum A. marinus

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Sr. #: 1. 2. 3. 4. 5. 6. 7.

19. 20. 21. 22. 23. 24. 25. 26.

A. veronii A. trota A. allosaccharophilia A. jandaei A. salmonicida A. bestiarum A. eucrenophilia A. popofii

27. 28. 29. 30.

A. hydrophila A. sobria A. schubertii A. caviae

31. A. hydrophila

Organs of Aborted Porcine Lamb Feces Pig Manure Bovine Aborted Foetus Amputation wound Healthy Juvenile Freshwater Eel Red-leg Frog Human Feces Diseased Elvers Human Feces Freshwater Infected Fish Human Drinking water production plant Ditch Water Sludge Skin Epizootic of Young Guinea Pigs River Water 27

Strain ID ATCC 49616 LMG 26154 ATCC 33309 LMG 25693 LMG 26155 LMG 25534 LMG 25770 LMG 24559 LMG 26156 LMG 25694 LMG 21996 ATCC BAA1022 LMG 24486 ATCC 51132 LMG 28516 NCTC 11885 ATCC 35625 ATCC 51929 ATCC 9071 ATCC 49658 ATCC 51208 ATCC 49568 CDC 0434-84 ATCC 51108 ATCC 11163 LMG BAA-243 ATCC 13444 ATCC 35994 ATCC 43700 ATCC 15468 ATCC 23211

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Marine Fish Environmental isolate Environmental isolate Environmental isolate Canine Human Feces Diarrheic Stool of Child Intestine of Swine Swine Cat Human Feces Human Feces Human Feces Human Feces

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A. media Pseudomonas shigelloides Escherichia coli O157:H7 E. coli O157:H7 E. coli Salmonella enterica subsp. houtenae S. enterica subsp. diarizonae S. enterica subsp. arizonae Campylobacter jejuni subsp. doylei C. jejuni subsp. jejuni C. hyointestinalis C. coli C. helveticus C. coli C. jejuni C. jejuni C. lari

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32. 33. 34. 35. 36. 37. 38. 39. 40. 41. 42. 43. 44. 45. 46. 47. 48.

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CDC 0435-84 ATCC 35218 ATCC 29932 ATCC 12325 ATCC 13314 ATCC 49349 ATCC 29428 ATCC 35217 ATCC 43136 ATCC 51210 ATCC BAA-371 ATCC 33291 ATCC 33292 ATCC 43675

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Table 2A: Oligonucleotide PCR primers for amplifications of ciaB and cj1349 virulenceassociated genes (VAGs) (Douidah et al., 2012) Target Oligonucleotide Length Product Sequences (5´-3´) genes primers (nt) size (bp) ciaB-F TGG GCA GAT GTG GAT AGA GCT TGG A 25 ciaB 284 ciaB-R TAG TGC TGG TCG TCC CAC ATA AAG 24 cj1349-F CCA GAA ATC ACT GGC TTT TGA G 22 cj1349 659 cj1349-R GGG CAT AAG TTA GAT GAG GTT CC 23

GTG GAA GTA CAA CGA TAG CAG GCT C GTC TGT TTT AGT TGC TCT GCA CTC

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Table 2B: Oligonucleotide PCR primers for amplifications of pldA, irgA and hecA VAGs (Douidah et al., 2012) Target Oligonucleotide Length Product Sequences (5´-3´) genes primers (nt) size (bp) pldA-F TTG ACG AGA CAA TAA GTG CAG C 22 pldA 293 pldA-R CGT CTT TAT CTT TGC TTT CAG GGA 24 irgA-F TGC AGA GGA TAC TTG GAG CGT AAC T 25 irgA 437 irgA-R GTA TAA CCC CAT TGA TGA GGA GCA 24 25 24

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Table 2C: Oligonucleotide PCR primers for amplifications of tlyA, mviN and hecB VAGs (Douidah et al., 2012) Target Oligonucleotide Length Product Sequences (5´-3´) genes primers (nt) size (bp) tlyA-F CAA AGT CGA AAC AAA GCG ACT G 22 tlyA 230 tlyA-R TCC ACC AGT GCT ACT TCC TAT A 22 mviN-F TGC ACT TGT TGC AAA ACG GTG 21 mviN 294 mviN-R TGC TGA TGG AGC TTT TAC GCA AGC 24 hecB-F CTA AAC TCT ACA AAT CGT GC 20 528 hecB hecB-R CTT TTG AGT GTT GAC CTC 18

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Isolates tlyA 66 (66) 7 (17)

89 (89)

A. cryaerophilus

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38 (90)

3 (7)

A. skirrowii

3

3 (100)

0 (0)

145

130 (90)

23 (16)

hecB

pldA

irgA hecA

14 (14)

58 (58)

9 (9)

9 (9)

29 (69)

1 (2)

7 (17)

0 (0)

0 (0)

0 (0)

2 (67)

0 (0)

0 (0)

0 (0)

0 (0)

73 (50)

112 (77)

15 (10)

65 (45)

9 (6)

9 (6)

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A. butzleri

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mviN

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ciaB

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No. (%) of isolates positive for VAGs Species

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ACCEPTED MANUSCRIPT Highlights: 1. Arcobacter, virulence-associated genes (VAGs) and multiplex PCR assay development

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2. These Assays are useful markers for rapid screening of 8 VAGs in Arcobacter spp. 3. The developed assays are specific, sensitive and economical for high-throughput analysis.

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4. The assays can help in identifying, tracking, and intervening against disease outbreaks.

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