Genetic diversity of Arcobacter isolated from bivalves of Adriatic and their interactions with Mytilus galloprovincialis hemocytes

Abstract The human food‐borne pathogens Arcobacter butzleri and A. cryaerophilus have been frequently isolated from the intestinal tracts and fecal samples of different farm animals and, after excretion, these microorganisms can contaminate the environment, including the aquatic one. In this regard, A. butzleri and A. cryaerophilus have been detected in seawater and bivalves of coastal areas which are affected by fecal contamination. The capability of bivalve hemocytes to interact with bacteria has been proposed as the main factor inversely conditioning their persistence in the bivalve. In this study, 12 strains of Arcobacter spp. were isolated between January and May 2013 from bivalves of Central Adriatic Sea of Italy in order to examine their genetic diversity as well as in vitro interactions with bivalve components of the immune response, such as hemocytes. Of these, seven isolates were A. butzleri and five A. cryaerophilus, and were genetically different. All strains showed ability to induce spreading and respiratory burst of Mytilus galloprovincialis hemocytes. Overall, our data demonstrate the high genetic diversity of these microorganisms circulating in the marine study area. Moreover, the Arcobacter–bivalve interaction suggests that they do not have a potential to persist in the tissues of M. galloprovincialis.


| INTRODUCTION
The genus Arcobacter has become increasingly important in recent years because species such as A. butzleri and A. cryaerophilus are considered emergent food-borne enteropathogens (Collado & Figueras, 2011). Arcobacter butzleri, in particular, has been classified as a serious hazard to human health by the International Commission on Microbiological Specifications for Foods (ICMSF 2002) and a significant zoonotic pathogen (Cardoen et al., 2009). In Europe, A. butzleri was often recovered from samples of patients with diarrhea in Belgium, France, and Italy (Prouzet-Mauleon, Labadi, Bouges, Menard, & Megraud, 2006;Vandamme et al., 1992;Vandenberg et al., 2004).
Moreover, the persistence of microorganisms in bivalves can be species or strain dependent (De Abreu Corrìa et al., 2007;Maalouf et al., 2011;Morrison et al., 2011). This is not surprising considering that phagocytosis requires specific surface interactions with bacteria/bivalve (Canesi et al., 2002). Phagocytic efficiency of bivalve hemocytes toward foreign particles can be assessed by studying the hemocyte ability to spread toward them (Malagoli & Ottaviani, 2004;Mosca, Narcisi, Cargini, Calzetta, & Tiscar, 2011;Mosca, Lanni et al., 2013; and degrade these by producing reactive oxygen species (ROS). This latter activity is defined respiratory burst (Gunderson & Seifert, 2015;Ordàs, Novoa, & Figueras, 2000;Versleijen et al., 2008). To our knowledge, only one previous work studied the dynamic of interaction of A. butzleri and bivalves showing that the type strain A. butzleri LMG 10828 T did not persist in Mytilus galloprovincialis (Ottaviani, Chierichetti et al., 2013). In order to acquire preliminary information about potential persistence of Arcobacter spp.
in host tissues, it is important to investigate the circulation of human pathogenic species in different marine environments and possible inter-and intraspecies differences in the interaction with the bivalves that are more commonly harvested in those areas. Despite this, reports on the genetic characterization of arcobacters isolated from the marine environment and pathogen-bivalve immunologic interaction are scarce (Collado, Jara, Vásquez, & Telsaint, 2014;Levican et al., 2014;Ottaviani, Chierichetti et al., 2013). In this study Arcobacter strains, isolated from bivalves collected from a restricted harvesting area of the Central Adriatic Sea of Italy, were molecularly identified and typed by enterobacterial repetitive intergenic consensus PCR (ERIC-PCR) analysis. Moreover, spreading activity and respiratory burst induced by these isolates on hemocytes of M. galloprovincialis, the most common bivalve harvested in the Central Adriatic Sea, were investigated. for 48 hr in aerobic conditions, 0.2 ml of the broth were inoculated by passive filtration with Millipore filters (0.45 μm, 47 mm) onto blood agar plates (trypticase soy agar supplemented with 5% sheep blood) and incubated for 48-72 hr at 30°C under aerobic conditions.

| Identification and molecular characterization of Arcobacter isolates
Presumptive colonies biochemically identified as Arcobacter spp.  were tested by PCR for the rpoB gene of Arcobacter spp.  with primers from Korczak et al. (2006). In brief, 4-5 colonies from each isolate were suspended in 500 μl of sterile distilled water and denatured at 95°C for 10 min, then centrifuged at 13.000 rpm for 1 min. The supernatant was re- and Milli-Q water. The PCR consisted of an initial denaturing at 94°C for 5 min followed by 40 cycles of 94°C for 1 min, 25°C for 1 min, and 72°C for 2 min, with a final extension at 72°C for 5 min. Arcobacter butzleri LMG 10828 T was used as the reference strain in all molecular reactions.

| Experimental animals and hemolymph collection for phagocytosis assays
Each phagocytosis assay was performed on hemolymph pooled from 10 organisms of M. galloprovincialis (40-50 mm shell length) harvested from the same coastal area from which bivalves sampling was carried out. Moreover, each phagocytosis assay was repeated five times on five different lots of mussels, monthly sampled from January to May 2013. The organisms were maintained in a closed recirculating system, equipped with a filtering apparatus and air supply. A multiparameter probe (mod. YSI 556; Technosea) was used to monitor the temperature (mean 18.1 ± 0.6°C), salinity (mean 33.1 ± 0.2‰), and dissolved oxygen (DO) (mean 6.8 ± 0.3 mg/L). The mussels were acclimated for 48 hr at least before the experiment and no feed was administered.
The hemolymph was extracted from the posterior adductor muscle, it was pooled and the cell concentration was adjusted to 1 × 10 6 /ml in ice-cold artificial sterile seawater (ASS). The pool was then subdivided into two aliquots for phagocytosis assays. For both assays, cell wall of Saccharomyces cerevisiae (Zymosan A; Sigma-Aldrich) was used as the gold standard for its very high ability to stimulate phagocytosis (Malagoli & Ottaviani, 2004;Mosca 2013a,b;Ordàs et al., 2000).
Moreover, not stimulated hemolymph represented the basal level. For the experiments on hemocytes, the Arcobacter strains, grown in tryptone soy broth (Oxoid) at 30°C for 48 hr, were centrifuged at 5000g for 20 min at 4°C. Pellet was resuspended in phosphate buffer saline (PBS, 10% w/v) and adjusted to the concentration of 5 × 10 9 CFU/ml after optical measurement (5 × 10 8 CFU/ml gave approximately 0.5 OD600 nm).

| Spreading activity
The spreading activity of hemocytes was investigated, evaluating changes in hemocyte shape from a rounded (inactive) to an ameboid (active) form. In particular was measured the shape factor (SF), a pure number quantifying the degree to which a cell deviates from circularity, by using a video imaging system integrated into a computerized analyzer of cells in suspension (Cell Viability Analyzer Beckman Coulter) (Malagoli & Ottaviani, 2004). An aliquot of each hemolymph sample was incubated for 30 min with bacterial suspension, corresponding to bacteria to hemocytes ratio of 80:1 (Mosca et al., 2011). Ninety μl of the mixture was then placed on a microscope slide within a chamber delimited by a vaseline ring. A coverslip was placed over the slide in order to partially cover the chamber and, following F I G U R E 1 Geographical location of sampling areas in the Central Adriatic coast of Italy (Marches Region): blue and red sites correspond to Mytilus galloprovincialis and Chamelea gallina sampling areas, respectively hemocyte adhesion (time 0), 10 μl of 10 1 μmol/L N-formyl-Meth-Leu-Phe (fMLP) was added to the uncovered part of the chamber.
The digital image of the hemocytes was acquired after 20 min and the parameters for SF evaluation, that is, perimeter length and area, were automatically obtained by electronically tracing the edges of the cell. The assay was assayed in parallel with Zymosan A using a yeast to hemocytes ratio of 80:1 and untreated hemolymph (Mosca et al., 2011). Global results from five independent experiments for each test and control group were expressed as SF mean values ± standard deviation (SD).

| Hemocyte respiratory burst
Luminol-enhanced chemiluminescence (CL) assay has been widely used to measure the production of ROS in bivalve hemocytes (Gunderson & Seifert, 2015;Mosca et al., 2011;Mosca, Lanni et al., 2013;Ordàs et al., 2000;Versleijen et al., 2008). The measurement of the luminescence intensity (y-axis) versus time (x-axis) generates a positive polynomial curve. So, the results can be reported as integral value of the area under curve (AUC) (Gunderson & Seifert, 2015;Versleijen et al., 2008). For CL assay, hemolymph samples were placed in triplicate in 96-well microplates. Following 15 min incubation with Luminol (Sigma Aldrich) at 1 mmol/L, the hemocytes were stimulated to phagocytosis with Arcobacter suspensions (bacteria to hemocytes ratio of 80:1) and Zymosan A (zymosan to hemocytes ratio of 80:1). The chemiluminescence intensity by microplate reader (Sinergy H1; Bio-Tek) was detected for 60 min by plotting the curve of the counts per min and then calculating the AUC by integration (Gunderson & Seifert, 2015;Versleijen et al., 2008). Global results from five independent experiments for each test and control group were expressed as AUC mean values ± standard deviation (SD).

| Statistical analysis
For the analysis of ERIC-PCR electrophoresis products, dendrograms were constructed by the use of the Dice coefficient (Dice, 1945) and the unweighted-pair group method using arithmetic averages (software Treeconw version: 1.3b, Copyright © Yves Van de Peer University of Konstanz). Genetic relatedness between the PCR electrophoresis products of the strains was interpreted according to the method of Tenover et al. (1995). Thus, isolates were designated as indistinguishable, closely related; possibly related, and different with 0, 1-3, 4-6, >7 band differences, respectively (Tenover et al., 1995). Clusters were defined on the basis of the 80% similarity cutoff . In the phagocytosis assays, the statistical significance of differences between means was determined by one-way ANOVA for paired samples (each strain against each other and each control). The level for accepted significance was p < .01.
Seven strains (58%) were isolated from C. gallina and 5 (42%) from M. galloprovincialis (Table 1). Six strains (50%) were isolated in April, 2 (17%) in each month of January and March, and 1 (8%) in each month of February and May (Table 1). However, the number of strains is too low to assess whether differences of prevalence are significant.

| Immune response
All data of spreading activity are summarized in Moreover, no significant intra-and interspecies differences were observed among strains. Nevertheless, the stimulation with Zymosan A induced AUC value significantly higher compared to those induced by Arcobacter strains (Table 2).

| DISCUSSION
Arcobacter species have been frequently isolated from seawater of the Mediterranean area (Fera et al., , 2010Gugliandolo et al., 2008;Maugeri et al., 2004), where they may survive for a long time (Collado et al., 2008). These results suggest that the marine environment, thus, the marine organisms used as a food, particularly bivalves, may represent a potential reservoir of Arcobacter for infection (Collado & Figueras, 2011). In agreement with previous studies carried out in different geographical areas, this study confirms that potentially pathogenic arcobacters are frequently found in bivalve samples and A. butzleri is the most prevalent species Levican et al., 2014;Mottola et al., 2016;Nieva-Echevarria et al., 2013). Previous studies in Spanish marine areas reported A. butzleri and A. cryaerophilus as the most prevalent species isolated from mussels and clams, respectively , while, with respect to the seasonality, A. butzleri predominated from June to October and A. cryaerophilus from January to May (Levican et al., 2014). In this study, in the monitored Adriatic Coast of Central Italy, A. butzleri was more frequently isolated in clams than in mussels and A. cryaerophilus more in mussels rather than in clams. Moreover, A. butzleri and A. cryaerophilus were isolated throughout the period of the study, from January to May. This study period covers even the coldest months of the year for this geographical area, that is, January-March when water temperatures ranges, usually, between 10 and 14°C. Our ERIC-PCR results demonstrated that different strains of A. butzleri and A. cryaerophilus circulated in the restricted marine environment object of the study in January-May 2013. This high variability of Arcobacter strains circulation is in agreement with recent studies from Chile and Spain Levican et al., 2014). Unfortunately, the short period of investigation allowed us to isolate a number of strains too limited to make any statistical evaluation of these data. For this reason, in a future investigation, we intend to extend the period of study in order to assess if in this marine ecosystem differences of isolation  (Balbi et al., 2013;Canesi et al., 2001Canesi et al., , 2002Parisi et al., 2008;Pruzzo et al., 2005). Therefore, the elucidation of the interactions between pathogenic bacteria and hemocytes is important to explain their clearance in bivalve tissues and to predict the consequent efficiency of their elimination by depuration strategies (Pruzzo et al., 2005). The functional approach used in this study to evaluate pathogen-bivalve interactions demonstrates that all A. butzleri and A. cryaerophilus strains, including A. butzleri type strain, induced phagocytic answers of hemocytes significantly higher with respect to the basal level and spreading activity not significantly different from that of Zymosan. Zymosan is a highly concentrated immunogenic compound represented by the cell wall of Saccharomyces cerevisiae and, for this, it is usually used as the gold standard in phagocytosis assays. On the contrary, our Arcobacter strains were assayed without any treatment that could concentrate their antigenic components. This could explain the higher respiratory burst induced by Zymosan rather than that induced by strains. These results are consistent with bioaccumulation experiments of A. butzleri LMG 10828 in M. galloprovincialis that demonstrated how Arcobacter type strain was quickly removed from the host tissues (Ottaviani, Chierichetti et al., 2013). To the light of these findings it can be assumed for these Arcobacter strains a rapid clearance from the M. galloprovincialis tissues. In future investigations, to fully elucidate how bacteria-bivalve interactions develop and persist in natural condition, it is our intention also to consider the environment in which these interactions occur and to evaluate its influence on both the expression of bacterial cell properties and bivalve health status. However, to our knowledge, this study, even with these limitations, represent the first that tested, at the laboratory scale, the Arcobacter-mussel interactions by studying the phagocytic capability of M. galloprovincialis hemocytes toward A. butzleri and A. cryaerophilus strains, thus providing preliminary information to predict the efficiency of their elimination by host tissues and the consequent impact on human health.

| CONCLUSIONS
Different strains of human pathogenic A. butzleri and A. crioaerophilus were found in M. galloprovincialis and C. gallina harvested from a restricted marine environment of the Central Adriatic Sea of Italy between January and May 2013. However, the interaction of these Arcobacter strains with M. galloprovincialis hemocytes suggests that they do not have a potential to persist in mussels tissues and therefore they could be efficiently removed with the conventional purification practices.

ACKNOWLEDGMENTS
This work was funded by a research project (IZSUM 10/2011) from the Italian Ministry of Health.