Italian Journal of Food Safety 2015; volume 4:4583

Detection of Arcobacter spp. 2014). Currently, the genus includes 18 char- acterized species (Levican et al., 2014; Nieva- Correspondence: Anna Mottola, Dipartimento di in Mytilus galloprovincialis Echevarria et al., 2013), among them, Medicina Veterinaria, Università di Bari, Strada samples collected from Arcobacter butzleri, Arcobacter cryaerophilus, provinciale per Casamassima km. 3, 70010 Apulia region and Arcobacter skirrowii are considered as Valenzano (BA), Italy. potential emerging food borne enteropa - Tel. +39.080.5443853 - Fax: +39.080.5443853. E-mail: [email protected] Elisabetta Bonerba,1 Anna Mottola,1 thogens (Levican and Figueras, 2013) and Antonio Parisi,2 Angela Di Pinto,1 have been associated with human and animal Key words: Arcobacter butzleri, Mytilus gallopro- disease (Tabatabei et al., 2014; Levican et al., Andrea Serraino,3 Giancarlo Bozzo,1 vincialis, Foodborne pathogen. 2014; Suelam, 2012). A. butzleri has been clas- Federica Giacometti,3 Edmondo Ceci,1 sified as a serious hazard to human health by Conflict of interests: the authors declare no Giuseppina Tantillo1 the International Commission on potential conflict of interests 1Dipartimento di Medicina Veterinaria, Microbiological Specifications for Foods Received for publication: 17 July 2014. Università di Bari, Valenzano (BA); (ICMFS, 2002) and as a significant zoonotic Accepted for publication: 14 October 2014. 2Istituto Zooprofilattico Sperimentale pathogen (Cardoen et al., 2009). Moreover, A. della Puglia e Basilicata, Putignano (BA); butzleri has been recognised as a cause of This work is licensed under a Creative Commons 3Dipartimento di Scienze Mediche traveller’s diarrhea (Jiang et al., 2010). Attribution 3.0 License (by-nc 3.0). Veterinarie, Alma Mater Studiorum - Potential routes of Arcobacter spp. infection ©Copyright E. Bonerba et al., 2015 Università di Bologna, Ozzano Emilia in human may be associated to the consump- tion and/or manipulation of contaminated raw Licensee PAGEPress, Italy (BO), Italy Italian Journal of Food Safety 2015; 4:4583 or poorly cooked food of animal origin (Collado doi:10.4081/ijfs.2015.4583 and Figueras, 2011; Gonzales and Ferrús, 2011; Hausdorf et al., 2011; Nieva-Echevarria et al., Abstract 2013). Furthermore, these bacteria are mem- bers of seawater microbiota, wastewater and 6887-3 standard procedure (ISO, 2003). For drinking water reservoirs (Collado et al., each sample, 10 g of meat and intervalvar liq- The aim of the study was to evaluate the 2008). Studies carried out by Fera et al. (2004) uid were homogenized with 90 mL (1:10, occurrence of Arcobacter spp. in 20 samples of suggest that A. butzleri arrives in seawater wt/vol) of Arcobacter enrichment broth supple- Mytilus galloprovincialis purchased at fish through polluted freshwater and that this mented with Cefoperazone, Amphotericin B markets in Apulia region. The detection of organism survives in the marine environment and Teicoplanin (CAT) (selective supplement Arcobacter spp. was performed, after selective by adhering to zooplankton. SR0174E; Oxoid, Basingstoke, UK) in stom- enrichment, on modified charcoal cefopera- Bivalve mollusks, due to their ability to con- acher bags. The bags were closed and incubat- zone deoxycholate (mCCD) agar supplement- centrate microorganisms from contaminated ed at 30°C under aerobic conditions for 48 h, ed with Cefoperazone, Amphotericin B and water during their filter-feeding activities, are and then 200 µL of the broth was inoculated by Teicoplanin (CAT). In 6 out of the 20 tested considered as an important health risk, passive filtration onto modified Charcoal samples the presence of Arcobacter spp. was because they are often eaten poorly cooked Cefoperazone Deoxycholate Agar (mCCDA) found and confirmed by genus-based poly- and/or raw (Collado et al., 2009; Levican et al., supplemented with CAT selective supplement, merase chain reaction. All the isolates were 2014; Ottaviani et al., 2013). Despite this following the procedure described by Collado et identified as belonging to the species important risk, worldwide only a few surveys al. (2009). Subsequently, presumptive Arcobacter butzleri using 16S rDNA sequenc- investigated the presence of Arcobacter spp. in Arcobacter colonies (small colourless or beige ing and BLAST online. The results represent these products. In Italy, the occurrence of to off-white, translucent, convex with an entire the first report in Italy of A. butzleri detection Arcobacter spp. in marketed shellfish has not edge, Gram negative) were selected from each in marketed Mytilus galloprovincialis. The sur- been investigated yet; only Maugeri and col- plate and transferred to blood agar at least vey underlines the epidemiological importance leagues (2000) detected A. butzleri and A. three times to obtain pure cultures. Purified of A. butzleri as an emerging pathogen, and nitrofigilis in water and mussels collected from isolates were further subjected to biochemical highlights that mussels should be considered two brackish lakes near Messina, but the iso- analysis (catalase, oxidase and urease tests), as a potential cause of foodborne disease out- lates were characterized only phenotypically. microscopic examination, and genus-specific break. The purpose of this study was to evaluate polymerase chain reaction (PCR). the presence of Arcobacter spp. in Mytilus gal- loprovincialis sampled at retail in Apulia DNA extraction and purification region (Italy). DNA was extracted by using DNeasy Blood & Introduction Tissue Kit (Qiagen, Valencia, CA, USA). Briefly, bacterial pellet was added to 50 µL ATL lysis Arcobacter spp. was proposed as a new buffer and 5.56 µL of Proteinase K (20 mg/mL) genus in 1991 by Vandamme and De Ley who Materials and Methods and incubated at 56°C for 2 h. After adding 55.6 defined it as aerotolerant campylobacter. This µL AL buffer and 55.6 µL ethanol, the resulting genus belongs to the class Sampling and sample processing mixture was applied to the DNeasy Mini spin Epsilonproteobacteria and to family A total of 20 Mytilus galloprovincialis sam- column. The DNA, adsorbed onto the QIAamp Campylobacteraceae (Phillips, 2001; Levican et ples of average size (5±7 cm length) were col- silica-gel membrane during subsequent cen- al., 2014). Arcobacter are rod, gram negative, lected between January and April 2014 from trifugation steps at 6000 g for 1 min, was microaerophilic, non-spore forming, motile, local fish markets of Apulia region, Italy. Each washed using 140 µL AW1 and 140 µL AW2 curved and occasionally straight organisms sample was individually packaged and kept in washing buffers. Finally, the DNA was eluted which can grow between 15 and 39°C coolers. Mussels were aseptically prepared for with 50 µL AE Elution Buffer (Qiagen). The type (González and Ferrús, 2011; Tabatabaei et al., analysis in accordance with the UNI EN ISO strains of A. butzleri (ATCC 49616) was used as

[Italian Journal of Food Safety 2015; 4:4583] [page 23] Article positive control. A negative extraction control Montage PCR filter units (Millipore, Billerica, Biomolecular analysis (no added tissue) was included to verify the MA, USA). Sequence reactions were carried The isolates were confirmed as Arcobacters purity of the extraction reagents. The DNA con- out using BigDye 3.1 ready reaction mix by genus-based PCR. Polymerase chain reac- centration and purity were established by eva- (Applied Biosystems, Carlsbad, CA, USA) tions performed on each bacterial pellet sam- luating the ratio A260 nm/A280 nm using a according to the manufacturer’s instructions. ples gave positive results for Arcobacter Beckman DU-640B spectrophotometer. The sequenced products were separated with a species in 6/10 (Table 1). Sequence analysis of 3130 Genetic Analyzer (Applied Biosystems). the amplified 16S rDNA revealed that all Oligonucleotide primers Sequences were imported and assembled with The oligonucleotide primers ARCOI (5’-AGA Arcobacters isolates have a complete (100%) the Bionumerics 7.1 software (Applied Maths, homology with A. butzleri. GAT TAG CCT GTA TTG TAT C-3’) and ARCOII Sint-Martens-Latem, Belgium) and submitted (5’-TAG CAT CCC CGT TTC GAA TGA-3’) report- to BLAST searches in GenBank (Altschul et al., ed by Harmon and Wesley (1996) and synthe- 1990) sized by Primm Srl (Milan, Italy) were used. Discussion Polymerase chain reaction assay The PCR reactions were performed in a final Results This is the first report of A. butzleri detec- volume of 25 µL, using 12.5 µL of HotStarTaq tion in Mytilus galloprovincialis marketed in Master Mix 2X (Qiagen), containing 2.5 units Microbiological analysis Apulia region. However, since only 20 samples of HotStarTaq DNA polymerase, 1.5 mM of Based on the phenotypic cultural character- were analysed, the reported results should be MgCl and 200 µL of each dNTP. Then, 1 µM of 2 istic, the morphological examination by the interpreted only as preliminary data and each oligonucleotide primer and 1 µL of DNA Gram staining, and the biochemical analysis require further sampling and analytical inves- were added. The amplification profile involved performed on each sample analysed, a total of tigations to determine the prevalence of an initial denaturation step at 95°C for 5 min, 10/20 presumptive Arcobacter species were Arcobacter spp. in Italian marketed mussels. followed by 30 amplification cycles. Each isolated by mCCDA supplemented with CAT The importance of the genus Arcobacter is amplification cycle consisted of 30 s at 94°C, (Table 1). The colonies showing white to due to some species defined as emerging 30 s at 51°C and 60 s at 72°C. A primer exten- whitish-grey, small (2-4 mm) diameter, convex enterophatogens and potential zoonotic sion step (72°C for 10 min) followed the final and opaque with entire edge, smooth, trans- agents. The current state-of-the-art on the amplification cycle. The positive and negative parent/translucent were suspected as transmission of Arcobacters to human sug- controls for the extraction were included. The Arcobacter spp. colonies. Moreover, oxidase gests that the potential routes are represented PCR reactions were processed in a and catalase positive and urease negative test by food and water contaminated. Mastercycler Personal (Eppendorf, Hamburg, revealed presumptive Arcobacter spp. isolates. Among Arcobacter spp. isolated from food Germany). All reactions were performed in duplicate. Detection of amplified products Polymerase chain reaction amplified prod- Table 1. Prevalence of Arcobacter butzleri isolated by cultural method. ucts were analyzed by electrophoresis on 1.5% (w/v) agarose NA (Pharmacia, Uppsala, Samples Positive samples Positive samples Species detected by Sweden) gel in 1X TBE buffer containing 0.089 on cultural media on genus-based PCR (n) sequencing M Tris, 0.089 M boric acid, 0.002 M EDTA, pH 1 - - 8.0 (USB, Cleveland, OH, USA), and stained 2 - - with Green gel Safe Nucleic Acid Stain, 10,000 3 + + A. butzleri X in water (Fisher Molecular Biology, Rome, Italy). A Gene Ruler™ 100 bp DNA Ladder Plus 4 + + A. butzleri (MBI Fermentas, Vilnius, Lithuania) was used 5 + + A. butzleri as the molecular weight marker. Image acqui- 6 - - sition was performed using UVITEC 7 + + A. butzleri (Eppendorf). 8 + - Polymerase chain reaction amplifi- 9 - - cation and sequencing of 16S rDNA 10 + - The universal 16S rDNA primers, forward 11 - - primer 8F (5AGTTGATCCTGGCTCAG3′), and 12 - - reverse primer 1492R (5ACCTTGTTAC- 13 - - GACTT3′) were used for PCR. The PCR ampli- fication was carried out in a reaction mixture 14 + + A. butzleri containing ~10 ng genomic DNA as template 15 - - in 25 mL reaction volumes containing 10 pmol 16 + - of each primer, 0.2 mM of each dNTP’s, 1 U 17 - - HotMaster Taq (Eppendorf), 2.5 mL of 10X 18 + + A. butzleri HotMaster Taq Buffer (Eppendorf). The reac- 19 - - tion conditions were an initial denaturation at 94°C for 2 min, followed by 35 cycles at 94°C 20 + - for 30 sec, 52°C for 1 min and 72°C for 1 min. Total 10 6 The PCR products were purified using PCR, polymerase chain reaction.

[page 24] [Italian Journal of Food Safety 2015; 4:4583] Article and water, A. butzleri is the most prevalent might be an important health risk when con- 57:447-52. species, followed by A. cryoaerophilus. sidering that these products are traditionally Ho HT, Lipman LJ, Gaastra W, 2006. Arcobacter, Arcobacter spp. have been frequently isolated eaten poorly cooked and/or raw. An effective what is known and unknown about a from products of animal origin (chicken, fol- national food control system is essential to potential foodborne zoonotic agent. Vet lowed by pork, beef meat, raw milk and dairy protect the health and safety of consumers, but Microbiol 115:1-3. products, lamb) (Ho et al., 2006; Fernandez et it must be accompanied by extensive research ICMFS, 2002. Microorganisms in food. al., 2001; Maugeri et al., 2000; Giacometti et. on emerging pathogens. Microbiological testing in food safety man- al., 2013). Recently Arcobacter spp. were agement. International Commission on detected in fresh vegetables (Gonzales and Microbiological Specifications for Foods- Ferrús, 2011), but these foods are generally Kluwer Academic Publ., New York, NY, considered as safe and Arcobacter contamina- References USA. tion levels seem to be rather lower than animal ISO, 2003. Microbiology of food and animal food products and waters. Arcobacters have Altschul SF, Gish W, Miller W, Myers EW, feeding stuffs. Preparation of test samples, been isolated from environmental waters, Lipman DJ, 1990. Basic local alignment initial suspension and decimal dilutions including surface waters, ground waters, search tool. J Mol Biol 215:403-10. for microbiological examination. Part 3: rivers, lakes, sea water, sewage and from Cardoen S, Van Huffel X, Berkvens D, Quoilin specific rules for the preparation of fish planktons (Fera et al., 2004; Collado et al., S, Ducoffre G, Saegerman C, Speybroeck and fishery products. ISO Norm 6887- 2008; Ghane, 2014). In Italy, Fera et al. (2004) N, Imberechts H, Herman L, Ducatelle R, 3:2003. International Standardization reported the detection of these microrganisms Dierick K, 2009. Evidence-based semi- Organization ed., Geneva, Switzerland. in seawater and plankton samples collected quantitative methodology for prioritization Jiang ZD, Dupont HL, Brown EL, Nandy K, from the Strait of Messina. Collado et al. of foodborne zoonoses. Foodborne Pathog Ramamurthy T, Sinha A, Ghosh S, Guin S, (2008) confirmed the association of Arcobacter Dis 6:1083-96. Gurleen K, Rodrigues S, Chen JJ, with faecally polluted waters. Collado L, Figueras MJ, 2011. Taxonomy, epi- McKenzie R, Steffen R, 2010. Microbial Comparing data on the rates of prevalence demiology and clinical relevance of the etiology of travelers’ diarrhea in Mexico, of the Arcobacters, isolated from different genus Arcobacter. Clin Microbiol Rev Guatemala, and India: importance of sources, is very difficult. 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