1491 Journal of Food Protection, Vol. 72, No. 7, 2009, Pages 1491–1495 Copyright ᮊ, International Association for Food Protection Research Note Specific PCR Detection of Arcobacter butzleri, Arcobacter cryaerophilus, Arcobacter skirrowii, and Arcobacter cibarius in Chicken Meat DANIELA PENTIMALLI,1 NICOLETTE PEGELS,2 TERESA GARCI´A,2 ROSARIO MARTI´N,2 Downloaded from http://meridian.allenpress.com/jfp/article-pdf/72/7/1491/1680203/0362-028x-72_7_1491.pdf by guest on 28 September 2021 AND ISABEL GONZA´ LEZ2* 1Dipartimento di Sanita` Pubblica, Facolta` di Agraria, Universita` degli studi di Parma, Parma, Italy; and 2Departamento de Nutricio´n, Bromatologı´a y Tecnologı´a de los Alimentos, Facultad de Veterinaria, Universidad Complutense, 28040 Madrid, Spain MS 08-536: Received 23 October 2008/Accepted 28 December 2008 ABSTRACT An enrichment PCR assay using species-specific primers was developed for the detection of Arcobacter butzleri, Arco- bacter cryaerophilus, Arcobacter skirrowii, and Arcobacter cibarius in chicken meat. Primers for A. cryaerophilus, A. skirrowii, and A. cibarius were designed based on the gyrA gene to amplify nucleic acid fragments of 212, 257, and 145 bp, respectively. The A. butzleri–specific primers were designed flanking a 203-bp DNA fragment in the 16S rRNA gene. The specificity of the four primer pairs was assessed by PCR analysis of DNA from a panel of Arcobacter species, related Campylobacter, Helicobacter species, and other food bacteria. The applicability of the method was then validated by testing 42 fresh retail- purchased chicken samples in the PCR assay. An 18-h selective preenrichment step followed by PCR amplification with the four Arcobacter primer sets revealed the presence of Arcobacter spp. in 85.7% of the retail chicken samples analyzed. A. butzleri was the only species present in 50% of the samples, and 35.7% of the samples were positive for both A. butzleri and A. cryaerophilus. A. skirrowii and A. cibarius were not detected in any of the chicken samples analyzed. The enrichment PCR assay developed is a specific and rapid alternative for the survey of Arcobacter contamination in meat. The genus Arcobacter belongs to the rRNA superfam- provide recovery and differentiation of arcobacters from re- ily VI of the Proteobacteria and was created in 1991 to lated organisms, but these techniques are cumbersome to accommodate organisms initially regarded as aerotolerant perform, time-consuming, and highly limited in specificity. campylobacters (37). Although the high prevalence of Arcobacter species are fastidious in growth requirements, Campylobacter in foods of animal origin has been docu- relatively biochemically inert, and morphologically similar mented as the main source of human gastrointestinal infec- to campylobacters, factors that may contribute to incorrect tion (6), data for Arcobacter species are much more limited. detection and identification of these organisms when rely- However, arcobacters are increasingly found on meats and ing on agar plating or phenotypic tests (33). In view of have been isolated from people with diarrhea, which en- culture failure and misidentification, nucleic acid approach- hances the significance of these bacteria as a potential food es, particularly PCR-based methods, are increasingly being safety concern (20, 39, 41). Arcobacter butzleri, Arcobacter considered for detection, identification, and monitoring of cryaerophilus (with two subgroups), Arcobacter skirrowii, arcobacters in foods. These methods include simplex and and Arcobacter cibarius are the Arcobacter species poten- multiplex PCR assays with species-specific primers (7, 15, tially associated with human disease (23, 39). Among these, 16, 19, 24, 30), PCR plus restriction fragment length poly- mainly A. butzleri, but also A. cryaerophilus and A. skir- morphism analyses (31), PCR plus random amplification of rowii, have been isolated from raw meats, with the highest polymorphic DNA (5, 22), PCR plus DNA sequencing prevalence in poultry followed by pork, beef, and lamb (6, (27), PCR plus enzyme-linked immunosorbent assays (3), 26, 34). These organisms also have been recovered from and real-time PCR assays (1, 8). untreated water, meat processing equipment surfaces, and The objective of the present investigation was to de- environmental samples (17, 40). A. cibarius was isolated in velop a rapid enrichment PCR assay for detection of the 2005 from the skin of broiler chicken carcasses (23) and, four emerging pathogenic Arcobacter species in poultry more recently, from piggery effluents (10). meat: A. butzleri, A. cryaerophilus, A. skirrowii, and A. ci- Conventional culture and phenotypic protocols may barius. The assay utilizes species-specific primers designed from gyrA and 16S rRNA gene sequences of these species * Author for correspondence. Tel: 34-91-3943751; Fax: 34-91-3943743; and was applied to screen for the presence of arcobacters E-mail: [email protected]. in 42 fresh retail-purchased chicken samples. 1492 PENTIMALLI ET AL. J. Food Prot., Vol. 72, No. 7 MATERIALS AND METHODS TABLE 1. Bacterial strains used in this study Bacterial strains, culture conditions, and DNA extraction. Species Straina The bacterial strains used in this study are listed in Table 1. A. butzleri, A. cryaerophilus, and A. skirrowii were grown on Muel- Arcobacter butzleri LMG 9910 T ler-Hinton agar supplemented with 5 to 10% defibrinated horse A. butzleri LMG 10828 blood (Oxoid, Basingstoke, UK). A. cibarius was grown on brain A. cryaerophilus subgroup 1 LMG 9863 heart infusion agar (Pronadisa, Madrid, Spain). All Arcobacter A. cryaerophilus subgroup 2 LMG 7537 T strains were incubated aerobically for 48 to 72 h at 30ЊC. Cam- A. skirrowii LMG 6621 pylobacter and Helicobacter spp. were grown in Mueller-Hinton A. skirrowii LMG 8538 blood agar (5 to 10%) and incubated for 48 h at 37ЊC microaerobi- A. cibarius CECT 7203 cally. After recovery of bacteria from pure cultures, DNA was A. cibarius LMG 21997 extracted following the method proposed by Sanz et al. (35). Campylobacter jejuni Clinical isolate C. jejuni subsp. jejuni LMG 6444T Primer design. Sequences from gyrA and 16S rRNA genes of C. coli Clinical isolate Downloaded from http://meridian.allenpress.com/jfp/article-pdf/72/7/1491/1680203/0362-028x-72_7_1491.pdf by guest on 28 September 2021 Arcobacter, Campylobacter, and Helicobacter strains available in the C. fetus subsp. fetus LMG 6442T National Center for Biotechnology Information database were aligned C. lari LMG 8845 and compared, and regions containing species-specific nucleotide dif- Helicobacter pylori LMG 18041T ferences were examined for the design of primers for A. butzleri, A. H. pullorum LMG 16318 cryaerophilus, A. skirrowii, and A. cibarius. A fifth oligonucleotide Escherichia coli CECT 515 primer pair was used as a control to avoid false-negative results in Salmonella Enteritidis CECT 4300 the PCRs. The sequences and description of every primer used in Yersinia enterocolitica CECT 559 this study and the length of the generated amplicons are listed in Listeria innocua CIP 103575 Table 2. EMBOSS software package version 2.2.0 and Primer Ex- Pseudomonas fluorescens B52b press 2.0 software (Perkin-Elmer, Applied Biosystems Division, Fos- ter City, CA) were used for primer design. a LMG, Laboratory for Microbiology, Gent, Belgium; CECT, Spanish Type Culture Collection, Valencia, Spain; CIP, Institute PCR setup. After a series of preliminary experiments, optimal Pasteur Collection, Paris, France. amplification conditions were established as follows. Each reaction b Isolate from milk origin supplied by Food Research Centre of ␮ ␮ (25 l) was set up to contain 2 l of extracted bacterial DNA, 2 Ottawa, Ottawa, Ontario. ␮ mM MgCl2, 200 M concentrations of each deoxynucleoside tri- phosphate, 15 pmol of primers 16SArcobutz, GyrArcocry, Gyr- Arcoski, and 16Bact, 25 pmol of primers GyrArcocib, and 1 U of cleanup system kit (Promega Corp., Madison, WI) with a vacuum Thermus thermophilus DNA polymerase (Biotools, Madrid, Spain) manifold according to the manufacturer’s instructions. in a reaction buffer containing 75 mM Tris-HCl, pH 9.0, 50 mM Growth and populations of Arcobacter were determined by KCl, 20 mM (NH ) SO , and 0.001% bovine serum albumin. The 4 2 4 incubation of seeded chicken samples in modified cefsulodin-ir- cycling conditions were 94ЊC for 3 min for denaturation, 40 cycles gasan-novobiocin (mCIN)–CAT agar at 30ЊC for 3 to 4 days under (Arcobacter primers 16SArcobutz, GyrArcocry, GyrArcoski, and aerobic conditions. The mCIN-CAT agar is a variant of the mCIN GyrArcocib) or 25 cycles (bacterial primers 16SBact) of amplification agar developed by Collins et al. (11). Unseeded samples were also at 94ЊC for 1 min, 55ЊC for 1 min, and 72ЊC for 1 min, and a final analyzed to confirm the absence of Arcobacter colonies. extension step at 72ЊC for 7 min. Analysis of retail chicken samples. Chilled fresh chicken Analysis of artificially contaminated chicken samples. samples (n ϭ 42) were purchased from several local supermarkets Batches of 40-g Arcobacter-free chicken muscle samples were and retail shops. Forty-gram portions (drumsticks, leg quarters, aseptically seeded with pure cultures of A. butzleri, A. cryaero- and breasts with the skin) were homogenized in a stomacher with philus, A. skirrowii, and A. cibarius to obtain bacterial counts of 120 ml (1:4 dilution) of Arcobacter broth, and 10-ml aliquots of approximately 10, 102,103, and 104 CFU/g. Negative uninocu- each homogenate were supplemented with CAT. A subsequent en-