Isolation and Identification of Zoonotic Species of Genus <i>Arcobacter</i> from Chicken Viscera Obtained from Retail Distributors of the Metropolitan Area of San JosÃ©, Costa Rica

Arcobacter is a genus of growing importance worldwide; some of its species are considered emerging enteropathogens and potential zoonotic agents. In Costa Rica, as well as in other countries, its isolation has been reported, so the objective of this project was to evaluate and identify the presence of Arcobacter in chicken viscera sold in the metropolitan area of San José, Costa Rica, as well as to determine the antimicrobial resistance patterns associated with it. One hundred fifty samples of chicken viscera including heart, liver, and other gastrointestinal organs were purchased from 15 supermarkets and 15 local retailers. De Boer and Houf broths were used as enrichment media; isolation was done with Arcobacter-selective medium and with membrane filtration with blood agar. Typical colonies were identified with genus-specific PCR, and species identification was made with multiplex PCR. Susceptibility to ampicillin, ciprofloxacin, chloramphenicol, erythromycin, gentamicin, and tetracycline was done with the Epsilometer test. The isolation frequency of Arcobacter genus obtained in this study was of 17.3%. A total of 33 isolates were obtained from the poultry samples, and according to the multiplex PCR methodology, 22 (66.7%) isolates were identified as Arcobacter butzleri, 8 (24.2%) as Arcobacter cryaerophilus, and 1 (3.1%) as Arcobacter skirrowii. Two strains were not identified. No statistical significant difference was found when the source of samples was compared. Resistance toward chloramphenicol was 68.75%, followed by ampicillin (43.75%) and ciprofloxacin (18.75%); all strains were susceptible to tetracycline. The genus Arcobacter, from known and unknown strains of Campylobacter, was proposed in 1991 by Vandamme and colleagues, including those (formerly known as) Campylobacter isolates capable of growing in the presence of oxygen. It is a genus of growing importance worldwide, since some of its species have been considered emerging enteropathogens and potentially zoonotic agents (3, 5). The genus comprises more than 15 different species, and from these, 3 have been associated both with human and animal diseases, although its pathogenic role is still not well understood. Arcobacter butzleri and Arcobacter cryaerophilus have been associated mainly with gastrointestinal outbreaks; although some cases have been associated with bacteremia (3, 6). Recently, A. butzleri was also classified as an agent associated with traveler’s diarrhea in Mexico, Guatemala, and India, with a prevalence rate of 8% (6). Scientific literature states that Arcobacter is highly prevalent in animal-origin food, being mainly isolated from poultry (8). Although it is well known that birds eliminate Arcobacter in their feces, no species has been associated with disease in these animals, suggesting that they could be a natural reservoir for this bacterium (3). Clinical symptoms of Arcobacter infections are quite similar to those produced by Campylobacter; however, persistent watery diarrhea is associated more frequently with Arcobacter infection (19). One of the major problems associated with this bacterium is the lack of standardized methods for its isolation and identification, a fact that could produce an underestimation of its real incidence worldwide (11). Identification has also been troublesome. Current phenotypic assays do not allow differentiation between the most common Arcobacter species. Molecular methods have been developed in order to minimize this problem, which offer more reliable results (6). Some studies were performed in industrialized countries in order to establish the susceptibility of this genus to several antimicrobial agents. Nevertheless, there is very little known about antibiotic resistance of Arcobacter in developing countries (18). In Costa Rica, the isolation of Campylobacter has been well documented (15); however, for Arcobacter, there is just one isolation report of which we are aware (1). The main objective of this project was to evaluate and identify the presence of some of the potentially pathogenic species of * Author for correspondence. Tel/Fax: 506-2511-4270; E-mail: maria.ariasechandi@ucr.ac. cr. 879 Journal of Food Protection, Vol. 76, No. 5, 2013, Pages 879–882 doi:10.4315/0362-028X.JFP-12-400 Copyright G, International Association for Food Protection D ow naded rom hp://m eridianenpress.com /jfp/article-pdf/76/5905/0362-028x_jfp-12-400.pdf by gest on 27 April 2020 Arcobacter present in chicken viscera sold in the metropolitan area of San José, Costa Rica, as well as to determine the antimicrobial resistance patterns associated with it. MATERIALS AND METHODS Samples. One hundred fifty samples of chicken viscera were purchased from various retailers located at the metropolitan area of San José, Costa Rica. Sample analysis was performed in less than 24 h at the Food Microbiology Laboratory, Microbiology Faculty, University of Costa Rica. Isolation of zoonotic species of Arcobacter. Twenty-five grams of each sample was diluted in 225 ml of sterile peptone water, and 1 ml of this dilution was inoculated into 10 ml of Arcobacterselective broth (4) and Houf Arcobacter broth. Enrichment media was incubated aerobically at 26uC for 48 h. Bacterial isolates were recovered with Arcobacter-selective medium and with the membrane filtration method (18), where 200 ml of enrichment broth was applied in small drops to a sterile cellulose filtration membrane (0.45 ml). Incubation was also performed aerobically at 26uC for 48 h. Pinpoint, translucent colonies were partially identified with Gram stain, wet mount observations under contrast microscopy, and catalase and oxidase tests. Identification of the Arcobacter genus. Isolated colonies were identified with a genus-specific PCR reaction (9). DNA extraction was performed by boiling a suspended typical Arcobacter colony in 150 ml of sterile distilled water. Genus PCR methodology included the use of primers and an oligonucleotide probe derived from the specific sequences of 16S rRNA of Arcobacter and A. butzleri (Table 1). PCR was performed with a thermal cycler (PerkinElmer, Norwalk, CT). Samples were subjected to an initial denaturation step (94uC for 2 min), followed by 25 amplification cycles consisting of 1 min at 94uC denaturation, 1 min at 56uC for Arco I and Arco II annealing or 52uC for Butz and Arco II, and 72uC for primer extension. A final step at 72uC for 7 min followed the final amplification cycle. Agarose electrophoresis was used to detect PCR reaction products; ethidium bromide staining was used for their visualization. Multiplex PCR for species identification. Definitive identification was made with the multiplex PCR proposed by Douidah et al. (5). Nucleotide sequences of the primers used are shown in Table 2. In short, a 50-ml final volume of reaction mixture was denaturalized at 94uC for 4 min, followed by 30 amplification cycles consisting of 45 s at 94uC for denaturation, 45 s at 58uC for hybridization, and 2 min at 72uC for extension. Final amplification was done at 72uC for 7 min. Electrophoresis with agarose gel and ethidium bromide dye was used for detecting the PCR fragments. In vitro susceptibility to antimicrobial drugs. Susceptibility to ampicillin, ciprofloxacin, chloramphenicol, erythromycin, gentamicin, and tetracycline was done with the Epsilometer test (Etest; AB Biodisk, Solna, Sweden). In brief, fresh culture colonies were suspended in 5 ml of Mueller-Hinton broth to a turbidity of a 0.5 McFarland standard. Sterile swabs were used to inoculate Mueller-Hinton agar plates supplemented with 5% sheep blood. Etest strips were applied to the agar plates, which were incubated at 26uC for 48 h. Inhibitory concentrations were read at the points where the elliptical zone of inhibition intersected the Etest strip. Staphylococcus aureus ATCC 25923 and Escherichia coli ATCC 25922 were used as control strains. The susceptibility criteria defined for Campylobacter jejuni by the German Institute for Standardization was used as reference in this study (7).