Prevalence and diversity of Arcobacter spp . in retail chicken meat in Turkey

Arcobacters are food and waterborne pathogens associated with human and animal infections. The objective of the present study was to investigate the prevalence and diversity of Arcobacter spp. in commercially sold chicken meat in İzmir region of Turkey. For this purpose, 100 samples including legs (n=40), 17 chicken quarters (n=17), drumstickers (n=16), breasts (n=11), wings (n=10), and carcasses (n=6) were collected from different retail markets. A total of 65 isolates were confirmed as Arcobacter spp. from 55 samples by genus-specific polymerase chain reaction (PCR). The prevalence of Arcobacter spp. was 32.5, 81.3, 64.7, 72.7, 83.3, and 50% for legs, drumstickers, chicken quarters, breasts, carcasses and wings, respectively. Based on the multiplex-PCR, most of the isolates were identified as A. butzleri (n=45, 80%), followed by A. cryaerophilus (n=2, 3.6%), A. skirrowii (n=1, 1.8%) and 17 isolates (30.9%) could not be identified at the species level.


Introduction
Contamination of poultry meat and its products with the emerging human pathogens such as Campylobacter and Arcobacter spp.poses a potential risk for the microbiological safety. 1 Arcobacters are characterized as Gram-negative, fastidious, microaerophilic, non-spore forming, usually motile, spiral-shaped bacteria.This genus belongs to the Campylobacteraceae family 2 and currently comprises 21 species. 3These organisms differ from closely related campylobacters by their ability to have aerotolerance and survival capacity at lower temperatures. 4,57][8] A. butzleri, A. cryaerophilus and A. skirrowii are known as the most important species causing infections in humans and animals. 6,9In the related literature, the highest prevalence has been observed for poultry, followed by pork and beef meat. 10If contaminated foods or water is consumed, the infection can occur. 11here are various studies about the isolation of Arcobacter spp.][22] Therefore, the main objective of this study was to determine the prevalence and diversity of Arcobacter spp. in chicken samples using molecular methods.

Genus-specific polymerase chain reaction
DNA was extracted using a commercial genomic DNA isolation kit (PureLink ® Kit, Invitrogen, Thermo Fisher Scientific, USA).The primers of Harmon and Wesley (1996) 23 were used for genus-specific polymerase chain reaction (PCR).The reaction was performed in a total volume of 25 µL containing 2 µL template DNA, 2.5 µL of 10×PCR buffer (750 mmol/L Tris-HCl (pH 8.8), 200 mmol/L (NH 4 ) 2 SO 4 , 0.1% (v/v) Tween 20, and 1.5 mmol/L MgCl 2 ), 10 µmol/L of each of the primers, 0.2 mmol/L each of the four dNTPs (Fermentas, Thermo Fisher Scientific, USA) and 1.5 U Taq DNA polymerase (Fermentas, Thermo Fisher Scientific, USA).The samples were subjected to an initial denaturation step (94°C for 5 min), followed by 35 amplification cycles.Each amplification cycle consisted of 1 min at 94°C (denaturation), 1 min at 56°C (primer annealing), and 1 min at 72°C (primer extension).A primer extension step (72°C for 7 min) followed the final amplification cycle.PCR experiments were repeated twice for each strain.The amplified products were resolved in 1% (w/v) Tris-acetate-EDTA (TAE) agarose gel and the band patterns were analyzed in the gel

Results and Discussion
Since it is difficult to identify Arcobacter species using cultural methods, molecular detection and identification methods have been developed for Arcobacter spp. 25 In the present study, we determined the prevalence and diversity of Arcobacter spp. in chicken samples collected from İzmir region of Turkey by PCR-based methods.
Based on the genus-specific PCR, 65 isolates were identified as Arcobacter spp.among 100 samples analyzed (Table 1).In total, 55 samples were positive for the presence of Arcobacter spp.(55%).The highest occurrence was obtained in carcasses (n=5, 83.3%), followed by drumstickers (n=13, 81.3%), breasts (n=8, 72.7%), leg quarters (n=11, 64.7%), wings (n=5, 50%) and legs (n=13, 32.5%).Studies indicated that the prevalence of Arcobacter spp. in poultry ranged from 38.4 to 90.6% and the overall prevalence (55%) obtained in this study was found within this range. 26The obtained variations in isolation rates may be related to plant conditions, processing procedure, geographical location, seasonal differences, experimental designs and analytical methods used to analyze the collected samples. 14,26As an example, Levican and colleagues 8 suggested that different conditions used for culturing can lead to different isolation rates.They found that the recovery rate of Arcobacter under aerobic incubation was higher (41.1%) than that of microaerobic conditions (23.2%).
Arcobacter spp.has been reported to be as a significant hazard for the public health. 26,27heir presence in food processing environments indicates possible persistence or crosscontamination. 281][32] In fact, Arcobacter spp.can persist and form biofilms on many pipe surfaces made of steel, copper and polyethylene resulting in colonization in water distribution systems. 33In a recent study, processing water and equipment used in the slaughterhouse were suggested as the main sources of broiler carcass contamination by Arcobacter. 34he isolated Arcobacter species were further examined by m-PCR to identify at the species level.Based on the m-PCR, the most prevalent species were A. butzleri (n=45), followed by A. cryaerophilus (n=2) and A. skirrowii (n=1).The remaining Arcobacter isolates (n=17) could not be identified using the available primers at the species level (Table 1).Collado and colleagues 35 also showed that these three species were found in the water and shellfish most probably due to sewage outlets and fecal contamination.In accordance with the results of previous studies, 13,23 A. butzleri was the most prevalent species on chicken meat. A. butzleri was isolated from all types of samples analyzed.Its isolation rates from wings, breasts, drumstickers, carcasses, legs, breasts, and quarters were 100, 87.5, 84.6, 80, 76.9, and 72.7%, respectively.On the other hand, A. cryaerophilus and A. skirrowii were detected on the wings and carcass samples and on the drumsticker, respectively.
In eight analyzed samples, both A. butzleri and other two species were isolated.Similar to this finding, the presence of more than one species in the same sample was reported by other researchers. 10,31It has been suggested that lower isolation rates for A. skirrowii may be due to its higher susceptibility to antimicrobials present in selective media used or growth competition favoring other microorganisms. 36][40] Since A. butzleri, A. cryaerophilus and A. skirrowii are emerging foodborne pathogens, effective control methods should be applied to prevent Arcobacter spp.contamination during food processing for human health.

Conclusions
Based on the results, the presence of Arcobacter spp. in chicken meat is a potential risk for human health because the consumption of these contaminated products may cause serious diseases.Therefore, effective control strategies should be applied to prevent their contamination during poultry processing in the food industry.
documentation system (Vilber Lourmat, France).DNA from reference strains were used as positive controls and sterile distilled water served as negative control.