Determination of the presence and antimicrobial resistance of Arcobacter species in broiler carcasses at different stages of slaughter line

Abstract In this study, to investigate Arcobacter spp. contamination post‐scalding and de‐feathering, post‐evisceration, post‐chilling, and packaged products, which are the most essential contamination stages of broiler slaughter, a total of 108 samples were taken from three different broiler slaughterhouses at different times. Isolates obtained by cultural methods in 104 of 108 samples were analyzed by mPCR method to identify pathogen Arcobacter spp. Arcobacter butzleri, Arcobacter cryaerophilus, and mixed contamination of both Arcobacter species were detected in 51 samples. Of the 51 isolates, 27 (52.9%) were A. butzleri, 16 (31.4%) were A. cryaerophilus, and 8 (15.7%) were mixed contamination of A. butzleri and A. cryaerophilus, while Arcobacter skirrowii was not detected. A. butzleri and A. cryaerophilus contamination was 59.2% post‐scalding and de‐feathering, 43.4% post‐evisceration, 44.4% and 48.1% post‐chilling and in packaged products, respectively. All A. butzleri strains were found to be 100% resistant to cefoperazone and penicillin and sensitive to tetracycline. A. cryaerophilus strains were 100% resistant to cefoperazone, penicillin, and cloxacillin and susceptible to tetracycline and erythromycin. In the study, it was determined that Arcobacter spp. caused a very intense contamination (85.18%–100%) and also contamination rates of identified pathogen strains (A. butzleri and A. cryaerophilus) were very high (59.2% and 43.4%) in broiler slaughtering stages. Considering that each step in broiler slaughter could contaminate the next stage, developing a safe slaughter and minimizing the risk toward the final product, it was concluded that critical control points could not be well managed in broiler slaughterhouses, and broiler meat may pose a significant risk to public health.


| INTRODUC TI ON
First isolated from aborted pig fetuses, Arcobacter has been initially described as an "aerotolerant Campylobacter-like" bacterium.Because it harbors two aerotolerant species (A.cryaerophilus and Arcobacter nitrofigilis), it was proposed to be separated from Campylobacter in 1991.The most important difference between Campylobacter and Arcobacter species is the ability of Arcobacter to grow at lower temperatures (15-30°C) (Vandamme et al., 1991(Vandamme et al., , 1992;;Sciortino et al., 2021).
Biochemical properties of Arcobacter species are insufficient to make a definitive identification (Diéguez et al., 2017;Levican et al., 2015;Vandamme et al., 1992).With molecular identification methods, difficulties encountered in the cultural identification of Arcobacter spp.have been overcome and the number of species has increased.Up to now, 26 Arcobacter species have been identified (Diéguez et al., 2017;Levican et al., 2015).Some species are known to be important enteropathogens for humans and animals (Collado & Figueras, 2011).
The difficulties in diagnosis of the pathogenic Arcobacter spp., especially A. butzleri, due to its live but nonculturable form (Fera et al., 2008;Zhang et al., 2021), and also its resistance to some antibiotics and its ability to form biofilms (Cervenka, 2007;Ferreira et al., 2013), it is reported to continue to pose a potential hazard to public health.In this context, A. butzleri has been accepted as a serious danger to human health and an important zoonotic pathogen by the International Commission on Microbiological Specifications for Foods (ICMSF) (Schönknecht et al., 2020).In a five-year prevalence study, it has been reported that Arcobacter spp. was the fourth most common gastrointestinal pathogen (1.3%) after Campylobacter spp.(5.6%), Salmonella spp.(2.0%), and Clostridium difficile (1.6%) (Nguyen et al., 2022).In a study conducted in Bangkok, Arcobacter spp. was reported to have a higher prevalence than other pathogens such as Salmonella and Campylobacter in raw products (e.g., retail meat, shellfish, vegetables) as well as in restaurant meals (Collado & Figueras, 2011).In a study conducted in Canada, it was reported that Arcobacter spp. was found to be 2-3 log higher in irrigation water than Campylobacter spp.and, therefore, posed a more significant threat to human health (Banting et al., 2016).
The acquisition of antibiotic resistance by bacteria through different mechanisms and contamination of foods with these resistant bacteria may pose severe problems for public health.Especially in developing countries, the prevalence of antibiotic resistance among foodborne pathogens is gradually increasing due to the irregular use of antibiotics (Jribi et al., 2020).In a meta-analysis study, resistance rates were between 69.3%-99.2%for penicillins and 30.5%-97.4% for cephalosporins.The same survey reported that the resistance rate to fluoroquinolones was between 4.3% and 14.0%, and the highest resistance rate was found against levofloxacin.However, resistance rates ranged between 10.7%-39.8%for macrolides, 1.8%-12.9%for aminoglycosides, and 0.8%-7.1% for tetracyclines (Ferreira et al., 2019).
Arcobacter spp.like Campylobacter spp.are known as foodborne microorganisms as they are isolated from broiler meat worldwide (Ferreira et al., 2019;Son et al., 2007).In studies conducted on the same broiler carcasses, it was reported that the isolation rates of Arcobacter spp.were higher than those of Campylobacter spp. in almost all samples (Houf et al., 2002b).Arcobacter spp.has been isolated from different stages of the broiler slaughter line (e.g., scalding and de-feathering, evisceration, pre-and post-chilling), equipment, and processing water (Ho et al., 2008).It was reported that Arcobacter spp.could not be isolated from the intestinal contents of broilers belonging to the same flock from which the carcasses were came (Atabay & Corry, 1997).This indicates that the source of Arcobacter contamination in carcasses may be the slaughterhouse environment and/or the water used during processing (Atabay & Corry, 1997;Ho et al., 2008).Three of the four reported Arcobacter outbreaks have been linked to the consumption of water contaminated with feces, and the other to the consumption of fried chicken (Collado & Figueras, 2011;Ferreira et al., 2016).An incidence ranging from 13.6% (Denmark) (Hsu & Lee, 2015) to 84.6% (Belgium) (Houf et al., 2002a) has been determined in poultry processing plants, depending on the country.
In this study, it was aimed to determine the presence and contamination sources of Arcobacter spp.and three pathogenic species (A. butzleri, A. skirrowii, A. creoaerophilus) at different stages of broiler slaughter line and to determine their antimicrobial resistance.

| MATERIAL S AND ME THODS
Three broiler slaughterhouses (approved by the government according to Regulation No. 28145 on the Official Control of Animal Food) were visited thrice.During each visit, 36 samples taken from four different stages of slaughter (post-scalding and de-feathering, post-evisceration, post-chilling, packaged product) were placed in sterile bags as whole carcasses and brought to the laboratory in a cold chain.At the end of the three visits, totally 108 samples were taken.Isolation and identification of Arcobacter spp.from a total of 108 samples were carried out using the cultural method; for confirmation and species-level identification of the isolates obtained, multiplex polymerase chain reaction (mPCR) was performed.The antibiogram test was used to determine antibiotic susceptibilities.
Broiler carcasses taken post-scalding and de-feathering were washed with a sponge in sterile bags using 400 mL Maximum Recovery Diluent (MRD-Merck-SK.M112535.0500-Germany)solution.The samples collected from the other stages were placed in sterile bags, 400 mL of MRD was added, and the internal and external surfaces of the carcass were thoroughly washed by shaking (approximately 35 rpm) for 1 min (Capita et al., 2004;Benli et al., 2015).
Ten milliliter of the washing water was transferred to sterile Pyrex bottles.
The method proposed by Lehmann et al. (2015) was used to isolate and identify Arcobacter spp.90 mL Arcobacter selective broth (ASB-HIMEDIA-M1894-India) with cefoperazone, amphotericin B, and teicoplanin (CAT Selective Supplement-HIMEDIA-FD145-India) was added to 10 mL rinse water.The broth was incubated at 30°C for 48 h under microaerobic conditions.After incubation, the broth was diluted 1/10 with Brucella Broth (BB-HIMEDIA-M348-India) and 0.3 mL was spread on the filter (Filter-Lab-MCE045047WGSN) on 5% sheep blood Mueller Hinton Agar (MHA-Merck-SK.M105437.0500-Germany)and incubated under aerobic conditions for 1 h, and the filter was removed with a sterile forceps.0.1 mL BB was added to MHA and incubated aerobically at 30°C for 48 h.Suspicious colonies were switched to MHA and incubated at 30°C for 48 h under microaerobic conditions.After incubation, Gram staining, oxidase, catalase, nitrate reduction, urease, and hippurate tests were performed.In addition, isolates of Arcobacter spp.were transferred to a BB storage medium containing 15% glycerin and stored at −86°C for molecular identification and antibiotic susceptibility tests.

| RE SULTS
In the study, to determine the contamination levels of Arcobacter spp.post-scalding and de-feathering, post-evisceration, postchilling, and packaged products, which have the highest contamination risk in broiler slaughtering processes, isolation, and identification procedures were carried out by cultural and molecular methods in 108 samples obtained from various slaughterhouses at different times.

| Cultural isolation and identification
After cultural isolation and identification, suspicious colonies of Arcobacter spp.were detected in 104 of 108 samples.The distribution of suspicious colonies according to the slaughter stages is given in Figure 1.

| Molecular identification
After mPCR and electrophoresis applied to the reference strains and the isolates obtained as Arcobacter spp.by cultural methods, A. butzleri was observed at 401 bp, A. cryaerophilus at 257 bp, and A. skirrowii at 641 bp (Figure 2).
After mPCR was applied to 104 isolates identified as Arcobacter spp.by cultural method 51 (49.03%) of the isolates were identified as Arcobacter spp.When the species-based distribution was analyzed, it was found that A. butzleri was more common (52.9%), followed by A. cryaerophilus (31.4%) and A. butzleri-A.cryaerophilus (15.7%) mixed contamination.
Statistical analysis on the distribution of pathogen Arcobacter spp.contamination detected at different slaughtering stages showed no correlation between pathogen Arcobacter spp.(p > .05)(Table 1).

| Antibiotic susceptibility test results
The six antibiotic types (cefoperazone, tetracycline, ampicillin, erythromycin, penicillin, and cloxacillin) used in the study were selected by considering the antibiotic sensitivities against Arcobacter species detected in different foods (Ferreira et al., 2019) and in poultry facilities (Jribi et al., 2020;Rahimi, 2014).All A. butzleri isolates were 100% resistant to cefoperazone and penicillin, 96.3% to cloxacillin, 66.6% to ampicillin, and 3.7% to erythromycin.However, all of the A. butzleri isolates were susceptible to tetracycline.
A. butzleri and A. cryaerophilus mixed isolates were found to be 100% resistant to cefoperazone, penicillin, and cloxacillin, 75% resistant to ampicillin, and 12.5% resistant to erythromycin, while all mixed isolates were susceptible to tetracycline (Figure 3).

| DISCUSS ION
In  In a study carried out by Khoshbakht et al. (2014), in which A.
butzleri was the most frequently detected species and the distribution was similar to the findings obtained at the slaughtering stages Similar to the findings of this study, Rahimi (2014) reported that 64 A. butzleri, 5 A. cryaerophilus, and 2 A. skirrowii isolated from poultry meat (chicken, turkey, quail, partridge, duck, ostrich) were all susceptible to tetracycline.In the same study, similar to our study, 1.6% and 0% resistance to erythromycin were found, respectively.

| CON CLUS ION
Although Arcobacter spp. is less known than other foodborne pathogens and there is no legal regulation regarding its presence in foods (Collado & Figueras, 2011), it has been reported that the isolation amounts of Arcobacter spp.are higher than Campylobacter spp. in studies conducted on broiler carcasses.In this study, as in many previous studies, 100% of the samples examined post-scalding and de-feathering, post-chilling, and packaged products were con-

ACK N OWLED G M ENTS
We express our gratitude to Selcuk University Scientific Research Projects Coordination.

FU N D I N G I N FO R M ATI O N
This research was supported by Selçuk University Scientific Research Projects Coordination Office with 21112001 project code and PhD Thesis project type.

CO N FLI C T O F I NTE R E S T S TATE M E NT
The authors declare that they do not have any conflicts of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
Data available on request due to confidentiality/ethical restrictions.

E TH I C S S TATEM ENT
This study was approved by the local ethics committee of the Isolates and reference strains (A.butzleri-DSM8739, A. cryaerophilus-DSM7289, A. skirrowi-DSM 7302) in glycerin BB storage medium were transferred to centrifuge tubes containing Nutrient Broth (HIMEDIA-M439SIndia).After incubation at 37°C, the isolates were suspended in centrifuge tubes containing 50 μL of sterile bidistilled water with the help of a pipette.Centrifuge tubes were kept in a heat block (Bioneer MyGenie 96 Thermal Block-Cycler) at 110°C for 15 min and then centrifuged (Nüve NF 1200 R) at 1200 g value for 15 min.The DNA supernatants obtained were placed in sterile centrifuge tubes and used in mPCR analysis.PCR Master Mix (2X) (Thermo Fisher-K0171) was used in mPCR analysis for molecular identification of Arcobacter spp.isolates.The mixture was prepared in sterile centrifuge tubes using 10 μL PCR master mix, 1 μL each of ARCO, BUTZ, CRY1, and CRY2, and 0.5 μL SKIR primers (Sentebiolab), 2 μL isolate DNA, and 12.55 μL sterile bidistilled water to a total volume of 25 μL.PCR amplification was performed at 95°C for 5 min (initial denaturation), 94°C for 1 min (denaturation), 55°C for 1 min annealing, 72°C for 2 min (primer extension), and 72°C for 15 min (final extension) for 40 cycles.The amplified PCR products were detected by electrophoresis in 1.5% agarose gel at 90 V, 70 mA, and 60 min.The resistance pattern of the isolates was determined by disk diffusion(Hudzicki, 2009) for cefoperazone (Bioanalyse-CEP200262, 75 μg-Turkey), tetracycline (Bioanalyse-TE201429, -ST00021, 1 μg-Turkey), and penicillin (Bioanalyse-P201153, 10 IU-Turkey).Since there is no standard zone range for Arcobacter spp., the zones formed on blood agar (BA-Merck-1.10886-Germany)containing 5% sheep blood were evaluated according to the criteria determined by the Clinical and Laboratory Standards Institute (CLSI, 2020).Statistical analyses of results obtained in the study were performed by Pearson chi-square and Fisher's exact chi-square tests using the SPSS package program (SPSS Version 21).
Atanassova et al. (2008) determined the contamination levels with Arcobacter spp. as 48% before and post-evisceration and 60% post-chilling.They reported that the presence of Arcobacter spp. in broiler slaughtering stages increased post-chilling.The increase determined by the researchers in the samples taken from the cooling stage applied post-evisceration is in parallel with the findings obtained in this study.In addition, similarly to our study, the researchers reported that A. butzleri was the most common species among Arcobacter spp. at different stages of broiler slaughtering, followed by A. skirrowii and A. cryaerophilus, respectively.Khoshbakht et al. (2014) determined the contamination levels with Arcobacter spp. as 30% before scalding, 48% post-scalding, 73% post-evisceration, and 18% at the 24th hour of cooling.The researchers' findings post-scalding and evisceration are similar to the values obtained in this study.It is thought that the difference before scalding and post-chilling may be due to the effectiveness of decontamination methods and the differences between the enterprises.In the same study, similar to the findings of this research, it was determined that the most common species among Arcobacter spp. in different stages of broiler slaughtering was A. butzleri, with 31.25%.Similar to the findings of this study, Ho et al. (2008) detected Arcobacter spp. in 95 of 100 broiler carcasses collected from different stages of broiler slaughtering.As a result of mPCR applied to 95 isolates, 58 samples (61.1%) were A. butzleri, 1 sample (1.1%) was A. cryaerophilus, 29 samples (30.5%) were A. butzleri and A. cryaerophilus mixed contamination, 7 samples (7.36%) were A. butzleri and A. F I G U R E 2 Results of molecular identification.M, marker (100 bp DNA Ladder 50μg Hibrigen MG-LDR-100-1 1-Turkey); N, negative control; PK1, A. skirrowii positive control; PK2, A. cryaerophilus positive control; PK3, A. butzleri positive control; 1, negative sample; 2, A. butzleri positive sample; 3, A. cryaerophilus and A. butzleri positive mixed contamination sample; 4, A. cryaerophilus positive sample.skirrowii mixed contamination.Atabay et al. (2006) isolated and identified Arcobacter spp. in 85 (85%) of 100 samples (30 whole carcasses, 70 cloacal swabs, and feces) collected from broiler slaughterhouses.Houf et al. (2002b) investigated the evisceration and post-chilling stages of 8 different broiler slaughterhouses.They found Arcobacter spp.contamination in 919 of 960 samples (95.7%).
taminated with Arcobacter spp., 85.15% of the samples examined post-evisceration, and 49.03% of the contaminated samples carried pathogenic Arcobacter spp.According to these results, it was concluded that the source of Arcobacter contamination in broiler carcasses may be the slaughterhouse environment and/or the water used during processing and that it is essential to pay utmost attention to hygiene rules inside and outside the slaughterhouse.Considering the application of HACCP rules in broiler slaughtering, each stage should be developed by considering the next step, and the risk should be minimized toward the final product.The findings obtained in the research concluded that HACCP could not be well managed in broiler slaughterhouses, and broiler meat may pose a significant risk to public health.It is essential to comply with the basic principles of HACCP and good production practices in slaughterhouses and to raise awareness by providing periodic training to the personnel.Considering the increase in the production, consumption, import, and export of broiler meat in the world and Turkey, it is thought that it is essential to carry out more studies on the subject and implement protective measures to consume health-Data curation (equal); formal analysis (equal); methodology (equal); resources (equal); writing -original draft (equal); writing -review and editing (equal).Ahmet Güner: Data curation (equal); formal analysis (equal); methodology (equal); resources (equal); writing -original draft (equal); writing -review and editing (equal).
the study, it was investigated the presence of Arcobacter spp. in broiler meat, the most consumed meat type in Turkey, at the slaugh- (Son et al., 2007)ness is that there are a limited number of studies examining different stages of broiler slaughter line in terms of Arcobacter spp., and this is the first study conducted in Turkey.Son et al. (2007)determined the contamination levels with Arcobacter spp. as 96.8% before scalding, 61.3% before chilling, and 9.6% postchilling.Similar to the findings of this study, the same researchers(Son et al., 2007)reported that A. butzleri was the most common species (79.1%), followed by A. cryaerophilus 1B (18.6%) and A. cryaerophilus 1A (2.3%) among Arcobacter spp.obtained at different stages of broiler slaughtering.