Aliarcobacter spp. are emerging pathogens that cause gastroenteritis in humans, so the spread and mechanisms of drug resistance, pathogenicity potential, and virulence mechanisms need to be fully understood (Brückner et al. 2020). According to our result, contamination with Aliarcobacter spp. in slaughterhouses wastewater samples was 33.3% and in swab samples taken from meat cutting boards, knives, carcasses, and walls were 10%, 20%, 6.6%, and 3.3%, respectively. Regarding wastewater samples, our results showed higher prevalence rates (33.3%) than those previously reported in Turkey (29.1%) (Elmali and Can 2017). Also, the isolation rate of Aliarcobacter in swab samples (knives 20% and cutting board 10%) was lower than a previous study in Turkey which showed a rate of 40% (Elmali and Can 2017). A higher rate of contamination has been documented in slaughterhouses of livestock meat from Spain and Chile with nearly 92% contamination rate (Collado and Figueras 2011). Differences of Aliarcobacter spp. contamination rates in various areas may be attributed to the differences in water resources, seasonal variations, hygiene and processing conditions, experimental designs and care in isolation, geographical locations and isolation methods (Ünver et al. 2013; Elmali and Can 2017).
In this study, A. butzleri was the most frequent species isolated from slaugterhouse, followed by A. cryaerophilus and A. skirrowii, which was similar with other studies (Elmali and Can 2017). The results are in accordance with other studies conducted on food samples collected at retail or supermarkets and from healthy animals (Shah et al. 2012; Ferreira et al. 2017; Vicente-Martins et al. 2018). The culture method has been defined as affecting the species and genetic diversity found in samples, with parameters such as media composition, pre-enrichment step, atmospheric conditions, or incubation time of enrichment and plating influencing the recovery selectivity and specificity of the process (Levican et al. 2016; Ferreira et al. 2017).
The presence of VAGs in isolates of Aliarcobacter spp. have been addressed by several studies (Douidah et al. 2012; Tabatabaei et al. 2014; Collado et al. 2014; Zacharow et al. 2015; Girbau et al. 2015). Among the nine putative virulence determinants, the virulence factor (mviN) and Campylobacter invasive antigen B gene (ciaB) were the most commonly identified in Aliarcobacter isolates (100%), followed by the gene encoding fibronectin-binding proteins (cadF: 95.45%), the iron-regulated outer membrane protein gene (irgA; 22.72%), and the phospholipase gene (pldA; 4.54%). These results show partial agreement with those of previous studies performed in Spain, India, Canada, South Korea, Czech Republic, and Iraq (Whiteduck-Léveillée et al. 2015; Girbau et al. 2015; Laishram et al. 2016; Šilha et al. 2019; Kim et al. 2019; Jasim et al. 2021).
These other studies reported that six putative virulence determinants (cadF, ciaB, cj1349, mviN, pldA, and tlyA) were identified more frequently than the others (hecA, hecB, and irgA) in Aliarcobacter isolated from various sources. Moreover, these studies report that almost all A. butzleri isolates harbored six frequent putative virulence genes (Douidah et al. 2012; Ferreira et al. 2013). Contrary to previous results, we found that six putative virulence genes do not always coexist, just as Kim et al. (2019) noted. This may indicate that Aliarcobacter harbors variable genotypic patterns according to various studied samples worldwide.
The virulence genes cadF, ciaB and mviN were detected in all but one of the A. butzleri isolates. These results show partial similarity with other studies (Tabatabaei et al. 2014; Brückner et al. 2020; Jasim et al. 2021). A study from Iraq revealed that among fresh raw cattle meat samples, all A. butzleri isolates contained the cadF gene (Jasim et al. 2021). Similarly, in a study conducted with human stool samples in Germany revealed that all the A. butzleri isolates contained ciaB and cadF genes with the rate of 100% and the rate of mviN gene was 83% (Brückner et al. 2020). Tabatabaei et al. (2014) previously highlighted the high prevalence of the ciaB and mviN genes, which are significantly more common than other virulence markers.
In this study, the pldA, cj1349, irgA, hecA, and hecB genes were not detected in any of the A. cryaerophilus isolates tested, which is in accordance with previous studies (Zacharow et al. 2015; Šilha et al. 2019). Also, consistently with our findings, Collado et al. (2014) reported that the occurrence of hecA and cj1394 in 14.3% of A. cryaerophilus isolates isolated from mollusks, but no hecB and irgA genes were found in any isolates. In general, more virulence-associatted genes have been detected in A. butzleri isolates compared to A. cryaerophilus. In accordance with earlier studies (Tabatabaei et al. 2014; Collado et al. 2014; Girbau et al. 2015), a higher incidence of virulence genes was found in A. butzleri compared to A. cryaerophilus. However, there is a theory in the literature that this difference could be due to the use of A. butzleri strain ATCC 49616 (Levican et al. 2013) for the design of primers for the detection of virulence genes. Therefore, it would be appropriate to use species-specific primers in future studies. Regarding A. skirrowii isolate, it harbored ciaB, cadF, and mviN genes. The present result shows partial agreement with Girbau et al. (2015) who reported that the genes cadF, ciaB, cj1349, mviN, pldA and tlyA were detected in A. skirrowii strain.
Aliarcobacter resistance to antibiotics used in clinical and veterinary practice has been reported. In this study, most strains were shown to be susceptible to tetracycline (81.8%), and high rates of resistance were found to ampicillin (95.4%), rifampin (90.9%), trimethoprim/sulfamethoxazole (72.7%), amoxicillin/clavulanic acid (59.09%), eritromycin (45.4%) and ciprofloxacin (40.9%). The overall antibiotic resistance rates across species show a higher frequency of resistance to A. butzleri, followed by A. cryaerophilus. The present results are consistent with those previously reported (Kabeya et al. 2004). In this study, A. butzleri strains were resistant to amoxicillin-clavulanic acid (AMC), erythromycin (E), tetracycline (TE), and ciprofloxacin (CIP) at the rate of 63.1%, 47.3%, 15.7%, and 42.1%, respectively. These antibiotic resistance rates were found to be not compatible with the study carried out on raw beef in Iraq. Jasim et al. (2021) found that most of A. butzleri were resistant to tetracycline (72%), erythromycin (67%) while 35% of them were resistant to ciprofloxacin.
Resistance to ampicillin has been reported previously for A. butzleri isolated from different sources (Ferreira et al. 2013; Yesilmen et al. 2014; Shirzad Aski et al. 2016; Rathlavath et al. 2016). A resistance rate of 84.1% was reported for A. butzleri isolates recovered from cattle in Iran Shirzad Aski et al. (2016), which is below the level of resistance observed in the present study (94.7%). However, in the same study, the resistance rate (100%) of A. cryaerophilus isolates against ampicillin was found to be compatible with our study. The present result is consistent with those previously reported ampicillin resistance rate of 97.7% in A. butzleri isolates recovered from poultry and environment of a Portuguese slaughterhouse (Ferreira et al. 2013).
Regarding erythromycin, of all strains, 45.4% were resistant to this antibiotic. This rate is higher than that reported by previous studies conducted with different sources (0- 18.1%) (Rahimi 2014; Šilha et al. 2019; Isidro et al. 2020). However, Zacharow et al. (2015) found that 63.3% of the A. butzleri and A. cryaerophilus isolated from chicken, pork and beef meat were resistant to erythromycin. Similarly, Rathlavath et al. (2016) found that 69.3% of the A. butzleri isolated from seafood and environmental sample were resistant to erythromycin. Resistant rates of 67% and 60% were reported for A. butzleri and A. cryaerophilus isolates recovered from raw cattle meat in Iraq (Jasim et al. 2021), which is higher than the level of resistance observed in the present study.
Concerning fluoroquinolones, A. butzleri and A. cryaerophilus were the only two species presenting resistance to this class of antibiotics. Most A. butzleri strains were susceptible to ciprofloxacin (57.89%), whereas half of the A. cryaerophilus isolates showed high-level resistance. The high prevalence of ciprofloxacin resistance was observed in our work, in contrast to low levels of resistance reported by others (Vandenberg et al. 2006; Van den Abeele et al. 2016). The present results are consistent with those previously reported for a poultry slaughterhouse in Portugal where a susceptible rate of 44.2% was described to ciprofloxacin (Ferreira et al. 2013).
In this study, it was determined that 6 (27.2%) isolates (4 were A. butzleri, and 2 were A. cryaerophilus) were positive for the gyrA resistance gene, but 9 (40.9%) isolates (8 were A. butzleri, and 1 was A. cryaerophilus) showed phenotypic resistance to ciprofloxacin. Many studies have reported finding the gyrA resistance gene in Aliarcobacter spp. isolates (Abdelbaqi et al. 2007; Sciortino et al. 2021). Sciortino et al. (2021) discovered a gyrA resistance gene in two strains that showed phenotypic resistance to ciprofloxacin in their study on water samples, but sequence analysis revealed that there was no mutation in the relevant gene's base sequence. To the best of our knowledge, Thr-85-Ile mutation of the gyrA gene of A. butzleri or other suggested mechanisms such as hydrophobic quinolones uptake are required for this resistance to occur (Abdelbaqi et al. 2007; Miller et al. 2007; Isidro et al. 2020). As a result, the fact that the isolate carries the gyrA gene does not mean that it will phenotypically show ciprofloxacin resistance. Furthermore, the absence of the gyrA gene does not rule out the possibility of phenotypic resistance, as the isolate may have other mechanisms for resistance development. For example, WGS was performed on 10 A. butzleri isolates obtained from milk samples in Italy, and it was determined that all of the strains harbored the adeF gene, which provides resistance to fluoroquinolone and tetracycline antibiotics (Parisi et al. 2019).
In terms of tetracycline susceptibility, the majority of A. butzleri strains (84.2%) were susceptible. However, one of the two strains of A. cryaerophilus developed resistance to this antibiotic. Kabeya et al. (2004), Zacharow et al. (2015), Elmali and Can (2017) found Aliarcobacter strains susceptible to tetracycline in contrast to Vicente-Martins et al. (2018) and Parisi et al., (2019). When the tetracycline resistance genes (tetW and tetO) were evaluated, it was determined that 7 (31.8%) A. butzleri isolates were positive for the tetW gene, while no isolates were positive for the tetO gene. According to Gungor et al., (2023), Twenty-four (85.7%) of 28 isolates were positive for the tetW gene, but none of the isolates harbour the tetO gene, similar to our findings. They were also noted that tetW gene had a distribution in 16 (94.1%) of A. butzleri, 7 (70%) of A. cryaerophilus, and 1 (100%) of A. skirrowii isolates. Furthermore, Sciortino et al. (2021) reported that they found tetW and tetO genes in Aliarcobacter spp. isolates isolated from water samples. The tetO and tetW genes are found more frequently than other tet genes in commensal bacteria isolated from stool and water samples (Yang et al. 2010). Apart from one isolate, the tetW gene identified in the samples did not phenotypically produce tetracycline resistance. This result may indicate the existence of other tet genes providing tetracycline resistance or other antibiotic resistance mechanisms (Parisi et al. 2019; Isidro et al. 2020). These results considered that there was no direct correlation between the presence of tetO and tetW genes, which are known to be coding for tetracycline resistance, and simultaneous antibiotic resistance (Gungor et al. 2023). It might be explained by the presence of other genetic elements playing an important role in antibiotic resistance (Hedayatianfard et al. 2014).
When considered that a strain is multidrug resistant (MDR) if it is resistant to three or more classes of antibiotics; 81.8% of the strains isolated in this study presented an MDR phenotype, of which 84.2% were among A. butzleri and 100% were among A. cryaerophilus and A. skirrowii isolates. MDR Aliarcobacter has been previously reported by other authors in isolates from various food samples (Ferreira et al. 2017; Gungor et al. 2023), with findings which show MDR phenotypes are more common in A. butzleri than in A. cryaerophilus (Kabeya et al. 2004; Jasim et al. 2021; Gungor et al. 2023). Due to the deterioration of treatment of serious infections, also to the possible transfer of resistance markers through the consumption of contaminated food with microorganisms resistant to different antibiotics, resistance to antibiotics can be assumed as a relevant public health problem (Rahimi 2014).
The ability to form biofilm plays a role in the pathogenesis, bacterial virulence, and antibiotic resistance of Aliarcobacter spp., as in many foodborne bacteria (Šilha et al. 2019; Martinez-Malaxetxebarria et al. 2022). In this study, one (50%) A. cryaerophilus isolate obtained from cutting board sample showed moderate biofilm ability and MDR. A. skirrowi isolate demonstrated a strong biofilm formation ability. Different biofilm profiles were found for the A butzleri isolates. It was observed that almost half of the isolates (47.3%) were able to produce biofilm in three different levels. In many studies on the biofilm ability of Aliarcobacter spp., it has been reported that they have different levels of biofilm production capacity (Girbau et al. 2017; Šilha et al. 2019; Chaves et al. 2021; Salazar-Sánchez et al. 2022; Martinez-Malaxetxebarria et al. 2022; Gungor et al. 2023). The ability to form biofilms can vary greatly between strains of the same species, depending on exogenous factors such as nutrient availability, environmental conditions, and surface properties (Girbau et al. 2017).
In the ERIC-PCR, a visual comparison of the banding patterns showed 1–10 DNA fragments ranging in size from 100 to 1500 bp. Cluster analysis showed that the ERIC-PCR patterns were divided into 7 (A-G) main clusters. One singleton and Aliarcobacter spp. isolated from slaughterhouses samples showed genetic diversity and heterogeneous populations despite their ancestry. Gungor et al. (2023) grouped Aliarcobacter spp. isolates obteined from edible giblets into three main clusters (A-C) and one singleton. In addition, they were stated that the highest number of A. butzleri (9/17) isolates were grouped in cluster B with varying antimicrobial resistance patterns and high genetic diversity. In our study, A. butzleri isolate was grouped mostly in C and F clusters, with four isolates each.
In conclusion, the findings of this study support the hypothesis that slaughterhouses can serve as a possible route for transmission of human enteropathogens. However, to establish the significance of this potential hazard, it is necessary to elucidate the roles played by these genes in each strain and the number of virulent species that harbor these virulence-associated genes. Therefore, promising approaches are required to detect virulent strains of Aliarcobacter spp. and to investigate their virulence (in vitro and in vivo) in detail. Furthermore, the results reveal that Aliarcobacter spp. exhibit resistance to various commonly used antibiotics, emphasizing the need for further research into the associated resistance mechanisms. Given their ability to form biofilms and their as-yet-unknown pathogenesis, appropriate hygiene practices and control measures in slaughterhouses are essential to prevent the spread of Aliarcobacter spp.