Genotyping, antibiotic resistance and prevalence of Arcobacter species in milk and dairy products

Abstract Background Arcobacter spp. has been considered an emerging foodborne pathogen and a hazard to human health. The dairy chain has been isolated from different sources; nevertheless, data on Arcobacter occurrence in raw milk and dairy products in Iran are still scant. Objective The present study investigates the prevalence, antimicrobial susceptibility and the presence of virulence genes of Arcobacters species isolated from milk and dairy products. Methods Then, a total of 350 raw milk samples and 400 dairy product samples were collected from dairy supply centers in Isfahan, Iran. Presumptive Arcobacter strains were obtained by enriching samples in Oxoid Arcobacter enrichment broth (AEB) followed by the filtration of enrichment product through 0.45‐μm pore size membrane filters laid onto non‐selective blood at 30°C under microaerophilic conditions. Molecular identification of Arcobacter cryaerophilus and A. butzleri was performed by Polymerase chain reaction (PCR) amplification of the 16S rRNA gene, followed by sequencing. The disc diffusion method was used to determine the antimicrobial susceptibility of isolates. Targeted resistance and virulence genes were detected using multiplex PCR. Results The results show a low recovery rate of Arcobacter spp. in milk. Arcobacters were found in all types of milk, except raw camel milk, but were absent from all dairy products. Arcobacter butzleri was the predominant species in raw milk. Detection of virulence genes shows that all virulence genes targeted were found among A. butzleri, and six (cadF, cj1349, irgA, mviN, pldA, tlyA) were found among A. cryaerophilus. All A. butzleri strains and some A. cryaerophilus strains isolated from milk were resistant to amoxicillin‐clavulanic acid and tetracycline. All A. cryaerophilus isolates from milk were susceptible to gentamycin, streptomycin, erythromycin and ciprofloxacin. The distribution of resistance genes in Arcobacter strains in milk shows that all isolates carried tet(O) and blaOXA‐61 genes. Conclusions In conclusion, the results indicate a low recovery rate of Arcobacter spp. in milk and milk products. However, a significant number of Arcobacter strains with putative virulence genes may be potential pathogens for humans and an overall increase in Arcobacter resistance to first‐line antibiotics. These results highlight the need for regular surveillance of Arcobacter strains in milk and milk products in Iran.

Regarding dairy animals, Arcobacters have been widely reported to be isolated (Piva et al., 2013;Shah et al., 2013) and have been found in various sources, including raw milk and fresh cheese (Ertas et al., 2010;Shah et al., 2012;Yesilmen et al., 2014). Due to the complexity of operations in the dairy production chain, Arcobacter contamination can occur in several ways . Indeed, Arcobacters have been found in bulk milk tanks (Elmali & Can, 2017;Ertas et al., 2010), milking equipment, barn floors, inline filters in milking machinery and cheese (Giacometti et al., , 2015Serraino et al., 2013). In Iran, the dairy sector is one of the leading traditional sectors, and economic activities and milk production have increased to a level of about 9 billion kg of milk per year (Beldman et al., 2017). With the high demand, the sale of raw milk for direct consumption may have increased human exposure to zoonotic agents (Haran et al., 2012). Numerous studies in Iran recovered Arcobacters species from animal products (Khodamoradi & Abiri, 2020;Rahimi, 2014;Shirzad Aski et al., 2016), but data about the occurrence of Arcobacters in milk and dairy products in Iran are scant. In addition, the isolation of resistant Arcobacter species from animal products with virulent and pathogenic determinants has been increasingly reported (Goojani et al., 2020.;Karadas et al., 2013;Sekhar et al., 2017;Tabata, 2014). In this respect, the present study investigates the prevalence, antimicrobial susceptibility and presence of virulence genes of Arcobacter species isolated from milk and dairy products collected from dairy supply centres in Isfahan, Iran.

Sampling
Samples analysed in the current study were collected randomly from dairy supply centres in Isfahan, Iran. The samples consisted of raw milk from various animals (bovine, ovine, caprine, buffalo and camel) and traditional dairy products (cheese, cream, butter and ice cream). All samples were aseptically collected in separate sterile plastic bags to avoid cross-contamination and were kept in a cooler with ice packs until they arrived at the laboratory for microbiological analysis. A total of 350 raw milk samples and 400 dairy product samples were collected.

Isolation of Arcobacters
Isolation of Arcobacters was performed following the method described by Atabay et al. (2003). Samples were mixed using a vortex mixer at room temperature.

Molecular identification of Arcobacters
Template DNA was extracted from presumptive Arcobacter isolates using PrepMan Ultra Reagent (Applied Biosystems) following the manufacturer's instructions. Molecular identification of A. cryaerophilus and A. butzleri was performed by amplification of the 16S rRNA gene using PCR, followed by sequencing. The resulting sequence was compared to known sequences of the 16S rRNA gene in GenBank by multiple sequence alignment (Lau et al., 2002).

F I G U R E 4 Antimicrobial resistance genes in A. butzleri strains in milk
milk was resistant to ampicillin, cephalothin, nalidixic acid and tetracycline. Figure 5 shows the distribution of resistance genes in A.
cryaerophilus strains in milk. All A. cryaerophilus isolates from milk carried the tet(O) and bla OXA-61 genes. All targeted resistance genes were found in isolates from bovine raw milk. None of the isolates from ovine raw milk had aphA-3 and aadE1. Figures 6 and 7 show the results of the PCR assay for the identification of 16S rRNA genes, virulence genes and resistance genes in Arcobacter isolates.

DISCUSSION
Arcobacter spp. is related to human and animal disease, and it is con- Arcobacters in milk but also in dairy products, including cheese Yesilmen et al., 2014). Numerous factors, including the experimental design, sample size and identification/isolation method used, influence the recovery rate in field studies of Arcobacter spp. or specific Arcobacter species in animals and animal products (Ho et al., 2006). Pasteurisation or sterilisation of milk before processing into dairy products may explain the absence of Arcobacters in the collected dairy products. The absence of was the most isolated species in raw milk and was found in each type of milk sample, while A. cryaerophilus was present in bovine and ovine raw milk. This finding is in line with some research showing that A. butzleri, followed by A. cryaerophilus, are the most commonly found species in milk and dairy products Yesilmen et al., 2014). Arcobacter butzleri is a pathogen responsible for diarrhoea and septicemia in humans and is frequently isolated from milk and dairy products (Parisi et al., 2019). It is the most recovered species because it has an inherent ability to survive in different environments and under extremely harsh conditions (Badilla-Ramírez et al., 2016;Giacometti et al., 2015;Ramees et al., 2017). In addition, Arcobacter species, including A. cryaerophilus and A. skirrowii, are more susceptible to antimicrobials and other components used in isolation media, making them more difficult to isolate (Atabay et al., 1998;Houf et al., 2001).
However, the contribution of these genes in each strain needs to be elucidated through both in vitro and in vivo approaches (Kim et al., 2019).
Determination of antimicrobial resistance patterns is vital for a better choice of antibiotic as a first-line drug for treating Arcobacter infection (Houf et al., 2004;Vandenberg et al., 2006). In the present study, all A butzleri strains and some A. cryaerophilus strains isolated from milk were resistant to amoxicillin-clavulanic acid and tetracycline. This is not the case in the study by Elmali and Can (2017), who found tetracycline to be the most effective antibiotic. Similar to our study, several authors found some isolates exhibiting resistance to gentamycin (Elmali & Can, 2017), streptomycin and tetracycline (Goojani et al., 2020), cephalothin (Atabay & Aydin, 2001;Rahimi, 2014), erythromycin and ciprofloxacin (Atabay & Aydin, 2001;Son et al., 2007) and ampicillin (Shah et al., 2013). All A. cryaerophilus isolates from milk were susceptible to gentamycin, streptomycin, erythromycin and ciprofloxacin. This result follows those obtained by Vidal-Veuthey et al. (2021), who reported that all Arcobacter strains were susceptible to four antibiotics evaluated in his study, including erythromycin and ciprofloxacin, tetracycline and gentamicin. Differences in the susceptibility patterns could be explained by the frequency of drugs in animals for treatment and/or prophylaxis, the lack of standardisation for Arcobacter antimicrobial susceptibility tests and the absence of established breakpoints (Rahimi, 2014). The distribution of resistance genes in Arcobacter strains in milk show that all isolates carried tet (O) and bla  genes. This indicates that tetracycline and beta-lactams are frequently used antibiotics in dairy animal production. The presence of the tet(o) gene in all Arcobacter strains isolated from milk is consistent with the resistance of these strains to the antibiotic tetracycline (Connell et al., 2003). The high resistance observed among Arcobacter strains to beta-lactam antibiotics, including amoxicillin and ampicillin, is confirmed by the presence of the blaOXA-61 genes encoding beta-lactamase production in all isolates (Forson et al., 2020). The CmeB gene present in some strains may confer the resistance observed in some isolates to several antibiotics by decreasing porin expression (Cagliero et al., 2006). The presence of the aadE gene in Arcobacters highlights the possibility of genetic transfer of information from grampositive to gram-negative bacteria, which explains the rarely observed resistance to antibiotics such as gentamycin (Pinto-Alphandary et al., 1990).
All targeted resistance genes in some isolates are due to the accumulation of many antibiotic resistance genes by Arcobacters species (Millar & Raghavan, 2017).

CONCLUSION
Arcobacter species are emerging human pathogens of animal origin.
The current study shows a low recovery rate of Arcobacter spp. in milk and their absence in dairy products. Pasteurisation or sterilisation of milk before processing into dairy products effectively reduces the occurrence of Arcobacters in these products. Antimicrobial susceptibility testing shows increasing resistance to first-line antibiotics used in clinical and veterinary settings. Detection of virulence and resistance genes showed that all targeted genes were found among Arcobacter strains. Then, handling raw milk and its direct consumption may expose humans to dangerous zoonotic agents such as Arcobacter species. These results also highlight the need for regular surveillance of Arcobacter strains in milk and milk products in Iran.

ACKNOWLEDGEMENT
We especially thank M. Momeni Shahraki for his support.

ETHICS STATEMENT
This study was approved by the Shahrekord Branch, Islamic Azad University Ethical Committee.

AUTHOR CONTRIBUTION
All authors read and approved the final manuscript.

CONFLICT OF INTEREST
The authors declare no conflict of interest.

DATA AVAILABILITY STATEMENT
All data generated or analysed during this study are included in this article. The datasets used and/or analysed during the current study are also available from the corresponding author.