Isolation, characterization, and antimicrobial resistance profiles of Campylobacter jejuni and Campylobacter coli from raw meat of large livestock in Iran

1. Introduction

Foodborne infections are caused by spoiled meals, especially red meat, including diseased meat or cadavers contaminated with harmful bacteria ( 1 , 2 ). Numerous foodborne organisms, including, Campylobacter spp., Salmonella enterica non-Typhi serovars, Shiga toxin-producing Escherichia coli isolates, and Listeria monocytogenes, have significant sources in food-producing livestock. Pathogenic large animals are responsible for millions of sporadic illnesses, chronic outcomes, and significant and difficult outbreaks in several countries ( 3 ). Pathogenic Campylobacter organisms are the most common foodborne pathogen, responsible for approximately 400-500 million illnesses per year ( 4 ). Various livestock, including camels, cattle, sheep, and goats, as well as wild animals, carry Campylobacter organisms in their digestive systems. Fecal matter is an important source of contamination that can enter cadavers through direct deposition ( 5 ). Campylobacteriosis can infect their meals when animals are killed and carcasses are dressed. Consumption of undercooked or cleaned meat, manipulation of raw items, cross-contamination of raw food with heated foods, bathing in natural waters, direct contact with contaminated animals or animal carcasses, and travel are ways that people can become ill ( 6 ). Campylobacter jejuni, Campylobacter rectus, Campylobacter hyointestinalis, Campylobacter insulaenigrae, Campylobacter sputorum, Campylobacter helveticus, Campylobacter lari, Campylobacter foetal, Campylobacter mucosalis, Campylobacter coli, Campylobacter upsaliensis, and Campylobacter ureolyticus are dangerous Campylobacter spp. associated with human illness. Campylobacter jejuni and C. coli are the most commonly observed zoonotic agents in humans and the most common agents of gastrointestinal infections worldwide ( 7 ).

Campylobacter jejuni is responsible for 90% of campylobacteriosis cases, followed by C. coli, accounting for 5-10% of cases ( 8 ). In addition, Campylobacter with antibiotic resistance has been associated with outbreaks throughout the world ( 9 ). The use of antimicrobials in meat animals has led to the establishment and spread of antibiotic-resistant bacteria, such as antimicrobial-resistant Campylobacter, which can be detrimental to human and animal health. In underdeveloped countries, where antimicrobial use is widespread and unregulated, the situation appears to be deteriorating even faster ( 10 ). In Iran, few studies have been conducted on the incidence and antibacterial tolerance of intestinal campylobacteriosis in humans ( 11 ) and animal-derived products. The lack of a national surveillance program, which limits the regular supply of cultures for Campylobacter spp. isolation in clinical practice and research, and the need for a selective medium and a specific growth environment make it difficult to accurately assess the impact of the disease in Iran ( 12 ).

The virulence of Campylobacter spp. depends on their virulome. Although relatively little is known about the virulence of Campylobacter spp., these microorganisms possess several virulence factors related to motility, adhesion, invasion, toxin activity, immune evasion, and iron uptake, among others ( 8 ).

This indicates that Campylobacter as a zoonotic disease is not receiving the attention it deserves, especially in the current research area. Therefore, the present study aimed to investigate the patterns of antimicrobial resistance, virulence genes, and genetic variation of thermophilic Campylobacter spp. obtained from a large livestock sample in Iran.

2. Materials and Methods

2.1. Ethical considerations

The Research Ethics Committee of the College of Veterinary Sciences, Islamic Azad University, Shahrekord Branch, Iran, reviewed and approved this work.

2.2. Research area and study design

The study was performed in Shahrekord City, Iran, between October 2020 and May 2021. Shahrekord is the capital of Chaharmahal and Bakhtiari province, where tens of thousands of large livestock from numerous districts of the region and surrounding areas can be slaughtered. The abundance and antibiotic resistance profiles of C. jejuni and C. coli were isolated, identified, and estimated from meat samples of large animals from slaughterhouses, butcher shops, and restaurants during a cross-sectional survey from October 2020 to May 2021.

2.3. Sample size and collection

A total of 550 fresh, ready-to-eat meat samples were collected from the slaughterhouses, butcher shops, and restaurants in the study region. The samples included meat from cattle (n=138), goats (n=102), camels (n=56), and sheep (n=254). To avoid spillage and cross-contamination, all samples were stored in polyethylene plastic packaging and immediately transferred to the molecular biology laboratory of the College of Veterinary Sciences, Islamic Azad University, Shahrekord Branch, using a refrigerator with ice packs.

2.4. Isolation and identification of Campylobacter spp.

The meat was placed on modified charcoal cefoperazone deoxycholate agar (mCCDA) (Oxoid Ltd., Basingstoke, Hampshire, England), including Campylobacter mCCDA selective additive, SR155E (Oxoid Ltd., Basingstoke, Hampshire, England) upon arrival at the laboratory. CampyGen^TM gas packets were used to create microaerophilic conditions, which were maintained at 37°C for 48 h (Oxoid, Basingstoke, England, United Kingdom). Campylobacter colonies, which are grayish, flat, moist, and readily spread, were subcultured on Mueller-Hinton agar enriched with 5% defibrinated horse blood and cultured for 48 h at 37°C under microaerophilic conditions. Campylobacter isolates were stored at 80°C in Mueller-Hinton broth containing 25% glycerol (v/v).

2.5. Extraction of DNA and identification of genus by polymerase chain reaction

The Qiagen QIAamp PowerFecal Kit (Qiagen, Hilden, Germany) was used to extract genomic DNA from pure cultures according to the instructions of the manufacturer. Multiplex polymerase chain reaction (PCR) was then performed using genus-specific primers (C412F and C1228R), C. jejuni cj0414 gene primers (C1 and C3), and C. coli ask gene primers (CC18F and CC519R) ( 8 ). The primers were selected for their ability to discriminate between Campylobacter genus and species. To prepare the PCR mixture (25 µl), 12.5 µl of 2X Master Mix (Thermo Fisher Scientific, Seoul, South Korea), 1 µl of primer (10 µM), 1.5 µl of template DNA (20 µg/ml), and 7 µl of sterile deionized water were used. The MiniAmp Plus Thermal Cycler (Applied Biosystems, MA, USA) was utilized to perform one cycle at 95°C for 5 min, 35 cycles at 94°C for 45 s, 55°C for 45 s, and 72°C for 45 s, and a final extension at 72°C for 5 min. The PCR products were stored at 4°C before analysis.

2.6. Antimicrobial susceptibility testing

The isolated Campylobacter spp. samples were tested for in vitro antibiotic susceptibility on Mueller-Hinton agar supplemented with 5% sheep blood (Oxoid Ltd., Basingstoke, Hampshire, England) using the standard agar disc diffusion method according to the recommendations of the Clinical and Laboratory Standards Institution (CLSI). The following 15 different antibiotic disks were used for antibiotic susceptibility testing with their concentrations indicated in parentheses: aphA-3-1, cmeB, tet(O), blaOXA-61, aadE1, GM10, CIP5, NA30, TE30, AM10, AMC30, E15, AZM15, CC2, and C30 (Oxoid Company, Hampshire, England). The size of the clear zones (zones of inhibition of bacterial growth around antibiotic discs, including the discs) was evaluated for each antibacterial agent and then classified as sensitive, intermediate, and resistant according to the CLSI interpretation table after 48 h of microaerophilic cultivation at 37°C.

2.7. Detection of antimicrobial resistance genes

Genes encoding antimicrobial resistance were determined by PCR experiments using the primers in table 1. The genes studied were: aphA-3-1 (gentamicin resistance), cmeB (efflux pump), blaOXA-61 (ampicillin (beta-lactam) resistance), tet(O) (tetracycline resistance), and aadE1 (aminoglycoside resistance) (Table 1) ( 9 ). After electrophoresis, bands of PCR products were observed under ultraviolet (UV) light using a dual UV transilluminator (Core BioSystem, Huntington Beach, CA, USA). The PCR procedure was performed as described above. After electrophoresis, the bands of PCR products were visualized under UV light using a dual UV transilluminator (Core BioSystem, Huntington Beach, CA, USA). The bands of amplification products were assessed by comparison with a 100-bp DNA ladder (Dyne bio, Seongnam-si, Republic of Korea). The antibiotic resistance gene PCR products underwent purification using AMPure XP beads (Beckman Coulter, Fullerton, CA, USA). These products were then sequenced through the Sanger technique at SolGent (Solutions for Genetic Technologies, Daejeon, Republic of Korea).

2.8. Detection of virulence genes

PCR was performed using specific primers for virulence-related genes (recR, wlaN, cdtB, cdtA, cdtC, virbll, flaA, pidA, cadF, ciaB, ceuE, cgtB, and dnaJ). The PCR mix (25 µl) was made by combining 12.5 µl of 2X Master Mix (Thermo Fisher Scientific, Seoul, South Korea), 1 µl of primer (10 µM), 1.5 µl of template DNA (20 µg/ml), and 7 µl of sterile deionized water. The MiniAmp Plus Thermal Cycler was used to run one cycle at 95°C for 5 min, 35 cycles at 94°C for 30 s, 55°C for 45 s, and 72°C for 45 s, and a final extension at 72°C for 5 min (Applied Biosystems, MA, USA).

2.9. Statistical analysis

All data were entered into a Microsoft Excel sheet (Microsoft Corp., Redmond, WA, USA) and analyzed in SPSS software (version 20). Associations were evaluated using the Chi-square test and logistic regression. A p-value of less than 0.05 was statistically significant for all experiments.

3. Results

3.1. Prevalence of Campylobacter spp. meat in large cattle

Two Campylobacter spp. (i.e., C. jejuni and C. coli) were found in 84 (15.27%) of the 550 meat samples. Cattle were much more likely to be infected with Campylobacter than with the other specimens. Cattle and camel samples were responsible for the highest (52.38%) and lowest (3.57%) frequency rates of infection with Campylobacter spp., respectively. As shown in figure 1 and table 2, there were significant differences in the prevalence of Campylobacter spp. in cattle compared to others (2=43.04 or odds ratio [OR]=7.68, confidence interval [CI]=3.40-17.30, P<0.01).

Figure 1. Prevalence of Campylobacter spp. among different sample types

3.2 Infection rates of Campylobacter jejuni and Campylobacter coli in different sample types

It was found that C. jejuni and C. coli accounted for 82.14% (n=69) of Campylobacter spp. isolated and characterized in the raw meat from cattle, goats, sheep, and camels. While C. jejuni was found in 39.28% of the samples (n=33), C. coli was found in 42.85% (n=36). Other Campylobacter spp. formed 17.85% (n=15) of the samples.

Based on the results, C. jejuni was detected in 59.09% (n=26), 11.11% (n=1), 21.42% (n=6), and 0% (n=0) of cattle, goat, sheep, and camel meat samples, respectively. The presence of C. coli was identified in 29.54% (n=13), 44.44% (n=4), 57.14% (n=16), and 100% (n=3) of cattle, goat, sheep, and camel meat samples, respectively (Table 2).

3.3 Polymerase chain reaction amplification results

The PCR products of the 84 samples showed that 39.28% (n=33) of the strains were C. jejuni (with a molecular size of 589 bp) and the rest 42.85% (n=36) were C. coli (with a molecular size of 462 bp) in terms of gene products. Campylobacter jejuni and C. coli accounted for 82.14% (n=69) of Campylobacter spp. isolated from raw meat. While C. jejuni was found in 39.28% of the samples (n=33), C. coli was detected in 42.85% (n=36). Other Campylobacter spp. accounted for 17.85% (n=15) (Figure 2).

Figure 2. Results of molecular analysis of Campylobacter isolates by PCR. L1: Negative control, L2-L5: Some C. coli samples; L6-L9: Some examples of C. jejuni; M: DNA marker 100 bp.

3.4 Distribution of genotype of Campylobacter spp. isolates

Tables 3 and 4 present the genotype pattern of Campylobacter spp. strains derived from different types of raw meat from large livestock. The most common genotypes observed in the collected C. jejuni bacteria were ciaB (100%), flaA (100%), recR (80.77%), cadF (76.93%), and dnaJ (76.93%), whereas wlaN (7.69%) and virbll (7.69%) were the least prevalent. Other genes were also detected in C. jejuni isolates from large livestock populations. There was a significant difference between the types of samples and the frequency of genotypes (P<0.05).

The genotyping pattern of the C. coli isolates is shown in table 4. Accordingly, ciaB (100%), flaA (100%), pidA (61.54%), and cadF (61.54%) were the most frequent genotypes found in C. coli bacteria from a series of large animal samples. On the other hand, dnaJ (0%), wlaN (0%), virbll (0%), and ceuE (0%) were detected with the lowest frequency in a series of large animal samples. Additional genes were identified in C. coli strains from large animal samples. There was a significant difference between the types of specimens and the prevalence of alleles (P<0.05).

3.5 Patterns of Campylobacter spp. isolates’ susceptibility to antimicrobials

Campylobacter spp. isolated from various sample types and sources were 100% sensitive to aphA-3-1 and GM10. The isolates were found to be resistant to E15 (76.93%), cmeB (69.24%), aadE1 (69.24%), CIP5 (69.24%), and AM10 (69.24%) (Table 5). As tabulated in table 5, 96.8% of the isolates showed resistance to two or more drugs.

According to the antibiogram results, the C. jejuni isolates demonstrated the highest sensitivity to aphA-3-1. In contrast, based on table 5, C. jejuni had the highest resistance to E15.

Sensitivity to antimicrobials was the highest in C. coli isolates aphA-3-1 (100%) and GM10 (100%) in cattle (Table 6). Moreover, C. coli isolates had the lowest susceptibility rate to E15 (23.07%). In addition, resistance to cmeB (69.24%), blaOXA-6 (69.24%), aadE1 (69.24%), CIP5 (69.24%), and AM10 (69.24%) were common. The results showed that most C. coli isolates from the samples of large livestock were resistant to at least three antibiotics. These isolates exhibited multiple drug resistance phenotypes. There was a statistical difference between the specimens and the frequency of antimicrobial resistance (P<0.05).

4. Discussion

In the current study, 15.27% of the 550 meat sample isolates were analyzed positive for Campylobacter species. Cattle meat samples were found to have the highest incidence (52.38%). Beef showed a twofold increased risk of Campylobacter compared to sheep, goat, and camel meat. The incidence of Campylobacter was found to vary significantly among meat samples (2=43.04 or OR=7.68, CI=3.40-17.30, P<0.01). The prevalence of Campylobacter spp. in meat samples was 52.38%, which was consistent with the values reported by Dabiri et al. (44%) ( 13 ), Rahimi et al. (56.1%) ( 14 ), and Habib et al. (48.02%) ( 15 ). This was more than the frequency of 1.93% reported by Marinou et al. ( 16 ). However, the current finding was lower than the prevalence of Campylobacter spp. reported by Rahimi et al. (61.7%) in Ahvaz, Iran, ( 17 ) and Pezzotti et al. (81.3%) in northern Italy ( 18 ). Large cattle were found to be a significant source of Campylobacter compared with other livestock, and cattle were reported to be strong gastrointestinal carriers of Campylobacter. In different countries, fresh meat showed a wide range of Campylobacter abundance (0-90%). These discrepancies in Campylobacter spp. abundance could be attributed to hygienic conditions, cross-contamination from de-feathering and excoriation, or some other environmental components.

In this study, the percentage of Campylobacter spp. in cattle meat was obtained at 52.38%. This was more than the results of studies performed in Nigeria (12.9%) ( 19 ) and Iran (10%) ( 20 ). However, it was more significant than the results in Ethiopia (6.2%), Morogoro, Tanzania (5.6%), and Australia (0.8%) ( 21 ). Food derived from animals is considered a major cause of Campylobacter infections in humans ( 22 ). Because raw beef is commonly used in this country, the presence of Campylobacter in meat increases the risk of infection in humans. The current result was lower than those of previous studies, which showed prevalence rates of 69.1% and 22%, respectively ( 23 ).

Differences in detection techniques for thermophilic Campylobacter, particularly the lack of an enrichment method for the separation of thermophilic Campylobacter in the work of Chen et al., are one of the most likely explanations for the discrepancies. These discrepancies could be caused by changes in sample collection methods, isolation and identification procedures, and sample size ( 24 ).

Campylobacter spp. was detected in 9.0% of goat meat. This result was consistent with those of previous studies reporting the prevalence of 7.6% and 6.4% ( 25 ). However, it was slightly higher than 4.4%, which was reported in the previous study. However, the current study had lower results than earlier studies, which showed 41.2% and 27.5%, respectively ( 26 - 28 ). Microbiological analysis and PCR identification of the isolated Campylobacter strains revealed that C. jejuni was more prevalent than C. coli in the present study. Campylobacter jejuni was identified as the most common spp. derived from animal-based foods, particularly beef ( 29 ). In previous studies, C. jejuni and C. coli were detected in 76% and 24% of beef, goat meat, and chicken, respectively ( 30 ). These results were consistent with those of a previous study demonstrating the detection of 78% of C. jejuni and 18% of C. coli ( 31 ). The incidence of C. jejuni in raw meat was consistent with previous studies from other countries ( 32 ).

Campylobacter antibiotic resistance is a worldwide problem that has already been identified by serval researchers and recognized as a public health problem by the World Health Organization. Antibiotic resistance to Campylobacter spp. (C. jejuni and C. coli) can be transmitted to humans in serval ways. This circumstance underscores the need for Campylobacter antibiotic susceptibility testing. The drug of choice for the treatment of foodborne campylobacteriosis are mainly macrolides and fluoroquinolones ( 33 , 34 ). Previous studies in Ethiopia found that 80%-100% of food animal strains were sensitive to antimicrobials. However, data from various parts of the world indicate that antibiotic resistance is increasing in both food animals and human isolates ( 35 , 36 ). In this study, the antibiotic susceptibility patterns of C. jejuni and C. coli strains were investigated. The percentage of Campylobacter strains with aphA-3-1 and GM10 susceptibility was 100%. This was consistent with the 97.2% and 83.3%, respectively, reported in the previous studies. In addition, Toledo et al. observed a C. coli resistance level of 100%, and reported that C. coli strains were often more resistant than C. jejuni strains ( 37 ). Despite international promises to reduce antibiotic resistance and ensure antimicrobial efficacy, most countries have failed to implement government policies to decrease the overuse and misuse of antibiotics ( 38 ). In countries such as Iran, where there is no uniform regulation or guidelines for therapeutic interventions, antibiotics can be purchased without medication for humans or animals, and antimicrobials are often overprescribed by healthcare workers and veterinarians and overused by the general public ( 39 ). In addition, new resistance mechanisms are emerging and spreading worldwide. As a result, antimicrobial resistance is rapidly increasing in all regions of the world.

The virulence of Campylobacter spp. depends on their virulome ( 8 ). The most common genotypes observed in C. jejuni bacteria collected from different types of large animal samples were ciaB (100%), flaA (100%), recR (80.77%), cadF (76.93%), and dnaJ (76.93%). On the other hand, wlaN (7.69%) and virbll (7.69%) were the C. jejuni strains with the lowest incidence reported in a variety of large livestock specimens. The most common genotypes found in C. coli bacteria from a variety of large animal samples were ciaB (100%), flaA (100%), pidA (61.54%), and cadF (61.54%). The diagnostic accuracy was consistent with that reported in a recent report from Korea ( 40 ), however, higher than that reported in South Africa ( 41 , 42 ) and Chile ( 43 ). The discrepancy may be due to the complexity of the colonization process, which involves several genes, as well as the use of isolates from a single sampling site ( 43 ).

In this study, Campylobacter isolates were characterized by the detection of specific resistance and virulence factors, which is limited to understanding the mechanisms of resistance and virulence. The results of whole genome sequencing analysis can determine the epidemiology and evolutionary pathways of Campylobacter spp. to better tailor measures to reduce campylobacteriosis cases in Iran.

In conclusion, Campylobacter spp. collected from raw meat of large livestock in this study showed significant antibiotic resistance and carried various virulence and antimicrobial resistance genes. These strains can pose a public health risk. The intensive use of antibiotics in large livestock farming is responsible for the increase in multidrug-resistant-Campylobacter isolates. Antibiotic resistance in pathogenic bacteria can be reduced by monitoring antibiotic resistance in Campylobacter and appropriate administration of antimicrobials in feed production.

Acknowledgment

The authors would like to thank the owners, managers, and employees of the numerous slaughterhouses, butcher shops, and restaurants in the study area for their keen interest and cooperation in collecting the meat samples. The authors would also like to appreciate Dr. Reza Sherafati for his assistance in the sampling process.

Authors' Contribution

ER and BM performed the sampling and culture method, carried out the molecular genetic studies, participated in primer sequence alignment, and drafted the manuscript. ER and AS participated in the design of the study, performed the statistical analysis, and wrote the manuscript. All authors read and approved the final manuscript.

Ethics

This research was conducted as part of a PhD thesis in food hygiene and was ethically approved by the Research Council of the Faculty of Veterinary Medicine, Shahrekord Branch, Islamic Azad University, Shahrekord, Iran. The review of this research project and licenses for sampling were approved by Professor Ebrahim Rahimi (Approval Ref Number MIC19817).

Conflict of Interest

The authors declare that they have no competing interests.

Funding statement

This research received no specific grants from public, commercial, or nonprofit entities.

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