High prevalence of antibiotic resistance in pathogenic foodborne bacteria isolated from bovine milk

This study aimed to investigate the prevalence of foodborne pathogenic bacteria in bovine milk, their antibiogram phenotype, and the carriage of antibiotic resistance genes. Raw bovine milk samples (n = 100) were randomly collected from different suppliers in the northwest of Iran. Antibiotic-resistant patterns and the presence of antibiotic resistance genes were evaluated in the isolates. Escherichia coli, Listeria monocytogenes, Staphylococcus aureus, and Salmonella spp. were isolated from 78%, 47%, 25%, and 21% of samples, respectively. All isolates showed high rates of resistance to amoxicillin, penicillin, and cefalexin. The blaTEM and blaSHV genes were detected in 50.0% and 6.4% of E. coli isolates, respectively. Also, 28.5% and 19.0% of Salmonella isolates were positive for blaTEM and blaSHV. The frequency of mecA and blaZ in S. aureus isolates was 20.0% and 12.0%, respectively. The high prevalence of bovine milk contamination with antimicrobial-resistant species in this study necessitates precise control on antibiotic prescription in veterinary medicine.

Isolation and detection of pathogenic bacteria. Eecherichia coli was isolated from samples according to the method of Feng et al. 25 and Ombarak et al. 26 Three to five presumptive colonies (dark centered and flat colonies with metallic green sheen) from Levine's Eosin Methylene Blue (L-EMB) agar plates were selected, transferred on tryptic soy agar (TSA), and incubated at 37 °C for 24 h. Biochemical confirmatory tests were performed according to the method of Feng et al. 25 and Quinn et al. 27 .
Staphylococcus aureus was detected in the samples using Baird-parker agar. After incubation of plates at 37 °C for 48 h, typical black colonies with a clear zone were considered as presumptive S. aureus. The isolates were confirmed by biochemical tests such as coagulase, catalase, DNase, lecithinase, oxidase, Lysostaphin sensitivity, VP, urease, glucose, and mannitol fermentation 28 .
For isolation and detection of L. monocytogenes, samples were enriched in Buffered Listeria enrichment broth (BLEB) at 30 °C for 48 h. The bacterial suspension was streaked onto PALCAM agar and incubated at 35 °C for 48 h. The isolates were confirmed by motility test, gram staining, and biochemical tests such as catalase, oxidase, hemolysis, nitrate reduction, carbohydrate fermentation, Christie-Atkins-Munch-Peterson test (CAMP), methyl red, and Voges-Proskauer (MR/VP) 29 .
For isolation and detection of Salmonella spp., the raw milk samples were cultured on Bismuth Sulphite agar (BSA), Brilliant Green, and Phenol-Red agar (BGA) for 24 h (BGA)/48 h (BSA) at 37 °C. The suspected colonies were transferred to Samonella-Shigella agar plates and incubated at 37 °C for another 24 h. The presumptive colonies on the plates were subjected to biochemical tests using Lysine Iron agar, Triple Sugar Iron Agar, Sulfide-Indole-Motility medium, and Christensen's Urea agar 30 . Antimicrobial susceptibility test. Antibiotic susceptibility tests of isolates were performed by the Kirby-Bauer disk diffusion method according to the guidelines of clinical laboratory standards 31 . Isolates were included in the study based on isolation rank (time criterion). Based on this criterion, the first isolate of a particular species isolated from a single sample was included in the analysis 32 . Briefly, bacterial suspensions were prepared in tubes containing 0.9% (w/v) phosphate-buffered saline with turbidity adjusted to 0.5 McFarland standard. Using a sterile cotton swab, bacterial suspension was streaked uniformly on the surface of Muller-Hinton agar. Antibiotic disks (Padtan Teb, Iran) including amoxicillin (25 μg/disk), azithromycin (15 μg), penicillin (10 IU), cephalexin (30 μg), ceftriaxone (30 μg), gentamicin(10 μg), chloramphenicol (30 μg), and tetracycline (30 μg) were placed on the surface of cultures. The selected antimicrobials were representative of the major classes of antibiotics commonly used in veterinary and human medicine in Iran. Finally, the diameter of the inhibition zone around the disks was measured after incubation of plates at 37 °C for 24 h.
Detection of bla TEM , bla SHV , mecA, and bla Z genes using multiplex-PCR. The genomic DNA was extracted by boiling method 33 . The primers used for the detection of target genes are listed in Table 1. The reaction contents for each 25 μL PCR consisted of 5.5 μL of deionized water, 12.5 μL RED-Extract-N-Amp master mix 2 × (containing buffer, salts, dNTPs, Taq polymerase, REDTaq dye, and JumpStart Taq antibody) (Sigma-Aldrich, USA), 1 μL of each primer and 3 μL of template DNA. The PCR program for bla TEM and bla SHV genes included initial denaturation for 5 min at 94 °C followed by 32 cycles of denaturation at 94 °C for 30 s, annealing step at 54 °C for 30 s, extension step at 72 °C for 60 s, and a final extension step at 72 °C for 10 min. The PCR condition for mecA and bla Z were as follows: initial denaturation at 95 °C for 4 min, 30 cycles of denaturation at 95 °C for 60 s, annealing step at 58 °C for 60 s, extension step at 72 °C for 60 sand final extension step at 72 °C for 4 min. PCR products were subjected to electrophoresis using 1.5% (w/v) agarose gel. The gel was stained with ethidium bromide. Ultraviolet transillumination (Biorad, USA) was applied for the visualization of DNA.

Results and discussion
Several studies have revealed that food products such as raw milk and dairy products made from raw milk may be the main sources for the outbreak of antibiotic-resistance pathogens which are known as a challenge for the safety of food products 38 . This problem is common in developing countries such as Iran, because of the poor food handling practices, inadequate food safety regulations, weak hygienic practices, insufficient financial resources to invest in food safety, weak regulatory systems, and inadequate education for food handlers. In the countries  39 . Numerous researchers previously reported the antimicrobial resistance of E. coli and Salmonella isolates from raw milk to the most common antibiotics in their studies [39][40][41][42] . Also, methicillin-resistant S. aureus as an emerging pathogen has become an important challenge for public health that has been isolated from raw milk 11,43 . The multidrug-resistant of L. monocytogenes isolates from raw milk to some commonly used antibiotics is reported in various countries such as Ethiopia 44 , Turkey 45 , Egypt 46 , and Pakistan 47 . So, the present study was designed to study the occurrence of the most common antibiotic-resistant foodborne pathogens from raw milk in Iran.
Totalmesophilic bacterial count, isolation, and identification of bacterial species. The mean total mesophilic bacterial count of the examined raw milk samples in this study was 5.75 ± 0.85 log 10 cfu mL −1 which was exceeded the permitted maximum value of raw milk contamination (5 log 10 cfu mL −1 ) 48 . Our findings of the high rate of contamination in raw milk are in agreement with that of the previous study conducted in Tabriz, indicating the poor microbial quality of raw milk delivered to pasteurized milk plants 4 . In another study which was conducted in Allahabad city (India), the total bacterial count of examined milk samples was reported between 4.79 log 10 cfu mL −1 by Yadav et al. 48 . Even, a higher level of contamination of about 6.32 ± 0.03 log 10 cfu mL −1 was found for the raw milk samples from the collection centers of Guwahati city in India 49 . In general, the total bacterial count of more than 6 log 10 cfu mL −1 reported by many countries is not desirable for raw milk supplies and is not usable for human consumption 50 .
The increased total bacterial count can be caused by the use of unsanitary equipment for milking, contamination of cow's udders, inadequate cooling of milk, and occasionally by the milking of cows with mastitis 51 .
In the present study, 78% of samples were contaminated with E. coli with a mean count of 3.41 ± 0.41 log 10 cfu mL −1 . High rates of raw milk contamination with E. coli have been reported in many developing and developed countries. It has been reported that 90.67% of raw milk samples in Arusha, Tanzania were contaminated with E. coli 52 as well as 76.4% of samples in Egypt 26 .
In our study, 25% of the raw milk samples were contaminated with S. aureus at an average level of 2.91 ± 0.80 log 10 cfu mL −1 . In agreement with our study, a study in California showed that 25.3% of the raw milk samples were contaminated with S. aureus 5 . In another study in Mansoura City, Egypt, the mean S. aureus counts were found to be 3.49 log 10 cfu g −1 in raw milk samples 43 , and 70.4% of raw milk samples in Brazil were contaminated with S. aureus 53 . These results indicate the different quality of milk samples in different regions of the world.
According to the results of the present study, L. monocytogenes was isolated from 47% of the raw milk samples. Over 70% of positive samples contained L. monocytogenes at a level of less than 10 cfu ml −1 . The mean count of this bacterium was detected at 0.60 ± 0.51 log 10 cfu mL −1 . Many studies in different countries reported the occurrence of L. monocytogenes by various rates of contamination in their raw milk supplies and related products. The occurrence of L. monocytogenes in raw milk has been reported in Kars city (Turkey) 45 . However, in research in Antakya, Turkey, L. monocytogenes was not detected in any of the raw milk samples 54 .
In the present study, Salmonella spp. was detected in 21% of the raw milk samples. After enrichment of samples followed by plating, the mean count of Salmonella spp. in the positive samples was detected at 0.26 ± 0.27 log 10 cfu mL −1 . Similar results have also been reported in different countries. The prevalence of Salmonella spp. in raw milk has also been reported in Arusha, Tanzania (37.33%) 52 , Egypt(44.44%) 55 , and Dhaka Metropolis, Bangladesh (25.71%) 56 .
Antimicrobial susceptibility of isolates to the used antibiotics and detection of bla TEM ,bla SHV , mecA, and bla Z genes in the isolates. In this study, it was shown that all strains of E. coli were highly resistant to penicillin (88.46%), cefalexin (82.05%), and amoxicillin (70.51%) ( Table 2). Fifty percent (50%) of E. coli isolates had bla TEM and 6.41%of them were positive for bla SHV (Table 3). Consistent with our study, another Table 1. PCR sets used for detection of target antibiotic-resistance genes in the selected foodborne bacterial isolated from bovine milk.  42 .
In the present study, bla TEM was the most common resistance gene in E. coli isolates. However, only 50% of the resistant isolates to both penicillin and amoxicillin harbored this gene. Also, bla SHV was present in five isolates of E. coli. All isolates containing this gene showed resistance to cephalexin, penicillin, and amoxicillin in phenotypic experiments.
The isolated strains of L. monocytogenes in our study were highly resistant to penicillin, cefalexin, and amoxicillin (97.87%) ( Table 4). Since ampicillin is an important first-choice antibiotic for the treatment of listeriosis 57 , the isolates of L. monocytogenes were evaluated for the presence of known genes responsible for resistance to beta-lactam antibiotics (bla TEM , bla SHV, mecA, bla Z ) using the specific primers. However, none of the resistance genes were detected in L. monocytogenes (Table 3). Similar results were found by Marian et al. 58 that showed 100% of L. monocytogenes strains in their study were resistant to ampicillin and penicillin, with no involvement of bla Z and mecA genes in their resistance. Also, Bertsch et al. 57 examined the antimicrobial susceptibility and antibiotic resistance genes in foodborne, clinical, and environmental isolates of L. monocytogenes that were negative for the presence of bla Z and mecA genes.  www.nature.com/scientificreports/ The results of antimicrobial resistance tests showed that the isolated strains of Salmonella were highly resistant to penicillin (100%), cefalexin (100%), and amoxicillin (71.42%) ( Table 5). High rates of antibiotic resistance for Salmonella spp. have been reported by many studies. In a study by Obaidat and Stringer (2019), more than 50% of S. enterica isolates in raw milk were resistant to kanamycin, streptomycin, amoxicillin, and tetracycline. In another study, the highest rate of antibiotic resistance for Salmonella was detected to ampicillin, chloramphenicol, streptomycin, sulfonamide, tetracycline, amoxicillin, ceftiofur, and ceftriaxone 41 . These results were consistent with the results obtained from the present study.
In this study, six (28.57%) and 4 (19.04%) isolates of Salmonella spp. were positive for bla TEM and bla SHV , respectively (Table 3). Four isolates with multidrug resistance to penicillin, ceftriaxone, amoxicillin, and cephalexin, carried both bla TEM and bla SHV genes. In a study by Ranjbar et al. 62 the frequency of Salmonella spp. with bla TEM and bla SHV genes was 29.9% and 2.89%, while the prevalence of these two genes in Salmonella in another study was reported 15.38% and 12.82%, respectively 63 . The results of these studies were in agreement with the present study.
The presence of the mecA gene was found in five (20%) isolates of S. aureus and the bla Z gene was positive in three (12.00%) isolates of S. aureus (Table 3). Notably, S. aureus isolates with phenotypic resistances to penicillin, amoxicillin, ceftriaxone, and cephalexin always harbored mecA and bla Z either individually or concurrently. These two genes are common genes involved in the antibiotic resistance of S. aureus strains. The electrophoresis pattern of the PCR products of the resistance genes in the bacteria under this study is shown in Figs. 1, 2 and 3.
The bla Z and mecA were identified as resistance genes in S. aureus isolated from subclinical mastitis in Egypt 65 . In another study that investigated the genomic profile of S. aureus isolates from bulk tank milk and dairy cows with clinical mastitis, the prevalence of bla Z gene was detected in 17.2% of isolates 66 .
Resistance to penicillin, amoxicillin, cephalexin, and ceftriaxone was more prevalent than the associated antibiotic resistance genes between isolates. The discrepancies between the phenotypic resistances and associated resistance genes in this study may be due to the fact that the entire suite of resistant genes, which could result  www.nature.com/scientificreports/ in phenotypic resistance, was not evaluated in this study. Also, it is possible that the antibiotic-resistant genes detected may be mutated and/or non-functional, inducible or not expressed. Other mechanisms of resistance such as multidrug efflux pumps, mutations in outer membrane porins, or other unknown resistance genes may be involved in the phenotypic resistance 67,68 .
In the present study, high resistance levels and multidrug resistances against up to 7 antibiotics were detected between the evaluated isolates, with a high proportion for beta-lactams. Since beta-lactams are the most commonly used antibiotics in veterinary medicine, the emergence of beta-lactam-resistant pathogenic bacteria can be a serious threat to the wide use of these drugs 69 .
The occurrence of antibiotic-resistance pathogens in raw milk can be directly affected by farm management and practices. Regular cleaning of the farm can decrease the prevalence of antibiotic resistance pathogens 70 . The types of animal breeding (intensive, semi-intensive, or free-ranging) can influence the occurrence of antibiotic resistance pathogens due to the inappropriate administration of antibiotics. Excessive use of antibiotics in therapeutic and sub-therapeutic levels in dairy cattle farms can result in the presence of antibiotic-resistant pathogens in raw milk. So, if raw milk is not heat-treated, the presence of antibiotic-resistant foodborne pathogens in raw milk may pose food safety hazards to humans 70,71 .  www.nature.com/scientificreports/

Conclusion
Our results show that raw milk has a great potential for transmission of antibiotic-resistant pathogens such as E. coli, S. aureus, L. monocytogenes, and Salmonella spp. In the present study, high levels of resistance were observed among the screened isolates to the most common beta-lactams such as amoxicillin, penicillin, and cefalexin. Also, the prevalence of beta-lactamase genes in E. coli, S. aureus, and Salmonella spp. provided evidence on the high risk of resistant food-borne pathogens to humans through raw milk.
Since antibiotics have extensive applications in dairy cattle farms in developing countries such as Iran; the microbiota of raw milk may contain relatively high levels of antibiotic-resistance bacteria. Therefore, enhancing the safety of milk and implementing good manufacturing practices are extremely important for the health of consumers. Pasteurization of raw milk, prevention of cross-contamination, storage of raw milk in cold temperature, appropriate authority supervision, and regulatory monitoring on the use of antibiotics in dairy cattle farms are necessary to ensure the safety of milk and dairy products.
The main route for the contamination of raw milk with resistant bacteria can be the subject of future studies to determine whether these bacteria get into the milk via cow's udder or mixed into the milk during or after milking. Since phylogenetic assays can be used to ensure the genetic variations of resistant bacteria; it is recommended that these assays be performed on foodborne pathogenic isolates in future studies.

Data availability
The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.  www.nature.com/scientificreports/ Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http:// creat iveco mmons. org/ licen ses/ by/4. 0/.