INTRODUCTION
The utilization of antimicrobial agents stands out as a highly economical approach to maintain or improve the health and feed efficiency of animals raised in the context of traditional agricultural methods (1). In intensive animal production, particularly within the pig production sector, antimicrobial usage rates rank among the highest, considering both absolute values and treatment incidence (2).
Excessive and improper utilization of antimicrobials in veterinary medicine has resulted in the rise of antimicrobial resistant bacteria (1). In light of the risk posed by the prevalence of infectious diseases stemming from non-treatable, multi-drug resistant bacteria to antimicrobials, considerable endeavors need to be undertaken to curb their emergence and dissemination (3). Hence, there is a necessity to enhance the management of bacterial infections through methods alternative to the use of antimicrobial agents.
All-In/All-Out (AIAO) is a management strategy in pig farming where all animals within a specific production group are introduced into a facility at the same time and are subsequently removed together when the production cycle is complete (4). Production systems employing AIAO practices enhance both growth performance and feed efficiency while concurrently mitigating the risk of disease transmission. Pigs are collectively moved to fresh housing as they progress through the production stages, ensuring adequate intervals between batches for thorough cleaning, disinfection, and drying of the facilities (4). AIAO enhances biosecurity practices by providing clear periods when facilities can be thoroughly cleaned and disinfected. This is crucial for preventing the introduction and transmission of pathogens that may contribute to the need for antimicrobial treatments (5).
AIAO reduces the mixing of animals from different groups, there by restricting interactions that could lead to the exchange of bacteria carrying antimicrobial resistance genes (6). This helps prevent the spread of resistant strains within the farm environment (5, 6). Also, AIAO contributes to reducing the overall environmental impact of use of antimicrobial agents (4, 5, 6). Effective disease control and management can reduce the frequency of antibiotic use, thereby lowing the environmental risk of antibiotic residues, which can contribute to reducing the risk of antibiotic resistance in environmental bacteria (5, 6, 7). The aim of this study was to analyzing whether the application of AIAO system in pig farm in Korea affects the reduction of antimicrobial resistance.
Materials and Methods
Experimental Design
To analyzing reduction in antimicrobial resistance through the application of the AIAO system, a total of five farms were recruited to apply the AIAO system, which sufficiently disinfects/dried the pig house before the next pig entry. All experimental farms housed about 500 sows, and AIAO system were applied at pigsty for weaned piglets (21 days-old to 70 days-old piglets).
Samples were collected from March 2022 to July 2022. Before the application of AIAO system, feces and dust samples were collected from pig farms. Samples were collected only once at 7 days before the pigs were out from the pigsty (March 2022). Also, three months after the application of AIAO system, feces and dust samples were collected from same space of pig farms, as the same manners (July 2022)
To gather feces and dust samples, a sterile surgical gauze swab was dampened using 10 mL of sterile phosphate-buffered saline solution. About 10 g of fecal samples were collected, and two distinct sections of pigsties were swabbed to obtain approximately 10 g of dust samples. All samples were transported aseptically to the laboratory with 4℃ for isolating Escherichia coli (E. coli) and Enterococcus species. All collected samples were transported to laboratory with ice-pack on the day of collection.
Isolation of E. coli
Aseptically collected feces and dust samples were individually inoculated into 5 mL of Modified Esculin (mEC; Becton-Dickinson, MD, USA) broth media at 37℃ for 24 h. After incubation, mEC media was streaked onto MacConkey (Becton-Dickinson) agar media and incubated at 37℃for 24 h. Typical pink colored-colonies were selected from each sample. Identification of E. coli was confirmed using polymerase chain reaction according to previously described study (8). Briefly, each selected colonies were heated at 95℃ for 5 min. The centrifuged supernatant was used as a DNA template. For identification of E. coli, 4 μM of forward primer (malB-F: 5’-GACCTCGGTTTAGTTCACAGA-3’) and reverse primer (malB-R: 5’-CACACGCTGACGCTGACCA-3’) were used. After amplification, the products were visualized by electrophoresis in 1.5% agarose gels stained with HiQ BlueMango (bioD, Seoul, Korea) under UV light, and products that showed size of 585 bp were identified as E. coli. All E. coli isolates were evenly isolated from each experimental farm (6 isolates before the application of AIAO, and 6 isolates after the application of AIAO from each of farms). A total of 60 E. coli isolates were tested in this study: 30 from “Before application of AIAO”, and “After application of AIAO”.
Isolation of Enterococcus species
Aseptically collected feces and dust samples were individually inoculated into 5 mL of Enterococcosel (Becton-Dickinson) broth media at 37℃ for 24 h. After incubation, Enterococcosel broth media was streaked onto Enterococcosel (Becton-Dickinson) agar media and incubated at 37℃ for 24 h. Typical black colored-colonies were selected from each sample. Identification of Enterococcus species was confirmed using polymerase chain reaction according to previously described study (8). Briefly, each selected colonies were heated at 95℃ for 5 min. The centrifuged supernatant was used as a DNA template. For identification of Enterococcus faecalis, 4 μM of forward primer (ddl-1-F: 5’-TGTTGTATGGCG GCAGAAGT -3’) and reverse primer (ddl-1-R: 5’-TCAGGTGTTTGTGCCCAAGT-3’) were used. Enterococcus faecium was identified using 4 μM of forward primer (ddl-2-F: 5’-ATGGACCCAAGTGGACAGA -3’) and reverse primer (ddl-2-R: 5’-ATTTCGCGCGCTTCAATTCC-3’). After amplification, the products were visualized by electrophoresis in 2.0% agarose gels stained with HiQ BlueMango (bioD, Seoul, Korea) under UV light, and products that showed size of 199 bp were identified as E. faecalis, and size of 474 bp were identified as E. faecium. All Enterococcus spp. isolates were evenly isolated from each experimental farm (6 isolates before the application of AIAO, and 6 isolates after the application of AIAO from each of farms). A total of 60 Enterococcus spp. isolates were tested in this study: 30 from “Before application of AIAO”, and “After application of AIAO”.
Antimicrobial Susceptibility Test
All E. coli and Enterococcus spp. isolates were tested for antimicrobial susceptibility using the disc diffusion test. The following antimicrobial agents were selected for testing E. coli isolates after referring to the Clinical and Laboratory Standards Institute (CLSI) guidance (9): ampicillin (10 μg), amoxicillin-clavulanic acid (20/10 μg), cefazolin (30 μg), cefoxitin (30 μg), ceftiofur (30 μg), ceftazidime (30 μg), cefepime (30 μg), gentamicin (10 μg), streptomycin (10 μg), kanamycin (30 μg), oxytetracycline (30 μg), tetracycline (30 μg), florfenicol (30 μg), chloramphenicol (30 μg), nalidixic acid (30 μg), ciprofloxacin (5 μg), sulfisoxazole (250 μg), trimethoprim-sulfamethoxazole (1.25/23.75 μg), and colistin (10 μg). Also, for testing antimicrobial susceptibility of Enterococcus spp. following antimicrobial agents were used: ampicillin (10 μg), penicillin (10 U), tylosin (30 μg), erythromycin (15 μg), doxycycline (30 μg), tetracycline (30 μg), tigecycline (15 μg), nalidixic acid (30 μg), ciprofloxacin (5 μg), vancomycin (30 μg), florfenicol (30 μg), chloramphenicol (30 μg), gentamicin (10 μg), kanamycin (30 μg), and streptomycin (10 μg).
The acquisition of all antimicrobial discs was made from Becton-Dickinson. Isolates exhibiting resistance to three or more CLSI subclasses were classified as multi-drug resistant strains (10).
Results
Antimicrobial resistance and Multi-drug resistance of E. coli
The antimicrobial resistance of E. coli isolated from pig farms were described in Table 1. The resistance rates to ampicillin, cefazolin, ceftiofur, streptomycin, oxytetracycline, tetracycline, florfenicol, chloramphenicol, nalidixic acid, and ciprofloxacin were more than 50.0%. We found that the resistance rates to cefazolin, cefoxitin, ceftazidime, cefepime, gentamicin, kanamycin, ciprofloxacin, sulfisoxazole, and trimethoprim-sulfamethoxazole were lower in E. coli isolates after the application of AIAO. Moreover, after the application of AIAO, there were significant decrease of resistance rates to cefoxitin (40.0% to 20.0%), cefepime (20.0% to 0.0%), gentamicin (50.0% to 20.0%), kanamycin (80.0% to 40.0%), sulfisoxazole (73.3% to 40.0%), and trimethoprim-sulfamethoxazole (86.7% to 40.0%). There was no difference of multi-drug resistance ratio between before and after the application of AIAO (Table 2) however, resistant to 9 antimicrobial subclasses were decreased (36.7% to 30.0%) after the application of AIAO.
Table 1.
Antimicrobial resistance phenotype of E. coli isolated from pig farms before and after all-in/all-out management
| Antimicrobial subclasses Antimicrobial agents |
No. of resistant isolates (Antimicrobial resistance %)* | |
|---|---|---|
|
Before all-in/all-out management (n=30) |
After all-in/all-out management (n=30) | |
| Aminopenicillins | ||
| Ampicillin | 26 (86.7%) | 27 (90.0%) |
| β-lactam / β-lactamase inhibitor combinations | ||
| Amoxicillin-clavulanic acid | 12 (40.0%) | 12 (40.0%) |
| 1st generation of cephalosporins | ||
| Cefazolin | 22 (73.3%) | 21 (70.0%) |
| 2nd generation of cephalosporins | ||
| Cefoxitin | 12 (40.0%)a | 6 (20.0%)b |
| 3rd generation of cephalosporins | ||
| Ceftiofur | 30 (100.0%) | 30 (100.0%) |
| Ceftazidime | 6 (20.0%) | 3 (10.0%) |
| 4th generation of cephalosporins | ||
| Cefepime | 6 (20.0%)a | 0 (0.0%)b |
| Aminoglycosides | ||
| Gentamicin | 15 (50.0%)a | 6 (20.0%)b |
| Streptomycin | 24 (80.0%)b | 27 (90.0%)a |
| Kanamycin | 24 (80.0%)a | 12 (40.0%)b |
| 1st generation of tetracyclines | ||
| Tetracycline | 26 (86.7%)b | 30 (100.0%)a |
| Oxytetracycline | 25 (83.3%)b | 30 (100.0%)a |
| Phenicol | ||
| Florfenicol | 22 (73.3%)b | 27 (90.0%)a |
| Chloramphenicol | 27 (90.0%)b | 30 (100.0%)a |
| Quinolones | ||
| Nalidixic acid | 24 (80.0%) | 24 (80.0%) |
| Fluoroquinolones | ||
| Ciprofloxacin | 23 (76.7%) | 21 (70.0%) |
| Sulfonamides | ||
| Sulfisoxazole | 22 (73.3%)a | 12 (40.0%)b |
| Trimethoprim-sulfamethoxazole | 26 (86.7%)a | 12 (40.0%)b |
| Lipopeptides | ||
| Colistin | 1 (3.3%) | 3 (10.0%) |
Table 2.
Multi-drug resistance of E. coli isolated from pig farms before and after all-in/all-out management
| No. of resistance |
No. of resistant isolates (Antimicrobial resistance %)* | |
|---|---|---|
|
Before all-in/all-out management (n=30) |
After all-in/all-out management (n=30) | |
| 0 subclass | 0 (0.0%) | 0 (0.0%) |
| 1 subclass | 0 (0.0%) | 0 (0.0%) |
| 2 subclasses | 0 (0.0%) | 0 (0.0%) |
| 3 subclasses | 1 (3.3%) | 1 (3.3%) |
| 4 subclasses | 2 (6.7%)a | 0 (0.0%)b |
| 5 subclasses | 5 (16.7%) | 5 (16.7%) |
| 6 subclasses | 4 (13.3%)b | 6 (20.0%)a |
| 7 subclasses | 2 (6.7%)b | 4 (13.3%)a |
| 8 subclasses | 5 (16.7%) | 5 (16.7%) |
| 9 subclasses | 11 (36.7%) | 9 (30.0%) |
|
Multi-Drug Resistance (≥ 3 subclasses) | 30 (100.0%) | 30 (100.0%) |
Antimicrobial resistance and Multi-drug resistance of Enterococcus spp.
Table 3 described the antimicrobial resistance of Enterococcus spp.isolated from pig farms. The resistance rates to tylosin, erythromycin, doxycycline, tetracycline, nalidixic acid, florfenicol, chloramphenicol, gentamicin, kanamycin, and streptomycin were more than 50.0%. After the application of AIAO, the antimicrobial resistance rates to ampicillin, penicillin, erythromycin, doxycycline, tetracycline, ciprofloxacin, florfenicol, and gentamicin were decreased. Also, we found that resistance rates to ampicillin (6.7% to 0.0%), penicillin (53.3% to 40.0%), ciprofloxacin (66.7% to 40.0%), and gentamicin (86.7% to 60.0%) were significantly decreased after the application of AIAO. Like the results of E. coli, there was no difference of multi-drug resistance ratio between before and after the application of AIAO (Table 4). However, resistant to 9 antimicrobial subclasses were significantly decreased (10.0% to 0.0%) after the application of AIAO.
Table 3.
Antimicrobial resistance phenotype of Enterococcus speciesisolated from pig farms before and after all-in/all-out management
| Antimicrobial subclasses Antimicrobial agents |
No. of resistant isolates (Antimicrobial resistance %)* | |
|---|---|---|
|
Before all-in/all-out management (n=30) |
After all-in/all-out management (n=30) | |
| Aminopenicillins | ||
| Ampicillin | 2 (6.7%)a | 0 (0.0%)b |
| Penicillins | ||
| Penicillin | 16 (53.3%)a | 12 (40.0%)b |
| Macrolides | ||
| Tylosin | 28 (93.3%)b | 30 (100.0%)a |
| Erythromycin | 28 (93.3%) | 27 (90.0%) |
| 1st generation of tetracyclines | ||
| Tetracycline | 28 (93.3%) | 27 (90.0%) |
| 2nd generation of tetracyclines | ||
| Doxycycline | 20 (66.7%) | 15 (50.0%) |
| 3rd generation of tetracyclines | ||
| Tigecycline | 0 (0.0%) | 0 (0.0%) |
| Quinolones | ||
| Nalidixic acid | 26 (86.7%)b | 30 (100.0%)a |
| Fluoroquinolones | ||
| Ciprofloxacin | 20 (66.7%)a | 12 (40.0%)b |
| Glycopeptides | ||
| Vancomycin | 0 (0.0%) | 0 (0.0%) |
| Phenicols | ||
| Florfenicol | 22 (73.3%) | 18 (60.0%) |
| Chloramphenicol | 22 (73.3%)b | 27 (90.0%)a |
| Aminoglycosides | ||
| Gentamicin | 26 (86.7%)a | 18 (60.0%)b |
| Kanamycin | 26 (86.7%)b | 30 (100.0%)a |
| Streptomycin | 28 (93.3%)b | 30 (100.0%)a |
Table 4.
Multi-drug resistance of Enterococcus speciesisolated from pig farms before and after all-in/all-out management
| No. of resistance |
No. of resistant isolates (Antimicrobial resistance %)* | |
|---|---|---|
|
Before all-in/all-out management (n=30) |
After all-in/all-out management (n=30) | |
| 0 subclass | 0 (0.0%) | 0 (0.0%) |
| 1 subclass | 0 (0.0%) | 0 (0.0%) |
| 2 subclasses | 1 (3.3%) | 1 (3.3%) |
| 3 subclasses | 2 (6.7%) | 2 (6.7%) |
| 4 subclasses | 4 (13.3%) | 3 (10.0%) |
| 5 subclasses | 2 (6.7%) | 3 (10.0%) |
| 6 subclasses | 4 (13.3%)b | 7 (23.3%)a |
| 7 subclasses | 6 (20.0%) | 6 (20.0%) |
| 8 subclasses | 8 (26.7%) | 8 (26.7%) |
| 9 subclasses | 3 (10.0%)a | 0 (0.0%)b |
|
Multi-Drug Resistance (≥ 3 subclasses) | 29 (99.7%) | 29 (99.7%) |
Discussion
In this study, we analyzed the antimicrobial resistance of E. coli and Enterococcus spp. isolated from 5 pig farms before and after the application of AIAO management system. For the analyzing the antimicrobial resistance of pig farms, we selected E. coli and Enterococcus spp. because those are intestinal commensal bacteria present in swine, and many countries’ antimicrobial surveillance systems were widely used E. coli and Enterococcus spp. as good indicators for monitoring the general level of antimicrobial resistance (1, 11, 12).
The effect on reducing antimicrobial resistance of AIAO management system was investigated in 5 pig farms. We found that when pig farms applicated AIAO management system, the antimicrobial resistance was significantly decreased. We found that there were significant decrease of resistance rates to cefoxitin (40.0% to 20.0%), cefepime (20.0% to 0.0%), gentamicin (50.0% to 20.0%), kanamycin (80.0% to 40.0%), sulfisoxazole (73.3% to 40.0%), and trimethoprim- sulfamethoxazole (86.7% to 40.0%) in E. coli, and ampicillin (6.7% to 0.0%), penicillin (53.3% to 40.0%), ciprofloxacin (66.7% to 40.0%), and gentamicin (86.7% to 60.0%) in Enterococcus spp. after the application of AIAO. In AIAO system, Pigs are collectively moved to fresh housing as they progress through the production stages, ensuring adequate intervals between batches for thorough cleaning, disinfection, and drying of the facilities (4). One of the important reasons for high antimicrobial resistance is the spread of resistance due to environmentally residual antimicrobial resistant bacteria, and it is thought that resistance had decreased due to the application of the AIAO management system that complies with clean and disinfect (7, 13, 14).
The World Health Organization classifies antimicrobial agents into highest priority critically important antimicrobials (HPCIA), critical important antimicrobials (CIA), high import antimicrobials (HIA), and important antimicrobials (IA) groups to ensure antimicrobial susceptibility according to their medical importance in humans (3). Also, the World Organization for Animal Health also defines antimicrobial agents according to their importance as veterinary critically important antimicrobials (VCIA), veterinary highly important antimicrobials (VHIA), and veterinary important antimicrobials (VIA) (3). The cefepime and ciprofloxacin are antimicrobial agents classified as HPCIA and VCIA. The cefepime which is the 4th generation of cephalosporins, is commonly used in pig farms to control enteric diseases and are known for their extended duration of action, which allows for less frequent dosing (15). And ciprofloxacin which is the fluoroquinolones, is commonly used to treat severe infections such as typhoid, paratyphoid fever. Thus, the risk management strategies for these antimicrobial agents are urgently needed (16).
Also, we found that resistance rates to gentamicin and kanamycin were significantly decreased after the application of AIAO management system. Those are aminoglycosides antimicrobial agents which is classified as VCIA (17). Owing to the unfavorable outcomes linked to aminoglycosides, including inner ear toxicity leading to sensorineural hearing loss and kidney damage resulting in chronic kidney disease, the utilization of aminoglycosides is restricted to the treatment of severe infections in human patients (17). However, in the case of pig, aminoglycosides may be employed for the control of enteric disease (11). Administering aminoglycosides to pigs holds the risk of inducing cross-resistance to critically important human antimicrobials, such as amikacin, posing a significant threat to human health (1).
In this study, we found that resistance rates to cefepime, ciprofloxacin, gentamicin, and kanamycin were significantly decreased after the application of AIAO management system. Through this, we assumed that the application of AIAO management system as the decreasing antimicrobial resistance model for pig farms.
The limitation of this study includes relatively short study period. For a significant reduction in antimicrobial resistance, approximately 6 to 12 months are required (18). Therefore, it is presumed that observed multi-drug resistance rates showed no significant differences because of the relatively short study period. The reduction of multi-drug resistant bacteria in pigs is crucial for preventing the dissemination of antimicrobial resistance from livestock to humans (19). Although there was no difference of multi-drug resistance between the application of AIAO management system, we found that there was significant decrease in Enterococcus spp. resistant to 9 antimicrobial subclasses, and significant increase in Enterococcus spp. resistant to 6 antimicrobial subclasses in this study. We presumed that increased rates of resistance patterns of 6 antimicrobial subclasses was caused because isolates resistant to 10 antimicrobial subclasses lost their resistance. Enterococcus spp. are known reservoirs of resistance genes, and their prevalence in pig populations raises concerns about the potential transfer of resistance to bacteria affecting human health (20). Addressing multi-drug resistance in pigs not only safeguards the efficacy of antimicrobial agents used in veterinary medicine but also plays a pivotal role in minimizing the risk of zoonotic transmission (5, 6). By mitigating the presence of resistant strains in pig farming, we contribute to the broader objective of preserving the effectiveness of antimicrobial treatments, promoting both animal welfare and public health (5).
Through this study, we confirmed that application of all-in/all-out management system lower antimicrobial resistance. This could contribute to the development of approaches for research on antimicrobial resistance and its effective management.
