In this study, we compared antimicrobial resistance and virulence genes of E. coli isolated from patients suffering from diarrhea and diarrheic pigs. It is noted that the overall prevalence of antimicrobial resistance in the isolates from pigs were higher than in those from humans, which was consistent with a previous study (14, 15). This suggests that the use of antimicrobial agents in pig farming may be more frequent than that in humans. Due to the lack of strict regulations on the use of antimicrobials in Korea (16), using antimicrobials indiscriminately by non-specialists, like livestock workers, could increase antimicrobial resistance. Isolates from pigs showed significantly higher resistance to aminoglycosides, fluoroquinolone, tetracyclines, nalidixic acid, trimethoprim / sulfamethoxazole, and chloramphenicol than in isolates from patients. The findings that the highest prevalence of resistance occurred among isolates from pigs and that resistance was seen to drug classes approved for use in swine (17) suggest that antimicrobial use in swine may be a factor in the emergence of antimicrobial resistance in E. coli. Because these agents are used for treating enteric infections in humans, decreasing resistance to these agents is crucial.
Fluoroquinolones are the primary option for controlling colibacillosis and have been widely used throughout the country for mass medication in pig farms (17, 18). The World Health Organization (WHO) and the World Organization for Animal Health (OIE) have classified fluoroquinolones as “critically important antimicrobial agents” because of their importance in both human and animal medicine (19). A previous study in Korea reported high resistance to ciprofloxacin (34.5%) in Korean pigs (20), which is in accordance with our study. The present study indicated that resistance of E. coli to ciprofloxacin (37.5%) from swine isolates was higher in Korea as compared to that in other countries with advanced swine farming (Netherland: 1.0%, Sweden: 0.0%, US: 0.0%) (21, 22), which restrict the use of antimicrobial agents. This is largely due to the massive use of fluoroquinolone in livestock for therapeutic and self-treatment purposes (quinolone sales: 44,380 kg) in Korea. Moreover, the resistance to ciprofloxacin (swine: 37.5%; patients: 10.0%) and norfloxacin (swine: 29.7%; patients: 8.0%) was higher in swine isolates than in patients’ samples. Since fluoroquinolone resistance can be transferred from pigs to humans (23), this becomes a public health hazard, and it is necessary to establish a strategy to reduce antimicrobial resistance. Therefore, additional regulations to reduce the resistance to ciprofloxacin is essential.
Introduced into clinical application in the mid-1990s, aminoglycosides are one class of antimicrobials that are used in veterinary and human medicine and have been used as the primary option for controlling colibacillosis (17, 24). In this study, we found higher resistance (51.6%) to gentamicin in Korea than in other countries (US: 0.0%; Australia: 7.4%) (22, 25). Gentamicin is no longer used in swine farming in advanced countries (25); however, in Korea, gentamicin is frequently used for the treatment of colibacillosis (5). This difference in the use of antimicrobial agents resulted in high resistance to gentamicin in Korea. Moreover, the resistance to some aminoglycoside antimicrobial agents (gentamicin, kanamycin, and amikacin) was significantly higher in isolates from swine than in those from patients. Due to the adverse events associated with aminoglycosides, such as inner ear toxicity (sensorineural hearing loss) and kidney damage (chronic kidney disease), the use of aminoglycosides is limited and administered for severe infections in humans (26). However, in pigs, aminoglycosides can be used to manage weaning pig scours caused primarily by E. coli (26, 27). The administration of aminoglycosides in pigs has the potential to generate cross-resistance to vitally important human antimicrobials like amikacin, which is a huge concern for human health (25).
In this study, we found high rates of multidrug resistance (swine: 93.8%; patients: 86.0%). Evidently, in our results, values obtained were higher than those obtained in other studies (Pig – Netherland: 34.2% (4), China: 84.2% (28), Thailand: 84.6% (14); Humans – Netherland: 7.1% (29), China: 15.2% (28), Thailand: 45.7% (14)) In Korea, antimicrobial use in veterinary (33.2 defined daily doses (DDD) per 1,000 inhabitants per day) and human medicine (31.7 DDD per 1,000 inhabitants per day) is relatively higher as compared to that in other member countries of the Organization for Economic Co-operation and Development (OECD) (21.3 and 23.7 DDD per 1,000 inhabitants per day, respectively) (30, 31). Besides the possible role of increased selective pressure by repeated exposure to therapeutic agents, this is a likely cofactor in the increased frequency of antimicrobial resistance observed among pathogens (32). The high level of antimicrobial resistance is directly linked to challenges in the treatment of diseases; therefore, it is important to manage antimicrobial resistance.
The clinical symptoms of colibacillosis vary depending on the pathotype of infective E. coli (33). The most predominant pathotype in Korea was EPEC (34); however, in this study, the most prevalent pathotype in patients was STEC (27 isolates, 54.0%), followed by ETEC (11 isolates, 22.0%). There was no clear reason why the pathotype of E. coli had temporal changes, but it is evident that the predominant pathotype has changed to ETEC and STEC from EPEC. STEC is the third most common zoonotic infection within the Europe (32). In this study, 28 isolates (43.8%) of swine contained STEC. Because STEC is a zoonotic food- and waterborne pathogen of a serious public health concern and it can cause outbreaks, hemorrhagic colitis, and potentially life-threatening complication, such as hemolytic-uremic syndrome (3, 35), careful attention should be paid to STEC’s pig–human cross-infection.
To produce enterotoxins and cause diseases, pathogenic E. coli needs to first attach to intestines (36). Fimbriae play an important role in allowing E. coli to attach to the intestinal mucosa and epithelial cells (37). In late 1990s, the most predominant fimbriae in Korean pigs was F6, which then changed to F5 in the mid-2000s (38, 39). However in this study, there was no fimbrial adhesin in isolates from patients, and the most prevalent virotype of E. coli in pigs encoded F4 (29 isolates, 45.3%) and F18 (35 isolates, 54.7%). In Korea, inactivated vaccines targeting F4 and F18 are being used nationwide (40). The use of these vaccines could cause the antigenic variations and would account for the prevalence of fimbriae or non-fimbrial adhesins, besides F4 and F18, in pigs.
The stx2 gene was detected both in isolates from pigs (15 isolates, 23.4%) and patients (19 isolates, 38.0%). The stx gene was known to be associated with edema disease in swine and hemolytic-uremic syndrome in human (3, 41, 42). The receptor for stx2 is globotriosyl ceramide which is seen in both swine and humans (43). Also, the LT and ST gene was detected both in isolates from swine (29 isolates, 45.3%) and patients (11 isolates, 22.0%), which is associated with neonatal or postweaning diarrhea in pigs and in traveler’s diarrhea in humans (44). Both enterotoxins elevate the extracellular levels of chloride and bicarbonate ion which could induce osmotic diarrhea (45). There was no common fimbrial adhesin in isolates from both swine and patients. However, several studies reported a high association between non-fimbrial adhesin AIDA-I and F18, which is the most prevalent fimbrial adhesin in the present study (46–48), and AIDA-I was detected in pigs (26.6%), and thus has the ability to cause cross-infection between pigs and humans (16). Furthermore, a recent study indicated that LT also could play a significant role in the enhancement of bacterial adherence (49). Thus, cross-infection of pathogenic E. coli between pigs and humans could occur sufficiently.
There was lower evidence on whether specific O-serogroup could cause diseases because a limited number of O-serogroups have been reported for specific disease (50, 51). In this study, the most prevalent O-serogroup in swine isolates was O149 (28 isolates, 43.8%), followed by O139 (13 isolates, 20.3%) and O157 (2 isolates, 3.1%). This is in accordance with the results obtained by Kusumoto et al. (52), which indicated that O139 was the predominant serogroup in Japan in 2010–2014. Kwon et al. indicated that O157 and O8 were the predominant O-serogroups in Korea from 1995 to 1997 (53). However, in this study, just two O157 isolates were detected from pigs. It is apparent that the predominant serogroup has shifted from O157 to O149 and O139 in Korean swine farms. In contrast to swine isolates, O157 (6 isolates, 12.0%) was the predominant serogroup in patients; O157 is known to be associated with eae, stx1 and/or stx2 gene (54). Interestingly, swine isolates had no stx2 gene while all isolates from patients encoded stx1 and/or stx2 gene. Because of the low number of O157 strains, it becomes hard to explain the cause of this phenomenon; however, we assumed that the relationship of O-serogroup with virotype has been changing over time.
MLST allows determining the phylogenetic relationships among deep lineages, providing a complementary view of the population structure (55). In this study, we analyzed 64 and 50 E. coli strains from swine and patients, respectively. There were only 10 STs in swine isolates while patients’ isolates showed more STs (28 STs). Most strains in pigs were ST 1 (21 isolates, 32.8%) and ST 100 (21 isolates, 32.8%), indicating that the cause of enteric colibacillosis in pigs had a similar origin. This is accordance with other studies which state that ST 1 and ST 100 isolates are the predominant ETEC type, and are important pig pathogens in the United States, Canada, Germany, and Thailand (http://mlst.warwick.ac.uk /mlst/dbs/Ecoli). In contrast to swine isolates, each ST of isolates from patients had small portions, which means that each strain from patients had been isolated from a different origin. Several studies reported ST 10 as one of the most common strains in human populations, and ST 10 strains commonly carry certain antimicrobial resistance genes (such as ampC-type beta lactamases and NDM-type carbapenemases) (56, 57). Also, ST 88 has been previously described in association with c-AmpC production in a French hospital (58). Interestingly, ST 10 (swine: 6 isolates; patients: 3 isolates) and ST 88 (swine 2 isolates; patients: 1 isolate) were detected simultaneously in swine and patients’ samples. Additionally, we found that isolates from different hosts (swine and patients) were closely related; ST 34 (patients: 1 isolate) – ST 10 (swine: 6 isolates, patients: 2 isolates), ST 88 (swine: 2 isolates, patients: 1 isolate) – ST 90 (swine: 2 isolates), ST 218 (patients: 3 isolates) – ST 10. In addition, five isolates from swine showing new ST were found that were phylogenetically closed with ST 641. Although, there was a weak relationship between patients’ isolates, the emergence of a pathogenic E. coli showing new ST may pose not only a problem in veterinary medicine but also a significant public health threat, and therefore is in need of urgent attention (59). We confirmed a phylogenetical association between swine and patients’ isolates. Similar ST indicates the risk of emergence of zoonotic disease (60) and a risk for cross-infection, and can even cause antimicrobial resistance to be transferred between pigs and humans in Korea.