Virulence-associated Genes and Antimicrobial Resistance of Escherichia coli Isolated From Post-weaning Piglets with Diarrhea in Korea

Abstract

Post-weaning diarrhea and/or porcine edema disease is caused by pathogenic Escherichia coli. In this study, 139 E. coli isolates were collected from post-weaning piglets with diarrhea and/or edema disease between 2013 and 2016 in Korea. Virulence (fimbria and toxin) genes and antimicrobial resistance were evaluated in the isolates. Among the 139 E. coli isolates, the F4ac fimbriae gene was observed in 7.2% of isolates, and the F18 variant fimbria genes were found to be F18 (12.9%), F18ab (33.1%), and F18ac (20.1%). The various fimbriae gene combinations were F4/F18/F41 (0.7%), F4ac/F18ab (0.7%), and F4ac/F18ac (1.4%) in the 139 E. coli isolates. A total of 111 enterotoxigenic E. coli (ETEC) isolates were detected, and the most prevalent toxin genes were identified as STa (35.9%) and STb (35.2%). Additionally, among 111 ETEC isolates, 79 isolates harbored the Stx2e gene. All 139 E. coli isolates were resistant to ceftiofur and sulphafurazole. In contrast, all isolates were susceptible to amikacin, vancomycin, lincomycin, and tircarcilin/clavulanic acid. All of the ampicillin, ceftiofur, chloramphenicol, imipenem, kanamycin, sulphafurazole, tetracycline, cephazolin, and ticarcillin-resistant isolates between 2013 and 2015, and the ampicillin, ceftiour, cephalothin, chloramphenicol, streptomycin, sulphafurazole, tetracycline, and ticacillin-resistant isolates from 2016 showed high resistance to the carbapenem, cephems, penicillins, phenicols, sulfas, and tetracyclines class and to the aminoglycosides, cephems, penicilins, phenicols, sulfas, and tetracyclines class. These results suggest that E. coli disease in piglets may not be associated with a single toxin or a major gene combination, but with more varied and complex toxins and their combinations.

Keywords: Antimicrobial Resistance; Escherichia coli; Piglets; Diarrhea

Introduction

Escherichia coli (E. coli) is an important pathogen in post-weaning diarrhea and/or edema disease, and is commonly found in pigs’ intestinal tracts on a wide variety of farms [1]. Various E. coli types adhere to the intestinal tracts via fimbriae, including F4, F5, F6, F18, and F41, and produce toxins, which include LT, STa, STb, and shiga toxin [2]. The fimbriae factor, F4⁺ E. coli, is associated with neonatal and post-weaning diarrhea, whereas F18⁺ E. coli causes post-weaning diarrhea and/or edema disease in piglets [3]. F4⁺ and F18⁺ E. coli exist as several variant types. Among the F4⁺ E. coli, there are three variants, F4ab, F4ac, and F4ad, in which fimbriae share a common ‘a’ epitope and type-specific epitope ‘b’, ‘c’, and/or ‘d’ determinants [4-7]. F18⁺ E. coli has two variants, namely F18ab and F18ac. Both fimbriae are colonization factors for enterocyte receptors, and F18ac is found mostly in pigs with edema diseases via enterotoxigenic E. coli [8-10]. Several studies have investigated the variant F4⁺ and F18⁺ fimbriae factors of E. coli isolated from pigs with diarrhea and/or edema disease [11-13], including those in Korea [14,15].

Most pathogenic E. coli causing porcine diarrhea and/or edema disease are characterized by antimicrobial resistance. The E. coli isolates from pigs, cattle and poultry are often resistant to two or more antimicrobials, including sulfamethoxazole (Sx), tetracycline (Te), streptomycin (S), ampicillin (Am), spectinomycin (Sp), apramycin (Ap), trimethoprim-sulfonamide (TMP/SMX), and neomycin (Nm) [16,17]. Recently, the frequency of antimicrobial resistance among E. coli has increased markedly, presumably owing to extensive and indiscriminate antimicrobial use, which has caused difficulty in treating pathogenic E. coli infections in animals and humans. Therefore, an increase in the incidence of outbreaks of severe E. coli-associated diarrhea and/or edema disease has been observed worldwide, and several studies have investigated antimicrobial resistance against E. coli isolates [2,18-21].

The present study’s objective was to evaluate the distribution of types of pathogenic E. coli isolates from pigs with diarrhea and/or edema disease, isolated between 2013 and 2016 in Korea, and examine the characteristics of their virulence genes, including fimbrial and toxin genes. We applied nucleotide primers and conditions for polymerase chain reaction (PCR) to detect these fimbriae and toxins using a previously reported method and/or modified method [4,10]. Another objective of the present study was to examine antimicrobial resistance and determine its genetic basis in the isolates.

Methods

Bacterial strains and growth conditions

A total of 139 pathogenic E. coli strains were isolated from fecal and intestinal samples taken from pigs diagnosed with diarrhea and edema disease at a Korean pig farm between 2013 and 2016. A 10-fold dilution of fecal and homogenized intestinal samples were inoculated on MacConkey agar (Oxoid, UK). The colonies were identified using standard biochemical procedures [22] and PCR analysis [15]. The 139 E. coli isolates were routinely grown in Luria-Bertani (LB) agar and/ or broth (Affymetrix USB, CA, USA). Mueller-Hinton agar (Becton, Dickinson and Company, NY, USA) was used for antimicrobial assessment of the 139 E. coli isolates.

Detection of virulence gene using PCR

The 139 E. coli isolates were grown on LB broth at 37°C for 24h. DNA extraction was performed by boiling at 100°C for 10min and centrifugation at 10,000xg for 5min. The supernatant was used as the DNA template for PCR amplification. Multiplex PCR to detect the E. coli virulence factor gene was performed according to methods described in a previous report [23]. In brief, Multiplex PCR was performed using the PCR premix kit (AccuPower^® Multiplex PCR PreMix, Daejeon, Bionner) and virulence gene-specific primers (Table 1). The PCR amplification process was performed with a thermal cycler (Applied Biosystems, CA, USA) under the following conditions: Initial denaturation (95°C, 15min); 25 cycles of denaturation (95°C, 30sec), annealing (63°C, 90sec), and extension (72°C, 90sec); final extension (72°C, 10min). In the reaction’s final step, the PCR mixture was cooled to 4°C. The PCR products were analyzed by electrophoresis on an 3% agarose gel containing Red SafeTM (iNtRON Biotechnology, Seongnam, Korea), and detected using an ultraviolet (UV) visualization system. According to the method of Zhang, et al [23], the virulence genes (F4, F5, F6, F18, F41, LT, STa, STb, and Stx2e) were confirmed by the band sizes of the PCR product (Table 1).

PCR for detection of F4⁺ or F18⁺ E. coli variants

To determine the variants of the fimbriae adhesin gene and variants type gene (F4, F4ac, F4ad, F18, F18ab, F18ac, and F41) of the 139 E. coli isolates, specific primers were used as described previously [4,10]. Multiplex PCR was performed using the PCR premix kit (AccuPower^® Multiplex PCR PreMix) with a total reaction volume of 20μl, which consisted of 3μl of template DNA, 1μl of 10pmol/l primers (F4, F4ac, F4ad, F18, F18ab, F18ac, and F41), and 17μl of distilled water. The F4 (K88) gene variants (F4ab and F4ad) primers and PCR conditions used were as described in a previously reported method [4]. We detected the F18 gene variants according to the modified PCR condition method and used F18 gene variant detection primers in accordance with the report by Cheng, et al [10]. In brief, we modified the PCR amplification process with a thermal cycler (Applied Biosystems, CA, USA) under the following conditions: initial denaturation (94°C, 3min); 25 cycles of denaturation (92°C, 30sec), annealing (50°C, 30sec), and extension (72°C, 60sec); final extension (72°C, 10min) for F18ab and F18ac (fedA1, fedA2, fedA3). In the final step of each PCR reaction, the mixture was cooled to 4°C, and the products were analyzed by electrophoresis on a 1.5% agarose gel containing Red SafeTM (iNtRON Biotechnology). The PCR products were detected using a UV visualization system.

Antimicrobial susceptibility testing

The antimicrobial susceptibilities of all E. coli isolates were assessed using the disk-agar method, as standardized by the Clinical and Laboratory Standards Institute (CLSI) [24]. The isolates were tested against a panel of 23 antimicrobials: Amikacin (An), ampicillin (Am), azithromycin (Azm), cefamandole (Ma), cefoxitin (Fox), ceftriaxone (Cro), cephalothin (Cf), chloramphenicol (C), ciprofloxacin (Cip), gentamicin (Gm), imipenem (Ipm), kanamycin (K), nanldixic acid (Na), streptomycin (S), tetracycline (Te), ticarcillin/clavulanic acid (Tim), ticarcillin (Tic), vancomycin (Va) (BD, NJ, USA) and ceftiofur (Ef), cephalfxin (Cl), cephazolin (Kz), lincomycin (My), sulphafurazole (Sf), and sulphamethoxazole (Sx) (Thermo Scientific™ Oxoid™, Hampshire, UK). The results were obtained after incubating the samples for 24h at 37°C and were interpreted according to the CLSI [24]. The resistant types were considered identical if they were similarly susceptible to all the agents tested. The quality control strains included E. coli ATCC25922 (American Type Culture Collection).

Results

Fimbrial adhesins and virulence genes

The fimbrial adhesin genes, F4, F6, F18, and F41, were detected in the 139 E. coli isolates, and the results are shown in Table 2. Among the 139 E. coli isolates collected between 2013 and 2016, 111 E. coli isolates (79.9%) exhibited one or more fimbrial adhesin genes. Among the 111 E. coli isolates, 10 (9.0%), two (1.8%), 18 (16.2%), 46 (41.4%), 28 (25.2%), and three (2.7%) isolates were detected as F4ac, F6, F18, F18ab, F18ac, and F41, respectively. Various fimbrial adhesion gene combinations were identified in 4/111 E. coli isolates: One (0.9%), one (0.9%), and two (1.8%) isolates were detected as F4/F18/F41, F4ac/ F18ab, and F4ac/F18ac, respectively of the 139 E. coli isolates, 28 E. coli (20.1%) were shown to have only a toxin gene, without fimbrial adhesin genes. Ten of the E. coli isolates with F4ac had several toxin genes, including LT (50%), STa (20%), STb (80%), and Stx2e (20%). Of the 92 E. coli isolates with the F18, F18ab, and F18ac adhesin genes, a number were positive for LT (27.8, 37.0, and 21.4 %), STa (33.3, 37.0, and 53.6 %), STb (11.1, 15.2, and 35.7 %), Stx2e (72.2, 67.4, and 71.4 %), and negative for the toxin gene (11.1, 8.7, 7.1 %). E. coli isolates carrying the F4/F18/F41 (1/139), F4ac/F18ab (1/139), and F4ac/F18ac (2/139) combinations were positive for LT (0, 0, and 50 %), STa (0, 100, and 100 %), and STb (100, 0, and 100 %), respectively.

Antimicrobial susceptibility

Among the 139 E. coli isolates, 60, 10, 51, and 18 strains were isolated in 2013, 2014, 2015, and 2016, respectively. The 139 E. coli isolates were analyzed for antimicrobial susceptibility (Table 3). Among the 139 E. coli isolates, all were resistant to one or more of the antimicrobials tested, regardless of the collection date. The antimicrobial susceptibility of the 60 E. coli isolates from 2013 exhibited higher resistance to Am, C, Ef, Ipm, Kz, S, Sf, and Te, and the 10 E. coli isolates from 2014 exhibited higher resistance to Am, C, Ef, Ipm, Cf, and Sf (resistance rate =85%) compared to the other antimicrobial agents (resistance rate =85%). The antimicrobial susceptibility of the 51 E. coli isolates collected in 2015 exhibited higher resistance to Ef, Ipm, Kz, Sf, and Tic, and the 18 E. coli isolates collected in 2016 exhibited higher resistance to Am, C, Ef, S, Sf, Te, and Tic (resistance rate =85%) compared to the other antimicrobial agents (resistance rate =85%). All E. coli isolates collected between 2013 and 2016 were shown to have 100% rates of resistance to Ef and Sf. By contrast, all isolates showed susceptibility to An, My, Tim, and Va (resistance rate 0-11.8%).

In total, 23 antimicrobial resistance patterns were observed in the 139 E. coli isolates (Table 4). Of the 121 E. coli isolates collected between 2013 and 2015, 28 (2.1% of 60) from 2013, five (2.0% of 10) from 2014, and 12 (4.1% of 51) from 2016 had the most frequent resistance phenotype, AmEfCIpmKSfTeKzTic (type 113). Among the 18 E. coli isolates from 2016, the most frequent resistance types were AmEfGmSSfSxTeClTic (type 116) and AmEfCfCSSfTeTic (type 119) (six isolates each; 3%).

Discussion

E. coli is distributed across a wide variety of farms and wild animals; it has been identified as the cause of disease and associated with outbreaks [25-27]. In this study, we isolated 139 E. coli strains from pigs with diarrhea and/or edema disease in Korea between 2013 and 2016, investigating the fimbriae adhesin, virulence genes, and antimicrobial susceptibility.

The toxin-producing E. coli strains with various fimbriae adhesin genes cause diarrhea and edema disease in post-weaning piglets. Therefore, E. coli infections can lead to severe economic losses in the pig industry, mostly because of medication costs, growth retardation, and mortality. Of the 139 E. coli isolates assessed, all isolates carried one or more of the virulence-associated genes. Among the 139 E. coli isolates, only 10 (7.19% of 139) had the F4ac fimbriae adhesin gene. In previous reports, the F4 E. coli strain was found to have high rates of prevalence, with rates of 23.4% in Canada and 44% in Denmark [28,29]. Among the 92 isolates carrying genes for fimbriae, the F18 gene variant was the most prevalent; F18 (19.5%), F18ab (50.0%), and F18ac (30.4%) were identified. In a previous report, among 63 PCR-positive isolates for 108 verotoxigenic E. coli and/ or enterotoxigenic E. coli, 53 isolates (49.07%) were detected with F18ab and 10 isolates (9.26%) were detected with F18ac [10]. Among the 139 isolates carrying genes for ETEC toxins, the most prevalent toxin genes were STa (35.9%) and STb (35.2%). These results show similarity to those of a previous study, which reported STa and STb were the most frequently isolated from pigs with diarrhea [29,30]. In another previous report in Korea, Kwon, et al [31], detected the prevalence rates of F4 and F18 E. coli isolates, which were 4.3% and 18.3%, respectively. Additionally, the STa, STb, and Stx2e virulence genes were detected with prevalence rates of 25.7%, 15.2%, and 15.6%, respectively, in the present study. The results of both F4 and F18 fimbriae in the present study are similar to those in previous Korean studies. However, the Stx2e gene results were found to have a high prevalence rate in the E. coli isolates in the present study. The most common combination of genes detected for ETEC fimbriae was F18ab/LT/STa. Among the 111 isolates of ETEC, the Stx2e gene was detected in 79 (71.1%) isolates. Of these 79 isolates, F4ac (20.0%), F18 (72.2%), F18ab (67.4%), and F18ac (71.4%) carried the Stx2e gene, respectively. These results showed the combined genes for fimbriae and virulence for E. coli isolates were more varied and complex than previous studies have indicated.

The prevalence of antimicrobial resistance in all E. coli isolates from pigs was high. All of the isolates were resistant to at least one antimicrobial, and the majority were resistant to at least seven antimicrobials. There were high rates of resistance to particular antimicrobials, notably S, Ipm, Ef, Kz, Am, Tic, Cip, Sx, and Te, regardless of the collection date. The major reason for the high resistance rates to high-level S, Ef, and Sf treatment is likely to be that these three antibiotics are commonly used as therapeutics and growth promotion agents in pig farms [16,17]. In a previous report, all E. coli isolates from Tibetan pigs exhibited high rates of resistance to Te, and a small number of isolates were resistant to Am, S, and Ef [32]. An Australian study reported [21] that 182 E. coli isolates collected from 200 pigs between 2003 and 2004 exhibited antimicrobial resistance to Am and florfenicol. A Korean study reported [33] that 744 E. coli isolates exhibited antimicrobial resistance to Te (96.3%), S (66.8%), Am (66.1%), and C (47.6%). Therefore, antimicrobial susceptibility results, obtained for E. coli from Korea and other countries, showed high levels of resistance to Te, S, and Am. The present study results were comparable to these observations, as the E. coli isolated from pigs showed a high prevalence of resistance to antimicrobials commonly used to treat livestock, including Te, S, Ef, and Am. Pig farms have increased the use of antimicrobials over time, which may have led to increased antimicrobial resistance among the isolates. The high prevalence of antimicrobial resistance to isolated E. coil strains in pig farms from Korea highlights the urgent need for measures to regulate the use of antimicrobials in pigs and other animals used for food production in Korea. By contrast, the high rates of resistance to the Ipm, Ma, and Kz of the E. coli isolates were decreased over the collection period. There was a high rate of susceptibility to particular antimicrobials, notably An, Cro, Va, My, and Tim, regardless of the collection date. Therefore, these antimicrobials remain potentially effective against E. coli species. The antimicrobial resistance patterns of 121 E. coli isolates were found to be AmEfCIpmKSfTeKzTic in the period between 2013 and 2015, and AmEfCfCSSfTeTic in the isolates collected in 2016; these were the most frequent resistance types. All of the AmEfCIpmKSfTeKzTic-resistant isolates collected between 2013 and 2015, and the AmEfCfCSSfTeTic-resistant isolates collected in 2016, showed high rates of resistance to the carbapenem, cephems, penicillins, phenicols, sulfas, and tetracyclines classes and the aminoglycosides, cephems, penicilins, phenicols, sulfas, and tetracyclines classes.

Conclusion

In the present study, the frequency of antimicrobial resistance and fimbriae-associated virulence genes were detected in E. coli isolates from pigs with diarrhea and/or edema disease between 2013 and 2016 in Korea. In the 139 E. coli isolates, variants of the fimbriae gene were detected, namely F4ac, F18, F18ab, and F18ac. Among 111 ETEC isolates, the most prevalent toxin genes, STa and STb, were detected. In addition, among these 111 ETEC isolates, 79 were detected with the presence of the Stxe2 gene. Overall, these results suggest that E. coli disease in piglets may not be associated with a single toxin or a major gene combination, but with more varied and complex toxins and their combinations. All the E. coli isolates were shown to have 100% rates of resistance to Ef and Sf. By contrast, all of the isolates showed susceptibility to An, Va, My, and Tim. Our results were comparable to this observation since E. coli isolated from pigs showed a high prevalence of resistance to antimicrobials commonly used in livestock, including Te, S, Ef, and Am.

Acknowledgement

This research was supported by Technology Development Program for Agriculture and Forestry; the Ministry for Food, Agriculture, Forestry, and Fisheries, Republic of Korea (no. 117031- 3).

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