Background

Colibacillosis caused by Escherichia coli (E. coli) in pigs is the most frequent enteric disease and an important cause of death in suckling piglets [1]. This disease may result in significant economic losses in the pig industry because of mortality, decreased weight gain, and costs for treatments, vaccinations and feed supplements [2]. The use of antimicrobial drugs such as β-lactams, aminoglycosides, and fluoroquinolones (FQs) has remained the primary option for controlling colibacillosis.

FQs are broad-spectrum antimicrobials agents and have been used for the treatment of various infections caused by E. coli or other gram-negative bacteria. The World Health Organization (WHO) has classified FQs as ‘‘critically important antimicrobials’’ because of their clinical importance in both human and animal medicine [3]. Because the importance of FQs in humans and animals is increasing, FQ-resistant bacteria are a major concern in the treatment of infectious diseases. Notably, FQ-resistant E. coli have emerged in Korea after enrofloxacin was licensed to be used for veterinary purposes [4].

Multiple mechanisms are involved in resistance to FQ in Enterobacteriaceae. The major mechanisms of resistance to FQ involve mutations of chromosomal genes encoding DNA gyrase and/or topoisomerase IV [5, 6]. In addition, three different plasmid-mediated quinolone resistance (PMQR) determinants have been described: qnr-mediated inhibition of quinolone binding to DNA [7, 8], qepA encoded efflux pump [9], and the aac(6’)-Ib-cr mediated FQ acetylation [10, 11]. The PMQR genes confer only low-level resistance to FQ; however, they can be spread horizontally among enterobacteria and facilitate the selection of resistant mutants following exposure to FQ [12]. Although studies from several countries have documented the prevalence and characteristics of FQ-resistance in healthy pigs [13,14,15], there is still limited information regarding the molecular characteristics of FQ-resistant and PMQR-positive E. coli isolated from suckling piglets with diarrhea. Therefore, the purpose of this study was to investigate the molecular characteristics of FQ-resistant E. coli isolates from suckling piglets with colibacillosis.

Materials and methods

Bacterial isolates

E. coli isolates collected from each colibacillosis clinical case in suckling piglets from 2007–2018 were tested. The farms consisted of 42 different pig herds (50 to 100 sows per each herd). The aseptically collected intestinal contents and feces were inoculated on MacConkey agar(BD Biosciences, Sparks, MD) containing 4 mg/mL of ciprofloxacin (CIP; Sigma-Aldrich, St.Louis, MO). Subsequently, suspected E. coli colonies were identified by VITEK II system (bioMéreiux, France). Thus, a total of 43 CIP-resistant E. coli were tested in this study.

Antimicrobial susceptibility testing

The disk diffusion method was performed according to the Clinical and Laboratory Standards Institute (CLSI) guidelines [16]. The 19 antimicrobial disks (BD Biosciences) used in this study were amikacin (30 μg), amoxicillin-clavulanate (20/10 μg), ampicillin (10 μg), aztreonam (30 μg), cefazolin (30 μg), cephalothin (30 μg), cefoxitin (30 μg), cefepime (30 μg), chloramphenicol (30 μg), colistin (10 μg), doxycycline (30 μg), gentamicin (10 μg), kanamycin (30 μg), nalidixic acid (30 μg), neomycin (30 μg), penicillin (10 U), streptomycin (10 μg), tetracycline (30 μg), and trimethoprim-sulfamethoxazole (1.25/23.75 μg). The minimum inhibitory concentration (MIC) for CIP, enrofloxacin (ENR), and norfloxacin (NOR) was determined by standard agar dilution methods using the Mueller–Hinton agar (BD Biosciences) method according to the guidelines of the CLSI [16]. The breakpoints of CIP, NOR, and ENR were also determined according to the guidelines of the CLSI [16]. E. coli ATCC 25,922 was included as a quality control. Multidrug-resistance (MDR) was defined as acquired resistance to at least one agent in three or more antimicrobial classes [17].

O-Serogroup typing

O-serogroup typing was performed using rabbit antisera purchased from SSI (Serum Staten Institute, Denmark) with the slide agglutination technique of the Animal and Plant Quarantine Agency (Gimcheon, Korea). A standard strain was obtained from Dr. J.M. Fairbrother (OIE E. coli Reference Laboratory, Quebec, Canada).

Identification of mutations in quinolone-resistance-determining regions and detection of plasmid-mediated quinolone-resistance genes

PCR was carried out to amplify quinolone resistance determining regions (QRDRs) of the target genes (gyrA, and parC) to identify mutations in 43 FQ-resistant E. coli isolates using primers and conditions described previously [18, 19]. The PCR products were purified using GFX PCR DNA and the gel band purification kit (Amersham Bioscience, Freiburg, Germany), and sequenced by an automatic sequencer (Cosmogenetech, Seoul, Korea). The sequences were aligned with those in the GenBank nucleotide database using the Basic Local Alignment Search Tool (BLAST) program available through the National Center for Biotechnology Information website (http://www.ncbi.nlm.nih.gov/BLAST). PMQR genes (qnrA, qnrB, qnrC, qnrD, qnrS, aac(6’)-Ib-cr, and qepA) were detected by PCR amplification and sequencing analysis, as described in Table S1.

PCR amplification and sequencing of antimicrobial resistance and virulence genes from PMQR-positive E. coli

For detection of antimicrobial resistance genes and virulence genes, PCR was performed using DNA extracted from PMQR-positive E. coli isolates. Primers used in the study are listed in Table S1. All PMQR-positive E. coli isolates were tested for resistance genes related to aminoglycosides (aac (3)-II, and ant(2’’)-I), β-lactam antimicrobials (blaCTX-M families, blaTEM, blaSHV, blaOXA, and pAmpC), chloramphenicols (cmlA and catA1), sulfonamides (sul1 and sul2), and tetracyclines (tetA, tetB, tetC, tetD, tetE and tetG). The virulence factor genes associated with the toxins (LT, STa, STb, Stx2e, and EAST-1), fimbriae (F4, F5, F6, F18, and F41), and non-fimbrial adhesins (AIDA-1, paa, eae) were also confirmed by PCR as previously described [20].

Plasmid replicon typing and detection of integrons and gene cassettes

For plasmid replicon typing and detection of integrons and gene cassettes, PCR was performed using DNA extracted from PMQR-positive E. coli isolates. The primers used in this study targeted 18 different replicons [21] and class 1 and 2 integrons (Table S1.). Gene cassettes were tested for integron-positive isolates. The PCR products of the gene cassettes were sequenced as described above.

Conjugation assay

To determine the transferability of PMQR and other genes, conjugation assays were performed using the broth mating method, with E. coli J53 used as the recipient as previously described [22]. Transconjugants were selected on MacConkey agar (BD Biosciences) plates containing sodium azide (100 µg/ml; Sigma-Aldrich) and ampicillin or tetracycline (100 μg/ml; Sigma-Aldrich). Transferability was confirmed by antimicrobial susceptibility tests and PCR for molecular analysis as described above.

Results

Antimicrobial resistance

MDR patterns of FQ-resistant E. coli isolates from colibacillosis are shown in Table 1. Among the 43 FQ-resistant E. coli, all isolates showed MDR against 6 to 11 classes of antimicrobial agents. The rates of resistance to the various antimicrobial classes were as follows: aminoglycosides (43/43, 100.0%), penicillins (43/43, 100.0%), quinolones (43/43, 100.0%), tetracyclines (42/43, 97.7%), phenicols (39/43, 90.7%), β-lactam/β-lactamase inhibitor combinations (32/43, 74.4%), folate pathway inhibitors (32/43, 74.4%), cephems (31/43, 72.1%), monobactams (7/43, 16.3%), and polypeptides (6/43, 14.0%). The rate of resistance to 9 antimicrobial classes was the highest at 39.5%, and one (2.3%) FQ-resistant E. coli isolate was identified resistance to 11 antimicrobial classes.

Table 1 Distribution of multi-drug resistance patterns among 43 fluoroquinolone-resistance E. coli isolates
Full size table

Molecular characteristics of FQ-resistant E. coli

The molecular characteristics of 43 FQ-resistant E. coli isolates are shown in Table 2. Among the isolates, 36 isolates were classified into 17 O-serogroups, and 6 isolates were ungrouped. The most common serogroup was O149 (12 isolates, 27.9%). The gyrA amino acid substitutions were S83L (43 isolates), D87N (22 isolates), D87G (5 isolates), and D87E (2 isolates), and the parC mutations were S80I (23 isolates), S80R (9 isolates), E84A (5 isolates), S80K (3 isolates), S80N (1 isolates), S80W (1 isolates), E84G (1 isolates), A56C (1 isolates), and S57Q (1 isolates). The MIC ranges for CIP, ENR, and NOR were 4–256 mg/mL, 8–512 mg/ml, and 8–512 mg/mL, respectively, and isolates with double mutations in gyrA were relatively higher than those of other isolates with single mutations in gyrA. In particular, FQ-resistant E. coli isolates with a high level of MICs range (≥ 64 mg/L for CIP, ≥ 128 mg/L for ENR, and ≥ 256 mg/L for NOR) were shown to carry double mutations in gyrA in combination with double mutations in parC. PMQR genes were detected in 12 (27.9%) of the 43 FQ-resistant E. coli isolates. The aac(6’)-Ib-cr, qnrS, and qepA genes were identified in seven, three, and two FQ-resistant E. coli isolates, respectively. Among 12 PMQR-positive E. coli isolates, one isolate that showed the highest MICs for CIP (256 mg/mL), ENR (512 mg/mL), and NOR (512 mg/mL), also carried the PMQR gene aac(6’)-Ib-cr.

Table 2 Amino acid changes in the QRDRs, MICs and PMQR determinants of 43 fluoroquinolone-resistance E. coli isolates
Full size table

Characterization of PMQR-positive E. coli

The prevalence of antimicrobial resistance genes is shown in Table 3. All PMQR-positive E. coli isolates carried the following β-lactamase encoding genes: blaCMY-2 (10 isolates, 83.3%), blaTEM-1 (7 isolates, 58.3%), blaOXA-1 (7 isolates, 58.3%), blaSHV-1 (3 isolates, 16.7%), and blaAAC-2 (1 isolate, 8.3%). Tetracycline-resistance genes were detected in all PMQR-positive E. coli isolates as follows: tetD (12 isolates, 100.0%), tetE (12 isolates, 100.0%), tetA (11 isolates, 91.7%), and tetB (1 isolate, 8.3%). Two types of aminoglycoside-modifying enzyme genes were examined, but aac(3)-II was found only in 2 (16.7%) PMQR-positive E. coli isolates. The sul1 and sul2 sulfonamide-resistance genes were detected in 9 isolates (75.0%) and 10 isolates (83.3%), respectively. The cmlA chloramphenicol-resistance gene was found in 10 isolates (83.3%). Distributions of the virotypes are shown in Table 3. Seven (58.3%) and two (16.7%) of the isolates contained fimbrial antigen F4 and F41, respectively. The most prevalent combinations of toxin genes were LT:STb:EAST1 (5 isolates, 41.7%), followed by EAST1 (3 isolates, 25.0%), LT:STa:STb (1 isolates, 8.3%), STa:STb:EAST1 (1 isolates, 8.3%), STa:STb (1 isolates, 8.3%), Sta (1 isolates, 8.3%). The paa and AIDA non-fimbrial adhesins genes were detected in 3 isolates (25.0%) and 2 isolates (16.7%), respectively. Among 12 PMQR-positive FQ-resistant E. coli isolates, all isolates were found to have class 1 integrons. Class 1 integrons contained four types of gene cassette arrangements, aadA1-dfrA1 (2 isolates), aadA1-aadA2- aadB (1 isolate), and aadA1-aadA2- aadB-cmlA6 (1 isolate). Eight isolates did not carry any of the gene cassettes. A total of 10 plasmid replicon types were identified in all 12 PMQR-positive E. coli isolates. The most common plasmid replicon was FIB (9 isolates, 75.0%), followed by FIC, HI1, and N (7 isolates, 58.3%), respectively. Transferability was only identified in 7 isolates among 12 PMQR-positive FQ-resistant E. coli isolates.

Table 3 Phenotypes and genotypes of 12 PMQR-positive E. coli isolates
Full size table

Discussion

Suckling piglets are vulnerable to colibacillosis for many reasons such as changes in environmental conditions, a decline in maternal antibody titers, and various stresses. Antimicrobials are used in intensive pig production systems to control infectious diseases. In particular, FQs are highly effective antimicrobial class with many advantages including high oral absorption, large volume of distribution, and broad-spectrum antimicrobial activity [23]. However, increasing use of these agents has led to rising rates of resistance to FQs in E. coli worldwide [24]; thus, the probability of treatment failure may be increased [25]. In this study, all FQ-resistant E. coli isolates showed were MDR (resistance to more than six antimicrobial agents) with high levels of resistance to several antimicrobials: aminoglycosides (100.0%), penicillins (100.0%), tetracyclines (97.7%), and phenicols (90.7%). In particular, five isolates showed resistance to more than 10 classes. These results were consistent with previous studies showing co-association of resistance to other classes of antimicrobials and high MDR rates among FQ-resistant E. coli [26, 27]. This occurs because PMQR genes and other antimicrobial-resistance genes are linked together on the plasmid [26].

The O-serogroup is considered one of the major virulence factors of E. coli and variety of O-serogroups has been associated with diarrhea [28,29,30]. In the present study, 17 O-serogroups were detected and O149 was most common. The O149 serogroup has been determined to be the dominant serogroup in cases of diarrhea in many countries including China and Canada [31, 32]. In addition, in this study, all isolates (100%) had gyrA mutations, and 29 isolates (67.4%) were double amino acid substitutions (S83L plus substitution in aspartic acid 87). These results were consistent with previous studies showing that DNA gyrase is the primary target of FQ in gram-negative bacteria, and gyrA mutations are dominant mutations in E. coli [33]. Moreover, 37 (86.0%) FQ-resistant E. coli isolates had mutations at codon 80 in parC in the QRDRs, and the most common type of amino-acid substitution was S80I in parC. Previous studies reported that the substitution S80I was most frequently observed among substitutions in the QRDR of parC [34, 35]. These results are consistent with those observed in E. coli isolates from humans [36, 37]. Also, the MICs ranges of isolates with double mutations in gyrA were relatively higher than those of other isolates with single mutations in gyrA. Notably, FQ-resistant E. coli isolates carrying double mutations in gyrA in combination with double mutations in parC were identified at high levels of MICs (≥ 64 mg/L for CIP, ≥ 128 mg/L for ENR, and ≥ 256 mg/L for NOR). These results are consistent with those of recent studies showing that the total number of point mutations in QRDR was positively correlated with the increased MIC.

In this study, 12 FQ-resistant E. coli isolates carried three types of PMQR genes, aac(6’)-Ib-cr (7 isolates), qnrS (3 isolates), and qepA (2 isolates). These PMQR variants have been previously detected in E. coli from livestock, including in healthy animals and retail meats in the Czech Republic [38], China [39], and the United States [40], as well as from swine in Korea [14]. Also, to acquire FQ resistance, bacteria usually require at least double mutations in QRDRs [41]. However, in this study, two isolates (4 mg/L for CIP, 8 mg/L for ENR, and 8 mg/L for NOR) had only a single mutation in gyrA and harbored qnrS in its plasmid. This result showed that PMQR genes play a role in FQ resistance [42].

The rise of antimicrobial resistance is thought to be closely associated with the widespread transfer of resistance genes between bacterial species. In this study, all 12 PMQR-positive E. coli isolates carried a variety of antimicrobial resistance genes such as blaCMY, blaTEM, blaOXA, blaSHV, blaAAC, tetA, tetB, tetD, tetE, aac(3)-II, sul1, sul2, and cmlA and harbored mobile elements such as integrons and gene cassettes at the same time. The bla genes hydrolyze the characteristic β-lactam ring and confer resistance to most β-lactam antimicrobials, including cephalosporins [43]. Previous studies reported that the PMQR genes in bla positive-E. coli were detected at high levels [44]. The presence of the PMQR genes may be significantly associated with the β-lactamase gene, perhaps due to common carriage on a plasmid in Enterobacteriaceae [45]. Also, all 12 PMQR-positive E. coli isolates harbored class 1 integrons and four isolates also contained gene cassettes aadA or dfrA or both genes. These genes are frequently detected in class 1 integrons isolated from animals and humans in Korea [46]. Therefore, integrons in PMQR-positive E. coli isolates from suckling piglets may have acquired the genetic determinants of drug resistance, which could become a concern.

Plasmids are small DNA molecules that are distinct from chromosomes and can provide beneficial effects to bacteria such as antibiotic resistance through horizontal gene transfer [21]. In this study, 10 plasmid replicon types were identified in all 12 PMQR-positive E. coli isolates. The most common plasmid replicon was IncF plasmids including FIB, and FIC. IncF plasmids were associated with the important role in the spread of virulence and resistance to important classes of antimicrobials including quinolones, β-lactams, TEs, sulfonamides, chloramphenicol, and aminoglycosides among Enterobacteriaceae [47].

The detection of E. coli virulence factors is important for diagnosing and establishing preventative measures for colibacillosis [48]. In this study, toxin genes LT, STb, and Sta were detected in nine (75.0%), nine (75.0%), and one (8.3%) PMQR-positive E. coli isolates, respectively. These LT, STa, and STb genes damage the vessel and cause edema and a high mortality in pigs [49]. Also, the most prevalent fimbriae antigen was F4 (7 isolates, 58.3%). F4 fimbriae have been frequently detected in piglets in several countries such as Japan, Europe, and the United States [50,51,52]. Previous studies reported that paa is known to have a high association with F4 [50, 53]. In this study, 6 (50.0%) PMQR-positive E. coli isolates were identified as having the paa gene and coexisting with the F4 gene. Although the specific role of the paa gene in the development of pathogenic E. coli has not yet been clearly defined, various virotypes may also appear as a result of horizontal gene transferability of the paa gene [54].

Conclusions

This study investigated the molecular characteristics of FQ-resistant E. coli isolated from suckling piglets with colibacillosis. All FQ-resistant E. coli isolates showed an MDR phenotype, and the most prevalent of the mutations were double point mutations in gyrA and a single mutation in parC. Also, FQ-resistant E. coli isolates with PMQR genes carried various antimicrobial genes and harbored mobile elements and plasmid replicons. Antimicrobial resistance may become a serious problem because many drugs are probably ineffective for the treatment of colibacillosis and resistance elements can be horizontally transferred on pig farms. Also, this represents a public health hazard because FQ-resistant E. coli can serve as a reservoir of FQ resistant genetic determinants that can be transferred to pathogenic bacteria in humans or pigs. These data support the critical need for comprehensive surveillance of antimicrobial resistance on pig farms.