Phenotypic and genotypic characteristics of Escherichia coli with non-susceptibility to quinolones isolated from environmental samples on pig farms

Background In the last decade, the growth of the pig-farming industry has led to an increase in antibiotic use, including several used in human medicine, e.g. (fluoro)quinolones. Data from several studies suggest that there is a link between the agricultural use of antibiotics and the prevalence of antibiotic-resistant bacteria in the pig farm environment, including (fluoro)quinolone resistance. This poses a threat to human and animal health. Our goal was to phenotypically and genotypically characterize 174 E. coli showing non-susceptibility to quinolones isolated from environmental samples from pig farms. Antimicrobial susceptibility testing (AST) was performed using the disk diffusion method. PCR and sequence analysis were performed to identify chromosomal mutations in the quinolone resistance-determining regions (QRDR) of gyrA and the isolates were screened for the presence of the plasmid-mediated quinolone resistance (PMQR) genes aac-(6')-Ib-cr, qepA, qnrA, qnrB, qnrC, qnrD and qnrS. Strain relatedness was assessed by phylogenetic classification and multilocus sequence typing (MLST). Results Of 174 isolates, 81% (n = 141) were resistant to nalidixic acid, and 19% (n = 33) were intermediately resistant. Overall, 68.4% (n = 119) were multidrug resistant. This study revealed a prevalence of 79.9% (n = 139) for gyrA QRDR mutations, and detected 21.8% (n = 38) isolates with at least one PMQR gene. The two most frequently detected PMQR genes were qnrB and qnrS (13.8% (n = 24) and 9.8% (n = 17, respectively). E. coli belonging to phylogenetic group A (48.3%/n = 84) and group B1 (33.3% /n = 58) were the most frequent. E. coli ST10 (n = 20) and ST297 (n = 20) were the most common STs. Conclusions E. coli with non-susceptibility to quinolones are widespread among the environment of Swiss pig farms and are often associated with an MDR phenotype. In several cases these isolates possess at least one PMQR gene, which could spread by horizontal gene transfer. E. coli from pig farms have diverse STs, some of which are associated with human and animal disease. Electronic supplementary material The online version of this article (10.1186/s40813-019-0116-y) contains supplementary material, which is available to authorized users.


Introduction
Bacterial diseases in pigs reared on production farms are responsible for high morbidity and mortality rates and subsequently also for increased economic losses [1,2]. The main indications for antibiotic therapy in pigs in Switzerland are gastrointestinal and respiratory diseases [3]. Therapy may include (fluoro)quinolones, which are categorized by the World Health Organization (WHO) as critically important antimicrobial agents (CIAs) [4]. Despite the worldwide growth of the pig-farming industry, monitoring systems on the use of antibiotics in veterinary medicine have led to reduced antibiotic use in many European countries, including Germany, Denmark, The Netherlands, and Sweden [5].
In pig husbandry, the usual way of drug application is oral, via the feed mixture. This is a practical way of drug application from a farmer's point of view, but the drawback is the release of antimicrobial substances into the farm environment [6]. Data from several studies suggest that there is a link between the agricultural use of antibiotics and the prevalence of antibiotic-resistant bacteria in the pig farm environment, including (fluoro)quinolone resistance [7][8][9][10][11].
The pathways of antibiotic residues into the environment after animal treatment are numerous. Moreover, certain antibiotics such as fluoroquinolones and tetracyclines are not fully metabolized in pigs and their residues may be detected in dust, manure, sewage, soil, groundand surface water and crops [6,[12][13][14][15]. These various antibiotic residue reservoirs are perfect breeding grounds for resistant bacteria, including (fluoro)quinolone resistant E. coli [16]. During a previous study aimed at analysing the use of fluoroquinolones in Swiss pig farms (von Ah, et al., manuscript under review), quinolone non-susceptible E. coli were isolated from environmental samples (dust, liquid manure and wipe samples of bay walls) of the farms. The goal of this study was to characterize these isolates with regard to the two main mechanisms of (fluoro)quinolone resistance in Enterobacteriaceae, i.e., the accumulation of mutations in the Quinolone Resistance Determining Region (QRDR) of gyrA encoding DNA gyrase, and the acquisition of plasmid-mediated quinolone resistance (PMQR) genes [11]. A further aim was to characterize the strains by phylogenetic grouping and multilocus sequence typing (MLST).

Strains and data collection
In this study, we analysed 174 E. coli isolates collected during 2016 by the Division of Swine Medicine of the Vetsuisse Faculty Zurich (von Ah, et al., manuscript under review). The collection consisted of strains obtained using both qualitative and semi quantitative methods to isolate quinolone resistant E. coli from dust, wipe and slurry samples from farm environments. For the qualitative method, an average of 1.91 g of sample was diluted 1:10 in Enterobacteriaceae Enrichment (EE) broth (Becton, Dickinson, Heidelberg, Germany) and incubated at 37°C overnight. The enrichment was then spread on RAPID'E. coli agar (Biorad, Munich, Germany), supplemented with 8 μg/ml nalidixic acid, and incubated overnight at 37°C. Using the semi-quantitative approach, an average of 1.97 g of sample, were diluted in a ratio of 1:10 in 0.85% saline solution and homogenized in a Stomacher sample blender (Seward Medical Ltd., London, UK). The homogenate was spread in dilution steps of 1:100 and 1:1000, respectively, on RAPID'E. coli agar plates supplemented with 8 μg/ml nalidixic acid. One E. coli isolate randomly selected from each positive sample was collected for further analysis. Isolates with questionable identity on RAPID'E. coli agar were confirmed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS, Bruker Daltronics, Bremen, Germany).
In total, the strains originated from dust (n = 48), wipes (n = 56), and slurry (n = 70), collected from 55 different farms (24 farrowing and rearing farms, 23 fattening farms and 8 mating and gestation farms) located in central and north-eastern Switzerland. All farms were part of a sow pool system. Of the 55 farms, 23 (41.8%) reported use of fluoroquinolones during the study period of 2016, including 16 (66.7%) of the farrowing and rearing, four (17.4%) of the fattening, and three (37.5%) of the mating and gestation farms.
Isolates displaying resistance to three or more classes of antimicrobials (counting β-lactams as one class) were defined as multidrug-resistant (MDR).

Molecular methods
Analysis of the quinolone resistance-determining region (QRDR) in gyrA All strains were examined for mutations in the quinolone resistance-determining regions (QRDRs) of gyrA, using PCR amplification and sequencing primers as described previously [18]. Synthesis of primers and DNA custom sequencing was carried out by Microsynth (Balgach, Switzerland). Nucleotide sequences were analyzed with CLC Main Workbench 8.0.1 and aligned with the sequence gyrA reference strain E. coli K-12, substrain MG1655 (GenBank: U00096). For database searches the BLASTN program of the National Centre for Biotechnology Information (NCBI) (http:// www.ncbi.nlm.nih.gov/blast/) was used.
The presence of qnrS confirmed and sequenced as described by Zurfluh et al., using E. coli OW95E1 as a positive control and the forward primer (qnrS_orf_F) [27]. Sequences were analysed as described for qnrB.

Phylogenetic characterization and multilocus sequence typing
Phylogenetic classification of the E. coli isolates into one of the eight groups A, B1, B2, C, D, E, F (E. coli sensu stricto), or Escherichia clade I, was performed as described by Clermont et al. [28].
Sequence type (ST) determination of the E. coli isolates was carried out as described by Wirth et al. [29]. Sequences were imported into the E. coli multilocus sequence type (MLST) database (http://enterobase.warwick.ac.uk) to determine MLST types and clonal complexes (CC).

Serotyping of E. coli ST301
Strains belonging to CC165 and ST301 frequently belong to the unusual O80 serogroup [30]. To test this possibility, all isolates belonging to ST301 were serotyped. The O80 serogroup was identified by O80-specific PCR using primers and conditions described previously [30]. The H2 type was determined by PCR targeting the flicH2 gene with primers described elsewhere [31]. The presence of the intestinal virulence genes stx, eae and of extra-intestinal virulence genes associated with plasmid pS88 [32] was evaluated as described previously [33].

Antimicrobial resistance phenotypes
Antimicrobial susceptibility testing by the disc diffusion method showed that 81% (n = 141) of the strains were resistant and 19% (n = 33) were intermediately resistant to nalidixic acid (Table 1). Furthermore, 36.2% (n = 63) of the isolates were also resistant to ciprofloxacin (Table 1).
Of the 174 isolates analysed in this study, 68.4% (n = 119) were resistant to three or more classes of antibiotics and therefore categorised as MDR. The most frequent MDR combinations detected were SXT-TE-STR (n = 15), AM-SXT-TE-STR (n = 10) and AM-SXT-STR-K (n = 8) ( Table 1). E. coli strains resistant to four and five antibiotics were the most prevalent (21.3 and 19.0%, respectively).
By contrast, of the 32 farms without a history of fluoroquinolone use during the study period, nine (28.1%) tested positive for E. coli harbouring PMQR genes. Thereof, five (55.6%) were fattening farms, four (44.4%) were mating and gestation farms, and none (0%) were farrowing and rearing farms (Table 1).

Characteristics of E. coli ST301 isolates
Three isolates belonged to ST301 (Table 1). Thereof, two (isolates 30 and 31, respectively) belonged to serotype O80:H2 and possessed the eae-ξ variant. None of the isolates harboured stx or any genes related to pS88. Both isolates were therefore classified as enteropathogenic E.coli (EPEC).

Resistance profiles
In the present study, we determined the prevalence of point mutations within the QRDR of gyrA and the presence of PMQR genes among 174 E. coli isolated from pig farm environments. All isolates were non-susceptible to quinolones and, using the disk diffusion method, were classified as intermediate or as resistant to nalidixic acid.
All resistant isolates except two, possessed at least one mutation in gyrA. Notably, both these isolates possessed a PMQR gene, (isolate 65 possessed qnrB, and isolate 106 qnrS, respectively). Since qnr genes alone are insufficient to confer resistance [34][35][36], further mechanisms are likely associated with the resistance phenotype of these strains, such as mutations in the QRDR regions of the gyrB, parC genes, or increased efflux pump activity (both not evaluated in this study).
Of the 70 nalidixic acid resistant strains possessing one mutation in gyrA, only one (0.7%) was resistant to ciprofloxacin. By contrast, of the isolates with double mutations in gyrA, the majority (89.9%) were also resistant to ciprofloxacin. These observations correlate with previous studies that link the number of quinolone resistance-associated mutations and the resistance phenotype of an isolate [37,38].
In this study, only a minority of the resistant strains carried PMQR genes, and, except for aac(6′)-Ib-cr, none of the PMQR genes were associated with ciprofloxacin resistance. Isolates with qnrS also possessed a Ser83 to   Table A1); PG phylogenetic group, PMQR plasmid-mediated quinolone resistance, QRDR quinolone resistance-determining region, R resistant, S susceptible, STR streptomycin, SL slurry sample, SXT sulfamethoxazole/trimethoprim, ST sequence type, TE tetracycline, W wipe sample; +, use of fluoroquinolone during study period 2016; −, no use of fluoroquinolone during study period 2016; −-, absence of point mutation, gene, CC, or additional resistance Leu substitution in the gyrA QRDR and showed decreased susceptibility to ciprofloxacin. Two further resistant isolates harbouring qnrS or qnrB lacked mutations in the gyrA QRDR and were susceptible to ciprofloxacin. In short, these data correlate with results from previous studies on the coexistence of different resistance mechanisms in one isolate [37][38][39].
Notably, the vast majority (90.9%) of strains with intermediate resistance to nalidixic acid were associated with the presence of qnrB, qnrS or a combination thereof. The three remaining intermediately resistant isolates lacking both gyrA mutations and aac(6′)-Ib-cr, qnrA, qnrB, qnrC, qnrD, qnrS, or qepA genes, are likely to possess other resistance-mechanisms that we did not screen for in the present study, e.g., point mutations within the QRDR of gyrB or in the topoisomerase genes parC or parE [11]. Four isolates possessed a combination of qnrB and qnrS genes but remained intermediately resistant to nalidixic acid. These results are in agreement with the findings of other studies [40,41], which demonstrate that the presence of two different qnr genes in the same strain has no additive effect on resistance levels. Further, our data correlate with previously mentioned studies concerning the coexistence of different resistance mechanisms in one isolate [37][38][39].
The predominance of PMQR among isolates that lack gyrA mutations is noteworthy, since these genes are known to facilitate the selection of resistant mutants. Data from other studies [42,43] suggest that, depending on which mutations are already present in a strain, the acquisition of further fluoroquinolone resistance genes could increase the strain's fitness.
In this study, the most frequent PMQR genes were qnrB and qnrS, respectively. Correlating with our data, it has been reported previously that qnrB is the most frequent PMQR gene, followed by qnrS. [11,36,44,45]. The presence of qnr genes in environmental E. coli indicates that selection could occur without exposure to inhibitory concentrations of fluoroquinolones. It has been demonstrated previously for environmental Klebsiella pneumoniae isolated from wastewater that qnr genes confer a selective advantage in the presence of residual subinhibitory fluoroquinolone concentrations present in wastewater [46] Accordingly, pig farms with a history of (in-feed) application of fluoroquinolones may represent environments containing residual concentrations of antibiotics which propagate PMQR genes. Our data suggest that this may hold true in particular for farrowing and rearing farms, where such genes were detected exclusively on farms with a history of use of fluoroquinolones. Environmental pollution with residual fluoroquinolones is facilitated by their poor degradability and strong potential for binding to sediments [47]. In the absence of solar radiation some fluoroquinolones (enrofloxacin) remain stable for at least 120 days [48]. In their long-term experimental study Xu and colleagues [49] stated that the application of swine manure lead to an increase of fluoroquinolone resistances in soil, including PMQR genes, which persisted at least five months. On farms without prior treatment of animals, strains harbouring quinolone resistance genes may be introduced during transfer of pigs from other locations, comparable to inter-farm transmissions of extendedspectrum ß-lactamase (ESBL) producing E. coli in pigs [50]. Our data suggest that this may especially be the case for mating and gestation farms and for fattening farms, where, in contrast to farrowing and rearing farms, environmental contamination with E. coli harbouring PMQR genes was detected irrespective of the history of fluoroquinolone treatment. These findings suggest that animal movement to and from farrowing and rearing farms with recent histories of treatment may promote the risk of transmission of resistant bacteria and of fluoroquinolone resistance genes among farms within sow pool systems.
Apart from direct transmission of resistant E. coli, PMQR genes can be transferred horizontally to other bacteria in the pig farm environment. Exposure to quinolones of bacteria containing qnr genes may increase their capacity to acquire point mutations in the gyrase and/or topoisomerase IV genes [46]. PMQR genes are often harboured on plasmids containing other resistance genes, e.g., ß-lactamases [51], thus, the use of non-fluoroquinolone antimicrobials enables their co-selection [52].
Overall, the resistance profiles of the isolates described in this study are in agreement with previous studies that have demonstrated that some of the most common antibiotic resistances (other than to quinolones) in E. coli in the pig environment are to tetracycline, ampicillin, streptomycin and sulfamethoxazole [53,54], and that E. coli strains from the pig environment often are resistant to four or five antibiotics simultaneously [55].

Phylogenetic grouping, MLST and serotyping of ST301
The majority of the isolates belonged to phylogenetic groups A or B1. The predominance of ST10 among the E. coli belonging to group A reflect previous observations regarding isolates from pigs, chicken faeces, as well as retail chicken and pork meat [56,57]. Furthermore, Araùjo et al. [58] observed that E. coli ST10 and ST297 were the predominant sequence types among MDR isolates isolated from irrigation water and vegetables in household farms, highlighting the wide dissemination of this sequence type and its association with MDR. Notably, E. coli ST10 (CC10), as well as E. coli CC23 also identified in this study, both including ciprofloxacin resistant and MDR strains, are also associated with urinary tract infection and sepsis in humans [59].
In addition, several other E. coli belonging to phylogenetic group A identified in this study have been associated with disease in food producing animals, such as avian pathogenic E. coli ST93 [60], E. coli ST744 isolated from diseased calves [61], and strains belonging to CC165 from food producing animals [62]. Notably, among the latter, we detected two EPEC O80:H2 isolates, which is considered an emerging pathogen among calves in Belgium [63]. This serotype has emerged among humans as a highly virulent extra-intestinal pathogenic Shiga-toxin producing E. coli (STEC) in France and Switzerland since 2015 [30,33]. As opposed to the STEC O80:H2 found in humans, the isolates from this study lacked pS88-associated extra-intestinal virulence genes. Therefore, the relationship between EPEC O80:H2 isolated from the farm environment and human STEC isolates needs to be assessed, e.g. by whole genome sequencing, and the prevalence of EPEC O80:H2 in pigs should be established.
E. coli strains belonging to the phylogenetic group B1 are for the most part commensal, with the ability to persist in the environment [64]. Most ST of this phylogroup, such as ST58, ST162, or ST453 have been frequently detected among healthy livestock [65]. However, E. coli ST297, which was one of most frequently observed ST in this study, has been associated with disease in both poultry and humans [66]. Likewise, E. coli ST453 is known to cause extraintestinal disease in humans (urinary tract infections) and metritis in cattle [67].
A minority of strains were assigned to the extraintestinal virulent phylogroups D and F. Among these, we detected two MDR strains belonging to ST117 which is a well-recognized avian pathogenic E. coli with zoonotic potential [68].

Conclusions
Quinolone non-susceptible E. coli are widespread in the environment of Swiss pig farms. In particular, isolates showing intermediate resistance to nalidixic acid frequently possess transmissible PMQR genes. This is worrisome, since the presence of qnr genes may increase the ability of bacteria to acquire point mutations in the gyrase and topoisomerase IV genes, resulting in high level resistance to (fluoro)quinolones. Furthermore, plasmids harbouring qnr genes may contribute to the horizontal spread of antibiotic resistance in livestock and in the environment. In pig farms which are part of sow pool systems, inter-farm measures that aim to reduce the risk of spreading resistant bacteria and resistance genes from one stage of production to the next need to be assessed and promoted. Our data further show that farm environments contain commensal MDR E. coli as well as E. coli with zoonotic potential. In particular, we demonstrate for the first time the presence of EPEC O80:H2 in an environmental sample from a pig farm.
In order to preserve the usefulness of fluoroquinolones and to protect animal and human health, surveillance of antimicrobial resistance is warranted. Measures for prudent use of (fluoro)quinolones as provided by the European Union's guidelines for use of antimicrobials in veterinary medicine are of utmost importance [69].

Additional file
Additional file 1: Table A1. Results of the MLST analysis for E. coli strains with new STs. (DOCX 26 kb)