Between May 2013 and December 2014, E. coli isolates were obtained from human clinical materials (urine, stool, sputum, blood, bile, endotracheal aspirate, samples from cannulas, and bronchoalveolar lavages) of patients hospitalized at the University Hospital Olomouc (UHO) in the Czech Republic and from urine samples from outpatients in the community of Olomouc Region. The community subjects had neither been hospitalized in the previous 3 months nor had been living in nursing homes.

In the same period (May 2013 and December 2014), E. coli isolates were obtained from environmental samples from chicken farms (n = 2628) and cloacal swabs from market-weight turkeys at slaughterhouses (n = 120) in the eastern part of the Czech Republic (Olomouc and South Moravian Region). Environmental samples were taken from bedding using gauze shoe covers worn by a worker who walked through a poultry house. Turkey cloacal swabs were collected at slaughterhouses and placed into Amies transport medium.

Wastewaters were taken during 2013–2015 in six sampling sites that included wastewater treatment plants (WWTP) from four hospitals located in three towns of the Olomouc Region (Olomouc, Prostejov and Sternberk) (WWTP1-4), the abattoir in Prerov (WWTP5) and WWTP near Henčlov (WWTP6). In total, 124 wastewater samples were examined including 21 samples from WWTP1 (in 2013-6 samples; 2014-13; 2015-2), 25 from WWTP2 (2014-20, 2015-5), 19 from WWTP3 (in 2013-6; 2014-11, 2015-2), 19 from WWTP4 (in 2013-6; 2014-11, 2015-2), 21 from WWTP5 (collected weekly for a period of 4 months in 2013/2014, plus 3 control samplings were made in May and August 2014) and 19 from WWTP6 (collected weekly for a period of 4 months in 2013/2014). Cellulose swabs were immersed into wastewater at the inflow and/or outflow of sampling points for 48 h according to the procedure described (Moore et al., 1952).

Faecal rook (Corvus frugilegus) samples (n = 595) were collected from roosting places used by rook flocks in the Olomouc Region. The samples were collected in Prerov in October, November and December 2012, in Tovacov from January till March 2013, and in Troubky in November 2013. The sampling method has been described previously (Literak et al., 2012). Smears from fresh feces were taken by cotton swabs tampons and inserted into Amies transport medium.

After delivery to the laboratory, collected samples were handled according to the following procedures in order to isolate E. coli.

Samples from the environment of chicken farms were placed into peptone water and incubated aerobically for 24 h at 37°C. Subsequently, the peptone water was inoculated onto MacConkey agar with ciprofloxacin (0.05 mg/L; MCA_CIP) and cultivated overnight. Swabs from retailed turkeys were subcultivated directly on MCA_CIP overnight. One isolate per sample was selected for further phenotypic and genetic analysis.

Swabs taken from wastewaters were inserted into a sterile bottle with peptone water and incubated at 37°C for 24 h. The enriched samples of peptone water were subcultivated on MCA_CIP overnight. One to ten colonies of lactose-positive colonies showing different morphology were taken from each MCA_CIP and subjected to further examination.

Faecal swabs from rooks were incubated in buffered peptone water at 37°C overnight and subsequently cultivated on MCA_CIP overnight. In this type of sample, also only one isolate per sample was selected.

Human clinical material was handled according to the types of material and cultivated aerobically for 24 h at 37°C. A total number of 3521 E. coli isolates were collected and from these, three hundred and three isolates were randomly selected for detection of PMQR genes. The collection analyzed included 270 resistant (MIC of ciprofloxacin >0.5 mg/L) and 33 sensitive (MIC of ciprofloxacin ≤ 0.5 mg/L) isolates according to the European Committee in Antimicrobial Susceptibility Testing (EUCAST) criteria.

The species identification of all isolates was confirmed using matrix-assisted laser desorption/ionization-time of flight mass spectrometer (MALDI-TOF MS) (Biotyper Microflex, Bruker Daltonik GmbH, Bremen, Germany).

Genomic DNA of all E. coli isolates obtained using heat lysis was used as a template for PCR detection of PMQR genes (aac(6′)-Ib-cr, qepA, qnrA, qnrB, qnrC, qnrD, qnrS, oqxAB), followed by sequencing of the amplicons. As positive controls, well-known characterized strains were included in each reaction (Table S1).

For each PMQR-positive E. coli isolate, susceptibilities to ampicillin (the ranges of tested concentrations for the antimicrobial substance were 0.5–64 mg/L, ampicillin/sulbactam (0.5–64), cefazolin (0.5–64), cefuroxime (0.5–64), gentamicin (0.25–32 or 0.5–64), trimethoprim/sulfamethoxazole (1–128), colistin (0.25–32), oxolinic acid (0.25–32 or 0.5–64), ofloxacin (0.125–16 or 0.25–32), tetracycline (0.25–32), aztreonam (1–64), piperacillin (2–256), piperacillin/tazobactam (2–256), cefoperazone (0.25–32), cefotaxime (0.125–16), ceftazidime (0.125–16), cefepime (0.125–16), cefoperazone/sulbactam (0.5–64), meropenem (0.125–16), ciprofloxacin (0.125–16 or 0.25–32), tigecycline (0.06–8), tobramycin (0.25–32), and amikacin (0.5–32) were tested using the standard microdilution method according to the EUCAST (European Committee on Antimicrobial Susceptibility Testing) breakpoint criteria (European Centre for Disease Prevention and Control, 2015). Microdilution antibiotic panels were prepared using dispensing instrument DYNAMIC 3000 automated system (DYNEX, Czech Republic). E. coli ATCC 25922 and E. coli ATCC 35218 were used as reference strains for quality control.

All PMQR-positive isolates of E. coli with the minimum inhibitory concentration (MIC) of the tested 3rd and 4th generation cephalosporins ≥1 mg/L were screened for extended-spectrum beta-lactamase production (ESBL) using Jarlier's double-disk synergy test (DDST) which was modified by including a disk with cefepime and another disk with ceftazidime and ceftazidime/clavulanic acid (Jarlier et al., 1988; Htoutou Sedlakova et al., 2011). Each ESBL-producing isolate was screened for bla_CTX-M, bla_TEM and bla_SHV genes by PCR followed by sequencing of amplicons of genes responsible for ESBL phenotype (Table S1).

From a group of 262 PMQR-positive E. coli isolates, 144 isolates from all of the studied areas were selected for further characterization that included the detection of specific mutations in gyrA, gyrB, parC and parE genes and multilocus sequence typing (MLST). Isolates were selected randomly in order to cover all the studied areas.

Total genomic DNA from E. coli strains was prepared from an overnight culture (16 h, 37°C) grown on meat-peptone agar using DNeasy Blood & Tissue kit (QIAGEN, Germany) according to the manufacturer's recommendations.

PCR amplification of the part of gyrA, gyrB, parC and parE genes that included the sequence of QRDRs was performed using previously described primers (Oram and Fisher, 1991; Vila et al., 1994, 1996; Ruiz et al., 1997). The reaction mixture contained complete reaction buffer with MgCl₂ (containing 100 mmol/L Tris-HCl (pH 8.8), 500 mmol/L KCl, 1% Triton X-100, 15 mmol/L MgCl₂ (Top-Bio, Czech Republic), 0.5 U of Taq DNA polymerase (Top-Bio), 0.4 μmol/L primer concentration for each primer, 40 μmol/L concentration of deoxynucleoside triphosphates and 1 μL of template DNA. The PCR was run in the Light Cycler96 instrument (Roche, USA) under the following conditions: initial denaturation at 95°C for min, 30 cycles of denaturation (95°C for 30 s), annealing (58°C for 30 s), extension (72°C for 60 s), and final extension at 72°C for 7 min.

MLST of seven housekeeping genes (adk, fumC, gyrB, icd, mdh, purA, recA) was performed according to the MLST protocol standardized for E. coli (http://mlst.warwick.ac.uk/mlst/).

For each of the 144 samples, 11 amplicons (4 topoisomerase and 7 MLST amplicons) were pooled in an equimolar ratio to generate one single sample and sent to the laboratories of the Institute of Applied Biotechnologies (Prague, Czech Republic) for sequencing. DNA libraries were constructed using the Nextera XT DNA Sample Preparation Kit (Illumina, Inc., San Diego, CA) and two multiplex sequencing assays (1 × 96 samples, 1 × 48 samples) were performed on the Illumina MiSeq platform (Illumina, Inc. San Diego, CA).

A quality control check of pair-end FASTQ files was performed as a first step. Bases with Phred Quality Score lower than defined threshold (threshold = 30) were filtered out. Quality control included check for possible adapter contamination. Then all variants of reference sequences of single housekeeping genes of E. coli (adk, fumC, gyrB, icd, mdh, purA, recA) in FASTA file were downloaded (http://mlst.warwick.ac.uk/mlst/dbs/Ecoli) and indexed by Burrows-Wheeler Aligner (BWA version 0.7.13; Li and Durbin, 2010). Paired-end FASTQ files were aligned against all indexed reference FASTA files using BWA-mem algorithm with minimum seed length = 19, matching score = 1, mismatch penalty = 4, gap open penalty = 6, and gap extension penalty = 1. Generated Sequence Alignment Map (SAM format) was converted into its binary form (BAM format) and sorted by coordinates. Unmapped sequences reads were filtered out from BAM files. Algorithms Samtools (samtools version 0.1.18) and VarScan (Koboldt et al., 2009) were applied for variant calling using the following parameters: minimum coverage = 10, minimum mapping quality = 10, minimum reads = 2, minimum variant frequency = 0.05, and p = 0.05 were used for variant calling. Generated Variant Calling Format (VCF format) was statistically analyzed and a minimum number of variants (required 0, this number means that our FASTQ files have 100% match with a variant of the reference gene) was evaluated like a gene form of selected housekeeping gene of E. coli. All bioinformatics analyses were performed on Operating System Linux (Ubuntu 14.04 LTS) and using programming language Python.

Single point mutations in selected part of gyrA, gyrB, parC and parE including QRDRs were evaluated using the Integrative Genomics Viewer IGV version 2.3. (Broad Institute, Cambrige, UK) (https://www.broadinstitute.org/software/igv). The genome sequence E. coli MG1655 (NC_000913.2) was selected as a reference sequence for alignment.

Sanger sequencing was performed with 11 samples to confirm the results of MLST analysis carried out by NGS. This was done because some problems occurred in the assignment of the appropriate allele in MLST analysis. Gene sequences were subsequently submitted to the E. coli MLST Database, University of Warwick (http://mlst.warwick.ac.uk/mlst/dbs/Ecoli/). Obtained MLST allele sequences were compared with the results generated by NGS.

E. coli isolates belonging to the same sequence type (ST) according to MLST analysis and originating from different sources (human, animal or wastewater) were subjected to examination of their epidemiological relationship using pulsed-field gel electrophoresis (PFGE). The total genomic DNA was isolated from overnight bacterial culture according to the procedure published (Husickova et al., 2012). All samples were digested with XbaI (30 U for 3 h) (Takara, Bio, Otsu, Shiga, Japan) and subjected to PFGE. The resulting restriction profiles were analyzed with the GelCompar II software (Applied Maths, Kortrijk, Belgium) using the Dice coefficient (1,5%) for comparing similarity and unweighted pair group method using arithmetic averages for cluster analysis. The results were interpreted according to criteria described by Tenover et al. (1995).

A collection of 1050 isolates of E. coli with reduced susceptibility or resistance to ciprofloxacin was obtained from humans, food producing, or wild animals and wastewater samples from the area of the Olomouc and South Moravian Regions, Czech Republic (Table 1). The highest occurrence of E. coli strains isolated from MCA_CIP of animal originwas detected from turkey samples (105 isolates per 120 samples), the lowest from chicken samples (156 isolates per 2628 samples). In human population, 303 E. coli isolates were selected according to the procedure described in material and methods section. From the total amount of 124 wastewater samples, 372 E. coli isolates were analyzed.

PMQR genes were identified in 262 (25%) E. coli isolates (Table 1). The gene qnrS1 was the most prevalent, found in 56% of PMQR-positive isolates from all studied areas. It was identified in 76% PMQR-positive isolates from poultry farms, turkey, rooks and wastewater. The second most common gene was aac(6′)-Ib-cr found in 82% of human PMQR-positive clinical isolates followed by qnrB19 that dominated in isolates from turkey and wastewater samples. The qnrA, qnrC, and qepA genes were not found.

Twenty-three isolates carried two different PMQR genes of the following combination: qnrB19+qnrS1 (12 isolates), qnrB1+aac(6′)-Ib-cr (7), qnrB4+aac(6′)-Ib-cr (2), qnrS1+oqxAB (1) qnrB10+qnrS1 (1).

In case of wastewaters where more than one E. coli colony per sample was examined, the occurrence of several E. coli isolates carrying different PMQR genes and originating the same water sample was detected. Six wastewater samples contained E. coli isolates carrying qnrS1 together with E. coli carrying qnrB19 were detected. In one sample E. coli harboring qnrB1 and E. coli with qnrS1 were found. The last sample contained two different types of E. coli, one carrying aac(6′)-Ib-cr and the other with qnrS1.

For each PMQR-positive E. coli isolate, susceptibility to 23 antimicrobial agents was tested (Table S2). Isolates from hospitalized patients showed the highest level of resistance including ampicillin (97%), piperacillin (96%), third generation cephalosporins (63% for cefotaxime and 43% for ceftazidime), tetracycline (83%), and tobramycin (86%). Isolates from the community displayed high level of resistance to ampicillin (93%), tetracycline (93%), piperacillin (93%), tobramycin (64%), and resistance to third generation cephalosporins (50.0% for cefotaxime and 43% for ceftazidime). On the other hand, wastewater isolates were less resistant to tested antibiotics. High level of resistance was detected only for ampicillin (79%), tetracycline (83%) and piperacillin (78%).

Resistance to ampicillin was common also among isolates from retailed turkeys (96%), chicken farms (71%) and rook feces (75%). Other frequent resistance phenotypes included tetracycline and piperacillin found in 77 and 59% of chicken, in 86 and 90% from turkey and in 75 and 70% from rook isolates, respectively. The majority of E. coli isolates from food-producing animals were susceptible to third generation cephalosporins while 20% of rook isolates showed resistance. Most isolates from the inpatients and the community showed resistance to ciprofloxacin. A total of 270 isolates were resistant (MIC of ciprofloxacin >0.5 mg/L) and 33 sensitive (MIC of ciprofloxacin ≤ 0.5 mg/L) according to the EUCAST criteria. However, this results is influenced by different selection criteria for human isolates since isolates with MIC of ciprofloxacin >0.5 mg/L were preferably included in the study. In contrast, only 50 of all PMQR-positive isolates from other studied areas showed resistance to ciprofloxacin with the highest level observed in isolates from chicken farms (41%). A total of 36% of animal E. coli isolates (chicken, turkey, and rook) were resistant to oxolinic acid, 32% to ofloxacin and 28% to ciprofloxacin. In the group of wastewater samples, 28% of isolates displayed resistance to oxolinic acid as well as to ciprofloxacin, and 34% to ofloxacin. The distribution of MIC values of tested quinolones in selected isolates (n = 144) and the comparison with the results of genetic detection is summarized in the Table 2.

A total number of 66 isolates was screened for ESBLs using phenotypic and genetic methods. Production of ESBL enzymes was found in 61 E. coli isolates. Subsequent genetic analysis revealed the presence of bla_CTX-M-15 in 51 isolates (hospital, community, wastewater). Two isolates from chicken and wastewater carried the gene bla_CTX-M-1, bla_CTX-M-14 was found in two isolates from hospital and wastewater and bla_SHV-12 was detected in three isolates from wastewater. Three isolates showing ESBL phenotype were negative for all tested bla genes.

Various mutations in gyrA, parC or parE genes were found in 60 out of 144 PMQR-positive isolates while no isolate with mutation in gyrB was observed (Table 2).

The most common GyrA substitution was Ser(83)Leu found in 59 isolates (41%), followed by Asp(87)Asn, detected in 49 isolates (34%). A total of 49 isolates carried double or triple substitution in GyrA.

Two aminoacid substitutions in ParC were found in 25 isolates (17%) with Ser(80)Ile and Glu(84)Val being the most common combination (n = 23). The most common substitution in ParE was Asp(475)Glu found in 18 isolates (13%).

All the E. coli isolates with a high level of ciprofloxacin resistance (>32 mg/L) were shown to carry double mutations in gyrA in combination with single or double mutations in parC. Substitutions at codon 83 and 87 in GyrA along with substitutions at codon 80 and 84 in parC gene were the most common. One isolate carried three mutations in gyrA with aminoacid changes at positions 83, 84, and 87. The majority of high-level ciprofloxacin resistant isolates were obtained from hospitalized patients and possessed the gene aac(6′)-Ib-cr. Other isolates with high level ciprofloxacin resistance originated from community and WWTP3 also carried aac(6′)-Ib-cr.

In most isolates susceptible to ciprofloxacin according to the EUCAST criteria (≤ 0.5 mg/L), no mutations in gyrA, gyrB, or parC were detected. However, five isolates possessed single aminoacid substitution at the codon 83 in GyrA (MIC of ciprofloxacin was 0.5 mg/L). All isolates were susceptible to almost all tested antimicrobial agents except ofloxacin (the MIC was 16–64 mg/L) and three of them were resistant to ofloxacin (MIC = 2 mg/L). Substitutions at position 378 [Arg(378)His)] or 475 [Asp(475) Glu] in ParE were found in 18 isolates susceptible to ciprofloxacin (MIC of ciprofloxacin was 0.2 or 0.5 mg/L). These strains displayed high level of resistance to ampicillin and piperacillin.

In the group of 144 isolates, analyzed by MLST, 48 different STs were identified (Table S3) including five major clones ST131 (n = 26), ST355 (19), ST48 (13), ST95 (10), and ST10 (5). Isolates of ST131 were obtained from various types of human clinical materials and from one wastewater sample, while the second most common clone ST355 carrying the qnrS1 or the combination of qnrS1+qnrB19 genes was found predominantly from retailed turkey. MLST analysis also showed the presence of identical STs in samples of different origin. Isolates of ST88 or ST226 were identified from chicken farms as well as from retailed turkeys. ST10, ST48, and ST95 were found in the poultry and wildlife (chicken farms, retailed turkey, and rooks) and in wastewater samples. Other sequence types identified in samples of different origin included ST428, ST533, and ST617. The determination of relevant ST failed in four samples. In these samples bioinformatics analysis did not assign appropriate allele variant and Sanger sequencing did not provide relevant sequences, suggesting the isolates belonged to atypical E. coli isolates untypable by MLST.

ST131 was most often associated with bla_CTX-M-15 and aac(6′)-Ib-cr, however one strain harbored the combination of bla_CTX-M-15 a qnrB1. The production of CTX-M-15 enzyme was also detected together with qnrB1 in one ST393 and two ST410 strains. In three ST405 and one ST617 a combination of bla_CTX-M-15 and aac(6′)-Ib-cr was identified. One ST48 E. coli isolate harbored bla_CTX-M-15, together with qnrB1 and aac(6′)-Ib-cr. In one qnrS1-postitive ST43 strain, bla_CTX-M-1 gene was detected. Two qnrS1-positive and SHV-12 producing strains, one belonging to ST540 and the other to ST58 were identified. The other CTX-M-14 or CTX-M-15 producing strains were not analyzed using MLST.

E. coli isolates belonging to the same ST and originating from different sources (human, animal or environmental) were subjected to PFGE to determine the level of their genetic relatedness (Figure 1). Based on these criteria a total of 68 isolates of the following STs were analyzed: ST10 (n = 5), ST48 (n = 13), ST58 (n = 2), ST88 (n = 3), ST95 (n = 10), ST131 (n = 26), ST226 (n = 2), ST428 (n = 2), ST533 (n = 2), and ST617 (n = 3). Overall, a high diversity of restriction patterns was observed in isolates of the same ST. No isolates with 100% identity of PFGE profiles originating from different areas (human, animal, or environment) were identified.

Globally disseminated ST131 was predominant in samples from hospitalized patients as well as from community subjects while no isolates of this clonal group were found in food-producing animals or rooks. The majority (69%) of E. coli ST131 isolates collected from these samples displayed high-level of ciprofloxacin resistance (>32 mg/L), harbored double or triple mutations in gyrA along with double mutations in parC gene and the PMQR gene aac(6′)-Ib-cr. The overall similarity of PFGE patterns of ST131 isolates was <50% confirming high genetic diversity of this clonal group. Only two E. coli ST131 isolates originating from the same patient from which the samples were taken 17 days apart shared 100% identity.

Significant clonal similarity was detected only in isolates originating from single collection areas, including ST10, ST48, ST88, and ST95 from retailed turkeys. In the ST10 group two isolates shared 100% similarity of their PFGE profiles. Both isolates were susceptible to ciprofloxacin, carried qnrS1, and did not contain gyrase or topoisomerase mutations. A group of six closely related strains was detected in ST48 group. All of them were collected from turkeys, displayed susceptibility or a low level of resistance to ciprofloxacin (MIC 1 - 2 mg/L), harbored qnrS1 and were originated from the same turkey farm. Another three ST48 isolates from WWTP5 displayed 100% identity of their PFGE patterns, originated from wastewater samples obtained from the same collection date and shared only 52% similarity of the PFGE patterns with ST48 isolates from turkey.

In the ST95 group, two and three strains from turkeys showed identical PFGE profiles. All isolates of the ST95 group were susceptible to ciprofloxacin, did not possess any mutations in gyrase or topoisomerase genes and most of them carried both qnrB19 and qnrS1 genes.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.