Broilers as a Source of Quinolone-Resistant and Extraintestinal Pathogenic Escherichia coli in the Czech Republic

Abstract

Extraintestinal Escherichia coli infections are associated with extraintestinal pathogenic E. coli (ExPEC) strains. A total of 114 E. coli isolates were characterized regarding their antimicrobial resistance in a prospective study of 319 broilers from 12 slaughterhouses in the Czech Republic, a European Union member, during 2008. PCR-based assays to define ExPEC-associated traits were performed in resistant strains. Consumption of antimicrobial drugs by poultry in the Czech Republic was also analyzed. Antibiotic resistance was detected in 82% of isolates. Resistance to nalidixic acid and ciprofloxacin was predominant. Plasmid-mediated quinolone resistance genes, qnrB19 and qnrS1, were detected in 1 and 3 of 93 resistant isolates, respectively. Twenty-three percent of resistant isolates were considered as ExPEC. In total, 972 kg of flumequine, enrofloxacin, and difloxacin were used in poultry in the Czech Republic during 2008. High prevalence of broilers with ciprofloxacin-resistant E. coli isolates was linked to consumption of quinolones in poultry. Broilers may comprise an important vehicle for community-wide dissemination of fluoroquinolone-resistant E. coli and ExPEC. Withdrawal of fluoroquinolones from use in chicken production should be seriously considered in the Czech Republic and the European Union as well.

Introduction

E scherichia coli causing extraintestinal diseases are known as extraintestinal pathogenic E. coli (ExPEC) and include the uropathogenic E. coli (UPEC), neonatal meningitis E. coli, and avian-pathogenic E. coli (APEC) subpathotypes.⁴⁴ A number of studies have shown that members of various ExPEC subpathotypes harbor similar virulence-associated genes. Characterization of ExPEC strains using various typing techniques has shown many similarities, despite their isolation from different host species, thus leading to the hypothesis that ExPEC may have zoonotic potential.⁴⁴

The use of fluoroquinolone agents in food animal production is suspected of selecting fluoroquinolone-resistant E. coli that can be transmitted to humans via the food chain.¹⁸ The epidemiological link between poultry consumption and human disease due to fluoroquinolone-resistant bacteria, combined with the high prevalence of fluoroquinolone-resistant bacteria in retail poultry products, has prompted the U.S. Food and Drug Administration to propose the withdrawal of fluoroquinolones from use in poultry.⁶ In the European Union, including the Czech Republic, there are currently, in place, recommendations for the prudent use of fluoroquinolones in food-producing animals.¹⁶

Retail poultry products are routinely contaminated heavily with avian fecal E. coli. Such E. coli extensively contaminate kitchen surfaces during meal preparation and can subsequently be isolated from the feces of a person preparing meals.³² Thus, the possibility of foodborne transmission of ExPEC and/or fluoroquinolone-resistant E. coli from poultry to humans is highly plausible.

As early as 1976, Levy et al. reported the transfer of tetracycline resistance genes between chicken E. coli strains, from chicken to chicken, and from chicken to humans.³¹ The aim of our study was to characterize fluoroquinolone- and cephalosporin-resistant E. coli and ExPEC isolates from broilers in the Czech Republic and the overall consumption of quinolones and cephalosporins used in poultry in the country during 2008.

Materials and Methods

Chicken meat sampling scheme and isolation of E. coli

Broilers were examined from April to December 2008 at 12 poultry slaughterhouses in the Czech Republic serving more than 300 Czech broiler farms. A total of 319 broilers were sampled. Each randomly sampled broiler originated from a group of broilers from one farm sent to a slaughterhouse on 1 day. The whole chilled broiler was placed individually into a sterile plastic bag and transported on ice to the laboratory. The skin of the broiler's body was detached from the rest of the body and a piece weighing 27 g was taken as a sample. The skin sample was homogenized for 1 min using a homogenizer in 243 ml of buffered peptone water (Oxoid). One-milliliter aliquots from individual suspension samples were frozen with a few drops of glycerin at −18°C until further examination. Individual samples were then enriched in the MacConkey broth and cultivated on MacConkey agar (MCA; Oxoid) for E. coli isolation. All E. coli strains were identified using the API 10S test (bioMerieux). One E. coli isolate per sample was randomly selected and further analyzed.

Antibiotic susceptibility testing

The disk-diffusion method⁸ was used to test susceptibilities of E. coli isolates to 12 antimicrobial substances: amoxicillin/clavulanic acid (30 μg), ampicillin (10 μg), cephalothin (30 μg), ceftazidime (30 μg), chloramphenicol (30 μg), ciprofloxacin (5 μg), gentamicin (10 μg), nalidixic acid (30 μg), streptomycin (10 μg), sulfamethoxazole/trimethoprim (25 μg), sulfonamide compounds (300 μg), and tetracycline (30 μg) (Oxoid). For the selective isolation of ESBL (extended-spectrum beta-lactamase)-positive E. coli, all samples enriched in the MacConkey broth were subcultivated on MCA with cefotaxime (2 mg/L) and the cefotaxime-resistant isolates should have been tested in the double-disk synergy test.⁴³

PCR and testing of antibiotic resistance genes

In E. coli isolates found to be resistant to one or more of the antibiotics listed above, antibiotic resistance genes and integrons were detected by PCR using specific primers as described previously.³³ These E. coli isolates were also tested for the plasmid-mediated quinolone resistance (PMQR) genes qnrA, qnrB, qnrC, qnrD, qnrS, aac(6')-Ib-cr, qepA, and oqxAB and the PCR products were further analyzed by sequencing.^{3,4,12,27,28,37,38,40,47} PMQR-positive isolates were examined for minimum inhibition concentrations of ciprofloxacin and nalidixic acid using the agar dilution method in accordance with CLSI standards.⁸

Detection of extraintestinal pathogenic E. coli

Lysates of each E. coli isolate resistant to antimicrobials were tested by PCR amplification for virulence factors or genes iutA, cvaC, kpsII, iss, tsh, papC, ibeA, and felA.^{11,13,17,22,30,35} The PCR cycle was 94°C for 5 min followed by 30 cycles at 94°C for 1 min, at the annealing temperature specific for each pair of primers for 1 min, and at 72°C for 1 min. As previously reported,²³ ExPEC was defined by detection of ≥2 of iutA, kpsII, tsh, papC, ibeA, and felA genes.

Serotyping

Somatic O-antigen typing was performed using a U-type microplate agglutination assay. E. coli strains were cultured in the nutrient broth (Imuna). After cultivation, viability staining of E. coli strains was made using triphenyltetrazolium chloride and heating at 120°C for 1 h followed. Agglutination was performed with a set of 70 types of O-antisera, encompassing the most common O-serogroups. O-antisera were prepared by immunization of rabbits.⁴¹

Pulse-field gel electrophoresis, determination of E. coli phylogenetic group, and transferability of PMQR genes by conjugation

PMQR-positive E. coli isolates were typed by XbaI pulse-field gel electrophoresis (PFGE).⁵ Macrorestriction patterns were analyzed using the BioNumerics 6.6 fingerprinting software (Applied Maths). Cluster analysis of the Dice similarity indices was done to generate a dendrogram describing the relationships among PFGE profiles. The level of similarity between patterns was defined at 85%. Identification of E. coli phylogenetic groups (A, B1, B2, and D) was performed using PCR.⁷ Transferability of PMQR genes was tested by conjugation and transformation.³⁶

Consumption of antimicrobial drugs in poultry in the Czech Republic

Sales data were collected from all wholesalers and feed mills licensed in the Czech Republic during the period monitored during 2008. Special attention was given to the groups of fluoroquinolones and cephalosporins of the 3rd and 4th generations, in relation to which detailed data have been collected, as these substances have been under a prudent use regimen in the Czech Republic since 1998.

Results

Antimicrobial resistance in chicken E. coli isolates

A total of 114 E. coli isolates was obtained from 319 broiler samples. Resistance to one or more antibiotics was detected in 93 (82%) isolates. In total, 33 (29%) of 93 isolates were resistant to only one antimicrobial agent and the remaining 60 isolates were resistant to more than one agent. The resistance patterns in individual isolates were highly variable. Resistance to nalidixic acid and ciprofloxacin was predominant, being found in 78 (68%) and 30 (26%, Table 1) isolates, respectively. Resistance to tetracycline was detected in 29 (25%) isolates, to ampicillin in 27 (24%), to sulfonamides in 22 (19%), to streptomycin in 18 (16%), to sulfamethoxazole/trimethoprim in 11 (10%), to cephalothin and chloramphenicol each in 4 (4%), and to amoxicillin-clavulanic acid and ceftazidime each in two (2%) isolates. None of the isolates was resistant to gentamicin. No ESBL-producing isolates were found in the Czech broilers.

Table 1.

Ciprofloxacin-Resistant Escherichia coli (Results of Disk Diffusion Method) from Broilers (n=319) Slaughtered in the Czech Republic

Antibiotic resistance phenotype	Antibiotic resistance genes and integrons	Number of isolates
CipNa	Not determined	10
CipNa	int1, class 1 integron 1 kb: aadA1	1
ACipNa	bla _TEM	3
CipNaS	aadA1	1
CipNaS	strA	1
CipNaT	tetA	2
ACipNaS	bla _TEM , strA	1
ACipNaSu	bla _TEM , sul2	1
CipNaSSu	strA, sul2	1
CipNaSuT	sul2, tetA	1
ACCipNaSu	bla_TEM, cat, sul1, int1, class 1 integron 1 kb: aadA1	1
ACipNaSSu	bla _TEM , strA, sul2	1
ACipNaST	bla _TEM , strA, tetB	1
CipNaSuSxtT	sul1, sul2, tetA	1
ACipNaSSuT	bla _TEM , strA, sul2, tetA	1
CipNaSSuSxtT	strA, aadA1, sul1, sul2, tetA	1
ACfCipNaSuSxtT	bla _TEM , sul2, tetB	1
ACCfCipNaSSuSxtT	bla_TEM, cmlA, aadA1, sul3, tetA,	1
Total		30

Antibiotic resistance phenotype: A, ampicillin; C, chloramphenicol; Cf, cephalothin; Cip, ciprofloxacin; Na, nalidixic acid; S, streptomycin; Su, sulfonamides cp.; Sxt, sulfamethoxazole/trimethoprim; T, tetracycline.

The following genes were identified in E. coli isolates: tetA, tetB (resistance to tetracycline), bla_TEM (beta-lactams), strA, aadA1 (streptomycin), sul1, sul2, sul3 (sulfonamides), cat and cmlA (chloramphenicol). All ampicillin-resistant isolates were positive for the presence of the bla_TEM gene. As regards to tetracycline resistance determinants, the tetA gene was detected in 22 and tetB in 7 of 29 tetracycline-resistant strains. Screening for sul-genes revealed the presence of at least one sul-type determinant in sulfonamide-resistant E. coli isolates. In 18 isolates, a single sul-type resistance determinant was found (sul1 in 5, sul2 in 11, and sul3 in two isolates), while in 5 isolates, 2 different sul genes (both sul1 and sul2) were found. Resistance to streptomycin was connected with the gene strA and/or aadA1. All chloramphenicol-resistant isolates were positive for presence of either the gene cat or clmA.

Eleven of 93 antibiotic-resistant E. coli isolates from the broiler samples yielded the intI1 gene and class 1 integron. A class 1 integron of 1 kb with the gene cassette aadA1 was present in seven E. coli isolates, and four isolates contained a 1.5-kb integron with the gene cassettes, dfr1-aadA1. Two of all the antibiotic-resistant isolates were positive for the intI2 and class 2 integron with the gene cassettes, estX-sat-aadA1 (Table 2).

Table 2.

Characteristics of Escherichia coli Isolates with Class 1 and 2 Integrons and Gene Cassettes

Isolate No.	Phylo-genetic group	O-antigen serotype	Size of class 1 and 2 integron (kb)	Gene cassettes	intI1	intI2	sul	Antibiotic resistance phenotype	Additional resistance genes	Virulence genes
O/67	A	O20	1	aadA1	+	−	—	Na	—	iutA
J/85	D	O25	1	aadA1	+	−	—	CipNa	—	iutA, cvaC, iss, kps
J/102	B2	nt	1	aadA1	+	−	sul3	ACSu	bla _TEM , cmlA	iutA, tsh
J/33	B2	O108	1	aadA1	+	−	—	NaST	tetA	iutA, cvaC
P/70	B2	nt	1	aadA1	+	−	sul2	ASSuSxt	bla _TEM , strA	iutA, cvaC, iss
J/43	B2	nt	1	aadA1	+	−	sul1	ANaSuT	bla _TEM , tetB	iutA, cvaC, tsh, papC
J/53	B2	O145	1	aadA1	+	−	sul1	ACCipNaSu	bla _TEM , cat	iutA
J/3	B2	O140	1.5	dfr1-aadA1	+	−	—	ANa	bla _TEM	cvaC
J/76	B2	nt	1.5	dfr1-aadA1	+	−	—	NaT	tetA	iutA, cvaC, kps
J/4	D	nt	1.5	dfr1-aadA1	+	−	sul1	NaSuSxtT	tetA	cvaC, tsh
P/64	B2	nt	1.5	dfr1-aadA1	+	−	sul1, sul2	ASSuSxtT	bla_TEM, strA, tetA	tsh
J/99	B2	O8	2.5	estX-sat-aadA1	−	+	—	ANaS	bla _TEM	iutA, cvaC
P/105	D	O86	2.5	estX-sat-aadA1	−	+	—	ANaS	bla _TEM	iutA

Antibiotic resistance phenotype: A, ampicillin; C, chloramphenicol; Cip, ciprofloxacin; Na; nalidixic acid; S, streptomycin; Su, sulfonamides cp.; Sxt, sulfamethoxazole/trimethoprim; T, tetracycline; nt, not typeable.

From a total of 93 antibiotic-resistant E. coli isolates obtained from the broiler samples, PMQR genes were detected in 4 (4%) strains. Three isolates were positive for the qnrS1 gene and one isolate was positive for the qnrB19 gene (Table 3). The genes qnrA, qnrC, qnrD, aac(6′)-Ib-cr, qepA, and oqxAB were not detected. Transformation and conjugation to the E. coli and Salmonella strains of plasmids harboring the qnr genes were successful.

Table 3.

Characterization of PMQR-Positive Escherichia coli from Broilers

Strain No.	Antibiotic resistance phenotype	PMQR genes	Additional resistance genes	MIC (mg/L) ^a Na/Cip	PFGE profile	Phylo-genetic group	O-antigen serotype	Virulence factor genes	Conjugation to E. coli	Salmonella
J/13	ANa	qnrS1	bla _TEM-1	256/1	a	A	nt	cvaC, iss, iut, kps, tsh	+	+
O/66	CfT	qnrB19	tetB	32/0.25	b	B1	nt	kps II	+	+
P/65	ANa	qnrS1	bla _TEM-1	512/4	c	B1	nt	iss, kps II	+	+
P/67	ANa	qnrS1	bla _TEM-1	512/4	c	B1	nt	iss, kps II	+	+

Minimum inhibitory concentration of nalidixic acid and ciprofloxacin to donor strains.

Antibiotic resistance phenotype: A, ampicillin; Cf, cephalothin; Cip, ciprofloxacin; Na, nalidixic acid; T, tetracycline; PMQR, plasmid-mediated quinolone resistance; MIC, minimum inhibition concentration; PFGE, Pulse-field gel electrophoresis.

Phylogenetic analysis, detection of virulence genes (ExPEC), O-antigen serotypes

PCR analysis of the 93 resistant isolates showed that phylogroup B2 was predominant (41%) followed by phylogroups A (26%) and D (24%), whereas phylogroup B1 (9%) was minor in our collection. In total, 65% of isolates belonged to pathogenic groups B2 and D. Two of the PMQR-positive isolates showed the same PFGE profile and belonged to the B1 phylogenetic group (Table 3).

At least one virulence gene was detected in 68 (73%) out of 93 resistant isolates. The genes iutA, cvaC, tsh, kpsII, iss, papC, ibeA, and felA occured in frequencies of 46%, 28%, 26%, 22%, 20%, 4%, 1%, and 0%, respectively. Among these 93 isolates, 21 (23%) carried two or more virulence genes iutA, kpsII, tsh, papC, and ibeA, and hence, were considered as ExPEC isolates.

The O-antigen serotypes were highly variable. A total of 48 out of 93 resistant isolates were successfully determined by agglutination. These strains belonged to the following serotypes (according Johnson et al.,²⁴ those strains in bold should be considered as associated with ExPEC: O2 (1 isolate, B2 phylogroup, with iutA, cvaC, tsh, kpsII, iss, and ibA genes), O4 (1, A, without virulence genes tested), O7 (1, A, without virulence genes tested), O8 (6), O9 (3), O11 (1, B2, cvaC, tsh, kpsII), O18 (1, A, iutA), O20 (2), O22 (2), O25 (1, D, iutA, cvaC, iss, kpsII), O51 (1), O54 (1), O69 (1), O71 (2), O75 (1, A, iss), O86 (6), O88 (1), O103 (2), O108 (2), O115 (1), O119 (2), O139 (4), O140 (1), and O145 (4).

Consumption of antimicrobial drugs in chickens and turkeys in the Czech Republic

In 2008, tetracyclines (15,200 kg), aminopenicillins (5,400 kg), and sulfonamides, including sulfonamides in combinations (2,660 kg) were the three groups of antimicrobials used most frequently in chickens and turkeys (for mass medication). One substance from the quinolone group (flumequine) and two from the fluoroquinolone group (enrofloxacin and difloxacin) are used in poultry in the Czech Republic. Totally, 972 kg of these substances were consumed in chickens and turkeys in 2008. This takes in ∼70% of the total consumption of all quinolones and fluoroquinolones used in animals for which veterinary medicinal products containing these substances are authorized in the Czech Republic. Veterinary medicinal products containing cephalosporins are not authorized for poultry in the Czech Republic.

Discussion

Antimicrobial resistance in selected bacteria from poultry was broadly reviewed by Gyles.¹⁹ APEC isolates were found to be often highly resistant, especially to tetracycline, streptomycin, and sulfonamides. The European data on antimicrobial resistance in commensal E. coli from poultry show remarkable differences in various countries. The situation in the Czech Republic is similar to those in the Netherlands, France, and UK regarding the resistance to old-generation antibiotics and similar to those in Spain, Slovakia, and Iran regarding the fluoroquinolones.

Surprisingly, a high percentage of E. coli strains resistant to such new quinolones as ciprofloxacin, norfloxacin, flumequine, and pefloxacin (7%–16%) was found in healthy chickens in Spain already in the 1990s and a high prevalence in chickens of E. coli resistant to nalidixic acid and ciprofloxacin and carrying a virulence factor specific to ExPEC has been found in Spain recently.^2,10 Moreover, 35% of the chicken E. coli isolates in Spain satisfied criteria for ExPEC.

It has been postulated that the most likely evolutionary source of human fluoroquinolone-resistant E. coli strains is fluoroquinolone-susceptible chicken and not fluoroquinolone-susceptible human strains.^9,25 Chicken E. coli strains are not species-specific. Fluoroquinolone-resistant isolates in humans were found to be similar to resistant isolates in chickens and quite different from susceptible human isolates.²⁵ It was indicated that fluoroquinolone use in food animals is an important source of the emerging fluoroquinolone resistance among ExPEC and that resistant E. coli should therefore be considered a foodborne pathogen.²⁵

Healthy broiler chickens from various farms in Slovakia were tested for the presence of E. coli isolates resistant to nalidixic acid and ciprofloxacin during 2006–2008.²⁹ High frequencies of resistance to nalidixic acid and ciprofloxacin were found. High quinolone resistance was considered to be a consequence of frequent therapeutic administrations of enrofloxacin on Slovak poultry farms. Moreover, broiler meat at retail originating from Slovakia, Czech Republic, and Poland was examined for the presence of ExPEC isolates.¹⁴ A total of 28 isolates were analyzed. Thirteen of these belonged to the B2 phylogroup and all of these B2 isolates harbored ≥2 of the virulence factors iutA, iss, cvaC, tsh, and papC. The majority of those isolates belonged to the pathogenic serotype O78.

High frequencies of E. coli isolates resistant to nalidixic acid and ciprofloxacin were observed in Iran, and this resistance pattern was presumed to be linked to flumequine and enrofloxacin commonly used in poultry flocks there.³⁴ The results of our study strongly support the hypothesis that the emergence and dissemination of fluoroquinolone-resistant E. coli is a consequence of fluoroquinolone use in chickens.

Regarding the high prevalence of fluoroquinolone-resistant E. coli in broilers presumably caused by high consumption of fluoroquinolones on Czech chicken farms, it is useful to mention the results of a similar nationwide study from 2009 on the prevalence of Campylobacter spp. including antibiotic-resistant isolates in the Czech Republic.¹ The patterns of antibiotic resistance in Campylobacter spp. and E. coli isolates from broilers in the Czech Republic are very similar, and they probably are caused by the same selection pressure of quinolones administered on farms.

We found broilers to be contaminated with 23% of E. coli isolates considered as ExPEC. Some of the APEC strains tested in a rat model of human neonatal meningitis were able to cause meningitis, supporting the hypothesis that APEC strains have a zoonotic potential.⁴⁴ E. coli isolates from broiler chicken meat in Denmark belonging to the B2 phylogroup and carrying ≥2 virulence factors were tested for their ability to cause urinary tract infection (UTI) in a murine model representative of UTI in humans.²¹ All isolates tested caused UTI in mice, and thereby in vivo evidence was provided for the first time that E. coli UTI is a zoonosis. Moreover, UPEC can cause significant diseases in poultry, as has been demonstrated in vivo using a chicken challenge model.⁴⁸ Comparing serotypes supposedly associated with ExPEC according Johnson et al.²⁴ with the presence of virulence genes tested, we have confirmed such an association in serotypes O2, O11, and O25, but not in O4, O7, O18, and O75.

The proportion of ExPEC isolates with resistance to fluoroquinolones increased from 8% in 2001 to 26% in 2008 in Czech human microbiology laboratories¹⁵ and it can be associated with the fact that chicken meat consumption was increasing simultaneously in the Czech Republic to 25 kg per person per year in 2008, which is above the EU average.

A quite different situation regarding fluoroquinolone-resistant E. coli isolates from poultry has been reported in the U.S. Only 1 of 62 retailed chicken products with nalidixic acid-resistant E. coli was found to be also resistant to ciprofloxacin.²³ In that study, 21% of the E. coli isolates satisfied the criteria for ExPEC.²³ In a later study from Minnesota and Wisconsin, only 7 out of 931 E. coli isolates of human and poultry origin were found to be resistant to ciprofloxacin.²⁶ Similarly, resistance to ciprofloxacin was found to be only exceptionally occurring in ExPEC in Canada, and no such isolate was isolated from chicken in that country.³⁹

Due to the increasing emergence of antimicrobial resistance, several measures have come into force within the EU during the past decade. Using of antimicrobials as growth promoters in food-producing animals has been wholly prohibited in the EU since 1 January 2006. Recommendations for the prudent use of quinolones/fluoroquinolones in food-producing animals were published by the European Medicines Agency.¹⁶ Comparing EU policy to, for example, the U.S., where growth promoters are allowed to be used to enhance production in food-producing animals, EU rules seems to be more restrictive. On the other hand, U.S. policies are more restrictive regarding the use of fluoroquinolones, especially in respect to utilizing this antimicrobial group in poultry. A ban is in force on using enrofloxacin in poultry and off-label use of fluoroquinolones also is prohibited.⁴⁵ These different approaches in the EU and U.S. can explain the great difference in the prevalence of ciprofloxacin-resistant E. coli in chicken in these areas. While the prevalence is high in Europe, in the U.S. it is quite substantially lower.

The three most frequently used groups of antimicrobials on poultry farms in the Czech Republic correspond well to the detected resistances to tetracyclines, aminopenicillins, and sulfonamides (including sulphonamide in combinations) in chicken E. coli in our study. The resistance determinants indicating the presence of ESBLs were not detected in the samples examined. This corresponds to the fact that cephalosporins are not approved for use in broilers in the Czech Republic.

Excessive use of antimicrobials on chicken and turkey farms in Tunisia resulted in a high percentage of resistance to some antimicrobials (up to 95%), including fluoroquinolones (20% resistance to ciprofloxacin) in E. coli isolates from meat samples.⁴² The presence of class 1 and 2 integrons was demonstrated in 52% (n=166) of E. coli isolates in that study. Even though we found some similar integrons and sul1, sul2, and sul3 genes in E. coli isolates from chicken reared in the Czech Republic, the prevalence of integron-positive isolates was much lower (only 14%). This could mean that the use of antimicrobials in poultry in the Czech Republic is not so excessive as is that in North African Tunisia.

We found the PMQR genes, qnrS1 and qnrB19. Salmonella and/or E. coli isolates with the qnrS1 and qnrB19 genes were found recently in food and food-producing animals in various European countries.^20,46 Our findings of these genes correspond with these reports.

In conclusion, our results provide support from the Czech Republic for the hypothesis that chicken products can be a source of fluoroquinolone-resistant ExPEC for humans and that selection of fluoroquinolone-resistant E. coli strains in chickens is a result of fluoroquinolone use in chickens. These are good reasons for efforts to eliminate such organisms from the food supply, including by withdrawing fluoroquinolones from use in chicken production, improving the hygienic aspects of chicken processing, and improving distribution practices. We agree that reducing fluoroquinolone use in food animals will improve human health and that widespread use of these critically important antimicrobial agents in food animals poses a needless additional risk to humans in both the community and the hospital.⁹

Footnotes

Acknowledgments

We thank Ivo Papousek, Marie Slavikova, Eva Suchanova, Dagmar Tausova, and Raluca Uricariu for their cooperation in the laboratory. Our thanks go to Lars Hansen (University of Copenhagen, Denmark), Lina Cavaco, and Henrik Hasman (National Food Institute, Copenhagen, Denmark) for providing control strains. This work was supported by the project “CEITEC—Central European Institute of Technology” (CZ.1.05/1.1.00/02.0068) from the European Regional Development Fund.

Disclosure Statement

No competing financial interests exist.

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