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Article

Comparative Studies of Antimicrobial Resistance in Escherichia coli, Salmonella, and Campylobacter Isolates from Broiler Chickens with and without Use of Enrofloxacin

by
Ke Shang
1,†,
Ji-Hyuk Kim
2,†,
Jong-Yeol Park
3,
Yu-Ri Choi
3,
Sang-Won Kim
3,
Se-Yeoun Cha
3,
Hyung-Kwan Jang
3,4,
Bai Wei
3,* and
Min Kang
3,4,*
1
College of Animal Science and Technology, Luoyang Key Laboratory of Live Carrier Biomaterial and Animal Disease Prevention and Control, Henan University of Science and Technology, Luoyang 471000, China
2
Department of Animal Resources Science, Kongju National University, Yesan 32439, Republic of Korea
3
Department of Avian Diseases, College of Veterinary Medicine and Center for Avian Disease, Jeonbuk National University, Iksan 54596, Republic of Korea
4
Bio Disease Control (BIOD) Co., Ltd., Iksan 54596, Republic of Korea
*
Authors to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Foods 2023, 12(11), 2239; https://doi.org/10.3390/foods12112239
Submission received: 28 April 2023 / Revised: 29 May 2023 / Accepted: 30 May 2023 / Published: 1 June 2023
(This article belongs to the Section Food Microbiology)
Versions Notes

Abstract

:
This study investigated the effect of enrofloxacin (ENR) administration on the prevalence and antimicrobial resistance of E. coli, Salmonella, and Campylobacter isolated from broiler chickens under field conditions. The isolation rate of Salmonella was significantly lower (p < 0.05) on farms that administered ENR (6.4%) than on farms that did not (11.6%). The Campylobacter isolation rate was significantly higher (p < 0.05) in farms that administered ENR (6.7%) than in farms that did not (3.3%). The ratio of resistance to ENR was significantly higher (p < 0.05) in E. coli isolates from farms that used ENR (88.1%) than farms that did not (78.0%). The respective ratio of resistance to ampicillin (40.5% vs. 17.9%), chloramphenicol (38.0% vs. 12.5%), tetracycline (63.3% vs. 23.2%), and trimethoprim/sulfamethoxazole (48.1% vs. 28.6%) and the ratio of intermediate resistance to ENR (67.1% vs. 48.2%) were significantly higher (p < 0.05) in Salmonella isolates from the farms that used ENR than farms that did not. In conclusion, the use of ENR at broiler farms was an important factor in decreasing the prevalence of Salmonella but not Campylobacter and caused ENR resistance among E. coli and Salmonella but not Campylobacter. Exposure to ENR could have a co-selective effect on antimicrobial resistance in enteric bacteria in the field.

1. Introduction

In many countries, antimicrobial resistance in foodborne pathogens is recognized as an accumulative problem [1,2]. Systemically administered antimicrobial agents may cause the emergence and spread of antimicrobial resistance among zoonotic foodborne pathogens, such as non-typhoid Salmonella serotypes and Campylobacter [3,4]. Salmonella enterica and Campylobacter infections are the major causes of foodborne gastroenteritis in humans (known as salmonellosis and campylobacteriosis, respectively) in many countries [4,5,6,7]. Salmonella causes gastroenteritis with clinical symptoms such as headache, abdominal pain, and diarrhea in both adults and children; campylobacteriosis frequently occurs in children younger than five years of age, and Campylobacter species cause a wide range of syndromes, from asymptomatic infections to severe systemic infections. In addition, the major source of Salmonella and Campylobacter infections in humans has been identified as poultry, as a reservoir of foodborne enteric pathogens and antimicrobial-resistant bacteria [8,9]. Fluoroquinolones (FQs) are the primary treatment against these bacteria [10].
Fluoroquinolones belong to a family of synthetic antimicrobials with a broad spectrum of activity. Nalidixic acid, which does not contain fluorine, is the parent compound of the quinolone family, which includes FQs. The FQs are one of the few therapeutics available for the treatment of campylobacteriosis, which is transmitted to humans through animal sources, such as poultry [11,12]. Given this importance, FQs are classified by the World Health Organization (WHO) as critically important antimicrobials (for human medicine) and veterinary critically important antimicrobial agents (for animals) that directly inhibit DNA replication and transcription [13,14]. Several mechanisms facilitate bacterial FQ resistance, including mutation(s) at the drug binding sites in the quinolone resistance determining region (QRDR) of gyrA, gyrB, parA, and parC [15], reduced accumulation of FQs in cells [16,17], and plasmid mediated quinolone resistance (PMQR) genes [qnr family, aac(6′)-Ib-cr, oqxAB, and qepA genes] [18]. Such resistance is usually associated with a single point mutation in the gyrA gene [19].
Fluoroquinolones are used in the treatment of animal and human diseases [20]. In veterinary medicine, especially in broiler fattening, they are ordinarily used to treat colibacillosis, with oral administration being the preferred route [21]. Despite their efficacy, the use of FQs in veterinary medicine is controversial. As such, the use of FQs intensifies FQ resistance in foodborne pathogens and thus poses a public health problem [22]. The escalating FQ resistance in foodborne pathogens may be caused by using antimicrobials in veterinary medicine, as food animals are the main reservoir for these pathogens [23]. FQ resistance (via oral administration) has been found in Salmonella, Campylobacter, and Escherichia coli (E. coli) in broilers [24,25]. Antimicrobial resistance is a major problem for the intestinal health of humans and other animals. In 2017, the WHO listed FQ-resistant Salmonella and Campylobacter jejuni among the top nine bacterial pathogens, for which new antimicrobials are urgently needed [26]. The use of FQs to treat diseases in food animals has led to the selection of FQ-resistant strains of Campylobacter, especially in poultry [15]. FQ resistance develops rapidly among bacteria in poultry treated with FQ [27,28,29,30]. Contamination of food with Salmonella and Campylobacter remains a significant food safety concern [31], and antimicrobial-resistant (AMR) Salmonella and Campylobacter strains pose an additional threat to food safety. FQ-resistant Campylobacter is found in poultry in most regions of the world [8]. In addition, the increasing use of antimicrobials, especially FQs, likely increases the incidence of foodborne infections with AMR Salmonella [1].
Enrofloxacin (ENR), a second-generation FQs, is commonly used in chickens because of its favorable pharmacokinetic profile and its excellent activity against gram-negative aerobic bacteria and some gram-positive bacteria. Prophylactic treatment of poultry with ENR, an FQ antibacterial used to treat infections in animals, has been associated with increasing resistance to ciprofloxacin, posing a risk to human health [28,32,33]. Despite its efficacy, some countries, such as the USA, Finland, and Denmark, have banned the use of FQ in poultry [34,35]. In Poland and most countries of the European Union, therapeutic use of FQ in poultry is still allowed [36]. Concerns about the entry of zoonotic AMR bacteria into the food chain and resulting human infections led the Food and Drug Administration (FDA) to ban the use of ENR in poultry in the USA in September 2005. The EU has established maximum residue limits (Regulation CE N. 2377/90) [37]. In Korea, ENR has been progressively utilized to treat bacterial infections in chickens since it was approved for veterinary use in 1987 [38]. In fact, ENR was the best-selling antimicrobial agent for chickens in Korea. In addition, the use of all antibiotic feed additives classified as growth promoters has been banned in Korea since July 2011, and the use of ENR in commercial laying hens has been prohibited since May 2017 [39,40].
The bacterial species selected for the present study (Campylobacter, Salmonella, and E. coli) are targets of FQs in human medicine or veterinary medicine, and data on FQ resistance in these bacterial species span several years [41]. Treatment of livestock with ENR leads to a rapid selection of Campylobacter, E. coli [42,43,44], and Salmonella [36] which are less susceptible to these antibiotics. Nevertheless, few studies have focused on the relationship between ENR use and ENR resistance in foodborne pathogens from broilers in the field [45]. In this field trial, we investigated the relationship between ENR use and the emergence of ENR resistance in Salmonella and Campylobacter, whereas E. coli was used as an indicator bacterium.

2. Materials and Methods

2.1. Studied Isolates

There were two groups of farms (Group 1—contained farms that use ENR; and Group 2—contained farms that do not use ENR). E. coli isolates (n = 516; 306 and 210 isolates from Group 1 and Group 2, respectively), Salmonella isolates (n = 255; 134 and 121 isolates from Group 1 and Group 2, respectively), and Campylobacter isolates (n = 165; 132 and 33 isolates from Group 1 and Group 2, respectively) were sequestered and assessed. All isolates were from 2418 clinical samples composed of fecal (1-day-old and 15–25-days-old broiler chickens) or from environmental source (litter, feed, and water) samples collected from broiler farms or meat samples collected from downstream retail markets (Table S1). All samples were collected between September and December in 2015. In Group 1, 0.5 g/L ENR was constantly added to the drinking water of 2–4-day-old chicks. Salmonella, E. coli, and Campylobacter were isolated using the standard method described previously [45,46,47,48].
No endangered population was involved, and no endangered species were used for the experiments. No chickens were touched or killed; fecal samples were collected by a veterinarian with prior approval from the farm managers. Therefore, no ethical approval was required for this study.

2.2. Antimicrobial Susceptibility Testing

For E. coli and Salmonella isolates, the minimum inhibitory concentrations (MICs) of 14 antimicrobial agents, including nalidixic acid (2–128 μg/mL), ciprofloxacin (0.12–16 μg/mL), neomycin (2–32 μg/mL), gentamicin (1–64 μg/mL), streptomycin (2–128 μg/mL), tetracycline (2–128 μg/mL), amoxicillin/clavulanic acid (2/1–64/32 μg/mL), cefoxitin (1–32 μg/mL), ceftiofur (0.5–8 μg/mL), ampicillin (2–32 μg/mL), trimethoprim/sulfamethoxazole (0.12/0.38–4/76 μg/mL), colistin (2–32 μg/mL), florfenicol (2–64 μg/mL), and chloramphenicol (2–64 μg/mL) were determined using the KRNV4F Sensititre Panel (TREK Diagnostic Systems, Incheon, Korea), and the MIC of ENR (0.125–128 μg/mL) was verified by the agar dilution method (Table S2) [49]. KRNV4F Sensititre panel made by Trek Diagnostic Systems is ready-to-use 96-well microplate coated with 15 kinds of antimicrobial substances with multiple dilution concentration using Mueller–Hinton broth (MHB) medium. E. coli (ATCC 25922) was used as a quality control strain. The susceptibility breakpoints of most antimicrobials were interpreted based on Clinical and Laboratory Standards Institute (CLSI) guidelines [50] or other references [49,51,52].
The susceptibility of all Campylobacter isolates to 11 antimicrobial agents was ascertained using the Campy Sensititre Panel (TREK) and the agar dilution method. Campy Sensititre Panel made by Trek Diagnostic Systems is ready-to-use 96-well microplate coated with nine kinds of antimicrobial substances detecting for Campylobacter. Cation-adjusted Mueller–Hinton broth with TES buffer with 5% lysed horse blood medium was used in this method. Susceptibility to two antimicrobial agents (not available in this plate), namely ENR (0.125–128 μg/mL) and ampicillin (8–128 μg/mL), were confirmed by the standard agar dilution method (Table S2) [49]. The remaining nine antimicrobials were tested with Sensititre plates containing azithromycin (0.015–64 μg/mL), erythromycin (0.03–64 μg/mL), telithromycin (0.015–8 μg/mL), nalidixic acid (4–64 μg/mL), ciprofloxacin (0.015–64 μg/mL), clindamycin (0.03–16 μg/mL), gentamicin (0.12–32 μg/mL), florfenicol (0.03–64 μg/mL), and tetracycline (0.06–64 μg/mL). Breakpoints were established according to the National Antimicrobial Resistance Monitoring System criteria [51]. Because there were no ampicillin and ENR breakpoints for Campylobacter, we used the breakpoints for Enterobacteriaceae from the CLSI criteria [49,50]. Campylobacter jejuni ATCC 33560 was used as the reference quality control. Isolates that were resistant to at least three antimicrobial classes were classified as multidrug-resistant (MDR).
The MIC50 and MIC90 values, as well as the range of values obtained, are important parameters for reporting the results of susceptibility testing when multiple isolates of a given species are investigated. The MIC50 represents the MIC value at which ≥50% of the isolates in a test population are inhibited; it corresponds to the mean MIC value. The MIC90 represents the MIC value at which ≥90% of the strains within a test population are inhibited [53].

2.3. Molecular Characterization of Antimicrobial Resistance

For molecular characterization of quinolone resistance genes, the quinolone-resistant isolates were screened for plasmid-mediated quinolone resistance (PMQR) genes by PCR and sequencing. These genes included qnrA, qnrB, qnrS, qnrD, qepA, oqxA, aac(6′)-lb-cr, and mutations in the quinolone resistance determining region (QRDR), and gyrA, gyrB, parC, and parE as previously described [54,55]. Positive controls were used in all PCR reactions (Table S3). PCR products were purified using a QIAquick Gel Extraction Kit (Qiagen, Hilden, Germany) and then directly sequenced (SolGent Co., Ltd., Daejeon, Republic of Korea) for protein sequence analysis and alignment using the program BLAST (www.ncbi.nlm.nih.gov/BLAST/) accessed on 1 March 2022.

2.4. Statistical Analysis

Statistical analysis was performed using the SPSS program version 19.0 for Windows. The Chi-square test was used to analyze significant differences in isolation rates of Salmonella and Campylobacter and AMR rates of E. coli, Salmonella, and Campylobacter between broilers from Groups 1 and 2. p-values < 0.05 were considered indicative of statistical significance. Results are reported as the mean with 95% confidence interval (CI).

3. Results

3.1. Prevalence of Salmonella and Campylobacter in the Presence or Absence of ENR Use

Salmonella isolation rates from 15–25-days-old broilers and from retail meat were significantly lower (p < 0.001) in Group 1 than in Group 2 (Table 1). There was no significant difference in the Salmonella isolation rate of 1-day-old broilers between Group 1 and Group 2. Campylobacter isolation rates of 15–25-days-old broilers and retail meat were significantly higher (p < 0.05) in Group 1 than in Group 2. The Campylobacter isolation rate of 1-day-old broilers was similar between Group 1 and Group 2.

3.2. Antimicrobial Resistance of E. coli, Salmonella, and Campylobacter Isolates in the Presence or Absence of ENR Use

The prevalence of antimicrobial resistance differed between Group 1 and Group 2 for 16 antimicrobials assessed (Table 2, Table 3 and Table 4). E. coli isolates from 15–25-days-old broilers from Group 1 were significantly more likely to be resistant to cephalothin (p = 0.038) and ENR (p = 0.048) (Table 2). The prevalence of multidrug resistance in E. coli isolates did not differ significantly between Group 1 and Group 2. A higher frequency of Salmonella isolates from 15–25-days-old broilers from Group 1 were resistant to ampicillin (p = 0.005), chloramphenicol (p = 0.001), tetracycline (p < 0.001), and intermediate resistance (IR) to ENR (p = 0.028) (Table 3). The prevalence of multidrug resistance in Salmonella isolates was also significantly higher in 15–25-days-old broilers in Group 1 (p = 0.016). Campylobacter isolates from 15–25-days-old broilers from Group 1 had significantly lower resistance to tetracycline (p < 0.001). The prevalence of multidrug resistance in Campylobacter isolates was also significantly lower on Group 1 (p = 0.005) (Table 4).

3.3. Distribution of the MIC50/MIC90 Values of (Fluoro)Quinolones among E. coli, Salmonella, and Campylobacter Isolates from Untreated or ENR-Treated Broilers

The MIC50/MIC90 values of E. coli, Salmonella, and Campylobacter isolates from the broilers with or without ENR treatment are illustrated in Tables S3–S5. The MIC50 of ciprofloxacin and ENR were 8 μg/mL and 16 μg/mL against E. coli isolates from both 1-day-old broilers and retail meat in Group 1, respectively, which was higher than the 4 μg/mL and 8 μg/mL, respectively, in Group 2 (Table S4). Similarly, the MIC50 of ENR against the Salmonella isolates from ENR-treated and untreated 15–25-days-old broilers were 0.5 and <0.25 μg/mL, respectively. The MIC90 of ciprofloxacin against Salmonella isolates from ENR-treated and untreated 15–25-days-old broilers was 0.5 and 0.25 μg/mL, respectively (Table S5). There was no enhanced MIC50/MIC90 value against Campylobacter isolates from broilers in Group 1 as compared with isolates from broilers in Group 2 (Table S6). On the contrary, the MIC50 of ciprofloxacin and ENR were 16 μg/mL and 8 μg/mL against E. coli isolates from retail meat of broilers in Group 2, which was higher than that of 8 μg/mL and 4 μg/mL against the isolates from in Group 1, respectively. The MIC90 of ENR were 32 μg/mL and 16 μg/mL against Campylobacter isolates from 15–25-days-old broilers and retail meat of broilers in Group 2 and 16 μg/mL and 8 μg/mL against broilers in Group 1, respectively.

3.4. Prevalence of PMQR and QRDR Mutations among the Selected E. coli, Salmonella, and Campylobacter Isolates from Untreated or ENR-Treated Broilers

The prevalence of PMQR and QRDR mutations in E. coli isolates (ENR, MIC ≥ 1) is displayed in Table 5. PMQR genes were not detected in any isolate; qnrB and qnrS1 genes were revealed in E. coli isolates from broilers with ENR treatment. The most frequent QRDR pattern in E. coli isolates (ENR MIC, 16–32 µg/mL) from broilers with ENR treatment was Ser-83-Leu and Asp-87-Asn in gyrA and Ser-80-Ile and Ser-83-Tyr in parC (8/28, 28.6%). The most common QRDR patterns observed in E. coli isolates (ENR MIC, 8–16 µg/mL) from broilers without ENR treatment were Ser-80-Ile and Ser-83-Tyr in parC (6/17, 35.3%). Alterations in the gyrA gene were frequently observed in 92.9% (26/28) and 76.5% (13/17) of E. coli isolates from broilers in Group 1 and Group 2, respectively. Alterations in the parC gene were noted in all E. coli isolates. Substitutions in the parE gene occurred less often, with 85.7% (24/28) and 82.4% (14/17) of isolates from broilers in Group 1 and Group 2, respectively, having wild type parC.
The gyrA gene sequences of targeted isolates were submitted to the GenBank database and are available under the accession numbers OR047806-OR047961; the parC gene sequences of targeted isolates were submitted to the GenBank database and are available under the accession numbers OR047672-OR047805.
The prevalence of PMQR and QRDR mutations in Salmonella isolates (ENR, MIC ≥ 0.25) is indicated in Table 6. PMQR genes were not observed in any Salmonella isolate. The most common QRDR pattern detected in Salmonella isolates (ENR MIC, 0.25–1.00 µg/mL) from broilers in Group 1 was Asp-87-Gly in gyrA and Tyr-57-Ser in parC (28/50, 56.0%); the same QRDR pattern was observed in Salmonella isolates (ENR MIC, 0.25–0.50 µg/mL) from broilers in Group 2 (33/49, 67.3%). Changes in the gyrA gene were observed in 90.0% (45/50) and 98.0% (48/49) of Salmonella isolates from broilers in Group 1 and Group 2, respectively. In Salmonella isolates from broilers in Group 1 and Group 2, 92.0% (46/50) and 98.0% (48/49) of the changes in the parC gene were observed, respectively.
The prevalence of PMQR and QRDR mutations among Campylobacter isolates (ENR, MIC ≥ 4) is presented in Table 7. PMQR genes were not detected in any Campylobacter isolate. The only QRDR pattern of Thr-86-Ile in gyrA was observed in all Campylobacter isolates (ENR MIC, 4–32 µg/mL) from broilers in Group 1 and Group 2.

4. Discussion

The development of antimicrobial resistance indicates a clear link between the use of antimicrobials in veterinary medicine and animal production [56]. Administration of FQs such as ENR to poultry can lead to rapid selection of E. coli, Salmonella, and Campylobacter, which are less susceptible to such antimicrobials. In the present study, the interaction between the use of ENR and antimicrobial resistance in E. coli, Salmonella, and Campylobacter of broiler chickens was investigated under field conditions.
As expected, the isolation rate of Salmonella from samples of 15–25-days-old broilers was significantly lower (p < 0.05) in Group 1 than in Group 2 (56/485, 11.6%) (Table 1). The isolation rate of Salmonella from broiler meat was also significantly lower (p < 0.05) in Group 1 (30/105, 28.6%) than in Group 2 (39/64, 60.9%). Therefore, the use of ENR was an important factor leading to a lower prevalence of Salmonella in both broiler farms and the retail market, which is consistent with a previous study [46]. In contrast, Campylobacter isolation rates from samples of 15–25-days-old broilers were significantly higher (p < 0.05) in Group 1 (83/1236, 6.7%) than in Group 2 (16/485, 3.3%). Similarly, Campylobacter isolation rates from broiler meat were significantly higher (p < 0.05) in Group 1 (48/105, 45.7%) than in Group 2 (17/64, 26.6%). Instead, the use of ENR increased Campylobacter prevalence in both broiler farms and the retail market. That could be because Campylobacter resistance to FQs has already reached a high level, and the use of ENR did not reduce prevalence as it did for Salmonella [55]. However, a previous study demonstrated a low prevalence of Campylobacter in retail chickens after the ENR ban [37]. In the present study, the use of ENR at the broiler stage had a different effect on the prevalence of Salmonella and Campylobacter in broilers and their meat products such that ENR use resulted in a high positive rate of Campylobacter but a low positive rate of Salmonella. The reasons for the above phenomena might be that the ENR resistance of Salmonella and Campylobacter isolates was different [47,55]. Moreover, low and high ENR resistance values were observed in Salmonella (Table 3) and Campylobacter isolates (Table 4), respectively. Therefore, the use of FQs in chickens rapidly leads to the selection of resistant Campylobacter organisms. However, FQ-resistant Campylobacter isolates are counter-selected under field conditions in the absence of selection pressure [25,29].
After administration of ENR, a significant change in resistance to ENR was observed in E. coli isolates from 15–25-days-old broilers. In the present study, we discovered a significant difference (p = 0.048) in resistance to ENR in E. coli isolates from 15–25-days-old broilers between Group 1 (89/101, 88.1%) and Group 2 (92/118, 78.0%) farms (Table 2). Therefore, ENR use may be an important factor in ENR resistance of E. coli in broiler farms. Similarly, antimicrobial resistance in E. coli is mainly drug-dependent [45]. Resistance to cefoxitin and tetracycline was significantly higher in E. coli isolates from broiler meat in Group 2 (45.0%; 100%) than in broilers in Group 1 (20.5%; 64.1%). This observation could be due to cross-contamination by E. coli from resistant carrier chickens slaughtered on the same day or contamination by resident flora with AMR in the slaughterhouse [47]. These data are consistent with previously published studies indicating that antimicrobial resistance in broilers not exposed to ENR showed changes over time [44,57].
There was no significant difference (p > 0.05) in resistance to FQs in Salmonella isolates from 15–25-days-old broilers between Group 1 (ciprofloxacin, 1.3%; ENR, 2.5%) and Group 2 (0%) farms (Table 3). This result could be due to low FQ resistance of Salmonella isolates from both Group 1 and Group 2, likely because of the short growth cycle of modern broilers, which does not allow sufficient time for resistance to develop, even after antimicrobial treatment. Salmonella isolates from 15–25-days-old broilers in Group 1 revealed a significant increase (p = 0.028) in intermediate resistance to ENR after ENR administration. A significant escalation (p < 0.05) in resistance to ampicillin, chloramphenicol, tetracycline, and trimethoprim/sulfamethoxazole was also observed in Salmonella isolates from 15–25-days-old broilers in Group 1. Moreover, the results of E. coli resistance to ampicillin, chloramphenicol, tetracycline, and trimethoprim/sulfamethoxazole were also consistent. The plausible explanations of the above phenomena were that the four antimicrobials were commonly used in broiler farms, and resistance to these antimicrobials was most frequently observed in isolates from chickens. In fact, selection pressure exerted by the antimicrobial agent contributed to the co-selection of this antimicrobial resistance pattern; the co-selection of resistance to more than one antimicrobial agent is a common feature of resistance acquired by horizontal gene transfer due to the genetic linkage of resistance genes [45,58,59].
There was no significant difference (p > 0.05) in resistance to FQs in Campylobacter isolates from broilers between Group 1 (78.4%) and Group 2 (100%) farms (Table 4). This may be due to the high FQ resistance of Campylobacter isolated from both the ENR-treated and untreated broilers, making it difficult to discern differences in FQ resistance between the two populations. A previous study demonstrated a low rate of ciprofloxacin resistance in Campylobacter in retail chickens after the ENR ban [36]. In other previous studies, therapeutic administration of ENR to mice did not result in FQ resistance in Campylobacter jejuni [60]. However, treatment of pigs with FQs selected and spread FQ-resistant Campylobacter in the house [61].
In general, PMQR genes confer low resistance to quinolones but may play an important role in the emergence of resistance mutations and the spread of antimicrobial resistance [62]. In the present study, the PMQR genes (qnrB and qnrS1) were detected in E. coli isolates from the broilers in Group 1, whereas these genes were absent in E. coli isolates from the broilers in Group 2 (Table 5). Accordingly, the qnrS1 gene was uncovered in E. coli isolates from chicken meat produced in integrated broiler operations in Korea [63]. In the present study, more amino-acid mutations in the gyrA, parC, and parE genes were discovered in broiler isolates from Group 1 than in isolates from Group 2. Thus, the use of ENR resulted in the emergence of PMQR genes and higher resistance to ENR with more mutations in QRDR genes.
Although PMQR has been extensively studied, QRDR mutations seem to represent the main mechanism of quinolone resistance in animal isolates [64]. Moreover, PMQR was commonly detected in Enterobacteriaceae, particularly in E. coli, and the prevalence of PMQRs in Salmonella remains extremely low [65]. In the present study, no PMQR gene was realized in all Salmonella (n = 99) and Campylobacter (n = 76) isolates tested (Table 6 and Table 7). The most common QRDR pattern, an exchange of a single amino acid in both gyrA (Asp-87-Gly) and parC (Tyr-57-Ser), was identical in Salmonella isolates from broilers in Group 1 and Group 2 (Table 6), and similar QRDR patterns were noted, except for the Ser-83-Ala exchange in the gyrA gene in Salmonella isolates from Group 1. Only one QRDR pattern (Thr-86-Ile in gyrA) was revealed in the Campylobacter isolates from both Group 1 and Group 2 (Table 7). This is consistent with our previous findings that the Thr-86-Ile substitution in the gyrA was the primary contributor to the high-level quinolone resistance in Campylobacter isolates from duck meats [66]. Therefore, the use of ENR may not lead to the emergence of PMQR genes and QRDR genes with amino acid exchange in Salmonella and Campylobacter isolates [67].

5. Conclusions

In conclusion, ENR use in broiler farms is an important factor in reducing the prevalence of Salmonella but not Campylobacter, as well as causing ENR resistance in E. coli but not in Salmonella or Campylobacter. ENR use in broiler farms may lead to the emergence of PMQR genes and high levels of QRDR mutations and ENR resistance in E. coli and less so in Salmonella and Campylobacter. Understanding the relationship between antimicrobial use in food-producing animals and the emergence of resistant commensal bacteria may facilitate the prudent use of antimicrobials in these animals. Therefore, because of its selective effect, ENR must be used judiciously.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/foods12112239/s1, Table S1: Sampling of feces and isolation information. Table S2: Specific guideline used for each antimicrobial substance; Table S3: Strains chosen as specific positive control with sequence accession numbers; Table S4: Distribution of the MIC50/MIC90 values of (fluoro)quinolones among E. coli isolates from broiler chickens with or without ENR treatment; Table S5: Distribution of the MIC50/MIC90 values of (fluoro)quinolones among Salmonella isolates from broiler chickens with or without ENR treatment; Table S6: Distribution of the MIC50/MIC90 values of (fluoro)quinolones among Campylobacter isolates from broiler chickens with or without ENR treatment.

Author Contributions

Conceptualization, B.W. and M.K.; methodology, K.S., J.-Y.P., Y.-R.C. and S.-W.K.; investigation, K.S. and J.-H.K.; formal analysis, K.S., H.-K.J. and S.-Y.C.; data curation, K.S., J.-H.K., B.W. and M.K.; writing—original draft, K.S. and J.-H.K.; writing—review and editing, K.S., J.-H.K., S.-Y.C., H.-K.J., B.W. and M.K.; supervision, S.-Y.C., H.-K.J. and M.K.; project administration, S.-Y.C., H.-K.J., B.W. and M.K.; funding acquisition, S.-Y.C., H.-K.J. and M.K. All authors have read and agreed to the published version of the manuscript.

Funding

This work was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (2017R1D1A1B03030883) and by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIT; No. 2020R1F1A1065136). In addition, this work was supported by the Technology Development Program (S3308313) funded by the Ministry of SMEs and Startups (MSS, Korea). This work was also supported by Korea Institute of Planning and Evaluation for Technology in Food, Agriculture and Forestry (IPET) through the Animal Disease Management Technology Advancement Support Program, funded by the Ministry of Agriculture, Food and Rural Affairs (MAFRA; 122014-2).

Data Availability Statement

Data is contained within the article or Supplementary Material.

Conflicts of Interest

Author Hyung-Kwan Jang and Min Kang were employed by the company of Bio Disease Control (BIOD) Co., Ltd. The remaining 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.

References

  1. Koningstein, M.; Simonsen, J.; Helms, M.; Mølbak, K.J. The interaction between prior antimicrobial drug exposure and resistance in human Salmonella infections. J. Antimicrob. Chemother. 2010, 65, 1819–1825. [Google Scholar] [CrossRef]
  2. D’Costa, V.M.; King, C.E.; Kalan, L.; Morar, M.; Sung, W.W.; Schwarz, C.; Froese, D.; Zazula, G.; Calmels, F.; Debruyne, R.J. Antibiotic resistance is ancient. Nature 2011, 477, 457–461. [Google Scholar] [CrossRef]
  3. Snary, E.L.; Kelly, L.A.; Davison, H.C.; Teale, C.J.; Wooldridge, M.J. Antimicrobial resistance: A microbial risk assessment perspective. J. Antimicrob. Chemother. 2004, 53, 906–917. [Google Scholar] [CrossRef]
  4. Ibrahim, J.; Eghnatios, E.; El Roz, A.; Fardoun, T.; Ghssein, G. Prevalence, antimicrobial resistance and risk factors for campylobacteriosis in Lebanon. J. Infect. Dev. Ctries. 2019, 13, 11–20. [Google Scholar] [CrossRef]
  5. Kaakoush, N.; Castaño-Rodríguez, N.; Mitchell, H.; Man, S. Global Epidemiology of Campylobacter Infection. Clin. Microbiol. Rev. 2015, 28, 687–720. [Google Scholar] [CrossRef]
  6. Popa, G.; Papa, M. Salmonella spp. infection—A continuous threat worldwide. Germs 2021, 11, 88–96. [Google Scholar] [CrossRef]
  7. Ghssein, G.; Awada, R.; Salami, A.; Bahmad, H.; Awad, A.; Joumaa, W.; El Roz, A. Prevalence, laboratory findings and clinical characteristics of campylobacteriosis agents among hospitalized children with acute gastroenteritis in lebanon. Pediatr. Gastroenterol. Hepatol. Nutr. 2021, 24, 346–356. [Google Scholar] [CrossRef]
  8. Smith, J.; Fratamico, P.M. Fluoroquinolone resistance in Campylobacter. J. Food Prot. 2010, 73, 1141–1152. [Google Scholar] [CrossRef]
  9. Gaffga, N.H.; Barton, B.C.; Ettestad, P.J.; Smelser, C.B.; Rhorer, A.R.; Cronquist, A.B.; Comstock, N.A.; Bidol, S.A.; Patel, N.J.; Gerner, S.P.; et al. Outbreak of salmonellosis linked to live poultry from a mail-order hatchery. N. Engl. J. Med. 2012, 366, 2065–2073. [Google Scholar] [CrossRef]
  10. Wieczorek, K.; Osek, J. Antimicrobial resistance mechanisms among Campylobacter. Biomed. Res. Int. 2013, 2013, 340605. [Google Scholar] [CrossRef]
  11. Andersson, M.; Macgowan, A. Development of the quinolones. J. Antimicrob. Chemother. 2003, 51 (Suppl. 1), 1–11. [Google Scholar] [CrossRef]
  12. Millanao, A.; Mora, A.; Villagra, N.; Bucarey, S.; Hidalgo, A. Biological effects of quinolones: A family of broad-spectrum antimicrobial agents. Molecules 2021, 26, 7153. [Google Scholar] [CrossRef]
  13. World Health Organization. Critically Important Antimicrobials for Human Medicine (6th Revision 2018): Ranking of Medically Important Antimicrobials for Risk Management of Antimicrobial Resistance Due to Non-Human Use; WHO: Geneva, Switzerland, 2018. [Google Scholar]
  14. International Epizootic Office. OIE List of Antimicrobial Agent of Veterinary Importance (July 2019); International Epizootic Office: Paris, France, 2020. [Google Scholar]
  15. Hopkins, K.L.; Davies, R.H.; Threlfall, E.J. Mechanisms of quinolone resistance in Escherichia coli and Salmonella: Recent developments. Int. J. Antimicrob. Agents 2005, 25, 358–373. [Google Scholar] [CrossRef]
  16. Blair, J.M.; Richmond, G.E.; Piddock, L.J. Multidrug efflux pumps in Gram-negative bacteria and their role in antibiotic resistance. Future Microbiol. 2014, 9, 1165–1177. [Google Scholar] [CrossRef]
  17. Randall, L.; Ridley, A.; Lemma, F.; Hale, C.; Davies, R.J. In vitro investigations into the use of antimicrobials in combination to maintain efficacy of fluoroquinolones in poultry. Res. Vet. Sci. 2016, 108, 47–53. [Google Scholar] [CrossRef]
  18. Robicsek, A.; Jacoby, G.A.; Hooper, D.C. The worldwide emergence of plasmid-mediated quinolone resistance. Lancet Infect. Dis. 2006, 6, 629–640. [Google Scholar] [CrossRef]
  19. Engberg, J.; Aarestrup, F.M.; Taylor, D.E.; Gerner, S.P.; Nachamkin, I.J. Quinolone and macrolide resistance in Campylobacter jejuni and C. coli: Resistance mechanisms and trends in human isolates. Emerg. Infect. Dis. 2001, 7, 24. [Google Scholar] [CrossRef]
  20. Mathew, A.G.; Cissell, R.; Liamthong, S.J. Antibiotic resistance in bacteria associated with food animals: A United States perspective of livestock production. Foodborne Pathog. Dis. 2007, 4, 115–133. [Google Scholar] [CrossRef]
  21. Landoni, M.F.; Albarellos, G.J. The use of antimicrobial agents in broiler chickens. Vet. J. 2015, 205, 21–27. [Google Scholar] [CrossRef]
  22. Takahashi, T.; Ishihara, K.; Kojima, A.; Asai, T.; Harada, K.; Tamura, Y.J. Emergence of fluoroquinolone resistance in Campylobacter jejuni in chickens exposed to enrofloxacin treatment at the inherent dosage licensed in Japan. Antimicrob. Agents Chemother. 2005, 52, 460–464. [Google Scholar] [CrossRef]
  23. Doyle, M.E. Multidrug-resistant pathogens in the food supply. Foodborne Pathog. Dis. 2015, 12, 261–279. [Google Scholar] [CrossRef]
  24. Chantziaras, I.; Smet, A.; Haesebrouck, F.; Boyen, F.; Dewulf, J.J. Studying the effect of administration route and treatment dose on the selection of enrofloxacin resistance in commensal Escherichia coli in broilers. J. Antimicrob. Chemother. 2017, 72, 1991–2001. [Google Scholar] [CrossRef]
  25. McDermott, P.F.; Bodeis, S.M.; English, L.L.; White, D.G.; Walker, R.D.; Zhao, S.; Simjee, S.; Wagner, D.D. Ciprofloxacin resistance in Campylobacter jejuni evolves rapidly in chickens treated with fluoroquinolones. J. Infect. Dis. 2002, 185, 837–840. [Google Scholar] [CrossRef]
  26. World Health Organization. WHO Publishes List of Bacteria for Which New Antibiotics Are Urgently Needed. Available online: https://www.who.int/news/item/27-02-2017-who-publishes-list-of-bacteria-for-which-new-antibiotics-are-urgently-needed (accessed on 27 February 2017).
  27. Luo, N.; Sahin, O.; Lin, J.; Michel, L.O.; Zhang, Q.J. In vivo selection of Campylobacter isolates with high levels of fluoroquinolone resistance associated with gyrA mutations and the function of the CmeABC efflux pump. Antimicrob. Agents Chemother. 2003, 47, 390–394. [Google Scholar] [CrossRef]
  28. Randall, L.P.; Eaves, D.J.; Cooles, S.W.; Ricci, V.; Buckley, A.; Woodward, M.J.; Piddock, L.J. Fluoroquinolone treatment of experimental Salmonella enterica serovar Typhimurium DT104 infections in chickens selects for both gyrA mutations and changes in efflux pump gene expression. J. Antimicrob. Chemother. 2005, 56, 297–306. [Google Scholar] [CrossRef]
  29. Stapleton, K.; Cawthraw, S.; Cooles, S.; Coldham, N.; La Ragione, R.; Newell, D.; Ridley, A.J. Selecting for development of fluoroquinolone resistance in a Campylobacter jejuni strain 81116 in chickens using various enrofloxacin treatment protocols. J. Appl. Microbiol. 2010, 109, 1132–1138. [Google Scholar] [CrossRef]
  30. Mourand, G.; Jouy, E.; Bougeard, S.; Dheilly, A.; Kérouanton, A.; Zeitouni, S.; Kempf, I.J. Experimental study of the impact of antimicrobial treatments on Campylobacter, Enterococcus and PCR-capillary electrophoresis single-strand conformation polymorphism profiles of the gut microbiota of chickens. J. Med. Microbiol. 2014, 63, 1552–1560. [Google Scholar] [CrossRef]
  31. MDPH. Foodborne Illness Investigation and Control Manual; Massachusetts Department of Public Health: Boston, MA, USA, 2017.
  32. Angulo, F.J.; Johnson, K.R.; Tauxe, R.V.; Cohen, M.L. Origins and consequences of antimicrobial-resistant nontyphoidal Salmonella: Implications for the use of fluoroquinolones in food animals. Microb. Drug Resist. 2000, 6, 77–83. [Google Scholar] [CrossRef]
  33. Li, J.; Hao, H.; Cheng, G.; Liu, C.; Ahmed, S.; Shabbir, M.A.; Hussain, H.I.; Dai, M.; Yuan, Z. Microbial shifts in the intestinal microbiota of Salmonella infected chickens in response to enrofloxacin. Front. Microbiol. 2017, 8, 1711. [Google Scholar] [CrossRef]
  34. FDA/CVM Proposes to Withdraw Poultry Fluoroquinolones Approval. 2000. Available online: https://www.fda.gov/animal-veterinary/recalls-withdrawals/withdrawal-enrofloxacin-poultry (accessed on 21 May 2007).
  35. Price, L.; Lackey, L.; Vailes, R.; Silbergeld, E. The persistence of fluoroquinolone-resistant Campylobacter in poultry production. Environ. Health Perspect. 2007, 115, 1035–1039. [Google Scholar] [CrossRef]
  36. Gbylik, S.M.; Posyniak, A.; Sniegocki, T.; Sell, B.; Gajda, A.; Sawicka, A.; Olszewska, T.M.; Bladek, T.; Tomczyk, G.; Zmudzki, J. Influence of enrofloxacin traces in drinking water to doxycycline tissue pharmacokinetics in healthy and infected by Mycoplasma gallisepticum broiler chickens. Food Chem. Toxicol. 2016, 90, 123–129. [Google Scholar] [CrossRef]
  37. Han, F.F.; Lestari, S.I.; Pu, S.H.; Ge, B.L. Prevalence and antimicrobial resistance among Campylobacter spp. in Louisiana retail chickens after the enrofloxacin ban. Foodborne Pathog. Dis. 2009, 6, 163–171. [Google Scholar] [CrossRef]
  38. Hong, J.; Kim, J.M.; Jung, W.K.; Kim, S.H.; Bae, W.; Koo, H.C.; Gil, J.; Kim, M.; Ser, J.; Park, Y.H. Prevalence and antibiotic resistance of Campylobacter spp. isolated from chicken meat, pork, and beef in Korea, from 2001 to 2006. J. Food Prot. 2007, 70, 860–866. [Google Scholar] [CrossRef]
  39. Kim, J.J.; Seo, K.W.; Mo, I.P.; Lee, Y.J. Genetic Characterization of fluoroquinolone resistance in Salmonella enterica serovar Gallinarum isolates from chicken in Korea. Avian Dis. 2019, 63, 584–590. [Google Scholar] [CrossRef]
  40. Park, H.; Kang, J.; Jang, Y.; Song, J.Y.; Lee, K.J. Studies on pharmacokinetics/pharmacodanymics of enrofloxacin in broiler chicken. FASEB J. 2016, 30, lb539. [Google Scholar]
  41. Perrin, G.A.; Jouy, E.; Urban, D.; Chauvin, C.; Granier, S.A.; Mourand, G.; Chevance, A.; Adam, C.; Moulin, G.; Kempf, I. Decrease in fluoroquinolone use in French poultry and pig production and changes in resistance among E. coli and Campylobacter. Vet. Microbiol. 2020, 243, 108637. [Google Scholar] [CrossRef]
  42. Delsol, A.A.; Sunderland, J.; Woodward, M.J.; Pumbwe, L.; Piddock, L.J.; Roe, J.M. Emergence of fluoroquinolone resistance in the native Campylobacter coli population of pigs exposed to enrofloxacin. J. Antimicrob. Chemother. 2004, 53, 872–874. [Google Scholar] [CrossRef]
  43. Van, B.M.; Veldman, K.T.; de Jong, M.C.; Mevius, D.J. Rapid selection of quinolone resistance in Campylobacter jejuni but not in Escherichia coli in individually housed broilers. J. Antimicrob. Chemother. 2003, 52, 719–723. [Google Scholar]
  44. Chantziaras, I.; Smet, A.; Filippitzi, M.E.; Damiaans, B.; Haesebrouck, F.; Boyen, F.; Dewulf, J. The effect of a commercial competitive exclusion product on the selection of enrofloxacin resistance in commensal E. coli in broilers. Avian Pathol. 2018, 47, 443–454. [Google Scholar] [CrossRef]
  45. Da Costa, P.M.; Belo, A.; Gonçalves, J.; Bernardo, F.J. Field trial evaluating changes in prevalence and patterns of antimicrobial resistance among Escherichia coli and Enterococcus spp. isolated from growing broilers medicated with enrofloxacin, apramycin and amoxicillin. Vet. Microbiol. 2009, 139, 284–292. [Google Scholar] [CrossRef]
  46. Shang, K.; Wei, B.; Kang, M. Distribution and dissemination of antimicrobial-resistant Salmonella in broiler farms with or without enrofloxacin use. BMC Vet. Res. 2018, 14, 257. [Google Scholar] [CrossRef]
  47. Shang, K.; Wei, B.; Jang, H.K.; Kang, M. Phenotypic characteristics and genotypic correlation of antimicrobial resistant (AMR) Salmonella isolates from a poultry slaughterhouse and its downstream retail markets. Food Control 2019, 100, 35–45. [Google Scholar] [CrossRef]
  48. Cavaco, L.; Aarestrup, F. Evaluation of quinolones for use in detection of determinants of acquired quinolone resistance, including the new transmissible resistance mechanisms qnrA, qnrB, qnrS, and aac(6′)Ib-cr, in Escherichia coli and Salmonella enterica and determinations of wild-type distributions. J. Clin. Microbiol. 2009, 47, 2751–2758. [Google Scholar]
  49. Clinical and Laboratory Standards Institute (CLSI). Performance Standards for Antimicrobial Disk and Dilution Susceptibility Tests for Bacteria Isolated from Animals, 4th ed.; CLSI: Wayne, PA, USA, 2018. [Google Scholar]
  50. CLSI. Performance Standards for Antimicrobial Susceptibility Testing, Approved Standard M100-S30; 30th informational supplement; Clinical Laboratory Standards Institute: Wayne, PA, USA, 2020. [Google Scholar]
  51. CDC. National Antimicrobial Resistance Monitoring System for Enteric Bacteria (NARMS): Human Isolates Surveillance Report for 2015 (Final Report); U.S. Department of Health and Human Services, CDC: Atlanta, GA, USA, 2018.
  52. Ojo, O.E.; Ogun Yinka, O.G.; Agbaje, M.; Okuboye, J.O.; Kehinde, O.O.; Oyekunle, M.A. Antibiogram of Enterobacteriaceae isolated from free-range chickens in Abeokuta, Nigeria. Vet. Arhiv. 2012, 82, 577–589. [Google Scholar]
  53. Schwarz, S.; Silley, P.; Simjee, S.; Woodford, N.; van Duijkeren, E.; Johnson, A.P.; Gaastra, W.J. Assessing the antimicrobial susceptibility of bacteria obtained from animals. J. Antimicrob. Chemother. 2010, 65, 601–604. [Google Scholar] [CrossRef]
  54. Jeong, H.S.; Kim, J.A.; Shin, J.H.; Chang, C.L.; Jeong, J.; Cho, J.H.; Kim, M.N.; Kim, S.; Kim, Y.R.; Lee, C.H. Prevalence of plasmid-mediated quinolone resistance and mutations in the gyrase and topoisomerase IV genes in Salmonella isolated from 12 tertiary-care hospitals in Korea. Microb. Drug Resist. 2011, 17, 551–557. [Google Scholar] [CrossRef]
  55. Park, M.; Kim, J.; Feinstein, J.; Lang, K.S.; Ryu, S.; Jeon, B. Development of fluoroquinolone resistance through antibiotic tolerance in Campylobacter jejuni. Microbiol. Spectr. 2022, 10, e0166722. [Google Scholar] [CrossRef]
  56. Pleydell, E.; Brown, P.; Woodward, M.J.; Davies, R.; French, N.J. Sources of variation in the ampicillin-resistant Escherichia coli concentration in the feces of organic broiler chickens. Appl. Environ. Microbiol. 2007, 73, 203–210. [Google Scholar] [CrossRef]
  57. Awada, R.; Ghssein, G.; Roz, A.; Farhat, M.; Nehme, N.; Hassan, H. Prevalence of Campylobacter spp. in broilers in North Lebanon. Vet. World 2023, 16, 322–328. [Google Scholar] [CrossRef]
  58. Andersson, D.I.; Hughes, D. Antibiotic resistance and its cost: Is it possible to reverse resistance? Nat. Rev. Microbiol. 2010, 8, 260–271. [Google Scholar] [CrossRef]
  59. Enne, V.I.; Bennett, P.M.; Livermore, D.M.; Hall, L.M. Enhancement of host fitness by the sul2-coding plasmid p9123 in the absence of selective pressure. J. Antimicrob. Chemother. 2004, 53, 958–963. [Google Scholar] [CrossRef]
  60. Inglis, G.D.; Zaytsoff, S.J.; Selinger, L.B.; Taboada, E.N.; Uwiera, R.R. Therapeutic administration of enrofloxacin in mice does not select for fluoroquinolone resistance in Campylobacter jejuni. Can. J. Microbiol. 2018, 64, 681–694. [Google Scholar] [CrossRef]
  61. Usui, M.; Sakemi, Y.; Uchida, I.; Tamura, Y.J. Effects of fluoroquinolone treatment and group housing of pigs on the selection and spread of fluoroquinolone-resistant Campylobacter. Vet. Microbiol. 2014, 170, 438–441. [Google Scholar] [CrossRef]
  62. Yang, H.; Chen, H.; Yang, Q.; Chen, M.; Wang, H.J. High prevalence of plasmid-mediated quinolone resistance genes qnr and aac (6′)-Ibra in clinical isolates of Enterobacteriaceae from nine teaching hospitals in China. Antimicrob. Agents Chemother. 2008, 52, 4268–4273. [Google Scholar] [CrossRef]
  63. Seo, K.W.; Lee, Y.J. Characterization of plasmid mediated quinolone resistance determinants in ciprofloxacin resistant-Escherichia coli from chicken meat produced by integrated broiler operations in Korea. Int. J. Food Microbiol. 2019, 307, 108274. [Google Scholar] [CrossRef]
  64. Hordijk, J.; Veldman, K.; Dierikx, C.; van Essen-Zandbergen, A.; Wagenaar, J.A.; Mevius, D. Prevalence and characteristics of quinolone resistance in Escherichia coli in veal calves. Vet. Microbiol. 2012, 156, 136–142. [Google Scholar] [CrossRef]
  65. Lin, D.; Chen, K.; Chan, E.W.-C.; Chen, S. Increasing prevalence of ciprofloxacin-resistant food-borne Salmonella strains harboring multiple PMQR elements but not target gene mutations. Sci. Rep. 2015, 5, 14754. [Google Scholar] [CrossRef]
  66. Kang, M.; Wei, B.; Choi, S.W.; Cha, S.Y.; Jang, H.K. Molecular characterization of fluoroquinolone resistance mechanisms of Campylobacter isolates from duck meats. J. Food Prot. 2017, 80, 2056–2059. [Google Scholar] [CrossRef]
  67. Goulart, D.; Beyi, A.; Wu, Z.; Adiguzel, C.; Wilson, S.; Xu, C.; Pang, J.; Dewell, R.; Dewell, G.; Plummer, P.; et al. Influence of single dose enrofloxacin injection on development of fluoroquinolone resistance in Campylobacter jejuni in calves. Antibiotics 2022, 11, 1407. [Google Scholar] [CrossRef]
Table
Table 1. Prevalence of Salmonella and Campylobacter in untreated or ENR-treated broiler chickens.
Table 1. Prevalence of Salmonella and Campylobacter in untreated or ENR-treated broiler chickens.
PathogensGroup1-Day-Old15–25-Days-OldRetail Meat
No. of SamplesNo. of IsolatesIsolation Rate (%)No. of SamplesNo. of IsolatesIsolation Rate (%)No. of SamplesNo. of IsolatesIsolation Rate (%)
Salmonella1308258.1 (5.1–11.2)1236796.4 (5.0–7.8)1053028.6 (19.9–37.2)
2 2202611.8 (7.6–16.1)4855611.6 (8.7–14.4)643960.9 (49.0–72.9)
p-value 0.114 <0.001 <0.001
Campylobacter130810.3 (0.0–1.0)1236836.7 (5.2–8.1)1054845.7 (36.2–55.2)
2 22000.0 (0.0–0.0)485163.3 (1.7–4.9)641726.6 (15.7–37.4)
p-value 0.720 0.006 0.013
Bold type indicates p-value < 0.05; ENR: enrofloxacin; results are expressed as the mean (95% confidence interval). Group 1—contained farms that use ENR; and Group 2—contained farms that do not use ENR. In Group 1, 0.5 g/L ENR was constantly added to the drinking water of 2–4-day-old chicks.
Table
Table 2. Antimicrobial resistance of E. coli isolates from untreated or ENR-treated broiler chickens.
Table 2. Antimicrobial resistance of E. coli isolates from untreated or ENR-treated broiler chickens.
Antimicrobial ClassAntimicrobialsAntimicrobial Resistance (%)
1-Day-Old15–25-Days-OldRetail Meat
Group 1 Group 2p-ValueGroup 1Group 2p-ValueGroup 1Group 2p-Value
CombinationsAmoxicillin/clavulanic acid9.19.11.0006.15.40.83823.145.00.083
PenicillinAmpicillin63.672.90.31688.682.20.18194.970.00.025
CephalosporinsCephalothin100.040.0<0.00154.739.60.03837.960.00.128
Cefoxitin9.18.01.0009.410.90.73420.545.00.049
Ceftiofur11.411.91.00020.016.90.55746.260.00.314
AmphenicolsChloramphenicol25.025.41.00061.052.50.20659.055.00.770
Florfenicol18.223.70.66255.250.40.48780.647.10.016
AminoglycosidesStreptomycin43.254.20.26767.666.90.91543.655.00.406
Gentamicin9.18.51.00026.017.80.14061.525.00.017
Neomycin2.38.50.36614.68.50.1660.00.0N/A
TetracyclineTetracycline75.062.70.18676.082.20.25264.1100.00.006
SulfonamidesTrimethoprim/sulfamethoxazole29.534.00.69240.640.00.93176.980.01.000
QuinolonesNalidixic acid100.081.40.00799.096.60.43982.185.01.000
Ciprofloxacin72.755.90.08181.973.70.14482.170.00.290
Enrofloxacin68.259.30.35788.178.00.04879.580.01.000
PolymyxinColistin0.00.0N/A1.00.81.0002.60.01.000
MDR 77.366.10.61295.289.80.763100.0100.01.000
N/A, not available; bold type indicates p-value < 0.05. Group 1—contained farms that use ENR; and Group 2—contained farms that do not use ENR. In Group 1, 0.5 g/L ENR was constantly added to the drinking water of 2–4-day-old chicks.
Table
Table 3. Antimicrobial resistance of Salmonella isolates from untreated or ENR-treated broiler chickens.
Table 3. Antimicrobial resistance of Salmonella isolates from untreated or ENR-treated broiler chickens.
Antimicrobial
Class		AntimicrobialsAntimicrobial Resistance (%)
1-Day-Old15–25-Days-OldRetail Meat
Group 1Group 2p-ValueGroup 1Group 2p-ValueGroup 1Group 2p-Value
CombinationsAmoxicillin/clavulanic acid0.00.0N/A1.30.01.0000.00.0NA
PenicillinAmpicillin8.00.00.45340.517.90.00556.735.90.140
CephalosporinsCephalothin0.00.0N/A2.51.81.0006.70.00.361
Cefoxitin20.011.50.6567.67.11.0000.02.61.000
Ceftiofur8.03.80.9726.33.60.7500.07.70.338
AmphenicolsChloramphenicol0.00.0N/A38.012.50.00156.735.90.140
Florfenicol0.00.0N/A22.812.50.13020.023.10.990
AminoglycosidesStreptomycin36.026.90.69249.444.60.58826.728.21.000
Gentamicin0.00.0N/A1.31.81.0000.00.00.055
Neomycin60.034.60.12541.844.60.74016.741.00.155
TetracyclineTetracycline24.011.50.42463.323.2<0.00153.333.30.404
SulfonamidesTrimethoprim/sulfamethoxazole0.03.81.00048.128.60.02256.743.61.000
QuinolonesNalidixic acid100.096.21.00089.9100.00.03796.797.41.000
Ciprofloxacin-R0.00.0N/A1.30.01.0003.30.01.000
Ciprofloxacin-IR48.019.20.06058.255.40.74066.787.20.080
Enrofloxacin-R0.00.0N/A2.50.00.6340.05.11.000
Enrofloxacin-IR48.026.90.12067.148.20.02873.369.20.710
PolymyxinColistin 0.00.0N/A6.31.80.4020.02.61.000
MDR 28.023.10.76569.632.10.01656.756.40.991
N/A, not available; bold type indicates p-value < 0.05; R, resistance; IR, intermediate resistance. Group 1—contained farms that use ENR; and Group 2—contained farms that do not use ENR. In Group 1, 0.5 g/L ENR was constantly added to the drinking water of 2–4-day-old chicks.
Table
Table 4. Antimicrobial resistance of Campylobacter isolates from untreated or ENR-treated broiler chickens.
Table 4. Antimicrobial resistance of Campylobacter isolates from untreated or ENR-treated broiler chickens.
Antimicrobial
Class		AntimicrobialsAntimicrobial Resistance (%)
1-Day-Old15–25-Days-OldRetail Meat
Group 1Group 2p-ValueGroup 1Group 2p-ValueGroup 1Group 2p-Value
PenicillinAmpicillin100.0-N/A66.756.30.44839.682.40.002
TetracyclineTetracycline0.0-N/A15.781.3<0.00158.376.50.183
MacrolidesAzithromycin0.0-N/A0.00.0N/A2.10.00.549
Erythromycin0.0-N/A0.00.0N/A0.00.0N/A
AmphenicolsFlorfenicol0.0-N/A0.00.0N/A2.10.0N/A
AminoglycosidesGentamicin0.0-N/A0.00.0N/A0.00.0N/A
KetolidesTelithromycin0.0-N/A0.00.0N/A0.00.0N/A
LincosamidesClindamycin0.0-N/A0.00.0N/A0.00.0N/A
QuinolonesNalidixic acid0.0-N/A78.4100.00.05495.888.20.263
Ciprofloxacin0.0-N/A78.4100.00.054100.0100.0N/A
Enrofloxacin0.0-N/A78.4100.00.054100.0100.0N/A
MDR 0.0-N/A11.843.80.00520.858.80.004
N/A, not available; -, indicates no isolate; bold type indicates p-value < 0.05. Group 1—contained farms that use ENR; and Group 2—contained farms that do not use ENR. In Group 1, 0.5 g/L ENR was constantly added to the drinking water of 2–4-day-old chicks.
Table
Table 5. Prevalence of PMQR and amino acid changes in the gyrA, parC, and parE genes of E. coli isolates (n = 45) from Group 1 and Group 2 or the corresponding MICs of ENR a.
Table 5. Prevalence of PMQR and amino acid changes in the gyrA, parC, and parE genes of E. coli isolates (n = 45) from Group 1 and Group 2 or the corresponding MICs of ENR a.
Group b (n)n (%) cENR MIC (µg/mL)PMQR (%)QRDR Pattern
gyrA parC parE
Group 1
(n = 28)		8 (28.6)16–320.0Ser-83-LeuAsp-87-Asne Ser-80-Ile Ser-83-Tyr wt
7 (25.0)4–16qnrB (14.3)wt d Ser-80-Ile Ser-83-Tyr wt
2 (7.1)40.0Ser-83-Leu Ser-83-Tyr wt
2 (7.1)320.0Ser-83-LeuAsp-87-Asn Ser-80-Ile Ser-83-Tyr Ser-458-Ala
2 (7.1)1–2qnrS1 (50.0)wt Ser-83-Tyr wt
1 (3.6)160.0Ser-83-Leu Asp-87-His Ser-80-Ile Ser-83-Tyr wt
1 (3.6)160.0Ser-83-Leu Asp-87-Ala Ser-80-Ile Ser-83-Tyr wt
1 (3.6)160.0Ser-83-Leu Asp-87-Tyr Ser-80-Ile Ser-83-Tyr wt
1 (3.6)160.0Ser-83-Leu Asp-87-GlySer-80-Ile Ser-83-Tyr wt
1 (3.6)320.0Ser-83-LeuAsp-87-Asn Ser-80-Ile Ser-83-Tyr Ile-464-Phe
1 (3.6)320.0Ser-83-LeuAsp-87-Asn Ser-83-TyrGlu-84-Lys wt
1 (3.6)> 320.0Ser-83-LeuAsp-87-Asn Ser-80-Ile Ser-83-Tyr Glu-84-GlySer-458-Ala
Group 2 (n = 17)6 (35.3)8–160.0 Ser-80-Ile Ser-83-Tyr wt
3 (17.6)160.0Ser-83-LeuAsp-87-Asn Ser-80-Ile Ser-83-Tyr wt
3 (17.6)> 320.0Ser-83-LeuAsp-87-Asn Ser-80-Ile Ser-83-Tyr Ser-458-Ala
3 (17.6)80.0wt Ser-83-Tyr wt
1 (5.9)320.0Ser-83-Leu Asp-87-His Ser-80-Ile Ser-83-Tyr wt
1 (5.9)320.0wt Ser-80-ArgSer-83-Tyr wt
a PMQR: plasmid-mediated quinolone resistance; QRDR: quinolone resistance determining region; b Group 1—contained farms that used enrofloxacin; and Group 2—contained farms that did not use enrofloxacin; c n (%), number of isolates (percentage); d wt, wild type without gene mutation; e Bank means no point mutation.
Table
Table 6. Prevalence of PMQR and amino acid changes in the gyrA and parC genes of Salmonella isolates (n = 99) from Group 1 and Group 2 and the corresponding MICs of ENR a.
Table 6. Prevalence of PMQR and amino acid changes in the gyrA and parC genes of Salmonella isolates (n = 99) from Group 1 and Group 2 and the corresponding MICs of ENR a.
Group b (n) n (%) cENR MIC (µg/mL)PMQR (%)QRDR Pattern
gyrA parC
1 (n = 50)28 (56.0)0.25–1.000.00d Asp-87-Gly Tyr-57-Ser
9 (18.0)0.25–0.500.00Ser-83-Phe Tyr-57-Ser
5 (10.0)0.25–1.000.00wt e Tyr-57-Ser
4 (8.0)0.25–0.500.00Ser-83-Phe wt
2 (4.0)0.12–0.250.00 Ser-83-Tyr Tyr-57-Ser
1 (2.0)0.500.00 Ser-83-AlaAsp-87-Gly Tyr-57-Ser
1 (2.0)0.250.00 Asp-87-AsnTyr-57-Ser
2 (n = 49)33 (67.3)0.25–0.500.00 Asp-87-Gly Tyr-57-Ser
9 (18.4)0.50–1.000.00Ser-83-Phe Tyr-57-Ser
4 (8.2)0.250.00 Asp-87-AsnTyr-57-Ser
1 (2.0)0.500.00Ser-83-Phe wt
1 (2.0)0.250.00wt Tyr-57-Ser
1 (2.0)0.250.00 Ser-83-Tyr Tyr-57-Ser
a PMQR: plasmid-mediated quinolone resistance; QRDR: quinolone-resistance determining region; b Group 1—contained farms that used enrofloxacin; and Group 2—contained farms that did not use enrofloxacin; c n (%), number of isolates (percentage); d Bank means no point mutation; e wt, wild type without gene mutation.
Table
Table 7. Prevalence of PMQR and amino acid changes in the gyrA gene of the Campylobacter isolates (n = 76) from Group 1 and Group 2 and the corresponding MICs of ENR a.
Table 7. Prevalence of PMQR and amino acid changes in the gyrA gene of the Campylobacter isolates (n = 76) from Group 1 and Group 2 and the corresponding MICs of ENR a.
Group b (n) n (%) cENR MIC (µg/mL)PMQR (%)QRDR Pattern
gyrA
Group 1 (n = 50)50 (100.0)4–320.0Thr-86-Ile
Group 2 (n = 26)26 (100.0)4–320.0Thr-86-Ile
a PMQR: plasmid-mediated quinolone resistance; QRDR: quinolone-resistance determining region; b Group 1—contained farms that used enrofloxacin; and Group 2—contained farms that did not use enrofloxacin; c n (%), number of isolates (percentage).
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MDPI and ACS Style

Shang, K.; Kim, J.-H.; Park, J.-Y.; Choi, Y.-R.; Kim, S.-W.; Cha, S.-Y.; Jang, H.-K.; Wei, B.; Kang, M. Comparative Studies of Antimicrobial Resistance in Escherichia coli, Salmonella, and Campylobacter Isolates from Broiler Chickens with and without Use of Enrofloxacin. Foods 2023, 12, 2239. https://doi.org/10.3390/foods12112239

AMA Style

Shang K, Kim J-H, Park J-Y, Choi Y-R, Kim S-W, Cha S-Y, Jang H-K, Wei B, Kang M. Comparative Studies of Antimicrobial Resistance in Escherichia coli, Salmonella, and Campylobacter Isolates from Broiler Chickens with and without Use of Enrofloxacin. Foods. 2023; 12(11):2239. https://doi.org/10.3390/foods12112239

Chicago/Turabian Style

Shang, Ke, Ji-Hyuk Kim, Jong-Yeol Park, Yu-Ri Choi, Sang-Won Kim, Se-Yeoun Cha, Hyung-Kwan Jang, Bai Wei, and Min Kang. 2023. "Comparative Studies of Antimicrobial Resistance in Escherichia coli, Salmonella, and Campylobacter Isolates from Broiler Chickens with and without Use of Enrofloxacin" Foods 12, no. 11: 2239. https://doi.org/10.3390/foods12112239

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