Identification of risk factors associated with antimicrobial resistance in equine fecal Escherichia coli isolates

Journal Pre-proof Identification of risk factors associated with antimicrobial resistance in equine fecal Escherichia coli isolates

Mohammad H. Gharaibeh, Sameeh M. Abutarbush, Farah G. Mustafa, Shawkat Q. Lafi, Motasem S. Halaiqa PII:

S1567-1348(20)30148-9

DOI:

https://doi.org/10.1016/j.meegid.2020.104317

Reference:

MEEGID 104317

To appear in:

Infection, Genetics and Evolution

Received date:

19 January 2020

Revised date:

19 March 2020

Accepted date:

7 April 2020

Please cite this article as: M.H. Gharaibeh, S.M. Abutarbush, F.G. Mustafa, et al., Identification of risk factors associated with antimicrobial resistance in equine fecal Escherichia coli isolates, Infection, Genetics and Evolution (2019), https://doi.org/ 10.1016/j.meegid.2020.104317

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© 2019 Published by Elsevier.

Journal Pre-proof Identification of risk factors associated with antimicrobial resistance in equine fecal Escherichia coli isolates

Mohammad H. Gharaibeh1, * , Sameeh M. Abutarbush 2 , Farah G. Mustafa2 , Shawkat Q. Lafi3 ,

Department of Basic Veterinary Medical Science, Faculty of Veterinary Medicine, Jordan

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Motasem S. Halaiqa2

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2

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University of Science and Technology, P. O. Box 3030 Irbid, 22110, Jordan.

Department of Clinical Veterinary Medical Sciences, Faculty of Veterinary Medicine, Jordan

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University of Science and Technology, P.O. Box 3030 Irbid, 22110 Jordan.

Department of Pathology and Public Health, Faculty of Veterinary Medicine, Jordan University

*

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of Science and Technology, P.O. Box 3030 Irbid, 22110 Jordan.

Corresponding author: Mohammad H. Gharaibeh

E-mail: [email protected]

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Journal Pre-proof Abstract Antimicrobial resistance is a growing global problem that will need a multinational collaborative effort to overcome this serious challenge. The aim of the study is to investigate the potential risk factors associated with the prevalence and distribution of antimicrobial-resistance genes (ARGs) of Escherichia coli isolates obtained from equine fecal samples. One hundred eighteen horses from different geographical locations and management systems were enrolled in the study and a

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questionnaire containing information about each individual horse was designed and filled.

The

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enrolled horses belonged to 2 main categories (Hospitalized horses (n=31), and Non hospitalized

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horses (n=87)). In total, 103 E. coli isolates were collected from the 118 fecal horse samples.

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Genes that are responsible for resistance to β-lactams, tetracyclines, aminoglycosides, and trimethoprim were detected using PCR. The prevalence of antimicrobial resistance was

against

(trimethoprim,

doxycycline,

to

non-hospitalized

oxytetracycline,

and

ones (p≤0.05),

amoxicillin-clavulanic

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particularly

horses compared

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significantly higher in hospitalized

acid). The most prevalent antimicrobial-resistant genes were aminoglycoside resistant genes

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(strA, strB, and aadA) in percentages; 89%, 85%, and 84%, respectively. Statistical analysis

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revealed a significant association between risk factors and occurrence of ARGs (p≤0.05). Significant risk factors include the last treatment and history of antimicrobial administration, breed of horses, use of horses, type of diet fed for horses, practice management and history of last illness. Tetracycline-resistance gene (tetA) was 22 times more likely to be found in the Arabian and local breeds of horses compared to English and warmblood breed. TetA is also 8 times more likely to be found in horses that were fed a natural diet compared to other horses that were fed manufactured/ processed feed. In conclusion, E. coli bacterium can harbor high resistance to different classes of antimicrobials which increases the risk of potential uncontrolled

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Journal Pre-proof transmission of the multi-drug resistant E. coli bacterium to veterinarians and horse handlers, as well as to the equine population itself.

Key words: Escherichia coli; risk factors; antibiotic resistance; resistance genes; horse; Jordan

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1. Introduction

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Antimicrobial resistance (AMR) is defined as the ability of the microorganisms to resist the

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antimicrobial drugs that once could successfully treat them (CDC, 2020). It is a serious growing

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problem in veterinary practice and this has resulted in increased rates of morbidity and hindered the ability to effectively treat infections in equine practice (Ahmed et al., 2010). Escherichia coli

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is an important part of the gastrointestinal tract ecosystem of most mammals, particularly in

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equines (van Duijkeren et al., 2000). Despite being an important normal microflora, E. coli pathogenic strains are capable of causing clinical diseases in both the gastrointestinal tract and

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extra-intestinal sites (Lanz et al., 2003). Pathogenic E. coli can also be found in fecal samples of

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diarrheic foals suggesting the implication of the microorganism in foal’s diarrhea (BROWNING et al., 1991). Furthermore, horses can act as a reservoir of antimicrobial-resistant E. coli and its genetic determinants that subsequently pose a risk to human public health through hindering the ability to treat infections (Scott Weese, 2008). Resistant bacteria can be introduced to animals through several ways including environment, such as the land application of manure of livestock origin as fertilizers (Marshall and Levy, 2011). The soil is one of the major and favorable environments for bacterial diversity. Hundred species of microorganisms can be contained in only one gram of soil (Dunbar et al., 1999). The

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Journal Pre-proof prevalence of antimicrobial resistance of Escherichia coli isolated from equine feces is much higher in hospitalized horses that were administered antimicrobial drugs, suggesting that hospitalization and current illness are major risk factors for harboring antimicrobial-resistant E. coli and its genetic determinants in feces (Dunowska et al., 2006). Indeed, animal's gastrointestinal microflora has acquired ARGs over the years of exposure to antimicrobial agents, growth promotors, heavy metal compounds and animals waste-contaminated water, fruits

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and vegetables (Woolhouse et al., 2015). In a cross-sectional study to identify risk factors for

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fecal carriage of antimicrobial-resistant E. coli isolates in horses, the type of premises was

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identified as risk factors, where being stabled on a farm was a significant risk factor for The farm environment represents an increased

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harboring multi-drug resistant (MDR) bacteria.

opportunity for acquisition of multi-drug resistant bacteria (Maddox et al., 2012b). New

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strategies have been developed to deal with AMR which can be achieved by understanding the

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molecular characteristics of bacterial resistance acquisition and transfer with special interest in MDR microorganism (Wassenaar, 2005). Many studies in different animals including horses

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have described antimicrobial susceptibility of E. coli bacterium. Antimicrobial-resistant studies

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vary among countries with regards to human related MDR E. coli bacterium (Ahmed et al., 2010; Dunowska et al., 2006; Maddox et al., 2015). As most other countries, Jordan is facing a serious challenge in terms of controlling the emergence of AMR bacteria. Failure to respond to antimicrobial treatment is not uncommon complaint by equine practitioners in Jordan and AMR studies on bacterial microorganisms of equine origin in Jordan are lacking. Therefore, the aims of this study are to explore the prevalence of AMR Escherichia coli in equine fecal isolates and to identify the risk factors associated with the prevalence and distribution of ARGs.

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2. Materials and methods

2.1. Ethics statement This study was reviewed and approved by the Animal Care and Use Committee (ACUC), Jordan

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University of Science and Technology (JUST- ACUC approval number: 111/2019).

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2.2. Sample collection

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Fecal samples were collected from different stables and riding centers located in different

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geographical region in Jordan; Amman, Zarqa, Irbid, and Jerash. A total of 118 horse fecal samples were collected; 31 fecal samples were collected from horses hospitalized in the facilities

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of The Society for the Protection of Animals Abroad (SPANA) located in Wadi Al Seer area in

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Amman governorate during the period from November 2018 to February 2019. The rest of the rest of the fecal samples (n=87) were collected from non-hospitalized horses (community group);

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42 of sampled horses from a riding center in Amman, 15 from a stable in Zarqa, 15 from a stable

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in Irbid and 15 from a breeding stud in Jerash during the period from January 2019 to February 2019. A questionnaire was designed to collect information about risk factors that are hypothesized to have a potential effect on antimicrobial-resistance and antimicrobial-resistant genes prevalence based on recent literature (Caudell et al., 2018; Dunowska et al., 2006; Maddox et al., 2012a, 2011). The questionnaire contains three sections and include; signalment and history of the animal, water and feed supplement, diseases and hospitalization and antimicrobials usage. The questionnaire was completed by the owners or the grooms working at the hospital during our visit by personnel interviews by the researchers before collecting the fecal samples.

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2.3. E. coli isolation and identification One gram of fecal sample was suspended in 9 ml of phosphate buffered saline (PBS) containing tubes. 50 μl of the suspension was added and cultured on Eosin Methylene Blue (EMB) agar (Oxoid, England). The agar plates were incubated at 37°C for 18–24 hr. those with no growth were left up to 48 h. Isolates were sub-cultured on MacConkey agar plates (Oxoid,

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England), to get pure cultures. The suspected colonies were streaked on the agar plates and

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incubated overnight at 37º C (Tonu et al., 2011).

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2.4. Antimicrobial susceptibility test using standard disc diffusion method E. coli isolates were subjected to disc diffusion testing according to the Clinical and Laboratory

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Standards Institute (CLSI) guidelines (CLSI, 2013). The isolates were tested for resistance for amoxicillin (AX. 25

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fifteen different antimicrobial agents (purchased from Oxoid, England):

µg), gentamicin (CN. 10 μg), trimethoprim (TRI. 5 μg), doxycycline (DO. 30 μg),

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oxytetracycline (OT. 30 μg), streptomycin (S. 10 μg), cefotaxime (CTX. 30 μg), azithromycin

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(AZM. 15 μg), enrofloxacin (ENR. 5 μg), ampicillin (AMP. 10 μg), amoxicillin-clavulanic acid (AML. 20/10 μg), erythromycin (E. 15 μg), amikacin (AK. 30 μg), ceftiofur (EFT. 30 μg), metronidazole (MET. 5 μg). To date, no clinical breakpoint for resistant in E. coli against azithromycin has been established. Instead, a halo diameter <12 mm is considered as the azithromycin resistant breakpoint in Enterobacteriaceae based on S. Typhi microorganism (Gomes et al., 2019).

2.5. Minimum inhibitory concentration (MIC) test

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Journal Pre-proof The MICs of resistant E. coli isolates were determined according to CLSI guidelines (CLSI, 2013) for each of the following antimicrobial agents : streptomycin, trimethoprim, erythromycin, ampicillin, amoxicillin, doxycycline and oxytetracycline using the broth microdilution method as described previously (Wiegand et al., 2008). All used powder antimicrobial agents in the current study were from (Dar Al Dawa, Jordan).

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2.6. Identification of antimicrobial resistant genes by PCR

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PCR was used to identify genes responsible for resistance to beta-lactams, aminoglycosides,

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tetracyclines and trimethoprim. In total, 14 antimicrobial-resistant genes were investigated (Table S1). The method for DNA extraction was adapted from a method described by (Kimata et

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al., 2005). DNA amplification was performed in a Thermal cycler (Bio-Rad, USA); initial

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denaturation at 94°C for 3 min then for 35 cycles at 94°C for 1 min for denaturation, annealing

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for 40 seconds, followed by extension 72°C for 1 min and final extension 72°C for 10 min. PCR products detected by electrophoresis on 1.5% agarose gel with 8 µl ethidium bromide to

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listed in Table S1).

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visualize under UV transilluminator. PCR target genes and primers sequences that were used are

2.7. Risk factors statistical analysis The statistical analysis was performed using SPSS version 24 (SPSS Corp., IBM, Armonk., NY, USA). Initially, associations between the fourteen antimicrobial-resistant genes prevalence among all 103 E. coli isolates (outcome variables) and the associated risk factors (based on the literature) were screened using the univariate analysis (Chi-square X² and Fisher exact test). Potential risk factors with p ≤ 0.020 (X²; two-sided test) and no collinearity (r ≤ 0.6) were considered in building the final multivariate logistic regression model using manual stepwise 7

Journal Pre-proof forward logistic regression analysis. Only variables with p ≤ 0.05 were considered statistically significant and kept in the final model. The final model was performed and tested to fit the Hosmer and Lameshow-of-fit test.

3. Results

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3.1. Antimicrobial Susceptibility Results

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Fecal E. coli isolates were obtained from both hospitalized and non-hospitalized. For the

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hospitalized horse’s group, the highest resistance was found against amoxicillin, erythromycin,

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doxycycline, oxytetracycline and streptomycin in the percentage of 100%, 86.9%, 86.9%, 82.6%, and 82.6%, respectively. And for the non-hospitalized horses, the highest resistance was found

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against amoxicillin, erythromycin, and streptomycin in the percentage of 100%, 96.25%, and

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93.75%, respectively. The results of the 15 antimicrobials used in this study are shown in (Table 1, Fig. 1). Results obtained through MIC were listed in (Table 2, Fig. 2). In general, the

non-hospitalized

ones

(p≤0.05),

particularly

against

(trimethoprim,

doxycycline,

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prevalence of antimicrobial resistance was significantly higher in hospitalized horses compared

oxytetracycline, and amoxicillin-clavulanic acid) (Table 1, Fig. 1) and (Table 2, Fig. 2). The results of the MIC test were in good agreement with those obtained by disc diffusion (Table S2).

3.2. Detection of Antimicrobial Resistant Genes by PCR In total, thirteen antimicrobial-resistant genes (ARGs) have been detected among E. coli isolates in both hospitalized and non-hospitalized horses’ groups (Table 3). The most prevalent detected genes among both horse’s groups were aminoglycoside-resistant genes (strA, strB, and aadA) in

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Journal Pre-proof percentages; 89%, 85%, and 84%, respectively. Of the β-lactamases resistant genes, blaCTX-M the most prevalent gene (30%), followed by blaTEM at 15% and blaSHV at 10%. While, Tetracyclines resistant isolates, tetA was the most prevalent gene 25%, followed by tetB at 17% and blaSHV at 10%. Finally, the prevalence of Trimethoprim -resistant genes was 22% for dfrA17 dfrA7, followed by 17% for dfr1and 7% for dfrA12 dfrA13 genes. Generally, the prevalence for antimicrobial-resistant was significantly higher in hospitalized horses compared to

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non-hospitalized ones (p≤0.05), particularly against (Tetracyclines (tetA) and Trimethoprim

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(dfr1and dfrA17 dfrA7) genes (Fig. 3) (Fig. S1-5, Table S3).

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3.3. Risk Factors Statistical Analysis

The results of the univariate analysis are shown in (Table 4). The most antimicrobial-resistant

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gene ARG (outcome variable) that was highly associated with the studied risk factors was the

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tetracycline-resistant gene (tetA). Six risk factors were found to be significantly associated (p < 0.05) with the distribution and fecal shedding of E. coli that harbor the tetracycline-resistant gene

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(tetA). Therefore, the tetA was used for the construction of the final logistic model (Table 5). The

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final logistic regression model revealed that only two risk factors (Breeds; Local breed and Arabians versus English and warmbloods, and natural feed diet vs processed feed diet) were statistically significant (p ≤ 0.05). The tetA was 22 times more likely to shed fecal E. coli in the local breeds and Arabians compared with English and warmbloods, and 8 times more likely to be detected in horses that were fed a natural feed diet compared to horsed fed processed or manufactured feed diet.

Discussion

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Journal Pre-proof In the current study, hospitalized horses were more likely to shed antimicrobial-resistant E. coli, in their feces and are more likely to harbor anti-microbial- resistant genes (ARGs) than horses in the community group (non-hospitalized). It is known that, in the last few years, infection of animals in veterinary hospitals is considered to be a major source of resistance genes. The significantly higher prevalence of antimicrobial resistance and antimicrobial-resistance genes (ARGs) found in hospitalized horses in this study is in agree with previous observations (Ahmed

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et al., 2010; Maddox et al., 2012a). In this study, all horses at the hospital are referrals from

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private veterinary practices, so many of the horses might have undergone treatment, which may

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include prior antibiotic administration. Thus, the higher prevalence of resistant E. coli in

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hospitalized horses could reflect conditions prior to arrival at the hospital. The current study targeted 14 antimicrobial-resistant genes that are commonly associated with antimicrobial

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resistance among E. coli bacterium. In the current study, the proportion of β-lactamase resistant

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genes was much higher in hospitalized group samples compared to the community group. This comparison is similar to other studies performed on E. coli bacterium isolated from hospitalized

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and non-hospitalized horses in North West England (Ahmed et al., 2010). In the current study,

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the TEM β-lactamase gene was the most prevalent among ampicillin-resistant isolates in percentage of 57.1% (16/28), followed by SHV β-lactamase gene in percentage of 39.3% (11/28). However, a lower percentage of the TEM β-lactamase gene was found in the current study compared with a previous study in North West England which was 91%. On the other hand, a much higher percentage of SHV β-lactamase genes was detected in the current study, while only one isolate was positive for the SHV β-lactamase gene in the previous study in North West England (Ahmed et al., 2010). Regarding tetracyclines-resistant genes, doxycycline resistant isolates contain at least one of the tetracycline-resistant genes tetA and tetB, with tetA

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Journal Pre-proof being the most prevalent 50% (26/52), followed by tetB 34.6% (18/52) and (tetA and tetB) at 3.8% (2/52). Unlike the previous study in North West England, where tetB was the most prevalent gene (71%), followed by tetA at 18% and (tetA and tetB) at 11%. The differences in the distribution of tetracycline-resistant genes between individual countries are largely linked to how heavily they use tetracyclines (Stine et al., 2007). For example, tetracycline therapeutic use is very restricted in the UK compared to Jordan. However, the higher percentage of tetB in the

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previous study might be linked on the bacterial mobile genetic element; in these cases, exposure

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to a single antimicrobial agent can give rise to co-selection of multiple ARGs (Ahmed et al.,

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2010). Streptomycin resistance was attributable to the aadA, strA, and strB genes. aadA

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aminoglycoside resistant gene was found in 94.6% of E. coli isolates (87/92) compared with a study in Portugal where none of the nine streptomycin isolates harbored the aadA gene (Moura et

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al., 2013). strA and strB are aminoglycoside-resistant genes responsible for resistance to

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streptomycin, in the current study strA and strB resistant genes were highly detected among E. coli isolates in hospitalized horses in percentage; 82.6% and 78.3% respectively, and in non-

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hospitalized horses in percentage; 91.3% and 87.5% respectively. the persistence of these genes

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in the bacterial population is attributable to the overuse of streptomycin in clinical medicine in both humans and animals (Sundin and Bender, 1996). In the current study, resistant to gentamicin was attributable to aac(3)-IIa and aadB aminoglycosides-resistant genes, aac(3)-IIa was found in the current study at 54.5% (6/11), and aadB at 18.1% (2/11) in E. coli isolates that resistant to gentamicin. Furthermore,

the aminoglycoside resistant gene aac(6’) that is

responsible for resistance to amikacin was not detected in any of E. coli isolates, probably because the amikacin is not available for veterinary use in Jordan. Resistance to trimethoprim was mediated by dfr genes. In the current study, dfr genes were detected among all

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Journal Pre-proof trimethoprim-resistant E. coli isolates at the following frequencies; drf1 41.7% (20/48), drfA12 16.7% (8/48) and dfrA17 47.9% (23/48), with dfrA17 being the most prevalent trimethoprimresistance gene detected among trimethoprim-resistant isolates. Unlike the study performed in North West England by Ahmad et. al (2010), where the most prevalent gene responsible for trimethoprim resistance was dfr1. The high resistance found in this study against trimethoprim is probably because of the extensive use of sulphonamide products that are combined with

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trimethoprim in the veterinary hospital (SPANA) as well as in the community group of horses

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(e.g. Sulfadiazine/trimethoprim) (Ahmed et al., 2010).

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A wide range of risk factors that may affect the presence and occurrence of E. coli antimicrobial-

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resistance genes were included in this study. The main antimicrobial-resistant gene that was highly associated with several risk factors was the tetracycline-resistant gene (tetA). tetA and

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trimethoprim-resistant genes (dfr1 and dfrA17) were found to be highly associated with the

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grouping of horses, where higher percentages were in the hospitalized group compared to the non-hospitalized group of horses. Based on the literature, hospitalization and current illness play

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a major role in developing an environment for the spreading of antimicrobial-resistant genes

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(Bryan et al., 2010; Dunowska et al., 2006; Maddox et al., 2011). The age and sex of the horses enrolled in this study do not appear to play a role in the distribution of the ARGs. None of the samples were significantly associated with the age groups and gender of the horses. A similar result was found in a previous study by Maddox et al. (Maddox et al., 2011), where the age and gender of the horses did not show any significant relationship with the resistance pattern of E. coli isolates (Maddox et al., 2011). Breed of horses in this study was found to be significantly associated with β-lactamase resistant gene (blaCTX-M) and tetracycline-resistant gene (tetA), where higher percentages were found in the Arabian, Arabian mix, local breed and mix breed

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Journal Pre-proof horses compared to English and warmblood breeds. Arabian and local breed horses were being treated with several types of antimicrobials without the appropriate guidance by veterinarians especially in the local breed, which is the breed of working animals. tetA was also found to be significantly associated with the use of horses, being highest in the working horses, tetA was 22 times more likely to be found in the local breeds of horses compared to English and warmblood breeds, this is explained by relativeness to the poor conditions in areas where these animals live,

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lack of education by the owners, in addition to the inappropriate treatment options that are

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available for the working animals and local breed (Byarugaba, 2004). The geographical location

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showed only a statistically significant difference with the tetB tetracycline-resistant gene, where

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a higher percentage was found in Amman compared to other governorates in Jordan. Practice management of animals was found to affect different type of antimicrobial-resistant genes

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occurrence, including tetA and trimethoprim-resistant genes (dfr1 and dfrA17), where tetA

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antimicrobial-resistant gene fecal shedding was higher in the horses that are housed in stables, while dfr1 and dfrA17 antimicrobial-resistant genes were higher in horses that are kept in

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backyard or farms, this result is consistent with the previous study by Maddox et. al (Maddox et

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al., 2012b), which showed the type of premises as a major risk factor for antimicrobial-resistance in E. coli in horses. In the current study, backyards, farm environment, or even stables that are in direct contact with farm animals represent an increased opportunity for acquisition of multi-drug resistant bacteria carrying the antimicrobial-resistant genes including (tetA, dfr1, and dfrA17) (Maddox et al., 2012a). Other potential risk factors such as type of feed was found to be significantly associated with tetA tetracycline-resistant gene and the aminoglycosides-resistant genes (aadA and strB). A higher percentage of tetA antimicrobial-resistant gene was found in horses that are fed natural feed. On the other hand, aadA and strB antimicrobial-resistant genes

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Journal Pre-proof were found higher in horses that are fed manufactured or processed feed. Based on the final regression model results, E. coli bacterium harboring tetracycline-resistant genes (tetA) is 8 times more likely to be shed in feces of natural diet-fed horses, because the natural diet is more subjected to air, dust, soil and natural fertilizers, which are the ideal environment for bacterial growth and development, especially the multi-drug (MDR) resistant organisms (BengtssonPalme et al., 2018). The history of illness in animals could be another risk factor affecting the

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distribution of antimicrobial-resistant genes. Tetracycline-resistant gene tetA was much higher in

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horses that had a history of musculoskeletal and nervous diseases. Also, the history of the last

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treatment received for the animals was significantly associated with the presence of β-lactamase-

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resistant genes (blaTEM and blaCTX-M) as well as the tetA antimicrobial-resistant gene. The number of animals in the study that has received antimicrobial treatment were at higher risk for

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fecal shedding of antimicrobial-resistant E. coli, compared to the animals that didn’t receive

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antimicrobial treatment. The tetracycline-resistant gene tetA was significantly associated with the administration of antibiotics, based on the literature the inappropriate and overuse use of

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antimicrobials are the principal causes of antimicrobial resistance widespread and distribution,

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that may have clinical health consequences on both humans and animals (Pruden et al., 2013).

Conclusion E. coli bacterium was found to harbor high resistance against different antimicrobial classes, where higher resistance was found in hospitalized horses when compared to the non-hospitalized group. E. coli bacterium was also found to harbor many of the genes which are responsible for resistance against common antimicrobials used in equine medicine including; beta-lactams, aminoglycosides, tetracyclines and trimethoprim, those antimicrobial-resistant genes ARGs are

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Journal Pre-proof commonly found in other domestic animals and humans. It is suggested that antimicrobial resistance found in horses originate and transmit from the same source and mechanism found in other animal species. Thus, horses are considered as both recipient and a source of zoonotic transmission of ARGs. In addition, many risk factors were statistically analyzed, and found to affect the distribution and occurrence of antimicrobial-resistant genes, these factors include; the last treatment and antimicrobial administration, breed of horses, type of use of horses, type of

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diet fed for horses, practice management and the history of last illness.

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Acknowledgments

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This work was supported by the Deanship of Research, Jordan University of Science and

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Technology, Irbid, Jordan (grant numbers 111/2019)

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Conflicts of Interest:

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Author contributions

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The authors declare no conflict of interest.

Mohammad Gharaibeh:

Conceptualization, Methodology, Writing - Review & Editing Sameeh

M. Abutarbush: Conceptualization, Resources, Writing - Review & Editing Farah Mustafa: Methodology, Formal analysis, Investigation, Writing - Original Draft Shawkat Hailat: Formal analysis, Methodology Motasem S. Halaiqa: Methodology, Investigation

Additional files Supplementary materials 15

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Dunbar, J., Takala, S., Barns, S.M., Davis, J.A., Kuske, C.R., 1999. Levels of bacterial community diversity in four arid soils compared by cultivation and 16S rRNA gene cloning. Appl. Environ. Microbiol. 65, 1662–1669. Dunowska, M., Morley, P.S., Traub-Dargatz, J.L., Hyatt, D.R., Dargatz, D.A., 2006. Impact of hospitalization and antimicrobial drug administration on antimicrobial susceptibility patterns of commensal Escherichia coli isolated from the feces of horses. J. Am. Vet. Med. Assoc. 228, 1909–1917. https://doi.org/10.2460/javma.228.12.1909 Gangoué-Piéboji, J., Bedenic, B., Koulla-Shiro, S., Randegger, C., Adiogo, D., Ngassam, P.,

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Journal Pre-proof Ndumbe, P., Hächler, H., 2005. Extended-spectrum-β-lactamase-producing Enterobacteriaceae in Yaounde, Cameroon. J. Clin. Microbiol. 43, 3273–3277. https://doi.org/10.1128/JCM.43.7.3273-3277.2005 Gibreel, A., Sköld, O., 1998. High-level resistance to trimethoprim in clinical isolates of Campylobacter jejuni by acquisition of foreign genes (dfr1 and dfr9) expressing druginsensitive dihydrofolate reductases. Antimicrob. Agents Chemother. 42, 3059–3064.

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levels and mechanisms in Escherichia coli 1–10. https://doi.org/10.1038/s41598-019-42423-

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2005. Rapid categorization of EC. Microbiol. Immunol. 49, 485–492.

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Lanz, R., Kuhnert, P., Boerlin, P., 2003. Antimicrobial resistance and resistance gene determinants in clinical Escherichia coli from different animal species in Switzerland. Vet.

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Microbiol. 91, 73–84. https://doi.org/10.1016/S0378-1135(02)00263-8

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Lee, J.C., Oh, J.Y., Cho, J.W., Park, J.C., Kim, J.M., Seol, S.Y., Cho, D.T., 2001. The prevalence of trimethoprim-resistance-conferring dihydrofolate reductase genes in urinary isolates of Escherichia coli in Korea. J. Antimicrob. Chemother. 47, 599–604. https://doi.org/10.1093/jac/47.5.599 Maddox, T.W., Clegg, P.D., Diggle, P.J., Wedley, A.L., Dawson, S., Pinchbeck, G.L., Williams, N.J., 2012a. Cross-sectional study of antimicrobial-resistant bacteria in horses. Part 1: Prevalence of antimicrobial-resistant Escherichia coli and methicillin-resistant Staphylococcus aureus. Equine Vet. J. 44, 289–296. https://doi.org/10.1111/j.2042-

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Journal Pre-proof 3306.2011.00441.x Maddox, T.W., Clegg, P.D., Williams, N.J., Pinchbeck, G.L., 2015. Antimicrobial resistance in bacteria from horses: Epidemiology of antimicrobial resistance. Equine Vet. J. 47, 756–765. https://doi.org/10.1111/evj.12471 Maddox, T.W., Pinchbeck, G.L., Clegg, P.D., Wedley, A.L., Dawson, S., Williams, N.J., 2012b. Cross-sectional study of antimicrobial-resistant bacteria in horses. Part 2: Risk factors for

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faecal carriage of antimicrobial-resistant Escherichia coli in horses. Equine Vet. J. 44, 297–

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Maddox, T.W., Williams, N.J., Clegg, P.D., O’Donnell, A.J., Dawson, S., Pinchbeck, G.L.,

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2011. Longitudinal study of antimicrobial-resistant commensal Escherichia coli in the faeces of horses in an equine hospital. Prev. Vet. Med. 100, 134–145.

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Marshall, B.M., Levy, S.B., 2011. Food animals and antimicrobials: Impacts on human health. Clin. Microbiol. Rev. https://doi.org/10.1128/CMR.00002-11

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Moura, I., Torres, C., Silva, N., Somalo, S., Igrejas, G., Poeta, P., 2013. Genomic description of

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antibiotic resistance in escherichia coli and enterococci isolates from healthy lusitano horses. J. Equine Vet. Sci. 33, 1057–1063. https://doi.org/10.1016/j.jevs.2013.04.002 Ng, L.K., Martin, I., Alfa, M., Mulvey, M., 2001. Multiplex PCR for the detection of tetracycline resistant genes. Mol. Cell. Probes 15, 209–215. https://doi.org/10.1006/mcpr.2001.0363 Pruden, A., Larsson, D.G.J., Amézquita, A., Collignon, P., Brandt, K.K., 2013. Management of Options for Reducing the Release of Antibiotics 878, 878–885. https://doi.org/10.1289/ehp.1206446 Ribeiro, V.B., Lincopan, N., Landgraf, M., Franco, B.D.G.M., Destro, M.T., 2011.

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Journal Pre-proof Characterization of class 1 integrons and antibiotic resistance genes in multidrugresistant salmonella enterica isolates from foodstuff and related sources. Brazilian J. Microbiol. 42, 685–692. https://doi.org/10.1590/S1517-83822011000200033 Scott Weese, J., 2008. Antimicrobial resistance in companion animals. Anim. Health Res. Rev. 9, 169–176. https://doi.org/10.1017/S1466252308001485 Stine, O.C., Johnson, J.A., Keefer-Norris, A., Perry, K.L., Tigno, J., Qaiyumi, S., Stine, M.S.,

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Morris, J.G., 2007. Widespread distribution of tetracycline resistance genes in a confined

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animal feeding facility. Int. J. Antimicrob. Agents 29, 348–352.

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Sundin, G.W., Bender, C.L., 1996. Dissemination of the strA-strB streptomycin-resistance genes among commensal and pathogenic bacteria from humans, animals, and plants. Mol. Ecol.

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Thong, K.L., Modarressi, S., 2011. Antimicrobial resistant genes associated with Salmonella from retail meats and street foods. Food Res. Int. 44, 2641–2646.

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https://doi.org/10.1016/j.foodres.2011.05.013

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Tonu, N., Sufian, M., Sarker, S., Kamal, M., Rahman, M., Hossain, M., 2011. Pathologicalstudy on Colibacillosis in Chickens and Detection of Escherichia Coli By Pcr. Bangladesh J. Vet. Med. 9, 17–25. https://doi.org/10.3329/bjvm.v9i1.11205 van Duijkeren, E., van Asten, A.J.A.M., Gaastra, W., 2000. Characterization of Escherichia coli isolated from adult horses with and without enteritis. Vet. Q. 22, 162–166. https://doi.org/10.1080/01652176.2000.9695048 Wassenaar, T.M., 2005. Use of antimicrobial agents in veterinary medicine and implications for human health. Crit. Rev. Microbiol. 31, 155–169.

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Journal Pre-proof https://doi.org/10.1080/10408410591005110 Wiegand, I., Hilpert, K., Hancock, R.E.W., 2008. Agar and broth dilution methods to determine the minimal inhibitory concentration (MIC) of antimicrobial substances. Nat. Protoc. 3, 163–175. https://doi.org/10.1038/nprot.2007.521 Woolhouse, M., Ward, M., Van Bunnik, B., Farrar, J., 2015. Antimicrobial resistance in humans, livestock and the wider environment. Philos. Trans. R. Soc. B Biol. Sci. 370.

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https://doi.org/10.1098/rstb.2014.0083

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Figure captions

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Figure 1: Percentage of antimicrobial resistance of E. coli isolates using disc diffusion

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method against 15 antimicrobial agents, *: statistically significant at P≤0.05 (two-sided

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test). data from the three sources are shown as follows, blue bars represent both horse’s group, red bars represent hospitalized group and green bars represent non-hospitalized

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group.

Figure 2: Percentage of antimicrobial resistance of E. coli isolates by minimum

ur

inhibitory concentration (MIC) test, * : statistically significant at P≤0.05 (two-sided

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test).data from the three sources are shown as follows, blue bars represent both horse’s group, red bars represent hospitalized group and green bars represent non-hospitalized group.

Figure 3: Percentage of antimicrobial resistance-genes of E. coli isolates detected by PCR, * : statistically significant at P≤0.05 (two-sided test). data from the three sources are shown as follows, blue bars represent both horse’s group, red bars represent hospitalized group and green bars represent non-hospitalized group.

22

Journal Pre-proof

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Fig. 1

100%

*

60%

*

*

lP

40%

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ur

na

20%

0%

*

re

80%

-p

120%

Both Hors e’s Groups

Hospitalized Group

23

Non-hospitalized Group

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ro

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Fig. 2

-p

120

re

100 80

*

lP

60

ur Jo

0

na

40 20

*

Both Hors e’s Groups

Hospitalized Group

24

Non-hospitalized Group

*

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-p

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Fig. 3

100%

re

90% 80%

*

lP

70% 60% 50%

10% 0%

ur

20%

*

Jo

30%

na

40%

Both Hors e’s Groups

Hospitalized Group

25

Non-hospitalized Group

*

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Journal Pre-proof

26

Journal Pre-proof Table 1: Percentage and number of antimicrobial resistant E. coli isolates from equine fecal samples using disc diffusion method.

Classification

β – lactams β - lactamase inhibitors Cephalosporins Tetracyclines Trimethoprim Fluoroquinolones Aminoglycosides

Macrolides Nitroimidazole

Disc content (μg)

Antimicrobials (abbreviations)

Amoxicillin (AX) Ampicillin (AMP) Amoxicillin clavulanic acid (AML) ª Ceftiofur (EFT) Cefotaxime (CTX) Doxycycline (DO) ª Oxytetracycline (OT) ª Trimethoprim (TMP) ª Enrofloxacin (ENR) Gentamicin (CN) Amikacin (AK) Streptomycin (S) Erythromycin (E) Azithromycin (AZM) * Metronidazole (MET)

% & Number of resistant E. coli isolates

100% (n=23) 65.2% (n=15)

31% (n=32)

47.8% (n=11)

26.3% (n=21)

0.048

0% (n=0) 39.8% (n=41) 53.4% (n=55)

0% (n=0) 47.8% (n=11) 87% (n=20)

0% (n=0) 37.5% (n=30) 43.8% (n=35)

-ᶜ 0.372 0.0003 ᵇ

≥ 19

38.8% (n=40)

82.6% (n=19)

26.3% (n=21)

0.000 ᵇ

S

Both Horse’s Groups (n=103)

25 μg 10 μg

≤ 22 ≤ 13

≥ 31 ≥ 17

100 % (n=103) 49.5% (n=51)

20/10 μg

≤ 13

≥ 18

30 μg 30 μg 30 μg

≤ 17 ≤ 23 ≤ 12

≥ 21 ≥ 30 ≥ 16

30 μg

≤ 14

l a

rn

Pvalue (X²) **

Nonhospitalized Group (n=80) 100% (n=80) 45% (n=36)

R

r P

f o

o r p

e

Hospitalized Group (n=23)

-ᶜ 0.087

5 μg

≤ 15

≥ 18

51.5 (n=53)

78.3% (n=18)

43.8% (n=35)

0.003

5 μg 10 μg 30 μg 10 μg 15 μg

≤ 15 ≤ 12 ≤ 15 ≤ 11 ≤ 13

≥ 21 ≥ 15 ≥ 18 ≥ 15 ≥ 18

6.8% (n=7) 10.7% (n=11) 0% (n=0) 91.3% (n=94) 100% (n=103)

30.4% (n=7) 21.7% (n=5) 0% (n=0) 82.6% (n=19) 100% (n=23)

0% (n=0) 7.5% (n=6) 0% (n=0) 93.8 (n=75) 100% (n=80)

-ᶜ 0.051 -ᶜ 0.095 -ᶜ

15 μg

≤ 14

≥ 19

1.9% (n=2)

8.7% (n=2)

0% (n=0)

-ᶜ

5 μg

≤ 21

-

100% (n=103)

100% (n=23)

100% (n=80)

-ᶜ

u o

J

Disc diffusion interpretive criteria (mm)

*To date, no clinical breakpoint for resistant in E. coli against azithromycin has been established. Instead, a halo diameter <12 mm is considered as the azithromycin resistant breakpoint in Enterobacteriaceae based on S. Typhi microorganism (Gomes et al., 2019). R: resistant isolates, S: sensitive isolates, **P-value: Chi-square value for the difference between hospitalized and the non-hospitalized 27

Journal Pre-proof resistant isolates (R), a: statistically significant at P≤0.05 (two-sided), b: Fischer-Exact test was performed instead of (X²) when variables had expected count less than 5 in one or more cells, c: Chi-square was not performed when there was zero number of samples.

f o

l a

e

o r p

r P

n r u

Jo

28

Journal Pre-proof Table 2: Minimal inhibitory concentration (MIC) test results and comparison between the hospitalized and the non-hospitalized horses for E. coli isolates, CLSI (2013).

Antimicrobial agents

MIC interpretive criteria

(%) and Number of E. coli isolates Both Horse’s Groups (n=103)

S

I

R

R

I

S

R

Streptomycin

≤ 16

-

≥ 32

87.4% (n=90)

1.9% (n=2)

10.7% (n=11)

82.6% (n=19)

Trimethoprimª

≤2

4 ≥ 16

42.7% (n=44)

3.9% (n=4)

53.4% (n=55)

≥8

85.4% (n=88)

14.6% (n=15) 12.6% (n=13)

Erythromycin

≤ 0.5

4

f o

Hospitalized Group (n=23)

o r p

0% (n=0)

60.9% (n=14)

(R+I)

I

S

17.4% (n=4)

88.8% (n=71)

2.5% (n=2)

8.8% (n=7)

0.258ᵇ

4.3% (n=1)

34.8% (n=8)

37.5% (n=30)

3.8% (n=3)

58.8% (n=47)

0.001

78.3% (n=18)

21.7% (n=5)

0% (n=0)

87.5% (n=70)

12.5% (n=10)

0% (n=0)

-ᶜ

rn

60.2% (n=62)

39.1% (n=9)

17.4% (n=4)

43.5% (n=10)

23.8% (n=19)

11.3% (n=9)

65% (n=52)

0.631

l a

S

P-value (X²) **

R

0% (n=0)

I

Non - Hospitalized Group (n=80)

e

r P

Ampicillin

≤8

16 ≥ 32

27.2% (n=28)

Amoxicillin

≤8

16 ≥ 32

35.9% (n=37)

64.1% (n=66)

0% (n=0)

73.9% (n=17)

26.1% (n=6)

0% (n=0)

25% (n=20)

75% (n=60)

0% (n=0)

-ᶜ

Doxycyclineª

≤4

8 ≥ 16

47.6% (n=49)

2.9% (n=3)

49.5% (n=51)

87% (n=20)

0% (n=0)

13% (n=3)

36.3% (n=29)

3.8% (n=3)

60% (n=48)

0.001ᵇ

J

u o

26.2% 10.7% 63.1% 78.3% 4.3% 17.4% 11.3% 12.5% 76.3% 0.001ᵇ (n=27) (n=11) (n=65) (n=18) (n=1) (n=4) (n=9) (n=10) (n=61) R: resistant isolates, S: sensitive isolates, I: intermediate isolates, **P-value: Chi-square value for the difference between hospitalized Oxytetracyclineª

≤4

8 ≥ 16

and the non-hospitalized resistant isolates (R+I), a: statistically significant at P≤0.05 (two-sided), b: Fischer-Exact test was performed

29

Journal Pre-proof instead of (X²) when variables had expected count less than 5 in one or more cells, c: Chi-square was not performed when there was zero number of samples.

f o

l a

e

o r p

r P

n r u

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30

Journal Pre-proof Table 3: Comparison of polymerase chain reaction PCR test results for antimicrobial-resistant genes in E. coli isolates between hospitalized and non-hospitalized horse’s groups of the study.

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Prevalence (%) and No. of isolates AntimicrobialBoth Antimicrobial P-value Nonresistance horse’s Hospitalized class (X²) ** hospitalized genes groups group (n=23) group (n=80) (n=103) 15.5% blaTEM 17.4% (n=4) 15% (n=12) 0.751 ᵇ (n=16) 10.7% β-lactamases blaSHV 13% (n=3) 10% (n=8) 0.705 ᵇ (n=11) 30.1% blaCTX-M 34.8% (n=8) 28.8% (n=23) 0.578 (n=31) 25.2% tetA ª 69.6% (n=16) 12.5% (n=10) 0.000 (n=26) Tetracyclines 17.5% tetB 17.4% (n=4) 17.5% (n=14) 1.00 ᵇ (n=18) aac(3)-IIa 5.8% (n=6) 8.7% (n=2) 5% (n=4) 0.613 ᵇ aac(6’) 0% (n=0) 0% (n=0) 0% (n=0) -ᶜ aadB 1.9% (n=2) 8.7% (n=2) 0% (n=0) -ᶜ 84.5% aadA 73.9% (n=17) 87.5% (n=70) 0.112 Aminoglycosides (n=87) 89.3% strA 82.6% (n=19) 91.3% (n=73) 0.258 ᵇ (n=92) 85.4% strB 78.3% (n=18) 87.5% (n=70) 0.268 (n=88) 19.4% dfr1 ª 34.8% (n=8) 15% (n=12) 0.034 (n=20) Trimethoprim dfrA12, dfrA13 7.8% (n=8) 8.7% (n=2) 7.5% (n=6) 1.00 ᵇ 22.3% dfrA7, dfrA17 ª 39.1% (n=9) 17.5% (n=14) 0.004 ᵇ (n=23) **P-value: Chi-square value for the difference between the prevalence of antimicrobialresistant genes ARGs in hospitalized

and the non-hospitalized

groups, a: statistically

significant at P≤0.05 (two-sided), b: Fischer-Exact test was performed instead of (X²) when variables had expected count less than 5 in one or more cells, c: Chi-square was not performed when there was zero number of samples.

31

Journal Pre-proof Table 4: Univariate analysis of potential risk factors associated with the presence of E. coli antimicrobial-resistance genes of the study.

blaTEM ª

Coding

Last treatment Supportive treatment, Antimicrobial therapy, anti-inflammatories, and others No treatment Breed Arabian & Arabian mix + Local breed & mix breed Warmblood & English (Thoroughbred) Last treatment Supportive treatment, Antimicrobial therapy, anti-inflammatories, and others No treatment Group Hospitalized Nom-hospitalized Breed Arabian & Arabian mix + Local breed & mix breed

-p

re

lP

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tetA ª

ur

na

Warmblood & English (Thoroughbred) Use Breeding & beauty show Working animals Pleasure, riding + Sport (Polo, racing and jumping) Practice management Stables Backyard, owned farms and others Type of diet Manufactured die / Processed feed Natural feed Last illness Colic, diarrhea and ulcers and (parasitism Dental problems) Musculoskeletal and nervous system (lameness, laminitis, limb injuries, skin conditions, nervous signs, ataxia, incoordination) and respiratory and ocular disease No illness Last treatment Supportive treatment, Antimicrobial therapy, anti-inflammatories, and others No treatment Administration of antimicrobials Yes

tetA ª

32

No. of No. of Positive PTotal samples harboring value samples the resistant genes (X²)

1

39

10 (25.6%)

0

64

6 (9.3%)

2

42

19 (45.2%)

0.027

0.010

1

61

13 (21.3)

1

39

17 (43.5)

0

64

15 (23.4%)

1 2

23 80

18 (78.2%) 11 (13.7%)

0.001

2

42

25 (59.5%)

0.001

1

61

4 (6.5%)

1 2

25 23

5 (20%) 18 (78.2%)

3

55

6 (10.9%)

1 2

23 80

18 (78.2%) 11 (13.7%)

0.001

1 2

49 54

5 (10.2%) 24 (44.4%)

0.001

1

18

5 (27.7%)

2

20

11 (55%)

0

65

13 (20%)

1

39

17 (43.6%)

0

64

22 (34.4%)

1

14

10 (71.4%)

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blaCTX-bM ª

Risk Factors

of

Outcome Variables (AntimicrobialResistant Genes)

0.032

0.001

0.010

0.007

0.001

Journal Pre-proof

aadA ª

strB ª

89

19 (21.3%)

1 2

64 39

15 (23.4%) 2 (5.1%)

0.015

1 2

23 80

8 (34.7%) 12 (15%)

0.035

1 2

23 80

8 (34.7%) 12 (15%)

0.035

1 2

23 80

9 (39.1%) 14 (17.5%)

0.028

1 2

23 80

9 (39.1%) 14 (17.5%)

0.028

1 2

49 54

45 (91.8%) 39 (72.2%)

0.010

1 2

49 54

47 (96%) 41 (76%)

0.004

of

dfrA17 ª

0

ro

dfr1 ª

-p

tetB ª

No Location Amman Others (Irbid, Jerash, Az Zarqaa') Group Hospitalized Nom-hospitalized Practice management Stables Backyard, owned farms and others Group Hospitalized Nom-hospitalized Practice management Stables Backyard, owned farms and others Type of diet Manufactured die / Processed feed Natural feed Type of diet Manufactured die / Processed feed Natural feed

re

** P-value: Chi-square value for potential risk factor and prevalence of antimicrobial-resistant

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genes ARGs, a: statistically significant at P≤0.05 (two-sided).

33

Journal Pre-proof Table 5: Final multivariate logistic regression model of antimicrobial-resistant genes tetA and associated with risk factors in equine fecal E. coli isolates of the study.

Outcome Variables (AntimicrobialResistant Genes)

No. of Positive samples harboring the resistant genes (%)

Risk Factors

Pvalue (X²)

OR Lower Upper

of

Breed Arabian & Arabian mix + Local breed & mix breed

OR 95% CI

ro

25 (59.5%)

Warmblood & English 4 (6.5%) (Thoroughbred) Type of diet Manufactured die / 5 (10.2%) 7.809 2.202 27.692 0.001 Processed feed Natural feed 24 (44.4%) Hosmer and Lameshow goodness of fit test p = 0.828, 95% CI; 95% confidence intervals, OR;

lP

re

-p

tetA

22.503 6.261 80.883 0.001

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odd ratio.

34

Journal Pre-proof Supplementary materials

Table S1. PCR target genes, primer sequence, PCR product size and annealing temperature. Classification

Targe t genes

Primer sets MultiT SO-T _f

Annealing

size (bp)

Temp.

800 bp

57

713 bp

57

(F) CAT T T CCGT GT CGCCCT T AT T C

blaTEM MultiT SO-T _r

(R) CGT T CAT CCAT AGT T GCCT GAC

MultiT SO-S_f

(F) AGCCGCT T GAGCAAAT T AAAC

blaSHV MultiT SO-S_r

(R) AT CCCGCAGAT AAAT CACCAC

blaCTX-M f

(F) CGCT T T GCGAT GT GCAG

ro

blaCTX-M blaCTX-M r

(Dallenne et al., 2010)

550 bp

(Gangoué-Piéboji et 49

(R) ACCGCGAT AT CGT T GGT

al., 2005)

-p

(F) GT GGAT GGCGGCCT GAAGCC-3 aadA

aadA

Re fe rences (Dallenne et al., 2010)

of

Beta–lactams

PCR product Primer Sequence 5'-3'

(Ribeiro et al., 2011) 526 bp

62

700 bp

54

546

55

509

55

439 bp

55

(R) AT T GCCCAGT CGGCAGCG-3

re

(F) T CCAGAACCT T GACCGAAC aadB

aadB

(Ribeiro et al., 2011)

(R) GCAAGACCT CAACCT T T T CC

lP

(F) CCT GGT GAT AACGGCAAT T C strA

strA

(Lanz et al., 2003)

(R) CCAAT CGCAGAT AGAAGGC Aminoglycosides strB

na

(F) AT CGT CAAGGGAT T GAAACC strB

(Lanz et al., 2003)

(R) GGAT CGT AGAACAT AT T GGC

aac(3)-11a

(F) CGGCCT GCT GAAT CAGT T T C

ur

aac(3)-11a

(T hong and

(R) AAAGCCCACGACACCT T CT C

tetA

aac(6’)

Jo

aac(6’)

Modarressi, 2011)

(F) T T GGACGCT GAGAT AT AT GA

(T hong and 476 bp

55

(R) GCT CCT T T T CCAGAAT ACT T

Modarressi, 2011)

(F) GCT ACA T CC T GC T T G CCT T C

tetA

(Ng et al., 2001) 210 bp

55

659 bp

55

254

58

(R) CAT AGA T CG CCG T GA AGA GG T etracyclines (F) T T G GT T AGG GGC AAG T T T T G tetB

tetB

(Ng et al., 2001)

(R) GT A AT G GGC CAA T AA CAG CG (F) ACGGAT CCT GGCT GT T GGT T GGACGC dfr1

dfr1

(Gibreel and Sköld,

(R) CGGAAT T CACCT T CCGGCT CGAT GT C

1998)

(F) GT CGCCCT AAAACAAAGT T A T rimethoprim

dfrA7, dfrA17

dfrA7, dfrA17

(Lee et al., 2001) 195

51

485

58

(R) CGCCCAT AGAGT CAAAT GT dfrA12,

(F) CCGT GGGT CGAT GT T T GAT G dfrA12, dfA13

dfrA13

(R) GCAT T GGGAAGAAGGCGT T CAC

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(Lee et al., 2001)

Journal Pre-proof Table S2. Resistance profiles of E. coli isolates and the proportion of isolates resistant by disc diffusion method, which were confirmed by MIC from the equine fecal sample in all 103 samples.

103 51

100% 49.5%

32

31%

103 41

100% 80.3%

NA

NA

0% 39.8% 53.39% 38.8% 51.4% 6.8% 10.6% 0% 91.2% 100%

NA NA 52 38 48 NA NA NA 92 103

NA NA 94.5% 95% 90.5% NA NA NA 97.9% 100%

2

1.9%

NA

NA

103

100%

NA

NA

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0 41 55 40 53 7 11 0 94 103

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NA: not applicable

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Amoxicillin (AX) Ampicillin (AMP) Amoxicillin β - lactamase clavulanic acid inhibitors (AML) Ceftiofur (EFT) Cephalosporins Cefotaxime (CTX) Doxycycline (DO) Tetracyclines Oxytetracycline (OT) Trimethoprim Trimethoprim (TMP) Fluoroquinol ones Enrofloxacin (ENR) Gentamicin (CN) Aminoglycosides Amikacin (AK) Streptomycin (S) Erythromycin (E) Macrolides Azithromycin (AZM) Metronidazole Nitroimidazole (MET) β - lactams

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Antimicrobial agents

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Antimicrobial class

No. of E. coli No. of E. coli The proportion of isolates isolates exhibiting Disc a resistant isolates resistant by disc diffusion diffusion phenotype resistant by method, which were method (% ) (Disk diffusion MIC test confirmed by MIC method)

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Journal Pre-proof Table S3. Summary of results showing the identification and distribution of antimicrobial resistant genes in E. coli isolates taken from fecal samples of hospitalized and non-hospitalized horses.

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Antibiotic (No. of % and (No. of Antibioticresistant isolates Not Source and number (n) isolates resistant gene investigated by MIC identified of resistant isolates identified by composition tested from both horses’ by PCR PCR) by PCR groups) Hospitalized (n=13) 53.8% (n=7) (n=6) Ampicillin (n=41) Non-hospitalized (n=28) 57.1% (n=16) (n=12) TEM, SHV βlactamase genes Hospitalized (n=23) 30.4% (n=7) (n=16) Amoxicillin (n=103) Non-hospitalized (n=80) 20% (n=16) (n=64) Hospitalized (n=11) 72.7% (n=8) (n=3) CTX-M β-lactamase Cefotaxime (n=41) * gene Non-hospitalized (n=30) 76.6% (n=23) (n=7) Hospitalized (n=20) 90% (n=18) (n=2) Doxycycline (n=52) tetA, tetB Non-hospitalized (n=32) 75% (n=24) (n=8) Oxytetracycline Hospitalized (n=19) 84.2% (n=16) (n=3) tetA (n=38) Non-hospitalized (n=19) 52.6% (n=10) (n=9) Hospitalized (n=15) 86.6% (n=13) (n=2) dfr1, dfrA12, Trimethoprim (n=48) Non-hospitalized (n=33) 84.8% (n=28) (n=5) dfrA17 Hospitalized (n=19) 100% (n=19) (n=0) Streptomycin (n=92) aada, strA, strB Non-hospitalized (n=73) 100% (n=73) (n=0) Hospitalized (n=5) 80% (n=4) (n=1) Gentamicin (n=11) * aac(3)-IIa & aadB Non-hospitalized (n=6) 66.6% (n=4) (n=2) * All data are based on Minimal Inhibitory Concentration (MIC) test results, except Cefotaxime

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and Gentamicin the data were based on the disk diffusion method.

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Journal Pre-proof

Figure S1: Electrophoresis analysis using 1.5 agarose gel of PCR results for detection of ESβLs producing E. coli isolates, amplified by specific primers, A: blaTEM (800 bp), B: blaSHV (713 bp), C: blaCTX-M (550 bp), where L 100bp DNA ladder; -ve is negative control; +ve is positive

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control; 1-3 lanes; E. coli isolates.

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Journal Pre-proof

Figure S2: Electrophoresis analysis using 1.5 agarose gel of PCR results for detection of tetracycline-resistance genes in E. coli isolates, amplified by specific primers, A: tetA(210 bp), B: tetB (659 bp), where L 100bp DNA ladder; -ve is negative control; +ve is positive control; 1-

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3 lanes; E. coli isolates.

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Journal Pre-proof

Figure S3: Electrophoresis analysis using 1.5 agarose gel of PCR results for detection of trimethoprim-resistance genes in E. coli isolates, amplified by specific primers, A: dfr1 (254 bp), B: dfrA12 (485 bp), C: dfrA17 (195 bp), where L 100bp DNA ladder; -ve is negative control;

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+ve is positive control; 1-3 lanes; E. coli isolates.

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Journal Pre-proof

Figure S4: Electrophoresis analysis using 1.5 agarose gel of PCR results for detection of aminoglycoside-resistance gene in E. coli isolates, amplified by specific primer, A: aac(3)-IIa (439 bp), where L 100bp DNA ladder; -ve is negative control; +ve is positive control; 1-3 lanes;

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E. coli isolates.

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Journal Pre-proof

Figure S5: Electrophoresis analysis using 1.5 agarose gel of PCR results for detection of aminoglycoside-resistance genes in E. coli isolates, amplified by specific primer, A: aadA (526 bp), B: aadB (700 bp), C: strA (546 bp), D: strB (509 bp), where L 100bp DNA ladder; -ve is

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negative control; +ve is positive control; 1-3 lanes; E. coli isolates.

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Journal Pre-proof Author contributions Mohammad Gharaibeh: Conceptualization, Methodology, Writing - Review & Editing Sameeh M. Abutarbush: Conceptualization, Resources, Writing - Review & Editing Farah Mustafa: Methodology, Formal analysis, Investigation, Writing - Original Draft Shawkat Hailat: Formal

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analysis, Methodology Motasem S. Halaiqa: Methodology, Investigation

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Journal Pre-proof Highlights 

The prevalence of antimicrobial resistance is significantly higher in hospitalized horses



Aminoglycoside resistant genes (strA, strB, and aadA) are the most prevalent antimicrobial-resistant genes



Last treatment and history of antimicrobial administration, breed of horses, use of horses,

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type of diet fed for horses, practice management and history of last illness are important risk factors

TetA is 22 times more likely to be found in the Arabian and local breeds of horses

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TetA is 8 times more likely to be found in horses that were fed a natural diet compared to

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other horses that were fed manufactured/ processed feed.

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compared to English and warmblood breed.

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

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Figure 1

Figure 2

Figure 3