Microarray-based detection of resistance and virulence factors in commensal Escherichia coli from livestock and farmers in Egypt

Journal Pre-proof Microarray-based detection of resistance and virulence factors in commensal Escherichia coli from livestock and farmers in Egypt Mayada Gwida, Amal Awad, Maged El-Ashker, Helmut Hotzel, Stefan Monecke, Ralf Ehricht, Elke Muller, ¨ Annett Reißig, Stefanie A. Barth, Christian Berens, Sascha D. Braun

PII:

S0378-1135(19)30751-5

DOI:

https://doi.org/10.1016/j.vetmic.2019.108539

Reference:

VETMIC 108539

To appear in:

Veterinary Microbiology

Received Date:

25 June 2019

Revised Date:

26 November 2019

Accepted Date:

28 November 2019

Please cite this article as: Gwida M, Awad A, El-Ashker M, Hotzel H, Monecke S, Ehricht R, Muller ¨ E, Reißig A, Barth SA, Berens C, Braun SD, Microarray-based detection of resistance and virulence factors in commensal Escherichia coli from livestock and farmers in Egypt, Veterinary Microbiology (2019), doi: https://doi.org/10.1016/j.vetmic.2019.108539

This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier.

Original article Microarray-based detection of resistance and virulence factors in commensal Escherichia coli from livestock and farmers in Egypt

Mayada Gwida1, Amal Awad2, Maged El-Ashker3, Helmut Hotzel4, Stefan Monecke5, 6, Ralf Ehricht5, 6,8, Elke Müller5, 6, Annett Reißig5, 6, Stefanie A. Barth7, Christian Berens7,

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Sascha D. Braun5, 6*

Department of Hygiene and Zoonoses, Faculty of Veterinary Medicine, Mansoura University,

35516 Mansoura, Egypt

Department of Bacteriology, Mycology and Immunology Faculty of Veterinary Medicine,

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Mansoura University, 35516 Mansoura, Egypt

Department of Internal Medicine and Infectious Diseases, Faculty of Veterinary Medicine,

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Friedrich-Loeffler-Institut,

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Mansoura University, 35516 Mansoura, Egypt Institute

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Bacterial

Infections

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Zoonoses, 07743 Jena, Germany

Leibniz Institute of Photonic Technology (IPHT), 07745 Jena, Germany

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INFECTOGNOSTICS Research Campus, 07745 Jena, Germany

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Friedrich-Loeffler-Institut, Institute of Molecular Pathogenesis, 07743 Jena, Germany

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Friedrich Schiller University Jena; Institute of Physical Chemistry, Jena, Germany

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Corresponding author details Dr. Sascha D. Braun; [email protected] Tel: +493641948389; Fax: +493641948302

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Highlights we collected 132 fecal samples from healthy farm animals and its contact farmers

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47 E. coli were isolated using standard methods

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resistance and virulence genes were analyzed by microarray-based assays

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20 isolates harboring several resistance genes were defined as multidrug-resistant

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6 isolates were identified as STEC harboring allelic variants of stx1 and stx2 genes

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we found evidence for transmission identifying one clone in both, farmer and buffalo

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Abstract

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The objective of our study was to provide a molecular analysis using DNA-microarray based assays of commensal E. coli populations from apparently healthy livestock and their attendants

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to assess the virulence potential as well as multidrug resistance (MDR) genotypes.

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We randomly collected 132 fecal samples from seemingly healthy smallholder´s food producing animals [buffalo (n = 32) and cattle (n = 50)] as well as from contacting farmers

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(n = 50). Bacterial isolation and identification were performed using standard protocols, while E. coli isolates were characterized using a DNA microarray system targeting 60 different

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virulence and 47 antibiotic resistance genes of clinical importance and allowing assignment to most common H and O types.

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From the fecal samples examined, 47 E. coli isolates were obtained. The array predicted serotypes for 14 out of the 47 E. coli isolates. Six E. coli isolates were identified as STEC since Shiga toxin genes were detected. In summary, 36 different virulence genes were identified; of which, hemL, lpfA and iss were most prevalent. Thirty-four E. coli isolates were found to carry at least one antimicrobial resistance gene. Of these, 20 did exhibit genes allowing strain classification as MDR. More than half of the isolates contained antimicrobial resistance genes 2

associated with beta lactam resistance 27/47 (57.5%). The 13 remaining isolates did not contain any resistance gene tested with the array. Our study demonstrated the presence of antimicrobial resistance genes and virulence genotypes among commensal E. coli of human and animal sources.

Keywords: MDR, Genetic diversity, Escherichia coli, human feces, livestock animals,

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Egypt, STEC

1. Introduction

Escherichia coli is a commensal bacterium that inhabits the alimentary tract of humans

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and most warm-blooded animals. The majority of E. coli strains remain harmless. However,

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when the gastrointestinal barriers are damaged, even such strains can cause infections. Among the enteric pathogens of worldwide major concern are Shiga toxin-producing Escherichia coli

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(STEC). STEC emerged as foodborne pathogens of increasing importance associated with life threatening syndromes, including hemorrhagic colitis (HC) and hemolytic uremic syndrome

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(HUS). Ruminants usually carry STEC for long periods of time and, thus, are considered to represent important reservoirs (Baranzoni et al., 2016). Animal feces are a major source of

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human exposure to such pathogens resulting in public health concern (Rice et al., 2006). In Egypt, small-scale livestock farming is common (Aidaros, 2005). Here the close

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proximity between the owners and their animals and the lack of an infrastructure to handle animal waste appropriately is an issue. Animal feces are commonly used as a fertilizer for crops and pastures providing effective and economic means of reintroducing organic matter to the soil to maintain its quality and fertility (Nicholson et al., 2000). They are also considered a vital source of solid fuel that can be used for cooking, heating and for biogas production (Amon et al., 2007). Hence, handling of untreated animal manure for agricultural purposes is considered 3

to be a primary risk factor for the transmission and spread of different foodborne diseases (Oliver et al., 2005). Another issue is an alarming increase in the rate of antimicrobial resistant (AMR) bacteria (ECDC, 2019). This could be attributed to an overuse of antibiotics in human and veterinary medicine and farming. Successful treatment of infections with resistant bacteria is becoming more and more difficult (Andersson, 2003). This situation is aggravated by a possible transfer of AMR bacteria between food producing animals and humans, either through direct

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contact or by consumption of contaminated food (Boxall et al., 2003). According to EFSA (EFSA, 2009), the prevalence of AMR among commensal E. coli from food producing animals should be regularly monitored. However, the co-existence of

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resistance genes and the ability of infectious agents to colonize the human intestine are not adequately considered. Currently, various laboratory methods are used to identify antimicrobial

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resistance and virulence genes harbored by bacteria such as E. coli. Among the most

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informative methods, DNA microarray technology emerges as an available, comparable, economic (compared to NGS) and effective tool due to its ability to screen for multiple markers simultaneously (Batchelor et al., 2008). The objectives of our study were to analyze the gene

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content of commensal E. coli populations from apparently healthy livestock and their attendants

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to assess the virulence potential and antibiotic resistance patterns using DNA microarrays.

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2. Material and Methods 2.1. Ethic Statement

All procedures were performed in accordance with the principles and specific guidelines

presented in the Guidelines for the Care and Use of Agricultural Animals in Research and Teaching (3rd ed.; http://www.fass.org/) and those of Mansoura University Animal Care. The study was approved by the Ethics Committee of Mansoura University. Informed consent for E.

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coli investigations on domestic animals was given by the owners and consent was also given by human participants. 2.2. Study Population In the present study, nearly ten grams of fecal sample were collected directly from each rectum of 82 seemingly healthy farm animals (cattle; n=50, buffalo; n=32) during May 2016. Furthermore, stool samples from contacting farmers (n=50) were taken in parallel by

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themselves using sterile containers. These containers were then collected from 15 rural-based farming communities at Meet EL-Amel village, Aga District in Dakahlia Governorate, Egypt. All samples were aseptically collected in sterile plastic cups and transferred into individual sterile bags to be transported, via insulated coolers containing cold packs, to the laboratory of

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Hygiene and Zoonoses, Faculty of Veterinary Medicine, Mansoura University for immediate

2.3. Bacterial Isolation and Resistance Phenotyping

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bacteriological processing.

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The procedure of bacterial isolation and identification was performed using standard protocols as defined in ISO 7251:2005 (ISO, 2005). Briefly, E. coli was isolated by adding 1 g

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of fecal sample to 9 ml of sterile normal saline. An aliquot of 100 μl was plated onto Eosin Methylene Blue (EMB) agar (Oxoid, Basingstoke, Hampshire, UK) and incubated for 24 h at

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37°C. Typical shiny colonies were then sub-cultured on blood agar (Oxoid, Basingstoke,

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Hampshire, UK) and incubated at 37°C for 24 h. The purified strains were further identified using Gram-staining and tested for biochemical reactions with API 20E (bioMérieux, Marcy l'Etoile, France). Colonies were stored in 30% sterile glycerol at -20°C until shipping to the Friedrich-Loeffler-Institut, Institute of Molecular Pathogenesis, Jena, Germany, for further genotyping using a DNA microarray based assay. The determination of the resistance phenotype of all isolates was performed using the VITEK2 system (bioMérieux, Nürtingen, Germany). Briefly, isolates were cultivated on tryptone yeast 5

agar and subsequently re-tested using an automated micro-dilution technique (VITEK-2, card: AST-248) that included the following antibiotics, imipenem, meropenem, cefepime, cefotaxime,

ceftazidime,

piperacillin,

piperacillin/tazobactam,

aztreonam,

amikacin,

gentamicin, tobramycin, ciprofloxacin, moxifloxacin, co-trimoxazole, tigecycline, fosfomycin and colistin. The criteria for the interpretation of the antimicrobial susceptibility testing was according to EUCAST or CLSI breakpoints determined automatically by the VITEK-2 Expert software (bioMerieux, VITEK-2 system version: 07.01, MIC guidelines: EUCAST 2014 +

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CLSI 2014 D). 2.4. DNA Extraction

The genomic DNA was extracted from overnight cultures at 37°C in lysogeny broth

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(LB) media using the High Pure PCR Template Preparation Kit (Roche Diagnostics,

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Mannheim, Germany) according to the manufacturer’s instructions. The concentration of DNA was determined photometrically using a Nano Drop ND-1000 UV-VIS spectrophotometer

Wilmington, DE, USA).

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according to manufacturer’s instructions at 260 / 280 nm (Nano-Drop Technologies,

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2.5. Characterization of Escherichia coli using a DNA Microarray based assay Genotyping was performed using the E. coli Pantype AS-1 kit [Abbott (Alere

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Technologies GmbH), Jena, Germany] according to the manufacturer´s instructions. This kit

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identifies 60 virulence genes (Anjum et al., 2007) and 47 resistance genes of clinical importance (Batchelor et al., 2008). It also facilitates DNA-based serotyping by targeting 24 of the epidemiologically most relevant O antigens and 47 different H antigens (Ballmer et al., 2007). A site specific labeling approach was used for labeling and biotinylation of the genomic DNA as previously published by (Monecke and Ehricht, 2005). Primer elongation, hybridization, washing, and staining of the array strips were performed as described earlier (Barth et al., 2017). Using the ShigaToxType AS-2 Kit, all stx genes detected were subtyped (Scheutz et al., 2012). 6

Array strips were automatically analyzed using an ArrayMate instrument [Abbott (Alere Technologies GmbH), Jena, Germany]. The intensities of mean signal (mean) and local background (lbg) were measured for each probe position after automated spot detection. All values were calculated by the following formula: v a lu e 

1  m ean lb g

Values below 0.1 were assigned as negative and above 0.3 were considered positive while

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values between 0.1 and 0.3 were regarded as ambiguous. The validation was performed using a collection of sequenced E. coli control strains [Gene Bank Accession numbers AE005174 (E. coli EDL933, O157:H7), FM180568 (E. coli E2348/69, O127:H6), U00096 (E. coli K-12 MG1655), AP009048 (E. coli K-12 W3110), CP000247 (E. coli 536, O6:K15:H31), CP001509

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2.6. Split network tree construction

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(E. coli BL21), AE014075 (E. coli CFT073, O6:K2:H1), and CP000946 (E. coli ATCC 8739)].

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A split network tree was used to visualize similarities (not phylogeny) between hybridization patterns. The results of all array hybridization experiments were arranged in a matrix where the columns represent the target genes and the rows represent the experiments.

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The hybridization results were converted into ‘sequences’ using ‘A’ for positive and ‘T’ for

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negative results. Thus, the matrix was converted into a series of ‘sequences’. These were used for tree construction using splits tree 4 (Huson and Bryant, 2006) software (characters

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transformation, uncorrected P; distance transformation, nearest neighbor; and variance, ordinary least squares). 3. Results

The distribution and frequency of virulence and antimicrobial resistance genes among all E. coli isolates from all sources were summarized (Supplementary Table 1). For 14 E. coli isolates, both, H and O types were identified. Two isolates from buffalo were assigned to 7

genoserotype O7:H21, one cattle isolate to O127:H40, while O6:H14 was found in five isolates (two from buffalo and three from humans). O103:H16 and O8:H21 were found twice (buffalo and cattle). Finally, O172:H23 was identified twice (in a buffalo and a human isolate). In the remaining isolates (n = 33), different flagellar genes were detected (Table 1). A total of 36 different virulence associated genes were identified among the E. coli isolates (Supplementary Table S1). Of all, hemL (glutamate-1-semialdehyde aminotransferase, 44/47 isolates), lpfA (fimbria adhesion gene, in 32/47 isolates) and iss (increased serum survival

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gene, 20/47 isolates) were found to be predominant among the recovered isolates. The rest of the virulence genes targeted by the array were only detected sporadically. Different genes belonging to SPATE (serin protease auto transporters), adhesion factors (lpfA, f17-A, f17-G,

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iha, saa), toxins (hylA, hylE, astA, cdtB, cnf1, cma, mchf, mcmA, mchB, mchC, celB, stx), and iron acquisition (iroN) or transport systems were sporadically present among the isolates (Table

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1).

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Six E. coli isolates harbored different alleles of the Shiga toxin genes 1 and 2: stx1a (HUM048), stx2a (BUF105), stx2b (CATT074), stx2c (CATT065, CATT074, CATT085 and CATT086), stx2d (CATT085 and BUF105) and stx2e (BUF105) (Table 1). These E. coli strains

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were categorized as Shiga toxin-producing E. coli (STEC). Six isolates (one of buffalo origin, two isolates from cattle and three from humans) contained the heat stable enterotoxin gene astA.

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Another six E. coli isolates (one from human and five isolate from animals) had the iha gene

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which is an important virulence factor of uropathogenic E. coli. Isolates belonging to the same serotype were nearly identical with respect to the virulence markers detected (Table 1). The distribution of antimicrobial resistance genes among the recovered E. coli isolates

is listed in the second part of Supplementary Table S1. Thirty-one different resistance genes and one integrase gene (intI1) known to confer antimicrobial resistance in E. coli were detected. Resistance determinants to several antibacterial classes occurred with different frequencies.

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Microarray results indicate the presence of different antimicrobial resistance genes of major importance, regardless of the isolate’s source. Twenty isolates (42.5%) carried more than one resistance gene (Table 1). Our findings showed that human and buffalo isolates carried more resistance genes compared to cattle isolates. More than half of the isolates contained genes conferring resistance to beta lactams (27/47; 57.5%). Genes associated with resistance to aminoglycosides 23/47 (48.9%), quinolones 17/47 (36.0%), tetracycline 17/47 (36.0%), chloramphenicol 5/47 (10.6%) and

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macrolides 2/47 (4.3%) were less frequent. Thirty-four isolates were identified as having at least one antimicrobial resistance gene. The thirteen remaining isolates (5 isolates from buffaloes, 4 from cattle and 4 from humans) did not harbor any resistance genes covered by the

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array. Multiple genes causing resistance towards the same class of antimicrobials were detected in most isolates (Table 1).

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The quinolone resistance genes were mostly present in buffalo and human isolates. The

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most frequently detected gene responsible for tetracycline resistance was tet(A) which was identified in 13/47 isolates (27.7%). The genes conferring resistance to beta-lactams were blaCMY (14.9%), blaMOX (19.1%), blaACT (31.9%), blaOXA-7 (10.7%), blaTEM (27.7%), blaSHV

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(10.2%) and blaCTX-M1/15 (4.3%).

The phenotypic analysis of all isolates showed that the detection of a resistance gene

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mostly resulted in the expected phenotype (Supplementary Table S2). Detection of genes like

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blaCTX-M1/15 or blaSHV corresponded to an ESBL phenotype. If only blaTEM was detected, the isolates were resistant only to piperacillin and therefore defined as a non-ESBL isolate. All ampC genes (blaACT or blaCMY) detected did not confer resistance to the beta-lactam antibiotics tested. Similar results were found for the detected aminoglycoside resistance genes aadA1, aadA2, strA, strB and aac(3’)-IVa. No resistance to amikacin, gentamicin and tobramycin was measured. A good correlation between geno- and phenotype was found for quinolone resistance. Isolates with qnrS shows resistance to moxifloxacin, but isolates containing only the 9

gene qnrA were not resistant against both fluoroquinolones tested. Resistance against cotrimoxazole (sulfamethoxazole/trimethoprim) is mediated by both sul and dfr alleles. Our data showed that only isolates harboring both genes were resistant against this antibiotic. No resistance to tigecycline was detected for isolates with tet genes (tet(A), tet(B) and tet(X)). Hybridization profiles were used to construct a split network tree to visualize similarities or relationships between the 47 isolates. This network tree is presented in Figure 1 and abbreviations are the same as in Table 1. Human isolates HUM024, HUM030, HUM036,

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HUM034, HUM038 and HUM004 differ significantly, but some of the human isolates are highly related to isolates from animals. Strain HUM037 seems to be the same strain as CATT064 differing only in one hybridization signal (blaACT was detected in CATT064).

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Another cluster includes the very similar strains HUM014, HUM023, BUF104, BUF126 and HUM019. All isolates belong to the O6:H14 serotype. Within this group, BUF126 differed only

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in two signals and HUM014 only in one. Two isolates were directly associated with samples

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from buffalo holders, HUM048 and HUM050. Both isolates closely clustered to isolates from buffalo BUF114 and BUF118, respectively. A closer look to the cluster HUM048/BUF114

4. Discussion

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shows that both strains shared the same hybridization pattern.

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The emergence of multiple antimicrobial resistance and virulence genes in commensal E. coli from apparently healthy small scale animal holders and their livestock has not been

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adequately addressed. In the present study, we have studied the antimicrobial resistance and virulence genotypes of commensal E. coli strains isolated from food producing animals and humans. Combinations of resistance genes conferring resistance to three or more antimicrobial classes were detected in 42.5% of all E. coli isolates. In short, the most frequently detected genes were associated with beta lactam (57.5%), aminoglycoside (48.8%), quinolone (36.0%) or tetracycline (36.2%) resistance. Up to now, there is insufficient information concerning the 10

distribution and genotypes of commensal E. coli recovered from humans as well as animals in Egypt (El-Gendy et al., 2013; Hussein et al., 2013). Comparing detection rates between these studies is difficult due to variations in methodology, particularly in molecular detection, isolation, and characterization. Results in the present study indicate that genotyping by microarray is suitable for correct and fast multiparameter analysis. In a short time with minimal costs compared to standard PCR analysis, we could detect genes encoding resistance to several important antimicrobial classes

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including aminoglycosides (aadA1, aadA2, strA, strB, aac(3’)-IVa, ant2), quinolones (qnrA1, qnrS), tetracyclines (tetA, tetB, tetX), sulphonamides (sul1, sul2, sul3), trimethoprim (dfrA12, dfrA14, dfrA15, dfrA5), rifampin (arr), macrolides (mphA, mrx, ereB) and chloramphenicol

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(cmlA1). We found blaCTX genes in two isolates, one of buffalo origin, and the other from a human source. Thus, blaCTX was surprisingly rare given that this gene family is normally highly

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prevalent among clinical and nonclinical isolates of E. coli isolated from animals and humans

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(Ho et al., 2015). Nevertheless, buffaloes were the most prevalent host harboring the beta lactams resistances genes. Genes conferring an ESBL phenotype (blaSHV and blaCTX-M1/15) were only detected in isolates from buffaloes and their keepers.

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Among antimicrobial agents used in human and veterinary medicine, the class of betalactam antibiotics has received particular interest. In the present study, E. coli ESBLs carried

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feature several point mutation variants including blaSHV and blaCTX-M1/M15 genes. In addition,

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nine MDR E. coli strains had different resistance gene cassettes and carried class 1 integrons. It seems likely that the emergence and wide dissemination of ESBLs among the E. coli isolates obtained could have a potential impact on public health and increase the odds of treatment failure. These findings were similar to previous studies on ESBL-producing E. coli in animals (Szmolka and Nagy, 2013; van Duijkeren et al., 2018). In those reports, the authors appraised the risk associated with the increase of abundance of ESBL genes in commensal E. coli in different environments along the food chain. 11

Globally, fluoroquinolones are widely used in the treatment of enterobacterial infections in both human and veterinary medicine due to their excellent activity against E. coli and other Gram-negative bacteria. It has been reported that the E. coli strains recovered from food animals could exhibit an increasing level of resistance towards fluoroquinolones (Webber and Piddock, 2001). Plasmid-mediated resistance to quinolones is usually due to the presence of the transferable Qnr proteins encoded by several genes, qnr(A), qnr(B), qnr(S), qnr(C), and/or qnr(D) (Hammerum and Heuer, 2009). Here, we detected qnr(A1) and qnr(S). This result was

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in agreement with that obtained by Strahilevitz and colleagues (Strahilevitz et al., 2009) who added that qnr(S) seems to be the most frequently detected qnr gene in isolates from both animal and human sources.

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In the current study, 29.8% (14/47) of the isolates carried genes associated with sulfonamide resistance (sul1, sul2, sul3). A high rate (16 out of 17) of sulfonamide resistance

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genes (sul) was detected among E. coli isolates recovered from healthy broilers in Egypt

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(Moawad et al., 2018). The same resistance genes were previously identified among E. coli of animal origins (Szmolka et al., 2012). Nevertheless, it is difficult to compare our results with these studies since the definition of MDR was different. Other reports from Europe revealed

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that approximately 10–30% of commensal E. coli, that had been isolated from food or food animals, were identified as being multidrug resistant (EFSA, 2012; Szmolka and Nagy, 2013).

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The phenotypic analysis of all E. coli strains recovered shows mostly the expected

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results regarding the correlation between genotype and phenotype. However, no correlation was detected for genes mediating aminoglycoside resistance. These findings were to be expected for the genes aadA1, strA and strB, as the aminoglycosides tested (amikacin, gentamicin and tobramycin) are not substrates for the enzymes they encode (Saenz et al., 2004). The main substrate for these enzymes is streptomycin-spectinomycin, which was not tested. Isolates in which an aac(3’)-IV gene was detected, which is described in the literature to mediate resistance to apramycin, gentamicin and tobramycin (Boerlin et al., 2005), were also susceptible 12

to all aminoglycosides tested. We assume that all strains, which were isolated in the same area of Egypt but from different hosts, harbor the same variant of the aac(3’)-IV gene that might be dysfunctional due to a point-mutation or frameshift event. To prove this statement, further investigation is needed (e.g., by PCR or sequencing). In the present study, the E. coli strains recovered showed highly variable antimicrobial resistance and virulence gene patterns, which is in accordance with published data (Boerlin et al., 2005). In that study, the authors found that virulence factors of E. coli isolates were

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frequently associated with MDR. A wide variety of genes associated with virulence was also detected, but by far the most commonly identified genes were those associated with adhesion of E. coli (lpfA, iha, f17G and f17A). lpfA was previously found to be prevalent in clinical and

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commensal E. coli isolated from human and bovine hosts (Szmolka et al., 2012). In another study, it was mentioned that lpfA, in combination with iss and astA, were the most common

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virulence factors in E. coli associated with mastitis (Blum and Leitner, 2013). In agreement

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with this observation, we found lpfA and iss to be more abundant in animal isolates than in human isolates. These genes seem to be important for colonization of E. coli in cattle. The gene astA, encoding the thermo-stable enterotoxin EAST1, is usually carried by enterotoxigenic E.

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coli, which causes diarrhea in humans, but is also present in bovine STEC and there significantly more often found in strains that persistently colonize the bovine gut (Barth et al.,

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2016). The study demonstrated a low prevalence of iha, f17G and f17A among the recovered

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isolates. Nevertheless, iha was detected in four STEC strains from cattle (CATT065, CATT074, CATT085 and CATT086). Previous reports demonstrated that the iha gene is commonly found in STEC strains associated with human cases of HUS (Baranzoni et al., 2016). In one bovine STEC strain, the eae gene was detected. It encodes the adherence factor intimin, which is part of the LEE locus and causes attaching and effacing lesions (McDaniel et al., 1995). E. coli positive for stx and eae might represent a high zoonotic risk, as these genes are often found in human pathogenic EHEC strains. The genes f17G and f17A were identified sporadically in 13

animal isolates (Johnson et al., 2005). Our data was in accordance with that previously presented by (Le Bouguenec and Bertin, 1999) who stated that f17G and f17A could be present specifically in E. coli isolates pathogenic to ruminant hosts. Hybridization profiles in combination with splits tree analyses is a good tool to point out similarities between strains isolated from different environmental, human and/or animal samples. One aim of this study was to show a relationship between isolates from human and animal samples. In our analysis we identified different strains which were very similar to each

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other. But in only one case we know exactly that one isolate was from a sample of a buffalo holder, who had direct contact to the animals (HUM048/BUF114). Due to the high similarity of both hybridization patterns we assume that both isolates are identical. We assume a direct

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transmission between human and animal or vice versa of this multi resistant strain.

Toxin-encoding genes were detected in only a few isolates. Similar data was previously

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reported from Tanzania (Madoshi et al., 2016). For the seven Shiga toxin (Stx) positive strains

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in our study, none were O157. The Stx positive strains mainly harbored the gene stx2. This is a potent toxin. There are five subtypes (stx2a, stx2b, stx2c, stx2d, stx2e) and three of them (stx2a, stx2c, stx2d) are more often associated with the development of HC and HUS in humans.

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Boerlin and colleagues (Boerlin et al., 1999) mentioned that stx2 was frequently associated with more severe human disease than stx1. Generally, Shiga toxins inhibit protein synthesis resulting

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in host cell death. The identification of different stx2 genes among apparently healthy animals

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was not surprising as such findings were already published (Barth et al., 2016; Hussein and Sakuma, 2005). We also detected one stx1 positive E. coli from an apparently healthy buffalo holder but there was no hint that this isolate originated from his livestock. 5. Conclusion Animal feces may contaminate the food chain and may be a source of major public health hazards. Antibiotic resistant organisms that are constantly being shed into the 14

environment through the feces of apparently healthy animals may cause serious antibiotic resistant infections in humans. Decisive efforts must be made to establish and maintain hygienic conditions at the small-scale animal holder’s level. In the current study, detection of the same gene pattern in E. coli isolates from humans and animals showed that some E. coli populations may colonize the intestine of different hosts in the same geographic area and, thus, may potentially be the source of exchange of genetic material between them. The majority of genes encoding a wide variety of resistance mechanisms are carried by mobile genetic elements such

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as plasmids or integrons. This facilitates a co-transfer of MDR phenotypes between commensals and pathogens, in and between both animal and human hosts. This study suggests that an increase of antibacterial resistance genes in human isolates may be the result of poor

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hygiene, irrational use of antibiotics and constant contact with animals. The presence of antimicrobial resistance and virulence genotypes among commensal E. coli may limit the

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chances for successful treatment of animal and human bacterial infections and assist in the

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emergence and dissemination of novel combinations and more dangerous antimicrobial resistance and virulence genotypes. The high prevalence of antimicrobial resistance genes among the strains analyzed here highlights the urgent need for strict measures to regulate the

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use of antimicrobials within food producing animals.

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Conflict of interest statement The authors declare that they have no conflict of interest.

Declarations of interest Nothing to declare

Acknowledgments

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During preparation of this manuscript, we were sorry to hear that our esteemed colleague Dr. Lutz Geue, who had the idea for the present project, died. We had the privilege to work

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together with him for several years and will always remember him.

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References Aidaros, H., 2005. Global perspectives--the Middle East: Egypt. Rev. Sci. Tech. 24, 589-596, PMID:16358510. Amon, T., Amon, B., Kryvoruchko, V., Zollitsch, W., Mayer, K., Gruber, L., 2007. Biogas production from maize and dairy cattle manure – influence of biomass composition on the methane yield. Agric. Ecosyst. Environ. 118, 173–182, DOI:

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10.1016/j.agee.2006.05.007.

Andersson, D.I., 2003. Persistence of antibiotic resistant bacteria. Curr. Opin. Microbiol. 6, 452-456, DOI: 10.1016/j.mib.2003.09.001.

-p

Anjum, M.F., Mafura, M., Slickers, P., Ballmer, K., Kuhnert, P., Woodward, M.J., Ehricht,

R., 2007. Pathotyping Escherichia coli by using miniaturized DNA microarrays. Appl.

re

Environ. Microbiol. 73, 5692-5697, DOI: 10.1128/AEM.00419-07.

lP

Ballmer, K., Korczak, B.M., Kuhnert, P., Slickers, P., Ehricht, R., Hachler, H., 2007. Fast DNA serotyping of Escherichia coli by use of an oligonucleotide microarray. J. Clin.

na

Microbiol. 45, 370-379, DOI: 10.1128/JCM.01361-06. Baranzoni, G.M., Fratamico, P.M., Gangiredla, J., Patel, I., Bagi, L.K., Delannoy, S., Fach, P., Boccia, F., Anastasio, A., Pepe, T., 2016. Characterization of shiga toxin subtypes and

ur

virulence genes in porcine shiga toxin-producing Escherichia coli. Front. Microbiol. 7,

Jo

574-581, DOI: 10.3389/fmicb.2016.00574. Barth, S.A., Menge, C., Eichhorn, I., Semmler, T., Pickard, D., Geue, L., 2017. Evaluation of applicability of DNA microarray-based characterization of bovine Shiga toxinproducing Escherichia coli isolates using whole genome sequence analysis. J. Vet. Diagn. Invest. 29, 721-724, 10.1177/1040638717700689. Barth, S.A., Menge, C., Eichhorn, I., Semmler, T., Wieler, L.H., Pickard, D., Belka, A., Berens, C., Geue, L., 2016. The accessory genome of Shiga Toxin-producing 17

Escherichia coli defines a persistent colonization type in cattle. Appl. Environ. Microbiol. 82, 5455-5464, 10.1128/AEM.00909-16. Batchelor, M., Hopkins, K.L., Liebana, E., Slickers, P., Ehricht, R., Mafura, M., Aarestrup, F., Mevius, D., Clifton-Hadley, F.A., Woodward, M.J., Davies, R.H., Threlfall, E.J., Anjum, M.F., 2008. Development of a miniaturised microarray-based assay for the rapid identification of antimicrobial resistance genes in Gram-negative bacteria. Int. J. Antimicrob. Agents 31, 440-451, DOI: 10.1016/j.ijantimicag.2007.11.017.

ro of

Blum, S.E., Leitner, G., 2013. Genotyping and virulence factors assessment of bovine mastitis Escherichia coli. Vet. Microbiol. 163, 305-312, DOI: 10.1016/j.vetmic.2012.12.037. Boerlin, P., McEwen, S.A., Boerlin-Petzold, F., Wilson, J.B., Johnson, R.P., Gyles, C.L.,

-p

1999. Associations between virulence factors of Shiga toxin-producing Escherichia coli and disease in humans. J. Clin. Microbiol. 37, 497-503, PMID: 9986802.

re

Boerlin, P., Travis, R., Gyles, C.L., Reid-Smith, R., Janecko, N., Lim, H., Nicholson, V.,

lP

McEwen, S.A., Friendship, R., Archambault, M., 2005. Antimicrobial resistance and virulence genes of Escherichia coli isolates from swine in Ontario. Appl. Environ. Microbiol. 71, 6753-6761, DOI: 10.1128/AEM.71.11.6753-6761.2005.

na

Boxall, A.B., Kolpin, D.W., Halling-Sorensen, B., Tolls, J., 2003. Are veterinary medicines causing environmental risks? Environ. Sci. Technol. 37, 286A-294A,

ur

PMID:12966963.

Jo

ECDC 2019. Surveillance of antimicrobial resistance in Europe 2018. (Stockholm, ECDC). EFSA, 2009. Joint Opinion on antimicrobial resistance (AMR) focused on zoonotic infections. EFSA Journal 7, 1372-1398, DOI: 10.2903/j.efsa.2009.1372.

EFSA, 2012. The European Union summary report on antimicrobial resistance in zoonotic and indicator bacteria from humans, animals and food in 2010. EFSA Journal 10, 2598-2620, DOI: 10.2903/j.efsa.2012.2598.

18

El-Gendy, A.M., Mansour, A., Shaheen, H.I., Monteville, M.R., Armstrong, A.W., El-Sayed, N., Young, S.Y., Klena, J.D., 2013. Genotypic characterization of Egypt enterotoxigenic Escherichia coli isolates expressing coli surface antigen 6. J. Infect. Dev. Ctries. 7, 90-100, DOI: 10.3855/jidc.2454. Hammerum, A.M., Heuer, O.E., 2009. Human health hazards from antimicrobial-resistant Escherichia coli of animal origin. Clin. Infect. Dis. 48, 916-921, DOI: 10.1086/597292.

ro of

Ho, P.L., Liu, M.C., Lo, W.U., Lai, E.L., Lau, T.C., Law, O.K., Chow, K.H., 2015. Prevalence and characterization of hybrid blaCTX-M among Escherichia coli isolates from livestock and other animals. Diagn. Microbiol. Infect. Dis. 82, 148-153, DOI:

-p

10.1016/j.diagmicrobio.2015.02.010.

Huson, D.H., Bryant, D., 2006. Application of phylogenetic networks in evolutionary studies.

re

Mol. Biol. Evol. 23, 254-267, DOI: 10.1093/molbev/msj030.

lP

Hussein, A.H., Ghanem, I.A., Eid, A.A., Ali, M.A., Sherwood, J.S., Li, G., Nolan, L.K., Logue, C.M., 2013. Molecular and phenotypic characterization of Escherichia coli isolated from broiler chicken flocks in Egypt. Avian Dis. 57, 602-611, DOI:

na

10.1637/10503-012513-Reg.1.

Hussein, H.S., Sakuma, T., 2005. Prevalence of shiga toxin-producing Escherichia coli in

ur

dairy cattle and their products. J. Dairy. Sci. 88, 450-465, DOI: 10.1637/10503-

Jo

012513-Reg.1.

ISO, 2005. Microbiology of food and animal feeding stuffs -- Horizontal method for the detection and enumeration of presumptive Escherichia coli -- Most probable number technique. ISO 7251:2005. Johnson, J.R., Owens, K., Gajewski, A., Kuskowski, M.A., 2005. Bacterial characteristics in relation to clinical source of Escherichia coli isolates from women with acute cystitis

19

or pyelonephritis and uninfected women. J. Clin. Microbiol. 43, 6064-6072, DOI: 10.1128/JCM.43.12.6064-6072.2005. Le Bouguenec, C., Bertin, Y., 1999. AFA and F17 adhesins produced by pathogenic Escherichia coli strains in domestic animals. Vet. Res. 30, 317-342, PMID:10367361. Madoshi, B.P., Kudirkiene, E., Mtambo, M.M., Muhairwa, A.P., Lupindu, A.M., Olsen, J.E., 2016. Characterisation of commensal Escherichia coli isolated from apparently healthy cattle and their attendants in Tanzania. PLoS One 11, e0168160, DOI:

ro of

10.1371/journal.pone.0168160. McDaniel, T.K., Jarvis, K.G., Donnenberg, M.S., Kaper, J.B., 1995. A genetic locus of

enterocyte effacement conserved among diverse enterobacterial pathogens. Proc. Natl.

-p

Acad. Sci. U S A 92, 1664-1668, 10.1073/pnas.92.5.1664.

Moawad, A.A., Hotzel, H., Neubauer, H., Ehricht, R., Monecke, S., Tomaso, H., Hafez, H.M.,

re

Roesler, U., El-Adawy, H., 2018. Antimicrobial resistance in Enterobacteriaceae from

lP

healthy broilers in Egypt: emergence of colistin-resistant and extended-spectrum betalactamase-producing Escherichia coli. Gut Pathog. 10, 39, DOI: 10.1186/s13099-0180266-5.

na

Monecke, S., Ehricht, R., 2005. Rapid genotyping of methicillin-resistant Staphylococcus aureus (MRSA) isolates using miniaturised oligonucleotide arrays. Clin. Microbiol.

ur

Infect. 11, 825-833, DOI: 10.1111/j.1469-0691.2005.01243.x.

Jo

Nicholson, F.A., Hutchison, M.L., Smith, K.A., Keevil, C.W., Chambers, B.J., Moore, A., 2000. A study on farm manure applications to agricultural land and an assessment of

the risks of pathogen transfer into the food chain. MAFF.

Oliver, S.P., Jayarao, B.M., Almeida, R.A., 2005. Foodborne pathogens in milk and the dairy farm environment: food safety and public health implications. Foodborne Pathog. Dis. 2, 115-129, DOI: 10.1089/fpd.2005.2.115.

20

Rice, J.M., Caldwell, D.F., Humenik, F.J. 2006. Pathogens in animal wastes and the impacts of waste management practices on their survival, transport and fate, In: Sobsey, M.D., Khatib, L.A., Hill, V.R., Alocilja, E.C., Pillai, S.D. (Eds.) Animal Agriculture and the Environment: National Center for Manure and Animal Waste Management White Papers. ASABE, St. Joseph, Michigan, 609-666, DOI: 10.13031/2013.20268. Saenz, Y., Brinas, L., Dominguez, E., Ruiz, J., Zarazaga, M., Vila, J., Torres, C., 2004. Mechanisms of resistance in multiple-antibiotic-resistant Escherichia coli strains of

ro of

human, animal, and food origins. Antimicrob. Agents Chemother 48, 3996-4001, 10.1128/AAC.48.10.3996-4001.2004.

Scheutz, F., Teel, L.D., Beutin, L., Pierard, D., Buvens, G., Karch, H., Mellmann, A.,

-p

Caprioli, A., Tozzoli, R., Morabito, S., Strockbine, N.A., Melton-Celsa, A.R.,

Sanchez, M., Persson, S., O'Brien, A.D., 2012. Multicenter evaluation of a sequence-

re

based protocol for subtyping Shiga toxins and standardizing Stx nomenclature. J. Clin.

lP

Microbiol. 50, 2951-2963, DOI: 10.1128/JCM.00860-12. Strahilevitz, J., Jacoby, G.A., Hooper, D.C., Robicsek, A., 2009. Plasmid-mediated quinolone resistance: a multifaceted threat. Clin. Microbiol. Rev. 22, 664-689, DOI:

na

10.1128/CMR.00016-09.

Szmolka, A., Anjum, M.F., La Ragione, R.M., Kaszanyitzky, E.J., Nagy, B., 2012.

ur

Microarray based comparative genotyping of gentamicin resistant Escherichia coli

Jo

strains from food animals and humans. Vet. Microbiol. 156, 110-118, DOI: 10.1016/j.vetmic.2011.09.030.

Szmolka, A., Nagy, B., 2013. Multidrug resistant commensal Escherichia coli in animals and its impact for public health. Front. Microbiol. 4, 258, DOI: 10.3389/fmicb.2013.00258.

21

van Duijkeren, E., Schink, A.K., Roberts, M.C., Wang, Y., Schwarz, S., 2018. Mechanisms of bacterial resistance to antimicrobial agents. Microbiol. Spectr. 6, 10.1128/microbiolspec.ARBA-0019-2017. Webber, M., Piddock, L.J., 2001. Quinolone resistance in Escherichia coli. Vet. Res. 32, 275-

Jo

ur

na

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re

-p

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284, DOI: 10.1051/vetres:2001124.

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Figure 1

---------------------------------------------------A------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------A-------

2 HUM024

---------------------------------------------------A---------------------A-----------------------------------------------------------------------A--------------------------------------------------------------------------------------------------------A----A--

3 HUM030

------------------------A--------------------------------------------------------------------------------------------------------A------------------------------------------------------------------------------------------------------------------------A-------

4 HUM036

------------------------------------A--------------------------------------------------------------------------------------------A------------------------------------------------------------------------------------------------------------------------A-------

5 HUM034

---------------------------------A---------------------------------------------------------------------------------------------------------------------A--------------------------------------------------------------------------------------------------A-------

6 HUM038

---------------------------AA--------------------------------------------------------------------------------------------------------------------------A--------------------------------------------------------------------------------------------------A-------

7 HUM004

------------------------A------------------------------------------------A---------------------A-A-----------------------------------------------A-------------A-----------------------------A-----------------------------A------------------------------A----A--

8 BUF128

------------------------------A------------------------------------------A--------------------AA-A---------------------------------A-------------A---A------------A--------------------------A----A-------------------------------------------------------A----A--

9 BUF129

----------------------------------------------------------------A--------A--------------------AA-A---------------------------------A-------------A---A------------A---------------------A----A----A-------------------------------------------------------A-------

10 BUF131

------------------------A------------------------------------------------A--------------------AA-A-----------------------------------------------A----------------A--------------------------A----A------------------------A------------------------------A----A--

11 CATT069

---------------------------------------------A----------------------------------A--------------A-A-------------------------------------------------------------------------------------------------------------------------A------------------------------A-------

12 HUM037

------------------------A--------------------------------------------------------------------------------------------------------------------------------------------------------------------A-----------------------------A------------------------------A----A--

13 CATT064

------------------------A---------------------------------------------------------------------A----------------------------------------------------------------------------------------------A-----------------------------A------------------------------A----A--

14 CATT068

--------------------------------------------A-------------------------------------------------A------------------------------------A------------------------------A---------------------------------------------------------------------------------------A----A--

15 CAL084

-------------------------------------------------------A--------------------------------------A------------------------------------------------------A---------------------------------------A------------------------------------------------------------A-------

16 BUF121

------------------------------A---------------------------------------------------------------A--A-------------------------------------------------------------------------------------------A------------------------------------------------------------A-------

17 BUF103

--A--------------------------------------A----------------------------------------------------A----------------------------------------------------------------------------------------------A------------------------------------------------------------A----A--

18 BUF107

-----------------------------------------A----------------------------------------------------A--------------------------------------------------A-------------------------------------------A------------------------------------------------------------A-------

19 HUM014

-A---------------------------------A----------------------------------------A------------------------------------A------------A--A------A--------A--AA-A--------A----------------------------A------------------------------------------------------------AA---A--

20 HUM023

-A---------------------------------A----------------------------------------A------------------------------------A------------A--A------A-----------AA-A--------A----------------------------A------------------------------------------------------------AA---A--

21 BUF104

-A---------------------------------A----------------------------------------A------------------------------------A------------A--A------A-----------AA-A--------A----------------------------A------------------------------------------------------------AA---A--

22 BUF126

-A---------------------------------A----------------------------------------AA----------------A------------------A------------A--A------A-----------AA-A--------A----------------------------A------------------------------------------------------------AA---A--

23 HUM019

-A---------------------------------A----------------------------------------A------------------------------------A------------A--A------A-----------AA-A--------A----------------------------A------------------------------------------------------------AA------

24 HUM016

----------------------------A------------------------------------------------A-------------A-------------------------------------A------------AA----A--A------AA----A-------------------------------------------------------------------------------------AA------

25 HUM018

-----------------------------------------A-------------------------------------------------AA-----A------------------------------A------------------A--A-------A------A-----------------------------------------------------------------------------------AA------

26 BUF123

-----------------------------------------A--------------------------------------A----------AA-AA-AA------------------------------A------------------A--A-------A------AA----------------A-----------------------------------------------------------------AA------

27 CATT057

-----------------------------------------A-------------------A------------------------------A-------------------------------------------------------A--A-------A------A-----------------------------------------------------------------------------------AA---A--

28 HUM027

-----------------------------------------------A-------------------------------------------AA----------------------------------------------------------A-------A------------------------------------------------------------------------------------------A-------

29 HUM048

---------------------------------------A---------------------------------------------------AA-----------------------------------------------------------A------A-----------------------------A-----A-A------------------------A-AA------A----------A------A---AA--

30 BUF114

---------------------------------------A---------------------------------------------------AA-----------------------------------------------------------A------A-----------------------------A-----A-A------------------------A-AA------A---------------------AA--

CATT 31 CAL099

-----------------------------------------------------------A-------------------------------AA------------------------------------A------------------A--A-----A-A-----------------------------A------------------------A-----------------A-----------------A---AA--

32 CATT058

---A-------------------------------------A-------------------------------------------------AA------------------------------------A----------------------A------------------------------------A------------------------------------------------------------A-------

33 BUF132

---A-------------------------------------A-------------------------------A-----------------AA-AA-A-------------------------------A---------------A------A---------A--------------------------A------------------------------------------------------------A-------

34 HUM006

----------------------A-------------------A------------------------------------------------------------------------------------------------------A------------------------------A---A--------A------------------------------------A----------------------------A--

35 BUF127

----------------------A-------------------A-------------------------------------------------------------------------------------------------------------------------------------A---A--------A----------A-------------------------A----------------------------A--

36 BUF105

---------------------------------------A-----------------------------------------------------------------------------------------------------------------------------------------------------A---------------------------------A--A-----A-----------A-A---A----A--

37 CATT065

----------------------A----------------------------------A------------------------------------A---------------------------------------------------------------------------------A-------AA--------------A-------------------------------------------A----AA-------

38 CATT074

-------------------------------------A--------------------------------------------------------A------------------------------------------------------A--------------------------A------------A----------A-----------------------------AAAA--------A-AA---AA----A--

39CATT CAL085

------------------------A------------------------------------------------A--------------------A--A-------------------------------------------AA---------------------------A-----A------------A----------------------------------------------------A-A----AA-------

40 CAL086

------------------------A-------A-----------------------------------------------------------------------------------------------------------------------------------------------A------------A------------------------------------------------------A----AA-------

41 HUM050

---------------------------------------A-----------------------------------------------------------------------------------------------------------------------------------------------------A--------------------A--------A------------------------------A-------

42 BUF118

--------------------------------------------------A---------------------------------------------------------------------------------------------------------------------------------------------------------------A---------------------------------------A-------

43 BUF106

--------------------------------A------------------------------------------------------------------------------------------------------------------------------------------------------------A------------------------------------------------------------A-------

44 HUM029

-----------------------------A---------------------------------------------------------------------------------------------------------------------------------------------------------------A------------------------------------------------------------A-------

45 CAL087

-----------------------------------------------A---------------------------------------------------------------------------------------------------------------------------------------------A------------------------------------------------------------A-------

46 CATT062

--------------A----------------------A-------------------------------------------------------------------------------------------------------------------------------------------------------A------------------------------------------------------------A-------

47 BUF110

--------------A----------------------A-------------------------------------------------------------------------------------------------------------------------------------------------------A------------------------------------------------------------A-------

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1 CATT056

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ur na

CATT

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Similarity analysis. We used a split network tree to visualize similarities (not phylogeny) between hybridization patterns, which were detected by microarray.

CATT

CATT

23

24

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Table 1 Genotypic characterization of E. coli isolates recovered from buffalo, cattle and human fecal samples Isolate

Source

O-type

H-type

HUM004

Human

n.d.

H02

HUM006

Human

O172

H23

HUM014

Human

O6

H14

HUM016

Human

n.d.

H06

HUM018

Human

n.d.

H21

HUM019

Human

O6

H14

HUM023

Human

O6

H14

HUM024

Human

n.d.

HUM027

Human

n.d.

HUM029

Human

n.d.

H07

HUM030

Human

n.d.

H02

blaTEM

HUM034

Human

n.d.

H11

tet(A)

HUM036

Human

n.d.

H15

blaTEM

HUM037

Human

n.d.

H02

HUM038

n.d.

H05/H06 tet(A)

n.d.

H19

n.d.

H19

CATT056

Human Human (Buffalo holder) Human (Buffalo holder) Cattle

n.d.

H32

CATT057

Cattle

n.d.

H21/H45 str(B), qnr(S), tet(A), sul2, dfrA14

hemL, intl1, iss

CATT058

Cattle

O8

H21

lpfA, hemL

CATT062

Cattle

O103

H16

CATT064

Cattle

n.d.

H02

blaACT

CATT065

Cattle

O172

H40

blaACT

CATT068

Cattle

n.d.

H25

blaACT, dfrA5

CATT069

Cattle

n.d.

H26

ant2, blaCMY, blaMOX-CMY9

CATT074

Cattle

n.d.

H16

blaACT, arr

CATT084

Cattle Cattle

n.d.

H38

n.d.

H02

blaACT, arr aac(3’)-IVa, blaACT, blaMOX-CMY9, ere(B), mph(A)

CATT086

Cattle

n.d.

H02/H10

CATT087

Cattle

n.d.

H28

n.d.

H42

Jo

CATT085

CATT099

str(A), str(B), tet(A), sul2

hemL

Cattle

lpfA, astA, hemL, iss iha, saa, lpfA, hlyA, iss lpfA, hemL, intl1, iss hemL, intl1,

of

hemL, intl1

lpfA, hemL, intl1

ro

lpfA, hemL, intl1, iss hemL, iss

-p

lpfA, hemL hemL

re

hemL

str(A), str(B), tet(B), sul2

lP

HUM050

H28

ur na

HUM048

Virulence markers

H32

Resistance markers aac(3’)-IVa, blaCMY, blaMOX-CMY9, qnrA1, sul2 qnrA1 aadA1, blaOXA-7, blaSHV, blaTEM, cmlA1, qnrA1, qnr(S), arr, tet(A), sul3 aadA2, str(A), blaTEM, mph(A), mrx, qnr(S), tet(A), sul1, sul2, dfrA12 str(A), str(B), blaCTXM1,blaCTXM15,blaTEM, qnr(S), tet(A), sul2, dfrA14 aadA1, blaOXA-7, blaSHV, blaTEM, cmlA1, qnr(S), arr, tet(A), sul3 aadA1, blaOXA-7, blaSHV, blaTEM, cmlA1, qnr(S), arr, tet(A), sul3 aac(3’)-IVa, qnrA1

str(A), str(B), blaTEM, tet(B)

hemL lpfA, hemL, iss, astA hemL stx1a, lpfA, espA, espF, cdtB, cma, cnf1, mchF, hemL, iroN lpfA, pic, astA, hemL hemL

lpfA, hemL lpfA, astA, hemL, iss stx2c, hemL, iha, f17-A, f17-G, espI hemL, iss astA, hemL stx2b, stx2c, iha, lpfA, espI, mchB, mchC, mchF, mcmA, virF, hemL, iss lpfA, hemL stx2c, stx2d, hemL, eae consensus, iha, lpfA, virF stx2c, iha, lpfA, hemL

lpfA, hemL str(A), str(B), blaTEM, qnr(S), tet(A), tet(X), lpfA, tsh, mchF, hemL, iroN, iss sul2

25

Isolate

Source

O-type

H-type

Resistance markers

Virulence markers

BUF103

Buffalo

O7

H21

lpfA, hemL, iss

BUF104

Buffalo

O6

H14

blaACT aadA1, blaOXA-7, blaSHV, blaTEM, cmlA1, qnr(S), arr, tet(A), sul3

BUF105

Buffalo

n.d.

H19

BUF106

Buffalo

n.d.

H10

BUF107

Buffalo

n.d.

H21

BUF110

Buffalo

O103

H16

BUF114

Buffalo

n.d.

H19

BUF118

Buffalo

n.d.

H31

BUF121

Buffalo

n.d.

H08

BUF123

Buffalo

n.d.

H21

BUF126

Buffalo

O6

H14

BUF127

Buffalo

O172

H23

BUF129

Buffalo

n.d.

H49

BUF131

Buffalo

n.d.

H02

BUF132

Buffalo

O8

H21

str(A), str(B), tet(B), sul2

lpfA, hemL lpfA, espA, espF, cdtB, cma, cnf1, mchF, iroN, iss pic, hemL lpfA, hemL

of

blaACT, blaMOX_CMY9 ant2, str(A), str(B), blaACT, blaCMY, blaMOXCMY9, blaCTX-M1/M15, blaTEM, qnr(S), tet(A), sul2, dfrA14, dfrA15 aadA1, aadA2, blaACT, blaOXA-7, blaSHV, blaTEM, cmlA1, qnr(S), arr, tet(A), sul3

f17-A, hemL, intl1

lpfA , hemL, intl1, iss

ro

H08

lpfA, hemL

iha, saa, lpfA, espI, hlyA, iss

aac(3’)-IVa, blaACT, blaCMY, blaMOX-CMY9, qnrA1, arr, dfrA5 aac(3’)-IVa, blaACT, blaCMY, blaMOX-CMY9, qnrA1, arr, dfrA5 aac(3’)-IVa, blaACT, blaCMY, blaMOX-CMY9, qnrA1, dfrA5 aac(3’)-IVa, str(A), str(B), blaACT, blaCMY, blaMOX-CMY9, blaTEM, qnrA1, tet(B), dfrA5

lpfA, espA, hemL, iss

-p

n.d.

blaACT, qnrA1

re

Buffalo

stx2a, stx2d, stx2e, lpfA, celB, hlyA, mchF, hemL, iss lpfA, hemL

Jo

ur na

lP

BUF128

lpfA, hemL, iss, int1,

26

f17-A, lpfA, espA, hemL lpfA, espA, astA, hemL, iss lpfA, hemL

27

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ro

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re

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ur na

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