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Advance Publication by J-STAGE Japanese Journal of Infectious Diseases Molecular typing of Escherichia coli O157 isolates from Romanian human cases

Mihaela Oprea, Adriana Simona Ciontea, Mădălina Militaru, Sorin Dinu, Daniela Cristea, and Codruţa-Romaniţa Usein

Received: March 23, 2018. Accepted: July 17, 2018 Published online: July 31, 2018 DOI:10.7883/yoken.JJID.2018.129

Advance Publication articles have been accepted by JJID but have not been copyedited or formatted for publication.

Title page

Molecular typing of Escherichia coli O157 isolates from Romanian human cases

Authors:

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Running head: Typing of Romanian Escherichia coli O157 isolates

Mihaela Oprea1, 2, Adriana Simona Ciontea1, Mădălina Militaru1, Sorin Dinu1, Daniela Cristea1, Codruţa-Romaniţa Usein1, 3* Affiliation:

Cantacuzino National Medical - Military Institute of Research and Development, Splaiul

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1

Independentei 103, sector 5, 050096, Bucharest, Romania 2

The Research Institute of the University of Bucharest, Bd. M. Kogalniceanu 36-46, sector 5,

050107, Bucharest, Romania

Carol Davila University of Medicine and Pharmacy, Dionisie Lupu 37, sector 1, Bucharest,

Romania

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3

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The work was done at: Molecular Epidemiology Laboratory, Cantacuzino National Institute

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of Medical - Military Research - Development, Splaiul Independentei 103, sector 5, 050096, Bucharest, Romania

Corresponding author: Codruţa-Romaniţa Usein Molecular Epidemiology Laboratory, Cantacuzino National Medical - Military Institute of Research and Development, Splaiul Independentei 103, sector 5, Bucharest, Romania e-mail: [email protected]; Tel: +40213069223 Keywords: Escherichia coli O157, molecular characterisation, whole genome sequencing 1   

Summary Verocytotoxin-producing Escherichia coli (VTEC) of serogroup O157 are among the most important causes of severe cases of foodborne disease and outbreaks worldwide. As little is known about the characteristic of these strains in Romania, we aimed to provide reference

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information on the virulence gene content, phylogenetic background, and genetic diversity of seven autochthonous O157 strains collected during 2016 and 2017 from epidemiologically non-related cases. These strains were typed by a combination of phenotypic and molecular methods routinely used by the national reference laboratory. Additionally, four of them were also subjected to whole-genome sequencing (WGS) and public web-based tools were used to

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extract information on virulence gene profiles, multilocus sequence types (MLST), and SNPbased phylogenetic relatedness. Molecular typing provided evidence of the circulation of a polyclonal

population

while

distinguishing

a

cluster

of

non-sorbitol-fermenting,

glucuronidase-negative, phylogenetic group E, MLST 1804 strains, representing lineage II

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and clade 7, which harbored vtx2c, eae-gamma, and ehxA genes. A good correlation between the routine typing methods and WGS data was observed. Yet SNP-based genotyping provided a higher resolution in depicting the relationships between the O157:H7 strains than

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that provided by PFGE. This study should be a catalyst for improved laboratory-based

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surveillance of autochthonous VTEC.

Introduction

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Diarrheagenic Escherichia coli (DEC) strains are extensively studied due to their clinical and epidemiological importance. Classified in more categories, based on the mechanisms that lie behind the distinct syndromes they generate, DEC are known as enteropathogenic E. coli (EPEC), enterotoxigenic E. coli (ETEC), enteroinvasive E. coli (EIEC), enteroaggregative E. coli (EAEC), diffusely adherent E. coli (DAEC), and

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verocytotoxin producing E. coli (VTEC) (1).

VTEC (also known as Shiga toxin-producing E. coli, STEC) are foodborne pathogens with worldwide distribution linked to illness outbreaks and severe pathology. They are defined by their capacity to produce verocytotoxins or Shiga toxins, bacteriophage-encoded cytotoxins considered the most important virulence trait that differentiate them from EPEC.

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Enterohemorragic E. coli (EHEC) are a subset of VTEC which are able to colonize the bowel usually by an intimate effacing adherence to the epithelial cell membrane. EHEC strains induce the formation of „attaching-and-effacing” (A/E) lesions, a histopathological feature also detected in EPEC colonization (2). Intimin is the main adherence factor demonstrated to

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play a role in the intestinal colonization, a complex process that requires mechanisms encoded by genes located on the so-called locus of enterocyte effacement (LEE) pathogenicity island (3). Commonly, EHEC are capable of producing severe bloody diarrhea

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and hemorrhagic colitis that can progress to complications such as hemolytic uremic

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syndrome (HUS). EHEC most commonly isolated from humans worldwide are representing a limited number of serogroups although hundreds of VTEC serotypes have been identified to date (4). O157:H7 is the most common and notorious EHEC serotype, known for causing foodborne outbreaks (5). Molecular typing facilitated the characterization and comparison of E. coli O157:H7 strains recovered from different isolation sources. Relevant genomic information supported

3   

their segregation into more distinct evolutionary lineages and clades which seemed to differ in host specificity and virulence characteristics (6-8). The public health importance of VTEC infections across Europe displays considerable national differences and apparently, they are quite rare in Romania (9). However, the recent HUS outbreak attributed to VTEC members of O26 serogroup registered among young

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children (10) was evidence that the burden caused by VTEC infections is significantly underestimated in Romania and a more consistent laboratory-based surveillance is needed for reassessing it. In this context, we employed a molecular approach to confirm the intrinsic virulence of autochthonous E. coli selected as presumptive VTEC by O157-serotyping

serotype. Materials and methods

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method and to provide a much thorough genotyping of representative members of O157:H7

Bacterial strains and phenotypic characterization

The E. coli O157 strains investigated in this study were received for confirmation

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tests in the national reference laboratory for VTEC infection from Cantacuzino National Institute of Medical-Military Research-Development. The strains originated from 7 apparently sporadic cases of acute diarrhea (5 strains) and HUS (2 strains) with no

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epidemiological linkage, diagnosed in 2016 and 2017 in children less than 2 years old,

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resided throughout Romania (Table 1). The strains were serotyped for the O157 and H7 antigens by slide agglutination with

commercially available antisera (SSI Diagnostica) and tested for sorbitol fermentation on sorbitol MacConkey agar (bioMérieux). For this study, the strains were given identification numbers (from RO_O157_1 to RO_O157_7) according to the chronological order of isolation. PCR-based characterization 4   

A multiplex PCR-based protocol targeting rfbEO157 and fliCH7 with previously published primers (11) was used for the molecular serotyping of the strains. The strains were also screened for the presence of EHEC virulence markers vtx1 (verocytotoxin 1), vtx2 (verocytotoxin 2), and eae (intimin) by using a commercially available multiplex PCR-based kit (DEC Primer Mix, SSI Diagnostica). Additionally,

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previously published primers (12) were used for the detection of EHEC enterohemolysin encoding gene ehxA.

Subtyping of vtx1 and vtx2 genes was performed according to a published protocol recommended by WHO Collaborating Centre for Reference and Research on Escherichia and Klebsiella from Statens Serum Institute, Denmark (13).

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Phylogenetic group assignment of the O157 E. coli strains was performed according to the method described by Clermont et al. (14). Pulsed-field gel electrophoresis

Pulsed-field gel electrophoresis (PFGE) was used to assess the genetic relatedness of

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the E. coli O157 strains. XbaI-digested genomic DNA was analyzed on a 1% SeaKem Gold agarose gel in 0.5 × TBE buffer using CHEF MAPPER (BioRad) (15). The run time was 18 h at 6 V cm−1, with initial and final switch times of 2.16 and 54.17 s, respectively. The gel was

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stained with 1 μg mL−1 ethidium bromide, visualized on the Gel Doc XR system (BioRad)

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and analyzed using Bionumerics fingerprinting software version 6.6 (Applied Maths). Pulsotype designation was assigned at the 100% profile similarity level. Whole genome sequencing The genomes of E. coli strains RO_O157_2, RO_O157_3, RO_O157_5 and RO_O157_7 were sequenced using an Ion Torrent PGM (Thermo Fisher Scientific) according to 400-bp protocols for library preparation through enzymatic shearing, Ion OneTouch2 emulsion PCR, enrichment, Hi-Q View sequencing kits (Thermo Fisher 5   

Scientific). The sequences obtained were subjected to de novo assembly by using the Assembler SPAdes plugin version 5.8 (16) installed on Ion Torrent Server. Analysis of WGS sequence data Whole genome sequencing (WGS) data were analyzed using the web-based tools, designed by the Center of Genomic Epidemiology (CGE), available on the public CGE server

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(at https://cge.cbs.dtu.dk/services/). VirulenceFinder tool was used for the detection of virulence genes included in the CGE database (17) SerotypeFinder tool was used for WGSbased O and H typing (18). Multilocus sequence typing (MLST) tool with the configuration set to E. coli scheme 1 (adk, fumC, gyrB, icd, mdh, purA, recA) was used for determination of the multilocus sequence types (19). Call SNPs & Infer (CSI) Phylogeny tool (20) with default

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parameters (a minimal depth at SNP positions of 10x, a minimal distance between SNPs of 10 bp, a minimal SNP quality score of 30, a minimal Z-score of 1.96) was used to perform a single nucleotide polymorphism (SNP)-typing phylogenetic analysis with E. coli EDL 933 strain as reference sequence (NZ_CP008957.1) and construct a maximum likelihood tree

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from the SNP alignment using Mega 7 (21). Published sequences available in the NCBI GenBank and Sequence Read Archive (SRA) were used for comparison (Table 2). For a better comparison with the Romanian sequenced isolates, the reference strains were also

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analyzed with VirulenceFinder tool.

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Additionally, intimin typing was performed in silico through a BLAST search (22) of the eae gene sequences from the WGS against the National Center for Biotechnology Information (NCBI) nucleotide repository with a threshold of 95% nucleotide sequence identity with existing intimin genes sequences. Also, a BLAST analysis for detection of the uidA gene mutations was performed in order to predict the beta-glucuronidase (GUD) activity. Besides, in order to confirm the non-sorbitol fermenting character of the strains, the annotated WGS sequences were investigated for the following supplementary markers: urease gene cluster, 6   

tellurite resistance and adherence-conferring pathogenicity island, and fimbrial sfp cluster (23, 24). The lineage assignment of the four O157:H7 strains sequenced was based on the investigation of the polymorphism of the six markers proposed by Yang et al. (6) using the Integrative Genomics Viewer (IGV) software (25) and EDL933 as reference strain. For this

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study, Sanger sequencing was used to confirm the insertions and deletions observed in the target loci at IGV inspection.

Clade typing relied on the identification of clade specific SNPs originally described by Manning et al. (8) targeting known informative positions (26).

The WGS reads of the four O157:H7 strains are part of the European Molecular

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Biology Laboratory’s (EMBL) European Nucleotide Archive (ENA) sequence database (accession number PRJEB25277). All assembled genomes were uploaded on NCBI Prokaryotic Genome Annotation Pipeline and received the following GenBank accession numbers: PUIS00000000 (RO_O157_2), PUIU00000000 (RO_O157_3), PUIT00000000

Results

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(RO_O157_5), and PUIR00000000 (RO_O157_7).

Phenotypic characterization

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Five O157 strains belonged to serotype O157:H7 and were sorbitol non-fermenters.

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The other two O157 strains, although motile, lacked the H7 flagellar antigen. Only one of them was able to ferment the sorbitol. PCR-based serotyping, virulotyping, and phylogenetic grouping Molecular serotyping confirmed all the strains to be of serogroup O157, distinguished the fliCH7 gene in all five O157:H7 strains, and resolved the two fliCH7 gene negative strains. Of the virulence genes for whose presence the O157 E. coli strains were investigated by PCR, vtx, eae, and ehxA were detected. Overall, six PCR-defined virulotypes were 7   

identified which qualified the strains as VTEC/EHEC (4 strains), EPEC (2 strains) or nonVTEC non-EPEC (1 strain) (Table. 1). The study strains were assigned to phylogenetic groups A (1 strain), B1 (1 strain), D (1 strain), and E (4 strains). The O157:H7 strains belonged to phylogroups D (1 strain) and E (4 strains) and harbored combinations of at least two of the virulence gene markers

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considered for DEC diagnostic identification (i.e. eae+ehxA+, vtx2c+eae+ehxA+, vtx1a+vtx2a+). PFGE analysis

All the E. coli O157 strains yielded interpretable XbaI PFGE profiles which extended to the 68% similarity level overall. However, the 100% similarity criterion resolved six pulsotypes of which one contained 2 strains. The phylogenetic group E O157:H7 strains

(Fig. 1). WGS-based typing

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(RO_O157_2, RO_O157_3 and RO_O157_5) formed a discrete cluster on the dendrogram

The VTEC O157:H7 strains of phylogenetic group E were subjected to a more

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thorough analysis performed by WGS. WGS assembly metrics are presented in Table 3. The serotype detection tool confirmed the accuracy of the PCR-based molecular serotyping indicating all 4 strains as serotype O157:H7.

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Sequence type ST1804 was assigned by the MLST tool to RO_O157_2, RO_O157_3,

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RO_O157_5, and ST 11 to RO_O157_7, respectively. Concerning the GUD encoding gene uidA, comparison of the WGS data with the

same sequence of K12 DH10B strain (GenBank access no. CP000948) revealed that the autochthonous strains displayed 4 mutations that resulted in the following amino acid changes: D31E, A64V, C253Y, and Q259R. Moreover, RO_O157_2, RO_O157_3, and RO_O157_5 strains but not RO_O157_7 had a GG insertion within uidA at nucleotide 686, resulting in a premature stop codon. 8   

The WGS annotated sequences showed that all the strains carried a complete urease cluster and tellurite resistance and adherence-conferring pathogenicity island and were negative for the sfp cluster. A 100% concordance between the virulence detection tool and conventional PCR results for vtx (type/subtype), ehxA, and eae genes was observed, in silico eae subtyping

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indicating a γ-type intimin. Strains RO_O157_2, RO_O157_3 and RO_O157_5 displayed almost identical WGS-based virulence profiles, the only difference being that RO_O157_2 lacked the gad gene encoding glutamate decarboxylase. RO_O157_7 strain exhibited a different profile (Table 1). All the strains analyzed carried a pO157-like plasmid which in the case of RO_O157_7 strain lacked espP and katP genes. CSI Phylogeny tool allowed SNPs

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identification from WGS data and further phylogenetic inference separated the four autochthonous strains from one another and from selected published genomes available in the NCBI GenBank. The topology of the SNP-based phylogenetic tree (Fig. 2) showed the clustering of the RO_O157_2, RO_O157_3, and RO_O157_5 strains and their separation

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from the ST11 RO_O157_7 strain, which resembled the topology observed on the PFGEbased tree (Fig. 1). The SNP difference matrix, calculated by CSI Phylogeny, indicated that the clustered strains were separated from one another by 30 to 75 SNPs and each of them

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harbored >2600 SNP differences compared to

RO_O157_7 strain. Overall, the

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autochthonous strains clustered with the published strains belonging to the same STs. On the basis of the WGS data used for lineage and clade typing, RO_O157_2,

RO_O157_3, and RO_O157_5 strains were assigned to lineage II (genotype 232123) and clade 7 and RO_O157_7 to lineage I/II (genotype 211111) and clade 9.

Discussion

9   

As the globalization process appears to be causing unpredictable changes in the epidemiology of infectious diseases, the local background of infectious agents warrants further attention in order to understand the risks they pose to both resident and visiting populations. The year 2016 was marked by the occurence of the first major event linked to VTEC in Romania and brought public awareness about how little was known about the

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autochthonous reservoir of these pathogens. Our aim was to give an insight into the features of a set of autochthonous E. coli strains belonging to O157 serogroup which is often assumed to be the source of the most virulent VTEC (2) and provide reference information about their intrinsic virulence, phylogenetic background, and genetic diversity.

The molecular approach commonly used at national reference laboratory level reliably

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distinguished O157:H7 from O157:nonH7 strains and detected associations of key virulence gene markers indicative of the capacity to produce the verocytotoxins, presence of the LEE pathogenicity island, and possession of the large enterohemolysin-encoding plasmid in the O157:H7 strains. With one exception, these strains carried vtx2, the gene possessed by most

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of the O157:H7 strains circulating now in Europe as well as in other regions of the world (2729). The vtx-negative O157:H7 strain found was not a surprise, knowing that spontaneous loss of the vtx phage(s) during in vitro subcultivation was reported for both VTEC O157 (30)

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and non-O157 strains (31). Yet, this finding was a matter of concern from the diagnostic

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point of view.Together with the evidence of O157 strains of other serotypes but H7 circulation. it indicated that presumptive DEC, selected solely on the basis of the slide agglutination with commercially available antisera, should be confirmed and further characterized in the national reference laboratory. Considering that WGS is the pinnacle for strain characterisation and epidemiological analyses, we took the opportunity to extract additional and important information about the autochthonous O157:H7 strains from WGS data. Due to budget constraints, we initially 10   

selected the strains which seemed to have circulated more frequently among the Romanian population during the study period, namely the O157:H7 strains belonging to phylogenetic group E. In order to facilitate our WGS approach we made use of open sources of web-based analysis tools to compensate our limited bioinformatics skills. As a matter of fact, the bioinformatics processing and analysis of WGS data was considered by the European Centre

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for Disease Prevention and Control (ECDC) as a great challenge for the strategy to harness WGS to strengthen outbreak investigations and public health surveillance (32). WGS typing allowed us not only to verify the accuracy of the routine typing approach but to expand the microbiological information that can be used for interlaboratory international comparisons and understand how the autochthonous O157:H7 strains fit among similar strains isolated

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elsewhere. VirulenceFinder confirmed that the investigated strains harboured vtx2c in association with intimin γ encoding eae, and ehxA genes, a virulence genotype commonly found in E. coli human strains belonging to this serotype (33, 34) and gave much more information about chromosomally and plasmidic encoded pathogenicity-associated gene

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content of the strains than the PCR-based routine typing. The WGS MLST performed with the E. coli scheme 1 that employed 7 house-keeping genes (35) showed that 3 of the Romanian O157:H7 strains studied were ST1804, a less

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common sequence type according to the data comprised in Escherichia coli EnteroBase (36)

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where only 31 such isolates from the United Kingdom (UK), the United States (US), Spain and Italy are registered. However, the largely spread ST11 clone was also represented by the only vtx-negative O157:H7 strain detected in this study, namely RO_O157_7 strain. Unlike the ST1804 strains, in silico prediction of the GUD activity for this strain showed only the changing amino acid mutations known not to alter the bacterial capacity but not the frameshift mutation caused by G-G insertion at +686 in the uidA gene responsible for the loss of

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GUD expression (37). This strain possessed a plasmid lacking the katP and espP. O157:H7 strains with similar characteristics were previously reported in US (38) and Japan (39). PFGE provided a better strain differentiation than MLST but when compared with WGS to evaluate the genetic relatedness of the O157:H7 strains, the SNP-based genotyping was able to separate the strains with indistinguishable PFGE profiles. When placed among

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other strains belonging to the O157:H7 lineage, used as references for genomic comparisons, the autochthonous ST1804 strains formed a more homogeneous cluster with human strains of the same sequence type isolated from other regions, namely from UK and US. The VirulenceFinder results revealed that with one exception, all these ST1804 strains were vtx2c-positive and shared eae, ehxA, espA, espB, espJ, espP, etpD, iha, iss, katP, nleA, nleB,

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nleC, tir, and toxB genes. The exception was a British vtx2a-positive strain that possessed the eae,  espA, espB, espJ, iha, iss,  nleA, nleB, nleC, and tir but not the plasmid-encoded genes ehxA, katP, espP, and toxB. Of note, type III secretion system effector encoding genes espF and tccP were less commonly present among the ST1804 members, these genes being absent

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from the Romanian strains and also from the ones originated from UK. Comparison of the Romanian ST11 strain with the reference ST11 strains placed the former closer to strains which share a large plasmid lacking katP and espP genes.

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Based on the sequencing data and in silico analyses, we were able to integrate the

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autochthonous strains into the global picture of the O157:H7 population and provide the first evidence of the presence of members of lineages II and I/II belonging to clades 7 and 9, respectively, among Romanian human O157:H7 isolates. Clade 7 was reported as one of most common clades in a study on US EHEC O157 isolates (8) with strains significantly more likely to carry vtx2c, the gene also detected in the Romanian clade 7 strains. The American study (8) as well as another one, conducted in Japan (40), showed that while strains of such clades as 6 and 8 were more frequently found in HUS patients, clade 7 strains 12   

originated predominantly from less severe cases of diarrhea or even from asymptomatic individuals. Only one of the clade 7 Romanian strains was associated with a HUS patient but the limited set of strains investigated prevented us from determining the clinical relevance of the autochthonous clade 7 pathogens. It is worth noting that the clade 9 strain identified in the present study, a ST11

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member, possessed an uidA gene without any base alteration that could affect its expression. Manning et al. previously reported that clade 9 included such strains with GUD positive and sorbitol-positive or negative phenotypes (8).

The low number of strains investigated and the lack of consistent patient information to better link the strain profile with disease outcome and epidemiology were perceived as

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main limitations of this study. However, the data it provided, resulted from an extensive molecular characterisation conducted up to WGS-level resolution, can be used as reference for future research and comparisons with similar human, food or animal isolates at national or international level. Moreover, given the public health importance of members of E. coli

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serogroup O157, accurate characterization and typing are critical for the laboratory-based surveillance and should be consistently performed at least at national reference laboratory level.

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Funding: This work was supported by The Research Institute of the University of

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Bucharest, Romania (ICUB Fellowships for Young Researchers awarded to Mihaela Oprea, November 2016) and by Cantacuzino National Medical - Military Institute of Research and Development, Bucharest, Romania. Conflict of interest: None to declare

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Available

at

Accessed March 23, 2018. 37. Monday SR, Whittam T, Feng P. Genetic and evolutionary analysis of mutations in the gusA gene that caused the absence of β-glucuronidase activity in Escherichia coli O157:H7. J Infect Dis. 2001;184:918–21.

17   

38. Hayes PS, Blom K, Feng P, et al. Isolation and characterization of a β-D-glucuronidaseproducing strain of Escherichia coli O157:H7 in the United States. J Clin Microbiol. 1995;33:3347–8. 39. Nagano H, Okui T, Fujiwara O, et al. Clonal structure of Shiga toxin (Stx)-producing and

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sporadic cases in Hokkaido, Japan. J Med Microbiol. 2002;51:405–16.

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β-D-glucuronidase-positive Escherichia coli O157:H7 strains isolated from outbreaks and

40. Iyoda S, Manning SD, Seto K, et al. Phylogenetic clades 6 and 8 of enterohemorrhagic Escherichia coli O157:H7 with particular stx subtypes are more frequently found in isolates from hemolytic uremic syndrome patients than from asymptomatic carriers. Open Forum Infect Dis. 2014;1(2):ofu061.

M an

Figure caption

Fig. 1. PFGE dendrogram showing the genetic relatedness of the Escherichia coli O157

pt ed

human strains investigated in this study.

Fig. 2. SNP-based phylogenetic tree separated Escherichia coli O157:H7 Romanian strains from one another and from selected published genomes.

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The evolutionary history was inferred by using the Maximum Likelihood method, with 1000

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bootstrap replicates. The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. There were 5674 positions in the final dataset. Evolutionary analyses were conducted in MEGA7 (21).

18   

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t

Table 1. Characteristics of the seven Escherichia coli O157 strains investigated in this study

RO_O157_1

RO_O157_2

RO_O157_3

RO_O157_4

RO_O157_5

RO_O157_6 RO_O157_7

Year of isolation

2016

2016

2016

2016

2016

2016

2017

Patient residence Arges

Neamt

Brasov

Iasi

Bucharest

Neamt

Brasov

Specimen

Stool sample

Stool sample

Stool sample

Stool sample

Stool sample

Underlying

HUS

D

D

D

HUS

Stool sample D

Stool sample D

negative

negative

negative

negative

negative

positive

negative

Serotype

O157:H7

O157:H7

O157:H7

O157:H-

O157:H7

O157:H-

O157:H7

Sequence type

ND2)

ST1804

ST1804

ND

ST1804

ND

ST11

Phylogenetic

D

E

E

A

E

B1

E

yes

ND

yes

ND

no

disease1) Sorbitol

GG insertion in uidA gene

ce p

Ac

group

ted

fermentation

ND

M an

Strain ID

yes

vtx2c

vtx2c

-

vtx2c

eae

+

+

+

+

+

ehxA

-

+

+

-

toxB

ND

+

+

katP

ND

+

espA

ND

espB

-

t

vtx1a, vtx2a

+

+

-

+

ND

+

ND

+

+

ND

+

ND

-

+

+

ND

+

ND

+

ND

+

+

ND

+

ND

+

espF

ND

-

-

ND

-

ND

+

espJ

ND

+

+

ND

+

ND

+

espP

ND

+

+

ND

+

ND

-

etpD

ND

+

+

ND

+

ND

+

iha

ND

+

+

ND

+

ND

+

iss

ND

+

+

ND

+

ND

+

gad

ND

-

+

ND

+

ND

+

nleA

ND

+

+

ND

+

ND

+

nleB

ND

+

+

ND

+

ND

+

M an

ted

ce p

us cr ip

-

Ac

Virulence genes3)

vtx

ND

+

+

ND

+

ND

+

tccP

ND

-

-

ND

-

ND

t

+

tir

ND

+

+

ND

+

ND

+

Clade

ND

7

7

ND

7

ND

9

Lineage

ND

II

II

ND

II

ND

I/II

HUS = hemolytic uremic syndrome, D = diarrhea

2)

not determined

M an

1)

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nleC

3) vtx, eae and ehxA genes were assessed by PCR and in silico WGS analysis and the remaining genes were assessed exclusively

Ac

ce p

ted

by in silico WGS analysis.

Serotype

beta-

sorbitol-

glucuronidas

fermenting

vtx

ST

e activity O157:H7

-

-

1, 2

11

Sakai

O157:H7

-

-

1, 2

11

Frik2000

O157:H7

-

-

1, 2

11

FDAARGOS_2

O157:H7

-

-

1, 2

Xuzhou21

O157:H7

-

-

FORC_044

O157:H7

-

-

-

E09/10

O157:N

+

M C1-057

ce p

O157:H7

Ac

343

O157:H7

-

Year

GenBank/SRA accession number

USA

1982

CP008957.1

Human

Japan

1996

NC_002695.1

Animal

USA

1991

CP015842.1

11

Human

USA

2014

CP022050.1

1, 2

11

Human

China

1999

NC_017906.1

1, 2

11

Human

South

2015

CP016755

ted

93

Country

Food

M an

EDL933

Source

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Strain

t

Table 2. Characteristics of the reference strains used for SNP-based phylogenetic analysis1).

Korea

-

2

11

Human

Holland

2013

LDOZ00000000

+

2

11

Human

Germany

2009

LGBK00000000

-

-

11

Animal

USA

2008

LAZO00000000

+

-

1, 2

11

Human

USA

EC96038

O157:H7

+

+

1, 2

11

Human

Japan

287

O157:N

-

-

-

11

Human

+

+

-

11

Human

+

+

-

11

M 393

O157:N

E09/224

O157:N M

AETX00000000

1996

AMUI00000000

Holland

2013

LGBL00000000

Holland

2013

LGBM0000000

Human

Germany

2009

LGBP00000000

M an

M

1995

t

O157:H7

us cr ip

G5101

O157:H7

-

-

2

1804

Human

UK

2016

SRR5006331

H130660198

O157:H7

-

-

2

1804

Human

UK

2012

SRR1981404

PNUSAE01204

O157:H7

-

-

2

1804

?

USA

?

SRR6667658

O157:H7

-

2

1804

Human

UK

2012

SRR1980991

Ac

H124580172

ce p

5

ted

271122

-

t

ce p

ted

M an

us cr ip

The characteristics of the reference strains were inferred from the information linked to the corresponding databases.

Ac

1)

Table 3. Quality assembly metrics for the sequenced Escherichia coli genomes      Strain ID 

No. of contigs

N50 

227  207  218  229 

146610  146871  142561  122706 

Total contigs  length  5442416  5423585  5344220  5338828 

No. of CDS  (total)  5838  5904  5854  5780 

Ac

ce

pt ed

M an

us cr ip

t

RO_O157_2  RO_O157_3  RO_O157_5  RO_O157_7   

Sequence  coverage  93,76x  108,7x  103,4x  99,57x 

M an

RO_O157_3

ted

RO_O157_2

ce p

t

us cr ip

100

90

80

Strain ID

RO_O157_5

Ac

70

Similarity (%)

RO_O157_7

RO_O157_6

RO_O157_4

RO_O157_1

58 99

us cr ip

t

100 100

271122 RO O157/5 H130660198 RO O157/2 RO O157/3 PNUSAE012045

99

H124580172

100

343 Frik2000

100

100

M an

C1-057 FDAARGOS 293 Xuzhou21 EDL933

100

pt ed

Sakai

100

ce

FORC 044 287

Ac

100

100

100

G5101 EC96038 RO O157/7 100 98

E09/10 393 E09/224

0.05