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Characterization of Pathogenic Escherichia coli in River Water by Simultaneous Detection and Sequencing of 14 Virulence Genes Ryota Gomi, Tomonari Matsuda, Yuji Fujimori, Hidenori Harada, Yasuto Matsui, and Minoru Yoneda Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.5b00953 • Publication Date (Web): 28 Apr 2015 Downloaded from http://pubs.acs.org on May 3, 2015

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Environmental Science & Technology

Characterization of Pathogenic Escherichia coli in River Water by Simultaneous Detection and Sequencing of 14 Virulence Genes Ryota Gomi,† Tomonari Matsuda,*,‡ Yuji Fujimori,§ Hidenori Harada,§ Yasuto Matsui,† and Minoru Yoneda† †

Department of Environmental Engineering, Graduate School of Engineering, Kyoto University, Katsura, Nishikyo-ku, 615-8540, Kyoto, Japan ‡

Research Center for Environmental Quality Management, Kyoto University, 1-2 Yumihama, Otsu, 520-0811, Shiga, Japan §

Graduate School of Global Environmental Studies, Kyoto University, Yoshida-honmachi, Sakyo-ku, 606-8501, Kyoto, Japan

ACS Paragon Plus Environment

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Abstract

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The occurrence of pathogenic Escherichia coli in environmental waters increases the risk of

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waterborne disease. In this study, 14 virulence genes in 669 E. coli isolates (549 isolates from the

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Yamato River in Japan, and 30 isolates from each of the following hosts: humans, cows, pigs,

5

and chickens) were simultaneously quantified by multiplex PCR and dual index sequencing to

6

determine the prevalence of potentially pathogenic E. coli. Among the 549 environmental

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isolates, 64 (12%) were classified as extraintestinal pathogenic E. coli (ExPEC) while eight

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(1.5%) were classified as intestinal pathogenic E. coli (InPEC). Only ExPEC-associated genes

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were detected in human isolates and pig isolates, and 11 (37%) and five (17%) isolates were

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classified as ExPEC, respectively. A high proportion (63%) of cow isolates possessed Shiga-

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toxin genes (stx1 or stx2) and they were classified as Shiga toxin-producing E. coli (STEC) or

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enterohemorrhagic E. coli (EHEC). Among the chicken isolates, 14 (47%) possessed iutA, which

13

is an ExPEC-associated gene. This method can determine the sequences as well as the

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presence/absence of virulence genes. By comparing the sequences of virulence genes, we

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determined that sequences of iutA were different among sources and may be useful for

16

discriminating isolates, although further studies including larger numbers of isolates are needed.

17

Results indicate that humans are a likely source of ExPEC strains in the river.

18 19

Introduction

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The occurrence of pathogenic bacteria in the aquatic environment is a global health concern.

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Among these microbes, pathogenic strains of Escherichia coli are a serious problem and increase


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the risk of waterborne disease.1 Conventionally, the microbial quality of water is monitored by

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detecting fecal indicator bacteria, including E. coli, using selective media.2, 3 However, detection

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based on metabolic phenotype of an organism does not take into account the genetic elements

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involved in pathogenesis.4 Therefore, detection of virulence genes in such organisms is needed to

26

accurately assess the health risks associated with aquatic environments.

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E. coli strains are generally characterized as commensals or harmless bacteria.5 However,

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certain strains may carry virulence genes and can cause intestinal infections such as diarrhea or

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hemorrhagic colitis, or extraintestinal infections such as urinary tract infections and

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sepsis/meningitis. Pathogenic E. coli strains can be classified as intestinal pathogenic E. coli

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(InPEC) or extraintestinal pathogenic E. coli (ExPEC) based on their virulence properties.5-7

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InPEC can be further divided into six well-described pathovars: enterohemorrhagic E. coli

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(EHEC), enteropathogenic E. coli (EPEC), enteroinvasive E. coli (EIEC), enteroaggregative E.

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coli (EAEC), enterotoxigenic E. coli (ETEC), and diffusely adherent E. coli (DAEC).5, 7 Shiga

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toxin-producing E. coli (STEC) is also the term used for any E. coli strain that produces Shiga

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toxin (Stx).5 ExPEC strains carry different combinations of virulence genes from those of InPEC

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strains, and thus cause different clinical symptoms.8 One previous study reported that there are

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genes specific to each pathovar.9 Polymerase chain reaction (PCR) and quantitative-PCR have

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been used to detect and quantify such genes in E. coli isolates.10-14 However, many studies

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analyzing E. coli strains in environmental water have neglected ExPEC strains and targeted only

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InPEC-associated genes4, 12, 15, 16 even though some studies found that almost all pathogenic E.

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coli strains in surface waters were ExPEC.17-19 ExPEC strains are responsible for many deaths

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and are an increasing public health concern.20-23 Therefore, including ExPEC as a target pathovar

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is needed to accurately assess the risk of waterborne disease.


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In a previous study, we developed a method employing multiplex PCR and dual index

46

sequencing for analyzing multiple host-specific genetic markers in multiple E. coli isolates, and

47

identified the likely sources of fecal contamination.24 However, we did not analyze virulence

48

genes in those isolates in the previous study. In order to conduct the risk assessment of

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waterborne disease, information on virulence potential of those isolates is needed. It also has to

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be noted that the protocol used in the previous study was not optimized for the detection of

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longer target sequences. In the present study, we improved and optimized the protocol, and

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applied the method to simultaneously quantify 14 virulence genes (stx1, stx2, eaeA, ipaH, aggR,

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StIb, LtI, daaE, afa/dra, kpsMT II, iutA, papA, papC, sfa/foc), which enabled the differentiation

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of all the pathovars mentioned above, in 669 E. coli isolates (549 isolates from the Yamato River

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in Japan, and 30 isolates from each of the following hosts: humans, cows, pigs, and chickens).

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Prevalence of pathogenic E. coli isolates in river water and known host sources was compared,

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and this information was combined with the source information obtained in our previous study to

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predict the potential sources of pathogenic E. coli in the river. This method can determine not

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only the presence/absence of virulence genes, but also the sequences of the target regions in

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those genes. Virulence genes are horizontally mobile and prone to undergo mutations compared

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with housekeeping genes because these genes are frequently under selective pressure from the

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immune system of the host. For this reason, although sequence information on virulence genes is

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not appropriate for phylogenetic analysis, it is useful for discriminating pathogenic E. coli.25 In

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the present study, sequences of target regions in virulence genes were compared among isolates

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to understand the potential of sequence differences in virulence genes to discriminate and

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characterize isolates. Further characterization of isolates classified as potential ExPEC was also


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performed by the whole genome sequencing of randomly selected isolates to determine the

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distribution of other ExPEC-associated genes and O-serogroups.

69 70

Materials and Methods

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E. coli Strains.

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E. coli strains from known host sources (humans, cows, pigs, and chickens) and the Yamato

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River were isolated between 2011 and 2013 as previously described.24 Locations of sampling

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sites within the Yamato River are shown in Supporting Information Figure S1. Thirty isolates

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from each known host source and 549 isolates from the Yamato River were used and analyzed in

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this study. The seven reference strains used as controls in the multiplex PCR and dual index

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sequencing included E. coli strains KH007 (kpsMT II positive, iutA positive), KCo002 (stx2

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positive, eaeA positive), KCo003 (stx1 positive, stx2 positive), KP003 (kpsMT II positive, papA

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positive, papC positive, sfa/foc positive), KKa001 (afa/dra positive, iutA positive), and KGu002

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(aggR positive). E. coli strain KP002 served as a negative control for virulence genes in the assay.

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Strains KH007, KCo002, KP002, and KP003 were chosen from isolates that were sequenced in

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our previous study (DDBJ accession no. DRP002307).24 Strains KCo003, KKa001, and KGu002

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were chosen from 17 isolates that we sequenced for the present study (DDBJ accession no.

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DRA003179). Sequencing of the 17 isolates was carried out as previously described,24 with a

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few minor modifications. Briefly, DNA was extracted from each E. coli isolate by using a

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DNeasy Blood and Tissue Kit (Qiagen, Hilden, Germany). Sequencing-ready libraries were

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prepared using a Nextera XT DNA Sample Preparation Kit (Illumina, San Diego, CA), and each

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library was sequenced for 500 cycles on the MiSeq (Illumina). Positive controls for ipaH, StIb,

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LtI, and daaE were prepared by mixing synthesized genes with E. coli strain KP002 (negative


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control strain). For example, a positive control for ipaH was prepared by mixing a synthesized

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gene containing the target sequence of ipaH with strain KP002. Positive controls for StIb, LtI,

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and daaE were prepared in the same manner. All genes were synthesized by Eurofins Genomics

93

(Ebersberg, Germany). All E. coli isolates were stored at -85 °C in 35% glycerol until analysis.

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Detection and Sequencing of Virulence Genes by Multiplex PCR and Dual Index

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Sequencing.

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A total of 669 E. coli isolates (549 from the Yamato River and 120 from known host sources)

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and three sets of controls were tested for virulence genes using multiplex PCR and dual index

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sequencing. By using a previously described barcoding strategy,24 all isolates used in our study

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could be analyzed in a single run on the MiSeq. The barcoding strategy consists of two PCRs: a

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multiplex PCR to amplify and add adapters to the target sequences, and a second PCR to add P5

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and P7 amplification primer sequences with dual indices to the adaptered amplicons. Primers for

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multiplex PCR were carefully selected and designed to avoid primer-dimer formations and

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amplify 14 virulence genes simultaneously in a single reaction (Table 1). In this study, forward

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adapter sequence (5’-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG-3’) was added to

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the

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GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAG-3’) was added to the 5’ end of each

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reverse primer in Table 1. First, multiplex PCR was performed on each E. coli isolate using the

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multiplex mixture of 14 sets of primers described above to amplify virulence genes and add

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adapter sequences. The primers were used at final concentrations of 0.1 µM except that the

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forward primer and reverse primer targeting daaE were used at final concentrations of 0.2 µM.

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The PCR mixture (15 µl) was composed of 7.5 µl of 2× Gflex PCR Buffer (Mg2+, dNTP plus)

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(Takara, Otsu, Japan), 0.3µl of Tks Gflex DNA Polymerase (1.25U/µl) (Takara), 5.2 µl of the

5’

end

of

each

forward

primer,

and

reverse

adapter

sequence

(5’-


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multiplex mixture of primers, and 2 µl of cell suspension (1:10 dilution of the glycerol stock).

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All PCRs were performed in the 96-well Hi-Plate for Real Time (Takara) with a Thermal Cycler

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Dice Real Time System 2 (Takara). The reactions were initiated by incubation at 94 °C for 1 min,

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and this was followed by 35 cycles of 98 °C for 10 s, annealing at 60 °C for 15 s, and elongation

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at 68 °C for 1 min. After the multiplex PCR, the PCR product was diluted 100-fold with water

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and used as the template for the second PCR. The primer sequences designed for the second PCR

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were

120

and

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Designing 30 kinds of primer containing different sequences for Index1 and 30 kinds of primer

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containing different sequences for Index2 enabled the differentiation of up to 900 samples. The

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length of each index was 8 bp. The PCR mixture for the second PCR (15 µl) was composed of

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7.5 µl of 2× Gflex PCR Buffer (Mg2+, dNTP plus) (Takara), 0.3µl of Tks Gflex DNA

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Polymerase (1.25U/µl) (Takara), 0.3 µl each of the outer primers (50 µM), 1µl of prepared

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template, and 5.6 µl of ultrapure water. The reactions were initiated by incubation at 94 °C for 1

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min, and this was followed by 10 cycles of 98 °C for 10 s, annealing at 60 °C for 15 s, and

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elongation at 68 °C for 1 min. After the second PCR, 3 µl of each PCR product was transferred

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to one tube and mixed well. A portion (18µl) of the mixture was electrophoresed in a 1.5%

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agarose gel, and agarose containing DNA fragments between 250 and 1000 bp in length was

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excised with a clean razor blade to remove non-specific PCR products such as primer dimers.

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DNA fragments in the excised agarose were purified using Quantum Prep Freeze ‘N Squeeze

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DNA Gel Extraction Spin Columns (Bio-Rad, Hercules, CA). The product was further purified

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using AMPure XP beads (Beckman Coulter Inc., Brea, CA). The final product was sequenced for

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500 cycles on the MiSeq according to the MiSeq System Quick Reference Guide.

5’-AATGATACGGCGACCACCGAGATCTACAC-(Index1)-TCGTCGGCAGCGTC-3’ 5’-CAAGCAGAAGACGGCATACGAGAT-(Index2)-GTCTCGTGGGCTCGG-3’.


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Data Analysis.

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Sequenced reads were sorted into each sample according to Index1 and Index2 sequences and

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analyzed with CLC Genomics Workbench (CLC Bio, Aarhus, Denmark). Reads were initially

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trimmed to remove low-quality or short sequence reads. Trimmed reads were mapped against 14

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virulence genes and the number of mapped reads was counted for each gene. The average

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number of mapped reads (ANMR) was calculated using the data of positive controls for each

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gene. A mapped read count of more than ANMR/10 was determined to be positive. The criteria

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for determination of E. coli pathotypes were defined as follows: the presence of stx1 or/and stx2

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and eaeA for EHEC,26 the presence of eaeA without stx1 or stx2 for EPEC,26 the presence of stx1

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or/and stx2 for STEC,26 the presence of ipaH for EIEC,27 the presence of aggR for EAEC,27 the

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presence of StIb or/and LtI for ETEC,28, 29 the presence of daaE for DAEC,30 and the presence of

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two or more of papA and/or papC; afa/dra; kpsMT II; iutA; and sfa/foc for ExPEC.31,

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Nucleotide sequences of the mapped reads were then extracted and aligned for each virulence

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gene. Primer sequences were removed from both the 5’ and 3’ ends before alignment because

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these sequences were identical among isolates.

32

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Environmental isolates were classified into each host according to the possession patterns of

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host-specific genetic markers developed in the previous study,24 with a few modifications.

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Briefly, isolates that had genetic markers specific to only one host were determined to be from

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the host. In the present study, isolates possessing genetic markers specific to more than one host

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were not classified to avoid the misclassification of strains that can colonize multiple host

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species.33, 34 In addition, isolates having P1, which is a pig-specific genetic marker and encodes a

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fimbrial usher protein, were not classified in this study. This was because sequences that encode

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F1C fimbrial usher protein and are identical to P1 were found in some uropathogenic E. coli


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isolates obtained from human,35,

36

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isolates obtained from human feces.37

and F1C fimbriae are known to be expressed by E. coli

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Further Characterization of Potential ExPEC Strains.

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Fourteen environmental isolates and seven human isolates were randomly selected from

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isolates that were classified as ExPEC, and sequenced on the MiSeq as described above (DDBJ

164

accession no. DRA003504). In total, sequence data of 24 potential ExPEC strains were analyzed

165

to determine the distribution of other virulence factors (VFs) and O-serogroups (one

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environmental isolate and two human isolates had already been sequenced by us and the

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BioSample

168

SAMD00013338). VF reference sequences were prepared based on the reference sequences

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identified by Salipante et al., with a slight modification.38 Reference sequences for O-antigen

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biosynthesis gene clusters (O-AGCs) were prepared based on a complete sequence set of the O-

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AGCs provided by Iguchi et al.39 The presence of VFs and O-serogroups were determined by

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mapping sequence reads against reference sequences and comparing assembled contigs with

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reference sequences by BLAST searches of CLC Genomics Workbench.

numbers

of

these

strains

are

SAMD00027193,

SAMD00013359,

and

174 175

Results and Discussion

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Assay Performance.

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A combination of multiplex PCR and dual index sequencing was used to simultaneously

178

quantify 14 virulence genes in 669 E. coli isolates. Three sets of control strains were tested for

179

virulence genes at the same time to evaluate the specificity and sensitivity of the method. Results

180

in SI Table S1 show the number of reads mapped against each target gene. The results show that

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the genes of positive control strains were precisely detected as sequence reads in all control sets


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and the positively mapped genes in SI Table S1 were consistent with the gene possession

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patterns of control strains. However, some reads (in most cases fewer than 10) were mapped

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against genes that control strains do not possess. This is because of the sequencing errors at bases

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in Index1 or Index2, which led to the misclassification of the sequence reads. In addition, we

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examined the differences of read count among target genes. For example, the average number of

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mapped reads against stx1 was about 600 times higher than that of papA. This was caused by

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differences in amplification efficiencies in the PCR steps. Therefore, we set a threshold, which is

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ANMR/10, for each gene to circumvent these problems. However, we observed false positive

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results in one human isolate after comparing the results with those of the whole genome

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sequencing, so further verification on the threshold may be needed.

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Distribution of Virulence Genes among the Tested E. coli Strains.

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In total, 669 E. coli isolates were tested for 14 virulence genes and classified into eight

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pathotypes according to the presence of different virulence genes (Table 2 and Table 3). Our

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results revealed relatively high numbers of ExPEC-associated genes in E. coli isolates from river

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water. Indeed, it was found that 149 (27%) environmental E. coli isolates possessed at least one

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ExPEC-associated gene. In particular, 113 (21%) environmental isolates possessed kpsMT II,

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which encodes group II capsular polysaccharide units. In our study, most environmental isolates

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that were considered to be pathogenic were classified as ExPEC. These results are consistent

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with those of previous studies, which demonstrated high percentages of ExPEC in surface

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waters.17-19 It is true that we cannot determine whether E. coli isolates that possess virulence

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genes are actually capable of expressing pathogenicity and causing disease without conducting

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an in vivo study.40 However, it should be noted that ExPEC isolates have potential to cause

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disease when they exit the gut and enter a sterile body site,21 and the percentage of potential


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ExPEC isolates in this river was not low considering the fact that the prevalence of E. coli

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isolates carrying virulence genes in environmental water is usually less than 10%.41 In contrast,

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only eight strains were classified as InPEC (one as EAEC and seven as EPEC). There were no

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environmental E. coli isolates that were classified as other InPEC pathotypes. In our study, we

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isolated E. coli strains based on beta-glucuronidase activity. However, E. coli O157:H7 strains

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are known to lack this activity.42, 43 Therefore, it is possible that we could not detect O157:H7

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strains and underestimated the number of EHEC, though the occurrence of O157:H7 strains in

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surface waters has been found to be low.44, 45

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Among the 14 virulence genes tested, only ExPEC-associated genes were detected in E. coli

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isolates from humans, and 11 (37%) of the human isolates were classified as ExPEC. One

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previous study also reported that ExPEC isolates can exist as commensals in the gut of healthy

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humans and constitute the predominant fecal E. coli type in some cases.21 Similarly, only

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ExPEC-associated genes were detected in pig isolates, and five (17%) were classified as ExPEC.

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A different observation was made for cow isolates. At least one of Shiga toxin gene (stx1 or stx2)

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was detected in 19 (63%) cow isolates, and they were classified as STEC. Four cow isolates were

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classified as EHEC because they had both Shiga-toxin genes and eaeA, which encodes intimin.

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These results support the report that ruminants, especially cattle, are known to be the main

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reservoirs of STEC, shed the strains in their feces, and have potential to be a primary source of

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environmental outbreaks of STEC infection in humans.46 Virulence gene eaeA was detected in

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three (10%) chicken isolates and those isolates were classified as EPEC. Although the ExPEC-

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associated gene iutA was detected in 14 (47%) chicken isolates, they were not classified as

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ExPEC because they did not meet the criteria (the presence of two or more of ExPEC-associated

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genes).


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Table 4 shows the prevalence of ExPEC-associated genes and ExPEC strains among identified

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sources of environmental E. coli isolates. Among 64 environmental isolates that were classified

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as ExPEC, 18 (28%) were classified as human, whereas only two isolates were classified as other

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sources (chicken), indicating that humans are a likely source of ExPEC strains in the river. In the

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present study, most pathogenic isolates from river water were classified as ExPEC, and 37% of

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isolates obtained from humans were classified as ExPEC, which also supports this prediction.

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However, the proportion is relatively low considering that 145 environmental isolates were

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classified as human. These results can be attributed to two reasons. First, wastewater treatment

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processes reduce the prevalence of pathogenic E. coli, including ExPEC strains.47 However, it

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should be noted that one previous study demonstrated that some E. coli strains carrying ExPEC-

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associated genes can survive all treatment processes of sewage treatment plants.48 Second, some

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pathogenicity islands are unstable and can be deleted from the genome by environmental

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stimuli.49 We observed environmental isolates that did not possess any host-specific genetic

241

markers but were classified as ExPEC. We also observed two ExPEC strains that did not have

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human-specific markers among 30 strains obtained from humans. Moreover, we did not consider

243

sources other than humans, cows, pigs, or chickens. Therefore, those ExPEC isolates may have

244

originated from humans or other sources that were not considered in this study. There is some

245

inconsistency between the virulence gene profiles of E. coli isolates obtained from actual hosts

246

(Table 2) versus assigned hosts (Table 4). This may be because a limited fraction of the diversity

247

within each host was sampled in our studies (30 isolates from each source) and, therefore, did not

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cover all possible gene possession patterns in those hosts.

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Sequence Analysis of Virulence Genes.


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The sequences of target regions in virulence genes were aligned and compared among isolates

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to determine whether a sequenced-based comparison of these genes could be applied to

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discriminate E. coli isolates. Among the virulence genes compared, we found that sequences of

253

the target region in iutA, which encodes aerobactin receptor, were different among hosts and can

254

be used for discriminating E. coli isolates (Table 5 and Figure 1). We observed three different

255

alleles (I, II, and III) in 31 iutA-positive isolates from known host sources, and two different

256

alleles (I and III) in 40 iutA-positive isolates from river water. Isolates from a specific source

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mainly had one allele. For example, all of the iutA-positive human isolates had iutA allele I,

258

while all of the iutA-positive chicken isolates had iutA allele II. Interestingly, we found that the

259

only sequence that had 100% identity to allele II was from avian pathogenic E. coli (APEC) by

260

using the BLAST tool at the National Center for Biotechnology Information (NCBI) Web site

261

(http://blast.ncbi.nlm.nih.gov/Blast.cgi). Moreover, three environmental isolates that had iutA

262

allele III, which was mainly shared among pig and cow isolates, also possessed cow-specific

263

markers developed in our previous study and classified as cow. On the other hand, APEC strains

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having the identical sequence to the iutA allele I were reported in previous studies,50, 51 and 10

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iutA-positive environmental isolates that possessed chicken-specific markers developed in our

266

previous study and were classified as chicken also had iutA allele I. These results indicate that,

267

although further studies including larger numbers of isolates and geographically diverse animal

268

hosts are needed for the verification, sequences of iutA may be useful for identifying sources of

269

E. coli isolates possessing this gene.

270

Further Characterization of Potential ExPEC Strains.

271

ExPEC strains have been defined as strains having two or more of papA and/or papC; afa/dra;

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kpsMT II; iutA; and sfa/foc.31, 32 However, it is important to analyze the presence of other VFs


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because the number of ExPEC-associated virulence genes in an isolate is proportional to its

274

pathogenic potential.41, 52 In total, 24 potential ExPEC isolates obtained from river water and

275

humans were randomly selected and further characterized by whole genome sequencing (SI

276

Table S2). We should point out that results in the table were obtained from a small number of

277

isolates, and a limited fraction of the diversity was sampled regarding human isolates. Actually,

278

we observed isolates that had identical or similar genome sequences among nine human isolates.

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Therefore, we did not calculate the significance such as similarities or differences between

280

environmental isolates and human isolates. Regardless of these facts, it is notable that some

281

virulence genes such as yersiniabactin-associated genes (e.g., fyuA, ybtE, ybtT, ybtU, irp1, and

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irp2) were detected from all 24 isolates. Among the toxin genes, hlyA (hemolysin), sat (secreted

283

autotransporter toxin), cnf1 (cytotoxic necrotizing factor 1), and vat (vacuolating autotransporter

284

toxin) were detected in 8 (53%), 3 (20%), 8 (53%), and 13 (87%) environmental isolates and in 0,

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6 (67%), 0, and 3 (33%) human isolates. These toxin genes are associated with uropathogenic E.

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coli (UPEC), meningitis-associated E. coli (MNEC), or septicemia-causing pathogenic E. coli

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(SEPEC), which are subtypes of ExPEC.47 Other VFs needed for adherence (e.g., papA, papC,

288

and papG) and iron acquisition (e.g., iutA, sitA, and sitD) were also detected in a relatively high

289

percentage of isolates, indicating that potential ExPEC isolates identified in this study may be

290

actually capable of expressing pathogenicity. We also determined O-serogroups of the 24

291

isolates because certain O-serogroups are frequently detected in and associated with ExPEC

292

strains.53-55 SI Table S3 shows O-serogroups among the 24 isolates. In total, 9 different O-

293

serogroups were detected in the analyzed isolates. Regarding the 22 isolates that could be

294

assigned to single O-serogroups, all belong to O-serogroups that are commonly associated with

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ExPEC.53-55 These results are consistent with the results of the VF analysis.


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In conclusion, we applied multiplex PCR and dual index sequencing to determine the

297

prevalence of 14 virulence genes in 669 E. coli isolates. We identified that most pathogenic E.

298

coli isolates obtained from the Yamato River were classified as ExPEC. ExPEC strains were also

299

prevalent among isolates obtained from humans and pigs. On the other hand, 63% of cow

300

isolates were classified as STEC, and no pathogenic E. coli, according to the criteria, was

301

detected from chicken isolates. Prevalence of pathogenic E. coli isolates in river water and

302

known host sources was compared, and this information was combined with the source

303

information obtained in our previous study to predict the sources of ExPEC strains in the river

304

water. Results obtained by the prevalence analyses indicate that humans are a likely source of

305

ExPEC strains in the river. Comparison of sequences of virulence genes revealed that sequences

306

of iutA were different among sources and may be useful for discriminating isolates. However,

307

further studies including larger numbers of isolates and geographically diverse animal hosts are

308

needed to validate the discriminating power of this marker.


15


Page 16 of 24

Figure 1. Nucleotide sequence alignment of three iutA alleles. Primer sequences were removed and target sequences of 259 bp were aligned. The dots indicate nucleotide identity to iutA allele I.

Table 1. Primer Sets Used in This Study gene

sequence (5’-3’)

product size (bp)

reference

stx1

F-ATAAATCGCCATTCGTTGACTAC

180

Paton et (1998)56

al.

255

Paton et (1998)56

al.

384

Paton et (1998)56

al.

171

This studya

254

Toma et (2003)27

al.

171

Muller et (2007)57

al.

322

Toma et (2003)27

al.

R-AGAACGCCCACTGAGATCATC stx2

F-GGCACTGTCTGAAACTGCTCC R-TCGCCAGTTATCTGACATTCTG

eaeA

F-GACCCGGCACAAGCATAAGC R-CCACCTGCAGCAACAAGAGG

ipaH

F-CCTTTTCGATAATGATACCG R-GTGGAGAGCTGAAGTTTCTCTGC

aggR

F-GTATACACAAAAGAAGGAAGC R-ACAGAATCGTCAGCATCAGC

StIb

F-TGTCTTTTTCACCTTTCGCTC R-CGGTACAAGCAGGATTACAACAC

LtI

F-TCTCTATGTGCATACGGAGC R-CCATACTGATTGCCGCAAT


16

Page 17 of 24


daaE

F-GAACGTTGGTTAATGTGGGGTAA

542

Vidal et 30 (2005)

594

Johnson et al. (2000)31

272


302


717


205


410


R-TATTCACCGGTCGGTTATCAGT afa/dra

F-GGCAGAGGGCCGGCAACAGGC R-CCCGTAACGCGCCAGCATCTC

kpsMT II F-GCGCATTTGCTGATACTGTTG R-CATCCAGACGATAAGCATGAGCA iutA

F-GGCTGGACATCATGGGAACTGG R-CGTCGGGAACGGGTAGAATCG

papA

F-ATGGCAGTGGTGTCTTTTGGTG R-CGTCCCACCATACGTGCTCTTC

papC

F-GTGGCAGTATGAGTAATGACCGTTA R-ATATCCTTTCTGCAGGGATGCAATA

sfa/foc

F-CTCCGGAGAACTGGGTGCATCTTAC R-CGGAGGAGTAATTACAAACCTGGCA

al.

a

Primers used for amplification of ipaH were designed from sequences obtained from the following GenBank accession numbers: AF386526, AL391753, AF348706, M76445, and M32063.

Table 2. Occurrence of Virulence Genes in 669 E. coli Isolates no. of isolates (%) gene

river water

human

cow

pig

chicken

(n=549)

(n=30)

(n=30)

(n=30)

(n=30)

stx1

0

0

6 (20)

0

0

stx2

0

0

16 (53)

0

0

eaeA

7 (1)

0

4 (13)

0

3 (10)

ipaH

0

0

0

0

0

aggR

1 (0.2)

0

0

0

0


17


Page 18 of 24

StIb

0

0

0

0

0

LtI

0

0

0

0

0

daaE

0

0

0

0

0

afa/dra

3 (0.5)

0

0

0

0

kpsMT II

113 (21)

11 (37)

2 (7)

3 (10)

0

iutA

40 (7)

11 (37)

3 (10)

3 (10)

14 (47)

papA

44 (8)

3 (10)

0

2 (7)

0

papC

50 (9)

3 (10)

0

2 (7)

0

sfa/foc

29 (5)

0

0

5 (17)

0

Table 3. Pathotype Assignments of 669 E. coli Isolates Based on Virulence Gene Content no. of isolates (%) pathotype

river water

human

cow

pig

chicken

(n=549)

(n=30)

(n=30)

(n=30)

(n=30)

EHEC

0

0

4 (13)

0

0

EPEC

7 (1)

0

0

0

3 (10)

STEC

0

0

19 (63)

0

0

EIEC

0

0

0

0

0

EAEC

1 (0.2)

0

0

0

0

ETEC

0

0

0

0

0

DAEC

0

0

0

0

0

ExPEC

64 (12)

11 (37)

2 (7)

5 (17)

0

Table 4. Distribution of ExPEC-Associated Genes among Identified Sources of Environmental E. coli Isolatesa


18

Page 19 of 24


bacterial trait number of traits within the identified source (%) or gene isolates isolates isolates isolates classified as classified as classified as classified human cow pig as chicken

unclassified (n=328)

(n=145)

(n=17)

(n=2)

(n=57)

afa/dra

1 (1)

0

0

0

2 (0.6)

kpsMT II

24 (17)

1 (6)

0

10 (18)

78 (24)

iutA

11 (8)

3 (18)

0

10 (18)

16 (5)

papA

13 (9)

0

0

0

31 (9)

papC

13 (9)

0

0

0

37 (11)

sfa/foc

0

0

0

0

29 (9)

ExPEC

18 (12)

0

0

2 (4)

44 (13)

a

Sources of environmental isolates were identified based on the possession patterns of hostspecific genetic markers developed in our previous study.24 Detailed source identification methods are provided in Materials and Methods.

Table 5. Distribution of iutA Alleles in iutA-Positive E. coli Isolates allele

no. of isolates (%) river water

Human

cow

pig

chicken

(n=40)

(n=11)

(n=3)

(n=3)

(n=14)

I

37 (92.5)

11 (100)

0

1 (33.3)

0

II

0

0

0

0

14 (100)

III

3 (7.5)

0

3 (100)

2 (66.7)

0

ASSOCIATED CONTENT Supporting Information. Figure S1 and Table S1−S3. This material is available free of charge via the Internet at http://pubs.acs.org.


19


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AUTHOR INFORMATION Corresponding Author *Phone: +81-77-527-6224 Fax: +81-77-524-9869. E-mail: [email protected]

ACKNOWLEDGEMENTS This research was supported by KAKENHI (23221006), Kyoto University’s Global Survivability Studies (GSS) program, and the River Fund in charge of The River Foundation, Japan. The authors declare no competing financial interest.

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Characterization of Pathogenic Escherichia coli in River Water by

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