Open Access

Draft genomes of four enterotoxigenic Escherichia coli (ETEC) clinical isolates from China and Bangladesh

  • Fei Liu1, 2,
  • Xi Yang1,
  • Zhiyun Wang1,
  • Matilda Nicklasson3,
  • Firdausi Qadri4,
  • Yong Yi5,
  • Yuying Zhu1,
  • Na Lv1,
  • Jing Li1,
  • Ruifen Zhang1,
  • Huijuan Guo1,
  • Baoli Zhu1, 2,
  • Åsa Sjöling3, 6Email author and
  • Yongfei Hu1, 2Email author
Gut Pathogens20157:10

https://doi.org/10.1186/s13099-015-0059-z

Received: 19 December 2014

Accepted: 19 March 2015

Published: 8 April 2015

Abstract

Background

Enterotoxigenic Escherichia coli (ETEC) is an important pathogen that causes childhood and travelers’ diarrhea. Here, we present the draft genomes of four ETEC isolates recovered from stool specimens of patients with diarrhea in Beijing, China and Dhaka, Bangladesh, respectively.

Results

We obtained the draft genomes of ETEC strains CE516 and CE549 isolated in China, and E1777 and E2265 isolated in Bangladesh with a length of 5.1 Mbp, 4.9 Mbp, 5.1 Mbp, and 5.0 Mbp, respectively. Phylogenetic analysis indicated that the four strains grouped with the classical Escherichia coli phylogenetic groups A and B1 and three of them including a multi drug-resistant Chinese isolate (CE549) belonged to two major ETEC lineages distributed globally. The heat stable toxin (ST) structural gene (estA) was present in all strains except in strain CE516, and the heat labile toxin (LT) operon (eltAB) was present in all four genomes. Moreover, different resistance gene profiles were found between the ETEC strains.

Conclusions

The draft genomes of the two isolates CE516 and CE549 represent the first genomes of ETEC reported from China. Though we revealed that ETEC is uncommon in Beijing, China, however, when it does occur, multi-drug resistance and ESBL positive isolates might pose a specific public health risk. Furthermore, this study advances our understanding of prevalence and antibiotic resistance of ETEC in China and adds to the number of sequenced strains from Bangladesh.

Keywords

ETEC Virulence factors Antibiotic resistance

Background

ETEC infections are an important cause of childhood diarrhea resulting in significant morbidity and mortality, primarily among children aged <5 years living in developing countries [1] as well as travelers visiting these countries [2]. ETEC is characterized by the presence of the heat-labile toxin (LT) and/or the heat-stable toxin (ST), both of which are plasmid encoded [3]. The presence of virulence factors such as enterotoxins and colonization factors differentiate ETEC from other categories of diarrheagenic E. coli. [4]. Colonization factors (CFs) enable ETEC bacteria to adhere to the intestinal epithelium [5]. At present more than 25 different CFs have been identified [5]. In addition to the CFs, other putative factors involved in ETEC pathogenesis were also identified, such as EtpA and EatA. EtpA can act as a bridge between the bacterial flagella and host epithelial cells [6] and EatA is a protein of the serine protease autotransporters of the Enterobacteriaceae (SPATE) family [7].

For a long time E. coli H10407 and E24377A were the only two ETEC strains infecting humans that have their genomes completely sequenced together with a draft genome of ETEC strain B7A [8,9]. Recently whole genome sequences of additional draft genomes were published [10]. A comprehensive analysis of 362 ETEC genomes from strains isolated globally over three decades identified that ETEC distribute into several conserved monophyletic lineages that have distrubuted globally [11] . In this study we analysed four additional ETEC strains with the aim to compare additional ETEC isolated in China and Bangladesh with the global collection and to better understand the dissemination of the pathogen. We also included two additional Bangladeshi strains to increase the number of sequenced genomes from Bangladeshi ETEC strains.

Methods

Strain selection

To assess the frequency of ETEC in Beijing, China, we investigated patients presenting with acute watery diarrhea at four hospitals between 2010 and 2011. This research was approved by the Research Ethics Committee of the Institute of Microbiology, Chinese Academy of Sciences. ETEC isolates were recovered after streaking diarrheal samples on to MacConkey agar followed by PCR confirmation for ETEC-specific enterotoxins [12]. In total, 880 cases were enrolled and tested for ETEC but ETEC was only recovered from three cases (0.3%). The two ETEC isolates CE516 and CE549 from China were recovered from stool of patients that tested negative for Vibrio cholerae, Shigella spp and Salmonella spp. CE549 expressed the heat-labile enterotoxin (LT) and the human heat-stabile enterotoxin (STh) in combination with CFs CS2, CS3 and CS21; CE516 expressed LT and CS6, CS8. Antimicrobial susceptibility was determined using the VITEK 2 Gram Negative Susceptibility Test Cards AST-GN04 and AST-GN 13 (Biomerieux, Marcy l’Etoile France). CE549 was resistant to 14 of the 22 antibiotics tested (cefuroxime axetil, sulfamethoxazole, ampicillin, tobramycin, ceftriaxone, aztreonam, piperacillin, cefuroxime, cefazolin, ceftazidime, cefepime, levofloxacin, gentamicin, ciprofloxacin, and extended spectrum beta lacatamase (ESBL) positive), while CE516 showed sensitive to all 22 antibiotics and was ESBL negative.

The two ETEC isolates E1777 and E2265 were collected from adult Bangladeshi patients that sought medical attention for severe diarrhea in hospital facilities in April 2005 and March 2006 during the bi-annual ETEC epidemic peaks in Dhaka, Bangladesh [13]. Stool samples were confirmed to be negative for Vibrio cholerae, Shigella ssp and Salmonella ssp. MacConkey agar plates were used for identification of lactose fermenting E. coli like colonies selection followed by PCR confirmation for ETEC [12]. The strains were further characterized by immunodiagnostic methods for toxins and colonization factors [12]. Both isolates expressed the common virulence factor combination of the enterotoxins heat labile toxin LT and heat stable toxin STh and the CFs CS5 and CS6.

Genome sequencing, assembly and annotation

DNA was extracted from bacterial cells cultured in Luria broth (LB) medium using the DNA Tissue and Blood kit (Qiagen, Duesseldorf, Germany). Genome sequencing work was carried out at the Microbial Genome Research Center, Institute of Microbiology, Chinese Academy of Sciences, Beijing. The genome sequences of each ETEC isolate were generated using paired-end libraries with 350 ~ 400 bp inserts on an Illumina GAIIX (Illumina, San Diego, CA, USA). The detailed methods for genome assembly were described in another paper [14]. Genome sequences were annotated by using Subsystem Technology (RAST) [15]. The functions of predicted protein-coding genes were then annotated through comparisons with the databases of NCBI-NR, and COG. To search the antibiotic resistance genes, the protein-coding sequences were aligned against Antibiotic Resistance Database (ARDB) [16], using similarity thresholds as recommended in ARDB.

Multiple locus sequence typing (MLST)

We used MLST system including the following seven housekeeping genes: adk, fumC, gyrB, icd, mdh, purA, and recA [17], which were extracted from draft genome sequences and were compared to allele profiles in the MLST database (http://mlst.warwick.ac.uk/mlst/dbs/Ecoli/documents/primersColi_html).

Comparative genomics

For comparative genomic analysis, genome sequences of 13 previously reported isolates including Escherichia coli B7A (GenBank accession number NZ_AAJT02000001.1), E24377A (NC_009786.1), H10407 (NC_017633.1), IAI39 (NC_011750.1), O127 H6 E2348 69 (NC_011601.1), O157 H7 EC4115 (NC_011350.1), O157 H7 EDL933 (NC_002655.2), O157 H7 TW14359 (NC_013008.1), O157 H7 Sakai (NC_002127.1), SMS 3 5 (NC_010485.1), TW10598 (NZ_AELA01000001.1), TW10722 (NZ_AELB01000001.1), and TW10828 (NZ_AELC01000001.1) were downloaded from the NCBI website (Table 1). Multiple sequence alignments of Escherichia coli genomes were performed with Mugsy [18]. The trees were constructed based on core SNPs (single nucleotide polymorphisms) from whole genome alignment by using the maximum-likelihood method in Phylogeny Inference Package (http://evolution.genetics.washington.edu/phylip.html). The map of ORF comparisons among E. coli genomes was constructed using Circos [19].
Table 1

Reference strains used for this study

Strain

GenBank BioSample

Accession number

Collection date

Isolation source

Genome size (bp)

GC content

B7A

SAMN02435852

NZ_AAJT02000001.1

\

\

5,300,242

50.7%

E24377A

SAMN02604038

NC_009786.1

\

\

5,249,288

50.6%

H10407

SAMEA2272237

NC_017633.1

prior to 1973

\

5,325,888

50.7%

IAI39

SAMEA3138234

NC_011750.1

\

\

5,132,068

50.6%

O127 H6 E2348/69

SAMEA1705959

NC_011601.1

1969

\

5,069,678

50.5%

O157 H7 EC4115

SAMN02603441

NC_011350.1

\

\

5,704,171

50.4%

O157 H7 EDL933

SAMN02604092

NC_002655.2

\

\

5,620,522

50.4%

O157 H7 TW14359

SAMN02604255

NC_013008.1

\

\

5,622,737

50.5%

O157 H7 Sakai

SAMN01911278

NC_002127.1

1996

Human feces

5,594,477

50.5%

SMS-3-5

SAMN02604066

NC_010485.1

\

\

5,215,377

50.5%

TW10598

SAMN02436015

NZ_AELA01000001.1

\

\

5,243,318

50.6%

TW10722

SAMN02435971

NZ_AELB01000001.1

\

\

5,689,893

50.5%

TW10828

SAMN02435898

NZ_AELC01000001.1

\

\

5,280,267

50.6%

Quality assurance

The genomic DNA was isolated from pure bacterial isolate and was further confirmed with 16S rRNA gene sequencing. Bioinformatic assessment of potential contamination of the genomic library by allochthonous microorganisms was done using PGAAP and RAST annotation system.

Initial findings

Genome characteristics

Through genome assembly, we obtained 99 scaffolds of 5,068,634 bp for CE516, 137 scaffolds of 4,859,890 bp for CE549, 150 scaffolds of 5,117,746 bp for E1777, and 142 scaffolds of 4,946,932 bp for E2265 (Table 2). RAST annotation of the whole genome indicated the presence of 611, 590, 605, and 605 SEED subsystems in CE516, CE549, E1777, and E2265, respectively. Table 3 shows the comparison of genomic features of the four sequenced ETEC genomes.
Table 2

Genomic characteristics of the 4 ETEC genomes

Sample name

Country

MLST

Colonization factors

ST

LT

Read length (bp)

Genome coverage

GC content

Scaffold number

CE516

China

1490

CS6, CS8

-

+

101

300x

50.5%

99

CE549

China

4

CS2, CS3, CS21

+

+

101

300x

50.6%

137

E1777

Bangladeshi

443

CS5, CS6

+

+

101

200x

50.4%

150

E2265

Bangladeshi

443

CS5, CS6

+

+

101

200x

50.3%

142

Table 3

Comparisons of subsystem features among the 4 ETEC genomes

Subsystem features

Number of CDS present in ETEC strains

CE516

CE549

E1777

E2265

Amino Acids and Derivatives

400

391

392

395

Carbohydrates

781

756

752

754

Cell Division and Cell Cycle

39

40

38

37

Cell Wall and Capsule

267

273

272

273

Cofactors, Vitamins, Prosthetic Groups, Pigments

285

285

287

284

DNA Metabolism

129

147

153

134

Dormancy and Sporulation

4

5

5

5

Fatty Acids, Lipids, and Isoprenoids

142

131

132

131

Iron acquisition and metabolism

22

22

22

22

Membrane Transport

291

190

268

270

Metabolism of Aromatic Compounds

44

5

30

30

Miscellaneous

67

63

66

64

Motility and Chemotaxis

80

130

80

80

Nitrogen Metabolism

77

75

77

77

Nucleosides and Nucleotides

146

150

147

144

Phages, Prophages, Transposable elements, Plasmids

130

32

160

146

Phosphorus Metabolism

53

53

53

53

Photosynthesis

0

0

0

0

Potassium metabolism

29

29

28

30

Protein Metabolism

299

290

298

300

Regulation and Cell signaling

160

156

160

163

Respiration

192

190

194

192

RNA Metabolism

248

251

250

250

Secondary Metabolism

27

26

26

26

Stress Response

184

181

186

184

Sulfur Metabolism

59

54

56

56

Virulence, Disease and Defense

109

108

110

130

Phylogenetic analysis

A maximum-likelihood tree of the sequenced 4 genomes and 13 publicly available Escherichia coli complete genomes which represent the classical phylogenetic groups (A, B1, B2, D, and E) were created based on core SNPs from whole genome alignment (Figure 1). The sequenced strains in this study grouped with the classical Escherichia coli phylogenetic groups A and B1. Specifically, strains CE549, H10407 and TW10598 which belong to group A were grouped together, while other sequenced strains which belong to group B1 as well as the previously sequenced strains formed a clade. Strains CE549 and TW10598 are closely related to each other, while strains E1777 and E2265 are closely related to each other. MLST analysis was used to compare the strains to a global collection of ETEC [11]. Three strains were found to belong to the major lineages described in ETEC [11]. Strains E1777 and E2265 belong to the global lineage L5 which express LT STh CS5 + CS5, while strain CE156, the multi drug-resistant isolate belongs to the conserved ETEC lineage L2 that is distributed globally [11]. The Chinese strain CS516 belonged to a MLST type previously identified in Bangladeshi and Egyptian ETEC strains [11].
Figure 1

Phylogenetic relationships of E. coli strains based on SNPs from whole genome sequences. The trees were constructed by the maximum-likelihood method. Scale bar indicates nucleotides substitutions per site.

Genomic variants among ETEC strains

We compared proteins from the 4 draft genomes and 6 references within groups A and B1 with that from H10407 using BLASTP and revealed many large variable regions (VR1 to VR10) (Figure 2). Among these VRs, VR3 and VR10 (regions of 5,072 to 5,121 kb) were predicted to be prophage loci which were highly variable among all strains. Interestingly, all strains within group B1 lack VR7 gene cluster encoding general secretory pathway associated genes. In addition, region 2,405 to 2,414 kb adjacent to VR4, which encoded ribitol metabolism related genes, was presented within group A but not detected within group B1.
Figure 2

ORF comparisons of E. coli genomes. Proteins from the 4 genomes and 6 references within groups A and B1 were aligned using H10407 as a reference. Track shows a plot of G + C contents. Circles from inside to outside are the BLASTP percent identities of H10407 against ORFs of H10407, TW10598, CE549, TW10722, E1777, E2265, E24377A, CE516, TW10828, B7A. Red is 90–100% identity, yellow is 60–89% identity, blue is 0–59% identity.

Virulence factors

The strains were analyzed for presence of known ETEC virulence factors. Strains E1777, E2265, and CE549 contained both LT and ST genes (Table 4). The ST structural gene (estA) was present in all strains except in strain CE516, while the LT structural gene (eltA) was present in all four genomes. In addition, genes clyA (cytolysin), eatA (serine protease autotransporter), and ecpA (pilus subunit) were also present in all of the 4 ETEC strains, but genes leoA (accessory protein for LT secretion), tibA (autotransporter), and tia (surface protein) were absent in all genomes. Only CE549 contained the complete ~14 kb operon encoding longus known as a type IV pilus [20]. The etpA gene, which mediates adhesion between ETEC flagella and host cells [6], was present only in CE549 but absent in other strains. These specific virulence factors present in CE549 may increase its virulence in humans, but their functional effects remain to be further determined.
Table 4

Virulence factors present or absent in the 4 ETEC genomes

Virulence factor

CE516

CE549

E1777

E2265

clyA

1

1

1

1

eatA

1

1

1

1

ecpA

1

1

1

1

eltA

1

1

1

1

estA

0

1

1

1

etpA

0

1

0

0

fimH

1

1

1

1

leoA

0

0

0

0

lngA

0

1

0

0

tia

0

0

0

0

tibA

0

0

0

0

“1” and “0” denotes the presence and absence of the corresponding virulence factors.

Antibiotic resistance genes

We compared all the protein-coding genes from the 4 ETEC strains with known antibiotic resistance genes [16] and found many kinds of antibiotic resistance genes, such as macrolide, tetracycline, fosmidomycin and polymyxin resistance genes (Table 5), most of which were annotated as Multidrug resistance efflux pump. Interestingly, strain CE549 has two tetracycline resistance genes that were not identified in the other 3 isolates. In addition, different resistance genes profiles were found between ETEC strains from different countries. For instance, the resistant type EmrE was only identified in the two strains isolated from China.
Table 5

Putative antibiotic resistance genes in the 4 ETEC strains determined using the antibiotic resistance genes database

Resistance type

Description

Resistance profile

CE516

CE549

E1777

E2265

acrA

Multidrug resistance efflux pump.

aminoglycoside, glycylcycline, beta_lactam, macrolide, acriflavin

*

*

*

*

acrB

*, *

*, *

*, *, *

*, *, *

arnA

The modified arabinose is attached to lipid A and is required for resistance to polymyxin and cationic antimicrobial peptides.

polymyxin

*

*

*

*

bacA

Undecaprenyl pyrophosphate phosphatase, which consists in the sequestration of Undecaprenyl pyrophosphate.

bacitracin

*

*

*

*

bcr

  

*

*

*

*

bl1_ec

Class C beta-lactamase.

cephalosporin

*

*

*

*

emrD

Multidrug resistance efflux pump.

 

*

*

*

*

emrE

aminoglycoside

*, *, *

*

  

ksgA

Its inactivation leads to kasugamycin resistance.

kasugamycin

*

*

*

*

macB

Macrolide-specific efflux system.

macrolide

*

*

*

*

mdfA

  

*

*

*

*

mdtE

Multidrug resistance efflux pump.

doxorubicin, erythromycin

*

*

*

*

mdtF

*

*

*

*

mdtG

Multidrug resistance efflux pump.

deoxycholate, fosfomycin

*

*

*

*

mdtH

*

*

*, *

*, *

mdtK

enoxacin, norfloxacin

*

*

*

*

mdtL

chloramphenicol

*

*

*

*

mdtM

chloramphenicol, acriflavine, norfloxacin

*

*

*

*

mdtN

Multidrug resistance efflux pump.

t_chloride, acriflavine, puromycin

*

*

*

*

mdtO

*

*

*

*

mdtP

*

*

*

*

rosB

Efflux pump/potassium antiporter system. RosB: Potassium antiporter.

fosmidomycin

*

*

*

*

tetC

Major facilitator superfamily transporter, tetracycline efflux pump.

tetracycline

 

*, *

  

tolC

Multidrug resistance efflux pump.

aminoglycoside, glycylcycline, beta_lactam, macrolide, acriflavin

*

*

*

*

“*” means one homolog of the antibiotic resistance gene is found.

Future directions

This study analyzed the prevalence of ETEC in Beijing, China and it was found that ETEC is not common. However the results reveal for the first time to our knowledge that a strain that belong to the globally distributed ETEC lineage L2 is multi resistant. This might have important implications for transmission of multi resistant ETEC strains as well as treatment of ETEC diarrhea and needs to be further addressed. The Chinese genomes presented here together with the two novel Bangladeshi ETEC genomes, will be valuable for future comparative genomic analysis of ETEC and will aid in molecular characterization of this important diarrheal pathogen.

Availability of supporting data

The genome sequences of ETEC strains CE516, CE549, E1777 and E2265 reported in this paper have been deposited in the GenBank under accession numbers JTGM00000000, JTGK00000000, JTHI00000000 and JUBB00000000, respectively.

Declarations

Acknowledgements

This work was supported by National Natural Science Foundation of China (grant 31270168 and 81401701), the National Basic Research Program of China (973 Program: grant 200CB504800), the Beijing Municipal Natural Science Foundation (5152019), the Swedish Research Council (grant no 521-2011-3435) and the Swedish Research Links (348-2011-7292) to ÅS and BLZ.

Authors’ Affiliations

(1)
CAS Key Laboratory of Pathogenic Microbiology & Immunology, Institute of Microbiology, Chinese Academy of Sciences
(2)
Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, The First Affiliated Hospital, College of Medicine, Zhejiang University
(3)
Department of Microbiology and Immunology, Institute of Biomedicine, University of Gothenburg
(4)
International Centre for Diarrhoeal Disease Research, Bangladesh
(5)
Clinical Diagnostic Center, 306nd Hospital of the People’s Liberation Army
(6)
Department of Microbiology, Tumor and Cell Biology, Karolinska Institutet

References

  1. Qadri F, Svennerholm AM, Faruque AS, Sack RB. Enterotoxigenic Escherichia coli in developing countries: epidemiology, microbiology, clinical features, treatment, and prevention. Clin Microbiol Rev. 2005;18:465–83.View ArticlePubMed CentralPubMedGoogle Scholar
  2. Black RE. Epidemiology of travelers’ diarrhea and relative importance of various pathogens. Rev Infect Dis. 1990;12 Suppl 1:S73–9.View ArticlePubMedGoogle Scholar
  3. Fleckenstein JM, Hardwidge PR, Munson GP, Rasko DA, Sommerfelt H, Steinsland H. Molecular mechanisms of enterotoxigenic Escherichia coli infection. Microbes Infect. 2010;12:89–98.View ArticlePubMedGoogle Scholar
  4. Nataro JP, Kaper JB. Diarrheagenic Escherichia coli. Clin Microbiol Rev. 1998;11:142–201.PubMed CentralPubMedGoogle Scholar
  5. Gaastra W, Svennerholm AM. Colonization factors of human enterotoxigenic Escherichia coli (ETEC). Trends Microbiol. 1996;4:444–52.View ArticlePubMedGoogle Scholar
  6. Roy K, Hilliard GM, Hamilton DJ, Luo J, Ostmann MM, Fleckenstein JM. Enterotoxigenic Escherichia coli EtpA mediates adhesion between flagella and host cells. Nature. 2009;457:594–8.View ArticlePubMed CentralPubMedGoogle Scholar
  7. Henderson IR, Cappello R, Nataro JP. Autotransporter proteins, evolution and redefining protein secretion. Trends Microbiol. 2000;8:529–32.View ArticlePubMedGoogle Scholar
  8. Crossman LC, Chaudhuri RR, Beatson SA, Wells TJ, Desvaux M, Cunningham AF, et al. A commensal gone bad: complete genome sequence of the prototypical enterotoxigenic Escherichia coli strain H10407. J Bacteriol. 2010;192:5822–31.View ArticlePubMed CentralPubMedGoogle Scholar
  9. Rasko DA, Rosovitz MJ, Myers GS, Mongodin EF, Fricke WF, Gajer P, et al. The pangenome structure of Escherichia coli: comparative genomic analysis of E. coli commensal and pathogenic isolates. J Bacteriol. 2008;190:6881–93.View ArticlePubMed CentralPubMedGoogle Scholar
  10. Sahl JW, Steinsland H, Redman JC, Angiuoli SV, Nataro JP, Sommerfelt H et al. A comparative genomic analysis of diverse clonal types of enterotoxigenic Escherichia coli reveals pathovarspecific conservation. Infect Immun. 2011; 79:950–60.View ArticlePubMed CentralPubMedGoogle Scholar
  11. von Mentzer A, Connor TR, Wieler LH, Semmler T, Iguchi A, Thomson NR, et al. Identification of enterotoxigenic Escherichia coli (ETEC) clades with long-term global distribution. Nat Genet. 2014;46:1321–6.View ArticleGoogle Scholar
  12. Sjoling A, Wiklund G, Savarino SJ, Cohen DI, Svennerholm AM. Comparative analyses of phenotypic and genotypic methods for detection of enterotoxigenic Escherichia coli toxins and colonization factors. J Clin Microbiol. 2007;45:3295–301.View ArticlePubMed CentralPubMedGoogle Scholar
  13. Nicklasson M, Sjoling A, von Mentzer A, Qadri F, Svennerholm AM. Expression of colonization factor CS5 of enterotoxigenic Escherichia coli (ETEC) is enhanced in vivo and by the bile component Na glycocholate hydrate. PLoS One. 2012;7:e35827.View ArticlePubMed CentralPubMedGoogle Scholar
  14. Liu F, Hu Y, Wang Q, Li HM, Gao GF, Liu CH, et al. Comparative genomic analysis of Mycobacterium tuberculosis clinical isolates. BMC Genomics. 2014;15:469.View ArticlePubMed CentralPubMedGoogle Scholar
  15. Aziz RK, Bartels D, Best AA, DeJongh M, Disz T, Edwards RA, et al. The RAST Server: rapid annotations using subsystems technology. BMC Genomics. 2008;9:75.View ArticlePubMed CentralPubMedGoogle Scholar
  16. Liu B, Pop M. ARDB–Antibiotic Resistance Genes Database. Nucleic Acids Res. 2009;37:D443–7.View ArticlePubMed CentralPubMedGoogle Scholar
  17. Wirth T, Falush D, Lan R, Colles F, Mensa P, Wieler LH, et al. Sex and virulence in Escherichia coli: an evolutionary perspective. Mol Microbiol. 2006;60:1136–51.View ArticlePubMed CentralPubMedGoogle Scholar
  18. Angiuoli SV, Salzberg SL. Mugsy: fast multiple alignment of closely related whole genomes. Bioinformatics. 2010;27:334–42.View ArticlePubMed CentralPubMedGoogle Scholar
  19. Krzywinski M, Schein J, Birol I, Connors J, Gascoyne R, Horsman D, et al. Circos: an information aesthetic for comparative genomics. Genome Res. 2009;19:1639–1645.View ArticlePubMed CentralPubMedGoogle Scholar
  20. Gomez-Duarte OG, Chattopadhyay S, Weissman SJ, Giron JA, Kaper JB, Sokurenko EV. Genetic diversity of the gene cluster encoding longus, a type IV pilus of enterotoxigenic Escherichia coli. J Bacteriol. 2007;189:9145–9.View ArticlePubMed CentralPubMedGoogle Scholar

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