Research Article Characteristics of Clinical Shiga Toxin ...downloads.hindawi.com/journals/bmri/2013/878956.pdfCharacteristics of Clinical Shiga Toxin-Producing Escherichia coli Isolated

Hindawi Publishing CorporationBioMed Research InternationalVolume 2013 Article ID 878956 11 pageshttpdxdoiorg1011552013878956

Research ArticleCharacteristics of Clinical Shiga Toxin-ProducingEscherichia coli Isolated from British Columbia

Kevin J Allen1 Chad R Laing2 Ana Cancarevic1 Yongxiang Zhang2 Lili R Mesak1

Hai Xu3 Ana Paccagnella4 Victor P J Gannon2 and Linda Hoang45

1 Food Nutrition and Health Program Faculty of Land and Food Systems University of British Columbia218-2205 East Mall Vancouver BC Canada V6T 1Z4

2 Laboratory for Foodborne Zoonoses Public Health Agency of Canada 225089 Township Road 9-1 (Box 640)Lethbridge AB Canada T1J 3Z4

3The State Key Laboratory of Microbial Technology School of Life Science Shandong University Jinan 250100 Shandong China4 BCCDC Public Health and Reference Microbiology Laboratory PHSA 655 West 12th Ave Vancouver BC Canada V5Z 4R45Department of Pathology and Laboratory Medicine University of British Columbia 2211 Wesbrook MallVancouver BC Canada V6T 2B5

Correspondence should be addressed to Kevin J Allen kevinallenubcca

Received 23 March 2013 Accepted 1 July 2013

Academic Editor Jacek Osek

Copyright copy 2013 Kevin J Allen et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Shiga toxin-producing Escherichia coli (STEC) are significant public health threats Although STECO157 are recognized foodbornepathogens non-O157 STEC are also important causes of human disease We characterized 10 O157H7 and 15 non-O157 clinicalSTEC derived from British Columbia (BC) Eae hlyA and stx were more frequently observed in STEC O157 and 80 and 100 ofisolates possessed 119904119905119909

1and 119904119905119909

2 respectively In contrast 119904119905119909

1and 119904119905119909

2occurred in 80 and 40 of non-O157 STEC respectively

Comparative genomic fingerprinting (CGF) revealed three distinct clusters (C) STEC O157 was identified as lineage I (LI LSPA-6111111) and clustered as a single group (C1) The cdi gene previously observed only in LII was seen in two LI O157 isolates CGFC2 strains consisted of diverse non-O157 STEC while C3 included only O103H25 O118 and O165 serogroup isolates With theexception of O121 and O165 isolates which were similar in virulence gene complement to STEC O157 C1 O157 STEC producedmore Stx2 than non-O157 STEC Antimicrobial resistance (AMR) screening revealed resistance or reduced sensitivity in all strainswith higher levels occurring in non-O157 STEC One STECO157 isolate possessed amobile 119887119897119886CMY-2 gene transferrable across genrevia conjugation

1 Introduction

Escherichia coli are Gram negative facultative anaerobic bac-teria found in mammalian gastrointestinal tracts Escherichiacoli possessing Shiga toxin genes (119904119905119909) (ie Shiga toxin-producing E coli [STEC]) pose serious health risks throughconsumption of contaminated food [1ndash4] Classical enterohe-morrhagic E coli encode 119904119905119909 plasmid pO157 (hlyA) and thelocus of enterocyte effacement (LEE) however LEE negativestrains may also cause severe disease [3] the most notableexample being E coli O104H4 [5]

In 2003 Karmali et al [6] grouped STEC into seropatho-types based on serogroup occurrence in human disease

the capacity to cause outbreaks and the association withhemolytic uremic syndrome (HUS) STEC O157H7 andO157NMwere identified as themost significant public healthrisk (seropathotype A) whereas non-O157 STEC are progres-sively of lower risk from groups B to E Recent estimates sug-gest that STEC O157 causes 50 to 70 of human infectionsmeaning that 30 to 50 are caused by non-O157 STEC [7ndash10] A US study examining high-risk non-O157 STEC in beefrevealed that 1006 of 4133 samples were positive for STEC byPCR though only 10 isolates possessed virulence gene com-binations of known pathogens [11] Outside North Americanon-O157 STEC arewell-recognized sources of human illness[12ndash14] In line with increasing concern for non-O157 STEC

2 BioMed Research International

the US Department of Agriculture has amended food safetyregulatory policy declaring STEC of O26 O45 O103 O111O121 and O145 serogroups as beef adulterants [15]

Reports of hypervirulent clades or lineages of STECO157 linked to human disease have been made SNP anal-ysis of 96 loci amongst 500 strains identified nine cladesincluding clade 8 linked with severe disease and designatedhypervirulent [16] Octamer-based genome scanning andlength polymorphisms have identified three lineages (L) ofO157 LI strains are represented in both cattle and humanclinical isolates and LII are predominantly from cattle [17ndash20] Subsequent research examining Stx2 production showeddifferences across and within lineages Sequencing of the 119904119905119909

2

flanking region demonstrates that LII has the transcriptionalactivator geneQ of L1 strains replaced by a pphA homologueFurther LI andLIII strains producedmore Stx2 than LII andLI strains of human origin produced more Stx2 than bovineLI [21]

In British Columbia (BC) Canada the rate of STECinfections have remained above the Canadian average since2004 ranging between 24 and 43 cases100000 individuals[22] However no data exist describing the salient geneticfeatures of STEC causing disease in BC or examined levelsof antimicrobial resistance (AMR) in clinical isolates To thisend we examined clinical STEC originating from BC usingmolecular and phenotypic methods to examine lineage 119904119905119909subtype toxin production presence of other virulence lociand plasmids and AMR

2 Materials and Methods

21 Strain Selection and Serotyping From the BCCDC Pub-lic Health Microbiology and Reference Laboratory EntericPathogen Monitoring Program during 2009-2010 we ran-domly selected 10 O157H7 and 15 non-O157 STEC forgenotypic and phenotypic characterization All strains werepropagated on Luria Bertani (LB) agar or broth (BectonDickinson [BD] Mississauga ON) and archived at minus80∘C inLB broth with 20 glycerol (Sigma Aldrich Oakville ON)Serotyping of all strains was performed using antisera fromthe Statens Serum Institute (Copenhagen Denmark)

22 Pulsed-Field Gel Electorphoresis Pulsed-field gel elec-trophoresis (PFGE) patterns were used to compare thegenetic relatedness of isolates belonging to the sameserogroup Whole-cell isolation of DNA for PFGE analysiswas prepared according to standard procedures [23] BrieflyDNA-containing agarose plugs were subjected to endonu-clease restriction using XbaI Resulting fragments were sep-arated using a CHEF DR III system (BioRad HerculesCalifornia) In each run Salmonella Branderup standardswere included every four lanes PFGE pattern analysis wasperformed using Bionumerics v60 software using standardcomparison criteria [24]

23 Plasmid Analysis Plasmid was extracted (QIAprep SpinMiniprep Qiagen Toronto ON) and 15120583L-electrophoresedusing Tris-acetate EDTA (TAE) buffer (70V 4-5 h)

Escherichia coli EDL933 was used as a control Plasmidsize markers included the BAC-Tracker Supercoiled DNALadder (Epicentre Markham ON) and the Supercoiled DNALadder (Invitrogen Burlington ON)

24 Virulence Typing Genomic DNA was isolated using theDNeasy Blood and Tissue kit (Qiagen) Multiplex PCR wasemployed to detect the presence of 119904119905119909

1 1199041199051199092 eaeA and hlyA

[25] All 119904119905119909 determinants were subtyped to identify 1199041199051199091

1199041199051199091c and 1199041199051199091d for 1199041199051199091 and 1199041199051199092 1199041199051199092c 1199041199051199092d 1199041199051199092-O118 1199041199051199092e

and 1199041199051199092g for 119904119905119909

2according to published methods [26 27]

25 Comparative Genomic Fingerprinting (CGF) PCRs of30 loci spanning the E coli O157H7 genome were usedto fingerprint isolates as previously described [28] Controlstrains included Sakai ECI-272 and ECI-1717 for E coli LIIII and II respectively andK-12 (MG1655) Seven additionalloci were used to increase genomic coverage and resolution(Table 1) For each locus PCRs were repeated twice with apositive result in either replicate being scored as presence(ldquo1rdquo) and a negative result in both replicates as absence (ldquo0rdquo)

PCR reactions were performed as previously described[28] Amplicons were visualized on a QIAxcel using theQIAxcel DNA Screening Kit (Qiagen) Binary PCR data wereanalyzed by constructing an Euclidean distance matrix andhierarchically clustering strains using complete linkageAnalyses were performed in R (httpwwwr-projectorg)using the heatmap2 method of the gplots package(httpcranr-projectorgwebpackagesgplotsindexhtml)The image was colored using the GNU Image ManipulationProgram v2611

26 Lineage Typing of STEC O157 STEC O157H7 EDL933and Sakai and FRIK 2001 and ECI-1717 were used as LI and IIcontrols respectively The LSPA was carried out according toYang et al [19] and analyzed as detailed by Sharma et al [29]

27 AMR Profiling AMR phenotypes were determined byKirby-Bauer disc diffusion assay A 5 120583L volume of 18 hculture grown inMueller Hinton broth (MH BD) was mixedwith 5mL of molten agar (44∘C) and overlaid on MH agarDiscs (BD) were placed on the agar surface and incubated(24 h 37∘C) and zones of inhibition measured to the nearestmillimeter Susceptibility was interpreted using CLSI guide-lines [30] In total 19 antimicrobials were screened amikacin(AMK 30 120583g) amoxicillinclavulanic acid (AMX 30120583g)ampicillin (AMP 10 120583g) cefoxitin (FOX 30120583g) ceftazidime(CAZ 30 120583g) ceftiofur (TIO 30 120583g) chloramphenicol (CHL30 120583g) ciprofloxacin (CIP 5 120583g) erythromycin (ERY 15120583g)gentamicin (GEN 10 120583g) imipenem (IPM 10 120583g) kanamycin(BCN 30 120583g) nalidixic acid (NAL 30 120583g) neomycin (NEO5 120583g) rifampicin (RIF 5 120583g) spectinomycin (SPT 100120583g)streptomycin (STR 10 120583g) tetracycline (TET 30 120583g) andtrimethoprim (TMP 5120583g)

28 Genotypic Characterization of AMR Isolates werescreened for the presence of class I II and III integrons [3132] A multiplex PCR assay was used to detect the presence

BioMed Research International 3

Table1Th

eseven

additio

nalcom

parativ

egenom

icfin

gerprin

tingprim

ersu

sedin

thisstu

dytheirgeno

miclocatio

nandfunctio

nof

targetregion

Thea

nnealin

gtemperature

used

fora

llprim

ersw

as55∘C

Prim

ername

Forw

ardsequ

ence

(51015840-31015840)

Reverses

equence(51015840-31015840)

O157H7straingenom

elocation(bp)

Functio

nA2

ACGGTT

TCGCG

CAGCT

CCTC

TTGCC

TGAT

GCG

CACG

GCA

TTCA

AEC

411528344

372834633

Phager

eplicationinitiationprotein

B2GGTG

CTCA

AGCA

GCG

CCAC

AAA

TGCC

GTT

GCT

TTGCC

TGCC

ATT

EC4115154285115

43021

Putativ

ecapsid

proteinof

prop

hage

C5TG

GGAG

GGTG

CATG

TAAG

GCG

TTG

GGGCA

TGAAC

TTGGGGGAG

TEC

4115329534332

95780

Predictedexcisio

nase

F1TC

GCA

GGTA

TGGGTG

CTGCT

GT

ACGAC

GAAG

CTTA

CCCT

GCT

GC

EC411551802055180308

Hypothetic

alprotein

E4TG

CAAAG

GCA

TGGGTC

CCAAC

GTG

ATGCG

GCA

GCT

ATGGTC

GCA

Sakai2

9330582933350

Transcrip

tionalregulatorX

REfamily

E1AG

TTCG

CCAG

TAGGCT

TGCG

CTTT

CGAC

GGGCA

ATTT

CTGCC

TGC

Sakai192952619

2960

6Pu

tativ

etranscriptio

nalregulator

C2AG

GCA

TGCG

ACCT

TTCT

AAC

TGGCA

TCTT

CAGCG

GCT

GCC

TGAT

ATGCT

Sakai193619019

36337

Hypothetic

alprotein


100908070

BC-19BC-17BC-16BC-24BC-23BC-25BC-21BC-18BC-20

Escherichia coliEscherichia coliEscherichia coliEscherichia coliEscherichia coliEscherichia coliEscherichia coliEscherichia coliEscherichia coliEscherichia coli

O157H7O157H7O157H7O157H7O157H7O157H7O157H7O157H7O157H7O157H7

BC-22

Figure 1 Clustering of STEC O157 by PFGE typing

of CMY-2 CTX-M OXA-1 SHV and TEM 120573-lactamases[33] DNA sequencing was used to confirm 119887119897119886CMY-2 identity

29 AMR Plasmid Association Approximately 15 ng of plas-mid was mixed with electrocompetent E coliDH5120572 (Invitro-gen) Following electroporation (MicroPulser BioRad) cellswere resuspended in SOC medium (Invitrogen) incubated(2 h 37∘C) plated on LB agar supplemented with AMP(100 120583gmL) CHL (30 120583gmL) or TET (30 120583gmL) andincubated (24 h 37∘C)

Plasmid mobility in MDR strains was evaluated byconjugation with E coli K802 (NALR) S TyphimuriumMSC001 (NALR) and Citrobacter rodentium MRS0026(AMPR NALR) Due to intrinsic resistance to ampicillina nonpolar bla deletion was generated in C rodentiumMRS0026 (lambda-red system) rendering it AMP sensitiveDonorsrecipients were grown for 18 h in LB broth (37∘C)containing appropriate antimicrobials For both 400120583L wascentrifuged (2000 rpm 10min) washed and resuspended in400120583L of fresh LB broth Matings were incubated for 5 hat 37∘C on a 400120583L agar slant within a 15mL microfugetubeThemixture was resuspended in 100120583L LB broth platedon LB agar with antimicrobials and incubated (24 h 37∘C)Transconjugants were streaked on LB agar and screened forAMR

210 Quantification of Stx The production of Stx2 wasquantified using polymixin lysis as described in Ziebell etal [34] with some modification Bacterial overnight culturesgrown at 37∘C with shaking (150 RPM) were diluted 1 250and used to inoculate 5mL of fresh brain heart infusion(BHI) broth in 50mL Falcon tubes Subsequently 05mgmLof polymixin was added and incubated at 37∘C for 1 hThree experimental replicates were used to assess Stx2 toxinproduction Total Stx was assessed using polymixin lysis[34] with slight modification Cells were incubated with05mgmL polymixin and incubated for 1 h at 37∘C Stx2production differences between clusters were assessed usingthe t-test function of R

3 Results

31 STEC Serotypes Clonality andVirulence Profiles In total10 serogroups and 12 unique serotypes were represented inthe STEC panel All O157 serogroup isolates displayed the H7

flagellar antigen Non-O157 serogroups included O26 O121and O165 NM variants the remaining isolates were uniqueserotypes (Table 2) PFGE of STEC O157 isolates showedtwo indistinguishable isolates (BC-20 and -21) with allstrains being distinguishable despite having ge88 similarity(Figure 1) In spite of similar pulsotypes BC-20 and -21 weredistinguishable based on differing plasmid profiles and AMRphenotypes Plasmid profiling revealed that 96 of isolatespossessed plasmids which varied in size and number Overall21 of 25 (84) strains possessed a plasmid similar in size topO157 with all STECO157 possessing it Twonon-O157 STEC(O26H11 and O111NM) were shown to have seven plasmids

All E coli O157H7 were eaeA and hlyA positive whilst13 and 12 of 15 non-O157 STEC were positive respectively(Table 2) In non-O157 STEC 119904119905119909

1was observed more fre-

quently than 1199041199051199092 with 12 of 15 isolates encoding it and only

six of 15 harboring 1199041199051199092 Only three non-O157 STEC (O8H16

and O165NM) possessed both Shiga toxin genes In contrasteight of 10 STEC O157 had both with all possessing 119904119905119909

2

Subtyping of 119904119905119909 revealed 19 of 20 STEC with Shiga toxin 1subtyped as 119904119905119909

1 the remaining isolate (O146H21) encoded

1199041199051199091c All STECwith Shiga toxin 2 had the 119904119905119909

2subtype LSPA

typing showed STEC O157 strains were LI (LSPA-6 111111)

32 Comparative Genomic Fingerprinting All strains hadunique CGF fingerprints with the exception of two O157H7(BC-23 and BC-24) and two non-O157 (BC-13 and BC-15) isolates (Figure 2) the O157H7 groupings mirroredthose obtained by PFGE in that they generated identicalstrain clusters The dendrogram in Figure 2 shows threeclusters of STEC with cluster (C) 1 consisting of O157H7LI strains C2 of non-O157 STEC of serogroups O26 O146O8 O121 O111 and O73 and the O103H3 strain (BC-3) C3including the O157H7 LII control strain all O165NM andO118H16 strains and O103H25 (BC-12) and C4 containingthe O157H7 LIII control C2 strains were positive for thefewest CGF loci followed in an increasing order by C3 C4and C1

33 Stx2 Production All O157H7 strains produced Stx2while only four of six non-O157 STEC encoding 119904119905119909

2did

including O121 (BC-1 BC-2) from C2 and O165NM (BC-8 BC-9) from C3 (Figure 2) Both O121 strains producedlevels of Stx2 similar to the STEC O157 Sakai strain Ofthe strains that produced Stx2 C1 and C2 strains producedsignificantly more than C3 strains (119875 = 0018 and 119875 =


fprn28 putative ferric enterobactin transport ATP-bindingfprn13 hypothetical proteinfprn11 hypothetical proteinfprn30 putative enzymefprn32 putative outer membrane proteinE4 transcriptional regulator XRE familyfprn35 putative resolvasefprn18 type III secretion protein EpaQfprn10 hypothetical proteinC2 hypothetical proteinfprn15 hypothetical proteinfprn29 putative repressor proteinE1 putative transcriptional regulatorfprn14 putative transcriptional regulatorfprn07 hypothetical proteinsfprn06 putative regulatory and binfing proteinsfprn34 hypothetical membrane proteinfprn02 putative sensor-type regulatorfprn33 putative transcriptional regulatorB01 replication protein ECI-1717 DNA adenine methylaseA12 unknown proteinfprn03 hypothetical proteinA2 phage replication initiation proteinF1 hypothetical proteinfprn24 putative repressor protein CIB2 putative capsid protein of prophagefprn16 putative prophage repressor CIC5 predicted excisionasefprn05 hypothetical proteinA0l contact-dependent inhibition gene

420

Stx2

pro

duct

ion

(ng

mL)

360300240180120600

Cluster 2 Cluster 1Cluster 3

ECI-

1717

O15

7H

7BC

-6O

118

H16

BC-1

2O

103

H25

BC-8

O16

5N

MBC

-9O

165

NM

BC-1

1O

73H

2BC

-7O

111

NM

BC-2

O12

1H

19BC

-1O

121

UT

BC-1

4O

26H

11BC

-4O

26N

MBC

-3O

103

H3

BC-1

3O

8H

16BC

-15

O14

6H

21M

G16

55K

12BC

-5O

26H

11BC

-10

O26

H11

ECI2

72O

157

H7

BC-2

0O

157

H7

Saka

iO15

7H

7BC

-16

O15

7H

7BC

-19

O15

7H

7BC

-22

O15

7H

7BC

-17

O15

7H

7BC

-21

O15

7H

7BC

-18

O15

7H

7BC

-25

O15

7H

7BC

-23

O15

7H

7BC

-24

O15

7H

7

Figure 2 Hierarchical clustering and Stx2 production of 25 clinical STEC strains

0005 respectively) There was no significant difference inStx2 production between C1 and C2 strains (119875 = 0072)

34 AMR All isolates were sensitive to AMK CIP GENIMP NAL RIF and TMP (Tables 2 and 3) However 11 of25 were resistant to at least one antibiotic while reducedsusceptibility (RSC) was observed in all remaining strainsparticularly to NEO (119899 = 16) SPT (119899 = 12) TET (119899 =5) BCN (119899 = 3) and STR (119899 = 3) The most commonresistance was to NEO (119899 = 6) STR (119899 = 5) and TET

(119899 = 4) CHL resistance and resistanceRSC to BCN wereinfrequently observed Three O157 and two non-O157 STECwere resistant to ge3 antibiotics though only O165NM (BC-8) possessed resistanceRSC to three different classes Sixmultidrug resistant (MDR) profiles were observed includingNEO-STR NEO-SPT and BCN-NEO-TET in non-O157STEC (Table 4) These were observed less frequently (0 to40) in the 10 O157H7 isolates Whilst four of 10 O157H7STEC were resistantRSC to one antibiotic only three of15 non-O157 were singularly resistant Interestingly BC-20


Table 2 Antimicrobial resistance serotypes PFGE plasmid and virulence profiles of clinical STEC isolated from British Columbia

Strainno Serotype

Virulence genesXbaI PFGE profile Plasmid profile (kb) AMR phenotype119904119905119909

1

a

(subtype)1199041199051199092

(subtype) 119890119886119890119860 ℎ119897119910119860

BC-13 O8H16 + + (1199041199051199092) minus + ECXA12261 100 16 12 8 7 BCNI NEOI SPTI STRI

BC-14 O26H11 + minus + + ECXA12513 93 14 7 NEO TETI

BC-5 O26H11 + minus + + ECXA12515 93 80 14 7 6 35 25 NEOI

BC-10 O26H11 + minus + + ECXA12280 93 14 7 NEOI SPTI

BC-4 O26NM + minus + + ECXA12516 93 14 7 NEOI SPTI

BC-11 O73H2 minus + (1199041199051199092) + + NDb 100 BCNI SPTI STR TETI NEOI

BC-3 O103H3 + minus + + ECXA12517 100 93 14 7 5 BCN NEO SPTI TETBC-12 O103H25 + minus + + ECXA12262 93 STR NEOI

BC-7 O111NM + minus + + ND 93 80 65 14 7 6 35 NEOI

BC-6 O118H16 + minus + + ND 93 14 7 6 BCNI KANI NEO SPT STR TETBC-2 O121H19 minus + (119904119905119909

2) + minus ECXA12518 None NEOI SPTI

BC-1 O121UT minus + (1199041199051199092) + + ECXA12518 93 NEO SPTI

BC-15 O146H21 + (1199041199051199091c) minus minus + ND 80 15 12 8 NEOI

BC-8 O165NM + + (1199041199051199092) + minus ECXA12514 93 AMPI NEOI SPT TETI

BC-9 O165NM + + (1199041199051199092) + minus ECXA12514 93 NEOI TETI

BC-16 O157H7 minus + (1199041199051199092) + + ECXA10023 93 NEO

BC-17 O157H7 + + (1199041199051199092) + + ECXA12426 93 SPTI

BC-18 O157H7 + + (1199041199051199092) + + ECXA12203 93 80 65 CHL NEOI SPTI STR TET

BC-19 O157H7 + + (1199041199051199092) + + ECXA10001 93 70 TETI

BC-20 O157H7 + + (1199041199051199092) + + ECXA12412 93 70 35 AMC AMP FOX CAZ TIO STRI

BC-21 O157H7 + + (1199041199051199092) + + ECXA12412 93 80 65 CHL NEOI STR TET

BC-22 O157H7 minus + (1199041199051199092) + + ECXA12203 93 80 50 30 NEOI

BC-23 O157H7 + + (1199041199051199092) + + ECXA10854 93 NEOI SPTI

BC-24 O157H7 + + (1199041199051199092) + + ECXA11107 93 NEO SPTI


aAll were subtype 1199041199051199091 with a single exception bnot determined Idenotes reduced susceptibility

possessed resistance to AMP CAZ and TIO suggesting thepresence of a beta-lactamase affording resistance to extendedspectrum cephalosporins (ESC)

35 Molecular AMR Characterization and Mobility No inte-grons were detected in any isolate In BC-20 the presenceof 119887119897119886CMY-2 was confirmed by PCR and DNA sequencingTransformants were shown to possess a similar 70 kb plasmidand resistance profile and were positive for 119887119897119886CMY-2 Matingexperiments showed that resistance was transferable to Ecoli STyphimurium andC rodentium through conjugationTransformations and conjugations were performed usingother MDR STEC (Table 5) With the exception of E coliO118H16 (BC-6) all strains readily transferred resistance

4 Discussion

Boerlin et al [35] reported an association between clinicalEHEC serotypes and 119904119905119909

2and eae and to a lesser extent hlyA

More recently it was reported that lineage and isolation origincorrelate with Stx2 production [21] Specifically human LI

isolates produce more toxin than cattle LI and LII strainsAlso high-toxin producing LI strains encode 119904119905119909

2whereas

LII strains possess 1199041199051199092c and LIII have both In this study

all E coliO157H7 isolates belonged to LI (LSPA-6 111111) andcarried the 119904119905119909

2subtype This is consistent with observations

made by Sharma et al [29] who reported 916 of clinicalstrains in Alberta typed as LSPA-6 111111 and elsewhere [17ndash19] However it was recently shown by Franz et al [36] andMellor et al [37] that the majority of clinical STEC O157 inThe Netherlands Argentina and Australia respectively areLIII strains As such our study provides further evidencedemonstrating that disease-causing STEC O157 in NorthAmerica differ from STEC causing disease on other conti-nents When Shiga toxin production was examined whilelevels of Stx2 associated withO157 strains were variable thesestrains clustered together by CGH and generally producedmore Stx2 than non-O157 STEC strains possessing 119904119905119909

2

Interestingly STEC O157 BC-17 produced higher levels oftoxin than the Sakai strain

Virulence profiles in non-O157 isolates displayed morevariability thanO157 STEC Buvens and Pierard [38] reporteda progressive decrease of O-island (OI) 122 components


Table 3 Antimicrobial resistance (AMR) amongst Shiga toxin-producing E coli

Antimicrobial agents STEC AMR susceptibility () AMRSusceptible Reduced susceptibility Resistant Non-O157 STEC (119899 = 15) O157 STEC (119899 = 10)

AminoglycosidesAmikacin 100 0 0 0 0Gentamicin 100 0 0 0 0Kanamycin 84 12 4 7 0Neomycin 0 76 24 27 20Streptomycin 72 8 20 20 20

PenicillinAmpicillin 92 4 4 0 10

CarbapenemImipenem 100 0 0 0 0

CephalosporinCeftazidime 96 0 4 0 10

MacrolideErythromycin 0 0 100 100 100

QuinolonesCiprofloxacin 100 0 0 0 0Nalidixic acid 100 0 0 0 0

PhenicolChloramphenicol 92 0 8 0 20

AnsamycinRifampicin 100 0 0 100 100

SpectinomycinSpectinomycin 44 48 8 13 0

TetracylinesTetracycline 64 20 16 13 20

SulfonamideTrimethoprim 100 0 0 0 0

Table 4 Multidrug resistant and reduced susceptibility STECphenotype patterns

Common antibiogramprofiles

AMRNo non-O157 STEC

()No O157 STEC

()NEO STR 10 (67) 2 (20)NEO SPT 9 (60) 4 (40)BCN NEO TET 3 (20) 0SPT STR TET 2 (13) 0CHL STR TET 0 2 (20)AMP CAZ AMC FOXCFT 0 1 (10)

(nleB nleE) when examining seropathotypes A to D FurtherOI-122 and the presence of 119904119905119909

2 eae and espP were present at

higher rates in non-O157 causing HUS Although we did notscreen for the presence of OI-122 only five non-O157 STECpossessed both 119904119905119909

2and eae Interestingly while three of these

isolates lacked hlyA four were the only non-O157 STEC toshow significant Stx2 production

This CGF scheme was developed for O157H7 STEC [28]Overall it performed well though in one case two STECfrom unrelated serogroups (BC-9 and BC-15) were indistin-guishable possessing only three of 30 loci Previous studiesreported low frequencies of O157H7-specific elements innon-O157 STEC suggesting independent acquisition of non-O157H7 traits and that these traits not be included inour CGF scheme [39ndash41] Generally non-O157H7 strainssharing more elements with O157H7 STEC grouped intoseropathotypes associated with more severe human disease

Seropathotyping [6] has been useful in assigning riskbut is broad in scope Here O26H11 (BC-14) was found todiffer at 10 of CGF loci from the other O26H11 strains(BC-5 BC-10) Thus similar to O157H7 non-O157 STECmay contain distinct lineages differing in genomic contentand their capacity to cause disease While the resolutionprovided by seropathotyping and the O157 CGF are helpfulin differentiating among strains complete genome sequenceanalysis of non-O157 STEC will be required to identifylineage-specific loci among them and the existence of uniqueldquogenopathotypesrdquo

Previous research found only LII O157H7 STEC pos-sessed cdi [42] which is common in uropathogenic E coli


Table 5 AMR phenotype of transformants and transconjugants derived from STEC plasmid DNA and matings respectively

Serotype (strain ID) Transferred AMR phenotypeTransformants Transconjugants

E coli DH5120572 E coliK802NR

C rodentiumDBS100

C rodentiumMCS026a

S TyphimuriumMCS001

O103H3 (C) BCN NEO TET + + + nab +O118H16 (F) SPT STR TET minus minus minus na minus

O157H7 (3) CHL STR TET + + minus na naO157H7 (5) AMP CAZ AMC FOX TIO +c +c na + +O157H7 (6) CHL STR TET + + minus na naaC rodentiumMCS026 was constructed by deleting bla in C rodentium DBS100bNot applicablecReduced susceptibility

[43] In this collection we observed two LI strains to bepositive for cdi This implies that these isolates have eitherrecently acquired this gene or the original study was notsufficiently broad to capture LI diversity

At this time antibiotic administration for human STECinfection is contraindicated in the treatment of STEC infec-tions in North America [44] though conflicting reports ofclinical outcomes and antibiotic administration have beenmade Antibiotic usage for treatment has been linked todiminished clinical outcomes [45 46] or had little influenceon patient outcomes [47 48] In contrast early administra-tion of fosfomycin was reported to improve clinical outcomes[49] Recent data from the E coli O104H4 outbreak supportadministration of antibiotics with fewer seizures deathsand surgeries required for antibiotic-treated patients [50]Further treated individuals experienced shorter symptomduration and shed the pathogen for significantly less timethus posing a lower risk of secondary disease transmission[51] For these reasons AMR data observed in clinicalSTEC strains is needed to provide appropriate therapeuticguidance to physicians should the current contraindicationbe rescinded

With the recognition of STEC as a significant source ofhuman disease increased reports of AMR have been made inrecent years [14 52ndash58] In this study though clinical STECwere sensitive to many drugs RSC in all examined strainsexamined or resistance in 11 of 25 strains to at least oneantibiotic was observed Similar sensitivity to AMK various120573-lactams CIP and TMP has been reported in STEC ofdiverse origin [53 54 56] Although the sample size in ourstudy is small levels of AMR were high considering theclinical origins of the strains For example in the US higherAMR levels were reported in cattle (34) and food (20)compared to clinical (10) isolates [59] Similarly in SpainSTEC O157 resistant to one or more antimicrobials wererecovered in 53 of bovine and 57 of beef isolates butonly 23 of clinical isolates were resistant [52] In AlbertaCanada whilst 34 of bovine isolates were resistant to oneof more antimicrobials only 10 of clinical isolates wereresistant with themost commonly observed resistances beingto STR sulfisoxazole and TET [29] Although resistance tosulfisoxazole was not screened in our study NEO STR andTET were the most frequently occurring AMR phenotypes

Notably BC-20 possessed a plasmidic 119887119897119886CMY-2 Consid-ering the highly promiscuous nature of plasmid harbouringobserved 119887119897119886CMY-2 in this study and previously reportedplasmids encoding it [60 61] it is not surprising that genericE coli and STECO157 possessing a similar plasmid have beenrecovered from cattle hides carcasses processing environ-ments and ground beef in Canada [62 63] Our observationof a clinical STEC possessing RSC to critically importanttherapeutic agents suggests caution in the administrationof ceftriaxone and other therapeutic ESCs that may beconsidered for treatment of STEC infections

5 Conclusions

We observed a small collection of clinical STEC in BCto be of variable genomic content STEC O157 were LIstrains producing significant amounts of Stx2 Based on CGFincreased genomic variationwas observed in non-O157 STECstrains with isolates clustering into two distinct groups CGFand Stx2 assays suggest that serogroups O121 and O165 weremore similar to STEC O157 in genetic content than othernon-O157 and that these isolates may produce high levels ofStx However further work examining clinical O121 andO165serogroup strains is required to substantiate this assertionAlthough this may make these serogroups of greater publichealth concern than other non-O157 STEC surveillance datais required to examine the frequency and disease severity thatthese serogroups cause in human STEC infections Despitethe clinical origins of the non-O157 strains the geneticvariability revealed by our CGF strategy highlights the needfor more detailed genetic information such as that offeredby whole genome sequencing Lastly we also observed highlevels of AMR and RSC in this clinical collection includinga highly mobile 119887119897119886CMY-2-encoding plasmid conferring resis-tance to clinically relevant treatment options Consideringrecent evidence suggesting that antimicrobial therapy maylead to reduced severity of clinical outcomes further dataexamining AMR in STEC seem prudent

References

[1] M A Karmali ldquoInfection by verocytotoxin-producing Escher-ichia colirdquo Clinical Microbiology Reviews vol 2 no 1 pp 15ndash381989


[2] Advisory Committee on Microbiological Safety of Food ldquoRe-port on Verocytotoxin-Producing Escherichia colirdquo 1995 httpwwwfoodgovukmultimediapdfsacmsfvtecreportpdf

[3] J P Nataro and J B Kaper ldquoDiarrheagenic Escherichia colirdquoClinical Microbiology Reviews vol 11 no 1 pp 142ndash201 1998

[4] J M Rangel P H Sparling C Crowe P M Griffin and DL Swerdlow ldquoEpidemiology of Escherichia coli O157H7 out-breaks United States 1982ndash2002rdquo Emerging Infectious Diseasesvol 11 no 4 pp 603ndash609 2005

[5] M Bielaszewska A Mellmann W Zhang et al ldquoCharac-terisation of the Escherichia coli strain associated with anoutbreak of haemolytic uraemic syndrome in Germany 2011a microbiological studyrdquo The Lancet Infectious Diseases vol 11no 9 pp 671ndash676 2011

[6] M A Karmali M Mascarenhas S Shen et al ldquoAssociationof genomic O island 122 of Escherichia coli EDL 933 withverocytotoxin-producing Escherichia coli seropathotypes thatare linked to epidemic andor serious diseaserdquo Journal ofClinical Microbiology vol 41 no 11 pp 4930ndash4940 2003

[7] P D Fey R S Wickert M E Rupp T J Safranek and S HHinrichs ldquoPrevalence of non-O157H7 Shiga toxin-producingEscherichia coli in diarrheal stool samples from NebraskardquoEmerging Infectious Diseases vol 6 no 5 pp 530ndash533 2000

[8] C H Park H J Kim and D L Hixon ldquoImportance oftesting stool specimens for Shiga toxinsrdquo Journal of ClinicalMicrobiology vol 40 no 9 pp 3542ndash3543 2002

[9] L H Thompson S Giercke C Beaudoin D Woodward andJ L Wylie ldquoEnhanced surveillance of non-O157 verotoxin-producing Escherichia coli in human stool samples from Man-itobardquo Canadian Journal of Infectious Diseases and MedicalMicrobiology vol 16 no 6 pp 329ndash334 2005

[10] J T Brooks E G Sowers J G Wells et al ldquoNon-O157 Shigatoxin-producing Escherichia coli infections in the United States1983ndash2002rdquo Journal of Infectious Diseases vol 192 no 8 pp1422ndash1429 2005

[11] J M Bosilevac and M Koohmaraie ldquoPrevalence and charac-terization of non-O157 shiga toxin-producing Escherichia coliisolates from commercial ground beef in the United StatesrdquoApplied andEnvironmentalMicrobiology vol 77 no 6 pp 2103ndash2112 2011

[12] O Andreoletti H Budka and S Buncic ldquoScientific opinionof the panel on biological hazards on a request from EFSAon monitoring of verotoxigenic Escherichia coli (VTEC) andidentification of human pathogenic VTEC typesrdquo The EFSAJournal vol 579 pp 1ndash61 2007

[13] K A Bettelheim ldquoThe non-O157 Shiga-toxigenic (verocy-totoxigenic) Escherichia coli under-rated pathogensrdquo CriticalReviews in Microbiology vol 33 no 1 pp 67ndash87 2007

[14] U Kappeli H Hachler N Giezendanner L Beutin andR Stephan ldquoHuman infections with non-o157 Shiga toxin-producing Escherichia coli Switzerland 2000ndash2009rdquo EmergingInfectious Diseases vol 17 no 2 pp 180ndash185 2011

[15] US Department of Agriculture ldquoShiga toxin-producingEscherichia coli in certain raw beef productsrdquo 2011 httpwwwfsisusdagovFrameFrameRedirectaspmain=httpwwwfsisusdagovOPPDErdadFRPubs2010-0023FRNhtm

[16] S D Manning A S Motiwala A C Springman et al ldquoVari-ation in virulence among clades of Escherichia coli O157H7associated with disease outbreaksrdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 105 no12 pp 4868ndash4873 2008

[17] J Kim J Nietfeldt and A K Benson ldquoOctamer-based genomescanning distinguishes a unique subpopulation of Escherichiacoli O157H7 strains in cattlerdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 96 no23 pp 13288ndash13293 1999

[18] J Kim J Nietfeldt J Ju et al ldquoAncestral divergence genomediversification and phylogeographic variation in subpopula-tions of sorbitol-negative 120573-glucuronidase-negative enterohe-morrhagic Escherichia coli O157rdquo Journal of Bacteriology vol183 no 23 pp 6885ndash6897 2001

[19] Z Yang J Kovar J Kim et al ldquoIdentification of commonsubpopulations of non-sorbitol-fermenting 120573-glucuronidase-negative Escherichia coli O157H7 from bovine production en-vironments and human clinical samplesrdquo Applied and Environ-mental Microbiology vol 70 no 11 pp 6846ndash6854 2004

[20] Y Zhang C LaingM Steele et al ldquoGenome evolution inmajorEscherichia coliO157H7 lineagesrdquoBMCGenomics vol 8 article121 2007

[21] Y Zhang C Laing Z Zhang et al ldquoLineage and host sourceare both correlated with levels of shiga toxin 2 production byEscherichia coli O157H7 strainsrdquo Applied and EnvironmentalMicrobiology vol 76 no 2 pp 474ndash482 2010

[22] British Columbia Centre for Disease Control ldquoBritishColumbia annual summary of reportable diseasesrdquo 2011 httpwwwbccdccautilaboutannreportdefaulthtm

[23] E M Ribot M A Fair R Gautom et al ldquoStandardization ofpulsed-field gel electrophoresis protocols for the subtyping ofEscherichia coliO157H7 Salmonella and Shigella for PulseNetrdquoFoodborne Pathogens and Disease vol 3 no 1 pp 59ndash67 2006

[24] F C Tenover R D Arbeit R V Goering et al ldquoInterpretingchromosomal DNA restriction patterns produced by pulsed-field gel electrophoresis criteria for bacterial strain typingrdquoJournal of Clinical Microbiology vol 33 no 9 pp 2233ndash22391995

[25] A W Paton and J C Paton ldquoDetection and characterization ofshiga toxigenic Escherichia coli by using multiplex PCR assaysfor stx1 stx2 eaeA enterohemorrhagic E coli hlyA rfb(O111)and rfb(O157)rdquo Journal of Clinical Microbiology vol 36 no 2pp 598ndash602 1998

[26] R Stephan and L E Hoelzle ldquoCharacterization of shigatoxin type 2 variant B-subunit in Escherichia coli strains fromasymptomatic human carriers by PCR-RFLPrdquo Letters in AppliedMicrobiology vol 31 no 2 pp 139ndash142 2000

[27] L Beutin A Miko G Krause et al ldquoIdentification of human-pathogenic strains of shiga toxin-producing Escherichia colifrom food by a combination of serotyping andmolecular typingof Shiga toxin genesrdquo Applied and Environmental Microbiologyvol 73 no 15 pp 4769ndash4775 2007

[28] C Laing C Pegg D Yawney et al ldquoRapid determination ofEscherichia coli O157H7 lineage types and molecular subtypesby using comparative genomic fingerprintingrdquo Applied andEnvironmental Microbiology vol 74 no 21 pp 6606ndash66152008

[29] R Sharma K Stanford M Louie et al ldquoEscherichia coliO157H7 lineages in healthy beef and dairy cattle and clinicalhuman cases in alberta Canadardquo Journal of Food Protection vol72 no 3 pp 601ndash607 2009

[30] Clinical and Laboratory Standards Institute ldquoPerformancestandards for antimicrobial susceptibility testing twentiethinformal supplement M100-S20rdquo Clinical and Laboratory Stan-dards Institute vol 30 pp 1ndash153 2010


[31] H Xu J Davies and V Miao ldquoMolecular characterization ofclass 3 integrons from Delftia spprdquo Journal of Bacteriology vol189 no 17 pp 6276ndash6283 2007

[32] H Xu K Broersma V Miao and J Davies ldquoClass 1 and class 2integrons in multidrugresistant gram-negative bacteria isolatedfrom the Salmon River British Columbiardquo Canadian Journal ofMicrobiology vol 57 no 6 pp 460ndash467 2011

[33] L F Mataseje E Bryce D Roscoe et al ldquoCarbapenem-resistant Gram-negative bacilli in Canada 2009-10 resultsfrom the CanadianNosocomial Infection Surveillance Program(CNISP)rdquo Journal of Antimicrobial Chemotherapy vol 67 no 6pp 1359ndash1367 2012

[34] K Ziebell M Steele Y Zhang et al ldquoGenotypic character-ization and prevalence of virulence factors among CanadianEscherichia coli O157H7 strainsrdquo Applied and EnvironmentalMicrobiology vol 74 no 14 pp 4314ndash4323 2008

[35] P Boerlin S A McEwen F Boerlin-Petzold J B Wilson RP Johnson and C L Gyles ldquoAssociations between virulencefactors of Shiga toxin-producing Escherichia coli and disease inhumansrdquo Journal of Clinical Microbiology vol 37 no 3 pp 497ndash503 1999

[36] E Franz A H A M Van Hoek F J Van Der Wal etal ldquoGenetic features differentiating bovine food and humanisolates of Shiga toxin-producing Escherichia coli O157 in TheNetherlandsrdquo Journal of Clinical Microbiology vol 50 no 3 pp772ndash780 2012

[37] G E Mellor E M Sim R S Barlow et al ldquoPhylogeneti-cally related Argentinean and Australian Escherichia coli O157isolates are distinguished by virulence clades and alternativeShiga toxin 1 and 2 prophagesrdquo Applied and EnvironmentalMicrobiology vol 78 no 13 pp 4724ndash4731 2012

[38] G Buvens and D Pierard ldquoVirulence profiling and diseaseassociation of verocytotoxin-producing Escherichia coli O157and non-O157 isolates in Belgiumrdquo Foodborne Pathogens andDisease vol 9 no 6 pp 530ndash535 2012

[39] S D Reid C J Herbelin A C Bumbaugh R K Selander andT S Whittam ldquoParallel evolution of virulence in pathogenicEscherichia colirdquo Nature vol 406 no 6791 pp 64ndash67 2000

[40] Y Ogura T Ooka A Asadulghani et al ldquoExtensive genomicdiversity and selective conservation of virulence-determinantsin enterohemorrhagic Escherichia coli strains of O157 and non-O157 serotypesrdquoGenome Biology vol 8 no 7 article R138 2007

[41] J L Kyle C A Cummings C T Parker et al ldquoEscherichiacoli serotype O55H7 diversity supports parallel acquisitionof bacteriophage at Shiga toxin phage insertion sites duringevolution of the O157H7 lineagerdquo Journal of Bacteriology vol194 no 8 pp 1885ndash1896 2012

[42] M Steele K Ziebell Y Zhang et al ldquoGenomic regions con-served in lineage II Escherichia coli O157H7 strainsrdquo Appliedand Environmental Microbiology vol 75 no 10 pp 3271ndash32802009

[43] S K Aoki R Pamma A D Hernday J E Bickham B ABraaten and D A Low ldquoMicrobiology contact-dependentinhibition of growth in Escherichia colirdquo Science vol 309 no5738 pp 1245ndash1248 2005

[44] P I Tarr C A Gordon and W L Chandler ldquoShiga-toxin-producing Escherichia coli and haemolytic uraemic syndromerdquoLancet vol 365 no 9464 pp 1073ndash1086 2005

[45] C S Wong S Jelacic R L Habeeb S L Watkins and P I TarrldquoThe risk of the hemolytic-uremic syndrome after antibiotictreatment of Escherichia coli O157H7 infectionsrdquo New EnglandJournal of Medicine vol 342 no 26 pp 1930ndash1936 2000

[46] S Dundas W T A Todd A I Stewart P S Murdoch AK R Chaudhuri and S J Hutchinson ldquoThe central ScotlandEscherichia coliO157H7 outbreak risk factors for the hemolyticuremic syndrome and death among hospitalized patientsrdquoClinical Infectious Diseases vol 33 no 7 pp 923ndash931 2001

[47] D L Martin K L MacDonald K E White J T Soler andM T Osterholm ldquoThe epidemiology and clinical aspects ofthe hemolytic uremic syndrome in Minnesotardquo New EnglandJournal of Medicine vol 323 no 17 pp 1161ndash1167 1990

[48] B P Bell PMGriffin P Lozano D L Christie JM Kobayashiand P I Tarr ldquoPredictors of hemolytic uremic syndrome inchildren during a large outbreak of Escherichia coli O157H7infectionsrdquo Pediatrics vol 100 no 1 article E12 1997

[49] K Ikeda O Ida K Kimoto T Takatorige N Nakanishi andK Tatara ldquoEffect of early fosfomycin treatment on preventionof hemolytic uremic syndrome accompanying Escherichia coliO157H7 infectionrdquo Clinical Nephrology vol 52 no 6 pp 357ndash362 1999

[50] J Menne M Nitschke R Stingele et al ldquoValidation oftreatment strategies for enterohaemorrhagic Escherichia coliO104H4 induced haemolytic uraemic syndrome case-controlstudyrdquo British Medical Journal vol 345 Article ID e4565 2012

[51] M Nitschke F Sayk C Hartel et al ldquoAssociation betweenazithromycin therapy and duration of bacterial sheddingamong patients with shiga toxin-producing enteroaggregativeEscherichia coli O104H4rdquo Journal of the American MedicalAssociation vol 307 no 10 pp 1046ndash1052 2012

[52] AMora J E Blanco M Blanco et al ldquoAntimicrobial resistanceof Shiga toxin (verotoxin)-producing Escherichia coli O157H7and non-O157 strains isolated from humans cattle sheep andfood in Spainrdquo Research inMicrobiology vol 156 no 7 pp 793ndash806 2005

[53] V Srinivasan L T Nguyen S I Headrick S E Murindaand S P Oliver ldquoAntimicrobial resistance patterns of Shigatoxin-producing Escherichia coli O157H7 and O157H7- fromdifferent originsrdquo Microbial Drug Resistance vol 13 no 1 pp44ndash51 2007

[54] P M Fratamico A A Bhagwat L Injaian and P J Fedorka-Cray ldquoCharacterization of Shiga toxin-producing Escherichiacoli strains isolated from swine fecesrdquo Foodborne Pathogens andDisease vol 5 no 6 pp 827ndash838 2008

[55] S P Gow and C L Waldner ldquoAntimicrobial resistance andvirulence factors stx1 stx2 and eae in generic Escherichiacoli isolates from calves in western Canadian cow-calf herdsrdquoMicrobial Drug Resistance vol 15 no 1 pp 61ndash67 2009

[56] G Buvens P Bogaerts Y Glupczynski S Lauwers and DPierard ldquoAntimicrobial resistance testing of verocytotoxin-producing Escherichia coli and first description of TEM-52extended-spectrum 120573-lactamase in serogroup O26rdquo Antimicro-bial Agents and Chemotherapy vol 54 no 11 pp 4907ndash49092010

[57] M C Cergole-Novella A C C Pignatari M Castanheiraand B E C Guth ldquoMolecular typing of antimicrobial-resistant Shiga-toxin-producing Escherichia coli strains (STEC)in Brazilrdquo Research in Microbiology vol 162 no 2 pp 117ndash1232011

[58] M-C Li FWang and F Li ldquoIdentification andmolecular char-acterization of antimicrobial-resistant shiga toxin-producingEscherichia coli isolated from retail meat productsrdquo FoodbornePathogens and Disease vol 8 no 4 pp 489ndash493 2011

[59] J Meng S Zhao M P Doyle and S W Joseph ldquoAntibioticresistance of Escherichia coli O157H7 and O157NM isolated


from animals food and humansrdquo Journal of Food Protectionvol 61 no 11 pp 1511ndash1514 1998

[60] K J Allen and C Poppe ldquoOccurrence and characterizationof resistance to extended-spectrum cephalosporins mediatedby 120573-lactamase CMY-2 in Salmonella isolated from food-producing animals in Canadardquo Canadian Journal of VeterinaryResearch vol 66 no 3 pp 137ndash144 2002

[61] L F Mataseje P J Baudry G G Zhanel et al ldquoComparisonof CMY-2 plasmids isolated from human animal and envi-ronmental Escherichia coli and Salmonella spp from CanadardquoDiagnosticMicrobiology and Infectious Disease vol 67 no 4 pp387ndash391 2010

[62] M Aslam and C Service ldquoAntimicrobial resistance and geneticprofiling of Escherichia coli from a commercial beef packingplantrdquo Journal of Food Protection vol 69 no 7 pp 1508ndash15132006

[63] M AslamM S Diarra C Service andH Rempel ldquoAntimicro-bial resistance genes in Escherichia coli isolates recovered froma commercial beef processing plantrdquo Journal of Food Protectionvol 72 no 5 pp 1089ndash1093 2009

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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PeptidesInternational Journal of


Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology


Molecular Biology International

GenomicsInternational Journal of


The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014


BioinformaticsAdvances in

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Signal TransductionJournal of


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Evolutionary BiologyInternational Journal of



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Genetics Research International


Advances in

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Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational



Enzyme Research



Microbiology


the US Department of Agriculture has amended food safetyregulatory policy declaring STEC of O26 O45 O103 O111O121 and O145 serogroups as beef adulterants [15]

Reports of hypervirulent clades or lineages of STECO157 linked to human disease have been made SNP anal-ysis of 96 loci amongst 500 strains identified nine cladesincluding clade 8 linked with severe disease and designatedhypervirulent [16] Octamer-based genome scanning andlength polymorphisms have identified three lineages (L) ofO157 LI strains are represented in both cattle and humanclinical isolates and LII are predominantly from cattle [17ndash20] Subsequent research examining Stx2 production showeddifferences across and within lineages Sequencing of the 119904119905119909

2

flanking region demonstrates that LII has the transcriptionalactivator geneQ of L1 strains replaced by a pphA homologueFurther LI andLIII strains producedmore Stx2 than LII andLI strains of human origin produced more Stx2 than bovineLI [21]

In British Columbia (BC) Canada the rate of STECinfections have remained above the Canadian average since2004 ranging between 24 and 43 cases100000 individuals[22] However no data exist describing the salient geneticfeatures of STEC causing disease in BC or examined levelsof antimicrobial resistance (AMR) in clinical isolates To thisend we examined clinical STEC originating from BC usingmolecular and phenotypic methods to examine lineage 119904119905119909subtype toxin production presence of other virulence lociand plasmids and AMR

2 Materials and Methods

21 Strain Selection and Serotyping From the BCCDC Pub-lic Health Microbiology and Reference Laboratory EntericPathogen Monitoring Program during 2009-2010 we ran-domly selected 10 O157H7 and 15 non-O157 STEC forgenotypic and phenotypic characterization All strains werepropagated on Luria Bertani (LB) agar or broth (BectonDickinson [BD] Mississauga ON) and archived at minus80∘C inLB broth with 20 glycerol (Sigma Aldrich Oakville ON)Serotyping of all strains was performed using antisera fromthe Statens Serum Institute (Copenhagen Denmark)

22 Pulsed-Field Gel Electorphoresis Pulsed-field gel elec-trophoresis (PFGE) patterns were used to compare thegenetic relatedness of isolates belonging to the sameserogroup Whole-cell isolation of DNA for PFGE analysiswas prepared according to standard procedures [23] BrieflyDNA-containing agarose plugs were subjected to endonu-clease restriction using XbaI Resulting fragments were sep-arated using a CHEF DR III system (BioRad HerculesCalifornia) In each run Salmonella Branderup standardswere included every four lanes PFGE pattern analysis wasperformed using Bionumerics v60 software using standardcomparison criteria [24]

23 Plasmid Analysis Plasmid was extracted (QIAprep SpinMiniprep Qiagen Toronto ON) and 15120583L-electrophoresedusing Tris-acetate EDTA (TAE) buffer (70V 4-5 h)

Escherichia coli EDL933 was used as a control Plasmidsize markers included the BAC-Tracker Supercoiled DNALadder (Epicentre Markham ON) and the Supercoiled DNALadder (Invitrogen Burlington ON)

24 Virulence Typing Genomic DNA was isolated using theDNeasy Blood and Tissue kit (Qiagen) Multiplex PCR wasemployed to detect the presence of 119904119905119909

1 1199041199051199092 eaeA and hlyA

[25] All 119904119905119909 determinants were subtyped to identify 1199041199051199091

1199041199051199091c and 1199041199051199091d for 1199041199051199091 and 1199041199051199092 1199041199051199092c 1199041199051199092d 1199041199051199092-O118 1199041199051199092e

and 1199041199051199092g for 119904119905119909

2according to published methods [26 27]

25 Comparative Genomic Fingerprinting (CGF) PCRs of30 loci spanning the E coli O157H7 genome were usedto fingerprint isolates as previously described [28] Controlstrains included Sakai ECI-272 and ECI-1717 for E coli LIIII and II respectively andK-12 (MG1655) Seven additionalloci were used to increase genomic coverage and resolution(Table 1) For each locus PCRs were repeated twice with apositive result in either replicate being scored as presence(ldquo1rdquo) and a negative result in both replicates as absence (ldquo0rdquo)

PCR reactions were performed as previously described[28] Amplicons were visualized on a QIAxcel using theQIAxcel DNA Screening Kit (Qiagen) Binary PCR data wereanalyzed by constructing an Euclidean distance matrix andhierarchically clustering strains using complete linkageAnalyses were performed in R (httpwwwr-projectorg)using the heatmap2 method of the gplots package(httpcranr-projectorgwebpackagesgplotsindexhtml)The image was colored using the GNU Image ManipulationProgram v2611

26 Lineage Typing of STEC O157 STEC O157H7 EDL933and Sakai and FRIK 2001 and ECI-1717 were used as LI and IIcontrols respectively The LSPA was carried out according toYang et al [19] and analyzed as detailed by Sharma et al [29]

27 AMR Profiling AMR phenotypes were determined byKirby-Bauer disc diffusion assay A 5 120583L volume of 18 hculture grown inMueller Hinton broth (MH BD) was mixedwith 5mL of molten agar (44∘C) and overlaid on MH agarDiscs (BD) were placed on the agar surface and incubated(24 h 37∘C) and zones of inhibition measured to the nearestmillimeter Susceptibility was interpreted using CLSI guide-lines [30] In total 19 antimicrobials were screened amikacin(AMK 30 120583g) amoxicillinclavulanic acid (AMX 30120583g)ampicillin (AMP 10 120583g) cefoxitin (FOX 30120583g) ceftazidime(CAZ 30 120583g) ceftiofur (TIO 30 120583g) chloramphenicol (CHL30 120583g) ciprofloxacin (CIP 5 120583g) erythromycin (ERY 15120583g)gentamicin (GEN 10 120583g) imipenem (IPM 10 120583g) kanamycin(BCN 30 120583g) nalidixic acid (NAL 30 120583g) neomycin (NEO5 120583g) rifampicin (RIF 5 120583g) spectinomycin (SPT 100120583g)streptomycin (STR 10 120583g) tetracycline (TET 30 120583g) andtrimethoprim (TMP 5120583g)

28 Genotypic Characterization of AMR Isolates werescreened for the presence of class I II and III integrons [3132] A multiplex PCR assay was used to detect the presence


Table1Th

eseven

additio

nalcom

parativ

egenom

icfin

gerprin

tingprim

ersu

sedin

thisstu

dytheirgeno

miclocatio

nandfunctio

nof

targetregion

Thea

nnealin

gtemperature

used

fora

llprim

ersw

as55∘C

Prim

ername

Forw

ardsequ

ence

(51015840-31015840)

Reverses

equence(51015840-31015840)

O157H7straingenom

elocation(bp)

Functio

nA2

ACGGTT

TCGCG

CAGCT

CCTC

TTGCC

TGAT

GCG

CACG

GCA

TTCA

AEC

411528344

372834633

Phager


B2GGTG

CTCA

AGCA

GCG

CCAC

AAA

TGCC

GTT

GCT

TTGCC

TGCC

ATT

EC4115154285115

43021

Putativ

ecapsid

proteinof

prop

hage

C5TG

GGAG

GGTG

CATG

TAAG

GCG

TTG

GGGCA

TGAAC

TTGGGGGAG

TEC

4115329534332

95780

Predictedexcisio

nase

F1TC

GCA

GGTA

TGGGTG

CTGCT

GT

ACGAC

GAAG

CTTA

CCCT

GCT

GC

EC411551802055180308

Hypothetic

alprotein

E4TG

CAAAG

GCA

TGGGTC

CCAAC

GTG

ATGCG

GCA

GCT

ATGGTC

GCA

Sakai2

9330582933350

Transcrip

tionalregulatorX

REfamily

E1AG

TTCG

CCAG

TAGGCT

TGCG

CTTT

CGAC

GGGCA

ATTT

CTGCC

TGC

Sakai192952619

2960

6Pu

tativ

etranscriptio

nalregulator

C2AG

GCA

TGCG

ACCT

TTCT

AAC

TGGCA

TCTT

CAGCG

GCT

GCC

TGAT

ATGCT

Sakai193619019

36337

Hypothetic

alprotein


100908070




BC-22






3 Results








2




2subtype LSPA




2did




420

Stx2

pro

duct

ion

(ng

mL)

360300240180120600


ECI-

1717

O15

7H

7BC

-6O

118

H16

BC-1

2O

103

H25

BC-8

O16

5N

MBC

-9O

165

NM

BC-1

1O

73H

2BC

-7O

111

NM

BC-2

O12

1H

19BC

-1O

121

UT

BC-1

4O

26H

11BC

-4O

26N

MBC

-3O

103

H3

BC-1

3O

8H

16BC

-15

O14

6H

21M

G16

55K

12BC

-5O

26H

11BC

-10

O26

H11

ECI2

72O

157

H7

BC-2

0O

157

H7

Saka

iO15

7H

7BC

-16

O15

7H

7BC

-19

O15

7H

7BC

-22

O15

7H

7BC

-17

O15

7H

7BC

-21

O15

7H

7BC

-18

O15

7H

7BC

-25

O15

7H

7BC

-23

O15

7H

7BC

-24

O15

7H

7







Strainno Serotype


1

a

(subtype)1199041199051199092

(subtype) 119890119886119890119860 ℎ119897119910119860
















BC-17 O157H7 + + (1199041199051199092) + + ECXA12426 93 SPTI


BC-19 O157H7 + + (1199041199051199092) + + ECXA10001 93 70 TETI










4 Discussion





2whereas





2



















AMRNo non-O157 STEC

()No O157 STEC














C rodentiumDBS100

C rodentiumMCS026a

S TyphimuriumMCS001







5 Conclusions


References











































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


Table1Th

eseven

additio

nalcom

parativ

egenom

icfin

gerprin

tingprim

ersu

sedin

thisstu

dytheirgeno

miclocatio

nandfunctio

nof

targetregion

Thea

nnealin

gtemperature

used

fora

llprim

ersw

as55∘C

Prim

ername

Forw

ardsequ

ence

(51015840-31015840)

Reverses

equence(51015840-31015840)

O157H7straingenom

elocation(bp)

Functio

nA2

ACGGTT

TCGCG

CAGCT

CCTC

TTGCC

TGAT

GCG

CACG

GCA

TTCA

AEC

411528344

372834633

Phager


B2GGTG

CTCA

AGCA

GCG

CCAC

AAA

TGCC

GTT

GCT

TTGCC

TGCC

ATT

EC4115154285115

43021

Putativ

ecapsid

proteinof

prop

hage

C5TG

GGAG

GGTG

CATG

TAAG

GCG

TTG

GGGCA

TGAAC

TTGGGGGAG

TEC

4115329534332

95780

Predictedexcisio

nase

F1TC

GCA

GGTA

TGGGTG

CTGCT

GT

ACGAC

GAAG

CTTA

CCCT

GCT

GC

EC411551802055180308

Hypothetic

alprotein

E4TG

CAAAG

GCA

TGGGTC

CCAAC

GTG

ATGCG

GCA

GCT

ATGGTC

GCA

Sakai2

9330582933350

Transcrip

tionalregulatorX

REfamily

E1AG

TTCG

CCAG

TAGGCT

TGCG

CTTT

CGAC

GGGCA

ATTT

CTGCC

TGC

Sakai192952619

2960

6Pu

tativ

etranscriptio

nalregulator

C2AG

GCA

TGCG

ACCT

TTCT

AAC

TGGCA

TCTT

CAGCG

GCT

GCC

TGAT

ATGCT

Sakai193619019

36337

Hypothetic

alprotein


100908070




BC-22






3 Results








2




2subtype LSPA




2did




420

Stx2

pro

duct

ion

(ng

mL)

360300240180120600


ECI-

1717

O15

7H

7BC

-6O

118

H16

BC-1

2O

103

H25

BC-8

O16

5N

MBC

-9O

165

NM

BC-1

1O

73H

2BC

-7O

111

NM

BC-2

O12

1H

19BC

-1O

121

UT

BC-1

4O

26H

11BC

-4O

26N

MBC

-3O

103

H3

BC-1

3O

8H

16BC

-15

O14

6H

21M

G16

55K

12BC

-5O

26H

11BC

-10

O26

H11

ECI2

72O

157

H7

BC-2

0O

157

H7

Saka

iO15

7H

7BC

-16

O15

7H

7BC

-19

O15

7H

7BC

-22

O15

7H

7BC

-17

O15

7H

7BC

-21

O15

7H

7BC

-18

O15

7H

7BC

-25

O15

7H

7BC

-23

O15

7H

7BC

-24

O15

7H

7







Strainno Serotype


1

a

(subtype)1199041199051199092

(subtype) 119890119886119890119860 ℎ119897119910119860
















BC-17 O157H7 + + (1199041199051199092) + + ECXA12426 93 SPTI


BC-19 O157H7 + + (1199041199051199092) + + ECXA10001 93 70 TETI










4 Discussion





2whereas





2



















AMRNo non-O157 STEC

()No O157 STEC














C rodentiumDBS100

C rodentiumMCS026a

S TyphimuriumMCS001







5 Conclusions


References











































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


100908070




BC-22






3 Results








2




2subtype LSPA




2did




420

Stx2

pro

duct

ion

(ng

mL)

360300240180120600


ECI-

1717

O15

7H

7BC

-6O

118

H16

BC-1

2O

103

H25

BC-8

O16

5N

MBC

-9O

165

NM

BC-1

1O

73H

2BC

-7O

111

NM

BC-2

O12

1H

19BC

-1O

121

UT

BC-1

4O

26H

11BC

-4O

26N

MBC

-3O

103

H3

BC-1

3O

8H

16BC

-15

O14

6H

21M

G16

55K

12BC

-5O

26H

11BC

-10

O26

H11

ECI2

72O

157

H7

BC-2

0O

157

H7

Saka

iO15

7H

7BC

-16

O15

7H

7BC

-19

O15

7H

7BC

-22

O15

7H

7BC

-17

O15

7H

7BC

-21

O15

7H

7BC

-18

O15

7H

7BC

-25

O15

7H

7BC

-23

O15

7H

7BC

-24

O15

7H

7







Strainno Serotype


1

a

(subtype)1199041199051199092

(subtype) 119890119886119890119860 ℎ119897119910119860
















BC-17 O157H7 + + (1199041199051199092) + + ECXA12426 93 SPTI


BC-19 O157H7 + + (1199041199051199092) + + ECXA10001 93 70 TETI










4 Discussion





2whereas





2



















AMRNo non-O157 STEC

()No O157 STEC














C rodentiumDBS100

C rodentiumMCS026a

S TyphimuriumMCS001







5 Conclusions


References











































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology



420

Stx2

pro

duct

ion

(ng

mL)

360300240180120600


ECI-

1717

O15

7H

7BC

-6O

118

H16

BC-1

2O

103

H25

BC-8

O16

5N

MBC

-9O

165

NM

BC-1

1O

73H

2BC

-7O

111

NM

BC-2

O12

1H

19BC

-1O

121

UT

BC-1

4O

26H

11BC

-4O

26N

MBC

-3O

103

H3

BC-1

3O

8H

16BC

-15

O14

6H

21M

G16

55K

12BC

-5O

26H

11BC

-10

O26

H11

ECI2

72O

157

H7

BC-2

0O

157

H7

Saka

iO15

7H

7BC

-16

O15

7H

7BC

-19

O15

7H

7BC

-22

O15

7H

7BC

-17

O15

7H

7BC

-21

O15

7H

7BC

-18

O15

7H

7BC

-25

O15

7H

7BC

-23

O15

7H

7BC

-24

O15

7H

7







Strainno Serotype


1

a

(subtype)1199041199051199092

(subtype) 119890119886119890119860 ℎ119897119910119860
















BC-17 O157H7 + + (1199041199051199092) + + ECXA12426 93 SPTI


BC-19 O157H7 + + (1199041199051199092) + + ECXA10001 93 70 TETI










4 Discussion





2whereas





2



















AMRNo non-O157 STEC

()No O157 STEC














C rodentiumDBS100

C rodentiumMCS026a

S TyphimuriumMCS001







5 Conclusions


References











































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology



Strainno Serotype


1

a

(subtype)1199041199051199092

(subtype) 119890119886119890119860 ℎ119897119910119860
















BC-17 O157H7 + + (1199041199051199092) + + ECXA12426 93 SPTI


BC-19 O157H7 + + (1199041199051199092) + + ECXA10001 93 70 TETI










4 Discussion





2whereas





2



















AMRNo non-O157 STEC

()No O157 STEC














C rodentiumDBS100

C rodentiumMCS026a

S TyphimuriumMCS001







5 Conclusions


References











































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

















AMRNo non-O157 STEC

()No O157 STEC














C rodentiumDBS100

C rodentiumMCS026a

S TyphimuriumMCS001







5 Conclusions


References











































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology





C rodentiumDBS100

C rodentiumMCS026a

S TyphimuriumMCS001







5 Conclusions


References











































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology










































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology












































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology














Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology








Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

Documents

Research Article Characteristics of Clinical Shiga Toxin ...downloads.hindawi.com/journals/bmri/2013/878956.pdfCharacteristics of Clinical Shiga Toxin-Producing Escherichia coli Isolated