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Avian pathogenic, uropathogenic, and newborn meningitis-causing

International Journal of Medical Microbiology 297(2007)163–176

Avian pathogenic,uropathogenic,and newborn meningitis-causing Escherichia coli :How closely related are they?

Christa Ewers a,?,Ganwu Li a ,Hendrik Wilking a ,Sabine Kie b ling a ,Katja Alt a ,

Esther-Maria Anta

o a ,Claudia Laturnus a ,Ines Diehl a ,Susanne Glodde a ,Timo Homeier a ,Ute Bo hnke a ,Hartmut Steinru ck b ,Hans-C.Philipp c ,Lothar H.Wieler a

a

Institut fu ¨r Mikrobiologie und Tierseuchen,Freie Universita ¨t Berlin,P.O.Box 040225,D-10061Berlin,Germany b

Bundesinstitut fu ¨r Risikobewertung,Berlin,Germany c

Lohmann Tierzucht GmbH,Cuxhaven,Germany

Received 27November 2006;received in revised form 19January 2007;accepted 23January 2007

Abstract

Avian pathogenic Escherichia coli (APEC),uropathogenic E.coli (UPEC),and newborn meningitis-causing E.coli (NMEC)establish infections in extraintestinal habitats (extraintestinal pathogenic E.coli ;ExPEC)of different hosts.As diversity,epidemiological sources,and evolutionary origins of ExPEC are so far only partially de?ned,we screened a collection of 526strains of medical and veterinary origin of various O-types for assignment to E.coli reference collection (ECOR)group and virulence gene patterns.Results of ECOR typing con?rmed that human ExPEC strains mostly belong to groups B2,followed by group D.Although a considerable portion of APEC strains did also fell into ECOR group B2(35.1%),a higher amount (46.1%)belonged to group A,which has previously been described to also harbour strains with a high pathogenic potential for humans.The number of virulence-associated genes of single strains ranged from 5to 26among 33genes tested and high numbers were rather related to K1-positive and ECOR B2strains than to a certain pathotype.With a few exceptions (iha ,afa/draB ,sfa/foc ,and hlyA ),which were rarely present in APEC strains,most chromosomally located genes were widely distributed among all ExPEC strains irrespective of host and pathotype.However,prevalence of invasion genes (ibeA and gimB )and K1capsule-encoding gene neuC indicated a closer relationship between APEC and NMEC strains.Genes associated with ColV plasmids (tsh ,iss ,and the episomal sit locus)were in general more prevalent in APEC than in UPEC and NMEC strains,indicating that APEC could be a source of ColV-located genes or complete plasmids for other ExPEC strains.Our data support the hypothesis that (a)poultry may be a vehicle or even a reservoir for human ExPEC strains,(b)APEC potentially serve as a reservoir of virulence-associated genes for UPEC and NMEC,(c)some ExPEC strains,although of different pathotypes,may share common ancestors,and (d)as a conclusion certain APEC subgroups have to be considered potential zoonotic agents.The ?nding of different evolutionary clusters within these three pathotypes implicates an independently and parallel evolution,which should be resolved in the future by thorough phylogenetic typing.r 2007Elsevier GmbH.All rights reserved.

Keywords:Escherichia coli ;UPEC;APEC;NMEC;Virulence-associated genes;Serotype;ECOR;Zoonosis;Colibacillosis

www.elsevier.de/ijmm

1438-4221/$-see front matter r 2007Elsevier GmbH.All rights reserved.doi:10.1016/j.ijmm.2007.01.003

?Corresponding author.Tel.:+493020936153;fax:+493020936067.

E-mail address:ewers.christa@vetmed.fu-berlin.de (C.Ewers).

Introduction

Knowledge on virulence traits of Escherichia coli pathotypes,which cause frequent infections in men and animals,has increased tremendously over the years. Based on anamnestic clinical reports and virulence features,veterinary and medical E.coli have been categorised into intestinal pathogenic,extraintestinal pathogenic(ExPEC),and commensal E.coli(Kaper et al.,2004).While intestinal pathotypes are well de?ned by the possession of distinct combinations of virulence-associated factors determining certain molecular path-ways,pathotypes have yet to be distinguished for extraintestinal diseases as they share a lot of common features including virulence-associated factors and serotypes(Kaper et al.,2004).ExPEC,which include uropathogenic(UPEC),new born meningitis-causing (NMEC),and avian pathogenic(APEC)E.coli,exhibit considerable genome diversity and possess a broad range of virulence-associated factors including adhesins, toxins,iron acquisition factors,lipopolysaccharides, polysaccharide capsules,and invasins,which are fre-quently encoded on islands and other mobile DNA elements(Johnson and Russo,2005;Rodriguez-Siek et al.,2005).One of the most relevant diseases caused by ExPEC in animals is systemic colibacillosis leading to signi?cant economical losses in the poultry industry worldwide(Dho-Moulin and Fairbrother,1999;Ewers et al.,2003).The disease,generally found in domes-ticated and wild birds,starts as a respiratory tract infection,leading to a systemic infection of internal organs with sepsis?nally setting in.A variety of serotypes have been identi?ed among APEC,and strains of the same O-types(O1,2,4,6,7,8,16,18,and75)are also noticeably involved in human diseases in adults and children,including urinary tract infections and menin-gitis in neonates(Johnson and Stell,2000;Bonacorsi and Bingen,2005).Phylogenetic analyses have shown that extraintestinal pathogenic strains of human source mainly belong to E.coli reference collection(ECOR) group B2and occasionally to group D,while groups B1 and A are rarely found(Johnson et al.,2001;Bingen-Bidois et al.,2002).

Limited experimental data on in vivo studies indicate that ExPEC strains exhibit a certain degree of host speci?city,although only recently it could be demon-strated that1-day-old chickens were killed by infection with an NMEC strain.It is becoming more and more apparent that the common presence of a set of virulence-associated genes among ExPEC strains as well as similar disease patterns and phylogenetic background indicate a signi?cant zoonotic risk of avian-derived E.coli isolates (Mokady et al.,2005a;Moulin-Schouleur et al.,2006; Ron,2006).

In order to de?ne the genetic relationship between APEC and isolates derived from urinary tract infections and cases of newborn meningitis,and to gain a deeper understanding of the APEC pathotype,we investigated a large collection of APEC,UPEC and NMEC strains for virulence features,O-types,and phylogenetic back-ground.A systematic comparison of the resulting data could probably unravel considerable overlaps between human and animal ExPEC strains with respect to the investigated traits as well as some kind of host-or pathotype-speci?c features.

Materials and methods

Bacterial strains

In this study,436E.coli isolates implicated in avian colibacillosis(APEC),65uropathogenic E.coli(UPEC), and25newborn meningitis-causing E.coli(NMEC) strains were investigated.APEC strains were obtained from several outbreaks in poultry?ocks as well as from single disease events in wild and zoo birds originating from representative geographical regions of Germany, while a smaller part of the strains was kindly allocated by colleagues from Brazil,France,Canada,USA,and The Netherlands.APEC isolates originated primarily from chickens including laying hens(211)and broilers (95)and from turkeys(97).Other avian hosts included ducks(16)and geese(3),the remaining14strains originated from single disease events in wild and zoo birds.Avian E.coli isolates were obtained from different sites within the hosts,including the trachea,air sacs, pericardium,spleen,liver,blood,bone marrow,and the salpinx.Some of these strains have been described previously(Ewers et al.,2004,2005).Human ExPEC isolates were kindly provided by other institutions located in different regions of Germany as well as from other countries while UPEC strains from dogs(10)and cats(3)were derived from diagnostic sample material collected in our laboratory.Controls used for molecular assays(Fig.1)were APEC strains IMT5155(O2:K1:H5) and IMT2470(O2:K1:H5),UPEC strains IMT7920 (O75:HM)and IMT1200(O18:H1),and NMEC strain BK658(O18:K1:H7).All strains were stored atà801C in brain heart infusion broth with10%glycerol until further use.

Serotyping

Serotyping was done by Hartmut Steinrueck at the Federal Institute for Risk Assessment(BfR),Berlin, Germany,by tube agglutination with rabbit anti-E.coli immune sera produced against a panel of antigenic test strains of E.coli containing E.coli O groups1–173and E.coli H groups1–56.

C.Ewers et al./International Journal of Medical Microbiology297(2007)163–176 164

Virulence genotyping

E.coli strains were investigated for various genes by multiplex and single PCR assays as well as by DNA–DNA hybridisation.Targeted genes and their descriptions as well as primer sequences for the ampli?cation procedures are given in Table 1.All primers used in the ampli?cation studies were obtained from Sigma Genosys (Steinheim,Germany).

Total DNA of E.coli strains for PCR analyses and probe synthesis was prepared using a Master Pure TM Genomic DNA Puri?cation Kit (Biozym Diagnostic GmbH,Hessisch Oldendorf,Germany)according to the manufacturer’s recommendations.Bacterial DNA for dot blot analyses was prepared by culturing bacteria overnight in Luria-Bertani broth at 371C.DNA was then released from whole organisms by boiling for 10min.After centrifugation,2.5m l of the supernatant was taken for DNA–DNA hybridisation.

Multiplex assay development

Primers for each virulence factor were ?rst validated individually by use of template DNA from appropriate

positive and negative control strains in single PCR assays.Primers were then sorted into four pools (Fig.1)according to primer compatibility and product size.Each primer pool was validated by use of control strains containing all the relevant virulence factors.

All multiplex PCR procedures were performed in a 25m l reaction mixture including 2.5m l 10?PCR buffer,2.0m l 50mM MgCl 2,2U Taq DNA polymerase (Rapidozym,Germany),0.5m l of each 10mM dNTP,0.1m l (100pmol)oligonucleotide primer pair (Sigma-Aldrich,Germany),and 4m l (40ng)template DNA,supplemented with appropriate volumes of double distilled water.Reaction mixtures were subjected to the following conditions in a thermal cycler (T1What-man Biometra,Germany):3min at 941C,25cycles of 30s at 941C,30s at 581C,and 3min at 681C,with a ?nal cycle of 10min at 721C,followed by a hold at 101C.

Single PCR analyses for genes where new primers had been designed were performed as described previously (Ewers et al.,2004).Brie?y,2m l (20ng)template DNA was added to the reaction mixture (25m l)containing 0.5m l (10pmol)of each primer pair,0.1m l of each 10mM dNTP,2.5m l 10?PCR buffer,1.25m l of 50mM MgCl 2,and 1U Taq polymerase.The samples were subjected to 25cycles of ampli?cation in the above-mentioned thermal cycler.

Horizontal gel electrophoresis was performed with 1.5%agarose for multiplex and 1.0%agarose (ROTI s GAROSE,Roth GmbH,Germany)for single PCR products.Amplicons were stained with ethidium bromide,photographed at UV exposure,and their size was determined by comparison to a 100-bp DNA marker (Invitrogen,Germany).

DNA–DNA hybridisation analyses

Dot-blot hybridisation of 526ExPEC strains was used to validate PCR assays for 29of the 33constituent primer sets given in Table 1;that is,all primers except those for the K1-speci?c gene (neuC )which were presumed to generate probes that would hybridise non-speci?cally with any group II kpsMT region,irrespective of the capsule type,and those for the chromosomal and epsiomal sit variant.

Probes were generated and labelled with digoxigenin (DIG)-dUTP (Roche Molecular Biochemicals,Mann-heim,Germany)by PCR.Blotting and detection was done as described previously (Ewers et al.,2004).Brie?y, 2.5m l of heat-boiled DNA was transferred onto positively charged nylon membranes.DNA was ?xed on the membrane by baking for 20min at 1201C.Hybridisation was then performed using the Roche Labelling and Detection Kit as recommended by the manufacturer.

2.072 bp 1.500 bp 1.000 bp 800 bp 600 bp 500 bp 400 bp 300 bp 200 bp 100 bp

1.200 bp M

M

M

M

MP I

MP II

MP III

MP IV

sfa /foc malX afa/dra neuC iha hrlA fimC pic hlyA kpsMTIIT

sit ep.ompA iroN gimB sit chr.traT ibeA chuA

vat tsh iucD cvi/cva papC irp2iss

EAST-1

mat fyuA sat tia cnf1/2ireA

crlA Fig. 1.Ampli?cation products from multiplex PCR (MP)assays,separately ampli?ed using primer pools I–IV.M:100-bp molecular weight marker.MP I:pooled DNA of NMEC strain IMT9267(BK658)and UPEC strain IMT7920(ampli-cons:kpsMTII [280bp],hlyA [352bp],pic [409bp],?mC [477bp],hra [537bp],iha [609bp],neuC [676bp],afa/draB [810bp],malX [922bp],and sfa/foc [1242bp]).MP II:APEC strain IMT5155(amplicons:chuA [278bp],ibeA [342bp],traT [430bp],sit chr.[554bp],gimB [736bp],iroN [847bp],ompA [919bp],and sit ep.[1052bp].MP III:APEC strain IMT2470(amplicons:EAST-1[116bp],iss [309bp],irp2[413bp],papC [501bp],cvi/cva [598bp],iucD [714bp],tsh [824bp],and vat [981bp].MP IV:pooled DNA of NMEC strain IMT9267(BK658)and UPEC strain IMT1200(amplicons:crlA [250bp],ireA [384bp],cnf1/2[446bp],tia [512bp],sat [667bp],fyuA [774bp],and mat [899bp].

C.Ewers et al./International Journal of Medical Microbiology 297(2007)163–176

165

Table 1.

Primers used for the detection of virulence-associated genes in ExPEC strains

Gene(s)or operon

Description

Primer sequence (50–30;s:sense;a:antisense)

Source of primer

Adhesins afa/draB A?mbrial/Dr antigen-speci?c adhesin s:TAAGGAAGTGAAGGAGCGTG Present study a:CCAGTAACTGTCCGTGACA crl Curli ?bre gene

s:TTTCGATTGTCTGGCTGTATG Maurer et al.(1998)

a:CTTCAGATTCAGCGTCGTC

?mC Type 1?mbriae (D -mannose-speci?c adhesin)

s:GGGTAGAAAATGCCGATGGTG Janssen et al.(2001)

a:CGTCATTTTGGGGGTAAGTGC hra Heat-resistant agglutinin

s:TCACTTGCAGACCAGCGTTTC Present study a:GTAACTCACACTGCTGTCACCT iha Iron-regulated-gene-homologue adhesin s:TAGTGCGTTGGGTTATCGCTC Present study a:AAGCCAGAGTGGTTATTCGC papC Pilus associated with pyelonephritis s:TGATATCACGCAGTCAGTAGC Janssen et al.(2001)

a:CCGGCCATATTCACATAAC

sfa/focCD S ?mbriae (sialic acid-speci?c)and F1C ?mbriae

s:GTCCTGACTCATCTGAAACTGCA Present study a:CGGAGAACTGGGTGCATCTTA tsh a Temperature-sensitive haemagglutinin s:ACTATTCTCTGCAGGAAGTC Ewers et al.(2004)

a:CTTCCGATGTTCTGAACGT mat

Meningitis-associated and temperature-regulated ?mbriae

s:TATACGCTGGACTGAGTCGTG Present study

a:CAGGTAGCGTCGAACTGTA Iron acquisition chuA Heme receptor gene (E.coli haem utilisation)

s:GACGAACCAACGGTCAGGAT Clermont et al.(2000)

a:TGCCGCCAGTACCAAAGACA fyuA Ferric yersinia uptake (yersiniabactin receptor)

s:GCGACGGGAAGCGATGACTTA Schubert et al.(1998)

a:CGCAGTAGGCACGATGTTGTA ireA Iron-responsive element (putative catecholate siderophore receptor)

s:ATTGCCGTGATGTGTTCTGC Present study a:CACGGATCACTTCAATGCGT iroN a Catecholate siderophore (salmochelin)receptor

s:ATCCTCTGGTCGCTAACTG Present study a:CTGCACTGGAAGAACTGTTCT irp2Iron-repressible protein (yersiniabactin synthesis)

s:AAGGATTCGCTGTTACCGGAC Schubert et al.(1998)

a:TCGTCGGGCAGCGTTTCTTCT iucD a Aerobactin synthesis

s:ACAAAAAGTTCTATCGCTTCC Janssen et al.(2001)

a:CCTGATCCAGATGATGCTC sitD chr.Salmonella iron transport system gene s:ACTCCCATACACAGGATCTG Present study a:CTGTCTGTGTCCGGAATGA sitD ep.a

Salmonella iron transport system gene

s:TTGAGAACGACAGCGACTTC Present study

a:CTATCGAGCAGGTGAGGA Protectins/serum resistance cvi/cva a Structural genes of colicin V operon

(microcin ColV)

s:TCCAAGCGGACCCCTTATAG Present study a:CGCAGCATAGTTCCATGCT iss a Increased serum survival s:ATCACATAGGATTCTGCCG Ewers et al.(2005)

a:CAGCGGAGTATAGATGCCA neuC K1capsular polysaccharide s:GGTGGTACATTCCGGGATGTC Watt et al.(2003)a:AGGTGAAAAGCCTGGTAGTGTG kpsMT II Group II capsule antigens s:CAGGTAGCGTCGAACTGTA

Johnson and Stell (2000)a:CATCCAGACGATAAGCATGAGCA ompA Outer membrane protein s:AGCTATCGCGATTGCAGTG Present study a:GGTGTTGCCAGTAACCGG traT a Transfer protein

s:GTGGTGCGATGAGCACAG

Present study

a:TAGTTCACATCTTCCACCATCG

Toxins astA

EAST1(heat-stable cytotoxin associated with enteroaggregative E.coli )s:TGCCATCAACACAGTATATCC

Yamamoto and Echeverria (1996)

a:TAGGATCCTCAGGTCGCGAGTGACGGC cnf1/2Cytotoxic necrotising factor s:TCGTTATAAAATCAAACAGTG Ewers et al.(2004)

a:CTTTACAATATTGACATGCTG sat

Secreted autotransporter toxin

s:TGCTGGCTCTGGAGGAAC

Present study

a:TTGAACATTCAGAGTACCGGG

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166

ECOR grouping

E.coli strains were classi?ed according to the ECOR system(Herzer et al.,1990)by use of the rapid phylogenetic grouping PCR technique described by Clermont et al.(2000).

Statistical analysis

Statistical testing was done by use of the Statistical Package for the Social Sciences(version10.0;SPSS). Comparisons of proportions for a particular character-istic in different populations were tested by use of w2 test.

Because of multiple comparisons,the threshold for statistical signi?cance was as indicated in the tables,with p o0.001re?ecting strong statistical signi?cance. Results

Serotyping

Among144serotyped APEC strains125(86.8%) could be classi?ed into a single O-type,while in13.2% of the strains the O-type could not be determined (Table2).Most of the typeable strains belonged to O-types O2(26.6%),O78(17.7%),O1(10.5%),and O18(4.8%),while40.8%were distributed among further34O-types.Among59UPEC,21(35.6%) isolates belonged to O-type O6,25(42.4%)were distributed among13further O-types while14(23.7%)strains could not be classi?ed into any O-type.The most common O-type in NMEC strains was O18(28.6%), followed by O7(19.0%).O-types shared by all pathotypes were O6and O18,while other antigens were either shared by APEC and UPEC(O2,O13,O21,O78, O86,and O114),APEC and NMEC(O1and O45),or by UPEC and NMEC isolates(O75).Of the125 typeable APEC strains53.6%overlapped with UPEC O-types and17.6%with NMEC,whereas73.3%of45 typeable UPEC strains overlapped with APEC O-types and61.9%with NMEC.All other O-types detected–a total of35–were unique to one of the three groups of ExPEC indicating that there is not only substantial overlap in O-types but also great difference across the three pathotypes.

Virulence genotyping

Validation of PCR assays

We present a set of four multiplex PCR assays amplifying a total of33virulence-associated genes (Fig.1).As con?rmed by DNA–DNA hybridisation, the PCR assays were100%sensitive for all individual virulence-associated genes except iroN(431/444,97.1%), cva/cvi(333/345,96.5%),and malX(228/240,95.0%). The speci?city was100%as all blot-negative results were correctly identi?ed by PCR.NeuC primers were validated by comparison of PCR results with serologi-cally determined K antigens,where available.K1 primers reacted with52of56K1-positive strains (sensitivity:92.9%),and with18strains,where no capsule was detected by means of serological typing.

Table1.(continued)

Gene(s)or

operon

Description Primer sequence(50–30;s:sense;a:antisense)Source of primer

vat Vacuolating autotransporter toxin s:TCCTGGGACATAATGGCTAG Ewers et al.

(2004)

a:GTGTCAGAACGGAATTGTC

hlyA Haemolysin A s:GTCCATTGCCGATAAGTTT Ewers et al.

(2004)

a:AAGTAATTTTTGCCGTGTTTT

Invasins

gimB Genetic island associated with newborn

meningitis s:TCCAGATTGAGCATATCCC Present study a:CCTGTAACATGTTGGCTTCA

ibeA Invasion of brain endothelium s:TGGAACCCGCTCGTAATATAC Present study

a:CTGCCTGTTCAAGCATTGCA

tia Toxigenic invasion locus in ETEC strains s:AGCGCTTCCGTCAGGACTT Present study

a:ACCAGCATCCAGATAGCGAT

Miscellaneous

pic Serin protease autotransporter s:ACTGGATCTTAAGGCTCAGG Present study

a:TGGAATATCAGGGTGCCACT

malX Pathogenicity-associated island marker

CFT073s:GGACATCCTGTTACAGCGCGCA Johnson and

Stell(2000) a:TCGCCACCAATCACAGCCGAAC

a Genes associated with large plasmids in APEC,like pAPEC-O2-ColV[NC_007675],pTJ100[AY553855],and pAPEC-O1-ColBM[DQ381420].

C.Ewers et al./International Journal of Medical Microbiology297(2007)163–176167

Virulence gene distribution and odds ratios

While a set of virulence features,including adhesion-related genes crl ,?mC ,and mat were detected in almost all strains (93.1%–100%)irrespective of their patho-type,others,like hra (23.4–53.8%)and papC (25.5–68.0%)were also frequently detected in the strains with APEC revealing the lowest prevalence compared to UPEC and NMEC (Table 3).The afa/draB genes were present in 1.2%of APEC,4.0%of NMEC,and in 6.2%of UPEC strains.Considerable differences in the prevalence data were present for iha ,encoding an iron-regulated adhesin originally identi?ed in E.coli O157:H7(Tarr et al.,2000).While 3.2%of APEC strains harboured this gene,it was detected in 23.1%of UPEC and 32.0%of NMEC strains.Similar data were obtained for genes sfa/foc ,which were present in half of the UPEC and 24.0%of the NMEC strains,while only 9.2%of APEC strains harboured these genes.Except for tsh that was highly present in APEC strains (ORs 25.84and 9.43compared to UPEC and NMEC,respectively),the remaining adhesion-related genes were more likely detected in UPEC and to a lesser extent in NMEC rather than in avian strains (ORs o 1).Plasmid-related genes were detected in the majority of APEC with cvi/cva ,sitD ep.,iucD ,iss ,traT ,and iroN present in 74.8–86.7%,and tsh present in 57.1%of 434isolates.Some of these genes were detected in a substantial number of UPEC and NMEC as well.IroN was present in 75.4%of UPEC and in 68.0%of NMEC,while traT occurred in 49.2%of UPEC and 80.0%of NMEC strains.IucD and iss were also present in more than half of the NMEC strains (68.0%/56.0%)investi-gated,while these genes occurred in a lower percentage in UPEC strains (33.8%/26.2%).Almost half of the NMEC strains harboured cvi/cva and the episomal sit gene (44.0%each)whereas these genes were a lot less prevalent in UPEC strains (12.3%/23.1%).Among all plasmid-associated genes tsh revealed the lowest pre-valence in NMEC (12.0%)and UPEC (4.6%)https://www.doczj.com/doc/776579904.html,paring APEC with UPEC there are notably high ORs for nearly all the plasmid-related genes,including tsh (27.5),cvi/cva (21.1),iss (17.7),sitD ep.(10.5),iucD (9.1),and traT (5.4)(Table 3).These genes were also more commonly present in APEC as compared to NMEC strains;however,the highest OR in this group was 9.8for tsh and the lowest 1.3for traT .

Table 2.Serotypes among ExPEC strains

Shared O-types

APEC (n ?144)UPEC (n ?59)NMEC (n ?21)Unshared O-types APEC (n ?144)UPEC (n ?59)NMEC (n ?21)No.No.No.No.No.No.O11303O29200O23330O32110O61212O35100O13110O39010O18626O46100O21220O55100O45202O64100O75021O71100O782220O76100O86110O83020O114

110O88400Total shared 82

35

14

O10110O103100Unshared O-types O113100O4020O119100O5200O125100O7004O131100O8300O147200O9200O149100O11100O152100O12002O153040O16001O160100O22100O161100O23400O166100O24

400

Total unshared

43

10

7

Non-typeable isolateses:APEC (n ?19);UPEC (n ?14).

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168

Chromosomally located virulence-associated genes were frequently found in all three E.coli groups.Iron acquisition genes,like the chromosomal sitD variant, chuA,fyuA,and irp2commonly occurred in APEC (33.0–70.9%),UPEC(56.9–83.1%),and NMEC (68.0–96.0%),while ireA was most often detected in APEC(42.7%),followed by NMEC(36.0%)and UPEC

(20.0%).

A feature clearly distinguishing UPEC from APEC and NMEC is the higher occurrence of neuC,which is involved in the synthesis of the K1capsule in the latter strains(ORs4.4and113.1).The gene was detected in nearly all NMEC(92.0%)and at least30.7%of the APEC,but only in9.2%of the UPEC strains.Group II capsule biosynthesis gene kpsMT II was present in most of the NMEC(96.0%),whereas only41.0–42.4%of UPEC and APEC strains possessed this gene.

Among the four toxin genes tested,only vat was frequently found in all three E.coli groups(41.5–55.4%) while the others were either commonly present in just one or two of these groups.In fact,cnf1/2occurred in 0.9%and sat in0.5%of the APEC strains,whereas

Table3.Prevalence of virulence-associated genes in ExPEC strains as detected by PCR and hybridisation analyses Gene(s)/categories Prevalence of virulence-associated genes(%)Odds ratio Phylogenetic groups

APEC (n?455)UPEC

(n?66)

NMEC

(n?26)

APEC

UPEC

APEC

NMEC

UPEC

NMEC

A B1B2D

Adhesins

afa/dra 1.3 6.1 3.80.210.33 1.61 3.50.00.9 1.0 crl92.797.096.20.400.51 1.2895.7100.091.591.7?mC94.9100.0100.0n.i.n.i.n.i.94.3100.095.3100.0 hra23.553.030.80.270.69 2.5423.511.124.145.8 iha 3.122.730.80.110.070.66 2.20.011.38.3 papC24.650.065.40.320.170.5319.133.341.527.1 sfa/foc8.850.026.90.100.26 2.71 2.211.133.5 3.1 tsh a54.9 4.511.525.849.430.3757.466.742.031.3 Iron

chuA50.381.896.20.230.040.180.00.0100.0100.0 fyuA66.456.169.2 1.560.890.5746.555.689.259.4 ireA41.319.734.6 2.87 1.330.4622.233.353.843.8 iroN a83.772.769.2 1.93 2.29 1.1979.666.792.963.5 irp268.881.896.20.490.090.1849.655.697.667.7 iucD a79.333.365.47.78 2.060.2672.688.972.675.0 sitB chr.31.656.169.20.360.210.57 1.30.083.519.8 sitB ep a73.221.242.310.14 3.720.3771.322.262.761.5 Protectins

cvi/cva a72.312.126.918.93 6.200.3766.577.864.649.0 iss a84.025.857.715.08 3.840.2578.788.977.862.5 neuC29.59.192.3 4.170.030.01 2.20.066.518.8 ompA99.192.4100.07.27n.i.n.i.97.8100.099.597.9 traT a81.350.076.9 4.35 1.310.3069.688.984.080.2 Toxins

EAST-120.0 6.10.0 3.82n.i.n.i.15.722.214.227.1 cnf1/20.931.823.10.020.03 1.560.411.111.3 5.2 sat0.421.234.60.020.010.51 1.30.0 6.68.3 vat39.854.550.00.550.66 1.2 2.211.183.549.0 Invasins

gimB23.79.161.5 3.110.190.06 1.311.157.5 4.2 ibeA26.218.238.5 1.590.570.36 3.522.251.921.9 tia23.731.846.20.670.360.5416.144.436.822.9 Miscellaneous

malX39.665.273.10.350.170.49 4.311.192.536.5 pic11.233.37.70.25 1.51 6.000.00.419.833.3 n.i.?not indicated,as at least one of the group harbours none or100%of the gene.

a Genes associated with large plasmids in APEC,like pAPEC-O2-ColV[NC_007675],pTJ100[AY553855],and pAPEC-O1-ColBM[DQ381420].

C.Ewers et al./International Journal of Medical Microbiology297(2007)163–176169

these toxin genes were found in 21.5–43.1%of the UPEC and NMEC strains.EAST-1,however,was more frequently found in APEC (20.4%)compared to UPEC (6.2%)but the gene was completely absent in NMEC strains.Consistent with these data,APEC showed strikingly low OR levels (0.01–0.02)for sat and cnf1/2compared to both UPEC and NMEC,while vat can be mostly expected in UPEC strains.

Invasion-related genes,including gimB and ibeA ,as well as toxigenic invasion locus tia were most frequently found in NMEC (36.0–60.0%),but were also present in 24.8–27.1%of APEC.GimB (9.2%)and ibeA (18.5%)were less common in UPEC while 32.3%of these strains harboured tia ,compared to 24.8%in APEC and 48.0%in NMEC.Also ORs for ibeA (1.6)and gimB (3.2)indicate a higher chance for APEC to harbour these genes as compared to UPEC strains,while similar to neuC ,these genes,particularly gimB ,were mostly distributed in NMEC strains (OR NMEC/UPEC:2.5and 14.8).Tia was relatively less distributed in APEC than in UPEC (OR 0.7)and NMEC (OR 0.4)strains.MalX ,a pathogenicity island (PAI)marker of UPEC strain CFTO73,occurred in similar percentages in UPEC (66.2%)and NMEC (72.0%),whereas 41.1%of the APEC strains harboured this island marker.The serine protease-encoding gene pic was most prevalent in UPEC (33.8%)and in comparable percentages in APEC (11.9%)and NMEC (8.0%).

Average number of virulence-associated genes among ExPEC and gene combinations

The number of virulence-associated genes detected in different strains ranged between 5and 26(in case neuC and kpsMTII are simultaneously detected they are

calculated as one factor)among the 33factors tested.We found highly heterologous gene combinations with 293different patterns among APEC,65among UPEC and 23among NMEC strains.Three NMEC and 11APEC strains had the same virulence gene pattern possessing 21identical genes and another two identical gene combinations were shared by one UPEC and four APEC strains per combination.Strains sharing the same gene combinations in all cases also shared the same phlyogenetic group but only partly the same O-antigen.However,one NMEC and three APEC strains shared the same virulence gene pattern (21virulence-associated genes),serogroup (O1:K1:H7),and phylogenetic group (B2).

The average number of virulence-associated genes in ExPEC strains was 15.1(Table 4).With 17.5genes NMEC strains revealed the highest value,followed by APEC and UPEC (both 15.0).Considerably high numbers of genes were present in strains belonging to the most common O-types O1(18.9),O2(19.7),and O18(19.7),whereas those of O-types O6(16.7)and O78(13.3)were closer to the average of the total number of strains (Table 4).Isolates belonging to the remaining O-types (n ?75)and non-typeable strains (n ?35)re-vealed an average number of 14.0and 14.3genes,respectively.

A considerably high number of virulence-associated genes was found in neuC (K1)-positive strains (19.4)compared to neuC -negative ones (13.2),which is consistent in all three pathogroups.Certain genes,including chuA (OR 46.4),gim

B (OR 34.6),sit chr .(OR 24.8),malX (OR 22.7),vat (OR 13.0),irp2(OR 12.7),ibeA (OR 8.3),and fyuA (OR 8.0)are more or less positively linked with the presence of neu

C ,as shown by odds ratio levels comparing neuC -positive with neuC -negative ExPEC strains.

Table 4.

Average number of virulence-associated genes among ExPEC related to somatic antigens and K1biosynthesis gene neuC

Total

APEC UPEC NMEC Isolates (n )

5264366525Average number of genes 15.1

15.0

15.0

17.5

Association to O-type O-types

Average number of genes/isolates O118.9/1619.1/13—18.0/3O219.7/3619.9/3317.0/3—O616.7/248.0/117.5/2112.5/2O1819.7/1418.7/621.0/219.3/6O78

13.3/2413.2/2215.0/2—

Less common O-types 14.0/7513.4/4813.9/1717.1/10Non-typeable strains 14.3/3514.8/2113.5/14—Association to K1antigen neuC -positive 19.4/16319.7/13418.2/617.9/24neuC -negative

13.2/363

12.9/302

14.7/59

12.5/2

C.Ewers et al./International Journal of Medical Microbiology 297(2007)163–176

170

ECOR typing and association of virulence-associated genes to phylogenetic groups

The distribution of ExPEC strains among the four phylogenetic groups is demonstrated in Table5.As determined by PCR most of the strains could be categorised into groups A(40.5%)and B2(40.1%), while another17.9%belonged to group D and the remaining strains(1.5%)to group B1.While APEC mainly follow this distribution in percentages,UPEC and NMEC strains most often belonged to group B2 (61.5%/72.0%)and group D(21.5%/24.0%).None of the UPEC and NMEC strains belonged to group B1, whereas one NMEC isolate and16.9%of UPEC strains were categorised into group A.

The mean number of virulence features differed with the highest number of genes being found in group B2 (18.7)and the lowest in group A isolates(11.6).With a few exceptions,genes positively linked to B2group strains were the same as those that were found to be signi?cantly associated with neuC(Fig.2,Table5). Discussion

With increasing knowledge of molecular genetics of ExPEC of human and animal sources,a categorisation of these strains into distinct pathotypes and thus the zoonotic potential of animal-derived strains has become a focus of current research.The data presented in this study reveal both a considerable overlap and remarkable differences between the so-far-determined pathotypes APEC,UPEC,and NMEC with respect to virulence features,O-types,and assignments to ECOR groups, hindering a clear de?nition of these three pathotypes.More than half of the serotyped APEC share the most commonly occurring somatic antigens in UPEC(e.g. O2,O6,O18,and O78)and almost20.0%those in NMEC isolates(e.g.O1,O2,O6,O18,and O45). Indeed,many serotypes detected among APEC strains have already been implicated in various human diseases including new born meningitis and urinary tract infections suggesting a possible transfer of avian strains to humans or vice versa and thus a potential zoonotic risk of avian strains(Johnson et al.,2001,2003a; Rodriguez-Siek et al.,2005).However,sound phyloge-netic typing of a representative collection of human-and animal-derived ExPEC strains that could verify APEC as putative zoonotic agents by unravelling the evolu-tionary descent of extrainestinal E.coli is yet missing. With75.0%of human and avian ExPEC strains yielding different virulence gene patterns a rather high intra-and inter-group variation was found.While there were considerable similarities between the three groups of isolates,some virulence features yielded a preferential association with certain pathotypes.Genes encoding temperature-sensitive haemagglutinin(tsh),aerobactin (iucD),and salmochelin(iroN)systems,ColV operon (cvi/cva),serum resistance protein(iss),and transfer protein(traT)are known for their frequent or exclusive localisation on large transmissible R plasmids or ColV plasmids in APEC(pAPEC-O1,pAPEC-O2-ColBM, and pTJ100)(Johnson and Russo,2002;Johnson et al.,2006b)and were clearly associated with APEC strains in our study.All of these mentioned episomal genes were found in more than57.0%of the APEC examined,suggesting that such plasmids,with a wide distribution among APEC,constitute a reservoir of plasmid-mediated virulence-associated genes transmis-sible to other bacteria.Indeed,similar plasmids have been identi?ed in UPEC strains,including pJS332,

Table5.Distribution of ECOR groups among ExPEC strains and mean number of virulence-associated genes in strains belonging to different ECOR groups

Pathogroup Phylogenetic group(%positive strains)

A B1B2D

APEC(n?436)46.1 1.835.117.0 Virulence genes per strain11.713.319.414.8

UPEC(n?65)16.90.061.521.5 Virulence genes per strain9.10.017.213.5

NMEC(n?25) 4.00.072.024.0 Virulence genes per strain12.00.018.814.3 Total(n?526)40.5 1.540.117.9 Virulence genes per strain11.613.318.714.6

Genes positively linked(p p0.001)to phylogenetic groups in ExPEC:ECOR A:papC,tsh,fyuA,ireA,irp2,tia;ECOR B1:none;ECOR B2:papC, sfa/foc,chuA,fyuA,ireA,iroN,irp2,sitD chr.,neuC,kpsMTII,cnf1/2,vat,hlyA,ibeA,gimB,tia,and malX;ECOR D:hra,chuA,iroN,sitD chr.,cvi/ cva,and pic.

C.Ewers et al./International Journal of Medical Microbiology297(2007)163–176171

harbouring the iro gene cluster and pRK100,a 145-kb big ColV plasmid,harbouring the aerobactin iron uptake system and the Tra operon (Ambrozic et al.,1998;Sorsa et al.,2003).However,according to the prevalence of cvi/cva operon genes,ColV plasmids are more frequently distributed in APEC and to a lesser extent in NMEC and UPEC strains underlining their contribution to virulence and ?tness of avian ExPEC strains particularly.Recently performed studies also indicated that genes contained within a portion of this putative virulence region are highly conserved among APEC and that they occur signi?cantly more often in APEC than in avian commensal E.coli (Johnson et al.,2006b ).It has also been shown that the acquisition of APEC plasmids by a commensal E.coli isolate enhances the ability of the strain to kill chicken embryos,grow in human urine,and colonise the murine kidney,under-lining the pathogenic importance of these big plasmids (Skyberg et al.,2006).

The inference of a closer genetic relationship between APEC and NMEC rather than with UPEC strains was further substantiated by the distribution of certain non-ColV plasmid-related genes.Although the presence of the K1capsule-speci?c gene neuC was strongly asso-ciated with NMEC,nearly one-third of APEC strains also harboured this gene,which was less commonly distributed in UPEC.Several epidemiological and experimental studies have clearly linked neonatal meningitis with E.coli strains expressing the K1antigen (Achtman et al.,1986;Kim et al.,1992).This link to pathogenicity has also been shown for APEC strain IMT5155(O2:K1:H5)where the virulence of kpsMT mutants was signi?cantly attenuated in a chicken model (Li et al.,2005).Moreover,chickens experimentally infected with K1-positive IMT5155develop neurologi-cal symptoms,and one NMEC strain (O18:K1:H7)has been shown to cause clinical symptoms typical of colisepticaemia in 5-week-old chickens (unpublished data).Although we found a considerably higher mean value of virulence-associated genes in neuC -positive strains (19.4)compared to neuC -negative ones (13.2),detailed experimental data have to be obtained to determine the role of those genes with high OR values,like chuA ,sit chr .,irp2,fyuA ,gimB ,ibeA ,malX ,and vat for the proposed higher virulence of K1-positive strains and to ascertain that a higher virulence is not solely based on the presence of the K1capsule.

With the exception of the vacuolating autotransporter gene vat ,which was nearly equally present in about half of the APEC,UPEC,and NMEC strains,all other toxin

a f a /d r a

c r l

f i m C

h r l A

i h a

p a p C

s f a /f o c

t s h

m a t

c h u A

f y u A

i r e A

i r o N

i r p 2

i u c D

s i t D e p

s i t D c h r

c v i /c v a

i s s

n e u C

k p s M T I I

o m p A

t r a T

E A S T -1

c n f

s a t

v a t

h l y A

i b e A

g i m B

t i a

R P a i

p i c

p ≤ 0.001 for APEC (+), UPEC (x), NMEC (#), APEC and UPEC (*), APEC and NMEC (°), APEC, UPEC, and NMEC (&); p ≥ 0.001 for APEC,UPEC, and NMEC (-)

Fig.2.Associations between 33virulence-associated genes among ExPEC.p p 0.001for APEC (+),UPEC (x),NMEC (#),APEC,and UPEC (*),APEC and NMEC (1),APEC,UPEC,and NMEC (&);p X 0.001for APEC,UPEC,and NMEC (à).

C.Ewers et al./International Journal of Medical Microbiology 297(2007)163–176

172

genes and the haemolysin gene hlyA showed clear discrepancies in their distribution among the three ExPEC groups.Almost none of the avian pathogenic strains possessed cnf1/2,encoding a cytonecrosis factor, as well as the secreted autotransporter toxin gene sat. Mutation of cnf1attenuates the virulence of UPEC in a murine model of urinary tract infection while the sat-encoded toxin contributes to vacuolating cytotoxicity in kidney and bladder epithelial cells(Rippere-Lampe et al.,2001;Guyer et al.,2002).While data concerning distribution among and pathogenic signi?cance of sat for APEC and NMEC are lacking,the low prevalence of cnf1/2in APEC is consistent with previously published data(Rodriguez-Siek et al.,2005).Thus,with the exception of vat extracellular toxins,most of them being located on PAIs described for UPEC do not appear to be a critical virulence factor in ExPEC strains in general but are probably characteristic for a certain subgroup colonising a distinct habitat(Dobrindt et al.,2002; Mokady et al.,2005b).Thus,the existence or absence of these genes does not contribute to a reliable distinction between avian and human strains,as some APEC still harbour toxin genes and human ExPEC strains possess them only infrequently.

Moreover,only a few APEC strains were positive for hlyA,and interestingly all UPEC and NMEC strains harbouring this gene displayed haemolysis on sheep blood agar plates while only two of the four positive APEC isolates showed this phenotype implicating an incomplete or non-functional operon or gene silencing through other mechanisms.Both cnf1and hly together with heat-resistant agglutinin gene hra are located on PAI II of UPEC strain J96(Dobrindt et al.,2002). Indeed,with the exception of two strains all UPEC strains yielded a signi?cant association of these three genes suggesting the presence of PAI II,which has to be con?rmed by macrorestriction and hybridisation ana-lyses.Although much less frequent in NMEC and especially in APEC strains with a few exceptions,cnf1/2, hlyA,and hra were detected in combination within these pathotypes,indicating an exchange of PAI II-related sequences or the whole island between the three ExPEC groups.

It is well known that human ExPEC strains often contain multiple PAIs,each with a distinctive combina-tion of virulence-associated genes that can disseminate horizontally between single E.coli strains(Dobrindt, 2005).Likewise,several PAIs and genetic islands have been identi?ed in APEC including PAI I(APEC-O1) harbouring ireA,pap operon genes and invasion locus tia(Kariyawasam et al.,2006).According to our prevalence data,combinations of PAI I(APEC-O1)-related genes occurred not only in strains belonging to the APEC pathotype(17.9%)but also in UPEC(10.7%) and NMEC(28.0%)isolates indicating a PAI I-like structure in human strains.Again,this assumption has to be con?rmed in future experiments by hybridisation or sequence analyses.

Our data also indicate the presence of further PAIs described for UPEC strains,like PAI III,containing iro and sfa,as well as the high-pathogenicity island(HPI), coding for the yersiniabactin system in APEC(Do-brindt,2005).While the presence and pathogenic relevance of the HPI in APEC has already been shown in several studies,it is not entirely known whether a PAI III-like structure exists in APEC.Data are available indicating the occurrence of two copies of the iro gene cluster in APEC strain MT512(Schouler et al.,2004), and we found signi?cant associations of iroN with several plasmid-related genes,including cvi/cva,tsh,and the episomal sit locus.

Further evidence of a remarkable genetic exchange between avian and human ExPEC is(1)the high prevalence of CFT073island marker malX in all three pathotypes and its predicted location on a metV genomic island in APEC strain IMT5155,(2)the distribution of ibeA,located on a genetic island associated with meninigitis(GimA),and(3)the presence of gimB not only in NMEC but also in APEC and UPEC strains(Huang et al.,2001;Germon et al.,2005; Li et al.,2005).

Our data show that although there are single genes or gene combinations with a greater linkage to a certain pathotype there is no host-or pathotype-speci?c virulence gene pattern.A categorisation of ExPEC strains into the pathotypes APEC,UPEC,or NMEC only by means of virulence gene typing is therefore not possible,as bacteria from the same host can have different patterns,just as bacteria from different hosts can share the same genes or gene combinations.A considerable variation in virulence gene patterns in human and avian ExPEC strains is consistent with extensive ongoing remodelling of PAIs and virulence-associated plasmids(Johnson et al.,2001;Bingen-Bidois et al.,2002).

Initial studies on the epidemiology of E.coli pathotypes led to the proposal of a clonal population structure in this bacterial species leading to the formulation of four different phylogenetic origins(A, B1,B2,and D)(Ochman and Selander,1984;Selander et al.,1987).In our study,human and avian ExPEC strains revealed discernible overlaps in their assignments to phylogenetic groups with some remarkable differ-ences.A gradient in the number of strains involved in neonatal meningitis and urinary tract infection was detected from strains belonging to the B2group(72.0% and61.5%)to the A group(4.0%and16.9%)via the D group(24.0%and21.5%).Although we also found a substantial number of avian strains in ECOR group B2 (35.1%),most of the strains were assigned to group A (46.1%).This group was underrepresented in NMEC strains,while a notable amount of UPEC strains

C.Ewers et al./International Journal of Medical Microbiology297(2007)163–176173

belonged to group A,con?rming published data(Bingen et al.,1998;Hilali et al.,2000;Johnson et al.,2001; Rodriguez-Siek et al.,2005).There are several studies suggesting that virulent clonal groups are derived primarily from phlyogenetic group B2,and to a lesser extent from group D,explaining the predominance of groups B2and D among clinical isolates(Johnson and Russo,2005).In the1990s,UPEC of clonal group A representing phylogenetic group D emerged as a prominent cause of drug-resistant urinary tract infection across the USA(Johnson and Russo,2002;Burman et al.,2003).There was evidence for a point-source spread within one community implicating contaminated food as a possible transmission vehicle for ExPEC (Manges et al.,2001).Indeed,it has previously been reported that retail food,especially from poultry,are commonly contaminated with avian E.coli strains that sometimes closely resemble human clinical ExPEC isolates(Johnson and Russo,2005).In another study, 24.0%of poultry-derived strains represented ExPEC and were derived from virulence-associated E.coli phylogenetic groups B2and D(42.0%)(Johnson et al.,2003b).Similarly our data suggest a potential human health threat by avian ExPEC strains as not just B2 isolates were found to yield comparable virulence gene pro?les to human ExPEC but also a large proportion of group A isolates among APEC were found that have to be considered to represent a potential zoonotic risk. However,in general the application of ECOR typing is somewhat limited concerning the determination of the evolutionary descent and relationship to each other,as the integration of ExPEC into a more holistic popula-tion-based background demands methods like multi-locus sequence typing(MLST)warranting a higher resolution(Johnson et al.,2006a;Wirth et al.,2006). The focus of current and future research,which is partly on its way,should therefore be a comprehensive investigation of human-and animal-derived strains considering a combination of the interaction between virulence determinants and the phylogenetic back-ground of bacteria.

In our study,we discovered strains of different hosts and pathotypes indistinguishable by the typing methods applied and strains widely differing from each other disregarding host and pathotype.A common origin of ExPEC strains of medical and veterinary source in general is thus questionable,although this has to be proven by sound phylogenetic typing methods.As APEC are epidemiologically highly abundant in con-trast to UPEC and NMEC,we speculate that some of these avian strains are the armoury of NMEC and UPEC suggesting that poultry may be a vehicle or even a reservoir for E.coli capable of causing urinary tract infection and newborn meningitis and that APEC therefore have to be considered potential zoonotic agents.Acknowledgements

We thank Ulrich Dobrindt,Helge Karch,Jo rg Enzenauer,and Kwang Sik Kim for providing uro-pathogenic and newborn meningitis-causing E.coli strains.We also thank Marc de Boer for allocating avian pathogenic E.coli from zoo birds,and Lisa K. Nolan and John M.Fairbrother for providing APEC reference strains.This work was supported by Grant WI 1436/5-1from the Deutsche Forschungsgemeinschaft (DFG)and by Lohmann Tierzucht GmbH,Cuxhaven, Germany.

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