JOURNAL OF CLINICAL MICROBIOLOGY,0095-1137/00/$04.0010

Sept. 2000, p. 3470–3473 Vol. 38, No. 9

Copyright © 2000, American Society for Microbiology. All Rights Reserved.

Cattle Can Be a Reservoir of Sorbitol-Fermenting ShigaToxin-Producing Escherichia coli O157:H2 Strains

and a Source of Human DiseasesMARTINA BIELASZEWSKA,1,2* HERBERT SCHMIDT,2 ALMUT LIESEGANG,3 RITA PRAGER,3

WOLFGANG RABSCH,3 HELMUT TSCHAPE,3 ALOIS CIZEK,4 JAN JANDA,5

KVETA BLAHOVA,5 AND HELGE KARCH2

Institute for Medical Microbiology, The 2nd Medical Faculty, Charles University,1 and 1st Clinic of Pediatrics,University Hospital Motol,5 150 06 Prague, and Faculty of Veterinary Medicine, University of Veterinary

and Pharmaceutical Sciences, 602 00 Brno,4 Czech Republic, and Institut fur Hygiene undMikrobiologie der Universitat Wurzburg, 97080 Wurzburg,2 and Robert Koch

Institut, Bereich Wernigerode, 38855 Wernigerode,3 Germany

Received 21 April 2000/Returned for modification 27 May 2000/Accepted 13 June 2000

Using the immunomagnetic separation procedure, we isolated sorbitol-fermenting (SF) Shiga toxin-produc-ing Escherichia coli (STEC) O157:H2 strains from two patients, one with hemolytic-uremic syndrome and theother with diarrhea, and from a dairy cow epidemiologically associated with the patients. The phenotypic andgenotypic characteristics of all isolates were identical or closely related. Moreover, the bovine isolate showeda clonal relatedness to SF STEC O157:H2 strains isolated from patients in Germany and the Czech Republicfrom 1988 to 1998. This is the first evidence that cattle can be a reservoir of SF STEC O157:H2 and a sourceof human diseases.

During the last 10 years, sorbitol-fermenting (SF) Shigatoxin (Stx)-producing Escherichia coli (STEC) strains of sero-type O157:H2 have emerged as important causes of hemolytic-uremic syndrome (HUS) and diarrhea in Germany (1, 8, 9). Inaddition to sporadic cases of human disease (8), two outbreaksof HUS caused by SF O157:H2 STEC strains have been iden-tified. The first outbreak, in 1988, lead to the discovery of thepathogen (9). The second outbreak occurred in the winter of1995 to 1996 and included 28 HUS cases, three of which werefatal (1). Analysis of phenotypic and molecular characteris-tics of SF STEC O157:H2 strains demonstrated that suchstrains represent a distinct clone within the E. coli O157 sero-group which shares virulence characteristics with non-SF(NSF) STEC O157:H7 (10). In 1995, strains belonging to theSF STEC O157:H2 clone were isolated from HUS patients inthe Czech Republic (2), suggesting the ability of this patho-gen to spread. Despite the increasing significance of SF STECO157:H2 in the etiology of HUS and diarrhea, the epidemi-ology of the infection, including reservoirs and routes of trans-mission, remains unknown. In contrast to NSF STEC O157:H7strains, which have cattle as their major reservoir (12), SFSTEC O157 strains have not been found in cattle (9, 10).

In this study, we isolated SF STEC O157:H2 strains fromtwo patients and an epidemiologically associated cow in theCzech Republic. We compared phenotypic and genotypic char-acteristics of the bovine and patients’ isolates to determinewhether the cow was the source of human infections. More-over, we determined the genetic relatedness of the bovine iso-late to SF STEC O157:H2 strains isolated previously frompatients in the Czech Republic and Germany in order to

investigate whether cattle can be reservoirs of SF STECO157:H2 strains.

The strains investigated are listed in Table 1. SF STECO157:H2 strains 258/98 and 269/98 were isolated from twosiblings who developed HUS and diarrhea, respectively, fol-lowing their visit to a dairy farm in Central Bohemia, CzechRepublic. During the farm visit on 12 January 1998, bothchildren were exposed to a herd of 32 dairy cows throughtouching and stroking some of the animals. The younger child(a 15-month-old boy) developed bloody diarrhea on 15 Janu-ary and was hospitalized for HUS on 20 January. His 6-year-old brother experienced a 2-day period of mild watery diarrheawhich began on 16 January. Investigation of fecal samples fromthe farm cows performed on 25 January 1998 yielded an SFSTEC O157:H2 strain (550/98) from 1 of the 32 animals. TheSF E. coli O157 strains from the patients and the cow wereisolated using the immunomagnetic separation (IMS) proce-dure (11), followed by slide agglutination with anti-O157 se-rum (ITEST, Hradec Kralove, Czech Republic) of up to 50 SFcolonies from each sorbitol-MacConkey agar (SMAC) plate.The colonies that displayed agglutination were biochemicallyconfirmed as E. coli and shown to produce Stx2 by using acommercial latex agglutination assay (Verotox-F; DenkaSeiken Co., Tokyo, Japan). Serotyping by standard proceduresidentified serotype O157:H2.

All of the isolates were tested for sorbitol fermentation,b-D-glucuronidase activity, and the production of enterohem-orrhagic E. coli (EHEC) hemolysin according to proceduresdescribed by Gunzer et al. (8) and Schmidt et al. (20). Phagetyping was performed according to the procedure described byKhakhria et al. (13). Since the isolates were nonmotile andtheir H antigens could not be determined by serotyping, thegene encoding the flagellin subunit (fliC) was detected andcharacterized using the fliC restriction fragment length poly-morphism (fliC-RFLP) method described by Fields et al. (7).The presence of stx1, stx2, and stx2c genes was investigated byPCR procedures described previously (8, 19). The location of

* Corresponding author. Mailing address: Institut fur Hygiene undMikrobiologie der Universitat Wurzburg, Josef-Schneider-Str. 2, 97080Wurzburg, Germany. Phone: 49-931/201 51 63. Fax: 49-931/201 51 66.E-mail: mbielaszewska@hygiene.uni-wuerzburg.de.

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stx2 was determined by hybridization of EcoRI-digested geno-mic DNA with digoxigenin-labeled stx2 probe (6). The intimin-encoding eae gene was detected using primers SK1 and SK2(19) and further characterized using primer pairs SK1-LP2,SK1-LP3, SK1-LP4, and SK1-LP5 (16), which are specific toeae types a, g, b, and ε, respectively (16). The P-gene profile,which reflects the number and positions of lambdoid phages inthe genome, was determined as described by Datz et al. (6).Pulsed-field gel electrophoresis (PFGE) was performed ac-cording to the PulseNet protocol of the Centers for DiseaseControl and Prevention (17), except that the gel running timewas increased to 40 h. PFGE patterns were analyzed using theRFLPscan software (Scanalytics; CSP Inc.). Plasmid profileswere determined as described previously (22). The presence ofplasmid-encoded putative virulence genes (EHEC hly, katP,espP, and etp) was investigated by PCR (4, 5, 20, 21) and byhybridization of plasmid DNA digested with BamHI or SmaIwith digoxigenin-labeled probes (22).

Four representative SF STEC O157:H2 strains isolatedfrom patients in Germany and the Czech Republic during 1988to 1998 (Table 1) and a control STEC O157:H7 strain, EDL933 (15), were investigated by the same procedures. Stx-nega-tive SF E. coli O157 strains 1083-36/91 (O157:H45) and 693/91(O157:H19) (2), isolated from infants with diarrhea, were usedas controls in fliC-RFLP and PFGE.

As shown in Fig. 1, the SF STEC O157:H2 isolates from thecow (lane 1) and from the two epidemiologically related pa-tients (lanes 2 and 3) and the four representative German andCzech SF STEC O157:H2 isolates from 1988 to 1998 (lanes 4to 7) shared a fliC-RFLP pattern which was identical to that ofE. coli O157:H7 strain EDL 933 (lane 8) but clearly differedfrom fliC-RFLP patterns of E. coli O157 strains possessing H45(lane 9) or H19 (lane 10). This demonstrated the presence ofthe H7-encoding fliC gene in all SF STEC O157:H2 strains.The other characteristics of the strains are summarized inTable 1. These results show that the epidemiologically relatedisolates from the cow (550/98) and the patients with HUS(258/98) and diarrhea (269/98) had identical phenotypic andgenotypic characteristics. Moreover, these three isolatesshared phenotypic features and chromosomal characteristics,including the presence of stx2 only, the presence of eae type g,

and closely related P-gene profiles, with the four representa-tive SF STEC O157:H2 strains isolated from HUS patients inGermany and the Czech Republic during 1988 to 1998 (Table1); in all strains, stx2 was localized to the same 18-kb EcoRIrestriction fragment of the genomic DNA (Table 1). In addi-tion, all seven STEC O157:H2 isolates had identical plasmidprofiles, possessing a single large plasmid of 90 to 100 kb.However, while the large plasmids of the bovine and two epi-demiologically related human isolates did not contain any ofthe putative virulence genes (Table 1), the other four SF STECO157:H2 strains possessed a combination of EHEC hly and etp(Table 1). This suggests the presence of two different large

TABLE 1. Characteristics of SF STEC O157:H2 strains isolated from a cow and patients and of control NSF STEC O157:H7 strain EDL 933

Straina Disease; countryb

(referencec)

Phenotypic characteristicsd Chromosomal characteristics Plasmid-encoded genesg

Serotype PT SF/GUD Stx EHEC Hly stxe eae P-gene profilef EHEC hly katP espP etp

258/98h HUS; CR O157:H2 88 1/1 2 2 stx2/18.0 g 17 2/2 2/2 2/2 2/2269/98h WD; CR O157:H2 88 1/1 2 2 stx2/18.0 g 17 2/2 2/2 2/2 2/2550/98h Cow; CR O157:H2 88 1/1 2 2 stx2/18.0 g 17 2/2 2/2 2/2 2/2703/88 HUS; G (10) O157:H2 88 1/1 2 2 stx2/18.0 g 17 1/48.5 2/2 2/2 1/3.9, 1.9i

221/95 HUS; CR (2) O157:H2 88 1/1 2 2 stx2/18.0 g 9 1/15.0 2/2 2/2 1/3.7, 1.71995/96 HUS; G (2) O157:H2 88 1/1 2 2 stx2/18.0 g 16 1/15.0 2/2 2/2 1/3.9, 1.93573/98 HUS; G O157:H2 88 1/1 2 2 stx2/18.0 g 16 1/15.0 2/2 2/2 1/3.9, 1.9EDL 933 HC; US (15) O157:H7 21 2/2 1 and 2 1 stx1/NP g 2 1/12.0 1/9.0 1/7.5 1/3.9, 1.9

stx2/4.7

a For the SF STEC O157:H2 strains, the last two numbers indicate the year of isolation.b WD, watery diarrhea; HC, hemorrhagic colitis; CR, Czech Republic; G, Germany; US, United States.c Strains for which no references are given are from this study.d PT, phage type; SF/GUD, sorbitol fermentation/b-D-glucuronidase activity; Stx, Stx phenotype; EHEC Hly, production of EHEC hemolysin; 1, positive result; 2,

negative result.e stx PCR result/size (in kilobases) of EcoRI restriction fragment of the genomic DNA hybridizing to the stx2 probe. NP, not performed.f P-gene profile 17 is characterized by three fragments of 20, 6.4, and 3.7 kb; the related P-gene profile 16 is characterized by two fragments of 20 and 3.7 kb, and

the other related P-gene profile 9 is characterized by two fragments of 6.4 and 3.7 kb. P-gene profile 2 consists of two fragments of 21.2 and 16.2 kb and is unrelatedto P-gene profiles 17, 16, and 9.

g Detection of the gene by PCR/size (in kilobases) of the plasmid DNA fragment hybridizing to the respective probe. 1, positive result; 2, no signal obtained.h Epidemiologically related strains; the other four SF STEC O157:H2 strains are representative isolates from 1988 to 1998 and are epidemiologically unrelated.i Fragments of two different sizes hybridized with the probe.

FIG. 1. Agarose gel electrophoresis of fliC PCR products of SF STECO157:H2 strains and of control E. coli O157 strains after restriction with RsaI.Lanes M, molecular size marker (1-kb DNA ladder; Gibco BRL). In lanes 1 to7, the following SF STEC O157:H2 strains are shown: lane 1, 550/98; lane 2,258/98; lane 3, 269/98; lane 4, 703/88; lane 5, 221/95; lane 6, 1995/96; and lane 7,3573/98. Lanes 8 to 10 contain control E. coli O157 strains as follows: lane 8,EDL 933 (O157:H7); lane 9, 1083-36/91 (O157:H45); and lane 10, 693/91 (O157:H19).

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plasmids in SF STEC O157:H2 strains. None of the SF STECO157:H2 strains harbored the full spectrum of the plasmid-encoded genes found in a prototype STEC O157:H7 strain,EDL 933 (Table 1).

The clonal relatedness of SF STEC O157:H2 isolates was

investigated using PFGE. As shown in Fig. 2, the PFGE pat-tern of the bovine isolate (lane 3) was closely related to thepatterns of the two epidemiologically related human isolates(lanes 1 and 2) as well as to the patterns of the four represen-tative, epidemiologically unrelated, human SF STEC O157:H2

isolates (lanes 4 to 7). In contrast, the PFGE patterns of thebovine and all human SF STEC O157:H2 isolates differedmarkedly from the patterns of all of the control E. coli O157strains, including NSF STEC O157:H7 strain EDL 933 (Fig. 2,lane 8) and two SF Stx-negative strains of serotypes O157:H45(Fig. 2, lane 9) and O157:H19 (Fig. 2, lane 10). Analysis of thePFGE patterns by the RFLPscan system (Fig. 3) demonstratedthat all seven SF STEC O157:H2 strains, including the bovineand human isolates, belonged to one cluster but were onlydistantly related to NSF STEC O157:H7 strain EDL933 and tothe SF Stx-negative E. coli O157:H19 and O157:H45 strains.Taking these findings together with the other chromosomalcharacteristics, it can be concluded that the SF STEC O157:H2

isolates of bovine and human origin belong to one clone com-plex. The observed differences in the gene composition of largeplasmids in strains of the same clone can be due to the loss,acquisition, or exchange of plasmid DNA during lateral trans-fer of these mobile elements.

By demonstrating the clonal relatedness between SF STECO157:H2 strains isolated from a cow and patients, we providethe first evidence that cattle can be a reservoir of SF STECO157:H2 strains and a source of the infection for humans.Importantly, the bovine SF STEC O157:H2 isolate containedboth stx2 and eae type g genes, which are the virulence char-acteristics possessed by NSF and SF STEC O157 strains iso-lated from patients (3, 8, 16) (Table 1). This strongly supportsthe pathogenic potential of the bovine isolate for humans. Onthe other hand, the bovine isolate and both epidemiologicallyrelated SF STEC O157:H2 strains isolated from patientslacked the plasmid-encoded putative virulence genes (Table1), suggesting that these genes may not be significant in thegenesis of human disease or that they have been lost duringinfection or storage.

Both patients in this study were likely to be infected throughdirect contact with the cow that shed the SF STEC O157:H2

strain in its feces. This observation is in agreement with pre-vious reports on direct transmission of STEC O157:H7 fromcattle to humans (12, 18) and contributes to the increasingbody of evidence that contact with farm animals, especially

FIG. 2. PFGE patterns of XbaI-digested genomic DNAs of SF STECO157:H2 strains and control E. coli O157 strains. Lane S, molecular size stan-dard (DNA from E. coli strain G5244 restricted with XbaI). In lanes 1 to 7, thefollowing SF STEC O157:H2 strains are shown: lane 1, 258/98; lane 2, 269/98;lane 3, 550/98; lane 4, 221/95; lane 5, 703/88; lane 6, 1995/96; and lane 7, 3573/98.In lanes 8 to 10, control E. coli O157 strains are shown as follows: lane 8, EDL933(NSF STEC O157:H7); lane 9, 1083-36/91 (SF, Stx-negative E. coli O157:H45);and lane 10, 693/91 (SF, Stx-negative E. coli O157:H19).

FIG. 3. Cluster analysis, derived from PFGE data, of SF STEC O157:H2 isolates from the cow and patients, NSF STEC O157:H7 strain EDL 933, and SFStx-negative strains O157:H45 and O157:H19 with the RFLPscan software.

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with cattle, is an important risk factor for acquiring STECinfection (14).

The failure to isolate SF STEC O157 strains from cattle inprevious studies that used molecular techniques (9, 10) wasprobably due to the fact that such techniques are 100- to1,000-fold less sensitive than the IMS procedure (11) that wasused for the detection of SF E. coli O157 strains in this study.However, despite the fact that we introduced the IMS enrich-ment step, subsequent identification of SF O157 colonies onSMAC plates was a laborious procedure that required slideagglutination of almost 50 colonies per plate. The widespreaddistribution of the SF STEC O157:H2 clone in central Europe(2, 8) and the emergence of cattle as a reservoir of such strainsthus place significant limitations on using SMAC as the solemethod for detecting STEC O157. This, combined with thefact that SF STEC O157 strains do not express EHEC he-molysin (2, 8) (Table 1) and thus cannot be detected onenterohemolysin agar (20), accentuates the need to developa selective medium for such strains. Additional studies usingappropriate diagnostic methods are necessary to determine thesignificance of cattle as a reservoir of SF STEC O157:H2 andto further investigate the epidemiology of the infection.

This study was supported by grant 4563-3 from the Ministry ofHealth of the Czech Republic, by grant 525/97/0373 from the CzechGrant Agency, and by grants 01 KI 9903 and 1368/343 from Bundes-ministerium fur Bildung und Forschung (BMBF), Germany.

We thank R. Ahmed and W. Demczuk (Health Canada, Winnipeg,Canada) for providing phages and reference strains for phage typingand L. Durso (University of Nebraska, Lincoln) for critical readingof the manuscript. The excellent technical assistance of B. Plaschke(Wurzburg), A. Reischelova (Prague), and G. Bartel, U. Siewert, andB. Tannert (Wernigerode) is highly appreciated.

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