Comparative Study of Five DifferentTechniques for EpidemiologicalTyping of Escherichia coli O157

Katharina Grif, Helge Karch,Christian Schneider, Franz D. Daschner,Lothar Beutin, Thomas Cheasty, Henry Smith,Bernard Rowe, Manfred P. Dierich, andFranz Allerberger

A set of 47 Austrian human, food, and veterinary Escherichiacoli O157:H7 isolates was used to evaluate five different epi-demiological typing methods. Ribotyping using an automatedmicrobial characterization system (RiboPrinter™) was notsuitable for detection of epidemiological relatedness. All butone E. coli strain were typeable by phage typing. Random am-plified polymorphic DNA-PCR fingerprinting was performedusing primer M13 containing the sequence 59-GAG GGTGGC GGT TCT-39 and primer 1247 (59-AAGAGCCCGT-39).

Although both methods recognized only two clusters, bothdendrograms grouped most of the EHEC O157 isolates intoepidemiologically related subgroups. Pulsed-field gel electro-phoresis of XbaI digested total DNA was a valuable subtypingsystem. We found that major differences can exist betweenresults of multiple subtyping methods. E. coli O157 isolatesshould not be classified as epidemiologically related or nonre-lated on the basis of a single typing method alone. © 1998Elsevier Science Inc.

INTRODUCTION

Shiga toxin (Stx)-producing Escherichia coli O157:H7is an important cause of infectious diarrhea and di-arrhea associated hemolytic uremic syndrome(HUS). An increasing variety of food sources andother vehicles for E. coli O157:H7 transmission havebeen documented in recent years (Armstrong et al.1996). Direct or indirect exposure to bovine or hu-

man feces account for most cases where the source isidentified.

Molecular subtyping, or fingerprinting, of E. coliO157:H7 makes it possible to create a molecular pro-file of a bacterium cultured from an ailing patient,which in turn can be compared with bacteria isolatedfrom other patients and from foods. If the patterns donot match, the isolates can be presumed to have nocommon link. A recent report from a World HealthOrganisation consultation recommends in-depth in-vestigation of enterohemorrhagic E. coli (EHEC) out-breaks to determine the point or process whereEHEC contamination has occurred so that effectivepreventative measures can be introduced (WHO1997). The value of molecular subtyping in enhanc-ing surveillance for E. coli O157:H7 infections hasbeen illustrated in previous outbreak investigations(Anonymous 1997). We used a set of Austrian iso-lates to evaluate five different typing methods.

From the Institute of Hygiene, University of Innsbruck (GK,MPD, FA), Innsbruck, Austria; the Institute of Hygiene and Mi-crobiology, University of Wurzburg (HK), Wurzburg, Germany;the Institute of Environmental Medicine and Hospital Epidemi-ology, University Hospital of Freiburg (CS, FDD), Freiburg, Ger-many; the Robert Koch Institute (LB), Berlin, Germany; and theLaboratory of Enteric Pathogens, Central Public Health Labora-tories (TC, HS, BR), London, United Kingdom

Address reprint requests to Univ. Prof. Dr. Franz Allerberger,Institute of Hygiene, Fritz Pregl Str. 3, A-6020 Innsbruck, Aus-tria.

Received 30 June 1998; revised and accepted 18 August 1998.

DIAGN MICROBIOL INFECT DIS 1998;32:165–176© 1998 Elsevier Science Inc. All rights reserved. 0732-8893/98/$19.00655 Avenue of the Americas, New York, NY 10010 PII S0732-8893(98)00103-5

MATERIALS AND METHODS

Microorganisms

Bacteria (47 Escherichia coli O157 and five Hafnia alvei)had been stored at the Institute of Hygiene (Inns-bruck, Austria) in vials containing Columbia agar(OXOID, Basingstoke, UK) at 4°C since initial isola-tion. Subcultures were grown on Columbia agarplates containing 5% defibrinated sheep blood at37°C for 48 h. One single colony was used to inocu-late a subset of five Columbia agar vials, sent to theparticipating centers after overnight incubation.

Human Isolates. Table 1 lists data on 17 clinicalisolates from patients in the Austrian provinces ofTyrol (n 5 15), Styria (n 5 1), and Upper Austria (n 51) from 1992 to 1996. The clinical isolates includedtwo strains of Hafnia alvei (isolates no. 7 and no. 17),stool isolates without pathogenic relevance. Hafniaalvei may resemble E. coli and therefore can pose aproblem in differential diagnosis. Isolates no. 1 to no.6 were from an EHEC outbreak in Tyrol, describedelsewhere (Solder et al. 1993). With the exception ofstrain no. 2, all isolates of this outbreak had beenconsidered indistinguishable previously (Solder etal. 1993). Isolate no. 2 harbored genes for Stx1 andStx2, while the remaining isolates of this 1992 out-break produced Stx2 and Stx2c. E. coli O157 isolatesno. 8 to no. 16 were collected during 1996; isolate no.8 originated from a patient from Styria, no. 13 fromUpper Austria. Isolates no. 11, no. 12, and no. 14affected members of one family. Isolate no. 15 wasfrom a patient with HUS. Isolate no. 16 was from apatient with hemorrhagic colitis hospitalized inTyrol, who contracted the EHEC infection during aholiday in Turkey. No microbiological confirma-tion of the causative agent from suspected foodwas possible. Altogether no fatalities were ob-served.

Food Isolates. Twenty-seven food isolates from26 samples of red minced meat and raw cow’s milktested in 1996 without any apparent relationship tohuman cases were used. Products originated fromthe Austrian states of Upper Austria (n 5 12),Tyrol (n 5 12), Carinthia (n 5 1) and Styria (n 51). The specimens included two strains of Hafniaalvei (no. 20 and no. 24) and one mixture of Hafniaalvei (no. 40a) plus E. coli O157:H7 (no. 40b). AgainHafnia alvei was included because it is often foundas background flora which must not be misread asE. coli. Three of the 24 E. coli O157:H7 isolates (no.41 to no. 43) were from one single batch of rawcows milk.

Veterinary Isolates. Eight E. coli O157:H7 strainsisolated from fecal samples of healthy Tyroleancalves in 1996 were used. Isolates no. 50 and no. 51

were from animals raised together on one farm.Veterinary isolates were provided by Dr. D.Khaschabi (Innsbruck, Austria).

Phage Typing

Strains were phage typed at the Laboratory of En-teric Pathogens (CPHL, London, UK) using thephage-typing scheme of Ahmed et al. (1987) andextended by Khakhria et al. (1990). The scheme com-prises 16 bacteriophages and recognizes 88 phage types(R. Khakhria, unpublished communication). Briefly,the phages were used at their routine test dilution(RTD; i.e., the highest dilution giving semi-confluentlysis on its first propagating strain). The test strainswere stored on Dorset’s egg medium at room temper-ature. Strains were inoculated into 5 mL of nutrientbroth (Difco, Detroit, MI, USA) and incubated withshaking at 37°C for 1.5 h. Nutrient agar plates (Difco)were inoculated with the broth by flooding the agarsurface, removing any excess liquid, and allowing thesurface to air dry. The sixteen typing phages (0.02-mLvolumes) were then applied to the surface of the plate.The plates were read after incubation at 37°C for 18 h.

Automated Ribotyping

All samples were ribotyped by the Institute of Hy-giene (Innsbruck, Austria) by use of an automatedmicrobial characterization system (RiboPrinter™,Qualicon, Wilmington, DE, USA) as described (Bruce1996). In brief, isolated colonies were plated ontobrain heart infusion agar (BBL, Cockeysville, MD,USA) plates. After overnight growth, each samplewas suspended in lysis buffer and heated at 80°C for10 min. The samples were then processed by use ofthe aforementioned automated microbial character-ization system. In the first step, samples were treatedwith a lysing agent to release the DNA. The DNAwas then digested to completion with a restrictionenzyme. The resulting DNA restriction fragmentswere transferred to an agarose gel cassette. Using aprocess known as direct blot electrophoresis, theDNA fragments were size-separated and transferredto a moving nylon membrane. After denaturation,each membrane was hybridized with a chemicallylabeled rRNA operon from E. coli. The membranewas washed and treated with blocking buffer and anantisulfonated DNA antibody/alkaline phosphateconjugate. Unbound conjugate was removed and achemiluminescent substrate applied. This step makeseach electrophoresis band containing genetic infor-mation from the rRNA genes visible to a custom

166 K. Grif et al.

TABLE 1 Summary of 17 Clinical Isolates from Patients with Diarrhea (Austria 1992–1996)a

Isolate

StoolSpecimensTaken on

Patients Ageand Sex

(f/m) CommentsDistrict of

Residence/State Enterohaemolysin Stx 1 Stx 2 Stx 2c

1 12.06.1992 2 years, f First Austrian outbreak Schwaz/Tyrol 1 2 1 12 29.06.1992 23 years, m First Austrian outbreak Schwaz/Tyrol 1 1 1 23 08.07.1992 54 years, f First Austrian outbreak Schwaz/Tyrol 1 2 1 14 09.07.1992 19 years, m First Austrian outbreak Schwaz/Tyrol 1 2 1 15 10.07.1992 20 years, f First Austrian outbreak Schwaz/Tyrol 1 2 1 16 16.07.1992 2.5 years, m First Austrian outbreak Schwaz/Tyrol 1 2 1 17 17.01.1995 4 years, f Hafnia alvei Innsbruck/Tyrol 2 2 2 28 03.05.1996 18 months, m Weiz/Styria 1 2 1 19 15.05.1006 61 years, f Schwaz/Tyrol 1 1 1 1

10 18.06.1996 20 years, m Reutte/Tyrol 1 1 1 111 08.07.1996 3 years, m Son of no. 12, brother of no. 14 Innsbruck area/Tyrol 1 2 1 112 15.07.1996 32 years, m Father of no. 11 and no. 14 Innsbruck area/Tyrol 1 2 1 113 18.07.1996 2.5 years, f Scharding/Upper Austria 2 1 1 214 19.07.1996 5 months, f Sister of no. 11, daughter of no. 12 Innsbruck area/Tyrol 1 2 1 115 20.08.1996 17 months, f Developed HUS Reutte/Tyrol 1 2 1 116 04.09.1996 48 years, m Contracted diarrhea during a holiday in Turkey Innsbruck area/Tyrol 1 1 1 217 30.12.1996 74 years, f Hafnia alvei Landeck/Tyrol 2 2 2 2

a All E. coli O157 isolates were sorbitol nonfermenting and expressed the eae-gene.

Epid

emiological

Typing

ofE

.coliO

157167

CCD camera in the RiboPrinter™ system. Throughcustomized software of this system, each samplewas characterized into a pattern (ribotype). Thesenormalized patterns were then compared with anexisting identification database (Bruce 1996) andarchived onto a dynamic library. Patterns that werenot statistically distinguishable (as determined byproprietary algorithms) were clustered into the sameribogroup. EcoR1 and PvuII were used as restrictionendonucleases.

Random Amplification of Polymorphic DNAAccording to Wurzburg (RAPD-PCR) Method

Random amplified polymorphic DNA-PCR (RAPD-PCR) fingerprinting was performed at the Institute ofHygiene and Microbiology (Wurzburg, Germany) bya method described elsewhere using primer 1247(59-AAGAGCCCGT-39) (Heuvelink et al. 1995). PCRwas performed in 50-mL reaction volumes containingapproximately 50 ng of bacterial DNA, 34.5 mL ofdistilled water, 6 mL of MgCl2 (2.5 mM), 5 mL 10 3PCR buffer (Pharmacia, Freiburg, Germany), 20pmol/mL of primer 1247, 10 mmol per base ofd-NTP, and 0.5 mL of Taq DNA polymerase (0.2U)(Pharmacia, Freiburg, Germany). A negative control,consisting of the same reaction mixture, but with notemplate DNA added, was included in each reaction.A Perkin Elmer thermal cycler (Gene Amp PCR Sys-tem 9600) was used for amplification under the fol-lowing conditions: 4 cycles of 95°C for 5 min, 36°Cfor 5 min, 72°C for 5 min, 30 cycles of 94°C for 1 min,36°C for 1 min, 72°C for 2 min, and then 72°C for 10min. Amplified DNA (9 mL) was mixed with 2 mL ofDNA-stop-mix solution (dextran blue, 5 mg/mL for-mamide), separated by gel electrophoresis in 1.5%agarose gel at constant voltage 140 V and visualizedby ethidium bromide staining (0.1 mL/mL) to esti-mate DNA purity and integrity. The results werevisualized by UV transillumination and photo-graphed. A molecular size marker (100 bp) was usedfor reference. If results of agarose gel electrophoresiswere satisfactory, the aliquote of 5.5 to 7 mL of targetDNA was mixed with 2 mL DNA-stop-mix-solutionand electrophoresed in polyacrylamide gel and pho-tographed after ethidium bromide staining. Dendro-grams for cluster analysis were constructed fromsimilarity matrices using Gel Compar Software forComparative Analysis of Electrophoresis Patterns(Applied Maths, Kortrijk, Belgium). This softwareallows cluster analysis and visualization as dendro-gram by using the Pearson correlation coefficient andUPGMA (Unweighted pair-group method using arith-metic averages) algorithm. Computer-assisted analysisand algorithms used in this study were carried outaccording to the instructions of the manufacturer of Gel

Compar. A tolerance in the position of 2% was appliedduring the comparison of the fingerprint patterns.

Random Amplification of Polymorphic DNAAccording to Freiburg (RAPD-ALFA) Method

Random amplified polymorphic DNA-PCR (RAPD-ALFA) fingerprinting was performed at the Instituteof Environmental Medicine and Hospital Hygiene(Freiburg, Germany) by a method described else-where using primer M13 containing the sequence59-GAG GGT GGC GGT TCT-39 (Grundmann et al.1995a, 1995b). Briefly, isolates were cultured on Co-lumbia blood agar plates (Heipha, Heidelberg, Ger-many) at 37°C overnight. DNA was prepared bysuspending 2 to 3 colonies in 100 mL of H2O andboiling for 15 min at 95°C. After short centrifugation,2 mL of the supernatant were used as PCR template.A single primer from the phage M13 core region wasused for the PCR. To optimize reaction conditionsand to increase reproducibility, PCR was performedwith pre-mixed RAPD reaction beads (Ready To GoRAPD Analysis Beads; Pharmacia, Freiburg, Ger-many). These are dried, temperature stable beadscontaining all salts for reaction buffer, dNTPs, and amixture of AmpliTAQ DNA Polymerase and Stoffelfragment, in appropriate concentrations. PCR wasdone in 25-mL reaction volumes containing 2.5 mgBSA, 3 mM MgCl2, 30 mM KCl, 10 mM Tris, pH 8.3,0.4 mM dNTP’s, 2 U Taq DNA Polymerase, 5 mMPrimer M13 and template. After initial denaturationfor 2 min at 94°C, 35 cycles of 20 s at 94°C, 60 s at50°C, and 20 s at 72°C followed. Final extension wasfor 5 min at 72°C. Amplification products were ana-lyzed with an automated DNA sequencer (ALFexpress;Pharmacia). DNA fragments were separated in de-naturating 5% acrylamide–7M urea gel (Long RangerPremix, FMC, Rockland, ME, USA) in 0.6 3 TBEbuffer (TBE: 89 mM Tris base, 89 mM Boric acid, 2mM EDTA, pH 8.0). Electrophoresis was run for 150min at 800 V, 50 mA, 50 W, 45°C. A fluorescence-labeled 100-bp ladder (Pharmacia) served as molec-ular weight standard. Two fluorescence-labeledDNA fragments, a 100-bp fragment of lambda DNA,and a 1064-bp product from E. coli small subunitrDNA, were applied as internal standards to eachsample. The fragment patterns were displayed ascurves representing fluorescence densitograms bythe sequencer. Distortions within or inconsistenciesbetween gels were normalized with the FragmentManager Software (Pharmacia) considering the inter-nal and external standards. The patterns were com-pared visually for grouping. Grouping was con-firmed by computerized analysis with Gel ComparSoftware for Comparative Analysis of Electrophore-sis Patterns (Applied Maths, Kortrijk, Belgium).

168 K. Grif et al.

Pulsed-Field Gel Electrophoresis (PFGE)

Pulsed-field gel electrophoresis (PFGE) of XbaI di-gested total DNA was performed at the Robert KochInstitute (Berlin, Germany) as described previously(Beutin et al. 1997). Results were interpreted accord-ing to criteria proposed by Tenover et al. (1995).Bacteria were grown at 37°C overnight from singlecolonies in 10 mL of 1% Bacto Tryptone (Difco, De-troit, MI, USA). Grown cultures were harvested bycentrifugation and resuspended in the same volumeof SE buffer (10 mM Tris-Cl, 25 mM EDTA, and 75mM NaCl (pH 7.5)). After a second centrifugation,the bacterial pellet was resuspended in 1 mL SEbuffer. A 300 mL aliquot of concentrated bacteria wasmixed rapidly in the same amount of molten 2%Rapid-Agarose (Gibco-BRL, Eggenstein, Germany)followed immediately by placing mixture in sampleinserts in a Bio-Rad sample mold (Bio-Rad Labora-tories, Richmond, CA, USA) as instructed by thesupplier. Lysis of bacteria embedded in agarose wasperformed by 15 h of incubation at 56°C in lysisbuffer (10 mM Tris-Cl, 1 mM EDTA, 1%N-laurylsarcosine sodium salt (pH 9.5)) containing0.5 mg of proteinase K (Boehringer Mannheim) permL. After lysis, agarose plugs were washed fivetimes in sterile TE buffer (10 mM Tris-Cl, 1 mMEDTA (pH 8.0) followed by a 1 h incubation at 37°Cin TE buffer containing RNase (final concentration,20 mg/mL). For XbaI restriction endonuclease cleav-age of genomic DNA, the agarose plugs were equil-ibrated for 1 h at 4°C in XbaI restriction enzymebuffer (Reac 2; Gibco-BRL) followed by an overnightincubation in Reac 2 containing 50 U of XbaI (Gibco-BRL) at 37°C. After enzymatic digestion, the agaroseplugs were washed in 10 mM Tris-Cl, 50 mM EDTA(pH 8.0) and were kept at 4°C in the same type ofbuffer for long-term storage. PFGE was performedwith the clamped homogeneous electric field(CHEF-DR II) system from Bio-Rad Laboratories asinstructed by the supplier. A slice of agarose plugwas sealed into a well of 1% horizontal agarose gel asdescribed in the Bio-Rad instruction manual.Lambda concatemers (Bio-Rad) were used as sizemarkers. Gels were run at 200 V for 25 h at 14°C witha pulse time increasing from 5 to 50 s. After electro-phoresis, gels were stained with ethidium bromidefor visualization of single bands and photographedunder UV light as described previously (Sambrook etal. 1995).

RESULTS

All five participating centers were able to differenti-ate Hafnia alvei from E. coli O157:H7. The phagetyping method and PFGE did not include the Hafniaalvei strains for typing.

Phage Typing

Phage typing yielded a total of 10 patterns for E. coliO157. One veterinary isolate of E. coli O157 (no. 48)yielded the result RDNC (reaction does not conformto standard patterns). Human E. coli isolates ac-counted for seven patterns; only one (PT 2) of themwas also found with food specimens. Food isolatesaccounted for a total of three patterns, none of whichwas found with veterinary isolates. Veterinary iso-lates yielded three patterns, two of them also presentin human strains, but none in food isolates. Phage-typing results are summarized in Table 2.

Ribotyping

By using a combination of the enzymes EcoR1 andPvuII ribotyping yielded a total of eight clusters. Thefive samples with Hafnia alvei, comprising three ofthe eight subgroups, gave RiboPrint™ patterns dif-ferent from E. coli O157. Of the 47 E. coli isolates, 46grouped into a single EcoRI ribogroup. This patternmatched that previously seen for E. coli O157:H7isolates from around the world. A second EcoR1RiboPrint™ pattern was seen with the E. coli isolateno. 12 (a strain which is epidemiologically relatedwith isolates no. 11 and no. 14). Isolate no. 12grouped with a large group of other E. coli O157:H7when PvuII was used as the restriction enzyme.When the 47 E. coli O157:H7 isolates were processedon the RiboPrinter™ system using PvuII as the re-striction enzyme, a total of four pattern types wasobtained. The combination of the enzymes EcoRI andPvuII allowed placement of EHEC O157 isolates intoa total of five subgroups (Table 2). Human E. coliisolates accounted for four pattern types, two ofwhich were also found with food isolates. Veterinaryisolates yielded two patterns; one of which wasfound neither in human nor in food isolates.

Random Amplification of Polymorphic DNAAccording to Wurzburg (RAPD-PCR) Method

Considering isolates with less than 75% of bandsmatching as different RAPD types, a total of fourclusters was identified (Figure 1). As with ribotypingHafnia alvei strains (no. 7, no. 17, no. 24, no. 40a) weregrouped in one cluster, clearly separated from Hafniaalvei strain no. 20. Of the 47 E. coli O157 strains, 34strains (cluster A) were separated from 13 other iso-lates (cluster B). Both clusters comprised isolates ofhuman, food, and veterinary origin. All but one (no.49) of the 8 veterinary E. coli isolates were foundconcentrated within cluster A. All but one of the 24 E.coli food isolates were found concentrated in clusterB. The two E. coli O157:H7 clusters showed 59%similarity.

Epidemiological Typing of E. coli O157 169

Random Amplification of Polymorphic DNAAccording to Freiburg (RAPD-ALFA) Method

Considering isolates with less than 75% of bandsmatching as different RAPD-ALFA types, again atotal of four clusters was identified (Figure 2). Aswith the previous three typing methods Hafnia alveistrains no. 7, no. 17, no. 24, no. 40 were grouped inone cluster and Hafnia alvei strain no. 20 was unique.Of the 47 E. coli O157 strains, 46 isolates formed onecluster (cluster C). E. coli isolate no. 8 was identifiedas distinct. Twenty of the 24 E. coli food isolates werelocated in a subcluster, which included also one hu-man isolate (no. 13). All but one (no. 49) of the eightE. coli veterinary isolates were located in a singlesubcluster (no. 44 to no. 48, no. 50, no. 51) of clusterC, including neither human nor veterinary strains.The two E. coli O157:H7 clusters showed 67% simi-larity.

PFGE

The results of PFGE are summarized in Table 2. Inhuman and food isolates nine clusters, named A to Iwere found (Table 2 and Figure 3). Veterinary iso-lates from cattle showed six clusters which wereunrelated to those of human and food isolates.Among groups A, B, D, and E XbaI profile variantswere found showing minor differences from theirmajor groups (The XbaI restriction patterns of theisolates differed from those of the group strains inthe position of less than seven DNA bands). Thevariants were designated by number following theletter indicating their group. Out of 39 E. coli isolatesfrom human and food origin, 23 (58.9%) were PFGEpattern A and B. Human E. coli isolates accounted foreight patterns; two of them (type A and B) werepresent among food isolates. Food isolates showed atotal of three different patterns: Type C was uniquefor three milk isolates (no. 41 to no. 43). Clusters D, E,F, G, H, and I consisted of sets of one up to fivestrains.

Comparison of the Various Typing Methods

The set of E. coli O157:H7 isolates from the 1992outbreak in the Tyrol consisted of six strains. Isolateno. 2 produced Stx1 and Stx2, contrary to strain no. 1and isolates no. 3 to no. 6, which only produced Stx2.Phage typing yielded four different patterns for theseoutbreak strains and showed a unique pattern forisolate no. 2 (PT54). Strain no. 1 had PT8, while theremaining isolates were of PT31 or PT32. By ribotyp-ing these outbreak strains had the most frequentlyseen pattern, as had another 24 E. coli O157 isolates.RAPD-PCR placed these outbreak strains into twodifferent clusters. Isolates no. 1 and no. 2 were one

TABLE 2 Distribution of Phage Types, Ribotypes,and PFGE Patterns

Isolates Phage Type

Ribotype

PFGE PatternEcoRI PvuII

Human1 8 1 1 G2 54 1 1 H3 31 1 1 E24 31 1 1 F5 32 1 1 E36 31 1 1 E7 Hafnia alvei Hafnia alvei Hafnia alvei8 31 1 8 E19 8 1 1 I10 8 1 1 D111 14 1 1 D12 14 4 1 D13 46 1 1 B114 14 1 1 D15 2 1 7 A116 8 1 1 D217 Hafnia alvei Hafnia alvei Hafnia alvei

Food18 2 1 7 A19 2 1 7 A20 Hafnia alvei Hafnia alvei Hafnia alvei21 2 1 7 A22 2 1 7 A23 21 1 1 B124 Hafnia alvei Hafnia alvei Hafnia alvei25 21 1 1 B126 2 1 7 A227 2 1 7 A28 2 1 7 A29 2 1 7 A30 2 1 7 A31 2 1 7 A32 21 1 1 B33 21 1 1 B35 21 1 1 B36 21 1 1 B37 21 1 1 B38 21 1 1 B39 2 1 7 A340a Hafnia alvei Hafnia alvei Hafnia alvei40b 2 1 7 A41 47 1 1 C42 47 1 1 C43 47 1 1 C

Veterinary44 8 1 1 M45 8 1 1 P46 32 1 1 N47 8 1 1 J48 RDNC 1 1 J49 8 1 1 K50 32 1 2 L51 32 1 2 K

RDNC, Reaction doesn’t conform to standard patterns.

170 K. Grif et al.

pair in cluster A; strains no. 3 to no. 6 formed asubcluster (consisting of a total of nine strains) incluster B. RAPD-ALFA placed these outbreak strainsinto two neighbouring subclusters of cluster C; iso-lates no. 1 and no. 2 forming a pair, strains no. 3 tono. 6 a quadruplet. By PFGE these outbreak-associated isolates from 1992 were of different pat-terns; isolate no. 2 again had a unique pattern (typeH). Isolate no. 1 had type G, while the remainingoutbreak strains were type E or F.

The set of E. coli O157:H7 isolated from a singlefamily in 1996 were considered indistinguishablefrom each other and different from all other strainsby phage typing. Ribotyping considered them notindistinguishable: strain no. 12 gave a unique EcoRIpattern not seen in other E. coli O157:H7 so far.RAPD-PCR placed them into one subcluster, but to-gether with 22 other isolates of human, food, andveterinary origin. RAPD-ALFA placed them into onesingle subcluster. This set from a single family wasfound to be indistinguishable by PFGE, showing pat-tern D. Variants of pattern D were found with twoother human isolates which differed in two bands(no. 10) and in four bands (no. 16) from pattern Dand were epidemiologically unrelated. While thefamily strains and strain no. 50 produced only Stx2,isolates no. 10 and no. 16 produced Stx1 plus Stx2. E.coli O157:H7 isolate no. 16 was acquired in Turkey.This strain was not considered to be different fromthe Tyrolean isolates by phage typing, ribotyping,RAPD-PCR, RAPD-ALFA and PFGE.

Three isolates originating from one single batch ofraw cow’s milk (no. 41, no. 42, no. 43) were consid-ered indistinguishable from each other and differentfrom all other isolates by phage typing. Ribotypingconsidered them to be identical isolates, but failed todifferentiate them from 27 other E. coli O157:H7strains. Isolates no. 42 and no. 43 formed a single pairin the dendrogram when tested by RAPD-PCR aswell as by RAPD-ALFA. RAPD-PCR placed all threeisolates into a subcluster together with 21 epidemio-logically unrelated isolates. RAPD-ALFA placed allthree isolates into a subcluster together with fiveepidemiologically unrelated isolates. PFGE (as didphage typing), considered them indistinguishablefrom each other and different from all other isolates.

Two of the eight E. coli O157:H7 originated fromcalves of one single herd. These isolates (no. 50 andno. 51) were indistinguishable from each other by alltyping methods except PFGE. Phage typing showedPT 32, the same phage type as a strain from a patientfrom the outbreak in 1992 (no. 5) and an isolate froma calf (no. 46) from another state. Ribotyping showeda pattern unique for these two strains. They werefound to form single pairs in the dendrograms givenby RAPD-PCR and RAPD-ALFA.

DISCUSSION

Our knowledge of the epidemiology of E. coliO157:H7 is incomplete and it is often difficult todetermine the source of the organism in cases ofhuman infection. Accordingly, many studies havebeen done to develop genotypic and phenotypic sub-typing schemes (Krause et al. 1996). It was specu-lated that enterohemorrhagic E. coli O157:H7 proba-bly emerged 20 to 30 years ago when E. coli O157:H7acquired bacteriophages that carried genes encodingtwo Shiga-like toxins (Konowalchuk et al. 1977;Rowe 1995). Therefore it is not surprising that thesephylogenetically young organisms have only a fewcharacteristics that can be used to distinguish onestrain from another.

Ribotyping uses restriction fragments of ribo-somal RNA genes for characterization of organisms(Webster et al. 1994). Using EcoRI and PvuII as re-striction enzymes, the discriminatory power of ourautomated ribotyping was insufficient to reliably dif-ferentiate epidemiologically unrelated E. coliO157:H7 isolates from each other. On the other hand,epidemiologically related strains (no. 11, no. 12, no.14) gave different results by ribotyping. Using theenzymes NcoI, BamHI, HindIII, and EcoRI, Martin etal. (1996) showed that nonautomated ribotyping wasalso not able to discriminate effectively within theO157:H7 serotype. It is not surprising that the phy-logenetically young enterohemorrhagic E. coliO157:H7 does not lend itself to ribotyping as theribosomal genes are phylogenetically highly con-served. We conclude that ribotyping is not suitablefor detection of epidemiologically relatedness in E.coli O157 strains.

Phage typing relies on the characterization ofstrains by their patterns of resistance or susceptibilityto a standard set of phages. Food isolates from asingle batch of milk, human isolates from a familyoutbreak, and veterinary isolates from calves of oneherd were correctly found to be indistinguishablefrom each other (within the respective group) byphage typing. According to Arbeit, this procedure“suffers from considerable experimental as well asbiologic variability” (Arbeit 1995). These differencescould be due to factors such as variation in phagereactions, variation of culture medium, or concentra-tion of phages. Changes in phage type by loss of aplasmid have been described by Bezanson et al.(1982) for Salmonella typhimurium. The set of E. coliO157 isolates from the first Austrian outbreakshowed four different phage types in our study, butonly two phage types when tested 5 years earlier(isolate no. 2: PT8; isolates no. 1 and no. 2 to no. 6:PT32) (Solder et al., 1993). The changes seen in pat-tern may well have occurred as a result of storage of

Epidemiological Typing of E. coli O157 171

strains since they were first isolated in 1992. Changesin phage type do not usually occur on storage buthave been documented (Frost et al. 1989). It is inter-esting to note that two of the phage reactions that

have changed with the “phage type 32” strains arewith phages 9 and 10 and these two phages areinvolved with previously observed changes (Frost etal. 1989). Nevertheless, phage typing is simple and

FIGURE 1 RAPD-PCR (Wurzburg) method analysis of 47 Austrian E. coli O157 strains (strain identification numbers asused in Tables 1 and 2).

172 K. Grif et al.

rapid and provides an excellent first level of straindiscrimination. Because of the need to maintainstocks of biologically active phages and controlstrains, phage typing is available only at reference

laboratories. Altogether, phage typing provided ad-ditional evidence to support the assignment of iso-lates to epidemiological related or epidemiologicalnonrelated categories.

FIGURE 2 RAPD-ALFA (Freiburg) method analysis of 47 Austrian E. coli O157 strains (strain identification numbers asused in Tables 1 and 2).

Epidemiological Typing of E. coli O157 173

PFGE allows the generation of simplified chromo-somal restriction fragment patterns without havingto resort to probe hybridization methods. PFGE in-volves embedding organisms in agarose, lysing theorganisms in situ, and digesting the chromosomalDNA with restriction endonucleases that cleave in-frequently. Motivated by the 1993 outbreak traced toJack in the Box, the CDC launched a program todevelop PFGE for typing of E. coli O157:H7 (Stephen-son 1997, Swaminathan 1998). Barrett et al. (1994)therefore recommend the use of XbaI as restrictionenzyme, on selected strains—for further discrimina-tion—the use of AvrII. Although PFGE patterns tendto be stable in outbreaks, single-band differenceswere described previously among isolates from epi-demiologically associated cases (Bender et al. 1997).Willshaw et al. (1997) suggested that the criteria pub-lished by Tenover et al. need modification for closelyrelated organisms such as EHEC O157. Using XbaI,our food isolates from a single batch of milk andhuman isolates from a family outbreak were cor-rectly found indistinguishable from each other byPFGE but not the veterinary isolates from calves ofone herd. However, Bohm and Karch (1992) foundthat PFGE (using XbaI, PacI, SfiI, and NotI as restric-tion enzymes) was unable to distinguish betweenepidemiologically unrelated isolates. Our data un-derline the value of PFGE as a subtyping system, butalso show the limitations of PFGE when phylogeneti-cally highly related strains are analyzed. It is difficultto unequivocally prove by PFGE alone that a givenstrain is responsible for the spread of an outbreak.The interpretation of PFGE data may be difficultwhen the patterns do not match exactly.

Random amplification of polymorphic DNA(RAPD) is based on the amplification of genomicDNA using a short primer with an arbitrarily chosensequence (Caetano-Anolles et al. 1991). There is noneed to know the exact nucleotide sequence of thespecies. This typing method is based on the primerannealing to the target sequence under low-stringency conditions that allow for multiple mis-matches (Venugopal et al. 1993). Using 75% homol-ogy as a criterion, RAPD ALFA as well as RAPD PCRwere able to recognize only two clusters. Neverthe-less, both dendrograms grouped most of the EHECO157 isolates into subgroups according to the kind ofsource of specimen and were able to recognize somestrains as epidemiologically related. Both methodsgrouped the two veterinary isolates from calves ofone herd as pairs. Two strains of the three foodisolates of a single batch of milk were grouped aspairs, with the third isolate being recognized ashighly related by being placed into a near subclusterof the dendrogram. The human isolates of the familyoutbreak were grouped as a triplet by RAPD-ALFA,but spread within one cluster by RAPD-PCR. RAPDsuffers from lack of standardization, with quite dif-ferent methods used in different laboratories. De-spite the use of different primers and quite differentmethods, both RAPD-procedures yielded similar re-sults. RAPD typing is increasingly applied for a largevariety of different pathogens (Birch et al. 1996, Grat-tard et al. 1995, Hermans et al. 1995, O’Donoghue etal. 1995, Yao et al. 1995). Like Birch et al. (1996),Kurazono and Makino (1997) and Bohm and Karch(1992), we conclude that this technique is also usefulin epidemiological studies of E. coli O157 infections.

While most of the epidemiologically related iso-lates were also considered highly related by our typ-ing methods, the typing results of the isolates fromthe first Austrian EHEC O157 outbreak in 1992showed a remarkable nonhomogeneity, indicatingthat these were of multiple origin. Ribotyping, withits insufficient power of discrimination, showed onepattern for all six strains. RAPD-ALFA showed onepattern, but showed two subclusters separating iso-lates no. 1 and no. 2 from strains no. 3 to no. 6.RAPD-PCR placed isolates no. 1 and no. 2 in onecluster, strains no. 3 to no. 6 in another cluster. Phagetyping and PFGE diagnosed four different patternseach; both recognized strains no. 1 and no. 2 asunique within this outbreak collection. Phage typingalso considered isolate no. 5 different from the re-maining strains, contrary to PFGE, which showedstrain no. 4 different from the remaining outbreakisolates. This example shows that major differencescan exist between results of multiple subtypingmethods. E. coli O157 isolates should not be classifiedas epidemiologically related or nonrelated on thebasis of a single typing method alone.

FIGURE 3 Major PFGE patterns of XbaI digested DNA ofEHEC O157 strains. The lane marked by an * contains aphage lambda concatemer DNA ladder used as size stan-dard (indicated in kb).

174 K. Grif et al.

Depending upon the methodology chosen, onecould prove or disprove the relatedness of organismsinvolved in a seemingly common source outbreak.Each method offers a different level of discriminationin the ability to distinguish apparently relatedgroups of organisms. In the case of O157 strains of E.coli, ribotyping appeared to be the most macroscopicof all typing methods. Other than being able to dis-tinguish H. alvei form E. coli, this method had atendency of placing most other strains of O157 into acommon heritage. PFGE and RAPD methods as wellas phage typing had higher levels of resolution andeach had a variety of desirable qualities includingease of performance and the ability to cluster appar-ently related strains (i.e., the milk, family outbreak,and herd-derived strains). The ability of differentmolecular techniques to categorize bacterial strainsdifferently has recently been recognized withpenicillin-resistant strains of Streptococcus pneu-moniae; the discriminatory power and the similarityvalues differed significantly among the five differentDNA fingerprint techniques investigated (Hermanset al. 1995). Karch et al. (1993) compared a collectionof sorbitol-fermenting EHEC O157 isolates with non-sorbitol-fermenting EHEC O157 by PFGE. Thesorbitol-fermenting EHEC O157 strains had identicalor closely related XbaI patterns that differed mark-edly from those for the other EHEC strains. Inves-tigating the length of time that EHEC O157 isexcreted after the onset of diarrhea, Karch et al.(1995) further observed that loss of the Stx-generesults in a new PFGE-pattern. Despite this im-

pressive capacity, typing methods should not beused as a substitute for biochemical or molecular-diagnostic methods.

Investigation of the epidemiology of EHEC O157infections should require a combined use of typingmethods. Ribotyping provides no additional evi-dence to support the assignment of isolates to epide-miologically related or nonrelated categories. In out-breaks well defined by epidemiologic studies, phagetyping and the identification of Stx1 and Stx2 genes,including Stx2 subtyping, are likely to be sufficient.PFGE gives a similar high level of discrimination, butis more time consuming and costly. RAPD-ALFAand RAPD-PCR, although less sensitive than phagetyping and PFGE when using 75% similarity as adecision criterion, nevertheless provided additionalevidence to support the assignment of isolates to therelated or nonrelated categories. In this study wemainly addressed discriminatory power. Intra- andinterlaboratory reproducibility, potential for stan-dardization and automation of the protocol, suitabil-ity for computer-assisted analysis, and robustnessand cost of the method are further parameters thathave to be considered when one is choosing a typingmethod. Despite the promise offered by these typingmethods, technology alone cannot prevent food-borne outbreaks unless investigators are available tocollect samples, interview patients, and track downthe source of contamination (Willshaw et al. 1997).Time will show whether upcoming new methodslike multilocus sequence typing will replace the pres-ently employed typing methods (Maiden et al. 1998).

REFERENCES

Ahmed R, Bopp A, Borczyk A, Kasatiya S (1987) Phage-typing scheme for Escherichia coli O157:H7. J Infect Dis155:806–809.

Anonymous (1997) Escherichia coli O157:H7 infections as-sociated with eating a nationally distributed commer-cial brand of frozen ground beef patties and burgers—Colorado, 1997. MMWR 46:777–778.

Armstrong GL, Hollingsworth J, Morris JG (1996) Emerg-ing foodborne pathogens: Escherichia coli O157:H7 as amodel of entry of a new pathogen into the food supplyof the developed world. Epidemiol Rev 18:29–51.

Arbeit RD (1995) Laboratory procedures for the epidemi-ologic analysis of microorganisms. In: Manual of ClinicalMicrobiology. Eds, Murray PR, Baron EJ, Pfaller MA,Tenover FC, Yolken RH. Washington, DC: ASM Press,pp. 190–208.

Asai Y, Murase T, Osawa R, Okitsu T, Suzuki R, Sata S,Yamai S, Wada A, Tamura K, Watanabe H (1997) Iso-lation of enterohemorrhagic Escherichia coli (O157:H7)by an immunomagnetic separation method. Kansenshog-aku Zasshi 71(1):46–55.

Barrett TJ, Lior H, Green JH, Khakhria R, Wells JG, Bell BP,Greene KD, Lewis J, Griffin PM (1994) Laboratory in-

vestigation of a multistate food-borne outbreak of Esch-erichia coli O157:H7 by using pulsed-field gel electro-phoresis and phage typing. J Clin Microbiol 32:3013–3017.

Bender JB, Hedberg CW, Besser JM, Boxrud DJ, Mac-Donald KL, Osterholm MT (1997) Surveillance for Esch-erichia coli O157:H7 infection in Minnesota by moleculartyping. N Engl J Med 337:388–394.

Beutin L, Geier D, Zimmerman S, Aleksic S, Gillespie HA,Whittam TS (1997) Epidemiolodical relatedness andclonal types of natural populations of Escherichia colistrains producing Shiga Toxins in separate populationsof cattle and sheep. Appl Environ Microbiol 63:2175–2180.

Bezanson G, Khakharia R, Lacroix R (1982) Involvement ofplasmids in determining bacteriophage sensitivity inSalmonella typhimurium: Genetic and physical analysis ofphagovar 204. Can J Microbiol 28:993–1001.

Birch M, Denning DW, Law D (1996) Rapid genotyping ofEscherichia coli O157 isolates by random amplification ofpolymorphic DNA. Eur J Clin Microbiol Infect Dis 15:297–302.

Bohm H, Karch H (1992) DNA fingerprinting of Escherichia

Epidemiological Typing of E. coli O157 175

coli O157:H7 strains by pulsed-field gel electrophoresis.J Clin Microbiol 30:2169–2172.

Bruce J (1996) Automated system rapidly identifies andcharacterizes microorganisms in food. Food Technol (Chi-cago) 50:77–81.

Caetano-Anolles G, Bassam BJ, Gresshoff PM (1991) DNAamplification fingerprinting using very short arbitraryoligonucleotide primers. Biotechnology 9:553–557.

Frost JA, Smith HR, Willshaw GA, Scotland SM, Gross RJ,Rowe B (1989) Phage-typing of Vero-cytotoxin (VT) pro-ducing Escherichia coli O157 isolates in the United King-dom. Epidemiol Infect 103:73–81.

Grattard F, Pozzetto B, Tabard L, Petit M, Ros A, GaudinOG (1995) Characterization of nosocomial strains ofEnterobacter aerogenes by arbitrarily primed PCR analy-sis and ribotyping. Infect Control Hosp Epidemiol 16:224–230.

Grundmann H, Schneider S, Daschner FD (1995a)Fluorescence-based DNA fingerprinting elucidates nos-ocomial transmission of phenotypically variable Pseudo-monas aeruginosa in intensive care units. Eur J Clin Mi-crobiol Infect Dis 14:1057–1062.

Grundmann H, Schneider S, Tichy HV, Simon R, Klare I,Hartung D, Daschner FD (1995) Automated laser fluo-rescence analysis of randomly amplified polymorphicDNA: A rapid method for investigation nosocomialtransmission of Acinetobacter baumanii. J Med Microbiol43:446–451.

Hermans PW, Sluijter M, Hoogenboezem T, Heersma H,van Belkum A, de Groot R (1995) Comparative study offive different DNA fingerprint techniques for moleculartyping of Streptococcus pneumoniae strains. J Clin Micro-biol 33:1606–1612.

Heuvelink AE, J van de Kar CA, Meis JFGM, MonnensLAH, Melchers WJG (1995) Characterization ofverotoxin-producing Escherichia coli O157 isolates frompatients with haemolytic-uraemic syndrome in WesternEurope, Epidemiol Infect 115:1–14.

Karch H, Bohm H, Schmidt H, Gunzer F, Aleksic S, Heese-mann J (1993) Clonal structure and pathogenicity ofShiga-like toxin-producing, sorbitol-fermenting Esche-richia coli O157:H7. J Clin Microbiol 31(5):1200–1205.

Karch H, Russmann H, Schmidt H, Schwarzkopf A, Heese-mann J (1995) Long-term shedding clonal turnover ofenterohemorrhagic Escherichia coli O157 in diarrheal dis-eases. J Clin Microbiol 33(6):1602–1605.

Khakhria R, Duck D, Lior H (1990) Extended phage-typingscheme for Escherichia coli O157:H7. Epidemiol Infect 105:511–520.

Khaschabi D, Beutin L, Schopf K, Schonbauer M (1997)Zum Vorkommen von Verotoxinbildenden Escherichiacoli O157:H7 in Kalberkotproben aus Tirol. Wien Tier-arztl Monatsschr 84(4):91–96.

Konowalchuk J, Speirs JL, Stavric S (1977) Vero response toa cytotoxin of Escherichia coli. Infect Immun 18:775–779.

Krause U, Thomson-Carter FM, Pennington TH (1996) Mo-lecular epidemiology of Escherichia coli O157:H7 bypulsed-field gel electrophoresis and comparison withthat by bacteriophage typing. J Clin Microbiol 34(4):959–961.

Kurazono T, Makino S (1997) Diversity of DNA sequencesamong enterohemorrhagic Escherichia coli O157:H7 de-

tected by PCR-based DNA fingerprinting. Nippon Rin-sho 55(3):671–674.

Maiden MC, Bygraves JA, Feil E, Morelli G, Russell JE,Urwin R, Zhang Q, Zhou J, Zurth K, Caugant DA,Feavers IM, Achtman M, Spratt BG (1998) Multilocussequence typing: A portable approach to the identifica-tion of clones within population of pathogenic microor-ganisms. Proc Natl Acad Sci USA 95(6):3140–3145.

Martin IE, Tyler SD, Tyler KD, Khakhria R, Johnson WM(1996) Evaluation of ribotyping as epidemiologic toolfor typing Escherichia coli serogroup O157 isolates. J ClinMicrobiol 34:720–723.

O’Donoghue K, Bowker K, McLauchlin J, Reeves DS, Ben-nett PM, MacGowan AP (1995). Typing of Listeria mono-cytogenes by random amplified polymorphic DNA(RAPD) analysis. J Food Microbiol 27:245–252.

Rowe PM (1995) Eradicating E. coli O157:H7. Lancet 345:117.

Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning:A Laboratory Manual. 2nd ed., vol. 2, p. 9.31–9.43. ColdSpring Harbor, NY: Cold Spring Harbor laboratoryPress, pp. 9.31–9.43.

Solder B, Allerberger F, Eigentler A, Kofler D, Larcher C,Dierich MP, Rowe B (1993) Vero cytotoxin-producingEscherichia coli O157 infections in Austria. Z Gastroen-terol 31:388–391.

Stephenson J (1997) New approaches for detecting andcurtailing foodborne microbial infections. JAMA277(17):1337–1340.

Swaminathan B (1998) National network for molecularsubtyping of Escherichia coli O157:H7 and other food-borne pathogens (abstr 164/y). Abstracts of the 98th Gen-eral Meeting of the American Society for Microbiology, At-lanta, May 17–21.

Tenover FC, Arbeit RD, Goering RV, Mickelsen PA, Mur-ray BE, Persing DH, Swaminathan B (1995) Interpretingchromosomal DNA restriction patterns produced bypulsed field gel electrophoresis: Criteria for strain typ-ing. J Clin Microbiol 33:2233–2239.

Venugopal G, Mohapatra S, Salo D, Mohapatro S (1993)Multiple mismatch annealing: Basis for random ampli-fied polymorphic DNA fingerprinting. Biochem BiophysRes Commun 197:1382–1387.

Webster JA, Bannerman TL, Hubner RJ, Ballard DN, ColeEM, Bruce JL, Fiedler F, Schubert K, Kloos WE (1994)The Staphylococcus sciuri species group described byEcoRI fragments containing ribosomal RNA sequenceswith recognition of Staphylococcus vitulus sp. Nov Int JSyst Bacteriol 44:454–460.

Willshaw GA, Smith HR, Cheasty T, Wall PG, Rowe B(1997) Vero cytotoxin-producing Escherichia coli O157outbreaks in England and Wales, 1995: Phenotypicmethods and genotypic subtyping. Emerg Infect Dis 3(4):561–565.

World Health Organisation (WHO). Prevention and Con-trol of Enterohaemorrhagic Escherichia coli (EHEC)(1997) Infections. Report of a WHO consultation, Geneva:WHO, 1997. WHO/FSF/FOS/97.6.

Yao JD, Conly JM, Krajden M (1995) Molecular typing ofStenotrophomonas (Xanthomonas) maltophilia by DNAmacrorestriction analysis and random amplified poly-morphic DNA analysis. J Clin Microbiol 33:2195–2198.

176 K. Grif et al.