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. 2006 Mar;72(3):2254–2259. doi: 10.1128/AEM.72.3.2254-2259.2006

Analysis of the Clonal Relationship of Shiga Toxin-Producing Escherichia coli Serogroup O165:H25 Isolated from Cattle

Lutz Geue 1,*, Thomas Selhorst 1, Christina Schnick 1, Birgit Mintel 1, Franz J Conraths 1
PMCID: PMC1393171  PMID: 16517683

Abstract

Variations in time and space of a clonal group of Escherichia coli O165:H25 on a cattle farm were monitored. The virulence marker pattern (stx genes, eae gene, hlyEHEC gene, katP gene, espP gene, efa gene) suggests that E. coli O165:H25 of bovine origin may represent a risk for human infection.

Shiga toxin-producing Escherichia coli (STEC) is a group of zoonotic enteric pathogens (29). Human infections with some STEC serotypes, also designated enterohemorrhagic Escherichia coli (EHEC), result in hemorrhagic or nonhemorrhagic diarrhea, which may be complicated by hemorrhagic colitis (HC) and several renal sequelae, including the hemolytic-uremic syndrome (HUS) (20, 34, 41, 52). The mechanisms by which EHEC strains cause disease are not completely understood. The virulence factors include production of two major phage-encoded toxins, Shiga toxin 1 (Stx1) and Shiga toxin 2 (Stx2), which can be produced alone or in combination. Stx1 and Stx2 are thought to cause the vascular endothelial damage observed in patients with HC and HUS (53). In addition to Stx, EHEC strains possess other virulence factors, such as the ability to cause attaching and effacing (eae) lesions in the large intestine (26). They often contain a plasmid that carries other potential virulence genes, such as an enterohemolysin gene (hlyEHEC), a catalase peroxidase gene (katP), and an extracellular serine protease gene (espP) (9, 10, 11, 45). The efa1 (E. coli factor for adherence) gene represents another intestinal colonization factor in an EHEC O111:H- strain (30). The efa1 gene of O111:H- is 99.9% homologous to the lifA gene in enteropathogenic E. coli. The efa1 locus is not physically linked to the locus for enterocyte effacement pathogenicity island (51).

Ruminants, especially cattle, are considered the primary reservoir for human EHEC infections (23). In this study, 38 bovine E. coli O165:H25 isolates were characterized to assess their potential to cause EHEC disease in humans. These isolates were detected over a 4-month period in eight different animals (Table 1) from a single group of beef cattle during a long-term study (19). Sporadic cases of human infections with O165:H25 and O165:H- in Europe and Canada have been described previously (6, 15, 17, 24). The four German O165:H25 and O165:H- strains of human origin (kindly supplied by H. Tschäpe, Robert Koch-Institute, Wernigerode, Germany) used in our study for comparison were associated with diarrhea in patients (54). Human HUS cases caused by O165 isolates have been reported from Denmark (15) and Germany (17).

TABLE 1.

Strains examined

Sampling day or yeara Source Strain Serotype Virulence profile
stx1 gene stx2 gene Stx1 (toxin) Stx2 (toxin) stx2- Subtype eae Subtype efa1/lifA gene hlyEHEC gene katP gene espP gene Plasmid(s) (bp) Genetic subcluster
Day 1 Cattle 17 02/17/009-9 O165:H25 − + − + stx2-EDL933 ɛ + + + + 70, 55 7
Cattle 24 02/24/007-1 O165:H25 − + − + stx2-EDL933 ɛ + + + + 70, 55 1
02/24/007-2 O165:H25 − + − + stx2-EDL933 ɛ + + + + 70, 55 1
02/24/007-3 O165:H25 − + − + stx2-EDL933 ɛ + + + + 70, 55 1
02/24/007-4 O165:H25 − + − + stx2-EDL933 ɛ + + + + 70, 55 1
02/24/007-5 O165:H25 − + − + stx2-EDL933 ɛ + + + + 70, 55 1
02/24/007-6 O165:H25 − + − + stx2-EDL933 ɛ + + + + 70, 55 3
02/24/007-7 O165:H25 − + − + stx2-EDL933 ɛ + + + + 70, 55 1
02/24/007-9 O165:H25 − + − + stx2-EDL933 ɛ + + + + 70, 55 1
02/24/007-10 O165:H25 − + − + stx2-EDL933 ɛ + + + + 70, 55 1
Cattle 26 02/26/006-1 O165:H25 − + − + stx2-EDL933 ɛ + + + + 70, 55 1
02/26/006-2 O165:H25 − + − + stx2-EDL933 ɛ + + + + 70, 55 1
02/26/006-3 O165:H25 − + − + stx2-EDL933 ɛ + + + + 70, 55 1
02/26/006-4 O165:H25 − + − + stx2-EDL933 ɛ + + + + 70, 55 9
02/26/006-5 O165:H25 − + − + stx2-EDL933 ɛ + + + + 70, 55 9
02/26/006-6 O165:H25 − + − + stx2-EDL933 ɛ + + − + 67, 55 9
02/26/006-7 O165:H25 − + − + stx2-EDL933 ɛ + − − − 55 4
02/26/006-8 O165:H25 − + − + stx2-EDL933 ɛ + + + + 70, 55 5
02/26/006-9 O165:H25 − + − + stx2-EDL933 ɛ + − − − 55 6
02/26/006-10 O165:H25 − + − + stx2-EDL933 ɛ + + + + 70, 55 1
Day 28 Cattle 25 02/25/007-1 O165:H25 − + − + stx2-EDL933 ɛ + + + + 70, 55 1
02/25/007-4 O165:H25 − + − + stx2-EDL933 ɛ + + + + 70, 55 1
02/25/007-5 O165:H25 − + − + stx2-EDL933 ɛ + − − − 55 1
02/25/007-6 O165:H25 − + − + stx2-EDL933 ɛ + + + + 70, 55 1
02/25/007-7 O165:H25 − + − + stx2-EDL933 ɛ + + + + 70, 55 1
02/25/007-8 O165:H25 − + − + stx2-EDL933 ɛ + + + + 70, 55 1
02/25/007-9 O165:H25 − + − + stx2-EDL933 ɛ + + + + 70, 55 1
02/25/007-10 O165:H25 − + − + stx2-EDL933 ɛ + + + + 70, 55 1
Day 56 Cattle 9 02/09/010-1 O165:H25 − + − + stx2-EDL933 ɛ + + + + 70, 55 8
02/09/010-2 O165:H25 − + − + stx2-EDL933 ɛ + + + + 70, 55 8
Cattle 18 02/18/011-1 O165:H25 − + − + stx2-EDL933 ɛ + + + + 70 10
02/18/011-5 O165:H25 − + − + stx2-EDL933 ɛ + + + + 70, 55 10
02/18/011-6 O165:H25 − + − + stx2-EDL933 ɛ + + + + 70, 55 10
Cattle 25 02/25/008-1 O165:H25 − + − + stx2-EDL933 ɛ + + + + 70, 55 1
02/25/007-3 O165:H25 − + − + stx2-EDL933 ɛ + + + + 70, 55 2
Day 119 Cattle 19 02/19/013-7 O165:H25 − + − + stx2-EDL933 ɛ − + + + 70, 55 11
02/19/013-8 O165:H25 − + − + stx2-EDL933 ɛ + + + + 70, 55 8
02/19/013-10 O165:H25 − + − + stx2-EDL933 ɛ + + + + 70, 55 8
1998 Human, Germany, diarrhea 98-4982b O165:H- − + − + stx2/2c ɛ + + + + 75
1998 Human, Germany, diarrhea 98-8419-1b O165:H- − + − + stx2/2c ɛ + + + + 95, 75
1999 Human, Germany, diarrhea 99-2258b O165:H25 − + − + stx2/2c ɛ + + + + 95, 75
2002 Human, Germany, diarrhea 02-11228b O165:H25 − + − + stx2/2c ɛ + + + + 95, 75
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a

On days −120 to 0 O165:H25 was not detected on eight occassions, on day 91 O165:H25 was not detected, and on days 147 to 511 O165:H25 was not detected by 17 investigations.

b

Kindly provided by H. Tschäpe.

Typing and subtyping of genes (stx1 and/or stx2, eae, hlyEHEC, katP, and espP) associated with STEC were performed by LightCycler fluorescence PCR (40) and different Block cycler PCRs (Tables 2 and 3). A complete pattern of virulence markers was detected in most bovine isolates examined. An stx2 gene, but not an stx1 gene, was present in all O165 strains (Table 1). EHEC strains with stx2 genes are significantly more frequently associated with HUS and other severe diseases than isolates with an stx1 gene, which are more often associated with uncomplicated diarrhea or healthy individuals (6, 36). Stx2 is closely related to a family of Stx2 variants or alleles, which includes Stx2c (48), Stx2d (36), Stx2e (56), and Stx2f (47), although Stx2c and Stx2d are produced by STEC strains isolated from humans (36, 38, 43, 44, 48, 50). Additional genetic variants of the stx2 gene have been described (5, 14, 28, 55). In contrast to STEC strains harboring stx2 gene variants, however, STEC strains with the stx2 genotype were significantly associated with HUS (17). An stx2 gene with the stx2-EDL933 genotype was found in all O165 isolates tested (Table 1). The nucleotide sequences of the A and B subunits of the stx2 gene of the bovine O165:25 strain 02/09/010-1 (GenBank accession number AY652745) were identical to the sequences of the stx2 gene of EHEC type strain EDL933 (35), a typical O157:H7 strain isolated from a HUS patient, with the sequences of the gene of bacteriophage 933W (37), and with the sequences of stx2 genes of other E. coli O157:H7 strains of human origin isolated from EHEC outbreaks (25, 27). All bovine O165:H25 strains produced an Stx2 with high cytotoxicity for Vero cells as determined by an Stx enzyme-linked immunosorbent assay and by a Vero cell neutralization assay (49).

TABLE 2.

Oligonucleotide primers and LightCycler hybridization probes used for LightCycler PCR

Target(s) Primer Sequence (5′-3′)a Reference
stx1 and stx2 STEC-1 GA(AG) C(AG)A AAT AAT TTA TAT GTG 40
STEC-2 TGA TGA TG(AG) CAA TTC AGT AT 33
stx1 STEC-I HP-1 TTT ACG TTT TCG GCA AAT ACA GAG GGG AT-(FL) 40
STEC-I HP-2 (Red 640)-TCG TAC AAC ACT GGA TGA TCT CAG TGG G-Ph
stx2 STEC-II HP-1 TCA GGC ACT GTC TGA AAC TGC TCC TGT GTA-(FL) 40
STEC-II HP-2 (Red 705)-ACC ATG ACG CCG GGA GAC GTG GAC CT-Ph
eae eaeAF GAC CCG GCA CAA GCA TAA GC 32
eaeAR CC ACCT GCA GCA ACA AGA GG
eae eae HP1 ACA GTT CTG AAA GCG AAA TGA TGA AGG c-(FL) 40
eae HP2 (Red 640)-CCT GGT CAG CAG ATC ATT TTG CCA CT-Ph
hlyEHEC hlyAF GCA TCA TCA AGC GTA CGT TCC 32
hlyAR AAT GAG CCA AGC TGG TTA AGC T
hlyEHEC hlyA HP1 GCA TGG CTC TTG ATG AAT TGC T-(FL) 40
hlyA HP2 (Red 705)-CAA CGG GAA GGA GAG GAT ATA AGT CAG-Ph
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a

FL, fluorescein; Red 640, LC Red 640-N-hydroxy-succinimide ester; Red 705, LC Red 705-phosphoramidite; Ph, 3-phosphate.

TABLE 3.

PCR primers used for detection and characterization of STEC by conventional PCR

Target Primer Sequence (5′-3′) PCR conditions
Product length (bp) Reference
Denaturation
Annealing
Extension
No. of cycles
Temp (°C) Time (s) Temp (°C) Time (s) Temp (°C) Time (s)
stxB2/2c GK3 ATG AAG AAG ATG TTT ATG 94 30 52 60 72 40 35 260 22
GK4 TCA GTC ATT ATT AAA CTG
stxB2d VT2-cm AAG AAG ATA TTT GTA GCG G 94 30 52 60 72 60 35 256 36
VT2-f TAA ACT GCA CTT CAG CAA AT
stxB2e FK1 CCG GAT CCA AGA AGA TGT TTA TAG 94 30 55 60 72 40 35 280 16
FK2 CCC GAA TTC TCA GTT AAA CTT CAC C
stxB2f 128-1 AGA TTG GGC GTC ATT CAC TGG TTG 94 30 57 60 72 60 35 428 47
128-2 TAC TTT AAT GGC CGC CCT GTC TCC
α-eae SK1 CCC GAA TTC GGC ACA AGC ATA AGC 94 30 55 60 72 120 30 2,807 31
LP2 CCC GAA TTC GGC ACA AGC ATA AGC
β-eae SK1 CCC GAA TTC GGC ACA AGC ATA AGC 94 30 55 60 72 120 30 2,287 31
LP4 CCC GTG AT ACCA GTA CCA ATT ACG GTC
γ-eae SK1 CCC GAA TTC GGC ACA AGC ATA AGC 94 30 45 60 72 120 3 2,792 31
LP3 CCC GAA TTC TTA TTC TAC ACA AAC CGC 94 30 52 60 72 120 28
δ-eae Int-d TAC GGA TTT TGG GGC AT 95 20 45 60 72 60 30 544 1
Int-Ru TTT ATT TGC AGC CCC CCA T
ɛ-eae SK1 CCC GAA TTC GGC ACA AGC ATA AGC 94 30 55 60 72 120 30 2,608 31
LP5 AGC TCA CTC GTA GAT GAC GGC AAG CG
katP wkat-B CTT CCT GTT CTG ATT CTT CTG G 94 30 56 60 72 150 30 2,125 10
wkat-F AAC TTA TTT CTC GCA TCA TCC
espP EspA AAA CAG CAG GCA CTT GAA CG 94 30 56 60 72 150 30 1,830 11
EspB GGA GTC GTC AGT CAG TAG AT
lifA/efa1 lifA1 AGC CAT TCC ATC AAT CCG AT 95 30 50 60 72 60 30 532 7
lifA2 TCC CTG CCA AAC TAC CGA CAC
lifA/efa1 lifA3 CAG CTA CAG GAG ACC GTT TTT 95 30 50 60 72 60 30 560 7
lifA4 CAA TAT CAG GCC AAT CAA
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Not only factors that influence basal and inducible Stx production are important in STEC pathogenesis. In previous studies, it has been suggested that the eae and hlyEHEC genes likely contribute to STEC pathogenicity (3, 6, 42). Ritchie et al. (42) found both of these genes in all HUS-associated STEC isolates that they analyzed. The stx2 genes were present in combination with eae genes in all O165 isolates that we obtained (Table 1). To date, 10 distinct variants of eae have been described (13, 21, 31, 39). Some serotypes were closely associated with a particular intimin variant (4, 12, 13, 55). Our study confirmed these associations. Like the O103 isolates, all bovine and human E. coli O165 strains were grouped into the ɛ-eae subgroup. Also, nucleotide sequencing of the bovine O165:H25 strain 02/09/010-1 (GenBank accession number AF479581) revealed a high level of sequence homology (99.7%) to the eae gene of an O103:H2 strain (31). E. coli O103:H2 strains have frequently been associated with human HUS cases in Europe. Like the eae gene, the hlyEHEC gene was found in association with severe disease in humans (45, 46). In our study, the hlyEHEC gene was detected in the O165 strains in which a 70-kb plasmid was also found (Table 1). The presence of a 70-kb plasmid was associated with the occurrence of additional virulence markers, such as the espP gene, and all but one isolate contained the katP gene (10, 11). The reason for the slightly smaller size (67 kb) of the plasmid in this isolate may be linked to fact that the katP gene was not present in this isolate (Table 1). The efa1 genes were detected in 37 of 38 bovine O165:H25 isolates with two DNA probes by colony hybridization, and the results were confirmed by Southern hybridization after pulsed-field gel electrophoresis (PFGE); in this analysis the DNA probes were labeled with digoxigenin (DIG), primers lifA1 and lifA2 and primers lifA3 and lifA4 (Table 3) were used with a PCR DIG probe synthesis kit (Roche Diagnostics, Mannheim, Germany), and DIG Easy Hyb solution (Roche) was used for prehybridization and hybridization. The efa-1 gene was located on an approximately 240-kb XbaI fragment or on an approximately 440-kb NotI fragment. These fragments were missing in the isolate that was negative as determined by colony hybridization (Fig. 1). To determine this, slices of the plugs were digested for 4 h with XbaI, NotI (New England Biolabs GmbH, Frankfurt am Main, Germany), BlnI (AvrII), or SpeI (Amersham Biosciences Inc., Buckinghamshire, United Kingdom). The resulting fragments were separated in a 1.0% agarose gel (SeaKem Gold agarose; Cambrex) in 0.5× Tris-borate-EDTA at 10°C with a CHEF Mapper XA system. The pulse times for XbaI and NotI digests were increased from 5 to 50 s (gradient, 6 V/cm) during 25 h at a constant angel of 120°. The switch time values for BlnI and SpeI were set using the Auto Algorithm function of the CHEF Mapper XA to separate fragments in the range from 50 to 450 kb (BlnI) or from 30 to 350 kb (SpeI). All fragments larger than 45 kb (up to 27 fragments with XbaI, up to 24 fragments with NotI, up to 25 fragments with BlnI, and up to 29 fragments with SpeI) were included in the clonal analysis of the isolates.

FIG. 1.

FIG. 1.

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(a) Dendrogram of DNA divergence (distance) generated by using the Dice coefficient for pairwise comparisons of banding patterns for XbaI, NotI, BlnI, and SpeI restriction of potential EHEC O165:H25 and O165:H- isolates. (b to e) Schematic representations of restriction patterns of bovine and human E. coli O165:H25 and O165:H- strains after digestion with XbaI (b), NotI (c), BlnI (d), and SpeI (e). Ellipses indicate the missing XbaI and NotI fragments in strain 02/19/013-7. The efa1 gene is located at this position in the other bovine strains.

We also analyzed the spatial and temporal behavior of the clonal group of O165:H25 strains in the herd by genomic typing with PFGE (Fig. 1). During a 3-year monitoring program on four cattle farms (19), the O165:H25 clone was detected on only one farm for 4 months. This serotype was not detected before or after this period, although many other potential EHEC strains belonging to other serotypes were found in this herd (19). Twenty O165:H25 isolates were found in four different cattle on the first date of detection. Different subclusters were already present and were even isolated from a single animal (Table 1). These results suggest that the O165 clone either had been introduced into the farm shortly before the first detection or was the result of recombination due to horizontal gene transfer (8, 18). The occurrence of different subclusters at the same time could have been the result of interactions between different bacteria or between bacteria and the host in the bovine intestine. At later sampling dates the number of O165:H25 isolates was reduced. The dominant subcluster, subcluster 1, could still be found, but many other subclusters were detected at the same time. The genetic distance between the O165:H25 isolates increased, and in some isolates plasmid-encoded virulence markers or complete virulence plasmids were missing. Variations in the O165:H25 clonal group might have been caused by increasing competition between the bacterial populations of various subtypes in the bovine intestine or by potential interactions between the O165:H25 EHEC and the host. The O165:H25 clonal group finally disappeared from the herd after it had persisted for 4 months. Perhaps the loss of the efa-1 gene in one isolate obtained on the last date when O165:H25 was isolated from the herd can help us understand why the clone disappeared. Efa1 is considered an E. coli factor for colonization of the bovine intestine by non-O157 STEC (51). Also, this O165:H25 strain exhibited the greatest genetic distance compared to the remaining strains in the clonal group (Fig. 1). The distances were calculated from the fragmentation patterns produced by each of the four PFGE enzymes by using the RAPDistance program, version 1.04 (2). The Euclidean distance in three dimensions was not calculated because all O165:H25 strains were isolated from the same farm (i.e., the geographic distance was zero for all isolates). A cluster analysis, using the unweighted pair group method with arithmetic averages, was performed for PFGE by using Statistica 6.1 for Windows (StatSoft Inc., Tulsa, OK). In any case, our results suggest that the persistence of a distinct clone of EHEC may be limited in time and space (i.e., in a cattle herd). This is apparently not a unique property of O165:H25, since we obtained similar results for clonal groups of other EHEC serotypes (O26:H11 and O157:H7) in cattle. The four human O165H25/H- isolates belonged to different other clonal groups (Fig. 1).

In conclusion, bovine O165:H25 isolates can carry virulence factors of EHEC that are associated with EHEC-related disease in humans, particularly HC and HUS. Therefore, strains of bovine origin may represent a considerable potential risk for human infection.

Acknowledgments

We thank J. Bockemühl, H. Tschäpe, T. Kuzius, and A. Fruth for serotyping the E. coli strains and H. Tschäpe for providing E. coli O165:H25 and O165:H- strains of human origin.

This study was funded by the German Federal Ministry of Consumer Protection, Food and Agriculture.

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