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. 2004 Aug;10(8):1482–1485. doi: 10.3201/eid1008.030784

Human Escherichia coli O157:H7 Genetic Marker in Isolates of Bovine Origin

Jeffrey T LeJeune *,✉, Stephen T Abedon *, Kaori Takemura *, Nicholas P Christie *, Srinand Sreevatsan *
PMCID: PMC3320398  PMID: 15496255

Abstract

The antiterminator Q gene of bacteriophage 933W (Q933) was identified upstream of the stx2 gene in 90% of human disease–origin Escherichia coli O157:H7 isolates and in 44.5% of bovine isolates. Shiga toxin production was higher in Q933-positive isolates than Q933-negative isolates. This genetic marker may provide a useful molecular tool for epidemiologic studies.

Keywords: E. coli O157, Shiga-toxin production, stx2-encoding phages, dispatch

Escherichia coli O157 is recognized worldwide as an important cause of diarrheal disease, which in some patients is followed by hemolytic uremic syndrome and death (1). A primary virulence factor of this pathogen is the prophage-encoded Shiga toxin (2). Greater Shiga toxin production per bacterium is associated with increasing severity of human disease (3,4). Because of its location in the phage genome, the stx-gene variant dubbed stx2 is under similar regulatory control as other phage late-genes, as it is governed by the interaction of the transcription antiterminator Q with the late promoter PR´ (5).

Although cattle and other ruminants appear to be the natural reservoir for E. coli O157 and other Shiga toxin–producing E. coli (STEC), only a small fraction of STEC serotypes routinely present in cattle are frequently isolated from human patients. Mounting evidence suggests that considerable genetic, phenotypic, and pathogenic diversity exists among these pathogens (6–8). Furthermore, genetic subtypes or lineages of E. coli O157 do not appear to be equally distributed among isolates of bovine and human origin (7). The purpose of this study was to examine the distribution of specific sequences upstream of the stx2 gene among E. coli O157:H7 of human and bovine origin, along with corresponding magnitudes of Shiga toxin production.

The Study

A total of 158 stx2-encoding E. coli O157:H7 isolates were assayed, 91 isolates of bovine origin and 67 originally isolated from ill persons (Tables A1 and A2). All isolates demonstrated unique banding patterns on pulsed-field gel electrophoresis (PFGE). For polymerase chain reaction (PCR) analysis, 5 µL of DNA obtained from boiled stationary-phase bacteria was added to a 50-µL PCR master mix containing a final concentration of 1.5 (Q933) or 2.5 (Q21) mmol MgCl2, 200 µmol/L each deoxynucleoside triphosphate, 1 U Taq polymerase, 0.6 pg/µL of primer 595 (5´-CCGAAGAAAAACCCAGTAACAG-3´) (9), and 0.6 pg/µL of either primer Q933 (5´-CGGAGGGGATTGTTGAAGGC-3´;QStxf) (9) or primer Q21 (5´-GAAATCCTCAATGCCTCGTTG-3´; this study). PCR consisted of an initial denaturation at 94°C for 5 min; 30 cycles of 94°C for 30 s, 52°C (Q933) or 55°C (Q21) for 1 min, and 72°C for 1 min; and a final 10-min extension step at 72°C. E. coli strain 933 or FAHRP88 was used as a positive control and master mix alone as a negative control. All PCR products were separated by gel electrophoresis (100 V) in 1% agarose gels, stained with ethidium bromide, and visualized by using UV illumination.

Shiga toxin production was determined by using a commercially available enzyme-linked immunosorbent assay (ELISA) kit (Premiere EHEC, Meridian Diagnostics, Cincinnati, OH). Briefly, log-phase cells from Luria-Bertani broth enrichments were diluted to 0.6 optical density (OD) at 600 nm, subsequently pelleted, resuspended in phosphate-buffered saline, and induced by exposure to UV light (240 nm) for 3 s (10). A 1:9 volume of a 10x concentrate of brain heart infusion broth was added to each culture and shaken at 37°C for 2.5 h. Replicate cultures that were not exposed to UV light (noninduced controls) were maintained at 4°C. Two hundred microliters of each induced and noninduced enrichment was subsequently used as the specimen in the EHEC ELISA, as described (11). OD results were recorded for each isolate both with and without UV induction. The relative change in Shiga toxin production after induction was calculated for each isolate; (ODinduced)/ODnoninduced). E. coli O157 (EDL933) and a toxin-negative control isolate were assayed as positive and negative controls each time the assay was repeated.

E. coli O157 isolates were classified on the basis of the presence or absence of bands of the predicted size on the Q933-595 and Q21-595 PCR reactions (Figure). A chi-square test was used to determine whether different PCR genotypes were equally distributed among isolates of bovine and human origin. Likewise, a chi-square test was used to assess the equality of distribution of PCR genotypes among bovine isolates from different countries. One-way analysis of variance for nonparametric data (Kruskal-Wallis test) was used to identify differences in ranked-transformed toxin production among noninduced and induced E. coli O157 isolates as well as to determine significant differences in the percent increase in toxin following induction.

Figure.

Figure

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Ethidium bromide–stained gel of the amplification products obtained from Q933-595 and Q21-595 polymerase chain reactions. aEDL933, human isolate (ATCC43895). Obtained from the STEC Center, Michigan State University. bFAHRP88, isolated from Ohio dairy cow. cFAHRP39, human isolate (E29962) (12).

Previously, Kim et al. described a nonrandom distribution of E. coli O157 subtypes among cattle and humans by using an octamer-based genome-scanning method (7). We tested several of the isolates that had been previously characterized. Nine had been previously identified as belonging to the lineage I genotype and seven isolates as belonging to the lineage II genotype. We found that all nine lineage I isolates consistently amplified the Q933 target, regardless of species of origin. All four bovine isolates classified as lineage II by Kim et al. amplified the Q21 target. One lineage II human isolate (NE015) amplified the Q933 target, and another lineage II isolate (NE037) produced no amplicons in either PCR reaction. One human isolate classified as lineage II (ATCC 43889) amplified both target sequences, presumably because of polylysogeny.

The distribution of the specific Q-gene alleles found upstream of the prophage stx region among bovine isolates may have a geographic component. The distribution of E. coli O157 phage genotypes collected from healthy cattle from diverse geographic areas is consistent with the variable incidences of human disease in different countries (Table 1). For example, six (75%) of eight Scottish bovine isolates examined amplified the Q933 target, the same target that is frequently present in human isolates of human disease origin. Scotland reports some of the highest incidence rates of human E. coli O157–related diseases and hemolytic uremic syndrome (13). In contrast, none of the seven Australian E. coli O157 bovine isolates amplified the 1750-bp fragment. Contrary to the situation in Scotland and the United States, E. coli O157 infection of humans is rarely reported in Australia (14).

Table 1. Distribution of polymerase chain reaction results from bovine Escherichia coli O157 isolates based on geographic origina.

Country of origin No. tested Q allele
933 21 Both
N (%) N (%) N (%)
USA 46 20 (44) 25 (54) 1 (2)
Scotland 8 – (0) 2 (25) 6 (75)
Australia 7 – (0) 7 (100) – (0)
Japan 17 3 (18) 14 (82) – (0)
Total 78 23 (29) 48 (62) 7 (9)
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a–, not detected. Percentages are read across rows, not down columns. Significant difference in proportion of Q alleles isolated from different countries (p < 0.05, chi-square test for homogeneity).

Conclusions

The Q933 gene target was more commonly identified in human disease–associated strains of E. coli O157 than from strains of bovine origin. Amplification of the Q933 target, either alone or in combination with amplification of the Q21 target from the same isolate, was identified in 60 (9%) of 66 (55/66 alone and 5/66 in combination with Q21; 1 isolate amplified neither target) compared to 40 (44%) of 91 (32/91 alone, and 8/91 in combination with Q21) of bovine isolates (p < 0.001). Furthermore, these genetic subtypes were nonrandomly distributed among the E. coli O157 isolates of bovine origin obtained from different countries (p < 0.05) (Table 1).

These limited data suggest that the distribution of E. coli O157 strains in cattle may differ between countries or regions, thereby providing an explanation for geographic differences in the incidence of human E. coli O157 infection. More isolates from cattle need to be analyzed with these methods to better characterize the E. coli O157 in the bovine reservoir of each country.

A positive reaction with the Q933 target was significantly associated with higher OD results on the Shiga toxin ELISA (both noninduced and induced) and higher-fold increases in toxin production following induction than isolates amplifying the Q21 target alone (p < 0.0001) (Table 2). Despite these differences, we did not identify any clinical associations between the magnitude of Shiga toxin production and severity of human disease could be identified in this study. Other, non–Shiga toxin–related virulence factors and host susceptibility are also believed to play essential roles in the outcome of clinical STEC infections. The Q933-negative isolates obtained from human disease might have lost this Q933-containing prophage by the time of isolation, or these isolates might have been recovered from patients also infected with STEC containing Q933-type prophage (15). Whether specific Q-gene alleles directly correlate with the magnitude of Shiga-toxin production or whether other (unstudied) factors within the phage lytic cascade genetically linked to specific Q alleles instead are responsible for the magnitude of toxin production is not known.

Table 2. Shiga toxin production by Escherichia coli O157:H7 by Q allele.

Assay Q allele Response
Median Minimum Maximum
OD600nm noninduced Q 933 0.442 0.153 2.814
Q 21 0.170 0.120 0.413
OD600nm induced Q 933 1.228 0.172 2.896
Q 21 0.165 0.084 1.210
Fold increase in OD600nm after inductiona Q 933 2.2 0.3 7.7
Q 21 0.9 0.4 5.1
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a(ODinduced)/(ODnoninduced). The maximum and minimum optical density readings at 600 nm listed in each row are not necessarily from the same isolate; therefore, the maximum- and minimum-fold increase cannot be calculated directly from the table.

The antiterminator Q, the protein product of the Q gene, and PR´, the late promoter, are reputed to be involved in regulating phage late-genes and, because of the location of PR´ in prophage genome, of Shiga toxin production as well (5). In E. coli O157 phage 933W (GenBank no. 9632466) and E. coli O157 stx2vhd (GenBank no. 15718404), the 359-bp sequence immediately upstream of the stx2 gene is nearly identical (>95% nucleotide identity). However, further upstream of this area of identity, DNA sequences differ significantly. In E. coli O157 933W, this gene is identified as the antiterminator Q gene. In contrast, in E. coli O157 stx2vhd this area is occupied by a gene with >95% sequence identity with the antiterminator Q gene of bacteriophage 21 (gi 4539472). The Q gene of bacteriophage 21 does not share DNA sequence homology with the Q gene of bacteriophage 933W, and only 36% predicted amino acid homology. Since the Q gene is reputed to play an important role in regulating toxin production, our results provide a plausible explanation (differential regulation of Shiga toxin production) of why certain E. coli O157 genotypes are more commonly isolated from human patients (7).

Acknowledgments

We thank the state departments of health of Ohio, Washington, and Idaho for many of the human isolates used in this study and the SETC Center, Michigan State University, for providing two of the strains we tested.

This project was funded by beef and veal producers and importers through their $1-per-head checkoff and was produced for the Cattlemen's Beef Board and state beef councils by the National Cattlemen's Beef Association. Research in S.S. and J.T.L. laboratories is also supported by state and federal funds appropriated to the Ohio Agricultural Research and Development Center.

Biography

Dr. LeJeune is an assistant professor in the Food Animal Health Research Program, in the Department of Veterinary Preventive Medicine, Ohio State University. His research interests include the epidemiology and ecology of foodborne pathogens in the preharvest stages of food production.

Table A1. Source of human isolates used in this studya.

FAHRP ID Source ID Country Year Clinical signs and symptoms References or source
6 FRIK 528 USA 1998 Diarrhea 16
7 FRIK 579 USA 1998 Diarrhea 16
8 93-001 USA 1999 Hemorrhagic colitis 17
9 ATCC 35150 USA 1999 Hemorrhagic colitis 17
16 91671 USA 1999 Hemorrhagic colitis 17
17 ATCC 43889 USA 1999 Hemorrhagic colitis 17
18 NE 037 USA 1999 Hemorrhagic colitis 17
19 NE 15 USA 1999 Hemorrhagic colitis 17
39 E29962 UK 1991 NR 18
54 CL56 Canada 1991 NR 18
60 E32511 USA 2002 HUS 19
58 EDL933 USA 1982 Hemorrhagic colitis 20
126 02 5225 USA 2002 NR Washingtonb
127 02 4857 USA 2002 NR Washington
128 02 6776 USA 2002 NR Washington
129 02 6579 USA 2002 NR Washington
130 02 6546 USA 2002 NR Washington
131 02 6722 USA 2002 NR Washington
132 02 6598 USA 2002 NR Washington
133 02 6696 USA 2002 NR Washington
134 02 6791 USA 2002 NR Washington
135 02 6829 USA 2002 NR Washington
136 02 6755 USA 2002 NR Washington
137 02 6644 USA 2002 NR Washington
138 06 781 USA 2002 Diarrhea Idahoc
139 06 852 USA 2002 NR Idaho
140 06 854 USA 2002 Watery diarrhea, vomiting Idaho
141 06 856 USA 2002 Diarrhea Idaho
142 06 855 USA 2002 NR
143 06 886 USA 2002 Diarrhea, abdominal pain Idaho
144 06 889 USA 2002 Abdominal pain Idaho
145 06 988 USA 2002 Gastrointestinal bleeding Idaho
146 07 004 USA 2002 Bloody stool Idaho
147 07 007 USA 2002 Bloody stool Idaho
148 07 023 USA 2002 Bloody stool Idaho
149 07 085 USA 2002 NR Idaho
150 07 147 USA 2002 NR Idaho
151 07 154 USA 2002 NR Idaho
152 O2191230 USA 2002 Diarrhea Ohiod
153 O2191229 USA 2002 Diarrhea Ohio
154 O2191231 USA 2002 Diarrhea Ohio
155 O2191294 USA 2002 Diarrhea Ohio
156 O2190819 USA 2002 Diarrhea Ohio
157 O2190864 USA 2002 Diarrhea Ohio
158 O2191309 USA 2002 Diarrhea Ohio
159 O2191311 USA 2002 Diarrhea Ohio
160 O2191313 USA 2002 Diarrhea Ohio
161 O2191361 USA 2002 Diarrhea Ohio
162 O2191602 USA 2002 Diarrhea Ohio
163 O2191624 USA 2002 Diarrhea Ohio
164 O2191541 USA 2002 Diarrhea Ohio
165 O2191546 USA 2002 Diarrhea Ohio
166 O2191423 USA 2002 Diarrhea Ohio
167 O2191509 USA 2002 Diarrhea Ohio
168 O2191363 USA 2002 Diarrhea Ohio
169 O2191364 USA 2002 Diarrhea Ohio
170 O2191365 USA 2002 Diarrhea Ohio
171 O2191366 USA 2002 Diarrhea Ohio
172 O2190889 USA 2002 Diarrhea Ohio
173 O2190893 USA 2002 Diarrhea Ohio
174 O2191176 USA 2002 Diarrhea Ohio
175 O2191177 USA 2002 Diarrhea Ohio
176 O2191623 USA 2002 Diarrhea Ohio
177 O2191625 USA 2002 Diarrhea Ohio
178 O2191645 USA 2002 Diarrhea Ohio
179 O2191675 USA 2002 Diarrhea Ohio
180 O2191765 USA 2002 Diarrhea Ohio
181 O2191831 USA 2002 Diarrhea Ohio
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aFAHRP, Food Animal Health Research Program, Ohio State University; NR, not reported; HUS, hemolytic uremic syndrome.
bWashington State Department of Health isolates.
cIdaho Department of Health and Welfare isolates.
dOhio Department of Health isolates.

Table A2. Source of bovine isolates used in this study.

FAHRPa ID Source ID Country Year References or source
1 FRIK 1986 USA 1991 21
2 FRIK 1997 USA 1991 21
3 FRIK 1994 USA 1991 21
4 FRIK 2002 USA 1991 21
5 FRIK 1987 USA 1991 21
10 FRIK 920 USA 1998 22
11 FRIK 1054 USA 1998 22
12 FRIK 1540 USA 1998 22
13 FRIK 1988 USA 1998 21
22 LCDC 87-2930 Canada 1991 23
27 OARDC1 USA 2002 FAHRP
29 OARDC2 USA 2002 FAHRP
31 OARDC3 USA 2002 FAHRP
35 P673 UK 1987 24
37 P277 UK 1987 24
47 c1526-77 Argentina 1991 23
50 CDC B9253-DMS1 USA 1991 23
51 A39 Canada 1991 23
52 A43 Canada 1991 23
56 LCDC 87-2924 Canada 1991 23
57 LCDC 87-1799 Canada 1991 23
62 CDC B6830-MS1/0 USA 1991 23
63 CDCB7205-MS1/0 USA 1991 23
64 CDC B8038-MS1/0 USA 1991 23
65 8832 USA 2002 25
66 EC66 USA 2002 FAHRP
67 EC 67 USA 2002 FAHRP
82 8833 USA 2002 25
83 EC 83 USA 2002 FAHRP
84 EC 84 USA 2002 FAHRP
85 8834 USA 2002 25
87 EC87 USA 2002 FAHRP
88 EC88 USA 2002 FAHRP
93 EC 93 USA 2002 FAHRP
94 EC94 USA 2002 FAHRP
95 EC95 USA 2002 FAHRP
96 EC96 USA 2002 FAHRP
97 EC97 USA 2002 FAHRP
98 EC98 USA 2002 FAHRP
99 EC99 USA 2002 FAHRP
100 EC100 USA 2002 FAHRP
102 EC102 USA 2002 FAHRP
103 EC103 USA 2002 FAHRP
104 EC104 USA 2002 FAHRP
113 8837 USA 2002 25
115 EC115 USA 2002 FAHRP
116 EC116 USA 2002 FAHRP
117 EC117 USA 2002 FAHRP
120 EC120 USA 2002 FAHRP
122 EC122 USA 2002 FAHRP
182 757 USA 1994 25
183 817 USA 1994 25
185 1104 USA 1994 25
186 1119 USA 1994 25
187 1124 USA 1994 25
188 1136 USA 1994 25
189 1273 USA 1994 25
190 3735 USA 1996 25
191 4048 USA 1996 25
192 7407 Japan 1996 25
193 7409 Japan 1996 25
194 7416 Japan 1996 25
195 7420 Japan 1996 25
196 7421 Japan 1996 25
197 7423 Japan 1996 25
198 7433 Japan 1996 25
199 7436 Japan 1996 25
200 7439 Japan 1996 25
201 7460 Japan 1996 25
202 7469 Japan 1996 25
203 7478 Japan 1996 25
204 7484 Japan 1996 25
205 7488 Japan 1996 25
206 7495 Japan 1996 25
207 7500 Japan 1996 25
208 7505 Japan 1996 25
209 7622 Scotland 1996 25
210 7630 Scotland 1999 25
211 7632 Scotland 1999 25
213 7637 Scotland 1999 25
214 7638 Scotland 1999 25
217 7648 Scotland 1999 25
218 7649 Scotland 1999 25
219 7653 Scotland 1999 25
220 8176 Australia 1999 25
221 8177 Australia 1996 25
222 8179 Australia 1997 25
223 8182 Australia 1997 25
224 8183 Australia 1997 25
225 8184 Australia 1998 25
226 8185 Australia 1999 25
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aFAHRP, Food Animal Health Research Program, Ohio State University.

Footnotes

Suggested citation for this article: LeJeune JT, Abedon ST, Takemura K, Christie NP, Sreevatsan S. Human Escherichia coli O157:H7 genetic marker in isolates of bovine origin. Emerg Infect Dis [serial on the Internet]. 2004 Aug [date cited]. http://dx.doi.org/10.3201/eid1008.030784

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