Escherichia coli O157:H7 and Other E. coli Strains Share Physiological Properties Associated with Intestinal Colonization

Escherichia coli isolates (72 commensal and 10 O157:H7 isolates) were compared with regard to physiological and growth parameters related to their ability to survive and persist in the gastrointestinal tract and found to be similar. We propose that nonhuman hosts in E. coli O157:H7 strains function similarly to other E. coli strains in regard to attributes relevant to gastrointestinal colonization.Escherichia coli is well known for its ecological versatility (15). A life cycle which includes both gastrointestinal and environmental stages has been stressed by both Savageau (15) and Adamowicz et al. (1). The gastrointestinal stage would be subjected to acid and detergent stress. The environmental stage is implicit in E. coli having transport systems for fungal siderophores (4) as well as pyrroloquinoline quinone-dependent periplasmic glucose utilization (1) because their presence indicates evolution in a location containing fungal siderophores and pyrroloquinoline quinone (1).Since its recognition as a food-borne pathogen, there have been numerous outbreaks of food-borne infection due to E. coli O157:H7, in both ground beef and vegetable crops (6, 13). Cattle are widely considered to be the primary reservoir of E. coli O157:H7 (14), but E. coli O157:H7 does not appear to cause disease in cattle. To what extent is E. coli O157:H7 physiologically unique compared to the other naturally occurring E. coli strains? We feel that the uniqueness of E. coli O157:H7 should be evaluated against a backdrop of other wild-type E. coli strains, and in this regard, we chose the 72-strain ECOR reference collection originally described by Ochman and Selander (10). These strains were chosen from a collection of 2,600 E. coli isolates to provide diversity with regard to host species, geographical distribution, and electromorph profiles at 11 enzyme loci (10).In our study we compared the 72 strains of the ECOR collection against 10 strains of E. coli O157:H7 and six strains of E. coli which had been in laboratory use for many years (Table ​(Table1).1). The in vitro comparisons were made with regard to factors potentially relevant to the bacteria''s ability to colonize animal guts, i.e., acid tolerance, detergent tolerance, and the presence of the Entner-Doudoroff (ED) pathway (Table ​(Table2).2). Our longstanding interest in the ED pathway (11) derives in part from work by Paul Cohen''s group (16, 17) showing that the ED pathway is important for E. coli colonization of the mouse large intestine. Growth was assessed by replica plating 88 strains of E. coli under 40 conditions (Table ​(Table2).2). These included two LB controls (aerobic and anaerobic), 14 for detergent stress (sodium dodecyl sulfate [SDS], hexadecyltrimethylammonium bromide [CTAB], and benzalkonium chloride, both aerobic and anaerobic), 16 for acid stress (pH 6.5, 6.0, 5.0, 4.6, 4.3, 4.2, 4.1, and 4.0), four for the ability to grow in a defined minimal medium (M63 glucose salts with and without thiamine), and four for the presence or absence of a functional ED pathway (M63 with gluconate or glucuronate). All tests were done with duplicate plates in two or three separate trials. The data are available in Tables S1 to S14 in the supplemental material, and they are summarized in Table ​Table22.

TABLE 1.

E. coli strains used in this study

Persistent colonization of sheep by Escherichia coli O157:H7 and other E. coli pathotypes

Shiga toxin-producing Escherichia coli (STEC) is an important cause of food-borne illness in humans. Ruminants appear to be more frequently colonized by STEC than are other animals, but the reason(s) for this is unknown. We compared the frequency, magnitude, duration, and transmissibility of colonization of sheep by E. coli O157:H7 to that by other pathotypes of E. coli. Young adult sheep were simultaneously inoculated with a cocktail consisting of two strains of E. coli O157:H7, two strains of enterotoxigenic E. coli (ETEC), and one strain of enteropathogenic E. coli. Both STEC strains and ETEC 2041 were given at either 10(7) or 10(10) CFU/strain/animal. The other strains were given only at 10(10) CFU/strain. We found no consistent differences among pathotypes in the frequency, magnitude, and transmissibility of colonization. However, the STEC strains tended to persist to 2 weeks and 2 months postinoculation more frequently than did the other pathotypes. The tendency for persistence of the STEC strains was apparent following an inoculation dose of either 10(7) or 10(10) CFU. One of the ETEC strains also persisted when inoculated at 10(10) CFU. However, in contrast to the STEC strains, it did not persist when inoculated at 10(7) CFU. These results support the hypothesis that STEC is better adapted to persist in the alimentary tracts of sheep than are other pathotypes of E. coli.     

Shiga Toxin and Shiga Toxin-Encoding Phage Do Not Facilitate Escherichia coli O157:H7 Colonization in Sheep

Isogenic strains of Escherichia coli O157:H7, missing either stx₂ or the entire Stx2-encoding phage, were compared with the parent strain for their abilities to colonize sheep. The absence of the phage or of the Shiga toxin did not significantly impact the magnitude or duration of shedding of E. coli O157:H7.     

Assessment of resistance to colicinogenic Escherichia coli by E. coli O157:H7 strains

AIMS: To assess a collection of 96 Escherichia coli O157:H7 strains for their resistance potential against a set of colicinogenic E. coli developed as a probiotic for use in cattle. METHODS AND RESULTS: Escherichia coli O157:H7 strains were screened for colicin production, types of colicins produced, presence of colicin resistance and potential for resistance development. Thirteen of 14 previously characterized colicinogenic E. coli strains were able to inhibit 74 serotype O157:H7 strains. Thirteen E. coli O157:H7 strains were found to be colicinogenic and 11 had colicin D genes. PCR products for colicins B, E-type, Ia/Ib and M were also detected. During in vitro experiments, the ability to develop colicin resistance against single-colicin producing E. coli strains was observed, but rarely against multiple-colicinogenic strains. The ability of serotype O157:H7 strains to acquire colicin plasmids or resistance was not observed during a cattle experiment. CONCLUSIONS: Escherichia coli O157:H7 has the potential to develop single-colicin resistance, but simultaneous resistance against multiple colicins appears to be unlikely. Colicin D is the predominant colicin produced by colicinogenic E. coli O157:H7 strains. SIGNIFICANCE AND IMPACT OF THE STUDY: The potential for resistance development against colicin-based strategies for E. coli O157:H7 control may be very limited if more than one colicin type is used.     

The lipopolysaccharide core type of Escherichia coli O157:H7 and other non-O157 verotoxin-producing E. coli

A mouse monoclonal antibody specific for the R3 lipopolysaccharide core type of Escherichia coli was used to determine the core type of E. coli O157:H7 and other non-O157 verotoxin-producing E. coli strains. Lipopolysaccharide extracts from 28 clinical isolates were examined by sodium dodecylsulfate-polyacrylamide gel electrophoresis and immunoblotting and all were found to have the R3 core. None of the core lipopolysaccharide from the strains tested reacted with the control R1 and R2 specific monoclonal antibodies. A common core type between all the verotoxin-producing E. coli strains tested may be significant when considering the immune response to these bacteria, and to the receptor for the VT bacteriophage.     

Escherichia coli O157:H7 Colonization at the Rectoanal Junction of Long-Duration Culture-Positive Cattle

Long-duration consistently Escherichia coli O157:H7 culture-positive cattle were euthanized and necropsied. Tissue and digesta from along the gastrointestinal tract (GIT) were cultured for the bacteria and examined histologically for lymphoid character. E. coli O157:H7 was detected only at the rectoanal junction mucosa and not at any other GIT location.     

Rectal Administration of Escherichia coli O157:H7: Novel Model for Colonization of Ruminants

Escherichia coli O157:H7 causes hemorrhagic colitis and life-threatening complications. Because healthy cattle are reservoirs for the bacterium, ruminant infection models have applications in analyzing the relationship between cattle and this human pathogen and in testing interventions to reduce or prevent bovine colonization with this bacterium. Current approaches often do not reliably mimic natural, long-term bovine colonization with E. coli O157:H7 in older calves and adult animals (ages that enter our food chain). Based on the recent identification of the bovine rectoanal junction mucosa as a site of E. coli O157:H7 colonization, we developed a novel rectal swab administration colonization model. We compared this method with oral dosing and direct contact transmission (Trojan) methods. E. coli O157:H7 carriage status was determined by fecal or rectoanal mucosa swab culture. High (~10¹⁰ CFU) and low (~10⁷ CFU) oral doses of E. coli O157:H7 in sheep and cattle resulted in variable infection with the bacterium. Some animals became colonized with the bacteria and remained culture positive for several weeks, and some animals did not become colonized and rapidly cleared the bacteria in a few days. Pen mates of E. coli O157:H7 culture-positive Trojan cattle had a low infection rate and variable colonization status. However, rectal swab administration of E. coli O157:H7 to cattle resulted in consistent long-term colonization in all animals. The surprising ease with which long-term infections resulted from a single application of bacteria to the rectoanal mucosa also strongly supported this location as a site of E. coli O157:H7 colonization in cattle.     

Electrophoretic mobilities of Escherichia coli O157:H7 and wild-type Escherichia coli strains.

The electrophoretic mobilities (EPMs) of a number of Escherichia coli O157:H7 and wild-type E. coli strains were measured. The effects of pH and ionic strength on the EPMs were investigated. The EPMs of E. coli O157:H7 strains differed from those of wild-type strains. As the suspension pH decreased, the EPMs of both types of strains increased.     

Reduction of Escherichia coli O157:H7 Populations in Cattle by Addition of Colicin E7-Producing E. coli to Feed

A cattle trial using artificially inoculated calves was conducted to determine the effect of the addition of colicinogenic Escherichia coli strains capable of producing colicin E7 (a 61-kDa DNase) to feed on the fecal shedding of serotype O157:H7. The experiment was divided into three periods. In period 1, which lasted 24 days, six calves were used as controls, and eight calves received 10⁷ CFU of E. coli (a mixture of eight colicinogenic E. coli strains) per g of feed. Both groups were orally inoculated with nalidixic acid-resistant E. coli O157:H7 strains 7 days after the treatment started. In periods 2 and 3, the treatment and control groups were switched, and the colicinogenic E. coli dose was increased 10-fold. During period 3, which lasted as long as period 1, both groups were reinoculated with E. coli O157:H7. The numbers of E. coli O157:H7 were consistently greater in the control groups during the three periods, but comparisons within each time period determined a statistically significant (P < 0.05) difference only at day 21 of period 1. However, when the daily average counts were compared between the period 1 control group and the period 3 treatment group that included the same six animals, an overall reduction of 1.1 log₁₀ CFU/g was observed, with a maximum decrease of 1.8 log₁₀ CFU/g at day 21 (overall statistical significance, P = 0.001). Serotype O157:H7 was detected in 44% of the treatment group's intestinal tissue samples and in 64% of those from the control group (P < 0.04). These results indicated that the daily addition of 10⁸ CFU of colicin E7-producing E. coli per gram of feed could reduce the fecal shedding of serotype O157:H7.     

Reduction of Escherichia coli O157:H7 populations in cattle by addition of colicin E7-producing E. coli to feed

A cattle trial using artificially inoculated calves was conducted to determine the effect of the addition of colicinogenic Escherichia coli strains capable of producing colicin E7 (a 61-kDa DNase) to feed on the fecal shedding of serotype O157:H7. The experiment was divided into three periods. In period 1, which lasted 24 days, six calves were used as controls, and eight calves received 10(7) CFU of E. coli (a mixture of eight colicinogenic E. coli strains) per g of feed. Both groups were orally inoculated with nalidixic acid-resistant E. coli O157:H7 strains 7 days after the treatment started. In periods 2 and 3, the treatment and control groups were switched, and the colicinogenic E. coli dose was increased 10-fold. During period 3, which lasted as long as period 1, both groups were reinoculated with E. coli O157:H7. The numbers of E. coli O157:H7 were consistently greater in the control groups during the three periods, but comparisons within each time period determined a statistically significant (P < 0.05) difference only at day 21 of period 1. However, when the daily average counts were compared between the period 1 control group and the period 3 treatment group that included the same six animals, an overall reduction of 1.1 log(10) CFU/g was observed, with a maximum decrease of 1.8 log(10) CFU/g at day 21 (overall statistical significance, P = 0.001). Serotype O157:H7 was detected in 44% of the treatment group's intestinal tissue samples and in 64% of those from the control group (P < 0.04). These results indicated that the daily addition of 10(8) CFU of colicin E7-producing E. coli per gram of feed could reduce the fecal shedding of serotype O157:H7.     

Measurements of Fitness and Competition in Commensal Escherichia coli and E. coli O157:H7 Strains

Although the main reservoirs for pathogenic Escherichia coli O157:H7 are cattle and the cattle environment, factors that affect its tenure in the bovine host and its survival outside humans and cattle have not been well studied. It is also not understood what physiological properties, if any, distinguish these pathogens from commensal counterparts that live as normal members of the human and bovine gastrointestinal tracts. To address these questions, individual and competitive fitness experiments, indirect antagonism assays, and antibiotic resistance and carbon utilization analyses were conducted using a strain set consisting of 122 commensal and pathogenic strains. The individual fitness experiments, under four different environments (rich medium, aerobic and anaerobic; rumen medium, anaerobic; and a minimal medium, aerobic) revealed no differences in growth rates between commensal E. coli and E. coli O157:H7 strains. Indirect antagonism assays revealed that E. coli O157:H7 strains more frequently produced inhibitory substances than commensal strains did, under the conditions tested, although both groups displayed moderate sensitivity. Only minor differences were noted in the antibiotic resistance patterns of the two groups. In contrast, several differences between commensal and O157:H7 groups were observed based on their carbon utilization profiles. Of 95 carbon sources tested, 27 were oxidized by commensal E. coli strains but not by the E. coli O157:H7 strains. Despite the observed physiological and biochemical differences between these two groups of E. coli strains, however, the O157:H7 strains did not appear to possess traits that would confer advantages in the bovine or extraintestinal environment.     

Measurements of fitness and competition in commensal Escherichia coli and E. coli O157:H7 strains

Although the main reservoirs for pathogenic Escherichia coli O157:H7 are cattle and the cattle environment, factors that affect its tenure in the bovine host and its survival outside humans and cattle have not been well studied. It is also not understood what physiological properties, if any, distinguish these pathogens from commensal counterparts that live as normal members of the human and bovine gastrointestinal tracts. To address these questions, individual and competitive fitness experiments, indirect antagonism assays, and antibiotic resistance and carbon utilization analyses were conducted using a strain set consisting of 122 commensal and pathogenic strains. The individual fitness experiments, under four different environments (rich medium, aerobic and anaerobic; rumen medium, anaerobic; and a minimal medium, aerobic) revealed no differences in growth rates between commensal E. coli and E. coli O157:H7 strains. Indirect antagonism assays revealed that E. coli O157:H7 strains more frequently produced inhibitory substances than commensal strains did, under the conditions tested, although both groups displayed moderate sensitivity. Only minor differences were noted in the antibiotic resistance patterns of the two groups. In contrast, several differences between commensal and O157:H7 groups were observed based on their carbon utilization profiles. Of 95 carbon sources tested, 27 were oxidized by commensal E. coli strains but not by the E. coli O157:H7 strains. Despite the observed physiological and biochemical differences between these two groups of E. coli strains, however, the O157:H7 strains did not appear to possess traits that would confer advantages in the bovine or extraintestinal environment.     

Dose-Response Envelope for Escherichia coli O157:H7

Escherichia coli O157:H7 is an emerging food and waterborne pathogen in the U.S. and internationally. The objective of this work was to develop a dose-response model for illness by this organism that bounds the uncertainty in the dose-response relationship. No human clinical trial data are available for E. coli O157:H7, but such data are available for two surrogate pathogens: enteropathogenic E. coli (EPEC) and Shigella dysenteriae. E. coli O157:H7 outbreak data provide an initial estimate of the most likely value of the dose-response relationship within the bounds of an envelope defined by beta-Poisson dose-response models fit to the EPEC and S. dysenteriae data. The most likely value of the median effective dose for E. coli O157:H7 is estimated to be approximately 190[emsp4 ]000 colony forming units (cfu). At a dose level of 100[emsp4 ]cfu, the median response predicted by the model is six percent.     

Experimental Escherichia coli O157:H7 carriage in calves.

Nine weaned calves (6 to 8 weeks of age) were given 10(10) CFU of a five-strain mixture of enterohemorrhagic Escherichia coli O157:H7 by oral-gastric intubation. After an initial brief period of pyrexia in three calves and transient mild diarrhea in five calves, calves were clinically normal throughout the 13- to 27-day study. The population of E. coli O157:H7 in the faces decreased dramatically in all calves during the first 2 weeks after inoculation. Thereafter, small populations of E. coli O157:H7 persisted in all calves, where they were detected intermittently in the feces and rumen contents. While withholding food increased fecal shedding of E. coli O157:H7 by 1 to 2 log10/g in three of four calves previously shedding small populations of E. coli O157:H7, the effect of fasting on fecal shedding of E. coli O157:H7 was variable in calves shedding larger populations. At necropsy, E. coli O157:H7 was not isolated from sites outside the alimentary tract. E. coli O157:H7 was isolated from the forestomach or colon of all calves at necropsy. Greater numbers of E. coli O157:H7 were present in the gastrointestinal contents than in the corresponding mucosal sections, and there was no histologic or immunohistochemical evidence of E. coli O157:H7 adhering to the mucosa. In conclusion, under these experimental conditions, E. coli O157:H7 is not pathogenic in weaned calves, and while it does not appear to colonize mucosal surfaces for extended periods, E. coli O157:H7 persists in the contents of the rumen and colon as a source for fecal shedding.     

Characterization of rpoS alleles in Escherichia coli O157:H7 and in other E. coli serotypes

The rpoS nucleotide and predicted amino acid sequences from three Escherichia coli O157:H7 isolates were compared with those from three other E. coli isolates, including the likely O157:H7 progenitor, E. coli O55:H7. These clinical and environmental isolates all had identical sigma S amino acid sequences, while laboratory strains K12 and DH1 had three and one amino acid alterations, respectively, in comparison with the majority sequence. To extend the analysis of sigma S sequence conservation to include other Gram-negative bacteria, the E. coli sigma S sequences were compared with those from diverse Gram-negative organisms; sigma S sequence identities ranged from 50.2 to 99.7% among the available sequences. The results further confirm the existence of rpoS alleles among different E. coli strains, although all strains were classified as acid-resistant with survival rates > 10% after 2 h exposure to pH 2.5. It was also found that all E. coli O157:H7 isolates tested had a unique nucleotide at position 543, thus differentiating these strains from other E. coli serotypes.