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

Abstract

Isogenic strains of Escherichia coli O157:H7, missing either stx 2 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.The major reservoirs for Shiga toxin-producing Escherichia coli (STEC) are cattle and other ruminants. These bacteria are carried in the gastrointestinal tract and do not cause disease in mature ruminants even when administered at very high doses (2). It is not known why the majority of ruminants are colonized by STEC in contrast to other animal species. One of the major virulence factors of STEC is Shiga toxin (Stx), an A 1 B 5 toxin which is encoded by lysogenic bacteriophages. The preferred receptor for the toxin is globotriaosylceramide (Gb 3 ). It has been shown that cattle lack Gb 3 receptors in their vascular endothelium, and it is thought that this may protect them from the systemic effects of Stx (9, 14). It has also been proposed that Stx may reduce the severities of viral infections such as bovine leukemia virus (4, 5) or modulate intestinal inflammation (12) in cattle.The objective of this study was to determine whether the Stx2-encoding bacteriophage or the Shiga toxin itself contributed to the magnitude or duration of colonization by E. coli O157:H7 in ruminants.(A preliminary report of this work was presented at the symposium Beyond Antimicrobials, the Future of Gut Microbiology, Aberdeen, Scotland, 2002.) The inoculum stains used in this study are listed in Table 1. Spontaneous nalidixic acid-, novobiocin-, and/or streptomycinresistant mutants were selected from parent strain 86-24 and its isogenic derivatives. Strain 86-24 ⌬933 was made as previously described by site-directed mutagenesis to delete stx 2 (15). Some mutants not only lost the stx 2 operon but suffered deletions in the region flanking the deleted stx 2 operon, suggesting that these mutants may have lost the entire 933 phage. Loss of the phage was confirmed by a PCR assay that utilized several sets of primers (designed based on the published nucleotide sequence for 933 [13]) to amplify approximately 1-kb portions from different regions of the 933 genome. The loss of 933 in strain 86-24 ⌬933 was also verified by challenging exponentially growing broth cultures of this strain and a 933-positive E. coli O157:H7 strain with subinhibitory amounts of norfloxacin (11). Inoculum strains were grown overnight in Trypticase soy broth and frozen in glycerol at Ϫ80°C until use.All experiments complied with the Iowa State University Animal Care and Use guidelines. Young adult sheep (4 to 12 months old, 80 to 120 lb) were purchased from a local source, housed two per pen under biosafety level 2 conditions, and acclimated to a diet of commercial sheep feed concentrate (1 lb/animal/day) and alfalfa/grass hay ad libitum for 2 weeks prior to inoculation as described previously (1). Sheep were randomly divided into three groups and inoculated wi...

Verotoxin 2 Enhances Adherence of Enterohemorrhagic Escherichia coli O157:H7 to Intestinal Epithelial Cells and Expression of β1-Integrin by IPEC-J2 Cells

“…Therefore, this study examined the effect of adherence of a wild-type EHEC O157 strain and its vt2-negative mutant on Gb3 expression in IPEC-J2 cells by investigating the expression of Gb3 synthase. Our data show that adherence of the wild-type bacteria resulted in an elevation of the level of expression of (1,2,21,22,25). The reasons for the conflicting results are not known, but they could be due to the different animal species, bacterial strains, and routes of infection.…”

Neutralizing antibodies to Shiga toxin type 2 (Stx2) reduce colonization of mice by Stx2-expressing Escherichia coli O157:H7

“…For example, introduction of the organism by a route that bypassed most of the gut [31] or the use of animals with no or reduced flora [33] could preclude detection of a role for toxin. Additionally, the conclusions in some of the reports are based on either a single time-point post-infection [29] or a limited temporal analysis [32, 33]. Finally, in one instance the authors excluded a role for Stx in gut adherence yet only measured colonization levels of an Stx-negative E. coli O157:H7 strain without inclusion of a wild-type toxigenic strain for comparison [30].…”

Adherence of Escherichia coli O157:H7 Mutants In Vitro and in Ligated Pig Intestines

“…VT is well established as a major virulence factor of EHEC O157:H7 that plays a central role as a toxin in HUS and HC (39), but its role in adherence is controversial (4). It is only recently that evidence supported a role for VT in adherence to HEp-2 cells and in colonization of the intestine of mice; this enhanced colonization correlated with a VT-induced increase in nucleolin, a receptor for intimin on the host epithelial cells (30).…”

“…Severe damage due to infection with EHEC is attributable to the cytotoxic verotoxin (VT), which damages epithelial and endothelial cells, leading to bloody diarrhea and HUS (16). Several investigators have reported that VT does not play a role in colonization of the intestine (2,4,34). However, Robinson et al (30) reported recently that VT enhances adherence to epithelial cells and colonization of the mouse intestine by E. coli O157:H7.…”