John W. Newland

Role of shiga-like toxin I in bacterial enteritis: Comparison between isogenic Escherichia coli strains induced in rabbits

BACKGROUND/AIMS Enteroadherent Escherichia coli that produce Shiga-like toxins are important causes of human disease, including enterohemorrhagic E. coli-induced colitis (EHEC). The role of Shiga-like toxins in these illnesses is unclear. The aim of this study was to establish an animal model for human EHEC and to determine the role of Shiga-like toxin I (SLT-I) in this model. METHODS E. coli strain RDEC-1 is an enteroadherent rabbit diarrheal pathogen. An isogenic variant of RDEC-1 (termed RDEC-H19A) producing high levels of SLT-I was obtained by infecting RDEC-1 with an SLT-I-converting bacteriophage. The effects of in vivo enteric infection produced in rabbits by RDEC-H19A were compared with those in uninfected and RDEC-1-infected animals. RESULTS SLT-I-producing RDEC-H19A induced a severe, noninvasive, enteroadherent infection in rabbits. Clinically, infection with RDEC-H19A was more severe than infection with RDEC-1 and caused more serious histological lesions including vascular changes, edema, and more severe inflammation. Interleukin 1 and platelet-activating factor appear to be important inflammatory mediators to this infection. CONCLUSIONS The illness induced by RDEC-H19A in rabbits resembled enterohemorrhagic E. coli-induced colitis of humans. SLT-I is an important virulence factor in the pathogenesis of EHEC.

Evaluation of the safety, immunogenicity, and efficacy in healthy adults of four doses of live oral hybrid Escherichia coli-Shigella flexneri 2a vaccine strain EcSf2a-2

In previous trials, live invasive Escherichia coli-Shigella flexneri 2a hybrid vaccine candidate EcSf2a-2, administered to adult volunteers as 3 doses of ca. 2 x 10(9) colony forming units (c.f.u.) spaced over one week, induced fever and/or diarrhea in 11% of subjects and provided only limited protection (36% efficacy) against illness following challenge with virulent S. flexneri 2a. We sought to improve the clinical safety of this vaccine by administering a lower inoculum, and to enhance protective immunity by administering additional booster doses at 2 weeks. Twenty-one healthy adults were immunized with ca. 7 x 10(8) c.f.u. of EcSf2a-2 on days 0, 3, 14, and 17. The vaccine consistently colonized the intestine without causing serious adverse reactions; mild diarrhea developed in one subject and low grade fever in another. Vaccination elicited an antibody secreting cell (ASC) response to lipopolysaccharide (LPS) in all subjects, which was highest on day 7 and notably diminished thereafter on days 10, 16, 21, and 24, suggesting that active mucosal immunity developed rapidly. The magnitude of the response was modest (geometric mean peak = 16 IgA ASC/10(6) peripheral blood mononuclear cells) and an IgG serological response to LPS was detected in only 19% of subjects. Following experimental challenge with virulent S. flexneri 2a administered with bicarbonate buffer, shigellosis (diarrhea, dysentery, or fever) developed in 10 of 16 vaccine recipients (63%) and in 12 of 14 unvaccinated controls (86%), resulting in a vaccine efficacy of 27% (95% confidence limits -197, 82, p = 0.15, 1-tailed).(ABSTRACT TRUNCATED AT 250 WORDS)

Shiga-like toxin converting phage of enterohemorrhagic Escherichia coli strain 933

Production of Shiga-like toxin (SLT) by enterohemorrhagic Escherichia coli (EHEC) is controlled by phage conversion, and specific phages carry either the SLT-I or SLT-II operon. EHEC strain 933 produces both SLT-I and SLT-II. Previous studies demonstrated that the vast majority of phages recovered from strain 993 have hexagonal heads with short tails and encode SLT-II. However, conflicting results were obtained concerning the properties of SLT-I converting phages from strain 933. The present study reexamined the recovery of phages from 933 by various methods and characterized the restriction fragments from strain 933 DNA that hybridized with radiolabeled DNA from the SLT-I converting phage 933J, which has an elongated head with a long tail, and the SLT-II converting phage 933W. In the present study, only SLT-II converting phages like 933W were recovered from strain 933. A set of restriction fragments that hybridized with DNA from phage 933J but not 933W was present both in wild type strain 933 and in the variant 933D, which produces only SLT-I and was shown here to be cured of phage 933W. The sizes of the restriction fragments in strain 933 that were homologous with phage 933J differed, however, from those of phage 933J. These data indicate that the phage we isolated and named 933J probably did not originate from strain 933 as we originally reported. The present evidence demonstrates that strain 933 contains both the SLT-II converting phage 933W and other sequences of DNA homologous with phage 933J that probably represent a defective SLT-I converting phage.

Construction, Characterization, and Animal Testing of WRSd1, a Shigella dysenteriae 1 Vaccine

ABSTRACT WRSd1 is a Shigella dysenteriae 1 vaccine containing deletions of the virG(icsA) gene required for intercellular spreading and a 20-kb chromosomal region encompassing the Shiga toxin genes (stxAB). WRSd1 was constructed from S. dysenteriae 1 strain 1617 that was originally isolated during the 1968 to 1969 epidemic of Shiga dysentery in Guatemala. The virG(icsA) deletion was constructed from a streptomycin-resistant (Strr) mutant of 1617 by a filter mating procedures using a virG(icsA) deletion derivative, pΔvirG2. A colony that was invasive for HeLa cells and negative for the virG(icsA) gene by Southern blotting was grown anaerobically on plates containing chlorate for selection of resistant colonies that had lost the entire Shiga toxin gene. A virG(icsA) stxAB Strr mutant selected from the chlorate plates was designated WRSd1. This candidate vaccine was evaluated for safety, immunogenicity, and protective efficacy using the guinea pig keratoconjunctivitis model. WRSd1 was Sereny negative, and two applications of this strain to the cornea elicited a significant protective immune response against the S. dysenteriae 1 O antigen. Vaccination with WRSd1 conferred protection against challenge with each of three virulent S. dysenteriae 1 strains. Since a vaccine protecting against multiple Shigella species is required for most areas where Shigella is endemic, protection studies using a combination vaccine of Shigella sonnei vaccine strain WRSS1, Shigella flexneri 2a vaccine strain SC602, and WRSd1 were also performed. Guinea pigs vaccinated with a mixture of equal amounts of the three vaccine strains were protected against challenge with each of the homologous virulent strains. Unlike WRSS1 and SC602, however, the level of protection afforded by WRSd1 in a combination vaccine was lower than the protection elicited by a pure culture. A current Good Manufacturing Practice product of WRSd1 given intragastrically to rhesus monkeys proved safe and immunogenic.

Genotypic and phenotypic characterization of an aroD deletion-attenuated Escherichia coli K12-Shigella flexneri hybrid vaccine expressing S. flexneri 2a somatic antigen

The construction and characterization of EcSf2a-2, an aroD-deleted Escherichia coli-Shigella hybrid vaccine carrying chromosomal and plasmid genes from Shigella flexneri and expressing S. flexneri 2a somatic antigen in association with E. coli K12 core are described. Expression of hybrid lipopolysaccharide and deletion of aroD resulted in the attenuation of phenotypic characteristics associated with pathogenicity. The addition of an aroD deletion results in a requirement for an aromatic precursor of para-aminobenzoic acid (PABA), an essential bacterial metabolite not present in mammalian tissues. The biosynthesis of hybrid somatic antigen prevents expression of a Sereny-positive reaction by invasive bacteria capable of expressing a plaque-positive phenotype. A functional kcpA gene is required for expression of the plaque-positive phenotype. The presence of an aroD deletion does not interfere with expression of an invasive phenotype; however, in bacteria containing a functional kcpA gene, replication and spread by invading bacteria are limited, preventing development of the plaque-positive phenotype.

Outpatient studies of the safety and immunogenicity of an auxotrophic Escherichia coli K-12-Shigella flexneri 2a hybrid vaccine candidate, EcSf2a-2

A phase II study was conducted in 244 volunteers at Fort Ord, CA, to determine the safety and immunogenicity of EcSf2a-2, a live, oral Shigella vaccine constructed by transfer of genes from Shigella flexneri to Escherichia coli K-12. In this placebo-controlled study, four doses of vaccine ranging from 2.3 to 9.0 x 10(8) colony-forming units were given on days 0, 3, 14 and 17. Vaccine shedding occurred from 1 to 3 days after each dose. The vaccine was well tolerated at every dose tested. Significant levels of IgA, IgG or IgM antibody-secreting cells (ASC) recognizing S. flexneri 2a lipopolysaccharide (LPS) were found in 94% of a volunteer subset tested 7 days after the first dose of EcSf2a-2. Seven days after the third dose, ASC were detected less often (57%), and were mainly IgA. Significant rises in serum antibody to LPS were detected in 37% of vaccine recipients.

Shiga and Shiga-Like Toxins: A Family of Related Cytotoxins

Shiga toxin is a potent cytotoxin produced by Shigella dysenteviae 1 that is also enterotoxic for ligated rabbit ileum and paralytic and lethal for mice and rabbits (1). Shiga toxin was first described in 1903 by Conradi (2), and it is the prototype toxin for a family of related toxins called Shiga-like toxins (SLT’s) or verotoxins. The members of the SLT family include cytotoxins produced by other Shigella species (3,4), Escherichia coli (5,6) two species of Vibrio (7) , as well as some strains of Salmonella typhimuriun (5,8), and Campylobactev jejuni (M. Moore, M. Blaser, G. Perez-Perez, and A. O’Brien, in preparation). The levels of cytotoxic activity produced by these organisms as measured by the number of 50% cytotoxic doses (CD50) per ml cell lysate (9) vary from low ( < 6 × 102) to moderate (1 × 103 to 1 × 104) to high (1 × 105) to 1 × 108). Production of moderate or high levels of SLT’s by E. coli is commonly associated with strains of E. coli that cause hemorrhagic colitis or the hemolytic uremic syndrome (9,10). The significance (if any) of low-level SLT production remains to be determined. The purpose of this report is to summarize our current understanding of the epidemiology, potential role in pathogenesis, structure, function and molecular genetics of the various members of the SLT family. Particular emphasis will be placed on Shiga toxin and the SLT’s produced at moderate to high levels by E. coli.