Shiga-like toxin-converting phages from Escherichia coli strains that cause hemorrhagic colitis or infantile diarrhea.

Escherichia coli K-12 acquired the ability to produce a high titer of Shiga-like toxin after lysogenization by either of two different bacteriophages isolated from a highly toxinogenic Escherichia coli O157:H7 strain that causes hemorrhagic colitis. One of these phages and another Shiga-like toxin-converting phage from an Escherichia coli O26 isolate associated with infantile diarrhea were closely related in terms of morphology, virion polypeptides, DNA restriction fragments, lysogenic immunity, and heat stability, although a difference in host range was noted. These phages are currently the best-characterized representatives from a broader family of Shiga-like toxin-converting phages.

Pathogenesis of salmonellosis. Studies of fluid secretion, mucosal invasion, and morphologic reaction in the rabbit ileum.

Strains of Salmonella typhimurium were studied in the ligated rabbit ileal loop model to gain insight into the mechanisms whereby bacteria which invade the gastrointestinal mucosa evoke fluid exsorption. The organisms employed differed in various biologic attributes including the ability to invade the ileal epithelium, multiply within the mucosa, elicit an acute inflammatory reaction, and disseminate across the intestinal wall. Some strains provoked small intestinal fluid exsorption although these did not elaborate enterotoxin. Only those strains which invaded the mucosa were accompanied by either mucosal inflammation or fluid exsorption. Noninvasive strains produced neither histologic abnormalities nor fluid secretion. While strains which invaded the mucosa caused an acute inflammatory reaction, not all such strains evoked fluid secretion. Furthermore, there was no correlation in ability of invasive organisms to evoke fluid secretion or in the intensity of mucosal inflammation, number of intramucosal salmonellae, or in ability to disseminate from the rabbit ileum. These observations suggest that, as is the case in shigellosis, mucosal invasion may be a necessary factor for the intestinal fluid loss in salmonellosis. A bacterial property or factor, in addition to invasion of the gastrointestinal mucosa, seems to be responsible for fluid exsorptin. However, it is unlikely that a salmonella enterotoxin comparable to that elaborated by Vibrio cholerae, toxigenic Escherichia coli, or Shigella dysenteriae 1 is related to fluid secretion in salmonellosis.

Cytotoxicity of Shigella dysenteriae 1 for cultured mammalian cells.

The cytotoxicity of an invasive toxigenic wild-type strain of Shigella dysenteriae 1 (3818T) was compared with that of noninvasive, toxigenic strain 38180 and hypotoxigenic strain 725. Cytolysis of HeLa or Henle 407 cells exposed to these strains was measured by release of (3H) uridine from prelabeled monolayers. HeLa cells exposed to noninvasive, toxigenic strain 38180, or to partially purified Shiga toxin were lysed only after a latent period of more than 8 hr. During this period, protein synthesis was inhibited. In contrast, Henle 407 cells that were exposed to strain 38180 or to exogenous Shiga toxin were unaffected. When either Henle 407 or HeLa cells were infected with invasive toxigenic strains, rapid lysis ensued. Quantitative microassay of cytosol toxicity showed that Shiga toxin was produced intracellularly by strain 3818T. The data suggest that cytolysis of infected mammalian cells is caused, at least in part, by intracellular Shiga toxin.

Shigellosis and Escherichia coli diarrhea: relative importance of invasive and toxigenic mechanisms.

Shigellae and dysentery-like Escherichia coli must invade the epithelium of the colon to cause disease which can present as dysentery, diarrhea, or both. This paper addresses the possible role of a Shigella dysenteriae-like (Shiga-like) toxin in the pathogenesis of shigellosis and E. coli diarrheal diseases. The possibility for such a role is suggested by the following observations: 1) diarrhea, considered to be a result of secretion of water by the small bowel, is frequently observed in shigellosis, a large bowel disease. 2) Even though shigellae do not invade the jejunum of monkeys fed Shigella flexneri, jejunal secretion is seen in animals with diarrhea. 3) The Shiga toxin of S. dysenteriae has enterotoxic activity and other serotypes of shigellae produce Shiga-like toxins. 4) E. coli 015 RDEC-1 causes a diarrheal disease and frequently death in young rabbits. This organism neither produces E. coli enterotoxins nor is it invasive, but it may produce low levels of a Shiga-like toxin.

Effects of intravenous and aerosol administration of crude Shigella toxin to rhesus macaques: preliminary study.

Experiments were conducted to determine the dose response, survival time, blood biochemical changes, and cardiohepatic responses to a single IV injection of crude Shigella toxin in rhesus macaques (Macaca mulatta). Circulatory shock, hepatic falure, respiratory depression, dyspnea, convulsions, and coma were observed before death. The survival time was inversely related to the administered toxic dose, which ranged from 20 to 200 microgram/kg of body weight. However, the severity of lesions in the heart and gastrointestinal tract was directly correlated with the amount of the injected dose. Macaques exposed to aerosols of crude Shigella toxin did not show any signs of toxicosis within 7 days after exposure and were considered permanent survivors. Seemingly, an IV crude preparation of Shigella toxin is lethal to rhesus macaques, but an aerosol crude preparation of Shigella toxin is not.

Protein synthesis in HeLa or Henle 407 cells infected with Shigella dysenteriae 1, Shigella flexneri 2a, or Salmonella typhimurium W118.

The incorporation of [14C]leucine into protein was studied in two mammalian cell lines which had been infected with strains of Shigella dysenteriae 1, Shigella flexneri 2a, or Salmonella typhimurium W118. These cell lines differed in susceptibility to the effects of exogenously applied Shiga cytotoxin. All invasive shigella strains (which synthesize this toxin to a greater or lesser degree) were found to inhibit protein synthesis in both cell lines with equal efficiency. Leucine accumulation continued in these cells, but the labeled amino acid was preferentially incorporated into bacterial protein. S. typhimurium W118, which has not been shown to elaborate a Shiga-like toxin, had little effect on protein synthesis in infected host cells.

Invasive Escherichia coli.

The present evidence indicates that Shigella-like pathogenicity is determined by a multiplacity of genes. Although deliberate attempts have been made to confer invasive virulence on E. coli strain K12 by employing classical procedures of recombination with virulent S. flexneri donor strains, they have not yet been successful. While we should, theoretically, be able to achieve this, the practical problem of testing for pathogenicity precludes screening larger numbers of hybrid clones for the acquisition of virulence. This increases the difficulty of successfully realizing that end. Nevertheless, since invasive-type pathogenicity is determined by multiple genetic loci, we consider it unlikely that random insertion of foreign DNA into the E. coli K12 genome could supply all of the genetic information necessary to convert this organism into an invasive enteric pathogen.

Restriction in genetic crosses between Escherichia coli and Shigella flexneri.

Shigella flexneri restricts Escherichia coli deoxyribonucleic acid (DNA) and can modify phage DNA so that it is restricted in E. coli.

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.

Effect of silica on the innate resistance of inbred mice to Salmonella typhimurium infection.

The role of macrophages in the innate immunity of (CBA/N female X DBA/2N male)F1 female mice to Salmonella typhimurium was assessed with silica, an agent which has been reported to selectively inactivate macrophages. Silica, administered intravenously to mice, markedly decreased the phagocytic capacity of splenic macrophages but had no effect on splenic responsiveness to the B-cell mitogen lipopolysaccharidide or the T-cell mitogen phytohemagglutinin, nor did it affect the frequency of surface immunoglobulin-positive cells (B cells). Silica given to mice 1 day before intraperitoneal challenge decreased the 50% lethal dose of S. typhimurium 100-fold. The incidence of survival of mice given silica up to 14 days before infection with a sublethal dose of organisms was also decreased. This susceptibility could also be demonstrated when silica was given 10 days, but not 20 days, after S. typhimurium infection. Poly-2-vinylpyridine-N-oxide, a lysosomal stabilizing agent, abrogated the silica effect. Deaths among silica-treated mice followed uncontrolled multiplication of the organism in the spleen. These results provide direct evidence that macrophages play an essential role in natural immunity to murine typhoid and demonstrate the efficacy of silica as a tool to analyze macrophage function.

Quantitation of the adherence of an enteropathogenic Escherichia coli to isolated rabbit intestinal brush borders.

Two assays were developed to quantitate the adherence of an Escherichia coli strain (RDEC-1) known to colonize the mucosal surface of the small intestine of rabbits to brush borders isolated from rabbit intestinal epithelial cells. In the first assay, the mean adherence per rabbit brush border was determined by counting the number of organisms adhering to each of 40 brush borders under phase microscopy. The mean adherence of RDEC-1 (11.5 +/- 0.7 per rabbit brush border) was significantly greater than adherence of two nonpathogenic strains: HS (2.7 +/- 0.4 per rabbit brush border) and 640 (0.8 +/- 0.1 per rabbit brush border). A similar distinction between the adherence of RDEC-1 and the control (nonadherent) organisms could be made more rapidly by determining the percentage of the total number of brush borders which had 10 or more adherent organisms; this second assay was used to define the optimum conditions for adherence. Maximum adherence was seen within 15 min. Adherence was temperature dependent, with adherence after 1 min at 37 degrees C being fourfold greater than that at 4 degrees C. The pH optimum for adherence was between 6.5 and 7.0, and adherence was abolished below pH 5.0. With the first, more sensitive assay, the effect of electrolytes and a number of hexoses and hexosamines on adherence was analyzed. RDEC-1 adherence was inhibited at high ionic strengths; however, adherence was not influenced at moderately high concentrations (20 mg/ml) by either d-mannose or l-fucose, in contrast to the case for other reported enteric pathogens. These two quantitative in vitro assays for adherence produce consistent results and have been used to partially characterize the adherence of RDEC-1 to rabbit brush borders.

Molecular characterization of plasmids from virulent and spontaneously occurring avirulent colonial variants of Shigella flexneri.

Spontaneous transition of Shigella flexneri from T- to O-type colonies with concomitant loss of virulence dose not appear to be accompanied by a change in any of the four plasmic species found in the virulent parent.

Immunization with Shigella dysenteriae type 1: evaluation of antitoxic immunity in prevention of experimental disease in rhesus monkeys (Macaca mulatta).

The role of serum antitoxic antibody in protection against the dysentery caused by Shigella dysenteriae type 1 (Shiga's bacillus) was studied in monkeys fed 10(10) virulent organisms after parenteral immunization with a formalin-inactivated Shiga toxoid preparation standardized in mice. Two 125-microgram doses of toxoid adsorbed on aluminum hydroxide adjuvant and given 14 days apart provided mice with a 54-fold increase in resistance to parenteral toxin. In rhesus monkeys (Macaca mulatta), the same regimen of toxoid permitted the safe parenteral administration of toxin in incremental doses ranging from 100 to 1,000 mouse 50% lethal doses and resulted in correspondingly high titers of antitoxin in serum. Nevertheless, the immunized monkeys responded to orally administered Shiga bacilli by development of diarrhea and dysentery that was as severe as (or more severe than) the response of unimmunized controls. The failure of extraordinarily high levels of circulating antibody to protect against experimental shigellosis suggests that the intestinal mucosal sites of toxinmediated response are beyond the reach of systemic antitoxin.

Shigella sonnei plasmids: evidence that a large plasmid is necessary for virulence.

Virulent form I Shigella sonnei strains contain a 120-megadalton plasmid that is absent in their form II derivatives, which are always avirulent and devoid of O side chains. In the present study, 165 biochemical and antibiotic traits were assessed, but no experimentally useful phenotype could be associated with this large form I plasmid. Therefore, the form I plasmids of several S. sonnei strains were tagged with the antibiotic resistance transposons Tn3, Tn5, or Tn10. Transposon-tagged form I plasmids were not self-transmissible, but could be mobilized by the plasmid R386. Form II S. sonnei transconjugants for the form I plasmid acquired both virulence and the ability to synthesize form I antigen, establishing that these properties are plasmid mediated. Further studies indicate that this 120-megadalton form I plasmid is physically unstable in any of several host bacteria and suggest that it is a member of the FI incompatibility group. Also, two commonly observed, small plasmids of S. sonnei, of 3.2 and 3.9 megadaltons, were shown to encode either colicin E1 production or resistance to streptomycin and sulfonamide, respectively.

Secretory immunoglobulin A response following peroral priming and challenge with Shigella flexneri lacking the 140-megadalton virulence plasmid.

This study evaluates the ability of noninvasive Shigella spp., lacking the 140-megadalton virulence plasmid, to elicit a mucosal immunoglobulin A immune response in the intestine. For these studies, we used Shigella flexneri M4243A1 (which lacks the plasmid and is Sereny test negative) to prime and challenge three groups of rabbits perorally. Both primary and immunoglobulin A memory responses were detectable in these secretions. These findings indicate that a mucosal memory response can be primed by nonpathogenic strains of Shigella which lack the virulence plasmid.