Protective efficacy and pharmacokinetics of human/mouse chimeric anti-Stx1 and anti-Stx2 antibodies in mice.

In the United States, Shiga toxin (Stx)-producing Escherichia coli (STEC) is the most frequent infectious cause of hemorrhagic colitis. Hemolytic uremic syndrome (HUS) is a serious sequela that may develop after STEC infection that can lead to renal failure and death in up to 10% of cases. STEC can produce one or more types of Stx, Stx1 and/or Stx2, and Stx1 and Stx2 are responsible for HUS-mediated kidney damage. We previously generated two monoclonal antibodies (MAbs) that neutralize the toxicity of Stx1 or Stx2. In this study, we evaluated the protective efficacy of human/mouse chimeric versions of those monoclonal antibodies, named cαStx1 and cαStx2. Mice given an otherwise lethal dose of Stx1 were protected from death when injected with cαStx1 either 1 h before or 1 h after toxin injection. Additionally, streptomycin-treated mice fed the mouse-lethal STEC strain B2F1 that produces the Stx2 variant Stx2d were protected when given a dose of 0.1 mg of cαStx2/kg of body weight administered up to 72 h post-oral bacterial challenge. Since many STEC strains produce both Stx1 and Stx2 and since either toxin may lead to the HUS, we also assessed the protective efficacy of the combined MAbs. We found that both antibodies were required to protect mice from the presence of both Stx1 and Stx2. Pharmacokinetic studies indicated that cαStx1 and cαStx2 had serum half-lives (t1/2) of about 50 and 145 h, respectively. We propose that cαStx1 and cαStx2, both of which have been tested for safety in humans, could be used therapeutically for prevention or treatment early in the development of HUS.

Enhanced virulence of the Escherichia coli O157:H7 spinach-associated outbreak strain in two animal models is associated with higher levels of Stx2 production after induction with ciprofloxacin.

Shiga toxin (Stx)-producing Escherichia coli (STEC) causes hemorrhagic colitis and the hemolytic-uremic syndrome (HUS). STEC strains may produce Stx1a and/or Stx2a or variants of either toxin. A 2006 spinach-associated outbreak of STEC O157:H7 resulted in higher hospitalization and HUS rates than previous STEC outbreaks. The spinach isolate, strain K3995, contains both stx2a and stx2c. We hypothesized that the enhanced virulence of K3995 reflects the combination of stx2 alleles (carried on lysogenic phages) and/or the amount of Stx2 made by that strain. We compared the virulence of K3995 to those of other O157:H7 isolates and an isogenic Stx2 mutant in rabbits and mice. We also measured the relative levels of Stx2 produced from those strains with or without induction of the stx-carrying phage. Some rabbits infected with K3995 exhibited intestinal pathology and succumbed to infection, while none of those infected with O157:H7 strain 2812 (Stx1a(+) Stx2a(+)) died or showed pathological signs. Rabbits infected with the isogenic Stx2a mutant K3995 stx2a::cat were not colonized as well as those infected with K3995 and exhibited no signs of disease. In the streptomycin-treated mouse model, more animals infected with K3995 died than did those infected with O157:H7 strain 86-24 (Stx2a(+)). Additionally, K3995 produced higher levels of total Stx2 and toxin phage DNA in cultures after phage induction than did 86-24. Our results demonstrate the greater virulence of K3995 compared to other O157:H7 strains in rabbits and mice. We conclude that this enhanced virulence is linked to higher levels of Stx2 expression as a consequence of increased phage induction.

Oral intoxication of mice with Shiga toxin type 2a (Stx2a) and protection by anti-Stx2a monoclonal antibody 11E10.

Shiga toxin (Stx)-producing Escherichia coli (STEC) strains cause food-borne outbreaks of hemorrhagic colitis and, less commonly, a serious kidney-damaging sequela called the hemolytic uremic syndrome (HUS). Stx, the primary virulence factor expressed by STEC, is an AB5 toxin with two antigenically distinct forms, Stx1a and Stx2a. Although both toxins have similar biological activities, Stx2a is more frequently produced by STEC strains that cause HUS than is Stx1a. Here we asked whether Stx1a and Stx2a act differently when delivered orally by gavage. We found that Stx2a had a 50% lethal dose (LD50) of 2.9 µg, but no morbidity occurred after oral intoxication with up to 157 µg of Stx1a. We also compared several biochemical and histological parameters in mice intoxicated orally versus intraperitoneally with Stx2a. We discovered that both intoxication routes caused similar increases in serum creatinine and blood urea nitrogen, indicative of kidney damage, as well as electrolyte imbalances and weight loss in the animals. Furthermore, kidney sections from Stx2a-intoxicated mice revealed multifocal, acute tubular necrosis (ATN). Of particular note, we detected Stx2a in kidney sections from orally intoxicated mice in the same region as the epithelial cell type in which ATN was detected. Lastly, we showed reduced renal damage, as determined by renal biomarkers and histopathology, and full protection of orally intoxicated mice with monoclonal antibody (MAb) 11E10 directed against the toxin A subunit; conversely, an irrelevant MAb had no therapeutic effect. Orally intoxicated mice could be rescued by MAb 11E10 6 h but not 24 h after Stx2a delivery.

Helmet-Cam: tool for assessing miners' respirable dust exposure.

Video technology coupled with datalogging exposure monitors have been used to evaluate worker exposure to different types of contaminants. However, previous application of this technology used a stationary video camera to record the worker's activity while the worker wore some type of contaminant monitor. These techniques are not applicable to mobile workers in the mining industry because of their need to move around the operation while performing their duties. The Helmet-Cam is a recently developed exposure assessment tool that integrates a person-wearable video recorder with a datalogging dust monitor. These are worn by the miner in a backpack, safety belt or safety vest to identify areas or job tasks of elevated exposure. After a miner performs his or her job while wearing the unit, the video and dust exposure data files are downloaded to a computer and then merged together through a NIOSH-developed computer software program called Enhanced Video Analysis of Dust Exposure (EVADE). By providing synchronized playback of the merged video footage and dust exposure data, the EVADE software allows for the assessment and identification of key work areas and processes, as well as work tasks that significantly impact a worker's personal respirable dust exposure. The Helmet-Cam technology has been tested at a number of metal/nonmetal mining operations and has proven to be a valuable assessment tool. Mining companies wishing to use this technique can purchase a commercially available video camera and an instantaneous dust monitor to obtain the necessary data, and the NIOSH-developed EVADE software will be available for download at no cost on the NIOSH website.

Characterization of a multi-component anthrax vaccine designed to target the initial stages of infection as well as toxaemia.

Current vaccine approaches to combat anthrax are effective; however, they target only a single protein [the protective antigen (PA) toxin component] that is produced after spore germination. PA production is subsequently increased during later vegetative cell proliferation. Accordingly, several aspects of the vaccine strategy could be improved. The inclusion of spore-specific antigens with PA could potentially induce protection to initial stages of the disease. Moreover, adding other epitopes to the current vaccine strategy will decrease the likelihood of encountering a strain of Bacillus anthracis (emerging or engineered) that is refractory to the vaccine. Adding recombinant spore-surface antigens (e.g. BclA, ExsFA/BxpB and p5303) to PA has been shown to augment protection afforded by the latter using a challenge model employing immunosuppressed mice challenged with spores derived from the attenuated Sterne strain of B. anthracis. This report demonstrated similar augmentation utilizing guinea pigs or mice challenged with spores of the fully virulent Ames strain or a non-toxigenic but encapsulated ΔAmes strain of B. anthracis, respectively. Additionally, it was shown that immune interference did not occur if optimal amounts of antigen were administered. By administering the toxin and spore-based immunogens simultaneously, a significant adjuvant effect was also observed in some cases. Thus, these data further support the inclusion of recombinant spore antigens in next-generation anthrax vaccine strategies.

Shiga Toxins: Potent Poisons, Pathogenicity Determinants, and Pharmacological Agents.

The Shiga toxins (Stxs), also known as Vero toxins and previously called Shiga-like toxins, are a family of potent protein synthesis inhibitors made by Shigella dysenteriae type 1 and some serogroups of Escherichia coli that cause bloody diarrhea in humans. Stxs act as virulence factors for both S. dysenteriae and E. coli and contribute to the disease process initiated by those organisms both directly and indirectly. A handful of methods exist for toxin purification, and the toxins can now even be purchased commercially. However, the Stxs are now classified as select agents, and specific rules govern the distribution of both the toxin and clones of the toxin. Toxin delivery into the host in S. dysenteriae type 1 is most likely aided by the invasiveness of that organism. Although the Stxs are made and produced by bacteria, they do not appear to act against either their host organism or other bacteria under normal circumstances, most likely because the A subunit is secreted from the cytoplasm as soon as it is synthesized and because the holotoxin cannot enter intact bacterial cells. The effectiveness of antibiotic therapy in patients infected with Stx-producing E. coli (STEC) such as O157:H7 as well as the potential risks of such treatment are areas of controversy. Several studies indicate that the course of the diarrhea stage of the disease is unaltered by antibiotic treatment. Several groups anticipate that a therapy that targets the Stxs is an important component of trying to alleviate disease caused by Stx-producing bacteria.

Cytotoxic necrotizing factor type 1-positive Escherichia coli causes increased inflammation and tissue damage to the prostate in a rat prostatitis model.

Infection of rat prostates with cytotoxic necrotizing factor type 1 (CNF1)-positive uropathogenic Escherichia coli caused more inflammation-mediated morphological and histological tissue damage than did infection with isogenic CNF1-negative mutants. These striking differences occurred despite the finding that bacterial counts for the strain pairs were indistinguishable. We conclude that CNF1 contributes to E. coli virulence in a model of acute prostatitis. To our knowledge, the results of this study provide the first demonstration of a role for any uropathogenic E. coli virulence factor in acute prostatitis.

Absence of all components of the flagellar export and synthesis machinery differentially alters virulence of Salmonella enterica serovar Typhimurium in models of typhoid fever, survival in macrophages, tissue culture invasiveness, and calf enterocolitis.

In this study, we constructed an flhD (the master flagellar regulator gene) mutant of Salmonella enterica serovar Typhimurium and compared the virulence of the strain to that of the wild-type strain in a series of assays that included the mouse model of typhoid fever, the mouse macrophage survival assay, an intestinal epithelial cell adherence and invasion assay, and the calf model of enterocolitis. We found that the flhD mutant was more virulent than its parent in the mouse and displayed slightly faster net growth between 4 and 24 h of infection in mouse macrophages. Conversely, the flhD mutant exhibited diminished invasiveness for human and mouse intestinal epithelial cells, as well as a reduced capacity to induce fluid secretion and evoke a polymorphonuclear leukocyte response in the calf ligated-loop assay. These findings, taken with the results from virulence assessment assays done on an fljB fliC mutant of serovar Typhimurium that does not produce flagellin but does synthesize the flagellar secretory apparatus, indicate that neither the presence of flagella (as previously reported) nor the synthesis of the flagellar export machinery are necessary for pathogenicity of the organism in the mouse. Conversely, the presence of flagella is required for the full invasive potential of the bacterium in tissue culture and for the influx of polymorphonuclear leukocytes in the calf intestine, while the flagellar secretory components are also necessary for the induction of maximum fluid secretion in that enterocolitis model. A corollary to this conclusion is that, as has previously been surmised but not demonstrated in a comparative investigation of the same mutant strains, the mouse systemic infection and macrophage assays measure aspects of virulence different from those of the tissue culture invasion assay, and the latter is more predictive of findings in the calf enterocolitis model.

Mutation of the gene encoding cytotoxic necrotizing factor type 1 (cnf(1)) attenuates the virulence of uropathogenic Escherichia coli.

Cytotoxic necrotizing factor type 1 (CNF1) is a 115-kDa toxin that activates Rho GTPases and is produced by uropathogenic Escherichia coli (UPEC). While both epidemiological studies that link CNF1 production by E. coli with urinary tract disease and the cytopathic effects of CNF1 on cultured urinary tract cells are suggestive of a role for the toxin as a UPEC virulence factor, few in vivo studies to test this possibility have been reported. Therefore, in this investigation, we evaluated the importance of CNF1 in a murine model of urinary tract infection (UTI) by comparing the degree of colonization and damage induced by three different CNF1-producing E. coli strains with isogenic CNF1-deficient derivatives. The data from single-strain challenge experiments with C3H/HeOuJ mice indicated a trend toward higher counts of the wild-type strains in the urine and bladders of these animals up to 3 days after challenge in two of three strain pairs. Furthermore, this difference was statistically significant at day 2 of infection with one strain pair, C189 and C189cnf(1). To control for the animal-to-animal variability inherent in this model, we infected C3H/HeOuJ mice with a mixture of CNF1-positive and -negative isogenic derivatives of CP9. The CNF1-positive strain was recovered in higher numbers than the CNF1-negative strain in the urine, bladders, and kidneys of the mice up to 9 days postinfection. These striking coinfection findings, taken with the trends observed in single-strain infections, led us to conclude that CNF1-negative strains were generally attenuated compared to the wild type in the C3H/HeOuJ mouse model of UTI. Furthermore, histopathological examination of bladder specimens from mice infected with CNF1-positive strains consistently showed deeper, more extensive inflammation than in those infected with the isogenic mutants. Lastly, we found that CNF1-positive strain CP9 was better able to resist killing by fresh human neutrophils than were CP9cnf(1) bacteria. From these data in aggregate, we propose that CNF1 production increases the capacity of UPEC strains to resist killing by neutrophils, which in turn permits these bacteria to gain access to deeper tissue and persist better in the lower urinary tract.

Flagellar phase variation of Salmonella enterica serovar Typhimurium contributes to virulence in the murine typhoid infection model but does not influence Salmonella-induced enteropathogenesis.

Although Salmonella enterica serovar Typhimurium can undergo phase variation to alternately express two different types of flagellin subunit proteins, FljB or FliC, no biological function for this phenomenon has been described. In this investigation, we constructed phase-locked derivatives of S. enterica serovar Typhimurium that expressed only FljB (termed locked-ON) or FliC (termed locked-OFF). The role of phase variation in models of enteric and systemic pathogenesis was then evaluated. There were no differences between the wild-type parent strain and the two phase-locked derivatives in adherence and invasion of mouse epithelial cells in vitro, survival in mouse peritoneal macrophages, or in a bovine model of gastroenteritis. By contrast, the locked-OFF mutant was virulent in mice following oral or intravenous (i.v.) inoculation but the locked-ON mutant was attenuated. When these phase-locked mutants were compared in studies of i.v. kinetics in mice, similar numbers of the two strains were isolated from the blood and spleens of infected animals at 6 and 24 h. However, the locked-OFF mutant was recovered from the blood and spleens in significantly greater numbers than the locked-ON strain by day 2 of infection. By 5 days postinfection, a majority of the mice infected with the locked-OFF mutant had died compared with none of the mice infected with the locked-ON mutant. These results suggest that phase variation is not involved in the intestinal stage of infection but that once S. enterica serovar Typhimurium reaches the spleens of susceptible mice those organisms in the FliC phase can grow and/or survive better than those in the FljB phase. Additional experiments with wild-type S. enterica serovar Typhimurium, fully capable of switching flagellin type, supported this hypothesis. We conclude that organisms that have switched to the FliC(+) phase have a selective advantage in the mouse model of typhoid fever but have no such advantage in invasion of epithelial cells or the induction of enteropathogenesis.