Mohamed A. Karmali

The Association Between Idiopathic Hemolytic Uremic Syndrome and Infection by Verotoxin-Producing Escherichia coli

Forty pediatric patients with idiopathic hemolytic uremic syndrome (HUS) were investigated for evidence of infection by Verotoxin-producing Escherichia coli (VTEC). Fecal VTEC (belonging to at least six different O serogroups including O26, O111, O113, O121, O145, and O157) or specifically neutralizable free-fecal Verotoxin (VT) or both were detected in 24 (60%) patients but were not detected in 40 matched controls. Ten of 15 of the former developed fourfold or greater rises in VT-neutralizing antibody titers, as did six other patients who were negative for both fecal VTEC and VT. A total of 30 (75%) patients had evidence of VTEC infection by one or more criteria. We concluded that a significant association exists between idiopathic HUS and infection by VTEC. The detection of free-fecal VT was the most important procedure for the early diagnosis of this infection because, in our study, VTEC were never isolated in the absence of fecal VT, whereas fecal VT was often present even when VTEC were undetectable.

A Severe Outbreak of Escherichia coli O157:H7–Associated Hemorrhagic Colitis in a Nursing Home

In September 1985, an outbreak of Escherichia coli O157:H7 enteritis affected 55 of 169 residents and 18 of 137 staff members at a nursing home. The outbreak was characterized by two phases: a primary wave whose source was probably a contaminated sandwich meal and a secondary wave compatible with person-to-person transmission of infection. Among the elderly residents, the incubation period was 4 to 9 days (mean, 5.7 +/- 1.2). Older age and previous gastrectomy increased the risk of acquiring the infection (P = 0.01 and 0.03, respectively). Antibiotic therapy during exposure was associated with acquiring a secondary infection (P = 0.001). Hemolytic uremic syndrome developed in 12 affected residents (22 percent), 11 of whom died. Overall, 19 (35 percent) of the affected residents died, 17 (31 percent) from causes attributable to their infection. Antibiotic therapy after the onset of symptoms was associated with a higher case fatality rate in the more severe cases, possibly because patients with more severe disease tended to be treated with antibiotics. There were no complications or deaths among the affected members of the staff. Evidence of infection by verotoxin-producing E. coli O157:H7 was detected in 30 of 70 cases on the basis of isolation of this organism or demonstration of free verotoxin in stools. All isolates belonged to the same phage type. The high morbidity and mortality associated with this condition emphasize the need for proper food hygiene, rapid identification of outbreaks of disease, and prompt institution of infection-control techniques among the institutionalized elderly.

Association of Genomic O Island 122 of Escherichia coli EDL 933 with Verocytotoxin-Producing Escherichia coli Seropathotypes That Are Linked to Epidemic and/or Serious Disease

ABSTRACT The distribution of EDL 933 O island 122 (OI-122) was investigated in 70 strains of Verocytotoxin-producing Escherichia coli (VTEC) of multiple serotypes that were classified into five “seropathotypes” (A through E) based on the reported occurrence of serotypes in human disease, in outbreaks, and/or in the hemolytic-uremic syndrome (HUS). Seropathotype A comprised 10 serotype O157:H7 and 3 serotype O157:NM strains. Seropathotype B (associated with outbreaks and HUS but less commonly than serotype O157:H7) comprised three strains each of serotypes O26:H11, O103:H2, O111:NM, O121:H19, and O145:NM. Seropathotype C comprised four strains each of serotypes O91:H21 and O113:H21 and eight strains of other serotypes that have been associated with sporadic HUS but not typically with outbreaks. Seropathotype D comprised 14 strains of serotypes that have been associated with diarrhea but not with outbreaks or HUS, and seropathotype E comprised animal VTEC strains of serotypes not implicated in human disease. All strains were tested for four EDL 933 OI-122 virulence genes (Z4321, Z4326, Z4332, and Z4333) by PCR. Negative PCRs were confirmed by Southern hybridization. Overall, 28 (40%) strains contained OI-122 (positive for all four virulence genes), 27 (38.6%) contained an “incomplete” OI-122 (positive for one to three genes), and 15 (21.4%) strains did not contain OI-122. The seropathotype distribution of complete OI-122 was as follows: 100% for seropathotype A, 60% for B, 36% for C, 15% for D, and 0% for E. The differences in the frequency of OI-122 between seropathotypes A, B, and C (associated with HUS) and seropathotypes D and E (not associated with HUS) and between seropathotypes A and B (associated with epidemic disease) and seropathotypes C, D, and E (not associated with epidemic disease) were highly significant (P < 0.0001).

Association of Campylobacter pylori on the gastric mucosa with antral gastritis in children

We investigated the presence of Campylobacter pylori colonization of the gastric mucosa and of histologic evidence of gastritis in a prospective study of 71 consecutive children undergoing upper gastrointestinal tract endoscopy and gastric biopsies because of gastrointestinal symptoms. Two tissue samples from the gastric antrum were obtained from 67 of the 71 children (mean age [+/- SD], 11.4 +/- 3.8 years). One sample was evaluated for evidence of gastritis and stained with silver to detect organisms morphologically resembling campylobacter. The second sample was cultured for C. pylori, and a portion was used to perform a urease-screening test for the presence of C. pylori. Antral gastritis was diagnosed histologically in 18 of 67 patients. C. pylori was identified by both culture and silver staining on the antral mucosa in 7 of 10 patients with unexplained gastritis (primary gastritis) but in none of 8 patients with gastritis associated with an identifiable underlying cause (secondary gastritis). C. pylori was not identified in any of the 49 cases with normal histologic features. The urease-screening test was positive in only three of six patients with a positive culture for C. pylori. Duodenal ulcers were diagnosed by endoscopy in five patients. Each of the five had C. pylori on the antral mucosa, but organisms were not identified on the duodenal mucosa. We conclude that the presence of C. pylori on the antral mucosa is specifically associated with primary antral gastritis and may also be associated with primary duodenal ulceration.

Growing concerns and recent outbreaks involving non-O157:H7 serotypes of verotoxigenic Escherichia coli

Verocytotoxin-producing E. coli (VTEC) of serotype O157:H7 have been shown to be important agents of foodborne disease in humans worldwide. While the majority of research effort has been targeted on this serotype it is becoming more evident that other serotypes of VTEC can also be associated with human disease. An increasing number of these non-O157:H7 VTEC have been isolated from humans suffering from HUS and diarrhea. Recently a number of foodborne outbreaks in the USA, Australia, and other countries have been attributed to non-O157:H7 VTEC serotypes. Surveys of animal populations in a variety of countries have shown that the cattle reservoir contains more than 100 serotypes of VTEC, many of which are similar to those isolated from humans. The diversity and complexity of the VTEC family requires that laboratories and public health surveillance systems have the ability to detect and monitor all serotypes of VTEC.

Infection by Shiga toxin-producing Escherichia coli: an overview.

Shiga toxin-producing Escherichia coli (STEC), especially of serotype O157:H7, cause a zoonotic food or waterborne enteric illness that is often associated with large epidemic outbreaks as well as the hemolytic uremic syndrome (HUS), the leading cause of acute renal failure in children. After ingestion, STEC colonize enterocytes of the large bowel with a characteristic attaching and effacing pathology, which is mediated by components of a type III secretion apparatus encoded by the LEE pathogenicity island. Shiga toxins are translocated from the bowel to the circularoty system and transported by leukocytes to capillary endothelial cells in renal glomeruli and other organs. After binding to the receptor globotriaosylceramide on target cells, the toxin is internalized by receptor-mediated endocytosis and interacts with the subcellular machinery to inhibit protein synthesis. This leads to pathophysiological changes that result in HUS. Specific therapeutic or preventive strategies are presently not available. The recent sequencing of genomes of two epidemic E. coli O157 strains has revealed novel pathogenicity islands which will likely provide new insights into the virulence of these bacteria.

Molecular Analysis as an Aid To Assess the Public Health Risk of Non-O157 Shiga Toxin-Producing Escherichia coli Strains

ABSTRACT Shiga toxin-producing Escherichia coli (STEC) strains are commensal bacteria in cattle with high potential for environmental and zoonotic transmission to humans. Although O157:H7 is the most common STEC serotype, there is growing concern over the emergence of more than 200 highly virulent non-O157 STEC serotypes that are globally distributed, several of which are associated with outbreaks and/or severe human illness such as hemolytic-uremic syndrome (HUS) and hemorrhagic colitis. At present, the underlying genetic basis of virulence potential in non-O157 STEC is unknown, although horizontal gene transfer and the acquisition of new pathogenicity islands are an expected origin. We used seropathotype classification as a framework to identify genetic elements that distinguish non-O157 STEC strains posing a serious risk to humans from STEC strains that are not associated with severe and epidemic disease. We report the identification of three genomic islands encoding non-LEE effector (nle) genes and 14 individual nle genes in non-O157 STEC strains that correlate independently with outbreak and HUS potential in humans. The implications for transmissible zoonotic spread and public health are discussed. These results and methods offer a molecular risk assessment strategy to rapidly recognize and respond to non-O157 STEC strains from environmental and animal sources that might pose serious public health risks to humans.

Type 2 diabetes mellitus and inflammation: Prospects for biomarkers of risk and nutritional intervention

Obesity is a major risk factor for type 2 diabetes mellitus (T2DM), which is a significant health problem worldwide. Active disease is associated with low-grade chronic inflammation resulting in part from the activation of the innate immune system. In obesity, this activation leads to the release of pro-inflammatory cytokines such as tumor necrosis factor-α, interleukin-1β and interleukin-6 that block major anabolic cascades downstream of insulin signaling and thus disrupt insulin homeostasis and action. Cytokines also trigger the production of acute-phase reactants such as C-reactive protein, plasminogen activator inhibitor-1, serum amyloid-A, and haptoglobin. The elevated synthesis of pro-inflammatory cytokines and acute-phase proteins (inflammatory network) characterizes the early (or pre-clinical) stages of T2DM and exhibits a graded increase with the disease progression. Current evidence suggests that understanding inflammatory networks can point to new biomarkers that may permit capturing the interaction between genetic and environmental risk factors in the pathogenesis of T2DM. Such biomarkers have a significant public health potential in the prediction of disease occurrence beyond risk factors presently monitored, such as family history, lifestyle assessment and standard clinical chemistry profiles. Furthermore, inflammatory markers may assist in the evaluation of novel strategies for prevention, particularly in relation to micronutrients. This review discusses the current knowledge linking T2DM risk to inflammatory signaling pathways interacting with the innate immunity system and the prospect of inflammatory markers serving as molecular targets for prevention and/or biomarkers for early risk prediction of T2DM. The potential of micronutrients replenishment to improve insulin action by attenuating inflammation is also evaluated in the context of the public health relevance of this approach.

Genome sequence of adherent-invasive Escherichia coli and comparative genomic analysis with other E. coli pathotypes

BackgroundAdherent and invasive Escherichia coli (AIEC) are commonly found in ileal lesions of Crohns Disease (CD) patients, where they adhere to intestinal epithelial cells and invade into and survive in epithelial cells and macrophages, thereby gaining access to a typically restricted host niche. Colonization leads to strong inflammatory responses in the gut suggesting that AIEC could play a role in CD immunopathology. Despite extensive investigation, the genetic determinants accounting for the AIEC phenotype remain poorly defined. To address this, we present the complete genome sequence of an AIEC, revealing the genetic blueprint for this disease-associated E. coli pathotype.ResultsWe sequenced the complete genome of E. coli NRG857c (O83:H1), a clinical isolate of AIEC from the ileum of a Crohns Disease patient. Our sequence data confirmed a phylogenetic linkage between AIEC and extraintestinal pathogenic E. coli causing urinary tract infections and neonatal meningitis. The comparison of the NRG857c AIEC genome with other pathogenic and commensal E. coli allowed for the identification of unique genetic features of the AIEC pathotype, including 41 genomic islands, and unique genes that are found only in strains exhibiting the adherent and invasive phenotype.ConclusionsUp to now, the virulence-like features associated with AIEC are detectable only phenotypically. AIEC genome sequence data will facilitate the identification of genetic determinants implicated in invasion and intracellular growth, as well as enable functional genomic studies of AIEC gene expression during health and disease.

Escherichia coli shiga-like toxins induce apoptosis and cleavage of poly(ADP-ribose) polymerase via in vitro activation of caspases

ABSTRACT Shiga-like toxin-producing Escherichia coli causes hemorrhagic colitis and hemolytic-uremic syndrome in association with the production of Shiga-like toxins, which induce cell death via either necrosis or apoptosis. However, the abilities of different Shiga-like toxins to trigger apoptosis and the sequence of intracellular signaling events mediating the death of epithelial cells have not been completely defined. Fluorescent dye staining with acridine orange and ethidium bromide showed that Shiga-like toxin 1 (Stx1) induced apoptosis of HEp-2 cells in a dose- and time-dependent manner. Stx2 also induced apoptosis in a dose-dependent manner. Apoptosis induced by Stx1 (200 ng/ml) and apoptosis induced by Stx2 (200 ng/ml) were maximal following incubation with cells for 24 h (94.3% ± 1.8% and 81.7% ± 5.2% of the cells, respectively). Toxin-treated cells showed characteristic features of apoptosis, including membrane blebbing, DNA fragmentation, chromatin condensation, cell shrinkage, and the formation of apoptotic bodies, as assessed by transmission electron microscopy. Stx2c induced apoptosis weakly even at a high dose (1,000 ng/ml for 24 h; 26.7% ± 1.3% of the cells), whereas Stx2e did not induce apoptosis of HEp-2 cells. Thin-layer chromatography confirmed that HEp-2 cells express the Stx1-Stx2-Stx2c receptor, globotriaosylceramide (Gb3), but not the Stx2e receptor, globotetraosylceramide (Gb4). Western blot analysis of poly(ADP-ribose) polymerase (PARP), a DNA repair enzyme, demonstrated that incubation with Stx1 and Stx2 induced cleavage, whereas incubation with Stx2e did not result in cleavage of PARP. A pan-caspase inhibitor (Z-VAD-FMK) and a caspase-8-specific inhibitor (Z-IETD-FMK) eliminated, in a dose-dependent fashion, the cleavage of PARP induced by Shiga-like toxins. Caspase-8 activation was confirmed by detection of cleavage of this enzyme by immunoblotting. Cleavage of caspase-9 and the proapoptotic member of the Bcl-2 family BID was also induced by Stx1, as determined by immunoblot analyses. We conclude that different Shiga-like toxins induce different degrees of apoptosis that correlates with toxin binding to the glycolipid receptor Gb3 and that caspases play an integral role in the signal transduction cascade leading to toxin-mediated programmed cell death.