Prevalence of Virulence Factors Among Hemolytic Escherichia coli

A polymerase chain reaction (PCR) assay was used to characterize 309 hemolytic E. coli isolates. The isolates were obtained from swine specimens presented to the diagnostic laboratory between August of 1996 and August of 1997. About one-half of the isolates contained genes for enterotoxin and/or Shiga toxin. Enterotoxigenic E. coli (ETEC), which cause diarrhea, were much more prevalent than Shigatoxigenic E. coli (STEC), which cause edema disease. K88 was the most common pilus type among ETEC and F18 was the only pilus type identified among STEC. These data are consistent with the notion that E. coli induced diarrheal disease is more prevalent than edema disease. However, they demonstrate that STEC persist in the swine population in spite of the low prevalence of clinical edema disease in recent years. The data suggest that vaccination and vaccine development based on K88 and F18 pilus antigens continue to be relevant for hemolytic E. coli infections. Some of the isolates that did not have genes for either enterotoxin or Shiga toxin, had genes for K88 or F18 pili. Such nontoxigenic isolates (NTEC) are probably not pathogenic and were speculated to act as naturally occurring K88 and F18 vaccines in some herds

Biosynthesis of amino acids by Oxalobacter formigenes: analysis using 13C-NMR

The gram-negative anaerobe Oxalobacter formigenes, grows on oxalate as the principal carbon and energy source, but a small amount of acetate is also required for growth. Experiments were conducted to determine the distribution and the position of label in cellular amino acids from cells grown on [13C]oxalate, [13C]acetate (1-13C, 2-13C, and U-13C), and 13CCO3. The labeling pattern (determined with NMR spectroscopy) of amino acids was consistent with their formation through common biosynthetic pathways. The majority of the carbons in the amino acids that are usually derived from pyruvate, oxaloacetate, α-ketoglutarate, 3-phosphoglycerate, and carbon in the aromatic amino acids were labeled by oxalate. Carbon from 13CO3 was assimilated primarily into amino acids expected to be derived from oxaloacetate and α-ketoglutarate. Approximately 60% of the acetate that was assimilated into amino acids was incorporated as a C2 unit into proline, arginine, glutamate, and leucine. The pattern of labeling from acetate in glutamate, arginine, and proline was consistent with acetate incorporation via citrate (si)-synthase and subsequent formation of α-ketoglutarate via the first third of the tricarboxylic acid pathway. Acetate was also assimilated into amino acids derived from pyruvate and oxaloacetate, but results indicated that this incorporation was as single carbon atoms. Based on these findings, cell-free extracts were assayed for several key biosynthetic enzymes. Enzymatic activities found included glutamate dehydrogenase, phosphoenolpyruvate carboxylase, and pyruvate carboxylase. These findings are consistent with proposed biosynthetic mechanisms

Colonization and Transmission of Escherichia Coli O157:H7 in Swine

Escherichia coli O157:H7 and other serogroups of Shiga toxin-producing E.coli (STEC) have emerged over the last several decades as a significant cause of food-borne illness in the United States. Approximately 5-10% of people clinically infected by these bacteria develop a systemic disease, hemolytic uremic syndrome, which has a fatality rate of approximately 5%. The Centers for Disease Control estimates that STEC cause some 110,000 illnesses and 90 deaths annually in the United States (Mead et al. 1999). In addition, the economic consequences of recalling large lots of food for public health reasons are significant. Cattle are considered to be the primary reservoir for STEC. Depending on the season, the methods used for bacterial culture and the age of the animals, the prevalence of E. coli O157:H7 in U.S. cattle ranges from 2-28% (Hancock et al. 1994; Elder et al. 2000). E.coli O157:H7 has also been recovered from other ruminants such as sheep (Kudva et al. 1996) and deer (Keene et al. 1997; Sargeant et al. 1999)

Biosynthetic pathways in Oxalobacter formigenes

Oxalate is the only substrate that supports the growth of the gram negative anaerobe, Oxalobacter formigenes. Oxalate is decarboxylated to formate plus CO[subscript]2. A small amount of acetate (0.5-1 mM) is required for biosynthetic reactions. Oxalate is reduced and assimilated into cell biomass by aerobic oxalate-degrading bacteria using either the glycerate pathway or the serine pathway. Oxalate is reduced to 3P-glycerate and assimilated as a C[subscript]3 unit. We detected the enzymatic activities of glycerate pathway but not those of the serine pathway in cell-free extracts of O. formigenes;Four potential sources of carbon for cell biomass are available to O. formigenes, oxalate, acetate, formate and CO[subscript]2. We grew the organism in [superscript]14 C labeled carbon sources and determined the contribution of each of these sources to cell carbon. O. formigenes derived at least 54% of its cell carbon from oxalate and at least 7% from acetate. The only other carbon source utilized was CO[subscript]3. Formate was not incorporated to a significant extent. Carbon from [superscript]14 C-oxalate and [superscript]14 CO[subscript]3 was detected in amino acids derived from [alpha]-ketoglutarate, oxaloacetate, pyruvate, 3P-glycerate and in the aromatic amino acids. Amino acids derived from [alpha]-ketoglutarate, oxaloacetate and pyruvate contained carbon derived from [superscript]14 C-acetate;When O. formigenes was grown on [superscript]13 C-labeled oxalate, acetate or CO[subscript]3,the labeling patterns of the amino acids were consistent with their formation through common biosynthetic pathways. [superscript]13 C from oxalate was detected in the majority of the carbons from all of the amino acids. Approximately 60% of the acetate was incorporated as a C[subscript]2 unit into four amino acids (glutamate, proline, arginine and leucine). The other 40% of the acetate was split and was detected in amino acids derived from oxaloacetate and pyruvate;Enzymatic activities detected in cell-free extracts included: glutamate dehydrogenase, phosphoenolpyruvate carboxylase, pyruvate carboxylase, citrate synthase and isocitrate dehydrogenase. These findings support the [superscript]14 C and [superscript]13 C data which indicate that O. formigenes assimilates acetate into protein using the first third of the TCA pathway and that C[subscript]4 units are formed from C[subscript]3 units by carboxylation of pyruvate or PEP

Shiga Toxin-Producing Escherichia coli Infection: Temporal and Quantitative Relationships among Colonization, Toxin Production, and Systemic Disease

Edema disease, a naturally occurring disease of swine caused by Shiga toxin-producing Escherichia coli (STEC), was used as a model for the sequence of events that occur in the pathogenesis of STEC infection. The mean time from production of levels of Shiga toxin 2e (Stx2e) detectable in the feces (day 1) to the onset of clinical disease (neurologic disturbances or death) was 5 days (range, 3–9). Bacterial colonization and titers of Stx2e in the ileum peaked at 4 days after inoculation in pigs without signs of clinical disease and at 6 days after inoculation in clinically affected pigs. Animals with the greatest risk of progressing to clinical disease tended to have the highest fecal toxin titers (⩾1 : 4096). Stx2e was detected in the red cell fraction from blood of some pigs showing clinical signs of edema disease but was not detected in the serum or cerebrospinal fluid

Prevalences of Some Virulence Genes among Escherichia Coli Isolates from Swine Presented to a Diagnostic Laboratory in Iowa

Escherichia coli strains that carry genes encoding for specific virulence attributes cause diarrhea and edema disease in swine. Enterotoxigenic E. coli (ETEC) have genes for enterotoxins that stimulate secretion of electrolytes and water by the small intestine. To colonize the small intestine and cause diarrhea, ETEC must also produce fimbriae (pili). Escherechia coli strains that cause edema disease produce E. coli Shiga toxin (Verotoxin) and are designated as STEC.Shiga toxin is absorbed from the intestine into blood and causes systemic vascular damage resulting in edema disease. STEC must also produce fimbriae to colonize the small intestine and cause disease. Some E. coli strains are designated as attaching/effacing E. coli (AEEC) because of their ability to attach intimately to the surface of intestinal epithelial cells and efface microvilli.10 The attaching/effacing attribute is encoded by a series of chromosomal genes located in a pathogenicity island called the locus of enterocyte effacement. ETEC, STEC, and AEEC are considered to be different pathotypes of E. coli. However, some of the virulence genes that characterize them can be located on mobile genetic elements (plasmids, transposons, bacteriophages), and combinations of pathotypes occur. For example, some AEEC such as the human pathogen E. coli O157:H7 also have genes for Shiga toxin production, and some strains associated with edema disease of swine have genes for both Shiga toxin and enterotoxin production

Therapeutic Use of a Receptor Mimic Probiotic Reduces Intestinal Shiga Toxin Levels in a Piglet Model of Hemolytic Uremic Syndrome

Hemolytic uremic syndrome (HUS) is a systemic and potentially fatal complication of gastroenteritis secondary to Shiga toxin-producing enterohemorrhagic Escherichia coli (EHEC) infection characterized by microangiopathic hemolytic anemia, thrombocytopenia, and acute renal damage. Shiga toxin (Stx), the toxin principle in HUS, is produced locally within the gut following EHEC colonization and is disseminated via the vasculature. Clinical development of HUS currently has no effective treatment and is a leading cause of renal failure in children. Novel post-exposure therapies are currently needed for HUS; therefore, the purpose of this study was to investigate the efficacy of a Stx receptor mimic probiotic in a porcine model of HUS. Edema disease, an infection of swine caused by host adapted Shiga toxin-producing Escherichia coli (STEC) and mediated by Shiga toxin 2e (Stx2e), shares many pathogenic similarities to HUS. In this study, three-week old piglets were inoculated with STEC and 24 hours later treated twice daily with a probiotic expressing an oligosaccharide receptor mimic for Stx2e to determine if the probiotic could reduce intestinal toxin levels

In Vitro Detection of Shiga Toxin Using Porcine Alveolar Macrophages

Porcine alveolar macrophages were found to be highly susceptible to the cytolytic effects of a toxin (Shiga toxin [Stx]) produced by certain strains of Escherichia coli and sometimes associated with clinical disease in pigs and other animals. In comparison with the cells that are most commonly used for Stx detection and titration in vitro (namely, Vero cells), porcine alveolar macrophages appeared to be generally more sensitive and test results could be obtained in less time. Moreover, unlike Vero cells, porcine alveolar macrophages need not be continuously propagated to ensure immediate availability. They can simply be removed from a low-temperature repository, thawed, seeded, and shortly thereafter exposed to the sample in question. These characteristics suggest that porcine alveolar macrophages may be useful in developing a highly sensitive and timely diagnostic test for Stx

Intervention with Shiga Toxin (Stx) Antibody after Infection by Stx-Producing Escherichia coli

Shiga toxins (Stxs) produced by Escherichia coli (STEC) cause systemic vascular damage, manifested as hemolytic uremic syndrome in humans and as edema disease in pigs. Edema disease, a naturally occurring disease of pigs, was used to determine whether Stx antibodies, administered after infection and after the onset of Stx production, could prevent the systemic vascular damage and clinical disease caused by Stxs. A total of 119 STEC-infected pigs were treated with low, medium, or high doses of Stx antibody or with placebo. After inoculation with STEC, antibodies or placebo was injected intraperitoneally at 2 days postinoculation (DPI; low dose) or 4 DPI (medium and high doses). Edema disease was prevented with the low- and high-dose Stx antibody treatments administered at 2 and 4 DPI, respectively. Highdose antibody treatment also reduced the incidence and extent of vascular lesions. The degree of protection depended on the dose of antibody and the time of administration

Lachnospira pectinoschiza sp. nov., an Anaerobic Pectinophile from the Pig Intestine

Pectinophiles are bacteria that utilize pectin and only a few related compounds as substrates. Obligately anaerobic pectinophiles have been isolated from the intestinal tracts and gingivae of humans and from the rumina of cattle. We isolated three strains of pectinophilic bacteria from colonic contents of pigs but were unable to isolate pectinophiles from the rumen contents of four sheep, even when the animals were fed a high-pectin diet. The pectinophiles isolated from pigs were strictly anaerobic, motile, gram-positive rods (0.36 to 0.56 by 2.4 to 3.1 μm). Pectin, polygalacturonic acid, and gluconate were the only substrates that supported rapid growth. All three strains grew slowly on either lactose or cellobiose and fermented fructose after a lag of several days. Pectin was degraded by means of an extracellular pectin methylesterase and a Ca2+-dependent exopectate lyase. A comparison of the 16S rRNA sequences of these isolates with the 16S rRNA sequences of other gram-positive bacteria revealed a specific relationship with Lachnospira multipara (level of similarity, 94%). The Gram reaction, formation of spore-like structures, and the utilization of lactose and cellobiose differentiated the pig isolates from previously described pectinophiles. The pig isolates represent a previously undescribed species of the genus Lachnospira, for which we propose the name Lachnospira pectinoschiza