Combinations of intervention treatments resulting in 5-log10-unit reductions in numbers of Escherichia coli O157:H7 and Salmonella typhimurium DT104 organisms in apple cider. (25/5328)

The U.S. Food and Drug Administration (FDA) recently mandated a warning statement on packaged fruit juices not treated to reduce target pathogen populations by 5 log10 units. This study describes combinations of intervention treatments that reduced concentrations of mixtures of Escherichia coli O157:H7 (strains ATCC 43895, C7927, and USDA-FSIS-380-94) or Salmonella typhimurium DT104 (DT104b, U302, and DT104) by 5 log10 units in apple cider with a pH of 3.3, 3.7, and 4.1. Treatments used were short-term storage at 4, 25, or 35 degrees C and/or freeze-thawing (48 h at -20 degrees C; 4 h at 4 degrees C) of cider with or without added organic acids (0.1% lactic acid, sorbic acid [SA], or propionic acid). Treatments more severe than those for S. typhimurium DT104 were always required to destroy E. coli O157:H7. In pH 3.3 apple cider, a 5-log10-unit reduction in E. coli O157:H7 cell numbers was achieved by freeze-thawing or 6-h 35 degrees C treatments. In pH 3.7 cider the 5-log10-unit reduction followed freeze-thawing combined with either 6 h at 4 degrees C, 2 h at 25 degrees C, or 1 h at 35 degrees C or 6 h at 35 degrees C alone. A 5-log10-unit reduction occurred in pH 4.1 cider after the following treatments: 6 h at 35 degrees C plus freeze-thawing, SA plus 12 h at 25 degrees C plus freeze-thawing, SA plus 6 h at 35 degrees C, and SA plus 4 h at 35 degrees C plus freeze-thawing. Yeast and mold counts did not increase significantly (P < 0.05) during the 6-h storage at 35 degrees C. Cider with no added organic acids treated with either 6 h at 35 degrees C, freeze-thawing or their combination was always preferred by consumers over pasteurized cider (P < 0.05). The simple, inexpensive intervention treatments described in the present work could produce safe apple cider without pasteurization and would not require the FDA-mandated warning statement.  (+info)

A trial comparing low-dose, short-course ciprofloxacin and standard 7 day therapy with co-trimoxazole or nitrofurantoin in the treatment of uncomplicated urinary tract infection. (26/5328)

The study was undertaken to compare the safety and efficacy of twice-daily ciprofloxacin for 3 days with standard 7 day therapy with either co-trimoxazole or nitrofurantoin in the treatment of women with acute, uncomplicated urinary tract infections (UTI). This multicentre, prospective, randomized, double-blind trial compared oral ciprofloxacin (100 mg bd) for 3 days with co-trimoxazole (160/800 mg bd) or nitrofurantoin (100 mg bd) for 7 days. Bacteriological and clinical evaluations were performed at study entry, during therapy and 4-10 days and 4-6 weeks after the completion of therapy. The primary efficacy parameter was eradication of the causative organism 4-10 days following treatment. Of 713 women enrolled and evaluable for safety, 521 were evaluable for efficacy (168 ciprofloxacin, 174 co-trimoxazole, 179 nitrofurantoin). Escherichia coli (83%) was the most frequently isolated pathogen in all treatment groups. Bacteriological eradication was reported in 88% of ciprofloxacin patients, 93% of co-trimoxazole patients and 86% of nitrofurantoin patients. At the 4-6 week follow-up, ciprofloxacin had statistically significantly higher eradication rates (91%) than co-trimoxazole (79%; 95% confidence limit (CL) = -20.6%, -3.9%) and nitrofurantoin (82%; 95% CL = -17.1%, -0.9%). Clinical resolution 4-10 days after therapy and at the 4-6 week follow-up was similar among the three treatment groups. The overall incidence of treatment-emergent adverse events was not significantly different (P = 0.093) among the three drug regimens, although co-trimoxazole was associated with a greater number of adverse events than ciprofloxacin (P < or = 0.05). Ciprofloxacin also caused fewer episodes of nausea than either of the other agents (P < or = 0.01).  (+info)

Identification and characterization of an Escherichia coli invasion gene locus, ibeB, required for penetration of brain microvascular endothelial cells. (27/5328)

Escherichia coli K1 is the most common gram-negative organism causing neonatal meningitis, but the mechanism by which E. coli K1 crosses the blood-brain barrier is incompletely understood. We have previously described the cloning and molecular characterization of a determinant, ibeA (also called ibe10), from the chromosome of an invasive cerebrospinal fluid isolate of E. coli K1 strain RS218 (O18:K1:H7). Here we report the identification of another chromosomal locus, ibeB, which allows RS218 to invade brain microvascular endothelial cells (BMEC). The noninvasive TnphoA mutant 7A-33 exhibited <1% the invasive ability of the parent strain in vitro in BMEC and was significantly less invasive in the central nervous system in the newborn rat model of hematogenous E. coli meningitis than the parent strain. The TnphoA insert with flanking sequences was cloned and sequenced. A 1,383-nucleotide open reading frame (ORF) encoding a 50-kDa protein was identified and termed ibeB. This ORF was found to be 97% identical to a gene encoding a 50-kDa hypothetical protein (p77211) and located in the 13-min region of the E. coli K-12 genome. However, no homology was observed between ibeB and other known invasion genes when DNA and protein databases in GenBank were searched. Like the TnphoA insertion mutant 7A-33, an isogenic ibeB deletion mutant (IB7D5) was unable to invade BMEC. A 7. 0-kb locus containing ibeB was isolated from a LambdaGEM-12 genomic library of E. coli RS218 and subcloned into a pBluescript KS vector (pKS7-7B). pKS7-7B was capable of completely restoring the BMEC invasion of the noninvasive TnphoA mutant 7A-33 and the ibeB deletion mutant IB7D5 to the level of the parent strain. More importantly, the ibeB deletion mutant IB7D5 was fully complemented by pFN476 carrying the ibeB ORF (pFN7C), indicating that ibeB is required for E. coli K1 invasion of BMEC. Taken together, these findings indicate that several E. coli determinants, including ibeA and ibeB, contribute to crossing of the blood-brain barrier.  (+info)

Shiga toxin 1 from Escherichia coli blocks activation and proliferation of bovine lymphocyte subpopulations in vitro. (28/5328)

Shiga toxin-producing Escherichia coli (STEC) is widespread in the cattle population, but the clinical significance of Shiga toxins (Stx's) for the bovine species remains obscure. Since Stx's exert immunomodulating effects in other species, we examined the effect of purified Stx1 on a bovine B lymphoma cell line (BL-3) and peripheral blood mononuclear cells (PBMC) isolated from adult bovine blood by viability assays and flow cytometry analysis. Stx1 markedly induced apoptosis in stimulated BL-3 cells. The susceptibility of this B-cell-derived cell line was induced only by either lipopolysaccharide (LPS) or pokeweed mitogen, while cultures stimulated with T-cell mitogens were unaffected by the toxin. In contrast, Stx1 did not induce cellular death-neither apoptosis nor necrosis-in primary cultures of PBMC but hindered the mitogen-induced increase in metabolic activity. The influence of Stx1 on single PBMC subpopulations varied with the type of mitogenic stimulus applied. Stimulation with phytohemagglutinin P particularly induced the proliferation of bovine CD8-expressing (BoCD8(+)) cells, and this proliferative response was blocked by Stx1. On the other hand, Stx1 reduced the portion of viable B cells in the presence of LPS. Modulation of activation marker expression (BoCD25 and BoCD71) by Stx1 indicated that the toxin hindered the proliferation of cells by blocking their activation. In conclusion, we assume that Stx1 contributes to the pathogenesis of STEC-associated diarrhea in calves by suppressing the mucosa-associated immune response. The usefulness of cattle as a model in which to study Stx-induced immunomodulation is discussed.  (+info)

Characterization of the Escherichia coli AF/R1 pilus operon: novel genes necessary for transcriptional regulation and for pilus-mediated adherence. (29/5328)

We isolated the genetic determinant of AF/R1 pilus production in attaching/effacing Escherichia coli RDEC-1 and identified seven genes required for pilus expression and function. DNA sequence analysis of the structural subunit gene afrA corrected an error in the published sequence and extended homology with the F18 pilus subunit of pig edema E. coli strains. AfrB and AfrC, encoded downstream from AfrA, were required for pilus expression. AfrB was related to the usher protein PefC of Salmonella typhimurium plasmid-encoded fimbriae, and AfrC was related to PefD, a chaperone protein. AfrD and AfrE, encoded downstream from AfrC, were not necessary for the expression of AF/R1 pili but were required for ileal adherence as assayed by ileal brush border aggregation. Thus, the adhesive subunit of the AF/R1 pilus is distinct from the structural subunit, as is the case for Pap pili and type 1 pili. AfrD was related to FedE of the F18 fimbrial operon of the E. coli strain that causes edema disease in pigs. AfrE was a novel protein. AfrR and AfrS are encoded upstream from AfrA, in the opposite orientation. AfrR is related to the AraC family of transcriptional regulators, and AfrR and AfrS interact to function in a novel mode of transcriptional activation of afrA. AF/R1 pili mediate the adherence to Peyer's patch M cells, ileal mucosa, and colonic mucosa in a rabbit model of diarrhea caused by enteropathogenic E. coli. Our observations will facilitate the further study of the phenomena of M-cell adherence.  (+info)

Enterohemorrhagic Escherichia coli O157:H7 produces Tir, which is translocated to the host cell membrane but is not tyrosine phosphorylated. (30/5328)

Intimate attachment to the host cell leading to the formation of attaching and effacing (A/E) lesions is an essential feature of enterohemorrhagic Escherichia coli (EHEC) O157:H7 pathogenesis. In a related pathogen, enteropathogenic E. coli (EPEC), this activity is dependent upon translocation of the intimin receptor, Tir, which becomes tyrosine phosphorylated within the host cell membrane. In contrast, the accumulation of tyrosine-phosphorylated proteins beneath adherent EHEC bacteria does not occur, leading to questions about whether EHEC uses a Tir-based mechanism for adherence and A/E lesion formation. In this report, we demonstrate that EHEC produces a functional Tir that is inserted into host cell membranes, where it serves as an intimin receptor. However, unlike in EPEC, in EHEC Tir is not tyrosine phosphorylated yet plays a key role in both bacterial adherence to epithelial cells and pedestal formation. EHEC, but not EPEC, was unable to synthesize Tir in Luria-Bertani medium but was able to secrete Tir into M9 medium, suggesting that Tir synthesis and secretion may be regulated differently in these two pathogens. EHEC Tir and EPEC Tir both bind intimin and focus cytoskeletal rearrangements, indicating that tyrosine phosphorylation is not needed for pedestal formation. EHEC and EPEC intimins are functionally interchangeable, but EHEC Tir shows a much greater affinity for EHEC intimin than for EPEC intimin. These findings highlight some of the differences and similarities between EHEC and EPEC virulence mechanisms, which can be exploited to further define the molecular basis of pedestal formation.  (+info)

Inverse relationship between severity of experimental pyelonephritis and nitric oxide production in C3H/HeJ mice. (31/5328)

The contribution of nitric oxide to host resistance to experimental pyelonephritis is not well understood. We examined whether the inhibition of nitric oxide synthesis alters the sensitivity of lipopolysaccharide (LPS) responder (C3H/HeN) and nonresponder (C3H/HeJ) mice to experimental Escherichia coli pyelonephritis. C3H/HeJ and C3H/HeN mice were implanted subcutaneously with minipumps containing an inhibitor of nitric oxide, NG-nitro-L-arginine methyl ester (L-NAME), or a corresponding vehicle. Ascending urinary tract infection by bladder catheterization with two strains of E. coli, an O75 strain bearing Dr fimbriae and an O75 strain bearing P fimbriae, was developed in tested animals. Twenty-four hours following bladder infection, the kidneys of C3H/HeN and C3H/HeJ mice were colonized at a similar rate. However, 5 weeks postinoculation, C3H/HeN mice cleared infection while C3H/HeJ mice showed persistent colonization. Twenty-four hours following infection, C3H/HeN mice treated with L-NAME showed no significant increase of renal tissue infection compared to the saline-treated control group. However, L-NAME-treated C3H/HeJ mice showed an approximately 100-fold increase in E. coli infection rate compared to the saline-treated controls in the Dr+ group but showed no change compared to those in the P+ group. Dissemination of Dr+ E. coli but not P+ E. coli to the liver and uterus was significantly enhanced with L-NAME treatment in C3H/HeJ mice only. Nitric oxide had no direct killing effect on E. coli in vitro. Nitrite production by various organs was found to be significantly lower in C3H/HeJ mice than in C3H/HeN mice. Alteration of nitric oxide and LPS responsiveness was significantly associated with the increased sensitivity of C3H/HeJ mice to experimental Dr+ but not to P+ E. coli pyelonephritis. These findings are consistent with the hypothesis that nitric oxide synthase activity in concert with LPS responsiveness may participate in the antibacterial defense mechanisms of the C3H mouse urinary tract. This phenomenon is strain dependent and possibly related to the invasive properties of E. coli.  (+info)

Host cell death due to enteropathogenic Escherichia coli has features of apoptosis. (32/5328)

Enteropathogenic Escherichia coli (EPEC) is a cause of prolonged watery diarrhea in children in developing countries. The ability of EPEC to kill host cells was investigated in vitro in assays using two human cultured cell lines, HeLa (cervical) and T84 (colonic). EPEC killed epithelial cells as assessed by permeability to the vital dyes trypan blue and propidium iodide. In addition, EPEC triggered changes in the host cell, suggesting apoptosis as the mode of death; such changes included early expression of phosphatidylserine on the host cell surface and internucleosomal cleavage of host cell DNA. Genistein, an inhibitor of tyrosine kinases, and wortmannin, an inhibitor of host phosphatidylinositol 3-kinase, markedly increased EPEC-induced cell death and enhanced the features of apoptosis. EPEC-induced cell death was contact dependent and required adherence of live bacteria to the host cell. A quantitative assay for EPEC-induced cell death was developed by using the propidium iodide uptake method adapted to a fluorescence plate reader. With EPEC, the rate and extent of host cell death were less that what has been reported for Salmonella, Shigella, and Yersinia, three other genera of enteric bacteria known to cause apoptosis. However, rapid apoptosis of the host cell may not favor the pathogenic strategy of EPEC, a mucosa-adhering, noninvasive pathogen.  (+info)