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Incidence of enterohemorrhagic Escherichia coli, Escherichia coli O157, Salmonella, and Listeria monocytogenes in retail fresh ground beef, sprouts, and mushrooms.

The objective of this study was to determine the prevalence of enterohemorrhagic Escherichia coli (EHEC), E. coli O157, Salmonella, and Listeria monocytogenes in retail food samples from Seattle, Wash. A total of 2,050 samples of ground beef (1,750 samples), mushrooms (100 samples), and sprouts (200 samples) were collected over a 12-month period and analyzed for the presence of these pathogens. PCR assays, followed by culture confirmation were used to determine the presence or absence of each organism. Of the 1,750 ground beef samples analyzed, 61 (3.5%) were positive for EHEC, and 20 (1.1%) of these were positive for E. coli O157. Salmonella was present in 67 (3.8%) of the 1,750 ground beef samples. Of 512 ground beef samples analyzed, 18 (3.5%) were positive for L. monocytogenes. EHEC was found in 12 (6.0%) of the 200 sprout samples, and 3 (1.5%) of these yielded E. coli O157. Of the 200 total sprout samples, 14 (7.0%) were positive for Salmonella and none were positive for L. monocytogenes. Among the 100 mushroom samples, 4 (4.0%) were positive for EHEC but none of these 4 samples were positive for E. coli O157. Salmonella was detected in 5 (5.0%) of the mushroom samples, and L. monocytogenes was found in 1 (1.0%) of the samples.

Agaricales↗

Competitive inhibition of adherence of enterotoxigenic Escherichia coli, enteropathogenic Escherichia coli and Clostridium difficile to intestinal epithelial cell line Lovo by purified adhesin of Bifidobacterium adolescentis 1027.

AIM: To observe competitive inhibition of adherence of enterotoxigenic Escherichia coli (ETEC), enteropathogenic Escherichia coli (EPEC) and Clostridium difficile (C. difficile) to intestinal epithelial cell line Lovo by purified adhesin of Bifidobacterium adolescentis 1027 (B. ado 1027). METHODS: The binding of bacteria to intestinal epithelial cell line Lovo was counted by adhesion assay. The inhibition of adherence of ETEC, EPEC and C. difficile to intestinal epithelial cell line Lovo by purified adhesin of B. ado 1027 was evaluated quantitatively by flow cytometry. RESULTS: The purified adhesin at the concentration of 10 microg/mL, 20 microg/mL and 30 microg/mL except at 1 microg/mL and 5 microg/mL could inhibit significantly the adhesion of ETEC, EPEC and C. difficile to intestinal epithelial cell line Lovo. Moreover, we observed that a reduction in bacterial adhesion was occurred with increase in the concentration of adhesin, and MFI (Mean fluorescent intensity) was decreased with increase in the concentration of adhesin. CONCLUSION: The purified adhesin of B. ado 1027 can inhibit the adhesion of ETEC, EPEC and C. difficile to intestinal epithelial cell line Lovo in a dose-dependent manner.

Adhesins, Bacterial↗

Development of a medium for differentiation between Escherichia coli and Escherichia coli O157:H7.

A new medium (Escherichia coli O157:H7 medium: EOH) was developed for differentiation between E. coli and E. coli O157:H7. The EOH medium was compared with sorbitol MacConkey agar (SMAC), which is the most popular medium to enumerate E. coli O157:H7. Several combinations of 35 dyes were evaluated to develop the new medium. Indigo carmine (0.03) g/liter) and phenol red (0.036 g/liter) were found as the best combination for differentiation between E. coli O157:H7 and E. coli and added to the basal agar medium (SMAC medium excluding neutral red and crystal violet) for EOH medium. On the dark blue EOH medium, E. coli produced a yellow color with clear zone, whereas E. coli O157:H7 produced a red color without clear zone. For differentiation between E. coli and E. coli O157:H7, EOH has much better potential than SMAC. Furthermore. the red color produced by normal E. coli in SMAC may mask the light gray color produced by E. coli O157: H7, whereas the yellow color with clear zone did not mask the red color without clear zone in the EOH medium. The recovery numbers of E. coli O157:H7 from inoculated ground beef, pork, and turkey were not significantly different between SMAC and EOH media (P > 0.05). The recovery rates of heat- and cold-injured E. coli O157:H7 also were not significantly different (P > 0.05).

Animals↗

Fishing new proteins in the twilight zone of genomes: the test case of outer membrane proteins in Escherichia coli K12, Escherichia coli O157:H7, and other Gram-negative bacteria.

We address the problem of clustering the whole protein content of genomes into three different categories-globular, all-alpha, and all-beta membrane proteins-with the aim of fishing new membrane proteins in the pool of nonannotated proteins (twilight zone). The focus is then mainly on outer membrane proteins. This is performed by using an integrated suite of programs (Hunter) specifically developed for predicting the occurrence of signal peptides in proteins of Gram-negative bacteria and the topography of all-alpha and all-beta membrane proteins. Hunter is tested on the well and partially annotated proteins (2160 and 760, respectively) of Escherichia coli K 12 scoring as high as 95.6% in the correct assignment of each chain to the category. Of the remaining 1253 nonannotated sequences, 1099 are predicted globular, 136 are all-alpha, and 18 are all-beta membrane proteins. In Escherichia coli 0157:H7 we filtered 1901 nonannotated proteins. Our analysis classifies 1564 globular chains, 327 inner membrane proteins, and 10 outer membrane proteins. With Hunter, new membrane proteins are added to the list of putative membrane proteins of Gram-negative bacteria. The content of outer membrane proteins per genome (nine are analyzed) ranges from 1.5% to 2.4%, and it is one order of magnitude lower than that of inner membrane proteins. The finding is particularly relevant when it is considered that this is the first large-scale analysis based on validated tools that can predict the content of outer membrane proteins in a genome and can allow cross-comparison of the same protein type between different species.

Bacterial Outer Membrane Proteins↗

Long-haul transport and lack of preconditioning increases fecal shedding of Escherichia coli and Escherichia coli O157:H7 by calves.

The effects of weaning and transport on fecal shedding of Escherichia coli and on E. coli O157:H7 were investigated using 80 Angus and 94 Charolais range steer calves blocked by breed and assigned to four treatments. The calves were or were not preconditioned before transport on commercial cattle liner to the feedlot via long (15 h) or short (3 h) hauling duration, yielding preconditioned long haul (P-L; n = 44), preconditioned short haul (P-S; n = 44), nonpreconditioned long haul (NP-L; n = 43), and nonpreconditioned short haul (NP-S; n = 43). Preconditioned calves were vaccinated and weaned 29 and 13 days, respectively, before transport. Nonpreconditioned calves were weaned 1 day before long or short hauling, penned for 24 h and hauled again for 2 h, and vaccinated on arrival at the feedlot. Fecal samples were collected from calves while on pasture, at weaning, at loading for transport, on arrival at the feedlot, twice in the first week, and on days 7, 14, 21, and 28 for enumeration of total E. coli (biotype 1) and detection of E. coli O157:H7. No calves were positive for E. coli O157:H7 before transport. Following transport, more (P < 0.005) NP-L calves (6 of 43) tested positive for E. coli O157:H7 than did P-L (1 of 44), NP-S (1 of 43), or P-S (0 of 44) calves, and on days 0, 1, 7, and 21, their levels of shedding of E. coli were higher (P < 0.005). The calves' susceptibility to infection from the environment (possibly the holding facilities or feedlot pens) was likely elevated by the stresses of weaning, transport, and relocation. Lack of preconditioning and long periods of transport (NP-L) increased fecal shedding of E. coli and E. coli O157:H7. Preconditioning may serve to reduce E. coli O157:H7 shedding by range calves on arrival at the feedlot.

Animals↗

Pathogenic effect of enterotoxigenic Escherichia coli and Escherichia coli causing infantile diarrhoea.

Macroscopic, light and electron microscopic alterations in ligated rabbit intestinal loops challenged with five standard enterotoxigenic Escherichia coli (ETEC) and twenty-three enteropathogenic E. coli (EEC-I) strains, freshly isolated from infantile enteritis cases, were investigated. Only two O26 : K60 : H11 strains produced enterotoxin. Their living cultures, sterile filtrates of the fluid medium and ultrasonic lysates of the bacteria resulted in pronounced hypersecretion of the intestinal epithelium followed by fluid accumulation and loop dilatation. These two E. coli strains, similarly as the other loop-negative EEC-I strains, were able to penetrate into the intestinal epithelium. In contrast to the standard ETEC strains, the EEC-I bacteria, adhering to the brush border, intruded into the microvilli, multiplied on the outer epithelial cell membrane making close contact with it and, causing, shedding of microvilli, penetrated into enterocytes becoming enclosed in membrane-bound phagosome-like vacuoles, appeared in the lamina propria and elicited mild focal polymorphonuclear infiltration.

Animals↗

Non-Escherichia coli versus Escherichia coli community-acquired urinary tract infections in children hospitalized in a tertiary center: relative frequency, risk factors, antimicrobial resistance and outcome.

BACKGROUND: Currently hospitalization for children with urinary tract infections (UTIs) is reserved for severe or complicated cases. Changes may have taken place in the characteristics and causative uropathogens of hospital-treated community-acquired UTI. OBJECTIVES: To study children hospitalized in a tertiary center with community-acquired UTI, compare Escherichia coli and non-E. coli UTI, define predictors for non-E. coli UTI and elucidate the appropriate therapeutic approach. PATIENTS AND METHODS: A prospective clinical and laboratory study from 2001 through 2002 in a tertiary pediatric medical center. Patients were divided by results of the urine culture into E. coli and non-E. coli UTI groups, which were compared. RESULTS: Of 175 episodes of culture-proved UTI, 70 (40%) were caused by non-E. coli pathogens. Non-E. coli UTI was more commonly found in children who were male (P = 0.005), who had underlying renal abnormalities (P = 0.0085) and who had received antibiotic therapy in the prior month (P = 0.0009). Non-E. coli uropathogens were often resistant to antibiotics usually recommended for initial therapy for UTI, including cephalosporins and aminoglycosides; 19% were initially treated with inappropriate empiric intravenous antibiotics (compared with 2% for E. coli UTI, P = 0.0001), with a longer hospitalization. CONCLUSIONS: Current treatment routines are often inappropriate for hospitalized children with non-E. coli UTI, which is relatively common in this population. The defined risk factors associated with non-E. coli UTIs and its antimicrobial resistance patterns should be considered to improve empiric antibiotic therapy for these infections.

Anti-Bacterial Agents↗

Acute airsacculitis in untreated and cyclophosphamide-pretreated broiler chickens inoculated with Escherichia coli or Escherichia coli cell-free culture filtrate.

Ninety commercial broiler chickens were divided into three equal groups; 30 were injected with brain-heart-infusion broth into the cranial thoracic air sacs (controls), 30 were similarly inoculated with a culture of Escherichia coli, and 30 were similarly inoculated with E. coli cell-free culture filtrate. Birds were examined from 0 to 6 hours post-inoculation. E. coli-inoculated and cell-free culture filtrate-inoculated chickens reacted similarly, with exudation of heterophils into the air sac. Microscopically, heterophils were present in low numbers perivascularly 0.5 hour after inoculation and became more numerous by 3 hours post-inoculation. By 6 hours post-inoculation, there was severe swelling of air sac epithelial cells and thickening of the air sac by proteinaceous fluid and heterophils. Ultrastructurally, air sac epithelial cells were swollen and vacuolated, and interdigitating processes were separated. Histologically and ultrastructurally, all features in control chickens were normal, with only rare heterophils in the air sac interstitium. In E. coli-inoculated and cell-free culture filtrate-inoculated chickens, cell counts (predominantly heterophils) in air sac lavage fluids increased markedly at 3 and 6 hours, with only slight increases in counts from lavages of controls. Heteropenia was observed in E. coli-inoculated chickens, whereas heterophilia was observed in cell-free filtrate chickens and controls. Ninety additional chickens were pretreated with cyclophosphamide, subdivided into three equal groups, and inoculated and examined similarly as above. Cyclophosphamide pretreatment reduced inflammatory changes in air sacs, lowered cell numbers in lavage fluids, and abolished hematologic changes; however, it did not prevent epithelial cell changes. These results indicate that cell-free culture filtrate of E. coli induces changes similar to those induced by cultures of E. coli.

Acute Disease↗

Development of a latex agglutination test for rapid identification of Escherichia coli.

Escherichia coli, one of the most important human pathogens, is usually identified by a battery of biochemical tests that require overnight incubation. For rapid identification of Escherichia coli, a latex agglutination test (LAT) was developed. Rabbits were immunized with cell-surface antigens extracted from Escherichia coli CCRC 15481 with 4 M urea, and the affinity-purified antibodies were used to coat latex particles for the identification of the bacterium. The following gram-negative bacteria were used to evaluate the LAT: Escherichia coli (n = 761), Enterobacteriaceae other than Escherichia coli (n = 632), Aeromonas spp. (n = 21), Pseudomonas spp. (n = 75), Vibrio spp. (n = 18), and other bacteria (n = 64). The LAT had a sensitivity and specificity of 99.2 and 93.3%, respectively. If the LAT was used in conjunction with the tests of indole production or lactose fermentation, the specificity values for the identification of Escherichia coli increased from 93.3 to 98.8 and 98.7%, respectively. If the LAT, indole production, and lactose fermentation were used together for the identification of Escherichia coli, the sensitivity and specificity were 94 and 99.7%, respectively. Lactose fermentation could be detected by observing the colonies grown on selective media (e.g. MacConkey agar), and indole production could be analyzed simply by the spot indole test. Strains producing negative reactions (i.e. not identified as Escherichia coli) should be processed by the conventional procedures for identification. The present protocol integrating the LAT, indole production, and lactose fermentation for the identification of Escherichia coli offers considerable savings of time, manpower, and cost.

Animals↗

Quinolone resistance in Escherichia coli.

Escherichia coli is an important pathogen of animals and humans that causes great financial cost in food production by causing disease in food animals. The quinolones are a class of synthetic antimicrobial agents with excellent activity against Escherichia coli and other Gram-negative bacteria used in human and veterinary medicine. Different quinolones are used to treat various conditions in animals in different parts of the world. All members of this class of drug have the same mode of action: inhibition of topoisomerase enzymes, DNA Gyrase and Topoisomerase IV. Escherichia coli can become resistant to quinolones by altering the target enzymes, reducing permeability of the cell to inhibit their entry, or by actively pumping the drug out of the cell. All these resistance mechanisms can play a role in high-level fluoroquinolone resistance, however target site mutations appear to be most important. As all quinolones act in the same way resistance to one member of the class will also confer decreased susceptibility to all members of the family. Quinolone resistant Escherichia coli in animals have increased in numbers after quinolone introduction in a number of different case studies. The resistance mechanisms in these isolates are the same as those in resistant strains found in humans. Care needs to be taken to ensure that quinolones are used sparingly and appropriately as highly resistant strains of Escherichia coli can be selected and may pass into the food chain. As these drugs are of major therapeutic importance in human medicine, this is a public health concern. More information as to the numbers of quinolone resistant Escherichia coli and the relationship between resistance and quinolone use is needed to allow us to make better informed decisions about when and when not to use quinolones in the treatment of animals.

Animals↗

The occurrence of ambient temperature-regulated adhesins, curli, and the temperature-sensitive hemagglutinin tsh among avian Escherichia coli.

Escherichia coli establishes a secondary respiratory tract infection in birds following inhalation of contaminated dust and litter particles. Escherichia coli express adhesins under conditions reflective of the ambient temperatures present in poultry houses. These microbial adhesins allow E. coli to attach to cell types that it initially encounters in the respiratory tract. Ambient temperature-regulated adhesins represent a new class of bacterial hemagglutinins that include pili and the thin, aggregative, flexible filaments known as "curli." This study examines the occurrence of the ambient temperature-regulated adhesins, curli (crl, csgA), and an avian-specific, temperature-sensitive hemagglutinin, tsh, among avian and mammalian E. coli isolates. The avian hemagglutinin gene tsh was present in approximately 46% of clinical avian E. coli isolates. This gene was not detected among commensal E. coli isolated from healthy broiler chickens. Unlike tsh, curli genes were ubiquitous among E. coli. However, curli were observed in only half of the avian E. coli examined by electron microscopy. Curli were not present among several nonavian E. coli positive for crl and csgA. Approximately 25% of avian E. coli isolates agglutinated chicken erythrocytes when bacteria were grown at room temperature. Hemagglutination was not specific to E. coli isolated from poultry. Presence of either tsh or curli genes was not indicative of an isolate's ability to agglutinate chicken red blood cells. No discernible structures were observed mediating attachment of the bacteria to chicken red blood cells. An additional avian-specific hemagglutinin appears to be present among avian E. coli.

Adhesins, Bacterial↗

Dermatitis in broilers caused by Escherichia coli: isolation of Escherichia coli from field cases, reproduction of the disease with Escherichia coli O78:K80 and conclusions under consideration of predisposing factors.

A dermatitis in broiler chickens, especially on the caudal back, thighs and around the cloaca is observed more frequently in the last years. The skin is swollen at sites of inflammation and a fibrineous plaque extends between muscle and subcutis. No clinical signs are visible in the living flock but the disease causes economical losses because of degrading and rejection of carcasses. Studies of literature and own field observations suggest that Escherichia (E.) coli is involved in the development of the dermatitis. The following serotypes were isolated from field cases: O78:K80 (3X), O2:K56 (2X), O127:K63 (3X), O9:K57 (1X), O140 (2X); two isolates could not be identified. The reproduction of the dermatitis was successful by infection via feather follicles with E. coli O78:K80. The density of broilers kept on farms may contribute to the outbreak of dermatitis by violation of the skin followed by infection of the injuries. Massage of the infected sites by close contact of birds and insufficient hygiene may support the development of the disease.

Animals↗

The effect of NAPRTase overexpression on the total levels of NAD, the NADH/NAD+ ratio, and the distribution of metabolites in Escherichia coli.

Escherichia coli (E. coli) maintains its total NADH/NAD+ intracellular pool by synthesizing NAD through the de novo pathway and the pyridine nucleotide salvage pathway. The salvage pathway recycles intracellular NAD breakdown products and preformed pyridine compounds from the environment, such as nicotinic acid (NA). The enzyme nicotinic acid phosphoribosyltransferase (NAPRTase; EC 2.4.2.11), encoded by the pncB gene, catalyzes the formation of nicotinate mononucleotide (NAMN), a direct precursor of NAD, from NA. This reaction is believed to be the rate-limiting step in the NAD salvage pathway. The current study investigates the effect of overexpressing the pncB gene from Salmonella typhimurium on the total levels of NAD, the NADH/NAD+ ratio, and the production of different metabolites in E. coli under anaerobic chemostat conditions and anaerobic tube experiments. In addition, this paper studies the effect of combining the overexpression of the pncB gene with an NADH regeneration strategy that increases intracellular NADH availability, as we have previously shown. (The effect of increasing NADH availability on the redistribution of metabolic fluxes in Escherichia coli chemostat cultures, Metabolic Eng. 4, 230-237; Metabolic engineering of Escherichia coli: Increase of NADH availability by overexpressing an NAD(+)-dependent formate dehydrogenase, Metabolic Eng. 4, 217-229.) Overexpression of the pncB gene in chemostat experiments increased the total NAD levels, decreased the NADH/NAD+ ratio, and did not significantly redistribute the metabolic fluxes. However, under anaerobic tube conditions, overexpression of the pncB gene led to a significant shift in the metabolic patterns as evidenced by a decrease in lactate production and an increase as high as two-fold in the ethanol-to-acetate (Et/Ac) ratio. These results suggest that under chemostat conditions the total level of NAD is not limiting and the metabolic rates are fixed by the system at steady state. On the other hand, under transient conditions (such as those in batch cultivation) the increase in the total level of NAD can increase the rate of NADH-dependent pathways (ethanol) and therefore change the final distribution of metabolites. The effect of combining overexpression of the pncB gene with the substitution of the native cofactor-independent formate dehydrogenase (FDH) with an NAD(+)-dependent FDH was also investigated under anaerobic tube conditions. This manipulation produced a metabolic pattern that combines a high Et/Ac ratio similar to that obtained with the new FDH with an intermediate lactate level similar to that obtained with the overexpression of the pncB gene. It was found that addition of the pncB gene to the FDH system does not increase further the production of reduced metabolites because the system for NADH regeneration already reached the maximum theoretical yield of approximately 4 mol NADH/mol of glucose.

Anaerobiosis↗

A new plasmidic cefotaximase in a clinical isolate of Escherichia coli.

Escherichia coli GRI was isolated from an ear exudate of a newborn. The strain was highly resistant to cefotaxime (MIC 128 mg/l). Resistance to cefotaxime and the majority of beta-lactam antibiotics was readily transferred to an Escherichia coli recipient strain. Both the wild type and the transconjugant strains are different in their resistance phenotype from TEM-3 beta-cefotaximase producers by higher MICs to the majority of beta-lactams and lower MICs to ceftazidime. The isoelectric point of the cefotaximase of E. coli GRI was 8.9 in comparison with 6.3 for TEM-3. Thus, the enzyme produced by E. coli GRI represents a new plasmidic (plasmid pMVP-3) broad spectrum beta-lactamase (CTX-M) which may not be closely related to either the TEM- oder SHV-family of extended broad spectrum beta-lactamases.

Cefotaxime↗

Comparative study of synthetic oligonucleotide and cloned polynucleotide enterotoxin gene probes to identify enterotoxigenic Escherichia coli.

Escherichia coli isolated from 2,126 children in Thailand and the Philippines was examined for enterotoxin production and for DNA hybridization with synthetic oligonucleotide and cloned polynucleotide enterotoxin gene probes. A total of 233 infections with E. coli that were detected by one or more of these assays were identified. Of the infections, 75% (164/233) were identified by all three methods. An additional 18% (43/233) were identified by two of three methods. Isolates from 10% (19/183) of infections with E. coli that hybridized with both the oligonucleotide and cloned enterotoxin gene probes were nontoxigenic, as determined by the Y1 adrenal cell and suckling mouse assays. Although synthetic oligonucleotide probes to detect enterotoxigenic E. coli are more uniform and easier to use than cloned enterotoxin gene probes, the heat-labile toxin oligo probe used in this study did not identify 13% (11/87) of infections with E. coli that produced heat-labile toxin, as identified with the Y1 adrenal cell assay and the cloned enterotoxin gene probe. Synthetic oligonucleotide probes enable laboratories with only minimal equipment to use DNA hybridization assays to identify enterotoxigenic E. coli.

Bacterial Toxins↗

Occurrence of genes for P and S fimbriae and hemolysin in urinary Escherichia coli.

Escherichia coli is the common causative agent of urinary tract infections. Sixty-one strains of E. coli isolated from children with urinary tract infections were tested by colony hybridization for the presence of genes determining P and S fimbriae and hemolysin. Of these strains, 46 possess a gene for hemolysin, 44 for P fimbriae and 28 for S fimbriae. Only 30 strains formed lytic zones around the colonies on plates with sheep erythrocytes. The results indicated that simultaneous occurrence of genes in urinary E. coli was highest for P fimbriae and hemolysin and lower for other combinations of the tested genes.

Bacterial Proteins↗

Practical mechanisms for interrupting the oral-fecal lifecycle of Escherichia coli.

Escherichia coli is a common gut inhabitant, but it is usually out numbered by strictly anaerobic bacteria. When fecal material is exposed to oxygen, fermentation acids can be respired, and E. coli numbers increase. E. coli can survive for long periods of time in feces, but subsequent proliferation is dependent on its ability to re-enter the gastrointestinal tract via contaminated water and food. The oral-fecal lifestyle of E. coli is facilitated by its ability to survive the low pH of the human gastric stomach. Most strains of E. coli do not cause human disease, but some strains produce toxins and other virulence factors. Mature cattle carry E. coli O157:H7 without showing signs of infection, and beef can be contaminated with cattle feces at slaughter. Cattle manure is often used as a fertilizer by the vegetable industry, and E. coli from manure can migrate through the soil into water supplies. Sanitation, cooking and chlorination have been used to combat fecal E. coli, but these methods are not always effective. Recent work indicates that cattle diets can be modified overcome the extreme acid resistance of E. coli. When cattle were fed have for only a few days, colonic volatile fatty acid concentrations declined, pH increased, and the E. coli were no longer able to survive a pH shock that mimicked the human gastric stomach. E. coli in stored cattle manure eventually become highly acid resistant even if the cattle were fed hay, but these bacteria could be killed by sodium carbonate (150 mM, pH 8.5). Because the diet manipulations and carbonate treatments affected E. coli in general rather than specific serotypes, there is an increased likelihood of successful field application.

Ammonium Chloride↗