Showing papers on "Escherichia coli published in 2004"
TL;DR: Few microorganisms are as versatile as Escherichia coli; it can also be a highly versatile, and frequently deadly, pathogen.
Abstract: Few microorganisms are as versatile as Escherichia coli. An important member of the normal intestinal microflora of humans and other mammals, E. coli has also been widely exploited as a cloning host in recombinant DNA technology. But E. coli is more than just a laboratory workhorse or harmless intestinal inhabitant; it can also be a highly versatile, and frequently deadly, pathogen. Several different E. coli strains cause diverse intestinal and extraintestinal diseases by means of virulence factors that affect a wide range of cellular processes.
4,583 citations
TL;DR: The current review presents the available genomics and biological data on prophages from bacterial pathogens in an evolutionary framework to demonstrate that the chromosomes from bacteria and their viruses (bacteriophages) are coevolving.
Abstract: Comparative genomics demonstrated that the chromosomes from bacteria and their viruses (bacteriophages) are coevolving. This process is most evident for bacterial pathogens where the majority contain prophages or phage remnants integrated into the bacterial DNA. Many prophages from bacterial pathogens encode virulence factors. Two situations can be distinguished: Vibrio cholerae, Shiga toxin-producing Escherichia coli, Corynebacterium diphtheriae, and Clostridium botulinum depend on a specific prophage-encoded toxin for causing a specific disease, whereas Staphylococcus aureus, Streptococcus pyogenes, and Salmonella enterica serovar Typhimurium harbor a multitude of prophages and each phage-encoded virulence or fitness factor makes an incremental contribution to the fitness of the lysogen. These prophages behave like “swarms” of related prophages. Prophage diversification seems to be fueled by the frequent transfer of phage material by recombination with superinfecting phages, resident prophages, or occasional acquisition of other mobile DNA elements or bacterial chromosomal genes. Prophages also contribute to the diversification of the bacterial genome architecture. In many cases, they actually represent a large fraction of the strain-specific DNA sequences. In addition, they can serve as anchoring points for genome inversions. The current review presents the available genomics and biological data on prophages from bacterial pathogens in an evolutionary framework.
1,499 citations
TL;DR: Recent advances in secretory and extracellular production of recombinant proteins using E. coli are discussed, including the twin-arginine translocation system, which has recently been employed for the efficient secretion of folded proteins.
Abstract: Escherichia coli is one of the most widely used hosts for the production of recombinant proteins. However, there are often problems in recovering substantial yields of correctly folded proteins. One approach to solve these problems is to have recombinant proteins secreted into the periplasmic space or culture medium. The secretory production of recombinant proteins has several advantages, such as simplicity of purification, avoidance of protease attack and N-terminal Met extension, and a better chance of correct protein folding. In addition to the well-established Sec system, the twin-arginine translocation (TAT) system has recently been employed for the efficient secretion of folded proteins. Various strategies for the extracellular production of recombinant proteins have also been developed. For the secretory production of complex proteins, periplasmic chaperones and protease can be manipulated to improve the yields of secreted proteins. This review discusses recent advances in secretory and extracellular production of recombinant proteins using E. coli.
628 citations
TL;DR: It is established that the pgaABCD locus affects biofilm development by promoting abiotic surface binding and intercellular adhesion, and proposed that PGA serves as an adhesin that stabilizes biofilms of E. coli and other bacteria.
Abstract: Production of a polysaccharide matrix is a hallmark of bacterial biofilms, but the composition of matrix polysaccharides and their functions are not widely understood. Previous studies of the regulation of Escherichia coli biofilm formation suggested the involvement of an unknown adhesin. We now establish that the pgaABCD (formerly ycdSRQP) locus affects biofilm development by promoting abiotic surface binding and intercellular adhesion. All of the pga genes are required for optimal biofilm formation under a variety of growth conditions. A pga-dependent cell-bound polysaccharide was isolated and determined by nuclear magnetic resonance analyses to consist of unbranched beta-1,6-N-acetyl-D-glucosamine, a polymer previously unknown from the gram-negative bacteria but involved in adhesion by staphylococci. The pga genes are predicted to encode envelope proteins involved in synthesis, translocation, and possibly surface docking of this polysaccharide. As predicted, if poly-beta-1,6-GlcNAc (PGA) mediates cohesion, metaperiodate caused biofilm dispersal and the release of intact cells, whereas treatment with protease or other lytic enzymes had no effect. The pgaABCD operon exhibits features of a horizontally transferred locus and is present in a variety of eubacteria. Therefore, we propose that PGA serves as an adhesin that stabilizes biofilms of E. coli and other bacteria.
586 citations
TL;DR: I2 and anti-Escherichia coli outer membrane porin C are associated with Crohn's disease phenotypes, and patients with the highest level of serum reactivity toward an increasing number of microbiota have the greatest frequency of strictures, internal perforations, and small bowel surgery.
508 citations
TL;DR: It is demonstrated that probiotic bacteria may stimulate the intestinal innate defense through the upregulation of inducible antimicrobial peptides such as hBD-2.
Abstract: Little is known about the defensive mechanisms induced in epithelial cells by pathogenic versus probiotic bacteria. The aim of our study was to compare probiotic bacterial strains such as Escherichia coli Nissle 1917 with nonprobiotic, pathogenic and nonpathogenic bacteria with respect to innate defense mechanisms in the intestinal mucosal cell. Here we report that E. coli strain Nissle 1917 and a variety of other probiotic bacteria, including lactobacilli--in contrast to more than 40 different E. coli strains tested--strongly induce the expression of the antimicrobial peptide human beta-defensin-2 (hBD-2) in Caco-2 intestinal epithelial cells in a time- and dose-dependent manner. Induction of hBD-2 through E. coli Nissle 1917 was further confirmed by activation of the hBD-2 promoter and detection of the hBD-2 peptide in the culture supernatants of E. coli Nissle 1917-treated Caco-2 cells. Luciferase gene reporter analyses and site-directed mutagenesis experiments demonstrated that functional binding sites for NF-kappaB and AP-1 in the hBD-2 promoter are required for induction of hBD-2 through E. coli Nissle 1917. Treatment with the NF-kappaB inhibitor Helenalin, as well as with SP600125, a selective inhibitor of c-Jun N-terminal kinase, blocked hBD-2 induction by E. coli Nissle 1917 in Caco-2 cells. SB 202190, a specific p38 mitogen-activated protein kinase inhibitor, and PD 98059, a selective inhibitor of extracellular signal-regulated kinase 1/2, were ineffective. This report demonstrates that probiotic bacteria may stimulate the intestinal innate defense through the upregulation of inducible antimicrobial peptides such as hBD-2. The induction of hBD-2 may contribute to an enhanced mucosal barrier to the luminal bacteria.
489 citations
TL;DR: The results of this systematic analysis of nutrients used by E. coli MG1655 to colonize the mouse intestine are intriguing in light of the nutrient-niche hypothesis, which states that the ecological niches within the intestine are defined by nutrient availability.
Abstract: Whole-genome expression profiling revealed Escherichia coli MG1655 genes induced by growth on mucus, conditions designed to mimic nutrient availability in the mammalian intestine. Most were nutritional genes corresponding to catabolic pathways for nutrients found in mucus. We knocked out several pathways and tested the relative fitness of the mutants for colonization of the mouse intestine in competition with their wild-type parent. We found that only mutations in sugar pathways affected colonization, not phospholipid and amino acid catabolism, not gluconeogenesis, not the tricarboxylic acid cycle, and not the pentose phosphate pathway. Gluconate appeared to be a major carbon source used by E. coli MG1655 to colonize, having an impact on both the initiation and maintenance stages. N-acetylglucosamine and N-acetylneuraminic acid appeared to be involved in initiation, but not maintenance. Glucuronate, mannose, fucose, and ribose appeared to be involved in maintenance, but not initiation. The in vitro order of preference for these seven sugars paralleled the relative impact of the corresponding metabolic lesions on colonization: gluconate > N-acetylglucosamine > N-acetylneuraminic acid = glucuronate > mannose > fucose > ribose. The results of this systematic analysis of nutrients used by E. coli MG1655 to colonize the mouse intestine are intriguing in light of the nutrient-niche hypothesis, which states that the ecological niches within the intestine are defined by nutrient availability. Because humans are presumably colonized with different commensal strains, differences in nutrient availability may provide an open niche for infecting E. coli pathogens in some individuals and a barrier to infection in others.
483 citations
TL;DR: Data demonstrate the variability of the host innate immune response to E. coli and S. aureus and suggest that the limited cytokine response to S.Aureus may contribute to the well-known ability of the bacterium to establish chronic intramammary infection.
Abstract: Staphylococcus aureus and Escherichia coli are among the most prevalent species of gram-positive and gram-negative bacteria, respectively, that induce clinical mastitis. The innate immune system comprises the immediate host defense mechanisms to protect against infection and contributes to the initial detection of and proinflammatory response to infectious pathogens. The objective of the present study was to characterize the different innate immune responses to experimental intramammary infection with E. coli and S. aureus during clinical mastitis. The cytokine response and changes in the levels of soluble CD14 (sCD14) and lipopolysaccharide-binding protein (LBP), two proteins that contribute to host recognition of bacterial cell wall products, were studied. Intramammary infection with either E. coli or S. aureus elicited systemic changes, including decreased milk output, a febrile response, and induction of the acute-phase synthesis of LBP. Infection with either bacterium resulted in increased levels of interleukin 1beta (IL-1beta), gamma interferon, IL-12, sCD14, and LBP in milk. High levels of the complement cleavage product C5a and the anti-inflammatory cytokine IL-10 were detected at several time points following E. coli infection, whereas S. aureus infection elicited a slight but detectable increase in these mediators at a single time point. Increases in IL-8 and tumor necrosis factor alpha were observed only in quarters infected with E. coli. Together, these data demonstrate the variability of the host innate immune response to E. coli and S. aureus and suggest that the limited cytokine response to S. aureus may contribute to the well-known ability of the bacterium to establish chronic intramammary infection.
482 citations
TL;DR: It is expected that BIM formation should not hinder the use of these phages as biocontrol agents, particularly since low levels of the pathogen are typically encountered in the environment.
Abstract: Escherichia coli O157:H7 is an endemic pathogen causing a variety of human diseases including mild diarrhea, hemorrhagic colitis, hemolytic-uremic syndrome, and thrombotic thrombocytopenic purpura. This study concerns the exploitation of bacteriophages as biocontrol agents to eliminate the pathogen E. coli O157:H7. Two distinct lytic phages (e11/2 and e4/1c) isolated against a human strain of E. coli O157:H7, a previously isolated lytic phage (pp01), and a cocktail of all three phages were evaluated for their ability to lyse the bacterium in vivo and in vitro. Phage e11/2, pp01, and the cocktail of all three virulent phages resulted in a 5-log-unit reduction of pathogen numbers in 1 h at 37°C. However, bacteriophage-insensitive mutants (BIMs) emerged following the challenge. All tested BIMs had a growth rate which approximated that of the parental O157 strain, although many of these BIMs had a smaller, more coccoid cellular morphology. The frequency of BIM formation (10−6 CFU) was similar for e11/2, pp01, and the phage cocktail, while BIMs insensitive to e4/1c occurred at the higher frequency (10−4 CFU). In addition, BIMs commonly reverted to phage sensitivity within 50 generations. In an initial meat trial experiment, the phage cocktail completely eliminated E. coli O157:H7 from the beef meat surface in seven of nine cases. Given that the frequency of BIM formation is low (10−6 CFU) for two of the phages, allied to the propensity of these mutants to revert to phage sensitivity, we expect that BIM formation should not hinder the use of these phages as biocontrol agents, particularly since low levels of the pathogen are typically encountered in the environment.
450 citations
TL;DR: Seventeen multiple-antibiotic-resistant nonpathogenic Escherichia coli strains of human, animal, and food origins showed a wide variety of antibiotic resistance genes, many of them carried by class 1 and class 2 integrons.
Abstract: Seventeen multiple-antibiotic-resistant nonpathogenic Escherichia coli strains of human, animal, and food origins showed a wide variety of antibiotic resistance genes, many of them carried by class 1 and class 2 integrons. Amino acid changes in MarR and mutations in marO were identified for 15 and 14 E. coli strains, respectively.
422 citations
TL;DR: The present study suggests that biofilm gene expression is strongly associated with environmental conditions and that stress genes are involved in E. coli JM109 biofilm formation.
Abstract: DNA microarrays were used to study the gene expression profile of Escherichia coli JM109 and K12 biofilms. Both glass wool in shake flasks and mild steel 1010 plates in continuous reactors were used to create the biofilms. For the biofilms grown on glass wool, 22 genes were induced significantly (p≤0.05) compared to suspension cells, including several genes for the stress response (hslS, hslT, hha, and soxS), type I fimbriae (fimG), metabolism (metK), and 11 genes of unknown function (ybaJ, ychM, yefM, ygfA, b1060, b1112, b2377, b3022, b1373, b1601, and b0836). The DNA microarray results were corroborated with RNA dot blotting. For the biofilm grown on mild steel plates, the DNA microarray data showed that, at a specific growth rate of 0.05/h, the mature biofilm after 5 days in the continuous reactors did not exhibit differential gene expression compared to suspension cells although genes were induced at 0.03/h. The present study suggests that biofilm gene expression is strongly associated with environmental conditions and that stress genes are involved in E. coli JM109 biofilm formation.
TL;DR: This study represents the first assessment of any E. coli pathotype's transcriptome in vivo and provides specific insights into the mechanisms necessary for urinary tract pathogenesis.
Abstract: A uropathogenic Escherichia coli strain CFT073-specific DNA microarray that includes each open reading frame was used to analyze the transcriptome of CFT073 bacteria isolated directly from the urine of infected CBA/J mice. The in vivo expression profiles were compared to that of E. coli CFT073 grown statically to exponential phase in rich medium, revealing the strategies this pathogen uses in vivo for colonization, growth, and survival in the urinary tract environment. The most highly expressed genes overall in vivo encoded translational machinery, indicating that the bacteria were in a rapid growth state despite specific nutrient limitations. Expression of type 1 fimbriae, a virulence factor involved in adherence, was highly upregulated in vivo. Five iron acquisition systems were all highly upregulated during urinary tract infection, as were genes responsible for capsular polysaccharide and lipopolysaccharide synthesis, drug resistance, and microcin secretion. Surprisingly, other fimbrial genes, such as pap and foc/sfa, and genes involved in motility and chemotaxis were downregulated in vivo. E. coli CFT073 grown in human urine resulted in the upregulation of iron acquisition, capsule, and microcin secretion genes, thus partially mimicking growth in vivo. On the basis of gene expression levels, the urinary tract appears to be nitrogen and iron limiting, of high osmolarity, and of moderate oxygenation. This study represents the first assessment of any E. coli pathotype's transcriptome in vivo and provides specific insights into the mechanisms necessary for urinary tract pathogenesis.
TL;DR: This strain's genome structure has been analyzed by sequence context screening of tRNA genes as a potential indication of chromosomal integration of horizontally acquired DNA, sequence analysis of 280 kb of genomic islands (GEIs) coding for important fitness factors, and comparison of Nissle 1917 genome content with that of other E. coli strains by DNA-DNA hybridization.
Abstract: Nonpathogenic Escherichia coli strain Nissle 1917 (O6:K5:H1) is used as a probiotic agent in medicine, mainly for the treatment of various gastroenterological diseases. To gain insight on the genetic level into its properties of colonization and commensalism, this strain's genome structure has been analyzed by three approaches: (i) sequence context screening of tRNA genes as a potential indication of chromosomal integration of horizontally acquired DNA, (ii) sequence analysis of 280 kb of genomic islands (GEIs) coding for important fitness factors, and (iii) comparison of Nissle 1917 genome content with that of other E. coli strains by DNA-DNA hybridization. PCR-based screening of 324 nonpathogenic and pathogenic E. coli isolates of different origins revealed that some chromosomal regions are frequently detectable in nonpathogenic E. coli and also among extraintestinal and intestinal pathogenic strains. Many known fitness factor determinants of strain Nissle 1917 are localized on four GEIs which have been partially sequenced and analyzed. Comparison of these data with the available knowledge of the genome structure of E. coli K-12 strain MG1655 and of uropathogenic E. coli O6 strains CFT073 and 536 revealed structural similarities on the genomic level, especially between the E. coli O6 strains. The lack of defined virulence factors (i.e., alpha-hemolysin, P-fimbrial adhesins, and the semirough lipopolysaccharide phenotype) combined with the expression of fitness factors such as microcins, different iron uptake systems, adhesins, and proteases, which may support its survival and successful colonization of the human gut, most likely contributes to the probiotic character of E. coli strain Nissle 1917.
TL;DR: Cell‐to‐cell signalling in prokaryotes that leads to co‐ordinated behaviour has been termed quorum sensing and several putative signalling systems have been identified, one that uses indole as a signal and another that releases what appears to be a peptide.
Abstract: Summary
Cell-to-cell signalling in prokaryotes that leads to co-ordinated behaviour has been termed quorum sensing. This type of signalling can have profound impacts on microbial community structure and host–microbe interactions. The Gram-negative quorum-sensing systems were first discovered and extensively characterized in the marine Vibrios. Some components of the Vibrio systems are present in the classical genetic model organisms Escherichia coli and Salmonella enterica. Both organisms encode a signal receptor of the LuxR family, SdiA, but not a corresponding signal-generating enzyme. Instead, SdiA of Salmonella detects and responds to signals generated only by other microbial species. Conversely, E. coli and Salmonella encode the signal-generating component of a second system (a LuxS homologue that generates AI-2), but the sensory apparatus for AI-2 differs substantially from the Vibrio system. The only genes currently known to be regulated by AI-2 in Salmonella encode an active uptake and modification system for AI-2. Therefore, it is not yet clear whether Salmonella uses AI-2 as a signal molecule or whether AI-2 has some other function. In E. coli, the functions of both SdiA and AI-2 are unclear due to pleiotropy. Genetic strategies to identify novel signalling systems have been performed with E. coli and Providencia stuartii. Several putative signalling systems have been identified, one that uses indole as a signal and another that releases what appears to be a peptide. The latter system has homologues in E. coli and Salmonella, as well as other bacteria, plants and animals. In fact, the protease components from Providencia and Drosophila are functionally interchangeable.
TL;DR: The unique features of the cspA gene are applied to develop a series of expression vectors, termed pCold vectors, that drive the high expression of cloned genes upon induction by cold-shock, and are highly complementary to the widely used pET vectors.
Abstract: Overexpression of proteins in Escherichia coli at low temperature improves their solubility and stability. Here, we apply the unique features of the cspA gene to develop a series of expression vectors, termed pCold vectors, that drive the high expression of cloned genes upon induction by cold-shock. Several proteins were produced with very high yields, including E. coli EnvZ ATP-binding domain (EnvZ-B) and Xenopus laevis calmodulin (CaM). The pCold vector system can also be used to selectively enrich target proteins with isotopes to study their properties in cell lysates using NMR spectroscopy. We have cloned 38 genes from a range of prokaryotic and eukaryotic organisms into both pCold and pET14 (ref. 3) systems, and found that pCold vectors are highly complementary to the widely used pET vectors.
TL;DR: Vaccination of cattle with proteins secreted by E. coli O157:H7 significantly reduced the numbers of bacteria shed in feces, the number of animals that shed, and the duration of shedding in an experimental challenge model, suggesting it is possible to vaccinate cattle to decrease the level of E. bacteria shed for the purpose of reducing the risk of human disease.
TL;DR: The panel of mini-Tn7 delivery plasmids expressing different fluorescent proteins (stable and unstable) from the Escherichia coli lac derived promoter, P(A1/04/03), or from the growth-rate-dependent EscherICHia coli promoter PrrnB P1 is expanded.
Abstract: The mini-Tn7 transposon system is a convenient tool for site-specific tagging of bacteria in which the tagging DNA is inserted at a unique and neutral chromosomal site. We have expanded the panel of mini-Tn7 delivery plasmids expressing different fluorescent proteins (stable and unstable) from the Escherichia coli lac derived promoter, P(A1/04/03), or from the growth-rate-dependent Escherichia coli promoter PrrnB P1. The mini-Tn7 transposons were inserted and tested in the soil bacterium, Pseudomonas putida KT2440. Successful and site-specific tagging was verified by Southern blots as well as by PCR. Furthermore, the effect of fluorescent protein expression on the cellular growth rate was tested by growth competition assays.
TL;DR: Findings suggest that the toxin secreted by the Shiga toxigenic Escherichia coli O113:H21 strain 98NK2 may contribute to the pathogenesis of human disease.
Abstract: The Shiga toxigenic Escherichia coli (STEC) O113:H21 strain 98NK2, which was responsible for an outbreak of hemolytic uremic syndrome, secretes a highly potent and lethal subtilase cytotoxin that is unrelated to any bacterial toxin described to date. It is the prototype of a new family of AB5 toxins, comprising a single 35-kilodalton (kD) A subunit and a pentamer of 13-kD B subunits. The A subunit is a subtilase-like serine protease distantly related to the BA_2875 gene product of Bacillus anthracis . The B subunit is related to a putative exported protein from Yersinia pestis , and binds to a mimic of the ganglioside GM2. Subtilase cytotoxin is encoded by two closely linked, cotranscribed genes ( subA and subB ), which, in strain 98NK2, are located on a large, conjugative virulence plasmid. Homologues of the genes are present in 32 out of 68 other STEC strains tested. Intraperitoneal injection of purified subtilase cytotoxin was fatal for mice and resulted in extensive microvascular thrombosis, as well as necrosis in the brain, kidneys, and liver. Oral challenge of mice with E. coli K-12–expressing cloned subA and subB resulted in dramatic weight loss. These findings suggest that the toxin may contribute to the pathogenesis of human disease.
TL;DR: The recombinant AtCCD7 protein catalyzes a specific 9-10 cleavage of β-carotene to produce the 10 ▾-apo-β- carotenal (C27) and β-ionone (C13) and the C18 product appears to result from a secondary Cleavage of the AtCCd7-derived C27 product.
TL;DR: This study has demonstrated the use of molecular techniques for determining relative proportions of bacterial species and monitoring pathogens in the chick gastrointestinal tract.
TL;DR: Findings support the idea that THP serves as a soluble receptor for type 1 fimbriated E. coli and helps eliminate bacteria from the urinary tract.
TL;DR: The crystal structure of Stx2 from E. coli O157:H7 was determined and it was found that, in contrast to Stx, the active site of the A-subunit of StX2 is accessible in the holotoxin, and a molecule of formic acid and a water molecule mimic the binding of the adenine base of the substrate.
TL;DR: 5'-nuclease PCR assays for detecting eight O-serogroups, H7 flagellar antigen and stx genes from the Shiga toxin-producing Escherichia coli (STEC) associated with the world's most frequent clinical cases indicate that these assays could serve as a basis for rapid specific stx, O and H7 typing of these major pathogenic serogroups of E. coli.
TL;DR: A comparison of anti-STEC activity among several Bifidobacterium strains with natural resistance to streptomycin revealed that strains such as B ifidobacteria bifidum ATCC 15696 and Bifinium catenulatum ATCC 27539T did not confer an anti-infectious activity, despite achieving high population levels similar to those of effective strains.
Abstract: The anti-infectious activity of probiotic Bifidobacteria against Shiga toxin-producing Escherichia coli (STEC) O157:H7 was examined in a fatal mouse STEC infection model. Stable colonization of the murine intestines was achieved by the oral administration of Bifidobacterium breve strain Yakult (naturally resistant to streptomycin sulfate) as long as the mice were treated with streptomycin in their drinking water (5 mg/ml). The pathogenicity of STEC infection, characterized by marked body weight loss and subsequent death, observed in the infected controls was dramatically inhibited in the B. breve-colonized group. Moreover, Stx production by STEC cells in the intestine was almost completely inhibited in the B. breve-colonized group. A comparison of anti-STEC activity among several Bifidobacterium strains with natural resistance to streptomycin revealed that strains such as Bifidobacterium bifidum ATCC 15696 and Bifidobacterium catenulatum ATCC 27539T did not confer an anti-infectious activity, despite achieving high population levels similar to those of effective strains, such as B. breve strain Yakult and Bifidobacterium pseudocatenulatum DSM 20439. The effective strains produced a high concentration of acetic acid (56 mM) and lowered the pH of the intestine (to pH 6.75) compared to the infected control group (acetic acid concentration, 28 mM; pH, 7.15); these effects were thought to be related to the anti-infectious activity of these strains because the combination of a high concentration of acetic acid and a low pH was found to inhibit Stx production during STEC growth in vitro.
TL;DR: The construction of a vector derived from pET3-His and pRSET plasmids for the expression and purification of recombinant proteins in Escherichia coli based on T7 phage RNA polymerase, which enables the expression of a fusion protein with a minimal amino-terminal hexa-histidine affinity tag.
Abstract: We report here the construction of a vector derived from pET3-His and pRSET plasmids for the expression and purification of recombinant proteins in Escherichia coli based on T7 phage RNA polymerase. The resulting pAE plasmid combined the advantages of both vectors: small size (pRSET), expression of a short 6XHis tag at N-terminus (pET3-His) and a high copy number of plasmid (pRSET). The small size of the vector (2.8 kb) and the high copy number/cell (200-250 copies) facilitate the subcloning and sequencing procedures when compared to the pET system (pET3-His, 4.6 kb and 40-50 copies) and also result in high level expression of recombinant proteins (20 mg purified protein/liter of culture). In addition, the vector pAE enables the expression of a fusion protein with a minimal amino-terminal hexa-histidine affinity tag (a tag of 9 amino acids using XhoI restriction enzyme for the 5'cloning site) as in the case of pET3-His plasmid and in contrast to proteins expressed by pRSET plasmids (a tag of 36 amino acids using BamHI restriction enzyme for the 5'cloning site). Thus, although proteins expressed by pRSET plasmids also have a hexa-histidine tag, the fusion peptide is much longer and may represent a problem for some recombinant proteins.
TL;DR: 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.
Abstract: 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
TL;DR: Stx-phages may be considered to represent highly mobile genetic elements that play an important role in the expression of Stx, in horizontal gene transfer, and hence in genome diversification.
TL;DR: A sensitive, specific, and rapid method for the detection of E. coli O157:H7 was demonstrated using quantum dots (QDs) as a fluorescence marker coupled with immunomagnetic separation using biotin-streptavidin conjugation.
Abstract: A sensitive, specific, and rapid method for the detection of E. coli O157:H7 was demonstrated using quantum dots (QDs) as a fluorescence marker coupled with immunomagnetic separation. Magnetic beads coated with anti-E. coli O157 antibodies were employed to selectively capture the target bacteria, and biotin-conjugated anti-E. coli antibodies were added to form sandwich immuno complexes. After magnetic separation, the immuno complexes were labeled with QDs via biotin−streptavidin conjugation. This was followed by a fluorescence measurement using a laptop-controlled portable device, which consisted of a blue LED and a CCD-array spectrometer. The peak intensity of the fluorescence emission was proportional to the initial cell concentration of E. coli O157:H7 in the range of 103−107 CFU/mL with a detection limit at least 100 times lower than that of the FITC-based method. The total detection time was less than 2 h. Neither E. coli K12 nor Salmonella typhimurium interfered with the detection of E. coli O157:H7.
TL;DR: Findings suggest that multiple-antimicrobial-resistant E. coli isolates, including fluoroquinolone-resistant variants, are commonly present among diseased swine and chickens in China, and they suggest the need for the introduction of surveillance programs in China to monitor antimicrobial resistance in pathogenic bacteria that can be potentially transmitted to humans from food animals.
Abstract: Escherichia coli isolates from diseased piglets (n = 89) and chickens (n = 71) in China were characterized for O serogroups, virulence genes, antimicrobial susceptibility, class 1 integrons, and mechanisms of fluoroquinolone resistance. O78 was the most common serogroup identified (63%) among the chicken E. coli isolates. Most isolates were PCR positive for the increased serum survival gene (iss; 97%) and the temperature-sensitive hemagglutinin gene (tsh; 93%). The O serogroups of swine E. coli were not those typically associated with pathogenic strains, nor did they posses common characteristic virulence factors. Twenty-three serogroups were identified among the swine isolates; however, 38% were O nontypeable. Overall, isolates displayed resistance to nalidixic acid (100%), tetracycline (98%), sulfamethoxazole (84%), ampicillin (79%), streptomycin (77%), and trimethoprim-sulfamethoxazole (76%). Among the fluoroquinolones, resistance ranged between 64% to levofloxacin, 79% to ciprofloxacin, and 95% to difloxacin. DNA sequencing of gyrA, gyrB, parC, and parE quinolone resistance-determining regions of 39 nalidixic acid-resistant E. coli isolates revealed that a single gyrA mutation was found in all of the isolates; mutations in parC together with double gyrA mutations conferred high-level resistance to fluoroquinolones (ciprofloxacin MIC, ≥8 μg/ml). Class 1 integrons were identified in 17 (19%) isolates from swine and 42 (47%) from chickens. The majority of integrons possessed genes conferring resistance to streptomycin and trimethoprim. These findings suggest that multiple-antimicrobial-resistant E. coli isolates, including fluoroquinolone-resistant variants, are commonly present among diseased swine and chickens in China, and they also suggest the need for the introduction of surveillance programs in China to monitor antimicrobial resistance in pathogenic bacteria that can be potentially transmitted to humans from food animals.
TL;DR: The entire DNA sequence of pLVPK, which is a 219-kb virulence plasmid harbored in a bacteremic isolate of Klebsiella pneumoniae, is determined and the presence of 13 insertion sequences located mostly at the boundaries of the aforementioned gene clusters suggests that pL VPK was derived from a sequential assembly of various horizontally acquired DNA fragments.