Showing papers on "Escherichia coli published in 2013"

Noncanonical Inflammasome Activation by Intracellular LPS Independent of TLR4

Abstract: Gram-negative bacteria including Escherichia coli, Citrobacter rodentium, Salmonella typhimurium, and Shigella flexneri are sensed in an ill-defined manner by an intracellular inflammasome complex that activates caspase-11. We show that macrophages loaded with synthetic lipid A, E. coli lipopolysaccharide (LPS), or S. typhimurium LPS activate caspase-11 independently of the LPS receptor Toll-like receptor 4 (TLR4). Consistent with lipid A triggering the noncanonical inflammasome, LPS containing a divergent lipid A structure antagonized caspase-11 activation in response to E. coli LPS or Gram-negative bacteria. Moreover, LPS-mutant E. coli failed to activate caspase-11. Tlr4(-/-) mice primed with TLR3 agonist polyinosinic:polycytidylic acid [poly(I:C)] to induce pro-caspase-11 expression were as susceptible as wild-type mice were to sepsis induced by E. coli LPS. These data unveil a TLR4-independent mechanism for innate immune recognition of LPS.

Host-Derived Nitrate Boosts Growth of E. coli in the Inflamed Gut

Abstract: Changes in the microbial community structure are observed in individuals with intestinal inflammatory disorders. These changes are often characterized by a depletion of obligate anaerobic bacteria, whereas the relative abundance of facultative anaerobic Enterobacteriaceae increases. The mechanisms by which the host response shapes the microbial community structure, however, remain unknown. We show that nitrate generated as a by-product of the inflammatory response conferred a growth advantage to the commensal bacterium Escherichia coli in the large intestine of mice. Mice deficient in inducible nitric oxide synthase did not support the growth of E. coli by nitrate respiration, suggesting that the nitrate generated during inflammation was host-derived. Thus, the inflammatory host response selectively enhances the growth of commensal Enterobacteriaceae by generating electron acceptors for anaerobic respiration.

Genomically Recoded Organisms Expand Biological Functions

Abstract: We describe the construction and characterization of a genomically recoded organism (GRO). We replaced all known UAG stop codons in Escherichia coli MG1655 with synonymous UAA codons, which permitted the deletion of release factor 1 and reassignment of UAG translation function. This GRO exhibited improved properties for incorporation of nonstandard amino acids that expand the chemical diversity of proteins in vivo. The GRO also exhibited increased resistance to T7 bacteriophage, demonstrating that new genetic codes could enable increased viral resistance.

The Epidemic of Extended-Spectrum-β-Lactamase-Producing Escherichia coli ST131 Is Driven by a Single Highly Pathogenic Subclone, H30-Rx

Abstract: The Escherichia coli sequence type 131 (ST131) clone is notorious for extraintestinal infections, fluoroquinolone re- sistance, and extended-spectrum beta-lactamase (ESBL) production, attributable to a CTX-M-15-encoding mobile element. Here, we applied pulsed-field gel electrophoresis (PFGE) and whole-genome sequencing to reconstruct the evolutionary history of the ST131 clone. PFGE-based cluster analyses suggested that bothfluoroquinolone resistance and ESBL production had been acquired by multiple ST131 sublineages through independent genetic events. In contrast, the more robust whole-genome- sequence-based phylogenomic analysis revealed thatfluoroquinolone resistance was confined almost entirely to a single, rapidly expanding ST131 subclone, designated H30-R. Strikingly, 91% of the CTX-M-15-producing isolates also belonged to a single, well-defined clade nested within H30-R, which was named H30-Rx due to its more extensive resistance. Despite its tight clonal relationship withH30Rx, the CTX-M-15 mobile element was inserted variably in plasmid and chromosomal locations within the H30-Rx genome. Screening of a large collection of recent clinical E. coli isolates both confirmed the global clonal expansion of H30-Rx and revealed its disproportionate association with sepsis (relative risk, 7.5; P<0.001). Together, these results suggest that the high prevalence of CTX-M-15 production among ST131 isolates is due primarily to the expansion of a single, highly vir- ulent subclone,H30-Rx. IMPORTANCE We applied an advanced genomic approach to study the recent evolutionary history of one of the most important Escherichia coli strains in circulation today. This strain, called sequence type 131 (ST131), causes multidrug-resistant bladder, kidney, and bloodstream infections around the world. The rising prevalence of antibiotic resistance in E. coli is making these infections more difficult to treat and is leading to increased mortality. Past studies suggested that many different ST131 strains gained resistance to extended-spectrum cephalosporins independently. In contrast, our research indicates that most extended- spectrum-cephalosporin-resistant ST131 strains belong to a single highly pathogenic subclone, called H30-Rx. The clonal nature ofH30-Rx may provide opportunities for vaccine or transmission prevention-based control strategies, which could gain impor- tance as H30-Rx and other extraintestinal pathogenic E. coli subclones become resistant to our best antibiotics.

High Prevalence of Mucosa-Associated E. coli Producing Cyclomodulin and Genotoxin in Colon Cancer

Abstract: Some Escherichia coli strains produce toxins designated cyclomodulins (CMs) which interfere with the eukaryotic cell cycle of host cells, suggesting a possible link between these bacteria and cancers. There are relatively few data available concerning the colonization of colon tumors by cyclomodulin- and genotoxic-producing E. coli. We did a qualitative and phylogenetic analysis of mucosa-associated E. coli harboring cyclomodulin-encoding genes from 38 patients with colorectal cancer (CRC) and 31 with diverticulosis. The functionality of these genes was investigated on cell cultures and the genotoxic activity of strains devoid of known CM-encoding gene was investigated. Results showed a higher prevalence of B2 phylogroup E. coli harboring the colibatin-producing genes in biopsies of patients with CRC (55.3%) than in those of patients with diverticulosis (19.3%), (p<0.01). Likewise, a higher prevalence of B2 E. coli harboring the CNF1-encoding genes in biopsies of patients with CRC (39.5%) than in those of patients with diverticulosis (12.9%), (p = 0.01). Functional analysis revealed that the majority of these genes were functional. Analysis of the ability of E. coli to adhere to intestinal epithelial cells Int-407 indicated that highly adherent E. coli strains mostly belonged to A and D phylogroups, whatever the origin of the strains (CRC or diverticulosis), and that most E. coli strains belonging to B2 phylogroup displayed very low levels of adhesion. In addition, 27.6% (n = 21/76) E. coli strains devoid of known cyclomodulin-encoding genes induced DNA damage in vitro, as assessed by the comet assay. In contrast to cyclomodulin-producing E. coli, these strains mainly belonged to A or D E. coli phylogroups, and exhibited a non significant difference in the distribution of CRC and diverticulosis specimens (22% versus 32.5%, p = 0.91). In conclusion, cyclomodulin-producing E. coli belonging mostly to B2 phylogroup colonize the colonic mucosa of patients with CRC.

Volatile Metabolites of Pathogens: A Systematic Review

Abstract: Ideally, invading bacteria are detected as early as possible in critically ill patients: the strain of morbific pathogens is identified rapidly, and antimicrobial sensitivity is known well before the start of new antimicrobial therapy. Bacteria have a distinct metabolism, part of which results in the production of bacteria-specific volatile organic compounds (VOCs), which might be used for diagnostic purposes. Volatile metabolites can be investigated directly in exhaled air, allowing for noninvasive monitoring. The aim of this review is to provide an overview of VOCs produced by the six most abundant and pathogenic bacteria in sepsis, including Staphylococcus aureus, Streptococcus pneumoniae, Enterococcus faecalis, Pseudomonas aeruginosa, Klebsiella pneumoniae, and Escherichia coli. Such VOCs could be used as biological markers in the diagnostic approach of critically ill patients. A systematic review of existing literature revealed 31 articles. All six bacteria of interest produce isopentanol, formaldehyde, methyl mercaptan, and trimethylamine. Since humans do not produce these VOCs, they could serve as biological markers for presence of these pathogens. The following volatile biomarkers were found for identification of specific strains: isovaleric acid and 2-methyl-butanal for Staphylococcus aureus; 1-undecene, 2,4-dimethyl-1-heptane, 2-butanone, 4-methyl-quinazoline, hydrogen cyanide, and methyl thiocyanide for Pseudomonas aeruginosa; and methanol, pentanol, ethyl acetate, and indole for Escherichia coli. Notably, several factors that may effect VOC production were not controlled for, including used culture media, bacterial growth phase, and genomic variation within bacterial strains. In conclusion, VOCs produced by bacteria may serve as biological markers for their presence. Goal-targeted studies should be performed to identify potential sets of volatile biological markers and evaluate the diagnostic accuracy of these markers in critically ill patients.

Basic and applied uses of genome‐scale metabolic network reconstructions of Escherichia coli

Abstract: The genome-scale model (GEM) of metabolism in the bacterium Escherichia coli K-12 has been in development for over a decade and is now in wide use. GEM-enabled studies of E. coli have been primarily focused on six applications: (1) metabolic engineering, (2) model-driven discovery, (3) prediction of cellular phenotypes, (4) analysis of biological network properties, (5) studies of evolutionary processes, and (6) models of interspecies interactions. In this review, we provide an overview of these applications along with a critical assessment of their successes and limitations, and a perspective on likely future developments in the field. Taken together, the studies performed over the past decade have established a genome-scale mechanistic understanding of genotype–phenotype relationships in E. coli metabolism that forms the basis for similar efforts for other microbial species. Future challenges include the expansion of GEMs by integrating additional cellular processes beyond metabolism, the identification of key constraints based on emerging data types, and the development of computational methods able to handle such large-scale network models with sufficient accuracy.

β-lactam antibiotics promote bacterial mutagenesis via an RpoS-mediated reduction in replication fidelity

Abstract: Regardless of their targets and modes of action, subinhibitory concentrations of antibiotics can have an impact on cell physiology and trigger a large variety of cellular responses in different bacterial species. Subinhibitory concentrations of β-lactam antibiotics cause reactive oxygen species production and induce PolIV-dependent mutagenesis in Escherichia coli. Here we show that subinhibitory concentrations of β-lactam antibiotics induce the RpoS regulon. RpoS-regulon induction is required for PolIV-dependent mutagenesis because it diminishes the control of DNA-replication fidelity by depleting MutS in E. coli, Vibrio cholerae and Pseudomonas aeruginosa. We also show that in E. coli, the reduction in mismatch-repair activity is mediated by SdsR, the RpoS-controlled small RNA. In summary, we show that mutagenesis induced by subinhibitory concentrations of antibiotics is a genetically controlled process. Because this mutagenesis can generate mutations conferring antibiotic resistance, it should be taken into consideration for the development of more efficient antimicrobial therapeutic strategies.

Escherichia coli in Europe: An overview

Abstract: Escherichia coli remains one of the most frequent causes of several common bacterial infections in humans and animals. E. coli is the prominent cause of enteritis, urinary tract infection, septicaemia and other clinical infections, such as neonatal meningitis. E. coli is also prominently associated with diarrhoea in pet and farm animals. The therapeutic treatment of E. coli infections is threatened by the emergence of antimicrobial resistance. The prevalence of multidrug-resistant E. coli strains is increasing worldwide principally due to the spread of mobile genetic elements, such as plasmids. The rise of multidrug-resistant strains of E. coli also occurs in Europe. Therefore, the spread of resistance in E. coli is an increasing public health concern in European countries. This paper summarizes the current status of E. coli strains clinically relevant in European countries. Furthermore, therapeutic interventions and strategies to prevent and control infections are presented and discussed. The article also provides an overview of the current knowledge concerning promising alternative therapies against E. coli diseases.

Photocatalytic antibacterial performance of TiO2 and Ag-doped TiO2 against S. aureus. P. aeruginosa and E. coli.

Abstract: This paper reports the structural and optical properties and comparative photocatalytic activity of TiO2 and Ag-doped TiO2 nanoparticles against different bacterial strains under visible-light irradiation. The TiO2 and Ag-doped TiO2 photocatalysts were synthesized by acid catalyzed sol–gel technique and characterized by X-ray diffraction (XRD), transmission electron microscopy (TEM), UV–vis spectroscopy and photoluminescence (PL). The XRD pattern revealed that the annealed sample of TiO2 has both anatase and rutile phases while only an anatase phase was found in Ag-doped TiO2 nanoparticles. The decreased band-gap energy of Ag-doped TiO2 nanoparticles in comparison to TiO2 nanoparticles was investigated by UV–vis spectroscopy. The rate of recombination and transfer behaviour of the photoexcited electron–hole pairs in the semiconductors was recorded by photoluminescence. The antimicrobial activity of TiO2 and Ag-doped TiO2 nanoparticles (3% and 7%) was investigated against both gram positive (Staphylococcus aureus) and gram negative (Pseudomonas aeruginosa, Escherichia coli) bacteria. As a result, the viability of all three microorganisms was reduced to zero at 60 mg/30 mL culture in the case of both (3% and 7% doping) concentrations of Ag-doped TiO2 nanoparticles. Annealed TiO2 showed zero viability at 80 mg/30 mL whereas doped Ag-TiO2 7% showed zero viability at 40 mg/30 mL culture in the case of P. aeruginosa only.

E. coli as an all-rounder: the thin line between commensalism and pathogenicity.

Abstract: Escherichia coli is a paradigm for a versatile bacterial species which comprises harmless commensal as well as different pathogenic variants with the ability to either cause intestinal or extraintestinal diseases in humans and many animal hosts. Because of this broad spectrum of lifestyles and phenotypes, E. coli is a well-suited model organism to study bacterial evolution and adaptation to different growth conditions and niches. The geno- and phenotypic diversity, however, also hampers risk assessment and strain typing. A marked genome plasticity is the key to the great variability seen in this species. Acquisition of genetic information by horizontal gene transfer, gene loss as well as other genomic modifications, like DNA rearrangements and point mutations, can constantly alter the genome content and thus the fitness and competitiveness of individual variants in certain niches. Specific gene subsets and traits have been correlated with an increased potential of E. coli strains to cause intestinal or extraintestinal disease. Intestinal pathogenic E. coli strains can be reliably discriminated from non-pathogenic, commensal, or from extraintestinal E. coli pathogens based on genome content and phenotypic traits. An unambiguous distinction of extraintestinal pathogenic E. coli and commensals is, nevertheless, not so easy, as strains with the ability to cause extraintestinal infection are facultative pathogens and belong to the normal flora of many healthy individuals. Here, we compare insights into phylogeny, geno-, and phenotypic traits of commensal and pathogenic E. coli. We demonstrate that the borderline between extraintestinal virulence and intestinal fitness can be blurred as improved adaptability and competitiveness may promote intestinal colonization as well as extraintestinal infection by E. coli.

Abrupt Emergence of a Single Dominant Multidrug-Resistant Strain of Escherichia coli

Abstract: Background. Fluoroquinolone-resistant Escherichia coli are increasingly prevalent. Their clonal origins—potentially critical for control efforts—remain undefined. Methods. Antimicrobial resistance profiles and fine clonal structure were determined for 236 diverse-source historical (1967–2009) E. coli isolates representing sequence type ST131 and 853 recent (2010–2011) consecutive E. coli isolates from 5 clinical laboratories in Seattle, Washington, and Minneapolis, Minnesota. Clonal structure was resolved based on fimH sequence (fimbrial adhesin gene: H subclone assignments), multilocus sequence typing, gyrA and parC sequence (fluoroquinolone resistance-determining loci), and pulsed-field gel electrophoresis. Results. Of the recent fluoroquinolone-resistant clinical isolates, 52% represented a single ST131 subclonal lineage, H30, which expanded abruptly after 2000. This subclone had a unique and conserved gyrA/parC allele combination, supporting its tight clonality. Unlike other ST131 subclones, H30 was significantly associated with fluoroquinolone resistance and was the most prevalent subclone among current E. coli clinical isolates, overall (10.4%) and within every resistance category (11%–52%). Conclusions. Most current fluoroquinolone-resistant E. coli clinical isolates, and the largest share of multidrug-resistant isolates, represent a highly clonal subgroup that likely originated from a single rapidly expanded and disseminated ST131 strain. Focused attention to this strain will be required to control the fluoroquinolone and multidrug-resistant E. coli epidemic.

Strategies for the Production of Recombinant Protein in Escherichia coli

Abstract: In the recent past years, a large number of proteins have been expressed in Escherichia coli with high productivity due to rapid development of genetic engineering technologies. There are many hosts used for the production of recombinant protein but the preferred choice is E. coli due to its easier culture, short life cycle, well-known genetics, and easy genetic manipulation. We often face a problem in the expression of foreign genes in E. coli. Soluble recombinant protein is a prerequisite for structural, functional and biochemical studies of a protein. Researchers often face problems producing soluble recombinant proteins for over-expression, mainly the expression and solubility of heterologous proteins. There is no universal strategy to solve these problems but there are a few methods that can improve the level of expression, non-expression, or less expression of the gene of interest in E. coli. This review addresses these issues properly. Five levels of strategies can be used to increase the expression and solubility of over-expressed protein; (1) changing the vector, (2) changing the host, (3) changing the culture parameters of the recombinant host strain, (4) co-expression of other genes and (5) changing the gene sequences, which may help increase expression and the proper folding of desired protein. Here we present the resources available for the expression of a gene in E. coli to get a substantial amount of good quality recombinant protein. The resources include different strains of E. coli, different E. coli expression vectors, different physical and chemical agents and the co expression of chaperone interacting proteins. Perhaps it would be the solutions to such problems that will finally lead to the maturity of the application of recombinant proteins. The proposed solutions to such problems will finally lead to the maturity of the application of recombinant proteins.

Multidrug resistant commensal Escherichia coli in animals and its impact for public health

Abstract: After the era of plentiful antibiotics we are alarmed by the increasing number of antibiotic resistant strains. The genetic flexibility and adaptability of E. coli to constantly changing environments allows to acquire a great number of antimicrobial resistance mechanisms. Commensal strains of E. coli as versatile residents of the lower intestine are also repeatedly challenged by antimicrobial pressures during the lifetime of their host. As a consequence, commensal strains acquire the respective resistance genes, and/or develop resistant mutants in order to survive and maintain microbial homeostasis in the lower intestinal tract. Thus, commensal E. coli strains are regarded as indicators of antimicrobial load on their hosts. This chapter provides a short historic background of the appearance and presumed origin and transfer of antimicrobial resistance genes in commensal intestinal E. coli of animals with comparative information on their pathogenic counterparts. The dynamics, development and ways of evolution of resistance in the E. coli populations differ according to hosts, resistance mechanisms and antimicrobial classes used. The most frequent tools of E. coli against a variety of antimicrobials are the efflux pumps and mobile resistance mechanisms carried by plasmids and/or other transferable elements. The emergence of hybrid plasmids (both resistance and virulence) among E. coli is of further concern. Co-existence and co-transfer of these “bad genes” in this huge and most versatile in vivo compartment may represent an increased public health risk in the future. Significance of multidrug resistant (MDR) commensal E. coli seem to be highest in the food animal industry, acting as reservoir for intra- and interspecific exchange and a source for spread of MDR determinants through contaminated food to humans. Thus, public health potential of MDR commensal E. coli of food animals can be a concern and needs monitoring and more molecular analysis in the future.

Nutritional Basis for Colonization Resistance by Human Commensal Escherichia coli Strains HS and Nissle 1917 against E. coli O157:H7 in the Mouse Intestine

Abstract: Escherichia coli is a single species consisting of many biotypes, some of which are commensal colonizers of mammals and others that cause disease. Humans are colonized on average with five commensal biotypes, and it is widely thought that the commensals serve as a barrier to infection by pathogens. Previous studies showed that a combination of three pre-colonized commensal E. coli strains prevents colonization of E. coli O157:H7 in a mouse model (Leatham, et al., 2010, Infect Immun 77: 2876–7886). The commensal biotypes included E. coli HS, which is known to successfully colonize humans at high doses with no adverse effects, and E. coli Nissle 1917, a human commensal strain that is used in Europe as a preventative of traveler's diarrhea. We hypothesized that commensal biotypes could exert colonization resistance by consuming nutrients needed by E. coli O157:H7 to colonize, thus preventing this first step in infection. Here we report that to colonize streptomycin-treated mice E. coli HS consumes six of the twelve sugars tested and E. coli Nissle 1917 uses a complementary yet divergent set of seven sugars to colonize, thus establishing a nutritional basis for the ability of E. coli HS and Nissle 1917 to occupy distinct niches in the mouse intestine. Together these two commensals use the five sugars previously determined to be most important for colonization of E. coli EDL933, an O157:H7 strain. As predicted, the two commensals prevented E. coli EDL933 colonization. The results support a model in which invading pathogenic E. coli must compete with the gut microbiota to obtain the nutrients needed to colonize and establish infection; accordingly, the outcome of the challenge is determined by the aggregate capacity of the native microbiota to consume the nutrients required by the pathogen.

A Moonlighting Enzyme Links Escherichia coli Cell Size with Central Metabolism

Abstract: Growth rate and nutrient availability are the primary determinants of size in single-celled organisms: rapidly growing Escherichia coli cells are more than twice as large as their slow growing counterparts. Here we report the identification of the glucosyltransferase OpgH as a nutrient-dependent regulator of E. coli cell size. During growth under nutrient-rich conditions, OpgH localizes to the nascent septal site, where it antagonizes assembly of the tubulin-like cell division protein FtsZ, delaying division and increasing cell size. Biochemical analysis is consistent with OpgH sequestering FtsZ from growing polymers. OpgH is functionally analogous to UgtP, a Bacillus subtilis glucosyltransferase that inhibits cell division in a growth rate-dependent fashion. In a striking example of convergent evolution, OpgH and UgtP share no homology, have distinct enzymatic activities, and appear to inhibit FtsZ assembly through different mechanisms. Comparative analysis of E. coli and B. subtilis reveals conserved aspects of growth rate regulation and cell size control that are likely to be broadly applicable. These include the conservation of uridine diphosphate glucose as a proxy for nutrient status and the use of moonlighting enzymes to couple growth rate-dependent phenomena to central metabolism.

Current state and recent advances in biopharmaceutical production in Escherichia coli , yeasts and mammalian cells

Abstract: Almost all of the 200 or so approved biopharmaceuticals have been produced in one of three host systems: the bacterium Escherichia coli, yeasts (Saccharomyces cerevisiae, Pichia pastoris) and mammalian cells. We describe the most widely used methods for the expression of recombinant proteins in the cytoplasm or periplasm of E. coli, as well as strategies for secreting the product to the growth medium. Recombinant expression in E. coli influences the cell physiology and triggers a stress response, which has to be considered in process development. Increased expression of a functional protein can be achieved by optimizing the gene, plasmid, host cell, and fermentation process. Relevant properties of two yeast expression systems, S. cerevisiae and P. pastoris, are summarized. Optimization of expression in S. cerevisiae has focused mainly on increasing the secretion, which is otherwise limiting. P. pastoris was recently approved as a host for biopharmaceutical production for the first time. It enables high-level protein production and secretion. Additionally, genetic engineering has resulted in its ability to produce recombinant proteins with humanized glycosylation patterns. Several mammalian cell lines of either rodent or human origin are also used in biopharmaceutical production. Optimization of their expression has focused on clonal selection, interference with epigenetic factors and genetic engineering. Systemic optimization approaches are applied to all cell expression systems. They feature parallel high-throughput techniques, such as DNA microarray, next-generation sequencing and proteomics, and enable simultaneous monitoring of multiple parameters. Systemic approaches, together with technological advances such as disposable bioreactors and microbioreactors, are expected to lead to increased quality and quantity of biopharmaceuticals, as well as to reduced product development times.

Uropathogenic Escherichia coli in Iran: Serogroup distributions, virulence factors and antimicrobial resistance properties

Abstract: Background Urinary tract infections (UTIs) are one of the most common bacterial infections with global expansion. These infections are predominantly caused by uropathogenic Escherichia coli (UPEC).

Screening for Enhanced Triacetic Acid Lactone Production by Recombinant Escherichia coli Expressing a Designed Triacetic Acid Lactone Reporter

Abstract: Triacetic acid lactone (TAL) is a signature byproduct of polyketide synthases (PKSs) and a valuable synthetic precursor. We have developed an endogenous TAL reporter by engineering the Escherichia coli regulatory protein AraC to activate gene expression in response to TAL. The reporter enabled in vivo directed evolution of Gerbera hybrida 2-pyrone synthase activity in E. coli . Two rounds of mutagenesis and high-throughput screening yielded a variant conferring ~20-fold increased TAL production. The catalytic efficiency (kcat/Km) of the variant toward the substrate malonyl-CoA was improved 19-fold. This study broadens the utility of engineered AraC variants as customized molecular reporters. In addition, the TAL reporter can find applications in other basic PKS activity screens.

Purification and Characterization of Biofilm-Associated EPS Exopolysaccharides from ESKAPE Organisms and Other Pathogens

Abstract: In bacterial biofilms, high molecular weight, secreted exopolysaccharides can serve as a scaffold to which additional carbohydrates, proteins, lipids, and nucleic acids adhere, forming the matrix of the developing biofilm. Here we report methods to extract and purify high molecular weight (>15 kDa) exopolysaccharides from biofilms of eight human pathogens, including species of Staphylcococcus, Klebsiella, Acinetobacter, Pseudomonas, and a toxigenic strain of Escherichia coli O157:H7. Glycosyl composition analysis indicated a high total mannose content across all strains with P. aeruginosa and A. baumannii exopolysaccharides comprised of 80–90% mannose, K. pneumoniae and S. epidermidis strains containing 40–50% mannose, and E. coli with ∼10% mannose. Galactose and glucose were also present in all eight strains, usually as the second and third most abundant carbohydrates. N-acetyl-glucosamine and galacturonic acid were found in 6 of 8 strains, while arabinose, fucose, rhamnose, and xylose were found in 5 of 8 strains. For linkage analysis, 33 distinct residue-linkage combinations were detected with the most abundant being mannose-linked moieties, in line with the composition analysis. The exopolysaccharides of two P. aeruginosa strains analyzed were consistent with the Psl carbohydrate, but not Pel or alginate. The S. epidermidis strain had a composition rich in mannose and glucose, which is consistent with the previously described slime associated antigen (SAA) and the extracellular slime substance (ESS), respectively, but no polysaccharide intracellular adhesion (PIA) was detected. The high molecular weight exopolysaccharides from E. coli, K. pneumoniae, and A. baumannii appear to be novel, based on composition and/or ratio analysis of carbohydrates.

Genetic mechanisms of antimicrobial resistance identified in Salmonella enterica, Escherichia coli, and Enteroccocus spp. isolated from U.S. food animals.

Abstract: The prevalence of antimicrobial resistance (AR) in bacteria isolated from U.S. food animals has increased over the last several decades as have concerns of AR foodborne zoonotic human infections. Resistance mechanisms identified in U.S. animal isolates of Salmonella enterica included resistance to aminoglycosides (e.g. alleles of aacC, aadA, aadB, ant, aphA, and StrAB), -lactams (e.g. blaCMY-2, TEM-1, PSE-1), chloramphenicol (e.g. floR, cmlA, cat1, cat2), folate pathway inhibitors (e.g. alleles of sul and dfr), and tetracycline (e.g. alleles of tet(A), (B), (C), (D), (G) and tetR). In the U.S., multidrug resistance (MDR) mechanisms in Salmonella animal isolates were associated with integrons, or mobile genetic elements (MGEs) such as IncA/C plasmids which can be transferred among bacteria. It is thought that AR Salmonella originates in food animals and is transmitted through food to humans. However, some AR Salmonella isolated from humans in the U.S. have different AR elements than those isolated from food animals, suggesting a different etiology for some AR human infections. The AR mechanisms identified in isolates from outside the U.S. are also predominantly different. For example the extended spectrum -lactamases (ESBLs) are found in human and animal isolates globally; however, in the U.S., ESBLs thus far have only been found in human and not food animal isolates. Commensal bacteria in animals including Escherichia coli and Enterococcus spp. may be reservoirs for AR mechanisms. Many of the AR genes and MGEs found in E. coli isolated from U.S. animals are similar to those found in Salmonella. Enterococcus spp. isolated from animals frequently carry MGEs with AR genes, including resistances to aminoglycosides (e.g. alleles of aac, ant, and aph), macrolides (e.g. erm(A), erm(B),and msrC), and tetracyclines (e.g. tet(K), (L), (M), (O), (S)). Continuing investigations are required to help understand and mitigate the impact of AR bacteria on human and animal health.

L-glutamine provides acid resistance for Escherichia coli through enzymatic release of ammonia

Abstract: Bacteria, exemplified by enteropathogenic Escherichia coli (E. coli), rely on elaborate acid resistance systems to survive acidic environment (such as the stomach). Comprehensive understanding of bacterial acid resistance is important for prevention and clinical treatment. In this study, we report a previously uncharacterized type of acid resistance system in E. coli that relies on L-glutamine (Gln), one of the most abundant food-borne free amino acids. Upon uptake into E. coli, Gln is converted to L-glutamate (Glu) by the acid-activated glutaminase YbaS, with concomitant release of gaseous ammonia. The free ammonia neutralizes proton, resulting in elevated intracellular pH under acidic environment. We show that YbaS and the amino acid antiporter GadC, which exchanges extracellular Gln with intracellular Glu, together constitute an acid resistance system that is sufficient for E. coli survival under extremely acidic environment.

Streptomycin-Induced Inflammation Enhances Escherichia coli Gut Colonization Through Nitrate Respiration

Abstract: Treatment with streptomycin enhances the growth of human commensal Escherichia coli isolates in the mouse intestine, suggesting that the resident microbial community (microbiota) can inhibit the growth of invading microbes, a phenomenon known as “colonization resistance.” However, the precise mechanisms by which streptomycin treatment lowers colonization resistance remain obscure. Here we show that streptomycin treatment rendered mice more susceptible to the development of chemically induced colitis, raising the possibility that the antibiotic might lower colonization resistance by changing mucosal immune responses rather than by preventing microbe-microbe interactions. Investigation of the underlying mechanism revealed a mild inflammatory infiltrate in the cecal mucosa of streptomycin-treated mice, which was accompanied by elevated expression of Nos2 , the gene that encodes inducible nitric oxide synthase. In turn, this inflammatory response enhanced the luminal growth of E. coli by nitrate respiration in a Nos2 -dependent fashion. These data identify low-level intestinal inflammation as one of the factors responsible for the loss of resistance to E. coli colonization after streptomycin treatment. IMPORTANCE Our intestine is host to a complex microbial community that confers benefits by educating the immune system and providing niche protection. Perturbation of intestinal communities by streptomycin treatment lowers “colonization resistance” through unknown mechanisms. Here we show that streptomycin increases the inflammatory tone of the intestinal mucosa, thereby making the bowel more susceptible to dextran sulfate sodium treatment and boosting the Nos2- dependent growth of commensal Escherichia coli by nitrate respiration. These data point to the generation of alternative electron acceptors as a by-product of the inflammatory host response as an important factor responsible for lowering resistance to colonization by facultative anaerobic bacteria such as E. coli.

10-Fold increase (2006–11) in the rate of healthy subjects with extended-spectrum β-lactamase-producing Escherichia coli faecal carriage in a Parisian check-up centre

Abstract: Objectives: In 2006, 0.6% of healthy subjects living in the Paris area had extended-spectrum b-lactamase (ESBL)-producing Escherichia coli in their gut. To assess the evolution of this rate, a study identical to that of 2006 was conducted in 2011. Participants and methods: Healthy adults who visited the IPC check-up centre in February–March 2011 and agreed to participate, provided stools and answered a questionnaire on the visit day. Stools were analysed to detect ESBL producers and to isolate the dominant E. coli population. ESBLs were molecularly characterized. For the subjects harbouring ESBL-producing E. coli, the phylogenetic group and sequence type (ST) were determined for both ESBL-producing and dominant E. coli isolates. PFGE profiles were also determined when two types of isolates had the same ST. Results: Among the 345 subjects included, 21 (6%) had ESBL-producing E. coli faecal carriage. None of the previously published risk factors was identified. CTX-M accounted for 86% and SHV-12 for 14%. Dominant and ESBL-producing E. coli were similarly distributed into phylogenetic groups (A, 52%–48%; B1, 5%; B2, 24%–14%; and D, 19% –33%). Dominant and ESBL-producing E. coli displayed a polyclonal structure (18 STs each). However, ST10 and ST131 were identified in dominant and ESBL-producing E. coli isolates from different subjects. Most (20/21) ESBL producers were subdominant and belonged (16/21) to STs different from that of the corresponding dominant E. coli. Conclusions: The 10-fold increase in the rate of healthy subjects with ESBL-producing E. coli faecal carriage over a 5 year period suggests wide dissemination of these isolates in the Parisian community.