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The selC tRNA gene is a common site for the insertion of pathogenicity islands in a variety of bacterial enteric pathogens. We demonstrate here that Escherichia coli that produces Shiga toxin 2d and does not harbor the locus of enterocyte effacement (LEE) contains, instead, a novel genomic island. In one representative strain ( E . coli O91:H − strain 4797/97), this island is 33,014 bp long and, like LEE in E . coli O157:H7, is integrated 15 bp downstream of selC . This E . coli O91:H − island contains genes encoding a novel serine protease, termed EspI; an adherence-associated locus, similar to iha of E . coli O157:H7; an E . coli vitamin B 12 receptor (BtuB); an AraC-type regulatory module; and four homologues of E . coli phosphotransferase proteins. The remaining sequence consists largely of complete and incomplete insertion sequences, prophage sequences, and an intact phage integrase gene that is located directly downstream of the chromosomal selC . Recombinant EspI demonstrates serine protease activity using pepsin A and human apolipoprotein A-I as substrates. We also detected Iha-reactive protein in outer membranes of a recombinant clone and 10 LEE-negative, Shiga toxin-producing E . coli (STEC) strains by immunoblot analysis. Using PCR analysis of various STEC, enteropathogenic E . coli , enterotoxigenic E . coli , enteroaggregative E . coli , uropathogenic E . coli , and enteroinvasive E . coli strains, we detected the iha homologue in 59 (62%) of 95 strains tested. In contrast, espI and btuB were present in only two (2%) and none of these strains, respectively. We conclude that the newly described island occurs exclusively in a subgroup of STEC strains that are eae negative and contain the variant stx 2d gene.

The exploitation of bacterial genome sequences has so far provided a wealth of new general information about the genetic diversity of bacteria, such as that of many pathogens. Comparative genomics uncovered many genome variations in closely related bacteria and revealed basic principles involved in bacterial diversification, improving our knowledge of the evolution of bacterial pathogens. A correlation between metabolic versatility and genome size has become evident. The degenerated life styles of obligate intracellular pathogens correlate with significantly reduced genome sizes, a phenomenon that has been termed "evolution by reduction". These mechanisms can permanently alter bacterial genotypes and result in adaptation to their environment by genome optimization. In this review, we summarize the recent results of genome-wide approaches to studying the genetic diversity of pathogenic bacteria that indicate that the acquisition of DNA and the loss of genetic information are two important mechanisms that contribute to strain-specific differences in genome content.

The sfa I determinant encoding the S-fimbrial adhesin of uropathogenic Escherichia coli strains was found to be located on a pathogenicity island of uropathogenic E. coli strain 536. This pathogenicity island, designated PAI III 536 , is located at 5.6 min of the E. coli chromosome and covers a region of at least 37 kb between the tRNA locus thrW and yagU . As far as it has been determined, PAI III 536 also contains genes which code for components of a putative enterochelin siderophore system of E. coli and Salmonella spp. as well as for colicin V immunity. Several intact or nonfunctional mobility genes of bacteriophages and insertion sequence elements such as transposases and integrases are present on PAI III 536 . The presence of known PAI III 536 sequences has been investigated in several wild-type E. coli isolates. The results demonstrate that the determinants of the members of the S-family of fimbrial adhesins may be located on a common pathogenicity island which, in E. coli strain 536, replaces a 40-kb DNA region which represents an E. coli K-12-specific genomic island.

Suppression subtractive hybridisation (SSH) was performed to identify genomic differences between the uropathogenic Escherichia coli strain 536 and the non-pathogenic E. coli K-12 strain MG1655. In total, 22 DNA fragments were isolated which were specific for strain 536. Five of these fragments showed homology to known virulence determinants and four fragments matched genes for lipopolysaccharide (LPS) or capsule biosynthesis and a siderophore receptor. Seven fragments did not show any homology to known genes. These fragments may represent parts of putative pathogenicity islands (PAIs). Whereas two fragments were highly specific for uropathogenic E. coli (UPEC), the other fragments could also be detected among the other tested wild-type strains.

The outer membrane protein ChuA responsible for hemin utilization has been recently identified in several pathogenic Escherichia coli strains. We report that the regulatory protein RfaH influences ChuA expression in the uropathogenic E. coli strain 536. In an rfaH mutant, the chuA transcript as well as the ChuA protein levels were significantly decreased in comparison with those in the wild-type strain. Within the chuA gene, a consensus motif known as the JUMPStart (just upstream of many polysaccharide associated gene starts) sequence was found, which is shared by RfaH-affected operons. Furthermore, the presence of two different subclasses of the chuA determinant and their distribution in E. coli pathogroups are described.

Various genetic mechanisms including point mutations, genetic rearrangements and lateral gene transfer processes contribute to the evolution of microbes. Long-term processes leading to the development of new species or subspecies are termed macroevolution, and short–term developments, which occur during days or weeks, are considered as microevolution. Both processes, macro– and microevolution need horizontal gene transfer, which is particularly important for the development of pathogenic microorganisms. Plasmids, bacteriophages and so–called pathogenicity islands (PAIs) play a crucial role in the evolution of pathogens. During microevolution, genome variability of pathogenic microbes leads to new phenotypes, which play an important role in the acute development of an infectious disease. Infections due to Staphylococcus epidermidis , Candida albicans and Escherichia coli will be described with special emphasis on processes of microevolution. In contrast, the development of PAIs is a process involved in macroevolution. PAIs are especially important in processes leading to new pathotypes or even species. In this review, particular attention will be given to the fact that the evolution of pathogenic microbes can be considered as a specific example for microbial evolution in general.

The uropathogenic Escherichia coli strain 536 (O6:K15:H31) carries four distinct DNA regions in its chromosome, termed pathogenicity islands (PAIs I536 to IV536). Each of these PAIs encodes at least one virulence factor. All four PAIs are associated with tRNA genes. PAI I536 and PAI II536 can be spontaneously deleted from the chromosome by homologous recombination between flanking direct repeats. The deletion of PAI II536 results in the truncation of the associated gene leuX encoding the tRNALeu. This tRNA influences the expression of various virulence traits. In order to get a deeper insight into the role of PAI I536/II536 and of the tRNA5LeU for the protein expression, the protein expression patterns of Escherichia coli 536 and different derivatives were studied. Differences in the protein expression patterns of the wild-type strain Escherichia coli 536, its mutants 536-21 (PAI I536-, PAI II536-, leuX-), 536delta102 (PAI I536+, PAI II536+, leuX-) as well as of the strain 536R3 (PAI I536-, PAI II536-, leuX+) were analyzed by two-dimensional polyacrylamide gel electrophoresis and MALDI-TOF mass spectrometry. We identified about 39 different intracellular proteins whose expression is markedly altered in the different strain backgrounds. These differences can be linked either to the presence or absence of the PAI I536 and PAI II536 or to that of the tRNA gene leuX. The identities of 34 proteins have been determined by MALDI-TOF-MS. The identification of five proteins was not possible. The results suggest that proteome analysis is an efficient approach to study differences in global gene expression. The comparison of protein expression patterns of the uropathogenic E. coli strain 536 and different derivatives revealed that in this strain the expression of various proteins including those encoded by many housekeeping genes is affected by the presence of PAI I536 and Pai II536 or by that of the tRNA5Leu.

The uropathogenic Escherichia coli strain 536 (O6:K15: H31) carries two large chromosomal pathogenicity islands (Pais). Both Pais are flanked by tRNA genes. Spontaneous deletion of Pai II results in truncation of the leuX tRNALeu5 gene. This tRNA is required for the expression of type 1 fimbriae (Fim) and other virulence factors. Transcription of fimA, encoding the major type 1 fimbrial subunit is controlled by an invertable DNA switch. The inversion is catalysed by two recombinases, FimB and FimE. FimB is able to turn the switch on, FimE only off. The fimB gene of strain 536 contains five TTG codons recognized by tRNALeu5, fimE contains only two. It was proposed that turning on the fim switch requires efficient translation of FimB, in turn requiring tRNALeu5. Strains in which the TTG codons in fimB were replaced with CTG codons at the wild-type locus were able to produce type 1 fimbriae in the absence of leuX. fimB transcription was influenced by the presence of leuX, but only slightly affected by the presence or absence of leuX codons in fimB. FimB translation was significantly higher from codon-replaced fimB genes than that of wild-type fimB genes in various strain backgrounds. The fim switch was shown to be switched off in leuX − derivatives of E. coli 536, but could be found in the on position when the codon-altered fimB gene was exchanged into the chromosome of these strains. From these data, it is apparent that tRNALeu5 is required for efficient translation of FimB, in turn, leading to type 1 fimbrial expression.

Asymptomatic bacteriuria established by intravesical inoculation of Escherichia coli 83972 is protective in patients with recurrent urinary tract infections. In this randomized, controlled crossover study a total of 3 symptomatic urinary tract infection episodes developed in 2 patients while they carried E. coli 83972. We examined whether virulence reacquisition by symptom isolates may account for the switch from asymptomatic bacteriuria to symptomatic urinary tract infection.We used E. coli 83972 re-isolates from 2 patients in a prospective study and from another 2 in whom symptoms developed after study completion. We phylogenetically classified the re-isolates, and identified the genomic restriction patterns and gene expression profiles as well as virulence gene structure and phenotypes. In vivo virulence was examined in the murine urinary tract infection model.The fim, pap, foc, hlyA, fyuA, iuc, iroN, kpsMT K5 and malX genotypes of the symptomatic re-isolates remained unchanged. Bacterial gene expression profiles of flagellated symptomatic re-isolates were unique to each host, providing no evidence of common deregulation. Symptomatic isolates did not differ in virulence from the wild-type strain, as defined in the murine urinary tract infection model by persistence, symptoms or innate immune activation.The switch from asymptomatic E. coli 83972 carriage to symptomatic urinary tract infection was not explained by reversion to a functional virulence gene repertoire.

International audience; The role of the post-transcriptional carbon storage regulator (Csr) system in nutrient utilization and in the control of the central metabolism in E. coli reference commensal strain Nissle 1917 was investigated. Analysis of the growth capabilities of mutants altered for various components of the Csr system (csrA51, csrB, csrC and csrD mutations) showed that only the protein CsrA - the key component of the system - exerts a marked role in carbon nutrition. Attenuation of CsrA activity in the csrA51 mutant affects the growth efficiency on a broad range of physiologically relevant carbon sources, including compounds utilized by the Entner-Doudoroff (ED) pathway. Detailed investigations of the metabolomes and fluxomes of mutants and wild-type cells grown on carbon sources representative of glycolysis and of the ED pathway (glucose and gluconate, respectively), revealed significant re-adjusting of central carbon metabolism for both compounds in the csrA51 mutant. However, the metabolic re-adjusting observed on gluconate was strikingly different from that observed on glucose, indicating a nutrient-specific control of metabolism by the Csr system.

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.

Heterogeneity of cell population is a key component behind the evolutionary success of Escherichia coli. The heterogeneity supports species adaptation and mainly results from lateral gene transfer. Adaptation may also involve genomic alterations that affect regulation of conserved genes. Here we analysed regulation of the mat (or ecp) genes that encode a conserved fimbrial adhesin of E. coli. We found that the differential and temperature-sensitive expression control of the mat operon is dependent on mat promoter polymorphism and closely linked to phylogenetic grouping of E. coli. In the mat promoter lineage favouring fimbriae expression, the mat operon-encoded regulator MatA forms a positive feedback loop that overcomes the repression by H-NS and stabilizes the fimbrillin mRNA under low growth temperature, acidic pH or elevated levels of acetate. The study exemplifies phylogenetic group-associated expression of a highly common surface organelle in E. coli.

Between Commensalism and Pathogenicity:Bacterial and Host Aspects.- E. coli as an all-rounder: The thin line between commensalism and pathogenicity.- What distinguishes non-pathogenic, from medium and highly pathogenic staphylococci?.- Microevolution of Pseudomonas aeruginosa to a chronic pathogen of the cystic fibrosis lung.- Lactobacillus: Host-Microbe Relationships.- Bacterial Moonlighting Proteins and Bacterial Virulence.- Symbionts and pathogens - what is the difference?.-Host-microbe Interaction in the Intestinal Tract.- Ecology and physiology of the intestinal tract.- The gut microflora and its variety of roles in health and disease.- Mammalian intestinal host-microbe relationships.- Contribution of the intestinal microbiota to human health - from birth to 100 years of age.- Subject index.

The normal flora furnishes the host with ecological barriers that prevent pathogen attack while maintaining tissue homeostasis. Urinary tract infections (UTIs) constitute a highly relevant model of microbial adaptation in which some patients infected with Escherichia coil develop acute pyelonephritis, while other patients with bacteriuria exhibit an asymptomatic carrier state similar to bacterial commensalism. It remains unclear if the lack of destructive inflammation merely reflects low virulence or if carrier strains actively inhibit disease-associated responses in the host. Here, we identify a new mechanism of bacterial adaptation through broad suppression of RNA polymerase II-dependent (Pol II-dependent) host gene expression. Over 60% of all genes were suppressed 24 hours after human inoculation with the prototype asymptomatic bacteriuria (ABU) strain E. coil 83972, and inhibition was verified by infection of human cells. Specific repressors and activators of Pol II-dependent transcription were modified, Pol II phosphorylation was inhibited, and pathogen-specific signaling was suppressed in cell lines and inoculated patients. An increased frequency of strains inhibiting Pol II was epidemiologically verified in ABU and fecal strains compared with acute pyelonephritis, and a Pol II antagonist suppressed the disease-associated host response. These results suggest that by manipulating host gene expression, ABU strains promote tissue integrity while inhibiting pathology. Such bacterial modulation of host gene expression may be essential to sustain asymptomatic bacterial carriage by ensuring that potentially destructive immune activation will not occur.

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.

Pertussis toxin (PTx), the major virulence factor of the whooping cough-causing bacterial pathogen Bordetella pertussis, permeabilizes the blood–brain barrier (BBB) in vitro and in vivo. Breaking barriers might promote translocation of meningitis-causing bacteria across the BBB, thereby facilitating infection. PTx activates several host cell signaling pathways exploited by the neonatal meningitis-causing Escherichia coli K1-RS218 for invasion and translocation across the BBB. Here, we investigated whether PTx and E. coli K1-RS218 exert similar effects on MAPK p38, NF-κB activation and transcription of downstream targets in human cerebral endothelial TY10 cells using qRT-PCR, Western blotting, and ELISA in combination with specific inhibitors. PTx and E. coli K1-RS218 activate MAPK p38, but only E. coli K1-RS218 activates the NF-κB pathway. mRNA and protein levels of p38 and NF-κB downstream targets including IL-6, IL-8, CxCL-1, CxCL-2 and ICAM-1 were increased. The p38 specific inhibitor SB203590 blocked PTx-enhanced activity, whereas E. coli K1-RS218’s effects were inhibited by the NF-κB inhibitor Bay 11-7082. Further, we found that PTx enhances the adherence of human monocytic THP-1 cells to human cerebral endothelial TY10 cells, thereby contributing to enhanced translocation. These modulations of host cell signaling pathways by PTx and meningitis-causing E. coli support their contributions to pathogen and monocytic THP-1 cells translocation across the BBB.

Nowhere is the relationship between microorganisms and eukaryotes as diverse, intimate and clinically relevant as in the gastrointestinal tract. An estimated 500–1000 mostly anaerobic species reside in the intestine, approaching enormous densities of 10 11 –10 12 organisms per gram colon content. The commensal interactions between intestinal microorganisms and animal hosts have been difficult to study in the past because of the diversity of microorganisms involved and because of the lack of culturability that accompanies many microbial consortia. However, recent work has provided new insights into these interactions.

Table of Contents Part I Bacterial Infections 1 Impact of Genome Plasticity on Adaptation of Escherichia coli during Urinary Bladder Colonization Ulrich Dobrindt, Jaroslaw Zdziarski, and Jorg Hacker 2 Genotypic Changes in Enterohemorrhagic Escherichia coli during Human Infection Alexander Mellmann, Martina Bielaszewska, and Helge Karch 3 Genomic Fluidity of the Human Gastric Pathogen Helicobacter pylori Niyaz Ahmed, Singamaneni Haritha Devi, Shivendra Tenguria, Mohammed Majid, Syed Asad Rahman, and Seyed E. Hasnain 4 Genome Structure and Variability in Coagulase-Negative Staphylococci Wilma Ziebuhr 5 Genome Plasticity in Legionella pneumophila and Legionella longbeachae: Impact on Host Cell Exploitation L. Gomez Valero, C. Rusniok, and C. Buchrieser 6 Genome Plasticity in Salmonella enterica and Its Relevance to Host-Pathogen Interactions Rosana B. Ferreira, Michelle M. Buckner, and B. Brett Finlay 7 Mechanisms of Genome Plasticity in Neisseria meningitidis: Fighting Change with Change Roland Schwarz, Biju Joseph, Matthias Frosch, and Christoph Schoen 8 Selfish Elements and Self-Defense in the Enterococci Kelli L. Palmer and Michael S. Gilmore Part II Viral Infections 9 Host-Driven Plasticity of the Human Immunodeficiency Virus Genome Stephen Norley and Reinhard Kurth 10 Genome Plasticity of Influenza Viruses Silke Stertz and Peter Palese 11 Plasticity of the Hepatitis C Virus Genome Joerg Timm and Michael Roggendor 12 Genome Diversity and Host Interaction of Noroviruses Eckart Schreier 13 Genome Diversity and Evolution of Rotaviruses Jelle Matthijnssens and Ulrich Desselberger 14 Genome Plasticity of Papillomaviruses Hans-Ulrich Bernard 15 Genome Plasticity of Herpesviruses: Conservative yet Flexible Mirko Trilling, Vu Thuy Khanh Le, and Hartmut Hengel Part III Parasitic and Fungal Infections 16 Genome Diversity, Population Genetics, and Evolution of Malaria Parasites Xin-zhuan Su and Deirdre A. Joy 17 The Fundamental Contribution of Genome Hypervariability to the Success of a Eukaryotic Pathogen, Trypanosoma brucei J. David Barry 18 Genome Plasticity in Candida albicans Claude Pujol and David R. Soll 19 Genome Plasticity of Aspergillus Species Thorsten Heinekamp and Axel A. Brakhage Part IV Host Susceptibility 20 DNA Polymorphisms and Their Relevance for Infections with Human Cytomegalovirus and Aspergillus fumigatus Markus Mezger, Hermann Einsele, and Juergen Loeffler 21 Host Genetic Variation, Innate Immunity, and Susceptibility to Urinary Tract Infection Bryndis Ragnarsdottir and Catharina Svanborg

Flagella provide advantages to Escherichia coli by facilitating taxis towards nutrients and away from unfavourable niches. On the other hand, flagellation is an energy sink to the bacterial cell, and flagella also stimulate host innate inflammatory responses against infecting bacteria. The flagellar assembly pathway is ordered and under a complex regulatory circuit that involves three classes of temporally regulated promoters as well as the flagellar master regulator FlhD(4)C(2). We report here that transcription of the flhDC operon from the class 1 promoter is under negative regulation by MatA, a key activator of the common mat (or ecp) fimbria operon that enhances biofilm formation by E. coli. Ectopic expression of MatA completely precluded motility and flagellar synthesis in the meningitis-associated E. coli isolate IHE 3034. Northern blotting, analysis of chromosomal promoter-lacZ fusions and electrophoretic mobility shift assays revealed an interaction between MatA and the flhDC promoter region that apparently repressed flagellum biosynthesis. However, inactivation of matA in the chromosome of IHE 3034 had only a minor effect on flagellation, which underlines the complexity of regulatory signals that promote flagellation in E. coli. We propose that the opposite regulatory actions of MatA on mat and on flhDC promoters advance the adaptation of E. coli from a planktonic to an adhesive lifestyle.

The mammalian gut harbors a dense microbial community interacting in multiple ways, including horizontal gene transfer (HGT). Pangenome analyses established particularly high levels of genetic flux between Gram-negative Enterobacteriaceae . However, the mechanisms fostering intraenterobacterial HGT are incompletely understood. Using a mouse colitis model, we found that Salmonella -inflicted enteropathy elicits parallel blooms of the pathogen and of resident commensal Escherichia coli . These blooms boosted conjugative HGT of the colicin-plasmid p2 from Salmonella enterica serovar Typhimurium to E. coli . Transconjugation efficiencies of ∼100% in vivo were attributable to high intrinsic p2-transfer rates. Plasmid-encoded fitness benefits contributed little. Under normal conditions, HGT was blocked by the commensal microbiota inhibiting contact-dependent conjugation between Enterobacteriaceae . Our data show that pathogen-driven inflammatory responses in the gut can generate transient enterobacterial blooms in which conjugative transfer occurs at unprecedented rates. These blooms may favor reassortment of plasmid-encoded genes between pathogens and commensals fostering the spread of fitness-, virulence-, and antibiotic-resistance determinants.

International audience; Oral administration of the probiotic bacterium Escherichia coli Nissle 1917 improves chronic inflammatory bowel diseases, but the molecular basis for this therapeutic efficacy is unknown. E. coli Nissle 1917 harbors a cluster of genes coding for the biosynthesis of hybrid nonribosomal peptide-polyketide(s). This biosynthetic pathway confers the ability for bacteria to induce DNA double strand breaks in eukaryotic cells. Here we reveal that inactivation of the clbA gene within this genomic island abrogated the ability for the strain to induce DNA damage and chromosomal abnormalities in non-transformed cultured rat intestinal epithelial cells but is required for the probiotic activity of E. coli Nissle 1917. Thus, evaluation of colitis severity induced in rodent fed with E. coli Nissle 1917 or an isogenic non-genotoxic mutant demonstrated the need for a functional biosynthetic pathway both in the amelioration of the disease and in the modulation of cytokine expression. Feeding rodents with a complemented strain for which genotoxicity was restored confirmed that this biosynthetic pathway contributes to the health benefits of the probiotic by modulating its immunomodulatory properties. Our data provide additional evidence for the benefit of this currently used probiotic in colitis but remind us that an efficient probiotic may also have side effects as any other medication.

Escherichia coli (E. coli) exhibits considerable physiological and metabolic versatility and includes a variety of non-pathogenic, commensal variants, which belong to the normal gut flora of humans and warm-blooded animals. Additionally, several pathogenic variants have been identified which cause various types of intestinal or extraintestinal infections in humans and animals. In contrast to intestinal pathogenic E. coli (IPEC), which are obligate pathogens, extraintestinal pathogenic E. coli (ExPEC) are facultative pathogens which belong to the normal gut flora of a certain fraction of the healthy population where they live as commensals. Comparative genomics and epidemiological studies have been applied to study genomic diversity, markers, and phenotypic traits that may support discrimination of different E. coli pathotypes. Whereas IPEC are often epidemiologically and phylogenetically distinct from ExPEC and non-pathogenic, commensal strains, many ExPEC and non-pathogenic E. coli share large genomic fractions. Furthermore, extraintestinal infections of elderly or immunocompromised patients can be caused by E. coli variants which differ in their geno- and phenotypes from archetypal ExPEC. Thus, strain typing based on the detection of a limited number of ExPEC virulence/fitness-related genes may be ambiguous. A limited number of ExPEC-dominated clonal complexes can be identified in the E. coli population by multi locus sequence typing. Nevertheless, ExPEC and non-pathogenic E. coli cannot be clearly discriminated by molecular epidemiological approaches. Increased knowledge of the phylogeny, virulence and fitness traits, and host factors contributing to host susceptibility of the different groups of ExPEC variants is required for a better understanding of the biological basis of ExPEC infections.

Mastitis represents one of the most significant health problems of dairy herds. The two major causative agents of this disease areEscherichia coliandStaphylococcus aureus. Of the first, its lipopolysaccharide (LPS) is thought to play a prominent role during infection. Here, we report the O-antigen (OPS, O-specific polysaccharide) structure of the LPS from bovine mastitis isolateE. coli1303. The structure was determined utilizing chemical analyses, mass spectrometry, and 1D and 2D NMR spectroscopy methods. The O-repeating unit was characterized as -[→4)-β-d-Quip3NAc-(1→3)-α-l-Fucp2OAc-(1→4)-β-d-Galp-(1→3)-α-d-GalpNAc-(1→]- in which theO-acetyl substitution was non-stoichiometric. The nucleotide sequence of the O-antigen gene cluster ofE. coli1303 was also determined. This cluster, located between thegndandgalFgenes, contains 13 putative open reading frames, most of which represent unknown nucleotide sequences that have not been described before. The O-antigen ofE. coli1303 was shown to substitute O-7 of the terminalld-heptose of the K-12 core oligosaccharide. Interestingly, the non-OPS-substituted core oligosaccharide represented a truncated version of the K-12 outer core – namely terminalld-heptose and glucose were missing; however, it possessed a third Kdo residue in the inner core. On the basis of structural and genetic data we show that the mastitis isolateE. coli1303 represents a new serotype and possesses the K-12 core type, which is rather uncommon among human and bovine isolates.

Tellurite (Tel) resistant enterohaemorrhagic Escherichia coli (EHEC) O157:H7 is a global pathogen. In strain EDL933 Tel resistance (Tel(R) ) is encoded by duplicate ter cluster in O islands (OI) 43 and 48, which also harbour iha, encoding the adhesin and siderophore receptor Iha. We identified five EHEC O157:H7 strains that differentiate into large (L) colonies and small (S) colonies with high and low Tel minimal inhibitory concentrations (MICs) respectively. S colonies (Tel-MICs ≤ 4 µg ml⁻¹) sustained large internal deletions within the Tel(R) OIs via homologous recombination between IS elements and lost ter and iha. Moreover, complete excision of the islands occurred by site-specific recombination between flanking direct repeats. Complete excision of OI 43 and OI 48 occurred in 1.81 × 10⁻³ and 1.97 × 10⁻⁴ cells in culture, respectively; internal deletion of OI 48 was more frequent (9.7 × 10⁻¹ cells). Under iron limitation that promotes iha transcription, iha-negative derivatives adhered less well to human intestinal epithelial cells and grew slower than did their iha-positive counterparts. Experiments utilizing iha deletion and complementation mutants identified Iha as the major factor responsible for these phenotypic differences. Spontaneous deletions affecting Tel(R) OIs contribute to EHEC O157 genome plasticity and might impair virulence and/or fitness.

Background The Enterobacteriaceae comprise a large number of clinically relevant species with several individual subspecies. Overlapping virulence-associated gene pools and the high overall genome plasticity often interferes with correct enterobacterial strain typing and risk assessment. Array technology offers a fast, reproducible and standardisable means for bacterial typing and thus provides many advantages for bacterial diagnostics, risk assessment and surveillance. The development of highly discriminative broad-range microbial diagnostic microarrays remains a challenge, because of marked genome plasticity of many bacterial pathogens. Results We developed a DNA microarray for strain typing and detection of major antimicrobial resistance genes of clinically relevant enterobacteria. For this purpose, we applied a global genome-wide probe selection strategy on 32 available complete enterobacterial genomes combined with a regression model for pathogen classification. The discriminative power of the probe set was further tested in silico on 15 additional complete enterobacterial genome sequences. DNA microarrays based on the selected probes were used to type 92 clinical enterobacterial isolates. Phenotypic tests confirmed the array-based typing results and corroborate that the selected probes allowed correct typing and prediction of major antibiotic resistances of clinically relevant Enterobacteriaceae, including the subspecies level, e.g. the reliable distinction of different E. coli pathotypes. Conclusions Our results demonstrate that the global probe selection approach based on longest common factor statistics as well as the design of a DNA microarray with a restricted set of discriminative probes enables robust discrimination of different enterobacterial variants and represents a proof of concept that can be adopted for diagnostics of a wide range of microbial pathogens. Our approach circumvents misclassifications arising from the application of virulence markers, which are highly affected by horizontal gene transfer. Moreover, a broad range of pathogens have been covered by an efficient probe set size enabling the design of high-throughput diagnostics.

Objectives To investigate clinical outomes, bacterial virulence factors, and antimicrobial resistance in E. coli from young men presenting with acute bacterial prostatitis. Methods Initial E. coli isolates from previously healthy young men with no factors compromising urinary tract anatomy or function were tested for virulence-associated genes by polymerase chain reaction (PCR) assays, phylogenetic grouping by triplex polymerase PCR, and antibiotic resistance. Results All 18 patients responded to treatment, including 2 who required long-term therapy. E. coli were allocated to phylogenetic groups B2 (13 strains) and D (5 strains). Prostatitis isolates belonged to clones mainly represented by extraintestinal pathogenic E. coli (ExPEC) and preferentially uropathogenic E. coli and displayed marked accumulation of virulence genes ( hly , cdt 1, clb , pap , sfa / foc , fyuA , iroN, kpsMT (II), and traT ) characteristic of highly virulent ExPEC. All phylogenetic group B2 strains coded for at least 1 toxin with carcinogenic potential (Colibactin, cytolethal distending toxins, or cytotoxic necrotizing factor). In contrast to their accumulation of virulence-associated traits, prostatitis strains were sensitive to standard antibiotics. Conclusions The phylogenetic background and accumulation of an exceptional repertoire of extraintestinal pathogenic virulence-associated genes indicate that these E. coli strains belong to a highly virulent subset of uropathogenic variants. In contrast, antibiotic resistance was minimal in these E. coli strains from previously healthy, young outpatients.

The mat (or ecp) fimbrial operon is ubiquitous and conserved in Escherichia coli, but its functions remain poorly described. In routine growth media newborn meningitis isolates of E. coli express the meningitis-associated and temperature-regulated (Mat) fimbria, also termed E. coli common pilus (ECP), at 20 °C, and here we show that the six-gene (matABCDEF)-encoded Mat fimbria is needed for temperature-dependent biofilm formation on abiotic surfaces. The matBCDEF deletion mutant of meningitis E. coli IHE 3034 was defective in an early stage of biofilm development and consequently unable to establish a detectable biofilm, contrasting with IHE 3034 derivatives deleted for flagella, type 1 fimbriae or S-fimbriae, which retained the wild-type biofilm phenotype. Furthermore, induced production of Mat fimbriae from expression plasmids enabled biofilm-deficient E. coli K-12 cells to form biofilm at 20 °C. No biofilm was detected with IHE 3034 or MG1655 strains grown at 37 °C. The surface expression of Mat fimbriae and the frequency of Mat-positive cells in the IHE 3034 population from 20 °C were high and remained unaltered during the transition from planktonic to biofilm growth and within the matured biofilm community. Considering the prevalence of the highly conserved mat locus in E. coli genomes, we hypothesize that Mat fimbria-mediated biofilm formation is an ancestral characteristic of E. coli.

Bacterial pathogens with the ability to cause persistent infection have different strategies to withstand the induction of host immune responses and to successfully establish long-term colonization. In case of asymptomatic bacteriuria and other persistent infections, prolonged growth in the host is accompanied with genomic alterations that result in e.g., bacterial attenuation thus contributing to bacterial adaptation to their host niche and a reduced activation of host immune responses. The accumulating amount of information regarding bacterial adaptation during persistent infection helps to increase our understanding of driving forces of bacterial adaptation in vivo as well as of factors that contribute to symptomatic infection.

Extraintestinal pathogenic Escherichia coli (ExPEC) are a common cause of disease in both mammals and birds. A vaccine to prevent such infections would be desirable given the increasing antibiotic resistance of these bacteria. We have determined the genome sequence of ExPEC IHE3034 (ST95) isolated from a case of neonatal meningitis and compared this to available genome sequences of other ExPEC strains and a few nonpathogenic E. coli . We found 19 genomic islands present in the genome of IHE3034, which are absent in the nonpathogenic E. coli isolates. By using subtractive reverse vaccinology we identified 230 antigens present in ExPEC but absent (or present with low similarity) in nonpathogenic strains. Nine antigens were protective in a mouse challenge model. Some of them were also present in other pathogenic non-ExPEC strains, suggesting that a broadly protective E. coli vaccine may be possible. The gene encoding the most protective antigen was detected in most of the E. coli isolates, highly conserved in sequence and found to be exported by a type II secretion system which seems to be nonfunctional in nonpathogenic strains.

The Escherichia coli genome consists of a conserved part, the so-called core genome, which encodes essential cellular functions and of a flexible, strain-specific part. Genes that belong to the flexible genome code for factors involved in bacterial fitness and adaptation to different environments. Adaptation includes increase in fitness and colonization capacity. Pathogenic as well as non-pathogenic bacteria carry mobile and accessory genetic elements such as plasmids, bacteriophages, genomic islands and others, which code for functions required for proper adaptation. Escherichia coli is a very good example to study the interdependency of genome architecture and lifestyle of bacteria. Thus, these species include pathogenic variants as well as commensal bacteria adapted to different host organisms. In Escherichia coli, various genetic elements encode for pathogenicity factors as well as factors, which increase the fitness of non-pathogenic bacteria. The processes of genome dynamics, such as gene transfer, genome reduction, rearrangements as well as point mutations contribute to the adaptation of the bacteria into particular environments. Using Escherichia coli model organisms, such as uropathogenic strain 536 or commensal strain Nissle 1917, we studied mechanisms of genome dynamics and discuss these processes in the light of the evolution of microbes.

The mucosal immune system identifies and fights invading pathogens, while allowing non-pathogenic organisms to persist. Mechanisms of pathogen/non-pathogen discrimination are poorly understood, as is the contribution of human genetic variation in disease susceptibility. We describe here a new, IRF3-dependent signaling pathway that is critical for distinguishing pathogens from normal flora at the mucosal barrier. Following uropathogenic E. coli infection, Irf3−/− mice showed a pathogen-specific increase in acute mortality, bacterial burden, abscess formation and renal damage compared to wild type mice. TLR4 signaling was initiated after ceramide release from glycosphingolipid receptors, through TRAM, CREB, Fos and Jun phosphorylation and p38 MAPK-dependent mechanisms, resulting in nuclear translocation of IRF3 and activation of IRF3/IFNβ-dependent antibacterial effector mechanisms. This TLR4/IRF3 pathway of pathogen discrimination was activated by ceramide and by P-fimbriated E. coli, which use ceramide-anchored glycosphingolipid receptors. Relevance of this pathway for human disease was supported by polymorphic IRF3 promoter sequences, differing between children with severe, symptomatic kidney infection and children who were asymptomatic bacterial carriers. IRF3 promoter activity was reduced by the disease-associated genotype, consistent with the pathology in Irf3−/− mice. Host susceptibility to common infections like UTI may thus be strongly influenced by single gene modifications affecting the innate immune response., Author Summary The host immune system must identify pathogens and defeat them through TLR-dependent signaling pathway activation, while distinguishing them from commensal flora. Contrary to current dogma, the host cannot solely use “pattern recognition” since the microbial molecules involved in such recognition are present on pathogens and commensals alike. We identify here a pathogen-specific mechanism of TLR4 activation and signaling intermediates in this pathway, leading to IRF3-dependent transcription of innate immune response genes. We show in knockout mice that Irf3 deficiency causes severe tissue pathology and that effector functions controlled by IFNβ are involved. Finally, in highly disease-prone pyelonephritis patients we found a high frequency of IRF3 promoter polymorphism compared to asymptomatic bacterial carriers or controls. The polymorphisms influenced promoter activity in reporter assays, suggesting that they are functionally important. Urinary tract infections are among the most common bacterial infections in man, and are a major cause of morbidity and mortality. A subset of disease-prone individuals is at risk for recurrent disease, severe renal dysfunction and end-stage renal disease. At present, there is no method to identify disease-prone infants and to prevent future morbidity and renal damage. The genetic and functional studies described here indicate that genetic variation in IRF3 influences individual susceptibility to kidney infection and might serve as a new tool for future risk assessment in this patient group.

International audience; Avian pathogenic Escherichia coli (APEC) and human extraintestinal pathogenic E. coli (ExPEC) cause various diseases in humans and animals and cannot be clearly distinguished by molecular epidemiology and genome content. We characterized traits of eight representative human ExPEC and APEC variants to either support the zoonotic potential or indicate factors involved in host specificity. These strains were very similar regarding phylogeny, virulence gene content and allelic variation of adhesins. Host- or serogroup-specific differences in type 1-, P-, S/F1C-fimbriae, curli, flagella, colicin and aerobactin expression or in vivo virulence were not found. Serogroup-dependent differences in genome content may depend on the phylogenetic background. To identify traits involved in host specificity, we performed transcriptome analysis of human ExPEC IHE3034 and APEC BEN374 in response to human (37 degrees C) or avian (41 degrees C) body temperature. Both isolates displayed similar transcriptional profiles at both temperatures. Transcript levels of motility/chemotaxis genes were repressed at 41 degrees C. The hdeAB and cadA genes involved in acid stress resistance, although often induced at 41 degrees C, could not be correlated with host specificity. Beside strain-specific effects, the common behavior of both strains at human or avian body temperature supports the idea of a potential zoonotic risk of certain human ExPEC and APEC variants.

Surface components are essential for bacterial pathogenicity. Adhesins, fimbrial and afimbrial, mediate direct physical contact between bacteria and host surfaces. Fimbrial adhesins are composed of several subunit proteins with the adhesin proper very often located at the tip of the pilus. In contrast, afimbrial adhesins usually present as monomers. Although the majority of fimbriae were identified in Gram-negative bacteria, the number of pili in Gram-positives is steadily increasing. Most of the fimbriae of Gram-negative bacteria are anchored in the outer membrane, the fimbrial subunits are noncovalently connected and these fimbriae bind to carbohydrate residues. Pili of Gram-positives are covalently linked to peptidoglycan and among themselves and in general they interact with host proteins. The only fimbrial type present in certain Gram-positive as well as Gram-negative bacteria is type IV pili. In both bacterial groups they originate in the cytoplasmic membrane. Other surface organelles important for bacterial pathogenicity are the flagella. They is responsible not only for motility, but might function also as adhesins and invasins which can depend on glycosylation of the subunit protein flagellin. In addition, a secreted protein (EtpA) which binds to the tip of the flagella can mediate adherence. Certain adhesins and specialized surface proteins are responsible for the ability of certain pathogenic bacteria to invade host cells. The best studied examples are the invasins of the Gram-negative species Yersinia enterocolitica and Y. pseudotuberculosis and the internalins of the Gram-positive species Listeria monocytogenes. Alternatively, bacteria such as Salmonella and Shigella force their internalization by host cells by injecting effector proteins via a type three secretion system directly into host cells. The intracellular location provides invasive bacteria with a relative safe niche similar to the location in a biofilm. Formation of biofilms involves surface associated proteins such as autotransporters, adhesins and flagella and the synthesis of an extracellular matrix composed of polysaccharides. This chapter presents an overview rather than a comprehensive paper regarding the great variety of cell envelope components and pathogenicity.

SummaryEnterohaemorrhagic Escherichia coli (EHEC) cause haemolytic uraemic syndrome (HUS),a thrombotic microangiopathy resulting from endothelial injury in the renal glomeruli and other organs. EHEC virulence factors that damage the microvascular endothelium play therefore major roles in the pathogenesis of HUS.We identified an EHEC strain that vacuolates and kills primary human glomerular microvascular endothelial cells (GMVECs) and a human brain microvascular endothelial cell (HBMEC) line. Because the vacuolating effect closely resembles those elicited on other cells by the vacuolating cytotoxin of Helicobacter pylori (VacA), we designated the factor responsible for this effect EHEC vacuolating cytotoxin (EHEC-Vac). EHEC-Vac (a secreted non-serine protease protein) binds to HBMECs rapidly and irreversibly, vacuolates within 30 min after exposure and the effect is maximally apparent at 48 h. Despite the vacuolisation, HBMECs survive for several days before they undergo necrosis. Electron and immunofluorescence microscopy demonstrate that the vacuoles induced by EHEC-Vac originate from lysosomes.Accordingly, they stain with neutral red indicating an acidic microenvironment. Similar to VacA, the EHEC-Vac-mediated vacuolisation is both prevented and reverted by the vacuolar proton pump inhibitor bafilomycin A1, suggesting a similar mechanism of vacuole formation by these toxins. Despite the similarity of phenotypes elicited by EHEC-Vac and VacA, genomic DNA from the EHEC-Vac-producing strain failed to hybridise to a vacA probe, as well as to probes derived from presently known E. coli vacuolating toxins.Through its microvascular endothelium-injuring potential combined with the ability to induce interleukin 6 release from these cells EHEC-Vac might contribute to the pathogenesis of HUS.

Escherichia coli K1 causes disease in humans and birds. Its polysialic acid capsule can be O-acetylated via phase-variable expression of the acetyltransferase NeuO encoded by prophage CUS-3. The role of capsule O-acetylation in ecological adaptation or pathogenic invasion of E. coli K1 is largely unclear. A population genetics approach was performed to study the distribution of neuO among E. coli K1 isolates from human and avian sources. Multilocus sequence typing revealed 39 different sequence types (STs) among 183 E. coli K1 strains. The proportion of the ST95 complex (STC95) was 44%. NeuO was found in 98% of the STC95 strains, but only in 24% of other STs. Grouping of STs and prophage genotypes revealed a segregation of prophage types according to STs, suggesting coevolution of CUS-3 and the E. coli K1 host. Within the STC95, which is known to harbour both human and avian pathogenic isolates, CUS-3 genotypes were shared irrespective of the host species. Functional analysis of a variety of strain pairs revealed that NeuO-mediated K1 capsule O-acetylation enhanced desiccation resistance. In contrast, NeuO expression led to a reduced biofilm formation in biofilm positive E. coli K1 isolates. These findings suggest a delicate ecological balance of neuO'on'/'off' switching.

It is a well-known observation and a long-standing hypothesis that pathogen genome dynamics are important in infectious disease processes. Recent achievements in large-scale genome sequencing, compara

Five types of cytolethal distending toxin (CDT-I to CDT-V) have been identified in Escherichia coli . In the present study we cloned and sequenced the cdt-IV operon and flanking region from a porcine extraintestinal pathogenic E. coli (ExPEC) strain belonging to serogroup O75. We confirmed that similar to other CDTs, CDT-IV induced phosphorylation of host histone H2AX, a sensitive marker of DNA double-strand breaks, and blocked the HeLa cell cycle at the G 2 -M transition. The cdt-IV genes were framed by lambdoid prophage genes. We cloned and sequenced the cdt-I operon and flanking regions from a human ExPEC O18:K1:H7 strain and observed that cdt-I genes were also flanked by lambdoid prophage genes. PCR studies indicated that a gene coding for a putative protease was always associated with the cdtC-IV gene but was not associated with cdtC genes in strains producing CDT-I, CDT-III, and CDT-V. Our results suggest that the cdt-I and cdt-IV genes might have been acquired from a common ancestor by phage transduction and evolved in their bacterial hosts. The lysogenic bacteriophages have the potential to carry nonessential “cargo” genes or “morons” and therefore play a crucial role in the generation of genetic diversity within ExPEC.

Antigen 43 (Ag43) represents an entire family of closely related autotransporter proteins in Escherichia coli and has been described to confer aggregation and fluffing of cells, to promote biofilm formation, uptake and survival in macrophages as well as long-term persistence of uropathogenic E. coli in the murine urinary tract. Furthermore, it has been reported that glycosylation of the Ag43 passenger domain (alpha(43)) stabilizes its conformation and increases adhesion to Hep-2 cells. We characterized the role of Ag43 as an adhesin and the impact of O-glycosylation on the function of Ag43. To analyze whether structural variations in the alpha(43) domain correlate with different functional properties, we cloned 5 different agn43 alleles from different E. coli subtypes and tested them for autoaggregation, biofilm formation, adhesion to different eukaryotic cell lines as well as to purified components of the extracellular matrix. These experiments were performed with nonglycosylated and O-glycosylated Ag43 variants. We show for the first time that Ag43 mediates bacterial adhesion in a cell line-specific manner and that structural variations of the alpha(43) domain correlate with increased adhesive properties to proteins of the extracellular matrix such as collagen and laminin. Whereas O-glycosylation of many alpha(43) domains led to impaired autoaggregation and a significantly reduced adhesion to eukaryotic cell lines, their interaction with collagen was significantly increased. These data demonstrate that O-glycosylation is not a prerequisite for Ag43 function and that the different traits mediated by Ag43, i.e., biofilm formation, autoaggregation, adhesion to eukaryotic cells and extracellular matrix proteins, rely on distinct mechanisms.

Extraintestinal pathogenic Escherichia coli (ExPEC) are able to colonize, invade and induce disease in body niches outside the gastrointestinal tract. The emergence of (multi)resistant ExPEC variants calls for alternative antimicrobial strategies with a reduced pressure on selection of antibiotic resistances. We report on recent research efforts to target such virulence traits or to interfere with ExPEC colonization of extraintestinal niches in order to prevent or treat ExPEC infections and reduce the risk of further emergence of antibiotic resistances.

We describe the expression and regulation of the gene sfaX(II) located near the Sfa(II) fimbrial determinant in the newborn meningitis Escherichia coli (NMEC) isolate IHE3034. sfaX(II) belongs to a gene family, the 17-kDa genes, typically located downstream (300-3000bp) of different fimbrial operons found in E. coli isolates of uropathogenic and newborn meningitis origin. Using transcriptional sfaX(II) reporter gene fusions we found that different environmental conditions commonly affecting expression of fimbrial genes also affected sfaX(II) expression. Analysis of the sfaX(II) transcripts showed that the gene is part of the main fimbrial operon as it is transcribed together with the rest of the fimbrial genes. In addition, the sfaX(II) gene can be expressed from a more proximal promoter and is found to be subject to strong down-regulation by the nucleoid protein H-NS. Studies with an sfaX(II) mutant derivative of IHE3034 did not reveal effects on Sfa(II) fimbrial biogenesis as monitored by e.g. immunofluorescence microscopy. Nevertheless, a mutation in sfaX(II) resulted in altered expression of other surface components. Moreover, we define a new gene, sfaY(II), coding for a putative phosphodiesterase that is located in between the sfaX(II) gene and the fimbrial biogenesis genes. Our studies by ectopic expression of sfaY(II) in Vibrio cholerae showed that the gene product caused reduced biofilm formation and it is proposed that sfaY(II) can influence cyclic-di-GMP turnover in the bacteria. Our findings demonstrate that the operons typical for S-fimbriae of extraintestinal pathogenic E. coli include previously unrecognized novel regulatory genes.

In uropathogenic Escherichia coil strain 536, six pathogenicity islands (PAls) encode key virulence factors. All PAls except PAI IV 536 are flanked by direct repeats and four of them encode integrases responsible for their chromosomal excision. To study recombination sites used for the integration by PAI II 536 and III 536 integrases, we measured site-specific recombination between the chromosomal integration site attB, and the PAI-specific attachment site attP. We show that PAI III 536 IntB, but not IntA, mediates PAI III 536 integration. Studies of integrative recombination sites of both PAls show that, when using a large cognate attP site (839 bp for PAI II 536 and 268 bp for PAI III 536 ), PAI II 536 and III 536 attB sites could be reduced to 16 bp and 20 bp, respectively, without affecting recombination. Further reduction to 14 bp for PAI II 536 and 13 bp for PAI III 536 diminished recombination efficiency. Surprisingly, attP sites could also be reduced to 14 bp (PAI II 536 ) and 20 bp (PAI III 536 ). The integration host factor (IHF) and the DNA-bending HU protein do not influence PAI II 536 recombination, but IHF enhances PAI-III 536 excision and negatively affects its integration. These data suggest that PAI intasomes differ from those of lambda and P4 integrase paradigms.

International audience; The emerging genomic technologies and bioinformatics provide novel opportunities for studying life-threatening human pathogens and to develop new applications for the improvement of human and animal health and the prevention, treatment, and diagnosis of infections. Based on the ecology and population biology of pathogens and related organisms and their connection to epidemiology, more accurate typing technologies and approaches will lead to better means of disease control. The analysis of the genome plasticity and gene pools of pathogenic bacteria including antigenic diversity and antigenic variation results in more effective vaccines and vaccine implementation programs. The study of newly identified and uncultivated microorganisms enables the identification of new threats. The scrutiny of the metabolism of the pathogen in the host allows the identification of new targets for anti-infectives and therapeutic approaches. The development of modulators of host responses and mediators of host damage will be facilitated by the research on interactions of microbes and hosts, including mechanisms of host damage, acute and chronic relationships as well as commensalisms. The study of multiple pathogenic and non-pathogenic microbes interacting in the host will improve the management of multiple infections and will allow probiotic and prebiotic interventions. Needless to iterate, the application of the results of improved prevention and treatment of infections into clinical tests will have a positive impact on the management of human and animal disease. The Pathogenomics Research Agenda draws on discussions with experts of the Network of Excellence "EuroPathoGenomics" at the management board meeting of the project held during 18-21 April 2007, in the Villa Vigoni, Menaggio, Italy. Based on a proposed European Research Agenda in the field of pathogenomics by the ERA-NET PathoGenoMics the meeting's participants updated the established list of topics as the research agenda for the future.

In some patients, Escherichia coli strains establish significant bacteriuria without causing symptoms of urinary tract infection (UTI). These asymptomatic-bacteriuria (ABU) strains have been shown to express fewer virulence factors than the uropathogenic E. coli (UPEC) strains that cause severe, symptomatic UTI. Paradoxically, ABU strains carry many typical UPEC virulence genes, and the molecular basis of their low virulence therefore remains unclear. This study examined whether ABU strains might evolve from UPEC by genome loss and virulence gene attenuation. The presence of conserved E. coli K-12 genes was examined using an E. coli K-12 strain MG1655-specific DNA array and the distribution of UPEC virulence-related genes was examined with the E. coli pathoarray. Two groups of strains could be distinguished. Several ABU strains were shown by multilocus sequence typing and by comparative genomic analyses to be related to UPEC but to have smaller genome sizes. There were significant alterations in essential virulence genes, including reductive evolution by point mutations, DNA rearrangements, and deletions. Other strains were unrelated to UPEC and lacked most of the virulence-associated genes. The results suggest that some ABU strains arise from virulent strains by attenuation of virulence genes while others are nonvirulent and resemble commensal strains. We propose that virulence attenuation might constitute a general mechanism for mucosal pathogens to evolve toward commensalism.

Extraintestinal pathogenic Escherichia coli (ExPEC) are usually harmless colonizer of the intestinal microflora. However, they are capable to translocate and cause life-threatening disease. Translocation of ExPEC isolates was quantified in colonic monolayers. Transepithelial resistance (R(t)) was monitored and local changes in conductivity analysed with conductance scanning. Confocal microscopy visualized the translocation route. Corroboratory experiments were performed on native rat colon. One translocating strain E. coli O4 was identified. This translocation process was associated with an R(t) decrease (36 +/- 1% of initial resistance) beginning only 2 h after inoculation. The sites of translocation were small defects in epithelial integrity (focal leaks) exhibiting highly increased local ion permeability. Translocation was enhanced by preincubation of monolayers with tumour necrosis factor-alpha or interleukin-13. Mutant strains lacking alpha-haemolysin lost the ability to induce focal leaks, while this effect could be restored by re-introducing the haemolysin determinant. Filtrate of a laboratory strain carrying the alpha-haemolysin operon was sufficient for focal leak induction. In native rat colon, E. coli O4 decreased R(t) and immunohistology demonstrated focal leaks resembling those in cell monolayers. E. coli alpha-haemolysin is able to induce focal leaks in colonic cell cultures as well as in native colon. This process represents a novel route of bacterial translocation facilitated by pro-inflammatory cytokines.

International audience; tRNA-encoding genes (tDNA) are known hot-spots for the integration of ecto-chromosomal DNA (ECDNA) including genomic islands. However, only a few loci are currently known to be targeted by such insertions in Escherichia coli. A PCR-based screening of tDNA integrity was therefore performed on a collection of E. coli strains in order to identify tDNA loci that are most frequently intact and those that are preferred ECDNA insertion sites. It was shown that only a subset of tDNAs were hot-spots for ECDNA insertions, and that the majority of loci were never targeted by such insertions. Polycistronic tDNAs, highly transcribed tDNAs or tDNAs encoding tRNAs recognizing frequently used codons were generally not targeted by ECDNA insertions. Most interestingly, strains of different ECOR groups showed different patterns of tDNA loci polymorphism. More subtle differences were also observed between strains of different pathotypes.

Background Adenomyomatosis of gallbladder (GB) is considered as benign condition, but several reports suggest association with malignancies, especially for segmental subtype. Because of this chance, patients suspected to have segmental or diffuse type adenomyomatosis on the imaging study may undergo cholecystectomy. But, in some cases, histopathology reveals only chronic inflammation. In this study, we aimed to analyze radiologic and pathologic features of chronic cholecystitis and adenomyomatosis for more accurate diagnosis. Methods Patients who performed cholecystectomy during recent four years in one medical center were reviewed retrospectively. Among those, patients who were suspected to have adenomyomatosis on their abdominal imaging but with no adenomyoma detected on the histopathologic examination were analyzed for their radiologic, pathologic and clinical features compared to patients who had consistent results between abdominal imaging and histopathologic examination. Results Among 3144 patients who underwent cholecystectomy, 358 patients were suspected to have GB adenomyomatosis on the abdominal imaging study. Among them (358 patients), only 92 patients had histopathologic findings compatible with adenomyomatosis. Remaining 266 patients (74.3%) did not have adenomyomatosis. Instead, many GB specimens showed chronic cholecystitis. In this group, diffuse subtype of adenomyomatosis was significantly frequent compared to 92 patients who had coherent adenomyoma finding for both imaging study and pathology. The frequency of accompanying GB stones was significantly higher in mismatching group. Radiologically, contour of GB wall thickening, intralesional cystic area and inner layer enhancement pattern was helpful in differentiating adenomyomatosis from chronic cholecystitis. Conclusion Chronic cholecystitis mimicking adenomyomatosis is difficult to diagnose solely based on the abdominal imaging, and this can lead to unnecessary cholecystectomy. Existence of gallstones and several radiologic features can help differentiate these challenging entities.