Use of zebrafish to probe the divergent virulence potentials and toxin requirements of extraintestinal pathogenic Escherichia coli.
ABSTRACT: Extraintestinal pathogenic E. coli (ExPEC) cause an array of diseases, including sepsis, neonatal meningitis, and urinary tract infections. Many putative virulence factors that might modulate ExPEC pathogenesis have been identified through sequencing efforts, epidemiology, and gene expression profiling, but few of these genes have been assigned clearly defined functional roles during infection. Using zebrafish embryos as surrogate hosts, we have developed a model system with the ability to resolve diverse virulence phenotypes and niche-specific restrictions among closely related ExPEC isolates during either localized or systemic infections. In side-by-side comparisons of prototypic ExPEC isolates, we observed an unexpectedly high degree of phenotypic diversity that is not readily apparent using more traditional animal hosts. In particular, the capacity of different ExPEC isolates to persist and multiply within the zebrafish host and cause disease was shown to be variably dependent upon two secreted toxins, alpha-hemolysin and cytotoxic necrotizing factor. Both of these toxins appear to function primarily in the neutralization of phagocytes, which are recruited in high numbers to sites of infection where they act as an essential host defense against ExPEC as well as less virulent E. coli strains. These results establish zebrafish as a valuable tool for the elucidation and functional analysis of both ExPEC virulence factors and host defense mechanisms.
Project description:In individuals with sepsis, the infecting microbes are commonly viewed as generic inducers of inflammation while the host background is considered the primary variable affecting disease progression and outcome. To study the effects of bacterial strain differences on the maladaptive immune responses that are induced during sepsis, we employed a novel zebrafish embryo infection model using extraintestinal pathogenic Escherichia coli (ExPEC) isolates. These genetically diverse pathogens are a leading cause of sepsis and are becoming increasingly dangerous because of the rise of multidrug-resistant strains. Zebrafish infected with ExPEC isolates exhibit many of the pathophysiological features seen in septic human patients, including dysregulated inflammatory responses (cytokine storms), tachycardia, endothelial leakage, and progressive edema. However, only a limited subset of ExPEC isolates can trigger a sepsis-like state and death of the host when introduced into the bloodstream. Mirroring the situation in human patients, antibiotic therapy reduced ExPEC titers and improved host survival rates but was only effective within limited time frames that varied, depending on the infecting pathogen. Intriguingly, we find that phylogenetically distant but similarly lethal ExPEC isolates can stimulate markedly different host transcriptional responses, including disparate levels of inflammatory mediators. These differences correlate with the amounts of bacterial flagellin expression during infection, as well as differential activation of Toll-like receptor 5 by discrete flagellar serotypes. Altogether, this work establishes zebrafish as a relevant model of key aspects of human sepsis and highlights the ability of genetically distinct ExPEC isolates to induce divergent host responses independently of baseline host attributes. IMPORTANCE Sepsis is a life-threatening systemic inflammatory condition that is initiated by the presence of microorganisms in the bloodstream. In the United States, sepsis due to ExPEC and other pathogens kills well over a quarter of a million people each year and is associated with tremendous health care costs. A high degree of heterogeneity in the signs and symptomology of sepsis makes this disease notoriously difficult to effectively diagnose and manage. Here, using a zebrafish model of sepsis, we find that similarly lethal but genetically distinct ExPEC isolates can elicit notably disparate host responses. These variances are in part due to differences in the levels and types of flagellin that are expressed by the infecting ExPEC strains. A better understanding of the variable impact that bacterial factors like flagellin have on host responses during sepsis could lead to improved diagnostic and therapeutic approaches to these often deadly infections. Podcast: A podcast concerning this article is available.
Project description:Extraintestinal pathogenic Escherichia coli (ExPEC) is one of the top pathogens responsible for bloodstream infection and severe, often fatal, sepsis. Although the virulence factors and host immune responses to ExPEC infection have been investigated, the responses to a particular ExPEC strain could be very different. In this study, we investigated the mechanisms of Cyclooxygenase-2 (COX-2) up-regulation in influencing the host defenses against infection of ExPEC XM O2:K1:H7. Our results demonstrated that ExPEC XM O2:K1:H7 infection in mouse and RAW264.7 macrophages leads to COX-2 up-regulation, and COX-2 inhibition significantly enhances ExPEC infection. The up-regulation of COX-2 in macrophages was mediated by Toll-like receptor 4 (TLR4) through the activation of p38 and extracellular signal-regulated kinase/Mitogen-activated protein kinase (ERK/MAPK) pathways. Further studies showed that COX-2 inhibition significantly decreased autophagy in macrophages during ExPEC XM O2:K1:H7 infection. Autophagy inhibition significantly enhanced, while induction reduced ExPEC XM O2:K1:H7 survival in macrophages. In addition, COX-2 inhibition significantly increased macrophage cell death during ExPEC XM O2:K1:H7 infection and increased the expression of anti-inflammatory cytokine interleukin-10 (IL-10). Our results indicate that COX-2 up-regulation benefits host defense against ExPEC XM O2:K1:H7 infection by increasing autophagy in macrophages and by reducing IL-10 expression and macrophage cell death during ExPEC infection.
Project description:Although dogs have been proposed as carriers of extraintestinal pathogenic Escherichia coli (ExPEC) with infectious potential for humans, presumed host species-specific differences between canine and human ExPEC strains have cast doubt on this hypothesis. The recent discovery that allele III of papG (the P fimbrial adhesin gene) predominates among human cystitis isolates and confers an adherence phenotype resembling that of canine ExPEC prompted the present reevaluation of the canine-human ExPEC connection. Sixteen paired pap-positive urine and rectal E. coli isolates from dogs with urinary tract infection were studied. papG (adhesin) and papA (pilin) allele type, agglutination phenotypes, virulence factor genotypes, and randomly amplified polymorphic DNA and pulsed-field gel electrophoresis fingerprints were analyzed and compared with those of human ExPEC controls. The 16 canine strains contained predominantly papG allele III. Agglutination phenotypes segregated strictly according to papG allele status and were homogeneous among strains with the same papG allele profile irrespective of their human versus canine origin. Canine and human PapG variant III peptide sequences were highly homologous, without host species-specific differences. The most prevalent canine papA allele was F48, a novel variant recently identified among human urosepsis isolates. In addition to pap, human ExPEC-associated virulence genes detected among the canine strains included sfa/focDE, sfaS, fyuA, hlyA, cnf1, cdtB, kpsMT-II and -III, rfc, traT, ompT, and a marker for a pathogenicity-associated island from archetypal human ExPEC strain CFT073. Molecular fingerprinting confirmed the fecal origin of all but one canine urine isolate and showed one pair of O6 canine urine and fecal isolates to be extremely similar to an O6 human urosepsis isolate with which they shared all other genotypic and phenotypic characteristics analyzed. These data demonstrate that canine ExPEC strains are similar to, and in some instances essentially indistinguishable from, human ExPEC strains, which implicates dogs and their feces as potential reservoirs of E. coli with infectious potential for humans.
Project description:To test the canine reservoir hypothesis of extraintestinal pathogenic Escherichia coli (ExPEC), 63 environmental canine fecal deposits were evaluated for the presence of ExPEC by a combination of selective culturing, extended virulence genotyping, hemagglutination testing, O serotyping, and PCR-based phylotyping. Overall, 30% of canine fecal samples (56% of those that yielded viable E. coli) contained papG-positive E. coli, usually as the predominant E. coli strain and always possessing papG allele III (which encodes variant III of the P-fimbrial adhesin molecule PapG). Multiple other virulence-associated genes typical of human ExPEC were prevalent among the canine fecal isolates. According to serotyping, virulence genotyping, and random amplified polymorphic DNA analysis, over 50% of papG-positive fecal E. coli could be directly correlated with specific human clinical isolates from patients with cystitis, pyelonephritis, bacteremia, or meningitis, including archetypal human ExPEC strains 536, CP9, and RS218. Five canine fecal isolates and (clonally related) archetypal human pyelonephritis isolate 536 were found to share a novel allele of papA (which encodes the P-fimbrial structural subunit PapA). These data confirm that ExPEC representing known virulent clones are highly prevalent in canine feces, which consequently may provide a reservoir of ExPEC for acquisition by humans.
Project description:Background:Extraintestinal pathogenic Escherichia coli (ExPEC) is an important bacterium and responsible for many bloodstream infections, including urinary tract infections and even fatal bacteremia. The aim of this research was to investigate whether ExPEC strains isolated from Turkish blood cultures have a relationship between 16S rRNA based phylogenetic clusters and antibiotic resistance profiles, virulence factors or clonal lineages. Methods:Phenotypically identified ExPEC blood culture isolates (n = 104) were included in this study. The 16S rRNA partial sequence analysis was performed for genotypic identification of ExPEC isolates. Antibiotic susceptibility and Extended-Spectrum ?-Lactamase testing of isolates were performed. Phylogenetic classification (A, B1, B2 and D), Multi Locus Sequence Typing analysis and virulence-associated genes were investigated. Results:Based on 16S rRNA partial sequence analysis, 97 out of 104 (93.26%) ExPEC isolates were confirmed as E. coli. Ampicillin (74.22%) and cefuroxime axetil (65.97%) resistances had the highest frequencies among the ExPEC isolates. In terms of phylogenetic classification of ExPEC, D (38.14%, 37/97) was the most prevalent group after A (29.89%, 29/97), B2 (20.61%, 20/97), and B1 (11.34%, 11/97). The sequence types of the 20 ExPEC isolates belonging to the B2 phylogenetic group were analyzed by Multi Locus Sequence Typing. Ten isolates out of 20 (50.0%) were identified as ST131. The other STs were ST95 (n = 1), ST14 (n = 1), ST10 (n = 1), ST69 (n = 1), ST1722 (n = 2), ST141 (n = 1), ST88 (n = 1), ST80 (n = 1), and ST998 (n = 1). Of the ST131 strains, six (60%, 6/10) represented serogroup O25. The most common virulence factor genes were serum resistance factor gene, traT (55.7%) aerobactin siderophore receptor and yersiniabactin encoding genes iutA (45.3%) and fyuA (50.5%), respectively. In addition, PAI (41.2%), iroN (23.7%), hlyA (15.4%), kpsII (13.4%), ompT (13.4%), papG (12.4%), iss (9.3%), cnf1 (7.2%), ibeA (2.06%), and sfaS (2.06%) genes were present in the ExPEC isolates. Conclusion:The 16S rRNA-based phylogenetic relationship tree analysis showed that a large cluster was present among 97 ExPEC isolates along with related reference strains. There were 21 main clusters with 32 closely related subclusters. Based on our findings, different clonal lineages of ExPEC can display different antibiotic susceptibilities and virulence properties. We also concluded that virulence factors were not distributed depending on phylogenetic groups (A, B1, B2, and D). The ExPEC isolates belonging to the same phylogenetic group and sequence type could display different resistance and virulence characteristics.
Project description:Swine extraintestinal pathogenic Escherichia coli (ExPEC) is an important pathogen that leads to economic and welfare costs in the swine industry worldwide, and is occurring with increasing frequency in China. By far, various virulence factors have been recognized in ExPEC. Here, we investigated the virulence genotypes and clonal structure of collected strains to improve the knowledge of phylogenetic traits of porcine ExPECs in China.We isolated 64 Chinese porcine ExPEC strains from 2013 to 14 in China. By multiplex PCR, the distribution of isolates belonging to phylogenetic groups B1, B2, A and D was 9.4%, 10.9%, 57.8% and 21.9%, respectively. Nineteen virulence-related genes were detected by PCR assay; ompA, fimH, vat, traT and iutA were highly prevalent. Virulence-related genes were remarkably more prevalent in group B2 than in groups A, B1 and D; notably, usp, cnf1, hlyD, papA and ibeA were only found in group B2 strains. Genotyping analysis was performed and four clusters of strains (named I to IV) were identified. Cluster IV contained all isolates from group B2 and Cluster IV isolates had the strongest pathogenicity in a mouse infection model. As phylogenetic group B2 and D ExPEC isolates are generally considered virulent, multilocus sequence typing (MLST) analysis was performed for these isolates to further investigate genetic relationships. Two novel sequence types, ST5170 and ST5171, were discovered. Among the nine clonal complexes identified among our group B2 and D isolates, CC12 and CC95 have been indicated to have high zoonotic pathogenicity. The distinction between group B2 and non-B2 isolates in virulence and genotype accorded with MLST analysis.This study reveals significant genetic diversity among ExPEC isolates and helps us to better understand their pathogenesis. Importantly, our data suggest group B2 (Cluster IV) strains have the highest risk of causing animal disease and illustrate the correlation between genotype and virulence.
Project description:Extraintestinal pathogenic E. coli (ExPEC) are facultative pathogens that are part of the normal human intestinal flora. The ExPEC group includes uropathogenic E. coli (UPEC), neonatal meningitis E. coli (NMEC), sepsis-associated E. coli (SEPEC), and avian pathogenic E. coli (APEC). Virulence factors (VF) related to the pathogenicity of ExPEC are numerous and have a wide range of activities, from those related to bacteria colonization to those related to virulence, including adhesins, toxins, iron acquisition factors, lipopolysaccharides, polysaccharide capsules, and invasins, which are usually encoded on pathogenicity islands (PAIs), plasmids and other mobile genetic elements. Mechanisms underlying the dynamics of ExPEC transmission and the selection of virulent clones are still poorly understood and require further research. The time shift between colonization of ExPEC and the development of infection remains problematic in the context of establishing the relation between consumption of contaminated food and the appearance of first disease symptoms. What appears to be most difficult is to prove that ExPEC strains cause disease symptoms and to examine the mechanism of transition from the asymptomatic colonization of the intestines to the spreading of the bacteria outside the digestive system. A significant problem for researchers who are trying to ascribe ExPEC transmission to food, people or the environment is to draw the distinction between colonization of ExPEC and infection. Food safety is an important challenge for public health both at the production stage and in the course of its processing and distribution. Examination of the genetic similarity of ExPEC strains will allow to determine their origin from different sources. Many levels of genotyping have been proposed in which the typing of strains, plasmids and genes is compared in order to obtain a more complete picture of this complex problem. The aim of our study was to characterize E. coli strains isolated from humans, animals and food for the presence of bacterial genes encoding virulence factors such as toxins, and iron acquisition systems (siderophores) in the context of an increasing spread of ExPEC infections.
Project description:Extraintestinal pathogenic Escherichia coli (ExPEC) is an important human and animal pathogen. Despite the apparent similarities in their known virulence attributes, some ExPEC strains can cross the host species barrier and present a zoonotic potential, whereas other strains exhibit host specificity, suggesting the existence of unknown mechanisms that remain to be identified. We applied a transposon-directed insertion site sequencing (TraDIS) strategy to investigate the ExPEC XM strain, which is capable of crossing the host species barrier, and to screen for virulence-essential genes in both mammalian (mouse) and avian (duck) models of E. coli-related septicemia. We identified 151 genes essential for systemic infection in both mammalian and avian models, 97 required only in the mammalian model, and 280 required only in the avian model. Ten genes/gene clusters were selected for further validation, and their contributions to ExPEC virulence in both mammalian and avian models or mammalian- or avian-only models were confirmed by animal tests. This represents the first comprehensive genome-wide analysis of virulence-essential genes required for systemic infections in two different host species and provides a further comprehensive understanding of ExPEC-related virulence, host specificity, and adaptation.
Project description:OBJECTIVE:Extraintestinal Pathogenic E. coli (ExPEC), are responsible for host diseases such as Neonatal Meningitis Escherichia coli (NMEC), the second-leading cause of neonatal bacterial meningitis, Avian Pathogenic E. coli (APEC), a cause of extraintestinal disease in poultry, and Uropathogenic E. coli (UPEC), the most common cause of urinary tract infections. Virulence factors associated with NMEC include outer membrane protein A (OmpA) and type I fimbriae (FimH), which also occur in APEC and UPEC. OmpA contributes to NMEC's ability to cross the blood-brain barrier, persist in the bloodstream and has been identified as a potential vaccine target for ExPEC, however the protein has amino acid variants, which may influence virulence of strains or alter vaccine efficacy. Although OmpA is present in virtually all E. coli, differences in its amino acid residues have yet to be surveyed in ExPEC. RESULTS:Here the ompA gene (n?=?399) from ExPEC collections were sequenced and translated in silico. Twenty-five different OmpA polymorphism patterns were identified. Seven polymorphism patterns were significantly associated with an ExPEC subpathotype, but chromosomal history most likely accounts for most differences found. The differences in OmpA protein sequences suggest that OmpA may influence variation in virulence and host specificity within ExPEC subpathotypes.
Project description:How extraintestinal pathogenic Escherichia coli (ExPEC) and antimicrobial-resistant E. coli disseminate through the population is undefined. We studied public restrooms for contamination with E. coli and ExPEC in relation to source and extensively characterized the E. coli isolates. For this, we cultured 1,120 environmental samples from 56 public restrooms in 33 establishments (obtained from 10 cities in the greater Minneapolis-St. Paul, MN, metropolitan area in 2003) for E. coli and compared ecological data with culture results. Isolates underwent virulence genotyping, phylotyping, clonal typing, pulsed-field gel electrophoresis (PFGE), and disk diffusion antimicrobial susceptibility testing. Overall, 168 samples (15% from 89% of restrooms) fluoresced, indicating presumptive E. coli: 25 samples (2.2% from 32% of restrooms) yielded E. coli isolates, and 10 samples (0.9% from 16% of restrooms) contained ExPEC. Restroom category and cleanliness level significantly predicted only fluorescence, gender predicted fluorescence and E. coli, and feces-like material and toilet-associated sites predicted all three endpoints. Of the 25 E. coli isolates, 7 (28%) were from phylogenetic group B2(virulence-associated), and 8 (32%) were ExPEC. ExPEC isolates more commonly represented group B2 (50% versus 18%) and had significantly higher virulence gene scores than non-ExPEC isolates. Six isolates (24%) exhibited ?3-class antibiotic resistance, 10 (40%) represented classic human-associated sequence types, and one closely resembled reference human clinical isolates by pulsed-field gel electrophoresis. Thus, E. coli, ExPEC, and antimicrobial-resistant E. coli sporadically contaminate public restrooms, in ways corresponding with restroom characteristics and within-restroom sites. Such restroom-source E. coli strains likely reflect human fecal contamination, may pose a health threat, and may contribute to population-wide dissemination of such strains.