Plasmid-Borne Virulence-Associated Genes Have a Conserved Organization in Virulent Strains of Avian Pathogenic Escherichia coli

Avian pathogenic Escherichia coli (APEC) is an important respiratory pathogen of poultry. Various virulence factors are responsible for determining the pathogenicity of these strains, and it is commonly believed they are encoded on large plasmids the strains carry. This study examined a series of strains, the pathogenicity of which had previously been determined by aerosol exposure, for possession of large plasmids and found all isolates carried at least one large plasmid, regardless of the level of virulence. Virulence-associated genes carried on these plasmids were also examined, and it was shown that highly virulent strains carried at least four virulence-associated genes on their largest plasmid. Two of the virulence-associated genes were shown to be chromosomally located in a strain of intermediate virulence, while no virulence-associated genes were carried by the low-virulence strain. The organization of the virulence-associated genes was shown to be highly conserved among APEC isolates of high virulence, supporting the concept of a conserved portion of the putative virulence region that contributes to the pathogenicity of APEC strains.Avian pathogenic Escherichia coli (APEC) strains cause respiratory disease and septicemia in poultry and are economically important worldwide, causing significant mortality (13). The carriage of large plasmids is considered characteristic of APEC isolates (8), and pathogenicity is thought to be determined by virulence-associated factors encoded by them (15). These factors include serum resistance, encoded by the iss gene (14), temperature-sensitive hemagglutination, encoded by tsh (10), adhesins, the production of colicin V (ColV) and the possession of iron-scavenging mechanisms, such as aerobactin production (encoded by the iucABCD operon), and the more recently identified putative iron transport system encoded by the etsABC operon (18).Another iron acquisition system found in APEC utilizes salmochelin, a catecholate siderophore. The chromosomal iroA gene cluster that encodes this system was first found in Salmonella enterica (2) and is absent from the corresponding region of the E. coli chromosome (32), although it has been found on a transmissible plasmid from a uropathogenic E. coli isolate (34). The iroA gene cluster has been found on multiple APEC virulence plasmids (9, 17, 18, 37), and deletion studies have shown that the iroA gene cluster is required for full virulence (9).A further iron transport system, designated the sitABCD system, was first identified on a pathogenicity island in Salmonella enterica serovar Typhimurium (39), and it has been shown that sitABCD is required for full virulence of Salmonella serovar Typhimurium (16). Genomic subtraction identified the plasmid-located sitA gene from the sitABCD operon as unique to an APEC strain (32), and the sitA gene was found to be more prevalent in APEC than in commensal E. coli (18, 29, 32).The sitABCD operon occurs on APEC virulence plasmids (17, 18, 30, 37), but a sitABCD deletion mutant was still pathogenic for birds, suggesting that other iron transport systems are able to compensate for the loss of sitABCD (30).The carriage of ColV plasmids has previously been thought to be essential for virulence (3, 33, 38). However, other studies have suggested it is not the presence of the ColV gene itself but other genes that these plasmids carry that are responsible for virulence (28, 35). The well-characterized APEC virulence plasmids pAPEC-O2-ColV (18) and pAPEC-1 (9) encode ColV, while carriage of the Australian APEC virulence plasmid pVM01 does not confer production of ColV (12). Despite various ColV statuses, all three of these virulence plasmids are F-type plasmids, and hence this is potentially another way to characterize APEC virulence plasmids.SopA and SopB, which have similarity to the ParA and ParB proteins of the P1 plasmid, are thought to be essential for F-plasmid partitioning (22, 24). Detection of the genes of the sopABC locus could thus indicate the presence of a putative virulence plasmid.Strain E3 is an O-nontypeable:H28 APEC field isolate (11) that carries the 151-kb virulence plasmid pVM01 (12), which contains a virulence region with the virulence-associated genes iucA, tsh, iss, iroN, and sitA, as well as hlyF, ompT, and the etsABC operon (37). The arrangement of the virulence-associated genes around pVM01 (37) is similar to that in the plasmids pAPEC-O2-ColV from APEC strain O2 (18), pAPEC-O1-ColBM from APEC strain O1 (17), and pAPEC-1 from APEC strain χ7122 (23). Identifying a specific region that is conserved in highly virulent APEC strains will facilitate diagnosis of colibacillosis by differentiation of pathogenic strains from commensal E. coli and will also enable surveillance for pathogenic isolates in the environment of poultry.This study examined six E. coli strains, some of which were isolated from diseased birds and some of which were recovered from healthy birds (11, 36). The pathogenicity of these strains has been determined using aerosol exposure (11, 36), making this the largest known collection of APEC strains fulfilling Koch''s postulates. The series of strains includes the highly virulent strains E3, E30, and E956 and the less-virulent strains E133, E1043, and E1292. The presence of the virulence-associated genes iucA, tsh, and iss in these strains has previously been elucidated by PCR amplification (36). However, while previous studies have found many of these virulence factors to be encoded by APEC strains associated with disease (29) and have suggested that they are encoded on virulence plasmids (18), they have not conclusively determined whether they are encoded on virulence plasmids or are chromosomally encoded. Similarly, although previous studies suggest that these virulence-associated genes are consistently present in isolates from diseased birds (1, 6, 18, 21, 26, 29), no study has yet determined if these genes are consistently associated with each other.The aim of this study was to examine a series of strains of known pathogenicities for the possession of large plasmids and to determine if known virulence-associated genes from the putative virulence region were carried on them. The second objective was to investigate any association between the virulence-associated genes.     

Similarity and Divergence among Adherent-Invasive Escherichia coli and Extraintestinal Pathogenic E. coli Strains

Adherent-invasive Escherichia coli (AIEC) pathovar strains, which are associated with Crohn''s disease, share many genetic and phenotypic features with extraintestinal pathogenic E. coli (ExPEC) strains, but little is known about the level of genetic similarity between the two pathovars. We aimed to determine the frequency of strains with the “AIEC phenotype” among a collection of ExPEC strains and to further search for a common phylogenetic origin for the intestinal and extraintestinal AIEC strains. The adhesion, invasion, and intramacrophage replication capabilities (AIEC phenotype) of 63 ExPEC strains were determined. Correlations between virulence genotype and AIEC phenotype and between intestinal/extraintestinal origin, serotype, and phylogroup were evaluated for the 63 ExPEC and 23 intestinal AIEC strains. Phylogenetic relationships between extraintestinal and intestinal AIEC strains were determined using multilocus sequence typing (MLST) and pulsed-field gel electrophoresis. Only four (6.35%) ExPEC strains, belonging to the O6:H1, O83:H1, and O25:H4 serotypes, were classified as having an AIEC phenotype. These strains were found to be genetically related to some intestinal AIEC strains of the same serotypes as revealed by MLST. No particular virulence gene sets correlated with the intestinal/extraintestinal origin of the strains or with the AIEC phenotype, whereas the gene sets did correlate with the serogroup. We identified two intestinal AIEC strains and one extraintestinal AIEC strain belonging to the O25:H4 serotype that also belonged to the emerging and virulent clonal group ST131. In conclusion, the ExPEC and AIEC pathovars share similar virulence gene sets, and certain strains are phylogenetically related. However, the majority of ExPEC strains did not behave like AIEC strains, thus confirming that the AIEC pathovar possesses virulence-specific features that, to date, are detectable only phenotypically.Members of the Enterobacteriaceae family, especially Escherichia coli, have been repeatedly suggested to play a role in the origin and/or perpetuation of Crohn''s disease (CD). In part, this suggestion was based on the higher abundance of this bacterium in CD patients than in control subjects (4, 10, 20, 23, 28, 29, 32, 41, 48, 51). Although considerable effort has been devoted to the search for intestinal pathogenic E. coli strains associated with CD, to date none of the six previously described pathovars (27) has been implicated in this condition. Darfeuille-Michaud et al. (18) observed that E. coli strains with adhesion and invasion properties colonized the ileal mucosae of CD patients more frequently than those of control subjects. Darfeuille-Michaud et al. further characterized these strains and proposed a new potential E. coli pathovar associated with CD, which was designated adherent-invasive E. coli (AIEC) (10). The implication of AIEC in CD is becoming increasingly relevant because several independent studies from different countries have reported a higher prevalence of invasive E. coli in CD patients (4, 17, 33, 34, 47).The main characteristics of AIEC are (i) the ability to adhere to and invade intestinal epithelial cells, (ii) the ability to survive and replicate expansively within macrophages without triggering host cell death and inducing the release of tumor necrosis factor alpha (21), and (iii) the lack of known invasive determinants (17). Recently, Glasser and Darfeuille-Michaud (22) proposed a model explaining the mechanism of pathogenesis for AIEC strains. The AIEC strains isolated to date are clonally diverse and belong to distinct serotypes. Moreover, despite the fact that they fall primarily into the B2 phylogroup, AIEC strains belonging to the A, B1, and D phylogroups have also been isolated (4, 33-35, 47). Although no specific virulence factors have been described for this pathovar, AIEC strains carry many virulence-associated genes characteristic of extraintestinal pathogenic E. coli (ExPEC) strains, which suggests that the AIEC pathovar could be closely related to the ExPEC pathovar (4, 17, 34).The aim of this work was to determine the frequency of strains with the “AIEC phenotype” among E. coli strains that cause extraintestinal infections, including uropathogenic E. coli (UPEC), septicemic E. coli, and neonatal meningitis E. coli strains. To achieve this objective, we determined the ability of a collection of ExPEC strains to adhere to and invade intestinal epithelial cells, as well as their capacity to survive and replicate within macrophages. In parallel, we compared the distributions of virulence-associated genes among ExPEC and AIEC strains. Furthermore, we searched for a common phylogenetic origin of the ExPEC strains that had an AIEC phenotype (referred to in this study as extraintestinal AIEC) and a collection of AIEC strains isolated mainly from the intestinal mucosae of CD patients (intestinal AIEC).     

Characterization of the Contribution to Virulence of Three Large Plasmids of Avian Pathogenic Escherichia coli χ7122 (O78:K80:H9)

Despite the fact that the presence of multiple large plasmids is a defining feature of extraintestinal pathogenic Escherichia coli (ExPEC), such as avian pathogenic E. coli (APEC), and despite the fact that these bacteria pose a considerable threat to both human and animal health, characterization of these plasmids is still limited. In this study, after successfully curing APEC of its plasmids, we were able to investigate, for the first time, the contribution to virulence of three plasmids, pAPEC-1 (103 kb), pAPEC-2 (90 kb), and pAPEC-3 (60 kb), from APEC strain χ7122 individually as well as in all combinations in the wild-type background. Characterization of the different strains revealed unique features of APEC virulence. In vivo assays showed that curing the three plasmids resulted in severe attenuation of virulence. The presence of different plasmids and combinations of plasmids resulted in strains with different pathotypes and levels of virulence, reflecting the diversity of APEC strains associated with colibacillosis in chickens. Unexpectedly, our results associated the decrease in growth of some strains in some media with the virulence of APEC, and the mechanism was associated with some combinations of plasmids that included pAPEC-1. This study provided new insights into the roles of large plasmids in the virulence, growth, and evolution of APEC by showing for the first time that both the nature of plasmids and combinations of plasmids have an effect on these phenomena. It also provided a plausible explanation for some of the conflicting results related to the virulence of ExPEC strains. This study should help us understand the virulence of other ExPEC strains and design more efficient infection control strategies.Escherichia coli strains are members of the normal intestinal microflora of most mammals and birds. They colonize their primary habitat, the lower intestinal tract of the host, within the first few hours of the host''s life (37, 54). E. coli strains are very versatile organisms, and the environment is considered their secondary habitat; approximately one-half of all living E. coli cells are actually living outside their hosts. Even though most E. coli strains are commensals and their presence provides a benefit to the host, a subset of these bacteria has acquired the ability to cause intestinal and extraintestinal diseases. These bacteria can be distinguished from commensals by their virulence factors (29, 37).Extraintestinal pathogenic E. coli (ExPEC), including avian pathogenic E. coli (APEC), pose a considerable threat to both human and animal health due to potential economic losses stemming from illness (30, 55, 62). ExPECs are responsible for a broad spectrum of infections in humans, including urinary tract infection (UTI), newborn meningitis (NBM), and septicemia. In addition, they are involved in animal diseases, such as avian colibacillosis, one of the most significant and widespread infectious diseases occurring in poultry and the cause of increased mortality, condemnations, and decreased production (3, 16). The most common disease syndromes associated with E. coli in birds are lower-respiratory-tract infections (air sacculitis), cellulites, meningitis, and septicemia (3).The different groups of E. coli have evolved mainly by acquisition of genes via horizontal gene transfer, a common phenomenon in bacteria that occurs even between very distantly related species (12, 45). This mechanism contributes to the evolution of E. coli variants, resulting in the development of novel strains and pathotypes. Conjugative plasmids are known to mediate transfer of genes between bacteria in diverse environments (42, 67). Acquisition of plasmids by bacteria is one of the fastest ways for survival in and adaptation to one or multiple hosts, as plasmids can encode multiple traits, including antibiotic and heavy metal resistance, virulence, and persistence in different environments (21).ExPEC strains (ExPECs) are differentiated from other pathotypes by the presence of specific virulence genes that allow them to spread systemically in hosts (62). ExPECs, particularly APEC isolates, carry multiple large plasmids (13, 32-35) belonging to different incompatibility groups (35), and the most prevalent plasmids in APEC strains (APECs) are the IncFIB, IncFIC, IncFIIA, IncI1, incP, incB/O, and IncN plasmids, some of which encode virulence factors. Additionally, plasmids encoding multiple drug resistance have been isolated from both APEC and uropathogenic E. coli (UPEC) strains. To date, few studies have undertaken sequencing and characterization of plasmids from avian isolates, particularly the ColV and ColBM plasmids from the IncFIB incompatibility group, which are considered common among ExPEC strains (22, 32, 33, 48, 66). Each of these plasmids has a conserved region harboring the FIB replicon, the ColV and/or ColBM operon, several known virulence genes, and iron acquisition and transport operons. According to recent studies the zoonotic risk seems to be related to the presence of large plasmids in APECs (48, 61).A fuller understanding of ExPEC virulence mechanisms is needed to develop treatments and preventative measures for use against ExPEC infections (55). Reductionism has been used for many years as a critical and powerful tool for identification of key genes responsible for microbial pathogenesis. However, the limitations of this approach for understanding the pathogenicity of bacteria include the multifactorial nature of virulence and the complex cross-regulation of gene expression. The ExPECs that cause diseases in humans and animals are very diverse, and although serotype and virulence factors are related to this diversity, the exact molecular mechanism behind the extensive diversity has not been elucidated yet.APEC strain χ7122 (O78:K80:H9) has been used for many years as a model strain to study the molecular mechanisms of APEC pathogenicity. The results of such studies have contributed greatly to increasing our understanding of the virulence of both human and animal ExPECs. This bacterium has three large plasmids, pAPEC-1 (103 kb), pAPEC-2 (90 kb), and pAPEC-3 (60 kb) (48). Most known virulence factors associated with APEC, including iron acquisition systems, tsh, and colicin V, are located on pAPEC-1, whereas the contents of pAPEC-2 and pAPEC-3 are completely unknown.Despite the fact that the presence of multiple large plasmids is a defining feature of the APEC pathotype (13, 32-35), characterization of these plasmids is still very limited. The exact role of many of them, as well the epistatic interactions between them, are unknown. The study of these plasmids has been complicated by their diversity and by the difficulty of curing them from the wild type. The few previous studies dedicated to understanding the role of the large plasmids of APEC in virulence were done in either E. coli K-12 (15, 31, 63) or avian commensal E. coli backgrounds (61, 70), which did not necessarily show the true functions of these plasmids in the wild-type background host strain.A plasmidless strain obtained from a wild-type APEC strain would provide a better background to evaluate the potential virulence of individual plasmids. In this study, after successfully curing APEC of its plasmids, we were able to investigate the contribution to virulence of each of the three large plasmids of APEC χ7122 by generating a plasmidless strain, strains with each plasmid individually, and strains with two plasmids in different combinations. We then determined the genetic locations of different virulence genes and compared the plasmid-containing derivative strains to the wild-type strain in terms of virulence, growth rate, serum resistance, iron uptake, and lipopolysaccharide (LPS) and iron-regulated outer membrane protein (IROMP) profiles. The results of this study provide new insights into the role of large plasmids in virulence, growth, and evolution of APEC by showing for the first time that both the nature of plasmids and combinations of plasmids have an effect on these factors. They also provide a plausible explanation for some conflicting results related to the virulence of ExPECs.     

Cyclomodulins in Urosepsis Strains of Escherichia coli

Determinants of urosepsis in Escherichia coli remain incompletely defined. Cyclomodulins (CMs) are a growing functional family of toxins that hijack the eukaryotic cell cycle. Four cyclomodulin types are actually known in E. coli: cytotoxic necrotizing factors (CNFs), cycle-inhibiting factor (Cif), cytolethal distending toxins (CDTs), and the pks-encoded toxin. In the present study, the distribution of CM-encoding genes and the functionality of these toxins were investigated in 197 E. coli strains isolated from patients with community-acquired urosepsis (n = 146) and from uninfected subjects (n = 51). This distribution was analyzed in relation to the phylogenetic background, clinical origin, and antibiotic resistance of the strains. It emerged from this study that strains harboring the pks island and the cnf1 gene (i) were strongly associated with the B2 phylogroup (P, <0.001), (ii) frequently harbored both toxin-encoded genes in phylogroup B2 (33%), and (iii) were predictive of a urosepsis origin (P, <0.001 to 0.005). However, the prevalences of the pks island among phylogroup B2 strains, in contrast to those of the cnf1 gene, were not significantly different between fecal and urosepsis groups, suggesting that the pks island is more important for the colonization process and the cnf1 gene for virulence. pks- or cnf1-harboring strains were significantly associated with susceptibility to antibiotics (amoxicillin, cotrimoxazole, and quinolones [P, <0.001 to 0.043]). Otherwise, only 6% and 1% of all strains harbored the cdtB and cif genes, respectively, with no particular distribution by phylogenetic background, antimicrobial susceptibility, or clinical origin.The bacterial species Escherichia coli comprises a wide diversity of strains belonging to the commensal intestinal flora of humans and warm-blooded animals. Among these strains, several pathogenic variants cause intestinal or extraintestinal infections in humans and animals (33). Population genetic studies based on multilocus enzyme electrophoresis and various DNA markers (10, 20, 44) classify the E. coli strains into four major phylogenetic groups (A, B1, B2, and D). The groups are diversely associated with certain ecological niches and propensities to cause disease.Extraintestinal pathogenic E. coli (ExPEC) strains are facultative pathogens that are not yet fully described. They are reported to belong mainly to phylogroups B2 and D, and they possess high numbers of virulence genes that belong to a flexible gene pool (43, 53). Among ExPEC strains, uropathogenic E. coli (UPEC) strains take advantage of host behavior and susceptibility by employing virulence factors that facilitate bacterial growth and persistence in the urinary tract (5, 28-30). Important virulence mechanisms are adhesion, invasion, subversion of host defenses, and direct interference with host cellular functions via secreted effectors (33, 69).These effectors include the cyclomodulins (CMs), a functional class of toxins that hijack the cell cycle, a fundamental host cell process (48). In the species E. coli, four kinds of CMs have been identified: the rho GTPase-targeting toxins CNF-1 to CNF-3 (cytotoxic necrotizing factors) (34), the cycle-inhibiting factor (Cif) (38), and two kinds of genotoxins, cytolethal distending toxins (CDTs) I to V (19) and the recently discovered colibactin (41). Colibactin is probably a hybrid polyketide-nonribosomal peptide toxin, whose activity is encoded by the genomic pks island (41). CDTs, Cif, and colibactin block mitosis, whereas CNFs promote DNA replication without cytokinesis. CM production can therefore be detected by the analysis of the cytopathic effects induced (41, 50).E. coli CMs are encoded by mobile elements (genomic islands, plasmids, and bacteriophages) that belong to the flexible gene pool of E. coli (22). The aim of the present study was to compare the prevalences of CMs in E. coli strains that differed in their clinical origins (community-acquired urosepsis or feces), phylogroups, and susceptibilities to antimicrobial agents.     

(CGG)4-Based PCR as a Novel Tool for Discrimination of Uropathogenic Escherichia coli Strains: Comparison with Enterobacterial Repetitive Intergenic Consensus-PCR

Urinary tract infections are one of the most frequent bacterial diseases in humans, and Escherichia coli is most often the relevant pathogen. A specific pathotype of E. coli, known as uropathogenic E. coli (UPEC), often causes serious and difficult-to-treat infections of the urinary tract. We propose a new single-tube screening tool that uses an (N)₆(CGG)₄ primer to generate fingerprint profiles that allow rapid discrimination and epidemiology of this group of bacteria. We found 71 different CGG-PCR profiles among 127 E. coli strains, while enterobacterial repetitive intergenic consensus (ERIC)-PCR of the same group yielded only 28 profiles. Additionally, the (CGG)₄-based PCR test turned out to be very effective for clustering UPEC strains exhibiting multiple virulence genes and usually belonging to the B2 phylogenetic group, and it separated these strains from E. coli strains lacking most of the UPEC-specific virulence factors. Since the reproducibility of the CGG-PCR screen is higher than that of ERIC-PCR, our test should be a valuable means of increasing the discriminatory power of current UPEC typing schemes.Gram-negative rods are the major etiological agents in urinary tract infections (UTIs) in humans, and Escherichia coli comprises most of these agents (20, 30, 32, 34, 38, 42). In some cases, UTI treatment is difficult because of persistent recurrences. Furthermore, UTIs are often asymptomatic at the beginning of the infection process. Particular phenotypic features of uropathogenic E. coli (UPEC) strains facilitate their persistence in urinary tracts and differentiate them from the other pathogenic and commensal E. coli strains (7, 29, 31). UPEC-specific virulence factors (VFs), which are mostly adhesins (P and S fimbriae), toxins (cytotoxic necrotizing factor type 1, α-hemolysin), bacteriocin (uropathogenic-specific protein), and siderophores (aerobactin and yersiniabactin), are important for colonization of the urinary tract (7, 8, 27). Also, type 1 fimbriae and afimbrial adhesin I are beneficial in this type of infection. Additionally, phylogenetic analyses have revealed that UPEC strains differ substantially from other E. coli strains (2, 10, 43). Pathogenic E. coli strains, including UPEC strains, belong mainly to groups B2 and D (2, 5, 14).In the case of E. coli, 16S rRNA gene sequence analysis, phylogenetic studies, and VF profiles are valuable for detailed genetic identification (4, 5, 11, 35). PCR-based methods are very efficient, inexpensive, and rapid (44). Previously, two distinct prokaryotic repetitive elements were used for gram-negative enterobacterial strain discrimination: repetitive extragenic palindromic (REP) elements and enterobacterial repetitive intergenic consensus (ERIC) sequences (16, 37, 40). Because the ERIC-PCR band patterns were less complex than the REP-PCR band patterns, differences within the analyzed species were easier to distinguish with ERIC-PCR.The goals of this work were to develop a novel genetic test (termed CGG-PCR) for the differentiation and epidemiological investigation of UPEC strains and to compare it to ERIC-PCR. In the comparison, the following factors were taken into consideration: cluster cutoff values, clustering capability with regard to virulence profiles, phylogenetic groups and quinolone susceptibility, reproducibility of band patterns, number of different profiles, and discriminatory indices. Our assay is based on the presence in bacterial genomes of microsatellites-trinucleotide repeat sequences. Since the different trinucleotide repeat sequence elements vary in copy number and distribution in bacterial genomes, they have the potential to serve as valuable markers for phylogenetic and epidemiological studies. A (CGG)₅ hybridization probe has been successfully used in conjunction with restriction fragment length polymorphism to type Mycobacterium tuberculosis (28). However, such hybridization techniques require the isolation of large amounts of genomic DNA and are time-consuming and expensive. Also, (GTG)₅-PCR was tested for its ability to track the origins of E. coli, Lactobacillus spp., and Enterococcus spp. isolated from various sources (12, 24, 25, 39). We propose an improved PCR methodology that employs an N₆(CGG)₄ primer with a high annealing temperature. Trinucleotide repeats are present on both DNA strands, enabling us to design a single PCR primer harboring the CGG motif that yields characteristic electrophoretic CGG-PCR band patterns. Considering the high reproducibility and specificity of the CGG-PCR profiles, this test has potential both as an alternative to and as an additional screening tool for the rapid and efficient genotyping of E. coli strains.     

Roles of the Extraintestinal Pathogenic Escherichia coli ZnuACB and ZupT Zinc Transporters during Urinary Tract Infection

Roles of the ZnuACB and ZupT transporters were assessed in Escherichia coli K-12 and uropathogenic E. coli (UPEC) CFT073. K-12 and CFT073 Δznu ΔzupT mutants demonstrated decreased ⁶⁵Zn²⁺ uptake and growth in minimal medium. CFT073Δznu demonstrated an intermediate decrease of ⁶⁵Zn²⁺ uptake and growth in minimal medium, whereas the CFT073ΔzupT mutant grew as well as CFT073 and exhibited a less marked decrease in ⁶⁵Zn²⁺ uptake. CFT073 mutants grew as well as the wild type in human urine. In competitive infections in CBA/J mice, the ΔzupT mutant demonstrated no disadvantage during urinary tract infection. In contrast, the UPEC Δznu and Δznu ΔzupT strains demonstrated significantly reduced numbers in the bladders (mean 4.4- and 30-fold reductions, respectively) and kidneys (mean 41- and 48-fold reductions, respectively). In addition, in single-strain infection experiments, the Δznu and Δznu ΔzupT mutants were reduced in the kidneys (P = 0.0012 and P < 0.0001, respectively). Complementation of the CFT073 Δznu ΔzupT mutant with the znuACB genes restored growth in Zn-deficient medium and bacterial numbers in the bladder and kidneys. The loss of the zinc transport systems decreased both motility and resistance to hydrogen peroxide, which could be restored by supplementation with zinc. Overall, the results indicate that Znu and ZupT are required for growth in zinc limited-conditions, that Znu is the predominant zinc transporter, and that the loss of Znu and ZupT has a cumulative effect on fitness during UTI, which may in part be due to reduced resistance to oxidative stress and motility.Escherichia coli is a versatile bacterial species comprised of innocuous and pathogenic strains. Pathogenic E. coli cause intestinal or extraintestinal infections in humans and other animals (28). Extraintestinal pathogenic E. coli (ExPEC) cause an array of diseases, including urinary tract infections (UTIs), neonatal meningitis, and septicemia. ExPEC associated with UTI is termed uropathogenic E. coli (UPEC). Compared to E. coli K-12 and other commensal strains, ExPEC strains encode in their genomes a greater variety of characterized, as well as putative, metal transport systems (11, 54). The importance of pathogen-specific iron transport systems for ExPEC has been established (20, 21, 26). However, the importance of other metal transport systems in ExPEC is less well characterized, and in particular nothing has been reported on the potential implication of the zinc transporters for the virulence of ExPEC.Zinc (Zn²⁺) is an essential micronutrient in all living cells, since this transition metal is a component of numerous metalloproteins and serves as an enzymatic cofactor or a structural element (5). In mammals, Zn²⁺ has an important immunomodulatory function and is critical for innate and acquired immunity (45). After exposure to lipopolysaccharide (LPS), zinc levels are decreased in the serum, and zinc is accumulated in the liver (38). In addition, during bacterial infection, the host protein calprotectin is released by neutrophils and may reduce bacterial numbers by restricting the availability of metals, including zinc and manganese (16, 39, 50). Also, although zinc concentrations are estimated to be in the millimolar range within host cells, available zinc may be mostly inaccessible to bacterial pathogens (3) and/or dramatically reduced after activation of innate immune defenses (45).In bacteria, the uptake of zinc is mediated by two major types of transporters: ZnuACB, which belongs to the cluster C9 family of (TroA-like) ATP-binding cassette (ABC) transporters (15, 27), and ZupT, which is a member of the ZIP (for ZRT/IRT-like protein) family of transporters that are also present in eukaryotes (27). Thus far, the Znu transport system has been shown to be important for virulence in a number of bacterial pathogens, including Salmonella enterica, Brucella abortus, and Haemophilus ducreyi (3, 12, 35, 56). Currently, the roles of the Znu and ZupT transporters for ExPEC virulence have not been investigated.The genomes of all E. coli strains that have been sequenced thus far contain genes encoding the two described zinc transporters: ZupT and ZnuACB. In addition to the transport of zinc, ZupT can also mediate the uptake of Co²⁺, Fe²⁺, and Mn²⁺ (24). The exact mechanism by which ZupT mediates metal uptake is currently unknown, although it may involve a chemiosmotic transmembrane gradient (24). Zinc uptake mediated by the Znu system requires hydrolysis of ATP by ZnuC to transport Zn²⁺ captured by the periplasmic binding protein ZnuA through the pore in the cytoplasmic membrane formed by a ZnuB dimer (36, 44). In ExPEC strains, the SitABCD transport system represents an additional ABC transporter belonging to the C9 (TroA-like) cluster (46). Sit transporters have thus far been characterized as manganese and iron transporters (8, 29, 47), although zinc has been shown to be an effective competitive inhibitor of manganese or iron uptake by SitABCD (8, 29). The SitA periplasmic binding protein also shares similarities to the TroA periplasmic binding protein of Treponema pallidum, which has been shown to bind both zinc and manganese with essentially equal affinities (19). It remains to be determined whether in addition to Znu and ZupT transporters, SitABCD or any other undefined transport systems may contribute to zinc transport or the virulence of ExPEC.In the present study, the roles of the Znu, ZupT, and SitABCD transport systems for zinc uptake and growth in zinc-restricted medium were compared in an E. coli K-12 Δznu ΔzupT mutant. In addition, the roles of the Znu and ZupT transporters for growth of ExPEC strain CFT073 in zinc-restricted conditions and for colonization in the murine ascending UTI model were assessed.     

Salmonella enterica Serovar Typhimurium Vaccine Strains Expressing a Nontoxic Shiga-Like Toxin 2 Derivative Induce Partial Protective Immunity to the Toxin Expressed by Enterohemorrhagic Escherichia coli

Shiga-like toxin 2 (Stx2)-producing enterohemorrhagic Escherichia coli (referred to as EHEC or STEC) strains are the primary etiologic agents of hemolytic-uremic syndrome (HUS), which leads to renal failure and high mortality rates. Expression of Stx2 is the most relevant virulence-associated factor of EHEC strains, and toxin neutralization by antigen-specific serum antibodies represents the main target for both preventive and therapeutic anti-HUS approaches. In the present report, we describe two Salmonella enterica serovar Typhimurium aroA vaccine strains expressing a nontoxic plasmid-encoded derivative of Stx2 (Stx2ΔAB) containing the complete nontoxic A2 subunit and the receptor binding B subunit. The two S. Typhimurium strains differ in the expression of flagellin, the structural subunit of the flagellar shaft, which exerts strong adjuvant effects. The vaccine strains expressed Stx2ΔAB, either cell bound or secreted into the extracellular environment, and showed enhanced mouse gut colonization and high plasmid stability under both in vitro and in vivo conditions. Oral immunization of mice with three doses of the S. Typhimurium vaccine strains elicited serum anti-Stx2B (IgG) antibodies that neutralized the toxic effects of the native toxin under in vitro conditions (Vero cells) and conferred partial protection under in vivo conditions. No significant differences with respect to gut colonization or the induction of antigen-specific antibody responses were detected in mice vaccinated with flagellated versus nonflagellated bacterial strains. The present results indicate that expression of Stx2ΔAB by attenuated S. Typhimurium strains is an alternative vaccine approach for HUS control, but additional improvements in the immunogenicity of Stx2 toxoids are still required.Shiga-like toxins (Stx) play a crucial role in the pathogenesis of enterohemorrhagic Escherichia coli (EHEC) strains, which may lead to hemorrhagic colitis, central nervous system disturbances, and hemolytic-uremic syndrome (HUS) (27, 33). HUS involves acute renal failure, thrombocytopenia, and microangiopathic hemolytic anemia, with mortality rates ranging from 1% to 4% (45, 50). EHEC strains may express different serotypes, including the widely distributed O157:H7 serotype, and infection correlates with the ingestion of contaminated ground beef and cow manure-contaminated water, vegetables, juices, and other products (13, 18). The incidence of EHEC-associated HUS cases is particularly high in developed countries, and high incidence rates have been recorded in Argentina, where cultural and diverse epidemiological factors contribute to the widespread dissemination of the disease among children and teenagers (38).EHEC strains may express two different Stx types. Stx1 is virtually identical to Stx produced by Shigella dysenteriae, while Stx2 shows only 56% homology to Stx1 at the amino acid sequence level (14, 33, 51). Both toxin types are formed by one A subunit and five B subunits, which bind to glycosphingolipid receptors, such as globotriaosyl ceramide (Gb3), on host cell membranes and promote retrograde toxin transport through the Golgi complex and endoplasmic reticulum. In the cell cytoplasm, Stx2 subunit A is processed into two fragments; one of them (A1) is endowed with N-glycosidase activity, which depurinates a specific adenine residue of the eukaryotic 28S rRNA, inhibits protein synthesis, and induces apoptosis of the target cell (18, 51).After ingestion and gut colonization, Stx molecules are released by the bacterial cells and translocate across the gut epithelium to reach, via the bloodstream, capillary endothelial cells at renal glomeruli, where the most relevant tissue damage occurs (33, 45, 50). Epidemiological data indicate that individuals infected with Stx2-producing bacterial strains, and some closely related variants, have a high probability of developing HUS (45, 50). In addition, Stx2 expression has been shown to increase gut colonization by bacterial cells due to induction of increased receptor expression by enterocytes (39).So far, there is no effective prophylactic or therapeutic approach for the prevention of HUS development among EHEC-infected individuals. The treatments available involve platelet transfusion in cases of severe anemia, hemodialysis, and supportive care (7, 50). A more direct anti-Stx treatment under clinical or preclinical evaluation involves the use of synthetic Stx glycolipid receptor analogs and humanized anti-Stx monoclonal antibodies (44, 52).Attempts to develop prophylactic anti-HUS vaccines are focused on the generation of Stx-neutralizing antibodies or the blockade of gut colonization. The vaccine strategies based on Stx2 that have been tested under experimental conditions have included DNA vaccines (5, 12), protein-conjugated polysaccharides (28), purified recombinant B subunits (24, 25, 29, 30, 47, 53, 55, 58), and B-subunit-derived synthetic peptides (19, 20). Anti-EHEC vaccine approaches based on the blockade of gut colonization have employed intimin and type III secreted proteins, such as EspA and EspB (3, 37, 54).Live bivalent anti-Stx vaccines based on genetically modified, attenuated Vibrio cholerae or Salmonella enterica serovar Typhimurium strains have been reported to induce anti-StxB antibody responses following oral administration to mice or rabbits (1, 10, 49). Attenuated Salmonella strains, used as orally administered vaccine vectors for the expression of heterologous antigens, show several advantages over conventional parenterally delivered cellular or acellular vaccine formulations (15, 16). Attenuated Salmonella strains are safe, are easily administered by untrained personnel, and, more relevantly, may induce systemic and secreted antigen-specific antibody and cell-based immune responses against self and heterologous antigens. In addition, whole bacterial cells carry on their surfaces several molecular structures known to activate both innate and adaptive immune responses. These molecules, such as lipopolysaccharide and flagellin, act as strong adjuvants, both systemically and at mucosal surfaces.Flagellins, the structural subunit of flagellar filaments, contribute both to the virulence of bacterial pathogens and to the activation of inflammatory responses in mammalian hosts. Bacterial flagellins have been shown to bind both extracellular and intracellular receptors of antigen-presenting cells, leading to inflammation and increased adaptive immune responses, including the generation of antigen-specific antibodies and T cells (2, 26). The strong adjuvant effects of Salmonella flagellins, either when admixed with purified antigens or when used as hybrid proteins genetically fused to the target antigens, have been demonstrated recently (4, 8, 22, 23, 36). However, there is no clear evidence that the expression of flagellin affects the immunogenicity of heterologous antigens expressed by attenuated Salmonella vaccine strains.In the present study, we generated new experimental anti-HUS vaccine formulations based on two recombinant attenuated S. Typhimurium aroA vaccine strains differing in the expression of flagellin. The two strains were genetically modified in order to express a nontoxic Stx2 derivative consisting of the whole Stx2 B subunit and a partially deleted A subunit encompassing the first amino acid of the A1 subunit genetically fused to the whole A2 subunit (Stx2ΔAB). The Stx2ΔAB protein was previously tested in mice immunized with a DNA vaccine (5). The results of the present study show that the S. Typhimurium vaccine strains express and secrete the recombinant toxin and induce both systemic and mucosal anti-StxB antibodies with anti-Stx2 neutralization activity, conferring partial protection against intravenous (i.v.) challenge with Stx2.     

Sharing of Escherichia coli Sequence Type ST131 and Other Multidrug-Resistant and Urovirulent E. coli Strains among Dogs and Cats within a Household

A multidrug-resistant (MDR) Escherichia coli strain from a human-associated pulsotype within sequence type ST131 (O25:H4) colonized three of five dogs and cats within a household. Of the four other colonizing strains identified, two were MDR and two colonized multiple hosts. The ST131 strain uniquely exhibited high resistance and virulence scores.Within-household sharing of Escherichia coli strains among humans and pets has been documented in multiple studies (8, 12, 13, 18, 24). This phenomenon, which likely reflects host-to-host transmission, may facilitate the dissemination of virulent and antimicrobial-resistant E. coli within the community.An extensively antimicrobial-resistant E. coli clonal group, sequence type ST131 (O25:H4), has been recognized as an important human pathogen only within the last several years, suggesting recent clonal dissemination and expansion (4, 7, 25). Currently best known for its association with extended-spectrum cephalosporin resistance, ST131 has contributed importantly to the global emergence of the CTX-M-15 extended-spectrum beta-lactamase (and, perhaps, vice versa) (4, 7, 25). E. coli ST131 also commonly occurs as a fluoroquinolone-resistant (FQ-R) but cephalosporin-susceptible pathogen (1, 15, 20).Although ST131 has been isolated from a companion animal (a dog) (27), this report did not address other hosts from the same household or the isolate''s relationship to human ST131 isolates. The recent finding by the Escherichia coli Reference Center (the Pennsylvania State University) of serotype O25:H4 for sequential FQ-R E. coli urine isolates from a dog with recurrent/persisting bacteriuria (see below) suggested E. coli ST131. The source household included multiple pets, providing an opportunity to investigate within-household sharing of the index FQ-R urine strain. We assessed whether (i) the dog''s urine strain represented ST131, (ii) this strain was shared with other pets in the household, (iii) the strain resembled known human ST131 isolates, and (iv) additional sharing of antimicrobial-resistant and/or virulent E. coli clones could be found among the household pets.     

Non-spa-Typeable Clinical Staphylococcus aureus Strains Are Naturally Occurring Protein A Mutants

Staphylococcus aureus is a major human pathogen responsible for increasing the prevalence of community- and hospital-acquired infections. Protein A (SpA) is a key virulence factor of S. aureus and is highly conserved. Sequencing of the variable-number tandem-repeat region of SpA (spa typing) provides a rapid and reliable method for epidemiological studies. Rarely, non-spa-typeable S. aureus strains are encountered. The reason for this is not known. In this study, we characterized eight non-spa-typeable bacteremia isolates. Sequencing of the entire spa locus was successful for five strains and revealed various mutations of spa, all of which included a deletion of immunoglobulin G binding domain C, in which the upper primer for spa typing is located, while two strains were truly spa negative. This is the first report demonstrating that nontypeability of S. aureus by spa sequencing is due either to mutation or to a true deficiency of spa.Staphylococcus aureus is an important human pathogen responsible for many community- and hospital-acquired infections (22). S. aureus can cause various diseases, ranging from superficial skin infections to severe and life-threatening diseases, such as pneumonia, osteomyelitis, endocarditis, and sepsis (36). Recently, the prevalence of community- and hospital-acquired methicillin (meticillin)-resistant S. aureus (MRSA) infections has increased (2). MRSA infections, especially, are responsible for enhanced mortality and significant increases in the length of hospitalization and hospital charges (4).Protein A (SpA) represents an important virulence factor for S. aureus (7, 8, 29) and has recently been shown to be involved in the pathogenesis of S. aureus pneumonia (10, 11), to be expressed in invasive diseases (21), and to play a role in proteinaceous biofilm formation (25). SpA is a protein of 42 kDa and comprises several regions with different functions: The signal sequence (S region) in the N-terminal part is followed by four or five highly homologous immunoglobulin G (IgG)-binding domains in tandem (the E, D, A, B, and C regions) (20). The C-terminal region, or X region, is divided into two domains: (i) the repeat region X_R, consisting of variable repeats with mostly octapeptide structures, which are used for spa typing, and (ii) the X_C region, consisting of a conserved sequence, which confers anchoring to the cell wall via an LPXTG-binding motif (31, 32). The best-studied function of SpA is the interaction with human IgG by binding to the F_c part, thereby compromising the host immune system (6, 13). Furthermore, SpA can bind to various host structures, such as the von Willebrand factor and the receptor gC1qR/p33 on platelets (15, 27), which promote adhesion to platelets.In recent years, spa (S. aureus protein A) typing has been used frequently as a typing method. As a single-locus sequence-based typing method, it combines a number of technical benefits, such as rapidity, reproducibility, and portability (33), thereby allowing easy interlaboratory comparability via the Internet and synchronization to a central server (http://www.ridom.de/spaserver/) (1, 14). spa typing uses the sequence of the polymorphic X region, which consists of a variable number of tandem repeats of 24 bp, as a genetic marker (9). This region has been shown to be rather stable and allows distinguishing of strains to a degree comparable to that of pulsed-field gel electrophoresis (PFGE) and whole-genome DNA microarray (19). Interestingly, mutations of the repeat region, including insertions, deletions, and point mutations, have been observed only after long-term persistence of S. aureus in vivo in the airways of cystic fibrosis patients, allowing calculation of the clock speed of this region (18) and to establish an algorithm for the analysis of this region (24).So far, no spa-deficient clinical strains have been described. Although some studies demonstrate 100% spa typeability (12), non-spa-typeable strains have been detected recently (23, 34). The reason for this is not known. Therefore, this study was performed to investigate the underlying mechanism of the nontypeability of eight invasive S. aureus strains, which were isolated from bacteremic patients with different infections. We sequenced the whole spa locus and investigated the expression of SpA by Western blot analysis and real-time PCR.     

Identification of Specific and Universal Virulence Factors in Burkholderia cenocepacia Strains by Using Multiple Infection Hosts

Over the past few decades, strains of the Burkholderia cepacia complex have emerged as important pathogens for patients suffering from cystic fibrosis. Identification of virulence factors and assessment of the pathogenic potential of Burkholderia strains have increased the need for appropriate infection models. In previous studies, different infection hosts, including mammals, nematodes, insects, and plants, have been used. At present, however, the extent to which the virulence factors required to infect different hosts overlap is not known. The aim of this study was to analyze the roles of various virulence factors of two closely related Burkholderia cenocepacia strains, H111 and the epidemic strain K56-2, in a multihost pathogenesis system using four different model organisms, namely, Caenorhabditis elegans, Galleria mellonella, the alfalfa plant, and mice or rats. We demonstrate that most of the identified virulence factors are specific for one of the infection models, and only three factors were found to be essential for full pathogenicity in several hosts: mutants defective in (i) quorum sensing, (ii) siderophore production, and (iii) lipopolysaccharide biosynthesis were attenuated in at least three of the infection models and thus may represent promising targets for the development of novel anti-infectives.The Burkholderia cepacia complex (BCC) comprises a group of the following 17 formally named bacterial species: Burkholderia cepacia, Burkholderia multivorans, Burkholderia cenocepacia, Burkholderia stabilis, Burkholderia vietnamiensis, Burkholderia dolosa, Burkholderia ambifaria, Burkholderia anthina, Burkholderia pyrrocinia, Burkholderia ubonensis, Burkholderia latens, Burkholderia diffusa, Burkholderia arboris, Burkholderia seminalis, Burkholderia metallica, Burkholderia lata, and Burkholderia contaminans (46, 69, 70, 71). Strains of the BCC are ubiquitously distributed in nature and have been isolated from soil, water, the rhizosphere of plants, industrial settings, hospital environments, and infected humans. Some BCC strains have enormous biotechnological potential and have been used for bioremediation of recalcitrant xenobiotics, plant growth promotion, and biocontrol purposes. At the same time, however, BCC strains have emerged as problematic opportunistic pathogens in patients with cystic fibrosis (CF) and in immunocompromised individuals (12, 19, 44, 46). The clinical outcomes of BCC infections range from asymptomatic carriage to a fulminant and fatal pneumonia, the so-called cepacia syndrome (30). Apart from acquisition from the environment, patient-to-patient transmission and indirect nosocomial acquisition from contaminated surfaces have caused several outbreaks within and between regional CF centers (55). Although all BCC species have been isolated from both environmental and clinical sources, B. cenocepacia and B. multivorans are most commonly found in clinical samples (12, 44).Members of the BCC not only are opportunistic pathogens of humans but also can cause infections in a diverse range of species, including animals, nematodes, and plants (59). This allowed the development of various infection models, using the mouse or rat, the nematode Caenorhabditis elegans, onions, or the alfalfa plant as an infection host. More recently, larvae of the wax moth Galleria mellonella have been used as infection hosts of BCC strains (58). These models have been employed to investigate the virulence of different BCC species as well as of mutants to understand the importance of specific genes in disease. These infection models have also been applied to studies of host response, gene therapy, antimicrobial delivery, and immunization for prevention of BCC lung disease (6, 32, 51).Previous work has identified several virulence factors that may play a role in infections caused by BCC strains. Some isolates have been demonstrated to be capable of surviving within eukaryotic cells, such as respiratory epithelial cells, macrophages, and amoebae (7, 50, 56). Other virulence factors that have been identified by the use of different infection models include the quorum-sensing system (40), biofilm formation (14), iron-chelating siderophores (16), proteases (15), type III and IV secretion systems (20, 24, 67), melanin production (77), catalase (38), lipopolysaccharide (LPS) (41), cable pili and flagella (57, 68), surface exopolysaccharides (11), a lysR regulator (4), capsule (29), and intrinsic antimicrobial resistance (10). Recently, a phenylacetic acid catabolic pathway was shown to be required for full pathogenicity of B. cenocepacia in the C. elegans infection model (37).At present, knowledge on the importance of these factors in different infection hosts is scarce. This study was initiated to identify both host-specific and conserved mechanisms of pathogenicity in C. elegans, G. mellonella, alfalfa, and murine infection models. We demonstrate that some virulence factors are important for pathogenicity in more than one infection model, while other factors were found to be host specific. N-Acyl homoserine lactone (AHL)-dependent quorum sensing (QS) was identified as a highly conserved regulatory mechanism for expression of pathogenic traits. Siderophore production and intact LPS were important for virulence in all animal models. However, we also identified several virulence factors that were required for pathogenesis in only one of the models.     

Shiga Toxin,Cytolethal Distending Toxin,and Hemolysin Repertoires in Clinical Escherichia coli O91 Isolates

Shiga toxin (Stx)-producing Escherichia coli (STEC) strains of serogroup O91 are the most common human pathogenic eae-negative STEC strains. To facilitate diagnosis and subtyping of these pathogens, we genotypically and phenotypically characterized 100 clinical STEC O91 isolates. Motile strains expressed flagellar antigens H8 (1 strain), H10 (2 strains), H14 (52 strains), and H21 (20 strains) or were H nontypeable (Hnt) (10 strains); 15 strains were nonmotile. All nonmotile and Hnt strains possessed the fliC gene encoding the flagellin subunit of the H14 antigen (fliC_H14). Most STEC O91 strains possessed enterohemorrhagic E. coli hlyA and expressed an enterohemolytic phenotype. Among seven stx alleles identified, stx_2dact, encoding mucus- and elastase-activatable Stx2d, was present solely in STEC O91:H21, whereas most strains of the other serotypes possessed stx₁. Moreover, only STEC O91:H21 possessed the cdt-V cluster, encoding cytolethal distending toxin V; the toxin was regularly expressed and was lethal to human microvascular endothelial cells. Infection with STEC O91:H21 was associated with hemolytic-uremic syndrome (P = 0.0015), whereas strains of the other serotypes originated mostly in patients with nonbloody diarrhea. We conclude that STEC O91 clinical isolates belong to at least four lineages that differ by H antigens/fliC types, stx genotypes, and non-stx putative virulence factors, with accumulation of virulence determinants in the O91:H21 lineage. Isolation of STEC O91 from patients'' stools on enterohemolysin agar and the rapid initial subtyping of these isolates using fliC genotyping facilitate the identification of these emerging pathogens in clinical and epidemiological studies and enable prediction of the risk of a severe clinical outcome.Shiga toxin (Stx)-producing Escherichia coli (STEC) strains cause diarrhea and a life-threatening hemolytic-uremic syndrome (HUS) worldwide (23, 44). STEC strains isolated from patients usually possess, in addition to one or more stx genes, the eae gene, encoding adhesin intimin (7, 11, 16, 25, 26, 41, 49). However, a subset of STEC strains associated with human disease lack eae, and among these, strains of serogroup O91 are the most common (2, 7, 35, 37, 47, 48). In Germany during the last 5 years, serogroup O91 accounted for 6.4% to 11.0% of all STEC strains reported from human infections and was therefore the fourth-most-common STEC serogroup (after O157, O26, and O103) isolated (47, 48; http://www.rki.de). However, in contrast to eae-positive STEC strains of the three leading serogroups, which cause disease mostly in young children (47), STEC O91 is the most common serogroup isolated from adult patients (48).Despite their association with human diseases worldwide (7, 9, 11, 13, 14, 30, 35, 37, 38, 40, 47, 48), the spectrum of serotypes of STEC O91 isolates from patients and the pathogenic traits of such strains are poorly understood. Moreover, characteristics of STEC O91 strains which could assist with their isolation from human stools and further subtyping in clinical microbiological laboratories have not been systematically investigated or reported. To gain insight into the serotype composition and putative virulence factors of STEC O91 strains causing human disease and to identify characteristics which can facilitate laboratory diagnosis of these organisms, we determined the motility and flagellar phenotypes, fliC types, stx genotypes, non-stx putative virulence loci, and diagnostically useful phenotypes of 100 clinical STEC O91 isolates. Moreover, we investigated possible associations between bacterial characteristics and clinical infection phenotypes.     

Lipopolysaccharide as an Antigen Target for the Formulation of a Universal Vaccine against Escherichia coli O111 Strains

A promising approach to developing a vaccine against O111 strains of diarrheagenic Escherichia coli that exhibit different mechanisms of virulence is to target either the core or the polysaccharide chain (O antigen) of their lipopolysaccharide (LPS). However, due to structural variations found in both these LPS components, to use them as antigen targets for vaccination, it is necessary to formulate a vaccine able to induce a humoral immune response that can recognize all different variants found in E. coli O111 strains. In this study, it was demonstrated that, despite differences in composition of oligosaccharide repeat units between O111ab and O111ac LPS subtypes, antibodies against one O111 subtype can recognize and inhibit the adhesion to human epithelial cells of all categories of O111 E. coli (enteropathogenic E. coli [EPEC], enterohemorrhagic E. coli [EHEC], and enteroaggregative E. coli [EAEC]) strains regardless of the nature of their flagellar antigens, mechanisms of virulence, or O111 polysaccharide subtypes. These antibodies were also able to increase the clearance of different strains of O111 E. coli by macrophages. PCR analyses of the pathways involved in O111 LPS core biosynthesis showed that all EAEC strains have core type R2, whereas typical EPEC and EHEC have core type R3. In contrast, atypical EPEC strains have core types R2 and R3. In summary, the results presented herein indicate that the O111 polysaccharide and LPS core types R2 and R3 are antigen targets for panspecific immunotherapy against all categories of O111 E. coli.Pathogenic strains of O111 Escherichia coli exist as three distinct categories of diarrheagenic organisms, namely, enteropathogenic E. coli (EPEC; typical and atypical), enterohemorrhagic E. coli (EHEC), and enteroaggregative E. coli (EAEC) (7). In developing countries, diarrhea induced by these pathogens is a serious illness that inflicts a huge health and economic burden on the population (46, 48). Despite the fact that sanitation and clean water can markedly reduce the cases of diarrhea in areas of endemicity, surveillance studies have demonstrated that in Latin America alone more than 80% of the population has no access to sewage systems or treated water (44). Different serotypes of Shiga toxin-producing E. coli pathogens (O111:H⁻, O111:H8, and O111:H2) are also a public health problem in developed countries worldwide, where they have been responsible for outbreaks of bloody diarrhea and cases of hemolytic-uremic syndrome (HUS) (4, 12, 14, 21, 28, 32, 35, 55). One of the worst outbreaks of O111 E. coli happened in August 2008 in Oklahoma, where 341 people become ill, 70 people were hospitalized, 17 people developed HUS, and 1 person died (5, 8). In addition, other pathogens such as Salmonella enterica subsp. enterica serovar Adelaide and Salmonella enterica subsp. enterica serovar 50:z:e,n,x also have the same lipopolysaccharide (LPS) polysaccharide structure as that found in O111 E. coli (29).Because of the impact that O111 E. coli strains have on public health, a lot of effort has been devoted to developing a safe, cheap, and effective vaccine to prevent diarrheagenic diseases caused by these pathogens.The best approach to constructing a vaccine capable of protecting against a wide range of different strains of O111 E. coli is to target the LPS polysaccharide chain (O antigen), since 75% of the outer membrane of all Gram-negative bacteria is covered by LPS (38, 50). This approach is supported by the fact that conjugated vaccines against polysaccharides have been used successfully against polysaccharide-encapsulated organisms such as Streptococcus pneumoniae and Haemophilus influenzae type b in clinical practice (42). However, to use the O111 polysaccharide chain as an antigen target for the construction of a universal vaccine against enteric O111 E. coli pathogens, the antigenic variation of O111 subtypes between different E. coli strains has to be taken into account (7, 33, 59). In addition, although the O111 polysaccharides that compose their capsules are identical to the ones present on their external membranes (17, 53, 54), it has been demonstrated by Goldman and coworkers that the capsules of O111 bacteria are poorly recognized by antibodies raised against O111 LPS derived from the bacterial membrane (17), indicating that immunization with capsulated bacteria induces antibody responses different from those induced by immunization with noncapsulated bacteria.In addition, the O111 E. coli strains can be either naked or capsulated, although the O111 polysaccharides that compose their capsules are identical to the ones present on their external membranes, except for the absence of a lipid A core (17, 53, 54).The LPS core can also be targeted for vaccination or immunotherapy (11, 19, 39). It is not considered a virulence factor, although its involvement in bacterial adhesion has been reported (24). Structural variations are also found in the external part of the LPS core (37), and they have to be considered in order to generate antibodies capable of identifying all antigenic variants encountered within O111 bacteria.Another element of the humoral immune response involved in clearance of pathogens is the complement system, which, independently of antibody, can be activated by pathogens in the initial stages of infection and, by itself, can kill pathogens directly. However, it is not effective in recognizing or eliminating all bacteria in samples (3, 30, 43, 45). The complement system can also promote bacterial uptake and destruction by macrophages by interacting with both the pathogen and the complement receptors present on the macrophage membrane. However, when complement activation is not enough to promote bacterial killing by macrophages, antibodies are required (25, 26, 34).To investigate whether the O111 LPS polysaccharide of E. coli is a good antigen candidate for the formulation of a universal vaccine capable of preventing infection by O111 pathogens, electrophoretic, molecular, serological, and immunological analyses were conducted in order to determine whether antibodies against O111 polysaccharides can recognize O111 EHEC, EPEC, and EAEC, can inhibit their adhesion to human epithelial cells, and can stimulate their clearance by macrophages.In addition, the compositions of the cores of 73 samples of all categories of O111 bacteria were characterized by PCR analysis of the enzymes responsible for the biosynthesis of all five types of LPS core: R1, R2, R3, R4, and K12.     

Selection of Polymorphic Peptides from GRA6 and GRA7 Sequences of Toxoplasma gondii Strains To Be Used in Serotyping

The evaluation of Toxoplasma gondii isolates obtained from geographical environments other than Europe and North America revealed the existence of atypical strains that are not included in the three archetypal clonal lineages (lineages I, II, and III). GRA6 and GRA7 are polymorphic genes that have been used for the genotyping of Toxoplasma. The coding regions of GRA6 and GRA7 from 49 nonarchetypal strains were sequenced and compared with the sequences of type I, II, and III reference strains. Eighteen and 10 different amino acid sequences were found for GRA6 and GRA7, respectively. The polymorphisms found between the different sequences were analyzed, with the objective of defining peptides to be used for the serotyping of Toxoplasma infections. Two peptides specific for clonal lineages I and III (peptides GRA7I and GRA7III, respectively) were selected from the GRA7 locus. Three peptides specific for some atypical strains (peptides Am6, Af6, and Am7) were selected from both the GRA6 and the GRA7 loci. Serum samples from humans infected with Toxoplasma strains of known genotypes were serotyped with the selected peptides. Peptide GRA7III seems to be a good candidate for the serotyping of infections caused by type III strains. Peptide GRA7I had a very low sensitivity. Peptides Am6 and Af6 had low specificities, since they reacted with serum samples from patients infected with strains belonging to the three archetypal lineages. Although peptide Am7 was specific, it had low sensitivity.The vast majority of Toxoplasma gondii isolates from human patients and domestic animals in Europe and North America belong to three archetypal clonal lineages, namely, types I, II, and III (1, 11, 30). However, nonarchetypal strains with atypical genotypes have recently been described in unusual hosts such as sea otters (10, 42, 43) and in tropical areas such as South America and Africa (2, 32, 39, 46, 54). Genotyping studies that distinguish different types of strains are important to gain knowledge of the biodiversity of the parasite in order to understand the molecular epidemiology of Toxoplasma and to highlight the correlation between the genotype of the parasite and the pathogenesis of human toxoplasmosis.The dense granules (GRA) are parasitic organelles involved in cell invasion and in the intracellular survival of the parasite. GRA proteins are expressed by the three stages of T. gondii: the tachyzoite, bradyzoite (38), and sporozoite (55) stages. GRA6 is a GRA antigen of 32 kDa described for the first time by Lecordier et al. (38). In extracellular parasites, GRA6 exists in dense secretor granules mostly as soluble proteins. Like the other GRA proteins, GRA6 is involved in host cell invasion. GRA6 is a glycine-rich protein and behaves like an integral membrane protein within the parasitophorous vacuole (36, 41). GRA6 is considered a good marker of acute infection (27, 28, 52). However, the immune response to GRA6 is very heterogeneous (25).GRA7 is a GRA antigen of 29 kDa (26, 31). Like GRA6, it is involved in host cell invasion. This protein is associated with the parasite membrane complex, the tubular elements of the intravacuolar network, and the parasitophorous vacuolar membrane. It migrates from the GRA to the parasitophorous vacuolar membrane through the intravacuolar network during host cell invasion (9). GRA7 is an antigen characteristic of the acute phase of the infection (27, 49, 51) and a target antigen in the intracerebral immune response during the chronic phase of infection (23, 45). These immunogenic properties make this antigen a good marker for serodiagnostic studies (5, 6, 7, 50).GRA6 and GRA7 are polymorphic loci. The coding region of the GRA6 locus has been used as a marker for the genotyping of Toxoplasma (8, 15-20, 24, 33, 34, 40, 43, 47, 53). Sequencing of GRA6 detected a high degree of polymorphism (24, 57). Single nucleotide polymorphisms (SNP) in the region encoding GRA6 can be detected by methods based on PCR followed by restriction fragment length polymorphism (PCR-RFLP): digestion of the amplification products with a single endonuclease (MseI) can differentiate genotypes I, II, and III (24). Another method based on GRA6 polymorphisms is pyrosequencing (21). This technique allows the analysis of short DNA sequences and SNP. Two SNP located at positions 162 and 171 of the GRA6 gene allow the differentiation of types I, II, and III. GRA7 has been less explored as a marker for the genotyping of Toxoplasma. Preliminary results (I. Villena, unpublished data) showed that GRA7 allows the discrimination between genotypes I, II, and III and some atypical strains by PCR-RFLP.Kong et al. (35) first proposed the use of the GRA6 and GRA7 proteins for serotyping. Basically, serotyping consists of a serological test with polymorphic peptides from Toxoplasma immunogens to detect strain-specific antibodies. Different peptides were proposed on the basis of the sequences of these antigens (35, 48, 53). However, the available peptides can differentiate only type II from non-type II infections. Infections due to nonarchetypal strains are misclassified as type II strains or type I or III strains, since GRA6 C-terminal peptides specific for type II and type I or III strains cross-react with serum samples from patients with infections caused by nonarchetypal strains (53).We proposed to determine polymorphic regions of GRA6 and GRA7 with the objective of defining possible polymorphic peptides that could be used to distinguish type I from type III infections and infections due to atypical strains by serotyping. Some of the defined peptides were tested in order to evaluate their utility as serotyping markers.     

Major Differences Exist in Frequencies of Virulence Factors and Multidrug Resistance between Community and Nosocomial Escherichia coli Bloodstream Isolates

Escherichia coli is a major cause of bloodstream infections and death due to sepsis. It is the most frequent Gram-negative bacterial pathogen recovered from cultures of blood from both community-acquired and nosocomial cases. We set out to determine the relationships between E. coli virulence factors (VFs), phylogenetic groups, and antibiotic resistance and whether bacteremia cases had a community, health care-associated. or nosocomial origin. Isolates from consecutive episodes of E. coli bacteremia in 303 patients presenting to a university hospital were screened for their VFs, phylogenetic group, and antibiotic resistance. The majority of VFs present in the collection were equally distributed between antibiotic-susceptible and multiple-drug-resistant (MDR) isolates, but the overall VF score was higher for isolates of community and health care-associated origin than those of nosocomial origin (P = 0.0002 and P = 0.0172, respectively); the papA, papG allele II, hlyA, and hek VFs were more prevalent in this cohort. Most isolates belonged to phylogenetic group B2, which harbored a greater proportion of antibiotic-susceptible isolates than MDR isolates (P = 0.04). The community, health care-associated, or nosocomial origin of E. coli bacteremia determines the virulence capacity of an isolate better than the phylogenetic group does. This study provides new insights into the relationships between the pathogenesis and epidemiology of E. coli bacteremia.Escherichia coli is a leading cause of bloodstream infections worldwide, and the associated rate of mortality is high (32). Extraintestinal pathogenic E. coli (ExPEC) is the predominant cause of these invasive infections, which often originate from the urinary tract. They possess a wide variety of specialized virulence factors (VFs) responsible for pathogenesis outside the gastrointestinal tract, including diverse adhesins, invasins, and protectins (48). ExPEC isolates are generally concentrated within phylogenetic group B2 and, to a lesser extent, group D, whereas less virulent and commensal E. coli isolates belong to groups A and B1 (25, 29, 37). However, the minimal requirements for bacterial invasion of the bloodstream have yet to be determined.Bacteremic isolates harbor a significantly greater repertoire of VFs than gastrointestinal tract commensal E. coli isolates (46), and where their molecular and epidemiological environments have been further analyzed, it appears that antibiotic-susceptible and -resistant ExPEC isolates are fundamentally different bacterial populations (19, 21, 23, 25, 27, 37, 38, 42, 43, 50). Antibiotic-susceptible strains mostly derive from phylogenetic group B2 and are associated with higher VF prevalences than antibiotic-resistant strains, which are typically associated with shifts toward groups D and A. This decreased prevalence of VFs within resistant strains has been suggested to be a possible trade-off between resistance and virulence in ExPEC (8). An important epidemiological consideration is whether community-acquired (CA) or health care-associated (HCA) E. coli strains causing bacteremia are distinct from strains that cause nosocomially acquired (NA) bacteremia. The only study to address this to date found that CA bacteremic isolates were significantly associated with papC and papG fimbrial VFs, phylogenetic group B2, and antibiotic susceptibility compared to the associations for their NA counterparts (21).In the study described here, we investigated a large collection of epidemiologically well documented E. coli bacteremia isolates to determine the relationships between VFs, phylogenetic group, and antibiotic resistance. We set out to draw a clearer distinction than has previously been achieved between fully antibiotic-susceptible isolates, those resistant to a limited set of antibiotics, and those resistant to multiple antibiotic classes on the basis of the fact that the last group would most closely represent a nosocomial population.     

Diverse Characteristics of the cagA Gene of Helicobacter pylori Strains Collected from Patients from Southern Vietnam with Gastric Cancer and Peptic Ulcer

The pathogenesis of gastroduodenal diseases is related to the diversity of Helicobacter pylori strains. CagA-positive strains are more likely to cause gastric cancer than CagA-negative strains. Based on EPIYA (Glu-Pro-Ile-Tyr-Ala) motifs at the carboxyl terminus corresponding to phosphorylation sites, H. pylori CagA is divided into East Asian CagA and Western CagA. The former type prevails in East Asia and is more closely associated with gastric cancer. The present study used full sequences of the cagA gene and CagA protein of 22 H. pylori strains in gastric cancer and peptic ulcer patients from Southern Vietnam to make a comparison of genetic homology among Vietnamese strains and between them and other strains in East Asia. A phylogenetic tree was constructed based on full amino acid sequences of 22 Vietnamese strains in accordance with 54 references from around the world. The cagA gene was found in all Vietnamese H. pylori strains. Twenty-one of 22 (95.5%) strains belonged to the East Asian type and had similar characteristics of amino acid sequence at the carboxyl terminus to other strains from the East Asian region. From evidence of East Asian CagA and epidemiologic cancerous lesions in Vietnam, H. pylori-infected Vietnamese can be classified into a high-risk group for gastric cancer, but further studies on the interaction among environmental and virulence factors should be done. Finally, phylogenetic data support that there is a Japanese subtype in the Western CagA type.Helicobacter pylori is a gram-negative bacterium that infects about 50% of the world''s population. Infections with H. pylori can result in chronic active gastritis and are a risk factor for peptic ulcers, gastric cancer and gastric mucosa-associated lymphoid tissue lymphoma (40, 52, 53, 56). The prevalence of H. pylori infections is not the same in different parts of the world. Recent studies reported that humans actually acquired H. pylori in the early days of their history, long before the migration of modern humans out of Africa, and the diverse distribution of H. pylori today is associated with waves of human migration in the past (19, 32, 36, 61, 62). The rate of H. pylori infections is high in Africa, East Asia, and South Asia; however, the incidence of gastric cancer is high in East Asia but not in South Asia or Africa; this may be explained partly by the diversity of H. pylori strains in these regions (62).In cases of gastroduodenal diseases, especially gastric cancer, the pathogenesis involves three major factors: H. pylori virulence factors, host factors, and environmental factors (1, 10, 14, 16, 21, 34, 57). Two H. pylori virulence factors that have been focused on in many studies all around the world are VacA and CagA. VacA is encoded by the vacA gene and found in all H. pylori strains. The vacA gene is classified into three major genotypes: s1/m1, s1/m2, and s2/m2. s1/m1 strains produce higher levels of cytotoxin than s1/m2 or s2/m2 strains (2, 3). CagA is encoded by cagA, located in the cagPAI (pathogenicity island) region of the H. pylori genome, and the presence of this protein is a marker of cagPAI. Unlike the vacA gene, the cagA gene has been found in only 50% to 70% of H. pylori strains infecting Western populations: however, 80% to 100% of H. pylori strains from East Asia have the cagA gene, with the cagPAI region, in their genome (17, 49, 55, 61, 64, 65). Patients infected with H. pylori strains possessing CagA were at greater risk of developing gastric adenocarcinoma than those uninfected or infected with CagA-negative strains (11, 41). CagA is injected directly from H. pylori into intragastric epithelial cells via the type IV secretion system. The injected CagA mimics eukaryotic adaptor proteins that recruit multiple host signaling factors into protein complexes that target cellular junctions, cell proliferation, and actin-cytoskeletal rearrangements (54). The translocated CagA undergoes tyrosine phosphorylation by Src and Abl family kinases and binds to SHP-2 in the human gastric mucosa (45, 47, 48). The repeated EPIYA (Glu-Pro-Ile-Tyr-Ala) motifs at the carboxyl-terminal end of CagA are the targets of tyrosine phosphorylation (8, 22, 24, 38, 63). CagA multimerization plays an important role in the pathophysiological activity of CagA in disturbing host cell functions via SHP-2 deregulation, and EPIYA polymorphisms of CagA greatly influence the magnitude and duration of phosphorylation-dependent CagA activity (37, 42). SHP-2, like its Drosophila melanogaster homolog Corkscrew, is known to play an important role in the mitogenic signal transduction that connects receptor tyrosine kinases and ras (20). It is possible that deregulation of SHP-2 by the translocation of CagA plays a role in the acquisition of a cellular-transformed phenotype at a relatively early stage in the carcinogenesis of gastric carcinoma. A recent study on generating CagA in transgenic mice has provided the first direct evidence of the role of CagA as a bacterium-derived oncoprotein that acts in mammals and further indicates the importance of tyrosine phosphorylation, which enables CagA to deregulate SHP-2, in the development of H. pylori-associated neoplasms (39). Based on characteristics of the EPIYA motif, the H. pylori CagA protein could be divided into a Western type and an East Asian type. The East Asian CagA protein exhibits stronger SHP-2-binding activity and so is more pathogenic than the Western CagA protein in H. pylori-infected patients (4, 7, 23, 37). Clinical data from East Asia, Japan, and South Korea indicated that the East Asian form of CagA was more closely related to persistent active inflammation, atrophic gastritis, and a higher risk of gastric cancer than the Western form (5, 6, 26, 44). It is quite clear that by studying the characteristics of H. pylori strains, especially the cagA gene and CagA protein, one can categorize H. pylori-infected patients into those at high risk of developing gastric cancer and those not.Vietnam is a developing country located in Southeast Asia. However, according to historical and migrational evidence, the Vietnamese are more closely related to people from East Asia than people from South Asia. Gastric cancer is one of the five most common cancers in Vietnam, including lung, stomach, liver, recto-colon and naso-pharynx cancer in males. In females, it ranks third behind breast and cervical-uterine cancer. The prevalence of gastric cancer in Northern Vietnam is as high as that in China or Korea. Its prevalence in Southern Vietnam is lower but still higher than that in Thailand and South Asia (46, 50). A few studies reported that cagA was found in nearly 100% of H. pylori-infected Vietnamese (60, 61), but no studies have examined the type of CagA protein or the full sequence of cagA in Vietnamese patients. The present study reports the diverse characteristics of cagA and classification of CagA in H. pylori-infected patients from Southern Vietnam based on the full genomic cagA sequence.     

A Multiepitope Subunit Vaccine Conveys Protection against Extraintestinal Pathogenic Escherichia coli in Mice

Infections due to extraintestinal pathogenic Escherichia coli (ExPEC) are common in humans and animals and include urinary tract infections (from uropathogenic E. coli [UPEC]), septicemia, and wound infections. These infections result in significant morbidity and mortality and in high health care costs. In view of the increasing number of ExPEC infections and the ever-growing antibiotic resistance capability of ExPEC isolates, preventive measures such as an effective vaccine against ExPEC are desirable. An ExPEC vaccine may be cost-effective for select patient groups. Previous vaccine candidates consisted of single target proteins or whole ExPEC cells. Here we describe a subunit vaccine against ExPEC which is based on immunodominant epitopes of the virulence-associated ExPEC proteins FyuA, IroN, ChuA, IreA, Iha, and Usp. Using a novel approach of computer-aided design, two completely artificial genes were created, both encoding eight peptide domains derived from these ExPEC proteins. The recombinant expression of these two genes resulted in a protein vaccine directed against ExPEC but not against commensal E. coli of the gut flora. In mice, the vaccine was highly immunogenic, eliciting both strong humoral and cellular immune responses. Nasal application resulted in high secretory immunoglobulin A (sIgA) production, which was detectable on the mucosal surface of the urogenital tract. Finally, it conveyed protection, as shown by a significant reduction of bacterial load in a mouse model of ExPEC peritonitis. This study provides evidence that a novel vaccine design encompassing distinct epitopes of virulence-associated ExPEC proteins may represent a means for providing a protective and pathogen-specific vaccine.Escherichia coli is among the most common bacterial species encountered in clinical microbiology laboratories. Although E. coli strains represent a significant part of the normal gut flora, distinct E. coli pathotypes may cause either diarrhea and gastroenteritis (intestinal pathogenic E. coli [IPEC]) or infections outside the gastrointestinal tract (extraintestinal pathogenic E. coli [ExPEC]) (41).ExPEC strains can reside in the gut as part of the normal intestinal flora and can be isolated from 10 to 20% of healthy individuals (12). However, their entry into and colonization of extraintestinal sites result in a wide variety of infections, which occur in patients from the ambulatory, long-term-care, and hospital settings (23, 39). Diverse organs and anatomical sites are affected. Typical extraintestinal infections due to ExPEC include urinary tract infections (UTIs), surgical site infections, soft tissue infections, newborn meningitis, diverse intra-abdominal infections, and pneumonia. Among these, ascending urinary tract infection (pyelonephritis) most commonly leads to severe sepsis, which ranks as the 10th overall cause of death in the United States (13, 23, 31, 42). Since ExPEC strains are the major cause of most types of extraintestinal infection due to Gram-negative bacteria, prevention of ExPEC infections is a desirable goal from both medical and economic viewpoints (39).In the past, ExPEC strains were usually highly susceptible to common antibiotics such as ampicillin and trimethoprim-sulfamethoxazole (SXT). However, in recent years, the prevalence of E. coli resistance to various classes of antibiotics has risen progressively, becoming a major concern in both hospitals and the community. For example, resistance to SXT, the traditional drug of choice for uncomplicated UTIs, has increased each year worldwide (17, 18). Moreover, many clinical ExPEC isolates have acquired genes encoding extended-spectrum β-lactamases (ESBLs), which confer resistance to extended-spectrum cephalosporins and aztreonam (50). ESBL-positive ExPEC strains frequently contain additional resistance determinants, e.g., for aminoglycosides and tetracyclines. Thus, emerging antimicrobial resistance likely will make the future management of extraintestinal E. coli infections more difficult and costly than ever. Furthermore, the incidence of serious extraintestinal infection due to E. coli increases with age (2, 30), and as the proportion of elderly patients increases, it is likely that so will the number of extraintestinal E. coli infections. Thus, a preventive strategy, such as vaccinations, is very desirable to counteract these infections.An ideal vaccine target should be (i) exposed on the bacterial surface and (ii) widely distributed among clinical ExPEC isolates but not among commensal E. coli strains of the gut flora. Furthermore, it should (iii) possess epitopes that are conserved across diverse ExPEC strains and (iv) elicit a protective immune response. Other desirable characteristics of vaccine targets include (v) increased expression at the site of infection and (vi) a role in the pathogenesis of disease.In the present study, we developed a novel multiepitope subunit vaccine against ExPEC infection which fulfils these criteria. We hypothesized that subunits of the E. coli outer membrane siderophore receptors FyuA, IroN, and IutA, the heme receptor ChuA, and the uropathogenic E. coli (UPEC)-specific protein UspA could be used as vaccine targets to prevent the majority of infections due to extraintestinal E. coli. The goals of this study were (i) to use computer-predicted immunogenic epitopes of the outer protein loops of these proteins in a rational vaccine approach to form two artificial chimeric polypeptides, (ii) to apply either of these two novel multiepitope subunit vaccines by the nasal route to elicit both strong humoral and cellular immune responses, and (iii) to provide a high degree of protection in a mouse model of ExPEC peritonitis.