Ancestral Lineages of Human Enterotoxigenic Escherichia coli

Enterotoxigenic Escherichia coli (ETEC) is a common cause of diarrhea among children living in and among travelers visiting developing countries. Human ETEC strains represent an epidemiologically and phenotypically diverse group of pathogens, and there is a need to identify natural groupings of these organisms that may help to explain this diversity. Here, we sought to identify most of the important human ETEC lineages that exist in the E. coli population, because strains that originate from the same lineage may also have inherited many of the same epidemiological and phenotypic traits. We performed multilocus sequence typing (MLST) on 1,019 ETEC isolates obtained from humans in different countries and analyzed the data against a backdrop of MLST data from 1,250 non-ETEC E. coli and eight ETEC isolates from pigs. A total of 42 different lineages were identified, 15 of which, representing 792 (78%) of the strains, were estimated to have emerged >900 years ago. Twenty of the lineages were represented in more than one country. There was evidence of extensive exchange of enterotoxin and colonization factor genes between different lineages. Human and porcine ETEC have probably emerged from the same ancestral ETEC lineage on at least three occasions. Our findings suggest that most ETEC strains circulating in the human population today originate from well-established, globally widespread ETEC lineages. Some of the more important lineages identified here may represent a smaller and more manageable target for the ongoing efforts to develop effective ETEC vaccines.Enterotoxigenic Escherichia coli (ETEC) infections are an important cause of childhood diarrhea and diarrheal deaths among young children in developing countries (59) and of diarrhea among travelers to these countries (6, 54). Human ETEC strains are E. coli that produce one or more of three plasmid-encoded protein enterotoxins called human heat-stable toxin (STh or STaII), porcine heat-stable toxin (STp or STaI), and heat-labile toxin (LT or LT-I). The enterotoxins induce secretion of salts and water into the intestinal lumen (29). Many ETEC strains also produce surface appendages, called colonization factors (CFs), which help anchor the bacteria to the small intestinal wall (20). The toxins and all but one known CF are plasmid encoded (20, 34).Human ETEC strains are phenotypically and epidemiologically diverse: more than 20 different CFs have thus far been described (20), the characterization of ETEC strains collected from different parts of the world has yielded 117 different serotypes (57), and some ETEC strains appear to be more pathogenic than others (9, 36, 46). This diversity poses a challenge for the ongoing efforts to develop effective ETEC vaccines (7). Many studies have shown that ETEC have emerged from E. coli on several occasions, probably through horizontal transfer of the enterotoxin-encoding virulence plasmids, and that some of these ETEC lineages appear to be widespread (4, 11, 31-33, 37, 38, 43, 47, 51). Because strains that originate from the same ETEC lineage may also have inherited many of the same epidemiological and phenotypic traits, identifying and defining these lineages may improve our understanding of the ETEC diversity and may lead to the identification of lineage-specific protective antigens that can be used in vaccines. To identify these lineages, we performed multilocus sequence typing (MLST) and phylogenetic analyses on a collection of ETEC strains that had been isolated from humans in different countries. We also estimated each lineage''s age as a measure of how stable and well established these lineages are in the E. coli population. If the ancestral origin of the human ETEC population changes frequently, it would complicate efforts to identify new, chromosomally encoded antigens capable of inducing protective immune responses against ETEC. In that case, today''s main vaccine development strategy of targeting plasmid-encoded virulence factors, such as the toxins and CFs, would probably continue to be the best approach for developing effective ETEC vaccines.     

Enterotoxigenic Escherichia coli Elicits Immune Responses to Multiple Surface Proteins

Enterotoxigenic Escherichia coli (ETEC) causes considerable morbidity and mortality due to diarrheal illness in developing countries, particularly in young children. Despite the global importance of these heterogeneous pathogens, a broadly protective vaccine is not yet available. While much is known regarding the immunology of well-characterized virulence proteins, in particular the heat-labile toxin (LT) and colonization factors (CFs), to date, evaluation of the immune response to other antigens has been limited. However, the availability of genomic DNA sequences for ETEC strains coupled with proteomics technology affords opportunities to examine novel uncharacterized antigens that might also serve as targets for vaccine development. Analysis of whole or fractionated bacterial proteomes with convalescent-phase sera can potentially accelerate identification of secreted or surface-expressed targets that are recognized during the course of infection. Here we report results of an immunoproteomics approach to antigen discovery with ETEC strain {"type":"entrez-nucleotide","attrs":{"text":"H10407","term_id":"875229","term_text":"H10407"}}H10407. Immunoblotting of proteins separated by two-dimensional electrophoresis (2DE) with sera from mice infected with strain {"type":"entrez-nucleotide","attrs":{"text":"H10407","term_id":"875229","term_text":"H10407"}}H10407 or with convalescent human sera obtained following natural ETEC infections demonstrated multiple immunoreactive molecules in culture supernatant, outer membrane, and outer membrane vesicle preparations, suggesting that many antigens are recognized during the course of infection. Proteins identified by this approach included established virulence determinants, more recently identified putative virulence factors, as well as novel secreted and outer membrane proteins. Together, these studies suggest that existing and emerging proteomics technologies can provide a useful complement to ongoing approaches to ETEC vaccine development.Infectious diarrhea substantially impacts human health in the developing world, where hundreds of millions of infections occur each year. Several pathogens, rotavirus, Shigella, Vibrio cholerae, and enterotoxigenic Escherichia coli (ETEC), each contribute significantly to this disease burden and collectively result in an estimated 2 million deaths due to diarrheal illness annually (52). Therefore, ETEC remains a high priority for vaccine development.Enterotoxigenic E. coli strains constitute a phenotypically and genetically diverse pathotype that have in common the production of enterotoxin heat-labile toxin (LT) and/or heat-stable toxin (ST). In the classic paradigm for ETEC pathogenesis, organisms must colonize the small intestine via fimbrial colonization factor antigens (CFAs) for effective toxin delivery and subsequent diarrhea (18). Since the early identification of colonization factors (CFs) as important virulence determinants (15), these structures have been a central focus of ETEC vaccine development, and significant inroads have been made into the identification of a broad array of CFs (22, 43), with over 25 antigens identified thus far. ETEC vaccines currently in development are designed to target the most prevalent CFs (56). Moreover, recent elegant structural characterization of the colonization factor antigen I (CFA/I) pilus has provided additional molecular details of pilus tip adhesin molecules that might be exploited (33) as more highly conserved vaccine targets.However, the remarkable plasticity of E. coli genomes (45) and studies demonstrating that many ETEC strains do not produce an identifiable CF (40, 54) suggest that additional antigens would likely need to be considered to produce a broadly protective vaccine. While much is known about the immunology of the CFs and LT following infection (44, 46, 63), very little is known about the nature of immune responses to ETEC in general, and there is no information regarding immunogenicity of more recently discovered putative virulence factors.Furthermore, large-scale epidemiologic studies have suggested that additional plasmid or chromosomally encoded factors contribute to the development of an effective protective immune response attributable to prior natural infections with ETEC (55). However, the identity of other antigens that might be involved in the development of protective immune responses to ETEC remains largely unexplored.The advent of high-throughput sequencing of multiple genomes and advances in proteomics permit avenues for discovery of novel antigens which might be useful in ETEC vaccine development. Two complete ETEC genomes, ETEC {"type":"entrez-nucleotide","attrs":{"text":"H10407","term_id":"875229","term_text":"H10407"}}H10407 and E24377A (45), and one draft genome sequence, B7A (45), as well as several plasmid sequences (21) are now publicly available. While it is anticipated that dozens if not hundreds of ETEC genome sequences will ultimately be made available, these existing genomes permit some initial antigen discovery and validation efforts that were not previously possible.Recent studies of mice have demonstrated that mice exposed to ETEC are protected from subsequent intestinal colonization (47). Therefore, these studies were undertaken to characterize the nature of protective immune responses afforded by prior exposures to ETEC in this model and to validate immune responses to selected antigens using sera from patients naturally infected with ETEC.     

Development of a DNA Microarray for Detection and Serotyping of Enterotoxigenic Escherichia coli

Enterotoxigenic Escherichia coli (ETEC) is a common pathogen worldwide causing infectious diarrhea, especially traveler''s diarrhea. Traditional physiological assays, immunoassays, and PCR-based methods for the detection of ETEC target the heat-labile enterotoxin and/or the heat-stable enterotoxin. Separate serotyping methods using antisera are required to determine the ETEC serogroup. In this study, we developed a DNA microarray that can simultaneously detect enterotoxin genes and the 19 most common O serogroup genes in ETEC strains. The specificity and reproducibility of this approach were verified by hybridization to 223 strains: 50 target reference or clinical strains and 173 other strains, including those belonging to other E. coli O serogroups and closely related species. The sensitivity of detection was determined to be 50 ng of genomic DNA or 10⁸ CFU per ml of organisms in pure culture. The random PCR strategy used in this study with minimal bias provides an effective alternative to multiplex PCR for the detection of pathogens using DNA microarrays. The assay holds promise for applications in the clinical diagnosis and epidemiological surveillance of pathogenic microorganisms.Enterotoxigenic Escherichia coli (ETEC) is the leading bacterial cause of infectious diarrhea in the developing world, causing infantile or cholera-like disease in all age groups (2). It is among the major etiologic agents, leading to an estimated 1.5 million deaths per year worldwide (13, 14). ETEC is also a major cause of traveler''s diarrhea (3, 8, 11) and the most common pathogen among the six recognized diarrheagenic categories of E. coli, especially in the developing world (18). ETEC strains produce one or both of the following two enterotoxins: heat-labile enterotoxin (LT) and heat-stable enterotoxin (ST). Two classes of STs—STa and STb—and two variants of STa—STp (initially discovered in isolates from pigs) and STh (initially discovered in isolates from humans)—have been described. The elt, estA, and estB genes encode the enterotoxins LT, STa, and STb, respectively (6, 23, 26).The O antigen comprises the outermost domain of the lipopolysaccharide molecule and is attached to the core oligosaccharide on the surfaces of Gram-negative bacteria (20). O antigens are among the most variable cellular constituents, imparting antigenic specificity. The composition of the O chain differs from strain to strain; more than 180 O-antigen structures are produced by different E. coli strains (25). The most common O serogroups reported in ETEC are O6, O8, O11, O15, O25, O27, O78, O85, O114, O115, O126, O128, O139, O148, O149, O159, O166, O167, and O173 (5, 18, 19, 31).Detection of ETEC has long relied on detection of the enterotoxins LT and/or ST by physiological assays and immunoassays, and serotyping has depended on assays using O-serogroup-specific antisera. These traditional approaches are slow and labor-intensive, and assays using antisera can be impeded by cross-reactivity. PCR assays, which are more rapid, sensitive, and specific, have also been widely used for ETEC diagnosis (15, 24). However, molecular methods for the serotyping of ETEC have not been developed.Molecular detection and typing by PCR and microarray techniques have many advantages over traditional methods. DNA microarrays provide an efficient approach for the parallel detection and analysis of a large number of pathogenic microorganisms. This technique has been applied to the detection of pathogens from all kinds of biological samples, including water, food, and soil (4, 7, 12, 17, 21).In this study, we developed a DNA microarray for the detection and typing of ETEC. The genes encoding the enterotoxins LT and ST were used for the detection of ETEC, and the serogroup-specific genes wzx and/or wzy were used for the typing of the 19 most common ETEC O serogroups. The microarray was examined for its specificity and sensitivity, and the findings of this study indicate that it is highly sensitive and reproducible.     

Genotypic and Phenotypic Characterization of Enterotoxigenic Escherichia coli Strains Isolated from Peruvian Children

Enterotoxigenic Escherichia coli (ETEC) is a major cause of childhood diarrhea. The present study sought to determine the prevalence and distribution of toxin types, colonization factors (CFs), and antimicrobial susceptibility of ETEC strains isolated from Peruvian children. We analyzed ETEC strains isolated from Peruvian children between 2 and 24 months of age in a passive surveillance study. Five E. coli colonies per patient were studied by multiplex real-time PCR to identify ETEC virulence factors. ETEC-associated toxins were confirmed using a GM1-based enzyme-linked immunosorbent assay. Confirmed strains were tested for CFs by dot blot assay using 21 monoclonal antibodies. We analyzed 1,129 samples from children with diarrhea and 744 control children and found ETEC in 5.3% and 4.3%, respectively. ETEC was more frequently isolated from children >12 months of age than from children <12 months of age (P < 0.001). Fifty-two percent of ETEC isolates from children with diarrhea and 72% of isolates from controls were heat-labile enterotoxin (LT) positive and heat-stable enterotoxin (ST) negative; 25% and 19%, respectively, were LT negative and ST positive; and 23% and 9%, respectively, were LT positive and ST positive. CFs were identified in 64% of diarrheal samples and 37% of control samples (P < 0.05). The most common CFs were CS6 (14% and 7%, respectively), CS12 (12% and 4%, respectively), and CS1 (9% and 4%, respectively). ST-producing ETEC strains caused more severe diarrhea than non-ST-producing ETEC strains. The strains were most frequently resistant to ampicillin (71%) and co-trimoxazole (61%). ETEC was thus found to be more prevalent in older infants. LT was the most common toxin type; 64% of strains had an identified CF. These data are relevant in estimating the burden of disease due to ETEC and the potential coverage of children in Peru by investigational vaccines.Enterotoxigenic Escherichia coli (ETEC) is one of the main causes of diarrhea in children from developing countries and in adult travelers from industrialized countries to the developing world (16, 21). According to the World Health Organization (WHO), ETEC is the second most common cause of diarrhea after rotavirus in children less than 5 years of age and is therefore an important target for vaccine development (11). Diarrhea due to ETEC develops between 8 and 72 h after initial infection, usually due to the ingestion of contaminated food and water (21). The disease varies from a mild illness to one of great severity, usually without leukocytes or fecal blood but often with vomiting and, potentially, dehydration (10).The ability of ETEC to adhere to and colonize the human intestinal mucosa has been correlated with the presence of specific antigenic fimbriae called colonization factors (CFs), which have been designated colonization factor antigens (CFAs), coli surface antigens (CSs), or putative colonization factors (PCFs), followed by a numeric designation. The CFs are mainly fimbrial or fibrillar proteins, although some are not fimbrial in structure (21). To date, over 25 human ETEC CFs have been described. In turn, these CFs have been divided into different families: (i) a CFA/I-like group including CFA/I, CS1, CS2, CS4, CS14, and CS17; (ii) a CS5-like group including CS5, CS7, CS18, and CS20; and (iii) a unique group including CS3, CS6, and CS10 to CS12 (8, 21, 33).Following CF-mediated mucosal adhesion, ETEC elaborates one or both of two enterotoxins: heat-labile toxin (LT), a protein multimer which shares many features with cholera toxin and which binds to intracellular adenylylcyclase, leading to increased cyclic AMP levels, and/or heat-stable toxin (ST), a small-peptide molecule that similarly activates guanylylcyclase and which produces increased intracellular cyclic GMP. For both toxins, the increased chloride secretion resulting from these toxins produces a watery diarrhea (10, 16). Both of these virulence factors are plasmid encoded. ST is encoded by two different genes: estA and st1, which produce STh (originally isolated from ETEC in humans) and STp (originally from a pig isolate), respectively. LT toxin is encoded by the eltA and eltB genes (12). The diagnosis of ETEC infection relies upon the detection of either the genes themselves or their gene products in clinical specimens.Currently, derivatives of LT and the CFs are targets for the development of vaccines against ETEC. However, the great variability of ETEC CFs requires determination of the CF types prevalent in different geographic locations (21, 33). The aims of this study were (i) to determine the clinical and epidemiological characteristics of ETEC diarrhea in Peruvian children, (ii) to determine the presence of ST and LT, (iii) to determine the presence and distribution of colonization factors in these strains, and (iv) to determine the antibiotic susceptibilities of these strains.     

Avian-Pathogenic Escherichia coli Strains Are Similar to Neonatal Meningitis E. coli Strains and Are Able To Cause Meningitis in the Rat Model of Human Disease

Escherichia coli strains causing avian colibacillosis and human neonatal meningitis, urinary tract infections, and septicemia are collectively known as extraintestinal pathogenic E. coli (ExPEC). Characterization of ExPEC strains using various typing techniques has shown that they harbor many similarities, despite their isolation from different host species, leading to the hypothesis that ExPEC may have zoonotic potential. The present study examined a subset of ExPEC strains: neonatal meningitis E. coli (NMEC) strains and avian-pathogenic E. coli (APEC) strains belonging to the O18 serogroup. The study found that they were not easily differentiated on the basis of multilocus sequence typing, phylogenetic typing, or carriage of large virulence plasmids. Among the APEC strains examined, one strain was found to be an outlier, based on the results of these typing methods, and demonstrated reduced virulence in murine and avian pathogenicity models. Some of the APEC strains tested in a rat model of human neonatal meningitis were able to cause meningitis, demonstrating APEC''s ability to cause disease in mammals, lending support to the hypothesis that APEC strains have zoonotic potential. In addition, some NMEC strains were able to cause avian colisepticemia, providing further support for this hypothesis. However, not all of the NMEC and APEC strains tested were able to cause disease in avian and murine hosts, despite the apparent similarities in their known virulence attributes. Thus, it appears that a subset of NMEC and APEC strains harbors zoonotic potential, while other strains do not, suggesting that unknown mechanisms underlie host specificity in some ExPEC strains.Escherichia coli strains causing extraintestinal disease are known as extraintestinal pathogenic E. coli (ExPEC) and include the uropathogenic E. coli (UPEC), neonatal meningitis E. coli (NMEC), and avian-pathogenic E. coli (APEC) subpathotypes. Recent studies have shown that members of various ExPEC subpathotypes harbor similar virulence-associated genes, despite their isolation from varied hosts and tissues (3, 8, 10, 20, 25, 27, 30, 32), and genomic sequencing of APEC O1 revealed that only 4.5% of the genome was not found in the other ExPEC strains sequenced (17). More recently, a cluster of isolates from human and avian hosts thought to represent potential zoonotic pathogens has been identified (20).Common among the isolates of this mixed cluster are genes associated with the conserved region of large virulence plasmids, which are a defining trait of the APEC subpathotype (15, 19, 24, 36, 37) and which are essential for APEC virulence (5, 23). Interestingly, a closely related plasmid that was associated with high-level bacteremia in a neonatal rat meningitis model has also been described in an NMEC isolate (30).Other virulence traits are also shared among ExPEC subpathotypes. Indeed, few traits, if any, appear to be exclusive to a particular ExPEC subpathotype, and in fact, some traits that were thought to be exclusive have been shown to contribute to the pathogenesis of more than one condition (8).Such similarities in the virulence traits found among APEC and other ExPEC subpathotypes have led to speculation that APEC has zoonotic potential (20, 25, 27) and may be a food-borne source of ExPEC causing disease in humans (10, 14, 18, 22). Indeed, ExPEC strains have been identified in retail foods and poultry products (7, 11, 12, 18), and at least one study has found avian isolates to be indistinguishable from human isolates (10). However, other studies showed that human ExPEC strains were clearly distinct from avian strains (6) and that the consumption of poultry or contact with poultry did not correlate with the colonization of antimicrobial-resistant E. coli (34).Here, we seek to further test the hypothesis that APEC strains have zoonotic potential. Of particular interest are O18 strains, which are common among human NMEC strains but which are also found among APEC strains (20, 26). In fact, it has been suggested that APEC O18:K1:H7 strains are potential human pathogens (27). Though it has been shown that human ExPEC strains can cause avian colibacillosis similar to that caused by APEC, suggesting that these ExPEC strains are not host specific (26), it has also been reported that E. coli strains from avian septicemia are more virulent to chicks than NMEC strains (33). However, the ability of APEC to cause disease in mammals has not yet been established.The aim of the present study was to explore the zoonotic potential of NMEC and APEC O18 strains by comparing their plasmid contents, genotypes, phylogenetic group assignments, pulsed-field gel electrophoresis (PFGE) patterns, and sequence types (ST), determined by multilocus sequence typing (MLST), and their abilities to cause disease in the rat model of human neonatal meningitis and chicken models of avian colisepticemia.     

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.     

(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.     

Genetic Fusions of Heat-Labile (LT) and Heat-Stable (ST) Toxoids of Porcine Enterotoxigenic Escherichia coli Elicit Neutralizing Anti-LT and Anti-STa antibodies

Enterotoxigenic Escherichia coli (ETEC) strains are a major cause of diarrheal disease in humans and farm animals. E. coli fimbriae, or colonization factor antigens (CFAs), and enterotoxins, including heat-labile enterotoxins (LT) and heat-stable enterotoxins (ST), are the key virulence factors in ETEC diarrhea. Unlike fimbriae or LT, STa has not often been included as an antigen in development of vaccines against ETEC diarrhea because of its poor immunogenicity. STa becomes immunogenic only after being coupled with a strongly immunogenic carrier protein. However, native or shorter STa antigens either had to retain toxic activity in order to become antigenic or elicited anti-STa antibodies that were not sufficiently protective. In this study, we genetically mutated the porcine LT (pLT) gene for a pLT_192(R→G) toxoid and the porcine STa (pSTa) gene for three full-length pSTa toxoids [STa_11(N→K), STa_12(P→F), and STa_13(A→Q)] and used the full-length pLT₁₉₂ as an adjuvant to carry the pSTa toxoid for pLT₁₉₂:pSTa-toxoid fusion antigens. Rabbits immunized with pLT₁₉₂:pSTa₁₂ or pLT₁₉₂:pSTa₁₃ fusion protein developed high titers of anti-LT and anti-STa antibodies. Furthermore, rabbit antiserum and antifecal antibodies were able to neutralize purified cholera toxin (CT) and STa toxin. In addition, preliminary data suggested that suckling piglets born by a sow immunized with the pLT₁₉₂:pSTa₁₃ fusion antigen were protected when challenged with an STa-positive ETEC strain. This study demonstrated that pSTa toxoids are antigenic when fused with a pLT toxoid and that the elicited anti-LT and anti-STa antibodies were protective. This fusion strategy could provide instructive information to develop effective toxoid vaccines against ETEC-associated diarrhea in animals and humans.Enterotoxigenic Escherichia coli (ETEC) strains, which colonize host small intestines and produce one or more enterotoxins, are the major cause of diarrheal disease in humans and farm animals. The virulence determinants of ETEC in diarrhea include fimbrial adhesins and enterotoxins (1, 6, 10, 27, 28, 37, 44, 48). Fimbrial adhesins mediate attachment of bacteria to host epithelium cells and facilitate subsequent bacteria colonization. Enterotoxins, including heat-stable enterotoxins (STa and STb) and heat-labile enterotoxins (LT) (19, 20, 35), disrupt intestinal fluid homeostasis and cause fluid and electrolyte hypersecretion through activation of adenyl cyclase (by LT) or guanylate cyclase (by STa) in small intestinal epithelial cells (21, 26). ETEC strains isolated from young pigs with diarrhea express LT, STa, STb, Stx2e, and enteroaggregative E. coli ST type 1 (EAST1), alone or combined (10, 15, 50). Recent experimental studies indicated that porcine ETEC strains expressing LT, STb, or STa alone are sufficiently virulent to cause diarrhea in young pigs (6, 48, 49).Porcine ETEC-associated diarrhea, especially postweaning diarrhea (PWD), causes a substantial economic loss to swine producers worldwide (18, 41). Currently, there are no vaccines available to effectively protect weaned pigs against ETEC infections. Experimental vaccines developed from fimbrial antigens alone showed only limited protection against ETEC strains (42). In addition, ETEC fimbriae are antigenically different. Thus, experimental vaccines developed from one specific fimbria could not provide protection against an ETEC strain expressing a different fimbria (42, 43). Moreover, recent evidence suggests that fimbriae may not function as protective antigens in the setting of naturally acquired infections and reinfections (7). Consequently, enterotoxin antigens have been reemphasized for ETEC vaccine development (43). Antitoxin vaccines currently under development largely use LT or its B subunit antigens because they are strongly immunogenic. The ST antigen cannot be used directly as a vaccine component because of its poor immunogenicity, unless it is coupled to a carrier protein and presented as a fusion or a chimeric protein (13, 22, 31, 38). Although a recent study suggested that anti-LT immunity may provide broader protection (14), experimental vaccine studies indicated that the induced anti-LT immunity provided protection only against LT-producing ETEC strains and not against ETEC strains that produce STa toxin (12, 13). As over two-thirds of human ETEC diarrhea cases and more than one-quarter of porcine ETEC diarrhea cases are caused by STa-producing ETEC strains (15, 16, 29, 30, 36, 45, 50), STa antigens must be included for developing broadly effective vaccines against ETEC infection.Porcine STa (pSTa), a protein that consists of 18 amino acids (human STa [hSTa] consists of 19 amino acids), is poorly immunogenic (35, 41). To include pSTa as a vaccine component, we need to enhance pSTa immunogenicity. In addition, a native pSTa is not suitable to be used in developing safe vaccines because it is sufficiently toxic to cause diarrhea. Therefore, we need to attenuate the pSTa toxicity. It has been reported that shorter synthetic hSTa peptides or hSTa with disulfide bonds disrupted showed toxicity reduction (4, 5, 19, 40, 46, 47). Moreover, several shorter synthetic hSTa peptides that had the 12th, 13th, or 14th amino acid residue replaced showed a great reduction in toxicity (46, 47). However, these shorter synthetic or disulfide bond-disrupted hSTa peptides either had not been characterized for immunogenicity or failed to induce protective immunity. Only when a shorter hSTa or an hSTa mutant (with disulfide bonds disrupted) was genetically fused to a carrier protein, such as the B subunit of cholera toxin (CT) or hLT, did the hSTa antigen become immunogenic (9, 31, 32, 33). However, anti-STa immunity from these fusion antigens was not sufficiently characterized, and retention of STa toxicity in these fusion proteins could cause safety concerns for their application in vaccine development (7, 9).No studies have been conducted to enhance pSTa immunogenicity and its potential application in vaccines against porcine ETEC infections. In this study, we mutated the porcine estA gene at nucleotides encoding the 11th, 12th, and 13th amino acids (which are homologous to the 12th, 13th, and 14th amino acids of hSTa) for three pSTa toxoids. These pSTa toxoids, which had all disulfide bonds retained, showed a great reduction in toxicity when examined in vitro (by cyclic GMP [cGMP] enzyme-linked immunosorbent assay [ELISA]) and in vivo (by porcine gut loop assay and challenge studies in a piglet model). We then genetically fused the mutated full-length porcine eltAB and estA genes for pLT₁₉₂:pSTa-toxoid fusion proteins. Purified toxoid fusion antigens were used to immunize adult rabbits to assess anti-LT and anti-STa antigenicity and antibody neutralization and to immunize a pregnant sow to preliminarily evaluate anti-STa immunity in protection against infection from an STa-producing ETEC strain.     

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).     

Genetic Fusions of Heat-Labile Toxoid (LT) and Heat-Stable Toxin b (STb) of Porcine Enterotoxigenic Escherichia coli Elicit Protective Anti-LT and Anti-STb Antibodies

Enterotoxigenic Escherichia coli (ETEC)-associated diarrhea causes a substantial economic loss to swine producers worldwide. The majority of ETEC strains causing porcine diarrhea, especially postweaning diarrhea (PWD), produce heat-labile toxin (LT) and heat-stable toxin b (STb). LT is commonly used in vaccine development, but STb has not been included because of its poor immunogenicity. As a virulence factor in porcine diarrhea, STb needs to be included as an antigen for development of broad-spectrum vaccines. In this study, we used an LT toxoid (LT_R192G [hereafter, LT₁₉₂]) derived from porcine ETEC to carry a mature STb peptide for LT₁₉₂-STb fusions to enhance STb immunogenicity for potential vaccine application. Anti-LT and anti-STb antibodies were detected in immunized rabbits and pigs. In addition, when challenged with an STb-positive ETEC strain, all 10 suckling piglets borne by immunized gilts remained healthy, whereas 7 out 9 piglets borne by unimmunized gilts developed moderate diarrhea. This study indicates that the LT₁₉₂-STb fusion enhanced anti-STb immunogenicity and suggests the LT₁₉₂-STb fusion antigen can be used in future vaccine development against porcine ETEC diarrhea.Enterotoxigenic Escherichia coli (ETEC) strains that produce heat-labile (LT) and heat-stable (ST) enterotoxins are a major cause of diarrheal disease (27, 32). Bacterial adhesins and enterotoxins are the virulence determinants in ETEC-associated diarrhea (1, 4, 19, 20, 26, 33, 34). Porcine ETEC-associated diarrhea, especially postweaning diarrhea (PWD), causes substantial economic loss to swine producers worldwide (15, 28). Currently, there are no effective vaccines available to protect young pigs against PWD. Antitoxin vaccines currently under development largely use LT antigens because they are strongly immunogenic, whereas STb antigens have not been included. However, STb is the toxin most commonly found in ETEC strains associated with PWD (36). Moreover, an ETEC strain expressing STb as the only toxin caused diarrhea in over half of the gnotobiotic pigs tested (34). Therefore, STb antigens need to be included for development of broadly effective vaccines against porcine diarrhea.The STb antigen cannot be used directly as a vaccine component because of the poor immunogenicity. Previous studies demonstrated that a small and poorly immunogenic molecule became more immunogenic when it was conjugated to a strongly immunogenic carrier protein (3, 8, 12, 13, 16, 22, 23, 37). A detoxified heat-labile toxin protein (hLT₁₉₂, where hLT₁₉₂ represents human-type LT_R192G) derived from the LT genes isolated from a human E. coli strain retains LT immunogenicity but has toxicity substantially reduced and has been commonly used as an antigen and/or an adjuvant in vaccine development against bacterial and viral pathogens. In this study, we used an analogous detoxified LT protein, designated LT₁₉₂, as the carrier to enhance STb immunogenicity. This LT₁₉₂ protein was produced by mutating the porcine-type LT genes (eltAB) isolated from a porcine E. coli strain. We fused the estB gene coding for the mature STb peptide to the mutated, full-length porcine-type LT₁₉₂ genes and examined LT₁₉₂-STb fusion proteins in enhancement of STb immunogenicity and potential vaccine application against porcine diarrhea.     

Diarrheagenic Escherichia coli Markers and Phenotypes among Fecal E. coli Isolates Collected from Nicaraguan Infants

We analyzed the prevalence of diarrheagenic Escherichia coli (DEC) markers and common phenotypes in 2,164 E. coli isolates from 282 DEC-positive samples. Enteropathogenic E. coli (EPEC) and enteroaggregative E. coli (EAEC) were very diverse and were not correlated with diarrhea. Enterotoxigenic E. coli (ETEC) estA and enterohemorrhagic E. coli (EHEC) belonged to a few phenotypes and were significantly correlated with diarrhea.In Nicaragua, diarrheagenic Escherichia coli (DEC) strains are considered to be common causes of diarrhea among infants, with enterotoxigenic E. coli (ETEC) and enteropathogenic E. coli (EPEC) the most common DEC types found (4, 6, 12). We previously performed a large screening study on stool samples from 526 infants with and without diarrhea to detect the prevalence of five different categories of DEC and to determine the phenotypic diversity of E. coli isolates from diarrheal and control infants. A one-step multiplex PCR using a mixture of eight primer pairs was applied to the primary streak of E. coli cultures, and eight isolates per sample were analyzed by biochemical fingerprinting (7, 12). As many as 54% of the diarrheal samples and 53% of the control samples were positive for one or more DEC markers, and no clear connection between biochemical phenotypes found in DEC-positive samples and diarrhea could be established.For the present report, we analyzed eight E. coli isolates from each of the 282 DEC-positive stool samples by PCR for the occurrence of the respective DEC markers found in the primary streak.     

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.     

Involvement of quorum sensing and heat-stable enterotoxin a in cell damage caused by a porcine enterotoxigenic Escherichia coli strain

Enterotoxigenic Escherichia coli (ETEC) strains with K88 fimbriae are often associated with the outbreaks of diarrhea in newborn and weaned piglets worldwide. In the present study, we observed that 10⁸ CFU/ml of K88⁺ ETEC strain JG280 caused more death of pig intestinal IPEC-J2 cells than did 10⁹ CFU/ml, suggesting that ETEC-induced cell death was cell density dependent and that quorum sensing (QS) may play a role in pathogenesis. Subsequent investigations demonstrated a positive correlation between autoinducer 2 (AI-2) activity of JG280 and death of IPEC-J2 cells during the infection for up to 3 h. However, there was a negative correlation between AI-2 activity and expression of the JG280 enterotoxin genes estA and estB when IPEC-J2 cells were exposed to the pathogen at 10⁸ CFU/ml. We therefore cloned the luxS gene (responsible for AI-2 production) from JG280 and overexpressed it in E. coli DH5α, because deletion of the luxS gene was retarded by the lack of suitable antibiotic selection markers and the resistance of this pathogen to a wide range of antibiotics. The addition of culture fluid from E. coli DH5α with the overexpressed luxS reduced cell death of IPEC-J2 cells by 10⁸ CFU/ml JG280. The addition also reduced the estA expression by JG280. Nonpathogenic K88⁺ strain JFF4, which lacks the enterotoxin genes, caused no death of IPEC-J2 cells, although it produced AI-2 activity comparable to that produced by JG280. These results suggest the involvement of AI-2-mediated quorum sensing in K88⁺ ETEC pathogenesis, possibly through a negative regulation of STa production.Enterotoxigenic Escherichia coli (ETEC) is an important pathogen causing severe watery diarrhea in farm animals, especially postweaning diarrhea in pigs (12). In humans, ETEC is the most common cause of traveler''s diarrhea and can be fatal for children under 5 years of age (6, 9, 16). ETEC strains are characterized by the production of adhesins that mediate bacterial adherence to the intestine and enterotoxins that elicit diarrhea. The most common and severe infections in pigs are caused by ETEC strains carrying F4 (K88) and F18 fimbrial adhesins (21). After binding of the fimbriae to enterocytes, ETEC strains proliferate rapidly to attain massive numbers and produce one or more types of enterotoxin, including heat-stable enterotoxins (STs) and heat-labile enterotoxin (LT) (22), which stimulate fluid and electrolyte secretion by intestinal cells, thus leading to diarrhea. Other virulence factors, including SepA (sepA), which belongs to a family of proteins from enteropathogenic (32) and enterohemorrhagic (7) E. coli, and Paa (paa), porcine attaching-effacing-associated protein (5), may also contribute to ETEC pathogenesis.Previous in vitro studies with pig intestinal epithelial (IPEC-J2) cells showed that infection with a wild-type (wt) ETEC strain (expressing LT and STb toxins) (3) significantly increased cell death and phosphatidylserine (PS) exposure on the outer leaflet of IPEC-J2 cells (14). In addition, infection with both the ΔeltAB (an isogenic mutant deficient in LT expression) and ΔeltAB/pLT (ΔeltAB mutant complemented by plasmid-based eltAB expression) ETEC strains also modestly increased death of IPEC-J2 cells (14). Another ETEC strain, B41M, that expresses STa toxin was also reported to induce IPEC-J2 cell damage (14). Previous in vivo studies also showed that enterotoxins LT and STb secreted by ETEC contributed significantly to the severity of diarrhea in gnotobiotic piglets, and deletion of estB and eltAB could prevent the development of electrolyte imbalances and dehydration of piglets (11). However, it is still unclear how much each of the enterotoxins contributes to ETEC pathogenesis.Diarrheagenic E. coli strains regulate their virulence gene expression in response to a variety of environmental factors and use quorum sensing (QS) to respond to their cell population to coordinate virulence gene expression (10). QS is a bacterial cell-to-cell communication mechanism, involving the production and detection of autoinducers (AIs). QS may be involved in the regulation of virulence factors, including the type III secretion system in enterohemorrhagic E. coli (EHEC) and enteropathogenic E. coli (EPEC) (29). A breakthrough discovery of a new signaling molecule, AI-3, whose synthesis is not dependent on LuxS (35), indicates that this molecule may be involved in EHEC cross talk with the epinephrine-norepinephrine host signaling system (30). QseBC (a two-component system) was reported to be activated by QS through the AI-3 system (31) and is responsible for the regulation of flagella and motility (31).To our knowledge, the role of QS in ETEC pathogenesis has not been reported. In the present study, we observed that K88⁺ ETEC strain-induced damage of cultured pig intestinal epithelial cells was bacterial cell density dependent, which suggests the involvement of QS. Further studies were carried out to determine possible roles of AI-2 of K88⁺ ETEC in causing the cell damage and regulating the expression of enterotoxin genes.     

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.     

Prevention of Escherichia coli K1 Penetration of the Blood-Brain Barrier by Counteracting the Host Cell Receptor and Signaling Molecule Involved in E. coli Invasion of Human Brain Microvascular Endothelial Cells

Escherichia coli meningitis is an important cause of mortality and morbidity, and a key contributing factor is our incomplete understanding of the pathogenesis of E. coli meningitis. We have shown that E. coli penetration into the brain requires E. coli invasion of human brain microvascular endothelial cells (HBMEC), which constitute the blood-brain barrier. E. coli invasion of HBMEC involves its interaction with HBMEC receptors, such as E. coli cytotoxic necrotizing factor 1 (CNF1) interaction with its receptor, the 67-kDa laminin receptor (67LR), and host signaling molecules including cytosolic phospholipase A₂α (cPLA₂α). In the present study, we showed that treatment with etoposide resulted in decreased expression of 67LR on HBMEC and inhibited E. coli invasion of HBMEC. Pharmacological inhibition of cysteinyl leukotrienes, lipoxygenated products of arachidonic acid released by cPLA₂α, using montelukast (an antagonist of the type 1 cysteinyl leukotriene receptor) also inhibited E. coli invasion of HBMEC. E. coli penetration into the brain was significantly decreased by etoposide as well as by montelukast, and a combination of etoposide and montelukast was significantly more effective in inhibiting E. coli K1 invasion of HBMEC than single agents alone. These findings demonstrate for the first time that counteracting the HBMEC receptor and signaling molecule involved in E. coli invasion of HBMEC provides a novel approach for prevention of E. coli penetration into the brain, the essential step required for development of E. coli meningitis.The mortality and morbidity associated with neonatal Gram-negative bacillary meningitis have remained significant despite advances in antimicrobial chemotherapy and supportive care. Inadequate knowledge of the pathogenesis has contributed to this mortality and morbidity (10-12). Escherichia coli is the most common Gram-negative organism that causes neonatal meningitis. Several lines of evidence from experimental animal models as well as human cases of E. coli meningitis indicate that E. coli penetrates into the brain initially in the cerebral vasculature (2, 13), but the underlying mechanisms contributing to E. coli penetration of the blood-brain barrier remain incompletely understood (10-12).We have developed an in vitro blood-brain barrier model by isolation and cultivation of human brain microvascular endothelial cells (HBMEC) (14, 20-22). Upon cultivation on collagen-coated Transwell inserts, the HBMEC exhibit morphological and functional properties of tight junction formation and a polarized monolayer. These properties are shown by our demonstrations of tight junction proteins (such as ZO-1), adherens junction proteins (such as β-catenin), and their spatial separation, limited permeability to propidium iodide (molecular mass, 668 Da) and inulin (molecular mass, 4,000 Da), and development of high transendothelial electrical resistance (14, 20-22). We have also developed an infant rat model of experimental hematogenous meningitis (8, 13). This animal model has important similarities to E. coli meningitis in humans, such as hematogenous infection of the meninges. Using these in vitro and in vivo models, we have shown that E. coli binding to and invasion of HBMEC are prerequisites for penetration into the brain (10-12).We have shown that E. coli K1 binding to and invasion of HBMEC require specific E. coli determinants (e.g., cytotoxic necrotizing factor 1 [CNF1] and OmpA), and these E. coli determinants contribute to HBMEC binding and invasion via interactions with their respective HBMEC receptors (10-12). For example, CNF1 contributes to E. coli K1 invasion of HBMEC via its interaction with 37-kDa laminin receptor precursor (37LRP)/67-kDa lamin receptor (67LR), while OmpA contributes to E. coli K1 binding to and invasion of HBMEC via its interaction with the HBMEC receptor, gp96 (3, 7, 9). We also showed that the E. coli determinants contributing to HBMEC binding and invasion exploit specific host signaling molecules for efficient invasion of HBMEC. For example, OmpA, NlpI, FliC, and IbeC (the E. coli structures contributing to HBMEC binding and invasion) are shown to exploit host cytosolic phospholipase A₂α (cPLA₂α) for E. coli invasion of HBMEC (4, 12, 25).We also showed that blockade of the HBMEC receptors and/or host signaling molecules was effective in preventing E. coli K1 invasion of HBMEC. For example, anti-67LR and -gp96 antibodies inhibited E. coli K1 invasion of HBMEC in a ligand-dependent manner (7, 9), and pharmacological inhibition of host cPLA₂α exhibited a dose-dependent inhibition of E. coli invasion of HBMEC (4). These findings suggest that inhibition of the HBMEC receptors and host signaling molecules involved in E. coli K1 invasion of HBMEC is likely to affect the ability of E. coli to penetrate into the brain.In screening drugs for their effects on the HBMEC receptors, we determined that etoposide (a topoisomerase inhibitor) decreased the expression of 67LR on HBMEC. cPLA₂α mediates agonist-induced release of arachidonic acid (6). We showed that the contribution of host cPLA₂α to E. coli invasion of HBMEC occurs via lipoxygenated products of arachidonic acid, cysteinyl leukotrienes (LTs), formed via LT biosynthetic pathways involving 5-lipoxygenase, and acting via the type 1 cysteinyl leukotriene receptor (CysLT1) (4, 12). More importantly, etoposide and montelukast (the CysLT1 antagonist) were additive in their prevention of E. coli K1 invasion of HBMEC and also efficient in preventing E. coli K1 penetration into the brain.     

Sab,a Novel Autotransporter of Locus of Enterocyte Effacement-Negative Shiga-Toxigenic Escherichia coli O113:H21, Contributes to Adherence and Biofilm Formation

Shiga-toxigenic Escherichia coli (STEC) strains cause serious gastrointestinal disease, which can lead to potentially life-threatening systemic complications such as hemolytic-uremic syndrome. Although the production of Shiga toxin has been considered to be the main virulence trait of STEC for many years, the capacity to colonize the host intestinal epithelium is a crucial step in pathogenesis. In this study, we have characterized a novel megaplasmid-encoded outer membrane protein in locus of enterocyte effacement (LEE)-negative O113:H21 STEC strain 98NK2, termed Sab (for STEC autotransporter [AT] contributing to biofilm formation). The 4,296-bp sab gene encodes a 1,431-amino-acid protein with the features of members of the AT protein family. When expressed in E. coli JM109, Sab contributed to the diffuse adherence to human epithelial (HEp-2) cells and promoted biofilm formation on polystyrene surfaces. A 98NK2 sab deletion mutant was also defective in biofilm formation relative to its otherwise isogenic wild-type parent, and this was complemented by transformation with a sab-carrying plasmid. Interestingly, an unrelated O113:H21 STEC isolate that had a naturally occurring deletion in sab was similarly defective in biofilm formation. PCR analysis indicated that sab is present in LEE-negative STEC strains belonging to serotypes/groups O113:H21, O23, and O82:H8. These findings raise the possibility that Sab may contribute to colonization in a subset of LEE-negative STEC strains.Shiga-toxigenic Escherichia coli (STEC) strains are prominent food-borne pathogens that cause watery or bloody diarrhea and hemorrhagic colitis, which can progress to the life-threatening hemolytic-uremic syndrome (HUS) (15, 21, 29). In order to establish and maintain an infection, STEC strains are equipped with a diverse array of virulence factors. Among these factors, Shiga toxin has been considered to be a sine qua non of virulence, as reviewed previously (21, 29). However, attachment of STEC to the human intestinal mucosa is a critical first step in pathogenesis. Many STEC strains, including those of the highly prevalent O157:H7 serotype, carry the locus of enterocyte effacement (LEE) pathogenicity island, which encodes the capacity to produce attaching and effacing (A/E) lesions on the intestinal epithelium, similarly to those produced by enteropathogenic E. coli strains (11, 35). These STEC strains are often referred to as enterohemorrhagic E. coli (EHEC), although this classification is ill defined. A/E lesions are characterized by ultrastructural changes including the remodeling of the host cell cytoskeleton and intimate attachment of the bacteria to the cell surface (11, 35). The process of the generation of A/E lesions involves the expression of the eae gene, which encodes intimin, an outer membrane surface adhesin, and the delivery of the intimin receptor Tir and several other effector proteins into host cells via the LEE-encoded type III secretion apparatus (reviewed in references 5 and 11).However, many STEC isolates from cases of severe disease, including HUS, lack the LEE locus yet are clearly capable of efficient colonization of the human gut (28, 29). Several candidate adhesins have been identified in these strains, including the megaplasmid-encoded autoagglutinating adhesin Saa (26), the long polar fimbriae encoded by the lpf operon (10) (two distinct homologues of which are also present in STEC O157:H7 strains [39, 40]), and the immunoglobulin-binding protein EibG, which contributes to a chain-like adherence phenotype on HEp-2 cells (18). Tarr et al. (38) also previously identified Iha, a homologue of Vibrio cholerae IrgA, which promotes the adherence of STEC O157:H7 to HeLa cells and is widely distributed in LEE-positive and LEE-negative strains. STEC O157:H7 strains also produce a type IV pilus, HCP (47), and an E. coli common pilus, ECP (30), both of which contribute to in vitro adherence to intestinal epithelial cells. Additional putative adhesins from LEE-positive STEC strains include Efa1, which mediates the attachment of O111:NM STEC strains to Chinese hamster ovary cells (23). In addition, Torres et al. (41) previously identified a calcium-binding and heat-extractable AT protein of EHEC, termed Cah, which mediates aggregation and participates in biofilm formation. Recently, Wells et al. (45) also characterized the EHEC-encoded AT protein EhaA, which contributes to adherence to primary bovine epithelial cells (but not HeLa cells) and promotes biofilm formation as well.The AT proteins referred to above belong to a rapidly growing family of gram-negative surface proteins that are exported across the periplasmic space and either attached to the external face of the outer membrane or released by proteolysis into the environment (13). These large proteins share a characteristic structure comprising three distinct domains, namely, an N-terminal signal peptide, a divergent functional passenger domain (α-domain), and a conserved C-terminal domain which forms a β-barrel pore in the outer membrane (13, 46). This unique protein structure provides all the information required for transport to the cell surface, with the N-terminal sequence directing the protein to the periplasm via the sec pathway and the C-terminal domain mediating the translocation of the passenger domain to the external surface (14). The various α-domains confer a broad range of functions and/or phenotypes including aggregation, biofilm formation, adherence, invasion, serum resistance, and protease or esterase activity (7, 12).Research in our laboratory has focused on the identification of novel virulence factors of LEE-negative STEC strains associated with human disease. In this study, we describe the identification and characterization of a member of the AT family produced by hypervirulent LEE-negative O113:H21 STEC strain 98NK2, which confers adherence to human epithelial cells and mediates biofilm formation.     

Virulence Potential of Escherichia coli Isolates from Skin and Soft Tissue Infections

Escherichia coli strains frequently are isolated from skin and soft tissue infections (SSTI); however, their virulence potential has not yet been extensively studied. In the present study, we characterized 102 E. coli SSTI strains isolated mostly from surgical and traumatic wounds, foot ulcers, and decubitus. The strains were obtained from the Institute of Microbiology and Immunology, University of Ljubljana, Slovenia. Phylogenetic backgrounds, virulence factors (VFs), and antibiotic resistance profiles were determined. Correlations between VFs and phylogenetic groups were established and analyzed with regard to patient factors. Further, the associations of the three most prevalent antibiotic resistance patterns with virulence potential were analyzed. Our results showed that the majority of the studied strains (65%) belonged to the B2 phylogenetic group. The most prevalent VF was ompT (80%), while toxin genes cnf1 and hlyA were found with prevalences of 32 and 30%, respectively. None of the investigated bacterial characteristics were significantly associated with patient gender, age, type of infection, or immunodeficiency. The most prevalent antibiotic resistance pattern was resistance to ampicillin (46%), followed by resistance to tetracycline (25%) and fluoroquinolones (21%). Strains resistant to ciprofloxacin exhibited a significantly reduced prevalence of cnf1 (P < 0.05) and usp (P < 0.01). Our study revealed that E. coli isolates from SSTIs exhibit a remarkable virulence potential that is comparable to that of E. coli isolates from urinary tract infections and bacteremia.Skin and soft tissue infections (SSTIs) are one of the most common infections in patients of all age groups. Infections mostly are self limited or can be treated with antibiotics. However, moderate or severe cases may require hospitalization and parenteral therapy (30). The most common causative agents are Staphylococcus aureus and aerobic streptococci (9, 10, 41, 43). However, several reports associating the enterobacterium Escherichia coli with SSTI have been published: E. coli was found to be the causative agent of neonatal omphalitis (7), cellulitis localized to lower or upper limbs (4, 6, 49), necrotizing fasciitis (1, 25, 28), surgical site infections (44), infections after burn injuries (37), and others. A study monitoring SSTIs during a 7-year period and encompassing three continents (Europe, Latin America, and North America) showed E. coli to be an important causative agent, since it was the third-most prevalent isolated species, preceded solely by S. aureus and Pseudomonas aeruginosa. The beta-hemolytic Streptococcus sp. group was only the 7th-ranked pathogen in North America and Europe and was 10th in Latin America in terms of prevalence (30). E. coli isolates from SSTI therefore merit detailed studies, especially taking into account the dramatic decline in antibiotic susceptibility of pathogenic E. coli strains in recent years. Despite the need for the characterization of E. coli strains from SSTI, to our knowledge only a single E. coli isolate from a deep surgical wound infection has been characterized (21). The aim of our study was to characterize a larger collection of E. coli isolates from SSTI. Our work was focused on their virulence potential: phylogenetic distribution, virulence factor (VF) profile, and the prevalence of antibiotic resistance patterns. Correlations between patient and strain characteristics were studied, as well as correlations between VF profiles and antibiotic resistance. As the strains were isolated from extraintestinal sites of infections, we screened for VFs that are typical of extraintestinal pathogenic E. coli (ExPEC).