Uropathogenic Escherichia coli (UPEC) strains, which cause the majority of uncomplicated urinary tract infections (UTIs), carry a unique assortment of virulence or fitness genes. However, no single defining set of virulence or fitness genes has been found in all strains of UPEC, making the differentiation between UPEC and fecal commensal strains of E. coli difficult without the use of animal models of infection or phylogenetic grouping. In the present study, we consider three broad categories of virulence factors simultaneously to better define a combination of virulence factors that predicts success in the urinary tract. A total of 314 strains of E. coli, representing isolates from fecal samples, asymptomatic bacteriuria, complicated UTIs, and uncomplicated bladder and kidney infections, were assessed by multiplex PCR for the presence of 15 virulence or fitness genes encoding adhesins, toxins, and iron acquisition systems. The results confirm previous reports of gene prevalence among isolates from different clinical settings and identify several new patterns of gene associations. One gene, tosA, a putative repeat-in-toxin (RTX) homolog, is present in 11% of fecal strains but 25% of urinary isolates. Whereas tosA-positive strains carry an unusually high number (11.2) of the 15 virulence or fitness genes, tosA-negative strains have an average of only 5.4 virulence or fitness genes. The presence of tosA was predictive of successful colonization of a murine model of infection, even among fecal isolates, and can be used as a marker of pathogenic strains of UPEC within a distinct subset of the B2 lineage.
Escherichia coli is the primary cause of urinary tract infections, the most common bacterial infection of humans. Virulence of a uropathogenic strain is typically defined by the clinical source of the isolate, the ability to colonize the bladder and kidneys in a murine model, the phylogenetic group of the bacterium, and virulence gene content. Here we describe a novel single gene, the repeat-in-toxin gene tosA, the presence of which predicts virulence of E. coli isolates regardless of source. Rapid identification of uropathogenic strains of E. coli may aid in the development of therapeutic and preventive therapies.
In order to obtain a better molecular understanding of recurrent urinary tract infection (RUTI), we collected 75 cases with repeatedly occurring uncomplicated UTI. The genetic relationships among uropathogenic Escherichia coli (UPEC) isolates were analyzed by pulsed-field gel electrophoresis. While 39 (52%) of the RUTI cases were defined as “persistence” of the same strain as the primary infecting strain, 36 (48%) were characterized by “reinfection” with a new strain that is different from the primary strain. We then examined the antimicrobial susceptibilities and phylogenetic backgrounds of 39 persistence and 86 reinfection UPEC isolates, and screened 44 virulence factor (VF) genes. We found that isolates had significant differences in the following: placement in phylogenetic group B2 (41% versus 21%; P = 0.0193) and the presence of adhesin genes iha (49% versus 28%; P = 0.0233) and papG allele I′ (51% versus 24%; P = 0.003), iron uptake genes fyuA (85% versus 58%; P = 0.0037), irp-2 (87% versus 65%; P = 0.0109), and iutA (87% versus 58%; P = 0.0014), and an aggregate VF score (median, 11 versus 9; P = 0.0030). In addition, 41% of persistence strains harbored three adhesin genes simultaneously, whereas 22% of reinfection isolates did (P = 0.0289). Moreover, 59% versus 29% (P = 0.0014) of persistence and reinfection isolates contained seven types of iron uptake genes. Taken together, the antimicrobial susceptibilities of UPEC isolates had little effect on the RUTI. Compared with reinfection strains, persistence UPEC isolates exhibited higher VF scores and carried more VF genes than may be involved in the development and progression of RUTI.
Uropathogenic Escherichia coli (UPEC) is responsible for the majority of uncomplicated urinary tract infections (UTI) and represents the most common bacterial infection in adults. UPEC utilizes a wide range of virulence factors to colonize the host, including the novel repeat-in-toxin (RTX) protein TosA, which is specifically expressed in the host urinary tract and contributes significantly to the virulence and survival of UPEC. tosA, found in strains within the B2 phylogenetic subgroup of E. coli, serves as a marker for strains that also contain a large number of well-characterized UPEC virulence factors. The presence of tosA in an E. coli isolate predicts successful colonization of the murine model of ascending UTI, regardless of the source of the isolate. Here, a detailed analysis of the function of tosA revealed that this gene is transcriptionally linked to genes encoding a conserved type 1 secretion system similar to other RTX family members. TosA localized to the cell surface and was found to mediate (i) adherence to host cells derived from the upper urinary tract and (ii) survival in disseminated infections and (iii) to enhance lethality during sepsis (as assessed in two different animal models of infection). An experimental vaccine, using purified TosA, protected vaccinated animals against urosepsis. From this work, it was concluded that TosA belongs to a novel group of RTX proteins that mediate adherence and host damage during UTI and urosepsis and could be a novel target for the development of therapeutics to treat ascending UTIs.
Extra-intestinal pathogenic Escherichia coli (ExPEC) strains are divided into uropathogenic E. coli (UPEC), strains causing neonatal meningitis and septicaemic E. coli. The most common pathotype of ExPEC is found among patients with urinary tract infection (UTI), defined as UPEC. These bacteria are responsible for >90% of cases of UTI and are often found amongst the faecal flora of the same host. E.coli strains are classified into four phylogenetic groups, A, B1, B2, and D. Groups A and B1 are commensal strains and carry few virulence-associated genes (VGs) while pathogenic group B2 and D usually possess VGs which enhance colonic persistence and adhesion in the urinary tract (UT). The gastrointestinal (GI) tract is widely accepted as a reservoir for UPEC and is believed that healthy humans have a reservoir of UPEC strains, belonging to phylogenetic group B2, and to a lesser extent, group D. These strains have superior ability to survive and persist in the gut of humans and can spread to cause extra-intestinal infections. ExPEC trains possess a range of VGs which are involved in their pathogenesis. These include adhesins, toxins, iron-acquisition systems (e.g. siderophores), and capsules. Evolutionary influences on the acquisition and main role of VGs amongst E. coli are widely debated, with some research holding that the prevalence of strains with VGs increases the likelihood of infections, whereas others believe that VGs provide a selective advantage for infection of extra-intestinal sites. This review is intended to present our existing knowledge and gaps in this area.
E.coli; Urinary tract infection; Gut; Virulence factors
In some patients, Escherichia coli strains establish significant bacteriuria without causing symptoms of urinary tract infection (UTI). These asymptomatic-bacteriuria (ABU) strains have been shown to express fewer virulence factors than the uropathogenic E. coli (UPEC) strains that cause severe, symptomatic UTI. Paradoxically, ABU strains carry many typical UPEC virulence genes, and the molecular basis of their low virulence therefore remains unclear. This study examined whether ABU strains might evolve from UPEC by genome loss and virulence gene attenuation. The presence of conserved E. coli K-12 genes was examined using an E. coli K-12 strain MG1655-specific DNA array and the distribution of UPEC virulence-related genes was examined with the E. coli pathoarray. Two groups of strains could be distinguished. Several ABU strains were shown by multilocus sequence typing and by comparative genomic analyses to be related to UPEC but to have smaller genome sizes. There were significant alterations in essential virulence genes, including reductive evolution by point mutations, DNA rearrangements, and deletions. Other strains were unrelated to UPEC and lacked most of the virulence-associated genes. The results suggest that some ABU strains arise from virulent strains by attenuation of virulence genes while others are nonvirulent and resemble commensal strains. We propose that virulence attenuation might constitute a general mechanism for mucosal pathogens to evolve toward commensalism.
Cytotoxic necrotizing factor 1 (CNF1) and hemolysin (HlyA1) are toxins produced by uropathogenic Escherichia coli (UPEC). We previously showed that these toxins contribute to the inflammation and tissue damage seen in a mouse model of ascending urinary tract infection. CNF1 constitutively activates small Rho GTPases by deamidation of a conserved glutamine residue, and HlyA1 forms pores in eukaryotic cell membranes. In this study, we used cDNA microarrays of bladder tissue isolated from mice infected intraurethrally with wild-type CP9, CP9cnf1, or CP9ΔhlyA to further evaluate the role that each toxin plays in the host response to UPEC. Regardless of the strain used, we found that UPEC itself elicited a significant change in host gene expression 24 h after inoculation. The largest numbers of upregulated genes were in the cytokine and chemokine signaling and Toll-like receptor signaling pathways. CNF1 exerted a strong positive influence on expression of genes involved in innate immunity and signal transduction and a negative impact on metabolism- and transport-associated genes. HlyA1 evoked an increase in expression of genes that encode innate immunity factors and a decrease in expression of genes involved in cytoskeletal and metabolic processes. Multiplex cytokine and myeloperoxidase assays corroborated our finding that a strong proinflammatory response was elicited by all strains tested. Bladders challenged intraurethrally with purified CNF1 displayed pathology similar to but significantly less intense than the pathology that we observed in CP9-challenged mice. Our data demonstrate substantial roles for CNF1 and HlyA1 in initiation of a strong proinflammatory response to UPEC in the bladder.
In this study, the association between virulence genotypes and phylogenetic groups among Escherichia (E.) coli isolates obtained from pet dogs and cats with cystitis was detected, and fingerprinting methods were used to explore the relationship among strains. Forty uropathogenic E. coli (UPEC) isolated from dogs (n = 30) and cats (n = 10) in Italy were analysed by polymerase chain reaction (PCR) for the presence of virulence factors and their classification into phylogenetic groups. The same strains were characterized by repetitive extragenic palindromic (REP)- and enterobacterial repetitive intergenic consensus (ERIC)-PCR techniques. We found a high number of virulence factors such as fimbriae A, S fimbriae (sfa) and cytotoxic necrotizing factor 1 (cnf1) significantly associated with phylogenetic group B2. We demonstrated a high correlation between α-hemolysin A and pyelonephritis C, sfa, and cnf1 operons, confirming the presence of pathogenicity islands in these strains. In addition, UPEC belonging to group B2 harboured a greater number of virulence factors than strains from phylogenetic groups A, B1, and D. REP- and ERIC-PCR grouped the UPEC isolates into two major clusters, the former grouping E. coli strains belonging to phylogenetic group B2 and D, the latter grouping those belonging to groups A and B1. Given the significant genetic variability among the UPEC strains found in our study, it can be hypothesized that no specific genotype is responsible for cystitis in cats or dogs.
cats; dogs; Escherichia coli; genetic variability; virulence factors
Escherichia coli is the primary cause of urinary tract infection (UTI) in the developed world. The major factors associated with virulence of uropathogenic E. coli (UPEC) are fimbrial adhesins, which mediate specific attachment to host receptors and trigger innate host responses. Another group of adhesins is represented by the autotransporter (AT) subgroup of proteins. In this study, we identified a new AT-encoding gene, termed upaH, present in a 6.5-kb unannotated intergenic region in the genome of the prototypic UPEC strain CFT073. Cloning and sequencing of the upaH gene from CFT073 revealed an intact 8.535-kb coding region, contrary to the published genome sequence. The upaH gene was widely distributed among a large collection of UPEC isolates as well as the E. coli Reference (ECOR) strain collection. Bioinformatic analyses suggest β-helix as the predominant structure in the large N-terminal passenger (α) domain and a 12-strand β-barrel for the C-terminal β-domain of UpaH. We demonstrated that UpaH is expressed at the cell surface of CFT073 and promotes biofilm formation. In the mouse UTI model, deletion of the upaH gene in CFT073 and in two other UPEC strains did not significantly affect colonization of the bladder in single-challenge experiments. However, in competitive colonization experiments, CFT073 significantly outcompeted its upaH isogenic mutant strain in urine and the bladder.
Uropathogenic Escherichia coli (UPEC) strains are responsible for the majority of uncomplicated urinary tract infections, which can present clinically as cystitis or pyelonephritis. UPEC strain CFT073, isolated from the blood of a patient with acute pyelonephritis, was most cytotoxic and most virulent in mice among our strain collection. Based on the genome sequence of CFT073, microarrays were utilized in comparative genomic hybridization (CGH) analysis of a panel of uropathogenic and fecal/commensal E. coli isolates. Genomic DNA from seven UPEC (three pyelonephritis and four cystitis) isolates and three fecal/commensal strains, including K-12 MG1655, was hybridized to the CFT073 microarray. The CFT073 genome contains 5,379 genes; CGH analysis revealed that 2,820 (52.4%) of these genes were common to all 11 E. coli strains, yet only 173 UPEC-specific genes were found by CGH to be present in all UPEC strains but in none of the fecal/commensal strains. When the sequences of three additional sequenced UPEC strains (UTI89, 536, and F11) and a commensal strain (HS) were added to the analysis, 131 genes present in all UPEC strains but in no fecal/commensal strains were identified. Seven previously unrecognized genomic islands (>30 kb) were delineated by CGH in addition to the three known pathogenicity islands. These genomic islands comprise 672 kb of the 5,231-kb (12.8%) genome, demonstrating the importance of horizontal transfer for UPEC and the mosaic structure of the genome. UPEC strains contain a greater number of iron acquisition systems than do fecal/commensal strains, which is reflective of the adaptation to the iron-limiting urinary tract environment. Each strain displayed distinct differences in the number and type of known virulence factors. The large number of hypothetical genes in the CFT073 genome, especially those shown to be UPEC specific, strongly suggests that many urovirulence factors remain uncharacterized.
Optical maps were generated for 33 uropathogenic Escherichia coli (UPEC) isolates. For individual genomes, the NcoI restriction fragments aligned into a unique chromosome map for each individual isolate, which was then compared with the in silico restriction maps of all of the sequenced E. coli and Shigella strains. All of the UPEC isolates clustered separately from the Shigella strains as well as the laboratory and enterohaemorrhagic E. coli strains. Moreover, the individual strains appeared to cluster into distinct subgroups based on the dendrogram analyses. Phylogenetic grouping of these 33 strains showed that 32/33 were the B2 subgroup and 1/33 was subgroup A. To further characterize the similarities and differences among the 33 isolates, pathogenicity island (PAI), haemolysin and virulence gene comparisons were performed. A strong correlation was observed between individual subgroups and virulence factor genes as well as haemolysis activity. Furthermore, there was considerable conservation of sequenced-strain PAIs in the specific subgroups. Strains with different antibiotic-resistance patterns also appeared to sort into separate subgroups. Thus, the optical maps distinguished the UPEC strains from other E. coli strains and further subdivided the strains into distinct subgroups. This optical mapping procedure holds promise as an alternative way to subgroup all E. coli strains, including those involved in infections outside of the intestinal tract and epidemic strains with distinct patterns of antibiotic resistance.
Urinary tract infections (UTI) are a major public health concern in developing countries. Most UTIs are caused by E. coli, accounting for up to 90% of community-acquired UTIs (CAUTI). Recurrent UTI is considered as a major risk factor for urolithiasis. Virulence factors like adhesins and biofilm have been extensively studied by authors on UPEC isolated from recurrent UTI. The studies on isolates from infection stones in kidney are scanty. In a prospective study, we aimed to determine the expression of Haemagglutinins, (Type 1 and P fimbriae), Biofilm production and resistance pattern to common antibiotics of Uropathogenic E.coli (UPEC) isolates from Community acquired Acute Urinary Tract Infection(CAUTI) and Urolithiasis.
Materials and Methods:
A total of 43 UPEC isolates, 23 mid-stream urine (MSU) samples from patients with CAUTI attending Out Patient Departments and 20 from renal calculi of urolithiasis patients at the time of Percutaneous nephrolithostomy (PCNL) were included in the study and the expression of Haemagglutinins,(Type 1 and P fimbriae), Biofilm production and resistance pattern to common antibiotics was assessed.
A total of 43 UPEC isolates 23 from CAUTI and 20 from renal calculi were tested for production of biofilm and hemagglutinins. In CAUTI, biofilm producers were 56.52% and hemagglutinins were detected in all isolates 100%. In urolithiasis, biofilm producers were 100% but hemagglutinins were detected only in 70% of isolates. All isolates were resistant to multiple antibiotics used. CAUTI isolates were susceptible to 3rd generation cephalosporins, whereas urolithiasis isolates were resistant to 3rd generation cephalosporins and 25% were Extended Spectrum Beta Lactamases ESBL producers.
HA mediated by type 1 fimbriae plays an important role in CAUTI (P < 0.001 highly significant), whereas, in chronic conditions like urolithiasis, biofilm plays an important role in persistence of infection and the role of hemagglutinins is less.
Biofilm; hemagglutination; mannose-resistant hemagglutination; mannose-sensitive hemagglutination; urolithiasis; uropathogenic Escherichia coli
To compare the genetic structures of uropathogenic and commensal Escherichia coli populations, a total of 181 urinary and rectal E. coli isolates were classified into intraspecies phylogenetic groups by PCR amplifications of phylogenetic markers. The genetic variability of these isolates within phylogenetic groups was further assessed by enterobacterial repetitive intergenic consensus (ERIC) typing. The distributions of 10 known virulence factors were also examined. In contrast with most reports, phylogenetic group B2 not only accounted for the majority of urinary isolates from young women with urinary tract infections (69%) but also was the dominant group among the rectal isolates from healthy young women (48%). Such difference may be explained by geographic variation, difference in host population characteristics, or differences in sampling method, or a combination of the three. Strains with known virulence factors most frequently belonged to phylogenetic groups B2 and D. Additionally, group B2 and D rectal isolates were more heterogeneous than urinary isolates. Two subclusters existed within group B2 strains by ERIC typing. These subclusters were not evenly distributed between rectal and urine isolates and differed in virulence gene distribution.
Many uropathogenic Escherichia coli (UPEC) strains produce both hemolysin (Hly) and cytotoxic necrotizing factor type 1 (CNF1), and the loci for these toxins are often linked. The conclusion that Hly and CNF1 contribute to urovirulence is supported by the results of epidemiological studies associating the severity of urinary tract infections (UTIs) with toxin production by UPEC isolates. Additionally, we previously reported that mouse bladders and rat prostates infected with UPEC strain CP9 exhibit a more profound inflammatory response than the organs from animals challenged with CP9cnf1 and that CNF1 decreases the antimicrobial activities of polymorphonuclear leukocytes. More recently, we created an Hly mutant, CP9ΔhlyA1::cat, and showed that it was less hemolytic and destructive for cultured bladder cells than CP9 was. Here we evaluated the relative effects of mutations in hlyA1 or cnf1 alone or together on the pathogenicity of CP9 in a mouse model of ascending UTI. To do this, we constructed an hlyA1-complemented clone of CP9ΔhlyA1::cat and an hlyA1 cnf1 CP9 double mutant. We found that Hly had no influence on bacterial colonization of the bladder or kidneys in single or mixed infections with the wild type and CP9ΔhlyA1::cat but that it did provoke sloughing of the uroepithelium and bladder hemorrhage within the first 24 h after challenge. Finally, we confirmed that CNF1 expression induces bladder inflammation and, in particular, as shown in this study, submucosal edema. From these data, we speculate that Hly and CNF1 may be largely responsible for the signs and symptoms of cystitis in humans infected with toxigenic UPEC.
Uropathogenic Escherichia coli (UPEC), a member of extraintestinal pathogenic E. coli, cause ∼80% of community-acquired urinary tract infections (UTI) in humans. UPEC initiates its colonization in epithelial cells lining the urinary tract with a complicated life cycle, replicating and persisting in intracellular and extracellular niches. Consequently, UPEC causes cystitis and more severe form of pyelonephritis. To further understand the virulence characteristics of UPEC, we investigated the roles of BarA-UvrY two-component system (TCS) in regulating UPEC virulence. Our results showed that mutation of BarA-UvrY TCS significantly decreased the virulence of UPEC CFT073, as assessed by mouse urinary tract infection, chicken embryo killing assay, and cytotoxicity assay on human kidney and uroepithelial cell lines. Furthermore, mutation of either barA or uvrY gene reduced the production of hemolysin, lipopolysaccharide (LPS), proinflammatory cytokines (TNF-α and IL-6) and chemokine (IL-8). The virulence phenotype was restored similar to that of wild-type by complementation of either barA or uvrY gene in trans. In addition, we discussed a possible link between the BarA-UvrY TCS and CsrA in positively and negatively controlling virulence in UPEC. Overall, this study provides the evidences for BarA-UvrY TCS regulates the virulence of UPEC CFT073 and may point to mechanisms by which virulence regulations are observed in different ways may control the long-term survival of UPEC in the urinary tract.
Uropathogenic Escherichia coli (UPEC) are the major cause of urinary tract infections (UTIs), and they have the capacity to induce the death and exfoliation of target uroepithelial cells. This process can be facilitated by the pore-forming toxin α-hemolysin (HlyA), which is expressed and secreted by many UPEC isolates. Here, we demonstrate that HlyA can potently inhibit activation of Akt (protein kinase B), a key regulator of host cell survival, inflammatory responses, proliferation, and metabolism. HlyA ablates Akt activation via an extracellular calcium-dependent, potassium-independent process requiring HlyA insertion into the host plasma membrane and subsequent pore formation. Inhibitor studies indicate that Akt inactivation by HlyA involves aberrant stimulation of host protein phosphatases. We found that two other bacterial pore-forming toxins (aerolysin from Aeromonas species and α-toxin from Staphylococcus aureus) can also markedly attenuate Akt activation in a dose-dependent manner. These data suggest a novel mechanism by which sublytic concentrations of HlyA and other pore-forming toxins can modulate host cell survival and inflammatory pathways during the course of a bacterial infection.
Uropathogenic Escherichia coli (UPEC) is a causative agent in the vast majority of urinary tract infections (UTIs), including cystitis and pyelonephritis, and infectious complications, which may result in acute renal failure in healthy individuals as well as in renal transplant patients. UPEC expresses a multitude of virulence factors to break the inertia of the mucosal barrier. In response to the breach by UPEC into the normally sterile urinary tract, host inflammatory responses are triggered leading to cytokine production, neutrophil influx, and the exfoliation of infected bladder epithelial cells. Several signaling pathways activated during UPEC infection, including the pathways known to activate the innate immune response, interact with calcium-dependent signaling pathways. Some UPEC isolates, however, might possess strategies to delay or suppress the activation of components of the innate host response in the urinary tract. Studies published in the recent past provide new information regarding how virulence factors of uropathogenic E. coli are involved in activation of the innate host response. Despite numerous host defense mechanisms, UPEC can persist within the urinary tract and may serve as a reservoir for recurrent infections and serious complications. Presentation of the molecular details of these events is essential for development of successful strategies for prevention of human UTIs and urological complications associated with UTIs.
Murine models of urinary tract infection (UTI) have provided substantial data identifying uropathogenic E. coli (UPEC) virulence factors and assessing their expression in vivo. However, it is unclear how gene expression in these animal models compares to UPEC gene expression during UTI in humans. To address this, we used a UPEC strain CFT073-specific microarray to measure global gene expression in eight E. coli isolates monitored directly from the urine of eight women presenting at a clinic with bacteriuria. The resulting gene expression profiles were compared to those of the same E. coli isolates cultured statically to exponential phase in pooled, sterilized human urine ex vivo. Known fitness factors, including iron acquisition and peptide transport systems, were highly expressed during human UTI and support a model in which UPEC replicates rapidly in vivo. While these findings were often consistent with previous data obtained from the murine UTI model, host-specific differences were observed. Most strikingly, expression of type 1 fimbrial genes, which are among the most highly expressed genes during murine experimental UTI and encode an essential virulence factor for this experimental model, was undetectable in six of the eight E. coli strains from women with UTI. Despite the lack of type 1 fimbrial expression in the urine samples, these E. coli isolates were generally capable of expressing type 1 fimbriae in vitro and highly upregulated fimA upon experimental murine infection. The findings presented here provide insight into the metabolic and pathogenic profile of UPEC in urine from women with UTI and represent the first transcriptome analysis for any pathogenic E. coli during a naturally occurring infection in humans.
Animal models of infection have been used extensively to study how bacteria and other pathogens cause disease. These models provide valuable information and have led to the development of numerous vaccines and antimicrobial therapies. However, it is important to recognize how these animal models compare to human infection and to understand how bacteria cause disease in humans. This study measured gene expression in E. coli, a major cause of urinary tract infection, immediately after collection from the urine of women with bladder infection symptoms. The data showed that E. coli gene expression in the urine from women with urinary tract infection was very often similar to what had been observed in a mouse model, but these studies also identified several potentially important differences, including a bacterial surface structure that is necessary for infection in mice but not detected in most E. coli in human urine. Although more precise measurements are still needed, these findings contribute to our understanding of bacterial infection in humans and will help in the development of vaccines and treatments for urinary tract infection.
Urinary tract infections continue to be among the most common extraintestinal diseases. Cystitis in women is by far the most common urinary tract infection; pyelonephritis in both sexes and prostatitis in men are more severe but less frequent complaints. Escherichia coli is by far the most common cause of urinary tract infection. It is believed that uropathogenic E. coli is adept at colonizing the urinary tract via the production of specific virulence factors. Recently, a novel virulence determinant, Vat, was described for the prototypical uropathogenic E. coli strain CFT073. Vat is a member of the SPATE (serine protease autotransporters of the Enterobacteriaceae) subfamily of the autotransporters. Previously, SPATEs have been described for all pathovars of E. coli, but until recently their presence had been noticeably absent in nonpathogenic E. coli. In this report we describe the prevalence and phylogenetic distribution of the SPATEs among uropathogenic E. coli and the ECOR collection, demonstrating an association between the presence of the SPATEs, including Vat, and uropathogenic E. coli phylogroups. In addition, we describe the distribution of SPATEs among nonpathogenic E. coli.
Entry into host cells is required for many bacterial pathogens to effectively disseminate within a host, avoid immune detection and cause disease. In recent years, many ostensibly extracellular bacteria have been shown to act as opportunistic intracellular pathogens. Among these are strains of uropathogenic Escherichia coli (UPEC), the primary causative agents of urinary tract infections (UTIs). UPEC are able to transiently invade, survive and multiply within the host cells and tissues constituting the urinary tract. Invasion of host cells by UPEC is promoted independently by distinct virulence factors, including cytotoxic necrotizing factor, Afa/Dr adhesins, and type 1 pili. Here we review the diverse mechanisms and consequences of host cell invasion by UPEC, focusing also on the impact of these processes on the persistence and recurrence of UTIs.
adhesion; bladder; cystitis; fimbriae; invasion; lipid rafts; pili; uroplakin
Background and Objectives
Urinary tract infection (UTI) is one of the most common infections in the world. The majority of UTIs are caused by Uropathogenic Escherichia coli (UPEC) strains. FimH and FliC are the most important virulence factors of UPEC. To date, any ideal vaccine against UTI has not been approved for human use and we need to test new targets to develop an ideal vaccine against UTI. In this study, we constructed fusion fimH/fliC of UPEC as a novel vaccine candidate against UTI.
Material and Methods
PCR amplification of fimH and fliC genes of the UPEC isolates was performed by specific primers designed for this purpose. Construction of fimH/fliC hybrid gene was performed by overlap PCR. The fimH, fliC and fimH/fliC were cloned in pET28a vector. The confirmation of expression of the proteins was done by SDS-PAGE and Western blot.
The fliC and fimH genes were amplified in all of the UPEC isolates tested. The fimH showed significant homology with the sequences in GenBank. We generated a fusion consisting of the fimH linked to the N-terminal end of fliC. Sequencing of the fusion fimH/fliC showed that fusion was constructed correctly. SDS-PAGE and western blot confirmed the expression of the proteins in optimized condition.
Urinary tract infection is a huge burden on healthcare system in many countries. UPEC is isolated in around 80% of UTI cases. Antibiotic therapy resulted in the emergence of antibiotic resistance in UPEC strains. This is the major cause for an increasing requirement for a vaccine to prevent UTI. This work describes for the first time the construction of a novel fusion protein from Iranian UPEC isolates. Further immunological studies are required for evaluation of this protein as a novel and safe vaccine candidate against UTI caused by UPEC.
urinary tract infection; Uropathogenic Escherichia coli; fimH; fliC; fusion protein expression
While in transit within and between hosts, uropathogenic Escherichia coli (UPEC) encounters multiple stresses, including substantial levels of nitric oxide and reactive nitrogen intermediates. Here we show that UPEC, the primary cause of urinary tract infections, can be conditioned to grow at higher rates in the presence of acidified sodium nitrite (ASN), a model system used to generate nitrosative stress. When inoculated into the bladder of a mouse, ASN-conditioned UPEC bacteria are far more likely to establish an infection than nonconditioned bacteria. Microarray analysis of ASN-conditioned bacteria suggests that several NsrR-regulated genes and other stress- and polyamine-responsive factors may be partially responsible for this effect. Compared to K-12 reference strains, most UPEC isolates have increased resistance to ASN, and this resistance can be substantially enhanced by addition of the polyamine cadaverine. Nitrosative stress, as generated by ASN, can stimulate cadaverine synthesis by UPEC, and growth of UPEC in cadaverine-supplemented broth in the absence of ASN can also promote UPEC colonization of the bladder. These results suggest that UPEC interactions with polyamines or stresses such as reactive nitrogen intermediates can in effect reprogram the bacteria, enabling them to better colonize the host.
Uropathogenic Escherichia coli (UPEC) is the predominant cause of urinary tract infection in both hospital and community settings. The recent emergence of multidrug-resistant clones like the O25b:H4-ST131 lineage represents a significant threat to health, and numerous studies have explored the virulence potential of these organisms. Members of the ST131 clone have been described as having variable carriage of key virulence factors, and it has been suggested that additional unidentified factors contribute to virulence. Here we demonstrated that ST131 isolates have high metabolic potential and biochemical profiles that distinguish them from isolates of many other sequence types (STs). A collection of 300 UPEC isolates recovered in 2007 and 2009 in the Northwest region of England were subjected to metabolic profiling using the Vitek2 Advanced Expert System (AES). Of the 47 tests carried out, 30 gave a positive result with at least one of the 300 isolates examined. ST131 isolates demonstrated significant association with eight tests, including those for peptidase, decarboxylase, and alkalinization activity. Metabolic activity also correlated with antibiotic susceptibility profiles, with resistant organisms displaying the highest metabolic potential. This is the first comprehensive study of metabolic potential in the ST131 lineage, and we suggest that high metabolic potential may have contributed to the fitness of members of the ST131 clone, which are able to exploit the available nutrients in both the intestinal and urinary tract environments.
We phylogenetically analyzed the Escherichia coli strains isolated from children with urinary tract infection (UTI) in 2 regions of Korea. Virulence factors (VFs) and antibiotic resistance of the strains were also determined to compare the possible differences.
A total of 138 E. coli strains were collected from the 2 regions; Gyeongin (78 strains) and Gyeongnam (60 strains). The phylogenetic groups were determined using the triplex polymerase chain reaction (PCR) method and multiplex PCRs were used to detect 7 VFs genes (fimH, papC, iutA, hlyA, sfa/focDE, afa/draBC, and kpsMT II). We also tested for antibiotic resistance.
Phylogenetic groups, B2 (61.6%) and D (26.8%), comprised the majority of all isolated strains. Regional comparisons revealed that more B2 strains and fewer non-B2 (A+B1+D) strains were found in Gyeongnam, than in the Gyeongin region (P=0.033), and certain VFs were predominantly detected in Gyeongnam (P<0.05). Neither regional nor phylogenetic differences, in antibiotic resistance of the strains, were significant.
We were able to confirm that the geographic location is an important determinant of the distribution of the phylogenetic groups and VFs among the E. coli strains that cause UTI in children.
Urinary tract infections; Escherichia coli; Phylogeny; Child
Strains of uropathogenic Escherichia coli (UPEC) are the primary cause of urinary tract infections, representing one of the most widespread and successful groups of pathogens on the planet. To colonize and persist within the urinary tract, UPEC must be able to sense and respond appropriately to environmental stresses, many of which can compromise the bacterial envelope. The Cpx two-component envelope stress response system is comprised of the inner membrane histidine kinase CpxA, the cytosolic response regulator CpxR, and the periplasmic auxiliary factor CpxP. Here, by using deletion mutants along with mouse and zebrafish infection models, we show that the Cpx system is critical to the fitness and virulence of two reference UPEC strains, the cystitis isolate UTI89 and the urosepsis isolate CFT073. Specifically, deletion of the cpxRA operon impaired the ability of UTI89 to colonize the murine bladder and greatly reduced the virulence of CFT073 during both systemic and localized infections within zebrafish embryos. These defects coincided with diminished host cell invasion by UTI89 and increased sensitivity of both strains to complement-mediated killing and the aminoglycoside antibiotic amikacin. Results obtained with the cpxP deletion mutants were more complicated, indicating variable strain-dependent and niche-specific requirements for this well-conserved auxiliary factor.
In many bacteria, the second messenger cyclic AMP (cAMP) interacts with the transcription factor cAMP receptor protein (CRP), forming active cAMP-CRP complexes that can control a multitude of cellular activities, including expanded carbon source utilization, stress response pathways, and virulence. Here, we assessed the role of cAMP-CRP as a regulator of stress resistance and virulence in uropathogenic Escherichia coli (UPEC), the principal cause of urinary tract infections worldwide. Deletion of genes encoding either CRP or CyaA, the enzyme responsible for cAMP synthesis, attenuates the ability of UPEC to colonize the bladder in a mouse infection model, dependent on intact innate host defenses. UPEC mutants lacking cAMP-CRP grow normally in the presence of glucose but are unable to utilize alternate carbon sources like amino acids, the primary nutrients available to UPEC within the urinary tract. Relative to the wild-type UPEC isolate, the cyaA and crp deletion mutants are sensitive to nitrosative stress and the superoxide generator methyl viologen but remarkably resistant to hydrogen peroxide (H2O2) and acid stress. In the mutant strains, H2O2 resistance correlates with elevated catalase activity attributable in part to enhanced translation of the alternate sigma factor RpoS. Acid resistance was promoted by both RpoS-independent and RpoS-dependent mechanisms, including expression of the RpoS-regulated DNA-binding ferritin-like protein Dps. We conclude that balanced input from many cAMP-CRP-responsive elements, including RpoS, is critical to the ability of UPEC to handle the nutrient limitations and severe environmental stresses present within the mammalian urinary tract.