Structural and sequence diversity of the pathogenicity island of uropathogenic Escherichia coli which encodes the USP protein

A total of 321 uropathogenic Escherichia coli (UPEC) strains and 12 strains of E. coli isolated from stool samples of healthy individuals, which were previously shown to be positive in colony hybridization test using the usp (encoding for the uropathogenic-specific protein) DNA probe, were examined by PCR amplification to determine the size of the usp gene and the pathogenicity island (PI). Three types of size variation were observed for the usp gene and four types for the PI. Sequencing analysis of the PIs from seven representative strains (six UPEC and one from a normal healthy individual) revealed that the usp genes can be classified into two groups, each having different sequences in the 3'-terminal region. The peptides encoded by the three open reading frames (ORFs) downstream of usp had identical 23 amino acid residues in the C-terminal region. The subregion encoding these small ORFs has a mosaic structure constituted of six segments. The positions of these segments vary from strain to strain, and in some strains, two to four segments are deleted. This indicates that rearrangements occur frequently in this region and the mosaic arrangement apparently contributes to the size variation observed in the PCR examination of the usp genes and PIs.     

PicU, a second serine protease autotransporter of uropathogenic Escherichia coli

Escherichia coli is the major aetiological agent of urinary tract infections (UTI). Like diarrhoeagenic strains of E. coli, uropathogenic isolates possess virulence determinants that distinguish them from commensal strains and allow them to produce the clinical manifestations associated with UTI. Several autotransporter proteins have been associated with the ability of E. coli, and other Gram-negative bacteria, to cause disease. Recently, we described the existence within uropathogenic E. coli (UPEC) strains of Sat, a toxin of the serine protease autotransporter of Enterobacteriaceae (SPATE) subfamily. Using features common to proteins secreted via the autotransporter pathway we have identified nine additional autotransporter proteins from the genomic sequence data of UPEC CFT073. Surprisingly, two additional members of the SPATE subfamily were identified. One protein, designated PicU, was homologous to the Pic protein identified in Shigella flexneri and enteroaggregative E. coli. The PicU protein was expressed and investigated for functional activity.     

Cytotoxic and hemolytic activities in uropathogenic Escherichia coli

Fifty nine Escherichia coli strains obtained from patients with upper or lower urinary tract infections (UTI) and 30 E. coli strains isolated from stools of healthy individuals were tested for hemolytic and cytotoxic activities. Forty four percent of uropathogenic E. coli (UPEC) and 3.3% of fecal E. coli were hemolytic. Among the hemolytic UPEC, 92% produced alpha-hemolysin. A cytotoxic activity was detected in culture filtrates of 71% of UPEC strains and 30% of fecal E. coli. No relationship was found between cytotoxic and hemolytic activities or between cytotoxic titers and UPEC origin (upper or lower UTI). E. coli cytotoxin has a cytocidal activity against some epithelioid cultured cell lines (Vero, HeLa and Hep-2) but was almost inactive for avian-fibroblast cells. Cytotoxin-affected cells appeared rounded, refractile and detached from the surface of the vessel. Some characteristics exhibited by the cytotoxin as the morphological response induced on cells, the increasing of cytopathic effect with time, its irreversible cytocidal activity and its heat-lability resemble the properties described for E. coli Verotoxin (VT). Adherence to uroepithelial cells is recognized as a virulence factor for UPEC. It is suggested that cell damage by cytotoxic and adhering UPEC might contribute to E. coli virulence to urinary tract.     

Insights into a multidrug resistant Escherichia coli pathogen of the globally disseminated ST131 lineage: genome analysis and virulence mechanisms

Escherichia coli strains causing urinary tract infection (UTI) are increasingly recognized as belonging to specific clones. E. coli clone O25b:H4-ST131 has recently emerged globally as a leading multi-drug resistant pathogen causing urinary tract and bloodstream infections in hospitals and the community. While most molecular studies to date examine the mechanisms conferring multi-drug resistance in E. coli ST131, relatively little is known about their virulence potential. Here we examined E. coli ST131 clinical isolates from two geographically diverse collections, one representing the major pathogenic lineages causing UTI across the United Kingdom and a second representing UTI isolates from patients presenting at two large hospitals in Australia. We determined a draft genome sequence for one representative isolate, E. coli EC958, which produced CTX-M-15 extended-spectrum β-lactamase, CMY-23 type AmpC cephalosporinase and was resistant to ciprofloxacin. Comparative genome analysis indicated that EC958 encodes virulence genes commonly associated with uropathogenic E. coli (UPEC). The genome sequence of EC958 revealed a transposon insertion in the fimB gene encoding the activator of type 1 fimbriae, an important UPEC bladder colonization factor. We identified the same fimB transposon insertion in 59% of the ST131 UK isolates, as well as 71% of ST131 isolates from Australia, suggesting this mutation is common among E. coli ST131 strains. Insertional inactivation of fimB resulted in a phenotype resembling a slower off-to-on switching for type 1 fimbriae. Type 1 fimbriae expression could still be induced in fimB-null isolates; this correlated strongly with adherence to and invasion of human bladder cells and bladder colonisation in a mouse UTI model. We conclude that E. coli ST131 is a geographically widespread, antibiotic resistant clone that has the capacity to produce numerous virulence factors associated with UTI.     

Prevalence of avian-pathogenic Escherichia coli strain O1 genomic islands among extraintestinal and commensal E. coli isolates

Escherichia coli strains that cause disease outside the intestine are known as extraintestinal pathogenic E. coli (ExPEC) and include pathogens of humans and animals. Previously, the genome of avian-pathogenic E. coli (APEC) O1:K1:H7 strain O1, from ST95, was sequenced and compared to those of several other E. coli strains, identifying 43 genomic islands. Here, the genomic islands of APEC O1 were compared to those of other sequenced E. coli strains, and the distribution of 81 genes belonging to 12 APEC O1 genomic islands among 828 human and avian ExPEC and commensal E. coli isolates was determined. Multiple islands were highly prevalent among isolates belonging to the O1 and O18 serogroups within phylogenetic group B2, which are implicated in human neonatal meningitis. Because of the extensive genomic similarities between APEC O1 and other human ExPEC strains belonging to the ST95 phylogenetic lineage, its ability to cause disease in a rat model of sepsis and meningitis was assessed. Unlike other ST95 lineage strains, APEC O1 was unable to cause bacteremia or meningitis in the neonatal rat model and was significantly less virulent than uropathogenic E. coli (UPEC) CFT073 in a mouse sepsis model, despite carrying multiple neonatal meningitis E. coli (NMEC) virulence factors and belonging to the ST95 phylogenetic lineage. These results suggest that host adaptation or genome modifications have occurred either in APEC O1 or in highly virulent ExPEC isolates, resulting in differences in pathogenicity. Overall, the genomic islands examined provide targets for further discrimination of the different ExPEC subpathotypes, serogroups, phylogenetic types, and sequence types.     

The genome sequence of avian pathogenic Escherichia coli strain O1:K1:H7 shares strong similarities with human extraintestinal pathogenic E. coli genomes

Escherichia coli strains that cause disease outside the intestine are known as extraintestinal pathogenic E. coli (ExPEC) and include human uropathogenic E. coli (UPEC) and avian pathogenic E. coli (APEC). Regardless of host of origin, ExPEC strains share many traits. It has been suggested that these commonalities may enable APEC to cause disease in humans. Here, we begin to test the hypothesis that certain APEC strains possess potential to cause human urinary tract infection through virulence genotyping of 1,000 APEC and UPEC strains, generation of the first complete genomic sequence of an APEC (APEC O1:K1:H7) strain, and comparison of this genome to all available human ExPEC genomic sequences. The genomes of APEC O1 and three human UPEC strains were found to be remarkably similar, with only 4.5% of APEC O1's genome not found in other sequenced ExPEC genomes. Also, use of multilocus sequence typing showed that some of the sequenced human ExPEC strains were more like APEC O1 than other human ExPEC strains. This work provides evidence that at least some human and avian ExPEC strains are highly similar to one another, and it supports the possibility that a food-borne link between some APEC and UPEC strains exists. Future studies are necessary to assess the ability of APEC to overcome the hurdles necessary for such a food-borne transmission, and epidemiological studies are required to confirm that such a phenomenon actually occurs.     

Asymptomatic bacteriuria Escherichia coli strains: adhesins, growth and competition

Urinary tract infections (UTIs) affect millions of people each year. Escherichia coli is the most common organism associated with asymptomatic bacteriuria (ABU) in humans. Persons affected by ABU may carry a particular E. coli strain for extended periods of time without any symptoms. In contrast to uropathogenic E. coli (UPEC) that cause symptomatic UTI, very little is known about the mechanisms by which these strains colonize the urinary tract. Here, we have investigated the growth characteristics in human urine as well as adhesin repertoire of nine ABU strains; the ability of ABU strains to compete against the UPEC strain CFT073 was also studied. The different ABU strains displayed a wide variety of the measured characteristics. Half of the ABU strains displayed functional type 1 fimbriae while only one expressed functional P fimbriae. A good correlation between the growth rate of a particular strain and the survival of the strain in competition against CFT073 was observed. Our results support the notion that for strains with reduced capacity to express fimbriae, the ability to grow fast in human urine becomes crucial for colonization of the urinary tract.     

O-antigen modulates infection-induced pain States

The molecular initiators of infection-associated pain are not understood. We recently found that uropathogenic E. coli (UPEC) elicited acute pelvic pain in murine urinary tract infection (UTI). UTI pain was due to E. coli lipopolysaccharide (LPS) and its receptor, TLR4, but pain was not correlated with inflammation. LPS is known to drive inflammation by interactions between the acylated lipid A component and TLR4, but the function of the O-antigen polysaccharide in host responses is unknown. Here, we examined the role of O-antigen in pain using cutaneous hypersensitivity (allodynia) to quantify pelvic pain behavior and using sacral spinal cord excitability to quantify central nervous system manifestations in murine UTI. A UPEC mutant defective for O-antigen biosynthesis induced chronic allodynia that persisted long after clearance of transient infections, but wild type UPEC evoked only acute pain. E. coli strains lacking O-antigen gene clusters had a chronic pain phenotype, and expressing cloned O-antigen gene clusters altered the pain phenotype in a predictable manner. Chronic allodynia was abrogated in TLR4-deficient mice, but inflammatory responses in wild type mice were similar among E. coli strains spanning a wide range of pain phenotypes, suggesting that O-antigen modulates pain independent of inflammation. Spinal cords of mice with chronic allodynia exhibited increased spontaneous firing and compromised short-term depression, consistent with centralized pain. Taken together, these findings suggest that O-antigen functions as a rheostat to modulate LPS-associated pain. These observations have implications for an infectious etiology of chronic pain and evolutionary modification of pathogens to alter host behaviors.     

Role of Capsule and O Antigen in the Virulence of Uropathogenic Escherichia coli

Urinary tract infection (UTI) is one of the most common bacterial infections in humans, with uropathogenic Escherichia coli (UPEC) the leading causative organism. UPEC has a number of virulence factors that enable it to overcome host defenses within the urinary tract and establish infection. The O antigen and the capsular polysaccharide are two such factors that provide a survival advantage to UPEC. Here we describe the application of the rpsL counter selection system to construct capsule (kpsD) and O antigen (waaL) mutants and complemented derivatives of three reference UPEC strains: CFT073 (O6:K2:H1), RS218 (O18:K1:H7) and 1177 (O1:K1:H7). We observed that while the O1, O6 and O18 antigens were required for survival in human serum, the role of the capsule was less clear and linked to O antigen type. In contrast, both the K1 and K2 capsular antigens provided a survival advantage to UPEC in whole blood. In the mouse urinary tract, mutation of the O6 antigen significantly attenuated CFT073 bladder colonization. Overall, this study contrasts the role of capsule and O antigen in three common UPEC serotypes using defined mutant and complemented strains. The combined mutagenesis-complementation strategy can be applied to study other virulence factors with complex functions both in vitro and in vivo.     

Defining genomic islands and uropathogen-specific genes in uropathogenic Escherichia coli

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.     

Impact of Host Age and Parity on Susceptibility to Severe Urinary Tract Infection in a Murine Model

The epidemiology and bacteriology of urinary tract infection (UTI) varies across the human lifespan, but the reasons for these differences are poorly understood. Using established monomicrobial and polymicrobial murine UTI models caused by uropathogenic Escherichia coli (UPEC) and/or Group B Streptococcus (GBS), we demonstrate age and parity as inter-related factors contributing to UTI susceptibility. Young nulliparous animals exhibited 10–100-fold higher bacterial titers compared to older animals. In contrast, multiparity was associated with more severe acute cystitis in older animals compared to age-matched nulliparous controls, particularly in the context of polymicrobial infection where UPEC titers were ∼1000-fold higher in the multiparous compared to the nulliparous host. Multiparity was also associated with significantly increased risk of chronic high titer UPEC cystitis and ascending pyelonephritis. Further evidence is provided that the increased UPEC load in multiparous animals required TLR4-signaling. Together, these data strongly suggest that the experience of childbearing fundamentally and permanently changes the urinary tract and its response to pathogens in a manner that increases susceptibility to severe UTI. Moreover, this murine model provides a system for dissecting these and other lifespan-associated risk factors contributing to severe UTI in at-risk groups.     

Biofilm formation by asymptomatic and virulent urinary tract infectious Escherichia coli strains

Escherichia coli is the most common organism associated with asymptomatic bacteriuria (ABU) in humans. In contrast to uropathogenic E. coli (UPEC) that cause symptomatic urinary tract infection, very little is known about the mechanisms by which these strains colonize the urinary tract. Here, we have investigated the biofilm-forming capacity on abiotic surfaces of groups of ABU strains and UPEC strains in human urine. We found that there is a strong bias; ABU strains were significantly better biofilm formers than UPEC strains. Our data suggest that biofilm formation in urinary tract infectious E. coli seems to be associated with ABU strains and appears to be an important strategy used by these strains for persistence in this high-flow environment.     

Identification of a type III secretion system in uropathogenic Escherichia coli

To determine virulence-related genes in uropathogenic Escherichia coli (UPEC) showing invasiveness to T-24 bladder cancer cells, genomic subtractive hybridization was performed between a highly invasive and a less invasive strain. Forty-nine DNA fragments were isolated from the invasive strain. One of them showed homology with Salmonella invA gene. By chromosomal walking of the strain, a type III secretion system that has been described in E. coli O157:H7 was identified on the genome of the invasive strains. Three strains out of 100 UPEC isolates had a type III secretion system inserted at 64 min of the chromosome, corresponding to E. coli K-12 MG1655. This finding suggested that the type III secretion system could play a part in uropathogenicity of UPEC.     

尿道致病性大肠杆菌HEC4株和禽源致病性大肠杆菌E058株毒力相关特性的比较

对人尿道致病性大肠杆菌(uropathogenic Escherichia coli,UPEC)HEC4株和禽致病性大肠杆菌(avian pathogen-ic Escherichia coli,APEC)E058株进行毒力基因和其他相关特性的比较,结果显示,它们具有一些共同的毒力基因,包括一些存在于APEC中一个大的可传递质粒上的基因;同时,它们也具有一些相似的生化特性。对SPF鸡的致病性试验显示,这两株分离株具有相似的致病力。因此,对于APEC和UPEC的相关性,以及APEC是否有可能导致人尿道感染或者成为UPEC的毒力基因贮主,有待进一步研究。     

Antigen-specific responses accelerate bacterial clearance in the bladder

Urinary tract infections (UTIs) cause patient morbidity and have a substantial economic impact. Half of all women will suffer a UTI at least once, and 25% of these women will have recurrent infections. That 75% of previously infected women do not become reinfected strongly suggests a role for an adaptive immune response. The goal of this study was to characterize the adaptive immune responses to uropathogenic Escherichia coli (UPEC), the predominant uropathogen. A novel murine model of UTI reinfection was developed using the prototypic cystitis UPEC isolate NU14 harboring a plasmid encoding OVA as a unique antigenic marker. Bacterial colonization of the bladder was quantified following one or more infections with NU14-OVA. Animals developed anti-OVA serum IgG and IgM titers after the initial infection and marked up-regulation of activation markers on splenic T cells. We observed a 95% reduction in bacterial colonization upon reinfection, and splenic leukocytes showed Ag-specific proliferation in vitro. Adoptive transfer of splenic T cells or passive transfer of serum from previously infected mice protected naive syngeneic mice from UPEC colonization. These findings support our hypothesis that adaptive immune responses to UPEC protect the bladder from reinfection and form the basis of understanding susceptibility to recurrent UTI in women.     

Prevalence of ColE1-like plasmids and colicin K production among uropathogenic Escherichia coli strains and quantification of inhibitory activity of colicin K

Colicin K exhibited pronounced inhibitory activity against uropathogenic Escherichia coli (UPEC) strains. Low prevalence of colicin K production and a relatively high prevalence of ColE1-like plasmids were determined among 215 UPEC strains from Slovenia. Sequencing of the colicin K-encoding pColK-K235 revealed a mosaic structure and the presence of the insertion sequence IS2.     

Pathogenic Escherichia coli found in sewage treatment plants and environmental waters

We previously demonstrated that some Escherichia coli strains with uropathogenic properties survived treatment stages of sewage treatment plants (STPs), suggesting that they may be released into the environment. We investigated the presence of such strains in the surrounding environmental waters of four STPs from which these persistent strains were isolated. In all, 264 E. coli isolates were collected from 129 receiving water sites in a 20-km radius surrounding STPs. We also included 93 E. coli strains collected from 18 animal species for comparison. Isolates were typed using a high-resolution biochemical fingerprinting method (the PhPlate system), and grouped into common (C) types. One hundred forty-seven (56%) environmental isolates were identical to strains found in STPs' final effluents. Of these, 140 (95%) carried virulence genes (VGs) associated with intestinal pathogenic E. coli (IPEC) or uropathogenic E. coli (UPEC) and were found in a variety of sites within areas sampled. Of the remaining 117 environmental strains not identical to STP strains, 105 belonged to 18 C types and 102 of them carried VGs found among IPEC or UPEC strains. These strains belonged mainly to phylogenetic groups A (A0 and A1) and B1 and to a lesser extent B2(2), B2(3), D1, and D2. Eight of 18 environmental C types, comprising 50 isolates, were also identical to bird strains. The presence of a high percentage of environmental E. coli in waters near STPs carrying VGs associated with IPEC and UPEC suggests that they may have derived from STP effluents and other nonpoint sources.     

Virulence properties of uropathogenic <Emphasis Type="Italic">Escherichia coli</Emphasis> isolated from children with urinary tract infection in Korea

Urinary tract infections (UTIs) are one of the most common types of bacterial infection in humans in various parts of the world and are caused mainly by uropathogenic Escherichia coli (UPEC). A total of 58 UPEC isolates from urine were characterized by serotyping and pulsed-field gel electrophoresis (PFGE). The majority of the UPEC strains belonged to serogroups O2 and O6. The UPEC strains were grouped under different pulsotypes and majority of them belonged to serogroups O2 and O6. Among the 14 virulence factors considered, 13 were present in various serogroups. The virulence genes fimH and sfa were present in all the isolates while none of the isolates carried lt-1. The strains exhibited 36 different virulence patterns, of which 11, referred to as UP (UPEC pattern) 1 to UP 11 were most common. Antibiotic resistance profiling of the UPEC isolates revealed that the serogroups O2 and O6 contain the highest number of resistant strains. The data from the current study depicting the distribution of UPEC strains among various serogroups and pulsotypes, and the occurrence of virulence genes and antibiotics resistance offer useful information on the epidemiological features of UPEC in Korea for the enhanced surveillance of potential emergence of UPEC.