A uropathogenic Escherichia coli strain CFT073-specific DNA microarray that includes each open reading frame was used to analyze the transcriptome of CFT073 bacteria isolated directly from the urine of infected CBA/J mice. The in vivo expression profiles were compared to that of E. coli CFT073 grown statically to exponential phase in rich medium, revealing the strategies this pathogen uses in vivo for colonization, growth, and survival in the urinary tract environment. The most highly expressed genes overall in vivo encoded translational machinery, indicating that the bacteria were in a rapid growth state despite specific nutrient limitations. Expression of type 1 fimbriae, a virulence factor involved in adherence, was highly upregulated in vivo. Five iron acquisition systems were all highly upregulated during urinary tract infection, as were genes responsible for capsular polysaccharide and lipopolysaccharide synthesis, drug resistance, and microcin secretion. Surprisingly, other fimbrial genes, such as pap and foc/sfa, and genes involved in motility and chemotaxis were downregulated in vivo. E. coli CFT073 grown in human urine resulted in the upregulation of iron acquisition, capsule, and microcin secretion genes, thus partially mimicking growth in vivo. On the basis of gene expression levels, the urinary tract appears to be nitrogen and iron limiting, of high osmolarity, and of moderate oxygenation. This study represents the first assessment of any E. coli pathotype's transcriptome in vivo and provides specific insights into the mechanisms necessary for urinary tract pathogenesis.Urinary tract infections (UTIs) are a serious health concern. Forty to 50% of women experience at least one UTI, leading to an estimated 8 million annual physician visits in the United States alone (39, 46). Uropathogenic Escherichia coli (UPEC) is by far the most common etiological agent of all UTIs. UPEC strain CFT073, derived from the clonal group O6:K2:H1 (26), was originally isolated from the blood and urine of a woman diagnosed with acute pyelonephritis (28). It is considered a prototype of the O6 serogroup, one of the most prevalent UPEC clonal lines (23,24). The virulence of this strain was reproduced in the well-established CBA mouse model of ascending UTI (28). In addition to numerous virulence studies, the genome of E. coli CFT073 has recently been sequenced and compared to that of enterohemorrhagic E. coli EDL933 and the nonpathogenic laboratory strain E. coli MG1655 (42).Mutations have been introduced into a number of candidate virulence genes in UPEC, leading to attenuated mutants in experimental UTI. These include fim, encoding type 1 fimbria (7, 13), sat, encoding secreted autotransporter toxin (14), cnf-1, encoding cytotoxic necrotizing factor (36), tonB, involved in iron transport (40), proP, involved in osmoprotectant transport (8), and degS (35). Large-scale screens for virulence factors of UPEC have also identified factors that aid UPEC during growth in urine (38) and have implicated capsule, lipopolysaccharide, ...

Enterococcus faecalis bacteria isolated from patients with bacteremia, endocarditis, and urinary tract infections more frequently express the surface protein Esp than do fecal isolates. To assess the role of Esp in colonization and persistence of E. faecalis in an animal model of ascending urinary tract infection, we compared an Esp ؉ strain of E. faecalis to its isogenic Esp-deficient mutant. Groups of CBA/J mice were challenged transurethrally with 10 8 CFU of either the parent or mutant strain, and bacteria in the urine, bladder, and kidneys were enumerated 5 days postinfection. Significantly higher numbers of bacteria were recovered from the bladder and urine of mice challenged with the parent strain than from the bladder and urine of mice challenged with the mutant. Colonization of the kidney, however, was not significantly different between the parent and mutant strains. Histopathological evaluations of kidney and bladder tissue done at 5 days postinfection did not show marked histopathological changes consistent with inflammation, mucosal hyperplasia, or apoptosis, and there was no observable difference between the mice challenged with the parent and those challenged with the mutant. We conclude that, while Esp does not influence histopathological changes associated with acute urinary tract infections, it contributes to colonization and persistence of E. faecalis at this site.

Acute pyelonephritis, a complication of Escherichia coli bacteriuria, must represent a bacterial invasion through the kidney epithelium. To study this process, we overlaid bacterial suspensions onto monolayers of cultured human kidney proximal tubular epithelial cells and measured cytotoxicity by release of lactate dehydrogenase (LDH). Thirty-four isolates cultured from patients with acute pyelonephritis were screened for the ability to cause pyelonephritis in CBA mice by transurethral challenge. The eight most virulent strains

Type 1 fimbriae and extracellular polysaccharides are preeminent uropathogenic Escherichia coli virulence determinants in the murine urinary tract the E. coli K-12 strain. Mutations in type 1 fimbrial genes resulted in severely attenuated colonization, even in the case of a mutant with an insertion upstream of the fim operon that affected the rate of fimbrial switching from the 'off' to the 'on' phase. Three mutants had insertions in a new type II capsule biosynthesis locus on a pathogenicity island and were impaired in the production of capsule in vivo. An additional mutant with an insertion in wecE was unable to synthesize enterobacterial common antigen. These results confirm the pre-eminence of type 1 fimbriae, establish the importance of extracellular polysaccharides in the pathogenesis of UTI and identify new urovirulence determinants. IntroductionThe urinary tract is a normally sterile system that is protected from the nearby colonic microflora by non-specific defences including the epithelial barrier, the antibacterial properties of the bladder mucosa and the flow of urine. Nevertheless, urinary tract infections (UTIs) are among the most common infectious diseases, affecting a wide range of individuals including preschool girls, women of childbearing age and the elderly (Warren, 1996). These infections usually result from the entry of periurethral microorganisms through the urethra into the bladder lumen. The bacteria may ascend further via the ureters into the kidneys and even breach the kidney parenchyma to enter the lymphatic system or the bloodstream. Therefore, the manifestations of UTIs can range from asymptomatic bacteriuria to urethritis, cystitis, pyelonephritis, bacteraemia and sepsis (Warren, 1996).Escherichia coli is by far the most common uropathogen. Reports of dramatic outbreaks of nosocomial and community-acquired pyelonephritis and cystitis involving single clones of E. coli (Tullus et al., 1984;Phillips et al., 1988;Kunin et al., 1993;Manges et al., 2001) emphasize the fact that certain strains are uniquely equipped to infect the urinary tract. Such uropathogenic E. coli (UPEC) strains may belong to specific lineages among specific serogroups (Arbeit et al., 1990;Johnson et al., 1997;Zhang et al., 1997). The concept that certain traits are more common among E. coli isolated from patients with UTIs than among other E. coli strains is well SummaryEscherichia coli is the leading cause of urinary tract infections (UTIs). Despite the association of numerous bacterial factors with uropathogenic E. coli (UPEC), few such factors have been proved to be required for UTI in animal models. Previous investigations of urovirulence factors have relied on prior identification of phenotypic characteristics. We used signature-tagged mutagenesis (STM) in an unbiased effort to identify genes that are essential for UPEC survival within the murine urinary tract. A library of 2049 transposon mutants of the prototypic UPEC strain CFT073 was constructed using mini-Tn5km2 carrying 92 unique tags and screened in ...

Proteus mirabilis, a common cause of urinary tract infections (UTI) in individuals with functional or structural abnormalities or with long-term catheterization, forms bladder and kidney stones as a consequence of urease-mediated urea hydrolysis. Known virulence factors, besides urease, are hemolysin, fimbriae, metalloproteases, and flagella. In this study we utilized the CBA mouse model of ascending UTI to evaluate the colonization of mutants of P. mirabilis HI4320 that were generated by signature-tagged mutagenesis. By performing primary screening of 2088 P. mirabilis transposon mutants, we identified 502 mutants that ranged from slightly attenuated to unrecoverable. Secondary screening of these mutants revealed that 114 transposon mutants were reproducibly attenuated. Cochallenge of 84 of these single mutants with the parent strain in the mouse model resulted in identification of 37 consistently out-competed P. mirabilis transposon mutants, 25 of which were out-competed >100-fold for colonization of the bladder and/or kidneys by the parent strain. We determined the sequence flanking the site of transposon insertion in 29 attenuated mutants and identified genes affecting motility, iron acquisition, transcriptional regulation, phosphate transport, urease activity, cell surface structure, and key metabolic pathways as requirements for P. mirabilis infection of the urinary tract. Two mutations localized to a approximately 42-kb plasmid present in the parent strain, suggesting that the plasmid is important for colonization. Isolation of disrupted genes encoding proteins with homologies to known bacterial virulence factors, especially the urease accessory protein UreF and the disulfide formation protein DsbA, showed that the CBA mouse model and mutant pools are a reliable source of attenuated mutants with mutations in virulence genes.