Escherichia coli global gene expression in urine from women with urinary tract infection.

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.

Genomic characterization of asymptomatic Escherichia coli isolated from the neobladder.

The replacement of the bladder with a neobladder made from ileal tissue is the prescribed treatment in some cases of bladder cancer or trauma. Studies have demonstrated that individuals with an ileal neobladder have recurrent colonization by Escherichia coli and other species that are commonly associated with urinary tract infections; however, pyelonephritis and complicated symptomatic infections with ileal neobladders are relatively rare. This study examines the genomic content of two E. coli isolates from individuals with neobladders using comparative genomic hybridization (CGH) with a pan-E. coli/Shigella microarray. Comparisons of the neobladder genome hybridization patterns with reference genomes demonstrate that the neobladder isolates are more similar to the commensal, laboratory-adapted E. coli and a subset of enteroaggregative E. coli than they are to uropathogenic E. coli isolates. Genes identified by CGH as exclusively present in the neobladder isolates among the 30 examined isolates were primarily from large enteric isolate plasmids. Isolations identified a large plasmid in each isolate, and sequencing confirmed similarity to previously identified plasmids of enteric species. Screening, via PCR, of more than 100 isolates of E. coli from environmental, diarrhoeagenic and urinary tract sources did not identify neobladder-specific genes that were widely distributed in these populations. These results taken together demonstrate that the neobladder isolates, while distinct, are genomically more similar to gastrointestinal or commensal E. coli, suggesting why they can colonize the transplanted intestinal tissue but rarely progress to acute pyelonephritis or more severe disease.

Genomic islands of uropathogenic Escherichia coli contribute to virulence.

Uropathogenic Escherichia coli (UPEC) strain CFT073 contains 13 large genomic islands ranging in size from 32 kb to 123 kb. Eleven of these genomic islands were individually deleted from the genome, and nine isogenic mutants were tested for their ability to colonize the CBA/J mouse model of ascending urinary tract infection. Three genomic island mutants (Delta PAI-aspV, Delta PAI-metV, and Delta PAI-asnT) were significantly outcompeted by wild-type CFT073 in the bladders and/or kidneys following transurethral cochallenge (P

Uropathogenic Escherichia coli Suppresses the host inflammatory response via pathogenicity island genes sisA and sisB.

Extraintestinal pathogenic Escherichia coli can successfully colonize the urinary tract of the immunocompetent host. In part, this is accomplished by dampening the host immune response. Indeed, the sisA and sisB genes (shiA-like inflammation suppressor genes A and B) of uropathogenic E. coli strain CFT073, homologs of the Shigella flexneri SHI-2 pathogenicity island gene shiA, suppress the host inflammatory response. A double deletion mutant (DeltasisA DeltasisB) resulted in a hyperinflammatory phenotype in an experimental model of ascending urinary tract infection. The DeltasisA DeltasisB mutant not only caused significantly more inflammatory foci in the kidneys of CBA/J mice (P = 0.0399), but these lesions were also histologically more severe (P = 0.0477) than lesions observed in mice infected with wild-type CFT073. This hyperinflammatory phenotype could be suppressed to wild-type levels by in vivo complementation of the DeltasisA DeltasisB mutant with either the sisA or sisB gene in trans. The DeltasisA DeltasisB mutant was outcompeted by wild-type CFT073 during cochallenge infection in the bladder (P = 0.0295) at 48 h postinoculation (hpi). However, during cochallenge infections, we reasoned that wild-type CFT073 could partially complement the DeltasisA DeltasisB mutant. Consistent with this, the most significant colonization defect of the DeltasisA DeltasisB mutant in vivo was observed during independent challenge relative to wild-type CFT073, with attenuation of the mutant observed in the bladder (P < 0.0001) and kidneys (P = 0.0003) at 6 hpi. By 24 and 48 hpi, the DeltasisA DeltasisB mutant was no longer significantly attenuated in the bladder or kidneys, suggesting that the sisA and sisB genes may be important for suppressing the host immune response during the initial stages of infection.

Identifying cloned Helicobacter pylori promoters by primer extension using a FAM-labelled primer and GeneScan analysis.

The transcriptional start sites of 27 promoters in Helicobacter pylori strain 4187E have been successfully identified using a non-radioactive primer extension protocol. The technique involves reverse transcribing mRNA with a sequence-specific FAM-labelled primer. The length of the FAM-labelled cDNA primer extension product can be analysed on a standard DNA sequencer using GeneScan software. This information can be used in conjunction with DNA sequencing data to identify the transcriptional start site of a promoter. Total bacterial RNA produced more specific primer extension products with stronger FAM signals than a population enriched for mRNA. Using this technology, it is not necessary to complete the DNA sequencing reactions in parallel with the primer extension experiments. The FAM-labelled primer extension products do not require a PCR amplification step prior to analysis on a sequencing gel, and no phenol/chloroform purifications are required at any stage of the procedure. Fluorescent-based primer extension methods have obvious advantages over the conventional radioactive protocols, and this report extends the currently used methodologies in this field.

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.

Role of phase variation of type 1 fimbriae in a uropathogenic Escherichia coli cystitis isolate during urinary tract infection.

Type 1 fimbrial phase-locked mutants of uropathogenic Escherichia coli cystitis isolate F11 were used to assess the role of the invertible element during urinary tract infection. Compared to the wild type, the phase-locked off mutant was attenuated, and constitutive production of type 1 fimbriae by the phase-locked on mutant did not provide a competitive advantage.

Uropathogenic Escherichia coli strains generally lack functional Trg and Tap chemoreceptors found in the majority of E. coli strains strictly residing in the gut.

The prevalence and function of four chemoreceptors, Tsr, Tar, Trg, and Tap, were determined for a collection of uropathogenic, fecal-commensal, and diarrheagenic Escherichia coli strains. tar and tsr were present or functional in nearly all isolates. However, trg and tap were significantly less prevalent or functional among the uropathogenic E. coli strains (both in 6% of strains) than among fecal-commensal strains (both in > or =50% of strains) or diarrheal strains (both in > or =75% of strains) (P < 0.02).