Identification of genes important for growth of asymptomatic bacteriuria Escherichia coli in urine

Escherichia coli is the most important etiological agent of urinary tract infections (UTIs). Unlike uropathogenic E. coli, which causes symptomatic infections, asymptomatic bacteriuria (ABU) E. coli strains typically lack essential virulence factors and colonize the bladder in the absence of symptoms. While ABU E. coli can persist in the bladder for long periods of time, little is known about the genetic determinants required for its growth and fitness in urine. To identify such genes, we have employed a transposon mutagenesis approach using the prototypic ABU E. coli strain 83972 and the clinical ABU E. coli strain VR89. Six genes involved in the biosynthesis of various amino acids and nucleobases were identified (carB, argE, argC, purA, metE, and ilvC), and site-specific mutants were subsequently constructed in E. coli 83972 and E. coli VR89 for each of these genes. In all cases, these mutants exhibited reduced growth rates and final cell densities in human urine. The growth defects could be complemented in trans as well as by supplementation with the appropriate amino acid or nucleobase. When assessed in vivo in a mouse model, E. coli 83972carAB and 83972argC showed a significantly reduced competitive advantage in the bladder and/or kidney during coinoculation experiments with the parent strain, whereas 83972metE and 83972ilvC did not. Taken together, our data have identified several biosynthesis pathways as new important fitness factors associated with the growth of ABU E. coli in human urine.

Molecular characterization of Escherichia coli strains that cause symptomatic and asymptomatic urinary tract infections

The differences between Escherichia coli strains associated with symptomatic and asymptomatic urinary tract infections (UTIs) remain to be properly determined. Here we examined the prevalence of plasmid types and bacteriocins, as well as genetic relatedness, in a defined collection of E. coli strains that cause UTIs. Comparative analysis identified a subgroup of strains with a high number of virulence genes (VGs) and microcins M/H47. We also identified associations between microcin genes, VGs, and specific plasmid types.

Contribution of siderophore systems to growth and urinary tract colonization of asymptomatic bacteriuria Escherichia coli

The molecular mechanisms that define asymptomatic bacteriuria (ABU) Escherichia coli colonization of the human urinary tract remain to be properly elucidated. Here, we utilize ABU E. coli strain 83972 as a model to dissect the contribution of siderophores to iron acquisition, growth, fitness, and colonization of the urinary tract. We show that E. coli 83972 produces enterobactin, salmochelin, aerobactin, and yersiniabactin and examine the role of these systems using mutants defective in siderophore biosynthesis and uptake. Enterobactin and aerobactin contributed most to total siderophore activity and growth in defined iron-deficient medium. No siderophores were detected in an 83972 quadruple mutant deficient in all four siderophore biosynthesis pathways; this mutant did not grow in defined iron-deficient medium but grew in iron-limited pooled human urine due to iron uptake via the FecA ferric citrate receptor. In a mixed 1:1 growth assay with strain 83972, there was no fitness disadvantage of the 83972 quadruple biosynthetic mutant, demonstrating its capacity to act as a “cheater” and utilize siderophores produced by the wild-type strain for iron uptake. An 83972 enterobactin/salmochelin double receptor mutant was outcompeted by 83972 in human urine and the mouse urinary tract, indicating a role for catecholate receptors in urinary tract colonization.

Intramacrophage survival of uropathogenic Escherichia coli : differences between diverse clinical isolates and between mouse and human macrophages

Uropathogenic E. coli (UPEC) are the primary cause of urinary tract infections. Recent studies have demonstrated that UPEC can invade and replicate within epithelial cells, suggesting that this bacterial pathogen may occupy an intracellular niche within the host. Given that many intracellular pathogens target macrophages, we assessed the interactions between UPEC and macrophages. Colonization of the mouse bladder by UPEC strain CFT073 resulted in increased expression of myeloid-restricted genes, consistent with the recruitment of inflammatory macrophages to the site of infection. In in vitro assays, CFT073 was able to survive within primary mouse bone marrow-derived macrophages (BMM) up to 24 h post-infection. Three additional well-characterized clinical UPEC isolates associated with distinct UTI symptomatologies displayed variable long-term survival within BMM. UPEC strains UTI89 and VR50, originally isolated from patients with cystitis and asymptomatic bacteriuria respectively, showed elevated bacterial loads in BMM at 24 h post-infection as compared to CFT073 and the asymptomatic bacteriuria strain 83972. These differences did not correlate with differential effects on macrophage survival or initial uptake of bacteria. E. coli UTI89 localized to a Lamp1+ vesicular compartment within BMM. In contrast to survival within mouse BMM, intracellular bacterial loads of VR50 were low in both human monocyte-derived macrophages (HMDM) and in human T24 bladder epithelial cells. Collectively, these data suggest that some UPEC isolates may subvert macrophage anti-microbial pathways, and that host species differences may impact on intracellular UPEC survival.

Experimental colonization of the canine urinary tract with the asymptomatic bacteriuria Escherichia coli strain 83972

Establishment of asymptomatic bacteriuria (ABU) with Escherichia coli 83972 is a viable prophylactic alternative to antibiotic therapy for the prevention of recurrent bacterial urinary tract infection in humans. Approximately 2 x 108 viable E. coli 83972 cells were introduced into the bladder of six healthy female dogs via a sterile urinary catheter. The presence of pyuria, depression, stranguria, pollakiuria and haematuria was documented for 6 weeks and urinalysis and aerobic bacterial cultures were performed every 24–72 h. Pyuria was present in all dogs on day 1 post-inoculation and 4/6 dogs (67%) had a positive urine culture on this day. Duration of colonization ranged from 0 to 10 days (median 4 days). Four dogs were re-inoculated on day 20. Duration of colonization following the second inoculation ranged from 1 to 3 days. No dog suffered pyrexia or appeared systemically unwell but all dogs initially exhibited mild pollakiuria and a small number displayed gross haematuria and/or stranguria. By day 3 of each trial all clinical signs had resolved. Persistent bacteriuria was not achieved in any dog but two dogs were colonized for 10 days following a single inoculation. Further research is required to determine whether establishment of ABU in dogs with recurrent urinary tract infection is a viable alternative to repeated doses of antimicrobial agents.