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

Abstract

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.

Fig 1

Growth mutants identified by transposon mutagenesis. (A) Identified insertion sites of the mariner transposon in the two E. coli strains 83972 (black triangles) and VR89 (white triangles). The illustrated gene order and orientations refer to the genomic context of E. coli MG1655. (B) Biosynthesis pathways for the amino acids arginine, methionine, leucine, valine, and isoleucine. The gene(s) encoding the enzyme responsible for each step is indicated. Genes that were mutated (knocked out) in E. coli strain 83972 are indicated in boldface.

Fig 2

Growth of E. coli 83972 and its isogenic mutants in human urine. (A) The curves are shown as means of four replicates, and error bars indicate standard deviations (σ_n−1). (B) Growth rates of E. coli 83972 in human urine with and without addition of amino acids and/or nucleobases (black) and growth rates of its mutants in urine without (white) and with (gray) addition of amino acids and/or nucleobases. Growth rates are displayed as means of triplicates of independent experiments relative to the growth rate of E. coli 83972 grown in human urine with no addition of amino acids. Error bars indicate standard errors.

Fig 3

Final cell density of E. coli 83972 and its isogenic mutants in human urine supplemented with the appropriate amino acids and/or nucleobases or complemented with plasmid expressing the defunct gene. Black bars, wild-type E. coli 83972 with and without addition of amino acids and/or nucleobases; white bars, isogenic mutants; light gray bars, isogenic mutants with addition of amino acids and/or nucleobases; dark gray bars, isogenic mutants complemented with plasmids expressing genes corresponding to the defunct gene. Growth characteristics of the isogenic mutants and the corresponding mutant expressing the control vector pACYC184 were identical. Values are means of at least triplicates, and error bars indicate standard errors.

Fig 4

Expression levels of E. coli 83972 in minimal medium (MOPS) and human urine (in vitro and in vivo) of all genes involved in the biosynthesis of amino acids, as determined by microarray analysis. Genes that were mutated in E. coli 83972 are indicated in boldface. Asterisks indicate expression levels in human urine that were significantly higher than those in MOPS (P < 0.05).

Fig 5

Competition experiment in human urine cultures between E. coli 83972 and its mutants, mixed 1:1 at the starting point. After 16 h of growth under hydrodynamic conditions at 37°C, the number of CFU of each strain in the mixed biofilm population was determined. The data shown are the means of three independent experiments in different batches of urine, and error bars indicate standard errors. wt, wild type.

Fig 6

Competitive colonization of mouse urine, bladder, and kidneys. E. coli 83972^AMP and each mutant were mixed at a 1:1 ratio and inoculated into a group of eight mice. Urine (A), bladders (B), and kidneys (C) were collected 48 h after inoculation and processed for quantitative colony counts. Asterisks indicate significant differences in colonization.