Rapid Growth of Uropathogenic Escherichia coli during Human Urinary Tract Infection

Abstract

Uropathogenic Escherichia coli (UPEC) strains cause most uncomplicated urinary tract infections (UTIs). These strains are a subgroup of extraintestinal pathogenic E. coli (ExPEC) strains that infect extraintestinal sites, including urinary tract, meninges, bloodstream, lungs, and surgical sites. Here, we hypothesize that UPEC isolates adapt to and grow more rapidly within the urinary tract than other E. coli isolates and survive in that niche. To date, there has not been a reliable method available to measure their growth rate in vivo Here we used two methods: segregation of nonreplicating plasmid pGTR902, and peak-to-trough ratio (PTR), a sequencing-based method that enumerates bacterial chromosomal replication forks present during cell division. In the murine model of UTI, UPEC strain growth was robust in vivo, matching or exceeding in vitro growth rates and only slowing after reaching high CFU counts at 24 and 30 h postinoculation (hpi). In contrast, asymptomatic bacteriuria (ABU) strains tended to maintain high growth rates in vivo at 6, 24, and 30 hpi, and population densities did not increase, suggesting that host responses or elimination limited population growth. Fecal strains displayed moderate growth rates at 6 hpi but did not survive to later times. By PTR, E. coli in urine of human patients with UTIs displayed extraordinarily rapid growth during active infection, with a mean doubling time of 22.4 min. Thus, in addition to traditional virulence determinants, including adhesins, toxins, iron acquisition, and motility, very high growth rates in vivo and resistance to the innate immune response appear to be critical phenotypes of UPEC strains.IMPORTANCE Uropathogenic Escherichia coli (UPEC) strains cause most urinary tract infections in otherwise healthy women. While we understand numerous virulence factors are utilized by E. coli to colonize and persist within the urinary tract, these properties are inconsequential unless bacteria can divide rapidly and survive the host immune response. To determine the contribution of growth rate to successful colonization and persistence, we employed two methods: one involving the segregation of a nonreplicating plasmid in bacteria as they divide and the peak-to-trough ratio, a sequencing-based method that enumerates chromosomal replication forks present during cell division. We found that UPEC strains divide extraordinarily rapidly during human UTIs. These techniques will be broadly applicable to measure in vivo growth rates of other bacterial pathogens during host colonization.

Keywords: ABU; ExPEC; PTR; UPEC; UTI; in vivo growth; plasmid segregation.

FIG 1

UPEC, fecal, and asymptomatic strains colonize the murine bladder at 6 hpi. (A) Bacterial recovery from bladders of mice transurethrally inoculated with 10⁸ CFU/ml UPEC (CFT073, UTI89, 536), fecal (EFC1, EFC2, EFC4, and EFC7), or ABU (PUTS37, PUTS58, PUTS59, and ABU83972) strains. Infections proceeded for 1 h (black circles), 2 h (dark gray circles), 4 h (light gray circles), or 6 h (open circles). (B and C) Bacterial recovery from the bladder (B) and kidneys (C) of mice transurethrally inoculated with 10⁸ (black circles), 10⁹ (gray circles), or 10¹⁰ (open circles) CFU/ml of fecal (EFC4 and EFC7) or UPEC (F3, F15, F11, F24, F54, CFT073, CFT269, CFT189, CFT204, and CFT325) isolates. Bladder and kidneys were harvested at 48 hpi. In panels A, B, and C, symbols represent individual mice and bars represent the median. n = 4 to 60. The limit of detection is 100 CFU/g. As an index of type 1 fimbrial expression, strain CFT073 agglutinated a suspension of yeast (Sacchoromyces cerevisiae) at a bacterial titer of 1:32. Strains ABU83972 and EFC7 failed to agglutinate yeast. Colonization and virulence gene data for each strain may be found in Fig. S1.

FIG 2

pGTR902 copy number is variable among EXPEC isolates. (A to G) Representative growth curves in LB diluted 1:100 (ABU83972) or 1:1,000 (CFT073, UTI89, 536, EFC2, EFC7, and PUTS37) from overnight cultures grown in LB supplemented with 1% l-arabinose and kanamycin (25 μg/ml). Cultures were grown at 37°C with aeration, and CFU per milliliter of pGTR902-containing bacteria and total bacteria (bacteria containing and those not containing pGTR902) were determined at 30-min or 1-h intervals by plating on LB agar containing 1% l-arabinose and kanamycin (25 μg/ml) (open symbols) and LB agar containing no antibiotic (closed symbols), respectively. (H) The copy number of pGTR902 in each isolate was calculated using the following equation: CFU/ml of pGTR902-containing bacteria at stationary phase/CFU/ml of pGTR902-containing bacteria in the inoculum. Values are mean ± standard deviation.

FIG 3

Growth rate is correlated with PTR in vitro. (A) E. coli CFT073 was inoculated into M9 supplemented with 0.4% glucose (blue), LB (yellow), and Terrific broth (gray) and grown to the mid-exponential phase (OD₆₀₀ of 0.25, 0.55, and 0.67, respectively). DNA sequencing reads from each sample were aligned to the CFT073 chromosome (x axis), and the average coverage across the chromosome was calculated for replicate pairs (y axis). (B) DNA sequencing reads obtained from CFT073 grown in human urine were aligned to the CFT073 genome as in panel A. Average coverage from paired replicates is shown. (C) Triplicate OD₆₀₀ measurements (black) at the corresponding time points from panel B. Growth rates (red) were calculated as the logarithm of the change in OD. PTR values (blue) from panel B are shown. Symbols represent the average, and bars represent the standard deviation. (D) A linear model was constructed using PTR and the growth rate of CFT073 in human urine.

FIG 4

UPEC strain CFT073 growth is rapid at early time points during murine UTI. Mice were transurethrally inoculated with 10⁸ CFU/ml of E. coli CFT073. At 6 h (blue), 24 h (yellow), or 30 h (gray) postinoculation, bladder (A) and pooled urine (B) samples were processed to enrich for bacterial genomic DNA. DNA sequencing reads were aligned to the CFT073 genome (x axis), and the coverage across the chromosome was calculated (y axis). n = 3 to 4. (C) Doubling times for CFT073 in bladder and urine (y axis) were extrapolated using the formula from Fig. 2D. n = 3 to 4. Twenty-four-hour and 30-h urine samples from four mice were pooled for PTR determination. (D) The percentage of sequence reads from urine or bladder samples aligned to the CFT073 genome is shown (y axis) for the samples in panel C.

FIG 5

PTR of UPEC during human UTI is increased compared to in vitro growth or growth during murine UTI. (A) E. coli CFT073 containing pGTR902 grown in vitro in LB with 1% l-arabinose and kanamycin (25 μg/ml) was pelleted, washed in PBS, and diluted 1:1,000 into pooled human urine. Samples were taken hourly (3 to 8 h) and subjected to Illumina sequencing, and PTRs were calculated. n = 2. To calculate in vivo PTRs, mice were transurethrally inoculated with 10⁸ CFU/ml E. coli CFT073 containing pGTR902. The PTR of the inoculum of murine infection was 1.3, and the doubling time was 119 min. At 6, 24, or 30 hpi, bacterial genomic DNA from urine was sequenced. n = 4. PTRs of human urinary tract infections were calculated from bacterial genomic DNA harvested directly from infected human urine. (B) Doubling time (in minutes) extrapolated from the PTR of E. coli strains isolated during human UTI. In panels A and B, stippled bars represent individuals with no history of UTI. Each bar represents the mean. Error bars represent standard deviation.