PMC

IBCs are clonal and derived from a single invasive bacterium. C3H/HeN mice were coinfected with UTI89 and UTI89::HKGFP and sacrificed at 6 hpi and 12 hpi. Bladders were aseptically removed, splayed, and imaged with confocal microscopy. (A and B) IBCs in whole mount at 6 hpi, counterstained with TO-PRO-3. Each image shows the merged red and green channel data. (C and D) Coresident IBCs inside a single superficial facet cell at 12 hpi. Representative images of over 500 independent IBCs are shown.

IBC number correlates with the number of tags present. C3H/HeN mice were infected as described and processed to enumerate IBCs and extract bacterial genomic DNA (see Materials and Methods). Black squares represent the number of IBCs and the number of tagged strains present per mouse bladder. The line is the median number of tags expected based on a multinomial distribution of IBC number (see Discussion and the supplemental material). Data represent 3 independent experiments, with 5 to 10 mice per experiment. *, bladders with a greater number of tags than IBCs.

Higher fraction of unique bacterial signatures remains in mice experiencing chronic cystitis. (A) Two weeks postinfection, C3H/HeN mice were sacrificed, and the bladders were removed aseptically, homogenized, and plated to enumerate CFU. (B) Urine samples were obtained by gentle suprapubic pressure and plated to enumerate CFU. The dashed line at 10⁴ CFU represents the limit for assessing the presence of UTI in clean-catch urine samples (). Urine titers above this point are considered bacteriuria indicative of a UTI, while bladder titers greater than this cutoff indicate chronic cystitis. (C) Genomic DNA was then obtained from the bacteria from each bladder sample, and multiplex PCR was conducted. Bars are median values. P values were calculated using a two-tailed Mann-Whitney nonparametric comparison.

Bacterial diversity decreased dramatically during the first 24 hpi. At the indicated times postinfection, C3H/HeN mice were sacrificed, the bladder and kidneys were removed aseptically and homogenized in PBS, and 6% of the homogenate was plated to enumerate CFU in the bladders (A) and kidney pairs (B). Genomic DNA was isolated from a lawn of UPEC isolated from the tissue homogenates, and multiplex PCR was conducted to determine the fraction of the 40 unique tags present in each bladder (C) and kidney pair (D). CFU data are presented as CFU/whole organ. Bars are median values. n = 1 to 2 experiments, with 5 to 8 mice each. P values were calculated using a two-tailed Mann-Whitney nonparametric comparison. An initial experiment conducted with 43 tags is also included in this analysis.

Niche-specific diversity over time demonstrates population dynamics throughout UTI. At the indicated times postinfection, urine was obtained via gentle suprapubic pressure and plated to enumerate CFU (A) and the fraction of tags remaining (E). A gentamicin protection assay was performed to enumerate luminal CFU (B) and the fraction of tags remaining (F) and gentamicin-protected CFU (C) and tags remaining (G). Kidneys were also plated to enumerate CFU (D) and tags remaining (H). The niche occupation of specific tags was determined and is represented as the average fraction of tags present in distinct and overlapping niches (15 total permutations) and displayed as a 4-set Venn diagram at 6 hpi (I), 24 hpi (J), 1 wpi (K), 2 wpi (L), and 4 wpi (M). Each percentage listed displays the fraction of tags present in that unique or shared niche out of the total number of tags in each murine urinary tract. Data for panels A to H represent 1 to 3 experiments, with 5 mice per time point. Data for panels I to M represent experiments where kidney information was available: 2 experiments with 4 or 5 mice for panel I and 1 experiment with 5 mice each for panels J to M. *, the niche combination with the greatest unique diversity present. Bars are median values. P values were calculated using a two-tailed Mann-Whitney nonparametric comparison.