Persistence of uropathogenic Escherichia coli in the face of multiple antibiotics

Abstract

Numerous antibiotics have proven to be effective at ameliorating the clinical symptoms of urinary tract infections (UTIs), but recurrent and chronic infections continue to plague many individuals. Most UTIs are caused by strains of uropathogenic Escherichia coli (UPEC), which can form both extra- and intracellular biofilm-like communities within the bladder. UPEC also persist inside host urothelial cells in a more quiescent state, sequestered within late endosomal compartments. Here, we tested a panel of 17 different antibiotics, representing seven distinct functional classes, for their effects on the survival of the reference UPEC isolate UTI89 within both biofilms and host bladder urothelial cells. All but one of the tested antibiotics prevented UTI89 growth in broth culture, and most were at least modestly effective against bacteria present within in vitro-grown biofilms. In contrast, only a few of the antibiotics, including nitrofurantoin and the fluoroquinolones ciprofloxacin and sparfloxacin, were able to eliminate intracellular bacteria in bladder cell culture-based assays. However, in a mouse UTI model system in which these antibiotics reached concentrations in the urine specimens that far exceeded minimal inhibitory doses, UPEC reservoirs in bladder tissues were not effectively eradicated. We conclude that the persistence of UPEC within the bladder, regardless of antibiotic treatments, is likely facilitated by a combination of biofilm formation, entry of UPEC into a quiescent or semiquiescent state within host cells, and the stalwart permeability barrier function associated with the bladder urothelium.

FIG. 1.

Antibiotic effects on intracellular UPEC and host cell cytotoxicity. (A) 5637 bladder epithelial cell monolayers infected with UTI89 were incubated for 14 h in media containing 10 μg/ml gentamicin to prevent extracellular bacterial growth and to allow time for the establishment of UPEC within the host bladder cells. Following washes with PBS²⁺, infected monolayers were incubated for an additional 12 h with two concentrations (μg/ml) of each of the different antibiotics being tested, as indicated. Surviving bacterial titers are presented relative to reference samples that were treated with 800 μg/ml gentamicin. Data represent the mean results ± SEM from three or more independent assays performed in triplicate. (B) Cytotoxic effects of each antibiotic (used at the higher of the two concentrations indicated in panel A) on 5637 bladder cells were determined after 12 h treatments by measuring LDH release. Cells treated with the pore-forming glycoside saponin were used as positive controls. Results indicate the means ± SEM from three independent experiments performed in duplicate or triplicate.

FIG. 2.

Antibiotic effects on UPEC biofilms grown in vitro at 37°C in M9 medium. Biofilm levels were quantified relative to untreated controls following 24-h treatments with the indicated antibiotics, which were used at the higher of the two concentrations specified in Fig. 1A. Data represent the mean results ± SEM from three independent experiments performed in triplicate.

FIG. 3.

Antibiotic susceptibility of UPEC within mouse bladders. (A) Starting at day 3 postinoculation with UTI89, infected CBA/J mice were treated for 3 consecutive days with the indicated antibiotics. Following an additional 3-day period, surviving bacterial titers present within the bladders were determined. The graph depicts the cumulative results from two independent assays (n = 11). (B) The graph shows bacterial titers in aliquots of tissue homogenates from mouse bladders (n = 9) recovered at 3 days postinoculation and subsequently treated for 4 h with the indicated antibiotics. Bars indicate median values for each group, while the solid lines denote the LOQ. P values of <0.05 (A) or <0.001 (B), relative to the water-treated controls, were determined using Mann-Whitney U tests and are labeled with asterisks. Sparfloxacin-fosfomycin, SPX/FOF.

FIG. 4.

Antibiotic susceptibility of IBCs. At 6 h postinoculation with UTI89/pGEN-GFP(LVA), bladders obtained from CBA/J mice were collected, splayed, and incubated in the presence or absence of the antibiotic combination sparfloxacin-fosfomycin. (A) Using fluorescence microscopy, IBCs were enumerated at the indicated times after addition of the antibiotics. Bars denote median values. P values were calculated using the Mann-Whitney U test (n = 8 mice per group). (B) Representative images showing IBCs in control and sparfloxacin-fosfomycin-treated bladder explants at the 1- and 18-h time points. Scale bar = 100 μm.