Spectrum of bacterial colonization associated with urothelial cells from patients with chronic lower urinary tract symptoms

Abstract

Chronic lower urinary tract symptoms (LUTS), such as urgency and incontinence, are common, especially among the elderly, but their etiology is often obscure. Recent studies of acute urinary tract infections implicated invasion by Escherichia coli into the cytoplasm of urothelial cells, with persistence of long-term bacterial reservoirs, but the role of infection in chronic LUTS is unknown. We conducted a large prospective study with eligible patients with LUTS and controls over a 3-year period, comparing routine urine cultures of planktonic bacteria with cultures of shed urothelial cells concentrated in centrifuged urinary sediments. This comparison revealed large numbers of bacteria undetected by routine cultures. Next, we typed the bacterial species cultured from patient and control sediments under both aerobic and anaerobic conditions, and we found that the two groups had complex but significantly distinct profiles of bacteria associated with their shed bladder epithelial cells. Strikingly, E. coli, the organism most responsible for acute urinary tract infections, was not the only or even the main offending pathogen in this more-chronic condition. Antibiotic protection assays with shed patient cells and in vitro infection studies using patient-derived strains in cell culture suggested that LUTS-associated bacteria are within or extremely closely associated with shed epithelial cells, which explains how routine cultures might fail to detect them. These data have strong implications for the need to rethink our common diagnoses and treatments of chronic urinary tract symptoms.

Fig 1

Venn diagrams showing the symptom spectrum for the first female patient group, which provided catheter specimens (A), and for the patient group providing midstream urine specimens (B).

Fig 2

Graphs showing bacterial isolates from catheter specimens from patients and control volunteers. (A) Dominant isolates as percentages of all isolates. (B) Full spectrum of all isolates cultured as percentages of all isolates identified.

Fig 3

Graph showing the relationship between routine culture results (with a threshold of 10⁵ CFU ml⁻¹) and colony counts for cultures of urinary sediments in a cohort of midstream urine (MSU) specimens from patients with lower urinary tract symptoms. Positive routine culture results were associated with higher bacterial counts.

Fig 4

Graph showing the relationship between bacterial counts (CFU ml⁻¹) and microscopic pyuria (>0 WBCs/ml) in the patient cohort for both MSU and CSU samples.

Fig 5

Graphs showing bacterial isolates from midstream urine (MSU) specimens from patients and control volunteers. (A) Dominant isolates as percentages of all isolates. (B) Full spectrum of all isolates cultured as percentages of all isolates identified.

Fig 6

Total bacterial counts at different stages of the antibiotic protection assay for LUTS patients and control volunteers, using the antibiotics gentamicin, amoxicillin, and linezolid at concentrations of 200 μg/ml. Start, total bacterial counts (CFU ml⁻¹) at the beginning of the assay; 24-hours, total bacterial counts after 24 h of incubation; Post-antibiotic, total bacterial counts after incubation with antibiotics plus three washes with saline; Post-lysis, total bacterial counts after the addition of Triton X-100.

Fig 7

(A to E) Graphs showing mean bacterial counts (CFU ml⁻¹) and standard deviations at different stages of the cell line invasion assay for five patient-derived uropathogens (A to D) and one control commensal strain (E). (F) Graph comparing mean bacterial counts (CFU ml⁻¹), with the standard error of the mean (SEM), for pooled uropathogens and the commensal strain. *, statistically significant difference of the means. Start, total bacterial counts at the beginning of the assay; 24 hours, total bacterial counts after 24 h of incubation with the cell line; Pre gent, total bacterial counts after washing with saline and prior to the addition of gentamicin (200 μl/ml); Post gent, total bacterial counts after incubation with gentamicin (200 μg/ml) overnight; Wash, total bacterial counts after washing with saline after incubation with gentamicin; Lysis, total bacterial counts after cell lysis with Triton X-100.