Intracellular bacteria are known to occur in up to 80% of women with urge incontinence [1]. These recent studies used Wright staining and immunofluorescence to show that select uropathogens are capable of invading the urothelial lining of the bladder, thus avoiding antibiotic treatment and expulsion by micturition [1]. In response to this invasion, the bladder sheds the infected cells in an attempt to rid itself of the bacterium. Our study aimed to determine whether a prolonged rotating course of antibiotics could diminish the internalized bacterium, and reduce the degree to which they appeared intracellular (on Wright stain) in the shed cells over time.

Women over 50 years of age with urge incontinence were recruited to a double-blind, placebo controlled, randomized trial. Women recruited to the trial had urodynamically proven DO that was refractory to treatment. Exclusion criteria were kidney or voiding dysfunction. Women underwent a 2.5 week washout to establish a baseline (0 weeks), then were randomised into 6 weeks of antibiotic (2 weeks each of Norfloxacin, Augmentin Duo and Nitrofurantoin) or placebo (2:1 ratio).  
Midstream urine (MSU) samples were collected at baseline (0 weeks), then at weeks 2, 4, and 6 (during antibiotic/ placebo treatment). Half of the MSU sample was sent to the Hospital Microbiology department for culture. A diagnosis of UTI was determined by the presence of a single bacterial species (>106 CFU/L). The remaining urine was centrifuged to concentrate exfoliated urothelial cells onto microscope slides and Wright stained.
Approximately 100 cells were counted by light microscopy and categorised according to the presence of bacteria, the location (attached to the cell membrane or appeared intracellular) and the bacterial density (low density (LD) or high density (HD)). The mean percentage (±SEM) of each of these categories was calculated. 
Interim analysis is now occurring to check for futility before continuing the study, this has enabled our scientist access to the treatment codes. T-tests were used to determine differences between antibiotic and placebo treatment groups.

Thus far 36 women have been recruited for the study and randomized into antibiotic (n=24) and placebo (n=12). In the women treated with antibiotics, the bacterial culture results show that UTIs are reduced during week 4 (following two weeks of treatment with Augmentin Duo) (Figure 1A), with an increase in the number of women with no growth on microbiology analysis. A similar result was not seen in the women taking the placebo tablets (Figure 1B).  

In terms of the microscopic analysis of the Wright stained urothelial cells, most cells were seen to be either free of any bacterial or have low densities of bacteria that were observed to appear intracellular. In the antibiotic group, there was a significant increase in the number of cells which are free of any bacteria during Week 4 of the trial, ie following treatment with Augmentin duo (Figure 2A, P 0.028). Also, there is a decrease in the percentage of cells that appear to contain a low density of intracellular bacteria (Figure 2B, P 0.061).

In this study, Augmentin Duo was the most effective of the three antibiotics in reducing the incidence of classical UTI in refractory DO. At the same time point there was also an increase in the percentage of cells that appear to be free of any bacteria on Wright staining. It has been suggested that intracellular bacteria may represent a reservoir for recurrent infections [2]. 
Of the three antibiotics used in this study, β-Lactam group antibiotics (such as Augmentin) have been reported by others to have the greatest intracellular action [3] and to increase in efficacy the longer the treatment period [3].

Treatment of women with refractory DO with antibiotics that are able to accumulate within the urothelium (such as β-Lactam group) may significantly decrease the presence of intracellular bacteria. This would subsequently decrease the likelyhood of an intracellular reservoir for recurrent infections (and presumably decrease the incidence of clinically significant bacteriuria).

Cheng Y, Chen Z, Gawthorne JA, Mukerjee C, Varettas K, Mansfield KJ, et al. (2016). Detection of intracellular bacteria in exfoliated urothelial cells from women with urge incontinence. Pathog Dis, 74(7). DOI: 10.1093/femspd/ftw1067.
Justice SS, Hung C, Theriot JA, Fletcher DA, Anderson GG, Footer MJ, and Hultgren SJ (2004). Differentiation and developmental pathways of uropathogenic Escherichia coli in urinary tract pathogenesis. Proc. Natl. Acad. Sci. U. S. A. 101:1333-1338.
Carryn S, Chanteux H, Searl C, Mingeot-Leclercq MP, Van Bambeke F, and Tulkens PM (2003). Intracellular pharmacodynamics of antibiotics. Infect Dis Clin N Am. 3:615-634