Robert J. C. McLean, a. h Joseph A. Downey, h Ann L. Lablans, h. Janet M. Clark, h ...... Norln, L. J., Ekelund, P., Hedelin, H. & Johansson, S. L. (1988). Effects of.
International Biodeterioration & Biodegradation 30 (1992) 201-216
Modelling Biofilm-Associated Urinary Tract Infections in A n i m a l s
Robert J. C. McLean, a. h Joseph A. Downey, h Ann L. Lablans, h Janet M. Clark, h Anita J. Dumanski" & J. Curtis Nickel h "Department of Microbiology and Immunology,/'Department of Urology. Queen's University, Kingston, Ontario, Canada K7L 3N6
ABSTRACT Chronic infections in the urina~ environment include medical device related infections, chronic cystitis, chronic prostat#is, and infected struvite stones. Many d!fferent uropathogens are associated w#h these infections, but their common .feature is adhesion, .followed by growth as microcolonies and biofilms. In acute infections, the disease runs its course b£fore being cleared hy antibiotics or host de.fence mechanisms present at the tissue surface (e.g. the mucous laver and the immune response) and within the urine itself(e.g, low iron levels and high osmolali(v due to urea) and micturition. Chronic urinao, tract infections (UTI) arise when the natural host d£fences are defective or obstructed by the presence of a foreign object such as a catheter or calculus. In the~e cases, adherent pathogens are not cleared or eradicated. Rather th£v coat themselves with an extracellular polysaccharide matrix, and upon growth .form encapsulated microcolonies and biofilms. This mode of growth enables" the biofilm to be notoriously resistant to both the immune re,wonse of the host and to antibiotics. In order to accurately mimic these chronic human UTIs in animals, it is essential that the uropathogens are encouraged to grow in their natural state as biofilms. In this report, several models used to e~camine chronic hiofilm-associated infections am described.
INTRODUCTION
Urinary tract infections ( U T I ) o f the lower urinary tract are a c o m m o n p r o b l e m and can cause significant morbidity in females and males 201 hlternational Biodeterioration & Biodegradation 0964-8305/92/$05.00 © 1992 Elsevier Science Publishers Ltd. England. Printed in Great Britain.
202 R. J. C. McLean, J. A. Downey, A. L. Lablans, J. M. Clark, A. J. DumanskL J. C. Nickel
(Nickel. 1990). The causative organisms, which are most often gramnegative enteric bacteria, first colonize the introitus and the periurethral area before entering the bladder or prostate. There is an indigenous population of gram-positive, acid-producing, lactobacilli in this environment which under normal circumstances appears to enhance the urinary defence mechanisms and inhibit the successful progression of the enterics into the urethra and bladder (Chan et al., 1985: Reid et al., 1987. 1988. 1990a). When this ecological balance between enteric uropathogens and host defences is upset, uropathogens ascend the urethra into the bladder, prostate (in males) and kidneys (less often) where they colonize and cause infection. Most commonly, only the bladder is infected giving rise to simple uncomplicated acute cystitis. This condition is readily treated by several standard antibiotic regimes (Nickel, 1990). Problems arise when the infection ascends further up the urinary tract to the kidneys and induces pyelonephritis, or when it spreads into the prostate, or induces the formation of calculi. In these cases, significant tissue damage may occur, possibly causing permanent damage to renal function, which can be life-threatening. Numerous studies have shown the importance of uropathogen adhesion to the tissue surface as a necessary prerequisite for infection ( S a n d b e r g e t a l . , 1988: S t a m m e t a l . . 1989: Svanborg Ed6netal.. 1989. 1990: Nickel. 1990). As explained below, a major aspect of the host defence strategy in this environment is geared towards prevention and control of initial bacterial adhesion and subsequent growth. The major rationale behind this strategy is that once the organisms have established a 'beach head' on urinary tissue, they adopt a microcolony and biofilm mode ot" growth that enables them to develop chronic infections (cystitis, prostatitis) and persist in spite of host defences and often 'heroic" doses of antibiotics. Experimental manipulation of host defences, and appropriate selection ofuropathogens, is essential for the duplication of these various infections in animal models. This review outlines the host defence mechanisms of the lower urinary tract, and briefly summarizes several experimental strategies which can be used to manipulate these mechanisms to study the onset and progression of chronic and acute UTIs.
HOST D E F E N C E M E C H A N I S M S IN THE LOWER URINARY TRACT The bladder resists infection in part through inhibition of bacterial adhesion to its tissue surface (Kaye, 1975: Nickel, 1990: Svanborg Ed6n et al.. 1990). Bladder defence mechanisms include the periodic voiding of
Modelling biofilm-associated urinal' tract infections in anirnals
203
urine a n d washing out of u n a t t a c h e d pathogens (McLean et al., 1988). sloughing of pathogen-colonized uroepithelial cells (Orikasa & H i n m a n , 1977), the glycosaminoglycan (GAG) m u c o u s layer (Shrom et al., 1978: Parsons et al., 1980; Parsons, 1982, 1988: Cornish et al., 1987, 1988, 1990) a n d the cellular a n d h u m o r a l i m m u n e responses (Fukushi & Orikasa. 1981; Gillon et al., 1984; H o p k i n s et al., 1987; Cornish et al., 1988). The overall defence strategy of the b l a d d e r is to prevent adhesion a n d growth of i n c o m i n g pathogens to the epithelial tissue through the production of a m u c u s layer, secretory IgA a n d urine c o m p o n e n t s such as the T a m m Horsfall glycoprotein (Fukushi & Orikasa, 1981: Gillon et al., 1984: Cornish et al., 1987, 1988, 1990; H o p k i n s et al., 1987; Dulawa et al., 1988: P a r k k i n e n et al., 1988z H a w t h o r n & Reid, 1990: Reinhart et al., 1990: H a w t h o r n et al., 1991; Sobel. 1991). Bladder-adherent organisms are removed through the sloughing of colonized epithelial cells (Orikasa & H i n m a n . 1977) a n d cellular mediated i m m u n i t y (Fukushi & Orikasa. 1981: Gillon etal., 1984). I n c o m i n g pathogens must also compete with the urogenital Lactobacillus flora for nutrients a n d attachment sites (Chan et al., 1985; Reid et al., 1987. 1988, 1990). Inhibition of bacterial growth in urine is mediated through the low availability of iron (Shand et al., 1985) a n d osmotic stress caused by high concentrations of urea (Kaye. 1975). U n a t t a c h e d organisms unable to overcome these defences are invariably removed through voiding. Although the i m m u n e response is certainly important, there is evidence that IgG a n d sIgA production in themselves do not prevent recurrent cystitis (Rene et al., 1982). Other bladder defence m e c h a n i s m s are now being recognized which may contribute even more significantly to bladder defence, n a m e l y the protective m u c u s layer (Nickel, J. C., Cornish. J. & McLean, R. J. C.. u n p u b l i s h e d data) (Hurst et al., 1987: Cornish et al., 1988~ 1990: Holm-Bentzen & Ammitzboll. 1989). The mucus layer is a very thin cover on the transitional cell epithelium of the bladder. It is thought to act as an important m e c h a n i s m in shielding the bladder surface from pathogens, microcrystals, proteins and even carcinogenic molecules (Cornish et al., 1988, 1990: Parsons et al., 1988, 1990). In the studies by Parsons et al. the bladder mucus was removed or disrupted (often by administering acid to the tissue), and an increase in i n f l a m m a t i o n or infection was noted. In a sense, the degradation or deterioration of the G A G layer allowed the organisms to infect the bladder tissue cells. To date, however, there has been no confirmation of Parsons' data in the literature. Direct ultrastructural examination of these p h e n o m e n a were not done, so it is unclear whether i n f l a m m a t i o n is due to the m u c u s disruption treatment itself or whether it is due to the selective interaction between bacteria a n d bladder tissue in the localized
204 1~ J. C McLean, J. A. Downey, A. L. Lablans, J. M. Clark, A. J. Dumanski, J. C. Nickel
m i c r o e n v i r o n m e n t s that have been deprived of mucus. It is also unclear whether this layer becomes disrupted during the course of infection, or what effect n o r m a l a n d a b n o r m a l urine chemistry has on the G A G layer. This knowledge is essential before any potentially protective role can be unequivocally stated for the G A G layer. Techniques employed in past UTI studies related to bladder mucus (Cornish et al., 1988, 1990; Parsons et al., 1988, 1990) generated values that necessarily reflected average microbiological a n d biochemical conditions in the bladder. While these approaches are very useful and necessary, it has been the authors' experience that chemical and biological conditions in microenvironments, such as those immediately adjacent to uroepithelial cells, significantly differ from average values a factor that is extremely important in m a n y infectious processes (Nickel et al., 1985a, b,c, 1986: Costerton et al,, 1987: M c L e a n et al.. 1989, 1991; McLean & Costerton, 1990). Previous ultrastructural investigations of h u m a n bladder urothelium, processed a n d e x a m i n e d by conventional techniques, show little evidence of the mucus layer (Balish et al., 1982: Dixon et al.. 1986) due to dehydration artifacts. These problems can be overcome through the use of antibody stabilization tissue processing techniques that enable this fragile mucus layer to be visualized (Cornish et al., 1987. 1990). In this m a n n e r we are therefore able to investigate the integrity of the G A G layer a n d the bladder surface microenvironment from a n u m b e r of sites during experimental infection.
A C U T E A N D C H R O N I C CYSTITIS Several animal models of cystitis are reported in the literature. In the majority of cases, these involve ascending models of cystitis in various animals (mice, rats, dogs, cats a n d even n o n - h u m a n primates). In these cases the uropathogens are administered to the periurethral area in anaesthetized animals or even administered into the bladder through urethral catheterization. The animals are then allowed to recover a n d are then observed a n d tested for overt behavioural signs of urinary infection (lethargy, frequency or urination, painfial voiding, etc.), bacterial concentrations in the urine, antibody concentrations in the blood or urine, or u p o n sacrifice, gross morphological or histological changes to the various c o m p o n e n t s of the urinary tract. Ultrastructural observations of urinary tissue experimentally-infected with a pyelonephritogenic strain o f E s c h e r i c h i a coli show that u r o p a t h o g e n adhesion to tissues, even in acute infections, is quickly followed (within 24 h) by microcolony tbrmation (Hagberg et al., 1986). It has been the authors" experience that
Modelling biofilm-associated urina~ tract infections in animals
205
these experimental models of cystitis, m i m i c acute cystitis. The infectious dose required is generally very high (ca. 108-109 E. c o l i / m l in SpragueDawley rats) a n d the success rate of infection in test animals is in the order of 50-70%. In the absence of any other experimental m a n i p u l a t i o n , these infections are rapidly cleared usually within 2 or 3 days, a n d further complications such as prostatitis, pyelonephritis a n d calculus formation are extremely rare. M u c h higher rates of chronic bladder colonization can be obtained if the bladder e n v i r o n m e n t is experimentally altered. One such a p p r o a c h is to insert a foreign object such as a zinc disc (Satoh etal., 1984; Nickel et al., 1987, Olson et al., 1989) or p o l y u r e t h a n e sponge (Miller et aL, 1987) into the bladder - - a process which mimics foreign object i m p l a n t a t i o n leading to UTI. In these cases, the best experimental approach is to surgically i m p l a n t the sterile foreign object into the l u m e n of the bladder a n d to allow the animal (usually rats) to recover from surgery for several days before introducing the pathogens via urinary catheterization. This protocol of surgical sterile foreign object implantation--~ convalescence introduction of infection, is very successful in that the onset of experimental chronic UTI approaches 100%, yet the stress and discomfort on the test animals is m i n i m i z e d (Satoh et al., 1984; Nickel et al., 1987). In the authors" experience, chronic UTI associated with foreign object implants generally last for a m o n t h or more. The foreign object acts as an "immunologically inert' surface in that i n c o m i n g organisms have a place on which to colonize where they do not have to c o m b a t the host defence m e c h a n i s m s present in the m u c u s layer a n d urothelium. Colonization a n d biofilm formation occurs rapidly (
