J Med Microbiol 56 (2007), 1549-1557; DOI: 10.1099/jmm.0.47395-0
© 2007 Society for General Microbiology
ISSN 1473-5644
Species interactions in mixed-community crystalline biofilms on urinary catheters
Sarah M. Macleod and
David J. Stickler
Cardiff School of Biosciences, Cardiff University, Cardiff CF10 3TL, UK
Correspondence
David J. Stickler
stickler{at}cardiff.ac.uk
Received 15 May 2007
Accepted 4 July 2007
Previous experimental investigations of the crystalline biofilms that colonize and block urinary catheters have focussed on their formation by pure cultures of Proteus mirabilis. In the urine of patients undergoing long-term catheterization, P. mirabilis is commonly found in mixed communities with other urinary tract pathogens. Little is known about the effect that the other species have on the rate at which P. mirabilis encrusts catheters. In the present study, a set of data on the nature of the bacterial communities on 106 catheter biofilms has been analysed and it was found that while species such as Providencia stuartii and Klebsiella pneumoniae were commonly associated with P. mirabilis, when Escherichia coli, Morganella morganii or Enterobacter cloacae were present, P. mirabilis was rarely or never found. The hypothesis that the absence of P. mirabilis from some biofilm communities could be due to its active exclusion by other species has also been examined. Experiments in laboratory models showed that co-infection of P. mirabilis with M. morganii, K. pneumoniae or E. coli had no effect on the ability of P. mirabilis to encrust and block catheters. Co-infection with Ent. cloacae or Pseudomonas aeruginosa, however, significantly increased the time that catheters took to block (P <0.05). The growth of Ent. cloacae, M. morganii, K. pneumoniae or E. coli in the model for 72 h prior to superinfection with P. mirabilis significantly delayed catheter blockage. In the case of Ent. cloacae, for example, the mean time to blockage was extended from 28.7 h to 60.7 h (P 
0.01). In all cases, however, P. mirabilis was able to generate alkaline urine, colonize the biofilms, induce crystal formation and block the catheters. The results suggest that although there is a degree of antagonism between P. mirabilis and some of the other urinary tract organisms, the effects are temporary and whatever the pre-existing urinary microbiota, infection with P. mirabilis is thus likely to lead to catheter encrustation and blockage.
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INTRODUCTION
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The care of patients undergoing long-term indwelling bladder catheterization is frequently complicated by the encrustation and blockage of their catheters (Kohler-Ockmore & Feneley, 1996). The problem stems from infection by urease-producing bacteria, particularly Proteus mirabilis (Mobley & Warren, 1987; Kunin, 1989). These organisms colonize the catheters, forming extensive biofilm communities. They generate ammonia from urea, elevating the pH of the urine and biofilm. Under the resulting alkaline conditions, crystals of calcium and magnesium phosphates form in the urine and the biofilm. The continued development of the crystalline biofilms blocks the flow of urine from the bladder. In some cases, urine then leaks around the outside of the catheter and patients become incontinent. In others, urine retention causes painful distension of the bladder. Reflux of infected urine to the kidneys can then precipitate serious symptomatic episodes such as pyelonephritis, septicaemia and endotoxic shock (Kunin, 1997; Morris et al., 1999). All types of Foley catheter are vulnerable to this problem and there are no effective procedures available for its control (Morris et al., 1999).
Previous experimental work investigating the formation of these crystalline bacterial biofilms has concentrated on their development by pure cultures of P. mirabilis (Winters et al., 1995; Morris et al., 1997; Morris & Stickler, 1998). While these single-species biofilms do occur on catheters, in long-term catheterized patients the urine and catheter biofilms are more commonly contaminated by polymicrobial communities frequently containing P. mirabilis along with other uropathogens (Clayton et al., 1982; Warren et al., 1987; Ohkawa et al., 1990; Ganderton et al., 1992; Kohler-Ockmore & Feneley, 1996). Little is known about the interaction of the different species in these biofilms.
Over the years, a database of the composition of biofilms recovered from patients' catheters has been built up by successive workers in the catheter research laboratory of the Cardiff School of Biosciences, UK. A summary of this information is presented in Table 1
. Cases in which identical communities were isolated from a series of catheters from the same patient were only included once in the analysis. Single organisms were found on 30 of the catheters; the remaining 76 were colonized by two or more species. Pseudomonas aeruginosa, Enterococcus faecalis, Escherichia coli and P. mirabilis were clearly the most commonly found organisms. Similar results have been reported by other groups (Ohkawa et al., 1990; Matsukawa et al., 2005).
In the present study, we have examined the associations between organisms in the biofilm communities on these catheters. It became clear that some organisms, e.g. Providencia stuartii or Klebsiella pneumoniae, were commonly present with P. mirabilis in the catheter biofilm communities. However, when Enterobacter cloacae or Morganella morganii was colonizing catheters, P. mirabilis was rarely present in the biofilm. The hypothesis that the absence of P. mirabilis from some biofilm communities could be due to its active exclusion by other species was then examined experimentally in laboratory models.
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METHODS
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Isolation of bacteria from catheters.
Catheters from patients being cared for in hospitals and the community in South Wales and the Bristol area, UK, were collected over several years. Sections 1–2 cm and 3–4 cm from the catheter tip were cut and suspended in quarter-strength Ringer's solution (10 ml) in sterile universal containers. Sonication for 5 min at 35 kHz in a Transsonic water bath (Camlab) and by vortex mixing for 2 min was used to remove and disrupt the colonizing biofilms. The resulting cell suspensions were cultured onto CLED, Chromogenic UTI and tryptone soya agars (Oxoid). After 24 h incubation, the resulting colonies were identified by Gram-staining and the use of the appropriate BBL Crystal identification kits (Becton Dickinson). All isolates were kept as stock cultures in a 5 % (v/v) glycerol solution and stored at –80 °C in Nalge cryogenic vials.
Bacterial test strains.
The test organisms used in the bladder model experiments, P. mirabilis B2, Ent. cloacae RB19, M. morganii SM18, E. coli SM1, Ps. aeruginosa SM15 and K. pneumoniae SDM3, were selected from the collection of catheter biofilm isolates. Prior to use they were subcultured onto CLED agar.
The bladder model.
The bladder model has been described previously (Stickler et al., 1999). In essence, it consists of a glass chamber maintained at 37 °C by a water jacket. Each model was sterilized by autoclaving and then a size 14 all-silicone catheter (Bard) was inserted into the chamber through an outlet at the base. The catheter retention balloons were inflated with 10 ml sterile water and the catheters were connected to drainage bags in the normal way. Sterile artificial urine was pumped into the chambers so that residual volumes collected below the catheter eye-holes before flowing through the drainage tube to the collecting bags. The artificial urine supplied to the models was based on that devised by Griffith et al. (1976). It contained calcium chloride (0.49 g l–1), magnesium chloride hexahydrate (0.65 g l–1), sodium chloride (4.6 g l–1), di-sodium sulphate (2.3 g l–1), tri-sodium citrate dihydrate (0.65 g l–1), di-sodium oxalate (0.02 g l–1), potassium dihydrogen phosphate (2.8 g l–1), potassium chloride (1.6 g l–1), ammonium chloride (1.0 g l–1), urea (25 g l–1) and gelatin (5.0 g l–1). The pH of the medium was adjusted to 6.1 and then sterilized by membrane filtration using a Sartobran P capsule filter (Sartorious). Tryptone soya broth (Oxoid) was prepared separately, autoclaved and added to the sterile basal medium to a final concentration of 1.0 g l–1.
Experimental protocol.
Sets of models were assembled and supplied with artificial urine (15 ml) up to the level just below that of the catheter eye-holes. The urine supply was then switched off. Cultures of the test bacteria that had been grown for 4 h at 37 °C in artificial urine were used as the inocula (1 ml) for the residual urine in the bladder chambers. The models were left for 1 h to allow the organisms to establish themselves in the bladder. The urine supply (0.5 ml min–1) was then resumed for various set times or until the catheters blocked. The times taken for the catheters to block were recorded. The urinary pH and the numbers of viable cells in the urine voided from the model were measured at intervals up to the time of catheter blockage.
Determination of the calcium and magnesium content of catheter biofilms.
The degree of encrustation on catheters was measured by determining the amount of calcium and magnesium deposited within the crystalline bacterial biofilms on the luminal surfaces. At the end of each experimental period, catheters were removed from the models, the inflation balloons were dissected away from the catheters and the whole length of the catheter was cut into 1 cm sections. These sections were placed into 100 ml 4 % (v/v) nitric acid in double-deionized water and sonicated at 35 kHz for 5 min in the Transsonic water bath to aid biofilm removal and disruption. Samples were stood for at least 48 h to allow the crystals to dissolve. The calcium and magnesium contents of the resulting solutions were then assayed using flame atomic absorption spectroscopy in a SpectrAA-100 spectrophotometer (Varian) calibrated with Spectrosol standards obtained from VWR International. Samples were aspirated into the flame (air–acetylene for magnesium and nitrous oxide–acetylene for calcium). Calcium was measured at 422.7 nm and magnesium at 285.2 nm.
Enumeration of the viable cell populations in catheter biofilms.
Sections (1 cm in length) were cut from the region just below the eye-holes of catheters that had been removed from the bladder models. Each section was placed into quarter-strength Ringer's solution (10 ml) in a sterile universal container. They were then sonicated at 35 kHz for 5 min in the Transsonic water bath and vortex-mixed for 2 min in order to disrupt and suspend the luminal biofilms. Viable cell counts were performed on the resulting suspensions using appropriate selective differential agars and the results were expressed for each species as c.f.u. (cm catheter)–1.
Scanning electron microscopy of catheter sections.
At the conclusion of experiments, catheters were removed from the models and a number of sections (1 cm in length) were cut from various points down the catheters' length. In some cases, these sections were examined directly at low power using the low-vacuum facility of a JEOL 5200 scanning electron microscope. Other sections were perfusion-fixed in 2.5 % glutaraldehyde in 0.05 M sodium cacodylate buffer (overnight at room temperature). Catheter sections were then washed in the buffer for 15 min before post-fixation using a 1 : 1 solution of 0.05 M buffer and 1 % osmium tetroxide for 1 h. This was followed by two 5 min washes in distilled water before samples were dehydrated in an ascending ethanol series (15 min each of a 70 % and 90 % solution and two 15 min washes using 100 % ethanol). Sections were then critically point dried using a Balzers CPD 030 critical point dryer. The samples were gold sputter-coated (Edwards S150P sputter-coater) and visualized using a Philips XL-20 scanning electron microscope at an accelerating voltage of 20–25 kv.
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RESULTS AND DISCUSSION
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Bacterial flora of catheter biofilms
An analysis of the incidence of catheter colonization by P. mirabilis in the presence of the 10 other most common species is presented in Table 2. The species are ranked on the basis of their frequency of co-colonization with P. mirabilis. It can be seen that P. mirabilis was recovered from nearly half of the catheters colonized by Prov. stuartii. It was also commonly found in biofilms together with K. pneumoniae and Ent. faecalis. In contrast, P. mirabilis was recovered from only one of the 14 catheters colonized by M. morganii and none of the nine Ent. cloacae-containing biofilms.
Crystalline biofilm formation by mixed cultures of urinary pathogens
It is interesting to speculate on the nature of the apparent aversion of P. mirablis to exist in biofilms with some of these other urinary tract pathogens. Ent. cloacae, for example, might be antagonistic to P. mirabilis and exclude it from catheter biofilms. To test this possibility, experiments were performed in which pairs of organisms (P. mirabilis and another species) were introduced into bladder models simultaneously and the effect on the ability of P. mirabilis to survive and block the catheters with crystalline biofilm was observed. If bacterial factors could be identified which prevent crystalline biofilm formation, a biological interference strategy might be feasible to control a complication which undermines the health and quality of life for many elderly and disabled people.
Sets of three models were assembled in parallel. In the first series of experiments, one was inoculated with Ent. cloacae RB19, the second with P. mirabilis B2 and the third inoculated with the two organisms together. The models were supplied with urine at 0.5 ml min–1 and the experiment was run until both models containing P. mirabilis had blocked. The urinary pH and the numbers of viable cells ml–1 of each species present in the residual urine were determined at the time that the urine supply was started, at 24 h intervals and at catheter blockage. The experiments were performed in quadruplicate. In three replicates, the extent of catheter encrustation by calcium and magnesium salts was determined by chemical analysis. The rate of encrustation was then expressed as the amount of calcium and magnesium deposited per catheter h–1. Catheters from the fourth replicate were examined for crystalline biofilm by scanning electron microscopy.
The mean±SEM time that the catheters took to block in models infected with P. mirabilis was 18.7±0.1 h compared to 30.8±6.1 h in models inoculated with the pair of test organisms. The models inoculated with Ent. cloacae alone drained freely for the duration of the experiment. The results of the chemical estimation of the rates of deposition of crystalline biofilm on the catheters are presented in Fig. 1. There was no significant difference in the rate of catheter encrustation by P. mirabilis and the P. mirabilis and Ent. cloacae mixed population (P >0.05). The electron micrographs (Fig. 2) provided visual confirmation of these results. The numbers of viable cells in the residual urine in each model together with the urinary pH during the course of these experiments are shown in Table 3. It can be seen that in model 1, Ent. cloacae grew well over the experimental period with cell density approaching 108 c.f.u. ml–1 by the end of the experimental period. Ent. cloacae is not a urease producer and the pH of the urine remained at 6.2. As a result, there was very little deposition of calcium or magnesium salts on the catheters. In model 2, the population of P. mirabilis declined slightly over the experimental period but the pH rose to a mean value of 8.7 by the time of catheter blockage. When grown together in model 3, the population of Ent. cloacae declined to a mean value of just below 107 c.f.u. ml–1 while the population of P. mirabilis remained stable. The pH of the urine in model 3 rose to a mean value of 8.9 at catheter blockage.
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Fig. 1. Effect of co-inoculation of Ent. cloacae RB19 on the rate of catheter encrustation by P. mirabilis B2. Catheters were removed for analysis when they blocked in models inoculated with P. mirabilis and co-infected with both species. Catheters in models inoculated with Ent. cloacae drained freely for the course of the experiments. They were removed for analysis at the time that catheters in both the models containing P. mirabilis had blocked. The data presented are mean values calculated from three replicate experiments.
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Fig. 2. Low-vacuum scanning electron micrographs of catheters removed at blockage from models inoculated with (a) P. mirabilis, (b) P. mirabilis and Ent. cloacae and (c) Ent. cloacae at the time that the other two catheters had blocked.
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Table 3. Growth of Ent. cloacae RB19 and P. mirabilis B2 in bladder models and their effects on urinary pH
The values quoted are means±SEM calculated from four replicate experiments.
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These experiments were repeated with P. mirabilis and each of the other four test species. A summary of the results showing the times that catheters took to block in these experiments is presented in Table 4. Only co-inoculation with Ent. cloacae or Ps. aeruginosa significantly increased the time that P. mirabilis took to block catheters (P <0.05). The effect of co-inoculation with the other four species on the rates of catheter encrustation was very similar to that demonstrated in Fig. 1
. In all cases, there was no significant difference between the rates of deposition of calcium and magnesium in the P. mirabilis controls and the models inoculated with each of the pairs of organisms (P >0.05). The rates of encrustation on catheters inoculated with the five non-Proteus test organisms were all negligible. The Ent. cloacae and E. coli were both urease-negative organisms and were not expected to generate alkaline conditions and encrustation. Whilst M. morganella SM18, Ps. aeruginosa SM15 and K. pneumoniae SDM3 all registered as urease-positive in standard laboratory identification tests, the mean rates of calcium and magnesium accumulation they produced on catheters were only 3.8, 7.9 and 1.7 µg per catheter h–1, respectively. In comparison, accumulation rates of >400 µg per catheter h–1 were recorded for P. mirabilis. The viable cell counts of all the test species decreased significantly on co-incubation with P. mirabilis in a similar fashion to that illustrated in Table 3 (P <0.05). The effect on the urinary pH was similar with each pair of organisms. As with Ent. cloacae (Table 3), each of the other four test species growing alone maintained acidic urine from start to finish. In contrast, the urinary pH rose well above 8 in all the P. mirabilis control and mixed cultures. There were no statistically significant differences between the mean pH values recorded in each mixed culture and the corresponding P. mirabilis control (P >0.05).
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Table 4. Summary of the mean times to catheter blockage from the simultaneous co-inoculation of bladder models with pairs of organisms
The mean values±SEM for times (h) to catheter blockage were calculated from four replicate experiments.
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The scanning electron micrographs shown in Fig. 2 confirm that catheters removed from models infected with Ent. cloacae were free of crystalline material. Similar micrographs were obtained from mono-cultures of the other four test organisms. In contrast, extensive encrustation was visible around the eye-holes and throughout the lumen of catheters from all models infected with P. mirabilis and mixed-community cultures containing P. mirabilis (Fig. 2).
These experiments suggest that when the catheterized urinary tract is infected by approximately equal numbers of P. mirabilis and another urinary tract pathogen, the P. mirabilis will out-compete the other organism. The urine will become strongly alkaline and extensive catheter encrustation and blockage will follow.
Ability of P. mirabilis to encrust catheters colonized by mature biofilms of other species
In another set of experiments to investigate whether the five test organisms could modulate the rate of catheter encrustation and blockage, the test organisms were allowed to establish biofilms on catheters in the models for 72 h before P. mirabilis was introduced. In these experiments, sets of four models were assembled in parallel. The test species was inoculated as usual into models 1–3. After 72 h incubation, the catheter from model 1 was removed for examination. The urine supply to model 3 was disconnected and the bladder chamber was inoculated with P. mirabilis B2 (1 ml of a 4 h urine culture). At the same time, model 4 was also inoculated with the P. mirabilis culture. Models 2–4 were supplied with urine until the catheters in both of the models inoculated with P. mirabilis had blocked. The urinary pH and the numbers of viable cells ml–1 of each species present in the urine were determined every 24 h until the end of the experiment. These experiments were performed in quadruplicate for each pair of species and the times that catheters took to block were recorded. The extent of the encrustation on the catheters was also visualized by scanning electron microscopy.
The times that catheters took to block in models infected with the five test species and then superinfected with P. mirabilis at 72 h are summarized in Table 5. It was clear that the catheters in models infected with mono-cultures of P. mirabilis blocked significantly more rapidly than those in models in which P. mirabilis was introduced 72 h after the test organisms, except in the case of Ps. aeruginosa. It was clear, however, that P. mirabilis was still capable of producing extensive encrustation under these conditions (Fig. 3).
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Table 5. Ability of P. mirabilis to encrust and block catheters colonized by 72 h biofilms of other species
The mean values±SEM were calculated from the results of four replicate experiments. The P values indicate the significance level of the differences between the mean times to blockage.
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Fig. 3. Low-vacuum scanning electron micrographs showing the eyelets and central channels of catheters removed at blockage from (a) a control model inoculated with P. mirabilis and from models where (b) Ent. cloacae, (c) M. morganii, (d) E. coli, (e) Ps. aeruginosa and (f) K. pneumoniae were allowed to form biofilms for 72 h before superinfection with P. mirabilis.
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The results of the viable cell counts and urinary pH values in the various models were similar for each pair of species. As an example, the results from models infected with Ent. cloacae and P. mirabilis are summarized in Table 6. It can be seen that while P. mirabilis grew well in both the control and the superinfection models, the mean numbers of Ent. cloacae in the urine decreased significantly (P <0.05) on the introduction of P. mirabilis. The pH of the urine in the mixed cultures was significantly lower than in the control P. mirabilis culture throughout the test period (P <0.05).
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Table 6. Effect of superinfection with P. mirabilis B2 on viable cell populations and pH of the residual urine in models that had been infected with Ent. cloacae RB19 for 72 h
The mean values±SEM were calculated from the data from four replicate experiments.
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A further series of experiments examined the ability of P. mirabilis to colonize the 72 h biofilms of the other urinary pathogens. Sets of four models were assembled as above. Catheters were removed from the models at various times and the numbers of viable cells of the various species were enumerated in the biofilms on sections (1 cm in length) taken from immediately below the eye-hole. The results are summarized in Table 7. The micrographs presented in Fig. 4 illustrate the extent of the biofilms formed by the five test species after 72 h incubation. Unlike the P. mirabilis biofilm, there is no evidence of crystal formation in these biofilms. Even on incubation until the times at which catheters inoculated with P. mirabilis blocked, there was little evidence of encrustation on these catheters (Fig. 5). In contrast, the micrographs of catheters removed from models containing P. mirabilis all showed extensive accumulation of crystalline material around the eye-holes and in the lumen (Fig. 3).
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Table 7. Ability of P. mirabilis to colonize biofilms of other urinary pathogens
The mean number of viable cells recovered from the catheter biofilms, expressed as log10 [c.f.u. (cm catheter)–1±SEM], was calculated from three replicate experiments.
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Fig. 4. Scanning electron micrographs showing the extent of biofilm formation around the eyelets and in the central channels of catheters removed at 72 h from models infected with (a) Ent. cloacae, (b) M. morganii, (c) E. coli, (d) Ps. aeruginosa and (e) K. pneumoniae.
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Fig. 5. Scanning electron micrographs showing the extent of biofilm formation on catheters removed from control models that had been incubated with mono-cultures of (a) Ent. cloacae, (b) M. morganii, (c) E. coli, (d) Ps. aeruginosa and (e) K. pneumoniae at the end of each experiment upon blockage of both P. mirabilis-containing models.
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The results of the experimental work presented in this study indicate that any antagonism against P. mirabilis by the other five urinary tract pathogens is minor and temporary. The most pronounced effects were seen between P. mirabilis and Ent. cloacae. While the inoculation of the residual urine in the models with Ent. cloacae at the same time as P. mirabilis significantly delayed catheter blockage, P. mirabilis was still capable of elevating the urinary pH to levels at which crystalline biofilm formed on the catheters. These results are in contrast to those of Anderson et al. (1979), who concluded that the survival of species in mixed populations was a feature of the relative bacterial growth rates. In experiments in a model of the normal bladder supplied with pooled human urine, they demonstrated that mixed cultures of the fast-growing E. coli with a slower grower such as P. mirabilis became nearly pure cultures of E. coli within 24 h. The presence of the catheter would thus seem to have a major effect on the ability of organisms to survive in the residual urine.
When other species have established themselves in the models and formed biofilms on the catheters, most were capable of slowing catheter encrustation by P. mirabilis. These effects, however, were again transient and in all cases P. mirabilis was able to raise the urinary pH, colonize the biofilms of the other species, induce crystal formation and block the catheters.
In conclusion, the results of the experimental work indicate that the presence of species such as Ent. cloacae or M. morganii in a catheterized urinary tract is not likely to prevent subsequent infection by P. mirabilis. The absence of P. mirabilis from catheter biofilms containing these species is thus probably because the patients concerned have just not been exposed to P. mirabilis. It also suggests that Ent. cloacae and M. morganii have different sources from P. mirabilis. Infection with P. mirabilis whatever the pre-existing urinary flora is thus likely to lead to catheter encrustation and blockage.
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REFERENCES
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TOP
INTRODUCTION
METHODS
