The resistance of urinary tract pathogens to chlorhexidine bladder washouts

Journal

of Hospital

Izzfectzon (1987)

10, 28-39

The resistance of urinary tract pathogens chlorhexidine bladder washouts D. J. Stickler*, “Department Technology,

C. L. Clayton*

to

and J. C. Chawlat

of Applied Biologqr, University of Wales Institute of Science and CardifJ, UK and TThe Spinal-Ir$uies Unit, Department of Rehabilitation, Rookwood Hospital, Cardiff, UK

Accepted for publication

11 August

1986

Summary:

Isolates of Procidezzcza stuartii, Pseudomonas aevuginosa, Proteus nzzrabilzs, Klebsiella pneumoniae and Streptococcus faecalis from urmary-tract infections in spinally-mjured patients together with Eschevichia coli 10418 \vere challenged with chlorhexrdine (200 mg 1-l) in a model of a cathetertzed bladder under conditrons whrch simulate the bladder washout technique. All species surrrved the antiseptic. Organisms growing on the wall of the bladder model appeared to be particularly resistant and electron microscopy showed that these cells were embedded m a protective glycocalyx. The effect of chlorhexrdine bladder washouts on the bacterial flora in the urme of patients \vas also observed and shown to be mimmal and temporary. Examination of urmary sediments from patients revealed the presence of micro-colonies of bacteria embedded in a polysaccharrde matrix. \Ve conclude that bladder washouts with chlorhexidine are not likely to eliminate established infections \vith orgamsms that occur in patients with indxvelling bladder catheters.

Introduction Bacterial resistance to chlorhexidine has been reported in Proteus mi~abilis (Gillespie et al., 1967; Stickler, 1974), Pseudomonas spp. (Bentley et al., 1968), Pseudomonas aeruginosa (Stickler & Thomas, 1980; Nakahara & Kozukue, 1982), Serratia marcescens (Marrie & Costerton, 1981) and Prozlidencia stuartii (Stickler & Thomas, 1976; McHale et al., 1981). In these studies organisms have been designated as resistant when tests in nutrient agar or broth have shown them to have MICs of chlorhexidine approximately loo-fold greater than the values originally quoted for Gram-negative bacilli (Davies et al., 1954). MIC determinations to an antibacterial agent are influenced by variations in the size of inoculum and in the nature of the growth medium and merely test the ability of an agent to inhibit bacterial growth in a nutrient medium over a 24 h period. In practice, the antiseptic is required to be bactericidal within a short period of time under a variety of environmental conditions. When chlorhexidine is incorporated into urine drainage bags for example (Blandy, 1970; Southampton Infection Control Team, 1982), the antiseptic has to be 019556701/87/040028+

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Chlorhexidine

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bactericidal within minutes against urine grown organisms in the presence of the urine growth medium. To test whether chlorhexidine resistance registered in MIC tests was manifested under these clinical circumstances, urine grown cultures of Thomas, Sykes & Stickler (1978), challenged Gram-negative bacilli with the antiseptic at a concentration achieved in the drainage bag prior to complete filling (500 mg l-i), and found that strains with high MIC values consistently retained their viability in urine. such as Washing the bladder with instillations of antiseptics chlorhexidine has been advocated as a method of controlling urinary-tract infections in patients with indwelling catheters (Blandy, 1970; Tyrell et al., 1979). While substantial reductions in the urinary flora and control of infections may sometimes be achieved (Garrod, Lambert & O’Grady, 1981), the rationale for the use of antiseptics in this way is not well-established. Recently, Slade & Gillespie (1985) commented that in their experience chlorhexidine washouts generally failed to eliminate infections and they concluded that the value of antiseptic washouts in long term catheterization should be further assessed in clinical trials before it is adopted routinely. The present study was performed to establish whether NIIC determinations are likely to provide an insight into the success of chlorhexidine bladder washouts and to observe whether chlorhexidine resistance is manifested under these circumstances. A simple physical model of the catheterized bladder has been devised. Bacteria of known nlIC of chlorhesidine were grown in urine in the model and challenged with the antiseptic. Methods

MIC

determinations

Strains were grown for 2-Ch at 37°C in broth (Oxoid Nutrient No. 2), and 5 ill of lo-’ dilutions of these cultures (c. lo3 viable cells) were dropped onto the surface of nutrient agar (Oxoid) plates containing doubling dilutions of chlorhexidine. The plates were examined for growth after overnight incubation at 37°C. The MIC was defined as the lowest concentration of antiseptic preventing colony formation. Tests were performed in duplicate and E. co15 10418 and P. mirabilis 61 (Stickler, 1974) were included as chlorhexidine sensitive and resistant control organisms respectively. The bladder

model

The catheterized bladder represents a continuous culture system. Urine flows into the bladder from the ureters and a reservoir of urine is maintained in the bladder by the catheter. In designing a model of this system it was assumed that the average residual volume of urine in the bladder of the catheterized patient was 20 ml. The mean output of urine from our spinally injured patients over 24 h was determined to be 2 ml min-‘, this figure resulting from their high fluid intake. A glass model was set up at half-scale

30

D. J. Stickler

et al.

with a 10 ml residual volume of urine, a 1 ml min- ’ urine dilution rate and a capacity of 90 ml. The bladder was represented by a small fermentation flask which was maintained at 37°C by means of a water jacket. The residual volume of urine was kept at 10 ml by a catheter drainage system connected to a vacuum pump via a Buchner flask (2 1) which represented the urine drainage bag. The urine in the bladder was mixed gently by a magnetic bar. An aspirator bottle (2 1) represented the kidney and this provided sterile pooled urine which was pumped into the bladder flask at 1 ml min-’ by a peristaltic pump (Schuco Ltd). Electron microscopy Bacteria from infected urines, cultures that had been grown in urine in the laboratory and cells resuspended from wall growth in the bladder model were examined for the presence of polysaccharide glycocalyxes by a modification of the ruthenium red staining method of Marrie et al. (1979). Cells were harvested by centrifugation (3000 g for 10 min) and fixed for 1 h at room temperature in a mixture containing 3.6% v/v glutaraldehyde (0.5 ml), 0.2 M cacodylate buffer pH 7.3 (0.5 ml) and 10 mg ml-’ ruthenium red (0.5 ml). Cacodylate buffer (0.15 M) was then used to wash the cells three times. The cells were then suspended in a solution containing O-5 ml of ruthenium red (10 mg ml-‘), 0.5 ml of 5% w/v osmium tetroxide in distilled water and 0.5 ml of the O-2 M cacodylate buffer and stained at room temperature for 3 h. The stained bacteria were then rinsed in the 0.15 M cacodylate buffer, dehydrated through a graded alcohol series and embedded in araldite epoxyresin. Sections of these preparations were mounted on copper grids and stained with one drop of uranyl acetate (20% w/v uranyl acetate in methanol) and one drop of lead citrate (04% w/v Pb (NO,),, 0.6% w/v sodium citrate in distilled water) for 10 min. Specimens were then examined in a Phillips 300 transmission electron microscope. Bladder washout technique The method used was essentially that described by Fairley et al. (1971). The catheter was clamped and a urine specimen was collected by aspiration from the catheter. The bladder was then emptied by applying pressure to the abdomen. The antibacterial solution (100 ml of chlorhexidine 200 mg 1-l) was introduced into the bladder through the catheter and retained in the bladder for 30 min by clamping the catheter. The bladder was then drained and a sterile Y-connector was inserted into the catheter and connected to a paediatric drip apparatus and to the drainage bag. The bladder was then washed out via the drip apparatus with 100-150 ml of normal saline by intermittent clamping and releasing of the catheter. The bladder was washed with a total of 1 1 of saline in this way and a specimen of the last washout was kept for culture. Urine samples were then collected at intervals up to 50min. Viable bacterial cell counts were then performed on each sample.

Chlorhexidine

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Results

The bactericidal effect of chlorhexidine on organisms grown in urine in the bladder model The growth characteristics of the test organisms were first examined in the model. It was found that when 0.1 ml samples of a log phase culture of bacteria growing in urine (1 x 10’ cells ml-‘) were inoculated into 10 ml of warm sterile urine and incubated with stirring for 2 h before switching on the supply of fresh urine, the strains used in this work readhed a steady state population (approx. 10’ cells ml-‘) after a further 9 h incubation. The bladder washout technique used in the spinal injury unit to clear debris from the catheter and bladder, and to treat bladder infections,

6-O

2.0

(d) I

I

6.0

I 200 Time

(min)

after

bladder

I 400

(f)

instillation

Figure 1. The effect of chlorhexidine washouts on urine cultures of urinary in a model of a catheterized bladder. (a) Escherichia coli, (b) Klebsiella Providencia stuartii, (d) Pseudomonas aeruginosa, (e) Proteus mirabilis, faecalis. 0, Saline; 0 chlorhexidine 200 pg ml-‘.

pathogens grown pneumoniae, (c) (f) Streptococcus

32

D. J. Stickler

et al.

involved injection of 100 ml of the chlorhexidine solution (200 mg 1-l) into the bladder through the catheter. The antiseptic is retained in the bladder by clamping the catheter for 30 min. This procedure was simulated in the model by growing bacteria in continuous culture for 14 h, i.e., until a steady population was well established. The antiseptic solution (50 ml) was then injected through the catheter into the bladder, and the catheter clamped for 30 min while the model filled with urine from the reservoir, causing all surfaces of the incubation chamber to be bathed in the antiseptic urine mixture. The clamp was then removed, the vacuum reapplied and the urine level in the model returned to its residual volume (10 ml). Incubation was continued for a further 6 h. Isolates of Procidencia stuartii, Pseudomonas aeruginosa, Proteus mirabilis, K. pneumoniae and S. faecalis from urinary-tract infections in spinally-injured patients and E. coli 10418 were challenged with the chlorhexidine washout. Samples were removed from the urine culture prior to instillation of the antiseptic and at various time intervals for a further 6 h after the instillation period. Viable cell counts were performed on CLED Agar (Oxoid) after an initial dilution in neutralizer broth (Tween 80, 3% v/v in peptone-saline 1% w/v). The effect of a control washout with sterile saline was also observed with each organism. In all cases, a visible film of bacteria developed on the wall of the glass vessel; this was resuspended in the urine, 405 min after the end of the instillation period and further viable cell counts performed on the resulting suspensions. These experiments were all performed in duplicate and the means are reported in Figure 1. The reductions in the viable counts produced by the chlorhexidine were calculated and are presented together with the MIC of chlorhexidine for each organism in Table I. Table

I.

The bactericidal

effect of chlorhexzdine the bladder model

on bacteria

grown

in urine in

*Log,,, reduction in viable bacteria/ml urine after 30 min exposure to bladder instillation of TOrganism

MIC of chlorhexidine 6-w 1-Y

Escherzchia coli 10418 Streptococcus faecalis Klebsiella pneumoniae Pseudomonas aeruginosa Providencia stuartii Proteus mirabilis *Mean of TWO separate experiments. TApart from the E. cok all the strains infectmns in catheterized patients.

4 10 40 80 640 1280 used were

saline

chlorhexidine (200 mg I-‘)

0.30 0.22 0.15 0.16 0.24 0.08 clm~cal

isolates

2.99 7.18 7.02 3.27 3.96 7.77 from

urinary-tract

Chlorhexidine

and urinary

-

tract pathogens -

Figure 2. Electron micrographs of ruthenium red stained cells of Escherichiu coli from (a) resuspended bladder model wall growth and (b) static urine culture. The arrows indicate the presence of a fibrous extra-cellular matrix. The bars represent 1 pm.

34

D. J. Stickler

et al.

The increase in viable cell count that occurred when the wall growth was resuspended in the urine (Figure 1) suggests that this material is the source of the inoculum for the regrowth of the urinary organisms after exposure to the antiseptic. This possibility was examined in experiments in which E. coli 10418 was grown in continuous culture for 14 h in one model and then transferred to a second, sterile pre-warmed model and immediately challenged with chlorhexidine (200 mg I-‘). Under these conditions, where there was no opportunity for wall growth to occur in the test vessel, sterilization of the bladder occurred and there was no evidence of regrowth after 6 h further incubation. The possibility that bacteria attached to the model wall were enclosed within a protective glycocalyx (Costerton, Irvin & Cheng, 1981) was examined by electron microscopy on ruthenium red stained E. coli 10418 resuspended from the wall growth and from static batch culture in urine. The fibrous matrix characteristic of the glycocalyx (Costerton, Irvin & Cheng, 1981) can be seen around the cells resuspended from wall growth, while no such layer was detected in ruthenium red stained cells grown in static urine culture (Figure 2). Bladder washouts in patients During the course of attempts to use a bladder washout technique (Fairley et al., 1971) to localize the site of urinary-tract infections in the spine-injured patients, chlorhexidine was employed to eliminate organisms from the bladder urine. This provided an opportunity to observe, in two instances, how the bacterial population in the bladders of patients responded to the antiseptic. The results of these washouts are presented in Figures 3 and 4. It can be seen that although the MICs of chlorhexidine for many of the organisms were low (< 80 mg I-‘), the effect of the antiseptic was minor and temporary. Electron microscopy on ruthenium red stained sediments of urine from patients revealed the presence of micro-colonies of bacteria . embedded in a fibrous matrix. Discussion

For some 30 years chlorhexidine has proved to be a valuable antibacterial agent, its non-toxicity to skin and mucous membranes and broad spectrum of activity has led to its widespread use in medical and veterinary antisepsis. The original report on this compound (Davies et al., 1954) showed that it had high potency against both Gram-positive and Gram-negative bacteria, the MICs for Gram-negative bacteria ranging from 5 mg 1-l for Proteus that vulgaris to 20 mg 1-’ for Pseudomonas aeruginosa. The first indication clinical isolates of Gram-negative species might be less sensitive to chlorhexidine than the original tests indicated came from Gillespie et al. (1967) who immobilized catheters by sponge collars which were smeared twice daily with chlorhexidine obstetric cream and used chlorhexidine to

Chlorhexidine

a .g 9

and

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a 7.c

I

I 4

I

4

30 min

50 min Time after lnstlllottion

Chlorhexldlne lOOmI 2OOpq ml-’ Instilled for 30 mln

I 24h

Figure 3. The effect of a chlorhexidine bladder washout on the urinary flora of patient A. 0, k’ebsiella pneumoniae, 4OpgmlV’ MIC of chlorhexidine; A , Providencia stuartii 160 /c gml-‘; A, Eschertchia coli, 5 ,ug ml-‘; 0 Pseudomonas aeruginosa, 80 pg ml-‘; n, Staphvloccus aureus, < 5 pg ml-‘; 0, Streptococcus faecalis, 5 pg ml-‘.

4

4

50 mln 50 ml” Time ofter lnstlllotion

Chlorhexldme 100 ml 2OOpg ml?nstllled for 30 mln

Figure 4. The effect of a chlorhexidine bladder washout on the urinary flora of patient B. 0, Providencia stuartii, 640 /g ml-’ MIC of chlorhexidme; 0, Streptococcusfaecalis, 10 pg ml-‘; n , Proteus mirabilis, 1280 pg ml-‘; III, Proteus morganii 320 pg ml-‘.

36

D, J. Stickler

et al.

disinfect the urethra at each catheterization. Proteus mirabilis with MICs of chlorhexidine in the range 125-500 mg 1-l were isolated and it was suggested that their insensitivity to chlorhexidine allowed them to penetrate the antiseptic barrier. Subsequently studies on the effect of cleansing the external genitalia with aqueous chlorhexidine (600 mg 1-l) in male patients undergoing intermittent catheterization in the early stages of paraplegia (Stickler, Wilmot & O’Flynn, 1971; O’Flynn & Stickler, 1972), found that for the first 2-3 days after injury the peri-urethral skin had a predominantly Gram-positive flora that was greatly reduced in numbers by the application of the antiseptic. Providencia stuartii, Proteus mirabilis and Pseudomonas aeruginosa colonizing the urethra then appeared and were unaffected by the antiseptic. Many of these bacteria were shown to have MICs of chlorhexidine of 800-l 600 mg 1-l (Stickler, 1974; Stickler & Thomas, 1976) and survived challenges of chlorhexidine simulating those found when the antiseptic is included in catheter urine drainage bags (Thomas, Sykes & Stickler, 1978) and in bladder washouts (Stickler et al., 1981). In this latter study bacteria were added to antiseptic solutions and incubated for 20 min at 37”C, but in the catheterized patient urine continues to flow into the bladder during the instillation period and in spine-injured patients with high fluid intakes, this will dilute the antiseptic. We considered that a model of this dilution factor might provide a more appropriate test system. The results presented in Figure 1 show that the range of species responsible for infections in spine-injured patients are unlikely to respond to bladder washouts with chlorhexidine. Even strains of E. coli and S. faecalis, with MICs of 4 and 10 mg 1-l respectively, exhibited resistance to 200 mg 1-l of the antiseptic in the bladder model. In all cases, the cultures recovered from the initial bactericidal effect. Resuspension of the wall growth gave a ten-fold increase in bacterial numbers in the urine, indicating that the bacterial cells present on the model wall were resistant to the antiseptic and probably served to re-inoculate the urine. Pallent et al. (1983) noted that the adsorption of P. cepacia onto glass surfaces was followed by the secretion of a glycocalyx which protected against chlorhexidine (500 mg I-‘). Marrie & Costerton (1981) showed that S. marcescens could survive high concentrations of chlorhexidine (up to 20,000 mg 1-l) when grown on the walls of glass bottles and embedded in a polysaccharide matrix. P. aeruginosa, E. coli and S. aureus survive the bactericidal action of p-lactam antibiotics due to adherence to the culture vessel and protection by a polysaccharide capsule material (Gwynn, Webb & Rolinson, 1981). Costerton (1984) presented microscopic evidence that P. aeruginosa was capable of growing in biofilms on urinary catheters and bladder walls and suggested that although antibiotic therapy might kill swarmer cells shed into the urine from the surfaces, bacteria within the adherent films survive the challenge and subsequently produce new swarmer cells that cause an

Chlorhexidine

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37

apparent recurrence of the infection. Recently, Nickel et al. (1985) confirmed that P. aeruginosa could grow as a thick adherent layer composed of bacteria and exopolysaccharides on urinary catheters and strains with MICs of tobramycin of O-4 mg 1-l survived exposure for 12 h in 1000 mg 1-l of the antibiotic in the biofilm. The results reported in Figure 1 indicate that a similar phenomenon is occurring in the bladder model. The glycocalyx coated wall growth (Figure 2) survives the application of chlorhexidine and serves as a source to re-inoculate the urine after the antiseptic has been released from the bladder. The success of chlorhexidine in eliminating E. coli from the model in experiments where no wall-growth was allowed to occur, provides further evidence for this interpretation. Bacterial wall growth can also occur in infected bladders, with organisms adhering to the epithelial cells and mucoid lining. Microscopic studies by Elliot, Slack & Bishop (1984) and Costerton (1984) have revealed micro-colonies of bacteria on bladder surfaces. The failure of chlorhexidine washouts to have any appreciable impact on the bladder flora (Figures 3 and 4) even though most of the organisms concerned were extremely sensitive to the antiseptic in laboratory t’ests, could well result from the protection afforded by the polysaccharide matrix that we found surrounds the bladder organisms. Apart from indicating that antiseptic bladder washouts with chlorhexidine are unlikely to be successful in controlling established infections in catheterized patients, our results also confirm that two distinct mechanisms of chlorhexidine resistance occur in bacteria; an intrinsic resistance manifested by cells in suspension which is probably due to some property of the outer envelop obf certain Gram-negative species (Thomas & Stickler, 1979; Ismaeel et al., 1986) and a more general phenomenon exhibited by cells growing on surfaces, resulting from the development of a protective polysaccharide matrix. The polysaccharide glycocalyx is also likely to be responsible for the consistent failure of antibiotic therapy in long-term catheterized patients (Brocklehurst & Brocklehurst, 1978; Clayton, Chawla 8t Stickler, 1982). The intrinsic resistance of some Gram-negative species to chlorhexidine is also associated with resistance to other antibacterials, e.g. other cationic antiseptics and antibiotics and it was suggested by Stickler & Thomas (1980) that in the context of the management of catheterized bladders the extensive use of cationic antiseptics could result in the selection of multi-drug resistant species such as Pseudomonas aeruginosa, Proaidencia stuartii and Proteus mirabilis. Walker & Lowes (3985) have reported that a strain of P. mil-abilis, responsible for an outbreak of catheter associated urinary-tract infection in a unit where chlorhexidine was being added to the drainage bags, was multi-drug resistant and was resistant in urine to 500 mg I-’ chlorhexidine. They recommended that routine addition of chlorhexidine to catheter bags be abandoned. Our results indicate that even strains of bacteria designated as chlorhexidine sensitive by standard

D. J. Stickler

38

et al.

laboratory tests survive bladder washouts with this antiseptic, and we suggest that chlorhexidine bladder washouts for infections in catheterized patients should also be abandoned. We wish to acknowledge the advice and technical assistance given by Ruth MacKenzie Department of Renal hledicine, Cardiff Royal Infirmary over electron microscopy.

of the

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110, 46~. Blandy, J. P. (1970). Catheterization. British Journal of Hospital Medicine 4, 179-182. Brocklehurst, J. C. & Brocklehurst, S. (1978). The management of indwelling catheters. British Journal of Urology 50, 102-10.5. Clayton, C. L., Chawla, J. C. & Stickler, D. J. (1982). Some observations on urmary-tract infections in patients undergoing long-term bladder catheterization. Journal of Hospital Infection 3, 39-47. Costerton, J. W. (1984). The aetiology and persistence of cryptic bacterial infections: a hypothesis. Reviews of Infectious Disease 6 (suppl. 3), S608-612. Costerton, J. W., Irvm, R. T. & Cheng, K. J. (1981). The bacterial glycocalyx m nature and disease. Annual Reviews of Microbiology! 35, 299-324. Davies, G. E., Francis, J., Martin, A. R., Rose, F. L. & Swain, G. (1954). 1:6-di-4’-chlorophenyldiguanidohexane (Hibitane): labatory investigation of a new antibacterial agent of high potency. British Journal of Pharmacology and Chemotherapy 9, 192-196. Elliot, T. S. J., Slack, R. C. B. & Bishop, M. C. (1984). Scannmg electron microscopy of human bladder mucosa in acute and chronic urinary-tract infection. British Journal of Urolow 56. 38-43. Fairley, K.-F., ‘Carson, N. E., Gutch, R. C., Leighton, P., Grounds, A. D., Laird, E. C., nlcCallum. P. H. G.. Sleeman. R. L. & O’Keefe. C. nI. (1971). Site of infection in acute urinary-tract infections in general practice. Lancet ii, 6i5-6i8. Garrod, L. P., Lambert, H. P. & O’Grady, F. (1981). Antibiotic and Chemotherapy 5th Ed. Churchill-Livingstone, Edinburgh. Gillespie, W. A., Lennon, G. G., Linton, K. B. & Phippen, G. A. (1967). Prevention of urinary infection by means of closed drainage into a sterile plastic bag. British Medical Journal 3,90-92. Gwynn, M. N., Webb, L. T. & Rolinson, G. N. (1981). Regrowth of Pseudomonas aeruginosa and other bacteria after the bactericidal action of carbenicillin and other p-lactam antibiotics. The Journal of Infectious Diseases 144, 263-269. Ismaeel, N., El-Moug, T., Furr, J. R. 8r Russell, A. D. (1986). Resistance of PTovidencia stuartii to chlorhexidine: a consideration of the role of the inner membrane. Journal of Applied Bacteriology 60, 361-367. Marrie, T. J. & Costerton, J. W. (1981). Prolonged survival of Serratia marcescens in chlorhexidine. Applied and Environmental Microbiology 42, 1093-I 102. Marrie, T. J., Harding, G. K. M., Ronald, A. R., Dikkemaj J., Lam, J., Hoban, S. & Costerton. I. W. (1979). Influence of mucoidv on antibiotic coating of Pseudomonas aeruginosa: journal of Infectious Diseases 139, 357-361. stuartii McHale, P. J., Walker, F., Scully, B., English, L. & K eane, C. T. (1981). Providencia infections: a review of 117 cases over an 8 year period. Journal of Hospital Infection 2, 1.55-165. Nakahara, H. & Kozukue, H. (1982). Isolation of chlorhexidme resistant Pseudomonas 15, 166-168. aeruginosa from clinical lesions. Journal of Clinical Microbiology Nickel, J. C., Ruseska, I., Wright, J. B. & Costerton, J. W. (1985). Tobramycin resistance of Pseudomonas aeruginosa cells growing as a biofilm on urinary catheter material. Antimicrobial Agents 3 Chemotherapy 27, 619-624.

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O’Flynn, J. D. & Stickler, D. J. (1972). Disinfectants and Gram-negatilre bacteria. Lanret i, 489-490. Pallent, L. J., Hugo, W. B., Grant, D. J. W. & Davies , A. (1983). Pseudomonas cepacia and Infections. Journal of Hospital Infectton 4, 9-13. Slade, N. & Gillespie, W. A. (1985). The Urinary Tract and the Catheter. J. Wiley & Sons Inc., lNew York. Southampton Control of Infection Team (1982). Evaluation of aseptic techniques and chlorhexidine on the rate of catheter-associated urinary-tract infection. Lamet i, 89-91. Stickler, D. J. (1974). Chlorhexidine resistance in Proteus mirabilzs. Journal of Clinical Patholog?! 27, 284-287. Stickler, D. J., Plant, S., Bunni, N. H. & Chawla, J. C. (1981). Some observations on the activitv of three antiseptics used as bladder irrigants in the treatment of urinary-tract infection in patients with indwelling catheters. Paraplegia 19, 325-333. Stickler, D. J. & Thomas, B. (1976). Sensitivity of Proaidencia to antiseptics and disinfectants. Journal of Clinical Pathology 29, 815-823. Stickler, D. J. & ‘I’homas, B. (1980). Antiseptic and antibiotic resistance in Gram-negative bacteria causing urinary-tract infection. Journal of Clinical Pathology 33, 288-296. Stickler, D. J., Wilmot, C. B. & O’Flynn, J. D. (1971). The mode of development of urinary infection in intermittently catheterized male paraplegics. Paraplegia 8, 243-252. Thomas, B. & Stickler, D. J, (1979). Chlorhexidine resistance and the lipids of Prootdencia stuartii. Microbios 24, 141-l 50. Thomas, B., Sykes, L. & Stickler, D. J. (1978). S ensitivity of urine grown cells of Prozidencia stuartii to antisepti,cs. Journal of Clinical Pathology 31, 929-932. Tyrell, D. A. J., Phillips, I., Goodwm, C. S. & Blolvers, R. (1979). Microbial Disease: the use of the laboratory in diugnosis therapy and control, pp. 114, Edirard Arnold, London. methods for the detection Walker, E. M. & Lowes, J. A. (1985). An investigation into in-aitro of chlorhexidine resistance. Journal of Hospital Infection 6, 389-397.