Rosamund J. Williams, J. M. T. Hamilton-Miller, W. Brumfitt
Microbiological Studies on Some Bladder Irrigation Fluids Summary: A 1.5% glycine solution has recently been introduced as a bladder irrigation fluid. In this stud1,', glycine solution was compared with two other recognised bladder irrigants (chlorhexidine and noxythiolin) for its inhibitory activity against common urinary tract pathogens. Glycine solution supported the growth of nearly 50°/0 of the bacterial strains tested whereas chlorhexidine and noxythiolin were completely inhibitory. In quantitative studies with selected strains it was found that growth could be initiated from as few as 200 organisms/ml. It was concluded that glycine solution was capable of supporting bacterial growth and thus, from a microbiological viewpoint, was unsuitable as a bladder irrigant.
Zusammenfassung: Mikrobiologische Untersuchungen einiger Blasenspiilfliissigkeiten. Vor kurzer Zeit wurde die Gtyzin16sung als Blasenspfilfliissigkeit eingefiihrt. In der vorliegenden Untersuchung wurde die Glyzint6sung mit zwei anderen bew~ihrten Blasenspfitfltissigkeiten (Chlorhexidin und Noxythiolin) hinsichtlich ihrer Hemmwirkung gegeniiber gew/~hnlichen Krankheitserregern der Harnwege verglichen. Die Glyzinl/fsung f6rderte das Wachstum yon nahezu 50°/0 der gepriiften Bakterienstiimme, w~ihrend Chlorhexidin und Noxythiolin vollst~indige Hemmwirkung zeigten. In quantitariven Versuchen mit ausgew~ihlten St~immen wurde festgestellt, dab sich Wachstum mit einer Konzentration yon nur 200 Erregern pro ml in Gang setzen tieB. Es wurde gefolgert, dab Glyzinl/~sung bakterielles Wachstum f6rdern kann und aus mikrobiologischer Sieht somit als Blasensp~lfliissigkeit ungeeignet ist.
rates when 1 in 5000 chlorhexidine diacetate was used as an "irrigating solution. As chlorhexidine is inhibitory to a wide range of organisms, with M I C ' s ranging from 1.0--100 ~g per ml, a concentration of 1 in 5000 (200 # g per ml) should suffice to inhibit the common urinary tract pathogens. McFadyen and Simmons (5) compared chlorhexidine and noxythiolin as bladder irrigants. Noxythiolin (Noxyflex®)--N-hydroxymethyl-N'-methyl-thiourea--has a brod spectrum of antibacterial activity which is thought to be due to the release of formaldehyde (6). Noxythiolin has been found (5, 7, 8) to be slightly superior to chlorhexidine in preventing post-operative urinary tract infection, but the difference was in no case dramatic. More recently, 1.5% glycine solution (Travenol) has been introduced as a bladder irrigant. Reports on the use of this solution were not available from the manufacturers. The only record we have been able to find of the use of glycine as a urinary irrigant is that by Nesbit and Glickman (9), who used a 1.10/0 solution. It seems unlikely that glycine solution has antibacterial properties, so this study was carried out in order to determine whether it can support the growth of some common urinary tract pathogens.
It is well-known that the use of an indwelling urinary catheter carries with it a high risk of urinary tract infection and, particularly in debilitated patients, the subsequent danger of gram-negative septicaemia has been shown to constitute a life-threatening problem (1). Systemic chemoprophylaxis, although widely used, has not been shown to be effective in preventing such infections, partly because in the hospital environment resistant strains are always a problem. Irrigation of the bladder, via the catheter, with an antibacterial agent, was proposed some years ago as a means of inhibiting bacterial growth in the urine. Kass and Sossen (2) recommended a 0.25% solution of acetic acid administered continuously through a "double-lumen" indwelling catheter. Such catheters actually have three lumens; one for distending the retaining balloon and two which lead to the interior of the bladder. Thus, the irrigating solution is introduced through one and the mixture of urine and acid leaves through the other. This "continuous flow" was considered to be vital for the efficiency of the method. According to the findings of Kass and Sossen (2), when the flow rate of acetic acid is sufficient, bacterial counts in the urine can be kept below 10~ bacteria per ml for up to 60 days. The only adverse observation was the development of haematuria in a few patients. Martin and Bookrafian (3) compared irrigation with 0.250/0 acetic acid with a solution containing both neomycin (40 #g/ml) and polymyxin (20 #g/ml) in isotonic saline. They found that the antibiotic rinse was superior in preventing urinary tract infection, but they did comment on the possibility that the use of antibiotics may encourage the selection of resistant strains. Paterson, Barr and MacDonald (4) reported a considerable reduction in post-operative urinary tract infection
Materials and Methods Irrigating solutions: 1. 1.50/0 glycine solution. This was manufactured and supplied in a sterile 3-1itre pack by Travenol Laboratories Ltd., and we subsequently distributed it aseptically into 100 ml containers. Received: 25 July 1975 Dr. R. Williams, Dr. J. Hamilton-Miller, Prof. W. Brumfitt, Department of Medical Microbiology, Royal Free Hospital, Pond Str., London NW3 2QG.
Infection 4 (1976) Nr. 1
R. J. Williams, I. M. T. Hamilton-Miller, W. Brumfitt: Microbiological Studies on Some Bladder Irrigation Fluids 2. Noxythiolin (Noxyflex ®) was supplied by Geistlich Sons Ltd., in a solid form. A 1% w/v solution (as recommended by the manufacturer) was freshly prepared. 3. Chlorhexidine, 1 in 5000 sterile solution, supplied by the Pharmacy of the Royal Free Hospital. Bacterial strains: Ten strains each of Klebsiella aerogenes, Streptococcus faecalis and Proteus mirabilis and nine strains each of Pseudomonas aeruginosa and Escherichia coli were tested. These strains had all been isolated from clinical sources and were stored on nutrient agar slopes in bijoux bottles and plated on to MacConkey agar before use. Microbiological investigations: The ability of the bacterial strains to survive and multiply in the irrigating solutions was examined by the following method: Separate 3 ml volumes of each irrigating solution and of water and nutrient broth were distributed into sterile bijoux bottles, each inoculated with one drop of an 8-hour broth culture of the bacterial strains and incubated at 37 ° C. The samples were examined for visible turbidity after 18 and 36 hours incubation and were streaked on to MacConkey agar to check for viability and purity. A duplicate set of samples was inoculated and incubated at room temperature (22 ° C) and examined after 18 and 54 hours. The growth of one bacterial strain from each of the five genera was examined in the different irrigating solutions in greater detail. Ten ml volumes of glycine, chlorhexidine and noxythiolin solutions and of distilled water were distributed into 50 ml bottles and inoculated with one drop of an overnight nutrient broth culture of strains: E. coli 011, Strep. faecalis 3, KI. aerogenes 002, Pr. mirabilis 047 and Ps. aeruginosa 2. Initial viable counts were estimated by sampling from the inoculated water at time 0. A final viable count was made on each sample after 30 hours static incubation at 37 ° C. The samples were mixed thoroughly immediately before the viable counts were made. Viable counts were performed by making ten-fold dilutions of the samples in distilled water and spreading 0.1 ml volumes of suitable dilutions on to Mac Conkey agar with a sterile glass spreader. The plates were incubated overnight at 37 ° C. The ability of small inocula of the test strains to grow and multiply in glycine solution was examined. Tenfold dilutions were prepared in glycine from 6-hour nutrient broth cultures of the test strains. Ten ml volumes of glycine solution in 50 ml bottles were inoculated with 0.1 ml of a 10--4 or 10--5 dilution of the test strain and mixed well. A 0.1 ml sample was withdrawn from each bottle and used to perform a viable count by the method described above. The cultures were then incubated statically at 37 ° C for 24 hours, after which a final viable count was made.
Results T h e results of e x a m i n i n g irrigating solutions f o r t u r b i d i t y a f t e r i n o c u l a t i o n w i t h t h e b a c t e r i a l strains are set o u t in
T a b l e 1. I m m e d i a t e l y a f t e r i n o c u l a t i o n , the average viable c o u n t of t h e s a m p l e s was 2 X 106 b a c t e r i a p e r ml. T h e s a m p l e s were n o t t u r b i d initially a n d since t u r b i d i t y r e q u i r e s at least 107 o r g a n i s m s p e r ml, the d e v e l o p m e n t of t u r b i d i t y a f t e r i n c u b a t i o n was a n i n d i c a t o r of b a c t e r i a l m u l t i p l i c a t i o n . H o w e v e r , t u r b i d i t y was n o t sufficient to allow q u a n t i t a t i o n b y s p e c t r o p h o t o m e t r i c m e a n s (_< 0.005 at 450 nm). G l y c i n e s o l u t i o n s u p p o r t e d t h e g r o w t h of m a n y of the b a c t e r i a l strains tested a n d all t h e strains w e r e r e c o v e r e d a f t e r 48 hours, i n c l u d i n g Strep. faecalis, t h e cultures of w h i c h h a d n o t b e c o m e visibly t u r b i d . S o m e of t h e chtorh e x i d i n e s u b - c u l t u r e s a p p e a r e d to s h o w slight t u r b i d i t y b u t n o n e of t h e strains c o u l d b e r e c o v e r e d after 48 hours. Similarly, n o x y t h i o l i n did n o t s u p p o r t g r o w t h of a n y of t h e strains tested, despite the s u b - c u l t u r e of o n e Strep. faecalis s h o w i n g turbidity. T h u s , since the g r o w t h of one c o l o n y r e p r e s e n t s 10 o r g a n i s m s / m l , t h e two antiseptic solutions killed at least 9 9 . 9 9 % of t h e i r i n o c u l u m . T h e results a f t e r i n c u b a t i o n a t r o o m t e m p e r a t u r e were similar to t h o s e at 37 ° C, e x c e p t t h a t slightly f e w e r strains s h o w e d t u r b i d i t y in glycine e v e n a f t e r 54 h o u r s incubation. T h e a v e r a g e viable c o u n t of the samples at the start of the first q u a n t i t a t i v e e x p e r i m e n t was 2.8 X 105 b a c t e r i a p e r ml. T h e final viable c o u n t s in the d i f f e r e n t irrigating solutions are given in T a b l e 2. I n c u b a t i o n was c o n t i n u e d f o r 30 h o u r s b e c a u s e earlier e x p e r i m e n t s s h o w e d t h a t in glycine s o l u t i o n t h e r e was n o killing a f t e r six hours. A p r e l i m i n a r y c o m p a r a t i v e study of g r o w t h of two strains of Ps. a e r u g i n o s a a n d Pr. mirabilis in glycine s o l u t i o n u n d e r static a n d s h a k e n i n c u b a t i o n s h o w e d t h a t t h e bacteria grew b e t t e r u n d e r static conditions. C h l o r h e x i d i n e a n d n o x y t h i o l i n a p p e a r e d to be bactericidal at t h e c o n c e n t r a t i o n s tested, w h e r e a s glycine solution did n o t greatly i n h i b i t t h e b a c t e r i a e v e n after 30 h o u r s i n c u b a t i o n . T h e test strains of E. coli, Strep. faecalis a n d K1. a e r o g e n e s survived b e t t e r in glycine s o l u t i o n t h a n in water. T h e n u m b e r o f Ps. a e r u g i n o s a i n c r e a s e d 100-fold d u r i n g t h e e x p e r i m e n t . T h e s e results c o n f i r m the earlier qualitative findings u s i n g t u r b i d i t y as a n i n d i c a t o r of b a c t e r i a l growth. T h e results of the initial a n d final viable c o u n t s of o r g a n isms g r o w n f r o m s m a l l i n o c u l a are g i v e n in T a b l e 3. T h e
Table 1: The production of turbidity by bacteria in various bladder irrigating fluids Bacteria
E. coli Strep. faecalis K1. aerogenes Pr. mirabilis Ps. aeruginosa
N u m b e r of strains tested 9 10 10 10 9
Infection 4 (1976) Nr. 1
Number of strains showing turbidity after incubation in: Glycine ChlorNoxyWater hexidine thiolin 37 ° C 22 ° C 37 22 37 22 37 22 2 0 5 10
1 2 1 10
3 1 7 5
1 0 1 0
0 0 0 0
0 0 0 0
9 0 10 10
8 0 10 10
Nutrient broth 37 22 9 10 10 10 9
9 10 10 10 9
R. J. Williams, J. M. T. Hamilton-Miller, W. Brumfitt: Microbiological Studies on Some Bladder Irrigation Fluids Table 2: Viable counts of organisms after growth in various irrigating solutions
Bacterial strain E. coli 011 Strep. faecalis 3 K1. aerogenes 002 Pr. mirabilis 047 Ps. aeruginosa 2
No. of organisms per ml after 30 hours incubation at 37 ° C in: Glycine ChlorNoxyWater hexidine thiolin 6.4 1.1 2.4 2.3 8.6
X X X X X
104 105 104 104 10r
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