DIAGN MICROBIOLINFECTDIS 1991;14:1-6
1
BACTERIOLOGY Effect of a Siliconized Latex Urinary Catheter on Bacterial Adherence and Human Neutrophil Activity Guillermo L6pez-L6pez, Alvaro Pascual, Luis Martfnez-Martinez, and Evelio J. Perea
The effect of a siliconized latex urinary catheter on the in vitro adherence and growth of Pseudomonas aeruginosa and Escherichia coli and on the activity of human polymorphonuclear leukocytes (PMNs) was assessed. The adherence of P. aeruginosa to latex catheters was significantly greater than that of E. coli, being 30 times higher at 6 hr of incubation. The survival of E. coli (103 CFU/ml) in phosphate-buffered saline (PBS) containing segments of siliconized latex catheters was lower than in the controls. This effect seems to be due to a toxic effect produced by substances eluted into the medium from the catheters, as the viability of E. coli (103 CFU/ml) in
eluates obtained from the incubation of catheters in PBS (24 hr; 37°C) was significantly lower than in the controls. These phenomena were not observed with P. aeruginosa. The incubation of human PMNs with either catheter segments or eluates did not affect the uptake of opsonized E. coli but significantly decreased the production of superoxide radicals by these phagocytes. It is concluded that the adherence of P. aeruginosa to these catheters is higher than that of E. coli and that the latex urinary catheter used herein elutes substances that are in vitro toxic for E. coli and the oxygen-dependent bactericidal mechanisms of human PMNs.
INTRODUCTION
A transient increase in the adherence of Gramnegative bacteria to bladder epithelial cells has been postulated as an important early event in the development of catheter-associated bacteriuria (Daifuku and Stamm, 1986). Less attention has been paid, however, to the effect of the plastic materials used to make urinary catheters on the adherence of bacteria and especially on the activity of h u m a n polymorphonuclear leukocytes (PMNs), which constitute the most important defensive barrier against invading bacteria. Because of their good mechanical properties, latex catheters are used for urinary catheterization. To make the surface smooth for insertion, most latex catheters are coated with a thin layer or silicone (Ruutu et al., 1985). In previous studies, different brands of these catheters have s h o w n marked cytotoxicity in vitro and cell growth inhibition in various h u m a n cell cultures (Ruutu et al., 1985). The purpose of this study is to evaluate the effect of a siliconized latex urinary catheter on the adherence and growth of Escherichia coli and Pseudomona aeruginosa. The effect of these catheters on phago-
Approximately 15% of hospitalized patients receive an indwelling urethral catheter. The development of catheter-associated bacteriuria has been associated with an increased duration and cost of hospitalization, as well as with increased morbidity and mortality in catheterized patients w h o acquire infection (Givens and Wenzel, 1980; Platt et al., 1982). In previous epidemiologic studies, the overall prevalence of bacteriuria during catheterization was 25% in general hospital settings, where the m e a n duration of catherization is 4-5 days (Garibaldi et al., 1974). From the, Department of Microbiology, School of Medicine, University of Seville, 41080-Seville, Spain. Present address (G.L.-L.):Department of Medicine, University of Petropolis, Petropolis, Brazil. Address reprint requests to: Dr. A. Pascual, Department of Microbiology, School of Medicine, University of Seville, Apdo. 914, 41080, Seville, Spain. Received April 18, 1990; revised and accepted June 22, 1990. © 1991 Elsevier Science Publishing Co., Inc. 655 Avenue of the Americas, New York, New York 10010 0732-8893/91/$3.50
2
cytosis and superoxide radical production by human PMNs is also assessed.
MATERIALS AND METHODS Bacterial Strains and Radioactive Labeling Three strains of E. coli and three strains of P. aeruginosa were used. All of the strains were isolated from the urine of patients with catheter-associated urinary tract infections. Identification was performed by standard methods, and the strains were kept in aliquots at -70°C until used. For adherence and phagocytosis assays, several colonies from a nutrient agar plate were inoculated into 5 ml Mueller-Hinton broth containing 10 ~I [3H] adenine (sp. act. 24 Ci/mmol; Amersham, UK), as described previously (Verhoef et al., 1977). Prior to use, bacteria were washed three times with phosphate-buttered saline (PBS) and resuspended in Hank's balanced salt solution (HBSS) to obtain a final concentration of 10s CFU/ml. For phagocytosis studies, bacteria were opsonized by incubation in a shaking water bath for 15 min at 37°C in 5% pooled human serum.
Phagocytic Cells PMNs were isolated from heparinized blood by dextran sedimentation and Fycoll-Hypaque gradient centrifugation as described previously (Verhoef et al., 1977). PMN preparations were 97% pure. The purified PMNs were resuspended in HBSS supplemented with 0.1% gelatin (GHBSS) at a final concentration of 5 x 106 cells/ml.
Urinary Catheters Latex (siliconized) two-way pediatric Foley catheters (8FR; Inmed USA; Malaysia) were used. The catheters were cut under sterile conditions into segments 1.0 cm in length and preincubated in 10 ml of sterile isotonic PBS at 37~C for 60 min (Ashkenazi and Mirelman, 1984). Eluates of the catheters were prepared by incubating 30 segments of latex catheters in 10 ml of HBSS for 24 hr at 37°C under sterile conditions.
Bacterial Adherence to Urinary Catheters For the adherence assays, 10 ml of radioactive bacterial suspension (108 CFU/ml) and 30 segments of urinary catheters were incubated in 25-ml screwcapped glass vials and placed in an incubator at 37°C. At determined times, two catheter segments were removed from the vials with a sterile forceps and washed five times in cold PBS to remove all nonadherent bacteria. The catheter pieces were then
G. L6pez-L6pez et al.
transferred to polypropylene biovials (Biovial, Beckman Instruments). After adding 2.5 ml of scintillation fluid (Aqua Luma Plus, Lumac/3M, Netherlands), the catheter-bound radioactivity was determined in a scintillation counter (1701 LS Beckman Instruments, Inc., UK). From the amount of catheter-bound radioactivity, the number of bacteria adherent per square centimeter of catheter was calculated by dividing the number of bacteria adherent to a catheter piece by the total surface area of the piece. To evaluate the survival of bacteria in the presence of the catheter, 10-~1 volumes were taken from the vials containing bacteria and catheters at different times, and the number of colony-forming units was calculated by dilution and pour plating in agar. Colonies were counted after 48 hr of incubation at 37°C. All of the experiments were performed in triplicate on different days.
Bacterial Growth in Catheter Eluates Each strain was inoculated into eluates obtained from siliconized latex catheters at a final concentration of 103 CFU/ml. At different times, 10-~1 volumes were diluted and pour plated in agar, and colonies were counted after 48 hr of incubation at 37°C.
Phagocytosis Assay The uptake of radiolabeled bacteria by PMNs was measured as described (Verhoef et al., 1977). Before use, PMNs (5 x 105 PMN/ml) were preincubated for 30 min at 37~C in the eluate obtained from the catheter, as was described above. The phagocytic mixture contained 5 x 105 PMNs and 5 x 106 opsonized bacteria in a total volume of 200 ~l. After incubation for 12 min at 37°C in a shaking water bath, phagocytosis was stopped by adding 2.5 ml of ice-cold PBS. Leukocyte-associated bacteria were separated from nonassociated bacteria by differential centrifugation (three washes at 160g and 4°C). Leukocyte-associated radioactivity was measured in a liquid scintillation counter and expressed as a percentage of the total amount of radioactivity added. This figure was obtained by counting the pellet after centrifugation for 15 min at 1600g. Every experiment was done in duplicate and repeated three times with PMNs from three different donors.
Superoxide Production by PMN Superoxide production by PMNs was measured in the presence of (1) catheter segments or (2) eluates prepared by incubation of the catheters in HBSS for 24 hr. After a 30-min incubation with the catheter
Catheters, Bacterial Adherence, and PMN Activity
segments or eluates, the PMNs were then stimulated with phorbol myristate acetate (200 nM PMA; Sigma Chemicals), and superoxide production was measured by using a modification of the ferricytochrome c reduction microassay described by Pick and Mizel (1981). Aliquots containing 2.5 x 105 PMNs in 120 t~1 of HBSS (control) or eluates from the catheter were pipetted into wells of a 96-well flat-bottom microfiter plate (Flow Laboratories, McLean, VA). Half of the cell suspensions contained 60 units of superoxide dimutase (Sigma). Plates were then incubated for 30 rain at 37°C in an atmosphere containing 5% CO2. Finally, 80 t~1 of a suspension containing PMA or HBSS (control) and cytochrome c at a final concentration of 80 nM (Sigma) was added to the corresponding wells, and the plates were incubated again for 5, 20, and 60 rain at 37°C (5% CO2). Absorbance was read at 550 nm on a Multiskan MC photometer (Flow Laboratories). When catheter segments were used, PMNs were preincubated at 37°C for 30 min in 4-ml polystyrene tubes containing three 1-cm length segments of each catheter. After addition of the stimulus and cytochrome c, the tubes were incubated for 5, 20, and 60 rain, the supernatants were transferred to the wells of a microtiter plate, and absorbance was measured on a photometer. Controls were established to evaluate the possible spontaneous activation of the cytochrome system in the presence or absence of the catheter. The amount of superoxide was expressed as nmoles of superoxide per milligram of protein. PMN protein was determined by the method of Lowry et al. (1951). Every experiment was done four times with PMNs from different donors, three replicates were performed with each test.
3
30-
•Eo20 "o _< 'Y I 0
Ih
6h
Id
2d
4d 7d
TIME
FIGURE 1. Adherence of three strains of E. coli (O) and three strains of P. aeruginosa (A) to segments of siliconized latex urinary catheters (n = 3).
than that of E. coli. After 4 days of incubation, the adherence of both mcroorganisms was similar. The survival of E. coli and P. aeruginosa in PBS containing segments of siliconized latex catheters is shown in Figure 2. By using an initial inoculum of 108 CFU/ml, the viability of E. coli decreases significantly faster in the presence of the catheters than in the controls. After 48 hr incubation there were no viable E. coli (p
1
BACTERIOLOGY Effect of a Siliconized Latex Urinary Catheter on Bacterial Adherence and Human Neutrophil Activity Guillermo L6pez-L6pez, Alvaro Pascual, Luis Martfnez-Martinez, and Evelio J. Perea
The effect of a siliconized latex urinary catheter on the in vitro adherence and growth of Pseudomonas aeruginosa and Escherichia coli and on the activity of human polymorphonuclear leukocytes (PMNs) was assessed. The adherence of P. aeruginosa to latex catheters was significantly greater than that of E. coli, being 30 times higher at 6 hr of incubation. The survival of E. coli (103 CFU/ml) in phosphate-buffered saline (PBS) containing segments of siliconized latex catheters was lower than in the controls. This effect seems to be due to a toxic effect produced by substances eluted into the medium from the catheters, as the viability of E. coli (103 CFU/ml) in
eluates obtained from the incubation of catheters in PBS (24 hr; 37°C) was significantly lower than in the controls. These phenomena were not observed with P. aeruginosa. The incubation of human PMNs with either catheter segments or eluates did not affect the uptake of opsonized E. coli but significantly decreased the production of superoxide radicals by these phagocytes. It is concluded that the adherence of P. aeruginosa to these catheters is higher than that of E. coli and that the latex urinary catheter used herein elutes substances that are in vitro toxic for E. coli and the oxygen-dependent bactericidal mechanisms of human PMNs.
INTRODUCTION
A transient increase in the adherence of Gramnegative bacteria to bladder epithelial cells has been postulated as an important early event in the development of catheter-associated bacteriuria (Daifuku and Stamm, 1986). Less attention has been paid, however, to the effect of the plastic materials used to make urinary catheters on the adherence of bacteria and especially on the activity of h u m a n polymorphonuclear leukocytes (PMNs), which constitute the most important defensive barrier against invading bacteria. Because of their good mechanical properties, latex catheters are used for urinary catheterization. To make the surface smooth for insertion, most latex catheters are coated with a thin layer or silicone (Ruutu et al., 1985). In previous studies, different brands of these catheters have s h o w n marked cytotoxicity in vitro and cell growth inhibition in various h u m a n cell cultures (Ruutu et al., 1985). The purpose of this study is to evaluate the effect of a siliconized latex urinary catheter on the adherence and growth of Escherichia coli and Pseudomona aeruginosa. The effect of these catheters on phago-
Approximately 15% of hospitalized patients receive an indwelling urethral catheter. The development of catheter-associated bacteriuria has been associated with an increased duration and cost of hospitalization, as well as with increased morbidity and mortality in catheterized patients w h o acquire infection (Givens and Wenzel, 1980; Platt et al., 1982). In previous epidemiologic studies, the overall prevalence of bacteriuria during catheterization was 25% in general hospital settings, where the m e a n duration of catherization is 4-5 days (Garibaldi et al., 1974). From the, Department of Microbiology, School of Medicine, University of Seville, 41080-Seville, Spain. Present address (G.L.-L.):Department of Medicine, University of Petropolis, Petropolis, Brazil. Address reprint requests to: Dr. A. Pascual, Department of Microbiology, School of Medicine, University of Seville, Apdo. 914, 41080, Seville, Spain. Received April 18, 1990; revised and accepted June 22, 1990. © 1991 Elsevier Science Publishing Co., Inc. 655 Avenue of the Americas, New York, New York 10010 0732-8893/91/$3.50
2
cytosis and superoxide radical production by human PMNs is also assessed.
MATERIALS AND METHODS Bacterial Strains and Radioactive Labeling Three strains of E. coli and three strains of P. aeruginosa were used. All of the strains were isolated from the urine of patients with catheter-associated urinary tract infections. Identification was performed by standard methods, and the strains were kept in aliquots at -70°C until used. For adherence and phagocytosis assays, several colonies from a nutrient agar plate were inoculated into 5 ml Mueller-Hinton broth containing 10 ~I [3H] adenine (sp. act. 24 Ci/mmol; Amersham, UK), as described previously (Verhoef et al., 1977). Prior to use, bacteria were washed three times with phosphate-buttered saline (PBS) and resuspended in Hank's balanced salt solution (HBSS) to obtain a final concentration of 10s CFU/ml. For phagocytosis studies, bacteria were opsonized by incubation in a shaking water bath for 15 min at 37°C in 5% pooled human serum.
Phagocytic Cells PMNs were isolated from heparinized blood by dextran sedimentation and Fycoll-Hypaque gradient centrifugation as described previously (Verhoef et al., 1977). PMN preparations were 97% pure. The purified PMNs were resuspended in HBSS supplemented with 0.1% gelatin (GHBSS) at a final concentration of 5 x 106 cells/ml.
Urinary Catheters Latex (siliconized) two-way pediatric Foley catheters (8FR; Inmed USA; Malaysia) were used. The catheters were cut under sterile conditions into segments 1.0 cm in length and preincubated in 10 ml of sterile isotonic PBS at 37~C for 60 min (Ashkenazi and Mirelman, 1984). Eluates of the catheters were prepared by incubating 30 segments of latex catheters in 10 ml of HBSS for 24 hr at 37°C under sterile conditions.
Bacterial Adherence to Urinary Catheters For the adherence assays, 10 ml of radioactive bacterial suspension (108 CFU/ml) and 30 segments of urinary catheters were incubated in 25-ml screwcapped glass vials and placed in an incubator at 37°C. At determined times, two catheter segments were removed from the vials with a sterile forceps and washed five times in cold PBS to remove all nonadherent bacteria. The catheter pieces were then
G. L6pez-L6pez et al.
transferred to polypropylene biovials (Biovial, Beckman Instruments). After adding 2.5 ml of scintillation fluid (Aqua Luma Plus, Lumac/3M, Netherlands), the catheter-bound radioactivity was determined in a scintillation counter (1701 LS Beckman Instruments, Inc., UK). From the amount of catheter-bound radioactivity, the number of bacteria adherent per square centimeter of catheter was calculated by dividing the number of bacteria adherent to a catheter piece by the total surface area of the piece. To evaluate the survival of bacteria in the presence of the catheter, 10-~1 volumes were taken from the vials containing bacteria and catheters at different times, and the number of colony-forming units was calculated by dilution and pour plating in agar. Colonies were counted after 48 hr of incubation at 37°C. All of the experiments were performed in triplicate on different days.
Bacterial Growth in Catheter Eluates Each strain was inoculated into eluates obtained from siliconized latex catheters at a final concentration of 103 CFU/ml. At different times, 10-~1 volumes were diluted and pour plated in agar, and colonies were counted after 48 hr of incubation at 37°C.
Phagocytosis Assay The uptake of radiolabeled bacteria by PMNs was measured as described (Verhoef et al., 1977). Before use, PMNs (5 x 105 PMN/ml) were preincubated for 30 min at 37~C in the eluate obtained from the catheter, as was described above. The phagocytic mixture contained 5 x 105 PMNs and 5 x 106 opsonized bacteria in a total volume of 200 ~l. After incubation for 12 min at 37°C in a shaking water bath, phagocytosis was stopped by adding 2.5 ml of ice-cold PBS. Leukocyte-associated bacteria were separated from nonassociated bacteria by differential centrifugation (three washes at 160g and 4°C). Leukocyte-associated radioactivity was measured in a liquid scintillation counter and expressed as a percentage of the total amount of radioactivity added. This figure was obtained by counting the pellet after centrifugation for 15 min at 1600g. Every experiment was done in duplicate and repeated three times with PMNs from three different donors.
Superoxide Production by PMN Superoxide production by PMNs was measured in the presence of (1) catheter segments or (2) eluates prepared by incubation of the catheters in HBSS for 24 hr. After a 30-min incubation with the catheter
Catheters, Bacterial Adherence, and PMN Activity
segments or eluates, the PMNs were then stimulated with phorbol myristate acetate (200 nM PMA; Sigma Chemicals), and superoxide production was measured by using a modification of the ferricytochrome c reduction microassay described by Pick and Mizel (1981). Aliquots containing 2.5 x 105 PMNs in 120 t~1 of HBSS (control) or eluates from the catheter were pipetted into wells of a 96-well flat-bottom microfiter plate (Flow Laboratories, McLean, VA). Half of the cell suspensions contained 60 units of superoxide dimutase (Sigma). Plates were then incubated for 30 rain at 37°C in an atmosphere containing 5% CO2. Finally, 80 t~1 of a suspension containing PMA or HBSS (control) and cytochrome c at a final concentration of 80 nM (Sigma) was added to the corresponding wells, and the plates were incubated again for 5, 20, and 60 rain at 37°C (5% CO2). Absorbance was read at 550 nm on a Multiskan MC photometer (Flow Laboratories). When catheter segments were used, PMNs were preincubated at 37°C for 30 min in 4-ml polystyrene tubes containing three 1-cm length segments of each catheter. After addition of the stimulus and cytochrome c, the tubes were incubated for 5, 20, and 60 rain, the supernatants were transferred to the wells of a microtiter plate, and absorbance was measured on a photometer. Controls were established to evaluate the possible spontaneous activation of the cytochrome system in the presence or absence of the catheter. The amount of superoxide was expressed as nmoles of superoxide per milligram of protein. PMN protein was determined by the method of Lowry et al. (1951). Every experiment was done four times with PMNs from different donors, three replicates were performed with each test.
3
30-
•Eo20 "o _< 'Y I 0
Ih
6h
Id
2d
4d 7d
TIME
FIGURE 1. Adherence of three strains of E. coli (O) and three strains of P. aeruginosa (A) to segments of siliconized latex urinary catheters (n = 3).
than that of E. coli. After 4 days of incubation, the adherence of both mcroorganisms was similar. The survival of E. coli and P. aeruginosa in PBS containing segments of siliconized latex catheters is shown in Figure 2. By using an initial inoculum of 108 CFU/ml, the viability of E. coli decreases significantly faster in the presence of the catheters than in the controls. After 48 hr incubation there were no viable E. coli (p
