Some bacterial parameters influencing the neutrophil oxidative burst response to Pseudomonas aeruginosa biofilms

APMIS ZOO: 727-733, 1992

Some bacterial parameters influencing the neutrophil oxidative burst response to

Pseudomonas aerugimsa biofilms

ELSEBETH TVENSTRUP JENSEN', ARSALAN KHARAZMI', NIELS H0IBY'-', and J. WILLIAM COSTERTON'

'Department of Clinical Microbiology, Rigshospitalet, Copenhagen, Denmark, 'Institute of Medical Microbiology, University of Copenhagen, Denmark, and 'Department of Biological Sciences, University of

Calgary, Calgary, Alberta, Canada

Jensen, E. T., Kharazmi, A., Hoiby, N. & Costerton, J. W. Some bacterial parameters influencing the neutrophil oxidative burst response to Pseudomonas aeruginosa biofilms. APMIS 100: 727-733, 1992.

Persistence of bacteria in spite of a normal host immune system and relevant antibiotic treatment is a key problem in many chronic infections, such as the bronchopulmonary P aeruginosa infection in cystic fibrosis patients. The capability of bacteria to establish themselves in microcolonies or biofilms is an important protective mechanism of the microorganisms. We examined the human PMN oxidative burst response to P aeruginosa in biofilm and in planktonic form. The PMN chemiluminescence response to tl aeruginosa in biofilms was reduced to 30.547.5% (p<0.04) and the superoxide response to 85.9% (p < 0.02) of the response to equivalent numbers of planktonic bacteria. Mechanical disruption of the biofilms before the assays elicited a significantly increased response in the chemiluminescence experiments and to nonopsonized biofilms in the superoxide anion experiments. We conclude that biofilm bacteria, although able to stimulate the PMN, result in a reduced, suboptimal response leading to lack of efficient eradication of the bacteria in the chronic infection.

Key words: Biofilm; Pseudotnonas aeruginosa; Cystic fibrosis; PMN.

Elsebeth Tvenstrup Jensen, Department of Clinical Microbiology 8223, Rigshospitalet, Juliane Maries Vej 28, DK-2100 Copenhagen 0, Denmark.

The lack of eradication of bacteria is the key problem in the management of chronic infections. In recent years the persistence of the bacteria has been increasingly attributed to the con- cept of bacterial biofilms (5, 7). The capability of bacteria to establish themselves in microcolonies or biofilms, where they are enmeshed in glycocalyx and escape elimination by host defenses as well as by antibiotics, has been docu- mented in clinical cases and under experimental conditions (4, 16, 24, 27-29, 34). The mechanisms of antibiotic resistance of these sessile bac-

Received September 14, 1991. Accepted March 16, 1992.

teria are currently being investigated (2, 12, 15, 19), as are the mechanisms by which they escape host defenses (21, 39). In the cystic fibrosis pa- tient, the chronic Pseudomonas aeruginosa infection persists despite the intact immune defense system of the host and in spite of frequent, relevant antibiotic treatment. It has been shown that neutrophils accumulate in the lungs of cystic fibrosis patients with chronic I! aeruginosa infection (20, 22, 37) but that the biofilm-like microcolonies are not eliminated (7). We have shown that I? aeruginosa growing in biofilms do not stimulate the oxidative burst of polymorphonuclear leukocytes (PMNs) to the same de- gree as planktonically grown bacteria (2 1). The aim of the present investigation was to examine

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some parameters influencing the interactions between P aeruginosa biofilms and human PMNs.

MATERIALS AND METHODS

Bacterial strain l? aeruginosa 6680185, a nonmucoid revertant of

an initially stable mucoid cystic fibrosis bronchopulmonary isolate, was used. The strain was polyaggluti- nable and serum-sensitive.

Planktonic bacteria An overnight beef broth culture was spun down at

1400 g and the bacteria were resuspended in buffer in appropriate dilutions.

Biofi’lms Biofilms were produced on silicone disks using a

modified Robbins device as previously described (21). Briefly, an aerated log-phase beef broth culture of ?! ueruginosa was pumped through the device at 40-60 ml per hour at room temperature. After 20-24 h the studs protruding into the flow channel were taken out, and the disks were aseptically removed and rinsed five times with 1 ml of physiological saline. They were kept under humid conditions before use in PMN assays within 45 min. In some experiments mechanical disintegration of the biofilm was performed by scraping the bacteria off the disks into the reaction buffer, vortex-mixing, and mild sonication for 10 min.

Disks The disks used were made of silicone (quality 2020,

shore 60), with a thickness of 2 mm and a diameter of 7 mm. They were normally washed for three days at 4°C in distilled water before sterilization by auto- claving or ethylene oxide.

Enumeration of bacteria Viable counts. Planktonic bacteria were diluted to

appropriate dilutions and 100 p1 aliquots were spread on lactose agar and 5% blood agar plates (SSI, Cop- enhagen, Denmark) to estimate the number of col- ony-forming units (CFU). Biofilm bacteria were scraped off the disks into 2 ml of saline, mildly sonicated (Bransonic@ ultrasonic bath, 60 W, Soest, Hol- land) for 10 min, vortexed, and viable counts were performed as described above.

Microscopy. To estimate the total number of bacteria, dead or alive, the dilutions of either biofilm or planktonic bacteria were fixed with 5% of a fixative solution containing 85% methanol, 10% formalde- hyde, and 5% glacial acetic acid (14), and counted in a Fuchs Rosenthal counting chamber using anoptral microscopy.

Opsonization of bacteria Planktonic bacteria or disks with biofilm bacteria

were rotated with serum (concentration 5-66‘1/0) for 15 min at 35”C, spun down at 1400 g , and either resuspended in buffer (planktonic bacteria) or used as they were (disks with biofilm). In the chemiluminescence experiments designed to detect differences between the response to intact and disrupted biofilms, normal human serum and immune sera were used; for the other experiments normal human serum was used.

PMN isolation Fresh citrated blood from healthy donors or buffy

coat was used. Cells were separated by dextran sedi- mentation and sodium metrizoat-Ficoll (Lympho- prep@, Nygird, Oslo, Norway) gradient centrifuga- tion (6). After hypotonic lysis of the erythrocytes the cells were resuspended in buffer and kept cold until use. Krebs-Ringer solution with 10 mM glucose was used, or in some experiments substituted with Hank’s buffered salt solution with 0.1% gelatine because of spontaneous chemiluminescence (40).

Chemiluminescence assay Disks with biofilm or the equivalent number of

planktonic bacteria with or without sterile disks were mixed with buffer, supplied with 50 pl of 5.6 x M luminol (5-amino-2,3-dihydro- 1,4-phthalazine-dione, Sigma Chemical Co., St. Louis, Mo., U.S.A.) and instilled in dark-adapted glass vials in a total volume of 4.55 ml. Various CFU:PMN ratios were examined, but for comparison of the response to biofilm and planktonic bacteria a ratio of 40: 1 was generally used. After registering background counts, 1 ml of PMN (1 x lo6 cells) was added under red light and 0.5 min registrations were made at 10-15 min intervals by a Beckman L8000/ LS5000 TD scintillation counter in the out-of-coincidence mode for up to 150 rnin (23). Opsonized zymosan (Sigma Chemical Co., St. Louis, Mo., U.S.A.) was always run as a positive control. Samples were examined in duplicate and results presented as median values and 25th-75th percentiles unless otherwise stated.

Superoxide anion production The reduction of cytochrome c was measured as

follows (23): Biofilms or planktonic bacteria were mixed with cytochrome c (12 mg per ml, Sigma Chemical Co., St. Louis, Mo., U.S.A.) in Krebs- Ringer solution with 10 mM glucose, and PMN (5 x lo6 per vial) were added in a total volume of 2.9 ml. Various CFU:PMN ratios were examined, but for comparison of the response to biofilm and planktonic bacteria a ratio of 40:l was generally used. After 30 min rotation at 37°C the reaction was stopped with cold buffer, centrifuged at 1400 g for 10 min, and the OD of the supernatant at 550 nm was read. In some experiments, superoxide dismutase (SOD, Sigma

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PMN AND PSEUDOMONAS BIOFlLMS

Chemical Co., St. Louis, Mo., U.S.A.) was used to show if the reaction was inhibitable. Samples were examined in duplicate and results presented as median values and 25th-75th percentiles unless otherwise stated.

Statistics

statistical analysis. Wilcoxon’s test for paired samples was used for

RESULTS

Biofirm stability during assays Kinetic studies were performed to examine

if sloughing off from the biofilm during the chemiluminescence and superoxide anion production assays would result in free biofilm bacteria that could elicit a PMN response. The number of free bacteria released from five biofilm disks was in the order of lo6 CFU in the two assays.

Chemiluminescence experiments There was no PMN response at a CFU:PMN

ratio below 1O:l - i.e. 1 x lo7 planktonic o r biofilm bacteria were needed to elicit a minimal response in 1 x 10‘ PMN. An increasing response was observed by increasing the CFU:PMN ratio.

The PMN response to biofilms was reduced to a median of 47.5 (23.8--122.1) %I of the response to planktonic bacteria (p < 0.04). When the bacteria were opsonized, a similar reduction of 30.5 (16.1-68.1) % was found (p<0.03) (Table 1).

Mechanical disruption of the biofilms immediately before the interaction with the PMN elicited a response greater than that to the intact biofilm, i.e. 164.7 (120.2-200.6) % (p < 0.03) and 126.6 (88.2-141.6) ’% (pC0.04) of the response

1000-

900. - 5 aoO. u 700.

0 61) 80 100 120 I40 20 40

TIME ( m i n ) Fig. 1. Kinetics of PMN chemiluminescence response to nonopsonized Ei aeruginosa in the form of biofilm (intact or mechanically disintegrated) and planktonic bacteria. CFU:PMN ratio approx. 40.

intact biofilm, o disintegrated biofilm, A planktonic bacteria, 0 control.

to intact biofilm, for nonopsonized and opsonized biofilms, respectively. An example of the kinetics of the response to mechanically disintegrated biofilms, intact biofilms and planktonic bacteria is shown in Fig. 1, and the mean increase (in 10 experiments) in PMN response upon disruption of the biofilms is shown in Fig. 2.

To examine the influence of the disks themselves, five disks were added to the planktonic bacteria immediately before interaction with the PMNs. At a CFU:PMN ratio of 36-1 lO:l, the PMN response to nonopsonized planktonic bacteria was slightly, but significantly, reduced to 80.7 (71.9-89.3) Yn (p<0.02), and to opsonized bacteria to 77.9 (75.7-96.6) (XI (n.s.). When the PMNs were preincubated with the disks, or in buffer that had contained the disks for 24 h, no effect on the PMNs was registered (data not shown).

TABLE 1. PMN cheniiluminescence response to P. aeruninosa in biofilm and planktonic form Peak in % of response to

Peak (kcpm) planktonic bacteria Percentiles 25th 50th 75th 50th A. Planktonic bacteria nonopsonized 158 253 852 100 B. Biofilm bacteria nonopsonized 89 176 343 47.5 C. Planktonic bacteria opsonized 406 499 985 100 D. Biofilm bacteria opsonized 128 249 329 30.5 Results are median and 25th-75th percentiles of 22 (A + B) and 9 (C + D) experiments. A vs B: p < 0.04; C vs D: p < 0.03.

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JENSEN et al,

1000 -

TABLE 2. PMN superoxide response to P. aeruginosa biofilms

Nonomonized ODsonized Intact 3.8 10.8 p<o.o1

Disrupted 6.0 11.9 p<O.Ol (2.0-4.4) (8.7-13.3) ( n = 10)

(3.5-8.6) (10.0-15.1) (n=9) p<O.Ol n.s. (n = 9) (n= 13)

Legend: The biofilms were mechanically disrupted or left intact, before they were opsonized (10% normal human serum or buffer control). Five disks with biofilms were rotated with cytochrome c and 5 x lo6 PMN per vial for 30 min at 37°C. Results are pre- T

INTACT DISRUPTED BlOFl LM Bl OF1 LM

Fig. 2. The influence of mechanical disruption of nonopsonized bacterial biofilms on peak PMN chemiluminescence response. The biofilms were scraped off the disks into reaction buffer, vortex- mixed and mildly sonicated for 10 min. The PMN response increased to 164.7 (120.2-200.6) O/o of the response to the intact biofilms (p < 0.03). The time to peak response was not statistically different in the two groups. Median values and 25-75th percentiles from 10 experiments are shown.

Superoxide anion production The superoxide anion production in response

to opsonized biofilms was 10.8 (8.7-13.3) nmol cytochrome c reduced per 5 x lo6 PMN, equal to 85.9 (74.4-95.3) '% of the response to equivalent numbers of planktonic bacteria (p < 0.02). When the biofilms were mechanically disintegrated before opsonization, the response was 11.9 (10.0-15.1) nmol cytochrome c reduced per 5 x lo6 PMN, equal to 96.3 (84.2-104.4) YO of the response to planktonic bacteria (n.s.). As shown in Table 2, the superoxide anion production in response to nonopsonized intact biofilms increased from 3.8 to 6.0 nmol cytochrome c reduced per 5 x lo6 PMN when the biofilms were disintegrated (p < 0.01), whereas the increase upon disruption of opsonized biofilms only went

730

sented-as nmol cytochrome c reduced per 5 x lo6 PMN. Median values and 25-75th percentiles (paren- theses) are given. The spontaneous cytochrome c reduction was 0.86 (0.67-1.0) nmol cytochrome c reduced per 5 x lo6 PMN.

from 10.8 to 11.9 nmol cytochrome c reduced per 5 x lo6 PMN (n.s.).

Addition of sterile disks to planktonic bacteria at a CFU:PMN ratio of 5-5O:l resulted in a reduction to 93.0 (91.0-96.0) YO of the response to planktonic bacteria alone (p < 0.03). The disks did not affect the response to opsonized zymosan (data now shown). In the superoxide anion production assay dose-response studies showed that 5 x lo5 opsonized planktonic bacteria per vial were needed to elicit a minimal PMN response. Sodium dismutase was found to inhibit almost totally the PMN response to biofilms as well as to planktonic bacteria (data not shown).

DISCUSSION

Our investigations of the human neutrophil chemiluminescence and superoxide response to I? aeruginosa growing as biofilms showed that there was a definite response to biofilm bacteria. Both the superoxide and the myeloperoxidase pathways were activated by the biofilm bacteria. The chemiluminescence response to biofilm bacteria was strongly reduced compared to the response to planktonic bacteria. The superoxide anion production was also, although not as strongly, reduced; however, our data from mechanical disruption of the nonopsonized biofilms indicate that the superoxide response to the intact bacterial biofilm was submaximal.

PMN AND PSEUDOMONAS BIOFILMS

One mechanism behind a reduced/submaxi- ma1 response to bacterial biofilm could be that the number of bacteria available for contact with the PMNs was suboptimal. Our data (shown in Figs. 1, 2, and Table 2) on the effect of mechanical disintegration strongly support this point. When disruption took place more free bacteria were available for PMN interaction and the oxidative burst was increased.

It could be argued that the responses observed to biofilm bacteria were the result of stimulation by free bacteria dislodged from the biofilm. Our studies of biofilm stability showed that the spontaneous release of free bacteria from the biofilms was too low to elicit a chemiluminescence response, whereas the superoxide anion assay was more sensitive, and the released bacteria would probably be responsible for a relatively small fraction of the response. It is possible that the lack of a significant increase in superoxide anion response to opsonized biofilms upon disintegration was partly due to some PMN stimulation by free bacteria released spontaneously and during the opsonization so that near-maximal stimulation had already been obtained by the nonmechanically disintegrated biofilm.

However, it could be argued that the reason for the discrepancy between the superoxide and chemiluminescence response to opsonized disintegrated biofilms is that there is a myeloperoxidase mobilization rather than an oxidative burst per se. Our superoxide anion production assay measures extracellular superoxide only, whereas the chemiluminescence assay measures both extra- and intracellular myeloperoxidase (8). The fact that we do observe an increase in superoxide anion production upon disintegration of nonopsonized biofilms indicates, however, that there is a definite extracellular response and thus a genuine oxidative burst. In order to determine the extent and relative distribution of extra- and intracellular events further investigation of degranulation and phagocytosis is needed.

Another important factor leading to a reduced response to biofilm bacteria might be the glycocalyx. In the f! aeruginosa biofilm this is assumed to be mainly composed of a highly hydrated alginate matrix (19, 32). Although using a nonmucoid revertant of a mucoid CF strain, we have earlier shown that alginate is present in the biofilm (21). Many investigators

have found that alginate might have a strong effect on the interaction of PMNs with bacteria. Hydrophilic capsular substance of bacteria has been shown to protect against phagocytosis unless intact specific antibodies are present (35, 38). In the cystic fibrosis lung this is often not the case since the immunoglobulins are cleaved by proteases derived from the PMNs and from Pseudomonas aeruginosa (10, 13, 37). Alginate was found to impede chemotaxis (31, 36), and to reduce phagocytosis and the bactericidal effect of PMNs (3) and phagocytosis by alveolar macrophages (30, 33). In itself alginate was found to have no effect on chemiluminescence, but it was possible to prime PMN for an enhanced response to f-Met-Leu-Phe (3 1). More- over, Eftekhar & Speert (1 l ) showed that the otherwise reduced phagocytosis of mucoid Pseu- domonas aeruginosa was increased by enzymatic disintegration of alginate. Besides its hydrophilic properties, the strongly anionic alginate might well act as a permeability barrier trapping me- diators and ions necessary for an effective response (19). However, working with a mucoid strain, Allison et al. ( I ) showed that at slow growth rates Pseudomonas aeruginosa in planktonic form or as dispersed biofilm cells was equally hydrophilic. In our study we used early stationary phase biofilms and planktonic bacteria. According to the study by Allison et al. ( I ) they should both be hydrophilic, but they might differ with regard to hydrophobicity since we used a nonmucoid (revertant) strain.

Others have looked at the PMN response to bacteria in the presence of foreign material. A reduced PMN response has been reported in the presence of Teflon (41), and Lbpez-Lbpez et al. (26) found that catheters made of polyvinyl chloride, Teflon, and siliconized latex impaired superoxide anion production but not phagocytosis by PMN. These effects of material could be due to frustrated phagocytosis or cytotoxicity to the cells. As our data showed slight or no effect of silicone on the PMN response, this is not the likely reason for our observations of a reduced response to bacteria in biofilms.

Other studies have suggested that PMN might move towards the foreign material in a “race for the surface” (17), and some investigators have reported an increased PMN response to bacteria associated with foreign material (9, 18, 25), where the bacteria are concentrated on the

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surfaces, offering good contact possibilities for the phagocytic cells. In contrast to our data, these surface-adherent bacteria could be looked at as the very early phase of biofilm formation, where barriers to PMN contact have not yet been formed and the bacteria are still in mono- layers not masked by glycocalyx. It would be interesting to further substantiate this point by studying the neutrophil response to Z? aeruginosa bacteria in the process of biofilm formation, i.e. in the form of planktonic, adherent, young and old biofilms.

In conclusion, we have shown that one important aspect of a reduced PMN response to bacteria grown in biofilm is the mode of presen- tation of the bacteria. Mechanically disrupted biofilms elicit an increased PMN response probably due to the increased possibility of contact between bacteria and PMN. Our extended chemiluminescence experiments and investigation of the superoxide response to bacteria grown in biofilm presented in this paper have confirmed our earlier observations (21) that bacteria in biofilms are capable of activating the respiratory burst of the PMNs, but the response is low. Thus the bacteria grown in biofilm might elicit a response so low that they would not be eradicated, but high enough to contribute to the chronic inflammation taking place in the infected lung tissue.

The skillful technical assistance of Tina Wassermann and Hanne Kubert is greatly appreciated. This work was supported by grant 12-8716 from the Danish Medical Research Council and by the Lundbeck Foundation.

REFERENCES

1. Allison, D. G., Brown, M . R. W, Evans, D. E. & Gilbert, P: Surface hydrophobicity and dispersal of Pseudomonas aeruginosa from biofilms. FEMS Microbiol. Letters 71: 101-104, 1990.

2. Anwar, H. & Costerton, J. W: Enhanced activity of combination of tobramycin and piperacillin for eradication of sessile biofilm cells of Pseudo- monas aeruginosa. Antimicrob. Ag. Chemother. 34; 16661671, 1990.

3. Buyer, A. S., Speert, D. P, Park, S. , Tu, J . , Witt, M. , Nust, C. C. & Norman, D. C.: Functional role of mucoid exopolysaccharide (alginate) in antibiotic-induced and polymorphonuclear leukocyte-mediated killing of Pseudomonus aeruginosa. Infect. Immun. 59: 302-308, 1991.

732

4. Bergamini, T. M. , Bandyk, D. R, Govostis, D., Kaebnick, H. W & Towne, J. B.: Infection of vascular prostheses caused by bacterial biofilms. J. Vasc. Surg. 7: 21-29, 1988.

5. Brown, M. R. W, Costerton, J. W & Gilbert, P: Extrapolating to bacterial life outside the test tube. J. Antimicrob. Chemother. 27: 565-567, 1991.

6. Bsyum, A.: Isolation of mononuclear cells and granulocytes from human blood. Scand. J. Clin. Lab. Invest. 21: 77-89, 1968.

7. Costerton, J, W , Cheng, K-J., Geesey, G. G., Ladd, 'I: I. , Nickel, J. C . . Dasgupta, M . & Marrie, T J,: Bacterial biofilms in nature and disease. Annu. Rev. Microbiol. 41: 435-464, 1987.

8. Duhlgren, C. & Stendahl, 0.: Role of myeloperoxidase in luminol-dependent chemiluminescence of polymorphonuclear leukocytes. Infect. Immun.

9. Devalon, M. L., Elliot, G. R. & Regelmann, W E.: Oxidative response of human neutrophils, monocytes, and alveolar macrophages induced by unopsonized surface-adherent Staphylococcus aureus. Infect. Immun. 55: 2398-2403, 1987.

10. Doring, G.. Goldstein, W, Roll, A . . Schistz, P O., Hsiby, N. & Botzenhart, K.: Role of Pseudomonus aeruginosa exoenzymes in lung infections of patients with cystic fibrosis. Infect. Immun. 49: 557-562, 1985.

11. Eftekhar, I;: & Speert, 0. P: Alginase treatment of mucoid Pseudomonas aeruginosa enhances phagocytosis by human monocyte-derived macrophages. Infect. Immun. 56: 2788-2793, 1988.

12. Evans, D. J. , Brown, M. R. W , Allison, D. G. & Gilbert, F!: Susceptibility of bacterial biofilms to tobramycin: role of specific growth rate and phase in the division cycle. J. Antimicrob. Chem- other. 25: 585-591, 1990.

13. Fick, R. B., Baltimore, R. S., Squier, S. U, & Reynolds, H. Y: IgG proteolytic activity of Pseu- domonas aeruginosa in cystic fibrosis. J. Infect. Dis. 151: 589-598, 1985.

14. Gaub, J.: Feulgen-Naphtol Yellow S cytophoto- metry of liver cells. Histochem. 49: 293--301, 1976.

5. Giwercman, B., Jensen, E. T., Hsiby, N . , Kharaz- mi, A . & Costerton, J. W: Induction of p-lactama- se production in Pseudomonas aeruginosa biofilm. Antimicrob. Ag. Chemother. 35: 1008-1010, 1991.

6. Gristina, A . G., Ogu, M., Webb, L. X. & Hobgood, C. D.: Adherent bacterial colonization in the pathogenesis of osteomyelitis. Science 228:

17. Gristina, A. G.: Biomaterial-centered infection: Microbial adhesion versus tissue integration. Science 237: 1588-1 595, 1987.

18. Hayashi, K., Lee, D. A . & Quie, f? G. : Chemilumi- nescent response of polymorphonuclear leuko-

39: 736-741, 1983.

990-993, 1985.

PMN AND PSEUDOMONAS BIOFILMS

cytes to Streptococcus pneumoniae and Haemo- philus influenzae in suspension and adhered to glass. Infect. Immun. 52: 397400, 1986.

19. Hoyle, B. D., Jass, J. & Costerton, J. W: The biofilm glycocalyx as a resistance factor. J. Anti- microb. Chemother. 26: 1-6, 1990.

20. Hoiby, N. & Schiotz, I? 0.: Immune complex mediated tissue damage in the lungs of cystic fibrosis patients with chronic Pseudomonas aeruginosa infection. Acta. Paediatr. Scand. Suppl.

21. Jensen, E. T , Kharazmi. A , , Lam. K.. Costerton, J. W & Hoiby, N.: Human polymorphonuclear leukocyte response to Pseudomonas aeruginosa grown in biofilms. Infect. Immun. 58: 2383-2385, 1990.

22. Kharazmi, A, , Schiotz, I? O., Hoiby, N . , Bek, L. & Diiring, G.: Demonstration of neutrophil chemotactic activity in the sputum of cystic fibrosis patients with Pseudomonas aeruginosa infection. Eur. J. Clin. Invest. 16: 143-148, 1986.

23. Kharazmi, A , , Rechnitzer, C., Schiotz, I? O., Jen- sen, T , Bek , L. & Hoiby, N.: Priming of neutrophils for enhanced oxidative burst by sputum from cystic fibrosis patients with Pseudomonas aeruginosa infection. Eur. J. Clin. Invest. 17:

24. Lam, J., Chan, R., Lam, K. & Costerton, J. W: Production of mucoid microcolonies by Pseudo- monas aeruginosa within infected lungs in cystic fibrosis. Infect. Immun. 28: 546-556, 1980.

25. Lee, D. A . , Hoidal, J. R., Clawson, C. C., Quie, I? G. & Peterson, I? K.: Phagocytosis by polymorphonuclear leukocytes of Staphylococcus aureus and Pseudomonas aeruginosa adherent to plastic, agar, or glass. J. Immunol. Methods 63: 103-1 14, 1983.

26. Lopez-Lopez, G., Pascual, A . & Perea, E. J,: Ef- fect of plastic catheters on the phagocytic activity of human polymorphonuclear leukocytes. Eur. J. Clin. Microbiol. Infect. Dis. 9: 324-328, 1990.

27. Marrie, 7: J. & Costerton, J. W: Scanning and transmission electron microscopy of in situ bacterial colonization of intravenous and intraarteri- a1 catheters. J. Clin. Microbiol. 19: 687-693, 1984.

28. Mills, J., Pulliam, L., Dall, L., Marzouk, J., Wil- son, W & Costerton, J. W: Exopolysaccharide production by viridans streptococci in experimental endocarditis. Infect. Immun. 43: 359-367, 1984.

29. Nickel, J. C., Gristina, A . G., & Costerton, J. W:

301: 63-73, 1982.

256-261, 1987.

Electron microscopic study of an infected Foley catheter. Can. J. Surg. 28: 50-52, 1985.

30. Oliver, A. M. & Weir, D. M.: The effect of Pseudo- monas alginate on rat alveolar macrophage phagocytosis and bacterial opsonization. Clin. Exp. Immunol. 59: 190-196, 1985.

31. Pedersen, S. S., Kharazmi, A. , Espersen, E & Hoiby, N. : Pseudomonas aeruginosa alginate in cystic fibrosis sputum and the inflammatory response. Infect. Immun. 58: 3363-3368, 1990.

32. Russell, N. J. & Gacesa, I?: Chemistry and bi- ology of the alginate of mucoid strains of Pseudo- moms aeruginosa in cystic fibrosis. Molec. As- pects Med. 10: 1-91, 1988.

33. Simpson, J. A . , Smith, S. A. & Dean, R. E.: Alginate inhibition of the uptake of Pseudomonas aeruginosa by macrophages. J. Gen. Microbiol.

34. Speer, A. G., Cotton, I? B., Rode, J., Seddon, A. M., Neal, C. R., Holton, J. & Costerton, J. W: Biliary stent blockage with bacterial biofilm. Ann. Int. Med. 108: 546-553, 1988.

35. Speert, D. I?, Loh, B. A., Cabral, D. A . & Salit, I. E.: Nonopsonic phagocytosis of nonmucoid Pseudomonas aeruginosa by human neutrophils and monocyte-derived macrophages is correlated with bacterial piliation and hydrophobicity. In- fect. Immun. 53: 207-212, 1986.

36. Stiver, H. G.. Zachidniak, K. & Speert, D. I?: Inhibition of polymorphonuclear leukocyte chemotaxis by the mucoid exopolysaccharide of Pseudomonas aeruginosa. Clin. Invest. Med. 11:

37. Suter, S., Schaad, U. B., Roux. L., Nydegger, U. E. & Waldvogel, I;: A . : Granulocyte neutral proteases and Pseudomonas elastase as possible causes of airway damage in patients with cystic fibrosis. J. Infect. Dis. 149: 523-531, 1984.

38. van Oss, C. J.: Phagocytosis as a surface phenom- enon. Annu. Rev. Microbiol. 32: 19-39, 1978.

39. Vaudaux, I? E.. Lew, D. I? & Waldvogel, B A.: Host factors predisposing to foreign body infections. In: Bisno, A. L. & Waldvogel, E A. (Eds.): Infections associated with indwelling medical de- vices. American Society for Microbiology 1989,

40. Yanai, M. & Quie, I? G.: Chemiluminescence by polymorphonuclear leukocytes adhering to surfaces. Infect. Immun. 32: 1181-1186, 1981.

41. Zimmerli, W, Lew, I? D. & Waldvogel, I;: A.: Pathogenesis of foreign body infection. Evidence for a local granulocyte defect. J. Clin. Invest. 73:

134: 29-36, 1988.

247-252, 1988.

pp. 3-26.

1191-1200, 1984.

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Some bacterial parameters influencing the neutrophil oxidative burst response to Pseudomonas aeruginosa biofilms