Human polymorphonuclear leukocyte response to Pseudomonas aeruginosa grown in biofilms

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INFECTION AND IMMUNITY, JUlY 1990, p. 2383-2385 0019-9567/90/072383-03$02.00/0 Copyright X) 1990, American Society for Microbiology

Vol. 58, No. 7

NOTES Human Polymorphonuclear Leukocyte Response to Pseudomonas aeruginosa Grown in Biofilms ELSEBETH TVENSTRUP JENSEN,'* ARSALAN KHARAZMI,l KAN LAM,2 J. WILLIAM COSTERTON,2 AND NIELS H0IBY13 Department of Clinical Microbiology, Statens Seruminstitut, Copenhagen, Denmark'; Department of Biological Sciences, University of Calgary, Calgary, Alberta, Canada2; and Institute of Medical Microbiology, University of Copenhagen, Copenhagen, Denmark3 Received 6 February 1990/Accepted 31 March 1990

The interaction of human neutrophils with Pseudomonas aeruginosa bioflms was examined by using a chemiluminescence assay. The bioffilms induced an oxidative burst response by polymorphonuclear leukocytes which was slow and only 25% of the response to planktonic bacteria. The reduced response to P. aeruginosa bioffilms could play a role in the persistence of bacteria in chronic infections. ments showed that sonication did not reduce the viability of the bacteria. For opsonization 66% normal human serum was used with the biofilm disks or the planktonic bacteria (10). PMNs were prepared from fresh human citrate-treated blood by dextran sedimentation and sodium metrizoateFicoll gradient centrifugation. More than 97% of the cells were PMNs, and the level of cell viability exceeded 98%. The chemiluminescence assay was performed as previously described (10). Five disks with biofilms or the appropriate number of planktonic bacteria with or without disks were mixed with 1 x 106 PMNs in 5.5 ml (total volume) of Krebs-Ringer solution supplemented with 10 mM glucose, and 50 ,ul of luminol was added to each vial. Sequential 0.5-min counts were determined by using a scintillation counter for 120 min. Opsonized zymosan was used as a positive control, and sterile disks and PMNs without bacteria were used as negative controls. The Wilcoxon test for paired differences was used for the statistical analysis. A comparison of the PMN chemiluminescence responses to biofilm and planktonic bacteria is shown in Fig. 1. The responses to biofilm bacteria were significantly reduced to 25.0% (nonopsonized) and 27.4% (opsonized) of the responses to corresponding numbers of planktonic bacteria (P c 0.05). To determine whether the PMN responses observed in the chemiluminescence assay were due to biofilm bacteria and not just to free bacteria that had become detached from the sessile population, the kinetics of release of free bacteria from the biofilms and the chemiluminescence response to these free bacteria were determined. During the chemiluminescence assay period 105 to 106 bacteria sloughed off; this number of bacteria was not sufficient to induce a chemiluminescence response. The PMNs showed no response to serum or to sterile disks alone. Addition of five disks to planktonic bacteria reduced the PMN response to opsonized bacteria to 88.9%o, whereas no decrease in PMN response was observed with nonopsonized bacteria. The presence of foreign material in itself might influence the PMN response (23). We found that

Certain bacterial infections become chronic although the bacteria are susceptible to the action of phagocytic cells and to antibiotics in vitro. Examples are Pseudomonas aeruginosa bronchopulmonary infections in cystic fibrosis patients (11), staphylococcal osteomyelitis (8), and device-related infections (1, 13, 14). This paradox may be explained by the ability of bacteria to establish themselves in biofilms (4), in which they grow in microcolonies or adhere to natural or artificial surfaces in the body. In biofilms the growth kinetics (2, 3), cellular metabolism (2), and outer membrane antigen profiles (3) are different from those of free-floating, planktonic bacteria. In previous studies workers have looked at the polymorphonuclear leukocyte (PMN) response to adherent bacteria (9, 12), but to our knowledge the interaction of PMNs with biofilm bacteria has not been studied previously. A serum-sensitive nonmucoid revertant of an initially mucoid P. aeruginosa isolate from a cystic fibrosis patient (strain 6680/85) was used in this study. Biofilms were produced by using a modified Robbins device (15). An aerated log-phase beef broth culture was pumped at a rate of 40 ml/h through the device at room temperature, and biofilms were established on 0.5-cm2 silicone disks protruding into the flow channel. After 20 to 24 h the disks with the biofilms were removed aseptically, rinsed five times with 1 ml of saline, and used in chemiluminescence experiments within 45 min. Early-stationary-growth-phase bacteria from the device infusion flask were used to evaluate the PMN response to planktonic P. aeruginosa. The alginate content in biofilms was evaluated by measuring the levels of an alginate component, uronic acid, in sonicated autoclaved scrapings from biofilm disks (17). The bacteria in biofilm or planktonic form were enumerated by determining viable counts and by performing anoptral microscopy in a counting chamber (5). After a disk was rinsed, the biofilm was scraped off, mixed in saline, and ultrasonically treated for 10 min at 60 W. Control experi*

Corresponding author. 2383




are capable of activating the oxidative burst response of human PMNs. However, the response appears to be significantly weaker than the response induced by a similar number of bacteria grown in planktonic form, whether they are opsonized or not. Furthermore, the neutrophil response to biofilms is slower, resembling the response to a lower number of planktonic bacteria. This may contribute to the chronicity of P. aeruginosa pulmonary infections in individuals such as cystic fibrosis patients.

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The skillful technical assistance of Hanne B0dker, Tina Wassermann, and Anne Asanovski is greatly appreciated. This work was supported in part by grant 12-8716 from the Danish Medical Research Council and by the Lundbeck Foundation.












FIG. 1. PMN chemiluminescence responses to P. aeruginosa in biofilm and planktonic forms. The responses of 1 x 106 PMNs to 4 x


CFU of biofilm

planktonic bacteria were examined (ratio of colony-forming units to PMNs, 40). Symbols: 0, opsonized biofilm bacteria; A, opsonized planktonic bacteria; 0, nonopsonized biofilm bacteria; nonopsonized planktonic bacteria.

1. 2.



sterile disks reduced the response to opsonized bacteria slightly, but there was still a significant difference between the response to planktonic bacteria with disks and the response to biofilms on disks. Although we used a P. aeruginosa strain having a nonmucoid phenotype in this study, this strain has been shown to produce alginate (17), and we found 2.5 jig of uronic acid per disk, indicating that the nonmucoid strain produced an alginate concentration in biofilms which was similar to the concentration of alginate found in mucoid colonies on agar


plates (S. S. Pedersen et al., unpublished data). A hydrophilic capsular substance produced by bacteria has been shown to protect against phagocytosis (19, 20, 22) unless intact specific antibodies are present. However, this is not always the case in the lungs of patients with cystic fibrosis, as proteolytic enzymes from PMNs may cleave the immunoglobulins (6, 7, 21). Alginate interferes with phagocytosis by mononuclear cells (16, 18) and inhibits PMN chemotaxis (20). Masking and enclosure of biofilm bacteria with alginate may prevent other bacterial factors that are known to


activate neutrophils from reaching these phagocytic cells (4), thereby explaining the weakened PMN response. The mean number of bacteria per disk was 8.1 x 106 CFU (standard error of the mean, 3.2 x 106 CFU). Counting by microscopy yielded twice as many bacterial cells as colonyforming units. Five biofilm disks were necessary to induce a PMN chemiluminescence response. Thus, the corresponding ratio of colony-forming units to PMNs was approximately 40. The number of biofilm bacteria which actually came into contact with PMNs during the period of the experiment could not be determined. The possibility that this number was suboptimal could be another explanation for the low PMN response to biofilms. In our in vitro assay more than 107 planktonic bacteria were required to activate the neutrophils, and it is likely that there were fewer than this critical number of bacterial cells in the superficial layer of the biofilm that were readily contacted by the phagocytes. However, on the basis of our data, we cannot conclude that the response to a single bacterium in a biofilm is weaker than the response to a single planktonic bacterium. Our data demonstrate that P. aeruginosa biofilm bacteria



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