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ost defence against Staphylococcus aureus biofilms infection: Phagocytosis ofiofilms by polymorphonuclear neutrophils (PMN)
rank Günthera, Guido H. Wabnitza, Petra Stroha, Birgit Priora, Ursula Obstb,vonne Samstaga, Christof Wagnerc, G. Maria Hänscha,∗
Institut für Immunologie, Universität Heidelberg, GermanyInstitut für Technische Chemie, Forschungszentrum Karlsruhe, GermanyKlinik für Unfallchirurgie und Orthopädie, Berufsgenossenschaftliche Unfallklinik Ludwigshafen, Germany
r t i c l e i n f o
rticle history:eceived 11 November 2008eceived in revised form 14 January 2009ccepted 25 January 2009vailable online 4 March 2009
eywords:
a b s t r a c t
Bacteria organised in biofilms are a common cause of relapsing or persistent infections, particularlyin patients receiving medical implants such as ventilation tubes, indwelling catheters, artificial heartvalves, endoprostheses, or osteosynthesis materials. Bacteria in biofilms are relatively resistant towardsantibiotics and biocides, and – according to the current dogma – towards the host defence mechanisms aswell. In that context, we addressed the question, how polymorphonuclear neutrophils (PMN), the “firstline defence” against bacterial infection, would interact with Staphylococcus aureus biofilms generatedin vitro. By time-lapse video microscopy and confocal laser scan microscopy we observed a migration
iofilmMNeutrophilsost defencehagocytosis
of PMN towards and into the biofilms, as well as clearance of biofilms by phagocytosis. By labelling thebacteria within the biofilm with 3H thymidine, and by cytofluorometry we could confirm and quantifyclearance and phagocytosis of biofilm as well. Of note, the extent of biofilm clearance depended on itsmaturation state: developing “young” biofilms were more sensitive towards the attack by PMN comparedto mature biofilms. In conclusion, contrary to the current dogma, S. aureus biofilms are not inherently
t defe
protected against the hos
. Introduction
Bacterial biofilms are increasingly recognised as a commonause of persistent infection (Costerton et al., 1999; Parsek andingh, 2003; Hall-Stoodley et al., 2004; Lynch and Robertson,008). Biofilms are defined as microbial communities thatolonise surfaces, for example the epithelium, but preferentiallymplanted medical devices including indwelling catheters, artificialeart valves, orthopaedic prostheses, or osteosynthesis materialsreviewed in Donlan, 2001; Gottenbos et al., 2002; Zimmerli etl., 2004). A hallmark of biofilm formation is the production ofslimy extracellular matrix, in which the bacteria are embed-
ed. Production of the extracellular matrix, also referred to asxtracellular polymer substance (EPS), is part of a genetically con-
rolled process that induces a multitude of additional functional andhenotypic alterations, including loss of motility, reduced growthate, and adhesion to surfaces (Davey and O’Toole, 2000; Watnicknd Kolter, 2000; Dunne, 2002 and references therein). Living
∗ Corresponding author at: Institut für Immunologie der Universität Heidelberg,m Neuenheimer Feld 305, 69120 Heidelberg, Germany. Tel.: +49 6221 564071;ax: +49 6221 565536.
as a biofilm appears to be advantageous for the bacteria, espe-cially under unfavourable conditions. Among others, the bacteriaacquire a relative resistance towards antibiotics and biocides, whichcomplicates the management of biofilm infections and limits thetherapeutic options (Mah and O’Toole, 2001; Stewart, 2002; Davies,2003 and references therein). Moreover, bacterial biofilms appearto be a challenge for the host defence (Leid et al., 2002; Jesaitis etal., 2002).
In our previous work, we addressed the question of thehost defence in patients with implant-associated osteomyelitis, apersistent and destructive inflammatory disease, caused by thecolonisation of endoprostheses or of osteosynthesis materials bystaphylococci species. In those patients, the therapy of choice isthe removal of the infected implant. Thus, bacteria and infiltratedimmune cells can be collected from the infected site for analysis exvivo. Among the infiltrated cells, we found predominantly polymor-phonuclear neutrophils (PMN) and T lymphocytes in co-existencewith the bacterial biofilm (Wagner et al., 2003, 2006), leading tothe question whether or not PMN would recognise and attack the
bacterial biofilm.
Therefore, in the present study we tested the interaction of PMNwith Staphylococcus aureus biofilms generated in vitro. We choseS. aureus because it is one of the prevalent pathogens in implant-associated biofilms infections (Arciola et al., 2005). We found that
. aureus biofilms are not entirely protected against the attack byMN, and that especially immature, developing biofilms are clearedy phagocytosis.
. Materials and methods
.1. Cultivation of bacteria and generation of biofilms
S. aureus isolates from patients were used, as was the commer-ially available strain “Seattle 1945” (ATCC 25923). The bacteria1 × 104) were diluted in 1 ml Difco Micro Inoculum Broth (Bectonickinson, Heidelberg, Germany) and incubated as specified for the
espective experiment. Polystyrol culture dishes were used (either6- or 24 well dishes from), chamber slides, or Lab-tek II Cham-ered Coverglass (all obtained from Nunc, Roskilde, Denmark).or phagocytosis assays, bacteria were grown in 5 ml polystyreneound-bottom tubes (BD Biosciences, Erembrodegem, Belgium).acteria were cultivated for 2, 6 or 15 days, respectively, at 37 ◦Cith continuous shaking.
.2. Isolation of PMN
Peripheral blood from healthy human volunteers was obtainedy venipuncture and collected heparin-NH4 coated tubes (Sarst-dt, Nürnbrecht, Germany). PMN were isolated by centrifugation onolymorphPrep (Axis-Shield PoC AS, Oslo, Norway) which yieldedn 85–95% pure PMN population. The PMN were suspended inanks balanced salt solution (HBSS) and used within 1 h.
.3. Time-lapse video microscopy
Biofilms were cultivated in chambered cover slides (8 chambers,unc) in the presence of BacLight red (Molecular Probes, Leiden,he Netherlands) (100 nmol final conc.). Isolated PMN, incubatedor 15 min with 5 nM Calcein AM (Sigma–Aldrich, Taufkirchen,ermany), were added (2 × 105 per chamber). For up to 60 min,icroscopic images were taken every 60 s (Zeiss Axiovert 200 M;
eiss, Göttingen, Germany). The images were analysed with theupplied Visitron Metamorph 6.2 Software.
.4. Confocal laser-scanning microscopy
S. aureus were cultivated in Lab-Tek II chamber slides (Nunc) for, 6 or 15 days at 37 ◦C. PMN were added for up to 60 min at 37 ◦C or◦C. The slides were then washed carefully, and fixed with 4% PFA
37 ◦C, 5 min). PMN were labelled with anti-CD66b (Immunotech,arseille, France) and Cy3-conjugated antibody to mouse IgG from
oat (Dako, Glostrup, Denmark). Biofilm and ingested bacteriaere stained with SytoBC as nucleic acid stain (500 nM, Molec-lar Probes, Eugene Oregon, USA). For confocal laser-scanningicroscopy, the slides were mounted and analysed with a LeicaMRBE confocal laser-scan microscope using Leica TCS as software
Leica, Wetzlar, Germany). For quantification of the biofilm-freeones, the laserscan-images were converted to black and white.or each time point, four arbitrarily chosen areas of the image werenalysed by three investigators in a blinded fashion, and biofilmepleted areas surrounding at least on PMN were estimated using
mageJ Software. The data, given as square units, were averaged andhe difference was calculated by ANOVA.
To detect S. aureus a monoclonal antibody recognising S. aureuseptidoglycane was used (abcam, Cambridge, UK).
.5. Phagocytosis of 3H thymidine labelled biofilms
To quantify the effect of PMN on bacterial biofilms, the bac-eria were labelled with 3H thymidine during culture. Following
ology 46 (2009) 1805–1813
exposure to PMN, the radioactivity associated with the residualbiofilms was used as a measure for biofilm destruction. To distin-guish between true phagocytic effects and loss of biofilm materialdue to excessive washing and handling, experiments were car-ried out at 37 ◦C or 4 ◦C, respectively. The data are expressed ascounts per minutes (cpm); calculated was the mean ± S.D. of 12parallel wells. S. aureus were cultivated in 96-well plates (PackardBioscience, Groningen, The Netherlands) for 2, 6, and 15 days at37 ◦C in Inoculum Broth containing 7.4 × 104 Bq/ml 3H-thymidine(Hartmann Analytic, Braunschweig, Germany). The biofilms werewashed carefully to remove excess radioactivity. PMN suspended inHBSS, containing 0.5% BSA were added (1 × 105 per well) and incu-bated for 1 h at 37 ◦C or 4 ◦C. The non-adherent PMN were removedby careful washing; then aqua bidest. containing 3% acetic acid wasadded to lyse adherent PMN. The remaining biofilms were washedwith PBS containing 0.5% sodium azide and 1% saponin. Finally thebiofilms were fixed with 97% aethanol at 4 ◦C, then desiccated at50 ◦C for 30 min Microscint 20 (PerkinElmer, Rodgau-Juegesheim,Germany) scintillation fluid was added (100 �l/well and the plateswere counted with TopCount Microplate Scintillation & Lumines-cence Counter (Packard BioScience).
2.6. Cytofluorometry
Biofilms were grown for 2, 6, and 15 days, then labelled withSytoBC (500 nM final concentration; 30 min at 37 ◦C with mild shak-ing in the dark). Then, the biofilm was washed, and isolated PMN(4 × 106/2 ml) were added and incubated for 45 min at 37 ◦C inthe dark. PMN were then transferred to a new tube; to removeadherent PMN, 2 ml PBS, containing 10% trypsin and 0.01 M EDTAwere added. The PMN fractions were combined, pelleted, and resus-pended in PBS containing 1% paraformaldehyde. An equal volumeof crystal violet (2 g/l in 0, 15 M NaCl) was added to quench thefluorescence due to biofilm material attached to the outside of thePMN. For cytofluorometry, the gate was set for the PMN populationand fluorescence associated with the cells was measured as meanfluorescence intensity (MFI). To prevent phagocytosis, experimentswere carried out at 4 ◦C, or with PMN having been preincubatedwith cytochalasin B (Serva, Heidelberg, Germany). The differencebetween the MFI obtained at 4 ◦C and 37 ◦C was used as a measurefor phagocytosis.
2.7. Migration “Transwell” chamber assay
Bacteria (1 × 108 in 1 ml) were suspended in DifcoTM MicroInoculum Broth, diluted 1 to 10 in Hank’s balanced salt solution(HBSS), and placed in a 24 well culture plate (Nunc, Nümbrecht,Germany) for biofilm formation. After 2 days 10 mm tissue cul-ture inserts equipped with a polycarbonate membrane (pore size3 �m) (Nunc) were placed into the wells and PMN (1.5 × 106/ml)were added. After 24 h, migration of PMN into the lower cham-ber well was determined by counting the cells microscopically.For comparison, wells without bacteria and with or without inter-leukin (IL-) 8 (25 ng/ml) (Immunotools, Friesoythe, Germany) aschemoattractant were used. For each experimental condition, sixparallel wells were prepared; the values represent the number ofPMN (mean ± S.D.).
2.8. Chemotaxis across a membrane filter
A modified Boyden chamber assay was used (Brenneis et al.,
1993), equipped with a nitrocellulose filter (5 �m pore size; 200 �mthick; Schleicher & Schuell GmbH Dassel, Germany). As bona fidechemokine IL-8 (8 ng/ml) were used. Random migration was deter-mined using HBSS in place of the chemoattractant. The cells (1 × 106
in 1 ml) were placed into the upper compartment, the chemokines
F. Günther et al. / Molecular Immunology 46 (2009) 1805–1813 1807
Fig. 1. Interaction of PMN with a S. aureus biofilm over time: PMN (green) were placed on the biofilm (red) (time 00), and their action was observed by time-lapse videomicroscopy for up to 60 min (the time in minutes is depicted in the upper corner of the images). Time zero is shown on the upper left panel, on the right panel the same areaa ictedt mulatt ntedi
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fter 58 min. A sequence of events of a selected area (marked by the asterisk) is depo those PMN is seen; biofilm-depleted areas increase with time. In parallel, an accuhe result of the red and green superimposition (a video of these events is supplemes referred to the web version of the article.)
n the lower. After 2 h, the cells migrated into the filters were fixedith propanol, and stained with haematoxylin. The filters were
nalysed microscopically using an Omnicon Alpha Image AnalyzerBausch and Lomb, Heidelberg, Germany). Chemotaxis was mea-ured as “leading front”, defined as the distance in �m from the topf the filter to a level where at least five cells could still be detected.wo parallel filters were prepared, and on each filter, 10 differentreas were evaluated at minimum.
. Results
.1. PMN on bacterial biofilms: direct observation by time-lapseideo microscopy
Biofilms were grown for 6 days in chambered slides in the pres-nce of Baclight RedTM, an efficient fluorescence label for bacteria.solated PMN, labelled with calcein, a green fluorescence dye foriving cells, were placed on these biofilms and their action wasbserved directly by time-lapse video microscopy for up to 60 min.he PMN moved on the biofilm. Within minutes, the formationf aggregates containing 5–10 cells was observed. In the wake ofhe moving PMN, biofilm-depleted areas became apparent (Fig. 1hows a sequence of these events; a video clip is supplemented as
1). When biofilm fragments were used, a migration of the PMNowards those fragments was seen, followed by a disruption, andventually uptake of the biofilm, the latter indicated by the yellowtaining of the PMN (Fig. 2 shows a sequence of these events; aideo clip as supplement S2).
in the lower panel: by 14 min two PMN appear. By 17 min depletion of biofilm nextion of red biofilm material is seen on and in the PMN, as well as a yellow staining asas S1). (For interpretation of the references to color in this figure legend, the reader
To assess whether PMN migrate into biofilms image, PMN wereplaced onto the biofilm for 60 min. Following fixation the bacteriawere stained with SytoBC, the PMN were labelled with an antibodyto CD66b conjugated with Cy3. The images were viewed by laserscan microscopy. For three-dimensional reconstruction, z-stackswere generated. The image of the biofilm alone is shown on the leftpanel; the biofilm with PMN on the right (Fig. 3). The upper panelshows the reconstruction with a flat angle (approximately 15◦), thelower panels a side view, which reveals the typical biofilm mor-phology with spikes and protrusions. When PMN were added, aninfiltration into the biofilm was observed within 20–60 min, as wasan uptake of biofilm material, particularly of the spikes. The cellsmoved as a front, and turned yellow, the latter indicative again ofbiofilm uptake (Fig. 3).
3.2. Migration of PMN towards bacterial biofilms
Because the time-lapse video (S2) suggested a migration of PMNtowards bacterial biofilms, we performed experiments to quan-tify the migration. Biofilms were grown in multiwell chambers andPMN were placed in inserts on top in a transwell assay. After 24 h,the number of PMN having migrated to the lower chamber wasdetermined. When biofilm was present – or as bona fide chemokine– IL-8, PMN were found in the lower chamber, but not in the absence
of biofilms or IL-8. On average, 7 × 104 PMN were found on thebiofilm, and 5.5 × 105 PMN in the IL-8 containing compartment(Fig. 4A).
In a further set of experiments, we tested whether the super-natants of biofilms contained the chemotactic activity by the use
1808 F. Günther et al. / Molecular Immunology 46 (2009) 1805–1813
Fig. 2. Interaction of PMN with a S. aureus biofilm fragment over time: As described above, PMN were placed on the biofilm – here large fragments were used – and the actionwas observed. The kinetics is shown for two selected areas (a and b). (a) After 8 min a PMN has moved towards a biofilm fragment, it snatches a piece of the film (10 min)and attaches it to its surface (12 min), and finally tears it off (14 min). Then the cell moves towards another fragment (37 min). (b) shows an analogue sequence of events. Theintense yellow staining of the cells is indicative of uptake of bacteria (the video is supplemented as S2). (For interpretation of the references to color in this figure legend, thereader is referred to the web version of the article.)
Fig. 3. PMN migrate into the bacterial biofilm: PMN were placed on a S. aureus biofilm for 30 min and than viewed by confocal laser scan microscopy. The three-dimensionalreconstruction shows the migration of the PMN (red) into the biofilm (green). On the left, the biofilm alone is shown, on the right, the biofilm with PMN added. The side view(lower panel) shows the spikes and protrusion of the biofilm and the PMN attacking them (> < marks the section plane; the arrows give the orientation of the image).
F. Günther et al. / Molecular Immunology 46 (2009) 1805–1813 1809
Fig. 4. (A) PMN migrate towards and into bacterial biofilms. (a) Biofilms were grown in tissue culture plates for 2 days. Then PMN were added to an insert (5 �m pore size),and placed onto the cultured biofilm. After 24 h, migration of PMN into the lower chamber was determined microscopically. (b) For comparison interleukin 8 (IL-8) as bonafide chemoattractant was added to the culture dish in the absence of a biofilm, (c) shows the experiments without biofilm. In (d), the mean values of four parallel experimentsare summarised. (B) Supernatants of biofilms were harvested at the times indicated and used as chemoattractant for PMN in a Boyden chamber assay. On the left, randomm s IL-8o of thet e medf
ofttcTI
igration (i.e. migration without chemoattractant) is shown, and migration towardf 2 parallel filters, where 10 different areas were measured (the box contains 50%he mean value and the horizontal bar the median). Compared to the time 0 (culturor the migration towards the supernatants were significantly different (p < 10−5).
f a Boyden chamber assay. We used supernatants of biofilmsrom different maturation states, i.e. from 2 h to 72 h, based on
he assumption that the properties of the biofilms change withime. PMN were clearly attracted to the biofilm supernatants, indi-ating that a soluble chemoattractant is released by the biofilm.he attractive capacity was in the same range as observed for IL-8.nterestingly, although biofilms mature over days, the maximum
as “positive” control. Chemotaxis was measured in �m and shown is the summaryvalues; the whiskers show the highest and the lowest values, respectively, the dotium removed from the biofilm without incubation time) the mean values obtained
attraction was already achieved with the supernatant of a 2 h oldbiofilm (Fig. 4B).
3.3. Phagocytosis of bacterial biofilms
The time-lapse video films suggested phagocytosis uptake anddeterioration of biofilm. To verify these observations, PMN were
1810 F. Günther et al. / Molecular Immunology 46 (2009) 1805–1813
F ria ino n PMN( n for 2P given
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ig. 5. PMN on a S. aureus biofilm viewed by confocal laser scan microscopy: Bactef the PMN). The PMN were identified by an antibody to CD66b, labelled red. Whearrows), but not when the cells were incubated at 4 ◦C (B). (C) Biofilms were growMN were quantified by planimetry (shown as arbitrary square units; the values are
dded to bacterial biofilms for 30 min at 37 ◦C. Then the specimensere fixed, SytoBC was added to label the DNA, and anti-CD66b to
dentify the PMN. As exemplified in Fig. 5A, uptake of the bacteria byhe PMN was seen, and in addition depletion of the biofilm aroundhe PMN. When the experiments were carried out at 4 ◦C, a condi-ion known to prevent phagocytosis of planktonic bacteria, neitherptake of bacteria nor biofilm-depleted areas around the PMN werebserved, an example is shown in Fig. 5B. In other experiments,acteria were identified with an antibody directed to S. aureus pep-idoglycane instead of DNA-labelling. Essentially similar data werebtained (not shown).
.4. Uptake and deterioration of biofilms depends on theaturation stage
The observation that protrusions and spikes were readily takenp by the PMN suggested that newly added biofilm material mighte especially sensitive to clearance by PMN. To assess the sensitivityf biofilms towards PMN systematically, S. aureus were cultivatedn cover slips for 2, 6, and 15 days. By day 2 biofilms were clearlyisible, they were, however, more fragile than the films grown foror 15 days. When PMN were added to the biofilms, bacteria-
epleted zones around the PMN became apparent after 30 min.he biofilm-depleted areas around the PMN of the 2- and 6-days-ld biofilms appeared larger compared to those of the 15-days-oldiofilms. By planimetry, a trend towards greater biofilm depletion
n less mature biofilms could be confirmed, with statistically signif-cant differences only between 2 and 15 days biofilms (Fig. 5C). Thexperiments shown were all performed with S. aureus derived frompatient. With a commercially available S. aureus strain, essentiallyimilar data were obtained.
.5. Quantification by 3H labelling of the bacteria
Because planimetry of the bacteria depleted areas is semi-uantitative at best, and does not take into account three-imensional structures, we established a new method for the
the biofilm (6-days-old) were labelled with SytoBC that stains the DNA (also thatwere incubated at 37 ◦C, areas depleted of biofilm around the PMN appeared (A)
, 6 and 15 days. PMN were added and after 30 min, the areas depleted around theas mean ± S.D. of four different areas viewed independently by three investigators).
quantification of the bacteria within the biofilm. The bacteria werecultured for 2, 6 or 15 days in the presence of 3H-thymidine, whichincorporates into the bacterial DNA. Then the films were care-fully washed, and freshly isolated PMN were added for 30 min toallow phagocytosis. Then PMN, and hence phagocytosed bacte-ria, were removed, and the radioactivity of the residual biofilmswas counted. Decline of radioactivity was interpreted as a measurefor biofilm deterioration. Compared were always biofilms that hadbeen exposed to PMN with those which had not (an example isshown Fig. 6A). In a parallel experiment, PMN were incubated withthe biofilm at 4 ◦C, which prevents phagocytosis. In that experi-ment, no loss of biofilm was seen.
With use of this method, we now compared phagocytosis byPMN of biofilms that had been cultivated for 2, 6 and 16 days(Fig. 6B). On average, loss of biofilm amounted to 51.5% for 2-days-old films, to 43.7% for 6-days-old films, and to 21.4% for 15-days-oldfilms (mean of three independent experiments with either patients’derived S. aureus or S. aureus type Seattle 1945; the differencesbetween 2-days-old and 15-days-old films were statistically sig-nificant with p = 0.02 using ANOVA). Again, incubation of biofilmswith PMN at 4 ◦C did not result in loss of biofilm.
3.6. Quantification of bacterial biofilm uptake by cytofluorometry
As a further method to quantify uptake of bacterial biofilms bythe PMN and to verify phagocytosis, as opposed to killing by exo-cytosed bactericidal compounds, cytofluorometry was performedusing SytoBC labelled biofilms. In these experiments, PMN wereplaced on the biofilms for 30 min at 37 ◦C or at 4 ◦C, respectively,then harvested and subjected to cytofluorometry. To differenti-ate between bacteria that were attached to the outside of thePMN and those inside, crystal violet was used for quenching the
outside-fluorescence. When the gate was set for the PMN, greenfluorescence was seen in the samples incubated at 37 ◦C, but notin those incubated at 4 ◦C (example in Fig. 7A). Compared to 15-day-old biofilms, the phagocytosis of 2- and 6-day-old biofilmswas considerably higher compared (Fig. 7B and C). Again, with S.
F. Günther et al. / Molecular Immunology 46 (2009) 1805–1813 1811
Fig. 6. Deterioation of 3H thymidine-labelled biofilms. (A) Bacteria were grown for 2 days in the presence of 3H thymidine under conditions allowing biofilm formation. PMNwere placed on the biofilms for 30 min at 37 ◦C, then removed and radioactivity associated with the residual biofilms was determined (the data of 12 parallel experiments areshown as box blots with the box containing 50% of the values, the horizontal bar shows the median value, and the “whiskers” the highest and the lowest values, respectively).Compared to biofilm alone, significant loss of radioactivity was seen after incubation of the biofilm with PMN at 37 ◦C (calculated by ANOVA). When the incubation with PMNwas carried out at 4 ◦C, there was no loss of biofilm. (B) The percentage of loss of biofilm was determined for 2-, 6-, and 15-days-old biofilms (here data of three independentexperiments are evaluated; the mean values are given).
Fig. 7. Phagocytosis of bacterial biofilms determined by cytofluoromertry. (A) To parallel tubes containing SytoBC labelled biofilm (grown for 6 days) PMN were added andincubated at either 4 ◦C (green line) or 37 ◦C (blue line) for 45 min. Fluorescence outside the cells was quenched with crystal violet and fluorescence associated with thecells was determined. (B) PMN were added to biofilms (for 45 min at 37 ◦C) that had been grown for 2 (red line), 6 (dark blue line), or 15 days (light blue line), respectively,and fluorescence was measured. (C) Data of 8 independent experiments are summarised for 2, 6, and 15 days old biofilms, respectively. Shown is the mean fluorescenceintensity as statistical box blot with the box containing 50% of the values, the horizontal bar shows the median value, and the “whiskers” the highest and the lowest values,respectively). Differences between the mean values were calculated by ANOVA.
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ureus from the patients and with the strain Seattle 1945 essentiallyimilar data were obtained.
. Discussion
Bacterial biofilms are increasingly recognised as a commonause of chronic persistent infections. Thus, the question arises,ow the host defence mechanisms react to bacterial biofilms, andhether or not biofilms can be cleared. Based on the facts that PMN
re the primary cells in the host defence against bacteria, and arequipped with numerous bactericidal and cytotoxic entities, andhat they infiltrate sites of biofilm infections, we assessed the inter-ctions of isolated human PMN with in vitro-generated S. aureusiofilms by a variety of methods. By directly observing the inter-ction by time-lapse video microscopy, we came to the followingonclusions: PMN move on intact biofilms, and biofilm-free areasppeared at the sites the PMN were vacating. Apparently, PMN takep biofilm as they move along. When biofilm fragments were used,migration of PMN towards the biofilm was seen, as was tearingp of biofilm material, and eventually removal of the biofilm. Somef the PMN turned yellow, most probably due an overlap of thereen and the red staining, which indicates close vicinity of biofilmnd PMN, presumably attachment and uptake. Of note, the PMNemained viable during the observation time (60 min), and were notmmobilised, as had been reported earlier for PMN on Pseudomonaseruginosa biofilms (Jesaitis et al., 2002).
By quantitative methods, we characterised the interaction ofMN with the biofilms. Firstly, we confirmed migration of PMNowards S. aureus biofilms and towards supernatants of the biofilmss well. The latter is in line with the fact that numerous bacte-ia, including S. aureus, produce and release into the environmentubstance(s) that attract PMN (Dürr et al., 2005; Rot et al., 1987).oreover, we demonstrated a migration of PMN into the biofilm.
he three-dimensional reconstruction suggested a massive infil-ration of the cells that was not confined to water channels, asemonstrated before by Leid et al. (2002). Moreover, the imageshow that especially spikes and protrusions on the surface of theiofilm were removed.
Because these data suggested that the newly formed biofilmaterial might be especially sensitive to phagocytosis by PMN, we
erformed a series of experiments with biofilms grown for dif-erent times. To quantify the effect of the PMN, areas devoid ofiofilms around PMN were measured by planimetry. Comparedo mature biofilms, young, immature biofilms (2- to 6-day-old)ppeared to be more sensitive compared to 15-day-old biofilms.ecause planimetry is two-dimensional only, and thus does notake into account holes in the biofilm, a further method, based onhe DNA-labelling of the bacteria was developed. After exposureo PMN and removal of the cells to exclude phagocytosed bacteria,he radioactivity of the remaining biofilm was counted as a mea-ure for biofilm loss. By this method, we could essentially confirmur previous data: loss of biofilm was most extensive in imma-ure biofilms. The method, however, did not allow differentiatingetween phagocytosis and other means of biofilm degradation.herefore, we used a cytofluorometry-based method to quantifyhagocytosis of planktonic bacteria. We could confirm uptake ofacteria, and again, we found that immature biofilms were moreensitive to phagocytosis compared to mature films, but that alsoature biofilms are not entirely protected against the attack by
MN. Taken together, our data show phagocytosis of bacterial
iofilms by the PMN. Additional means of biofilm deterioration,uch as exocytosis of bactericidal substances, however, cannot beuled out.
Our data seem to contradict an earlier report by Leid et al. (2002),ho described adherence of leukocytes to S. aureus biofilms, their
ology 46 (2009) 1805–1813
migration into the film, but a failure to phagocytose the bacteria. Inthese studies, the mononuclear cell fraction was used, that containspredominantly monocytes, T- and B-cells, but not the PMN, whichcould account for the differences seen. The “halos” described by Leidet al., however, are reminiscent of the biofilm-depleted areas seenin our studies, and could be due to cytotoxic substances releasedfrom the PMN.
Why immature biofilms are more sensitive to the PMN attackcompared to mature biofilms is still a matter of speculation. Underour experimental conditions, the number of bacteria incorporatedinto the biofilm did not increase during biofilm formation after day2 (data not shown). Moreover, also, the thickness of the biofilmdid not drastically increase with maturation, but the mechanicalstability did. We assume that the architecture and the composi-tion of the extracellular polymer matrix changes with time. To date,methods to characterise and to analyse the extracellular polymersubstances (EPS) are still being developed. The EPS is a highly intri-cate and dynamic structure, consisting of complex carbohydrates,proteins, lipids, nucleic acid, and exoenzymes as well (Branda et al.,2005; Flemming et al., 2007). Whether or not the EPS of S. aureusinteracts with PMN has not yet been analysed. Data derived fromexperiments with P. aeruginosa EPS, however, show effects of EPS orEPS-derived compounds such as quorum-sensing molecules, algi-nates or rhamnolipids on PMN (Pedersen et al., 1990; Zimmermannet al., 2006; Jensen et al., 2007; Hänsch et al., 2008).
Accepting the fact that PMN can phagocytose and clear biofilmsthe question arises how biofilms can persist in vivo. The paradigm“too many–too late” might also apply here, and we presumethat the host defence against bacterial biofilms is subject to thesame restriction as the defence against planktonic bacteria: thewithin-host-population dynamics, the health status of the host,and conditions favouring the colonisation with bacteria. The latteris particularly relevant for the so-called implant-associated infec-tions, when biofilms form on medical devices such as indwellingcatheters, artificial heart valves, osteosynthesis materials or endo-prostheses (Donlan, 2001; Gottenbos et al., 2002). Presumably,these foreign materials provide a readily colonisable surface, andthe “the race for the surface”, as it was proposed by Gristina et al.(1989), might be a decisive factor in the formation of biofilms. Anattractive notion is that artificial surfaces – in contrast to epithelialcells – are without defence against colonisation and biofilm forma-tion. While data on S. aureus biofilms are lacking, for P. aeruginosathe neutralisation by epithelial cells of quorum sensing moleculesrequired for biofilm formation has been described, which in turnmight result in the prevention of biofilm formation on the epithe-lium (Chun et al., 2004; Hastings, 2004).
Another observation that might explain the persistence ofbiofilms was made when we analysed the infiltrated cells. The PMNat the site of biofilm infections were highly activated, as seen by theup-regulation of surface receptors and priming for superoxide gen-eration, their chemotactic activity, however, was reduced. Thus, itis possible that the PMN cannot infiltrate into the biofilm, and con-sequently, phagocytosis and clearing of biofilms is restricted to thesurface, and hence remains insufficient (Wagner et al., 2004).
In conclusion, S. aureus biofilms can be invaded and phagocy-tosed by PMN. Therefore, they are not inherently protected againstthe host defence. Whether or not an infection becomes clinicallyapparent depends on numerous factors, especially the health statusof the host, and conditions favouring biofilm formation.
Acknowledgment
The work was supported by a grant from the DeutscheForschungsgemeinschaft (WA1623/1-5).
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ppendix A. Supplementary data
Supplementary data associated with this article can be found, inhe online version, at doi:10.1016/j.molimm.2009.01.020.
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