The formation of bacterial biofilms is increasingly recognised as the leading cause of chronic infections. It limits the application of implant materials including catheters, heart valves, or orthopaedic prostheses. It is generally assumed that the infection persists because bacteria organised as biofilms escape the host defence mechanisms. Nevertheless, when studying patients with infected implants, we found a massive infiltration of leukocytes particularly polymorphonuclear neutrophils, PMN, into the site of infection, which led to the question, whether the PMN interact with the bacterial biofilm or not.

The interaction of human PMN with Staphylococcus aureus biofilms was studied in vitro.

S.aureus was cultivated on glass cover slips for various times under conditions allowing formation of biofilms. Adherence of PMN to biofilms and phagocytosis of the bacteria were observed by confocal laser scan microscopy and time lapse video microscopy.

Migration of PMN on and into the biofilm was identified as being phagocytosis, apparent as uptake of bacteria into the cell. Concominantly, in the wake of migrating PMN bacteria depleted zones appeared, which increased in size with time. In addition to phagocytosis, release from PMN of DNA and also of elastase was seen, suggesting the formation of neutrophil extracellular traps (NETs). So far, the signal for DNA release and NET formation has not been identified; of note is, however, that they occurred preferentially on established “old” biofilms and in the absence of the opsonising human serum, while phagocytosis was most efficient with developing “young” biofilms.

Taken together, our data provide evidence that bacteria in biofilms are not entirely protected against host defence but that phagocytosis is still possible, especially when the biofilm is opsonised with human serum. Whether NET formation also contributes to bacteria killing in biofilms cannot be decided as yet but remains an attractive alternative.

P.aeruginosa causes acute and chronic-destructive infections, particularly wound infections, or device-associated infections by colonising respiratory tubes, catheters, or implants. The pathogenicity of P.aeruginosa is largely attributed to the relative resistance towards host defence. Especially when organised as biofilms, the bacteria evade phagocytosis and killing by polymorphonuclear neutrophils (PMN).

To elucidate the evasion mechanisms, the migration of PMN towards and through P.aeruginosa biofilms was studied. Migration of PMN towards P.aeruginosa biofilms was tested using various in vitro techniques.

We found that PMN migrated towards developing P.aeruginosa biofilms, attracted by the quorum-sensing molecule N-3-oxododecanoyl homoserine lactone (3OC12-HSL). Mature biofilms which no longer produced 3OC12-HSL did not attract PMN. Addition of interleukin 8, a potent chemokine, restored the migratory capacity. Once arrived at the biofilms, PMN readily attached with no important difference between developing and mature biofilms. Migration into and penetration of the films, however, was only seen with developing films. By mass spectroscopy it became obvious that a major difference between developing and mature biofilms was the composition of the extracellular polymer substance, of which alginate is a prominent component. A series of experiments with isolated alginate showed that PMN did not migrate on or into alginate-containing matrices, but remained affixed at the contact site just as they did on mature biofilms. The mechanism of this firm attachment is still under investigation; prominent up-regulation of various adhesion molecules was seen, which could provide possible explanation.

Mature biofilms, most probably due to the composition of the extracellular polymer substance, do not allow the penetration of PMN. Consequently, bacteria embedded in deeper layers of the biofilm are protected against the host response. Due to the restricted movement of PMN, the bactericidal activity of PMN is only efficient against bacteria in the immediate vicinity, explaining the inefficient host defence.

Introduction: Posttraumatic osteitis is a localised inflammatory process leading to tissue destruction and eventually osteolysis. The molecular mechanisms underlying the disease progress are not yet fully understood. In a previous study we demonstrated infiltration of polymorphonuclear neutrophils (PMN) into the site of infection; the PMN were highly activated as seen by upregulation of the activation-associated surface receptors CD14 and CD64. In this study we analysed the superoxide generation by the infiltrated PMN as possible pathomechanism of the local tissue destruction.

Material and Methods: Ten patients with device-associated osteomyelitis requiring surgery were recruited into the study. When removing the infected implant the site was rinsed intraoperatively. The leukocytes were recovered, then activation-associated surface receptors were determined by cytofluorometry as was superoxide generation by reduction of cytochrome C.

Results: 1–2 x 10⁷ leukocytes were recovered from the «lavage» fluid; 80 to 90% were identified as PMN. The PMN were highly activated as seen by an upregulation of CD14 and CD64, and a concomitant downregulation of the selectin CD62L. In response to phorbol ester (PMA) the superoxide production of the infiltrated PMN was enhanced when compared to peripheral PMN of the same patient. The infiltrated PMN, but not the PMN of the peripheral blood, responded to the bacterial peptide f-Met-Leu-Phe (f-MLP) with superoxide production, indicating an enhanced responsiveness of the cells. The underlying molecular mechanisms were analysed in vitro using PMN of healthy donors: only the induction of superoxide production by f-MLP, but not by PMA, required a «priming» of the cells, for example by low doses of lipoploysaccharide (LPS) or cytokines (e.g. TNFa, IL-8).

Conclusions: In posttraumatic osteomyelitis PMN infiltrate the infected site; they are locally activated as seen by an upregulation of the appropriate receptors and by “priming” for superoxide generation. Priming of local PMN could on one hand potentiate the bactericidal activity, on the other hand contribute to tissue destruction. The occurrence of viable bacteria and activated «armed» PMN at the same site points to an esacpe mechanism, possibly due to biofilm formation. Due to their cytotoxic and proteolytic potential PMN might participate in local tissue destruction and osteolysis.