30845 Suppl Giot.pdf - Giornale Italiano di Ortopedia e Traumatologia

G.I.O.T. 2010;36(suppl. 1):S60-S62
Bacterial biofilms: the ultimate cause of implant-associated infections
Film batterico: causa primitiva delle infezioni negli impianti protesici
E. Meyle, G. M. Hänsch
Bacterial infections occurring after osteosynthesis or implantation
of prostheses are serious complications in orthopaedic surgery.
In the majority of cases, an antibiotic therapy is not successful.
Infections therefore persist and can give rise to a progressive,
destructive inflammatory process with massive tissue injury and
osteolysis, an entity known as implant-associated posttraumatic
osteomyelitis (for review see 1-5 ). In most cases the removal of the
implant, which might require extensive debridement and intricate,
costly reconstructive surgery, is the only option. Therefore, in the
recent years, considerable effort was put into unravelling the pathogenesis
of implant-associated osteomyelitis as a prerequisite for an
effective and eventually also causal therapy.
It became apparent that a specialised life-form of bacteria, the
so-called “biofilm” was responsible for the persistent infection.
Biofilms are defined as bacterial communities, where the bacteria
are embedded in an extracellular polymeric substance, morphologically
apparent as slime or film. In the case of implant-infection, the
biofilm is attached rather firmly to the implant. The formation of
the film is genetically programmed, with production of slime as a
hallmark (reviewed in 6-8 ).
Biofilm formation has been demonstrated for numerous bacteria
species. In implant-associated infection, staphylococci are prevalent,
and experimental data show that staphylococci readily attach
to metal surfaces, especially in the presence of serum or plasma,
and form biofilms there 9-11 . In vitro data imply that rough surfaces,
as they are generated by handling for example – as shown in
Fig. 1A – by clipping a wire favours the attachment of bacteria and
hence biofilm formation (Fig. 1A).
The slimy matrix protects the bacteria against unfavourable
environmental conditions, among others against antibiotics or
biocides.
How the immune system deals with biofilm infection is not yet
well understood. Most of our knowledge on bacteria-host defence
interactions is derived from studying single, free swimming “planktonic”
bacteria. Whether similar interactions occur also in biofilm
infection is unknown. Moreover, the link between biofilm infection
and the destructive inflammatory reaction is not yet understood,
nor the pathomechanism of the ensuing osteolysis, that is a serious
complication of implant-associated osteomyelitis.
To gain insight into the host defence against biofilm infections, the
main object of our experimental research is focussed on systemic
and local immune reaction in patients with implant-associated
osteomyelitis. In that context, we assessed interactions between
cells of the host defence with biofilms in vitro.
Institut für Immunologie der Universität Heidelberg, Germany
S60
In the following, we will briefly summarise our findings:
1. Implant-associated osteomyelitis is a localised infection.
Accordingly, systemic markers of infections, including fever
or increased leukocyte counts are not regularly seen, but
enhanced serum concentration of C-reactive protein (CRP)
are found in approximately 85 % of the patients (our own data
comprising 92 patients). An activation of phagocytic cells was
also seen in the majority of patients, particularly of neutrophils,
the so-called “first-line defence” against bacterial infection.
Moreover, neutrophils, and to a lesser extent, T-lymphocytes,
infiltrate the infected site, and can be recovered during surgery
for functional analysis ex vivo. We found that these cells were
highly activated, and “primed” for bactericidal and cytotoxic
activity 12-15 (Fig. 1C; 1D).
2. The presence of neutrophils at the site of infection leads to
the question whether these cells would be able to destroy biofilms.
In order to analyse the interactions between PMN and
biofilms of the staphylococci species we generated biofilms in
vitro. Afterwards neutrophils from healthy donors were added
and the effects were observed by means of time-lapse video
microscopy. We saw that PMN migrated towards and across
biofilms, and cleared areas of biofilm as they moved along.
By laser scan microscopy and cytofluorometry we could detect
and measure uptake of the bacteria by phagocytosis, similar to
the process known for free swimming “planktonic” bacteria.
After phagocytosis, the neutrophils died by a process known as
”programmed cell death” (apoptosis) 16 (Fig. 1E-H).
3. Immediately after contact with the biofilm, the neutrophils also
released lactoferrin, that in addition to its bactericidal activity
is also known to inhibit biofilm formation what we could confirm
in our experiments 17 (Fig. 1).
In Summary, our data indicate that biofilm infections are readily
recognised by the immune system; Moreover, the appropriate
immune cells infiltrate the infected site and are activated there; and
in addition the biofilms are not inherently protected against the
defence by neutrophils – at least in vitro.
Whether biofilms are attacked and cleared by PMN in vivo is difficult
to prove, because a localised immune defence is likely to
occur unnoticed by the patients. Only unsuccessful attempts of the
host defence become apparent, particularly in late stages, when the
tissue damage is already in an advanced state and the implant is
loosening.
So the questions arise why in those patients the host defence is
insufficient and how the infection is linked to the tissue damage.
With regard to the first question, the classical paradigm for infection
“too little – too late” might hold true also for biofilm infection.