• confocal laser scanning microscopy
• chronic rhinosinusitis
• bacterial biofilms
• isothermal microcalorimetry
• antibiotic susceptibility testing

Chronic rhinosinusitis (CRS), the most common illness among chronic disorders, affects persons of all age groups all over the world, determining a reduced quality-of-life and sense of health for the patients and consequently a socioeconomic burden on the community. An inflammatory process that involves the paranasal sinuses and persists for twelve weeks or longer characterises CRS. Despite being widespread, little is known about the aetiology of the disease. CRS is considered a heterogeneous multifactorial inflammatory disorder; however, recent evidence showing biofilms of microorganisms within the paranasal sinuses, suggests a role for biofilm in the pathogenesis.
Biofilms are structured and specialised communities of adherent/sessile microorganisms (bacteria/fungi) embedded in a matrix rich in polysaccharides, nucleic acids, and proteins. The biofilm state potentially provides a continuous presence of microbial antigens resulting in a prolonged mucosal inflammatory response (chronic inflammation).
Biofilm-associated infections are rarely resolved by the host defences and are characterised by acute exacerbations. The biofilm-forming microbial community is often difficult to define and culture. Moreover, microorganisms in biofilms are refractory to conventional drugs and their eradication results difficult or impossible to obtain with current antimicrobial therapy, despite the fact that the responsible microorganisms are susceptible to killing in the “free floating” planktonic state.
In order to evaluate the role of biofilms in the pathogenesis of CRS this thesis was focused on: i) identification of bacteria potentially involved in the pathogenesis of CRS; ii) investigation of the presence of biofilms on the paranasal sinus mucosa of CRS patients; iii) evaluation of the in vitro biofilm-forming ability of CRS strains isolated from infection sites; and iv) determination of antibiotic susceptibility profiles of bacteria in biofilm state in comparison to standard antibiograms.
The study group included eight patients with CRS and two controls who underwent nasal surgery for other pathologies. In collaborations with otorhinolaryngologists from Pisa University Hospital the following samples were collected: i) nasal swabs before the surgical intervention as an indicator of the “normal” bacterial flora, ii) swabs from the specific infection site, iii) biopsies at the time of functional endoscopic sinus surgery and iv) sinus aspirates (collected only from patients with rhinorrhea). The identification and drug sensitivity tests of the isolated bacteria in planktonic form were performed by routine microbiology techniques. In addition, confocal laser scanning microscopy (CLSM) was used to detect the presence of biofilms on biopsies. In vitro biofilm forming capacity of bacterial isolates was assessed by microtiter plate assays and crystal violet staining after 24h culture. The susceptibility profiles to antibiotics of S. aureus and P. mirabilis biofilms were also obtained using a new method based on isothermal microcalorimetry.
Staphylococci represented the majority of the isolates obtained from the infection site. Other isolates were represented by Enterobacteriaceae or by species present in the oral flora. CLSM of the mucosal biopsies taken from patients with CRS revealed the presence of monomicrobial or polymicrobial biofilms in majority of the specimens. In concordance, almost all bacteria isolated from the specific infection site of the CRS patients were able to form biofilm in vitro. No biofilm was observed by CLSM in the biopsies from control patients, although the same biopsies were positive for staphylococci in microbiological culture analysis. Drug-susceptibility tests have shown that the antibiogram profile of planktonic bacteria differs from that of sessile bacteria in biofilms. Bacteria in biofilm exhibited much higher antibiotic resistance levels, up to 1000 fold compared to those observed in planktonic form.
The consistent demonstration of structured sessile bacteria on mucosa biopsies and the presence of in vitro biofilm-forming bacteria in CRS patients suggest that biofilms may play a significant role in the pathogenesis/persistence of the disease. The results obtained also suggest that the choice of an eventual antimicrobial therapy to treat/prevent CRS could benefit from evaluation of the antibiotic sensitivity profiles of sessile bacteria.