• anti-inflammatory activity
• anti-virulence
• antimicrobial peptide
• biofilm
• catheter-related
• central venous catheter infections
• chronic infection
• cytokines
• dendrimeric peptide
• EDTA
• human immune response to biofilm
• implanted catheter
• LPS
• microbiological diagnosis
• natural killer cells
• peripheral blood mononuclear cells
• planktonic cells
• port
• primary ciliary dyskinesia
• Pseudomonas aeruginosa
• sputum
• Staphylococcus epidermidis
• virulence factor

Biofilms are one of the most widely distributed and successful form of microbial life and are associated to a significant amount of human infections. They typically contain aggregates of microorganisms adhering to a substrate and embedded in a self-produced matrix of extracellular polymeric substances (EPS) and exhibiting an altered phenotype with respect to growth rate and gene transcription as compared to their planktonic counterparts. Biofilm ability to resist antimicrobial treatments and host immune responses renders biofilm-associated infections as one of the major threats of the modern medicine. These types of infections commonly occur on medical devices and/or host tissues, are typically chronic and may result in serious complications such as sepsis, highly contributing to morbidity and mortality. Despite the extensive biofilm-research carried out in the last decades, many critical aspects of the biofilm physiology and biofilm-associated infections remains to be elucidated. By employing a number of in vitro or ex vivo experimental approaches, the present thesis focused on different areas of biofilm research including: i) the identification of methods to improve the microbiological diagnosis of biofilm infections associated with use of totally implanted venous access ports (TIVAP) that often result in life-threatening blood stream infections (BSI); ii) the dissection of the human immune response to planktonic and biofilm forms of two major biofilm-forming pathogens, Pseudomonas aeruginosa and Staphylococcus epidermidis; iii) the evaluation of the antimicrobial and the immune-modulating activity of a recently described antimicrobial peptide (AMP) as preclinical evaluation of its potential in the therapy of biofilm-associated infections. The results obtained were published in international peer-reviewed journals and contributed to shed light on some of the still poorly investigated areas of biofilm research as further addressed in the following sections.
Impediments to the laboratory diagnosis of biofilm-associated infections are mainly due to a lack of standard and reliable diagnostic tools. Standard culture methods often fail to detach microorganisms from biotic or abiotic surfaces due to their entrapment within the EPS, yielding false negative results. A definitive diagnosis of TIVAP-related blood stream infections (BSI) in particular, requires catheter culture, but there is no consensus on microbiological culture methods to be adopted. A prompt diagnosis of TIVAP-related infections is important to reduce unnecessary catheter removals which leads economic and social burden.
In order to contribute to the improvement of the microbiological diagnosis of TIVAP-related BSI, in this thesis a total of 53 TIVAPs removed because of suspected infection (n=36) or end of use (n=17) were studied. Different parts of the TIVAPs (e.g. reservoir, septum and catheter tip) were subjected to different culture treatments. These included: i) vigorous flushing and/or vortexing of the catheter and septum surfaces as a simple and low time-consuming method; 2) sonication followed by flushing and/or vortexing, as a stronger method for mechanical detachment of bacterial cells from the surfaces; 3) chemical treatment with DL-dithiothreitol (DTT), a method that have been reported to be highly efficient in detaching bacterial cells from infected prostheses, but it has never been applied to silicon catheters. The three methods were also evaluated in an in vitro catheter infection model with Staphylococcus epidermidis. Culture results were compared to those obtained from paired blood cultures drawn from TIVAP and peripheral vein and to the relative differential time to positivity (DTP) as a diagnostic tool before TIVAP removal. The results obtained demonstrated that vigorous flushing/vortexing of the catheter lumen/septum, allows the recovery of a number of microorganisms comparable to that of more complex procedures such as sonication or chemical treatment. Among 24 positive TIVAP cultures, nine were tip culture negative, whereas the corresponding reservoirs and septa were culture positive, supporting the evidence that sending the port reservoir in addition to the catheter tip to the microbiology laboratory may increase the sensitivity and the accuracy of diagnosis. Moreover, a good correlation was observed between DTP and TIVAP cultures (P<0.001), demonstrating that DTP is a useful diagnostic tool to decide device removal or a conservative approach.
During the course of an intravascular catheter-related infection, biofilms might be exposed to immune blood cells with consequences that have yet to be clarified. The majority of studies of the immune response against bacteria focus on planktonic bacteria whereas the immune response against infectious biofilms has been far less investigated. Simultaneous activation of both the innate and the adaptive immune responses in certain circumstances have been reported to exacerbate the clinical course of a biofilm-associated infection. Therefore, understanding the interactions between biofilm bacteria and the immune system may help in identifying new strategies of immune intervention against biofilm-associated infections. In order to lightening these interactions, in this thesis, the response of human peripheral blood mononuclear cells (PBMC) to planktonic and biofilm forms of two major biofilm-forming pathogens, P. aeruginosa and S. epidermidis, was dissected.
Firstly, an in vitro host cell-biofilm interaction model suitable to investigate the PBMC response to P. aeruginosa biofilms was established. The cytotoxic effect exerted by P. aeruginosa on host cells is one of the main hurdles that hampers in vitro study. In order to create an environment simulating the in vivo conditions, mature P. aeruginosa biofilms were obtained in RPMI 1640, a medium normally used for the growth of eukaryotic cells. Optimization of different parameters allowed to obtain well-structured mature biofilms and acceptable rates of cell-vitality in 24 h bacteria:cell co-culture. Interestingly, the results demonstrated that not only P. aeruginosa biofilms induced marked activation and response of PBMC, but also that PBMC or supernatants derived from biofilm:PBMC caused a statistically significant increase in biofilm-associated P. aeruginosa, suggesting a reciprocal complex interaction between host cells and bacterium.
Successively, the same experimental system optimized for P. aeruginosa was employed to compare the PBMC response to planktonic and biofilm forms of both P. aeruginosa and S. epidermidis. As compared to PBMC incubated without bacteria, after 24 h of co-culture with P. aeruginosa, approximately 80% of PBMC were alive while the percentage reached 90% in the case of S. epidermidis, with no statistically significant difference between biofilms and planktonic bacteria for both species. In response to both biofilms and planktonic cells of P. aeruginosa, around 20% of PBMC were activated. In contrast, in the case of S. epidermidis, the planktonic cells induced a statistically higher degree of activation than the biofilms (25% versus 15%; P <0.01). Furthermore, P. aeruginosa-biofilms stimulated pro-inflammatory (TNF-α, IL-1β, IFN-γ, IL-6) and anti-inflammatory (IL-10) cytokine-production at statistically higher levels than their planktonic counterparts, while an opposite trend was observed with S. epidermidis. Differences in the architecture of the biofilms between the two bacterial species could at least partially explain these findings. The results showed that marked differences in the host-cell response depending on the species and/or the mode of growth (biofilms versus planktonic), allowing dissertations on the possible influence of different life-styles in the pathogenesis of P. aeruginosa and S. epidermidis infections.
Since the immune system might be friend or foe against biofilm related infections, novel molecules endowed with anti-biofilm and immune-modulating activities (e.g. AMPs) have been suggested as promising therapeutic leads. In contrast to standard antibiotics, AMPs are generally effective against actively growing bacteria, display rapid killing kinetics, and exhibit anti-inflammatory effects, all properties potentially useful to target biofilm-associated infections. However, in the presence of biological fluids AMPs may lose part of their activity, requiring extensive optimization to improve their therapeutic potential.
In this thesis, a semi-synthetic AMPs, lin-SB056-1 was investigated for its anti-biofilm and anti-inflammatory ability in ex vivo and in vitro models. In combination with the cation-chelator EDTA, lin-SB056-1 was able to significantly reduce ex vivo the load of P. aeruginosa endogenous in the sputum of patients affected by primary ciliary dyskinesia (PCD). In addition, EDTA markedly reduced in vitro the production of relevant calcium-dependent virulence factors of P. aeruginosa (e.g., pyocyanin, proteases, LasA). These findings point to lin-SB056-1/EDTA as a combinatorial strategy endowed with a dual antimicrobial and anti-virulence action against P. aeruginosa, suggesting its therapeutic potential against chronic airway infections.
Recent studies have shown that the anti-microbial and anti-biofilm activity of lin-SB056-1 are enhanced in its further optimized dendrimeric derivative (lin-SB056-1)2-K. In the present thesis, lin-SB056-1 and (lin-SB056-1)2-K were evaluated in terms of anti-inflammatory properties by demonstrating the ability of both peptides to inhibit LPS-elicited production of pro-inflammatory cytokines. In addition to exerting a considerable anti-bacterial and anti-biofilm activity, (lin-SB056-1)2-K and, in minor extent, lin-SB056-1 were found to efficiently bind P. aeruginosa LPS and reduce LPS-stimulated pro-inflammatory cytokine release, thereby providing considerable potential for the control of pathological inflammatory responses associated with P. aeruginosa infections.
In conclusion, the results obtained in the present thesis contributed to shed light on several issues related to biofilm-infections. In particular they helped to: i) disclose evidence-based methods to improve the microbiological diagnosis of biofilm-associated and catheter-related bloodstream infections; ii) add important building blocks in our understanding of the reciprocal interaction between human peripheral blood mononuclear cells and biofilms of two major clinically important bacteria (P. aeruginosa and S. epidermidis); iii) evaluate, at a preclinical level, the therapeutic potential of recently described peptides in dampening the excessive pro-inflammatory response that is an hallmark of many biofilm-associated infections.