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The human immune cell response against bacterial biofilms is a crucial, but still poorly investigated area of research. Herein, we aim to establish an in vitro host cell-biofilm interaction model suitable to investigate the peripheral blood mononuclear cell (PBMC) response to Pseudomonas aeruginosa biofilms. P. aeruginosa biofilms were obtained by incubating bacteria in complete RPMI 1640 medium with 10% human plasma for 24 h. PBMC obtained from healthy donors were added to preformed P. aeruginosa biofilms. Following a further 24 h incubation, we assessed (i) PBMC viability and activation; (ii) cytokine profiles in the supernatants; and (iii) CFU counts of biofilm forming bacteria. Cell-death was

In primary ciliary dyskinesia (PCD) patients, Pseudomonas aeruginosa is a major opportunistic pathogen, frequently involved in chronic infections of the lower airways. Infections by this bacterial species correlates with a worsening clinical prognosis and recalcitrance to currently available therapeutics. The antimicrobial peptide, lin-SB056-1, in combination with the cation chelator ethylenediaminetetraacetic acid (EDTA), was previously demonstrated to be bactericidal against P. aeruginosa in an artificial sputum medium. The purpose of this study was to validate the anti-P. aeruginosa activity of such a combination in PCD sputum and to evaluate the in vitro anti-virulence effects of EDTA. In combination with EDTA, lin-SB056-1 was able to significantly reduce the load of endogenous P. aeruginosa ex vivo in the sputum of PCD patients. In addition, EDTA markedly reduced the production of relevant bacterial virulence factors (e.g., pyocyanin, proteases, LasA) in vitro by two representative mucoid strains of P. aeruginosa isolated from the sputum of PCD patients. These results indicate that the lin-SB056-1/EDTA combination may exert a dual antimicrobial and anti-virulence action against P. aeruginosa, suggesting a therapeutic potential against chronic airway infections sustained by this bacterium.

Antimicrobial peptides (AMPs) are promising templates for the development of novel antibiofilm drugs. Despite the large number of studies on screening and optimization of AMPs, only a few of these evaluated the antibiofilm activity in physiologically relevant model systems. Potent in vitro activity of AMPs often does not translate into in vivo effectiveness due to the interference of the host microenvironment with peptide stability/availability. Hence, mimicking the complex environment found in biofilm-associated infections is essential to predict the clinical potential of novel AMP-based antimicrobials. In the present study, we examined the antibiofilm activity of the semisynthetic peptide lin-SB056-1 and its dendrimeric derivative (lin-SB056-1)(2)-K against Pseudomonas aeruginosa in an in vivo-like three-dimensional (3-D) lung epithelial cell model and an in vitro wound model (consisting of an artificial dermis and blood components at physiological levels). Although moderately active when tested alone, lin-SB056-1 was effective in reducing P aeruginosa biofilm formation in association with 3-D lung epithelial cells in combination with the chelating agent EDTA. The dimeric derivative (Iin-SB056-1)(2)-K demonstrated an enhanced biofilm-inhibitory activity as compared to both lin-SB056-1 and the lin-SB056-1/EDTA combination, reducing the number of biofilm-associated bacteria up to 3-Log units at concentrations causing less than 20% cell death. Biofilm inhibition by (lin-SB056-1)(2)-K was reported both for the reference strain PAO1 and cystic fibrosis lung isolates of P aeruginosa. In addition, using fluorescence microscopy, a significant decrease in biofilm-like structures associated with 3-D cells was observed after peptide exposure. Interestingly, effectiveness of (lin-SB056-1)(2)-K was also demonstrated in the wound model with a reduction of up to 1-Log unit in biofilm formation by P aeruginosa PAO1 and wound isolates. Overall, combination treatment and peptide dendrimerization emerged as promising strategies to improve the efficacy of AMPs, especially under challenging host-mimicking conditions. Furthermore, the results of the present study underlined the importance of evaluating the biological properties of novel AMPs in in vivo-like model systems representative of specific infectious sites in order to make a more realistic prediction of their therapeutic success, and avoid the inclusion of unpromising peptides in animal studies and clinical trials.

Pseudomonas aeruginosa is a major cause of chronic lung infections in cystic fibrosis (CF) patients. The ability of the bacterium to form biofilms and the presence of a thick and stagnant mucus in the airways of CF patients largely contribute to antibiotic therapy failure and demand for new antimicrobial agents able to act in the CF environment. The present study investigated the anti-P. aeruginosa activity of lin-SB056-1, a recently described semi-synthetic antimicrobial peptide, used alone and in combination with the cation chelator ethylenediaminetetraacetic acid (EDTA). Bactericidal assays were carried out in standard culture conditions and in an artificial sputum medium (ASM) closely resembling the CF environment. Peptide’s structure and interaction with large unilamellar vesicles in media with different ionic strengths were also investigated through infrared spectroscopy. Lin-SB056-1 demonstrated fast and strong bactericidal activity against both mucoid and non-mucoid strains of P. aeruginosa in planktonic form and, in combination with EDTA, caused significant reduction of the biomass of P. aeruginosa mature biofilms. In ASM, the peptide/EDTA combination exerted a strong bactericidal effect and inhibited the formation of biofilm-like structures of P. aeruginosa. Overall, the results obtained highlight the potential of the lin-SB056-1/EDTA combination for the treatment of P. aeruginosa lung infections in CF patients.

The frog skin-derived peptide Temporin 1Tb (TB) has gained increasing attention as novel antimicrobial agent for the treatment of antibiotic-resistant and/or biofilm-mediated infections. Nevertheless, such a peptide possesses a preferential spectrum of action against Gram-positive bacteria. In order to improve the therapeutic potential of TB, the present study evaluated the antibacterial and antibiofilm activities of two TB analogs against medically relevant bacterial species. Of the two analogs, TB_KKG6A has been previously described in the literature, while TB_L1FK is a new analog designed by us through statistical-based computational strategies. Both TB analogs displayed a faster and stronger bactericidal activity than the parental peptide, especially against Gram-negative bacteria in planktonic form. Differently from the parental peptide, TB_KKG6A and TB_L1FK were able to inhibit the formation of Staphylococcus aureus biofilms by more than 50% at 12 μM, while only TB_KKG6A prevented the formation of Pseudomonas aeruginosa biofilms at 24 μM. A marked antibiofilm activity against preformed biofilms of both bacterial species was observed for the two TB analogs when used in combination with EDTA. Analysis of synergism at the cellular level suggested that the antibiofilm activity exerted by the peptide-EDTA combinations against mature biofilms might be due mainly to a disaggregating effect on the extracellular matrix in the case of S. aureus, and to a direct activity on biofilm-embedded cells in the case of P. aeruginosa. Both analogs displayed a low hemolytic effect at the active concentrations and, overall, TB_L1FK resulted less cytotoxic toward mammalian cells. Collectively, the results obtained demonstrated that subtle changes in the primary sequence of TB may provide TB analogs that, used alone or in combination with adjuvant molecules such as EDTA, exhibit promising features against both planktonic and biofilm cells of medically relevant bacteria.

In search of new antimicrobials with anti-biofilm potential, in the present study activity of the frog-skin derived antimicrobial peptide temporin 1Tb (TB) against Staphylococcus epidermidis biofilms was investigated. A striking ability of TB to kill both forming and mature S. epidermidis biofilms was observed, especially when the peptide was combined with cysteine or EDTA, respectively. Kinetics studies demonstrated that the combination TB/EDTA was active against mature biofilms already after 2-4-h exposure. A double 4-h exposure of biofilms to TB/EDTA further increased the therapeutic potential of the same combination. Of note, TB/EDTA was able to eradicate S. epidermidis biofilms formed in vitro on silicone catheters. At eradicating concentrations, TB/EDTA did not cause hemolysis of human erythrocytes. The results shed light on the anti-biofilm properties of TB and suggest a possible application of the peptide in the lock therapy of catheters infected with S. epidermidis.