Alternative tools and new molecules proposed to face the antibiotic resistance crisis

Multidrug- or pandrug-resistant microorganisms and bacterial biofilms are increasing issue of concern and represent two sides of the same coin, since threaten the very core of modern medicine and the sustainability of an effective, global public health response to the difficult-to-treat infections [Høiby N, 2015; Olsen L, 2015; WHO 2014]. The increasing awareness of the reduced efficacy of antibiotic has prompted the renewing of antibiotic discovery and development programs to fight the antibiotic resistance crisis. On this basis, strengthening behaviors have been aimed at reducing the dissemination of multidrug resistant pathogens (e.g. surveillance, infection control practices and adoption of antimicrobial stewardship programs) [Rossolini GM, 2014; Yokoe DS, 2014]. This PhD project approached this intriguing issue from different points of view. With the purpose of evaluating the efficacy of recently introduced antimicrobial agents, the bactericidal activity of ceftaroline, a new broad-spectrum cephalosporin, was analyzed against mature Staphylococcus aureus biofilms. The detected activity of long-term treatment against mature staphylococcal biofilms reinforces previous findings and encourages further studies to consolidate ceftaroline within the armamentarium for the treatment of biofilm-associated staphylococcal infections. Moreover, the evaluation of antimicrobial and anti-biofilm activity of well-known mucolytic agent N-acetylcysteine (NAC) have led to highlight promising efficacy against some clinically relevant respiratory pathogens (i.e. Pseudomonas aeruginosa, S. aureus, Stenotrophomonas maltophilia, Burkholderia cepacia complex, and Haemophilus influenzae). Obtained results bolstering foregoing findings and demonstrating for the first time the efficacy of NAC against emergent respiratory pathogens such as S. maltophilia and B. cepacia complex. In addition, no modulatory effect of NAC was observed on the activity of antibiotics with the exception of carbapenems, thus paving the way to further evaluation about a possible synergistic interaction between NAC and antibiotics against cells both in planktonic phase and biofilm. The infection control practices were dealt by evaluating antimicrobial coatings composed of nanoparticles synthetized by supersonic cluster beam deposition apparatus (i.e. innovative deposition method which produces clusters without chemical synthesis). A broad-spectrum bactericidal activity of pure silver nanoparticles films deposited on microscope slides was demonstrated for the first time. Furthermore, the bactericidal activity of bi-metal nanoparticles (i.e. 50% silver and 50% titanium) on commercial chrome-plated support were analyzed, demonstrating that titanium remarkably enhances the adhesion of nanoparticles upon the supports and provides a comparable antimicrobial activity of pure silver nanoparticles, saving at the same time more than 85% of the expensive metal. Further exploitation of the supersonic cluster beam deposition apparatus would aim to generate nanoparticles coating with broader antimicrobial spectrum and anti-biofilm activity. Several worldwide surveillance studies have shown the clonal dissemination of KPC-type carbapenemase-producing Klebsiella pneumoniae belonging to clonal complex 258 (CC258), characterized by extensively drug resistant phenotypes and ability to rapidly disseminate in healthcare settings. In order to deeply investigate the two differentially distributed lineages (i.e. clade I and clade II) belonging the CC258, genome analysis and biofilm forming assays were carried out. The prevalent distribution of clade II has been awarded to difference in the mrk mannose-resistant fimbrial gene cluster, since non-silent mutation on mrkABCDFHIJ cluster genes detected on clade I genome determined reduced fimbrial synthesis and biofilm forming ability on abiotic surfaces. Further studies aimed to evaluate the role of this mutation in the adhesion on biotic surfaces are needed to deeply explain such widespread dissemination. Among novel techniques proposed to go beyond the antibiotic resistance crisis, the laser therapy has been considered an alternative tool. On this basis, the anti-biofilm activity of λ 808-nm diode laser was assessed. Mature staphylococcal biofilms grown on titanium discs (surface of the osseous interface of dental implants), however, were not eradicated by λ 808-nm diode laser, even if a significant reduction of microbial load was highlighted. λ 808-nm diode laser could be considered a valuable tool for increasing the odds of successful control of peri-implant diseases, probably combined with conventional therapy. In conclusion, alternative tools and new molecules to fight difficult-to-treat infection (i.e. ceftaroline, N-acetylcysteine, and laser therapy), to contain and limit the indirect transmission of pathogens (i.e. antimicrobial coating composed of mono- or bi-metal nanoparticles), and to get novel insights on clonal dissemination of high-risk clone, are shown. Present results suggest encouraging outcome, but more efforts should be carried out in order to avoid the return to “pre-antibiotic era”.