Antimicrobial Activity of Natural and Synthetic Compounds Against Candida spp., Gram-Positive and Gram-Negative Bacteria

Antibiotics resistant bacteria have become over the last few decades one of the greatest threats to global health. Infections caused by multi-drug-resistant pathogens are generally difficult to treat and are often fatal. Biofilm formation has been recognized as one of the most important, and most difficult to treat, among the mechanisms conferring antibiotics tolerance to pathogens. There is currently an urgent need for novel and effective approaches to fight the global health challenge of antibiotic resistance and in particular biofilm based tolerance. In this context, antimicrobial peptides (AMP) and antimicrobial plant extracts are among the most promising solutions. This thesis work was aimed at the discovery, characterization and evaluation of the antimicrobial, and especially anti-biofilm, activity of novel AMPs and novel plant extracts. A set of analyses integrating bioinformatics, synthetic biology and biochemical approaches has been employed to identify new antimicrobial compounds. Clinical phenotype as well as reference fungal and bacterial strains were used to test the antimicrobial and anti-biofilm potential of these new compounds. Antimicrobial activity assays were performed by microdilution method and time kill assays, while anti-biofilm activity was evaluated with biomass quantification, biofilm vitality assessment and by means of confocal laser scanner microscopy. Finally, cytotoxic experiments were performed against human cells to determine if the discovered compounds could be considered promising candidates for the development of topical antimicrobial agents. As a first step, a cryptic AMP-like peptide named VLL-28, identified in the sequence of an archaeal protein was analyzed and characterized with efficient antifungal and anti-biofilm activities against all tested clinical strains. Then, a second cryptic AMP-like peptide (PAP-A3), derived by in silico screening of human proteins, was identified in the Pepsinogen A activation peptide along with two other fragments (IMY25 and FLK22). These three peptides exhibited considerable antimicrobial and anti-biofilm properties against a range of pathogenic bacteria, including foodborne organisms infecting the stomach and biofilm producing strains. Furan-motifs and lignan-motifs were than employed to identify new molecules exhibiting antimicrobial properties. This activity led to the characterization of fourteen synthetic arylfurans and lignan-like arylbenzylfurans, that in turn were tested against Gram-negative (Pseudomonas aeruginosa, Escherichia coli) and Gram-positive bacteria (Staphylococcus aureus and S. epidermidis). One of these compounds, methyl 4-(2-hydroxybenzyl)-2-phenylfuran-3-carboxylate, was found to be active against S. aureus and S. epidermidis. This latter showed also a good anti-biofilm activity and was found to be nontoxic in human cells. Finally, Abietic acid, a tricyclic diterpenoid derived from the resin of pine trees, was tested against Staphylococcus pseudintermedius strains and was found to be able to increase the susceptibility to Methicillin in Methicillin-resistant isolates of S. pseudintermedius (MRSP) by modulating the expression of mec genes. Moreover, Abietic acid also displayed a good anti-biofilm activity by killing almost all cells embedded in biofilm already at very low concentrations. In conclusion, new possible sources were explored in this work to find novel antimicrobial compounds to fight the emerging antimicrobial resistance to conventional drugs. As a result, a series of new and effective antimicrobial agents were detected, characterized and tested against a wide range of pathogens

Antifungal and anti-biofilm activity of the first cryptic antimicrobial peptide from an archaeal protein against Candida spp. clinical isolates

Candida species cause cutaneous and systemic infections with a high mortality rate, especially in immunocompromised patients. The emergence of resistance to the most common antifungal drugs, also due to bioflm formation, requires the development of alternative antifungal agents. The antimicrobial peptide VLL-28, isolated from an archaeal transcription factor, shows comparable antifungal activity against 10 clinical isolates of Candida spp. Using a fuoresceinated derivative of this peptide, we found that VLL-28 binds to the surface of planktonic cells. This observation suggested that it could exert its antifungal activity by damaging the cell wall. In addition, analyses performed on bioflms via confocal microscopy revealed that VLL-28 is diferentially active on all the strains tested, with C. albicans and C. parapsilosis being the most sensitive ones. Notably, VLL-28 is the frst example of an archaeal antimicrobial peptide that is active towards Candida spp. Thus, this points to archaeal microorganisms as a possible reservoir of novel antifungal agent

PYED-1 Inhibits Biofilm Formation and Disrupts the Preformed Biofilm of Staphylococcus aureus

Pregnadiene-11-hydroxy-16α,17α-epoxy-3,20-dione-1 (PYED-1), a heterocyclic corticosteroid derivative of deflazacort, exhibits broad-spectrum antibacterial activity against Gram-negative and Gram-positive bacteria. Here, we investigated the effect of PYED-1 on the biofilms of Staphylococcus aureus, an etiological agent of biofilm-based chronic infections such as osteomyelitis, indwelling medical device infections, periodontitis, chronic wound infections, and endocarditis. PYED-1 caused a strong reduction in biofilm formation in a concentration dependent manner. Furthermore, it was also able to completely remove the preformed biofilm. Transcriptional analysis performed on the established biofilm revealed that PYED-1 downregulates the expression of genes related to quorum sensing (agrA, RNAIII, hld, psm, and sarA), surface proteins (clfB and fnbB), secreted toxins (hla, hlb, and lukD), and capsular polysaccharides (capC). The expression of genes that encode two main global regulators, sigB and saeR, was also significantly inhibited after treatment with PYED-1. In conclusion, PYED-1 not only effectively inhibited biofilm formation, but also eradicated preformed biofilms of S. aureus, modulating the expression of genes related to quorum sensing, surface and secreted proteins, and capsular polysaccharides. These results indicated that PYED-1 may have great potential as an effective antibiofilm agent to prevent S. aureus biofilm-associated infections

Bacterial biofilm formation is variably inhibited by different formulations of antibiotic-loaded bone cement in vitro

PURPOSE: The aim of the present study was to quantitatively assess biofilm growth on the surface of bone cements discs containing different antibiotics, including colistin and linezolid. Biofilms of methicillin-resistant Staphylococcus aureus (MRSA), Pseudomonas aeruginosa, and Staphylococcus epidermidis were grown on bone cement discs for 96 h. METHODS: Biofilm amounts were measured by confocal laser microscopy using live/dead staining and dedicated software at different time intervals (48, 72, and 96 h). RESULTS: Bone cement containing vancomycin was not effective at reducing MRSA biofilm formation 96 h following bacterial inoculation. At a comparable time interval, linezolid-, clindamycin-, and aminoglycoside-loaded cement was still active against this biofilm. At the 72- and 96-h observations, S. epidermidis biofilm was present only on tobramycin and gentamicin discs. P. aeruginosa biofilms were present on cement discs loaded with colistin at all time intervals starting from the 48-h observation, whereas no biofilms were detected on tobramycin or gentamicin discs. CONCLUSION: Bone cements containing different antibiotics have variable and time-dependent windows of activity in inhibiting or reducing surface biofilm formation. The effectiveness of bone cement containing vancomycin against MRSA biofilm is questionable. The present study is clinically relevant, because it suggests that adding the right antibiotic to bone cement could be a promising approach to treat periprosthetic infections. Indeed, the antibiofilm activity of different antibiotic-loaded bone cements could be preoperatively assessed using the current methodology in two-stage exchange procedures

Isolation of Enterobacter aerogenes carrying blaTEM-1 and blaKPC-3 genes recovered from a hospital Intensive Care Unit.

Enterobacter aerogenes has recently emerged as an important hospital pathogen. In this study, we showed the emergence of E. aerogenes isolates carrying the blaKPC gene in patients colonized by carbapenem-resistant Klebsiella pneumoniae strains. Two multiresistant E. aerogenes isolates were recovered from bronchial aspirates of two patients hospitalized in the Intensive Care Unit at the "Santa Maria della Scaletta" Hospital, Imola. The antimicrobial susceptibility test showed the high resistance to carbapenems and double-disk synergy test confirmed the phenotype of KPC and AmpC production. Other investigation revealed that ESBL and blaKPC genes were carried on the conjugative pKpQIL plasmid. This is a relevant report in Italy that describes a nosocomial infection due to the production of KPC beta-lactamases by an E. aerogenes isolate in patients previously colonized by K. pneumoniae carbapenem-resistant. In conclusion, it's necessary a continuous monitoring of multidrug-resistant strains for the detection of any KPC-producing bacteria that could expand the circulation of carbapenem-resistant pathogens

Photodynamic and antibiotic therapy in combination to fight biofilms and resistant surface bacterial infections

Although photodynamic therapy (PDT), a therapeutic approach that involves a photosensitizer, light and O₂, has been principally considered for the treatment of specific types of cancers, other applications exist, including the treatment of infections. Unfortunately, PDT does not always guarantee full success since it exerts lethal effects only in cells that have taken up a sufficient amount of photosensitizer and have been exposed to adequate light doses, conditions that are not always achieved. Based on our previous experience on the combination PDT/chemotherapy, we have explored the possibility of fighting bacteria that commonly crowd infected surfaces by combining PDT with an antibiotic, which normally does not harm the strain at low concentrations. To this purpose, we employed 5-aminolevulinic acid (5-ALA), a pro-drug that, once absorbed by proliferating bacteria, is converted into the natural photosensitizer Protoporphyrin IX (PpIX), followed by Gentamicin. Photoactivation generates reactive oxygen species (ROS) which damage or kill the cell, while Gentamicin, even at low doses, ends the work. Our experiments, in combination, have been highly successful against biofilms produced by several Gram positive bacteria (i.e., Staphylococcus aureus, Staphylococcus epidermidis, etc.). This original approach points to potentially new and wide applications in the therapy of infections of superficial wounds and sores

N-Nonyloxypentyl-l-Deoxynojirimycin Inhibits Growth, Biofilm Formation and Virulence Factors Expression of Staphylococcus aureus

Staphylococcus aureus is one of the major causes of hospital- and community-associated bacterial infections throughout the world, which are difficult to treat due to the rising number of drug-resistant strains. New molecules displaying potent activity against this bacterium are urgently needed. In this study, d- and l-deoxynojirimycin (DNJ) and a small library of their N-alkyl derivatives were screened against S. aureus ATCC 29213, with the aim to identify novel candidates with inhibitory potential. Among them, N-nonyloxypentyl-l-DNJ (l-NPDNJ) proved to be the most active compound against S. aureus ATCC 29213 and its clinical isolates, with the minimum inhibitory concentration (MIC) value of 128 μg/mL. l-NPDNJ also displayed an additive effect with gentamicin and oxacillin against the gentamicin- and methicillin-resistant S. aureus isolate 00717. Sub-MIC values of l-NPDNJ affected S. aureus biofilm development in a dose-dependent manner, inducing a strong reduction in biofilm biomass. Moreover, real-time reverse transcriptase PCR analysis revealed that l-NPDNJ effectively inhibited at sub-MIC values the transcription of the spa, hla, hlb and sea virulence genes, as well as the agrA and saeR response regulator genes

Novel Antimicrobial Peptide from Temporin L in The Treatment of Staphylococcus pseudintermedius and Malassezia pachydermatis in Polymicrobial Inter-Kingdom Infection

Interkingdom polymicrobial diseases are caused by different microorganisms that colonize the same niche, as in the case of yeast-bacteria coinfections. The latter are difficult to treat due the absence of any common therapeutic target for their elimination, both in animals and humans. Staphylococcus pseudintermedius and Malassezia pachydermatis belong to distinct kingdoms. They can colonize the same skin district or apparatus being the causative agents of fastidious pet animals' pathologies. Here we analysed the antimicrobial properties of a panel of 11 peptides, derived from temporin L, against Malassezia pachydermatis. Only peptide 8 showed the best mycocidal activity at 6.25 μM. Prolonged application of peptide 8 did not cause M. pachydermatis drug-resistance. Peptide 8 was also able to inhibit the growth of Staphylococcus pseudintermedius, regardless of methicillin resistance, at 1.56 μM for methicillin-susceptible S. pseudintermedius (MSSP) and 6.25 μM for methicillin-resistant S. pseudintermedius (MRSP). Of interest, peptide 8 increased the susceptibility of MRSP to oxacillin. Oxacillin MIC value reduction was of about eight times when used in combination with peptide 8. Finally, the compound affected the vitality of bacteria embedded in S. pseudintermedius biofilm. In conclusion, peptide 8 might represent a valid therapeutic alternative in the treatment of interkingdom polymicrobial infections, also in the presence of methicillin-resistant bacteria