Synthesis and Antimicrobial Activity of Short Analogues of the Marine Antimicrobial Peptide Turgencin A: Effects of SAR Optimizations, Cys-Cys Cyclization and Lipopeptide Modifications.

We have synthesised short analogues of the marine antimicrobial peptide Turgencin A from the colonial Arctic ascidian Synoicum turgens. In this study, we focused on a central, cationic 12-residue Cys-Cys loop region within the sequence. Modified (tryptophan- and arginine-enriched) linear peptides were compared with Cys-Cys cyclic derivatives, and both linear and Cys-cyclic peptides were N-terminally acylated with octanoic acid (C8), decanoic acid (C10) or dodecanoic acid (C12). The highest antimicrobial potency was achieved by introducing dodecanoic acid to a cyclic Turgencin A analogue with low intrinsic hydrophobicity, and by introducing octanoic acid to a cyclic analogue displaying a higher intrinsic hydrophobicity. Among all tested synthetic Turgencin A lipopeptide analogues, the most promising candidates regarding both antimicrobial and haemolytic activity were C12-cTurg-1 and C8-cTurg-2. These optimized cyclic lipopeptides displayed minimum inhibitory concentrations of 4 µg/mL against Staphylococcus aureus, Escherichia coli and the fungus Rhodothorula sp. Mode of action studies on bacteria showed a rapid membrane disruption and bactericidal effect of the cyclic lipopeptides. Haemolytic activity against human erythrocytes was low, indicating favorable selective targeting of bacterial cells.

Amphipathic sulfonamidobenzamides mimicking small antimicrobial marine natural products; investigation of antibacterial and anti-biofilm activity against antibiotic resistant clinical isolates.

There is an urgent need for novel antimicrobial agents to address the threat of bacterial resistance to modern society. We have used a structural motif found in antimicrobial marine hit compounds as a basis for synthesizing a library of antimicrobial sulfonamidobenzamide lead compounds. Potent in vitro antimicrobial activity against clinically relevant bacterial strains was demonstrated for two compounds, G6 and J18, with minimal inhibitory concentrations (MIC) of 4-16 µg/ml against clinical methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococcus faecium (VRE). The two compounds G6 and J18, together with several other compounds of this library, also caused ≥90% eradication of pre-established biofilm of methicillin-resistant S. epidermidis (MRSE) at 40 µg/ml. Using a luciferase assay, the mechanism of action of G6 was shown to resemble the biocide chlorhexidine by targeting the bacterial cell membrane.

Synthesis and antimicrobial activity of small cationic amphipathic aminobenzamide marine natural product mimics and evaluation of relevance against clinical isolates including ESBL-CARBA producing multi-resistant bacteria.

A library of small aminobenzamide derivatives was synthesised to explore a cationic amphipathic motif found in marine natural antimicrobials. The most potent compound E23 displayed minimal inhibitory concentrations (MICs) of 0.5-2µg/ml against several Gram-positive bacterial strains, including methicillin resistant Staphylococcus epidermidis (MRSE).E23 was also potent against 275 clinical isolates including Staphylococcus aureus, Enterococcus spp., Escherichia coli, Pseudomonas aeruginosa, and Klebsiella pneumoniae, as well as methicillin-resistant S. aureus (MRSA), vancomycin-resistant enterococci (VRE), and ESBL-CARBA producing multi-resistant Gram-negative bacteria. The study demonstrates how structural motifs found in marine natural antimicrobials can be a valuable source for making novel antimicrobial lead-compounds.

Dominance Between Plasmids Determines the Extent of Biofilm Formation.

Bacterial biofilms have an impact in medical and industrial environments because they often confer protection to bacteria against harmful agents, and constitute a source from which microorganisms can disperse. Conjugative plasmids can enhance bacterial ability to form biofilms because conjugative pili act as adhesion factors. However, plasmids may interact with each other, either facilitating or inhibiting plasmid transfer. Accordingly, we asked whether effects on plasmid transfer also impacts biofilm formation. We measured biofilm formation of Escherichia coli cells harboring two plasmid types, or when the two plasmids were present in the same population but carried in different cells. Using eleven natural isolated conjugative plasmids, we confirmed that some indeed promote biofilm formation and, importantly, that this ability is correlated with conjugative efficiency. Further we studied the effect of plasmid pairs on biofilm formation. We observed increased biofilm formation in approximately half of the combinations when both plasmids inhabited the same cell or when the plasmids were carried in different cells. Moreover, in approximately half of the combinations, independent of the co-inhabitation conditions, one of the plasmids alone determined the extent of biofilm formation - thus having a dominant effect over the other plasmid. The molecular mechanisms responsible for these interactions were not evaluated here and future research is required to elucidate them.

Coagulase-negative staphylococci in Southern Brazil: looking toward its high diversity.

INTRODUCTION: Coagulase-negative staphylococci (CoNS) are the most prevalent pathogens in nosocomial infections and may serve as a reservoir of mobile genetic elements such as the staphylococcal cassette chromosome mec (SCCmec) encoding methicillin resistance. Molecular characterization of SCCmec types combined with advanced molecular typing techniques may provide essential information for understanding the evolution and epidemiology of CoNS infections. We therefore aimed to investigate the SCCmec distribution, multidrug-resistance (MDR), and biofilm formation in CoNS blood culture isolates from a hospital in Southern Brazil. METHODS: We analyzed 136 CoNS blood culture isolates obtained during 2002-2004 from patients admitted to a tertiary care hospital in Brazil. SCCmec types I to V were determined using multiplex PCR. The clonal relationship of Staphylococcus epidermidis was determined using pulsed field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). Molecular epidemiological data were interpreted along with data on biofilm formation, presence of the icaD gene, and MDR. RESULTS: The most prevalent species were S. epidermidis, Staphylococcus haemolyticus, and Staphylococcus hominis harboring mainly SCCmec types II, III, and V. Overall, the presence of multiple SCCmec was associated with non-MDR, except for S. epidermidis. S. epidermidis isolates showed a high prevalence of icaD, but had low phenotypic biofilm formation. PFGE and MLST revealed high genetic diversity in the S. epidermidis population. CONCLUSIONS: Our results suggest a major shift in SCCmec types within a short period and reveal a different behavior of S. epidermidis with regard to the association between the presence of multiple SCCmec types and MDR profile.

Staphylococcus epidermidis polysaccharide intercellular adhesin activates complement.

Staphylococcus epidermidis is a frequent cause of nosocomial infections. The central virulence factor of S. epidermidis is biofilm formation. Polysaccharide intercellular adhesin (PIA) constitutes the major biofilm matrix-component. PIA and biofilm have been implicated in S. epidermidis evasion of host immune defence. We examined the effects of S. epidermidis PIA on the inflammatory response with focus on complement activation. We used a human whole-blood ex vivo model of infection and compared the effects of a PIA-positive S. epidermidis strain (SE1457) and its PIA-negative isogenic mutant (M10). The independent effect of purified PIA on complement activation was investigated. In glucose-rich media, the mutant formed a proteinacious DNA-rich biofilm, whereas SE1457 formed a thick PIA-biofilm. In biofilm growth, SE1457 induced a stronger activation of the complement system compared with M10. We verified that purified PIA was independently responsible for a strong activation of the complement system. In contrast, M10 induced higher granulocyte activation by expression of CD11b and higher secretion of cytokines. We conclude that PIA has potent pro-inflammatory properties by activating the complement system. However, in a complex balance of the immune response, the decreased activation of granulocytes and cytokines by a PIA biofilm may limit host eradication of S. epidermidis.

Coagulase-negative staphylococci in Southern Brazil: looking toward its high diversity

Abstract: INTRODUCTION: Coagulase-negative staphylococci (CoNS) are the most prevalent pathogens in nosocomial infections and may serve as a reservoir of mobile genetic elements such as the staphylococcal cassette chromosome mec (SCCmec) encoding methicillin resistance. Molecular characterization of SCCmec types combined with advanced molecular typing techniques may provide essential information for understanding the evolution and epidemiology of CoNS infections. We therefore aimed to investigate the SCCmec distribution, multidrug-resistance (MDR), and biofilm formation in CoNS blood culture isolates from a hospital in Southern Brazil. METHODS: We analyzed 136 CoNS blood culture isolates obtained during 2002-2004 from patients admitted to a tertiary care hospital in Brazil. SCCmec types I to V were determined using multiplex PCR. The clonal relationship of Staphylococcus epidermidis was determined using pulsed field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). Molecular epidemiological data were interpreted along with data on biofilm formation, presence of the icaD gene, and MDR. RESULTS: The most prevalent species were S. epidermidis, Staphylococcus haemolyticus, and Staphylococcus hominis harboring mainly SCCmec types II, III, and V. Overall, the presence of multiple SCCmec was associated with non-MDR, except for S. epidermidis. S. epidermidis isolates showed a high prevalence of icaD, but had low phenotypic biofilm formation. PFGE and MLST revealed high genetic diversity in the S. epidermidis population. CONCLUSIONS: Our results suggest a major shift in SCCmec types within a short period and reveal a different behavior of S. epidermidis with regard to the association between the presence of multiple SCCmec types and MDR profile.