A series of new hybrid structures containing fluoroquinolone (ciprofloxacin) and aminoglycoside (neomycin) antibiotics linked via 1,2,3-triazole moiety were designed and synthesized, and their antibacterial activities were determined against both Gram-negative and Gram-positive bacteria, including resistant strains. The nature of spacers in both the ciprofloxacin and neomycin parts greatly influenced the antibacterial activity. The majority of hybrids was significantly more potent than the parent neomycin and overcame most prevalent types of resistance associated with aminoglycosides. Selected hybrids inhibited bacterial protein synthesis with the potencies similar to or better than that of neomycin and were up to 32-fold more potent inhibitors than ciprofloxacin for the fluoroquinolone targets, DNA gyrase and toposiomerase IV, indicating a balanced dual mode of action. Significant delay of resistance formation was observed in both E. coli and B. subtilis to the treatment with ciprofloxacin-neomycin hybrid in comparison to that of each drug separately or their 1:1 mixture.

The approach of dual-acting hybrid antibiotics holds significant current promise in overcoming existing resistance mechanisms, as three of such compounds are entering clinical trials. However, the key challenge in this area should be a broader experimental demonstration of whether the "synergistic effect" or the "antagonistic effect" of the developed hybrid drug is better at preventing/reducing the evolution of resistance. This fundamental challenge must be overcome before yielding a successful drug.

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Background: IcaB is a poly-␤-1,6-N-acetyl-D-glucosamine (PNAG) deacetylase required for polysaccharide intercellular adhesion-dependent biofilm formation by staphylococci. Results: The structure of Ammonifex degensii IcaB has been determined and its catalytic mechanism and localization characterized. Conclusion: IcaB is a membrane-associated PNAG deacetylase that uses an altered catalytic mechanism relative to other family 4 carbohydrate esterases. Significance: First structural characterization of a Gram-positive PNAG deacetylase.

A polymer of partially de-N-acetylated β-1,6-linked N-acetylglucosamine (dPNAG), also known as the polysaccharide intercellular adhesin (PIA), is an important component of many bacterial biofilm matrices. In Staphyloccocus epidermidis, the poly-N-acetylglucosamine polymer is partially de-N-acetylated by the extracellular protein IcaB. To understand the mechanism of action of IcaB, the enzyme was overexpressed and purified. IcaB demonstrates metal-dependent de-N-acetylase activity on β-1,6-linked N-acetylglucosamine oligomers with a broad preference for divalent metals. Steady-state kinetic analysis reveals the low catalytic efficiency (pentasaccharide kcat/KM 0.03 M(-1) s(-1)) of the enzyme toward the oligomeric substrates. While IcaB displays similar rates of de-N-acetylation with tri- through hexasaccharide PNAG oligomers, position specific de-N-acetylation was only observed with penta- and hexasaccharides. The enzyme preferentially de-N-acetylates the second residue from the reducing terminus in the pentasaccharide and second and third residues from the reducing terminus in the hexasaccharide. The data described here represent an important step toward a detailed understanding of dPNAG biosynthesis in S. epidermidis, an important nosocomial pathogen, as well as in other Gram-positive bacteria. The low catalytic activity of IcaB is consistent with reports of other enzymes which act on biofilm-related polysaccharides, and this emerging trend may indicate a common feature among this group of polysaccharide processing enzymes.