Human commensals producing a novel antibiotic impair pathogen colonization.

The vast majority of systemic bacterial infections are caused by facultative, often antibiotic-resistant, pathogens colonizing human body surfaces. Nasal carriage of Staphylococcus aureus predisposes to invasive infection, but the mechanisms that permit or interfere with pathogen colonization are largely unknown. Whereas soil microbes are known to compete by production of antibiotics, such processes have rarely been reported for human microbiota. We show that nasal Staphylococcus lugdunensis strains produce lugdunin, a novel thiazolidine-containing cyclic peptide antibiotic that prohibits colonization by S. aureus, and a rare example of a non-ribosomally synthesized bioactive compound from human-associated bacteria. Lugdunin is bactericidal against major pathogens, effective in animal models, and not prone to causing development of resistance in S. aureus. Notably, human nasal colonization by S. lugdunensis was associated with a significantly reduced S. aureus carriage rate, suggesting that lugdunin or lugdunin-producing commensal bacteria could be valuable for preventing staphylococcal infections. Moreover, human microbiota should be considered as a source for new antibiotics.

Secretion of and Self-Resistance to the Novel Fibupeptide Antimicrobial Lugdunin by Distinct ABC Transporters in Staphylococcus lugdunensis.

Lugdunin is the first reported nonribosomally synthesized antibiotic from human microbiomes. Its production by the commensal Staphylococcus lugdunensis eliminates the pathogen Staphylococcus aureus from human nasal microbiomes. The cycloheptapeptide lugdunin is the founding member of the new class of fibupeptide antibiotics, which have a novel mode of action and represent promising new antimicrobial agents. How S. lugdunensis releases and achieves producer self-resistance to lugdunin has remained unknown. We report that two ABC transporters encoded upstream of the lugdunin-biosynthetic operon have distinct yet overlapping roles in lugdunin secretion and self-resistance. While deletion of the lugEF transporter genes abrogated most of the lugdunin secretion, the lugGH transporter genes had a dominant role in resistance. Yet all four genes were required for full-level lugdunin resistance. The small accessory putative membrane protein LugI further contributed to lugdunin release and resistance levels conferred by the ABC transporters. Whereas LugIEFGH also conferred resistance to lugdunin congeners with inverse structures or with amino acid exchange at position 6, they neither affected the susceptibility to a lugdunin variant with an exchange at position 2 nor to other cyclic peptide antimicrobials such as daptomycin or gramicidin S. The obvious selectivity of the resistance mechanism raises hopes that it will not confer cross-resistance to other antimicrobials or to optimized lugdunin derivatives to be used for the prevention and treatment of S. aureus infections.

Synthetic Lugdunin Analogues Reveal Essential Structural Motifs for Antimicrobial Action and Proton Translocation Capability.

Lugdunin, a novel thiazolidine cyclopeptide, exhibits micromolar activity against methicillin-resistant Staphylococcus aureus (MRSA). For structure-activity relationship (SAR) studies, synthetic analogues obtained from alanine and stereo scanning as well as peptides with modified thiazolidine rings were tested for antimicrobial activity. The thiazolidine ring and the alternating d- and l-amino acid backbone are essential. Notably, the non-natural enantiomer displays equal activity, thus indicating the absence of a chiral target. The antibacterial activity strongly correlates with dissipation of the membrane potential in S. aureus. Lugdunin equalizes pH gradients in artificial membrane vesicles, thereby maintaining membrane integrity, which demonstrates that proton translocation is the mode of action (MoA). The incorporation of extra tryptophan or propargyl moieties further expands the diversity of this class of thiazolidine cyclopeptides.

High Frequency and Diversity of Antimicrobial Activities Produced by Nasal Staphylococcus Strains against Bacterial Competitors.

The human nasal microbiota is highly variable and dynamic often enclosing major pathogens such as Staphylococcus aureus. The potential roles of bacteriocins or other mechanisms allowing certain bacterial clones to prevail in this nutrient-poor habitat have hardly been studied. Of 89 nasal Staphylococcus isolates, unexpectedly, the vast majority (84%) was found to produce antimicrobial substances in particular under habitat-specific stress conditions, such as iron limitation or exposure to hydrogen peroxide. Activity spectra were generally narrow but highly variable with activities against certain nasal members of the Actinobacteria, Proteobacteria, Firmicutes, or several groups of bacteria. Staphylococcus species and many other Firmicutes were insusceptible to most of the compounds. A representative bacteriocin was identified as a nukacin-related peptide whose inactivation reduced the capacity of the producer Staphylococcus epidermidis IVK45 to limit growth of other nasal bacteria. Of note, the bacteriocin genes were found on mobile genetic elements exhibiting signs of extensive horizontal gene transfer and rearrangements. Thus, continuously evolving bacteriocins appear to govern bacterial competition in the human nose and specific bacteriocins may become important agents for eradication of notorious opportunistic pathogens from human microbiota.

Cytotoxicity Assays as Predictors of the Safety and Efficacy of Antimicrobial Agents.

The development of safe antimicrobial agents is important for the effective treatment of pathogens. From a multitude of discovered inhibitory compounds, only a few antimicrobial agents are able to enter the market. Many antimicrobials are, on the one hand, quite effective in killing pathogens but, on the other hand, cytotoxic to eukaryotic cells. Cell health can be monitored by various methods. Plasma membrane integrity, DNA synthesis, enzyme activity, and reducing conditions within the cell are known indicators of cell viability and cell death. For a comprehensive overview, methods to analyze cytotoxic and hemolytic effects, e.g., lactate dehydrogenase release, cell proliferation analysis, cell viability analysis based on the activity of different intracellular enzymes, and hemolysis assay of antimicrobial compounds on human cells, are described in this updated chapter.

Cytotoxicity Assays as Predictors of the Safety and Efficacy of Antimicrobial Agents.

The development of safe antimicrobial agents is important for the effective treatment of pathogens. From a multitude of discovered inhibitory compounds only few antimicrobial agents are able to enter the market. Many antimicrobials are, on the one hand, quite effective in killing pathogens but, on the other hand, cytotoxic to eukaryotic cells. Cell health can be monitored by various methods. Plasma membrane integrity, DNA synthesis, enzyme activity, and reducing conditions within the cell are known indicators of cell viability and cell death. For a comprehensive overview, methods to analyze cytotoxic and hemolytic effects, e.g., lactate dehydrogenase release, cell proliferation analysis, cell viability analysis, and hemolysis assay of antimicrobial compounds on human cells, are described in this chapter.

The commensal lifestyle of Staphylococcus aureus and its interactions with the nasal microbiota.

Although human colonization by facultative bacterial pathogens, such as Staphylococcus aureus, represents a major risk factor for invasive infections, the commensal lifestyle of such pathogens has remained a neglected area of research. S. aureus colonizes the nares of approximately 30% of the human population and recent studies suggest that the composition of highly variable nasal microbiota has a major role in promoting or inhibiting S. aureus colonization. Competition for epithelial attachment sites or limited nutrients, different susceptibilities to host defence molecules and the production of antimicrobial molecules may determine whether nasal bacteria outcompete each other. In this Review, we discuss recent insights into mechanisms that are used by S. aureus to prevail in the human nose and the counter-strategies that are used by other nasal bacteria to interfere with its colonization. Understanding such mechanisms will be crucial for the development of new strategies for the eradication of endogenous facultative pathogens.