Commensal production of a broad-spectrum and short-lived antimicrobial peptide polyene eliminates nasal Staphylococcus aureus.

Antagonistic bacterial interactions often rely on antimicrobial bacteriocins, which attack only a narrow range of target bacteria. However, antimicrobials with broader activity may be advantageous. Here we identify an antimicrobial called epifadin, which is produced by nasal Staphylococcus epidermidis IVK83. It has an unprecedented architecture consisting of a non-ribosomally synthesized peptide, a polyketide component and a terminal modified amino acid moiety. Epifadin combines a wide antimicrobial target spectrum with a short life span of only a few hours. It is highly unstable under in vivo-like conditions, potentially as a means to limit collateral damage of bacterial mutualists. However, Staphylococcus aureus is eliminated by epifadin-producing S. epidermidis during co-cultivation in vitro and in vivo, indicating that epifadin-producing commensals could help prevent nasal S. aureus carriage. These insights into a microbiome-derived, previously unknown antimicrobial compound class suggest that limiting the half-life of an antimicrobial may help to balance its beneficial and detrimental activities.

Detection and evaluation of the antimicrobial activity of Micrococcin P1 isolated from commensal and environmental staphylococcal isolates against MRSA.

BACKGROUND: Bacteriocins (of different origins) have been proposed as promising alternatives to face antimicrobial resistance-associated health problems. Isolates of the Staphylococcus genus are well-known bacteriocin producers, especially coagulase-negative species. METHODS: Twenty-eight bacteriocin-producing staphylococcal isolates were selected from a previous study for in-depth characterisation. The antimicrobial activities (AA) of the producing isolates were studied by the spot-on-lawn method and their crude cell-free supernatants (CFS) and butanol extracts (BT) were evaluated by agar diffusion assays against six indicator bacteria, including multidrug-resistant and zoonotic isolates (such as Listeria monocytogenes or methicillin-resistant Staphylococcus aureus [MRSA]). RESULTS: Six bacteriocin-producing isolates showed AA in their CFS, whereas all staphylococcal BT extracts inhibited at least one of the tested indicator bacteria. Micrococcin P1 (MP1) bacteriocin was detected by mass spectrometry in four producing isolates: Staphylococcus aureus-C5802, Staphylococcus hominis-C5835, Staphylococcus sciuri-X3041, and -X3011. Growth curves performed with CFS and BT extracts of the four MP1 producers revealed a strong AA profile against MRSA and Listeria monocytogenes, even when considerably diluted. Moreover, synergism between the BT extract of MP1-producing Staphylococcus sciuri-X3041 and several antibiotics against an MRSA indicator was observed: BT-clindamycin (> 80%) and BT-oxacillin (30%) combinations. For the BT-chloramphenicol combination, synergism and near synergism values were observed in 37% of the combinations. Competition studies revealed potent inhibitory effects of the MP1-producing isolates against the MRSA indicator. CONCLUSION: These results help to identify Staphylococcus isolates or their bacteriocins as interesting candidates for potential future applications.

Genomic Analysis of Bacteriocin-Producing Staphylococci: High Prevalence of Lanthipeptides and the Micrococcin P1 Biosynthetic Gene Clusters.

Bacteriocins are antimicrobial peptides produced by bacteria. This study aimed to in silico analyze the presence of bacteriocin gene clusters (BGCs) among the genomes of 22 commensal Staphylococcus isolates from different origins (environment/human/food/pet/wild animals) previously identified as bacteriocin producers. The resistome and plasmidome were studied in all isolates. Five types of BGC were detected in 18 genomes of the 22 bacteriocin-producing staphylococci included in this study: class I (Lanthipeptides), class II, circular bacteriocins, the non-ribosomal-peptide lugdunin and the thiopeptide micrococcin P1 (MP1). A high frequency of lanthipeptides was detected in this collection: BGC variants of BSA, bacCH91, and epilancin15X were identified in two Staphylococcus aureus and one Staphylococcus warneri isolates from food and wild animals. Moreover, two potentially new lanthipeptide-like BGCs with no identity to database entries were found in Staphylococcus epidermidis and Staphylococcus simulans from food and wild animal, respectively. Interestingly, four isolates (one S. aureus and one Staphylococcus hominis, environmental origin; two Staphylococcus sciuri, food) carried the MP1 BGC with differences to those previously described. On the other hand, seven of the 22 genomes (~32%) lacked known genes related with antibiotic or disinfectant-acquired resistance mechanisms. Moreover, the potential carriage of plasmids was evaluated, and several Rep-proteins were identified (~73% of strains). In conclusion, a wide variety of BGCs has been observed among the 22 genomes, and an interesting relationship between related Staphylococcus species and the type of bacteriocin has been revealed. Therefore, bacteriocin-producing Staphylococcus and especially coagulase-negative staphylococci (CoNS) can be considered good candidates as a source of novel bacteriocins.

Horizontal Transfer of Bacteriocin Biosynthesis Genes Requires Metabolic Adaptation To Improve Compound Production and Cellular Fitness.

Biosynthetic gene clusters (BGCs) encoding the production of bacteriocins are widespread among bacterial isolates and are important genetic determinants of competitive fitness within a given habitat. Staphylococci produce a tremendous diversity of compounds, and the corresponding BGCs are frequently associated with mobile genetic elements, suggesting gain and loss of biosynthetic capacity. Pharmaceutical biology has shown that compound production in heterologous hosts is often challenging, and many BGC recipients initially produce small amounts of compound or show reduced growth rates. To assess whether transfer of BGCs between closely related Staphylococcus aureus strains can be instantly effective or requires elaborate metabolic adaptation, we investigated the intraspecies transfer of a BGC encoding the ribosomally synthesized and posttranslationally modified peptide (RiPP) micrococcin P1 (MP1). We found that acquisition of the BGC by S. aureus RN4220 enabled immediate MP1 production but also imposed a metabolic burden, which was relieved after prolonged cultivation by adaptive mutation. We used a multiomics approach to study this phenomenon and found adaptive evolution to select for strains with increased activity of the tricarboxylic acid cycle (TCA), which enhanced metabolic fitness and levels of compound production. Metabolome analysis revealed increases of central metabolites, including citrate and α-ketoglutarate in the adapted strain, suggesting metabolic adaptation to overcome the BGC-associated growth defects. Our results indicate that BGC acquisition requires genetic and metabolic predispositions, allowing the integration of bacteriocin production into the cellular metabolism. Inappropriate metabolic characteristics of recipients can entail physiological burdens, negatively impacting the competitive fitness of recipients within natural bacterial communities. IMPORTANCE Human microbiomes are critically associated with human health and disease. Importantly, pathogenic bacteria can hide in human-associated communities and can cause disease when the composition of the community becomes unbalanced. Bacteriocin-producing commensals are able to displace pathogens from microbial communities, suggesting that their targeted introduction into human microbiomes might prevent pathogen colonization and infection. However, to develop probiotic approaches, strains are needed that produce high levels of bioactive compounds and retain cellular fitness within mixed bacterial communities. Our work offers insights into the metabolic burdens associated with the production of the bacteriocin micrococcin P1 and highlights evolutionary strategies that increase cellular fitness in the context of production. Metabolic adaptations are most likely broadly relevant for bacteriocin producers and need to be considered for the future development of effective microbiome editing strategies.

A rare loss-of-function genetic mutation suggest a role of dermcidin deficiency in hidradenitis suppurativa pathogenesis.

Hidradenitis suppurativa (HS) is a chronic inflammatory skin disease with a multifactorial aetiology that involves a strict interplay between genetic factors, immune dysregulation and lifestyle. Familial forms represent around 40% of total HS cases and show an autosomal dominant mode of inheritance of the disease. In this study, we conducted a whole-exome sequence analysis on an Italian family of 4 members encompassing a vertical transmission of HS. Focusing on rare damaging variants, we identified a rare insertion of one nucleotide (c.225dupA:p.A76Sfs*21) in the DCD gene encoding for the antimicrobial peptide dermcidin (DCD) that was shared by the proband, his affected father and his 11-years old daughter. Since several transcriptome studies have shown a significantly decreased expression of DCD in HS skin, we hypothesised that the identified frameshift insertion was a loss-of-function mutation that might be associated with HS susceptibility in this family. We thus confirmed by mass spectrometry that DCD levels were diminished in the affected members and showed that the antimicrobial activity of a synthetic DCD peptide resulting from the frameshift mutation was impaired. In order to define the consequences related to a decrease in DCD activity, skin microbiome analyses of different body sites were performed by comparing DCD mutant and wild type samples, and results highlighted significant differences between the groins of mutated and wild type groups. Starting from genetic analysis conducted on an HS family, our findings showed, confirming previous transcriptome results, the potential role of the antimicrobial DCD peptide as an actor playing a crucial part in the etio-pathogenesis of HS and in the maintenance of the skin's physiological microbiome composition; so, we can hypothesise that DCD could be used as a novel target for personalised therapeutic approach.

Sensitizing Staphylococcus aureus to antibacterial agents by decoding and blocking the lipid flippase MprF.

The pandemic of antibiotic resistance represents a major human health threat demanding new antimicrobial strategies. Multiple peptide resistance factor (MprF) is the synthase and flippase of the phospholipid lysyl-phosphatidylglycerol that increases virulence and resistance of methicillin-resistant Staphylococcus aureus (MRSA) and other pathogens to cationic host defense peptides and antibiotics. With the aim to design MprF inhibitors that could sensitize MRSA to antimicrobial agents and support the clearance of staphylococcal infections with minimal selection pressure, we developed MprF-targeting monoclonal antibodies, which bound and blocked the MprF flippase subunit. Antibody M-C7.1 targeted a specific loop in the flippase domain that proved to be exposed at both sides of the bacterial membrane, thereby enhancing the mechanistic understanding of bacterial lipid translocation. M-C7.1 rendered MRSA susceptible to host antimicrobial peptides and antibiotics such as daptomycin, and it impaired MRSA survival in human phagocytes. Thus, MprF inhibitors are recommended for new antivirulence approaches against MRSA and other bacterial pathogens.

Secondary Metabolites Governing Microbiome Interaction of Staphylococcal Pathogens and Commensals.

Various Staphylococcus species colonize skin and upper airways of warm-blooded animals. They compete successfully with many other microorganisms under the hostile and nutrient-poor conditions of these habitats using mechanisms that we are only beginning to appreciate. Small-molecule mediators, whose biosynthesis requires complex enzymatic cascades, so-called secondary metabolites, have emerged as crucial components of staphylococcal microbiome interactions. Such mediators belong to a large variety of compound classes and several of them have attractive properties for future drug development. They include, for instance, bacteriocins such as lanthipeptides, thiopeptides, and fibupeptides that inhibit bacterial competitor species; signaling molecules such as thiolactone peptides that induce or inhibit sensory cascades in other bacteria; or metallophores such as staphyloferrins and staphylopine that scavenge scant transition metal ions. For some secondary metabolites such as the aureusimines, the exact function remains to be elucidated. How secondary metabolites shape the fitness of Staphylococcus species in the complex context of other microbial and host defense factors remains a challenging field of future research. A detailed understanding will help to harness staphylococcal secondary metabolites for excluding the pathogenic species Staphylococcus aureus from the nasal microbiomes of at-risk patients, and it will be instrumental for the development of advanced anti-infective interventions.

The microbiome-shaping roles of bacteriocins.

The microbiomes on human body surfaces affect health in multiple ways. They include not only commensal or mutualistic bacteria but also potentially pathogenic bacteria, which can enter sterile tissues to cause invasive infection. Many commensal bacteria produce small antibacterial molecules termed bacteriocins that have the capacity to eliminate specific colonizing pathogens; as such, bacteriocins have attracted increased attention as potential microbiome-editing tools. Metagenome-based and activity-based screening approaches have strongly expanded our knowledge of the abundance and diversity of bacteriocin biosynthetic gene clusters and the properties of a continuously growing list of bacteriocin classes. The dynamic acquisition, diversification or loss of bacteriocin genes can shape the fitness of a bacterial strain that is in competition with bacteriocin-susceptible bacteria. However, a bacteriocin can only provide a competitive advantage if its fitness benefit exceeds the metabolic cost of production, if it spares crucial mutualistic partner strains and if major competitors cannot develop resistance. In contrast to most currently available antibiotics, many bacteriocins have only narrow activity ranges and could be attractive agents for precision therapy and prevention of infections. A common scientific strategy involving multiple disciplines is needed to uncover the immense potential of microbiome-shaping bacteriocins.

Staphylococcus epidermidis clones express Staphylococcus aureus-type wall teichoic acid to shift from a commensal to pathogen lifestyle.

Most clonal lineages of Staphylococcus epidermidis are commensals present on human skin and in the nose. However, some globally spreading healthcare-associated and methicillin-resistant S. epidermidis (HA-MRSE) clones are major causes of difficult-to-treat implant or bloodstream infections. The molecular determinants that alter the lifestyle of S. epidermidis have remained elusive, and their identification might provide therapeutic targets. We reasoned that changes in surface-exposed wall teichoic acid (WTA) polymers of S. epidermidis, which potentially shape host interactions, may be linked to differences between colonization and infection abilities of different clones. We used a combined epidemiological and functional approach to show that while commensal clones express poly-glycerolphosphate WTA, S. epidermidis multilocus sequence type 23, which emerged in the past 15 years and is one of the main infection-causing HA-MRSE clones, contains an accessory genetic element, tarIJLM, that leads to the production of a second, Staphylococcus aureus-type WTA (poly-ribitolphosphate (RboP)). Production of RboP-WTA by S. epidermidis impaired in vivo colonization but augmented endothelial attachment and host mortality in a mouse sepsis model. tarIJLM was absent from commensal human sequence types but was found in several other HA-MRSE clones. Moreover, RboP-WTA enabled S. epidermidis to exchange DNA with S. aureus via siphovirus bacteriophages, thereby creating a possible route for the inter-species exchange of methicillin resistance, virulence and colonization factors. We conclude that tarIJLM alters the lifestyle of S. epidermidis from commensal to pathogenic and propose that RboP-WTA might be a robust target for preventive and therapeutic interventions against MRSE infections.

Distinct Lugdunins from a New Efficient Synthesis and Broad Exploitation of Its MRSA-Antimicrobial Structure.

A new solid-phase peptide synthesis and bioprofiling of the antimicrobial activity of lugdunin, a fibupeptide, enable a comprehensive structure-activity relationship (SAR) study (MRSA Staphylococcus aureus). Distinct lugdunin analogues with variation of the three important amino acids Val2, Trp3, and Leu4 are readily available based on the established high-output synthesis. This efficient synthesis concept takes advantage of the presynthesized thiazolidine building block. To gain further knowledge of SAR, d-Val2, and d-Leu4 were replaced with aliphatic amino acids. For l-Trp3 derivatization, a set of non-natural aromatic amino acids with manifold substitution and annulation patterns precisely shows structural imperatives, starting from the exchange of d-Val6 → d-Trp6 with a 2-fold improved biological activity. d-Trp6-lugdunin analogues with additional variation of d-Val2 and d-Leu4 residues were designed and synthesized followed by antimicrobial profiling. For the first time, these SAR studies deliver valuable information on the tolerance of other amino acids to d-Val2, l-Trp3, and d-Leu4 in the sequence of lugdunin.

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.

Lugdunin amplifies innate immune responses in the skin in synergy with host- and microbiota-derived factors.

Recently our groups discovered lugdunin, a new cyclic peptide antibiotic that inhibits Staphylococcus aureus epithelial colonization in humans and rodents. In this work, we analyzed its immuno-modulatory and antimicrobial potential as a single agent or in combination with other microbiota- or host-derived factors. We show that pretreatment of primary human keratinocytes or mouse skin with lugdunin in combination with microbiota-derived factors results in a significant reduction of S. aureus colonization. Moreover, lugdunin increases expression and release of LL-37 and CXCL8/MIP-2 in human keratinocytes and mouse skin, and results in the recruitment of monocytes and neutrophils in vivo, both by a TLR/MyD88-dependent mechanism. Interestingly, S. aureus elimination by lugdunin is additionally achieved by synergistic antimicrobial activity with LL-37 and dermcidin-derived peptides. In summary, our results indicate that lugdunin provides multi-level protection against S. aureus and may thus become a promising treatment option for S. aureus skin infections in the future.

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.

Staphylococcus aureus Colonization of the Human Nose and Interaction with Other Microbiome Members.

Staphylococcus aureus is usually regarded as a bacterial pathogen due to its ability to cause multiple types of invasive infections. Nevertheless, S. aureus colonizes about 30% of the human population asymptomatically in the nares, either transiently or persistently, and can therefore be regarded a human commensal as well, although carriage increases the risk of infection. Whereas many facets of the infection processes have been studied intensively, little is known about the commensal lifestyle of S. aureus. Recent studies highlight the major role of the composition of the highly variable nasal microbiota in promoting or inhibiting S. aureus colonization. Competition for limited nutrients, trace elements, and epithelial attachment sites, different susceptibilities to host defense molecules and the production of antimicrobial molecules by bacterial competitors may determine whether nasal bacteria outcompete each other. This chapter summarizes our knowledge about mechanisms that are used by S. aureus for efficient nasal colonization and strategies used by other nasal bacteria to interfere with its colonization. An improved understanding of naturally evolved mechanisms might enable us to develop new strategies for pathogen eradication.

Lantibiotic production is a burden for the producing staphylococci.

Lantibiotics are antimicrobial peptides that contain non-proteinogenic amino acids lanthionine and 3-methyllanthionine and are produced by Gram-positive bacteria. Here we addressed the pros and cons of lantibiotic production for its producing strains. Two staphylococcal strains, S. gallinarum Tü3928 and S. epidermidis Tü3298 producing gallidermin and epidermin respectively were selected. In each of these parental strains, the structural genes gdmA and epiA were deleted; all the other biosynthetic genes including the immunity genes were left intact. Comparative analysis of the lantibiotic-producing strains with their non-producing mutants revealed that lantibiotic production is a burden for the cells. The production affected growth, caused release of ATP, lipids and increased the excretion of cytoplasmic proteins (ECP). The epidermin and gallidermin immunity genes were insufficient to protect the cells from their own product. Co-cultivation studies showed that the ΔgdmA mutant has an advantage over the parental strain; the latter was outcompeted. On the one hand, the production of staphylococcal lantibiotics is beneficial by suppressing competitors, but on the other hand they impose a burden on the producing-strains when they accumulate in higher amounts. Our observations explain why antibiotic-producing strains occur as a minority on our skin and other ecological niches, but retain corresponding antibiotic resistance.

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.

Keratinocytes as sensors and central players in the immune defense against Staphylococcus aureus in the skin.

Healthy human skin provides an effective mechanical as well as immunologic barrier against pathogenic microorganisms with keratinocytes as the main cell type in the epidermis actively participating and orchestrating the innate immune response of the skin. As constituent of the outermost layer encountering potential pathogens they have to sense signals from the environment and must be able to initiate a differential immune response to harmless commensals and harmful pathogens. Staphylococci are among the most abundant colonizers of the skin: Whereas Staphylococcus epidermidis is part of the skin microbiota and ubiquitously colonizes human skin, Staphylococcus aureus is only rarely found on healthy human skin, but frequently colonizes the skin of atopic dermatitis (AD) patients. This review highlights recent advances in understanding how keratinocytes as sessile innate immune cells orchestrate an effective defense against S. aureus in healthy skin and the mechanisms leading to an impaired keratinocyte function in AD patients.

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.

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.