In the fight against bacterial infections, particularly those caused by multi-resistant pathogens known as "superbugs", the need for new antibacterials is undoubted in scientific communities and is by now also widely perceived by the general population. However, the antibacterial research landscape has changed considerably over the past years. With few exceptions, the majority of big pharma companies has left the field and thus, the decline in R&D on antibacterials severely impacts the drug pipeline. In recent years, antibacterial research has increasingly relied on smaller companies or academic research institutions, which mostly have only limited financial resources, to carry a drug discovery and development process from the beginning and through to the beginning of clinical phases. This review formulates the requirements for an antibacterial in regard of targeted pathogens, resistance mechanisms and drug discovery. Strategies are shown for the discovery of new antibacterial structures originating from natural sources, by chemical synthesis and more recently from artificial intelligence approaches. This is complemented by principles for the computer-aided design of antibacterials and the refinement of a lead structure. The second part of the article comprises a compilation of antibacterial molecules classified according to bacterial target structures, e.g. cell wall synthesis, protein synthesis, as well as more recently emerging target classes, e.g. fatty acid synthesis, proteases and membrane proteins. Aspects of the origin, the antibacterial spectrum, resistance and the current development status of the presented drug molecules are highlighted.

With the rise of antimicrobial resistance, there is high demand for novel antimicrobials to combat multi-drug resistant pathogens. The bacterial genus Pantoea produces a diversity of antimicrobial natural products effective against a wide range of bacterial and fungal targets. These antimicrobials are synthesized by specialized biosynthetic gene clusters that have unique distributions across Pantoea as well as several other genera outside of the Erwiniaceae. Phylogenetic and genomic evidence shows that these clusters can mobilize within and between species and potentially between genera. Pantoea antimicrobials belong to unique structural classes with diverse mechanisms of action, but despite their potential in antagonizing a wide variety of plant, human, and animal pathogens, little is known about many of these metabolites and how they function. This review will explore the known antimicrobials produced by Pantoea: agglomerins, andrimid, D-alanylgriseoluteic acid, dapdiamide, herbicolins, pantocins, and the various Pantoea Natural Products (PNPs). It will include information on the structure of each compound, their genetic basis, biosynthesis, mechanism of action, spectrum of activity, and distribution, highlighting the significance of Pantoea antimicrobials as potential therapeutics and for applications in biocontrol.

Moiramide B is a peptide-polyketide hybrid with a bacterial origin and interesting antibiotic activity. Besides its structurally conserved peptide part, it contains a highly variable fatty acid side chain. We modified this part of the molecule by introducing a terminal alkyne, and we then subjected it to click reactions and Sonogashira couplings. This provided a library of moiramide B derivatives with high and selective in vivo activities against S. aureus.

Antimicrobial resistance (AMR) is one of the leading global health issues that demand urgent attention. Very soon the world will have to bear the consequences of increased drug resistance if new anti-infectives are not pumped into the clinical pipeline in a short period. This presses on the need for novel chemical entities, and the marine environment is one such hotspot to look for. The Ocean harbours a variety of organisms, of which from this aspect, "Sponges (Phylum Porifera)" are of particular interest. To tackle the stresses faced due to their sessile and filter-feeding lifestyle, sponges produce various bioactive compounds, which can be tapped for human use. The sponges harbour several microorganisms of different types and in most cases; the microbial symbionts are the actual producers of the bioactive compounds. This review describes the alarming need for the development of new antimicrobials and how marine sponges can contribute to this. Selected antimicrobial compounds from the marine sponges and their associated bacteria have been described. Additionally, measures to tackle the supply problem have been covered, which is the primary obstacle in marine natural product drug discovery.

Antimicrobial resistance is a worldwide health crisis for which new antibiotics are needed. One strategy for antibiotic discovery is identifying unique antibiotic biosynthetic gene clusters that may produce novel compounds. The aim of this study was to demonstrate how an integrated approach that combines genome mining, comparative genomics, and functional genetics can be used to successfully identify novel biosynthetic gene clusters that produce antimicrobial natural products. Secondary metabolite clusters of an antibiotic producer are first predicted using genome mining tools, generating a list of candidates. Comparative genomic approaches are then used to identify gene suites present in the antibiotic producer that are absent in closely related non-producers. Gene sets that are common to the two lists represent leading candidates, which can then be confirmed using functional genetics approaches. To validate this strategy, we identified the genes responsible for antibiotic production in Pantoea agglomerans B025670, a strain identified in a large-scale bioactivity survey. The genome of B025670 was first mined with antiSMASH, which identified 24 candidate regions. We then used the comparative genomics platform, EDGAR, to identify genes unique to B025670 that were not present in closely related strains with contrasting antibiotic production profiles. The candidate lists generated by antiSMASH and EDGAR were compared with standalone BLAST. Among the common regions was a 14 kb cluster consisting of 14 genes with predicted enzymatic, transport, and unknown functions. Site-directed mutagenesis of the gene cluster resulted in a reduction in antimicrobial activity, suggesting involvement in antibiotic production. An integrated approach that combines genome mining, comparative genomics, and functional genetics yields a powerful, yet simple strategy for identifying potentially novel antibiotics.

Intense competition between microbes in the environment has directed the evolution of antibiotic production in bacteria. Humans have harnessed these natural molecules for medicinal purposes, magnifying them from environmental concentrations to industrial scale. This increased exposure to antibiotics has amplified antibiotic resistance across bacteria, spurring a global antimicrobial crisis and a search for antibiotics with new modes of action. Genetic insights into these antibiotic-producing microbes reveal that they have evolved several resistance strategies to avoid self-toxicity, including product modification, substrate transport and binding, and target duplication or modification. Of these mechanisms, target duplication or modification will be highlighted in this review, as it uniquely links an antibiotic to its mode of action. We will further discuss and propose a strategy to mine microbial genomes for these genes and their associated biosynthetic gene clusters to discover novel antibiotics using target directed genome mining.

Covering: up to 2017The overwhelming majority of antibiotics in clinical use originate from Gram-positive Actinobacteria. In recent years, however, Gram-negative bacteria have become increasingly recognised as a rich yet underexplored source of novel antimicrobials, with the potential to combat the looming health threat posed by antibiotic resistance. In this article, we have compiled a comprehensive list of natural products with antimicrobial activity from Gram-negative bacteria, including information on their biosynthetic origin(s) and molecular target(s), where known. We also provide a detailed discussion of several unusual pathways for antibiotic biosynthesis in Gram-negative bacteria, serving to highlight the exceptional biocatalytic repertoire of this group of microorganisms.

Infections due to multidrug-resistant bacteria represent a major global health challenge. To combat this problem, new antibiotics are urgently needed and some plant-associated bacteria are a promising source. The rhizobacterium Serratia plymuthica A153 produces several bioactive secondary metabolites, including the anti-oomycete and antifungal haterumalide, oocydin A and the broad spectrum polyamine antibiotic, zeamine. In this study, we show that A153 produces a second broad spectrum antibiotic, andrimid. Using genome sequencing, comparative genomics and mutagenesis, we defined new genes involved in andrimid (adm) biosynthesis. Both the expression of the adm gene cluster and regulation of andrimid synthesis were investigated. The biosynthetic cluster is operonic and its expression is modulated by various environmental cues, including temperature and carbon source. Analysis of the genome context of the adm operon revealed a gene encoding a predicted LysR-type regulator, AdmX, apparently unique to Serratia strains. Mutagenesis and gene expression assays demonstrated that AdmX is a transcriptional activator of the adm gene cluster. At the post-transcriptional level, the expression of the adm cluster is positively regulated by the RNA chaperone, Hfq, in an RpoS-independent manner. Our results highlight the complexity of andrimid biosynthesis - an antibiotic with potential clinical and agricultural utility.

After years of inadequate use and the emergence of multidrug resistant (MDR) strains, the efficiency of "classical" antibiotics has decreased significantly. New drugs to fight MDR strains are urgently needed. Bacteria hold much promise as a source of unusual bioactive metabolites. However, the potential of marine bacteria, except for Actinomycetes and Cyanobacteria, has been largely underexplored. In the past two decades, the structures of several antimicrobial compounds have been elucidated in marine Proteobacteria. Of these compounds, polyketides (PKs), synthesised by condensation of malonyl-coenzyme A and/or acetyl-coenzyme A, and non-ribosomal peptides (NRPs), obtained through the linkage of (unusual) amino acids, have recently generated particular interest. NRPs are good examples of naturally modified peptides. Here, we review and compile the data on the antimicrobial peptides isolated from marine Proteobacteria, especially NRPs.

Vibrio coralliilyticus is a putative coral pathogen in tropical oceans, but also possesses antagonistic traits. We previously reported antibacterial activity in Vibrio coralliilyticus strain S2052 based upon the antibiotic andrimid. The purpose of the present study was to determine whether V. coralliilyticus S2052 produces the antibiotic under conditions mimicking natural habitats of vibrios. S2052 synthesized andrimid with both chitin and macroalgal extracts as sole nutrient source. With chitin, the biosynthesis of metabolites other than andrimid was largely abolished, and the yield of the antibiotic per cell was twofold higher. In cultures with Artemia as live chitin model system, S2052 reached up to 10(8)  cells ml(-1) , produced andrimid and showed attachment to the exoskeleton and chitinous exuviae. The metabolic focus on andrimid production with chitin indicates that the antibiotic could serve an ecophysiological function. S2052 was compared with two related V. coralliilyticus strains (LMG20984(T) and LMG10953). Despite overall similar secondary metabolomes, LMG20984(T) and LMG10953 did not produce andrimid, and their optimum biosynthetic temperature was 30 as compared with 25°C for S2052. In addition, S2052 appeared less pathogenic towards Artemia than reported for the type strain. Different physiologies of S2052 and closely related strains indicated that V. coralliilyticus subspecies may be adapted to different niches.

Bacteria belonging to the Vibrionaceae family are widespread in the marine environment. Today, 128 species of vibrios are known. Several of them are infamous for their pathogenicity or symbiotic relationships. Despite their ability to interact with eukaryotes, the vibrios are greatly underexplored for their ability to produce bioactive secondary metabolites and studies have been limited to only a few species. Most of the compounds isolated from vibrios so far are non-ribosomal peptides or hybrids thereof, with examples of N-containing compounds produced independent of nonribosomal peptide synthetases (NRPS). Though covering a limited chemical space, vibrios produce compounds with attractive biological activities, including antibacterial, anticancer, and antivirulence activities. This review highlights some of the most interesting structures from this group of bacteria. Many compounds found in vibrios have also been isolated from other distantly related bacteria. This cosmopolitan occurrence of metabolites indicates a high incidence of horizontal gene transfer, which raises interesting questions concerning the ecological function of some of these molecules. This account underlines the pending potential for exploring new bacterial sources of bioactive compounds and the challenges related to their investigation.

Many lead compounds in the search for new drugs derive from peptides and polyketides whose similar biosynthetic enzymes have been difficult to engineer for production of new derivatives. Problems with generating multiple analogs in a single experiment along with lack of high-throughput methods for structure-based screening have slowed progress in this area. Here, we use directed evolution and a multiplexed assay to screen a library of >14,000 members to generate three derivatives of the antibacterial compound, andrimid. Another limiting factor in reengineering these mega-enzymes of secondary metabolism has been that commonly used hosts such as Escherichia coli often give lower product titers, so our reengineering was performed in the native producer, Pantoea agglomerans. This integrated in vivo approach can be extended to larger enzymes to create analogs of natural products for bioactivity testing.

On a global research expedition, over 500 bacterial strains inhibitory towards pathogenic bacteria were isolated. Three hundred of the antibacterial strains were assigned to the Vibrionaceae family. The purpose of the present study was to investigate the phylogeny and bioactivity of five Vibrionaceae strains with pronounced antibacterial activity. These were identified as Vibrio coralliilyticus (two strains), V. neptunius (two strains), and Photobacterium halotolerans (one strain) on the basis of housekeeping gene sequences. The two related V. coralliilyticus and V. neptunius strains were isolated from distant oceanic regions. Chemotyping by LC-UV/MS underlined genetic relationships by showing highly similar metabolite profiles for each of the two V. coralliilyticus and V. neptunius strains, respectively, but a unique profile for P. halotolerans. Bioassay-guided fractionation identified two known antibiotics as being responsible for the antibacterial activity; andrimid (from V. coralliilyticus) and holomycin (from P. halotolerans). Despite the isolation of already known antibiotics, our findings show that marine Vibrionaceae are a resource of antibacterial compounds and may have potential for future natural product discovery.

To create a drug, nature's blueprints often have to be improved through semisynthesis or total synthesis (chemical postevolution). Selected contributions from industrial and academic groups highlight the arduous but rewarding path from natural products to drugs. Principle modification types for natural products are discussed herein, such as decoration, substitution, and degradation. The biological, chemical, and socioeconomic environments of antibacterial research are dealt with in context. Natural products, many from soil organisms, have provided the majority of lead structures for marketed anti-infectives. Surprisingly, numerous "old" classes of antibacterial natural products have never been intensively explored by medicinal chemists. Nevertheless, research on antibacterial natural products is flagging. Apparently, the "old fashioned" natural products no longer fit into modern drug discovery. The handling of natural products is cumbersome, requiring nonstandardized workflows and extended timelines. Revisiting natural products with modern chemistry and target-finding tools from biology (reversed genomics) is one option for their revival.

Changes in global climate have raised concerns about the emergence and resurgence of infectious diseases. Vibrio cholerae is a reemerging pathogen that proliferates and is transported on marine particles. Patterns of cholera outbreaks correlate with sea surface temperature increases, but the underlying mechanisms for rapid proliferation of V. cholerae during ocean warming events have yet to be fully elucidated. In this study, we tested the hypothesis that autochthonous marine bacteria impede the spread of V. cholerae in the marine environment. It was found that some marine bacteria are capable of inhibiting the growth of V. cholerae on surfaces and that bacterial isolates derived from pelagic particles show a greater frequency of V. cholerae inhibition than free-living bacteria. Vibrio cholerae was less susceptible to antagonism at higher temperatures, such as those measured during El Niño-Southern Oscilliation and monsoonal events. Using a model system employing green fluorescent protein-labeled bacteria, we found that marine bacteria can directly inhibit V. cholerae colonization of particles. The mechanism of inhibition in our model system was linked to the biosynthesis of andrimid, an antibacterial agent. Antibiotic production by the model antagonistic strain decreased at higher temperatures, thereby explaining the increased competitiveness of V. cholerae under warmer conditions. These findings suggest that bacterium-bacterium antagonism is a contributing mechanism in regulating the proliferation of V. cholerae on marine particles.

To identify the gene (s) related to the antagonistic activity of Enterobacter cloacae B8 and to elucidate its antagonistic mechanism.

Transposon-mediated mutagenesis and tagging method and cassette PCR-based chromosomal walking method were adopted to isolate the mutant strain(s) of B8 that lost the antagonistic activity and to clone DNA fragments around Tn5 insertion site. Sequence compiling and open reading frame (ORF) finding were done with DNAStar program and homologous sequence and conserved domain searches were performed with BlastN or BlastP programs at www.ncbi.nlm.nih.gov. To verify the gene involved in the antagonistic activity, complementation of a full-length clone of the anrF gene to the mutant B8F strain was used.

A 3 321 bp contig around the Tn5 insertion site was obtained and an ORF of 2 634 bp in length designated as anrF gene encoding for a 877 aa polyketide synthase-like protein was identified. It had a homology of 83% at the nucleotide level and 79% ID/87% SIM at the protein level, to the admM gene of Pantoea agglomerans andrimid biosynthetic gene cluster (AY192157). The Tn5 was inserted at 2 420 bp of the gene corresponding to the COG3319 (the thioesterase domain of type I polyketide synthase) coding region on B8F. The antagonistic activity against Xanthomonas oryzae pv. oryzae was resumed with complementation of the full-length anrF gene to the mutant B8F.

The anrF gene obtained is related to the antagonistic activity of B8, and the antagonistic substances produced by B8 are andrimid and/or its analogs.

The multisubunit acetyl-CoA carboxylase, which catalyzes the first committed step in fatty acid biosynthesis, is broadly conserved among bacteria. Its rate-limiting role in formation of fatty acids makes this enzyme an attractive target for the design of novel broad-spectrum antibacterials. However, no potent inhibitors have been discovered so far. This report describes the identification and characterization of highly potent bacterial acetyl-CoA carboxylase inhibitors with antibacterial activity for the first time. We demonstrate that pseudopeptide pyrrolidine dione antibiotics such as moiramide B inhibit the Escherichia coli enzyme at nanomolar concentrations. Moiramide B targets the carboxyltransferase reaction of this enzyme with a competitive inhibition pattern versus malonyl-CoA (K(i) value = 5 nm). Inhibition at nanomolar concentrations of the pyrrolidine diones is also demonstrated using recombinantly expressed carboxyltransferases from other bacterial species (Staphylococcus aureus, Streptococcus pneumoniae, and Pseudomonas aeruginosa). We isolated pyrrolidine dione-resistant strains of E. coli, S. aureus, and Bacillus subtilis, which contain mutations within the carboxyltransferase subunits AccA or AccD. We demonstrate that such mutations confer resistance to pyrrolidine diones. Inhibition values (IC(50)) of >100 microm regarding an eukaryotic acetyl-CoA carboxylase from rat liver indicate high selectivity of pyrrolidine diones for the bacterial multisubunit enzyme. The natural product moiramide B and synthetic analogues show broad-spectrum antibacterial activity. The knowledge of the target and the availability of facile assays using carboxyltransferases from different pathogens will enable evaluation of the antibacterial potential of the pyrrolidine diones as a promising antibacterial compound class acting via a novel mode of action.