Lipopeptide Antibiotic Research Articles

Diversity, enzyme production and antibacterial activity of Bacillus strains isolated from sesame-flavored liquor Daqu.

Daqu provides enzymes and precursors for liquor fermentation, and is the core of liquor fermentation. In this study, 11 Bacillus strains were isolated from sesame-flavored liquor Daqu, which can not only produce protease and amylase, but also have antagonistic effects on common pathogens Escherichia coli and Staphylococcus aureus. According to the gyrA gene phylogeny analysis, these 11 Bacillus strains belong to three species, B1, Y14, Y15, and YPDW9 belong to Bacillus mojavensis, W7, W13, YPDW6, and YPDW12 belong to Bacillus subtilis, and W14, Y5, and YPDW1 belong to Bacillus velezensis. According to the results of random amplified polymorphic DNA (RAPD) typing, there are three strains in Bacillus mojavensis, among which Y14 and Y15 are the same ones. All four Bacillus subtilis strains and three Bacillus velezensis strains are different. The specific primers were used to randomly amplify the biological control genes expressing lipopeptide antibiotics (bioA, bmyB, ituC, fenD, srfAA, srfAB, yngG,and yndJ), and the results showed that antagonistic genes other than fenD gene were amplified in four Bacillus mojavensis strains; Bacillus subtilis amplification was significantly different, but srfAA, bmyB and yndJ genes were all present; All genes were amplified in Bacillus velezensis except YPDW1 without ituC. This research provides new ideas for strengthening Daqu and lays a foundation for improving the quality of liquor.

Polymyxins, the last-resort antibiotics: Mode of action, resistance emergence, and potential solutions

Infections caused by multi-drug resistant (MDR) bacterial pathogens are a leading cause of mortality and morbidity across the world. Indiscriminate use of broad-spectrum antibiotics has seriously affected this situation. With the diminishing discovery of novel antibiotics, new treatment methods are urgently required to combat MDR pathogens. Polymyxins, the cationic lipopeptide antibiotics, discovered more than half a century ago, are considered to be the last-line of antibiotics available at the moment. This antibiotic shows a great bactericidal effect against Gram-negative bacteria. Polymyxins primarily target the bacterial membrane and disrupt them, causing lethality. Because of their membrane interacting mode of action, polymyxins cause nephrotoxicity and neurotoxicity in humans, limiting their usability. However, recent modifications in their chemical structure have been able to reduce the toxic effects. The development of better dosing regimens has also helped in getting better clinical outcomes in the infections caused by MDR pathogens. Since the mid-1990s the use of polymyxins has increased manifold in clinical settings, resulting in the emergence of polymyxin-resistant strains. The risk posed by the polymyxin-resistant nosocomial pathogens such as the Enterobacteriaceae group, Pseudomonas aeruginosa, and Acinetobacter baumannii, etc. is very serious considering these pathogens are resistant to almost all available antibacterial drugs. In this review article, the mode of action of the polymyxins and the genetic regulatory mechanism responsible for the emergence of resistance are discussed. Specifically, this review aims to update our current understanding in the field and suggest possible solutions that can be pursued for future antibiotic development. As polymyxins primarily target the bacterial membranes, resistance to polymyxins arises primarily by the modification of the lipopolysaccharides (LPS) in the outer membrane (OM). The LPS modification pathways are largely regulated by the bacterial two-component signal transduction (TCS) systems. Therefore, targeting or modulating the TCS signalling mechanisms can be pursued as an alternative to treat the infections caused by polymyxin-resistant MDR pathogens. In this review article, this aspect is also highlighted.

Binding Studies Reveal Phospholipid Specificity and Its Role in the Calcium-Dependent Mechanism of Action of Daptomycin.

Multidrug-resistant bacteria pose a serious global health threat as antibiotics are increasingly losing their clinical efficacy. A molecular level understanding of the mechanism of action of antimicrobials plays a key role in developing new agents to combat the threat of antimicrobial resistance. Daptomycin, the only clinically used calcium-dependent lipopeptide antibiotic, selectively disrupts Gram-positive bacterial membranes to illicit its bactericidal effect. In this study, we use isothermal titration calorimetry to further characterize the structural features of the target bacterial phospholipids that drive daptomycin binding. Our studies reveal that daptomycin shows a clear preference for the phosphoglycerol headgroup. Furthermore, unlike other calcium-dependent lipopeptide antibiotics, calcium binding by daptomycin is strongly dependent on the presence of phosphatidylglycerol. These investigations provide new insights into daptomycin’s phospholipid specificity and calcium binding behavior.

Effects of mixed culture fermentation of Bacillus amyloliquefaciens and Trichoderma longibrachiatum on its constituent strains and the biocontrol of tomato Fusarium wilt.

To study the effects of mixed culture fermentation (MCF) of Bacillus amyloliquefaciens and Trichoderma longibrachiatum on its constituent strains and the application values for agricultural production, with the intention of developing efficient and environmentally friendly biocontrol agents. In this study, an in vitro antifungal growth experiment showed that the inhibitory rate of the MCF broth on pathogenic fungi (Fusarium oxysporum f. sp. lycopersici, Botrytis cinerea, Trichothecium roseum and Colletotrichum gloeosporioides) was less than that of B. amyloliquefaciens culture fermentation (BCF). Moreover, the content and gene expression of lipopeptide antibiotics were also lower than that in the BCF group. However, the pot experiments based on irrigation with appropriately diluted fermentation broth showed that the biocontrol effect of MCF on tomato Fusarium wilt was significantly higher than that of TCF (T. longibrachiatum culture fermentation) and BCF, and was approximately 15.79% higher than that of the BTF group which made by mixing equivalent amounts of BCF and TCF. In MCF broth, two micro-organisms antagonized and coexisted, and the growth of T. longibrachiatum was inhibited. Using transcriptomic analysis, we speculated that MCF can upregulate the expression of genes related to carbon and nitrogen metabolism, oxidation-reduction activity, sporulation, environmental information response and chemotaxis, and biosynthesis of secondary metabolites of B. amyloliquefaciens, which might enhance the nutrient substances metabolism and competitiveness, survival ability, colonization and adaptability to the environment to increase its biocontrol potential. Mixed culture fermentation could promote the more reasonable and effective utilization of biocontrol micro-organisms though improving biocontrol effect, enhancing strains survival and competitiveness, increasing beneficial metabolites, combined with resistance induction or synergistic control. Using MCF agronomically utilizes biocontrol agents in an efficient way, which has a good potential for commercial implementation and could reduce production costs.

A Concise Synthetic Strategy Towards the Novel Calcium-dependent Lipopeptide Antibiotic, Malacidin A and Analogues.

Malacidin A is a novel calcium-dependent lipopeptide antibiotic with excellent activity against Gram-positive pathogens. Herein, a concise and robust synthetic route toward malacidin A is reported, employing 9-fluorenylmethoxycarbonyl solid-phase peptide synthesis of a linear precursor, including late-stage incorporation of the lipid tail, followed by solution-phase cyclization. The versatility of this synthetic strategy was further demonstrated by synthesis of a diastereomeric variant of malacidin A and a small library of simplified analogues with variation of the lipid moiety.

Daptomycin exerts bactericidal effect through induction of excessive ROS production and blocking the function of stress response protein Usp2

AbstractDaptomycin, as a lipopeptide antibiotic, exhibits high potency in the treatment of infections caused by clinically relevant drug‐resistant Gram‐positive pathogens, such as methicillin‐resistant Staphylococcus aureus and vancomycin‐resistant Enterococci. However, its bactericidal mechanism of action remains controversial. In this study, we report that daptomycin kills bacteria through triggering overproduction of deleterious reactive oxygen species (ROS). This outcome is attributed to daptomycin binding to the universal stress response protein (Usp2) and subsequent blocking its function, triggering stress, and anti‐ROS response. Based on these findings, we conclude that daptomycin causes bacterial cell death by simultaneously triggering ROS production through inflicting cell membrane damages and inhibiting antioxidant defense by blocking Usp2 function. This study depicts molecular mechanisms underlying the bactericidal effect of daptomycin, a combination of triggering ROS production and inhibiting anti‐ROS response.Key points Novel bactericidal mechanism of daptomycin by ROS Identification of first bacterial protein target, Usp2, for daptomycin Deciphering the dual role of Usp2 on daptomycin mediated bacterial killing

Removal of peptidoglycan and inhibition of active cellular processes leads to daptomycin tolerance in Enterococcus faecalis.

Daptomycin is a cyclic lipopeptide antibiotic used in the clinic for treatment of severe enterococcal infections. Recent reports indicate that daptomycin targets active cellular processes, specifically, peptidoglycan biosynthesis. Within, we examined the efficacy of daptomycin against Enterococcus faecalis under a range of environmental growth conditions including inhibitors that target active cellular processes. Daptomycin was far less effective against cells in late stationary phase compared to cells in exponential phase, and this was independent of cellular ATP levels. Further, the addition of either the de novo protein synthesis inhibitor chloramphenicol or the fatty acid biosynthesis inhibitor cerulenin induced survival against daptomycin far better than controls. Alterations in metabolites associated with peptidoglycan synthesis correlated with protection against daptomycin. This was further supported as removal of peptidoglycan induced physiological daptomycin tolerance, a synergistic relation between daptomycin and fosfomycin, an inhibitor of the fist committed step peptidoglycan synthesis, was observed, as well as an additive effect when daptomycin was combined with ampicillin, which targets crosslinking of peptidoglycan strands. Removal of the peptidoglycan of Enterococcus faecium, Staphylococcus aureus, and Bacillus subtilis also resulted in significant protection against daptomycin in comparison to whole cells with intact cell walls. Based on these observations, we conclude that bacterial growth phase and metabolic activity, as well as the presence/absence of peptidoglycan are major contributors to the efficacy of daptomycin.

Shipworm symbiosis ecology-guided discovery of an antibiotic that kills colistin-resistant Acinetobacter

Teredinibacter turnerae is an intracellular bacterial symbiont in the gills of wood-eating shipworms, where it is proposed to use antibiotics to defend itself and its animal host. Several biosynthetic gene clusters are conserved in T.turnerae and their host shipworms around the world, implying that they encode defensive compounds. Here, we describe turnercyclamycins, lipopeptide antibiotics encoded in the genomes of all sequenced T.turnerae strains. Turnercyclamycins are bactericidal against challenging Gram-negative pathogens, including colistin-resistant Acinetobacter baumannii. Phenotypic screening identified the outer membrane as the likely target. Turnercyclamycins and colistin operate by similar cellular, although not necessarily molecular, mechanisms, but turnercyclamycins kill colistin-resistant A.baumannii, potentially filling an urgent clinical need. Thus, by exploring environments that select for the properties we require, we harvested the fruits of evolution to discover compounds with potential to target unmet health needs. Investigating the symbionts of shipworms is a powerful example of this principle.

Dosing Considerations for Combination Antistaphylococcal β-Lactam and Glyco/lipopeptide Salvage Therapy for Resistant Gram-Positive Infections: A Systematic Review.

The objective of this systematic review is to evaluate dosing regimens of combination salvage regimens used as part of infectious disease pharmacotherapy. A systematic review was conducted on PubMed, MEDLINE, Scopus, ProQuest Central, and CINAHL through March 2021 using the following terminology: "combination" OR "Seesaw" OR "see-saw" OR "salvage" AND "infection" OR "resistant infection" OR "Gram-positive" AND "beta-lactam" OR "cephalosporin" OR "carbapenem" OR "monobactam" OR "glycopeptide" OR "lipopeptide." Following the application of inclusion and exclusion criteria, 8 pieces of literature were ultimately included in this review. Vancomycin in combination with another agent was most commonly prescribed as initial or empirical therapy. The most common combination salvage therapy regimen consisted of daptomycin in doses up to 12 mg/kg IV every 24 hours with ceftaroline 200 to 600 mg IV every 8 to 12 hours. Although the duration of combination salvage therapy varied drastically, blood culture clearance was typically observed within 24 hours. Antimicrobial-resistant Gram-positive organisms have posed an emergent threat to antimicrobial stewardship initiatives. Utilizing either a glycopeptide or lipopeptide antibiotic in combination with an antistaphylococcal β-lactam antibiotic has demonstrated efficacy in treating resistant bacteria. This work describes the heterogeneity of dosing regimens and seeks to define an optimal dose, duration, and combination of antibiotics. Combination salvage therapy has demonstrated efficacy and safety in treatment of resistant Gram-positive infections. It appears the combination of daptomycin and ceftaroline can clear resistant infections expeditiously.

Comparative Transcriptome Analysis Demonstrates the Positive Effect of the Cyclic AMP Receptor Protein Crp on Daptomycin Biosynthesis in Streptomyces roseosporus.

Daptomycin, which is produced by Streptomyces roseosporus, has been characterized as a novel cyclic lipopeptide antibiotic that is effective against Gram-positive bacteria. The biosynthesis of daptomycin is regulated by various factors. In the present study, we demonstrated that the cyclic AMP receptor protein (Crp) plays an important role in producing daptomycin in the S. roseosporus industrial strain. We found that daptomycin production from the crp deletion strain decreased drastically, whereas production from the crp overexpression strain increased by 22.1%. Transcriptome and qPCR analyses showed that some genes related to the daptomycin biosynthetic gene cluster (dpt) and the pleiotropic regulator (adpA) were significantly upregulated. RNA-seq also shows Crp to be a multifunctional regulator that modulates primary metabolism and enhances precursor flux to secondary metabolite biosynthesis. These results provide guidance for the development and improvement of potential natural products.

Fabrication of an allyl-β-cyclodextrin based monolithic column with triallyl isocyanurate as co-crosslinker and its application in separation of lipopeptide antibiotics by HPLC

A novel microporous structure monolithic polymer was prepared via free radical polymerization within a stainless steel column (4.6 mm [i.d.] × 50 mm) for separation of lipopeptide antibiotics. The monolithic column was synthesized using allyl-β-cyclodextrin and methyl methacrylate as co-monomers, triallyl isocyanurate and ethylene glycol dimethacrylate as co-crosslinkers, and azobisisobutyronitrile as an initiator. The optimized monolithic columns were evaluated by scanning electron microscopy, a nitrogen absorption-desorption apparatus, and mercury intrusion porosimetry. The homemade monolithic columns not only had a relatively uniform porosity structure, low back pressure, and good permeability, but also show good separation performance. For low molecular weight substances, six aromatic compounds were separated in 6 min and the column efficiency of naphthalene achieved 35, 660 plates m−1. The enantiomers of ibuprofen and ephedrine, which were used as standard samples, were separated by the homemade monolithic column. The application potential of the monolithic column for the separation of Chinese medicine bioactive substances was illustrated by the ephedrine and pseudoephedrine, which were successfully detected in a crude extract of ephedra. Based on the excellent separation performance of the column for the aforementioned low molecular weight substances, we assumed that there are many forces, such as hydrogen bonds, π-π bonds, and dipole-dipole interactions, that are involved between enantiomers and allyl-β-cyclodextrin. Thus, natural lipopeptide antibiotics in the fermentation broth were used as samples to verify the assumption. Eighteen peaks were precisely separated from the fermentation broth. Among the 18 peaks, 4 lipopeptide antibiotics were identified by mass spectrometry. Methodologic validation was confirmed using two enantiomers of ibuprofen, and all parameters meet the testing requirements.

Vegetable associated Bacillus spp. suppress the pea (Pisum sativum L.) root rot caused by Fusarium solani

F. solani causing root rot is the most devastating pathogen of pea (P. sativum L). Biological control using plant growth promoting rhizobacteria has great potential in plant disease management. In the current study, 41 bacteria were isolated from the rhizosphere of different vegetables grown under variable cropping practices. Only two out of 41 rhizobacteria antagonized the root rot pathogen (45–65%) until the 15th day of dual culture assay. The antagonistic bacteria produced one or more of the antifungal metabolites viz siderophores (discolourization zone = 3.8 mm), protease (solubilization index = 2.2–2.3), glucanases (solubilization index = 4.6–5.2) and solubilized the zinc (solubilization index = 4.0–4.6) from the insoluble ore. The antagonistic bacteria were identified as Bacillus subtilis FZV-1 and B. halotolerans FZV 34 by 16S rRNA, pta, glp F, and pyr C gene analysis. The rhizobacteria also indicated putative production of lipopeptide antibiotics surfactin and fengycin based on the presence of sfp and fen D genes. In pea inoculation assays, rhizobacteria decreased the germination time over control (4–35%) and enhanced the shoot length (29–64%), root length (23–78%) and fresh weight (28–70%). The rhizobacteria also reduced the root rot severity over control (25–89%) in the pea varieties meteor and climax. The versatile plant growth promoting potential of these strains emphasizes their potential use in the biological management of pea root rot.

Acinetobacter baumannii Antibiotic Resistance Mechanisms.

Acinetobacter baumannii is a Gram-negative ESKAPE microorganism that poses a threat to public health by causing severe and invasive (mostly nosocomial) infections linked with high mortality rates. During the last years, this pathogen displayed multidrug resistance (MDR), mainly due to extensive antibiotic abuse and poor stewardship. MDR isolates are associated with medical history of long hospitalization stays, presence of catheters, and mechanical ventilation, while immunocompromised and severely ill hosts predispose to invasive infections. Next-generation sequencing techniques have revolutionized diagnosis of severe A. baumannii infections, contributing to timely diagnosis and personalized therapeutic regimens according to the identification of the respective resistance genes. The aim of this review is to describe in detail all current knowledge on the genetic background of A. baumannii resistance mechanisms in humans as regards beta-lactams (penicillins, cephalosporins, carbapenems, monobactams, and beta-lactamase inhibitors), aminoglycosides, tetracyclines, fluoroquinolones, macrolides, lincosamides, streptogramin antibiotics, polymyxins, and others (amphenicols, oxazolidinones, rifamycins, fosfomycin, diaminopyrimidines, sulfonamides, glycopeptide, and lipopeptide antibiotics). Mechanisms of antimicrobial resistance refer mainly to regulation of antibiotic transportation through bacterial membranes, alteration of the antibiotic target site, and enzymatic modifications resulting in antibiotic neutralization. Virulence factors that may affect antibiotic susceptibility profiles and confer drug resistance are also being discussed. Reports from cases of A. baumannii coinfection with SARS-CoV-2 during the COVID-19 pandemic in terms of resistance profiles and MDR genes have been investigated.

Genome mining for drug discovery: cyclic lipopeptides related to daptomycin.

The cyclic lipopeptide antibiotics structurally related to daptomycin were first reported in the 1950s. Several have common lipopeptide initiation, elongation, and termination mechanisms. Initiation requires the use of a fatty acyl-AMP ligase (FAAL), a free-standing acyl carrier protein (ACP), and a specialized condensation (CIII) domain on the first NRPS elongation module to couple the long chain fatty acid to the first amino acid. Termination is carried out by a dimodular NRPS that contains a terminal thioesterase (Te) domain (CAT-CATTe). Lipopeptide BGCs also encode ABC transporters, apparently for export and resistance. The use of this mechanism of initiation, elongation, and termination, coupled with molecular target-agnostic resistance, has provided a unique basis for robust natural and experimental combinatorial biosynthesis to generate a large variety of structurally related compounds, some with altered or different antibacterial mechanisms of action. The FAAL, ACP, and dimodular NRPS genes were used as molecular beacons to identify phylogenetically related BGCs by BLASTp analysis of finished and draft genome sequences. These and other molecular beacons have identified: (i) known, but previously unsequenced lipopeptide BGCs in draft genomes; (ii) a new daptomycin family BGC in a draft genome of Streptomyces sedi; and (iii) novel lipopeptide BGCs in the finished genome of Streptomyces ambofaciens and the draft genome of Streptomyces zhaozhouensis.

Combining Colistin and Fluconazole Synergistically Increases Fungal Membrane Permeability and Antifungal Cidality.

The increasing emergence of drug-resistant fungal pathogens, together with the limited number of available antifungal drugs, presents serious clinical challenges to treating systemic, life-threatening infections. Repurposing existing drugs to augment the antifungal activity of well-tolerated antifungals is a promising antifungal strategy with the potential to be implemented rapidly. Here, we explored the mechanism by which colistin, a positively charged lipopeptide antibiotic, enhances the antifungal activity of fluconazole, the most widely used orally available antifungal. In a range of susceptible and drug-resistant isolates and species, colistin was primarily effective at reducing fluconazole tolerance, a property of subpopulations of cells that grow slowly in the presence of a drug and may promote the emergence of persistent infections and resistance. Clinically relevant concentrations of colistin synergized with fluconazole, reducing fluconazole minimum inhibitory concentration 4-fold. Combining fluconazole and colistin also increased survival in a C. albicans Galleria mellonella infection, especially for a highly fluconazole-tolerant isolate. Mechanistically, colistin increased permeability to fluorescent antifungal azole probes and to intracellular dyes, accompanied by an increase in cell death that was dependent upon pharmacological or genetic inhibition of the ergosterol biosynthesis pathway. The positive charge of colistin is critical to its antifungal, and antibacterial, activity: colistin directly binds to several eukaryotic membrane lipids (i.e., l-α-phosphatidylinositol, l-α-phosphatidyl-l-serine, and l-α-phosphatidylethanolamine) that are enriched in the membranes of ergosterol-depleted cells. These results support the idea that colistin binds to fungal membrane lipids and permeabilizes fungal cells in a manner that depends upon the degree of ergosterol depletion.

Isolation and yield optimization of lipopeptides from Bacillus subtilis Z-14 active against wheat take-all caused by Gaeumannomyces graminis var. tritici.

Wheat take-all, caused by the soil-borne fungus Gaeumannomyces graminis var. tritici, is one of the major constraints on wheat production worldwide. Bacillus subtilis Z-14 exerts significant biocontrol activity against wheat take-all, and lipopeptide antibiotics are the main antifungal substances. Herein, lipopeptide antibiotics C14-C15 iturin A, C14-C16 fengycin A, and C15-C17 fengycin B from B. subtilis Z-14 culture filtrates were separated and identified by high-performance liquid chromatography, matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry, and mass spectrometry/mass spectrometry, respectively. The optimal medium components for Z-14 lipopeptide antibiotic production were 3.85g/L corn flour, 1.57g/L soybean meal, 0.03g/L FeSO4 ·7H2 O, 0.2g/L NaH2 PO4 ·2H2 O, and 0.4g/L Na2 HPO4 ·2H2 O. Quantification analysis by high-performance liquid chromatography showed that fengycins played a main role in antifungal activity against Gaeumannomyces graminis var. tritici. Quantitative reverse transcription polymerase chain reaction showed that lipopeptide synthesis genes fenD and ituC reached maximum expression levels after 48 h of fermentation. The strongest control of wheat take-all by Z-14 was achieved by adding 30mL of culture filtrate per 350g of soil in pot experiments, during which disease reduction reached 88.15%. This study provides theoretical support and a material basis for the prevention and treatment of wheat take-all disease.

The Use of Daptomycin in the Treatment of Persistent Coagulase-Negative Staphylococcal Sepsis in Premature Infants: A Case Series.

Daptomycin is a lipopeptide antibiotic with rapid bactericidal activity against Gram-positive bacteria. Reports regarding the use of daptomycin in infants are still limited. Thus, the objective of this report is to describe the safety and efficacy of daptomycin in premature infants with persistent coagulase-negative staphylococci (CoNS) infection. This was a retrospective chart review of 10 premature infants with persistent CoNS infection who received daptomycin therapy between January 2018 and September 2019. Four patients had endocarditis and 1 had bacterial meningitis and infectious endocarditis. The other 5 patients had persistent CoNS bacteraemia only. Daptomycin treatment was successful for 5 patients. The others died owing to multiple factors such as prematurity, sepsis, and chronic lung disease. Adverse drug reactions, including elevation of creatine phosphokinase and/or hepatotoxicity, were noted in 4 patients. Large and randomized studies are necessary to ensure daptomycin's safety and efficacy for the treatment of infants with persistent sepsis caused by Gram-positive bacteria.

Conformers of a novel lipopeptide antibiotic, Kannurin inhibits SARS-Cov2 replication via interfering with RNA-dependent-RNA polymerase activation and function

The key component 'nsp12' of RNA-dependent-RNA polymerase of Corona virus is considered as a primary target for drug intervention purposes. The broad spectrum antimicrobial lipopeptide Kannurin rev...

Conformers of a novel lipopeptide antibiotic, Kannurin inhibits SARS-Cov2 replication via interfering with RNA-dependent-RNA polymerase activation and function

Conformers of a novel lipopeptide antibiotic, Kannurin inhibits SARS-Cov2 replication via interfering with RNA-dependent-RNA polymerase activation and function

Strain Improvement Program of Streptomyces roseosporus for Daptomycin Production.

Daptomycin is a cyclic lipopeptide antibiotic with potent activity against gram-positive bacteria. It has a calcium-dependent mechanism of action that disrupts multiple features of the bacterial membrane function. This antibiotic is highly demanded due to its effectiveness against to microorganisms resistant to other antibiotics, including vancomycin-resistant Staphylococcus aureus (VRSA) and methicillin-resistant S. aureus (MRSA). Daptomycin is produced by fermentation of Streptomyces roseosporus, currently identified as Streptomyces filamentosus. However, low fermentation yields and high production costs are reported. This chapter describes a method of strain improvement involving random mutagenesis, rational screening by bioassay, and flask fermentation. The ultimate objective is to select mutants of S. roseosporus overproducing daptomycin in order to design a more cost-effective daptomycin production.