Metabolomics and Genomics Approach for the Discovery of Serrawettin W2 Lipopeptides from Serratia marcescens NP2.

A metabolomics/peptidomics and genomics approach, using UPLC-MSE, molecular networking, and genome mining, was used to describe the serrawettin W2 lipopeptide family produced by Serratia marcescens NP2. Seven known serrawettin W2 analogues were structurally elucidated along with 17 new analogues, which varied based on the first (fatty acyl length of C8, C10, C12, or C12:1), fifth (Phe, Tyr, Trp, or Leu/Ile), and sixth (Leu, Ile, or Val) residues. Tandem MS results suggested that the previously classified serrawettin W3 may be an analogue of serrawettin W2, with a putative structure of cyclo(C10H18O2-Leu-Ser-Thr-Leu/Ile-Val). Chiral phase amino acid analysis enabled the distinction between l/d-Leu and l-Ile residues within nine purified compounds. 1H and 13C NMR analyses confirmed the structures of four purified new analogues. Additionally, genome mining was conducted using Serratia genome sequences available on the NCBI database to identify the swrA gene using the antiSMASH software. NRPSpredictor2 predicted the specificity score of the adenylation-domain within swrA with 100% for the first, second, and third modules (Leu-Ser-Thr), 60-70% for the fourth module (Phe/Trp/Tyr/Val), and 70% for the fifth module (Val/Leu/Ile), confirming MSE data. Finally, antibacterial activity was observed for compounds 6 and 11 against a clinical Enterococcus faecium strain.

Poly(N-vinylpyrrolidone) Antimalaria Conjugates of Membrane-Disruptive Peptides.

The concepts of polymer-peptide conjugation and self-assembly were applied to antimicrobial peptides (AMPs) in the development of a targeted antimalaria drug delivery construct. This study describes the synthesis of α-acetal, ω-xanthate heterotelechelic poly(N-vinylpyrrolidone) (PVP) via reversible addition-fragmentation chain transfer (RAFT)-mediated polymerization, followed by postpolymerization deprotection to yield α-aldehyde, ω-thiol heterotelechelic PVP. A specific targeting peptide, GSRSKGT, for Plasmodium falciparum-infected erythrocytes was used to sparsely decorate the α-chain ends via reductive amination while cyclic decapeptides from the tyrocidine group were conjugated to the ω-chain end via thiol-ene Michael addition. The resultant constructs were self-assembled into micellar nanoaggregates whose sizes and morphologies were determined by dynamic light scattering (DLS) and transmission electron microscopy (TEM). The in vitro activity and selectivity of the conjugates were evaluated against intraerythrocytic P. falciparum parasites.

Correction to: Genetic and Phenotypic Characteristics of a Multi-strain Probiotic for Broilers.

The authors would like to correct the errors in the publication of the original article.

Genetic and Phenotypic Characteristics of a Multi-strain Probiotic for Broilers.

Bacteria isolated from different segments of the gastro-intestinal tract (GIT) of healthy free-range broilers were screened for probiotic properties. Six strains were selected and identified as Lactobacillus gallinarum, Lactobacillus johnsonii, Lactobacillus salivarius, Lactobacillus crispatus, Enterococcus faecalis and Bacillus amyloliquefaciens based on 16S rRNA, gyrB and recA gene sequence analyses. All six strains produced exopolysaccharides (EPS) and formed biofilms under conditions simulating the broiler GIT. Lactobacillus johnsonii DPN184 and L. salivarius DPN181 produced hydrogen peroxide, and L. crispatus DPN167 and E. faecalis DPN94 produced bile salt hydrolase (BSH) and phytase. Bacillus amyloliquefaciens DPN123 produced phytase, amylase, surfactin and iturin A1. No abnormalities were observed when broilers were fed the multi-strain combination, suggesting that it could be used as a probiotic.

Tyrocidine A interactions with saccharides investigated by CD and NMR spectroscopies.

Tyrocidines are a family of cyclic decapeptides produced by the soil bacterium, Brevibacillus parabrevis. These antibiotic peptides can be used to prevent infections in agriculture and food industry but also to prepare antimicrobial lozenges, creams, and dressings for medical applications. It has been observed that the tyrocidines interact with saccharides such as cellulose from their soil environment, as well as sugars in culture media and glycans in fungal cell walls. Here, we investigated the interactions of tyrocidines with glucose, sucrose, and cellotetraose (as cellulose model) in a quantitative fashion utilising CD and NMR spectroscopy. The CD and NMR spectra of tyrocidine A (TrcA) were analysed as a function of solvent composition, and the spectral properties agree with the formation of oligomeric structures that are governed by ß-sheet secondary structures once the acetonitrile content of the solvent is increased. Saccharides seem to also induce TrcA spectral changes reverting those induced by organic solvents. The CD spectral changes of TrcA in the presence of glucose agree with new ordered H-bonding, possibly ß-sheet structures. The amides involved in intramolecular H-bonding remained largely unaffected by the environmental changes. In contrast, amides exposed to the exterior and/or involved in TrcA intermolecular association show the largest 1 H chemical shift changes. CD and NMR spectroscopic investigations correlated well with TrcA-glucose interactions characterized by a dissociation constant around 200 µM. Interestingly, the association of cellotetraose corresponds closely to the additive effect from four glucose moieties, while a much higher dissociation constant was observed for sucrose. Similar trends to TrcA for binding to the three saccharides were observed for the analogous tyrocidines, tyrocidine B, and tyrocidine C. These results therefore indicate that the tyrocidine interactions with the glucose monosaccharide unit are fairly specific and reversible.

Synergistic activity of the tyrocidines, antimicrobial cyclodecapeptides from Bacillus aneurinolyticus, with amphotericin B and caspofungin against Candida albicans biofilms.

Tyrocidines are cationic cyclodecapeptides from Bacillus aneurinolyticus that are characterized by potent antibacterial and antimalarial activities. In this study, we show that various tyrocidines have significant activity against planktonic Candida albicans in the low-micromolar range. These tyrocidines also prevented C. albicans biofilm formation in vitro. Studies with the membrane-impermeable dye propidium iodide showed that the tyrocidines disrupt the membrane integrity of mature C. albicans biofilm cells. This membrane activity correlated with the permeabilization and rapid lysis of model fungal membranes containing phosphatidylcholine and ergosterol (70:30 ratio) induced by the tyrocidines. The tyrocidines exhibited pronounced synergistic biofilm-eradicating activity in combination with two key antifungal drugs, amphotericin B and caspofungin. Using a Caenorhabditis elegans infection model, we found that tyrocidine A potentiated the activity of caspofungin. Therefore, tyrocidines are promising candidates for further research as antifungal drugs and as agents for combinatorial treatment.

Inhibition of agronomically relevant fungal phytopathogens by tyrocidines, cyclic antimicrobial peptides isolated from Bacillus aneurinolyticus.

The tyrocidines, a complex of analogous cyclic decapeptides produced by Bacillus aneurinolyticus, exhibited noteworthy activity against a range of phytopathogenic fungi, including Fusarium verticillioides, Fusarium solani and Botrytis cinerea. The activity of the tyrocidine peptide complex (Trc mixture) and purified tyrocidines exhibited minimum inhibition concentrations below 13 µg ml(-1) (~10 µM) and was significantly more potent than that of the commercial imidazole fungicide, bifonazole. Although the tyrocidines' activity was negatively influenced by the presence of Ca(2+), it remained unaffected by the presence of Mg(2+), Na(+) and K(+). Microscopic analysis revealed significant impact on the morphology of F. solani and Bot. cinerea including retarded germination and hyperbranching of hyphae. Studies with membrane-impermeable dyes, SYTOX green and propidium iodide suggested that the main mode of action of tyrocidines involves the disruption of fungal membrane integrity. Because of the tyrocidines' broad spectrum and potent antifungal activity, possible multiple targets reducing the risk of overt resistance and general salt tolerance, they are promising candidates that warrant further investigation as bio-fungicides.

Antibacterial efficacy and membrane mechanism of action of the Serratia-derived non-ionic lipopeptide, serrawettin W2-FL10.

The study aimed to investigate the antibacterial activity, cytotoxicity, and mechanism of action of the non-ionic, cyclic lipopeptide, serrawettin W2-FL10 against Staphylococcus aureus. W2-FL10 exhibited potent activity against the Gram-positive bacteria S. aureus, Enterococcus faecalis, Enterococcus faecium, Listeria monocytogenes, and Bacillus subtilis, with minimum inhibitory concentration (MIC) values ranging from 6.3 to 31.3 µg/mL, while no activity was observed against Gram-negative bacteria. Broth microdilution assays showed that W2-FL10 interacted with key cell membrane components, such as lipid phosphatidyl glycerol and lipoteichoic acid of S. aureus. Upon membrane interaction, W2-FL10 dissipated membrane potential within 12 min and increased S. aureus membrane permeability within 28-40 min, albeit at slower rates and higher concentrations than the lytic peptide melittin. The observed membrane permeability, as detected with propidium iodide (PI), may be attributed to transmembrane pores/lesions, possibly dependent on dimer-driven lipopeptide oligomerization in the membrane. Scanning electron microscopy (SEM) imaging also visually confirmed the formation of lesions in the cell wall of one of the S. aureus strains, and cell damage within 1 h of exposure to W2-FL10, corroborating the rapid time-kill kinetics of the S. aureus strains. This bactericidal action against the S. aureus strains corresponded to membrane permeabilization by W2-FL10, indicating that self-promoted uptake into the cytosol may be part of the mode of action. Finally, this lipopeptide exhibited low to moderate cytotoxicity to the Chinese hamster ovarian (CHO) cell line in comparison to the control (emetine) with an optimal lipophilicity range (log D value of 2.5), signifying its potential as an antibiotic candidate. IMPORTANCE: Antimicrobial resistance is a major public health concern, urgently requiring antibacterial compounds exhibiting low adverse health effects. In this study, a novel antibacterial lipopeptide analog is described, serrawettin W2-FL10 (derived from Serratia marcescens), with potent activity displayed against Staphylococcus aureus. Mechanistic studies revealed that W2-FL10 targets the cell membrane of S. aureus, causing depolarization and permeabilization because of transmembrane lesions/pores, resulting in the leakage of intracellular components, possible cytosolic uptake of W2-FL10, and ultimately cell death. This study provides the first insight into the mode of action of a non-ionic lipopeptide. The low to moderate cytotoxicity of W2-FL10 also highlights its application as a promising therapeutic agent for the treatment of bacterial infections.

ß-sheet structures and dimer models of the two major tyrocidines, antimicrobial peptides from Bacillus aneurinolyticus.

The structures of two major tyrocidines, antibiotic peptides from Bacillus aneurinolyticus, in an aqueous environment were studied using nuclear magnetic resonance spectroscopy, restrained molecular dynamics (MD), circular dichroism, and mass spectrometry. TrcA and TrcC formed ß-structures in an aqueous environment. Hydrophobic and hydrophilic residues were not totally separated into nonpolar and polar faces of the peptides, indicating that tyrocidines have low amphipathicity. In all the ß-structures, residues Trp(4)/Phe(4) and Orn(9) were on the same face. The ability of the peptides to form dimers in aqueous environment was studied by replica exchange MD simulations. Both peptides readily dimerize, and predominant complex structures were characterized through cluster analysis. The peptides formed dimers by either associating sideways or stacking on top of each other. Dimers formed through sideways association were mainly stabilized by hydrogen bonding, while the other dimers were stabilized by hydrophobic interactions. The ability of tyrocidine peptides to form different types of dimers with different orientations suggests that they can form larger aggregates, as well.

Manipulation of the tyrothricin production profile of Bacillus aneurinolyticus.

A group of non-ribosomally produced antimicrobial peptides, the tyrocidines from the tyrothricin complex, have potential as antimicrobial agents in both medicine and industry. Previous work by our group illustrated that the more polar tyrocidines rich in Trp residues in their structure were more active toward Gram-positive bacteria, while the more non-polar tyrocidines rich in Phe residues had greater activity toward Plasmodium falciparum, one of the major causative pathogens of malaria in humans. Our group also found that the tyrocidines have pronounced antifungal activity, dictated by the primary sequence of the tyrocidine. By simply manipulating the Phe or Trp concentration in the culture medium of the tyrothricin producer, Bacillus aneurinolyticus ATCC 10068, we were able to modulate the production of subsets of tyrocidines, thereby tailoring the tyrothricin complex to target specific pathogens. We optimized the tailored tyrothricin production using a novel, small-scale, high-throughput deep 96-well plate culturing method followed by analyses of the peptide mixtures using ultra-performance liquid chromatography linked to mass spectrometry. We were able to gradually shift the production profile of the tyrocidines and analogues, as well as the gramicidins between two extremes in terms of peptide subsets and peptide hydrophobicity. This study demonstrated that tyrothricin peptide subsets with targeted activity can be efficiently produced by simple manipulation of the aromatic amino acid profile of the culture medium.

Self-assembling organo-peptide bolaphiles with KLK tripeptide head groups display selective antibacterial activity.

In keeping with recent efforts to generate compounds for antibiotic and microbicide development, we focused on the creation of non-natural organo-peptide hybrids of antimicrobial peptide amides (KLK(L)n KLK-NH2 ) derived from sapecin B and a self-assembling oligoglycine organo-peptide bolaphile containing an ω-amino fatty acid residue. The hybrid organo-peptide bolaphiles with two cationic KLK tripeptide motifs linked with an ω-amino acid residue (penta-, octa- or undecamethylene chain) maintained the self-assembling properties of the root oligoglycine bolaphile. Electron microscopy clearly revealed complex supramolecular architectures for both sapecin B-derived peptides and the hybrid analogues. FT-IR spectroscopy indicated that the supramolecular structures were composed primarily of ß-sheets. CD revealed that the hybrid bolaphiles did not share the same secondary structures as the sapecin B peptides in solution. However, although secondary structures of antimicrobial peptides are central in the activity, the organo-peptide bolaphiles also retained the potent antimicrobial activity of the leader sapecin B-derived peptide against both Gram-positive and Gram-negative bacteria. In general, the hybrids were more selective than the sapecin B peptides, as they displayed little or no appreciable haemolytic activity. The results obtained herald a new approach for the design of purpose-built hybrid organo-peptide bolaphiles.

Secondary metabolic profiling of Serratia marcescens NP10 reveals new stephensiolides and glucosamine derivatives with bacterial membrane activity.

Secondary metabolic profiling, using UPLC-MSE and molecular networking, revealed the secondary metabolites produced by Serratia marcescens NP10. The NP10 strain co-produced cyclic and open-ring stephensiolides (i.e., fatty acyl chain linked to Thr-Ser-Ser-Ile/Leu-Ile/Leu/Val) and glucosamine derivatives (i.e., fatty acyl chain linked to Val-glucose-butyric/oxo-hexanoic acid), with the structures of sixteen new stephensiolides (L-Y) and three new glucosamine derivatives (L-N) proposed. Genome mining identified sphA (stephensiolides) and gcd (glucosamine derivatives) gene clusters within Serratia genomes available on NBCI using antiSMASH, revealing specificity scores of the adenylation-domains within each module that corroborates MSE data. Of the nine RP-HPLC fractions, two stephensiolides and two glucosamine derivatives exhibited activity against Staphylococcus aureus (IC50 of 25-79 µg/mL). 1H NMR analysis confirmed the structure of the four active compounds as stephensiolide K, a novel analogue stephensiolide U, and glucosamine derivatives A and C. Stephensiolides K and U were found to cause membrane depolarisation and affect the membrane permeability of S. aureus, while glucosamine derivatives A and C primarily caused membrane depolarisation. New members of the stephensiolide and glucosamine derivative families were thus identified, and results obtained shed light on their antibacterial properties and mode of membrane activity.

Antimicrobial nano-assemblies of tryptocidine C, a tryptophan-rich cyclic decapeptide, from ethanolic solutions.

Tryptocidine C (TpcC), a Trp-rich cyclodecapeptide is a minor constituent in the antibiotic tyrothricin complex from Brevibacillus parabrevis. TpcC possesses a high tendency to oligomerise in aqueous solutions and dried TpcC forms distinct self-assembled nanoparticles. High-resolution scanning electron microscopy revealed the influence of different ethanol:water solvent systems on TpcC self-assembly, with the TpcC, dried from a high concentration in 15% ethanol, primarily assembling into small nanospheres with 24.3 nm diameter and 0.05 polydispersity. TpcC at 16 µM, near its CMC, formed a variety of structures such as small nanospheres, large dense nanospheroids and facetted 3-D-crystals, as well as sheets and coarse carpet-like structures which depended on ethanol concentration. Drying 16 µM TpcC from 75% ethanol resulted in highly facetted 3-D crystals, as well as small nanospheres, while those in 10% ethanol preparation had less defined facets. Drying from 20 to 50% ethanol led to polymorphic architectures with a few defined nanospheroids and various small nanoparticles, imbedded in carpet- and sheet-like structures. These polymorphic surface morphologies correlated with maintenance of fluorescence properties and the surface-derived antibacterial activity against Staphylococcus aureus over time, while there was a significant change in fluorescence and loss in activity in the 10% and 75% preparations where 3-D crystals were observed. This indicated that TpcC oligomerisation in solutions with 20-50% ethanol leads to metastable structures with a high propensity for release of antimicrobial moieties, while those leading to crystallisation limit active moieties release. TpcC nano-assemblies can find application in antimicrobial coatings, surface disinfectants, food packaging and wound healing materials.

Temperature-Induced Effects on the Structure of Gramicidin S.

We report on the structure of Gramicidin S (GS) in a model membrane mimetic environment represented by the amphipathic solvent 1-octanol using one-dimensional (1D) and two-dimensional (2D) IR spectroscopy. To explore potential structural changes of GS, we also performed a series of spectroscopic measurements at differing temperatures. By analyzing the amide I band and using 2D-IR spectral changes, results could be associated to the disruption of aggregates/oligomers, as well as structural and conformational changes happening in the concentrated solution of GS. The ability of 2D-IR to enable differentiation in melting transitions of oligomerized GS structures is attributed to the sensitivity of the technique to vibrational coupling. Two melting transition temperatures were identified; at Tm1 in the range 41-47 °C where the GS aggregates/oligomers disassemble and at Tm2 = 57 ± 2 °C where there is significant change involving GS ß-sheet-type hydrogen bonds, whereby it is proposed that there is loss of interpeptide hydrogen bonds and we are left with mainly intrapeptide ß-sheet and ß-turn hydrogen bonds of the smaller oligomers. Further analysis with quantum mechanical/molecular mechanics (QM/MM) simulations and second derivative results highlighted the participation of active GS side chains. Ultimately, this work contributes toward understanding the GS structure and the formulation of GS analogues with improved bioactivity.

Direct surfactin-gramicidin S antagonism supports detoxification in mixed producer cultures of Bacillus subtilis and Aneurinibacillus migulanus.

Antibiotic production as a defence mechanism is a characteristic of a wide variety of organisms. In natural evolutionary adaptation, cellular events such as sporulation, biofilm formation and resistance to antibiotics enable some micro-organisms to survive environmental and antibiotic stress conditions. The two antimicrobial cyclic peptides in this study, gramicidin S (GS) from Aneurinibacillus migulanus and the lipopeptide surfactin (Srf) from Bacillus subtilis, have been shown to affect both membrane and intercellular components of target organisms. Many functions, other than that of antimicrobial activity, have been assigned to Srf. We present evidence that an additional function may exist for Srf, namely that of a detoxifying agent that protects its producer from the lytic activity of GS. We observed that Srf producers were more resistant to GS and could be co-cultured with the GS producer. Furthermore, exogenous Srf antagonized the activity of GS against both Srf-producing and non-producing bacterial strains. A molecular interaction between the anionic Srf and the cationic GS was observed with circular dichroism and electrospray MS. Our results indicate that the formation of an inactive complex between GS and Srf supports resistance towards GS, with the anionic Srf forming a chemical barrier to protect its producer. This direct detoxification combined with the induction of protective stress responses in B. subtilis by Srf confers resistance toward GS from A. migulanus and allows survival in mixed cultures.

Bidirectional solid phase synthesis of a model oligoglycine bolaamphiphile and purification by rapid self-assembly.

We utilised a simple bidirectional (N→C and C→N) solid phase synthesis strategy entailing conventional solid phase peptide synthesis and fragment condensation with a water-soluble carbodiimide to synthesise a model anionic glycylglycine bolaamphiphile containing a suberic acid linker moiety, namely N,N'-suberoyldiglycylglycine. The synthetic suberoyldiglycylglycine was purified using its inherent ability to rapidly self-assemble in an aqueous acidic solution (0.1% trifluoroacetic acid). Monitoring of the rapid assembly process corroborated our visual observation and confirmed packing-directed self-assembly rather than non-specific aggregation or precipitation. The progress of suberoyldiglycylglycine self-assembly was observed to be via the formation of oligomers in the solution, which then self-assembled to form layered ß-sheet type macrostructures. Within 24 h, nanotubes grew from these macrostructures and eventually combined to formed microtubes, which we isolated after 5-7 days.

Creating Robust Antimicrobial Materials with Sticky Tyrocidines.

Modified antimicrobial and antifouling materials and surfaces can be used to limit the propagation of microorganisms on various surfaces and minimise the occurrence of infection, transfer, and spoilage. Increased demand for 'green' solutions for material treatment has pushed the focus towards to naturally produced antimicrobials. Tyrocidines, cyclo-decapeptides naturally produced by a soil bacterium Brevibacillus parabrevis, have a broad spectrum of activity against Gram-positive and Gram-negative bacteria, filamentous fungi, and yeasts. Continual losses in tyrocidine production highlighted the possible association of peptides to surfaces. It was found in this study that tyrocidines readily associates with many materials, with a selectivity towards polysaccharide-type materials, such as cellulose. Peptide-treated cellulose was found to remain active after exposure to a broad pH range, various temperatures, salt solutions, water washes, and organic solvents, with the sterilising activity only affected by 1% SDS and 70% acetonitrile. Furthermore, a comparison to other antimicrobial peptides showed the association between tyrocidines and cellulose to be unique in terms of antimicrobial activity. The robust association between the tyrocidines and various materials holds great promise in applications focused on preventing surface contamination and creating self-sterilising materials.

Direct Detection of Antibacterial-Producing Soil Isolates Utilizing a Novel High-Throughput Screening Assay.

The ever-increasing global threat of common infections developing resistance to current therapeutics is rapidly accelerating the onset of a primitive post-antibiotic era in medicine. The prevention of further antimicrobial resistance development is unlikely due to the continued misuse of antibiotics, augmented by the lack of discovery of novel antibiotics. Screening large libraries of synthetic compounds have yet to offer effective replacements for current antibiotics. Due to historical successes, discovery from large and diverse natural sources and, more specifically, environmental bacteria, may still yield novel alternative antibiotics. However, the process of antibiotic discovery from natural sources is laborious and time-consuming as a result of outdated methodologies. Therefore, we have developed a simple and rapid preliminary screening assay to identify antibacterial-producing bacteria from natural sources. In brief, the assay utilizes the presence or absence of luminescence in bioluminescent reporter bacteria and test bacterium co-cultures in a 96-well plate format to determine the absence or presence of antibacterial compound production. Our assay, called the bioluminescent simultaneous antagonism (BSLA) assay, can accurately distinguish between known antibacterial-producing and non-producing test bacteria. The BSLA assay was validated by screening 264 unknown soil isolates which resulted in the identification of 10 antibacterial-producing isolates, effectively decreasing the pool of isolates for downstream analysis by 96%. By design, the assay is simple and requires only general laboratory equipment; however, we have shown that the assay can be scaled to automated high-throughput screening systems. Taken together, the BSLA assay allows for the rapid pre-screening of unknown bacterial isolates which, when coupled with innovative downstream dereplication and identification technologies, can effectively fast-track antimicrobial discovery.

Antiplasmodial Cyclodecapeptides from Tyrothricin Share a Target with Chloroquine.

Previous research found that the six major cyclodecapeptides from the tyrothricin complex, produced by Brevibacillus parabrevis, showed potent activity against chloroquine sensitive (CQS) Plasmodium falciparum. The identity of the aromatic residues in the aromatic dipeptide unit in cyclo-(D-Phe1-Pro2-(Phe3/Trp3)-D-Phe4/D-Trp4)-Asn5-Gln6-(Tyr7/Phe7/Trp7)-Val8-(Orn9/Lys9)-Leu10 was proposed to have an important role in activity. CQS and resistant (CQR) P. falciparum strains were challenged with three representative cyclodecapeptides. Our results confirmed that cyclodecapeptides from tyrothricin had significantly higher antiplasmodial activity than the analogous gramicidin S, rivaling that of CQ. However, the previously hypothesized size and hydrophobicity dependent activity for these peptides did not hold true for P. falciparum strains, other than for the CQS 3D7 strain. The Tyr7 in tyrocidine A (TrcA) with Phe3-D-Phe4 seem to be related with loss in activity correlating with CQ antagonism and resistance, indicating a shared target and/or resistance mechanism in which the phenolic groups play a role. Phe7 in phenycidine A, the second peptide containing Phe3-D-Phe4, also showed CQ antagonism. Conversely, Trp7 in tryptocidine C (TpcC) with Trp3-D-Trp4 showed improved peptide selectivity and activity towards the more resistant strains, without overt antagonism towards CQ. However, TpcC lead to similar parasite stage inhibition and parasite morphology changes than previously observed for TrcA. The disorganization of chromatin packing and neutral lipid structures, combined with amorphous hemozoin crystals, could account for halted growth in late trophozoite/early schizont stage and the nanomolar non-lytic activity of these peptides. These targets related to CQ antagonism, changes in neural lipid distribution, leading to hemozoin malformation, indicate that the tyrothricin cyclodecapeptides and CQ share a target in the malaria parasite. The differing activities of these cyclic peptides towards CQS and CQR P. falciparum strains could be due to variable target interaction in multiple modes of activity. This indicated that the cyclodecapeptide activity and parasite resistance response depended on the aromatic residues in positions 3, 4 and 7. This new insight on these natural cyclic decapeptides could also benefit the design of unique small peptidomimetics in which activity and resistance can be modulated.

High throughput method to determine the surface activity of antimicrobial polymeric materials.

Surface colonization by microorganisms, combined with the rise in antibiotic resistance, is the main cause of production failures in various industries. Self-sterilising materials are deemed the best prevention of surface colonization. However, current screening methods for these sterilising materials are laborious and time-consuming. The disk diffusion antimicrobial assay and the Japanese industrial standard method for antimicrobial activity on solid surfaces, JIS Z 2801, were compared to our modified solid surface antimicrobial assay in terms of detecting the activity of antibiotic-containing cellulose disks against four bacterial pathogens. Our novel assay circumvents the long incubation times by utilising the metabolic active dye, resazurin, to shorten the time in which antibacterial results are obtained to less than 4 h. This assay allows for increased screening to identify novel sterilising materials for combatting surface colonisation.•Disk diffusion assay could only detect the activity of small compounds that leached from the material over 20-24 h.•JIS Z 2801 was also able to detect the surface activity of non-polar compounds, thought to be inactive based on the disk diffusion results.•The resazurin solid surface antimicrobial assay could obtain the same results as the JIS Z 2801, within a shorter time and in a high-throughput 96-well plate setup.