Drug-resistant Gram-positive Pathogens Research Articles

Unlocking roles of cationic and aromatic residues in peptide amphiphiles in treating drug-resistant gram-positive pathogens

Multidrug resistance (MDR) is a rising threat to global health because the number of essential antibiotics used for treating MDR infections is increasingly compromised. In this work we report a group of new amphiphilic peptides (AMPs) derived from the well-studied G3 (G(IIKK)3I-NH2) to fight infections from Gram-positive bacteria including susceptible Staphylococcus aureus and methicillin-resistant Staphylococcus aureus (MRSA), focusing on membrane interactions. Time-dependent killing experiments revealed that substitutions of II by WW (GWK), II by FF (GFK) and KK by RR (GIR) resulted in improved bactericidal efficiencies compared to G3 (GIK) on both S. aureus and MRSA, with the order of GWK > GIR > GFK > GIK. Electronic microscopy imaging revealed structural disruptions of AMP binding to bacterial cell walls. Fluorescence assays including AMP binding to anionic lipoteichoic acids (LTA) in cell-free and cell systems indicated concentration and time-dependent membrane destabilization associated with bacterial killing. Furthermore, AMP’s binding to anionic plasma membrane via similar fluorescence assays revealed a different extent of membrane depolarization and leakage. These observations were supported by the penetration of AMPs into the LTA barrier and the subsequent structural compromise to the cytoplasmic membrane as revealed from SANS (small angle neutron scattering). Both experiments and molecular dynamics (MD) simulations revealed that GWK and GIR could make the membrane more rigid but less effective in diffusive efficiency than GIK and GFK through forming intramembrane peptide nanoaggregates associated with hydrophobic mismatch and formation of fluidic and rigid patches. The reported peptide-aggregate-induced phase-separation emerged as a crucial factor in accelerated membrane disintegration and fast bacterial killing. This work has demonstrated the importance of membrane interactions to the development of more effective AMPs and the relevance of the approaches as reported in assisting this area of research.

An antibiotic from an uncultured bacterium binds to an immutable target

Antimicrobial resistance is a leading mortality factor worldwide. Here, we report the discovery of clovibactin, an antibiotic isolated from uncultured soil bacteria. Clovibactin efficiently kills drug-resistant Gram-positive bacterial pathogens without detectable resistance. Using biochemical assays, solid-state nuclear magnetic resonance, and atomic force microscopy, we dissect its mode of action. Clovibactin blocks cell wall synthesis by targeting pyrophosphate of multiple essential peptidoglycan precursors (C55PP, lipid II, and lipid IIIWTA). Clovibactin uses an unusual hydrophobic interface to tightly wrap around pyrophosphate but bypasses the variable structural elements of precursors, accounting for the lack of resistance. Selective and efficient target binding is achieved by the sequestration of precursors into supramolecular fibrils that only form on bacterial membranes that contain lipid-anchored pyrophosphate groups. This potent antibiotic holds the promise of enabling the design of improved therapeutics that kill bacterial pathogens without resistance development.

Robust Chemoenzymatic Synthesis of Keratinimicin Aglycone Analogues Facilitated by the Structure and Selectivity of OxyB.

The emergence of multidrug-resistant pathogens poses a threat to public health and requires new antimicrobial agents. As the archetypal glycopeptide antibiotic (GPA) used against drug-resistant Gram-positive pathogens, vancomycin provides a promising starting point. Peripheral alterations to the vancomycin scaffold have enabled the development of new GPAs. However, modifying the core remains challenging due to the size and complexity of this compound family. The recent successful chemoenzymatic synthesis of vancomycin suggests that such an approach can be broadly applied. Herein, we describe the expansion of chemoenzymatic strategies to encompass type II GPAs bearing all aromatic amino acids through the production of the aglycone analogue of keratinimicin A, a GPA that is 5-fold more potent than vancomycin against Clostridioides difficile. In the course of these studies, we found that the cytochrome P450 enzyme OxyBker boasts both broad substrate tolerance and remarkable selectivity in the formation of the first aryl ether cross-link on the linear peptide precursors. The X-ray crystal structure of OxyBker, determined to 2.8 Å, points to structural features that may contribute to these properties. Our results set the stage for using OxyBker broadly as a biocatalyst toward the chemoenzymatic synthesis of diverse GPA analogues.

Transcriptional Regulator DasR Represses Daptomycin Production through Both Direct and Cascade Mechanisms in Streptomyces roseosporus.

Daptomycin, produced by Streptomyces roseosporus, is a clinically important cyclic lipopeptide antibiotic used for the treatment of human infections caused by drug-resistant Gram-positive pathogens. In contrast to most Streptomyces antibiotic biosynthetic gene clusters (BGCs), daptomycin BGC has no cluster-situated regulator (CSR) genes. DasR, a GntR-family transcriptional regulator (TR) widely present in the genus, was shown to regulate antibiotic production in model species S. coelicolor by binding to promoter regions of CSR genes. New findings reported here reveal that DasR pleiotropically regulates production of daptomycin and reddish pigment, and morphological development in S. roseosporus. dasR deletion enhanced daptomycin production and morphological development, but reduced pigment production. DasR inhibited daptomycin production by directly repressing dpt structural genes and global regulatory gene adpA (whose product AdpA protein activates daptomycin production and morphological development). DasR-protected regions on dptEp and adpAp contained a 16 nt sequence similar to the consensus DasR-binding site dre in S. coelicolor. AdpA was shown to target dpt structural genes and dptR2 (which encodes a DeoR-family TR required for daptomycin production). A 10 nt sequence similar to the consensus AdpA-binding site was found on target promoter regions dptAp and dptR2p. This is the first demonstration that DasR regulates antibiotic production both directly and through a cascade mechanism. The findings expand our limited knowledge of the regulatory network underlying daptomycin production, and will facilitate methods for construction of daptomycin overproducers.

Time-Resolved Atomistic Imaging and Statistical Analysis of Daptomycin Oligomers with and without Calcium Ions.

Daptomycin (DP) is effective against multiple drug-resistant Gram-positive pathogens because of its distinct mechanism of action. An accepted mechanism includes Ca2+-triggered aggregation of the DP molecule to form oligomers. DP and its oligomers have so far defied structural analysis at a molecular level. We studied the ability of DP molecule to aggregate by itself in water, the effects of Ca2+ ions to promote the aggregation, and the connectivity of the DP molecules in the oligomers by the combined use of dynamic light scattering in water and atomic-resolution cinematographic imaging of DP molecules captured on a carbon nanotube on which the DP molecule is installed as a fishhook. We found that the DP molecule aggregates weakly into dimers, trimers, and tetramers in water, and strongly in the presence of calcium ions, and that the tetramer is the largest oligomer in homogeneous aqueous solution. The dimer remains as the major species, and we propose a face-to-face stacked structure based on dynamic imaging using millisecond and angstrom resolution transmission electron microscopy. The tetramer in its cyclic form is the largest oligomer observed, while the trimer forms in its linear form. The study has shown that the DP molecule has an intrinsic property of forming tetramers in water, which is enhanced by the presence of calcium ions. Such experimental structural information will serve as a platform for future drug design. The data also illustrate the utility of cinematographic recording for the study of self-organization processes.

Magnitude of Drug-Resistant Gram-Positive Bacterial Pathogens, and Its Associated Factors from External Ocular Infected Patients Attending at Jinka General Hospital Ophthalmic Clinic, Southern Ethiopia.

BackgroundThe emergence and the incidence of antimicrobial-resistant Gram-positive bacteria increase the risk of treatment failure of ocular infection. Thus, this study aimed to determine the magnitude of drug-resistant Gram-positive bacterial pathogens and associated factors among patients attending Jinka General Hospital Ophthalmic Clinic in Southern Ethiopia.MethodsA facility-based cross-sectional study was conducted on 347 external ocular infected patients attending Jinka General Hospital Ophthalmic Clinic from 15th March to 20th June 2021. Study participants were selected by a systematic random sampling technique. Required data were collected by using structured questionnaires. Swabs of the external eye were obtained with a sterile cotton swab and processed in the Jinka branch Public Health Laboratory. Each sample was inoculated on blood agar, chocolate agar, and mannitol salt agar and incubated aerobically and micro-aerobically at 37°C for 48 hrs. Identification was done by standard microbiological protocols and antimicrobial resistance testing by Kirby Bauer’s disk diffusion technique. Logistic regression was used to identify the associated factors with Gram-positive bacterial external-ocular infection.ResultsThe overall prevalence of Gram-positive bacterial pathogens among external ocular samples was 119/347 (34.3%). S. aureus 57/119 (47.39%), followed by coagulase-negative Staphylococcus spp. 38/119 (31.9%), and S. pneumoniae 13/119 (10.9%) were predominantly isolated. Overall, multi-drug resistance was observed in 72/119 (60.5%) of the bacteria isolates. The 78.9% of isolated S. aureus were MDR. Methicillin-resistant S. aureus (MRSA) and Methicillin-resistant CoNS (MR-CoNS) were accounted for 45.6% and 36.8%, respectively. Previous use of ocular antibiotics was statistically associated to external-ocular Gram-positive bacterial infection [AOR= 1.624, 95% CI: (1.037–2.542)].ConclusionHigh levels of drug resistance were observed for commonly prescribed antibiotics, which attracted the attention of an ophthalmic clinic. Thus, for the effective treatment and management of bacterial eye infections, regular monitoring of drug resistance trends is essential.

Visualizing the mode of action and supramolecular assembly of teixobactin analogues in Bacillus subtilis.

Teixobactin has been the source of intensive study and interest as a promising antibiotic, because of its excellent activity against drug-resistant Gram-positive pathogens and its novel but not yet fully understood mechanism of action that precludes drug resistance. Recent studies have demonstrated that the mode of action of teixobactin is more complicated than initially thought, with supramolecular assembly of the antibiotic appearing to play a critical role in the binding process. Further studies of the interactions of teixobactin with bacteria and its molecular targets offer the promise of providing deeper insights into its novel mechanism of action and guiding the design of additional drug candidates and analogues. The current study reports the preparation and study of teixobactin analogues bearing a variety of fluorophores. Structured illumination microscopy of the fluorescent teixobactin analogues with B. subtilis enables super-resolution visualization of the interaction of teixobactin with bacterial cell walls and permits the observation of aggregated clusters of the antibiotic on the bacteria. Förster resonance energy transfer (FRET) microscopy further elucidates the supramolecular assembly by showing that fluorescent teixobactin molecules co-localize within a few nanometers on B. subtilis. Fluorescence microscopy over time with a fluorescent teixobactin analogue and propidium iodide in B. subtilis reveals a correlation between cell death and binding of the antibiotic to cellular targets, followed by lysis of cells. Collectively, these studies provide new insights into the binding of teixobactin to Gram-positive bacteria, its supramolecular mechanism of action, and the lysis of bacteria that follows.

Design, Synthesis and Evaluation of Antimicrobial Database-Derived Peptides Against Drug-Resistant Gram-Positive and Gram-Negative Pathogens

Bacterial drug resistance has become a serious problem worldwide as a consequence of long-term use and misuse of antibiotics. Current research trends indicate the development of anti-microbial peptides (AMPs) as a promising alternative to conventional antibiotics and to tackle antibiotic resistance. In the present work, novel short AMPs containing less than 15 amino acids with potential broad-spectrum activity are de-novo designed. A computational database-guided pipeline of rational analysis and modification is developed and utilized based on established principles of AMP activity such as charge, hydrophobicity, length, amino-acid frequency, charge density and hydrophobic moments. Potential broad-spectrum AMP sequences are designed based on a consecutive refinement strategy in which seed AMP sequences with desirable properties are predicted from scratch and then modified for possible improvement inactivity. Finally, four AMP sequences were designed, synthesized and tested for their efficacy in-vitro for broad spectrum anti-microbial activity against major drug resistant pathogens namely two Gram-positive organisms Staphylococcus aureus, Bacillus cereus and two Gram-negative pathogens Klebsiella aerogenes and Klebsiella pnuemoniae. Based on the inhibitory concentrations, two among the four designed peptides namely PEP01 (GKIMYILTKKS) and PEP03 (FGIKLRSVWKR) showed best results as broad-spectrum AMPs among which the sequence of PEP03 is further projected as a better candidate in terms of its predicted cell-penetrating and non-toxic nature.

Lincolnenins A-D: Isomeric Bactericidal Bianthracenes from Streptomyces lincolnensis.

Cultivation profiling followed by chemical analysis of Streptomyces lincolnensis yielded four new isomeric bianthracenes, lincolnenins A-D (1-4), with relative stereostructures assigned on the basis of detailed spectroscopic analysis. Lincolnenins A (1) and B (2) exhibit restricted rotation about alternate bianthracene 9-9' and 9-8' bridges, respectively, and exist as single atropisomers, whereas C (3) and D (4) are thermally interconvertible atropisomers sharing a common 8-8' bianthracene bridge. Absolute configurations were assigned to 1-4 on the basis of diagnostic ROESY correlations and ECD calculations, whereas acid-mediated dehydration of 1 led to formation and revision of the absolute configuration of the biosynthetically related known Streptomyces antibiotic, setomimycin (5). Lincolnenin A (1) exhibited significant bactericidal activity against multiple susceptible and drug-resistant Gram-positive pathogens (MIC99 < 2.0 μM), including Mycobacterium tuberculosis H37Ra (MIC99 = 0.9 μM).

Phytochemical Screening and In Vitro Antibacterial Activities of Leaf Extract Acacia etbaica Schweinf Against Multidrug Resistant Enterobacteriaceae Human Pathogens

Acacia etbaica Schweinf belongs to the family Fabaceae widely distributed in Africa and various parts of this plant such as bark, leaves, flowers and roots are widely used as a folk medicine for curing of various ailments.This study was aimed to screen the phytoconstituents and evaluate the antibacterial activity of leaf extract of A. etbaica against selected multidrug resistant Enterobacteriaceae family. Leaves of A. etbaica were extracted with petroleum ether, chloroform, acetone, and ethanol by sequential soxhlet extraction. Phytochemical screening of organic leaf extract of A.etbaica was carried out for the detection of phytoconstituents accountable for antibacterial activity. In vitro antibacterial activities of A.etbaica leaf extracts against selected Enterobacteriaceae family gram‑positive bacteria such as (B.subtilis, E.faecalis,S.aureus)and gram-negative (E. coli, K. pneumonia, V. cholera) were evaluated by agar well diffusion. The antibacterial potential of acetone and ethanol leaf extracts of A.etbaica were determined by 96 well plate broth dilution assay. Among the tested organic leaf extracts, both acetone and ethanolic leaf extract of A.etbaica showed a potentially broad spectrum of in vitro antibacterial activity against tested multiple drug resistant Enterobacteriaceae family gram-positive pathogens such as B.subtilis, E. faecalis , and S.aureus and gram-negative bacterial strains E. coli, K. Pneumonia and V. Cholera with significant MIC values. The significant antibacterial activity of both acetone and ethanolic leaf extracts of A.etbaica was due to the subsistence of secondary metabolites phytoconstituents such as flavonoids and phenolic compounds.

Dimeric Stilbene Antibiotics Target the Bacterial Cell Wall in Drug-Resistant Gram-Positive Pathogens.

The prevalence of antibiotic resistance has been increasing globally, and new antimicrobial agents are needed to address this growing problem. We previously reported that a stilbene dimer from Photorhabdus gammaproteobacteria exhibits strong activity relative to its monomer against the multidrug-resistant Gram-positive pathogens methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococcus faecalis. Here, we show that related dietary plant stilbene-derived dimers also have activity against these pathogens, and MRSA is unable to develop substantial resistance even after daily nonlethal exposure to the lead compound for a duration of three months. Through a systematic deduction process, we established the mode of action of the lead dimer, which targets the bacterial cell wall. Genome sequencing of modest resistance mutants, mass spectrometry analysis of cell wall precursors, and exogenous lipid II chemical complementation studies support the target as being lipid II itself or lipid II trafficking processes. Given the broad distribution of stilbenes in plants, including dietary plants, we anticipate that our mode of action studies here could be more broadly applicable to multipartite host-bacterium-plant interactions.

Pharmacodynamics of ClpP-Activating Antibiotic Combinations against Gram-Positive Pathogens.

It is often difficult to cure endocarditis, osteomyelitis, and device-associated infections caused by Gram-positive pathogens, despite therapy with clinically appropriate antibiotics. This may be due to antibiotic tolerance or resistance development. Acyldepsipeptides (ADEPs) are a class of bactericidal compounds active against a variety of clinically important Gram-positive bacteria, including staphylococci, streptococci, and enterococci. ADEPs activate caseinolytic protease P (ClpP), killing high-density, nondividing cultures of bacteria that are tolerant to approved classes of antibiotics. Acyldepsipeptide analog 4 (ADEP4) was active against a panel of drug-resistant Gram-positive pathogens in MIC assays, with no preexisting resistance detected. Killing of stationary-phase cultures was observed when ADEP4 was combined with multiple classes of approved antibiotics. Additionally, a hollow-fiber infection model was used to assess the effects of ADEP4 antibiotic combinations on bacterial killing and resistance development. These studies were performed on high-density cultures of methicillin-resistant S. aureus (MRSA), methicillin-susceptible S. aureus (MSSA), and vancomycin-resistant Enterococcus faecalis (VRE). None of the approved antibiotics linezolid, ampicillin, and oxacillin tested alone had bactericidal activity under these conditions. ADEP4 initially caused killing, but regrowth of the culture was apparent within 96 h due to resistance. Combinations of ADEP4 with linezolid or oxacillin caused substantially improved killing of MRSA or MSSA cultures, respectively, and no regrowth due to resistance was observed. The combination of ADEP4 and ampicillin eradicated cultures of VRE to the limit of detection within 52 h. These data suggest that combining ClpP activators with traditional antibiotics may be a good strategy to treat complicated Gram-positive infections.

The plant-derived chalcone Xanthoangelol targets the membrane of Gram-positive bacteria

Xanthoangelol is a geranylated chalcone isolated from fruits of Amorpha fructicosa that exhibits antibacterial effects at low micromolar concentration against Gram-positive bacterial pathogens such as methicillin-resistant Staphylococcus aureus (MRSA), Enterococcus faecium and Enterococcus faecalis. We demonstrate that Xanthoangelol treatment of Gram-positive bacteria affects bacterial membrane integrity and leads to a leakage of intracellular metabolites. This correlates with a rapid collapse of the membrane potential and results in a fast and strong bactericidal effect. Proteomic profiling of Xanthoangelol-treated cells revealed signatures of cell wall and/or membrane damage and oxidative stress. Xanthoangelol specifically disturbs the membrane of Gram-positive bacteria potentially by forming pores resulting in cell lysis. In contrast, Xanthoangelol treatment of human cells showed only mildly hemolytic and cytotoxic effects at higher concentrations. Therefore, geranylated chalcones such as Xanthoangelol are promising lead structures for new antimicrobials against drug-resistant gram-positive pathogens.

Low cationicity is important for systemic in vivo efficacy of database-derived peptides against drug-resistant Gram-positive pathogens

As bacterial resistance to traditional antibiotics continues to emerge, new alternatives are urgently needed. Antimicrobial peptides (AMPs) are important candidates. However, how AMPs are designed with in vivo efficacy is poorly understood. Our study was designed to understand structural moieties of cationic peptides that would lead to their successful use as antibacterial agents. In contrast to the common perception, serum binding and peptide stability were not the major reasons for in vivo failure in our studies. Rather, our systematic study of a series of peptides with varying lysines revealed the significance of low cationicity for systemic in vivo efficacy against Gram-positive pathogens. We propose that peptides with biased amino acid compositions are not favored to associate with multiple host factors and are more likely to show in vivo efficacy. Thus, our results uncover a useful design strategy for developing potent peptides against multidrug-resistant pathogens.

A FASII Inhibitor Prevents Staphylococcal Evasion of Daptomycin by Inhibiting Phospholipid Decoy Production

Daptomycin is a treatment of last resort for serious infections caused by drug-resistant Gram-positive pathogens, such as methicillin-resistant Staphylococcus aureus We have shown recently that S. aureus can evade daptomycin by releasing phospholipid decoys that sequester and inactivate the antibiotic, leading to treatment failure. Since phospholipid release occurs via an active process, we hypothesized that it could be inhibited, thereby increasing daptomycin efficacy. To identify opportunities for therapeutic interventions that block phospholipid release, we first determined how the host environment influences the release of phospholipids and the inactivation of daptomycin by S. aureus The addition of certain host-associated fatty acids to the growth medium enhanced phospholipid release. However, in serum, the sequestration of fatty acids by albumin restricted their availability to S. aureus sufficiently to prevent their use in the generation of released phospholipids. This finding implies that in host tissues S. aureus may be completely dependent upon endogenous phospholipid biosynthesis to generate lipids for release, providing a target for therapeutic intervention. To test this, we exposed S. aureus to AFN-1252, an inhibitor of the staphylococcal FASII fatty acid biosynthetic pathway, together with daptomycin. AFN-1252 efficiently blocked daptomycin-induced phospholipid decoy production, even in the case of isolates resistant to AFN-1252, which prevented the inactivation of daptomycin and resulted in sustained bacterial killing. In turn, daptomycin prevented the fatty acid-dependent emergence of AFN-1252-resistant isolates in vitro In summary, AFN-1252 significantly enhances daptomycin activity against S. aureus in vitro by blocking the production of phospholipid decoys, while daptomycin blocks the emergence of resistance to AFN-1252.

Antibacterial Efficacy of Commercially Available Essential Oils Tested Against Drug-Resistant Gram-Positive Pathogens

The potential antibacterial activity of basil (Ocimum basilicum), chamomile (Matricaria chamomilla), origanum (Thymus capitatus), tea tree (Melaleuca alternifolia) and thyme (Thymus vulgaris) essential oils, was investigated against 29 Gram-positive bacterial strains isolated from wastewater treatment plants, clinical samples (n = 25) and American Type Culture Collection (ATCC) reference strains (n = 4). Wild bacterial strains included methicillin-resistant Staphylococcus aureus (n = 16) and vancomycin-resistant Enterococcus spp. (n = 9). The antimicrobial activity of the selected oils was studied using the broth macrodilution method. The Minimal Inhibitory Concentration (MIC) values for S. aureus ranged from 0.06 to 0.5% (v/v) for origanum oil, 0.06 to 1% (v/v) for thyme oil, 0.12 to 1% (v/v) for tea tree oil, 0.25 to 4% (v/v) for basil oil and 2 to &gt;4% (v/v) for chamomile oil. For enterococci the MIC values were significantly higher ranging from 0.25 to 1% (v/v), 0.5 to 2% (v/v), 1 to 4% (v/v), 4 to &gt;4% (v/v) and &gt;4% (v/v) for the above-mentioned oils, respectively. The main compounds of the tested essential oils were: estragole (Ocimum basilicum), bisabolol and trans-b-farnesene (Matricaria chamomilla), carvacrol and thymol (Thymus capitatus), terpinen-4-ol and p-cymene (Melaleuca alternifolia), thymol, linalool, and p-cymene (Thymus vulgaris). Origanum essential oil yielded the best antimicrobial results followed by thyme, tea tree, and basil oil, while chamomile oil exhibited weak antibacterial properties.

Design, Synthesis and Antibacterial Evaluation of 1-[(1R,2S)-2-Fluorocyclopropyl]ciprofloxacin-1,2,4-triazole-5(4H)-thione Hybrids.

A new class of 1-[(1R,2S)-2-fluorocyclopropyl]ciprofloxacin (CPFX)-1,2,4-triazole-5(4H)-thione hybrids 6a-6o was designed, synthesized and evaluated for their invitro antibacterial activities against a panel of clinically important drug-sensitive and drug-resistant Gram-positive and Gram-negative pathogens. Our results revealed that all hybrids 6a-6o had great potency against the tested strains, especially Gram-negative pathogens. The synthesized hybrids were more potent than the parent 1-[(1R,2S)-2-fluorocyclopropyl]CPFX (1) and comparable to CPFX and levofloxacin against the majority of the tested pathogens, worth to be further investigated.

Bioinformatic Expansion and Discovery of Thiopeptide Antibiotics.

Thiopeptides are members of the ribosomally synthesized and post-translationally modified peptide family of natural products. Most characterized thiopeptides display nanomolar potency toward Gram-positive bacteria by blocking protein translation with several being produced at the industrial scale for veterinary and livestock applications. Employing our custom bioinformatics program, RODEO, we expand the thiopeptide family of natural products by a factor of four. This effort revealed many new thiopeptide biosynthetic gene clusters with products predicted to be distinct from characterized thiopeptides and identified gene clusters for previously characterized molecules of unknown biosynthetic origin. To further validate our data set of predicted thiopeptide biosynthetic gene clusters, we isolated and characterized a structurally unique thiopeptide featuring a central piperidine and rare thioamide moiety. Termed saalfelduracin, this thiopeptide displayed potent antibiotic activity toward several drug-resistant Gram-positive pathogens. A combination of whole-genome sequencing, comparative genomics, and heterologous expression experiments confirmed that the thioamide moiety of saalfelduracin is installed post-translationally by the joint action of two proteins, TfuA and YcaO. These results reconcile the previously unknown origin of the thioamide in two long-known thiopeptides, thiopeptin and Sch 18640. Armed with these new insights into thiopeptide chemical-genomic space, we provide a roadmap for the discovery of additional members of this natural product family.

Molecular mechanisms underlying daptomycin resistance

Due to potent bactericidal activity and low rate of drug-resistance, daptomycin is recognized as first line antibiotic to treat serious infections caused by drug-resistant Gram-positive pathogens. However, the incidence of daptomycin resistance is increasing due to its widespread application. Alteration of cell wall homeostasis and membrane phospholipid metabolism is involved in daptomycin resistance. The unique mode of action underlying daptomycin resistance in important pathogens, including Staphylococcus aureus and Enterococci, is presented in this paper.

N-(1,3,4-oxadiazol-2-yl)benzamide analogs, bacteriostatic agents against methicillin- and vancomycin-resistant bacteria

Various reports of multidrug-resistant bacteria that are immune to all available FDA-approved drugs demand the development of novel chemical scaffolds as antibiotics. From screening a chemical library, we identified compounds with antibacterial activity. The most potent compounds, F6-5 and F6, inhibited growth of various drug-resistant Gram-positive bacterial pathogens at concentrations ranging from 1 μg/mL to 2 μg/mL. Both compounds were active against clinical isolates of methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-intermediate and vancomycin-resistant S. aureus (VISA and VRSA respectively) and vancomycin-resistant Enterococcus faecalis (VRE). Resistance generation experiments revealed that MRSA could develop resistance to the antibiotic ciprofloxacin but not to F6. Excitingly, F6 was found to be non-toxic against mammalian cells. In a mouse skin wound infection model, F6 was equipotent to the antibiotic fusidic acid in reducing MRSA burden.