Search

Ramoplanin is a glycolipodepsipeptide antibiotic active against Gram-positive bacteria including vancomycin-resistant enterococci. Ramoplanin inhibits bacterial cell wall biosynthesis by a mechanism different from that of glycopeptides and hence does not show cross-resistance with these antibiotics. The systemic use of ramoplanin has been so far prevented because of its low local tolerability when injected intravenously. To overcome this problem, the fatty acid side chain of ramoplanin was selectively removed and replaced with a variety of different carboxylic acids. Many of the new ramoplanin derivatives showed antimicrobial activity similar to that of the natural precursor coupled with a significantly improved local tolerability. Among them the derivative in which the 2-methylphenylacetic acid has replaced the di-unsaturated fatty acid side chain (48) was selected as the most interesting compound and submitted to further in vitro and in vivo characterization studies. more...

Ramoplanin and enduracidin are lipopeptide antibiotics effective against Gram-positive pathogens, which share close similarity in structure and biosynthetic pathway. Both compounds have chlorine atoms attached to 4-hydroxyphenylglycine (Hpg) but with different chlorinating sites and levels. Here, to probe the factor affecting the site and level of halogenation, gene inactivation and heterologous expression were carried out in Actinoplanes sp. ATCC33076 by homologous recombination. Metabolite analysis confirmed that ram20 encodes the only halogenase in ramoplanin biosynthetic pathway, and enduracidin halogenase End30 could heterologously complement the ram20-deficient mutant. Additionally, the mannosyltransferase-deficient mutant produces a dichlorinated ramoplanin aglycone with the halogenation site at Hpg(13). This study has refined our understanding of how halogenation occurs in ramoplanin biosynthetic pathway, and lays the foundation for further exploitation of ramoplanin and enduracidin halogenase in combinatorial biosynthesis. more...

Staphylococcus aureus is a major human pathogen and ramoplanin is an antimicrobial attributed for effective treatment. The goal of this study was to examine the transcriptomic profiles of ramoplanin sensitive and resistant S. aureus to identify putative modules responsible for virulence and resistance-mechanisms and its characteristic novel genes. The dysregulated genes were used to reconstruct protein functional association networks for virulence-factors and resistance-mechanisms individually. Strong link between metabolic-pathways and development of virulence/resistance is suggested. We identified 15 putative modules of virulence factors. Six hypothetical genes were annotated with novel virulence activity among which SACOL0281 was discovered to be an essential virulence factor EsaD. The roles of MazEF toxin-antitoxin system, SACOL0202/SACOL0201 two-component system and that of amino-sugar and nucleotide-sugar metabolism in virulence are also suggested. In addition, 14 putative modules of resistance mechanisms including modules of ribosomal protein-coding genes and metabolic pathways such as biotin-synthesis, TCA-cycle, riboflavin-biosynthesis, peptidoglycan-biosynthesis etc. are also indicated. more...

The lipoglycodepsipeptide antibiotic ramoplanin is proposed to inhibit bacterial cell wall biosynthesis by binding to intermediates along the pathway to mature peptidoglycan, which interferes with further enzymatic processing. Two sequential enzymatic steps can be blocked by ramoplanin, but there is no definitive information about whether one step is inhibited preferentially. Here we use inhibition kinetics and binding assays to assess whether ramoplanin and the related compound enduracidin have an intrinsic preference for one step over the other. Both ramoplanin and enduracidin preferentially inhibit the transglycosylation step of peptidoglycan biosynthesis compared with the MurG step. The basis for stronger inhibition is a greater affinity for the transglycosylase substrate Lipid II over the MurG substrate Lipid I. These results provide compelling evidence that ramoplanin's and enduracidin's primary cellular target is the transglycosylation step of peptidoglycan biosynthesis. more...

OBJECTIVE: To review the pharmacology, antimicrobial activity, pharmacokinetics, clinical applications, and safety of ramoplanin, a lipoglycodepsipeptide antibiotic. DATA SOURCES: Information was obtained from MEDLINE and BIOSIS databases (1984–August 2004) and Oscient Pharmaceu ticals using the key words ramoplanin, A 16686, A 16686A, and MDL 62198. STUDY SELECTION AND DATA EXTRACTION: Available English-based articles and abstracts were reviewed, along with information from Oscient Pharmaceuticals. DATA SYNTHESIS: Ramoplanin exerts its bactericidal activity against gram-positive aerobic and anaerobic bacteria by blocking peptidoglycan synthesis via lipid II. In vitro susceptibility reports have demonstrated efficacy against antibiotic-resistant gram-positive pathogens. Cross-resistance has not been documented with vancomyin and other glycopeptides. Clinical trials are investigating ramoplanin's oral administration for treatment of Clostridium difficile–associated diarrhea. Previous clinical trials had evaluated the suppression of colonization of vancomycin-resistant Enterococcus with ramoplanin. Adverse effects are minimal, and drug—drug interactions have not been documented. CONCLUSIONS: The completion of clinical trials will determine whether ramoplanin has a promising role as a treatment option for diarrhea due to C. difficile. more...

Ramoplanin is a glycolipodepsipeptide antibiotic with activity against gram-positive bacteria that is in clinical trials for prevention of vancomycin-resistant Enterococcus (VRE) bloodstream infections and treatment of Clostridium difficile diarrhea. Orally administered ramoplanin suppresses VRE intestinal colonization, but recurrences after discontinuation of treatment have frequently been observed. We used a mouse model to examine the efficacy of ramoplanin for inhibition of VRE colonization and evaluated the etiology of recurrences of colonization. Eight days of treatment with ramoplanin (100 μg/ml) in drinking water suppressed VRE to undetectable levels, but 100% of mice developed recurrent colonization; a higher dose of 500 μg/ml in water was associated with recurrent colonization in 50% of mice. Two of eight (25%) mice treated with the 100-μg/ml dose of ramoplanin had low levels of VRE in their cecal tissues on day 8 despite undetectable levels in stool and cecal contents. Mice that received prior ramoplanin treatment did not develop VRE overgrowth when challenged with 10 7 CFU of oral VRE 1, 2, or 4 days later. In communal cages, rapid cross-transmission and overgrowth of VRE was observed among clindamycin-treated mice; ramoplanin treatment effectively suppressed VRE overgrowth in such communal cages. Ramoplanin treatment promoted increased density of indigenous Enterobacteriaceae and overgrowth of an exogenously administered Klebsiella pneumoniae isolate. These results demonstrate the efficacy of ramoplanin for inhibition of VRE colonization and suggest that some recurrences occur due to reexpansion of organisms that persist within the lining of the colon. Ramoplanin treatment may be associated with overgrowth of gram-negative bacilli. more...

By using an agar dilution method, the in vitro activities of ramoplanin, teicoplanin, vancomycin, linezolid, and five other agents were determined against 300 gram-positive and 54 gram-negative strains of intestinal anaerobes. Ramoplanin was active at ≤2 μg/ml against 287 of 300 (95.7%) gram-positive organisms, including 18 strains of Clostridium difficile for which MICs of ramoplanin were 0.25 to 0.5 μg/ml; for 3 of these, linezolid MICs were 8 to 16 μg/ml. Nineteen Clostridium innocuum strains for which the vancomycin MIC at which 90% of strains were inhibited was 16 μg/ml were susceptible to ramoplanin at 0.06 to 0.25 μg/ml and to teicoplanin at 0.125 to 1.0 μg/ml. All strains of Eubacterium , Actinomyces , Propionibacterium , and Peptostreptococcus spp. were inhibited by ≤0.25 μg of ramoplanin per ml and ≤1 μg of vancomycin per ml. Ramoplanin was also active at ≤4 μg/ml against 15 of 22 of the Prevotella and Porphyromonas strains tested, but ramoplanin MICs for all 31 strains of the Bacteroides fragilis group, the Fusobacterium mortiferum-Fusobacterium varium group, and Veillonella spp. were ≥256 μg/ml. Ramoplanin displays excellent activity against C. difficile and other gram-positive enteric anaerobes, including vancomycin-resistant strains; however, it has poor activity against most gram-negative anaerobes and thus potentially has a lesser effect on the ecological balance of normal fecal flora. more...

The prevention of vancomycin-resistant Enterococcus (VRE) colonization and infection continues to be a high priority for clinicians. An oral antimicrobial agent that reduces or eliminates VRE gastrointestinal colonization could be useful for preventing VRE infection in selected patients. Ramoplanin, a glycolipodepsipeptide, is the first in a new class of antimicrobials. It has excellent in vitro activity against vancomycin-resistant Enterococcus faecium and Enterococcus faecalis. It is orally administered, and not absorbed systemically. In clinical trials, VRE gastrointestinal colonization was reduced to undetectable levels in 80-90% of patients during receipt of ramoplanin. A randomized, double-blinded, placebo-controlled multicentre study is currently being conducted to determine whether ramoplanin will prevent VRE bloodstream infection in oncology patients who are neutropenic due to treatment for a haematological malignancy or a bone marrow/stem cell transplant. more...

Ramoplanin is a glycolipodepsipeptide antibiotic obtained by fermentation of the Actinoplanes sp. ATCC 33076, isolated as a complex of three closely related components A1, A2 and A3, which differ in their fatty acid moiety. We have investigated the influence of L-leucine and L-valine, the biosynthetic precursors of the fatty acids in A2 and A3 factors, on the complex composition and antibiotic productivity. Addition of 5 g/litre of L-leucine at the time of inoculation increases antibiotic production and improves the production of A2 factor, which represents the active principle component under clinical development. Addition of L-valine in the same conditions modifies the composition of the complex towards the A3 factor but does not improve total antibiotic productivity. A possible explanation for the different actions of the two amino acids is presented. more...

The peptide antibiotic ramoplanin factor A2 is a promising clinical candidate for treatment of Gram-positive bacterial infections that are resistant to antibiotics such as glycopeptides, macrolides, and penicillins. Since its discovery in 1984, no clinical or laboratory-generated resistance to this antibiotic has been reported. The mechanism of action of ramoplanin involves sequestration of peptidoglycan biosynthesis Lipid intermediates, thus physically occluding these substrates from proper utilization by the late-stage peptidoglycan biosynthesis enzymes MurG and the transglycosylases (TGases). Ramoplanin is structurally related to two cell wall active lipodepsipeptide antibiotics, janiemycin, and enduracidin, and is functionally related to members of the lantibiotic class of antimicrobial peptides (mersacidin, actagardine, nisin, and epidermin) and glycopeptide antibiotics (vancomycin and teicoplanin). Peptidomimetic chemotherapeutics derived from the ramoplanin sequence may find future use as antibiotics against vancomycin-resistant Enterococcus faecium (VRE), methicillin-resistant Staphylococcus aureus (MRSA), and related pathogens. Here we review the chemistry and biology of the ramoplanins including its discovery, structure elucidation, biosynthesis, antimicrobial activity, mechanism of action, and total synthesis. more...

NAI-603 is a ramoplanin derivative designed to overcome the tolerability issues of the parent drug as a systemic agent. NAI-603 is highly active against aerobic and anaerobic Gram-positive bacteria, with MICs ranging from 0.008 to 8 μg/ml. MICs were not significantly affected by pH (range, 6 to 8), by inoculum up to 10 8 CFU/ml, or by addition of 50% human serum. Against staphylococci and enterococci, NAI-603 was rapidly bactericidal, with minimum bactericidal concentration (MBC)/MIC ratios never exceeding 4. The frequency of spontaneous resistance was low at 2× to 4× MIC (≤1 × 10 −6 to ≤1 × 10 −8 ) and below the detection limit (about ≤1 × 10 −9 ) at 8× MIC. Serial subcultures at 0.5× MIC yielded at most an 8-fold increase in MICs. In a systemic infection induced by methicillin-resistant Staphylococcus aureus (MRSA), the 50% effective dose (ED 50 ) of intravenous (i.v.) NAI-603 was 0.4 mg/kg, lower than that of oral (p.o.) linezolid (1.4 mg/kg) and subcutaneous (s.c.) teicoplanin (1.4 mg/kg) or vancomycin (0.6 mg/kg). In neutropenic mice infected with vancomycin-resistant enterococci (VRE), the ED 50 s for NAI-603 were 1.1 to 1.6 mg/kg i.v., compared to 0.5 mg/kg i.v. of ramoplanin. The bactericidal activity was confirmed in vivo in the rat granuloma pouch model induced by MRSA, where NAI-603, at 40 mg/kg i.v., induced about a 2- to 3-log 10 -reduction in viable bacteria in the exudates, which persisted for more than 72 h. The pharmacokinetic (PK) profiles of NAI-603 and ramoplanin at 20 mg/kg show similar half-lives (3.27 and 3.80 h, respectively) with the maximum concentration ( C max ) markedly higher for NAI-603 (207 μg/ml versus 79 μg/ml). The favorable pharmacological profile of NAI-603, coupled with the absence of local tolerability issues, supports further investigation. more...

The chemoenzymatic deacylation of ramoplanin A2 is described for the first time: ramoplanin A2 was Boc-protected and hydrogenated to Boc-protected tetrahydroramoplanin, which was subsequently deacylated using an acylase from Actinoplanes utahensis NRRL 12052. The chemoenzymatic process proceeded with 80% overall yield, which favourably compares with the previously described chemical deacylation. more...

The in-vitro activities of ramoplanin, vancomycin, and teicoplanin against Gram-positive organisms isolated from cancer patients were determined. Ramoplanin was the most active agent tested inhibiting all isolates at a concentration of < or = 0.5 mg/L. Although all isolates were also susceptible to vancomycin and teicoplanin, their activities were surpassed by that of ramoplanin. The activity of teicoplanin was moderately better than that of vancomycin against Bacillus cereus, Enterococcus faecium, Listeria monocytogenes, methicillin-resistant Staphylococcus aureus, and most streptococcal isolates, and was comparable to vancomycin for the remainder of the isolates tested. more...

The 3D structure of ramoplanin was studied by NMR spectroscopy in aqueous solution. A total of 320 interproton distances were determined from a NOESY spectrum and were used as restraints in distance geometry calculations. A structural refinement was carried out by molecular dynamics calculations in a solvent box. The structure of ramoplanin is characterized by two antiparallel beta-strands which are formed by the residues 2-7 and 10-14, respectively. The beta-strands are connected by six intramolecular hydrogen bonds and a reverse beta-turn which is formed by Thr8 and Phe9 (in positions i+1 and i+2, respectively). Residues 2 and 14 are connected by a loop consisting of Leu15, Ala16, Chp17, and the side chain of Asn2. Although residues 14-17 show the formation of a beta-turn, only the N-terminal end of the turn is directly connected to one of the beta-strands (Gly14), whereas the C-terminal end (Chp17) is linked via the side chain of Asn2. The 3D conformation of ramoplanin is also stabilized by a hydrophobic cluster of the aromatic sidechains of the residues 3, 9, and 17. This hydrophobic collapse leads to a U-shaped topology of the beta-shee: with the beta-turn at one end and the loop at the other end. The structure found for ramoplanin differs corsiderably from the published structure of ramoplanose which might be due to a smaller number of NOE distance restraints used in the previous study. more...

In this paper unmodified gold nanoparticles (AuNPs) were used as a sensing element to detect ramoplamin. Detection relies on the fact that the dispersed AuNPs solution is red due to the intense surface plasmon absorption band at 530 nm whereas the AuNPs solution in the presence of ramoplanin is blue. Upon aggregation, there is a significant change in absorbance intensity at 620 nm. Based on the aggregation of AuNPs induced by ramoplanin, a simple colorimetric method was developed for determining the of ramoplanin concentration. Experimental conditions influencing the analytical performance such as particle size, amount of AuNPs, incubation time and pH were evaluated. Under the optimized experimental conditions, this method could detect ramoplanin in a linear range from 0.30 to 1.30 ppm with a detection limit of 0.01 ppm and exhibited good reproducibility, selectivity and recovery. Analysis time of this assay was only 2 min. To investigate its potential applicability, this assay was successfully applied for the determination of ramoplanin in urine samples without costly instruments. more...

Vancomycin-resistant Enterococcus faecium (VREF) collected between July 1991 and February 1994 were tested in vitro against RP 59500 and ramoplanin using agar dilution and standard macro broth dilution procedures. Colony counts were determined at 0, 4, and 24 h. RP 59500 had an MIC range ofor = 0.5-8 micrograms/ml with an MIC90 of 2 micrograms/ml and a MBC range ofor = 0.5-16 micrograms/ml with an MBC90 of 16 micrograms/ml. Ramoplanin had an MIC range ofor = 0.125-1 microgram/ml with an MBC range ofor = 0.125-4 micrograms/ml. The MIC90 for ramoplanin was 1 microgram/ml and the MBC90 was 4 micrograms/ml for the tested isolates. Against these isolates of E. faecium, RP 59500 was bactericidal at 8x MIC, a potentially achievable level using a high drug dosage. Ramoplanin was bactericidal at 2x MIC. more...

Ramoplanin is a potent antibiotic, first disclosed in 1984, that acts by inhibiting bacterial cell-wall biosynthesis. The original ramoplanin complex was shown to consist of a mixture of three closely related compounds, ramoplanin A1–A3, of which ramoplanin A2 is the most abundant. The structure of ramoplanin A2 was unambiguously established first through a series of extensive spectroscopic studies, allowing complete stereochemical assignments and subsequently providing a minor reassignment of the side-chain double-bond stereochemistry and, most recently, through total synthesis of authentic material. Here we report the total syntheses of the aglycons of the minor components of the ramoplanin complex, A1 and A3, which unambiguously establish their structure and provide an expected structural revision for the lipid side-chain double-bond stereochemistry. more...

Due to the ongoing problem of recurrence of Clostridium difficile -associated diarrhea following antibiotic treatment, there is an urgent need for alternative treatment options. We assessed the MICs of five antimicrobials singly and in combinations against a range of C. difficile clinical isolates. Ramoplanin-actagardine combinations were particularly effective, with partial synergistic/additive effects observed against 61.5% of C. difficile strains tested. more...

The lipopeptides ramoplanin from Actinoplanes sp. ATCC 33076 and enduracidin produced by Streptomyces fungicidicus are effective antibiotics against a number of drug-resistant Gram-positive pathogens. While these two antibiotics share a similar cyclic peptide structure, comprising 17 amino acids with an N-terminal fatty acid side chain, ramoplanin has a di-mannose moiety that enduracidin lacks. The mannosyl substituents of ramoplanin enhance aqueous solubility, which was important in the development of ramoplanin as a potential treatment for Clostridium difficile infections. In this study we have determined the function of the putative mannosyltransferase encoded by ram29 from the ramoplanin biosynthetic gene cluster. Bioinformatics revealed that Ram29 is an integral membrane protein with a putative DxD motif that is suggested to bind to, and activate, a polyprenyl phosphomannose donor and an extracytoplasmic C-terminal domain that is predicted to bind the ramoplanin aglycone acceptor. The ram29 gene was cloned into the tetracycline inducible plasmid pMS17 and integrated into the genome of the enduracidin producer S. fungicidicus. Induction of ram29 expression in S. fungicidicus resulted in the production of monomannosylated enduracidin derivatives, which are not present in the WT strain. Tandem MS analysis showed that mannosylation occurs on the Hpg11 residue of enduracidin. In addition to confirming the function of Ram29, these findings demonstrate how the less common, membrane-associated, polyprenyl phosphosugar-dependent glycosyltransferases can be used in natural product glycodiversification. Such a strategy may be valuable in future biosynthetic engineering approaches aimed at improving the physico-chemical and biological properties of bioactive secondary metabolites including antibiotics. more...

The susceptibilities of 316 gram-positive bacteremic isolates to ramoplanin, vancomycin, and teicoplanin and seven other antibiotics were tested. Ramoplanin demonstrated MICs of < or = 0.25 microgram/ml for at least 99% of Staphylococcus aureus isolates and 100% of coagulase-negative staphylococci tested. For both oxacillin-susceptible and oxacillin-resistant S. aureus and coagulase-negative staphylococci, the activity of ramoplanin surpassed those of both vancomycin and teicoplanin. Ramoplanin and teicoplanin had comparable activities against enterococci and Streptococcus pneumoniae and were superior to vancomycin. more...

The total synthesis and evaluation of three key ramoplanin aglycon analogues are detailed. The first (5a) represents replacement of the labile depsipeptide ester with a stable amide (HAsn2 → Dap2) with removal of the HAsn pendant carboxamide, and it was found to be slightly more potent than the natural aglycon in antimicrobial assays providing a new lead structure with an improved profile and a more stable and accessible macrocyclic template on which to conduct structure−function studies. In contrast, a second amide analogue 5b which contains a single additional methylene relative to 5a (HAsn2 → Dab2) was found to be inactive in antimicrobial assays (>100-fold loss in activity). The third key analogue 5c in which the Asn1 lipid side chain was replaced with an acetyl group revealed that it contributes significantly to the antimicrobial activity (16-fold) of the ramoplanins, but is not essential. more...

A method has been developed and validated for the quantification of ramoplanin, a 2554 Da peptide antibiotic, in human dried blood spots using high-performance liquid chromatography with tandem mass spectrometric detection. The validation data meet FDA acceptance criteria for bioanalytical assays and cover the quantification of ramoplanin over the range 10–5000 ng/mL. The assay determines ramoplanin at the same lower limit of quantification as conventional liquid sample methods. Dried blood spot analysis provides an approach for quantification of peptide therapeutics and delivers significant benefits for sample collection and handling and also sample cleanup over conventional plasma and serum assays. Copyright © 2010 John Wiley & Sons, Ltd. more...

Ramoplanin is a potent lipoglycodepsipeptide antibiotic that is active against a wide range of Gram-positive bacteria, including methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant enterococcus (VRE). It acts as an inhibitor of peptidoglycan (PG) biosynthesis that disrupts glycan chain polymerization by binding and sequestering Lipid II, a PG precursor. Herein, we report the functional antimicrobial activity (MIC, S. aureus) and fundamental biochemical assessments against a peptidoglycan glycosyltransferase (Escherichia coli PBP1b) of a set of key alanine scan analogues of ramoplanin that provide insight into the importance and role of each of its individual amino acid residues. more...

Vancomycin, teicoplanin, and ramoplanin are potent glycopeptide antibiotics that act by inhibiting bacterial cell wall biosynthesis. The former are used clinically as the antibiotics of last resort for the treatment of methicillin-resistant Staphylococcus aureus and the latter is a promising new antibiotic that is not susceptible to the emerging bacterial resistance to vancomycin and teicoplanin. A summary of our recent total synthesis of the vancomycin aglycon, our first and second generation total syntheses of the teicoplanin aglycon, and our progress on the total synthesis of the ramoplanins is presented. This work lays the foundation for ongoing structure-function studies on the antibiotics that may clarify or define their site and mechanism of action leading to the development of improved or reengineered antibiotics. more...

Ramoplanin is a lipoglycodepsipeptide produced by Actinoplanes sp. ATCC 33076 and active on bacterial cell wall biosynthesis by binding to Lipid II. A screening of an actinomycetes collection was performed to select enzymatic activities able to introduce specific modifications in the ramoplanin molecule. An extracellular mannosidase from Streptomyces GE 91081 was found to selectively remove one mannose unit from ramoplanin and tetrahydroramoplanin to give the corresponding mannosyl aglycones. These molecules show an improved microbiological activity versus some resistant staphylococci and streptococci, and are useful intermediates in the synthesis of novel ramoplanin-like antibiotics. The biotransformation of ramoplanin has been optimised to improve molar conversion and the transformation reaction rate. more...

Ramoplanin, a novel antibiotic with activity against aerobic and anaerobic gram-positive bacteria, acts to prevent cell wall peptidoglycan formation by binding to a key intermediate moiety, lipid II. It has been fast-tracked by the US FDA for the prevention of enterococcal infections and the treatment of Clostridium difficile. The minimum inhibitory concentration(90s) have been < or = 1.0 microg/ml against gram-positive organisms examined. In carriers of vancomycin-resistant enterococci, a double-blind, placebo-controlled Phase II trial of two doses of ramoplanin versus placebo showed proof of concept. A second Phase II trial also demonstrated the equivalence of ramoplanin compared with vancomycin for the treatment of C. difficile colitis. The clinical value and place in therapy of ramoplanin is dependent upon the results of Phase III trials addressing its utility in suppressing carriage of target organisms in the gastrointestinal tract or in the nares. more...

We investigated the effect of ramoplanin coating on Staphylococcus aureus colonization of catheter segments in mice. Segments (1 cm in length) were inserted subcutaneously, 10(7) cfu of S. aureus were inoculated nearby at different times and segments were removed 24 or 48 h later. Uncoated segments were colonized with 10(4) to >10(5) cfu, whereas ramoplanin-coated segments had mean counts of more...

Objectives: Treatment of Clostridium difficile infection (CDI) is limited primarily to either metronidazole or vancomycin. We compared vancomycin and a novel glycolipodepsipeptide, ramoplanin, in both hamster and in vitro gut models of clindamycin-induced CDI. Methods: We used an in vitro triple-stage chemostat model that simulates the human gut, and an in vivo hamster model, both primed with clindamycin. Results: Clindamycin exposure elicited symptomatic disease in the hamster model, and promoted C. difficile germination and toxin production in the gut model. C. difficile germination and toxin production were not associated with depletion of gut microflora in the gut model, but were temporarily associated with subinhibitory concentrations of clindamycin. Both ramoplanin and vancomycin were associated with rapid symptom resolution in the hamster model, and rapid toxin titre decrease in the in vitro gut model. In both models of CDI, vancomycin was associated with greater persistence of C. difficile spores. C. difficile spores were recovered significantly more often from the caecal contents of vancomycin-treated (n = 19/23) compared with ramoplanin-treated (n = 6/23) hamsters (P < 0.05). Conclusions: Results from the in vitro gut and hamster models were concordant. Ramoplanin and vancomycin were similarly effective at reducing cytotoxin production in the gut CDI model and in resolving symptoms in the hamster model. Ramoplanin may be more effective than vancomycin at killing spores and preventing spore recrudescence. These findings suggest a potential therapeutic role for ramoplanin in CDI that requires further clinical investigation. more...

We evaluated the in vitro activity of ramoplanin, an antimicrobial compound that inhibits cell wall synthesis by acting at the level of lipid intermediate formation, against Clostridium difficile. We included strains with reduced susceptibilities to vancomycin (vancomycin-intermediate [Van i ] strains) or with resistance to metronidazole (Mtz r ), in order to assess the potential utility of ramoplanin for the treatment of C. difficile -associated diarrhea. We tested the activity of ramoplanin against a total of 105 nonduplicate clinical isolates of toxigenic C. difficile , including 8 Van i isolates and 6 Mtz r isolates, obtained from our laboratory. Ramoplanin was active against all strains tested at concentrations ranging from 0.03 to 0.5 μg/ml (MICs at which 50 and 90% of isolates were inhibited, 0.25 μg/ml; geometric mean MIC, 0.22 μg/ml). All isolates, independently of their levels of susceptibility to vancomycin or metronidazole, were considered susceptible to ramoplanin (MICs, ≤0.5 μg/ml). more...

Ramoplanin is a lipglycodepsipeptide antibiotic that inhibits peptidoglycan biosynthesis. Its mechanism of action has been the subject of debate. It was originally proposed to inhibit the MurG step of peptidoglycan synthesis by binding Lipid I. In this paper, we report that ramoplanin inhibits bacterial transglycosylases by binding to Lipid II, the substrate for these enzymes. The inhibition curves reveal that the inhibitory species has a stoichiometry of 2:1 ramoplanin:Lipid II. A Job titration confirms that ramoplanin binds as a dimer to Lipid II. The apparent dissociation constant is in the nanomolar range, which is unusually low given the nature of the interacting species. We show that Lipid II binding is coupled to the formation of a higher order species, which may explain the tight binding. We also present a testable model for the binding-competent dimeric conformation of ramoplanin. more...