Presentation on theme: "“A new antibiotic kills pathogens without detectable resistance”"— Presentation transcript:
1 “A new antibiotic kills pathogens without detectable resistance” Published in Jan. 7, 2015 Nature onlineNature, 517 (2015) 455L.L. Ling1 et al. (1NovoBiotic Pharmaceuticals, Cambridge, USA)792,000 google hits (Feb. 4) with a keyword “teixobactin”A screen of 10,000 bacterial strains, cultured in their normal soil, has uncovered an antibiotic “teixobactin” with broad and potent activity.Teixobactin is an inhibitor of cell wall synthesis that acts by binding to lipid II, which is a precursor to peptidoglycan, similar to the action of vancomycin.
2 Introduction (1)Widespread introduction of antibiotics in the 1940s, with penicillin and streptomycin, transformed medicine.Resistance development limits the useful lifespan of antibiotics and results in the requirement for a constant introduction of a new one.However, antibiotic drug discovery is uniquely difficult and overmining of cultivable soil organisms by 1960s brought an end to the initial era of antibiotic discovery.
3 Introduction (2)Approximately 99% of all microorganisms in external environments are uncultured and are promising source of new antibiotics.Treatment of uncultured organism:Cultivation in their natural environmentUsing specific growth factors such as iron-chelating siderophores
4 Identification of teixobactin (1) iChip was used to simultaneously isolate and grow uncultured bacteria. A sample of soil is diluted so that approximately one bacterial cell is delivered to a given channel, after which the device is covered with two semi-permeable membranes and placed back in the soil.The growth recovery by iChip approaches 50%, as compared to 1% of cells that will grow on a nutrient Petri dish.Once a colony is formed, a substantial number of uncultured isolates are able to grow in vitro.Extracts from 10,000 isolates in iChips were screened for antimicrobial activity on plates overlaid with S.aureus.
6 Identification of teixobactin (3) Based on 16S rDNA and in silico DNA/DNA hybridization, this organism is found to belong to a new genes related to Aquabacteria.This group of Gram-negative organism is not known to produce antibiotics.A partially purified active fraction contained a compound with molecular mass of 1,242 Da (Vancomycin: 1,449.3).
10 16S rRNA gene phylogeny of Eleftheria terrae. The phylogenetic position within the class b-proteobacteria
11 16S rRNA gene phylogeny of Eleftheria terrae. The phylogenetic position among its closest relatives
12 Resistance and mechanism (1) Teixobactin has strong activity against Gram-positive pathogens, including drug resistant strains and difficult-to-treat enterococci and M. tuberculosis. (below 1mg ml-1)Teixobactin is exceptionally active against Clostridium and Bacillus anthracis.Teixobactin has excellent activity against S.aureus, superior to vancomycin in killing late exponential phase, and retain activity against VISA.Teixobactin is ineffective against most Gram-negative bacteria, but effective against E.coli asmBI with defective outer membrane permeability barrier.
13 Teixobactin MIC (µg ml−1) Organism and genotypeTeixobactin MIC (µg ml−1)The MIC was determined by broth microdilution.S. aureus (MSSA)0.25S. aureus + 10% serumS. aureus (MRSA)Enterococcus faecalis (VRE)0.5Enterococcus faecium (VRE)Streptococcus pneumoniae (penicillinR)≤ 0.03Streptococcus pyogenes0.06Streptococcus agalactiae0.12Viridans group streptococciB. anthracis≤ 0.06Clostridium difficile0.005Propionibacterium acnes0.08M. tuberculosis H37Rv0.125Haemophilus influenzae4Moraxella catarrhalis2Escherichia coli25Escherichia coli (asmB1)2.5Pseudomonas aeruginosa>32Klebsiella pneumoniae
16 Time-dependent killing of pathogens by teixobactin S.aureus are grown to early (a) and late (b) exponential phase and challengedwith antibiotics. Teixobactin treatment results in lysis (c).(d) Resistance acquisition during serial passage of serial passaging in the presenceof sub-MIC levels of antimicrobials. The y-axis is the highest concentration ofthe cells grew in during passaging.
17 S.aureus are grown to early (a) and late (b) exponential phase and challenged with antibiotics.
18 Resistance and mechanism (2) It is unable to obtain mutants of S.aureus NCTC or M.tuberculosis resistat to teixobactin. [1010 c.f.u. are plated onto MHA containing 2x, 4x, and 10xMIC of teixobactin. After 48h of incubation at 37C, no resistant colonies are detected, giving the calculated frequency of resistance of <10-10]Serial passage of S.aureus in the presence of sub-MIC levels of teixobactin over a period of 27days failed to produce resistant mutants as well.Essential lack of resistance development through mutation might suggest that the target is not a protein.Like a case of vancomycin, binding to lipid II, a precursor of peptidoglycan could be a target of teixobactin.
20 Resistance and mechanism (3) Teixobactin strongly inhibits synthesis of peptidoglycan, but had virtually no effect on label incorporation to DNA, RNA and protain. (3a)Treatment of whole cells of S.aureus with teixobactin (1-5 x MIC) resulted in significant accumulation of the soluble cell wall precursor (UDP-MurNAc-pentapeptide), similar to vancomycin- treated control cells, showing membrane-associated steps of peptidoglycan biosnthesis is blocked. (3b)Teixobactin inhibits peptidoglycan biosynthesis reactions in vitro in a dose-dependent manner, and an almost complete inhibition at a two-fold molar excess of teixobactin with respect to the lipid substrate. (3c)
22 Impact of teixobactin (TEIX) on macromolecular biosyntheses in S Impact of teixobactin (TEIX) on macromolecular biosyntheses in S.aureus.Incorporation of 3H-thymidine (DNA), 3H-uridine (RNA), 3H-glucosamine(peptidoglycan) was determined in cells treated with teixobactin at1xMIC (grey bars). Ciprofloxacin (8xMIC), rifampicin (4xMIC), vancomycin(2xMIC) and erythromycin (2xMIC) are controls.
23 Analysis of cytoplasmic peptidoglycan nucleotide precursor pool of S. aureus ATCC29213 grown in 25ml MHB.Identification of UDP-MurNAc-pentapeptide is done by mass spectroscopy.
24 The effect of teixobactin on precursor consuming reactions.
25 Resistance and mechanism (4) In order to evaluate the minimal motif required for high affinity binding of teixobactin, purified precursors are incubated with teixobactin at different molar ratios, followed by extraction and subsequent thin-layer chromatography analysis. Lipid I and lipid II are fully trapped in a stable complex at molar ratio 2:1, forming a stoichiometric complex.Teixobactin is active against VRE (vancomycin-resistant enterococci) that has modified lipid II (lipid II-D-Ala-D-Lac and lipid II-D-Ala-D- Ser instead of lipid II-D-Ala-D-Ala) by aqurring vanA gene.Teixobactin retains bactericidal activity against VISA(SA1287) .
27 Complex formation of teixobactin with cell wall precursor variants as formed by VRE. Purified lipid intermediates withaltered stem peptides were incubated with teixobactin at a molecular ratio of 2:1. Binding of teixobactin is indicated by its absence of on the thin-layer chromatogram.
28 VISA(SA1287) are grown to late exponential phase and challenged with vancomycin orteixobactin
30 In vivo efficacyTeixobactin retains it potency in the presence of serum, is stable, and low toxicity.Pharmacokinetic parameters : After i.v. injection of 20mg per kg dose in mice, the teixobactin level is kept above the MIC for 4h.An animal efficacy study in a mouse septicemia model : Mice are injected with MRSA at a dose that leads to 90% death. One hour post-infection, teixobactin is introduced i.v. at single doses ranging 1 to 20 mg per kg. All treated animals survived and in a subsequent experiment the PD50 (protective dose at which half of the animals survive) is determined to be 0.2 mg per kg, which compares favourably to the 2.75 mg per kg Pd50 of vancomycin.A thigh model of infection with S.aureus, mice infection with Streptococcus pneumoniae, also show good efficacy.
34 Single dose treatment with teixobactin and vancomycin in neutropenic mouse thigh infection model using MRSA ATCC Thigh colony forming units are determined after 26h
35 Two dose treatment with teixobactin (i. v Two dose treatment with teixobactin (i.v., 24h and 36h post-infection) andsingle dose treatment with amoxicillin (subcutaneous, 24h post-infection)in immunocompetent lung infection model using S. pneumoniae ATCC6301for mice. Lung c.f.u. are determined at 48h post infection.
36 DiscussionThis study suggests that new organism such as uncultured bacteria are likely to harbour new antimicrobials.Binding of teixobactin to WTA precursor Lipid III contributes to efficient lysis and killing, due to digestion of the cell wall by liberated autolysin.Producer is a gram-negative bacterium whose outer membrane will protect it from re-entry of the compound, without having any antibacterial resistance mechanism. (In case of vancomycin, its producer Amycolatopsis orientalis is probably ? responsible to the occurrence of resistance)Teixobactin is the first member of a new class of lipid II binding antibiotics, structurally distinct from glycopeptides, lantibiotics, and defensins.
37 Remaining questionsCell wall peptidoglycan of a single S.aureus possesses about 6.0x106 D-alanyl-D-alanine residue (DDRs) of lipid II precursors. Binding of teixobactin to DDRs while penetrating the peptidoglycan layers should be checked.Authors expect that resistance to teixobactin is not easy to develop, because there are two targets (lipid II and lipid III) for teixobactin. How about the thicknning of cell wall which causes the vancomycin intermediate S. aureus, together with the clogging mechanism?
42 MBC (Minimum bactericidal concentration) MICMIC was determined by broth micro-dilution according to CLSI guidelines. The test medium for most species is cation-adjusted Mueller-Hinton broth. After 20h of incubation at 37C. MIC is defined as the lowest concentration of antibiotic with no visible growth.MBC (Minimum bactericidal concentration)S. aureus NCTC cells from the wells from an MIC microbroth plate that have been incubated for 20h at 38C are pelleted. An aliquot of the initial inoculum for the MIC plate is similarly processed. The cells are resuspended in fresh media, plated onto MHA (Muller-Hinton agar), and the colonies enumerated after the incubation for 24h at 37C. The MBC is defined as the first drug dilution which resulted in a 99.9% decrease from the initial bacterial titre of the starting inoculum, and was determined to be 2xMIC for teixobactin.Time-dependent killingAn overnight culture of cells (S. aureus HG003; VISA 1287) is diluted 1:10,000 in MHB and incubated at 37C with aeration at 225 r.p.m. for 2h (early exponential) or 5h(late exponential). Bacteria are then challenged with antibiotics at 10xMIC in culture tube at 37C and 225r.p.m.Resistance studiesFor single step resistance, S. aureus NCTC at 1010 c.f.u. are plated onto MHA containing 2x, 4x, and 10xMIC of teixobactin. After 48h of incubation at 37C, no resistant colonies are detected, giving the calculated frequency of resistance of <10-10.