Download S. aureus

“A new antibiotic kills pathogens
without detectable resistance”

Published in Jan. 7, 2015 Nature online
◦ Nature, 517 (2015) 455
◦ L.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.
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.
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 environment
◦ Using specific growth factors such as iron-chelating siderophores
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.
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).
Identification of teixobactin (2)

An extract from a new species of bproteobacteria provisionally named Eleftheria
terrae showed good activity.
NMR assignment of teixobactin structure
16S rRNA gene phylogeny of Eleftheria terrae.
The phylogenetic position within the class b-proteobacteria
16S rRNA gene phylogeny of Eleftheria terrae.
The phylogenetic position among its closest relatives
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.
Organism and genotype
Teixobactin MIC (µg ml−1)
The MIC was determined by broth microdilution.
S. aureus (MSSA)
0.25
S. aureus + 10% serum
0.25
S. aureus (MRSA)
0.25
Enterococcus faecalis (VRE)
0.5
Enterococcus faecium (VRE)
0.5
Streptococcus pneumoniae (penicillinR)
≤ 0.03
Streptococcus pyogenes
0.06
Streptococcus agalactiae
0.12
Viridans group streptococci
0.12
B. anthracis
≤ 0.06
Clostridium difficile
0.005
Propionibacterium acnes
0.08
M. tuberculosis H37Rv
0.125
Haemophilus influenzae
4
Moraxella catarrhalis
2
Escherichia coli
25
Escherichia coli (asmB1)
2.5
Pseudomonas aeruginosa
>32
Klebsiella pneumoniae
>32
S. aureus (MSSA)
S. aureus + 10% serum
S. aureus (MRSA)
Enterococcus faecalis (VRE)
Enterococcus faecium (VRE)
Streptococcus pneumoniae
(penicillinR)
Streptococcus pyogenes
Streptococcus agalactiae
0.25
0.25
0.25
0.5
0.5
≤ 0.03
0.06
0.12
Viridans group streptococci
B. anthracis
Clostridium difficile
Propionibacterium acnes
M. tuberculosis H37Rv
Haemophilus influenzae
Moraxella catarrhalis
Escherichia coli
Escherichia coli (asmB1)
Pseudomonas aeruginosa
Klebsiella pneumoniae
0.12
≤ 0.06
0.005
0.08
0.125
4
2
25
2.5
>32
>32
Time-dependent killing of pathogens by teixobactin
S.aureus are grown to early (a) and late (b) exponential phase and challenged
with antibiotics. Teixobactin treatment results in lysis (c).
(d) Resistance acquisition during serial passage of serial passaging in the presence
of sub-MIC levels of antimicrobials. The y-axis is the highest concentration of
the cells grew in during passaging.
S.aureus are grown to early (a) and late (b) exponential phase and challenged
with antibiotics.
Resistance and mechanism (2)

It is unable to obtain mutants of S.aureus NCTC8325-4 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.
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 vancomycintreated 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)
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 at
1xMIC (grey bars). Ciprofloxacin (8xMIC), rifampicin (4xMIC), vancomycin
(2xMIC) and erythromycin (2xMIC) are controls.
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.
The effect of teixobactin on precursor consuming reactions.
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-DSer instead of lipid II-D-Ala-D-Ala) by aqurring vanA gene.

Teixobactin retains bactericidal activity against VISA(SA1287) .
Complex formation of teixobactin with cell wall precursor
variants as formed by VRE. Purified lipid intermediates with
altered 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.
VISA(SA1287) are grown to late exponential
phase and challenged with vancomycin or
teixobactin
In vivo efficacy

Teixobactin 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.
Single dose treatment with teixobactin and vancomycin in neutropenic mouse thigh
infection model using MRSA ATCC33591. Thigh colony forming units are
determined after 26h
Two dose treatment with teixobactin (i.v., 24h and 36h post-infection) and
single dose treatment with amoxicillin (subcutaneous, 24h post-infection)
in immunocompetent lung infection model using S. pneumoniae ATCC6301
for mice. Lung c.f.u. are determined at 48h post infection.
Discussion

This 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.
Remaining questions

Cell 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?
Antibiotic activity of teixobactin and known drugs against contemporary clinical isolates
Antibiotic spectrum of

MIC
◦ MIC 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 NCTC8325-4 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 killing
◦ An 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 studies
◦ For single step resistance, S. aureus NCTC8325-4 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.

Lipid III : undecaprenyl-PP-GlcNac, wall teichoic acid (WTA)
precursor

C55-P : undecaprenyl-phosphate
C55-PP : undecaprenyl-pyrophosphate

culture : 培養する
tuberculosis : 結核
pneumonia : 肺炎
septicemia : 敗血症
entecococci : 腸球菌
pharmacokinetic : 薬物動態学的
thigh : 大腿部
i.v. : into a vein : 静脈注射
intraperitoneally : 腹腔内の i.p.
lantibiotic : 抗菌性ペプチド
defensin : 好中球の抗菌ペプチド
i.e. : that is
Streptococcus pneumoniae : 肺炎連鎖球菌
siderophores : 親鉄剤
clostridium difficile : クロストリジウム・ディフィシレ
Bacillus anthracis : 炭疽菌