Structure-activity relationship study of novel iminothiadiazolo- pyrimidinone antimicrobial agents

Article abstract of DOI:10.1038/ja.2013.69

An iminothiadiazolo-pyrimidinone derivative, 0002-04-KK, harboring a furan moiety, acts as an antimicrobial agent with a minimum inhibitory concentration (MIC) against Staphylococcus aureus of 25 μg ml -1. Several derivatives of 0002-04-KK were synthesize

Full text of DOI:10.1038/ja.2013.69

The Journal of Antibiotics (2013) 66, 663–667
2013 Japan Antibiotics Research Association All rights reserved 0021-8820/13
www.nature.com/ja
&
ORIGINAL ARTICLE
Structure–activity relationship study of novel
iminothiadiazolo-pyrimidinone antimicrobial agents
Atmika Paudel1, Keiichi Kaneko2, Ayako Watanabe1, Matsunaga Shigeki2,3, Kanai Motomu2,
Hiroshi Hamamoto1 and Kazuhisa Sekimizu1
An iminothiadiazolo-pyrimidinone derivative, 0002-04-KK, harboring a furan moiety, acts as an antimicrobial agent with a
minimum inhibitory concentration (MIC) against Staphylococcus aureus of 25 lg mlꢀ1. Several derivatives of 0002-04-KK were
synthesized and among them 0026-59-KK, harboring a nitrofuran moiety, had the most potent antimicrobial activity with an
MIC of 6 lg mlꢀ1. Both 0002-04-KK and 0026-59-KK inhibited the biosynthesis of DNA, RNA and proteins. Peptidoglycan
biosynthesis was inhibited by 0026-59-KK, and slightly inhibited by 0002-04-KK. Derivative 0002-04-KK showed bactericidal
activity in contrast to the bacteriostatic activity of 0002-04-KK. Derivative 0002-04-KK had less toxicity in silkworms (lethal
dose fifty (LD50): 4230 lg gꢀ1) than 0002-04-KK (LD50: 100 lg gꢀ1). The bactericidal activity against S. aureus was
because of the nitrofuran moiety. These findings suggest that iminothiadiazolo-pyrimidinone compounds could be used as
lead molecules to develop antimicrobial agents.
The Journal of Antibiotics (2013) 66, 663–667; doi:10.1038/ja.2013.69; published online 3 July 2013
Keywords: bactericidal; macromolecule; nitrofuran; novel antimicrobial; structure–activity relationship
INTRODUCTION
25 mgmlꢀ1. As the structure of the compound was promising, further
The search for antimicrobial agents against human pathogens has studies of the structure function relationship were performed. First,
been ongoing for centuries and remains a challenging task. Anti- we confirmed the antimicrobial activity by the synthesis of 1. Then, we
microbials launched into the clinical field have led to the rapid evaluated the toxicity of 1 using silkworms. The median lethal dose
emergence of more microbes resistant to these antimicrobials. The (LD50) value of the compound in the silkworm was 100 mggꢀ1
.
emergence of strains resistant to recently introduced antimicrobials
enhances the threat of infectious diseases spreading around the globe.
Many notorious resistant pathogens, such as methicillin-resistant
Staphylococcus aureus,1,2 vancomycin-intermediate S. aureus,3,4
vancomycin-resistant Enterococcus faecalis,5 multidrug-resistant
Pseudomonas aeruginosa,6 and multidrug-resistant Mycobacterium
tuberculosis7 cause serious problems for health-care facilities as well
as the community. Hence, the development of new antimicrobial
agents continues to be an urgent issue. To combat resistant strains,
new structural antimicrobial agents are required. Here we identified a
novel antimicrobial agent and modified its structure to reduce toxicity
and enhance antimicrobial activity.
Structure–activity relationship of 1
To enhance the antibacterial activity and reduce the toxicity of 1,
we examined the structure–activity relationship (SAR). Toxicity was
tested using silkworms as a model animal.8 Iminothaidiazolo-
pyrimidinone (Table 1) was used as the mother compound and
structures at the R1 and R2 positions were modified to obtain nine
different compounds. Figure 1 summarizes the synthesis of derivatives
of 1. Condensation of 2-amino-5-substituted-1,3,4-thiadiazole and
ethyl cyanoacetate gave 5-imino-2-(substituted)-5H-(1,3,4)thiadia-
zolo(3,2–a)pyrimidin-7(6H)-one. Further reaction with aldehyde
(R2CHO) in the presence of a catalytic amount of Et3N gave products
of 28–70% yield. Table 1 shows the results of the SAR analysis.
Compound 0026-59-KK (2) exhibited the strongest antibacterial
activity against S. aureus and had the least toxicity in silkworms.
Hence, this compound was selected for further evaluation.
RESULTS
Chemical library screening for antibacterial compounds against
S. aureus
Among the 103 873 compounds in the chemical library, we identified
3383 candidates (3.3%) with antimicrobial activity against the S. aureus Antimicrobial spectrum of 1 and 2
MSSA1 strain at a concentration of 100 mM. We selected 0002-04- Antimicrobial activities of 1 and 2 were tested with various micro-
KK (1), whose antibacterial activity against S. aureus MSSA1 was organisms including Gram-positive bacteria, Gram-negative bacteria
1Laboratory of Microbiology, Graduate School of Pharmaceutical Sciences, The University of Tokyo, Tokyo, Japan; 2Graduate School of Pharmaceutical Sciences, The University
of Tokyo, Tokyo, Japan and 3JST, ERATO, Kanai Life Science Catalysis Project, Hongo, Bunkyo-ku, Tokyo, Japan
Correspondence: Professor K Sekimizu, Laboratory of Microbiology, Graduate School of Pharmaceutical Sciences, The University of Tokyo, Tokyo 113-0033, Japan.
E-mail: sekimizu@mol.f.u-tokyo.ac.jp
Received 23 March 2013; revised 28 May 2013; accepted 30 May 2013; published online 3 July 2013

SAR study of novel iminothiadiazolo-pyrimidinone
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Table 1 Structural activity relationship of the compounds
R1
R2
Compound name
MIC (mg mlꢀ1
)
LD50 (mg gꢀ1
)
Mode of action
0001-03-KK
25
60
H
O
0002-04-KK (1)
0017-22-KK
0021-27-KK
25
100
140
50
Bacteriostatic
CF3
O
12.5
25
O
O
N
O
S
N
0027-56-KK (4)
25
4230
Bactericidal
O2N
R1
N
R2
NH
0005-08-KK
4400
4230
H
O
0006-09-KK (3)
25
160
Bacteriostatic
C6H5
O
0019-25-KK
0023-37-KK
12.5
400
30
4230
O
O
0026-59-KK (2)
6.3
4230
Bactericidal
O2N
Abbreviation: MIC, minimum inhibitory concentration.
and some fungi. Both compounds had antimicrobial activity Mechanism of antibacterial action of 1 and 2
against Gram-positive bacteria, including antibiotic-resistant strains Pulse-labeling experiments for 30min were performed. Both 1 and 2
such as methicillin-resistant S. aureus and vancomycin-resistant inhibited the incorporation of radiolabeled precursors, [3H]thymi-
E. faecalis. Compound
2
showed more potent activity than dine, [3H]uridine and [35S]methionine into acid-insoluble fractions
1
(Table 2). Neither compounds exerted any antimicrobial in exponentially growing S. aureus (Figure 2). Incorporation of
activity against Gram-negative bacteria. Compound 1 also had [3H]N-acetyl glucosamine was inhibited by 2 and slightly inhibited
weak activity against some fungi, such as Candida and Crypto- by 1 (Figure 2). Interestingly, 2 showed bactericidal activity, whereas
coccus, but not Aspergillus niger, whereas 2 exhibited no antifungal 1 did not; 1 merely exhibited bacteriostatic activity (Figure 3).
activity.
The bactericidal activity of 2 was potent: it killed 499.9% of bacteria
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LD50 values of cytotoxic compounds in the silkworm model are
similar to those in mammalian models.9 Thus, in mammalian models
the toxicity of synthesized compounds such as 2 would likely be
reduced compared to that of the original screened 1. Antimicrobial
activity of the mother structure has not yet been reported.
SAR studies are necessary to develop antimicrobial agents for
clinical purposes. Our preliminary SAR study suggested that 2
harboring a benzene ring and a nitrofuran moiety in the R1 and R2
positions, respectively, was the most effective against S. aureus and less
toxic to silkworm larvae. Nitrofuran-containing compounds are
reported to have cytotoxic and carcinogenic activities.10,11 Although
2 has less acute toxicity, its long-term toxicity and carcinogenicity are
unknown. The presence of hydrogen (0005-08-KK) and a furan ring
(0023-37-KK) led to a decreased antibacterial activity (Table 1). In
other cases, antibacterial activity was not affected, whereas toxicity
Figure 1 Scheme of synthesis of iminothiadiazolo-pyrimidinone derivatives.
Table 2 Minimum inhibitory concentration (MIC) of the compounds was higher in trifluoro-methane containing groups. Closer compar-
against microorganisms
ison of the structures revealed that the trifluoro-methane moiety was
responsible for the toxicity, except in 0019-25-KK (Table 1). The
combination of trifluoro-methane and methylfuran moieties might be
responsible for abolishing the toxicity owing to the counter action of
the methylfuran moiety. The toxicity of the trifluoro-methane moiety
was also verified by the antifungal activity possessed by 2. These
findings further support the usefulness of the silkworm model for
identifying a low toxic modification based on the SAR. In the
conventional method, the cytotoxicity of synthesized compounds is
tested using an in vitro cell culture system, but this method does not
reflect the pharmacokinetics of the tested compounds. Thus, the
toxicity of compounds between individual and cultured cells does not
always correspond. Testing the toxicity of many synthesized com-
pounds using mammals, however, is not only costly but is also
associated with ethical problems. The silkworm model solves these
problems and accelerates the development of novel antimicrobial
agents.
We found that 1 was bacteriostatic, whereas 2, which is a
chemically modified version of 1, had potent bactericidal activity
that killed S. aureus within 30min. Inhibition of the incorporation of
radiolabeled precursors into the macromolecules revealed that both 1
and 2 inhibited the biosynthesis of all the macromolecules: DNA,
RNA and proteins. Although peptidoglycan synthesis was inhibited by
2, it was only slightly inhibited by 1. Therefore, the potency of
peptidoglycan synthesis inhibition was enhanced. The fact that 3 was
bacteriostatic, whereas 4 was bactericidal, revealed that the nitrofuran
moiety was responsible for the bactericidal activity. In the present
study, SAR analysis led to the modification of a weak antimicrobial
agent to a compound with potent antimicrobial activity; bacteriostatic
activity was further improved to bactericidal activity. Further deriva-
tization based on these results is required.
MIC (mg mlꢀ1
)
Strain
1
2
S. aureus MSSA1
50
6.3
3.1
S. aureus RN4220
50
50
S. aureus NCTC8325-4
3.1
S. aureus Smith
50
6.3
Methicillin-resistant S. aureus MRSA3
Methicillin-resistant S. aureus MRSA4
Methicillin-resistant S. aureus MRSA6
Methicillin-resistant S. aureus MRSA8
Methicillin-resistant S. aureus MRSA9
Methicillin-resistant S. aureus MRSA11
Methicillin-resistant S. aureus MRSA12
Enterococcus faecalis EF1
50
6.3
50
6.3
50
6.3
50
6.3
25
6.3
50
3.1
50
6.3
100
100
100
100
4100
4100
4100
50
25
Vancomycin-resistant Enterococcus faecalis EF5 (VRE)
Bacillus subtilis JCM2499
12.5
6.3
Bacillus cereus JCM20037
Streptococcus sanguinis JCM5708
S. pyogenes SS1-9
3.1
25
25
S. agalactiae JCM5671
12.5
1.5
S. pneumoniae JCSC6523
Serratia marcescens 98-130-37
Escherichia coli W3110
4100
4100
4100
100
100
100
4100
4100
4100
4100
4100
4100
4100
4100
Pseudomonas aeruginosa PAO1
Candida albicans ATCC10231
C. tropicalis pK233
Cryptococcus neoformans H99
Aspergillus niger
within 30min. The killing activity was comparable to that of
daptomycin. To gain more insight into which moiety was responsible
METHODS
Microbial strains and culture conditions
for the killing action, two other compounds, 0006-09-KK (3) and Gram-positive bacteria, including some resistant strains, Gram-negative
bacteria and some fungi were used as test pathogens (Table 3). Bacterial
cultures were prepared in either Luria Bertani medium (tryptone 10g lꢀ1
0027-56-KK (4) were selected and a killing assay was performed.
Compound 4 exhibited bactericidal activity, whereas 3 showed
bacteriostatic activity (Figure 3).
,
yeast extract 5 g lꢀ1, NaCl 10g lꢀ1, pH 7.0) or Muller–Hinton Broth (MHB;
Difco, Franklin Lakes, NJ, USA). Cation-adjusted MHB was used for
antimicrobial susceptibility tests. For streptococcus species, cation-adjusted
MHB with 2.5% lysed horse blood (Nippon Biotest Laboratories Inc, Tokyo,
Japan), was used. Minimum inhibitory concentration (MIC) was determined
as per the Clinical and Laboratory Standards Institute,12 and Roswell Park
Memorial Institute medium (Sigma Aldrich, St Louis, MO, USA) was used for
testing antifungal susceptibility. Antifungal susceptibility was determined as
DISCUSSION
In this study, we found novel iminothaidiazolo-pyrimidinone anti-
microbial agents screened by an in vitro antimicrobial test from a
chemical library. We successfully produced compounds with more
potent antimicrobial activity against Gram-positive bacteria and less
toxicity in a silkworm model. We previously demonstrated that the per the Clinical and Laboratory Standards Institute.9
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Norfloxacin
200000
25000
20000
15000
10000
5000
0
Vancomycin
1 (5×MIC)
2 (5×MIC)
No drug
1 (5×MIC)
2 (5×MIC)
Vancomycin
No drug
150000
100000
50000
0
0
10
20
Time (minutes)
30
40
0
10
20
Time (minutes)
30
40
Chloramphenicol
20000
15000
10000
5000
0
20000
15000
10000
5000
0
Rifampicin
1 (5×MIC)
2 (5×MIC)
Ampicillin
No drug
1 (5×MIC)
2 (5×MIC)
Ampicillin
No drug
0
10
20
Time (minutes)
30
40
0
10
20
Time (minutes)
30
40
Figure 2 Effect of antimicrobial agents and comparator antibiotics on the incorporation of radiolabeled precursors in Staphylococcus aureus.
1010
109
1 (5×MIC)
Table 3 Microbial strains used in this study
2 (5×MIC)
3 (5×MIC)
Strain
Characteristics
108
4 (5×MIC)
Daptomycin (5µg ml-1
)
Staphylococcus aureus MSSA1
S. aureus RN4220
MET-susceptible, clinical isolate
MET-susceptible
107
No drug
106
S. aureus NCTC8325-4
S. aureus Smith
MET-susceptible
105
MET-susceptible
<104
S. aureus MRSA3
OFXA, KAN, TET, ERM-resistant, clinical
isolate
0
50
100
Time (minutes)
150
200
1440
S. aureus MRSA4
S. aureus MRSA6
S. aureus MRSA8
S. aureus MRSA9
S. aureus MRSA11
S. aureus MRSA12
OFXA, KAN, CHL, CYP-resistant, clinical
isolate
Figure 3 Effect of the compounds on Staphylococcus aureus viability.
OFXA, FLX, KAN, TET, CYP, IM/CS-resistant,
clinical isolate
OFXA, FLX, KAN, ERM, CYP, IM/CS-resistant,
clinical isolate
Chemicals and antibiotics
OFXA, FLX, TET, ERM, CYP, IM/CS-resistant,
clinical isolate
All chemicals used were of analytical grade. Vancomycin, daptomycin and
norfloxacin were purchased from Wako Pure Chemicals (Tokyo, Japan),
Sequoia Research Products (Oxford, UK) and Sigma Aldrich, respectively.
Rifampicin and chloramphenicol were obtained from Nacalai Tesque (Kyoto,
Japan). Radiolabeled [3H]N-acetylglucosamine was obtained from American
Radiolabeled Chemicals (St Louis, MO, USA), [methyl-3H]thymidine and
[3H]uridine were obtained from Moravek Biochemical (Brea, CA, USA) and
[35S]methionine was obtained from the Institute of Isotopes (Budapest,
Hungary).
OFXA, KAN, ERM, CYP, IM/CS-resistant,
clinical isolate
OFXA, FLX, KAN, ERM, IM/CS-resistant,
clinical isolate
Enterococcus feacalis EF1
E. feacalis EF5
VM-susceptible
VM-resistant
Streptococcus sanguinis JCM 5708
S. pyogenes SS1-9
S. pneumoniae JCSC6523
S. agalactiae JCM5671
Bacillus subtilis JCM2499
Bacillus cereus JCM20037
Pseudomonas aeruginosa PAO1
Escherichia coli W3110
Serratia marcescens 98-130-37
Candida albicans ATCC10231
C. tropicalis pK233
Chemical library and screening method
The chemical library of the Open Innovation Center for Drug Discovery at the
University of Tokyo was screened for antibacterial activity against S. aureus
MSSA1. Screening was based on MIC values against methicillin-susceptible
S. aureus determined by broth dilution assay. Chemical compounds were
screened with a criterion to obtain compounds with MIC values of o100 mM.
Synthesis of iminothiadiazolo-pyrimidinone derivatives
Synthesis of 5-imino-2-(substituted)-5H-(1,3,4)thiadiazolo(3,2–a)pyrimidin-
7(6H)-one (0001-03-KK and 0005-08-KK):
Cryptococcus neoformans H99
Aspergillus niger
Sodium (140mg) was dissolved into anhydrous ethanol (20 ml), and
2-amino-5-substituted-1,3,4-thiadiazole (5.9mmol) and ethyl cyanoacetate
(5.9mmol) were added to the solution. The resulting solution was refluxed
for 8 h, and then cooled to room temperature. The solution was poured onto
Abbreviations: CHL, chloramphenicol; CYP, cyprofloxacin; ERM, erythromycin; FLX, flomoxef;
IM/CS, imipenem/cilastatin sodium; KAN, kanamycin; MET, methicillin; OFXA, ofloxacin;
TET, tetracycline; VM, vancomycin.
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ice water and acidified with acetic acid. The precipitate was collected and 371C in MHB was diluted 1000 times with MHB medium and cultured for 2 h
washed with water to afford 0001-03-KK in 64% yield as a yellow solid and at 371C. For daptomycin, MHB was supplemented with 50mg lꢀ1 Ca2 þ
0005-08-KK in 97% yield as a colorless solid.
Synthesis of compounds 0002-04-KK, 0019-25-KK, 0021-27-KK, 0027-56- added to 1 ml of the culture and incubated at 371C for 0–24 h. Culture
.
Compounds 1, 2 (5ꢁ MIC), or daptomycin (5ꢁ MIC; MIC: 1 mg mlꢀ1) was
KK, 0006-09-KK, 0017-22-KK, 0023-37-KK and 0026-59-KK:
Aldehyde (0.86 mmol) and one drop of Et3N were added to a solution of
aliquots were collected at the indicated times, diluted and spread on Luria
Bertani agar plates and incubated at 371C for 24h. Cell viability was
thiadiazolopyrimidine 0001-03-KK or 0005-08-KK (0.82 mmol) in dimethyl- determined by the colony-forming units per ml. The lower limit of detection
formamide (0.82 ml) and ethanol (0.82 ml). The reaction mixture was heated was 104 colony-forming units per ml.
at 50–601C for 3 h and then kept standing at 251C for 12 h to obtain a
precipitate. The resulting precipitate was collected by filtration and washed
with dry diethyl ether to give products (0002-04-KK (1), 0019-25-KK, 0021-
27-KK, 0027-56-KK (4), 0006-09-KK (3), 0017-22-KK, 0023-37-KK and 0026-
Toxicity tests in silkworms
Fifth instar silkworm larvae were injected into the hemolymph with 0–400mg
of each compound and observed for 3 days. The LD50 was determined as the
amount of chemical (mg gꢀ1) that killed 50% of the larvae.
59-KK (2)) of 28–70% yield. The physicochemical properties and structure
elucidation details for the compounds are shown in the Supplementary
Information.
Incorporation of [3H]N-acetylglucosamine into cell wall
peptidoglycans
CONFLICT OF INTEREST
The authors declare no conflict of interest.
Measurement of incorporated [3H]N-acetylglucosamine was performed as
previously described by Paudel et al.12 Briefly, S. aureus NCTC8325-4 was
cultured at 37 1C overnight in CGPY broth (Na2HPO4, 6 g; NaCl, 3 g;
MgCl2.6H2O, 0.1 g; NH4Cl, 2 g; Na2SO4, 0.15g; KH2PO4, 3 g; bactopeptone,
10 g; yeast extract, 0.1 g and glucose, 5 g lꢀ1, pH 7.0). The culture was diluted
100-fold with the same medium and further cultured until an optical density
(OD)660 of 0.2 was reached. The culture was centrifuged at 8000 g for 10min
and the pellet was suspended in modified cell wall synthesis medium
(KH2PO4, 6 g; K2HPO4, 6 g; NH4Cl, 2 g; MgSO4. 7H2O, 5 mg; FeSO4, 5 mg;
glucose, 100mg; uracil, 40mg; L-alanine, 50 mg; L-glutamic acid, 120 mg;
L-lysine, 50 mg and chloramphenicol, 100 mglꢀ1), pH 6.8) to obtain
OD660 ¼ 0.1. In the presence of 35mCi [3H]N-acetyl glucosamine, 1, 2,
vancomycin, or norfloxacin (5ꢁ MIC) was added to 1 ml of the cell
suspension at time zero and incubated at 37 1C with shaking at 150 r.p.m.
Samples were collected at the indicated times and an equal volume of 10%
trichloroacetic acid was added. The mixture was incubated at 901C for 15min,
placed on ice for 30 min and filtered with a membrane filter (0.45mm HA,
Millipore, County Cork, Ireland) followed by washing with 5% trichloroacetic
acid. Radioactivity was counted on a liquid scintillation counter (LS6000SE,
Beckman Coulter, Brea, CA, USA) and expressed as c.p.m.
ACKNOWLEDGEMENTS
We thank the Chemical Library in the Open Innovation Center for Drug
Discovery at the University of Tokyo for providing the compounds used in this
study. We also thank Ms Matsuzawa, Ms Noguchi (Yoshino) and Ms
Kyougoku for technical assistance. This study was supported by the Program
for Promotion of Fundamental Studies in Health Sciences of the National
Institute of Biomedical Innovation (NIBIO) and partly supported by JSPS
Grant-in-Aid for Young Scientists (A) (24689008) from JSPS (HH) and
Grant-in-Aid for Scientific Research on Innovative Areas (24102510) from
MEXT (KS), and Platform for Drug Discovery, Informatics (MS).
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Antifungal Susceptibility Testing of Yeasts: Approved Standard M27-A (Clinical and
Laboratory Standards Institute, Wayne, PA, USA, 1997).
Incorporation of radiolabeled thymidine, uridine and methionine
Incorporation of thymidine, uridine, and methionine was measured as
described previously.10 Briefly, S. aureus NCTC8325-4 was grown overnight
in Luria Bertani medium at 371C. The culture was diluted 200-fold in Luria
Bertani medium and incubated at 371C until an OD600 of 0.3 was reached.
Either 7 mCi [3H]uridine, 70mCi [3H]thymidine or 20 mCi [35S]methionine was
added to the culture. Rifampicin, norfloxacin and chloramphenicol at a
concentration of 5 ꢁ MIC were used to inhibit RNA, DNA and protein
synthesis, respectively; and vancomycin or ampicillin (5ꢁ MIC) was used
as a negative control. Compounds 1 and 2 (5ꢁ MIC) were used for all the
assays. The MICs of rifampicin, norfloxacin, chloramphenicol, vancomycin
and ampicillin were 0.004 mg mlꢀ1, 0.5 mg mlꢀ1, 4 mg mlꢀ1, 1 mg mlꢀ1 and
0.03 mg mlꢀ1, respectively. At time zero, agents were added to exponentially
growing cultures. Aliquots were collected at the indicated times, diluted twice
with 5% trichloroacetic acid and the acid-insoluble fraction was obtained by
filtration through glass fiber filters (Whatman, GE Healthcare, Maidstone,
Kent, UK). Radioactivity retained on the filters was measured on a liquid
scintillation counter and expressed as c.p.m.
5
6
7
8
9
10 Paudel, A et al. Identification of novel deoxyribofuranosyl indole antimicrobial agents.
J. Antibiot. (Tokyo) 65, 53–57 (2012).
11 National Committee for Clinical Laboratory Standards. Methods for Determining
Bactericidal Activity of Antimicrobial Agents; Approved Guideline NCCLS Document
M26-A, Vol. 19. (National Committee for Clinical Laboratory Standards, Wayne, PA,
USA, 1999).
12 Clinical and Laboratory Standards Institute. Methods for Dilution Antimicrobial
Susceptibility Tests for Bacteria that Grow Aerobically; Approved Standard, CLSI
document M07-A8, 8th edn, Vol. 29 (Clinical and Laboratory Standards Institute,
Wayne, PA, USA, 2009).
Killing assay
The killing assay was performed as per the National Committee for Clinical
Laboratory Standards.11 Briefly, an overnight culture of S. aureus MSSA1 at
Supplementary Information accompanies the paper on The Journal of Antibiotics website (http://www.nature.com/ja)
The Journal of Antibiotics