Evaluation of amphiphilic aminoglycoside-peptide triazole conjugates as antibacterial agents

Article abstract of DOI:10.1016/j.bmcl.2010.03.116

The solid- and solution-phase synthesis of amphiphilic aminoglycoside-peptide triazole conjugates (APTCs) accessed by copper(I)-catalyzed 1,3-dipolar cycloaddition reaction between a hydrophobic and ultrashort peptide-based alkyne and a neomycin B- or kanamycin A-derived azide is presented. Antibacterial evaluation demonstrates that the antibacterial potency is affected by the nature of the peptide component. Several APTCs exhibit superior activity against neomycin B- and kanamycin A-resistant strains when compared to their parent aminoglycoside while displaying reduced activity against neomycin B- and kanamycin A-susceptible strains.

Full text of DOI:10.1016/j.bmcl.2010.03.116

Bioorganic & Medicinal Chemistry Letters 20 (2010) 3031–3035
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Evaluation of amphiphilic aminoglycoside–peptide triazole conjugates
as antibacterial agents
Smritilekha Bera a, George G. Zhanel b, Frank Schweizer a,b,
*
a Department of Chemistry, University of Manitoba, Winnipeg, MB, Canada R3T 2N2
b Department of Medical Microbiology, University of Manitoba, Health Sciences Centre MS673-820 Sherbrook St. Winnipeg, MB, Canada R3A 1R9
a r t i c l e i n f o
a b s t r a c t
Article history:
The solid- and solution-phase synthesis of amphiphilic aminoglycoside–peptide triazole conjugates
(APTCs) accessed by copper(I)-catalyzed 1,3-dipolar cycloaddition reaction between a hydrophobic and
ultrashort peptide-based alkyne and a neomycin B- or kanamycin A-derived azide is presented. Antibac-
terial evaluation demonstrates that the antibacterial potency is affected by the nature of the peptide com-
ponent. Several APTCs exhibit superior activity against neomycin B- and kanamycin A-resistant strains
when compared to their parent aminoglycoside while displaying reduced activity against neomycin
B- and kanamycin A-susceptible strains.
Received 22 February 2010
Revised 29 March 2010
Accepted 31 March 2010
Available online 3 April 2010
Keywords:
Aminoglycosides
Peptides
Ó 2010 Elsevier Ltd. All rights reserved.
Cationic amphiphiles
Dipolar cycloaddition
MRSA
Aminoglycoside antibiotics (AAs) constitute a large family of
clinically important drugs used in the treatment of Gram-positive
and Gram-negative bacterial infections. AAs bind to specific sites
in prokaryotic ribosomal RNA affecting the fidelity of protein syn-
thesis.1 Although AAs exhibit potent bactericidal activity, their
widespread use has been compromised by toxicity and the world-
wide emergence of resistant strains.2 Cationic antimicrobial pep-
tides (AMPs) form another large class of antibacterials that are
currently studied for clinical use. Naturally occurring AMPs typi-
cally contain 10–50 amino acids are amphiphilic and are rich in
lysine or arginine and hydrophobic amino acids.3 However, short-
er, amphiphilic peptide sequences as short as di- or tri- or hexa-
peptides with antibacterial activity are known.4–6 Numerous
studies with linear, cyclic and diastereomeric AMPs have strongly
supported the hypothesis that their physicochemical properties,
rather than any precise sequence, are responsible for their
activities.3 It is generally believed that the amphiphilic topology
is essential for insertion into and disruption of the cytoplasmic
membrane.3 In particular, the ability to rapidly kill bacteria and
the relative difficulty with which bacteria develop resistance
in vitro, make AMPs attractive targets for drug development.3a
Herein, we describe the synthesis and antibacterial properties
of a new class of amphiphilic aminoglycoconjugates resembling
ultrashort cationic peptide antibacterials termed aminoglyco-
side–peptide triazole conjugates (APTCs). The motivation behind
this approach is based on the observation that cationic amphi-
philes such as cationic lipids, cationic peptides, and lipopeptides,
chlorhexidine, and quaternary ammonium salts have been used
for several decades with little or no occurrence of resistance.7,8
Our working hypothesis is that conversion of aminoglycoside anti-
biotics into cationic amphiphiles may enhance antibacterial activ-
ity against resistant strains in this old class of antibiotics. As
method of ligation we selected the click-based copper-catalyzed
azide–alkyne cycloaddition reaction9–12 between a terminal pep-
tide-based alkyne introduced in the form of propargylglycine
(Pra) and the azides 113 and 214 easily prepared from neomycin B
and kanamycin A (Fig. 1). Click-based cycloaddition reaction of
azides 1 and 2 to nonpeptide-based alkynes have previously been
reported in solution and on the solid phase13,14 but no reports on
the synthesis and antibacterial activity of amphiphilic APTCs exist.
Neomycin B and kanamycin A were selected due to their commer-
cial availability in multi-gram quantities, low price and low
susceptibility against multi-drug resistant bacteria. The C500–
(C600)-position of the azide group in 1 and 2 was based on easy syn-
thetic accessibility and previous reports that have indicated that
modified C500-analogs of neomycin B retain high binding affinity
to RNA15 and antimicrobial activities.16 To be compatible with
solution- and solid-phase peptide chemistry using the Fmoc-strat-
egy the remaining amino groups in 1 and 2 were protected as
tert-butylcarbamate (Boc). CuI-catalyzed cycloaddition of azides
1 and 2 to peptides 3–8 ( Fig. 1) afforded APTCs 9–11, 13, 14,
16–20 (Fig. 2).
Peptides 3–8 were selected as representative examples of solu-
ble or immobilized ultrashort and hydrophobic peptide sequences.
* Corresponding author. Tel./fax: +1 204 474 7012.
E-mail address: schweize@cc.umanitoba.ca (F. Schweizer).
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.03.116

3032
S. Bera et al. / Bioorg. Med. Chem. Lett. 20 (2010) 3031–3035
short peptide sequence
peptide
deprotection
(resin cleavage)
(in solution or immobilized)
N3
APTC
libraries
N
"click"
CuI
aminoglycoside
peptide
N
N
aminoglycoside
NHBoc
O
O
C
CO2-tBu
H
N
FmocHN
O
HO
HO
C
C
O
N
H
NHBoc
OH
FmocHN
NH
BocHN
O
NHBoc
O
N3
CO2Bn
4
O
BocHN
HO
3
OH
O
O
OH
NHBn
Boc
O
N
O
BocHN
C
FmocHN
N
Boc
H
(Boc)6neomycin B-C-5"-N3
N
O
FmocHN
O
O
NH
NH NHBn
1
BocN
O
HN
5
6
N3
O
OH
OH
O
HO
HO
O
BocHN
O
HO
O
H
O
HO
NHBoc
H
N
O
N
NHBoc
NHFmoc
NH
NHFmoc
BocHN
NH
N
H
O
O
(Boc)4kanamycin A-C6''-N3
8
7
2
Figure 1. Click-based glycoconjugation of neomycin B- and kanamycin A-based azides 1 and 2 to hydrophobic alkyne-based peptides 3–8 that form a novel class of ultrashort
antibacterial peptides termed aminoglycoside–peptide triazole conjugates (APTCs).
Short peptide sequences were selected to reduce the risk of
metabolic proteolytic cleavage while at the same time reducing
the production costs. Soluble peptides 3–6 are hydrophobic pep-
tides in which hydrophobicity is gradually increased. Dipeptide 5
and tetrapeptide 6 containing tryptophan units were selected
based on the preference for this hydrophobic amino acid in short
and cationic peptide antibiotics.17,18 The Fmoc-protecting group
was retained in these peptides because previous reports have
shown that Fmoc-protection in cationic peptide antibacterials en-
hances antibacterial activity.5 Tetrapeptide 6 containing two al-
kyne moieties was selected to explore divalency effects while
immobilized peptides 7 and 8 were chosen to study solid-phase
ligation with azide 1. Guanidinylation of the amino groups in con-
jugates 11 and 14 using N,N0-diBoc-N0-triflylguanidine19 produced
guanidine conjugates 12 and 15. These analogs were selected to
explore basicity affects in APTCs. In addition, it is known that
replacement of lysine by arginine in ultrashort cationic peptide
antibacterials enhances antibacterial activity.4 The antibacterial
potency of all APTCs were determined by measuring the minimal
inhibitory concentration (MIC) against a broad selection of Gram-
positive and Gram-negative pathogens including three AA-resis-
tant strains obtained from a national surveillance study assessing
antimicrobial resistance in Canadian intensive care units
(CAN-ICU)20 (Table 1). Our data demonstrate that the antibacterial
activity of APTCs is affected by the nature of the peptide chain. For
instance, APTCs 13 and 16 containing a carboxylate display weak
antibacterial activity against most bacterial strains while APTCs
14 and 17 exhibit relatively potent antimicrobial activity against
Gram-positive and most Gram-negative strains. More importantly,
APTCs 11, 12, 14, 15 and 17, 19 and 20 can overcome the acquired
resistance of MRSA against neomycin B and kanamycin A. Simi-
larly, kanamycin A–dipeptide conjugate 15 shows a 16-fold lower
minimal inhibitory concentration against MRSE when compared
to kanamycin A. In addition, kanamycin A–dipeptide conjugate
14 exhibits a P32-fold lower MIC against kanamycin A-resistant
Pseudomonas aeruginosa and a eightfold lower MIC (compared to
gentamicin) against Gentamicin-resistant P. aeruginosa. Further-
more, enhancement of the amphiphilicity in APTC via increase in
hydrophobicity enhances antibacterial activity. For instance,
Fmoc-protected APTC 11 displays a eightfold lower MIC against
MRSA when compared to unprotected peptide 10 suggesting that
the amphiphilic nature of APTCs is crucial for their antibacterial
activity. It is noteworthy that the guanidinylated APTCs 12 and
15 lead to a 2–4-fold enhancement in Gram-positive activity when
compared to their nonguanidinylated analogs 11 and 14, respec-
tively. This is in contrast to observations with nonamphiphilic gua-
nidinylated AAs that exhibit reduced antibacterial activity.1b,21
Moreover, our results show that amphiphilic peptides 17 and 18
containing two aminoglycoside units do not improve antibacterial
activity. In vitro toxicity studies show that most APTCs exhibit lit-
tle to no toxicity against mammalian erythrocytes at a concentra-
tion of 100 lg/mL (Table 1). However, substantial hemolytic
activities (>20%) are seen at higher concentrations (500–1000
lg/
mL).
In summary, we have prepared a new class of amphiphilic
aminoglycoconjugates via click-based glycoconjugation of neomy-
cin B- and kanamycin A-derived azides 1 and 2 to short hydropho-
bic peptides in solution and on the solid phase. Our results
demonstrate that APTCs display enhanced antibacterial activity
against neomycin B-, kanamycin A-resistant MRSA, kanamycin
A-resistant MRSE and gentamicin-resistant P. aeruginosa. However,
most APTCs display reduced antibacterial activity against neomy-
cin B- and kanamycin A-susceptible strains. In vitro toxicity
measurements demonstrate that most APTCs are selective for

S. Bera et al. / Bioorg. Med. Chem. Lett. 20 (2010) 3031–3035
3033
BnO
NH
O
BnHN
NH
NHR1
O
NHR1
O
HO
NHR1
O
H
N
O
O
HO
HO
HO
HO
NHR1
OH
HO
HO
NHR1
OH
NHR1
OH
NH
O
R1HN
R1NH
R1HN
NHR1
N
H
NHR1
O
O
NHR1
O
O
O
R2HN
O
O
O
N
O
N
N
N
R1HN
R2HN
O
N
O
O
N
FmocHN
N
N
N
R1HN
R1HN
OH
OH
HO
R1HN
O
HO
OH
O
OH
O
O
O
OH
R1HN OH
O
R1HN OH
10: R1 = H x TFA; R2 = H x TFA;
13: R1 = H x TFA; R2 = Fmoc
11: R1 = H x TFA; R2 = Fmoc;
9: R1 = H x TFA
12: R1 = C=NH(NH2) x TFA; R2 = Fmoc
H
N
O
H
N
O
O
NHBn
OH
NH
NH
O
C
C
N
N
O
N
N
N
N
OH
R2HN
OH
R2HN
HO
HO
O
OH
O
O
HO
OH
O
HO
NHR1
R1HN
HO
R1HN
HO
O
NHR1
HO
O
NHR1
O
HO
O
NHR1
R1HN
R1HN
14: R1 = H x TFA; R2 = Fmoc
16: R1 = H x TFA; R2 = Fmoc;
15: R1 = C=NH(NH2) x TFA; R2 = Fmoc
NHR1
O
H
HO
HO
N
H
NHR2
NH
N
N
R1HN
NHR1
NHR1
O
N
O
N
FmocHN
O
O
R1HN
O
O
O
O
NHBn
OH
NH
NH
OH
O
C
N
C
N
O
O
OH
OH
H
N
NHR1 OH
HO
N
C
HN
NHR1
O
HO
O
OH
NHR1
O
O
O
HO
R1HN
N
H
BnHN
C
HO
N
HO
N
N
HO
O
H
O
R1HN
N
NH
NHR1
N
OH
R1HN
OH
NHR1
N
O
N
HO
O
O
HO
R1HN
O
1
O
HO
NHR1
R HN
HO
R1HN
R1HN
OH
O
HO
O
O
R1HN
O
OH
NHR1
HO
17: R1 = H x TFA; R2 = Fmoc
18: R1 = H x TFA; R2 = Fmoc
NH2
NHR1
O
H2N
NH
O
O
O
NHR1
O
HN
HO
HO
NHR1
HO
O
NHR1
R1HNO
O
N
HO
HO
R1HN
NHR1
O
N
NH
NHR1
O
R1HN
N H
O
O
N
N
N
HO
N
O
R2HN
N
N
R1HN
N H
R2HN
HO
N
O
R1HN
O
HO
R1HN
H
N
OH
O
H
N
OH
O
O
OH
O
N
H
N
H
R1HN OH
NHR2
NHR2
O
O
21: R2 = Fmoc
20: R1 = H x TFA; R2 = Fmoc
22: R1 = H x TFA; R2 = Fmoc
19: R1 = H x TFA; R2 = Fmoc
Figure 2. Amphiphilic neomycin B- and kanamycin A-based peptide triazole conjugates (APTCs) prepared by click-based glycoconjugation of azides 1 and 2 to hydrophobic
alkyne-modified peptides 3–8. Peptides 21 and 22 are negative controls.
bacterial cells and show little hemolytic activity against mamma-
lian erythrocytes at MIC concentration although significant hemo-
lytic activity occurs at higher concentrations.
neomycin–lipid conjugates are able to synergistically enhance
the antibacterial activity of antibiotics interacting with intracellu-
lar targets such as amikacin and neomycin.22 In addition, we and
others have recently shown that the antibacterial activity of C500-
modified neomycin–lipid conjugates against MRSA is strongly
dependent on the length of the lipid moiety.23,16c For instance,
C500-modified neomycin–C6 lipid does not inhibit the growth of
The physicochemical similarities between APTCs and AMPs sug-
gest a membranolytic mode of action. Further evidence for a mem-
branolytic mode of action of APTCs is supported by (a) the
observed concentration-dependent hemolytic activity of all APTCs;
(b) the requirement for a strongly hydrophobic peptide segment to
induce potent antibacterial activity; (c) the precedence of a pleth-
ora of cationic antibacterial amphiphiles with membranolytic
mode of action and (d) a recent study of C500-modified neomy-
cin–lipid amphiphiles that have demonstrated that polycationic
MRSA (MIC >512
l
g/mL) while C500-modified neomycin–C16 lipid
g/mL). This is consis-
displays potent anti-MRSA activity (MIC = 4
l
tent with observations obtained with other cationic amphiphiles
with membranolytic mode of action that have clearly indicated
that the presence of a hydrophobic moiety above a certain thresh-

Table 1
Minimal inhibitory concentration (MIC) in
l
g/ml of APTCs 9–20, gentamicin, neomycin B, kanamycin A (positive controls) and 21 and 22 (negative controls) against Gram-positive and Gram-negative organisms
Organism
Compound
k
k
k
k
k
l
k
k
Genta-micin
Neo-mycin B
Kana-mycin A
21m
22
9 N
10 N
11 N
12 N
13 N
14 K
15 Kl
16 Kl
17 N
18 Kl
19 N
20 Nl
S. aureusa
1
2
0.25
32
4
1
128
8
8
128
n.d.
1
4
>512
2
128
8
8
16
32
>512
>512
<0.5
128
128
128
64
128
128
128
128
128
128
n.d
512
512
256
512
>512
512
512
n.d.
16
64
4
8
>512
32
32
64
512
512
1.0
32
128
8
8
>512
32
8
16
4
4
8
1
2
64
32
64
64
128
128
n.d
16
256
8
8
128
32
16
32
8
4
8
2
8
64
64
64
64
128
128
13.7
16
>256
4
64
8
128
32
>256
>256
>256
n.d
8
16
2
32
32
8
32
32
16
16
>128
64
128
128
128
64
32
32
8
16
>256
64
64
MRSAb
256
0.25
0.25
8
2
4
S. epidermidisc
MRSEd
8
16
64
32
32
32
128
16
3.9
4
16
64
64
64
32
128
64
10.2
S. pneumoniaee
E. colif
64
16
32
n.d.
128
64
9.9
64
32
64
16
128
32
1.5
E. colig (Gent-R)
E. colih (Amik-R)
P. aeruginosai
P. aeruginosaj (Gent-R)
(%) Hemolysisn at 100
32
32
32
n.d.
512
256
1.0
256
256
256
0.8
64
512
512
<0.5
>512
>512
n.d
>256
>256
2.2
l
g/ml
1.0
n.d. = not determined.
a
ATCC 29213.
b
Methicillin-resistant S. aureus ATCC 33592.
S. epidermidis ATCC 14990.
Methicillin-resistant S. epidermidis Canadian Intensive Care Unit (CAN-ICU) 61589.
ATCC 49619.
ATCC 25922.
CAN-ICU 61714.
CAN-ICU 63074.
ATCC 27853.
CAN-ICU 62308.
Neomycin B-based conjugate.
Kanamycin A-based conjugate.
Dissolved in DMSO and diluted with H2O.
Human erythrocytes.
c
d
e
f
g
h
i
j
k
l
m
n

S. Bera et al. / Bioorg. Med. Chem. Lett. 20 (2010) 3031–3035
3035
old is critical for antibacterial activity.3c,24–26 However, RNA-med-
iated interactions leading to additive or synergistic effects when
combined with cell permeation cannot be ruled out.27 In addition,
it is noteworthy that the hemolytic activity of the highly potent
neomycin–C16 lipid conjugate is significantly elevated (56% hemo-
9. Kolb, H. C.; Finn, M. G.; Sharpless, K. B. Angew. Chem., Int. Ed. 2001, 40, 2004.
10. Meldal, M.; Tornoe, C. W. Chem. Rev. 2008, 108, 2952.
11. Tornoe, C. W.; Christensen, C.; Meldal, M. J. Org. Chem. 2002, 67, 3057.
12. Rostovtsev, V. V.; Green, L. G.; Fokin, V. V.; Sharpless, K. B. Angew. Chem., Int. Ed.
Engl. 2002, 41, 2596.
13. Disney, M. D.; Barrett, O. J. Biochemistry 2007, 46, 11223.
14. Quader, S.; Boyd, S. E.; Jenkins, I. D.; Houston, T. A. J. Org. Chem. 2007, 72, 1962.
15. Asensio, J. L.; Hidalogo, A.; Bastida, A.; Torrado, M.; Corzana, F.; Chiara, J. L.;
Garcia-Junceda,
lysis at 100 lg/mL) when compared to peptide-based APTCs sug-
gesting that APTCs may exhibit reduced in vitro toxicity.28
E.; Canada, J.; Jimenez-Barbero, J. J. Am. Chem. Soc. 2005, 127,
8278.
16. (a) Hanessian, S.; Masse, R.; Capmeau, M. L. J. Antibiot. 1977, 30, 893; (b)
Bastida, A.; Hidalog, A.; Chiara, J. L.; Torrada, M.; Corzna, F.; Perez-Canadillas, J.
M.; Groves, P.; Garcia-Junceda, E.; Gonzalez, C.; Jimenez-Barbero, J.; Asensio, J.
L. J. Am. Chem. Soc. 2006, 128, 100; (c) Zhang, J.; Chiang, F.-I.; Wu, L.; Czyryca, P.
G.; Li, D.; Chang, C.-W. T. J. Med. Chem. 2008, 51, 7563; (d) Pokrovskaya, V.;
Belakhov, V.; Hainrichson, M.; Yaron, S.; Baasov, T. J. Med. Chem. 2009, 52,
21243.
Acknowledgements
This research was supported by the Canadian Institutes of
Health Research (CIHR), Manitoba Health Research Council
(MHRC), Natural Sciences and Engineering Research Council of
Canada (NSERC) and the Canadian Foundation for Innovation (CFI).
17. Fjell, C. D.; Jenssen, H.; Hilpert, K.; Cheung, W. A.; Pante, N.; Hancock, R. E. W.;
Cherkasov, A. J. Med. Chem. 2009, 52, 2006.
18. Liu, Z.; Brady, A.; Young, A.; Rasimick, K.; Chen, C.; Kallenbach, N. R. Antimicrob.
Agents Chemother. 2007, 51, 597.
Supplementary data
19. Baker, T. J.; Luedtke, N. W.; Tor, Y.; Goodman, M. J. Org. Chem. 2000, 65, 9054.
20. Zhanel, G. G.; DeCorby, M.; Laing, N.; Weshnoweski, B.; Vashisht, R.; Tailor, F.;
Nichol, K. A.; Wierzbowski, A.; Baudry, P. J.; Karlowsky, J. A.; Lagacé-Wiens, P.;
Walkty, A.; McCracken, M.; Mulvey, M. R.; Johnson, J.; the Canadian
Antimicrobial Resistance Alliance (CARA); Hoban, D. J. Antimicrob. Agents.
Chemother. 2008, 52, 1430.
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2010.03.116.
References and notes
21. Hoshi, H.; Aburaki, S.; Yamasaki, T.; Naito, T.; Kawaguchi, H. J. Antibiot. 1991,
44, 680.
1. (a) Davis, B. D. Microbiol. Rev. 1987, 51, 341; (b) Aminoglycoside Antibiotics: From
Chemical Biology to Drug Discovery; Arya, D. P., Ed.; John Wiley & Sons, 2007, p 1.
2. Vakulenko, S. B.; Mobashery, S. Clin. Microbiol. Rev. 2003, 16, 430.
3. (a) Hancock, R. E. W.; Sahl, H.-G. Nat. Biotechnol. 2006, 24, 1551; (b) Hancock, R.
E. W.; Lehrer, R. Nature 2002, 415, 389; (c) Makovitzki, A.; Avrahami, D.; Shai, Y.
Proc. Natl. Acad. Sci. U.S.A. 2006, 103, 15997.
4. Strom, M. B.; Haug, B. E.; Skar, M. L.; Stiberg, T.; Svendsen, J. S. J. Med. Chem.
2003, 46, 1567.
5. Blondelle, S. E.; Takahashi, E.; Dinh, K. T.; Houghten, R. A. J. Appl. Bacteriol. 1995,
78, 39.
22. Zhang, J.; Keller, K.; Takemoto, J. T.; Bensaci, M.; Litke, A.; Czyryca, P. G.; Chang,
C.-W. T. J. Antibiot. 2009, 44, 539.
23. Bera, S.; Zhanel, G. G.; Schweizer, F. J. Med. Chem. 2008, 51, 6160.
24. Andrä, J.; Lohner, K.; Blondelle, S. E.; Jerala, R.; Moriyon, I.; Koch, M. H. J., et al
Biochem. J. 2005, 385, 135.
25. Japelj, B.; Zorko, M.; Majerle, A.; Pristovšek, P.; Sanchez-Gomez, S.; Martinez de
Tejada, G., et al J. Am. Chem. Soc. 2007, 129, 1022.
26. Malina, A.; Shai, Y. Biochem. J. 2005, 390, 695.
27. Shelburne, S. A.; Musher, D. M.; Hulten, K.; Ceasar, H.; Lu, Y. M. Antimicrob.
Agents Chemother. 2004, 48, 4016.
6. Haug, B. E.; Stensen, W.; Stiberg, T.; Svendsen, J. S. J. Med. Chem. 2004, 47, 4159.
7. Gilbert, P.; Moore, L. E. J. Appl. Microbiol. 2005, 99, 703.
8. Chen, L. F.; Kaye, D. Infect. Dis. Clin. N. Am. 2009, 23, 1053.
28. Bera, S.; Zhanel, G. G.; Schweizer, F. J. Antimicrob. Chemother., in press.