13-(2-methylbenzyl) berberine is a more potent inhibitor of MexXY-dependent aminoglycoside resistance than berberine

Article abstract of DOI:10.3390/antibiotics8040212

We previously showed that berberine attenuates MexXY efflux-dependent aminoglycoside resistance in Pseudomonas aeruginosa. Here, we aimed to synthesize berberine derivatives with higher MexXY inhibitory activities. We synthesized 11 berberine derivatives, of which 13-(2-methylbenzyl) berberine (13-o-MBB) but not its regiomers showed the most promising MexXY inhibitory activity. 13-o-MBB reduced the minimum inhibitory concentrations (MICs) of various aminoglycosides 4- to 128 fold for a highly multidrug resistant P. aeruginosa strain. Moreover, 13-o-MBB significantly reduced the MICs of gentamicin and amikacin in Achromobacter xylosoxidans and Burkholderia cepacia. The fractional inhibitory concentration indices indicated that 13-o-MBB acted synergistically with aminoglycosides in only MexXY-positive P. aeruginosa strains. Time-kill curves showed that 13-o-MBB or higher concentrations of berberine increased the bactericidal activity of gentamicin by inhibiting MexXY in P. aeruginosa. Our findings indicate that 13-o-MBB inhibits MexXY-dependent aminoglycoside drug resistance more strongly than berberine and that 13-o-MBB is a useful inhibitor of aminoglycoside drug resistance due to MexXY.

Full text of DOI:10.3390/antibiotics8040212

antibiotics
Article
13-(2-Methylbenzyl) Berberine Is a More Potent
Inhibitor of MexXY-Dependent Aminoglycoside
Resistance than Berberine
Kenta Kotani 1, Mio Matsumura 2, Yuji Morita 3, Junko Tomida 1, Ryo Kutsuna 1,
Kunihiko Nishino 4, Shuji Yasuike 2 and Yoshiaki Kawamura 1,
*
1
Department of Microbiology, School of Pharmacy, Aichi Gakuin University, Nagoya 560-0043, Japan;
ag163a01@dpc.agu.ac.jp (K.K.); jtomida@dpc.agu.ac.jp (J.T.); kutsuna@dpc.agu.ac.jp (R.K.)
Department of Organic and Medicinal Chemistry, School of Pharmacy, Aichi Gakuin University, Nagoya
560-0043, Japan; m-matsu@dpc.agu.ac.jp (M.M.); s-yasuik@dpc.agu.ac.jp (S.Y.)
Department of Microbial Science and Host Defense, Meiji Pharmaceutical University, Noshio, Kiyose, Tokyo
560-0043, Japan; morita@my-pharm.ac.jp
2
3
4
Department of Biomolecular Science and Regulation, Institute of Scientific and Industrial Research, Osaka
University, Osaka 560-0043, Japan; nishino@sanken.osaka-u.ac.jp
*
Correspondence: kawamura@dpc.agu.ac.jp
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
Received: 24 September 2019; Accepted: 2 November 2019; Published: 6 November 2019
ꢀꢁꢂꢃꢄꢅꢆ
Abstract: We previously showed that berberine attenuates MexXY efflux-dependent aminoglycoside
resistance in Pseudomonas aeruginosa. Here, we aimed to synthesize berberine derivatives with higher
MexXY inhibitory activities. We synthesized 11 berberine derivatives, of which 13-(2-methylbenzyl)
berberine (13-o-MBB) but not its regiomers showed the most promising MexXY inhibitory activity.
13-o-MBB reduced the minimum inhibitory concentrations (MICs) of various aminoglycosides 4- to 128
fold for a highly multidrug resistant P. aeruginosa strain. Moreover, 13-o-MBB significantly reduced the
MICs of gentamicin and amikacin in Achromobacter xylosoxidans and Burkholderia cepacia. The fractional
inhibitory concentration indices indicated that 13-o-MBB acted synergistically with aminoglycosides
in only MexXY-positive P. aeruginosa strains. Time-kill curves showed that 13-o-MBB or higher
concentrations of berberine increased the bactericidal activity of gentamicin by inhibiting MexXY in
P. aeruginosa. Our findings indicate that 13-o-MBB inhibits MexXY-dependent aminoglycoside drug
resistance more strongly than berberine and that 13-o-MBB is a useful inhibitor of aminoglycoside
drug resistance due to MexXY.
Keywords: Pseudomonas aeruginosa; efflux; MexXY; aminoglycoside resistance; berberine
1. Introduction
Pseudomonas aeruginosa is a major cause of nosocomial infections. Treatment of P. aeruginosa
infections with antimicrobial concentrations insufficient to inhibit P. aeruginosa growth results in the
emergence of new multidrug resistant P. aeruginosa strains [1] that are difficult to eradicate and may
increase mortality [2].
Drug efflux is a major mechanism leading to antimicrobial resistance in P. aeruginosa [
Four resistance-nodulation-division (RND)-type multidrug efflux pumps (MexAB-OprM [
MexCD-OprJ [ ], MexEF-OprN [ ] and MexXY-OprM/OprA [
efflux systems involved in the drug resistance of P. aeruginosa. Of these, only MexXY contributes to
aminoglycoside drug resistance [ ]. The MexXY-OprM system comprises a cytoplasmic membrane
3].
4],
5
6
7,8]) have been reported as drug
8,9
antibiotic-proton antiporter (MexY), an outer membrane porin (OprM), and a periplasmic membrane
fusion protein (MexX) [10]. MexXY has multiple functions, including the expulsion of antibiotics.
Antibiotics 2019, 8, 212; doi:10.3390/antibiotics8040212
www.mdpi.com/journal/antibiotics

Antibiotics 2019, 8, 212
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Wild-type P. aeruginosa expresses low MexXY levels but elevated MexXY has been detected in
aminoglycoside-resistant P. aeruginosa strains [11 12]. Therefore, the development of MexXY inhibitors
,
would allow the use of lower concentrations of aminoglycoside drugs that can cause severe side effects
such as kidney damage [13].
There have been various reports of inhibitors of RND-type multidrug efflux pumps, but no clinical
applications have been published to date [14]. Phenyl-arginine-β-naphthylamide (PAβN, MC-207,110),
a well-known efflux pump inhibitor, does not inhibit aminoglycoside resistance due to MexXY [15].
We previously reported that berberine attenuates MexXY-dependent aminoglycoside resistance in
P. aeruginosa [15], consistent with a recent report that berberine has high affinity to a MexXY model
protein in silico [16].
Berberine is an isoquinoline quaternary alkaloid isolated from many kinds of medicinal plants
such as Coptis chinensis, Coptis rhizome, Coptis japonica and Phellondendron amurense [17] and has weak
antibacterial activity against Gram-negative bacteria such as P. aeruginosa [18]. Various derivatives of
berberine have been developed and studied for their anti-hyperglycemic, anti-cancer, anti-inflammatory,
anti-Alzheimer’s disease and anti-microbial activities [19]. Derivatives with multidrug resistance
pump inhibitory activity against Staphylococcus aureus [20] and that reduce fluconazole resistance
against Candida albicans [21] have been reported. In addition, quaternary ammonium compounds
inhibit the biofilm formation in P. aeruginosa and C. albicans have been reported [22].
The optimum concentration of berberine to inhibit MexXY in P. aeruginosa cells is more than
512 µg/mL [15], which is too high for clinical application. In this study, we aimed to synthesize
berberine derivatives with higher MexXY inhibitory activities.
2. Results
2.1. Antibacterial Activity of Berberine Derivatives toward P. aeruginosa
We first measured the minimum inhibitory concentrations (MICs) of 11 berberine derivatives
(Figure 1) synthesized against P. aeruginosa mutants PAGUg1927, which expresses MexXY, and
PAGUg1931, which does not express MexXY. A difference in the activity of a derivative toward
the two strains indicates that the MexXY activity is not masked by the other four pumps (MexAB,
MexCD, MexEF and MexVW) [15]. The MIC values of the berberine derivatives were lower in
both strains compared to berberine (Table 1), suggesting that these berberine derivatives had higher
anti-pseudomonas activity compared with berberine. These berberine derivatives showed similar MIC
values that differed no more than, 4-fold. Their MIC values against PAGUg1927 were 2-fold greater
than against PAGUg1931, indicating that the derivatives are MexXY substrates.
Figure 1. Structure of berberine derivatives.

Antibiotics 2019, 8, 212
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Table 1. Antibacterial activities of berberine derivatives against PAGUg1931 and PAGUg1927.
MIC of (µg/mL)
Compound
-R
PAGUg1927
PAGUg1931
GM2
-
-
1024
>512
256
256
128
512
256
256
512
256
256
512
128
8
>512
128
128
64
256
128
128
256
128
128
256
64
Ber3
1
-H
o-Br
p-Br
o-F
o-Cl
p-Cl
o-CH3
m-CH3
p-CH3
o-NO2
2,6-Cl
2
3
4
5
6
7
8
9
10
11
Note: R, side chain of the benzyl group of 13-benzylberberine derivatives; GM, gentamicin; Ber, berberine.
2.2. Inhibition of Drug Resistance in P. aeruginosa Using Combined Berberine Derivatives
We investigated the MexXY inhibitory activities of the berberine derivatives by measuring the
MICs of gentamicin in the presence of the derivatives against P. aeruginosa mutants PAGUg1927 and
PAGUg1931 (Table 2). The concentrations of the berberine derivatives were 1/2, 1/4, or 1/8 that of the
MICs for PAGUg1931.
The MIC of gentamicin for PAGUg1927 in the presence of 256
µg/mL berberine was 128 µg/mL
(Table 2), which is one-eighth that of gentamicin alone (Table 1). Compounds 1–5 and 7 exhibited
apparently increased MexXY inhibitory activity, with compound 7 reducing the MIC of gentamicin
64-fold. Compound 7 was named 13-o-MBB. Compounds 8 and 9, which are regioisomers of 13-o-MBB,
increased sensitivity to gentamicin by up to 4-fold and were the weakest MexXY inhibitors.
Table 2. Increase in sensitivity to gentamicin by combination with berberine derivatives.
GM MIC with Berberine Derivative (µg/mL)
Concomitant
Compound
PAGUg1927
PAGUg1931
-R
256 *
128
64
32
16
8
256 128 64 32 16
8
Ber
1
2
3
4
5
6
7
8
−
128
−
256
−
256
32
32
512
64
64
128
128
64
256
64
256
256
256
128
-
−
−
8
8
−
−
−
4
−
−
4
−
−
8
−
8
8
4
−
8
4
4
8
4
4
8
−
8
8
8
4
8
8
8
8
8
8
8
4
−
−
−
−
8
−
−
−
−
−
−
−
8
-H
128
64
−
−
−
−
−
−
−
−
−
−
−
8
o−Br
−
−
−
8
8
p−Br
−
−
−
256
−
512
−
o−F
−
32
−
64
−
o−Cl
−
32
256
32
128
256
−
−
8
p−Cl
−
−
−
8
o−CH3
m−CH3
p−CH3
o−NO2
2,6−Cl
−
16
−
−
−
−
256
256
128
−
512
512
−
−
8
9
10
11
−
−
−
8
−
128
−
−
−
−
256
512
8
Note: R, side chain of the benzyl group of 13
combined concentration (µg/mL).
−
benzyl
−
berberine derivatives; GM, gentamicin; Ber, berberine, *;
We also examined changes in sensitivity to drugs other than gentamicin by combination
with the berberine derivatives (Table 3). The combined use of 13-o-MBB 128 g/mL reduced
µ
the MIC values of various substrate drugs (amikacin, tobramycin, kanamycin, gentamicin,
spectinomycin, norfloxacin, ciprofloxacin, erythromycin, carbenicillin, ethidium bromide, tetracycline,
chloramphenicol, azithromycin and cefepime) targeting MexXY by 2-fold to 16-fold (Table 3).
The regiosomer 13-(3-methylbenzyl) berberine bromide (13-m-MBB) increased the spectinomycin
sensitivity of PAGUg1927 8-fold and that of cefepime 4-fold at 64
µg/mL, whereas the other derivatives

Antibiotics 2019, 8, 212
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did not change the sensitivity to spectinomycin more than 2-fold. In addition, the combined use
of 64 g/mL of 13-(4-methyl-benzyl)-berberine bromide (13-p-MBB) increased sensitivity t cefepime
µ
4-fold for PAGUg1927. Moreover, the sensitizing action of 13-(3-methyl-benzyl)-berberine bromide
and 13-(4-methyl-benzyl)-berberine bromide did not exceed that of 13-o-MBB for PAGUg1927. Taken
together, these results suggest that the o-methyl group of 13-o-MBB increases antimicrobial sensitivity
in a MexXY-dependent manner.
Table 3. Increase in sensitivity to antibiotic resistance due to 13−o−MBB and its regioisomers.
MIC in the Presence of Berberine Derivative (µg/mL)
Drug
PAGUg1927
PAGUg1931
Ber
13−o−MBB 13−m−MBB 13−p−MBB
−
Ber
13−o−MBB 13−m−MBB 13−p−MBB
−
1
0.25
64
64
0.015625
16
1
64
0.25
2
AMK
TOB
KM
SPCM
NLFX
EM
CBPC
EtBr
Tc
4
0.25
128
256
0.25
128
1
512
2
4
2
0.5
64
64
0.0625
64
4
0.5
256
128
1
4
0.5
256
128
1
8
0.5
512
1024
1
1
0.25
64
64
0.015625
16
1
64
0.125
2
0.5
0.5
0.5
0.25
0.5
0.125
32
32
64
8
8
32
0.015625
0.015625
0.015625
512
1
256
1
512
1
256
16
16
1
8
16
1
8
8
1
16
1
128
1
256
8
256
8
0.125
2
0.125
2
0.125
1
Cp
2
8
8
8
AZM
CEF
64
1
64
0.25
256
0.125
256
2
512
8
8
8
8
4
8
0.0625
0.125
0.125
0.125
0.125
Note: Ber, combined berberine 256
µ
g/mL; 13
−
o
−
MBB, combined 13
−
o
−
MBB 128
µ
g/mL; 13
−
m MBB, combined
−
13 MBB 64 g/mL; 13 MBB, combined 13
−
m
−
µ
−
p
−
−
p
−
MBB 64 µg/mL; AMK, amikacin; TOB, tobramycin; KM,
kanamycin; SPCM, spectinomycin; NLFX, norfloxacin; EM, erythromycin; CBPC, carbenicillin; EtBr, ethidium
bromide; Tc, tetracycline; Cp, chloramphenicol; AZM, azithromycin; CEF, cefepime.
We investigated whether the inhibitory action of 13-o-MBB against MexXY-dependent drug
resistance can be observed in PAGU 1606, a multidrug resistant P. aeruginosa clinical strain, and its
MexXY-deficient strain PAGUg1659 (Table 4). The MIC of amikacin alone against PAGU 1606 was
256
µg/mL and 64
µg/mL when combined with berberine. In contrast, the combined use of 13-o-MBB
and amikacin decreased the MIC to 16
µg/mL. Thus, 13-o-MBB inhibits amikacin resistance 4-fold more
effectively than berberine in a MexXY-dependent drug resistant strain. Another aminoglycoside drug,
13-o-MBB, inhibited drug resistance two to four times stronger than berberine but had no greater effect
on the drug resistance of PAGU 1606 than the other aminoglycosides. However, the MICs of norfloxacin,
erythromycin and azithromycin were increased towards PAGUg1659, a pump-deficient strain.
Table 4. Inhibited resistance to aminoglycoside-based drugs by 13-o-MBB in PAGU 1606
MIC (µg/mL)
PAGU 1606
PAGUg1659
Ber
Drug
13-o-
MBB
(256)
13-o-
MBB
(128)
13-o-
MBB
(64)
13-o-
MBB
(256)
13-o-
MBB
(128)
13-o-
MBB
(64)
Ber
Ber
(128)
Ber
(64)
Ber
(256)
Ber
(128)
-
-
(256) 1
(64)
AMK
TOB
KM
256
256
>2048
64
>2048
256
64
64
64
1024
4
>2048
256
64
128
>512
>512
16
128
128
1024
8
>2048
256
64
256
>512
>512
16
128
128
2048
16
>2048
256
64
256
>512
>512
16
16
16
256
2
2048
256
32
128
>512
>512
16
32
32
512
4
>2048
256
32
256
>512
>512
16
32
32
1024
4
>2048
256
32
256
>512
>512
16
16
8
256
0.5
2048
64
64
128
>512
>512
16
8
8
8
8
8
8
256
0.5
2048
256
64
256
>512
>512
16
8
8
8
8
8
8
256
0.5
2048
128
32
256
>512
>512
16
256
0.25
2048
256
64
256
>512
>512
16
256
0.25
2048
256
64
256
>512
>512
16
256
0.25
1024
128
32
256
>512
>512
16
256
0.25
2048
128
32
256
>512
>512
16
GM
SPCM
NLFX
CPFX
EM
CBPC
EtBr
Tc
256
>512
>512
32
Cp
AZM
CEF
128
256
512
64
64
512
128
64
512
128
128
512
64
64
512
64
64
512
64
128
512
128
32
512
128
256
512
128
256
512
128
256
512
128
128
512
128
128
512
128
128
512
Note: 1, values in parentheses are combined concentrations (
µg/mL); Ber, berberine; AMK, amikacin; TOB,
tobramycin; KM, kanamycin; GM, gentamicin; SPCM, spectinomycin; NLFX, norfloxacin; CPFX, ciprofloxacin;
EM, erythromycin; CBPC, carbenicillin; EtBr, ethidium bromide; Tc, tetracycline; Cp, chloramphenicol; AZM,
azithromycin; CEF, cefepime.

Antibiotics 2019, 8, 212
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The sensitizing action of 13-o-MBB for various aminoglycosides was compared with that of
berberine at the same concentrations as tested against P. aeruginosa clinical strains but using Burkholderia
cepacia PAGU 0013 and Achromobacter xylosoxidans PAGU 0002 (Table 5). The two non-P. aeruginosa
strains are naturally resistant to aminoglycosides due to the presence of MexXY orthologs [7,23].
13-o-MBB at the same concentration as berberine increased the sensitivity to the aminoglycosides more
than 4-fold over that of berberine. In addition, comparison of the MICs of the aminoglycosides in
combination with 13-o-MBB towards a clinical strain of P. aeruginosa and its mexXY-deficient strain
provided similar MIC values. 13-o-MBB greatly increased the sensitivity to aminoglycoside drugs for
P. aeruginosa, B. cepacia, and A. xylosoxidans, increasing the sensitivity to amikacin more than 128-fold
and to gentamycin more than 512-fold for A. xylosoxidans.
Table 5. Inhibition by 13−o−MBB of aminoglycoside resistance in P. aeruginosa clinical strains.
MIC of Aminoglycoside (µg/mL)
AMK
Ber
GM
Ber
0.5
TOB
KM
Ber
SPEC
Ber
Strain
13-o-
MBB
13-o-
MBB
13-o-
MBB
13-o-
MBB
13-o-
MBB
−
−
−
Ber
−
−
PAGU 0974
PAGUg 0975
PAGU 1498
PAGUg1565
PAGU 1569
PAGUg1627
*PAGU 0013
PAGU 0002
4
1
1
0.5
8
0.5
0.5
1
4
0.25
1024
8
0.25
0.5
0.125 0.125 128
32
32
32
32
512
32
128
32
32
32
0.125 0.25 0.25 0.25 0.125
64
32
2
128
8
8
8
8
8
4
4
256
8
32
8
8
8
4
4
1
4
>2048
512
512
256
256
256
1024
1024
2
512
32
128
32
32
1
1
32
256
32
128
64
32
32
32
32
4
256
8
32
8
16
8
8
>2048 >2048
512
128
1024
256
128
128
128
128
16
8
1024
64
512
8
128
>2048
32
32
64
512
8
>2048 256
16
16
>2048
2048
256
>2048 512
64
2.3. Interaction between 13-o-MBB and Aminoglycoside Drugs
The fractional inhibitory concentration (FIC) values were determined using 13-o-MBB or berberine
and gentamicin or amikacin in combination with P. aeruginosa strains PAGU 1606 and PAGUg1927 and
their MexXY-defective mutants PAGUg1659 and PAGUg 1931 (Table 6). The combination of 13-o-MBB
and amikacin or gentamicin showed a synergistic effect in the MexXY-expressing strain, showing
that the MexXY-dependent aminoglycoside resistance inhibitory action of 13-o-MBB is synergistic. In
addition, the MICs of 13-o-MBB and berberine were reduced only in combination with amikacin or
gentamicin and only in the MexXY-expressing strain, showing that the combination of amikacin or
gentamicin in the presence of MexXY increases the accumulation of 13-o-MBB and berberine in the cell.
Table 6. Antibacterial activities of berberine derivatives against P. aeruginosa.
MIC (µg/mL) for AMK in
MIC (µg/mL) for 13-o-MBB
Mode of
Interaction
the Presence of:
in the Presence of:
Strain
FIC
−
13-o-MBB
−
AMK
PAGUg1931
PAGUg1927
PAGUg1659
PAGU 1606
1
8
16
256
1
2
8
256
512
>512
>512
256
128
>512
64
2.0
0.5
>1.5
<0.5
Indifferent
Synergy
Indifferent
Synergy
16

Antibiotics 2019, 8, 212
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Table 6. Cont.
MIC (µg/mL) for AMK in
MIC (µg/mL) for Berberne
Mode of
Interaction
the Presence of:
in the Presence of:
Strain
FIC
−
1
8
Berberine
−
AMK
PAGUg1931
PAGUg1927
PAGUg1659
PAGU 1606
1
4
>512
>512
>512
>512
>512
512
>512
512
>2.0
<1.0
1.5
Indifferent
Synergy or Addition
Indifferent
<0.75 Synergy or Addition
16
256
8
64
MIC (µg/mL) for GM in the
MIC (µg/mL) for 13-o-MBB
Mode of
FIC
Presence of:
in the Presence of:
Strain
Interaction
−
8
1024
0.5
13-o-MBB
−
256
512
GM
PAGUg1931
PAGUg1927
PAGUg1659
PAGU 1606
8
32
0.5
2
256
4
2.0
Indifferent
Synergy
0.04
>1.0
<0.5
>512
>512
>512
8
Indifferent
Synergy
64
MIC (µg/mL) for GM in the
MIC(µg/mL) for Berberine
Mode of
Interaction
Presence of:
in the Presence of:
Strain
FIC
−
Berberine
−
GM
PAGUg1931
PAGUg1927
PAGUg1659
PAGU 1606
8
1024
0.5
8
128
0.5
8
>512
>512
>512
>512
>512
8
>512
256
>1.0
<0.5
>1.0
<0.5
Indifferent
Synergy
Indifferent
Synergy
64
Note: GM, gentamicin; AMK, amikacin; FIC, fractional inhibitory concentration index.
2.4. Time-Killing Assay
The bactericidal activity of gentamicin together with berberine and 13-o-MBB against P. aeruginosa
was investigated using PAGUg1933 and PAGUg1929. PAGUg1933 was killed after 4 h treatment with
gentamicin at 2 µg/mL whereas the growth of PAGUg1929 was suppressed but no bactericidal action
was observed (Figure 2). Treatment of PAGUg1929 for 4 h with 2
with 256
of PAGUg1929 with a combination of 2
µ
g/mL gentamicin in combination
g/mL berberine reduced the number of colonies about 100-fold. In addition, treatment
g/mL gentamicin and 64 g/mL 13-o-MBB enhanced the
µ
µ
µ
bactericidal action of gentamicin more than 10-fold over that of 256 µg/mL berberine.
(a)
Figure 2. Cont.

Antibiotics 2019, 8, 212
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(b)
Figure 2.
PAGUg1933, closed squares, control; closed triangles, berberine 256
64 g/mL; open squares, gentamicin 2 g/mL; open triangles, gentamicin 2
256 g/mL; open diamonds, gentamicin 2 g/mL with 13-o-MBB 64 g/mL; ( ) Time-kill curves by the
combination of gentamicin with berberine or 13-o-MBB against PAGUg1929, closed squares, control;
closed triangles, berberine 256 g/mL; closed diamonds, 13-o-MBB 64 g/mL; open squares, gentamicin
g/mL; open triangles, gentamicin 2 g/mL with berberine 256 g/mL; open diamonds, gentamicin
2 µg/mL with 13-o-MBB 64 µg/mL.
(a
) Time-kill curves by the combination of gentamicin with berberine or 13-o-MBB against
g/mL; closed diamonds, 13-o-MBB
g/mL with berberine
µ
µ
µ
µ
µ
µ
µ
b
µ
µ
2
µ
µ
µ
3. Discussion
The addition of 128
8-fold in comparison with 256
µ
g/mL 13-o-MBB increased the sensitivity to aminoglycosides by 2-fold to
µ
g/mL berberine in the MexXY-positive P. aeruginosa strain PAGUg1927
(Tables 2 and 3). The antimicrobial activity of 13-o-MBB was not significantly different from that of the
13-o-MBB regioisomers 13-(3-methylbenzyl) berberine bromide and 13-(4-methylbenzyl) berberine
bromide, although the drug resistance inhibitory action of 13-o-MBB on the MexXY system is greater
than that of these two regioisomers. This indicates that 13-o-MBB has greater inhibitory action against
MexXY-dependent drug resistance than berberine and the other berberine derivatives we synthesized.
The deletion of mexXY from PAGU 1606 strain generated the PAGUg1659 strain. The addition
of 13-o-MBB increased PAGUg1659 resistance towards norfloxacin, erythromycin and azithromycin
2-fold to 4-fold. Norfloxacin, erythromycin and azithromycin are substrates for MexCD-OprM and
increased resistance towards norfloxacin, erythromycin and azithromycin may be due to the induction
of MexCD-OprJ [24].
The addition of 13-o-MBB 256
Clinical and Laboratory Standards Institute (CLSI) breakpoint (amikacin is 64
16 g/mL) in a clinical strain of P. aeruginosa highly resistant to aminoglycosides. Amino acid residue
µg/mL increased the efficacies of azithromycin and gentamicin to a
µg/mL, gentamycin is
µ
Y613 within the loop of the drug binding pocket of MexY is directly involved in the recognition of
aminoglycoside drugs, based on a decrease in sensitivity to aminoglycoside drugs upon mutation of
Y613 have been reported [25]. Tobramycin and berberine have been reported to compete for Y613 on
the docking simulations of tobramycin or berberine on MexY [16]. Furthermore, they claimed that the
results of a combined berberine/tobramycin assay on different clinical isolates of P. aeruginosa were
consistent with the in silico findings [16]. The results of our combination assay using berberine and
13-o-MBB with aminoglycosides are consistent with this report [16] and substantiate that the main
mechanism of action of berberine and 13-o-MBB is competition for MexY inhibition. Another possible
mechanism is suppression of MexY expression. However, Berberine decreased MexY mRNA only 0.8
to 0.9-fold have been reported [26]. Another reported that the MIC of amikacin and gentamicin was
increased only up to 4-fold even in a strain P. aeruginosa that expresses 10–21 times more MexY mRNA
than the PAO1 strain [12]. Our study of inhibited resistance by berberine showed that the gentamicin

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MIC for PAGU1606 was reduced 4-fold to 16-fold by berberine (Table 4), suggesting that the inhibition
of MexY expression is not the main mechanism of action of berberine and 13-o-MBB.
13-o-MBB showed cytotoxicity against Caco-2 cells, a human epithelial colorectal adenocarcinoma
cell line, at 30 µg/mL (data not shown). Thus, a concentration of 256 µg/mL 13-o-MBB could be toxic to
human cells. There is thus a need to synthesize a compound that exhibits inhibitory action against
MexXY system-dependent drug resistance at a lower concentration than 13-o-MBB and that is non-toxic
to human cells.
4. Materials and Methods
4.1. Bacterial Strains and Growth Conditions
The bacterial strains used in this study are described in Table 7. Bacterial cells were grown in
Luria (L) broth and on L agar (1.5%) under aerobic conditions at 37 ◦C, as previously described [27].
Table 7. Bacterial strains and gene properties.
Strain Name
Relevant Characteristics
Pseudomonas aeruginosa
Reference
PAGU 0974
PAGUg0975
PAGU 1498
PAGUg1565
PAGU 1569
PAGU 1606
PAGUg1627
PAGUg1659
PAGUg1927
PAGUg1929
PAGUg1931
PAGUg1933
PAO1 (K. Poole Lab), wild type
PAGU 0974∆mexXY
PA7 Non-respiratory clinical isolate
[28]
[29]
[8]
PA7∆mexXY-oprA
[8]
K2162 Pan-aminoglycoside-resistant clinical isolate
NCGM2. S1 Multidrug-resistant clinical isolate
K2162∆mexXY
[30]
[31]
[30]
[8]
[15]
[15]
[15]
[15]
PAGU 1606∆mexXY
YM34 ∆mexZ, mexVW:: gfp-aacC1
YM34 ∆mexZ, mexVW
PAGUg1927::∆mexXY
PAGUg1929::∆mexXY
Others
PAGU 0002
PAGU 0013
ATCC 27061 Achromobacter xylosoxidans subsp. xylosoxidans
ATCC 25416 Burkholderia cepacia
[32]
[33]
4.2. Antibiotic Susceptibility Assay
MICs were assessed in cation-adjusted Mueller–Hinton (MH) broth after about 18–22 h of
incubation at 37 ◦C (for P. aeruginosa) or after about 20–24 h of incubation at 35 ◦C (for A. xylosoxidans
and B. cepacia) using the two-fold serial micro-titer broth dilution method described previously [15].
The categorization as susceptible, intermediate, and resistant was performed according to the
interpretive standards of the CLSI.
The FIC index was calculated as described previously [15]. The effects of the drugs were interpreted
to be indicative of synergy when the index was ≤0.5.
4.3. Time-Killing Assay
We examined the bactericidal activity of gentamicin monotherapy or combination therapy with
berberine or berberine derivatives towards PAGUg1929 and PAGUg1933. Each measurement was
started by inoculating between 5
×
106 to 2
×
107 CFU/mL in cation-adjusted MH broth and incubating
at 150 rpm at 37 ◦C on a shaker. Samples were withdrawn to measure the survival counts on MH agar
plates at 0, 1, 2, 3 and 4 h. The MH agar plates were incubated at 37 ◦C for 16–18 h. The concentrations
of drugs tested were gentamicin 2
µg/mL, berberine 256 µg/mL, and 13-o-MBB 64 µg/mL. The fraction
surviving vs. the control for each sample was determined by taking the average CFU/mL values of the

Antibiotics 2019, 8, 212
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treated samples and dividing by the value for the same sample at 0 h. Each experiment was repeated
at least three times, and a representative experiment is shown.
4.4. Synthesis
4.4.1. General Synthesis Information
Melting points were measured on a Yanagimoto micro melting point hot-stage apparatus (MP-S3)
and are reported as uncorrected values. 1H-NMR (TMS:
δ
: 0.00 ppm as an internal standard) and
13C-NMR (CDCl3:
δ: 77.00 or DMSO-d6: 39.52 ppm as an internal standard) spectra were recorded
on JEOL JNM-AL400 (400 MHz and 100 MHz) spectrometers in CDCl3 or DMSO-d6. Mass spectra
were obtained on a JEOL JMP-DX300 instrument (70 eV, 300 mA). Chromatographic separations were
accomplished using silica gel 60N (Kanto Chemical Co., Inc., Tokyo, Japan) or aluminum oxide 90
standardized (Merck KGaA., Inc., Darmstadt, Germany). Thin-layer chromatography (TLC) was
performed using silica gel 60F254 and aluminum oxide 60F254 neutral (Merck KGaA, Inc., Darmstadt,
Germany). All reagents were purchased from Wako Pure Chemical Industry, Osaka, Japan. Kanto
Chemical Co., Inc., Tokyo, Japan. Tokyo Chemical Industry Co., Ltd., Tokyo, Japan. Kishida Chemical
Co., Ltd., Osaka Japan and Sigma-Aldrich Co., LLC. St. Louis, MO, USA. Dihydroberberine was
synthesized by the reduction of berberine according to the reported procedure [21].
4.4.2. 13-Benzylberberine Derivatives; General Procedure
Each benzyl bromide (1.0 mmol) was added in a dropwise manner to a stirred solution of KI
(310 mg, 1.86 mmol, 1.86 equiv) and dihydroberberine (337 mg, 1.0 mmol, 1 equiv) in CH3CN (40 mL),
and the resulting mixture was held at reflux for 4 h. The reaction mixture was then filtered, and
the filtrate was collected and evaporated to dryness in vacuo to give the crude residue. The residue
was purified by column chromatography over neutral alumina using CHCl3/CH3OH (50:1 to 20:1)
as eluent and recrystallization to give the final compounds 1–11. Compounds 1–10 were known
compounds and their characterisation data were identical to those given in the literature. Their
◦
melting points (m.p.) were as follows: Compound 1; m.p. 198–200 C [21], Compound 2; m.p.
179–180 ◦C [21]◦, Compound 3; m.p. 235–240 ◦C [21], Compound 4; m.p. 214–216 ◦C [21], Compound 5;
◦
◦
m.p. 210–211 C [34], Compound 6; m.p. 218–220 C [35], Compound 7; m.p. 216–220 C [36],
◦
◦
Compound 8; m.p. 222–225 C [21], Compound 9; m.p. 204–207 C [36] and Compound 10; m.p.
22–230 ◦C [21].
4.4.3. Characterisation Data of 13-(2,6-Dichlorobenzyl)berberine Bromide (11)
Compound 11 is a yellow solid. Yield: 41%. 1H-NMR (DMSO-d6)
J = 9.3 Hz), 7.84 (1H, d, J = 9.3 Hz), 7.56 (1H, s), 7.37 (2H, d, J = 7.8 Hz), 7.23 (1H, t, J = 8.3 Hz), 7.15 (1H,
s), 6.18 (2H, s), 5.16 (2H, s), 4.84 (2H, br), 4.09 (3H, s), 4.01 (3H, s), 3.08 (2H, br). 13C-NMR (DMSO-d6)
δ: 9.95 (1H, s), 8.11 (1H, d,
δ
:
150.0 (s), 149.3 (s), 146.5 (s), 144.2 (d), 144.1 (s), 138.2 (s), 134.9 (s), 134.7 (s), 133.6 (s), 131.9 (s), 131.3 (s),
129.5 (d), 129.2 (d), 125.8 (d), 121.0 (s), 120.5 (d), 120.4 (s), 110.9 (d), 108.1 (d), 102.0 (t), 62.0 (q), 56.9 (q),
56.7 (t), 32.9 (t), 27.4 (t). MS m/z: 494 (M–Br)+, 119, 85. m.p. 228–231 ◦C.
5. Conclusions
Eleven berberine derivatives were synthesized and tested for MexXY-dependent inhibition of
gentamicin resistance using a Pseudomonas aeruginosa positive-MexXY strain and a negative-MexXY
strain. 13-o-MBB showed the greatest inhibitory effect on MexXY-dependent gentamicin resistance.
Regioisomers of 13-o-MBB exhibited no greater MexXY-dependent inhibition of gentamicin resistance
than berberine. 13-o-MBB inhibited resistance to aminoglycosides 4-fold to 16-fold compared with
berberine against the four tested P. aeruginosa clinical strains, and Achromobacter xylosoxidans and
Burkholderia cepacia. These results indicate that 13-o-MBB inhibits the resistance to aminoglycosides

Antibiotics 2019, 8, 212
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in a MexXY-dependent manner more strongly than berberine. 13-o-MBB is thus a useful inhibitor of
aminoglycoside drug resistance due to MexXY.
Author Contributions: K.K. and Y.M. have both significantly contributed to the creation of this manuscript.
All authors read and approved the final manuscript. conceptualization, K.K. and Y.M.; methodology, K.K., M.M. and
S.Y.; Investigation, K.K. and Y.M.; writing—original draft preparation, J.T. and R.K.; writing—review and editing,
K.K.; visualization, Y.K. and K.N.; supervision, Y.K.; project administration, Y.K. and Y.M.; funding acquisition.
Funding: This research was funded by a research grant from Nagai Memorial Research Scholarship from the
Pharmaceutical Society of Japan (to K.K.) and by the Japan Society for the Promotion of Science (JSPS) KAKENHI
Grant Number JP26460080.
Acknowledgments: This work was performed under the Research Program of “Dynamic Alliance for Open
Innovation Bridging Human, Environment and Materials” in “Network Joint Research Center for Materials and
Devices”.
Conflicts of Interest: The authors declare no conflict of interest. The funders had no role in the design of the
study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to
publish the results.
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