Aquaculture Research, 2010, 41, 1095^1100 doi:10.1111/j.1365-2109.2009.02399.

In vitro antibacterial effect of berberine hydrochloride

and enrofloxacin to fish pathogenic bacteria

Defeng Zhang1,2, Aihua Li1, Jun Xie3 & Cheng Ji1,2

1
State Key Laboratory of Freshwater Ecology and Biotechnology, Institute of Hydrobiology, Chinese Academy of Sciences,
Wuhan, China
2
Postgraduate School of Chinese Academy of Sciences, Beijing, China
3
Ministry of Agriculture, Freshwater Fisheries Research Center, Chinese Academy of Fishery Sciences,Wuxi, China

Correspondence: A Li, Institute of Hydrobiology, Chinese Academy of Sciences,Wuhan 430072, China. E-mail: liaihua@ihb.ac.cn

Abstract gencies associated with infectious disease epidemics.

The use of chemicals and antibiotics, however, has
Berberine the main antibacterial substance in the rhi-
the potential to increase the frequencies of bacterial
zome of Coptis chinensis Franch, is used as an antimi-
resistance and this would have a negative impact on
crobial agent for a long time. This study was carried
the subsequent use of these agents to control infec-
out to investigate the potential of berberine alone or
tious disease in aquaculture. In addition, there is also
in combination with enro£oxacin against six common
a possibility that the enrichment of resistant bacteria
¢sh pathogens for use in ¢sh disease management in
or genes encoding resistance could have an adverse
aquaculture. The minimum inhibitory concentrations
impact on the use of antimicrobial agents to control
(MICs) of berberine hydrochloride against Aeromonas
diseases in humans and other land-based animals.
hydrophila, Pseudomonas £uorescens, Vibrio vulni¢cus,
Therefore, the use of chemicals and antibiotics is fa-
Edwardsiella ictaluri, Escherichia coli and Streptococcus
cing greater restriction in the aquaculture industry.
agalactiae were determined and to be 4500, 4500,
China is a major aquaculture country, accounting
4500, 300, 400 and 100 mg mL  1 respectively. The
for nearly 70% of the world’s total aquaculture output;
minimal bactericidal concentrations (MBCs) of berber-
however, there are so many kinds of ¢sh diseases
ine hydrochloride to E. coli, E. ictaluri and S. agalactiae
caused by pathogenic bacteria, such as Aeromonas hy-
were 300^500 mg mL  1. In combination with enro-
drophila, Pseudomonas £uorescens, Streptococcus spp.,
£oxacin, the MICs and MBCs of berberine hydrochlor-
Edwardsiella ictaluri and Vibrio spp., which caused se-
ide signi¢cantly decreased against E. coli and E. ictaluri
vere economical loss in aquaculture throughout Chi-
but not against Streptococcus dysgalactiae. These results
na. Looking for alternative treatments to ¢sh diseases
demonstrated that the berberine hydrochloride en-
caused by various bacteria, such as the use of Chinese
hanced the bactericidal e¡ect of enro£oxacin and vice
herb medicines or plant extracts, to replace, or reduce
versa. The synergistic bactericidal e¡ect of berberine
the reliance on antibiotics is thought as a solution to
hydrochloride and enro£oxacin suggest its potential
this problem.
use in ¢sh disease management in aquaculture. This
Traditional Chinese medicines in combination
is the ¢rst study on the inhibitory e¡ect of berberine
with chemical medicines has been given special at-
hydrochloride against ¢sh pathogenic bacteria.
tentions in the area of human medicine, veterinary
medicine and ¢sh disease control in China because
Keywords: berberine hydrochloride, enro£oxacin,
of the low price and low toxicity of traditional
¢sh pathogenic bacteria, synergistic e¡ect
Chinese drugs (Lu, Ding & Chen 2008).
Berberine [18,5,6-dihydro-9,10-dimethoxybenzo(g)-
1,3-benzodioxolo (5,6-a) quinolizinium], a naturally
Introduction
occurring isoquinoline alkaloid, has been found in
Appropriate antimicrobial therapy represents one of roots and bark of various Berberis species [e.g., Ber-
the most e¡ective management responses to emer- beris vulgaris (barberry), Berberis aquifolium (Oregon

r 2009 Aihua Li
Journal Compilation r 2009 Blackwell Publishing Ltd 1095
Antibacterial e¡ect of berberine hydrochloride and enro£oxacin D Zhang et al.
grape), Berberis aristata (tree turmeric) and Tinospora
cordifolia]. The extracts of these species have been
used as an antidiarrhoeal drug and an antibacterial
agent, and now it has been synthesized. Berberine
has a wide range of pharmacological e¡ects. It can in-
hibit fungi, such as Candida albicans, Candida tropicalis
and Candida glabrata (Nakamoto, Sadamori & Hamada
1990). Berberine sulphate has been demonstrated to
reduce the infectivity of bacteria, fungi and protozoa
in both animals and humans (Subbaiah & Amin 1967;
Amin, Subbaiah & Abbasi 1969). Berberine hydro-
chloride, the most commonly used form of berberine,
in combination with £uconazole can result in potent
synergistic action against £uconazole-resistant C. al-
bicans in vitro (Quan, Cao, Xu, Zhao, Gao, Qin & Jiang
2006). Furthermore, berberine is the best starting
structure to look for new cationic alkaloids with dou-
ble isoquinolinoid skeleton, acting as antifungal
agents (Enriz & Freile 2006).
The aims of this study are to explore the potential
use of berberine alone and together with chemical
antimicrobials and antibiotics, in order to substitute
or cut down the wide use of these chemical antimi-
crobials and antibiotics in aquaculture, which had
caused a series of environmental and food safety pro-
blems. For this purpose, Escherichia coli, E. ictaluri,
A. hydrophila, P. £uorescens,Vibrio harveyi and Strepto-
coccus dysgalactiae were selected to test because they
are the most common ¢sh bacterial pathogens in Chi-
nese aquaculture practices. This work will be the
starting point to study the underlying mechanism of
the activity of berberine and berberine combined
with antibiotics to the pathogens.
Materials and methods
Bacterial strains and growth conditions
Five archived strains of ¢sh pathogenic bacteria and
one strain of E. coli listed in Table 1 were used in the
present study. All strains except E. coli were isolated
from diseased ¢sh and originally con¢rmed to be
Table 1 Strains of bacteria tested in this study
Strains Host fish
Streptococcus dysgalactiae (0722XY) Acipenser schrenckii
Edwardsiella ictaluri (HSN-1) Pelteobagrus fulvidraco
Pseudomonas fluorescens (56-12-10) Ctenopharyngodon idella
Aeromonas hydrophila (XS9141) Hypothalmichthys molitrix
Vibrio harveyi (Ecgy0204) Lutjanus erythopterus
Escherichia coli (ATCC25922) A American Type Cultures Collection (ATCC) strain
1096 Journal Compilation r 2009 Blackwell Publishing Ltd, Aquaculture Research, 41, 1095^1100
Aquaculture Research, 2010, 41, 1095^1100
pathogenic to ¢sh. All bacterial strains were stored
at  80 1C as usual. Before testing, the archived bac-
teria were removed from the ultra freezer, allowed to
thaw to room temperature, cultured in broth and in-
cubated at 25 1C for 24 h to reach the log growth
phase of the isolates.
In this study, brain heart infusion media (BHI, Ox-
oid, Basingstoke, UK) for E. ictaluri and S. dysgalac-
tiae, trypticase soy broth (TSB) and tryptonic soy
agar (Difco Laboratories, Detroit, MI, USA) for the
other bacteria were used to culture bacteria in this
study. For the culture of V. harveyi, a ¢nal concentra-
tion of 2% of sodium chloride was supplemented to
the media (Donovan & van Netten 1995).
Antimicrobial agents
The antimicrobials tested in this study included arti-
¢cially synthesized berberine hydrochloride powder
(purity, 97.1%) and enro£oxacin powder (purity,
99.1%). Both chemicals were obtained from Wuhan
Jiuzhou Shengnong Pharmaceutical (Wuhan, Hubei
province, China). They were dissolved in appropriate
solvents (PBS, pH7.4) to make stock solutions, and
then further diluted in BHI or TSB. After ¢ltration
through sterile a 0.22 mm membrane, stock solutions
were frozen at  20 1C until use. The concentrations
of stock solutions of berberine hydrochloride and
enro£oxacin were 1000 mg mL  1 (pH 6.4) and
1000 mg mL  1 (pH 7.0) respectively.
Minimal inhibitory concentration (MIC)
and minimal bactericidal concentration
(MBC) assays
The MIC was determined by a broth microdilution
method with Mueller^Hinton broth (Becton Dickin-
son Italia, Milan, Italy), according to the procedures
outlined by the National Committee for Clinical
Laboratory Standards (Clinical and Laboratory
Standards institute/National Committee for Clinical
Year of isolation Location of isolation
2006 Hubei province, China
2006 Hubei province, China
1956 Hubei province, China
1991 Hubei province, China
2002 Guangdong province, China
r 2009 Aihua Li
Aquaculture Research, 2010, 41, 1095^1100 Antibacterial e¡ect of berberine hydrochloride and enro£oxacin D Zhang et al.

Table 2 Results of MIC test of berberine hydrochloride by a checkerboard titration method using 96-well
against six strains of bacteria polypropylene microtitre plates. The ranges of drug
dilutions used were shown as listed in Tables 4^6.
Berberine hydrochloride concentrations The media, inocula and conditions were the same as
(lg mL  1)
those for MIC tests. The fractionary inhibitory con-
Strains 500 400 300 200 150 100 70 40 centration (FIC) index for combinations of two anti-
microbials was calculated by the formula
XS9141 1 1 1 1 1 1 1 1
56-12-10 1 1 1 1 1 1 1 1 FIC index ¼ partial FIC berberine
0722XY       1 1 þ partial FIC enrofloxacin
HSN-1    1 1 1 1 1
Ecgy0204 1 1 1 1 1 1 1 1 where partial FIC is the concentration of drug in com-
ATCC25922   1 1 1 1 1 1 bination/MIC of single drug.
1, represents visible bacterial growth; , represents no visible The result of the sum allows to know if the
bacterial growth. combination resulted in synergism, indi¡erence or
ATCC, American Type Cultures Collection; MIC, minimum inhi- antagonism, according to following criteria:  1,
bitory concentration.
synergistic; 1^2, indi¡erence;  2, antagonistic (Ca-
solari, Rossi, Baggio, Coppi, Zandomeneghi, Ruberto,
Laboratory Standards 2006), and an initial inoculum Farina, Fabio, Zanca & Castelli 2004).
of 5  105 CFU mL  1 was used. In brief, 50 mL BHI or
TSB was added to each well of 96-well plates, 50 mL of
drug solution with appropriate concentration was
Results
added to the ¢rst well, and then a serial twofold dilu-
tion was processed (it was not true for berberine hy- Antimicrobial activity
drochloride, the detailed concentrations of the series
Minimal inhibitory concentrations of berberine hy-
wells can be seen in the following tables), giving a
drochloride against E. coli, E. ictaluri and S. dysgalac-
series of tested concentrations for berberine hydro-
tiae were 300, 400 and 150 mg mL  1, respectively,
chloride and enro£oxacin, as listed in Table 2. Fifty
and were shown in Table 1. These ¢ndings demon-
microlitres of inocula with a concentration of
strate that berberine possesses good activity against
106 CFU mL  1 logarithmic organisms was added to
these organisms, especially against S. dysgalactiae.
each well, mixed thoroughly and incubated at 25 1C
Table 2 shows the MICs of enro£oxacin against six
for 24 h in a moist chamber before the visual determi-
strains of bacteria, A. hydrophila, P. £uorescens, S. dys-
nation of MICs. Wells without antimicrobial agents
galactiae, E. coli, V. harveyi and E. ictaluri; they were
and wells without bacteria were set up and incubated
0.4,1.6, 0.8, 0.025, 0.8 and 0.025 mg mL  1 respectively.
as controls. The ¢nal concentrations of bacteria
These ¢ndings show that enro£oxacin was much
adopted were 5  105 CFU mL  1. Minimal inhibi-
more e¡ective against E.coli and E. ictaluri than
tory concentration was de¢ned as the lowest drug
against the others.
concentration at which observable growth was
The results of antibacterial testing of berberine hy-
inhibited.
drochloride in combination with enro£oxacin
Minimal bactericidal concentrations of berberine
against E. coli, S. dysgalactiae and E. ictaluri are
hydrochloride and enro£oxacin alone or in combina-
shown in Tables 3^5.
tion were determined by subculturing 10 mL of the
The MICs of berberine hydrochloride in combina-
test dilutions without visible growth in MIC experi-
tion with enro£oxacin against E. coli or E. ictaluri is
ments on to a fresh drug-free solid medium and incu-
lower than that of berberine hydrochloride alone or
bating further for 18^24 h. The lowest concentration
enro£oxacin alone against the two organisms. The re-
of each drug that resulted in 499.9% reduction in
sults shown inTable 3 indicate that sub-MIC of berber-
the initial inocula was taken as MBC.
ine hydrochloride increased the antibacterial e¡ect of
enro£oxacin against E. coli and E. ictaluri. Minimal in-
hibitory concentration of enro£oxacin against E. coli
Combination antimicrobial susceptibility
decreased from 0.025 to 0.0125 mg mL  1 when there
testing
was 50 or 100 mg mL  1 of berberine hydrochloride in
In interaction studies, E. ictaluri, E. coli and S. dysga- microtitre plate wells. When 150 or 200 mg mL  1 of
lactiae were used to test the antibiotic combinations berberine hydrochloride was used, the MIC of enro-

r 2009 Aihua Li
Journal Compilation r 2009 Blackwell Publishing Ltd, Aquaculture Research, 41, 1095^1100 1097
Antibacterial e¡ect of berberine hydrochloride and enro£oxacin D Zhang et al. Aquaculture Research, 2010, 41, 1095^1100

Table 3 Results of MIC test of enro£oxacin against six Table 5 Inhibitory test of berberine hydrochloride com-
strains of bacteria bined with enro£oxacin to Streptococcus dysgalactiae
(0722XY)
Enrofloxacin concentrations (lg mL  1)
Enrofloxacin (lg mL  1)
Stains 1.6 0.8 0.4 0.2 0.1 0.05 0.1/4 0.1/8
Agents and concentration 1.6 0.8 0.4 0.2 0.1 0.05 0
XS9141    1 1 1 1 1
56-12-10  1 1 1 1 1 1 1 Berberine hydrochloride (mg mL  1)
0722XY   1 1 1 1 1 1 300       
HSN-1        1 200       
Ecgy0204   1 1 1 1 1 1 150       
ATCC25922        1 100   1 1 1 1 1
70   1 1 1 1 1
1, represents visible bacterial growth; , represents no visible
40   1 1 1 1 1
bacterial growth.
0   1 1 1 1 1
ATCC, American Type Cultures Collection; MIC, minimum inhi-
bitory concentration. 1, represents visible bacterial growth; , represents no visible
bacterial growth.

Table 4 Inhibitory test of berberine hydrochloride com-

bined with enro£oxacin to Escherichia coli (ATCC25922) Table 6 Inhibitory test of berberine hydrochloride com-
bined with enro£oxacin to Edwardsiella ictaluri (HSN-1)
Enrofloxacin (lg mL  1)

Agents and Concentration 0.1/2 0.1/4 0.1/8 0.1/16 0.1/32 0 Enrofloxacin (lg mL  1)

Berberine hydrochloride (mg mL  1) Agents and concentration 0.1/2 0.1/4 0.1/8 0.1/16 0.1/32 0
400      
Berberine hydrochloride (mg mL  1)
300      
400      
200     1 1
300      1
150     1 1
200     1 1
100    1 1 1
150    1 1 1
50    1 1 1
100    1 1 1
0   1 1 1 1
50    1 1 1
1, represents visible bacterial growth; , represents no visible 0   1 1 1 1
bacterial growth.
1, represents visible bacterial growth; , represents no visible
ATCC, American Type Cultures Collection.
bacterial growth.

£oxacin against E. coli decreased further to
0.00625 mg mL  1. In the same way, sub-MIC of enro-
£oxacin also enhanced the antibacterial e¡ect of ber-
berine hydrochloride, lowered its MIC against E. coli
from 300 to 50 mg mL  1 when 1/2MIC or 1/4MIC of
enro£oxacin was used. The calculated FIC was 0.67, in-
dicating a synergism e¡ect.
A similar synergistic action against E. ictaluri be-
tween berberine hydrochloride and enro£oxacin
was observed (Table 6). Sub-MIC (450 mg mL  1) of
berberine hydrochloride can reduce the MIC of enro-
£oxacin on E. ictaluri two to eight times, i.e., from
0.025 to 0.003125 mg mL  1. In the same way, sub-
MIC of enro£oxacin can enhance the antibacterial
activity of berberine hydrochloride, and allows the
MIC against E. ictaluri to decrease from 300 mg mL  1
go down to 50 mg mL  1. The FIC calculated was
0.625.
Berberine hydrochloride did not increase the inhi-
bitory e¡ect of enro£oxacin against S. dysgalactiae
(Table 5). The FIC calculated was 1.
The MBCs of berberine hydrochloride for E. coli,
S. dysgalactiae, E. ictaluri were 400, 300 and
500 mg mL  1 respectively. The MBCs of enro£oxacin
for E. coli, S. dysgalactiae and E. ictaluri were 0.2, 3.2
and 0.1 mg mL  1 respectively. The MBCs of berberine
hydrochloride in combination with enro£oxacin for
E. coli, S. dysgalactiae and E. ictaluri were 0.00625
mg mL  1 of enro£oxacin plus 300 mg mL  1 of berber-
ine hydrochloride, 0.4 mg mL  1 of enro£oxacin plus
200 mg mL  1 of berberine hydrochloride and 0.00625
mg mL  1 of enro£oxacin plus 300 mg mL  1 of ber-
berine hydrochloride respectively. These results
demonstrated that the enro£oxacin enhanced the
bactericidal e¡ect of berberine hydrochloride and
vice versa.

r 2009 Aihua Li
1098 Journal Compilation r 2009 Blackwell Publishing Ltd, Aquaculture Research, 41, 1095^1100
Aquaculture Research, 2010, 41, 1095^1100 Antibacterial e¡ect of berberine hydrochloride and enro£oxacin D Zhang et al.
Discussion
The MICs of berberine hydrochloride for theA. hydro-
phila, P. £uorescens, V. harveyi, E. coli, E. ictaluri and
S. dysgalactiae stains were  500,  500,  500,
400, 300 and 150 mg mL  1, respectively, which are
much higher than those of commonly used antibio-
tics. S. dysgalactiae was most sensitive to berberine
hydrochloride among the organisms tested in the
present study. This ¢nding is in coincidence with a
previous study, which showed that the antibacterial
e¡ect of berberine on Staphylococcus aureus, Bacillus
subtilis were stronger than on E. coli (Wang, Yan &
Gao 2006). This results show that berberine
hydrochloride can inhibit the growth of E. ictaluri,
E. coli and S. dysgalactiae at a concentration
o400 mg mL  1, but berberine hydrochloride
alone did not show e¡ective inhibition against
A. hydrophila, P. £uorescens andV. harveyi in vitro even
at a concentration of 500 mg mL  1. Combination of
berberine hydrochloride and enro£oxacin showed a
signi¢cant synergistic action against E. ictaluri and
E. coli (FIC 5 0.625 and 0.67 respectively), but did not
show such a e¡ect on S. dysgalactiae (FIC 51). Consid-
ering S. dysgalactiae is a Gram-positive bacterium,
and E. ictaluri and E. coli are Gram-negative bacteria,
does this mean that the synergistic action between
berberine hydrochloride and enro£oxacin can
only occur on Gram-negative bacteria? Of course, it
needs a more detailed study. This result clearly indi-
cated that berberine hydrochloride has signi¢cant
antibacterial activity against E. ictaluri and E. coli
when used together with enro£oxacin. The synergis-
tic action, however, has not been demonstrated
in vivo studies.
The ¢nding that the antibacterial e¡ect of berber-
ine hydrochloride on Gram-positive bacteria (S. aur-
eus, B. subtilis and S. dysgalactiae) was stronger than
on the Gram-negative bacteria tested (A. hydrophila,
P. £uorescens, V. harveyi, E. ictaluri and E. coli) prob-
ably owes to the di¡erence of structure and function
of bacterial cell wall partly.
Whether the combination of berberine hydro-
chloride and enro£oxacin can reduce the risk of
development of resistant bacteria to both antimicro-
bials also need further investigations.
Our results show that E. ictaluri and E. coli had
higher susceptibility to enro£oxacin than A. hydro-
phila, P. £uorescens, S. dysgalactiae and V. harveyi had.
The MIC value of enro£oxacin against E. coli
(ACTT25922) was in accordance with that reported
previously (Lykkeberg, Halling-Srensen & Jensen
r 2009 Aihua Li
Journal Compilation r 2009 Blackwell Publishing Ltd, Aquaculture Research, 41, 1095^1100
2007). This result can be taken as a quality control
for MIC assay.
There were several researchers exploring the me-
chanism behind the antimicrobial activity of berber-
ine. There was a report that berberine sulphate was
bacteriostatic for streptococci and that sub-MICs of
berberine blocked the adherence of streptococci to
host cells (Sun, Courtney & Beachey 1988). Berberine
speci¢cally blocks the synthesis and assembly of
Pap ¢mbriae on the surface of E. coli CI6 cell in the
presence of 200 mg mL  1 berberine for 18 h (Sun,
Abraham & Beachey 1988). In the presence of
1^50 mg mL  1 berberine, adhesion and intracellular
invasion ability of methicillin-resistant S. aureus were
notably decreased (Yu, Kim, Cha, Kim, Lee, Choi &
You 2005). Berberine was also found to have e¡ects
on toxins and plasmids of bacteria. Berberine might
not allow the formation of active and intact cholera
toxins (Modak, Modak & Venkatraman 1970). Berber-
ine also markedly inhibited the secretory response of
E. coli heat-stable enterotoxin in the infant mouse
model (Sack & Froehlich 1982). Berberine sulphate
has some e¡ects to eliminate the plasmids of E. coli
(Li, Wang & Hu 1994). In conclusion, berberine is a
multifunctional antimicrobial agent, which has a po-
tential to be used in aquaculture. Further studies are
de¢nitely needed to determine the exact underlying
mechanism of the antibacterial e¡ect of berberine
and the combination with antibiotics. Coptis chinen-
sis Franch, which is an important Chinese herb and
is an important source of berberine, has been used
widely in Chinese aquaculture to treat ¢sh diseases
and to promote ¢sh immune system with di¡erent le-
vels of success (Commission of Chinese Veterinary
Pharmacopoeia 2005).
Disease problem is a major threaten to aquaculture
in China. Unfortunately, antimicrobial treatment is
currently the major control method. However, as in
many of other countries, antibiotics are facing tough-
er control on the use on food-producing animals, in-
cluding on aquaculture ¢sh in China. Products from
plants are receiving greater attention for the substi-
tution of chemical antimicrobials for this purpose.
The ¢ndings of the present study proved that a Chi-
nese herb extract, berberine, was e¡ective in control-
ling some ¢sh pathogenic bacteria, suggesting its
potential as an antimicrobial agent for aquaculture
use, especially when used in combination with anti-
biotics. By this way, it will reduce the usage of anti-
biotics to a certain extent.
To our knowledge, this is the ¢rst study on the
inhibitory e¡ect of berberine hydrochloride against
1099
Antibacterial e¡ect of berberine hydrochloride and enro£oxacin D Zhang et al.
¢sh pathogenic bacteria when in combination with
antibiotics.
Acknowledgments
We thank W. M.Yang, C. Ji, J.Y. Liu and X. N. Gong for
their technical assistance. This study was supported
by the National Science and Technology Pillar
Programs (Grants No. 2006BAD03B04 and No.
2006BAK02A22). The authors are indebted to the
National Science Foundation of China for their sup-
port of the project (No.30670112).
References
Amin A.H., Subbaiah T.V. & Abbasi K.M. (1969) Barbering
sulfate: antimicrobial activity, bioassay and mode of ac-
tion. Canadian Journal of Microbiology 15,1067^1076.
Casolari C., Rossi T., Baggio G., Coppi A., Zandomeneghi G.,
Ruberto A.I, Farina C., Fabio G., Zanca A. & Castelli M.
(2004) Interaction between saquinavir and antimycotic
drugs on C. albicans and C. neoformans strains. Pharmaco-
logical Research 50, 605^610.
Commission of Chinese Veterinary Pharmacopoeia. (2005)
Veterinary Pharmacopoeia of The People’s Republic of
China,Vol.2 (pp.311^317. China Agriculture Press, Beijing,
China.
Clinical and Laboratory Standards Institute/National Com-
mittee for Clinical Laboratory Standards. (2006) Methods
forAntimicrobial Disk SusceptibilityTesting of Bacterial Iso-
lated from Aquatic Animals, Approved Guideline (CLSI/
NCCLS Document M42-A). CLSI/NCCLS,Wayne, PA, USA.
Donovan T.J. & van Netten P. (1995) Culture media for the
isolation and enumeration of pathogenicVibrio species in
foods and environmental samples. International Journal of
Food Microbiology 26,77^91.
Enriz R.D. & Freile M.L. (2006) Structure-activity relation-
ship of berberine and derivatives acting as antifungal
compounds. The Journal of the Argentine Chemical Society
94, 113^119.
1100 Journal Compilation r 2009 Blackwell Publishing Ltd, Aquaculture Research, 41, 1095^1100
Aquaculture Research, 2010, 41, 1095^1100
Li L.J., Wang Z. & Hu W.Z. (1994) Elimination of drug resis-
tance plasmids from Shigella by nor£oxacin and berber-
ine. ChineseJournal of Infectious Diseases 12, 4^8.
Lu A.P., Ding X.R. & Chen K.J. (2008) Current situation and
progress in integrative medicine in China. ChineseJournal
of Integrative Medicine 14, 234^240.
Lykkeberg A.K., Halling-Srensen B. & Jensen L.B. (2007)
Susceptibility of bacteria isolated from pigs to tiamulin
and enro£oxacin metabolites. Veterinary Microbiology
121,116^124.
Modak M.J., Modak S. & Venkatraman A. (1970) E¡ect of ber-
berine on the fatty acid composition of Vibrio cholerae and
Vibrio cholerae El tor. The IndianJournal of Medical Research
58,1523^1525.
Nakamoto K., Sadamori S. & Hamada T. (1990) E¡ects of
crude drugs and berberine hydrochloride on the activities
of fungi. The Journal of Prosthetic Dentistry 64, 691^694.
Quan H., CaoY.Y., Xu Z., Zhao J.X., Gao P.H., Qin X.F. & Jiang
Y.Y. (2006) Potent in vitro synergism of £uconazole and
berberine chloride against clinical isolates of Candida albi-
cans resistant to £uconazole. Antimicrobial Agents and
Chemotherapy 50,1096^1099.
Sack R.B. & Froehlich J.L. (1982) Berberine inhibits intestinal
secretory response of Vibrio cholerae and E. coli enterotox-
ins. Infection and Immunity 35, 471^475.
Subbaiah T.V. & Amin A.H. (1967) E¡ect of berberine sulfate
on Entamoeba histolytica. Nature 215, 527^528.
Sun D.X., Abraham S.N. & Beachey E.H. (1988) In£uence of
berberine sulfate on synthesis and expression of Pap ¢m-
brial adhesin in uropathogenic E. coli. Antimicrobial
Agents and Chemotherapy 32, 1274^1277.
Sun D.X., Courtney H.S. & Beachey E.H. (1988) Berberine
sulfate blocks adherence of Streptococcus pyogenes to
epithelial cell, ¢bronectin, and hexadecane. Antimicrobial
Agents and Chemotherapy 32, 1370^1374.
Wang J.T.,YanY.M. & Gao Q.Z. (2006) Study on the bacteriosta-
sic activity of berberine by micocalorimetry. Journal of Qufu
Normal University (NATURAL SCIENCE) 32, 99^103.
Yu H.H., Kim K.J., Cha J.D., Kim H.K., LeeY.E., Choi N.Y. & You
Y.O. (2005) Antimicrobial activity of berberine alone and
in combination with ampicillin or oxacillin against
methicillin-resistant Staphylococcus aureus. Journal of
Medicinal Food 8, 454^461.
r 2009 Aihua Li