148 2010, Vol. 31, No.

13

A Mechanism of Antibacterial Activity of Chitosan against

Gram-negative Bacteria
LI Xiao-fang FENG Xiao-qiang YANG Sheng *
(College of Biology and Chemistry, Tianshui Normal University, Tianshui 741001, China)

Abstract The antibacterial activities of chitosan against Pseudomonas aeruginosa (P. aeruginosa), Proteus mirabilis (P.
mirabilis) and Escherichia coli (E. coli) were evaluated by optical density method. Meanwhile, bacterial cell surface hydrophilicity
and negative charge analysis were investigated to illustrate the relationship between surface characteristics of Gram-negative
bacterial cell wall and antibacterial activity of chitosan. To investigate the action mode of chitosan towards Gram-negative
bacterial, E. coli was selected to be representative of bacteria. The permeability of the outer membrane was investigated by
determining the changes in the fluorescence intensity of cells treated with the fluorescent probe of 1-N-phenylnaphthylamine
(NPN). Furthermore, the interaction of chitosan with E. coli was studied by Fourier-transform infrared spectroscopy (FT-IR).
Results showed that the Gram-negative bacteria with better hydrophilicity and more negatively charged cell surfaces exhibited
greater interaction with chitosan and accordingly chitosan had preferable antibacterial activity against them. Chitosan increased
the permeability of outer membrane and ultimately disrupted bacterial cell membranes. This damage was likely caused by the
electrostatic interaction between NH3+ groups of chitosan and carbonyl or phosphoryl groups of phospholipid components
of cell membranes.
Key words chitosan Gram-negative bacteria antibacterial activity mechanism

*
( 741001)

1-N- (NPN)
(FT-IR)

Q935 A 1002-6630(2010)13-0148-06

Chitosan is among the most promising biomaterials in possesses a higher antibacterial activity, a broader spec-
the world, not only because of its abundance but also trum of activity, a higher killing rate, and lower toxicity
due to its various properties. It has attracted consider- towards mammalian cells. However, the actual mechanism
able interest due to its unique biological activity, such as of inhibition activity of chitosan and its derivatives is not
[1-7] [8-9]
antimicrobial activity , antitumor activity , and im- yet fully understood.
mune enhancing effect [10]. Moreover, chitosan has several The most feasible hypothesis is a change in cell perme-
advantages over other types of disinfectants, that is, it ability due to interactions between the positively charged
2009-04-01
(1983 ) E-mail lix_f06@lzu.cn
* (1963 ) E-mail ysh@mail.tsnc.edu.cn

Ê®ÈýÆÚ-»ù´¡-ÈýУÏÂ.p65 148 2010-7-27, 15:35


chitosan and negatively charged microbial cell membranes,
which prevents the transport of essential solutes into the
cell and results in leakage of proteinaceous and other intra-
cellular components, thus killing the bacterial cell [11-13].
Helander et al. [12] reported that the binding of chitosan
to the outer membrane of E. coli formed a vesicular structure,
caused disruption and extensive alteration in the outer mem-
brane surface and resulted in the loss of its barrier property.
Liu et al. [14] showed that the antibacterial activity of chitosan
oligomers (Mw 8000) to E. coli was caused mainly by the
inhibition of the transcription from DNA. Liu et al [11] demon-
strated that chitosan increased the permeability of the outer
membrane and inner membrane, and ultimately disrupted
bacterial cell membranes, with the release of cellular contents.
Tokura et al. [13] demonstrated the stacking of chitosan molecules
over the microbial cell surface, thus blocking the transport
of nutrients. However, little evidence has been provided to
demonstrate the relationship between antibacterial activity
of chitosan and surface characteristics of Gram-negative
bacterial cell wall.
This paper reports the antibacterial activity of chitosan
with different molecular weight against P. aeruginosa,
P. mirabilis and E. coli, and effects of hydrophilicity and
negative charge on the bacterial cell surface on antibacterial
activity of chitosan. Besides, the permeability of the outer
membrane of E. coli was dealt with by determining the
changes in the fluorescence intensity of cells treated with
the fluorescent probe of NPN. FT-IR analysis was described
for the chitosan-treated strains of E. coli, which can provide
a feasible antibacterial mechanism of chitosan for advanced
studies and even for wider application in the pharmaceutical
and food industries. So far, however, few related investiga-
tions have been reported.
1.1 Materials, strains and reagents
chitosan, from a shrimp shell with relative molecular
weights of 3 kD (yellow, water-soluble), 50 kD (yellow, acid-
soluble), and 1000 kD (white, acid-soluble), and deacetylation
degree of 95%, were purchased from Yuhuan Ocean Bio-
chemical Co., Ltd (Zhejiang province, China). Strains of
E. coli (ATCC 35218), P. aeruginosa (ATCC 27853) and
P. mirabilis (ATCC 12453) were provided by the Chinese
medicine hospital of Tianshui, Gansu province, China. Fresh
inoculants cultured in nutrient agar at 37 for 24 h were
used for the experimental assessment. Stock solution of
Ê®ÈýÆÚ-»ù´¡-ÈýУÏÂ.p65 149
2010, Vol. 31, No. 13 149
0.5 g/100mL chitosan (m/V) was prepared in 1% (V/V) acetic
acid. All other chemicals were of analytical grade and were
used without further purification.
1.2 Assessment of antibacterial activity
The antibacterial activity of chitosan was evaluated by
measuring OD610nm, as described by Jeon[15]. Bacteria were
grown in a nutrient agar culture medium (peptone 10 g, beef
extract 3 g, NaCl 3 g, distilled water 1000 mL, pH 7.4) and
incubated overnight at 37 . Then, a culture where bacteria
grew in a logarithmic growth phase (18 h) was prepared for
an antibacterial test. A representative colony was picked off
with a wire loop and placed in sterile physiological saline.
The final cell suspension was adjusted to an absorbance at
610 nm (OD610nm) of 0.5. One milliliter of cultured bacterial
suspension was added to the mixture which consisted of 4
mL of 0.5 g/100mL chitosan solution and 15 mL of nutrient
broth medium and autoclaved at 121 for 15 min. Then, the
plates were incubated with shaking at 37 for 24 h. The anti-
bacterial activity was estimated periodically by measuring the
turbidity of the culture medium at 610 nm with UV-vis spectro-
photometer (Rayleigh, UV-9200, Beijing, China). In the control
group, acetic acid at a concentration of 1% (V/V) was in place
of chitosan. Each batch experiment was carried out in
triplicate, and results were reported as an average of three
replicates.
1.3 Hydrophilicity analysis of cell surface
Cell suspension was harvested from the one-day culti-
vation broth and added to different volumes of cyclohexane.
Then, they were mixed for 5 min and balanced for 15 min [16].
The absorption of the water phase was measured at 610 nm
by UV-vis spectrophotometer (Rayleigh, UV-9200, Beijing,
China). The hydrophilicity was expressed as determined value
divided by control value. Each batch experiment was carried
out in triplicate.
1.4 Negative charge analysis of cell surface
Anion exchange resin was washed five times with
deionized water and balanced with 0.1 mol/L hydrochloric
acid in a burette for 6 h. Six milliliters of cell suspension
(OD610nm= 0.35) were sequentially added to the burette and
mixed completely. After poured from the burette, the cell sus-
pension was measured for the absorption at 610 nm with UV-
vis spectrophotometer. The relative adsorption ratio was
expressed as the difference of determined value and control
value divided by control value [16]. Each batch experiment was
carried out in triplicate.
1.5 Outer membrane permeabilization assays
2010-7-27, 15:35
 150 2010, Vol. 31, No. 13

The outer membrane permeabilization activity of chitosan
was determined by the NPN assay as described by Liu et al. [11]
E. coli cultures were harvested, washed and resuspended in
0.5 g/100mL NaCl solution. The final cell suspension was
adjusted to OD610nm of 0.6. One milliliter of chitosan solution
or distilled water was mixed with 25 L of 1 mmol/L NPN. The
fluorescence intensity, with an excitation wavelength of 350
against P. mirabilis. In the presence of 3 kD chitosan
oli g o m e r s , h i g h e r i n h i b i t i o n w a s o b s e r v e d a g a inst
P. aeruginosa and P. mirabilis than against E. coli. However,
high molecular weight chitosan (1000 kD) inhibited more
effectively P. mirabilis than other two bacterial species.
0.40

nm and an emission wavelength of 420 nm and both of slit 0.35

widths of excitation and emission raster of 5 nm was recorded

OD610nm
0.30 Control
with a RF-5301PC fluorescence spectrophotometer, from im- 3kD
50kD
mediately after the addition of 1 mL of a cell suspension until 0.25 1000kD
there was no further increase in the emission intensity. All
experiments were duplicated. 0 2 4 6 8 10
Time/h
1.6 FT-IR spectroscopic analysis
IR spectra (4000 400 cm-1) of E. coli, chitosan (50 kD)
and E. coli/ chitosan complex were taken on KBr pellets with
a Spectrum One FT-IR 360 spectrophotometer(Perkin Elmere)
under dry air at room temperature. 0.45
0.40
E. coli was washed with 1% (V/V) hydrochloric acid 0.35
OD610nm

0.30
solution, and centrifuged at 10000 r/min and 0 for 5 min,
0.25 Control
washed twice with deionized water and placed into a vacuum 0.20 3kD
0.15 50kD
oven at 40 for 3 h drying. 0.10 1000kD
Exactly 1.0 mL of E. coli suspension was mixed with 10 0 2 4 6 8 10
mL of 0.5 g/100mL chitosan (50 kD) solution. The mixture Time/h
was incubated with shaking at 37 for 1 h and filtrated, and
the filtration residues were then washed two times and placed
into a vacuum oven at 40 for 3 h drying.
1.7 Statistical analysis
0.75
All experiments were carried out in triplicate, and results 0.70
were reported as an average value of three replicates. Mean 0.65
OD610nm

0.60 Control
separation and significance were analyzed using the SPSS 0.55 3kD
0.50 50kD
(Statistical Package for Social Sciences, SPSS, Chicago, IL) 0.45 1000kD
software package. 0.40
0.35
0 2 4 6 8 10
Time/h

2.1 Antibacterial activity of chitosan

The antibacterial activities of chitosan against
P. aeruginosa, P. mirabilis and E. coli are shown in Fig. 1
respectively. As can be seen, the viable population of the
chitosan treated microorganisms was much less than that of
the control (non-chitosan treated), which indicated that
chitosan could markedly inhibit bacterial growth. However,
the molecular weight of chitosan and the species of bacteria
made a big difference to the antibacterial activity. Chitosan
with a molecular weight of 50 kD exhibited higher antibacte-
rial activity against P. aeruginosa and E. coli than
3,
Higher antibacterial activity of chitosan compared with
chitosan oligomers has been reported by several workers.
Cho et al. [17] reported that the antibacterial activity of chitosan
against E. coli increased with decreasing viscosity from 1000
to 10 cP. In the present study, the growth of E. coli and
P. aeruginosa also was inhibited more effectively by chitosan
of 50 kD than by chitosan of 1000 kD, except for P. mirabilis.
T h e m o l e c u l a r w e i g h t d e p e n d e n c e o f a n t i bacte-

Ê®ÈýÆÚ-»ù´¡-ÈýУÏÂ.p65 150 2010-7-27, 15:35

 2010, Vol. 31, No. 13 151

rial activity of chitosan oligomers has been reported by cyclohexane is shown in Fig. 5, which reveals that the
numerous investigators. Jeon et al [15]
reported that the Mw hydrophilicity of the bacterial cell was increased gradually
(10 1 kD) of chitosan oligomers was critical for microorgan- with the increasing time.
ism inhibition and the efficacy increased with increasing Mw. To investigate the relationship between antibacterial
[18]
Sekiguchi et al. investigated antibacterial activities of activity of chitosan and hydrophilicity of bacterial cell surface,
chitosan oligomers (Mw ranging from 2350 to 21600 D) for the antibacterial activity of chitosan in two-phase mixture
various bacteria. The growth of B. cereus on agar plates was with various ratios of cyclohexane to water were studied.
suppressed by 0.2% 0.3% chitosan oligomer with Mw 11 The results prove that bacterial hydrophilicity is proportional
[19]
kD. Uchida et al. reported that chitosan oligomer II (a to the antibacterial efficiency of chitosan, which can account
mixture of triose and tetraose) failed to display antibacte- for the effect of bacterial species on antibacterial activity of
rial activity against E. coli at a concentration of 0.5 chitosan. The exponential relationship between hydrophilic-
while chitosan oligomer I (a mixture of tetraose and heptaose) ity and antibacterial activity towards E. coli is shown in Fig. 6.
possessed antibacterial activity. In the present study,
50

Antibaoterial efficiency/%
chitosan oligomers of 3 kD showed relatively higher antimi- 45
crobial activity against E. coli than chitosan of 1000 kD. 40
35
Therefore, chitosan reveals a promising prospect as a 30
25
potential antibacterial agent. 20
15
2.2 Hydrophilicity analysis of cell surface 10
5
The hydrophilicity analysis of cell surface shown in Fig. 0
50 60 70 80 90 100 110 120
4 indicated the residual amounts of P. aeruginosa, P.
Hydrophilicity/%
mirabilis and E. coli in water phase were gradually de-
creased with the increasing volume of cyclohexane. The hydro-
philicity was decreased in the following order: P. aeruginos
P. mirabilis E. coli. The effect of culture time on hydrophi- 2.3 Negative charge analysis of cell surface
licity of E. coli in biphasic partition with different volumes of A key feature of chitosan is its positive charge of the
amino group at C2 below its pKa (pH 6.3). This creates a
Pseadomonas aeruginosa polycationic structure, which can be expected to interact
100 Bacillus mirabtlis
with the predominantly anionic components
Hydrophilicity/%

90 Eschenchia coli
80 (lipopolysaccharides, proteins) of the Gram-negative surface.
70
So, interactions between positively charged chitosan mol-
60
50
ecules and negatively charged residues on the cell surface
40 are considered to play an important role in the antibacterial
0 1 2 3 4 [20]
activity of chitosan . It has also been suggested that the
Cyclohexane/mL
difference in cell surface electro-negativity, which changes
as the growth phase changes, might lead to the difference in
the susceptibility of microbial cells toward chitosan [21]. Thus,
the different surface charge on cells of P. aeruginosa,
120 0.5h P. mirabilis and E. coli may result in the variation in the
110 4h
Hydrophilicity/%

100 8h susceptibility of test.

90 12h
Relative adsorption ratio (RAR) is commonly used to
80
70 represent the adsorption amount of cells in the suspension
60 when mixed with anion exchange resin. Higher RAR repre-
50
sents higher negatively charged density distributed over
0 1 2 3 4
the cell surface. RAR is defined as follows:
Cyclohexane/mL
RAR (%) = (1 cell density in the suspension with
anion exchange resin treatment /cell density in the suspen-

Ê®ÈýÆÚ-»ù´¡-ÈýУÏÂ.p65 151 2010-7-27, 15:36

 152 2010, Vol. 31, No. 13

sion without anion exchange resin treatment)
The negative charge analysis of cell surface showed
that the RARs were 89.5% for P. aeruginos, 58.5% for
P. mirabilis and 50.1% for E. coli, respectively. Obviously,
P. aeruginos had the highest negatively charged density on
its cell surface.
Moreover, the effect of RAR on antibacterial activity
of chitosan was studied, and the results indicated that the
higher bacterial RAR, the stronger antibacterial activity of
chitosan.
2.4 Permeability analysis of bacterial outer membrane
The addition of chitosan to E. coli suspensions in the
presence of 1-N-phenylnaphthylamine (NPN) caused a time-
dependent increase in fluorescence intensity (Fig. 7). As can
be clearly seen from Fig. 7, the fluorescence intensity in-
creased to a maximum value within 10 min at each addition
level of chitosan. In addition, higher concentration of
chitosan corresponded to higher fluorescence intensity at
the same time. These results suggeste that chitosan rapidly
increase the permeability of bacterial outer membrane.
100. interaction with E. coli.
Characteristic bands at 1665.30 cm-1 and 1541.07 cm-1
assigned to amide and bands, respectively are shown
in the FT-IR spectrum of E. coli, which also were shifted to
1657.18 cm-1 and 1535.64 cm-1 in E. coli-chitosan complex. The
N C stretching and N H stretching vibrations can
be characterized by the broad peak in the region of 1236.33 cm-1,
and might be the contribution of P O stretching vibra-
tion or C O stretching vibrations of carboxyl. At 1665.30
cm -1 is assigned to the absorption peak of C O; the
phosphoryl group asymmetric and symmetric bands, which
appear around 1240 1220cm-1 and 1085 cm-1, respectively [23].
In this study, the absorption bands at 1080.02 cm-1 and 1236.33
cm-1 were aroused by the PO2 symmetric stretching vibrations
of RNA, DNA or carbohydrates existing in cell wall, or stretch-
ing vibrations of C O, which disappeared in E.coli-
chitosan complex and were shifted to 1070.9 and 1232.43 cm-1,
respectively. These results indicate the involvement of both
C O and P O in E. coli in the interaction with
chitosan.
CTS

1385.43 893.39

1665.30
180 2905.39

1598.60

1152.71
Fluorescence intensity

3447.97

1082.96
150
120
E.coli+CTS
90 Control
0.25 g/100mL 1 3 8 5 . 2 41 4 0 0 . 2 3
1453.36
2925.82

60 3283.31 1232.43 559.93

1070.90
1535.64
1657.18

0.125 g/100mL 1236.33

30
0.025 g/100mL E.coli
1453.54
1541.07

0 524.56
1665.30

1080.02
3581.52
0 2 4 6 8 10 3266.08 2924.12

Time/min 4000 3200 2400 1800 1400 1000 600

Wave number/cm-1

2.5 FT-IR spectroscopic analysis
The above results indicate that chitosan with a molecular
weight of 50 kD possesses the best antibacterial activity
among three different molecular weight chitosans.
Consequently, it was selected to investigate the action mode
of chitosan towards E. coli.
Chitosan of 50 kD, E. coli and their complex were
subjected to structural characterization by FT-IR
spectroscopy, and the spectra are shown in Fig. 8. It was
found that the FT-IR spectrum of chitosan gave a distinct
-1
amide I band at 1665.30 cm and an intense
band at 1598.60 cm-1 [22], which disappeared in E. coli-chitosan
complex and shifted to 1535.64 cm-1. These results demon-
strate that NH3+ groups in chitosan are involved in the
Our study demonstrated excellent antibacterial activity
of chitosan against three species of Gram-negative bacteria,
which exhibited a difference because of the molecular weight
of chitosan and the bacterial species. The antibacterial ac-
tivity of chitosan was closely related to the surface charac-
teristics of cell wall and revealed an increase with increasing
hydrophilicity or negative charge of cell surface. Chitosan
increased the permeability of outer membrane and ultimately
disrupted bacterial cell membranes. FT-IR analysis proved
the interactions between NH3+ in chitosan and C O
NH2 stretching or P O groups in E. coli.
References:
[1] LI Yanyong, CHEN Xiguang, LIU Chengsheng, et al. Effect of the

Ê®ÈýÆÚ-»ù´¡-ÈýУÏÂ.p65 152 2010-7-27, 15:36


molecular mass and degree of substitution of oleoylchitosan on the
structure, rheological properties, and formation of nanoparticles[J]. Jour-
nal of Agricultural and Food Chemistry, 2007, 55: 4842-4847.
[2] RABEA E I, BADAWY M E T, STEVENS C V, et al. Chitosan as
antimicrobial agent: applications and mode of action[J].
Biomacromolecules, 2003, 4(6):1457-1465.
[3] LI Yan, CHEN Xiguang, LIU Nan, et al. Physicochemical characteriza-
tion and antibacterial property of chitosan acetates[J]. Carbohydrate
Polymers, 2007, 67: 227-232.
[4] LI Bin, WANG Xiao, CHEN Ruoxia, et al. Antibacterial activity of
chitosan solution against Xanthomonas pathogenic bacteria isolated from
Euphorbia pulcherrima[J]. Carbohydrate Polymers, 2008, 72: 287-292.
[5] KONG Ming, CHEN Xinguang, LIU Chengsheng, et al. Antibacterial
mechanism of chitosan microspheres in a solid dispersing system against
E. coli[J]. Colloids and Surfaces B: Biointerfaces, 2008, 65: 197-202.
[6] CHUNG Y C, CHEN C Y. Antibacterial characteristics and activity of
acid-soluble chitosan[J]. Bioresource Technology, 2008, 99: 2806-2814.
[7] SEYFARTH F, SCHLIEMANN S, ELSNER P, et al. Antifungal effect
of high- and low-molecular-weight chitosan hydrochloride, carboxym-
ethyl chitosan, chitosan oligosaccharide and N-acetyl-d-glucosamine
against Candida albicans, Candida krusei and Candida glabrata[J].
International Journal of Pharmaceutics, 2008, 353: 139-148.
[8] MITRA S, GAUR U, GHOSH P C, et al. Tumour targeted delivery of
encapsulated dextran-doxorubicin conjugate using chitosan nanoparticles
as carrier[J]. Journal of Controlled Release, 2001,74: 317-323.
[9] QIN Caiqin, ZHOU Bo, ZENG Lintao, et al. The physicochemical
properties and antitumor activity of cellulase-treated chitosan[J]. Food
Chemistry, 2004, 84: 107-115.
[10] JEON Y J, KIM S K. Potential immuno-stimulating effect of antitu-
moral fraction of chitosan oligosaccharides[J]. Journal of Chitin and
Chitosan, 2001(6): 163-167.
[11] LIU Hui, DU Yumin, WANG Xiaohui, et al. Chitosan kills bacteria
through cell membrane damage[J]. International Journal of Food
Microbiology, 2004, 95: 147-155.
[12] HELANDER IM, NURMIAHO-LASSILA E L, AHVENAINEN R, et al.
Chitosan disrupts the barrier properties of the outer membrane of Gram-
Ê®ÈýÆÚ-»ù´¡-ÈýУÏÂ.p65 153
2010, Vol. 31, No. 13 153
negative bacteria[J]. International Journal of Food Microbiology, 2001,
71: 235-244.
[13] TOKURA S K, UENO S, MIYAZAKI S, et al. Molecular weight
dependent antimicrobial activity of chitosan[J]. Macromol Symp, 1997,
120: 1-9.
[14] LIU Xiaofei, GUAN Yunlin, YANG Dongzhi, et al. Antibacterial action
of chitosan and carboxymethylated chitosan[J]. Journal of Applied Poly-
mer Science, 2001, 79: 1324-1335.
[15] JEON Y J, PARK P J, KIM S K. Antimicrobial effect of
chitooligosaccharides produced by bioreactor[J]. Carbohydrate Polymers,
2001, 44: 71-76.
[16] CHUNG Y C, SU Y P, CHEN C C, et al. Relationship between
antibacterial activity of chitosan and surface characteristics of cell wall[J].
Acta Pharmacol Sin Jul, 2004, 25 (7): 932-936.
[17] CHO Y I, NO H K, MEYERS S P. Physicochemical characteristics and
functional properties of various commercial chitin and chitosan
produchitosan[J]. J Agric Food Chem, 1998, 46: 3839-3843.
[18] SEKIGUCHI S, MIURA Y, KANEKO H, et al. Molecular weight
dependency of antimicrobial activity by chitosan oligomers[M]//
NISHINARI K. Food hydrocolloids: structures, properties, and functions.
New York: Kluwer Academic Pub, 1994: 71-76.
[19] UCHIDA Y, IZUME M, OHTAKARA A. Preparation of chitosan oligo-
mers with purified chitosanase and its application[M]// SKJAK-BRAEK
G, ANTHONSEN T, SANDFORD P, et al. Chitin and chitosan: sources,
chemistry, biochemistry, physical properties and applications. London:
Elsevier, 1989: 373-382.
[20] SUDARSHAN N R, HOOVER D G, KNORR D. Antibacterial action of
chitosan[J]. Food Biotechnol, 1992(6): 257-272.
[21] TSAI G J, SU W H. Antibacterial activity of shrimp chitosan against
Escherichia coli[J]. J Food Prot, 1999, 62: 239-243.
[22] WILLIAMS D H, FLEMING I. Spectroscopic methods in organic
chemistry[M]. New York: Mc Graw-Hill, 1980: 35-73.
[23] GOMEZ-FERNANDEZ J C, VILLALAIN J. The use of FT-IR for
quantitative studies of the apparent pKa of lipid carboxyl groups and the
dehydration degree of the phosphate group of phospholipids[J]. Chemis-
try and Physichitosan of Lipids, 1998, 96: 41-52.
2010-7-27, 15:36