Carbohydrate Polymers 86 (2011) 969 974

Contents lists available at ScienceDirect

Carbohydrate Polymers

jo u rn al hom epa ge: www.elsev ier .com

Effect o andPseudo reu

Yan Tao

College of Food

a r t i c l

Article history:Received 6 ApReceived in reAccepted 25 MAvailable onlin

Keywords:ChitosanAntibacterial aAntibacterial mMembrane pe

deacreus wteria ase (A

indieria city of

thatEM) oorks

n Co

1. Introduction

Chitosans antimicrobial activity has been well documented.Muzzarelli et al. (1990) examined N-carboxybutyl chitosansantimicrobiparticularlytested as dN-carboxybcocci were kagainst mostericidal actenterococciis likely thstatic, and eof chitosanthesized chamino-grou(q-CPCs) wcoccus aureGram-negaof about 0.these optimthe both baet al. (2011

CorresponE-mail add

quaternized chitosan lm against S. aureus and E. coli weresuperior to that of the virgin chitosan lm. The other literaturementioned that chitosan displays antibacterial activities, with min-imum inhibitory concentrations (MICs) reported to range from

1

0144-8617/$ doi:10.1016/j.al activity against 298 pathogens, nding that it was active against candidae and Gram-positive bacteriaetermined by the agar dilution. When a thin pad ofutyl chitosan was used, all candidae and most staphylo-illed, whereas percentages of the bactericidal activitiest Gram-negative species were lower. However, no bac-ivity was observed with Gram-positive streptococci and, and three Gram-negative Salmonella strains. Thus, itat chitosan might be bactericidal or simply bacterio-ach strain may be vulnerable/indifferent to the presence. Recently, Wu et al. (2011) reported that they syn-itosan-poly(-caprolactone) (CPC) copolymers via anp-protection method. The optimized quaternized CPCsere able to completely prevent growth of Staphylo-us and Escherichia coli, as model Gram-positive andtive bacteria, respectively, at different concentrations2% and 0.25%, respectively. At lower concentrations,al q-CPCs had higher antibacterial activities against

cteria as compared to chitosan. Simultaneously, Vallapa) reported that the antibacterial activity of the surface-

ding author. Tel.: +86 21 61900384; fax: +86 21 61900365.ress: ytao@shou.edu.cn (Y. Tao).

100 to 10,000 mg L against Gram-negative bacteria (Helander,Nurmiaho-Lassila, Ahvenainen, Rhoades, & Roller, 2001), and from100 to 1250 mg L1 against Gram-positive bacteria (Bae, Jun, Lee,Paik, & Kim, 2006; Jeon, Park, & Kim, 2001; No, Park, Lee, & Meyers,2002; Vishu Kumar, Varadaraj, Lalitha, & Tharanathan, 2004).

It has been demonstrated that chitosans antibacterial activitydepends on the strain examined and on its growth phase, besideschitosan concentration, molecular weight, degree of deacetyla-tion, and pH, temperature, and composition of the medium (Chen& Chou, 2005; Chung, Wang, Chen, & Li, 2003; Liu et al., 2006;Muzzarelli et al., 1990; No et al., 2002). Because it has beentested under widely varied conditions, it is hard to compare chi-tosans antibacterial effect among results obtained by differentresearchers.

Several antimicrobial mechanisms have been proposed for chi-tosan and its soluble derivatives. In the rst, the cationic chitosansinteract with anionic moieties at the cell surface thereby alteringcell permeability and resulting in material being prevented fromentering the cell and/or material being leaked from the cell (Chen,Liau, & Tsai, 1998; Jung, Kim, Choi, Lee, & Kim, 1999). The secondmechanism involves the binding of chitosan with DNA to inhibitRNA synthesis, which occurs through chitosan penetration towardthe nuclei of the microorganisms and interference with the syn-thesis of mRNA and proteins (Rabea, Badawy, Stevens, Smagghe,

see front matter. Crown Copyright 2011 Published by Elsevier Ltd. All rights reserved.carbpol.2011.05.054f chitosan on membrane permeability monas aeruginosa and Staphyloccocus au

, Li-Hong Qian, Jing Xie Science and Technology, Shanghai Ocean University, Shanghai 201306, China

e i n f o

ril 2011vised form 19 May 2011ay 2011e 6 June 2011

ctivityechanism

rmeability

a b s t r a c t

The mechanism of action of over 85%and Gram-positive Staphylococcus autosan, the electric conductivity of bacaverage release for alkaline phosphatPAGE and agarose gel electrophoresisdisappeared in chitosan-treated bactfunction via increasing the permeabilthe cell membrane of S. aureus than onTransmission electron microscopy (Tthan P. aeruginosa to chitosan. Our wregarded as a natural bactericide.

Crow/ locate /carbpol

cell morphology ofs

etylated chitosan on Gram-negative Pseudomonas aeruginosaas investigated. Results showed that after treated with chi-

suspensions increased, followed by increasing of the units ofLP) and glucose-6-phosphate dehydrogenase (G6PDH). SDS-

cated that the soluble proteins and intact DNAs decreased orells, demonstrating that chitosan performed its antibacterial

cell membranes. Moreover, chitosan had a stronger effect on of P. aeruginosa due to the differences in their cell structures.bservations further veried that S. aureus is more sensitive

provide additional evidences in support of chitosan being

pyright 2011 Published by Elsevier Ltd. All rights reserved.

970 Y. Tao et al. / Carbohydrate Polymers 86 (2011) 969 974

& Steurbaut, 2003; Wang, Du, & Liu, 2004). In another proposedmechanism, chitosan also acts as a chelating agent that selectivelybinds trace transition metals and thereby inhibits the produc-tion of toxins and microbial growth (Cuero, Osuji, & Washington,1991). Varioto investigaKumar, VarHowever, uantibacteriaria in the fapsychrotropas the mostfood (Jay, domonas aeinfections. antibiotics, tides and chAbrini, ZhirGaqn, & BSakharkar, 2for the mod

The objinteraction addition, Grto compare

2. Materia

2.1. Materi

Chitosanlation > 85%(Qingdao, aureus (ATC(ATCC1911cereus (ATCCollection (methyleneband glutarapany (St. Loavailable co

2.2. Determand minimu

MICs welowing the inocula of ttures to coof 50 L weand adjustefold dilutioshaking at tration of chexaminatiowith no groMBC was decolony was

2.3. Electric

After incwere harvelets were w7.4). The nmixed with

chitosan concentrations of 150 and 300 mg L1, and then the mix-tures were incubated at room temperature and measured for theirelectric conductivity every 10 min for 2 h. The experiment withoutchitosan was used as blank control.

kalinogen

chit5% (CFU mat 37awn

actiy anonta(pH 8

deny watrophDH ay an

desce. G6

mL)e-6-pThe sity

y wa-equ

ner m

er m relee cuoto, t br

11,0n. Th

at 4g L1

mMas d

usint des

mM

S-PA

arithCFU mn (6tratiliquous an

g fcord4% st

wit/v) o

DNAove pd subus techniques have been applied to S. aureus and E. coli,te and compare chitosans antibacterial modes (Vishuadaraj, Gowda, & Tharanathan, 2005; Xing et al., 2009).p to the present, there are few reports on chitosansl activity and antibacterial mechanism against bacte-

mily Pseudomonadaceae. These bacteria are consideredhs, growing well at 015 C, thereby being regarded

important spoilage bacteria originating in refrigerated2000; Vela, 1997). Pseudomonads, especially Pseu-ruginosa, can also cause outbreaks of intramammarySo far, although the activity and interactions of someinnate defense molecules such as antimicrobial pep-emicals against P. aeruginosa were reported (Bouhdid,

i, Espuny, & Manresa, 2009; Bellemare, Vernoux, Morin,ourbonnais, 2010; Jayaraman, Sakharkar, Lim, Tanq, &010; Uccelletti et al., 2010), there is no direct evidencee of action of chitosan on P. aeruginosa.ectives of the present study were to elucidate thebetween chitosan and Gram-negative P. aeruginosa. Inam-positive S. aureus was also selected as a tested strain

the modes of action of chitosan on the both strains.

ls and methods

als and microorganisms

(source: crab shell, high MW, degree of deacety-) was purchased from Li Zhong Chitin CompanyChina). Strains of P. aeruginosa (ATCC27853), S.C27853), E. coli (ATCC25922), Listeria monocytogenes

5), Salmonella enteritidis (ATCC13076) and BacillusC14579) were obtained from the American Type CultureATCC). Sodium dodecyl sulfate (SDS), acrylamide, N, N-isacrylamide, o-nitrophenyl--d-galactoside (ONPG)ldehyde were purchased from Sigma Chemicals Com-uis, Mo). All other reagents were of the highest grademmercially.

ination of minimum inhibitory concentrations (MICs)m bactericidal concentrations (MBCs)

re determined by microtiter broth dilution method, fol-guidelines in the literature (Andrews, 2001). In brief,hose strains were prepared by adjusting overnight cul-ntaining 2 105 CFU mL1 in nutrient broth. Aliquotsre mixed with chitosan (dissolved in 0.5% acetic acid,d to pH 6.0 with 0.1 M NaOH) of 50 L of serial two-ns in a 96-well plate, and the plate was incubated with37 C for 18 h. MIC was dened as the lowest concen-itosan where no growth was observed by microscopic

n. On the other hand, 10 L mixtures from the wellswth were spread on agar plates for MBC determination.ned as the lowest concentration of chitosan where no

observed on agar plates after 48 h incubation at 37 C.

conductivity assay

ubated at 37 C for overnight, S. aureus and P. aeruginosasted by centrifugation at 11,000 g for 10 min. The pel-ashed and resuspended in 0.1 M phosphate buffer (pHal cell suspensions were adjusted to 108 CFU mL1 and

chitosan (600 mg L1 in 0.5% acetic acid, pH 6.0) to nal

2.4. Aldehydr

Theto a 0.8at 107

bated withdr

ALPMalam1 mL) cbuffer opticalactivitof p-ni

G6PMalamsion asmixturume: 1glucos(TPN). cal denactivitof TPN

2.5. In

Inning theinto thSugimnutriention atsolutioof 1.2(600 mand 30time w420 nmaliquoand 30

2.6. SD

Log(107chitosaconcenture. AS. aure11,000ysis acwith a stained25% (v

Forthe ab8 h, ane phosphatase (ALP) and glucose-6-phosphatease (G6PDH) assay

osan (600 mg L1 in 0.5% acetic acid, pH 6.0) was addedw/w) NaCl solution containing S. aureus or P. aeruginosa

L1 to a nal concentration of 300 mg L1. After incu-C for 8 h, a 0.2-mL aliquot of the cell suspension was

and added to the reaction mixture.vity was determined using the method described byd Horecker (1964). The reaction mixture (total volume:ined 0.1 mg p-nitrophenylphosphate in 0.5 M TrisHCl). The reaction was followed at 28 C by measuring the

sity of the suspension at 420 nm. A unit of released ALPs dened as the amount of enzyme that produced 1 Menol-equivalent in 1 min at 28 C.ctivity was determined using the method described byd Horecker (1964). A 0.2-mL aliquot of the cell suspen-ribed in the above paragraph was added to the reactionPDH activity was determined in a solution (total vol-

containing 0.05 M Tris-HCl (pH 8), 0.01 M CaCl2, 1.0 Mhosphate and 0.4 M triphosphopyridine nucleotidereaction was followed at 28 C by measuring the opti-of the suspension at 340 nm. A unit of released G6PDHs dened as the amount of enzyme that reduced 1 Mivalent in 1 min at 28 C.

embrane (IM) permeabilization assay

embrane permeabilization was determined by measur-ase of cytoplasmic -galactosidase activity from E. colilture medium using ONPG as the substrate (Ibrahim,& Aoki, 2000). Logarithmic-phase bacteria grown inoth containing 2% lactose was harvested by centrifuga-00 g for 10 min, washed and resuspended in 0.5% NaCle nal cell suspension was adjusted to an absorbance20 nm. A 1.6-mL aliquot was mixed with chitosanor 300 mg L1 in 0.5% acetic acid, pH 6.0) of 1.6 mL

ONPG of 150 L. The production of o-nitrophenol overetermined by monitoring the increase in absorbance atg a spectrophotometer. The control contained a 1.6-mL

cribed as above and 0.5% acetic acid (pH 6.0) of 1.6 mL ONPG of 150 L.

GE and DNA fragmentation

mic phase cells of S. aureus and P. aeruginosaL1) in nutrient broth were mixed, respectively with

00 mg L1 in 0.5% acetic acid, pH 6.0) to a nal chitosanon of 300 mg L1, and then incubated at room tempera-ts of 5 mL were withdrawn every 1 h for 3 h and 6 h ford P. aeruginosa, respectively, and then centrifuged ator 10 min. The pellet was subjected to SDS-PAGE anal-ing to Laemmli (1970). The SDS-PAGE was performedacking gel and a 10% separating gel. Proteins bands wereh 0.1% Coomassie Brilliant Blue R-250 and destained inf ethanol and 9% (v/v) of acetic acid.

fragmentation analysis, aliquots of 5 mL as described inaragraph were withdrawn after incubated for 1 h andjected to centrifugation at 11,000 g for 10 min. DNAs

Y. Tao et al. / Carbohydrate Polymers 86 (2011) 969 974 971

Table 1Minimum inhibitory concentration (MIC) and minimum bactericidal concentration(MBC) of chitosan against the six types of strain.

Bacteria

S. aureus E. coliL. monocytogS. enteritidis B. cereus P. aeruginasa

were extraChina) and

2.7. Transm

TEM analiterature wOvernight cand the pellphate buffesuspension300 mg L1)11,000 g series of treto perform

2.8. Statisti

All expeues with stabetween thdenoted the

3. Results

3.1. Minimubactericidalof strain

The antassessed agbacteria byin the casewas 3 mg m0.38 mg mLteria, MIC P. aeruginosagainst six tChitosan wpH becauseOur resultsacetic acid (inhibited thnegative ba3 mg mL1. to 6.0) was for chitosangrowth. Madifferent raconditions iis difcult tresults obta

0

1

2

3

4

5

6

7

8

9

cond

uctiv

ity c

hang

e pe

rcen

tage

(%) 150mg L300mg L

150mg L300mg L

S. aureus

P. aeruginasa

lectricsa.

ectricus an

deteureun. Aepentivitease, andity inr P.

L1

otheity beect oeater

fect oaerug

effeus ane relreus

P. aeetic d (des rey annzymns ofLP was the rst molecule to be released into the medium,ed by G6PDH.

S-PAGE patterns of cellular soluble proteins fromn-treated S. aureus and P. aeruginosa

hown in Fig. 2, the normal cell protein electrophoresis bandsMIC (mg mL1) MBC (mg mL1)

0.38 0.750.75 0.75

enes 3 >33 3003 >3

3 >3

cted from the pellets using Kit (Tiangen Co., Beijing,were run on a 1% agarose gel.

ission electron microscopy (TEM)

lysis was performed following the guidelines in theith slight modication (Li, Wu, Wang, & Sun, 2002).ultures of S. aureus and P. aeruginosa were centrifugedets were washed and suspended in 0.1 M sodium phos-r (pH 7.4) to an absorbance of 0.4 at 600 nm. The cells were incubated with chitosan (nal concentration

at 37 C for 20 min and 2 h, and then centrifuged atfor 10 min. The resulting pellets were subjected to aatments according to the guidelines in the literatureTEM analysis.

c analysis

riments were carried out in triplicate, and average val-ndard deviation were revealed. Signicant differencese 2 groups were examined using t-test. A P value < 0.05

presence of a statistically signicant difference.

and discussion

m inhibitory concentration (MIC) and minimum concentration (MBC) of chitosan against the six types

imicrobial activity of chitosan was quantitativelyainst three Gram-positive and three Gram-negative

determining the MIC and MBC. As shown in Table 1, of Gram-positive bacteria, MIC value of chitosanL1 against both L. monocytogenes and B. cereus and

1 against S. aureus. In the case of Gram-negative bac-value was 3 mg mL1 against both S. enteritidis anda and 0.75 mg mL1 against E. coli. MBCs of chitosanypes of strain were 0.75 mg mL1, 3 mg mL1, or higher.as thought to have antibacterial activities only at acidic

of its poor solubility at pH > 6.5 (Rabea et al., 2003). using over 85% deacetylated chitosan, soluble in 0.5%pH 6.0), indicated that chitosan at acidic pH signicantlye growth of three Gram-positive and three Gram-cteria in 18 h at concentrations ranged from 0.38 to

Fig. 1. Eaerugino

3.2. ElS. aure

Wefor S. achitosatime-dconducto incr10 minductiv6.6% fo300 mgOn theductivthe effwas gr

3.3. Efand P.

TheS. aureaveragof S. ausion of0.5% acreleaseenzymMalamlular elocatiowhy Afollow

3.4. SDchitosa

As s

Inhibition with 5000 ppm acetic acid (pH was adjustednegligible. Thus, using acetic acid at low concentration

did not have any additional adverse effect on bacterialny literatures reported the IMC values of chitosan withnges. Because the bacterial strains and the experimentaln this study are different from those in the literature, ito directly compare chitosans antibacterial effect withined by other researchers.

of S. aureu

Table 2Effect of chito

S. aureus P. aeruginasa0 20 40 60 80 100 120incubation time (min)

conductivity of cell suspensions for chitosan-treated S. aureus and P.

conductivity of cell suspensions for chitosan-treatedd P. aeruginosa

rmined electric conductivity of the cell suspensionss and P. aeruginosa treated with 150 and 300 mg L1

s shown in Fig. 1, the electric conductivity showed adent increasing manner. It was found that the electricy of suspensions for S. aureus and P. aeruginosa began

after treated with 150 and 300 mg L1 chitosan for when the incubation time was 2 h, the electric con-creased by 6.1% and 8.3% for S. aureus, and 5.1% andaeruginosa compared with the controls, showing thatchitosan was more effective than 150 mg L1 chitosan.r hand, differences in the changes of the electric con-tween S. aureus and P. aeruginosa also suggested thatf chitosan on the membrane permeability of S. aureus

than on that of P. aeruginosa.

f chitosan on the leakage of enzymes from S. aureusinosa

cts of chitosan on the leakage of ALP and G6PDH fromd P. aeruginosa were shown in Table 2. The units ofease for both ALP and G6PDH in the cell suspension

were higher compared with those in the cell suspen-ruginosa. The control, which consisted of bacteria in aacid solution, showed zero units of the both enzymesata not shown). We also observed that the release ofached a plateau in 2 h for ALP and in 5 h for G6PDH.d Horecker (1964) reported that ALP was an extracel-e, while G6PDH was found in the cell membrane. The

these two enzymes clearly provided the answer as tos and P. aeruginosa appeared strong and clear. After

san on the leakage of enzymes from S. aureus and P. aeruginosa.

Alkaline phosphatase (Units) G-6-P dehydrogenase (Units)

128.4 13.6 185.7 17.498.2 11.7 147.8 14.3

972 Y. Tao et al. / Carbohydrate Polymers 86 (2011) 969 974

n-treated S. aureus (A) and P. aeruginosa (B).

treated withweight prodisappearincontents oftion time indistinct. Onnatant increnot shown)content of ing cell mempermeabilitaeruginosa. ited proteinof protein b

3.5. Agarosaureus and

The relecation of maeruginosa analyzed byDNA degraprominent chitosan fowas observ1 h. These sthat chitosaP. aeruginos

3.6. Releasewith chitosa

The innphatidyl gly-galactosimembrane it is knowninner memgalactosidamedium. Adifferent cowas followgalactosidaafter treate

garose gel electrophoresis of DNAs extracted from chitosan-treated S.A) and P. aeruginosa (B).

0.12 con trolFig. 2. SDS-PAGE patterns of cellular soluble proteins from chitosa

300 mg L1 chitosan for 13 h, bands of all-molecular-teins for S. aureus appeared obviously shallow, eveng altogether. In the case of P. aeruginosa, the protein

chitosan-treated cells reduced, and as the incuba-creased (after 3 h), these characteristics became more

the other hand, protein contents in the cell-free super-ased compared with control culture supernatants (data. These results suggested that chitosan decreased thecellular soluble proteins by permeating and disrupt-branes. However, the effect of chitosan on membrane

y of S. aureus was more obvious than on that of P.Cui et al. (2009) proposed that chitosan probably inhib-

synthesis or control gene expression. The mechanismreakdown remains unclear.

e gel electrophoresis of DNAs from chitosan-treated S.P. aeruginosa

ase of the intracellular component, DNA, is also an indi-embrane damage. DNAs isolated from S. aureus and P.treated with 300 mg L1 chitosan for 1 h and 8 h were

agarose gel electrophoresis. As shown in Fig. 3, specicdative smearing typical of necrotic degeneration wasin P. aeruginosa cells, especially for cells treated withr 8 h. In contrast, the occurrence of DNA disappearinged with chitosan-treated S. aureus cells even at time ofhifts occurring on S. aureus and P. aeruginosa promptedn seems to be more active against S. aureus than againsta.

Fig. 3. Aaureus ( of cytoplasmic -galactosidase from E. coli treatedn

er layer of Gram-negative bacteria consists of phos-cerol and cardiolipin (Je & Kim, 2006). The cytoplasmicdase is released as a consequence of change in innerpermeability. In this study, we used model E. coli since

that the ability of chitosan to permeate the E. colibrane can be veried as a function of cytoplasmic -se release, with bacteria grown in lactose-containings shown in Fig. 4, when cells were treated with twoncentrations of chitosan, a lag time of about 10 mined by a progressive release of the cytoplasmic -se for up to 90 min to reach a steady state. However,d for 50 min, the effect of 300 mg L1 chitosan on

0 20 40 60 80 10 0 12 0

0.00

0.02

0.04

0.06

0.08

0.10

Abs

orba

nce

at 4

20 n

m

incubation ti me (min)

150 mg L 300 mg L

Fig. 4. Release of cytoplasmic -galactosidase from E. coli treated with chitosan.

Y. Tao et al. / Carbohydrate Polymers 86 (2011) 969 974 973

Fig. 5. Transm0 min (A), 20 m

the membrchitosan. Thgalactosidawhich was and Sun (2consider thmeability o

3.7. Transmaeruginosa t

The effeP. aeruginosshowed thawith compaand notablission electron micrographs of S. aureus treated with chitosan forin (B) and 2 h (C).

ane permeability was greater than that of 150 mg L1

ese results indicated that the release of cytoplasmic -se caused by chitosan was time- and dose-dependant,coincident with the results reported by Liu, Du, Wang,004) and Xing et al. (2009). Thus, it is reasonable toat chitosan can also increase the inner membrane per-f the Gram-negative P. aeruginosa.

ission electron micrographs (TEM) of S. aureus and P.reated with chitosan

cts of chitosan on the cell morphology of S. aureus anda were investigated by TEM. The electron micrographst normal cells were surrounded by the cell membranesct surface, without release of intracellular componentse ruptures on the cell surfaces (Figs. 5A and 6A). As

Fig. 6. Transm0 min (A), 20 m

compared tbacterial ce300 mg L1

suspensionwhich couldtents, wereaeruginosa,of treatmencells complthat the adaeruginosa

The abointeract moGram-negareported byission electron micrographs of P. aeruginosa treated with chitosan forin (B) and 2 h (C).

o the untreated controls, remarkable modications ofll membranes were found after a period of exposure tochitosan. As shown in Figs. 5B and 6B, when bacterials were exposed to chitosan for 20 min, some ruptures,

cause some slight leakages of cellular cytoplasmic con- observed on the cell membranes of S. aureus and P.

but the morphous of cells still were regular. Two hourst later, the cytoplasmic membranes of chitosan-treatedetely collapsed (Figs. 5C and 6C). However, it was notedhering of chitosan to S. aureus was greater than to P.in amount.ve results effectively demonstrated that chitosan couldre strongly with Gram-positive S. aureus than with

tive P. aeruginosa. Similar observations have also been other researchers for chitosan-treated E. coli and S.

974 Y. Tao et al. / Carbohydrate Polymers 86 (2011) 969 974

aureus. Such results are probably attributed to the differences intheir membrane structures. All Gram-negative bacteria possess anouter membrane (OM) due to the presence of lipopolysaccharide(LPS) molecule, and outer membrane plays a role as a drug bar-rier (Nikaido, 1996). But the peptidoglycan layer of the cell wallof Gram-positive S. aureus is composed of networks with plentyof pores, so foreign molecules can enter the cell without difculty(Xing et al., 2009). Jeon et al. (2001) reported that 89% deacetylatedchitosan had more effective activity against pathogens than againstnon-pathogens expect in the case of lactic acid bacteria. Here ourdata supported this conjecture.

4. Conclusions

The results reported here demonstrated that chitosans antibac-terial activities against S. aureus and P. aeruginosa are due to theinteractionsincreasing ring on theinclusions (and P. aeruga stronger of P. aerugimembranesaeruginosa provide addas a natural

Acknowled

This woShanghai Sogy CommiLeading Acacation Com

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Effect of chitosan on membrane permeability and cell morphology of Pseudomonas aeruginosa and Staphyloccocus aureus1 Introduction2 Materials and methods2.1 Materials and microorganisms2.2 Determination of minimum inhibitory concentrations (MICs) and minimum bactericidal concentrations (MBCs)2.3 Electric conductivity assay2.4 Alkaline phosphatase (ALP) and glucose-6-phosphate dehydrogenase (G6PDH) assay2.5 Inner membrane (IM) permeabilization assay2.6 SDS-PAGE and DNA fragmentation2.7 Transmission electron microscopy (TEM)2.8 Statistic analysis

3 Results and discussion3.1 Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of chitosan against the six types ...3.2 Electric conductivity of cell suspensions for chitosan-treated S. aureus and P. aeruginosa3.3 Effect of chitosan on the leakage of enzymes from S. aureus and P. aeruginosa3.4 SDS-PAGE patterns of cellular soluble proteins from chitosan-treated S. aureus and P. aeruginosa3.5 Agarose gel electrophoresis of DNAs from chitosan-treated S. aureus and P. aeruginosa3.6 Release of cytoplasmic -galactosidase from E. coli treated with chitosan3.7 Transmission electron micrographs (TEM) of S. aureus and P. aeruginosa treated with chitosan

4 ConclusionsAcknowledgementsReferences