Quaternized chitosan inhibits icaA transcription and biofilm formation by Staphylococcus on a titanium surface.
ABSTRACT Our previous study (Z. X. Peng et al., Carbohydr. Polym. 81:275-283, 2010) demonstrated that water-soluble quaternary ammonium salts, which are produced by the reaction of chitosan with glycidyl trimethylammonium chloride, provide chitosan derivatives with enhanced antibacterial ability. Because biofilm formation is believed to comprise the key step in the development of orthopedic implant-related infections, we further evaluated the efficacy of hydroxypropyltrimethyl ammonium chloride chitosan (HACC) with different degrees of substitution (DS; referred to as HACC 6%, 18%, and 44%) in preventing biofilm formation on a titanium surface. We used a tissue culture plate method to quantify the biomass of Staphylococcus epidermidis and Staphylococcus aureus biofilms and found that HACC, especially HACC 18% and 44%, significantly inhibited biofilm formation compared to the untreated control, even at concentrations far below their MICs (P < 0.05). Scanning electron microscopy showed that inhibition of biofilm formation on titanium increased dramatically with increased DS and HACC concentrations. Confocal laser scanning microscopy indicated that growth of a preexisting biofilm on titanium was inhibited by concentrations of HACC 18% and 44% below their minimum biofilm eradication concentrations. We also demonstrated that HACC inhibited the expression of icaA, which mediates the production of extracellular polysaccharides, both in new biofilms and in preexisting biofilms on titanium. Our results indicate that HACC may serve as a new antibacterial agent to inhibit biofilm formation and prevent orthopedic implant-related infections.
- [show abstract] [hide abstract]
ABSTRACT: Over the last 15 years, with the advent of modern standards in the control of sterility within the operating room environment and adequate protocols of peri-operative antibiotic prophylaxis, the incidence of infections associated to orthopedic implants has become very low. Nevertheless, the event of infection still represents one of the most serious and devastating complications which may involve prosthetic devices. It leads to complex revision procedures and, often, to the failure of the implant and the need for its complete removal. In orthopedics, for the enormous number of surgical procedures involving invasive implant materials, even if nowadays rare, infections have a huge impact in terms of morbidity, mortality, and medical costs. The difficult battle to prevent and fight bacterial infections associated to prosthetic materials must be played on different grounds. A winning strategy requires a clear view of the pathogenesis and the epidemiology of implant-related infections, with a special attention on the alarming phenomenon of antibiotic resistance. In this regard staphylococci are the prevalent and most important causative pathogens involved in orthopedic implant-related infections, and, thus, the main enemy to defeat. In this paper, we offer an overview of the complexity of this battleground and of the current and new, in our opinion most promising, strategies in the field of biomaterials to reduce the risks and counteract the establishment of implant infections.Biomaterials 05/2006; 27(11):2331-9. · 7.60 Impact Factor
- [show abstract] [hide abstract]
ABSTRACT: Surfaces coated with the naturally-occurring polysaccharide chitosan (partially deacetylated poly N-acetyl glucosamine) resisted biofilm formation by bacteria and yeast. Reductions in biofilm viable cell numbers ranging from 95% to 99.9997% were demonstrated for Staphylococcus epidermidis, Staphylococcus aureus, Klebsiella pneumoniae, Pseudomonas aeruginosa and Candida albicans on chitosan-coated surfaces over a 54-h experiment in comparison to controls. For instance, chitosan-coated surfaces reduced S. epidermidis surface-associated growth more than 5.5 (10)log units (99.9997%) compared to a control surface. As a comparison, coatings containing a combination of the antibiotics minocycline and rifampin reduced S. epidermidis growth by 3.9 (10)log units (99.99%) and coatings containing the antiseptic chlorhexidine did not significantly reduce S. epidermidis surface associated growth as compared to controls. The chitosan effects were confirmed with microscopy. Using time-lapse fluorescence microscopy and fluorescent-dye-loaded S. epidermidis, the permeabilization of these cells was observed as they alighted on chitosan-coated surfaces. This suggests chitosan disrupts cell membranes as microbes settle on the surface. Chitosan offers a flexible, biocompatible platform for designing coatings to protect surfaces from infection.Journal of Biomaterials Science Polymer Edition 01/2008; 19(8):1035-46. · 1.70 Impact Factor
- [show abstract] [hide abstract]
ABSTRACT: Antimicrobial peptides are found in both myeloid cells and mucosal tissues of many vertebrates and invertebrates. These peptides are predicted to operate as a first-line host defense mechanism exerting broad-spectrum activity against pathogenic bacteria, fungi, parasites, and enveloped viruses. We report the characterization of a novel 25-residue linear antimicrobial peptide found in the skin mucous secretions of the winter flounder (Pleuronectes americanus). This peptide was purified through multiple chromatographic methods to obtain a single peak by reversed-phase high performance liquid chromatography. This purified peptide, which we named pleurocidin, exhibited antimicrobial activity against Escherichia coli in a bacterial cell lysis plate assay. Mass spectrometry and amino acid sequence analysis indicated that it is 25 amino acids in length. Pleurocidin is predicted to assume an amphipathic alpha-helical conformation similar to many other linear antimicrobial peptides. There is a high degree of homology between pleurocidin and two antimicrobial peptides, ceratotoxin from the Mediterranean fruit fly and dermaseptin from the skin of a hylid frog. The minimal inhibitory concentration and minimal bactericidal concentration of pleurocidin were determined against 11 different Gram-negative and Gram-positive bacteria. Immunohistochemistry locates pleurocidin in the epithelial mucous cells of flounder skin. Pleurocidin represents a novel antimicrobial peptide found in fish and may play a role in innate host defense.Journal of Biological Chemistry 06/1997; 272(18):12008-13. · 4.65 Impact Factor
ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Feb. 2011, p. 860–866
Copyright © 2011, American Society for Microbiology. All Rights Reserved.
Vol. 55, No. 2
Quaternized Chitosan Inhibits icaA Transcription and Biofilm
Formation by Staphylococcus on a Titanium Surface?
Zhao-Xiang Peng,1† Bing Tu,1† Yang Shen,1Lin Du,1Ling Wang,2
Sheng-Rong Guo,2and Ting-Ting Tang1*
Shanghai Key Laboratory of Orthopedic Implant, Department of Orthopedic Surgery, Shanghai Ninth People’s Hospital,
Shanghai Jiao Tong University School of Medicine, Shanghai 200011, China,1and School of Pharmacy,
Shanghai Jiao Tong University, Shanghai 200240, China2
Received 21 July 2010/Returned for modification 1 September 2010/Accepted 29 November 2010
Our previous study (Z. X. Peng et al., Carbohydr. Polym. 81:275–283, 2010) demonstrated that water-soluble
quaternary ammonium salts, which are produced by the reaction of chitosan with glycidyl trimethylammonium
chloride, provide chitosan derivatives with enhanced antibacterial ability. Because biofilm formation is be-
lieved to comprise the key step in the development of orthopedic implant-related infections, we further
evaluated the efficacy of hydroxypropyltrimethyl ammonium chloride chitosan (HACC) with different degrees
of substitution (DS; referred to as HACC 6%, 18%, and 44%) in preventing biofilm formation on a titanium
surface. We used a tissue culture plate method to quantify the biomass of Staphylococcus epidermidis and
Staphylococcus aureus biofilms and found that HACC, especially HACC 18% and 44%, significantly inhibited
biofilm formation compared to the untreated control, even at concentrations far below their MICs (P < 0.05).
Scanning electron microscopy showed that inhibition of biofilm formation on titanium increased dramatically
with increased DS and HACC concentrations. Confocal laser scanning microscopy indicated that growth of a
preexisting biofilm on titanium was inhibited by concentrations of HACC 18% and 44% below their minimum
biofilm eradication concentrations. We also demonstrated that HACC inhibited the expression of icaA, which
mediates the production of extracellular polysaccharides, both in new biofilms and in preexisting biofilms on
titanium. Our results indicate that HACC may serve as a new antibacterial agent to inhibit biofilm formation
and prevent orthopedic implant-related infections.
The development of infection is one of the most serious and
devastating complications associated with orthopedic implants.
However, significant progress in preventing infections has been
made at many individual treatment centers, and the incidence
of this complication has dropped considerably in the range of
5 to 10% in the past few years as a result of using antibiotic
prophylaxis during the perioperative period (15). Unfortu-
nately, the dependence on antibiotics as a treatment for asso-
ciated complications has emerged as an inevitable conse-
quence, and antibiotic resistance and the prevalence of
antibiotic-resistant strains continue to be problematic.
According to Trampuz et al., orthopedic-implant-associated
infection is primarily caused by bacterial growth in biofilms
(45). A biofilm consists of cells embedded in a self-synthesized
matrix of extracellular substances, which facilitates the adher-
ence of microorganisms and firmly attaches the bacterial clus-
ters to the underlying surface (13, 44). Biofilm formation is
considered to be an important virulence mechanism, because
the biofilm impairs the activity of antibiotics, prevents normal
immune responses, and complicates the eradication of infec-
tions (23, 25). As shown by Monzon et al., vancomycin exhibits
decreased antimicrobial activity as the biofilm progresses from
6 h to 2 days (32). Pathogenic bacteria are capable of persisting
in a biofilm in the presence of antibiotics at levels that are
1,000-fold higher than those necessary to eradicate a plank-
tonic population (7). Furthermore, once an infection has been
established and a well-organized biofilm has formed on the
implant surface, antibiotic therapies are less efficacious, and
removal and substitution of the implant are often the only way
to eradicate the problem (31, 36). Bacterial adherence to or-
thopedic implant surfaces occurs in two essential steps (29). Its
adherence to the implanted surface is followed by an accumu-
lation process and the production of extracellular substances,
such as polysaccharide intercellular adhesin (PIA) (28, 47).
The production of PIA is mediated by the intercellular adhesin
(ica) locus, which comprises four core genes (icaA, icaB, icaC,
and icaD) and a regulatory gene (icaR) (2, 19, 24). Potential
virulence-associated genes, such as icaADBC, aap, altE, bhp,
fbe, embp, and mecA, and phenotypic biofilm formation have
been investigated to identify pathogenic Staphylococcus epider-
midis strains (23, 40).
Our study builds on the observation that water-soluble qua-
ternary ammonium salts formed by the reaction of chitosan
(CS) with glycidyl trimethylammonium chloride (GTMAC)
have been reported as a chitosan derivative with enhanced
antibacterial ability against S. epidermidis (37). Quaternized
chitosan has been described to have significant antibacterial
activity (41). A large proportion of all implant-related infec-
tions are caused by staphylococci (roughly four out of five), and
two single Staphylococcus species, S. aureus and S. epidermidis,
account for two-thirds of infection isolates (5). Due to its
ability to form biofilms on indwelling medical devices, the
* Corresponding author. Mailing address: Shanghai Key Laboratory
of Orthopedic Implant, Department of Orthopedic Surgery, Shanghai
Ninth People’s Hospital, Shanghai Jiao Tong University School of
Medicine, Zhizaoju Road 639, Shanghai 200011, China. Phone: 86-21-
23271133. Fax: 86-21-63137020. E-mail: firstname.lastname@example.org.
† Both authors contributed equally to this work.
?Published ahead of print on 6 December 2010.
opportunistic human pathogen S. epidermidis has become the
most important cause of nosocomial infections in recent years
(14, 47), and its adherence was not significantly different
among implant materials (42). While prior studies have been
performed on quaternized chitosan, these studies have not
specifically tested the effect of chitosan on biofilm formation
on titanium. Titanium-based biomaterials are currently the
best and most widely used materials in the manufacture of
orthopedic and dental implants because of their good biocom-
patibility and mechanical properties (17, 34). The biocompat-
ibility of titanium implants can be attributed to a surface pro-
tein layer formed under physiological conditions that actually
makes the surface suitable for bacterial colonization and bio-
film formation (49). In this study, we address this issue by
evaluating the effect of hydroxypropyltrimethyl ammonium
chloride chitosan (HACC) on biofilm formation by a standard
strain of S. epidermidis, ATCC 35984, and two clinical isolates,
S. aureus 376 and S. epidermidis 389, on a titanium surface.
Biofilm prevention and biofilm susceptibility assays were used
to evaluate the potential of HACC to prevent and treat ortho-
pedic implant-associated infections.
MATERIALS AND METHODS
Materials. HACC with differing degrees of substitution (DS) of quaternary
ammonium (referred to as HACC 6%, 18%, and 44%) was prepared by com-
bining chitosan and glycidyl trimethylammonium (GTMAC) as previously re-
ported (37). Chitosan with a molecular weight of 20.0 ? 104or 3.0 ? 104and
N-deacetylation of 91.83% was purchased from Zhejiang Yuhuan Ocean Bio-
chemistry Co., Ltd. (China). GTMAC was purchased from Shandong Sangong
Chemical Co., Ltd., with a purity of 96%. Other chemicals were of analytical
Preparation of bacteria. S. epidermidis ATCC 35984 was kindly provided by Di
Qu (Laboratory of Medical Molecular Virology, Shanghai Medical College,
Fudan University, Shanghai, China). The clinical isolates S. aureus 376 and S.
epidermidis 389 were kindly provided by Saïd Jabbouri (Universite ´ du Littoral
Co ˆte d’Opale, Boulogne sur mer, France). Both these strains were coagulase-
negative staphylococcal (CoNS) strains (9). These strains were stored at ?80°C
as glycerol stocks. To obtain inocula for examination, we cultured the strains
overnight on BBL Trypticase soy agar (TSA; BD Biosciences, Franklin Lakes,
NJ) medium at 37°C. After two successive transfers of the test organism on TSA
medium at 37°C for 24 h, the activated culture was inoculated into 10 ml BBL
Trypticase soy broth (TSB) supplemented with 0.5% glucose (TSBG) and cul-
tured at 37°C for 12 h. Cells were then harvested by centrifugation (8,000 ? g for
MIC assays. MICs were determined by a microtiter broth dilution method as
described by Cole et al. (11) and modified by Beckloff et al. (3). In brief, 100 ?l
of bacteria at a density of 5 ? 105CFU/ml in Mueller-Hinton broth (BD
Biosciences) was inoculated into the wells of 96-well assay plates (tissue culture-
treated polystyrene; Costar 3595, Corning Inc., Corning, NY) in the presence of
CS (3.0 ? 104), GTMAC, and HACC 6%, 18%, or 44% at different final
concentrations (0, 1, 2, 4, 8, 16, 32, 64, 128, 256, 512, or 1,024 ?g/ml). Because the
water solubility of chitosan with a molecular weight of 20.0 ? 104is very low,
chitosan with a molecular weight of 3 ? 104was used as a control. GTMAC, the
free quaternary ammonium, also served as a control. The inoculated microplates
were incubated at 37°C for 24 h before analysis.
MBEC assay. The minimum biofilm eradication concentration (MBEC) phys-
iology and genetic assay (MBEC BioProducts Inc., Edmonton, Alberta, Canada)
was previously described by Ceri et al. (8). In brief, each of three strain suspen-
sions (200 ?l, 5 ? 105CFU/ml) was inoculated into the wells of an MBEC device.
The peg lids were then inserted into the microplates containing the inocula.
These devices were placed on a gyrorotary shaker at 110 rpm for 12 h in an
incubator at 37°C. The peg lids with biofilm were rinsed twice with 0.9% saline
(by placing the lid in a microplate containing 200 ?l of saline in each well) to
remove loosely adherent planktonic cells. The peg lids with biofilm were then
transferred to 96-well assay plates (tissue culture-treated polystyrene; Costar
3595) containing 200 ?l TSBG in the presence of CS (3.0 ? 104), GTMAC, and
HACC 6%, 18%, or 44% at different final concentrations (0, 1, 2, 4, 8, 16, 32, 64,
128, 256, 512, or 1,024 ?g/ml). These plates were incubated at 37°C for another
12 h. Subsequently, the peg lids were rinsed twice with 0.9% saline, and the biofilms
were disrupted in 200 ?l TSBG in 96-well assay plates using a water table sonicator
on the high setting for a period of 5 min. After sonication, the peg lids were removed
from the plates and the original lids of the original 96-well assay plates were re-
placed. These plates were then incubated at 37°C for 24 h. To determine the MBEC
values, a microtiter plate reader was used to determine the absorbance at 650 nm
(A650). Clear wells (A650? 0.1) were indicative of biofilm eradication.
Biofilm formation assay by the tissue culture plate (TCP) method. The TCP
assay method has been described in detail elsewhere (10, 30). It is the most
widely used biofilm formation assay and is considered the standard test to detect
biofilm formation. In brief, each of three strain suspensions was diluted to 1 ?
106CFU/ml in fresh TSBG. Then 200-?l aliquots of the diluted cultures were
added to 96-well flat-bottom tissue culture plates. Broth alone served as the
control. Wells containing only broth and different concentrations (0, 4, 32, 64,
128, or 256 ?g/ml) of HACC 6%, 18%, or 44% were incubated for 24 h at 37°C.
After incubation, the content of each well was gently removed by tapping of the
plates. The wells were washed twice with 200 ?l of deionized water to remove
free-floating planktonic bacteria. Biofilms in plates were dried at 60°C for 1 h,
and adherent bacteria were stained at room temperature with 200 ?l of a 0.1%
(wt/vol) aqueous solution of crystal violet (CV) for 5 min. The plates were rinsed
twice with deionized water to remove excess stain. After the plates were dried at
?l of 30% (wt/vol) glacial acetic acid for 10 min with shaking at 300 rpm. The
concentration of CV was determined using a Synergy HT multidetection microplate
reader at a wavelength of 492 nm (21). The mean absorbance obtained from the
medium control well was deducted from the test absorbance values.
Biofilm prevention assay on titanium by scanning electron microscopy (SEM).
Titanium plates (1 mm thick and 5 mm in diameter; Sh-puwei, China) were
sterilized, placed into the wells of 24-well microtiter plates, and washed twice
with phosphate-buffered saline (PBS). S. epidermidis ATCC 35984 cells were
resuspended at a density of 1.0 ? 106CFU/ml in fresh TSBG containing different
concentrations (0, 4, 32, 64, 128, or 256 ?g/ml) of HACC 6%, 18%, or 44%.
Aliquots (1 ml) of the cell suspensions were seeded into each well of the plates.
Titanium plates with cells grown in HACC-free medium were utilized as a
control. The titanium plates were incubated at 37°C for 24 h and then gently
washed three times with PBS to remove nonadherent bacteria. The adherent
bacteria were fixed and dehydrated. Briefly, the plates were gently rinsed twice
with 0.01 M PBS and fixed with 2.5% glutaraldehyde for 2 h at 4°C. The surfaces
were washed twice with 0.01 M PBS for 1 h and subsequently fixed with 0.1%
TABLE 1. MICs and minimum biofilm eradication concentrations (MBECs) of CS, GTMAC, and HACC 6%, 18%, or 44% on three strains
MIC (?g/ml)MBEC (?g/ml)
S. aureus 376
S. aureus 376
aMolecular weight, 3.0 ? 104.
VOL. 55, 2011 QUATERNIZED CHITOSAN INHIBITS BIOFILM FORMATION861
osmium tetraoxide for 1 h. The bacteria were then dehydrated by replacing the
buffer with increasing concentrations of ethanol (30%, 50%, 70%, 80%, 90%,
95%, and 100%) for 10 min each. After critical-point drying and coating by gold
sputter, samples were examined using a scanning electron microscope (JEOL
JSM-6360LV, JEOL Ltd., Tokyo, Japan).
Biofilm susceptibility assay on titanium by confocal laser scanning micros-
copy (CLSM). Prior to seeding, titanium plates (1 mm thick and 5 mm in
diameter; Sh-puwei, China) were sterilized and placed into the wells of 24-well
microtiter plates and washed twice with 1? PBS. S. epidermidis ATCC 35984
cells were resuspended at a density of 1.0 ? 106CFU/ml in fresh TSBG, and
aliquots (1 ml) of these cell suspensions were seeded onto titanium plates in wells
of the 24-well microtiter plates. These plates were incubated at 37°C for 12 h.
The titanium plates were gently washed three times with PBS to remove non-
adherent bacteria. The titanium with preexisting biofilms was incubated in 1 ml
fresh TSBG containing different concentrations (0, 4, 32, 64, 128, or 256 ?g/ml)
of HACC 6%, 18%, or 44% in new 24-well microtiter plates. These plates were
incubated at 37°C for another 12 h, nonadherent bacteria were removed by
gently washing the plates three times in PBS, and adherent bacteria were stained
using the LIVE/DEAD BacLight viability kit (Molecular Probes, Eugene, OR)
for 15 min at room temperature in the dark, followed by three PBS washes to
remove nonspecific stain. Fluorescence-adherent bacteria were visualized by
confocal laser scanning microscopy (Leica TCS SP2, Leica Microsystems, Hei-
delberg, Germany). Leica confocal software was used to analyze the biofilm
images. Images were acquired from random locations within the biofilm formed
on the upper side of the titanium plates.
Reverse transcription-PCR (RT-PCR) analysis of icaA transcription. In the
biofilm prevention assay, S. epidermidis strain ATCC 35984 was incubated in
TSBG with different concentrations of HACC 6%, 18%, or 44% (0, 4, 32, 64, 128,
or 256 ?g/ml) on the surface of titanium for 24 h. In the biofilm susceptibility
assay, S. epidermidis strain ATCC 35984 was incubated in TSBG for 12 h fol-
lowed by TSBG with different concentrations of HACC 6%, 18%, or 44% (0, 4,
32, 64, 128, or 256 ?g/ml) on the surface of titanium for another 12 h. The
bacteria were then harvested from the plates by scraping into RNAprotect
bacterial reagent (Qiagen, Germantown, MD) to ensure RNA integrity. The bac-
terial pellets were resuspended in 200 ?l TE buffer (10 mM Tris-HCl, 1 mM
EDTA, pH 7.0) containing 100 ?g/ml lysostaphin (Sigma) and incubated at 37°C
for 10 min. Total RNA was isolated using an RNeasy minikit (Qiagen) according
to the manufacturer’s instructions, with an additional step of treatment with
DNase I (Invitrogen) to eliminate residual genomic DNA. One microgram of
total RNA was reverse transcribed using a first-strand cDNA synthesis kit (MBI).
One microliter of cDNA was amplified by PCR using the following primers (12):
for gyrB transcripts, 5?-TTATGGTGCTGGACAGATACA-3? and 5?-CACCGT
GAAGACCGCCAGATA-3?; for icaA transcripts, 5?-AACAAGTTGAAGGCA
TCTCC-3? and 5?-GATGCTTGTTTGATTCCCT-3?. gyrB was used as an inter-
nal standard. Each reaction was performed in triplicate with RNA isolated on
separate occasions. Aliquots of the amplified products were separated on a 1.5%
agarose gel and visualized with ethidium bromide.
Statistical analysis. Differences between groups were examined for statistical
significance using analysis of variance (ANOVA). P values of ?0.05 were con-
sidered to indicate statistical significance. All experiments were performed in
triplicate and repeated three times.
MIC and MBEC determination. The MICs and MBECs of
the samples are given in Table 1. The MICs of HACC 6%, 18%,
and 44% against S. epidermidis ATCC 35984 were 512 ?g/ml, 64
?g/ml, and 32 ?g/ml, respectively. The respective MICs were 256
?g/ml, 64 ?g/ml, and 32 ?g/ml against S. aureus 376. For S.
epidermidis 389, the respective MICs were 256 ?g/ml, 32 ?g/ml,
and 16 ?g/ml. The MBECs of HACC 44% were 512 ?g/ml
against S. epidermidis ATCC 35984 and S. aureus 376 and 256
?g/ml against S. epidermidis 389. The MBECs of HACC 18%
were 1,024 ?g/ml against S. epidermidis ATCC 35984 and 512
?g/ml against S. epidermidis 389. The MBECs of HACC 18%
against S. aureus 376 and the MBECs of HACC 6% against all
three bacterial species were greater than 1,024 ?g/ml, which is the
highest tested concentration. These MBECs are expected to ex-
ceed 1,024 ?g/ml.
FIG. 1. Effect of HACC 6%, 18%, and 44% on biofilm formation
of three strains, S. epidermidis ATCC 35984 (A), S. aureus 376 (B), and
S. epidermidis 389 (C), as detected by the tissue culture plate method.
TSBG was used as the negative control. The data are representative of
results from three independent experiments and are expressed as the
means ? standard deviations. Biofilm formation of three strains
treated with different concentrations of HACC 6% (*), 18% (#), or
44% ($) was significantly inhibited compared with that of bacteria
without HACC treatment (P ? 0.05).
862PENG ET AL.ANTIMICROB. AGENTS CHEMOTHER.
Biofilm formation assayed by the tissue culture plate
method. The activities of HACC 6%, 18%, and 44% tested at
different concentrations against the total biomass of three
strains of biofilm are shown in Fig. 1. Following treatment with
4 ?g/ml HACC 6%, the A492values of the three tested strains
were not significantly different from that of the control group
(P ? 0.05). Similarly, a statistically significant difference in the
A492value was not observed following treatment of S. aureus
376 with 32 ?g/ml HACC 6% (P ? 0.05). At all other concen-
trations of HACC 6% and every concentration of HACC 18%
and 44%, the A492values of the three tested strains were
significantly decreased (P ? 0.05). These findings indicate that
HACC significantly inhibited biofilm formation compared with
that of samples without HACC treatment (P ? 0.05), even at
concentrations far below the MICs.
Biofilm prevention analysis by scanning electron micros-
copy. SEM was used to further examine the biofilm formed by
S. epidermidis ATCC 35984 on titanium plates following 24 h of
incubation (Fig. 2). After colonization with the strain for 24 h,
the control contained multiple small, spherical bacteria, con-
sistent with staphylococci. The bacteria on the titanium plates
conglomerated in a thick, heterogeneous layer with columnar
clusters, which are characteristic of staphylococci (Fig. 2, 0
?g/ml). On the titanium plates that had been treated with
HACC 6%, 18%, or 44% at concentrations of 256 ?g/ml (Fig.
2, a5), 64 ?g/ml (Fig. 2, b3), or 64 ?g/ml (Fig. 2, c3), respec-
tively, the cells grew only into isolated individual colonies.
When the concentrations were raised further, only a few bac-
terial microcolonies could be seen, and no biofilm formed on
the surface of the titanium plates (Fig. 2, b4, b5, c3, c4, and c5).
Biofilm susceptibility following HACC treatment. A biofilm
susceptibility assay was preformed to assess the susceptibility of a
preexisting (12-h growth) biofilm on a titanium surface to treat-
ment with HACC for 12 h. The results showed that thick green
clusters formed on most of the titanium surfaces (Fig. 3). Red
fluorescence began to appear on the titanium plates that had
been treated with HACC 6% at 256 ?g/ml (Fig. 3, a5), HACC
18% at 64 ?g/ml (Fig. 3, b3), and HACC 44% at 32 ?g/ml (Fig.
3, c2). For all concentrations of HACC 6%, the biofilm had intact
cell membranes. For HACC 18% at 128 ?g/ml (Fig. 3, b4) and
HACC 44% at 64 ?g/ml (Fig. 3, c3), the biofilm had a discontin-
uous area and more red fluorescence. Overall, biofilm suscepti-
bility was DS and concentration dependent.
Transcription of icaA. Fig. 4 shows the results of RT-PCR
analysis of icaA expression. IcaA has been reported to a play a
significant role in biofilm formation by S. epidermidis (2, 19,
24). The gyrB gene, which is constitutively expressed in Staph-
ylococcus, was used as an internal standard in these tests (20).
Because little biofilm was formed on the titanium plates
treated with HACC 18% and 44% at 128 and 256 ?g/ml, the
amount of RNA was insufficient to permit RT-PCR detection
of icaA and gyrB gene expression in these samples. Similarly,
the preexisting biofilms treated with 256 ?g/ml HACC 18%
and 44% did not yield sufficient RNA to permit RT-PCR
analysis. No significant changes in gyrB expression were ob-
served in any of the biofilms. The expression of icaA decreased
with increasing HACC concentrations in both new and preex-
isting biofilms treated with HACC 6%, 18%, or 44%.
As a natural antibacterial biopolymer, chitosan has been
investigated for its antimicrobial activity in the prevention of
orthopedic implant infection. Chitosan has been reported to
FIG. 2. Scanning electron micrographs of biofilms formed by S. epidermidis strain ATCC 35984 incubated on a titanium surface for 24 h with
HACC 6% (a), 18% (b), or 44% (c) at the following concentrations: 0 ?g/ml, 4 ?g/ml (1), 32 ?g/ml (2), 64 ?g/ml (3), 128 ?g/ml (4), or 256 ?g/ml
(5). Magnification, ?3,000. Scale bars, 5 ?m.
VOL. 55, 2011QUATERNIZED CHITOSAN INHIBITS BIOFILM FORMATION863
reduce the infection rate of experimentally induced S. aureus
osteomyelitis in rabbits (16) and offers a flexible, biocompat-
ible platform for the design of coatings to protect surfaces from
infection (6). In this study, we found that the MICs against S.
epidermidis ATCC 35984, S. aureus 376, and S. epidermidis 389
decreased with an increasing DS of HACC, which is in accor-
dance with previous results (38, 41). The MBECs of HACC
against the three strains were significantly higher than the
respective MICs, which suggests that a higher concentration is
needed to eradicate a preexisting biofilm on an orthopedic
implant. For CS and GTAMC, the MICs and MBECs against
the three strains exceeded the highest tested concentration
FIG. 3. Confocal laser scanning microscopy (CLSM) analysis of bacterial viability in a biofilm on a titanium surface. S. epidermidis strain ATCC
35984 was incubated in TSBG for 12 h, followed by incubation for another 12 h in TSBG supplemented with HACC 6% (a), 18% (b), or 44% (c)
at the following concentrations: 0 ?g/ml, 4 ?g/ml (1), 32 ?g/ml (2), 64 ?g/ml (3), 128 ?g/ml (4), or 256 ?g/ml (5). Bacteria were stained with green
fluorescent SYTO 9 and red fluorescent propidium iodide, resulting in live cells appearing green and dead cells appearing red under CLSM.
FIG. 4. Reverse-transcription PCR analysis of icaA transcription. (A) Expression of icaA, relative to gyrB, in S. epidermidis strain ATCC 35984
incubated on a titanium surface for 24 h in TSBG supplemented with different concentrations (0, 4, 32, 64, 128, or 256 ?g/ml) of HACC 6%, 18%
or 44%. (B) Expression of icaA, relative to gyrB, in a biofilm formed by S. epidermidis strain ATCC 35984. Bacteria were incubated in TSBG for
12 h and then grown on a titanium surface for another 12 h in TSBG supplemented with different concentrations (0, 4, 32, 64, 128, or 256 ?g/ml)
of HACC 6%, 18%, or 44%. Expression of gyrB was used as an internal control.
864 PENG ET AL.ANTIMICROB. AGENTS CHEMOTHER.