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.

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). Unfortunately, the dependence on antibiotics as a treatment for associated complications has emerged as an inevitable consequence, 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 adherence of microorganisms and firmly attaches the bacterial clusters 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 infections (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 planktonic 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 orthopedic implant surfaces occurs in two essential steps (29). Its adherence to the implanted surface is followed by an accumulation 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 epidermidis strains (23, 40).

Our study builds on the observation that water-soluble quaternary 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 infections 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 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 biocompatibility and mechanical properties (17, 34). The biocompatibility of titanium implants can be attributed to a surface protein layer formed under physiological conditions that actually makes the surface suitable for bacterial colonization and biofilm 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 orthopedic 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 combining chitosan and glycidyl trimethylammonium (GTMAC) as previously reported (37). Chitosan with a molecular weight of 20.0 × 10⁴ or 3.0 × 10⁴ and N-deacetylation of 91.83% was purchased from Zhejiang Yuhuan Ocean Biochemistry Co., Ltd. (China). GTMAC was purchased from Shandong Sangong Chemical Co., Ltd., with a purity of 96%. Other chemicals were of analytical grade.

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 (Université du Littoral Côte 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 cultured at 37°C for 12 h. Cells were then harvested by centrifugation (8,000 × g for 10 min).

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 μl of bacteria at a density of 5 × 10⁵ CFU/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 μg/ml). Because the water solubility of chitosan with a molecular weight of 20.0 × 10⁴ is very low, chitosan with a molecular weight of 3 × 10⁴ was 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) physiology and genetic assay (MBEC BioProducts Inc., Edmonton, Alberta, Canada) was previously described by Ceri et al. (8). In brief, each of three strain suspensions (200 μl, 5 × 10⁵ CFU/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 μl 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 μl 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 μg/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 μl 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 replaced. 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 × 10⁶ CFU/ml in fresh TSBG. Then 200-μl 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 μg/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 μl 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 μl 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 37°C for 2 h, biofilm formation was quantified by solubilization of the CV stain in 200 μl 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 × 10⁶ CFU/ml in fresh TSBG containing different concentrations (0, 4, 32, 64, 128, or 256 μg/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% 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 microscopy (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 × 10⁶ CFU/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 nonadherent bacteria. The titanium with preexisting biofilms was incubated in 1 ml fresh TSBG containing different concentrations (0, 4, 32, 64, 128, or 256 μg/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, Heidelberg, 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 μg/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 followed by TSBG with different concentrations of HACC 6%, 18%, or 44% (0, 4, 32, 64, 128, or 256 μg/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 bacterial pellets were resuspended in 200 μl TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 7.0) containing 100 μg/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′-CACCGTGAAGACCGCCAGATA-3′; for icaA transcripts, 5′-AACAAGTTGAAGGCATCTCC-3′ and 5′-GATGCTTGTTTGATTCCCT-3′. gyrB was used as an internal 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 considered to indicate statistical significance. All experiments were performed in triplicate and repeated three times.

RESULTS

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 μg/ml, 64 μg/ml, and 32 μg/ml, respectively. The respective MICs were 256 μg/ml, 64 μg/ml, and 32 μg/ml against S. aureus 376. For S. epidermidis 389, the respective MICs were 256 μg/ml, 32 μg/ml, and 16 μg/ml. The MBECs of HACC 44% were 512 μg/ml against S. epidermidis ATCC 35984 and S. aureus 376 and 256 μg/ml against S. epidermidis 389. The MBECs of HACC 18% were 1,024 μg/ml against S. epidermidis ATCC 35984 and 512 μg/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 μg/ml, which is the highest tested concentration. These MBECs are expected to exceed 1,024 μg/ml.

TABLE 1.

MICs and minimum biofilm eradication concentrations (MBECs) of CS, GTMAC, and HACC 6%, 18%, or 44% on three strains

Compound	MIC (μg/ml)			MBEC (μg/ml)
	S. epidermidis ATCC 35984	S. aureus 376	S. epidermidis 389	S. epidermidis ATCC 35984	S. aureus 376	S. epidermidis 389
HACC
6%	512	256	256	>1,024	>1,024	>1,024
18%	64	64	32	1024	>1,024	512
44%	32	32	16	512	512	256
CSa	>1,024	>1,024	>1,024	>1,024	>1,024	>1,024
GTMAC	>1,024	>1,024	>1,024	>1,024	>1,024	>1,024

^aMolecular weight, 3.0 × 10⁴.

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 μg/ml HACC 6%, the A₄₉₂ values 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 A₄₉₂ value was not observed following treatment of S. aureus 376 with 32 μg/ml HACC 6% (P > 0.05). At all other concentrations of HACC 6% and every concentration of HACC 18% and 44%, the A₄₉₂ values 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.

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).

Biofilm prevention analysis by scanning electron microscopy.

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, consistent 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 μg/ml). On the titanium plates that had been treated with HACC 6%, 18%, or 44% at concentrations of 256 μg/ml (Fig. 2, a5), 64 μg/ml (Fig. 2, b3), or 64 μg/ml (Fig. 2, c3), respectively, the cells grew only into isolated individual colonies. When the concentrations were raised further, only a few bacterial microcolonies could be seen, and no biofilm formed on the surface of the titanium plates (Fig. 2, b4, b5, c3, c4, and c5).

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 μg/ml, 4 μg/ml (1), 32 μg/ml (2), 64 μg/ml (3), 128 μg/ml (4), or 256 μg/ml (5). Magnification, ×3,000. Scale bars, 5 μm.

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 treatment 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 μg/ml (Fig. 3, a5), HACC 18% at 64 μg/ml (Fig. 3, b3), and HACC 44% at 32 μg/ml (Fig. 3, c2). For all concentrations of HACC 6%, the biofilm had intact cell membranes. For HACC 18% at 128 μg/ml (Fig. 3, b4) and HACC 44% at 64 μg/ml (Fig. 3, c3), the biofilm had a discontinuous area and more red fluorescence. Overall, biofilm susceptibility was DS and concentration dependent.

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 μg/ml, 4 μg/ml (1), 32 μg/ml (2), 64 μg/ml (3), 128 μg/ml (4), or 256 μg/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. Magnification, ×400.

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 Staphylococcus, 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 μg/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 μg/ml HACC 18% and 44% did not yield sufficient RNA to permit RT-PCR analysis. No significant changes in gyrB expression were observed in any of the biofilms. The expression of icaA decreased with increasing HACC concentrations in both new and preexisting biofilms treated with HACC 6%, 18%, or 44%.

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 μg/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 μg/ml) of HACC 6%, 18%, or 44%. Expression of gyrB was used as an internal control.

DISCUSSION

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 reduce the infection rate of experimentally induced S. aureus osteomyelitis in rabbits (16) and offers a flexible, biocompatible 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 accordance 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 (1,024 μg/ml). Based on the known MICs, we chose HACC concentrations of 0 μg/ml, 4 μg/ml, 32 μg/ml, 64 μg/ml, 128 μg/ml, and 256 μg/ml as the test concentrations to evaluate biofilm formation. The TCP method has been found to be the most sensitive and accurate method for detecting biofilm formation by staphylococci and has the advantage of being a quantitative model to study the adherence of staphylococci on biomedical devices (30). Our TCP results indicate that HACC significantly inhibited biofilm formation by the three strains compared with that of samples without HACC treatment (P < 0.05), even at concentrations far below the MICs.

We utilized S. epidermidis ATCC 35984, a standard strain for biofilm formation experiments, both to evaluate the ability of HACC to prevent biofilm development and to test the susceptibility of a preexisting biofilm to HACC on a titanium surface. In the biofilm prevention test, the SEM findings indicated that biofilm formation was significantly prevented by treatment with an increased DS and concentration of HACC. In the biofilm susceptibility test, CLSM revealed that biofilm formation and viability were inhibited only at high concentrations of HACC 18% or HACC 44%. These results indicate that precoating of orthopedic implants with HACC has the potential to block bacterial colonization and biofilm formation. Chitosan has been investigated as a potential coating material for the prevention of orthopedic infections (4, 16, 22). Although it has an improved ability to inhibit biofilm formation, coating of metal implants with HACC is challenging because of its high water solubility. To address this problem, the stable HACC implant surface should be covalently linked, as suggested by Antoci et al. (1).

A more feasible application of HACC might be its integration into bone cement (46). Incorporation of chitosan in bone cement has the potential to stimulate bone formation (18, 39, 48) and prevent bacterial colonization and biofilm formation on the bone cement surface (43). With its improved antimicrobial activity, HACC represents an attractive option for the impregnation of bone cement to provide a promising new strategy for combating orthopedic implant infections.

Biofilm formation on the surface of implants is influenced by multiple factors (27). Olson et al. reported that PIA appears to play a critical role in the adherence of S. epidermidis to biomaterials (35). PIA, encoded by the icaADBC locus, is a major matrix component of S. epidermidis biofilms (26, 33). We assessed icaA transcription as an index of biofilm formation on a titanium surface by RT-PCR. Our results showed that HACC can inhibit icaA expression, and the level of inhibition increased with higher HACC concentrations in the biofilm prevention and susceptibility assays. This effect was more significant for HACC 18% and 44%, which blocked the transcription of icaA at concentrations of 128 μg/ml and 256 μg/ml in the biofilm prevention assay and at 256 μg/ml in the biofilm susceptibility assay. The results suggest that the change in icaA transcription is more sensitive than the MBEC and CLSM assay to HACC. We postulate that this inhibition of icaA transcription results in reduced biofilm formation and increased susceptibility to HACC. Because the biofilm protects and supports the growth of bacteria on the surface of implants, inhibition of biofilm formation further impairs bacterial viability.

Overall, our results reveal that HACC 18% and 44% significantly prevent biofilm formation and bacterial survival on titanium surfaces and reduce bacterial viability in preexisting biofilms. Combined with our previous results (37), which indicate that HACC 44% is cytotoxic and that HACC 18% is both noncytotoxic to L-929 cells and biocompatible with osteogenic cells, we believe that HACC 18% represents a new antibacterial agent for the prevention and treatment of orthopedic implant-related infections.