- Email: [email protected]
Evaluation and insights into chitosan antimicrobial activity against anaerobic oral pathogens
Anaerobe 18 (2012) 305e309
Contents lists available at SciVerse ScienceDirect
Anaerobe journal homepage: www.elsevier.com/locate/anaerobe
Clinical microbiology
Evaluation and insights into chitosan antimicrobial activity against anaerobic oral pathogens E.M. Costa a, S. Silva a, C. Pina b, F.K. Tavaria a, M.M. Pintado a, * a b
CBQF, Escola Superior de Biotecnologia, Universidade Católica Portuguesa, Rua Dr. António Bernardino de Almeida, P-4200-072 Porto, Portugal Faculty of Health Sciences, Universidade Fernando Pessoa, Rua Carlos da Maia, 296, 4200-150 Porto, Portugal
a r t i c l e i n f o
a b s t r a c t
Article history: Received 18 January 2012 Received in revised form 21 March 2012 Accepted 19 April 2012 Available online 27 April 2012
The objective of this study was to assess the antimicrobial capability of non-chemically altered chitosan as an alternative to traditional antimicrobials used in the treatment of oral infections. The action mechanism of chitosan was also ascertained. High and low molecular weight chitosan showed antimicrobial activity at low concentrations for all tested bacteria with the MICs varying between 1 and 7 mg/ ml with a drop of efficacy relatively to the action of LMW chitosan. In addition chitosan showed also to be an effective bactericidal presenting bactericidal effect within 8 h at the latest. Additionally the evaluation of chitosan’s action mechanism showed that both MWs acted upon the bacterial cell wall and were not capable of interacting with the intracellular substances, as showed by the inefficacy obtained in the flocculation assay. Ó 2012 Elsevier Ltd. All rights reserved.
Keywords: Anaerobic pathogens Antimicrobial Chitosan Action mechanism
1. Introduction Chitosan is a high molecular weight (HMW), linear, polycationic heteropolysaccharide derivate from chitin, consisting of N-acetyl-2amino-2-deoxy-ᴅ-glucopyranose and 2-amino-2-deoxy-ᴅ-glucopyranose linked together by a b e (1/4) glycosidic bond. Chitosan molecules may vary widely originating samples of different degree of deacetylation (DD) (between 75 and 95%), molecular weights (MW) (between 50 and 2000 kDa), viscosity and pKa [1]. The high percentage of amino groups (6.89%), which provide chelating capability, in conjunction with antitumoral activity, immunoadjuvant activity, acceleration of wound healing, antimicrobial activity, biodegradability and biocompatibility make chitosan a high sought biomaterial [1e4]. Chitosan, however, possesses some limitations. It has limited use due to its insolubility in water, since it is only soluble in some organic acids e such as acetic and formic acids, possesses high viscosity and tendency to coagulate proteins at high pH [3,5]. The antimicrobial activity of chitosan was observed against a wide variety of microorganisms including fungi, algae and bacteria, being more active against Gram positive than Gram negative bacteria [1], in particular if it possess high MW. The
* Corresponding author. Tel. þ351 22 558 0000. E-mail addresses: [email protected], (M.M. Pintado).
[email protected]
1075-9964/$ e see front matter Ó 2012 Elsevier Ltd. All rights reserved. doi:10.1016/j.anaerobe.2012.04.009
reported minimum inhibitory concentrations (MICs) of chitosan vary widelydfrom 0.005% to 1.5% (w/v) for Staphylococcus aureus [6] or from 0.025% to 1.0% (w/v) for Escherichia coli [7], and its antibacterial effects seem to be closely related to MW and degree of deacetylation [8]. However few to none results have been published regarding the action of chitosan against anaerobic bacteria, and the few existing reported contradictory results or used chemically altered chitosan [9e12]. The oral cavity is a complex system composed by soft and hard tissue requiring adherence for the survival of microorganisms [13]. However, the occurrence of plaque formation will allow the formation of an anaerobic atmosphere that will potentiate the development of anaerobic bacteria, most of which are pathogenic. This will trigger an inflammatory response by the immune system that will result in tissue damage [13]. Plaque formation also contributed for a protective environment for bacterial growth, namely from the action of saliva and accumulation of acid produced by bacteria, which will lead to teeth lesions [14]. The routine usage of antibiotics in the treatment of oral diseases is not advisable, as such method of treatment must be only used in serious or relapsing cases, and no antibiotic will be capable of replacing tooth debridement and proper control of supragingival plaque formation [15,16]. However, systemic administration has great disadvantages e inability to control the quantity of antibiotic that reaches the active site, and consequently not guaranteeing an effective treatment [15], the incomplete elimination of bacteria in the oral cavity and finally
306
E.M. Costa et al. / Anaerobe 18 (2012) 305e309
all undesirable side effects due to interactions with another medicines [16]. On the other hand, topic administration requires specialized knowledge and personnel in their application and the time required to apply the techniques [16]. In view of the above data, this research effort was aimed at evaluating the antimicrobial activity of chitosan against oral anaerobic bacteria, in order to access the possibility of using chitosan as an alternative to local conventional antimicrobials, as well as the influence of metabolic differences between aerobic and anaerobic bacteria on chitosan’s action mechanism.
2.4. Time-inhibition curves
2. Material and methods
The antimicrobial activity at MBC for LMW and HMW chitosan were tested as previously described by Fernandes et al. [18] with minor modifications. Inocula were adjusted to 0.5 in the Macfarland scale (1 108 CFU/ml). Control tests were prepared with sterile deionised water instead of chitosan. Viable counts of different concentrations of chitosan solutions were determined at 0, 2, 4, 6, 8, 12, 24, 48 and 72 h by the Miles, Misra and Irwin [19] technique (1938) in WCA. Plates were incubated under anaerobic conditions, at 37 C for 24 h. Inhibition curves were constructed by plotting log CFU versus time. All assays were done in duplicate.
2.1. Sources of chitosan and microorganisms
2.5. Cell membrane integrity
High and low molecular weight chitosan were obtained from SigmaeAldrich (St. Louis, USA). High molecular weight chitosan was characterized by a DD > 75% and a MW of 624 kDa. Low molecular weight chitosan was characterized by a DD between 75 and 85% and a MW of 107 kDa. This study used 5 anaerobic bacteria (4 Gram negative and one Gram positive) and one Gram negative microaerophilic bacteria as model microorganism for oral diseases. Four of the six microorganisms used in this study were obtained from the culture collection of the Göteburg University (CCUG) (Sweden) e Prevotella buccae (CCUG 15401) an isolate from periodontitis; Tannarella forsythensis (CCUG 51269) an isolate from human deep periodontal pockets; Aggregatibacter actinomycetemcomitans (CCUG 13227) origin not discriminated; Streptococcus mutans (CCUG 45091) origin not discriminated; Porphyromonas gingivalis was obtained from Institute Pasteur (France) (9704 CIP 103683T) and Prevotella intermedia is a clinical isolate obtained from periodontal disease patients.
Bacteria cell membrane integrity was performed as described by Li et al. [20]. Briefly, the cell membrane integrity was examined by determination of the intracellular material released at 260 nm (nucleotides) and at 280 (proteins). Bacteria were incubated for 72 h at 37 C in WCB with chitosan at bactericidal concentrations and 1 ml samples were removed at 0, 2, 4, 6, 8, 12, 24, 48 and 72 h. Samples were then immediately filtered through 0.2 mm filters to remove bacteria followed by OD measurement. Control assay was done with sterile deionised water. Results were given as the ratio release (chitosan vs. control) for each assay. All assays were performed in triplicate.
2.2. Preparation of chitosan solutions Chitosan solutions were prepared in 1%(v/v) solution of glacial acetic acid 99% (Panreac, Barcelona, Spain). Chitosan was added to 1% acetic acid to the desired concentration. Afterwards, the solution was stirred overnight at 50 C to promote complete dissolution of chitosan. The pH was adjusted with 10 M NaOH (Merck, Darmstad, Germany) to a final value of 5.6e5.8 and solutions were stored at refrigerated temperature. 2.3. Determination of minimal inhibitory and bactericidal concentrations Determination of MIC was performed according to Clinical and Laboratory Standards Institute guidelines [17]. Briefly, an inoculum of 0.5 MacFarland (1.5 108 CFU/ml) of each bacteria was prepared from overnight cultures and inoculated in Wilkins Chalgrens Broth (WCB) (Oxoid, Hampshire, England) with chitosan concentrations ranging from 0.1 mg/ml to 7 mg/ml. Two controls were simultaneously assessed: one with 0.1 mg/ml chitosan but without inoculum, and another where chitosan was replaced by sterile water and with added inoculum. The MIC was determined by observing the lowest concentration of chitosan which inhibited bacterial growth. All assays were performed in duplicate. Determination of MBC was performed as described by Fernandes et al. [18]. Briefly, the MBC were determined as the lowest concentration of chitosan at which bacterial growth was prevented, and the initial viability was reduced by at least 99.9% within 24 h; it was determined by inoculation of 100 ml aliquots of negative tubes (absence of turbidity in MIC determination) on Wilkins Chalgrens Agar (WCA) (Oxoid, Hampshire, England), using the plate spread technique. All assays were performed in duplicate.
2.6. Flocculation assay Bacteria were grown in 250 ml flask at 37 C in WCB after which were centrifuged at 6026 g for 5 min. Bacteria were then washed twice and suspended in phosphate buffer (pH 6.2). The flocculation assays were done as described by Strand et al. [21]. Briefly, in 15 ml graduated polystyrene tubes with conical bottoms, 8 ml of bacterial suspension were added to 1 ml of chitosan under stirring to assure a homogeneous solution. Control was made through addition of 8 ml of bacterial suspension to 1 ml of 0.1 M sodium chloride solution. Tubes were shaken for 10 min on an orbital shaker (Boeco, Germany) at 200 rpm and allowed to stand for 120 min. First, a sample of supernatant for OD measurement, at 620 nm, was taken from the top layer. Tubes were then allowed to rest overnight for completion of the sedimentation process and the second supernatant sample was removed from the middle of the tube. Results were expressed as being a decrease in turbidity in relation to the control tube and were given using the following equation:
i h flocculation % ¼ 1 ODcontrol =ODsample 100 All assays were done in triplicate. 3. Results and discussion 3.1. MIC and MBC determination The MIC and MBC of HMW and LMW chitosan upon several anaerobic oral pathogens were determined and results are reported in Table 1. As described by various authors [1,17,22] the antimicrobial activity of chitosan is strongly related with its MW, with low MW, namely chitooligosaccharides, acting better in Gram negative bacteria and vice-versa. However, as can be seen in Table 1, analysis of the results obtained for the MIC showed a mixed behaviour with MIC values obtained ranging between 1 and 5 mg/ml in both MWs, with LMW exhibiting better results for A. actinomycetemcomitans
E.M. Costa et al. / Anaerobe 18 (2012) 305e309 Table 1 Minimum inhibitory concentrations (MIC) and minimum bactericidal concentrations (MBC) of HMW and LMW chitosan upon the studied bacteria. Values in mg/mL. All assays were done in duplicate.
P. gingivalis T. forsythensis P. buccae A. actinomycetemcomitans P. intermedia S. mutans
HMW (mg/mL)
LMW (mg/mL)
MIC
MBC
MIC
MBC
1 1 3 5 1 3
5 5 7 7 5 5
1 3 1 3 3 5
3 7 3 7 7 7
and P. buccae and HMW showed better results for S. mutans and T. forsythensis. On the other hand, P. gingivalis and P. intermedia showed equal MIC values for both MWs. This apparent decrease of activity for LMW chitosan relatively to Gram negative bacteria when comparing with other studies involving chitosan and aerobic Gram negatives may be a result of the differences in metabolism between strict anaerobic and aerobic bacteria. As one of the targets of LMW chitosan is electronegative substances present in the interior of the cell [22] and anaerobic bacteria do not possess a respiratory chain that may produce these substances [23], LMW chitosan may possess a constrained action capability under these conditions. Previous studies reported antimicrobial activity of chitosan against P. gingivalis, S. mutans and A. actinomycetemcomitans, however the concentrations and efficacy reported varied greatly. It has been reported, for S. mutans, MIC values ranging from 0.5 mg/ ml to 2.5 mg/ml [9e12]. However, these results are due to the higher DD and MW of the chitosan’s used or to chemical alterations made to the chitosan molecule. Nevertheless, it is possible to compare some values. For example, the MIC value of 2.5 mg/ml reported by Ji et al. [11] for HMW chitosan is only slightly lower than the one we obtained. Considering that Ji et al. [11] used a chitosan with almost the double of the MW of ours (1080e624 kDa) the 0.5 mg/ml difference in MIC is not significant. The values previously reported for P. gingivalis, P. intermedia and A. actinomycetemcomitans showed chitosan’s MIC ranging from 0.082 to 3.638 mg/ml for the first and 1e2.5 mg/ml for P. intermedia and A. actinomycetemcomitans [10,11,24]. These values are in the same range of one’s obtained in our study, but far from those reported by Ikinci et al. [24], who claim that chitosan, for P. gingivalis, had a greater antimicrobial activity with the increase of the MW. However, those contradictory results may be related to the differences in HMW used (1400 kDa) when compared to the other studies (624 kDa). On the other hand, the MIC values obtained for A. actinomycetemcomitans were higher than those presented by Choi et al. [10] and Ji et al. [11]. However, the first study was performed with chitooligosacharides of higher DD and the latter was performed with chemically altered chitosan’s of higher MW and DD. Additionally, should be highlighted that strain differences for same species may also affect antimicrobial susceptibility. For the remainder microorganisms there is no previous work. The MBC values obtained were higher than those obtained for the MIC, as expected, since in general higher concentrations of chitosan are required to kill the bacteria, with S. mutans and T. forsythensis presenting lower MBC’s for HMW (5 mg/ml both) and P. gingivalis, P. buccae and P. intermedia having lower MBC’s for LMW (7 mg/ml for the P. buccae, 5 mg/ml for the other two) chitosan. Aggregatibacter actionomycetemcomitans presented equal MBC for both MWs. Regarding these values in line with those obtained for the MIC one can see that, with the exception of P. intermedia, P. gingivalis and A. actinomycetemcomitans, bacteria which
307
presented lower MIC values for one MW presented lower MBC for the same MW. P. gingivalis and P. intermedia, which had presented equal MIC for both MW presented now lower MBC for LMW chitosan probably due to the interaction of LMW chitosan with the bacterial DNA [25]. 3.2. Time-inhibition curves The effects of chitosan upon cell-viability using MBC concentrations of both MW are presented in Fig. 1. Both chitosans presented bactericidal action (defined as reduction of 3 log cycles in CFUs [18]) and no significant differences (p > 0.05) between MW were observed. In addition, both MW chitosans presented rapid bactericidal activity, with efficient bactericidal reduction obtained after 2 h, at the earliest (P. buccae, S. mutans and Tannarella forsythia), and 8 h at the latest (A. actinomycetemcomitans and P. gingivalis). While both MW were capable of completely inhibiting the studied bacteria there were significant differences (p < 0.05) in behaviour between MW for A. actinomycetemcomitans, P. intermedia and P. gingivalis. In these cases LMW chitosan showed faster bactericidal activity than HMW chitosan. This is particularly relevant for A. actinomycetemcomitans, which presented the same MBC for HMW and LMW chitosan. Despite what has been reported by various authors regarding chitosans action mechanism and the relevance of MW concerning the efficacy of the same against Gram positive or Gram negative bacteria, in this case there is a clear decrease in efficiency of LMW chitosan antimicrobial activity, with both MW presenting very similar MICs and MBC for Gram positive and negative bacteria. Though this may be due to the different metabolism of anaerobic bacteria. 3.3. Cell membrane integrity The bacterial membrane serves as a structural component which may be compromised during exposure to a cationic biocide, such as chitosan. Therefore, the release of intracellular components is a good indicator of membrane integrity. Small ions such as potassium and phosphate tend to leach out first, followed by large molecules such as DNA, RNA and other materials. Since nucleotides have strong UV absorption at 260 nm, they are easy to detect using a UVeVis spectrophotometer [20,26]. As previously reported, the contact between chitosan and the cell wall, which is essentially a negatively charged phospholipidic bilayer, may alter the cell wall permeability. This will lead to destabilization of the cell wall and leakage of intracellular substances [27]. Considering that at a l 280 nm the results obtained are related to the release of intracellular proteins [20] we can, in conjunction with the reading at 260 nm, ascertain when chitosan only interacts with the cell wall leading to pore formation (results of OD280 nm) and when chitosan causes complete disruption of the membrane leading to cell death (results of OD260 nm). The results obtained (Fig. 1) showed a direct relation between cellular death and leakage of intracellular substances, with bactericidal action and complete inhibition being superimposed with high peaks of OD at both wavelengths. For all studied bacteria there was a clear pattern with peaks at OD280 nm, signifying that leakage of intracellular proteins occurs first. However, there were two exceptions; In A. actinomycetemcomitans leakage of nucleotides (OD260nm) happens earlier than leakage of intracellular proteins for LMW chitosan, while in P. gingivalis the release is simultaneous for both MW. This interaction was first reported for aerobic bacteria with previous studies showing through OD measurement that the interaction between chitosan and the cell membrane was responsible for cellular death [20,28,29].
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E.M. Costa et al. / Anaerobe 18 (2012) 305e309
Fig. 1. Time-inhibition curves obtained by viable cell enumeration represented on the left axis (A e high molecular weight chitosan; - e low molecular weight chitosan; D e positive control without chitosan) and cell membrane integrity results obtained by optical density (OD) ratio represented on the right axis (- 260 nm HMW; - 260 nm LMW; , 280 nm HMW; - 280 nm LMW) for : a e S. mutans; b e P. buccae; c e T. forsythensis; d e A. actinomycetemcomitans; e P. intermedia; f e P. gingivalis. Values in log (cfu/ml) correspond to the average of four replicates. Method detection limit >2.9 log CFU/ml.
3.4. Flocculation assay Chitosan’s flocculative capability has been well documented [21,30]. The cationic nature of chitosans and its interaction with negatively charged bacterial cell wall has been the main factor ascribed to substantiate this capability. Additionally, other factors may influence said capability, namely chitosan’s MW, differences in cell surface hidrophobicity, which may lead to different action mechanisms involved in chitosan’s flocculative effect [21,30]. However all these studies have been performed with aerobic bacteria. The results obtained for the two chitosans upon the studied bacteria showed little to none flocculation capability after 2 h (data not shown) and reduced flocculation percentages after 24 h (Fig. 2). Visible flocculation was only observed after 24 h and in low percentages for both MWs. In addition, the values obtained were significantly lower than those obtained for aerobic bacteria by above mentioned authors. As can be seen, the absence of intracellular electronegative charged particles in anaerobic bacteria [23] may be the possible explanation for the low flocculation percentages. These results are in accordance with the explanation previously given regarding the MIC values obtained and the small difference obtained between LMW and HMW chitosan MIC values for Gram negative bacteria.
Fig. 2. Flocculation percentages obtained after 24 h in contact with LMW and HMW chitosans relatively to control (bacteria without chitosan) for different Gram positive and Gram negative anaerobic bacteria.
E.M. Costa et al. / Anaerobe 18 (2012) 305e309
Flocculation, mediated by interaction with electronegative charged particles, was not possible due to the natural structure of anaerobic bacteria. However we still recorded flocculation values that may be explained by the “bridging mechanism”, which is basically an interaction between the positively charged chitosan and the negatively charged bacterial cell wall, as proposed by Strand et al. [21,30]. Nevertheless, LMW chitosan presented higher flocculation percentages than HMW chitosan, probably due to the higher adsorption rate for chitosan’s with lower molecular weights and its impact on the bridging mechanism reported by Strand et al. [30]. 4. Conclusions Chitosan shows antimicrobial activity against the studied anaerobic bacteria with low MICs and quick and efficient bactericidal activity, compared to other reported aerobic bacteria. LMW showed a decrease in efficiency against anaerobic bacteria probably due to the lack of intracellular electronegative charged particles in anaerobic cells, as demonstrated by the low flocculation percentages obtained. Chitosan showed capability to interact and damage the cell wall, probably by pore formation or membrane disruption leading to cellular lysis, however at present time more studies will be necessary to confirm this hypothesis. Therefore chitosan shows great promise as an alternative natural antimicrobial for anaerobic pathogens in particular against anaerobic pathogens involved in oral infections. Acknowledgements The author hereby gratefully acknowledges the Agency of Innovation (Agência de Inovação, ADI, Portugal) and Quadro de Referência Estratégico Nacional (QREN, Portugal) which through the project “QUITORAL e Desenvolvimento de novas formulações de quitosanos com aplicação em medicina oral” (QREN-ADI 3474) provided funding for the realization of this work. References [1] Raafat D, Sahl H-G. Chitosan and its antimicrobial potential e a critical literature survey. Microbiol Biotechnol 2009;2(2):186e201. [2] Avadi M. Diethylmethyl chitosan as an antimicrobial agent: synthesis, characterization and antibacterial effects. Eur Polym J 2004;40(7):1355e61. [3] Ravi Kumar MNV. A review of chitin and chitosan applications. React Funct Polym 2000;46(1):1e27. [4] Kurita K. Chemistry and application of chitin and chitosan. Polym Degrad Stabil 1998;59(4):117e20. [5] Rabea EI, Badawy ME-T, Stevens CV, Smagghe G, Steurbaut W. Chitosan as antimicrobial agent: applications and mode of action. Biomacromolecules 2003;4(6):1457e65. [6] Wang GH. Inhibition and inactivation of five species of foodborne pathogens by chitosan. J Food Protect 1992;55(11):916e9. [7] Gerasimenko DV, Avdienko ID, Bannikova GE, Zueva OI, Varlamov VP. Antibacterial effects of water-soluble low-molecular-weight chitosans on different microorganisms. Appl Biochem Microbiol 2004;40(3):253e7.
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[8] Lee H-W, Park Y-S, Jung J-S, Shin W-S. Chitosan oligosaccharides, dp 2-8, have prebiotic effect on the Bifidobacterium bifidium and Lactobacillus sp. Anaerobe 2002;8(6):319e24. [9] Bae K, Jun EJ, Lee SM, Paik DI, Kim JB. Effect of water-soluble reduced chitosan on Streptococcus mutans, plaque regrowth and biofilm vitality. Clin Oral Investig 2006;10(2):102e7. [10] Choi BK, Kim KY, Yoo YJ, Oh SJ, Choi JH, Kim CY. In vitro antimicrobial activity of a chitooligosaccharide mixture against Actinobacillus actinomycetemcomitans and Streptococcus mutans. Int J Antimicrob Agents 2001;18(6):553e7. [11] Ji QX, Zhong DY, Lü R, Zhang WQ, Deng J, Chen XG. In vitro evaluation of the biomedical properties of chitosan and quaternized chitosan for dental applications. Carbohydr Res 2009;344(11):1297e302. [12] Tarsi R, Muzzarelli R, Guzman C, Pruzzo C. Inhibition of Streptococcus mutans adsorption to hydroxyapatite by low-molecular-weight chitosans. J Dent Res 1997;76(2):665e72. [13] Arosa F, Cardoso F, Pacheco F. Fundamentos de Imunologia. Lisboa: Lidel; 2007. [14] Prescott LM, Harley JP, Klein DA. Microbiology. 6th ed. New York: McGrawHill; 2005. [15] Schwach-Abdellaoui K, Vivien-Castioni N, Gurny R. Local delivery of antimicrobial agents for the treatment of periodontal diseases. Eur J Pharm Biopharm 2000;50(1):83e99. [16] Seymour RA. Pharmacological control disease. II. Antimicrobial of periodontal agents. J Dent 1995;23(1):5e14. [17] Hecht DW, Citron DM, Cox M, Jacobus N, Jenkins SG, Onderdonk A, et al. Methods for antimicrobial susceptibility testing of anaerobic bacteria: approved standard e seventh edition. Clin Lab Stand Inst 2007;27(2): 16e20. [18] Fernandes JC, Tavaria FK, Soares JC, Ramos OS, João Monteiro M, Pintado ME, et al. Antimicrobial effects of chitosans and chitooligosaccharides, upon Staphylococcus aureus and Escherichia coli, in food model systems. Food Microbiol 2008;25(7):922e8. [19] Miles AA, Misra SS, Irwin JO. The estimation of the bactericidal power of the blood. J Hyg-Cambridge 1938;38(6):732e49. [20] Li X, Feng X, Yang S, Fu G, Wang T, Su Z. Chitosan kills Escherichia coli through damage to be of cell membrane mechanism. Carbohydr Polym 2010;79(3): 493e9. [21] Strand SP, Vandvik MS, Vårum KM, Østgaard K. Screening of chitosans and conditions for bacterial flocculation. Biomacromolecules 2001;2(1): 126e33. [22] Zheng L. Study on antimicrobial activity of chitosan with different molecular weights. Carbohydr Polym 2003;54(4):527e30. [23] Unden G, Bongaerts J. Alternative respiratory pathways of Escherichia coli: energetics and transcriptional regulation in response to electron acceptors. Biochim Biophys Acta 1997;1320(3):217e34. [24] Ikinci G, Senel S, Akincibay H, Kas¸ S, Ercis¸ S, Wilson CG, et al. Effect of chitosan on a periodontal pathogen Porphyromonas gingivalis. Int J Pharm 2002; 235(1e2):121e7. [25] Eaton P, Fernandes JC, Pereira E, Pintado ME, Xavier Malcata F. Atomic force microscopy study of the antibacterial effects of chitosans on Escherichia coli and Staphylococcus aureus. Ultramicroscopy 2008;108(10): 1128e34. [26] Chen CZ, Cooper SL. Interactions between dendrimer biocides and bacterial membranes. Biomaterials 2002;23(16):3359e68. [27] Kong M, Chen XG, Xing K, Park HJ. Antimicrobial properties of chitosan and mode of action: a state of the art review. Int J Food Microbiol 2010;144(1): 51e63. [28] Liu H, Du Y, Wang X, Sun L. Chitosan kills bacteria through cell membrane damage. Int J Food Microbiol 2004;95(2):147e55. [29] Tao Y, Qian L-H, Xie J. Effect of chitosan on membrane permeability and cell morphology of Pseudomonas aeruginosa and Staphylococcus aureus. Carbohydr Polym 2011;86(2):969e74. [30] Strand Sabina P, Va KM, Østgaard K. Interactions between chitosans and bacterial suspensions : adsorption and flocculation. Colloid Surf 2003;27(1): 71e81.
Contents lists available at SciVerse ScienceDirect
Anaerobe journal homepage: www.elsevier.com/locate/anaerobe
Clinical microbiology
Evaluation and insights into chitosan antimicrobial activity against anaerobic oral pathogens E.M. Costa a, S. Silva a, C. Pina b, F.K. Tavaria a, M.M. Pintado a, * a b
CBQF, Escola Superior de Biotecnologia, Universidade Católica Portuguesa, Rua Dr. António Bernardino de Almeida, P-4200-072 Porto, Portugal Faculty of Health Sciences, Universidade Fernando Pessoa, Rua Carlos da Maia, 296, 4200-150 Porto, Portugal
a r t i c l e i n f o
a b s t r a c t
Article history: Received 18 January 2012 Received in revised form 21 March 2012 Accepted 19 April 2012 Available online 27 April 2012
The objective of this study was to assess the antimicrobial capability of non-chemically altered chitosan as an alternative to traditional antimicrobials used in the treatment of oral infections. The action mechanism of chitosan was also ascertained. High and low molecular weight chitosan showed antimicrobial activity at low concentrations for all tested bacteria with the MICs varying between 1 and 7 mg/ ml with a drop of efficacy relatively to the action of LMW chitosan. In addition chitosan showed also to be an effective bactericidal presenting bactericidal effect within 8 h at the latest. Additionally the evaluation of chitosan’s action mechanism showed that both MWs acted upon the bacterial cell wall and were not capable of interacting with the intracellular substances, as showed by the inefficacy obtained in the flocculation assay. Ó 2012 Elsevier Ltd. All rights reserved.
Keywords: Anaerobic pathogens Antimicrobial Chitosan Action mechanism
1. Introduction Chitosan is a high molecular weight (HMW), linear, polycationic heteropolysaccharide derivate from chitin, consisting of N-acetyl-2amino-2-deoxy-ᴅ-glucopyranose and 2-amino-2-deoxy-ᴅ-glucopyranose linked together by a b e (1/4) glycosidic bond. Chitosan molecules may vary widely originating samples of different degree of deacetylation (DD) (between 75 and 95%), molecular weights (MW) (between 50 and 2000 kDa), viscosity and pKa [1]. The high percentage of amino groups (6.89%), which provide chelating capability, in conjunction with antitumoral activity, immunoadjuvant activity, acceleration of wound healing, antimicrobial activity, biodegradability and biocompatibility make chitosan a high sought biomaterial [1e4]. Chitosan, however, possesses some limitations. It has limited use due to its insolubility in water, since it is only soluble in some organic acids e such as acetic and formic acids, possesses high viscosity and tendency to coagulate proteins at high pH [3,5]. The antimicrobial activity of chitosan was observed against a wide variety of microorganisms including fungi, algae and bacteria, being more active against Gram positive than Gram negative bacteria [1], in particular if it possess high MW. The
* Corresponding author. Tel. þ351 22 558 0000. E-mail addresses: [email protected], (M.M. Pintado).
[email protected]
1075-9964/$ e see front matter Ó 2012 Elsevier Ltd. All rights reserved. doi:10.1016/j.anaerobe.2012.04.009
reported minimum inhibitory concentrations (MICs) of chitosan vary widelydfrom 0.005% to 1.5% (w/v) for Staphylococcus aureus [6] or from 0.025% to 1.0% (w/v) for Escherichia coli [7], and its antibacterial effects seem to be closely related to MW and degree of deacetylation [8]. However few to none results have been published regarding the action of chitosan against anaerobic bacteria, and the few existing reported contradictory results or used chemically altered chitosan [9e12]. The oral cavity is a complex system composed by soft and hard tissue requiring adherence for the survival of microorganisms [13]. However, the occurrence of plaque formation will allow the formation of an anaerobic atmosphere that will potentiate the development of anaerobic bacteria, most of which are pathogenic. This will trigger an inflammatory response by the immune system that will result in tissue damage [13]. Plaque formation also contributed for a protective environment for bacterial growth, namely from the action of saliva and accumulation of acid produced by bacteria, which will lead to teeth lesions [14]. The routine usage of antibiotics in the treatment of oral diseases is not advisable, as such method of treatment must be only used in serious or relapsing cases, and no antibiotic will be capable of replacing tooth debridement and proper control of supragingival plaque formation [15,16]. However, systemic administration has great disadvantages e inability to control the quantity of antibiotic that reaches the active site, and consequently not guaranteeing an effective treatment [15], the incomplete elimination of bacteria in the oral cavity and finally
306
E.M. Costa et al. / Anaerobe 18 (2012) 305e309
all undesirable side effects due to interactions with another medicines [16]. On the other hand, topic administration requires specialized knowledge and personnel in their application and the time required to apply the techniques [16]. In view of the above data, this research effort was aimed at evaluating the antimicrobial activity of chitosan against oral anaerobic bacteria, in order to access the possibility of using chitosan as an alternative to local conventional antimicrobials, as well as the influence of metabolic differences between aerobic and anaerobic bacteria on chitosan’s action mechanism.
2.4. Time-inhibition curves
2. Material and methods
The antimicrobial activity at MBC for LMW and HMW chitosan were tested as previously described by Fernandes et al. [18] with minor modifications. Inocula were adjusted to 0.5 in the Macfarland scale (1 108 CFU/ml). Control tests were prepared with sterile deionised water instead of chitosan. Viable counts of different concentrations of chitosan solutions were determined at 0, 2, 4, 6, 8, 12, 24, 48 and 72 h by the Miles, Misra and Irwin [19] technique (1938) in WCA. Plates were incubated under anaerobic conditions, at 37 C for 24 h. Inhibition curves were constructed by plotting log CFU versus time. All assays were done in duplicate.
2.1. Sources of chitosan and microorganisms
2.5. Cell membrane integrity
High and low molecular weight chitosan were obtained from SigmaeAldrich (St. Louis, USA). High molecular weight chitosan was characterized by a DD > 75% and a MW of 624 kDa. Low molecular weight chitosan was characterized by a DD between 75 and 85% and a MW of 107 kDa. This study used 5 anaerobic bacteria (4 Gram negative and one Gram positive) and one Gram negative microaerophilic bacteria as model microorganism for oral diseases. Four of the six microorganisms used in this study were obtained from the culture collection of the Göteburg University (CCUG) (Sweden) e Prevotella buccae (CCUG 15401) an isolate from periodontitis; Tannarella forsythensis (CCUG 51269) an isolate from human deep periodontal pockets; Aggregatibacter actinomycetemcomitans (CCUG 13227) origin not discriminated; Streptococcus mutans (CCUG 45091) origin not discriminated; Porphyromonas gingivalis was obtained from Institute Pasteur (France) (9704 CIP 103683T) and Prevotella intermedia is a clinical isolate obtained from periodontal disease patients.
Bacteria cell membrane integrity was performed as described by Li et al. [20]. Briefly, the cell membrane integrity was examined by determination of the intracellular material released at 260 nm (nucleotides) and at 280 (proteins). Bacteria were incubated for 72 h at 37 C in WCB with chitosan at bactericidal concentrations and 1 ml samples were removed at 0, 2, 4, 6, 8, 12, 24, 48 and 72 h. Samples were then immediately filtered through 0.2 mm filters to remove bacteria followed by OD measurement. Control assay was done with sterile deionised water. Results were given as the ratio release (chitosan vs. control) for each assay. All assays were performed in triplicate.
2.2. Preparation of chitosan solutions Chitosan solutions were prepared in 1%(v/v) solution of glacial acetic acid 99% (Panreac, Barcelona, Spain). Chitosan was added to 1% acetic acid to the desired concentration. Afterwards, the solution was stirred overnight at 50 C to promote complete dissolution of chitosan. The pH was adjusted with 10 M NaOH (Merck, Darmstad, Germany) to a final value of 5.6e5.8 and solutions were stored at refrigerated temperature. 2.3. Determination of minimal inhibitory and bactericidal concentrations Determination of MIC was performed according to Clinical and Laboratory Standards Institute guidelines [17]. Briefly, an inoculum of 0.5 MacFarland (1.5 108 CFU/ml) of each bacteria was prepared from overnight cultures and inoculated in Wilkins Chalgrens Broth (WCB) (Oxoid, Hampshire, England) with chitosan concentrations ranging from 0.1 mg/ml to 7 mg/ml. Two controls were simultaneously assessed: one with 0.1 mg/ml chitosan but without inoculum, and another where chitosan was replaced by sterile water and with added inoculum. The MIC was determined by observing the lowest concentration of chitosan which inhibited bacterial growth. All assays were performed in duplicate. Determination of MBC was performed as described by Fernandes et al. [18]. Briefly, the MBC were determined as the lowest concentration of chitosan at which bacterial growth was prevented, and the initial viability was reduced by at least 99.9% within 24 h; it was determined by inoculation of 100 ml aliquots of negative tubes (absence of turbidity in MIC determination) on Wilkins Chalgrens Agar (WCA) (Oxoid, Hampshire, England), using the plate spread technique. All assays were performed in duplicate.
2.6. Flocculation assay Bacteria were grown in 250 ml flask at 37 C in WCB after which were centrifuged at 6026 g for 5 min. Bacteria were then washed twice and suspended in phosphate buffer (pH 6.2). The flocculation assays were done as described by Strand et al. [21]. Briefly, in 15 ml graduated polystyrene tubes with conical bottoms, 8 ml of bacterial suspension were added to 1 ml of chitosan under stirring to assure a homogeneous solution. Control was made through addition of 8 ml of bacterial suspension to 1 ml of 0.1 M sodium chloride solution. Tubes were shaken for 10 min on an orbital shaker (Boeco, Germany) at 200 rpm and allowed to stand for 120 min. First, a sample of supernatant for OD measurement, at 620 nm, was taken from the top layer. Tubes were then allowed to rest overnight for completion of the sedimentation process and the second supernatant sample was removed from the middle of the tube. Results were expressed as being a decrease in turbidity in relation to the control tube and were given using the following equation:
i h flocculation % ¼ 1 ODcontrol =ODsample 100 All assays were done in triplicate. 3. Results and discussion 3.1. MIC and MBC determination The MIC and MBC of HMW and LMW chitosan upon several anaerobic oral pathogens were determined and results are reported in Table 1. As described by various authors [1,17,22] the antimicrobial activity of chitosan is strongly related with its MW, with low MW, namely chitooligosaccharides, acting better in Gram negative bacteria and vice-versa. However, as can be seen in Table 1, analysis of the results obtained for the MIC showed a mixed behaviour with MIC values obtained ranging between 1 and 5 mg/ml in both MWs, with LMW exhibiting better results for A. actinomycetemcomitans
E.M. Costa et al. / Anaerobe 18 (2012) 305e309 Table 1 Minimum inhibitory concentrations (MIC) and minimum bactericidal concentrations (MBC) of HMW and LMW chitosan upon the studied bacteria. Values in mg/mL. All assays were done in duplicate.
P. gingivalis T. forsythensis P. buccae A. actinomycetemcomitans P. intermedia S. mutans
HMW (mg/mL)
LMW (mg/mL)
MIC
MBC
MIC
MBC
1 1 3 5 1 3
5 5 7 7 5 5
1 3 1 3 3 5
3 7 3 7 7 7
and P. buccae and HMW showed better results for S. mutans and T. forsythensis. On the other hand, P. gingivalis and P. intermedia showed equal MIC values for both MWs. This apparent decrease of activity for LMW chitosan relatively to Gram negative bacteria when comparing with other studies involving chitosan and aerobic Gram negatives may be a result of the differences in metabolism between strict anaerobic and aerobic bacteria. As one of the targets of LMW chitosan is electronegative substances present in the interior of the cell [22] and anaerobic bacteria do not possess a respiratory chain that may produce these substances [23], LMW chitosan may possess a constrained action capability under these conditions. Previous studies reported antimicrobial activity of chitosan against P. gingivalis, S. mutans and A. actinomycetemcomitans, however the concentrations and efficacy reported varied greatly. It has been reported, for S. mutans, MIC values ranging from 0.5 mg/ ml to 2.5 mg/ml [9e12]. However, these results are due to the higher DD and MW of the chitosan’s used or to chemical alterations made to the chitosan molecule. Nevertheless, it is possible to compare some values. For example, the MIC value of 2.5 mg/ml reported by Ji et al. [11] for HMW chitosan is only slightly lower than the one we obtained. Considering that Ji et al. [11] used a chitosan with almost the double of the MW of ours (1080e624 kDa) the 0.5 mg/ml difference in MIC is not significant. The values previously reported for P. gingivalis, P. intermedia and A. actinomycetemcomitans showed chitosan’s MIC ranging from 0.082 to 3.638 mg/ml for the first and 1e2.5 mg/ml for P. intermedia and A. actinomycetemcomitans [10,11,24]. These values are in the same range of one’s obtained in our study, but far from those reported by Ikinci et al. [24], who claim that chitosan, for P. gingivalis, had a greater antimicrobial activity with the increase of the MW. However, those contradictory results may be related to the differences in HMW used (1400 kDa) when compared to the other studies (624 kDa). On the other hand, the MIC values obtained for A. actinomycetemcomitans were higher than those presented by Choi et al. [10] and Ji et al. [11]. However, the first study was performed with chitooligosacharides of higher DD and the latter was performed with chemically altered chitosan’s of higher MW and DD. Additionally, should be highlighted that strain differences for same species may also affect antimicrobial susceptibility. For the remainder microorganisms there is no previous work. The MBC values obtained were higher than those obtained for the MIC, as expected, since in general higher concentrations of chitosan are required to kill the bacteria, with S. mutans and T. forsythensis presenting lower MBC’s for HMW (5 mg/ml both) and P. gingivalis, P. buccae and P. intermedia having lower MBC’s for LMW (7 mg/ml for the P. buccae, 5 mg/ml for the other two) chitosan. Aggregatibacter actionomycetemcomitans presented equal MBC for both MWs. Regarding these values in line with those obtained for the MIC one can see that, with the exception of P. intermedia, P. gingivalis and A. actinomycetemcomitans, bacteria which
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presented lower MIC values for one MW presented lower MBC for the same MW. P. gingivalis and P. intermedia, which had presented equal MIC for both MW presented now lower MBC for LMW chitosan probably due to the interaction of LMW chitosan with the bacterial DNA [25]. 3.2. Time-inhibition curves The effects of chitosan upon cell-viability using MBC concentrations of both MW are presented in Fig. 1. Both chitosans presented bactericidal action (defined as reduction of 3 log cycles in CFUs [18]) and no significant differences (p > 0.05) between MW were observed. In addition, both MW chitosans presented rapid bactericidal activity, with efficient bactericidal reduction obtained after 2 h, at the earliest (P. buccae, S. mutans and Tannarella forsythia), and 8 h at the latest (A. actinomycetemcomitans and P. gingivalis). While both MW were capable of completely inhibiting the studied bacteria there were significant differences (p < 0.05) in behaviour between MW for A. actinomycetemcomitans, P. intermedia and P. gingivalis. In these cases LMW chitosan showed faster bactericidal activity than HMW chitosan. This is particularly relevant for A. actinomycetemcomitans, which presented the same MBC for HMW and LMW chitosan. Despite what has been reported by various authors regarding chitosans action mechanism and the relevance of MW concerning the efficacy of the same against Gram positive or Gram negative bacteria, in this case there is a clear decrease in efficiency of LMW chitosan antimicrobial activity, with both MW presenting very similar MICs and MBC for Gram positive and negative bacteria. Though this may be due to the different metabolism of anaerobic bacteria. 3.3. Cell membrane integrity The bacterial membrane serves as a structural component which may be compromised during exposure to a cationic biocide, such as chitosan. Therefore, the release of intracellular components is a good indicator of membrane integrity. Small ions such as potassium and phosphate tend to leach out first, followed by large molecules such as DNA, RNA and other materials. Since nucleotides have strong UV absorption at 260 nm, they are easy to detect using a UVeVis spectrophotometer [20,26]. As previously reported, the contact between chitosan and the cell wall, which is essentially a negatively charged phospholipidic bilayer, may alter the cell wall permeability. This will lead to destabilization of the cell wall and leakage of intracellular substances [27]. Considering that at a l 280 nm the results obtained are related to the release of intracellular proteins [20] we can, in conjunction with the reading at 260 nm, ascertain when chitosan only interacts with the cell wall leading to pore formation (results of OD280 nm) and when chitosan causes complete disruption of the membrane leading to cell death (results of OD260 nm). The results obtained (Fig. 1) showed a direct relation between cellular death and leakage of intracellular substances, with bactericidal action and complete inhibition being superimposed with high peaks of OD at both wavelengths. For all studied bacteria there was a clear pattern with peaks at OD280 nm, signifying that leakage of intracellular proteins occurs first. However, there were two exceptions; In A. actinomycetemcomitans leakage of nucleotides (OD260nm) happens earlier than leakage of intracellular proteins for LMW chitosan, while in P. gingivalis the release is simultaneous for both MW. This interaction was first reported for aerobic bacteria with previous studies showing through OD measurement that the interaction between chitosan and the cell membrane was responsible for cellular death [20,28,29].
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Fig. 1. Time-inhibition curves obtained by viable cell enumeration represented on the left axis (A e high molecular weight chitosan; - e low molecular weight chitosan; D e positive control without chitosan) and cell membrane integrity results obtained by optical density (OD) ratio represented on the right axis (- 260 nm HMW; - 260 nm LMW; , 280 nm HMW; - 280 nm LMW) for : a e S. mutans; b e P. buccae; c e T. forsythensis; d e A. actinomycetemcomitans; e P. intermedia; f e P. gingivalis. Values in log (cfu/ml) correspond to the average of four replicates. Method detection limit >2.9 log CFU/ml.
3.4. Flocculation assay Chitosan’s flocculative capability has been well documented [21,30]. The cationic nature of chitosans and its interaction with negatively charged bacterial cell wall has been the main factor ascribed to substantiate this capability. Additionally, other factors may influence said capability, namely chitosan’s MW, differences in cell surface hidrophobicity, which may lead to different action mechanisms involved in chitosan’s flocculative effect [21,30]. However all these studies have been performed with aerobic bacteria. The results obtained for the two chitosans upon the studied bacteria showed little to none flocculation capability after 2 h (data not shown) and reduced flocculation percentages after 24 h (Fig. 2). Visible flocculation was only observed after 24 h and in low percentages for both MWs. In addition, the values obtained were significantly lower than those obtained for aerobic bacteria by above mentioned authors. As can be seen, the absence of intracellular electronegative charged particles in anaerobic bacteria [23] may be the possible explanation for the low flocculation percentages. These results are in accordance with the explanation previously given regarding the MIC values obtained and the small difference obtained between LMW and HMW chitosan MIC values for Gram negative bacteria.
Fig. 2. Flocculation percentages obtained after 24 h in contact with LMW and HMW chitosans relatively to control (bacteria without chitosan) for different Gram positive and Gram negative anaerobic bacteria.
E.M. Costa et al. / Anaerobe 18 (2012) 305e309
Flocculation, mediated by interaction with electronegative charged particles, was not possible due to the natural structure of anaerobic bacteria. However we still recorded flocculation values that may be explained by the “bridging mechanism”, which is basically an interaction between the positively charged chitosan and the negatively charged bacterial cell wall, as proposed by Strand et al. [21,30]. Nevertheless, LMW chitosan presented higher flocculation percentages than HMW chitosan, probably due to the higher adsorption rate for chitosan’s with lower molecular weights and its impact on the bridging mechanism reported by Strand et al. [30]. 4. Conclusions Chitosan shows antimicrobial activity against the studied anaerobic bacteria with low MICs and quick and efficient bactericidal activity, compared to other reported aerobic bacteria. LMW showed a decrease in efficiency against anaerobic bacteria probably due to the lack of intracellular electronegative charged particles in anaerobic cells, as demonstrated by the low flocculation percentages obtained. Chitosan showed capability to interact and damage the cell wall, probably by pore formation or membrane disruption leading to cellular lysis, however at present time more studies will be necessary to confirm this hypothesis. Therefore chitosan shows great promise as an alternative natural antimicrobial for anaerobic pathogens in particular against anaerobic pathogens involved in oral infections. Acknowledgements The author hereby gratefully acknowledges the Agency of Innovation (Agência de Inovação, ADI, Portugal) and Quadro de Referência Estratégico Nacional (QREN, Portugal) which through the project “QUITORAL e Desenvolvimento de novas formulações de quitosanos com aplicação em medicina oral” (QREN-ADI 3474) provided funding for the realization of this work. References [1] Raafat D, Sahl H-G. Chitosan and its antimicrobial potential e a critical literature survey. Microbiol Biotechnol 2009;2(2):186e201. [2] Avadi M. Diethylmethyl chitosan as an antimicrobial agent: synthesis, characterization and antibacterial effects. Eur Polym J 2004;40(7):1355e61. [3] Ravi Kumar MNV. A review of chitin and chitosan applications. React Funct Polym 2000;46(1):1e27. [4] Kurita K. Chemistry and application of chitin and chitosan. Polym Degrad Stabil 1998;59(4):117e20. [5] Rabea EI, Badawy ME-T, Stevens CV, Smagghe G, Steurbaut W. Chitosan as antimicrobial agent: applications and mode of action. Biomacromolecules 2003;4(6):1457e65. [6] Wang GH. Inhibition and inactivation of five species of foodborne pathogens by chitosan. J Food Protect 1992;55(11):916e9. [7] Gerasimenko DV, Avdienko ID, Bannikova GE, Zueva OI, Varlamov VP. Antibacterial effects of water-soluble low-molecular-weight chitosans on different microorganisms. Appl Biochem Microbiol 2004;40(3):253e7.
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