International Journal of Biological Macromolecules 64 (2014) 302–305
Contents lists available at ScienceDirect
International Journal of Biological Macromolecules journal homepage: www.elsevier.com/locate/ijbiomac
Short communication
Size and pH effects of chitooligomers on antibacterial activity against Staphylococcus aureus Kecheng Li, Ronge Xing, Song Liu, Yukun Qin, Huahua Yu, Pengcheng Li ∗ Institute of Oceanology, Chinese Academy of Sciences, Qingdao 266071, China
a r t i c l e
i n f o
Article history: Received 8 October 2013 Received in revised form 26 November 2013 Accepted 29 November 2013 Available online 7 December 2013 Keywords: Chitooligomers Size Antibacterial activity
a b s t r a c t The antimicrobial activity of chitooligomers (COSs) has attracted considerable interest but there are few reports on antibacterial activity of each individual COS. This study focuses on investigating the size and pH effects of COS on its antibacterial activity against Staphylococcus aureus. Minimum inhibitory concentrations (MICs) of five single COSs and five COS fractions with narrow degrees of polymerization (DP) were determined at different pH mediums. The results revealed that the antibacterial activity of COS required structural essential with a DP of at least 5 and the inhibitory effect increased with increasing DP. Lower pH value could enhance the antibacterial activity of COS. The COS with DP > 12 showed a MIC value of 62.5 mg/mL at pH 6.0, while the MIC value increased to 500 and 1000 mg/mL at pH 6.5 and 7.0, respectively. The cell membrane integrity assay and SEM suggested that the COS with high DP could cause bacteria clustering and further lysis of cell. © 2013 Elsevier B.V. All rights reserved.
1. Introduction Chitooligomers (COS) has been reported to possess versatile biological functions including antitumor, antioxidant, antimicrobial activity, wound healing and immune-enhancing effects [1–3] and these oligomers are safe for human body [4]. Recently tailor-made COS with well-defined degree of polymerization (DP) has drawn considerable attention in order to determine the active ingredient and better understand biological action of COS. Several COSs with single or narrow DP have been prepared and their bioactivities were evaluated. Chitohexaose has been found to exhibit the highest inhibitory effect on the proliferation and migration of tumorinduced ECV304 cells among five COSs (dimers to hexamers) [5]. Antioxidant activity of a fully deacetylated COS series with several well-defined DPs has been investigated and the function–structure relationship was revealed [6,7]. However, the antimicrobial activity of each individual chitooligomers remains unknown. As a natural and potential antimicrobial agent, COS has a wide inhibitory spectrum for not only Gram-positive and Gram-negative bacteria but also yeasts and moulds [8–10]. Antimicrobial activity of COS is closely related to its molecular weight (MW) [11,12]. However, the antimicrobial assays in prior studies were performed using heterogeneous COS with various MWs, which resulted in some controversial conclusions [13–15]. Therefore, in order to make it clear,
antimicrobial activity of COS with well-define DP is required. In this study, ten well-defined COSs including five single COSs (chitobiose to chitohexaose) and five COS fractions with narrow DPs (>6) were used for antibacterial assays against Staphylococcus aureus (S. aureus) and Escherichia coli (E. coli). In addition, the effect of pH on the antibacterial activity of COS was investigated, and cell integrity assay and scanning electron microscope (SEM) observation were carried out to probe the possible antibacterial mechanism against S. aureus. 2. Experiments 2.1. Materials Ten fully deacetylated COS fractions with well-defined DP were prepared according to our previous studies [16,17], including five single COSs, chitobiose (≥98%), chitotriose (≥98%), chitotetraose (≥98%), chitopentaose (≥98%) and chitohexaose (≥98%), and five COS fractions with narrow DP which mainly contained chitooligomers with DP6–7 (41.3%, 50.2%), DP7–8 (22.5%, 70.1%), DP9–10 (53.1%, 28.0%), DP10–12 (18.4%, 49.4%, 22.3%), and DP > 12, respectively. All other chemicals and reagents were of analytical grade and not further purified unless otherwise specified. 2.2. Antibacterial activity assay
∗ Corresponding author. Tel.: +86 532 82898707; fax: +86 532 82968951. E-mail addresses: [email protected], [email protected], xueqinwang [email protected] (P. Li). 0141-8130/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.ijbiomac.2013.11.037
The antibacterial activity of the COS series was evaluated by the minimum inhibitory concentration (MIC). MIC was determined using a turbidity-based microdilution assay in Muller Hinton broth
K. Li et al. / International Journal of Biological Macromolecules 64 (2014) 302–305
(MHB) according to the method of Meng et al. [18] with slight modification. In brief, S. aureus in the midlogarithmic growth phase was adjusted to the turbidity of Mcfarland standard 0.5 (approximately 1–2 × 108 CFU/mL). Subsequently, the suspension was diluted 1:100 and 50 L suspension was mixed with serial dilutions of COS samples stock solution (50 L) in each well of a 96-well polypropylene microplate (Corning # 3359). MIC of each sample was determined after incubating for 24 h at 37 ◦ C as the lowest oligomers concentration that completely inhibited bacteria growth. Triplicate analyses of each sample were performed and each experiment was carried out in duplicate. For the tests of antibacterial activity at different pHs, the MHB medium was adjusted to pH 6.0, 6.5, 7.0, and 8.0 respectively using 6.0 M HCl or 6.0 M NaOH. Then the corresponding MHB medium was employed to dilute bacterial suspension and to dissolve COS samples. 2.3. Assay for cell membrane integrity The cell membrane integrity of S. aureus was examined by monitoring the release of material absorbing at 260 nm as described by Je et al. [19]. 2 mL COS samples were mixed with 2 mL bacterial suspension (2 × 108 CFU/mL) in 0.1 mol/L phosphate buffer solution (PBS) to get a final concentration of 1 mg/mL. The OD values at 260 nm were determined at different times. 2.4. SEM observation SEM assay was performed as follows. The bacterial suspension was incubated with 1 mg/mL COS samples at 37 ◦ C for different times and then was transported onto a cover slip. Slideimmobilized cells were fixed with 2.5% (w/v) glutaraldehyde in 0.1 mol/L phosphate buffer solution overnight at 4 ◦ C and then washed with the same buffer. The dehydration of cells was performed with a graded ethanol series. After critical-point drying and gold coating, the samples were subjected to SEM. 2.5. Statistics Statistical evaluation was carried out using SPSS 18.0 package. Data were presented as mean ± S.D. and statistical comparisons between groups were performed using t-test. p < 0.05 was considered to be statistically significant.
303
Table 1 MIC values of chitooligomers with different DPs against S. aureus at different pHs. Sample DP
2 3 4 5 6 6–7 7–8 9–10 10–12 >12 a
MIC (g/mL) pH 6.0
pH 6.5
pH 7.0
pH 8.0
–a – – 4000 2000 2000 2000 2000 500 62.5
– – – 8000 8000 4000 4000 2000 2000 500
– – – 8000 8000 4000 4000 4000 2000 1000
– – – – – – – – – –
The MIC could not be read.
been reported that, for the chitosan with MW below 300 kDa, the antibacterial effect on S. aureus was strengthened as MW increased [21]. In agreement with previous study, our results revealed that COS with high DP showed enhanced antibacterial effect on S. aureus but a DP of at least 5 is required for the structural essential of antibacterial activity. Furthermore, it is worth noting that the inhibitory effects of COSs against S. aureus and E. coli are significantly different. It might be suggested that COS generally shows stronger bactericidal effects on Gram-positive bacteria than Gramnegative bacteria, which is consistent with prior study reported by No et al. [22]. On the other hand, our results indicated that pH played a significant role in the antibacterial action of COS. Under the basic condition, all the COS samples showed no inhibitory effect on the growth of S. aureus. In comparison, lower pH value was favorable to enhance the antibacterial activity of COS. The COS with DP > 12 shows a MIC value of 62.5 mg/mL at pH 6.0, while the MIC values increased to 500 and 1000 mg/mL at pH 6.5 and 7.0, respectively. Cationic charges have been reported to help antimicrobial agents to bind with the anionic lipid components of bacterial membrane [23]. In this study, all the samples were fully deacetylated COSs with free amino groups. MIC values of COS with DP > 12 at pH 6.0 was reduced by 16-fold compared with that at pH 7.0. This might result from the protonation of amines on the sugar chains in acid medium, which played an important role in the binding process and enhanced the antibacterial activity.
3. Results and discussion 3.1. MIC of each chitooligomers fractions The MIC values of COS with different DPs were determined using different pH MHB mediums via miniaturization of the broth dilution susceptibility test, which was employed to evaluated the size and pH effects on the antibacterial activity of COS. Table 1 showed the MIC results of COS fractions against S. aureus. Antibacterial activity of the COS with DP 4 was not observed. It seems that antibacterial responses of COS against S. aureus generally need an essential chemical structure, and a DP higher than 4 is required. Under the same pH, for those COS with DP > 4, MIC displayed a decreasing trend with increasing DP. The MIC of COS with DP > 12 decreased more than 8-fold at pH 6.0 compared with those low DP oligomers. Additionally, the antibacterial activity of these COSs against E. coli was investigated in the same way. However, no obvious inhibitory effect on E. coli was observed for most tested chitooligomers except the COS of DP > 12, with high MIC values of 1000, 8000 and 8000 mg/mL at pH 6.0, 6.5 and 7.0, respectively. Recent studies have reported that antibacterial activity of chitosan and their derivates depended on their MWs [13,20]. It has
Fig. 1. Release of cell materials absorbing at 260 nm from S. aureus suspensions treated with 1 mg/mL of chitotriose (䊉), chitohexaose () and chitooligomers with DP > 12 (), respectively. Data were presented as mean ± S.D. (n = 3) and statistical comparisons of the data between groups at 180 min were very significant (p < 0.01).
304
K. Li et al. / International Journal of Biological Macromolecules 64 (2014) 302–305
Fig. 2. SEM observation of S. aureus untreated and treated with chitooligomers with different times of incubation: (a) control bacteria, 30 min; (b) control bacteria, 2 h; (c, d) S. aureus cells treated with 1 mg/mL of chitohexaose for 30 min and 2 h; (e, f, g) S. aureus cells treated with 1 mg/mL of chitooligomers with DP > 12 for 30 min, 2 h and 24 h.
3.2. Interaction of well-defined chitooligomers with bacteria When cell membrane is damaged, cytoplasmic constituents including DNA and RNA having strong UV absorption at 260 nm will be released [24]. In this case, cell membrane integrity against
three representative COSs with well-defined DPs was examined by 260 nm release assay. As is shown in Fig. 1, chitotriose and chitohexaose could promote the release of 260 nm absorbing materials in S. aureus compared with the untreated bacterial suspension of the same CFU and chitohexaose showed a higher level. The
K. Li et al. / International Journal of Biological Macromolecules 64 (2014) 302–305
release increased at the beginning stage and ultimately reached the plateau. For the group treated by chitotriose, the release is too weak to observe an MIC value mentioned above. In contrast, upon addition of the COS with DP > 12, the OD of S. aureus suspension displayed a different and interesting trend. The curve hardly showed any changes within 1 h. However, after about 70 min, a fast release of 260 nm absorbing materials was observed. At 180 min, the OD of bacterial suspension treated with the COS of DP > 12 ultimately exceeded that of the group treated with chitohexaose. This suggested that the COS of DP > 12 should have stronger antibacterial activity compared with chitohexaose, which is in agreement with the MIC determination result. In order to further understand the mechanism, SEM study was conducted to observe directly the cell morphology change and membrane damage. As is presented in Fig. 2, the control bacteria remained intact and dispersed and the cell surfaces were smooth and integrated. After being treated with chitohexaose, no gross morphological differences were seen except that a few irregular structures on the surface of some cells appeared after 2 h (during the time of plateau in Fig. 1). We inferred that chitohexaose might increase the permeability of the cell membranes and led to the increasing release of 260 nm absorbing materials but did not kill the cells. In contrast, after being exposed to COS with DP > 12 for 30 min, the cells existed as small clusters and convex cell surfaces were visible. At 2 h, the clusters observed became even larger and much viscous substances among microbial cells appeared, which should be cytoplasmic content resulting from lysis of the cell. These results were consistent with the 260 nm release assay. After being exposed to COS with DP > 12 for 24 h, the cell collapse was observed (Fig. 2g). Previous studies reported that cationic chitosan could cover the bacterial cell, prevent the nutrition transport and finally result in cell apoptosis [25,26]. In our experiments, conglutination between bacterial cells was observed in high-DP COS treated group and after a certain time of incubation, a fast release of 260 nm absorbing materials was presented. It seemed that the cationic oligomers could “glue” negatively charged cells and led to the formation of large bacteria cluster, which might block the nutrition transport of bacterial cell and resulted in impairment of vital bacterial activity. Subsequently, with time of incubation increasing, the cell lysis occurred and much 260 nm absorbing materials were released. However, the “glue” interaction seemed to require an enough size of COS. 4. Conclusion In this paper, antibacterial activity of ten COSs with single or narrow DP was investigated and the related mechanism was explored using cell integrity assay and SEM observation. Size and pH of COS played significant roles in its antibacterial activity. It is concluded as follows: (i) lower pH value could enhance the antibacterial activity of each well-defined COS fraction against S. aureus; (ii)
305
antibacterial activity of COS required structural essential with a DP of at least 5 and the MICs of high-DP COSs, especially those oligomers with DP > 12, were obviously lower than those of low-DP COSs; (iii) cell membrane integrity assay and SEM suggested that the high-DP COS could cause the bacteria clustering and further led to lysis of cell. Acknowledgments The study was supported by the National High Technology Research and Development Program (“863”Program) of China (2011AA09070405), the commonweal item of State Oceanic Administration People’s Republic of China (201305016), National Key Technologies R&D Program (2011BAE06B04), the Action Plan of CAS to Support China’s New and Strategic Industries with Science and Technology, Guangdong Province & Chinese Academy of Sciences overall strategy cooperative project (2011A090100035). References [1] B.B. Aam, E.B. Heggset, A.L. Norberg, M. Sørlie, K.M. Vårum, V.G.H. Eijsink, Mar. Drugs 8 (2010) 1482–1517. [2] S. Kim, N. Rajapakse, Carbohyd. Polym. 62 (2005) 357–368. [3] R.A.A. Muzzarelli, Carbohyd. Polym. 76 (2009) 167–182. [4] C.Q. Qin, J.N. Gao, L.S. Wang, L.T. Zeng, Y. Liu, Food Chem. Toxicol. 44 (2006) 855–861. [5] C. Xiong, H. Wu, P. Wei, M. Pan, Y. Tuo, I. Kusakabe, Y. Du, Carbohyd. Res. 344 (2009) 1975–1983. [6] K. Li, R. Xing, S. Liu, R. Li, Y. Qin, X. Meng, P. Li, Carbohyd. Polym. 88 (2012) 896–903. [7] A.S. Chen, T. Taguchi, K. Sakai, K. Kikuchi, M.W. Wang, I. Miwa, Biol. Pharm. Bull. 26 (2003) 1326–1330. [8] D. Campaniello, A. Bevilacqua, M. Sinigaglia, M. Corbo, Food Microbiol. 25 (2008) 992–1000. [9] A.B.V. Kumar, M.C. Varadara, L.R. Gowda, R.N. Tharanathan, Biochem. J. 391 (2005) 167–175. [10] R. Avila-Sosa, E. Palou, M.T. Jiménez Munguía, G.V. Nevárez-Moorillón, A.R. Navarro Cruz, A. López-Malo, Int. J. Food Microbiol. 153 (2012) 66–72. [11] J.C. Fernandes, F.K. Tavaria, J.C. Soares, Ó.S. Ramos, M. João Monteiro, M.E. Pintado, F. Xavier Malcata, Food Microbiol. 25 (2008) 922–928. [12] E.I. Rabea, M.E.T. Badawy, C.V. Stevens, G. Smagghe, W. Steurbaut, Biomacromolecules 4 (2003) 1457–1465. [13] H.K. No, N.Y. Park, S.H. Lee, S.P. Meyers, Int. J. Food Microbiol. 74 (2002) 65–72. [14] C. Qin, H. Li, Q. Xiao, Y. Liu, J. Zhu, Y. Du, Carbohyd. Polym. 63 (2006) 367–374. [15] X.F. Liu, Y.L. Guan, D.Z. Yang, Z. Li, K. De Yao, J. Appl. Polym. Sci. 79 (2001) 1324–1335. [16] K. Li, R. Xing, S. Liu, Y. Qin, B. Li, X. Wang, P. Li, Int. J. Biol. Macromol. 51 (2012) 826–830. [17] K. Li, S. Liu, R. Xing, H. Yu, Y. Qin, R. Li, P. Li, J. Sep. Sci. 36 (2013) 1275–1282. [18] X. Meng, R. Xing, S. Liu, H. Yu, K. Li, Y. Qin, P. Li, Int. J. Biol. Macromol. 50 (2012) 918–924. [19] J.Y. Je, S.K. Kim, J. Agr. Food Chem. 54 (2006) 6629–6633. [20] Y. Peng, B. Han, W. Liu, X. Xu, Carbohyd. Res. 340 (2005) 1846–1851. [21] L.Y. Zheng, J.F. Zhu, Carbohyd. Polym. 54 (2003) 527–530. [22] H.K. No, N.Y. Park, S.H. Lee, S.P. Meyers, Int. J. Biol. Macromol. 74 (2002) 65–72. [23] R.M. Epand, R.F. Epand, J. Pept. Sci. 17 (2011) 298–305. [24] C.Z.S. Chen, S.L. Cooper, Biomaterials 23 (2002) 3359–3368. [25] I.M. Helander, E.L. Nurmiaho-Lassila, R. Ahvenainen, J. Rhoades, S. Roller, Int. J. Food Microbiol. 71 (2001) 235–244. [26] P. Eaton, J.C. Fernandes, E. Pereira, M.E. Pintado, F.X. Malcata, Ultramicroscopy 108 (2008) 1128–1134.
Contents lists available at ScienceDirect
International Journal of Biological Macromolecules journal homepage: www.elsevier.com/locate/ijbiomac
Short communication
Size and pH effects of chitooligomers on antibacterial activity against Staphylococcus aureus Kecheng Li, Ronge Xing, Song Liu, Yukun Qin, Huahua Yu, Pengcheng Li ∗ Institute of Oceanology, Chinese Academy of Sciences, Qingdao 266071, China
a r t i c l e
i n f o
Article history: Received 8 October 2013 Received in revised form 26 November 2013 Accepted 29 November 2013 Available online 7 December 2013 Keywords: Chitooligomers Size Antibacterial activity
a b s t r a c t The antimicrobial activity of chitooligomers (COSs) has attracted considerable interest but there are few reports on antibacterial activity of each individual COS. This study focuses on investigating the size and pH effects of COS on its antibacterial activity against Staphylococcus aureus. Minimum inhibitory concentrations (MICs) of five single COSs and five COS fractions with narrow degrees of polymerization (DP) were determined at different pH mediums. The results revealed that the antibacterial activity of COS required structural essential with a DP of at least 5 and the inhibitory effect increased with increasing DP. Lower pH value could enhance the antibacterial activity of COS. The COS with DP > 12 showed a MIC value of 62.5 mg/mL at pH 6.0, while the MIC value increased to 500 and 1000 mg/mL at pH 6.5 and 7.0, respectively. The cell membrane integrity assay and SEM suggested that the COS with high DP could cause bacteria clustering and further lysis of cell. © 2013 Elsevier B.V. All rights reserved.
1. Introduction Chitooligomers (COS) has been reported to possess versatile biological functions including antitumor, antioxidant, antimicrobial activity, wound healing and immune-enhancing effects [1–3] and these oligomers are safe for human body [4]. Recently tailor-made COS with well-defined degree of polymerization (DP) has drawn considerable attention in order to determine the active ingredient and better understand biological action of COS. Several COSs with single or narrow DP have been prepared and their bioactivities were evaluated. Chitohexaose has been found to exhibit the highest inhibitory effect on the proliferation and migration of tumorinduced ECV304 cells among five COSs (dimers to hexamers) [5]. Antioxidant activity of a fully deacetylated COS series with several well-defined DPs has been investigated and the function–structure relationship was revealed [6,7]. However, the antimicrobial activity of each individual chitooligomers remains unknown. As a natural and potential antimicrobial agent, COS has a wide inhibitory spectrum for not only Gram-positive and Gram-negative bacteria but also yeasts and moulds [8–10]. Antimicrobial activity of COS is closely related to its molecular weight (MW) [11,12]. However, the antimicrobial assays in prior studies were performed using heterogeneous COS with various MWs, which resulted in some controversial conclusions [13–15]. Therefore, in order to make it clear,
antimicrobial activity of COS with well-define DP is required. In this study, ten well-defined COSs including five single COSs (chitobiose to chitohexaose) and five COS fractions with narrow DPs (>6) were used for antibacterial assays against Staphylococcus aureus (S. aureus) and Escherichia coli (E. coli). In addition, the effect of pH on the antibacterial activity of COS was investigated, and cell integrity assay and scanning electron microscope (SEM) observation were carried out to probe the possible antibacterial mechanism against S. aureus. 2. Experiments 2.1. Materials Ten fully deacetylated COS fractions with well-defined DP were prepared according to our previous studies [16,17], including five single COSs, chitobiose (≥98%), chitotriose (≥98%), chitotetraose (≥98%), chitopentaose (≥98%) and chitohexaose (≥98%), and five COS fractions with narrow DP which mainly contained chitooligomers with DP6–7 (41.3%, 50.2%), DP7–8 (22.5%, 70.1%), DP9–10 (53.1%, 28.0%), DP10–12 (18.4%, 49.4%, 22.3%), and DP > 12, respectively. All other chemicals and reagents were of analytical grade and not further purified unless otherwise specified. 2.2. Antibacterial activity assay
∗ Corresponding author. Tel.: +86 532 82898707; fax: +86 532 82968951. E-mail addresses: [email protected], [email protected], xueqinwang [email protected] (P. Li). 0141-8130/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.ijbiomac.2013.11.037
The antibacterial activity of the COS series was evaluated by the minimum inhibitory concentration (MIC). MIC was determined using a turbidity-based microdilution assay in Muller Hinton broth
K. Li et al. / International Journal of Biological Macromolecules 64 (2014) 302–305
(MHB) according to the method of Meng et al. [18] with slight modification. In brief, S. aureus in the midlogarithmic growth phase was adjusted to the turbidity of Mcfarland standard 0.5 (approximately 1–2 × 108 CFU/mL). Subsequently, the suspension was diluted 1:100 and 50 L suspension was mixed with serial dilutions of COS samples stock solution (50 L) in each well of a 96-well polypropylene microplate (Corning # 3359). MIC of each sample was determined after incubating for 24 h at 37 ◦ C as the lowest oligomers concentration that completely inhibited bacteria growth. Triplicate analyses of each sample were performed and each experiment was carried out in duplicate. For the tests of antibacterial activity at different pHs, the MHB medium was adjusted to pH 6.0, 6.5, 7.0, and 8.0 respectively using 6.0 M HCl or 6.0 M NaOH. Then the corresponding MHB medium was employed to dilute bacterial suspension and to dissolve COS samples. 2.3. Assay for cell membrane integrity The cell membrane integrity of S. aureus was examined by monitoring the release of material absorbing at 260 nm as described by Je et al. [19]. 2 mL COS samples were mixed with 2 mL bacterial suspension (2 × 108 CFU/mL) in 0.1 mol/L phosphate buffer solution (PBS) to get a final concentration of 1 mg/mL. The OD values at 260 nm were determined at different times. 2.4. SEM observation SEM assay was performed as follows. The bacterial suspension was incubated with 1 mg/mL COS samples at 37 ◦ C for different times and then was transported onto a cover slip. Slideimmobilized cells were fixed with 2.5% (w/v) glutaraldehyde in 0.1 mol/L phosphate buffer solution overnight at 4 ◦ C and then washed with the same buffer. The dehydration of cells was performed with a graded ethanol series. After critical-point drying and gold coating, the samples were subjected to SEM. 2.5. Statistics Statistical evaluation was carried out using SPSS 18.0 package. Data were presented as mean ± S.D. and statistical comparisons between groups were performed using t-test. p < 0.05 was considered to be statistically significant.
303
Table 1 MIC values of chitooligomers with different DPs against S. aureus at different pHs. Sample DP
2 3 4 5 6 6–7 7–8 9–10 10–12 >12 a
MIC (g/mL) pH 6.0
pH 6.5
pH 7.0
pH 8.0
–a – – 4000 2000 2000 2000 2000 500 62.5
– – – 8000 8000 4000 4000 2000 2000 500
– – – 8000 8000 4000 4000 4000 2000 1000
– – – – – – – – – –
The MIC could not be read.
been reported that, for the chitosan with MW below 300 kDa, the antibacterial effect on S. aureus was strengthened as MW increased [21]. In agreement with previous study, our results revealed that COS with high DP showed enhanced antibacterial effect on S. aureus but a DP of at least 5 is required for the structural essential of antibacterial activity. Furthermore, it is worth noting that the inhibitory effects of COSs against S. aureus and E. coli are significantly different. It might be suggested that COS generally shows stronger bactericidal effects on Gram-positive bacteria than Gramnegative bacteria, which is consistent with prior study reported by No et al. [22]. On the other hand, our results indicated that pH played a significant role in the antibacterial action of COS. Under the basic condition, all the COS samples showed no inhibitory effect on the growth of S. aureus. In comparison, lower pH value was favorable to enhance the antibacterial activity of COS. The COS with DP > 12 shows a MIC value of 62.5 mg/mL at pH 6.0, while the MIC values increased to 500 and 1000 mg/mL at pH 6.5 and 7.0, respectively. Cationic charges have been reported to help antimicrobial agents to bind with the anionic lipid components of bacterial membrane [23]. In this study, all the samples were fully deacetylated COSs with free amino groups. MIC values of COS with DP > 12 at pH 6.0 was reduced by 16-fold compared with that at pH 7.0. This might result from the protonation of amines on the sugar chains in acid medium, which played an important role in the binding process and enhanced the antibacterial activity.
3. Results and discussion 3.1. MIC of each chitooligomers fractions The MIC values of COS with different DPs were determined using different pH MHB mediums via miniaturization of the broth dilution susceptibility test, which was employed to evaluated the size and pH effects on the antibacterial activity of COS. Table 1 showed the MIC results of COS fractions against S. aureus. Antibacterial activity of the COS with DP 4 was not observed. It seems that antibacterial responses of COS against S. aureus generally need an essential chemical structure, and a DP higher than 4 is required. Under the same pH, for those COS with DP > 4, MIC displayed a decreasing trend with increasing DP. The MIC of COS with DP > 12 decreased more than 8-fold at pH 6.0 compared with those low DP oligomers. Additionally, the antibacterial activity of these COSs against E. coli was investigated in the same way. However, no obvious inhibitory effect on E. coli was observed for most tested chitooligomers except the COS of DP > 12, with high MIC values of 1000, 8000 and 8000 mg/mL at pH 6.0, 6.5 and 7.0, respectively. Recent studies have reported that antibacterial activity of chitosan and their derivates depended on their MWs [13,20]. It has
Fig. 1. Release of cell materials absorbing at 260 nm from S. aureus suspensions treated with 1 mg/mL of chitotriose (䊉), chitohexaose () and chitooligomers with DP > 12 (), respectively. Data were presented as mean ± S.D. (n = 3) and statistical comparisons of the data between groups at 180 min were very significant (p < 0.01).
304
K. Li et al. / International Journal of Biological Macromolecules 64 (2014) 302–305
Fig. 2. SEM observation of S. aureus untreated and treated with chitooligomers with different times of incubation: (a) control bacteria, 30 min; (b) control bacteria, 2 h; (c, d) S. aureus cells treated with 1 mg/mL of chitohexaose for 30 min and 2 h; (e, f, g) S. aureus cells treated with 1 mg/mL of chitooligomers with DP > 12 for 30 min, 2 h and 24 h.
3.2. Interaction of well-defined chitooligomers with bacteria When cell membrane is damaged, cytoplasmic constituents including DNA and RNA having strong UV absorption at 260 nm will be released [24]. In this case, cell membrane integrity against
three representative COSs with well-defined DPs was examined by 260 nm release assay. As is shown in Fig. 1, chitotriose and chitohexaose could promote the release of 260 nm absorbing materials in S. aureus compared with the untreated bacterial suspension of the same CFU and chitohexaose showed a higher level. The
K. Li et al. / International Journal of Biological Macromolecules 64 (2014) 302–305
release increased at the beginning stage and ultimately reached the plateau. For the group treated by chitotriose, the release is too weak to observe an MIC value mentioned above. In contrast, upon addition of the COS with DP > 12, the OD of S. aureus suspension displayed a different and interesting trend. The curve hardly showed any changes within 1 h. However, after about 70 min, a fast release of 260 nm absorbing materials was observed. At 180 min, the OD of bacterial suspension treated with the COS of DP > 12 ultimately exceeded that of the group treated with chitohexaose. This suggested that the COS of DP > 12 should have stronger antibacterial activity compared with chitohexaose, which is in agreement with the MIC determination result. In order to further understand the mechanism, SEM study was conducted to observe directly the cell morphology change and membrane damage. As is presented in Fig. 2, the control bacteria remained intact and dispersed and the cell surfaces were smooth and integrated. After being treated with chitohexaose, no gross morphological differences were seen except that a few irregular structures on the surface of some cells appeared after 2 h (during the time of plateau in Fig. 1). We inferred that chitohexaose might increase the permeability of the cell membranes and led to the increasing release of 260 nm absorbing materials but did not kill the cells. In contrast, after being exposed to COS with DP > 12 for 30 min, the cells existed as small clusters and convex cell surfaces were visible. At 2 h, the clusters observed became even larger and much viscous substances among microbial cells appeared, which should be cytoplasmic content resulting from lysis of the cell. These results were consistent with the 260 nm release assay. After being exposed to COS with DP > 12 for 24 h, the cell collapse was observed (Fig. 2g). Previous studies reported that cationic chitosan could cover the bacterial cell, prevent the nutrition transport and finally result in cell apoptosis [25,26]. In our experiments, conglutination between bacterial cells was observed in high-DP COS treated group and after a certain time of incubation, a fast release of 260 nm absorbing materials was presented. It seemed that the cationic oligomers could “glue” negatively charged cells and led to the formation of large bacteria cluster, which might block the nutrition transport of bacterial cell and resulted in impairment of vital bacterial activity. Subsequently, with time of incubation increasing, the cell lysis occurred and much 260 nm absorbing materials were released. However, the “glue” interaction seemed to require an enough size of COS. 4. Conclusion In this paper, antibacterial activity of ten COSs with single or narrow DP was investigated and the related mechanism was explored using cell integrity assay and SEM observation. Size and pH of COS played significant roles in its antibacterial activity. It is concluded as follows: (i) lower pH value could enhance the antibacterial activity of each well-defined COS fraction against S. aureus; (ii)
305
antibacterial activity of COS required structural essential with a DP of at least 5 and the MICs of high-DP COSs, especially those oligomers with DP > 12, were obviously lower than those of low-DP COSs; (iii) cell membrane integrity assay and SEM suggested that the high-DP COS could cause the bacteria clustering and further led to lysis of cell. Acknowledgments The study was supported by the National High Technology Research and Development Program (“863”Program) of China (2011AA09070405), the commonweal item of State Oceanic Administration People’s Republic of China (201305016), National Key Technologies R&D Program (2011BAE06B04), the Action Plan of CAS to Support China’s New and Strategic Industries with Science and Technology, Guangdong Province & Chinese Academy of Sciences overall strategy cooperative project (2011A090100035). References [1] B.B. Aam, E.B. Heggset, A.L. Norberg, M. Sørlie, K.M. Vårum, V.G.H. Eijsink, Mar. Drugs 8 (2010) 1482–1517. [2] S. Kim, N. Rajapakse, Carbohyd. Polym. 62 (2005) 357–368. [3] R.A.A. Muzzarelli, Carbohyd. Polym. 76 (2009) 167–182. [4] C.Q. Qin, J.N. Gao, L.S. Wang, L.T. Zeng, Y. Liu, Food Chem. Toxicol. 44 (2006) 855–861. [5] C. Xiong, H. Wu, P. Wei, M. Pan, Y. Tuo, I. Kusakabe, Y. Du, Carbohyd. Res. 344 (2009) 1975–1983. [6] K. Li, R. Xing, S. Liu, R. Li, Y. Qin, X. Meng, P. Li, Carbohyd. Polym. 88 (2012) 896–903. [7] A.S. Chen, T. Taguchi, K. Sakai, K. Kikuchi, M.W. Wang, I. Miwa, Biol. Pharm. Bull. 26 (2003) 1326–1330. [8] D. Campaniello, A. Bevilacqua, M. Sinigaglia, M. Corbo, Food Microbiol. 25 (2008) 992–1000. [9] A.B.V. Kumar, M.C. Varadara, L.R. Gowda, R.N. Tharanathan, Biochem. J. 391 (2005) 167–175. [10] R. Avila-Sosa, E. Palou, M.T. Jiménez Munguía, G.V. Nevárez-Moorillón, A.R. Navarro Cruz, A. López-Malo, Int. J. Food Microbiol. 153 (2012) 66–72. [11] J.C. Fernandes, F.K. Tavaria, J.C. Soares, Ó.S. Ramos, M. João Monteiro, M.E. Pintado, F. Xavier Malcata, Food Microbiol. 25 (2008) 922–928. [12] E.I. Rabea, M.E.T. Badawy, C.V. Stevens, G. Smagghe, W. Steurbaut, Biomacromolecules 4 (2003) 1457–1465. [13] H.K. No, N.Y. Park, S.H. Lee, S.P. Meyers, Int. J. Food Microbiol. 74 (2002) 65–72. [14] C. Qin, H. Li, Q. Xiao, Y. Liu, J. Zhu, Y. Du, Carbohyd. Polym. 63 (2006) 367–374. [15] X.F. Liu, Y.L. Guan, D.Z. Yang, Z. Li, K. De Yao, J. Appl. Polym. Sci. 79 (2001) 1324–1335. [16] K. Li, R. Xing, S. Liu, Y. Qin, B. Li, X. Wang, P. Li, Int. J. Biol. Macromol. 51 (2012) 826–830. [17] K. Li, S. Liu, R. Xing, H. Yu, Y. Qin, R. Li, P. Li, J. Sep. Sci. 36 (2013) 1275–1282. [18] X. Meng, R. Xing, S. Liu, H. Yu, K. Li, Y. Qin, P. Li, Int. J. Biol. Macromol. 50 (2012) 918–924. [19] J.Y. Je, S.K. Kim, J. Agr. Food Chem. 54 (2006) 6629–6633. [20] Y. Peng, B. Han, W. Liu, X. Xu, Carbohyd. Res. 340 (2005) 1846–1851. [21] L.Y. Zheng, J.F. Zhu, Carbohyd. Polym. 54 (2003) 527–530. [22] H.K. No, N.Y. Park, S.H. Lee, S.P. Meyers, Int. J. Biol. Macromol. 74 (2002) 65–72. [23] R.M. Epand, R.F. Epand, J. Pept. Sci. 17 (2011) 298–305. [24] C.Z.S. Chen, S.L. Cooper, Biomaterials 23 (2002) 3359–3368. [25] I.M. Helander, E.L. Nurmiaho-Lassila, R. Ahvenainen, J. Rhoades, S. Roller, Int. J. Food Microbiol. 71 (2001) 235–244. [26] P. Eaton, J.C. Fernandes, E. Pereira, M.E. Pintado, F.X. Malcata, Ultramicroscopy 108 (2008) 1128–1134.
