In vitro activity of water-soluble quaternary chitosan chloride salt against E. coli
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- Silva, L.P.d., de Britto, D., Seleghim, M.H.R. et al. World J Microbiol Biotechnol (2010) 26: 2089. doi:10.1007/s11274-010-0378-7
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The antibacterial effect of the N,N,N-trimethyl chitosan chloride salt (TMC) was evaluated against the bacterium Escherichia coli. The derivative was prepared via reaction of chitosan with dimethyl sulfate in the presence of NaOH and the Minimum Inhibitory Concentration was assessed by measuring the changes in turbidity and by counting of Colony Forming Units (c.f.u.). The results indicated good antibacterial activity against E. coli for all concentration of TMC tested (20.0; 7.5 and 3.5 mg/l) over 7 h of incubation. From the data, the ideal lethal concentration was determined as 20.0 mg/l.
KeywordsEscherichia coliChitosanWater-soluble chitosanMinimum inhibitory concentration
Chitosan is an unbranched homopolymer of glucosamine that occurs naturally or can be derived from chitin, an abundant by-product of seafood processing, via deacetylation in the presence of alkali. The term chitosan is usually referred to a family of copolymers with predominant fractions of deacetylated units with structure consisting of poly(2-amino-2-deoxy-D-glucopyranose) bonded by β(1 → 4) linkages, although, a completely deacetylated material is rarely obtained and small fractions of 2-acetamido-2-deoxy-D-glucopyranose units are generally present. Since chitosan is a biocompatible, biodegradable, antimicrobial and noncytotoxic polymer, it has been extensively evaluated as a potential material for uses in food, agriculture and biomedical applications (Kumar 2000; Marques et al. 2008).
One of the advantages of TMC is that this derivative is a cationic polyelectrolyte soluble over a wide pH range, while the parent chitosan is a weak base soluble only in dilute aqueous acidic solutions below its pKa (~6.3), i.e., it is insoluble in water. TMC has good biocompatibility and shows excellent intestinal absorption, being suggested for uses as a carrier for oral drug delivery (Bowman and Leong 2006) and for support for intranasal vaccine (Amidi et al. 2007). The antibacterial effect of methylated chitosan has been also reported to be more pronounced than the parent chitosan against a broad spectrum of bacteria (Jia et al. 2001; Sadeghi et al. 2008).
Several mechanisms have been suggested for the antimicrobial activity of these polysaccharides, but the most accepted model is that related to their polycationic nature (Shahidi et al. 1999; Goy et al. 2009). In this sense, the electrostatic interaction between positively charged quaternary R–N(CH3)3+ sites and negatively charged microbial cell membranes is the driving force for this antimicrobial activity (Tsai and Su 1999). Also, this complexation blocks Ca2+ absorption by the electronegative sites of the membrane surface, suppressing nutrients essential to microbial growth (Jia et al. 2001; Goy et al. 2009).
These properties make quaternized water-soluble chitosan widely feasible for applications in the biomedical fields such as for tissue and bone engineering (Shi et al. 2006; Ji et al. 2009), wound healing and also for food applications (Britto and Assis 2007b; Belalia et al. 2008).
The aim of the present investigation was to estimate the Minimum Inhibitory Concentration (MIC) of the TMC, synthesized via reaction with dimethyl sulfate, against the gram-negative bacterium E. coli, obtaining useful information for medical and food uses of this derivative.
Materials and methods
Methylation of chitosan
Chitosan from Sigma Aldrich® Co (medium molecular weight, 80% deacetylated) was used as starting material for the preparation of TMC. The methylation was carried out at 70 °C with dimethyl sulfate (from Vetec, R. Janeiro, Brazil). The basic reaction sequence consisted in the suspension of 1 g of chitosan (0.005 mol) in 16 mL of dimethyl sulfate and 4 mL of deionized water. About 1.2 g of NaOH (0.015 mol) and 0.88 g of NaCl (0.015 mol) were added and the dispersion kept under magnetic stirring. The resulting derivative was submitted to dialysis in a cellophane membrane (cut-off 12,000–14,000 g/mol) and the final product obtained by precipitation was washed with acetone. Details of the process and the derivative characterization can be found elsewhere (Britto and Assis 2007a).
Assays for antimicrobial activity
Escherichia coli (ATCC 8739, provided by Fundação Tropical André Tosello, Campinas, Brazil), was used to study the antimicrobial activity of methylated chitosan derivative. The E. coli pre-culture was incubated under aerobiosis and moderate shaking at 37 °C and transferred to an Erlenmeyer (500 mL) containing 142.5 mL of a synthetic broth medium. The bacterial suspensions used in the MIC tests were prepared using a twofold dilution series from suspensions of 9 mL of sterile distilled water plus (PBS) incorporating aliquots of 1 mL of bacterial broth. PBS (Phosphate-buffered saline), pH 7.4, consisted of Na2HPO4 (1.23 g), NaH2PO4–H2O (0.4 g), NaCl (8.5 g), and distilled water to 1 L.
The inhibitory effects of the TMC on the growth of E. coli were estimated both by turbidity and by colony-forming units (c.f.u.). In the first case, the growth of bacteria was determined by optical density, as measured at 600 nm every 30 min using a Pharmacia Biotech, model Novaspec II spectrophotometer.
For c.f.u. determination, the Drop Count Method was used (Collins et al. 2004). For this procedure, 1 mL of the mixture solution was diluted by 9 mL-fold and added to agar-based culture substrate. Six micro drops (5 μL) were plated on each single Petri dishes and colonies grown at 37 °C for 24 h. TMC was added to agar medium to give a final concentration of 20.0, 7.5 and 3.5 mg/L. Three replicate plates were used for each concentration. After incubation, the residual colonies were counted every hour to estimate bactericidal activity and expressed as c.f.u./mL.
Results and discussion
The TMC addition of 3.5 mg/L resulted in little inhibitory effect, with similar behavior to the control, but with turbidity decline after around 3 h of the stationary phase. From the turbidity assays, it is suggested that the best inhibitory concentration and the most probable MIC of TMC for E. coli was between 20.0 and 7.5 mg/L.
Although the turbidity method offered the possibility of a rapid detection of microbial growth in medium, it does not assure the absolute count of microorganisms. This method is useful for characterizing the initial growth (Lag and Log phases) but introduce errors in the subsequence measurements since dead cells remain in the medium, interfering in the optical density reading.
The decline phase (where the number of dying cells far exceeds the number of new cells formed) is clearly observed from the c.f.u. counts for all TMC concentrations while for culture medium with no addition of TMC, the bacterial growth was sustained during the period analysed.
In order to quantify the reduction and/or the stimulation to E. coli growth under the effect of different TMC concentrations and find numerically the best inhibitory concentration, the absorbance and c.f.u. curves can be mathematically derived as a function of the time. Thus, MIC can be expressed as the concentration as which the bacterial population (N) neither grows nor decreases, i.e., a steady state with dN/dt = 0. Otherwise, the maximum specific growth can be derived by calculating the first derivative at the inflection point (dN/dt = maximum).
A reduction of about 50% was found in the If value for the sample with TMC at 20.0 mg/L. On the other hand, for the sample with TMC at 7.5 mg/L the If was very small, only 2%. For the culture with the lowest addition of TMC (3.5 mg/L), a contrary effect is recorded. In such concentration, an increase in bacterial growth greater than 50% was observed. This can be explained considering that at this concentration the polysaccharide underwent transformation in nutrient medium which supported initial growth instead of inhibiting it. It is worth noticing, however, that after 7 h exposure, for all TMC concentrations tested, the number of living cells was reduced to almost zero (Fig. 3).
According to these results, the better MIC value for TCM is effectively around 20.0 mg/L, what is about 5–20 times more effective against E. coli than those reported in the literature for commercial medium molecular weight chitosan (Tsai et al. 2002; Seo et al. 2008). Since the main accepted mechanism is based on electrostatic interaction, it suggests that the greater the number of quaternary amino sites, the higher is the antimicrobial activity. It is important to highlight the influence on the antibacterial activity of the methyl moieties in the amino site. As pointed out by Jia et al. (2001), the alkyl chain length strongly affects the antibacterial activity. In fact, mechanisms such as changes in the membrane wall permeability (Shahidi et al. 1999) and hydrolysis of the peptidoglycans of the microorganism wall are undoubtedly affected by net positive charge and the spatial distribution of these charges alongside the polymeric chain and the arrangement of mono, di and trimethylated amino sites.
N,N,N-trimethyl chitosan (TMC) was shown to have an important inhibitory action against Gram-negative bacteria in tests conducted on E. coli. The results of this study indicate that the apparent minimum inhibitory concentrations of TMC range around 20.0 mg/L. Such satisfactory activity at a very low concentration serves as an indicator to the use of chitosan derivatives in treatment of infectious diseases and enhances potential uses, such as the development of active packaging materials and food applications.
The authors gratefully acknowledge the support of Embrapa (Rede AgroNano), FAPESP and CNPq (Brazilian Science Agencies).