Correspondence: Tao Zhou, School of Food Science and Biotechnology, Zhejiang Gongshang University, Hangzhou, Zhejiang 310012, China. Tel.: +86 571 8807 1024, ext. 7587; fax: +86 571 8890 5733; e-mail: firstname.lastname@example.org
The antimicrobial activity of the iron(III)-selective 3-hydroxypyridin-4-one chelators, CP251(1) and CP252(2), was evaluated in comparison with that of diethylenetriamine-penta acetic acid (3). CP251 was found to exhibit an inhibitory effect on the growth of both Gram-positive and Gram-negative bacteria. CP251 may find application in the treatment of external infections such as those associated with wounds.
Iron is an essential element for the growth of virtually all bacteria and fungi. Thus, limiting the amount of available iron should in principle inhibit microbial growth (Bezkorovainy, 1980; Lewin, 1984). Most microorganisms have developed efficient methods of absorbing iron from the environment and many microorganisms secrete siderophores in order to scavenge iron (Hider & Kong, 2010). Such methods of uptake can be circumvented by the introduction of high-affinity iron-selective chelating agents. However, the affinity of these agents for iron must be extraordinarily high, enabling them to compete efficiently with siderophores.
One problem with the concept of using iron chelators as antimicrobial agents is that they may interfere with the immunodefence centred on endothelial cells and phagocytes. The presence of a low level of relatively accessible iron is essential for the formation of hydroxyl radicals during the respiratory burst of such cells (Baggiolini, 1984; Halliwell & Gutteridge, 1984). Thus iron-chelating strategies are not likely to be successful with systemic application of chelators. However topical application of chelators will not suffer from such a disadvantage and may find cosmetic application, use in wound healing and in the treatment of nail infections.
8-Hydroxyquinoline and related compounds were first demonstrated to possess antimicrobial properties over 50 years ago (Albert et al., 1947; Lowe & Phillips, 1962). More recently, the hexadentate chelator N,N′-ethylenebis[2-(2-hydroxyphenyl)-glycine] has been found to exhibit moderate-to-good activity against isolates of pathogenic bacteria and fungi, whereas EDTA and diethylene-triamine pentaacetic acid (DTPA) revealed weaker activity (Bergan et al., 2001). Chew et al. (1985) reported that EDTA possessed strong activity against Gram-positive bacteria but was much less effective against Gram-negative bacteria. Indeed, DTPA was more growth inhibitory than EDTA against the Gram-negative bacteria. The antimicrobial activity of iron(III) chelators has been investigated by a number of groups over the past decade, but the majority of this effort has been directed to bidentate chelators (Jain et al., 2005; Banin et al., 2006; Gademann et al., 2007; Zhang et al., 2007), which generally possess a lower affinity for iron than their hexadentate analogues (Liu & Hider, 2002).
The chelating moiety, 3-hydroxypyridin-4-one, by virtue of possessing a high affinity and selectivity for iron(III), has been considered for several therapeutic applications (Liu & Hider, 2002). Its bidentate form, deferiprone, is an effective orally active iron chelator and has been widely used for the treatment of iron overload associated with β-thalassaemia (Balfour & Foster, 1999; Maggio, 2007; Porter, 2009). Hexadentate analogues of deferiprone possess a much higher affinity for iron(III) (Piyamongkol et al., 2005; Zhou et al., 2006), and thus, are predicted to inhibit the growth of a wide range of bacteria. Recently, we reported the synthesis of two such molecules: CP251 and CP252. CP251 was found to possess a very high affinity for iron(III) (Piyamongkol et al., 2005). Herein, we wish to report the inhibitory activity of these two compounds against several bacterial species.
Materials and methods
Preparation of antimicrobial agents
Hydrochloride salts of CP251 and CP252 were synthesized from methyl maltol as described in our previous publication (Piyamongkol et al., 2005). DTPA was purchased from Sigma. All compounds were tested in triplicate at several appropriate concentrations for their antimicrobial effects against major putrefaction bacteria. The solution of these compounds was prepared by dissolving the chelators in deionized water. CP251·4HCl was easily dissolved in deionized water, while DTPA solution was obtained only with heating, and the CP252·3HCl solution was obtained by suspending the compound in deionized water followed by exposure to ultrasound for 10 min. The solutions were stored at 4 °C.
The chemical structures of compounds 1, 2 and 3 are shown in Figure 1.
Pseudomonas aeruginosa, Staphyloccocus aureus and Escherichia coli were purchased from CGMCC. Bacillus subtilis, Bacillus cereus and Vibrio parahaemolyticus were separated from mussels. All bacteria were inoculated in a tube containing an inclined plane of brain–heart Infusion (BHI) agar and cultured at 37 °C for 24 h. This gel was then used to inoculate into 5 mL of BHI broth and incubated at 37 °C for 24 h before transferring 50 μL into another tube of fresh BHI broth. This transfer was incubated at 37 °C to an OD of P. aeruginosa, S. aureu, V. parahaemolyticus, and E. coli of approximately 104 CFU mL−1, B. subtilis and B. cereus to approximately 107 CFU mL−1.
Isolation and identification of B. subtilis, B. cereus and V. parahaemolyticus
Mytilus edulis linne was obtained from a local fishing company and was transported to the laboratory on ice. Samples of 25 g muscle were homogenized in 250 mL of 0.1% physiological peptone salt [PFZ 0.85%NaCl (w/v) and 0.1% peptone (w/v)] for 60 s in a stomacher bag. Suitable decimal dilutions were pour-plated on modified plate count agar (PCA) for bacteria species. PCA agar plates were incubated for 48 h at 30 °C. Representative colonies were picked up randomly and purified by repeatedly streaking on appropriate agar medium. The isolates were identified following the criteria outlined in Bergey's Manual of Systematic Bacteriology (Holt & Krieg, 1994). Further characterization and confirmation was carried out using a 6850 automated identification method (MIDI) and PCR identification method.
All assays were cultured at 37 °C for 24 h in 15 × 75-mm tubes. The incubation medium was BHI broth. All tubes contained 80 μL of antimicrobial agent (except for controls, which contained 80 μL of sterilized water), 20 μL of bacterial inoculum, with a total volume of 100 μL. After incubation, 900 μL of sterilized water was added to each tube to make a 10−1 dilution (Fig. 2). Other dilutions were carried out in sterilized water and ranged from 10−2 to 10−6, depending on the degree of bacterial growth. The number of viable bacteria in each tube was determined in triplicate. They were plated on BHI agar using 50 μL volumes in triplicate. The number of colonies on agar was counted on a light board after incubation at 37 °C for 24 h. The antimicrobial effects of the tested compounds with different concentrations were compared with the appropriate controls by anova. Similar comparisons were also made among different compounds within each concentration tested. The bactericidal rate is calculated as follows:
where R is the bactericidal rate, X0 the number of bacteria before the treatment with chelator, and Xt the number of bacteria after the treatment with chelator.
Inhibition of the three Gram-positive bacteria S. aureus, B. subtilis and B. cereus by the three chelators is illustrated in Fig. 3. As shown in Fig. 3a, CP251 completely inhibited the growth of S. aureus at 500 μg mL−1, indicating that CP251 can be bactericidal against S. aureus at this concentration, while at the same concentration, DTPA decreased the growth of S. aureus from 3.2 × 104 to 8.5 × 102 CFU mL−1, yielding a bactericidal rate of 97.3%. CP252 decreased the growth of S. aureus to 8.75 × 103 CFU mL−1, indicating a bactericidal rate of 72.7%. DTPA exhibited marked inhibition against B. subtilis isolated from mussel, decreasing the growth of B. subtilis from 4.5 × 107 to 2.2 × 106 CFU mL−1 at 1000 μg mL−1 (the bactericidal rate was 95.1%) and to 1.4 × 103 CFU mL−1 at 1500 μg mL−1 (the bactericidal rate was almost 100.0%). The inhibitory effects of CP251 and CP252 were found to be much weaker at 1500 μg mL−1. However, at a concentration of 3000 μg mL−1, CP251 and CP252 both showed a marked inhibitory effect on the growth of the bacterium, decreasing the growth of B. subtilis from 4.5 × 107 to 8.1 × 103 and 4.2 × 104 CFU mL−1, respectively. The bactericidal rate of both compounds at this concentration was close to 100.0% (Fig. 3b). However, all three chelators were found to have only a weak inhibitory influence against B. cereus. CP251, DTPA and CP252, respectively, decreased the growth of B. cereus from 7.45 × 107 to 1.35 × 107, 1.64 × 107 and 1.89 × 107 CFU mL−1 at 2000 μg mL−1, the corresponding bactericidal rates being 81.9%, 78.0% and 74.6% (Fig. 3c).
Inhibition of the chelators against three Gram-negative bacteria P. aeruginosa, V. parahaemolyticus and E. coli is illustrated in Fig. 4. CP251 completely inhibited the growth of P. aeruginosa at a concentration of 100 μg mL−1, indicating that CP251 is bactericidal against P. aeruginosa, while DTPA decreased the growth of P. aeruginosa from 2.75 × 104 to 3.8 × 103 CFU mL−1 at 100 μg mL−1, indicating a bactericidal rate of 86.2%. CP252 decreased the growth of P. aeruginosa from 2.75 × 104 to 8.45 × 103 CFU mL−1 at 100 μg mL−1, generating a bactericidal rate of 69.3% (Fig. 4a). Compared with S. aureus, the chelators inhibited the growth of P. aeruginosa more effectively. CP251 strongly inhibited the growth of V. parahaemolyticus (Fig. 4b). At a concentration of 500 μg mL−1, CP251 decreased the number of bacteria from 7.84 × 104 to 3.60 × 102 CFU mL−1 (the bactericidal rate was 99.5%). While the DTPA decreased the growth of V. parahaemolyticus from 7.84 × 104 to 8.90 × 103 CFU mL−1 (the bactericidal rate was 87.4%), and CP252 caused a decrease in growth of V. parahaemolyticus from7.84 × 104 to 2.21 × 103 CFU mL−1 (the bactericidal rate was 95.9%) at the same concentration. CP251 also effectively inhibited the growth of E. coli, decreasing the number of bacteria from 3.76 × 104 to 1.62 × 102 CFU mL−1 (the bactericidal rate was 99.6%) at a concentration of 250 μg mL−1. However, DTPA decreased the growth of E. coli from 3.76 × 104 to 5.60 × 102 CFU mL−1 (the bactericidal rate was 98.5%), and CP252 decreased the growth of E. coli from 3.76 × 104 to 7.90 × 103 CFU mL−1 (the bactericidal rate was 79.0%) (Fig. 4c).
In each case, CP251 was found to be the most effective inhibitor with the Gram-negative bacteria.
It is generally accepted that the iron chelators inhibit microbial growth by reducing iron absorption by microorganisms. Based on this concept, the higher the iron-binding constant for the iron chelator, the stronger the predicted antimicrobial activity. However, N,N′-bis(2-hydroxybenzyl)ethylenediamine-N,N′-diacetic acid, possessing very high affinity for iron (logK=40), was found to be a relatively weak inhibitor of bacteria (Chew et al., 1985), clearly indicating that the composition of the microorganism's cell wall and the physical nature of the iron chelator also affect the inhibition of bacterial growth.
A broad range of structurally different siderophores are produced by Gram-positive and Gram-negative bacteria (Hider & Kong, 2010). Siderophores can extract iron from various other soluble and insoluble iron compounds, such as ferric citrate, ferric phosphate, Fe-transferrin, ferritin, iron bound to sugars and glycosides or even from synthetic chelators such as EDTA and nitrilotriacetate. Catecholate siderophores predominate in certain Gram-negative genera, such as Enterobacteria and the genus Vibrio, the reasons for this being manifold, including complex stability, high environmental pH and a weak capability for nitrogen metabolism (Winkelmann, 2002).
The Gram-positive Streptomycetes produces hydroxamate-type ferrioxamines and the ascomycetous and basidiomycetous fungi synthesize ester- and peptide-containing hydroxamate siderophores that are acid-stable and well-suited for environmental iron solubilization. Both the Streptomycetes and fungi show a versatile nitrogen metabolism with active N-oxygenases (Winkelmann, 2002). Because of structurally different siderophores and different cell wall types, it can be expected that iron(III)-selective chelators will have a differential influence on a range of bacteria.
Of the three iron(III)-selective chelators investigated, CP251 was found to possess the strongest antimicrobial activity, followed by DTPA and CP252. This is primarily due to the fact that CP251 has a higher affinity for iron(III) (logK=30.7, pFe=30.5) than DTPA (logK=28.6) (Sohnle et al., 2001). CP252 had a lower inhibitory effect against bacteria probably because of its poor water solubility. The lower activity of CP251 against Gram-negative bacteria is consistent with the notion that the outer membrane of Gram-negative bacteria limits the penetration of compounds with molecular weight above the cutoff point of 500–600 (Hancock & Nikaido, 1978). The molecular weight of CP251 is 557. The iron(III)-selective chelators were found to possess a lower activity against the two Bacillus species studied. This finding is almost certainly related to the ability of Bacillus to utilize a wide range of iron complexes including haem (Heinrichs et al., 2004).
Surprisingly, in the case of B. subtilis, DTPA exhibited the strongest inhibitory activity among the three chelators. This was probably caused by the fact that DTPA is not a selective chelator, binding not only iron but bivalent ions including Ca2+. Calcium is essential for the membrane integrity of Bacillus species. CP251 and CP252 are iron(III)-selective and do not bind Ca2+ ions.
In summary, CP251 possesses strong inhibitory activity against the growth of both Gram-positive and Gram-negative bacteria and therefore has potential as an antimicrobial agent, particularly in the treatment of external infections and with food preservation.
The financial support by National Natural Science Foundation of China (No. 20972138), Scientific Research Foundation for the Returned Overseas Chinese Scholars, State Education Ministry of China and Qianjiang Scholars Fund, Zhejiang Province (No. 2010R10051) is gratefully acknowledged.