antibacterial activity of ag-, zn-, cd-, ba- modified ...comptes rendus de l’acad emie bulgare des...
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Доклади на Българската академия на наукитеComptes rendus de l’Academie bulgare des Sciences
Tome 67, No 11, 2014
BIOLOGIE
Biochimie
ANTIBACTERIAL ACTIVITY OF Ag-, Zn-, Cd-, Ba-MODIFIED NATURAL CLINOPTILOLITE AGAINST
ESCHERICHIA COLI
Louiza Dimowa, Stela Atanassova-Vladimirova∗, Iskra Piroeva∗,Bogdan Ranguelov∗, Boris L. Shivachev
(Submitted by Academician I. Gutsov on September 19, 2014)
Abstract
This study investigates the antibacterial effect of Ag-, Cs-, Zn-, Cd-, Ba-ion exchanged zeolite on Escherichia coli. A natural tuff rich in Clinoptilolitewith common formulae (Na2,K2,Ca)3Al6Si30O72 · 24H2O (from the Beli Plastore deposit Kardzhali, Bulgaria) was treated by heavy liquid separation toyield a nearly pure Clinoptilolite form. Modified Ag- Cs- Zn-, Cd-, Ba Clinop-tilolite was obtained by ion exchange and structurally characterized by X-raydiffraction (XRD) and Scanning electron microscopy (SEM). The antibacterialactivity of the modified forms was assessed by testing against Escherichia coliBL21 bacterial strain using a disc diffusion assay. The results show that onlyAg- and Cd- exchanged Clinoptilolite forms inhibit the growth of E. coli inconcentration below 10 µg ml−1 (corresponding to ∼ 0.093 and ∼ 0.048 µgml−1 for Ag and Cd Clinoptilolite forms). It is well known that Ag and Baions possess antibacterial properties, and that they are especially suited fordisinfection purposes.
Key words: clinoptilolite, ion exchange, antibacterial activity, SEM
Introduction. Natural zeolites are microporous materials and their struc-ture consists of a framework (alumosilicate), cations balancing the framework
The authors thank for the financial support by the Bulgarian National Science Fund throughcontract DRNF 02/1. The authors are grateful for the financial support of Project BG051PO001-3.3.06-0038 and BG051PO001-3.3.06-0027 funded by OP Human Resources Development 2007–2013 of EU Structural Funds.
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charge excess and water molecules [1]. Their pores (forming channels and cavi-ties) [2] contain various metal ions (like Na, K, Ca, Cs, etc.) that compensate thecharge of the framework. Zeolites are of special interest due to the variety of waysin which these ions can be exchanged [3,4] and synthetic analogues can be thusdesigned and synthesized [5]. Although there is a great diversity of metal ionspresent in natural zeolites they are not harmful to adjacent biological species [6].However, it is known that some metal ions have specific biological activity andin higher concentrations are even toxic [7]. This can be exploited by exchangingthe zeolite cations with a specific metal. The aim of the study is to exchangenatural Clinoptilolite cations with metals that will show antimicrobial effect. In-deed infections caused by multidrug resistant bacteria are spread worldwide andare an emerging medical problem, particularly amongst immune compromisedpatients and those hospitalized in intensive care units. Both Gram positive andGram negative bacteria have developed high level resistance to multiple classesof antibacterial agents. These include methicillin resistant Staphylococcus aureus(MRSA), Pseudomonas aeruginosa, clinical isolates of Acinetobacter baumannii,Escherichia coli and vancomycin resistant enterococci (VRE) [8–11]. Few avail-able drugs such as linezolid [12], tedizolid [13] and some newer glyco peptides areactive against MRSA and VRE, but the respective rates are variable [14]. Assuch, there is a need to explore other sources of effective antibacterial compoundsto augment the limited choice of medicines for therapeutic treatment.
In the present study, alkaline and transition metal ion exchange has beenperformed with natural Clinoptilolite. The thus obtained Ba-, Cs-, Ag-, Zn-,Cd- Clinoptilolite samples were examined for potential antibacterial activities byusing disc diffusion assays.
Materials and methods. Ion exchange. The starting material is atuff from Beli Plast (Kardzhali, Bulgaria) ore deposit. The tuff was subjectedto a purification procedure in order to remove additional minerals [15]. Theremaining material is high purity natural Clinoptilolite (Cp). The ion exchangewas conducted in Teflon autoclaves using double distilled water (ddH2O). For thepreparation of the exchanged Clinoptilolite forms, 1 M solutions of BaCl2, CsCl,AgNO3, ZnCl2, CdCl2 (Sigma-Aldrich) were used.
Cation exchange. Typically 0.1 g of Cp was added to 25 ml of a 1 Mcation solution and thus prepared mixture was heated to 100 ◦C. After 48 h thecation solution was removed and replaced with a fresh one. This procedure (sup-plying fresh 1 M solutions) was performed twice for a total exchange time of192 h (8 days). To the end of the procedure the solution was removed and theexchanged Clinoptilolite was centrifuged and washed several times with ddH2O.The resulting cation exchanged forms were dried at room temperature.
Scanning electron microscopy (SEM) and energy dispersive X-rayspectroscopy (EDS). SEM analyses were performed on a JSM 6390 electronmicroscope (JEOL, Japan) coupled with energy dispersive X-ray spectroscopy
1532 L. Dimowa, S. Atanassova-Vladimirova, I. Piroeva et al.
(EDS, Oxford INCA Energy 350) equipped with an ultrahigh resolution scanningsystem (ASID-3D) in regimes of secondary electron image (SEI). The sample(cover slip) was mounted on a double coated conductive carbon tape that holdsthe sample firmly to the stage surface and can be used as a ground strap fromthe sample surface to the sample holder. The samples were gold coated (time ofcoating ∼ 30 sec). This necessary thickness of gold layer resulted in a decent imagequality without causing electric charging. With thinner gold films (decrease ofcoating time below 30 sec) electric charging was observed [16]. When the coatingtime was longer (more than 40 s), the gold layer was thicker but no improvementof image quality was observed. The accelerating voltage was adjusted to 20 kV,I ∼ 65 µA. Lower voltages (e.g. 10 kV) resulted in loss of contrast while highervoltages led to rapid degradation of the imaging [17]. The pressure was of theorder of 10−4 Pa.
Powder X-ray diffraction. The XRD investigations were performed onan X-ray powder diffractometer D2 Phaser, Bruker AXS using Cu Kα radiation,with a step size 0.04 ◦C and a collection time of 2 s per step.
Antibacterial testing. The synthesized compounds were screened for po-tential antibacterial activity by testing against Escherichia coli BL21 bacterialstrain using a disc diffusion assay [18]. Briefly, a 100 µl of an overnight bacterialculture of BL21 strain was suspended in sterile LB media (approximate concen-tration OD 0.9) before being inoculated evenly on the entire surface with 1.5%agar LB with a sterile inoculation spreader. Sterilized paper discs (4 mm diame-ter) were impregnated with 2 µl of CpAg, CpCd, CpCs, CpBa and CpZn solutions(360 µg µl−1) and were placed on the inoculated agar plate. DMSO discs wereused as a negative control in the test. The diameter of the inhibition zone aroundthe impregnated paper disc was measured after 18 h of incubation at 37 ◦C. Alltests were performed in duplicate.
The SEM/EDS analyses were performed in the laboratory of Electron mi-croscopy at IPC – BAS while all remaining experiments were carried out at IMC –BAS.
Results and discussion. The most employed technique used to modifyzeolites is ion exchange. Ion-exchange properties of natural Clinoptilolite havealso been well studied [1,3]. As some of the 1M Me solutions (Me = BaCl2,CsCl, AgNO3, ZnCl2, CdCl2) have a slightly acidic character (pH around 4.7 atroom temperature) the duration of the ion exchange stage cannot be prolongedindeterminately due to the damage and destruction of the Clinoptilolite crystalphase. The selectivity of ion exchangers (in our case Clinoptilolite), is usuallydefined as the selection by the ion exchanger of one counter ion in preference tothe other. The selectivity of Clinoptilolite for different metal cations is reportedin several studies [19–22]. The reason for the observed differences in the selectivitymay be attributed not only to the different experimental conditions but also tothe chemical composition of the Clinoptilolite employed.
Compt. rend. Acad. bulg. Sci., 67, No 11, 2014 1533
Fig. 1. XRD patterns of natural and exchanged samples
The XRD powder patterns of the studied ion exchanged Clinoptilolites arepresented in Fig. 1. They do not show noticeable structural degradation of theClinoptilolite crystal phase after the ion exchange process. The differences in peakintensity observed on the diffractograms indicate that the cation exchange hasoccurred. This observation is further corroborated by performed SEM observation(Fig. 2) and EDS chemical composition analyses (Table 1).
According to the EDS data the conducted cation exchange (with 1 M solu-tions) of the natural Clinoptilolite form resulted in an almost complete displace-ment of the Na and Ca cations by Cs, Ag, Cd and Ba ions. In addition Cd and Bacations also displace the K+ ion. The small Mg2+ cation remains in the Cp struc-
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Fig. 2. SEM morphology of natural Clinoptilolite of Ag, Cd, Cs, Ba and Zn exchanged forms
T a b l e 1
EDS data for natural and cation exchanged forms
Sample Exchanged cation Chemical composition
Cp — (Na0.91Ca1.60 K1.17 Mg0.57) Al6.45 Si29.55 O72
CpCs Cs+ (Cs4.95K0.4Mg0.54) Al6.52 Si29.48 O72
CpAg Ag+ (Ag5.53K0.91) Al6.48 Si29.52 O72
CpCd Cd2+ (Cd2.75Mg0.47) Al6.40 Si29.60 O72
CpBa Ba2+ (Ba3.11K0.25) Al6.55 Si29.45 O72
CpZn Zn2+ (Zn2.44Na0.84Ca0.11K0.18Mg0.29) Al6.50 Si29.50 O72
ture, probably its location inside the Cp framework does not suit its replacementby a greater cation [23].
As a first level of antibacterial screening all exchanged forms were testedagainst E. coli bacterial strain with the disc diffusion assay (Fig. 3). Expectedly,the natural Clinoptilolite form did not show antibacterial activity. Only theCpAg and CpCd exchanged forms showed inhibitions zones (Table 2). The lackof inhibition zone of ZnCp may be due to its lower total concentration whencompared with CpAg and CpCd forms. From the EDS data one can see that thecation concentration in the conducted antibacterial screening was highest for Ag+
(∼ 0.093 µg ml−1) and Cs+ (∼ 0.100 µg ml−1), and lowest for Zn2+ (∼ 0.025 µgml−1) and Cd2+ (∼ 0.048 µg ml−1) and thus was not a governing factor. Thisantibacterial activity of the CpAg and CpCd forms is certainly due to the releaseof the Ag and Cd cations from the channels.
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Fig. 3. Photograph showing the antibacterial screening
T a b l e 2
Antibacterial activity of investigated samples after 18 h
Samples Inhibition diameter (E. Coli BL21)
CpAg 26 mm
CpCd 10 mm
CpBa/CpCs/CpZn/ No inhibition observed
The antimicrobial effect is perhaps a result of elevated cation concentration(Ag and Cd). Having in mind that only compounds with minimum inhibitoryconcentration (MIC) value lesser than 10 µg ml−1 are considered potentially activeagainst the bacterial species, one can say that the values (0.093 and 0.048 µg ml−1)of CpAg and CpCd are satisfactory. Further tests against a collection of resistantbacterial strains are envisaged.
Conclusions. In conclusion Ag- ion exchanged Clinoptilolite showed an-tibacterial activity against E. coli bacteria. Having in mind that the total con-centration of the cation in the Clinoptilolite exchanged forms is below 10 µg ml−1
natural exchanged Clinoptilolite could be used as potential antibacterial addi-
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tive to eliminate bacterial growth in the production of pharmaceutical straps orprevent infections in hospital premises by spraying and slow release.
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Institute of Mineralogy and CrystallographyBulgarian Academy of SciencesAcad. G. Bonchev St, Bl. 107,
1113 Sofia, Bulgariae-mail: [email protected]
∗Academician Rostislaw KaischewInstitute of Physical ChemistryBulgarian Academy of Sciences
Acad. G. Bonchev St, Bl. 111113 Sofia, Bulgaria
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