Microporous and Mesoporous Materials 169 (2013) 148–152

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Microporous and Mesoporous Materials

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Antibacterial activity of modified natural clinoptilolite against clinical isolatesof Acinetobacter baumannii

Jasna Hrenovic a,⇑, Jelena Milenkovic b, Ivana Goic-Barisic c, Nevenka Rajic b

a University of Zagreb, Faculty of Science, Division of Biology, Zagreb, Croatiab University of Belgrade, Faculty of Technology and Metallurgy, Belgrade, Serbiac University of Split, Split University Hospital and School of Medicine, Department of Clinical Microbiology, Split, Croatia

a r t i c l e i n f o

Article history:Received 11 September 2012Received in revised form 25 October 2012Accepted 29 October 2012Available online 10 November 2012

Keywords:BacteriaBenzalkoniumCopperSilverZeolite

1387-1811/$ - see front matter � 2012 Elsevier Inc. Ahttp://dx.doi.org/10.1016/j.micromeso.2012.10.026

⇑ Corresponding author. Tel.: +385 16189700; fax:E-mail address: jasna.hrenovic@biol.pmf.hr (J. Hre

a b s t r a c t

The antibacterial activity of natural zeolite (NZ) containing 2.03 wt.% of Cu2+ (CuNZ), 5.07 of Ag+ (AgNZ)or 8.50 of benzalkonium ion (BCNZ) were tested against clinical isolates of Acinetobacter baumanniibelonging to European clone I and II. Isolate from clone II was more sensitive to CuNZ and AgNZ withminimum bactericidal concentration (MBC) 125 and 31.2 mg L�1, respectively than isolate from clone Iwith MBC of 250 mg L�1 for both CuNZ and AgNZ. Isolate from clone I was more sensitive to BCNZ withMBC 250 mg L�1 than isolate from clone II with MBC 500 mg L�1. The minimum inhibitory concentration(MIC) values were the same or slightly lower than the MBC values. The antibacterial activity of CuNZ,AgNZ and BCNZ against the A. baumannii European clone I and II was due to the activity of cations loadedonto NZ while not to the NZ used as support material. The antibacterial activity of CuNZ, AgNZ and BCNZopens a possibility of future clinical investigations and its application.

� 2012 Elsevier Inc. All rights reserved.

1. Introduction

Acinetobacter baumannii is a Gram-negative coccobacillus thathas emerged over last decade as a leading cause of hospital ac-quired infections, particularly in the intensive care units. Infectionwith A. baumannii is usually complex, with the coexsistence of epi-demic and endemic infections [1]. This organism has an increasingability to develop antimicrobial resistance to commonly used anti-microbial agents, leading to limited options for antibiotic treat-ment [2]. Problems caused by A. baumannii in the hospital settingare emphasized by high degree of resistance to drying and disinfec-tants, leading to long-term persistence and the occurrence of out-breaks in the hospital environment [3]. The resistance which A.baumannii shows towards antibiotics as well as towards some hea-vy metals has been found to be in relation with beta-lactamaseproduction [1,4]. Beta-lactamase acts like a ‘‘sponge’’ which pre-vents both antibiotics and metal ions to access the target sites [4].

Because of their chemical inertness and high thermal stability,zeolites have found various commercial applications. Their usesare closely connected to their unique structural features which al-low ions to be readily exchanged and/or that different active speciesbe temporary or permanent immobilized inside the porous alumi-nosilicate framework. Recently, various natural and synthetic zeo-lites have been extensively studied as promising materials for

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+385 14826260.novic).

hosting ions with antimicrobial activity [5–7]. The antimicrobialactivity usually involves the slow release of antimicrobial metalions (such as Cu2+, Zn2+ or Ag+) from the zeolite lattice and the inac-tivation of some pathogenic viruses has been ascribed to metal ionheld by aluminosilicate lattice [6]. Moreover, natural clinoptilolitehas been recently extensively investigated for medical purposes be-cause of its exchange, adsorptive and biocatalytic activities [8] andnon-toxic behavior [9]. The modified clinoptilolite has been re-ported to exhibit antidiarrhetic [10], antacidic [11] and antiviralproperties [12]. Also, it has been shown that natural clinoptiloliteis a promising material for anticancer therapy [13–15].

We have previously reported the antibacterial activity of copper-[16] and surfactant-modified clinoptilolite [17,18] towards Acineto-bacter junii, Escherichia coli and Staphylococcus aureus. Also, goodantibacterial activity of silver-modified zeolite has been recently re-ported [19,20]. Taking this into account we continue to investigatethe antibacterial activity of the natural zeolite (NZ) in particularclinoptilolite modified with Cu2+, Ag+ or benzalkonium ions againstclinical isolates of A. baumannii isolated from the same hospital andbelonging to two different European clones (EU I and II).

2. Materials and methods

2.1. Preparation and characterization of the zeolites

The NZ containing 70 wt.% of clinoptilolite (quartz and feldsparwere major impurities) was obtained from the sedimentary depos-

J. Hrenovic et al. / Microporous and Mesoporous Materials 169 (2013) 148–152 149

it Zlatokop, Serbia [21]. The NZ (with particle size being 0.063–0.1 mm) was firstly converted into the Na-rich form in order to im-prove the clinoptilolite cation exchange capacity and NaNZ wasthen used for the preparation of modified zeolites. The modifiedzeolites were prepared by using 1.0 g of the NaNZ and 100 mL of6 mM aqueous solution of CuSO4, AgNO3 or benzalkonium chloride(BC). The suspensions were shaken for 24 h in a thermostatedwater bath (Memmert, WNB22) at 25 �C, separated by filtrationand obtained products were dried over-night. The modified zeo-lites contained 20.33 mg Cu2+, 50.65 mg Ag+ or 85.00 mg BC pergram of dry weight and denoted as CuNZ, AgNZ and BCNZ, respec-tively. The details of their characterization have been previouslypublished [18,21]. The AgNZ and BCNZ were tested for antibacte-rial activity without previous sterilization in order to avoid the dis-integration of loaded cations. However, no contamination of purecultures was observed during the experiments. The CuNZ and NZwere sterilized by autoclaving (121 �C, 15 min) prior to theexperiments.

2.2. Bacterial strains

We compared the antibacterial activity of the modified NZ totwo different isolates of A. baumannii from Split University Hospitalwith affiliation to European clone I (EU I) and II (EU II). Clinical iso-lates of A. baumannii were collected during two different out-breaks, first from 2002 to 2007 (EU I) and second from 2009 to2010 (EU II) in Clinical Hospital Center Split, Croatia [22,23]. It isa 1651-bed university teaching hospital and the only hospital inthe region. It serves a population of about 500,000 and acts as areferral hospital for a wider area of southern Croatia. Isolation ofA. baumannii was performed in routine work on blood agar plates(Bio Rad). Initial identification was made using the ATB 32GNand Vitek 2 systems (bioMerieux, Marcy-l0Etoile France), as a stan-dard procedure in microbiological laboratory according Clinicaland Laboratory Standards Institute [24].

2.3. Antibacterial activity tests

The antibacterial activity of CuNZ, AgNZ and BCNZ was testedagainst isolates of A. baumannii belonging to the European clone Iand II. The bacteria were pre-grown on nutrient agar (Biolife, Italy)for 16 h at 36.0 ± 0.1 �C to obtain the cultures in log phase ofgrowth. The bacterial biomass was then suspended in the sterile0.85% NaCl solution. In a 10 mL of bacterial suspension in the tubea 1000 mg L�1 of CuNZ, AgNZ or BCNZ were added. Serial dilutionsof modified zeolites were made according to the standard dilutionmethod [24]. The control tubes were left without addition of thezeolites. The tubes were sealed and incubated in a dark during24 h at 36.0 ± 0.1 �C with shaking at 120 rpm to assure the com-plete mixing. To confirm the bactericidal action of examined cat-ions which were loaded onto NZ, the experiments withCuSO4 � 5H2O, AgNO3 and BC were set up in the same way as de-scribed above. To confirm the absence of the antibacterial activityof Na form of NZ, the experimental bottles containing 1000 mg L�1

of the autoclaved NZ were set up.The number of viable cells was determined at the beginning of

experiment, after 5 min (for BCNZ and BC only), 1, 3, 5 and 24 h ofcontact. Tubes were vigorously shaken (40 Hz/3 min, Kartell TK3S).A 0.1 mL of sample was plated (spread plate method) directly ontothe nutrient agar and another sample was serially diluted (10�1–10�8) and inoculated onto the nutrient agar plates in triplicate.The inoculated plates were incubated at 36.0 ± 0.1 �C for 24 h. Afterthe incubation period, the bacterial colonies were counted and thenumber of viable cells was reported as CFU mL�1. The minimuminhibitory concentration (MIC) and minimum bactericidal concen-tration (MBC) values were determined after 24 h of experiments

according to the CLSI directions [24]. The numbers of CFU were log-arithmically transformed. The antibacterial activity of the modifiedzeolites was expressed as the percent reduction of log CFU as com-pared to the corresponding control.

After 24 h of experiments in all tubes the pH values were mea-sured with the WTW 330 pH-meter and dissolved oxygen concen-tration with the WTW Oxi 330i set. In the tubes showing the MICand MBC the leaching of Cu2+, Ag+ and BC from CuNZ, AgNZ andBCNZ, respectively was determined. The suspension was filteredthrough 0.20 lm Sartorius syringe filters and the solutions wereanalyzed for Cu and Ag by atomic absorption spectrophotometer(AAS Varian, Spectra AA 55b) and for BC using high-performanceliquid chomatography mass spectrometry (HPLC/MS) method(Waters Alliance�, Waters Corporation, Milford, MA, USA)equipped with Waters Micromass� ZQ™ (Waters Corporation, Mil-ford, MA, USA). The presence of extracellular substances in A. bau-mannii European clone I and II was confirmed by alcian bluestaining of 24 h old cultures cultivated in nutrient broth.

3. Results and discussion

3.1. Antibacterial activity of modified zeolites

Antibacterial activity of CuNZ against isolates of A. baumanniibelonging to European clone I and II is shown in Fig. 1. The antibac-terial activity of CuNZ increased by increasing the concentration ofmaterial and time of contact. The A. baumannii from clone I withMBC of 250 mg L�1 was more resistant to CuNZ than isolate fromclone II with MBC of 125 mg L�1. The MIC values were one orderof examined concentrations lower than the MBC values for bothclones (Fig. 1.). Good antibacterial activity was also reported forCuNZ at concentration of 10 g L�1 against E. coli and S. aureus in dif-ferent water media [16].

Antibacterial activity of AgNZ against isolates of A. baumannii isshown in Fig. 2. The antibacterial activity of AgNZ increased byincreasing the concentration of material and time of contact. TheA. baumannii from clone I with MBC of 250 mg L�1 was much moreresistant to AgNZ than isolate from clone II with MBC of31.2 mg L�1. The MIC values were two orders of examined concen-trations lower than the MBC values for clone I and at the lowestexamined concentration for clone II (Fig. 2.). The average antibac-terial activity was comparable to those reported for zeolite X con-taining the 5.8 wt.% of Ag, which inhibited the growth of E. coli, S.aureus and P. aeruginosa in less than 1 h at concentration of150 mg L�1 [20].

Antibacterial activity of BCNZ against isolates of A. baumannii isshown in Fig. 3. The A. baumannii from clone II with MBC of500 mg L�1 was more resistant to BCNZ than isolate from clone Iwith MBC of 250 mg L�1. The MIC values were the same as theMBC values for both clones (Fig. 3.). The bactericidal activity ofBCNZ sharply decreased by decrease of BCNZ concentration. Forclone I at BCNZ concentration of 125 mg L�1 and lower the antibac-terial activity obtained after 5 h of contact decreased by prolongingthe contact for 24 h. For clone II decrease of the antibacterial activ-ity of BCNZ was evident after 5 h of contact at concentration of250 mg L�1, 3 h of contact at concentration of 125 mg L�1 and only5 min of contact at concentration of 62.5 mg L�1 and lower. How-ever, the numbers of A. baumannii in the tubes with BCNZ werenot higher than the corresponding control, suggesting that bacteriadid not use BC as the source of carbon. Rapid decrease of bacterici-dal activity by decrease of BCNZ concentration and decrease of theantibacterial activity after 5 h of contact was also described forE. coli, while not for S. aureus [18].

After 24 h of contact the differences in pH values (data notshown) between control and experimental tubes were not higher

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FU (%

)

1 h 3 h 5 h 24 h

CuNZ, clone 1 MBC

MIC

15.6 31.2 62.5 125.0 250.0 500.0 1000.0

15.6 31.2 62.5 125.0 250.0 500.0 1000.0Concentration (mg/L)

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CuNZ, clone 2 1 h 3 h 5 h 24 h

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MIC

Fig. 1. Percent reduction in the number of A. baumannii isolates belonging toEuropean clone I and II during 24 h of contact with CuNZ as compared to thecorresponding control. c0 A. baumannii clone I (106 CFU mL�1) = 8.80 ± 0.17; c0 A.baumannii clone II (107 CFU mL�1) = 1.40 ± 0.17.

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FU(%

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AgNZ, clone 1 MBC

MIC

15.6 31.2 62.5 125.0 250.0 500.0 1000.0

15.6 31.2 62.5 125.0 250.0 500.0 1000.0Concentration (mg/L)

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AgNZ, clone 2 1 h 3 h 5 h 24 h MBC

MIC

Fig. 2. Percent reduction in the number of A. baumannii isolates belonging toEuropean clone I and II during 24 h of contact with AgNZ as compared to thecorresponding control. c0 A. baumannii clone I (106 CFU mL�1) = 8.80 ± 0.17; c0 A.baumannii clone II (107 CFU mL�1) = 1.40 ± 0.17.

150 J. Hrenovic et al. / Microporous and Mesoporous Materials 169 (2013) 148–152

than 1.1 and 0.7 pH units for clone I and II, respectively and min-imum values were not lower than 5.6 while maximum valuesnot higher than 6.8. Acinetobacter sp. grows in the pH range 5.0–8.0 [25]. The pH values showed significantly positive correlation(r = 0.45) with the percent reduction of log CFU. Therefore, thechanges in pH values were not the reason for the antibacterialactivity of modified zeolites. After 24 h of contact the dissolvedoxygen concentration in all tubes were not lower than 5.4 mgO2 L�1, suggesting that the antibacterial activity was observed inaerobic conditions.

3.2. Leaching of cations from modified zeolites

Some studies reported that the cations leached from modifiedzeolites are responsible for its antibacterial action. Bacteria cancome into contact with modified zeolites and active take up cat-ions, which consequently damage the cells [20,26]. The leachingof cations from CuNZ, AgNZ and BCNZ at MBC and MIC into solu-tion is shown in Table 1. The concentrations of Cu leached formCuNZ were four times higher in experiments with clone I whencompared to clone II, while the concentrations of Ag leached formAgNZ were similar for both clones of A. baumannii. Our results areconsistent with the report that 0.1 mg L�1 of Ag leached from Ag-zeolite possesses a bactericidal activity [26]. The concentrationsof BC leached form BCNZ were two times higher in experiments

with clone II when compared to clone I. By one 24 h exposure a rel-atively low leaching of Cu and Ag (0.7–8.2 wt.% of the metal cationsloaded onto NZ) was observed at MBC. The leaching of BC at MBC/MIC was about the half (48.4–52.9 wt.%) of the BC loaded onto NZ(Table 1). However, the time needed to obtain MBC with BCNZ was1 and 5 h for clone II and I, respectively. These suggest the possibil-ity of reuse of modified zeolites if used in water media [20,27].

3.3. Antibacterial activity of cations

The BC at concentration of 850 mg L�1 and Cu at concentrationof 260 mg L�1 resulted in 100% reduction of E. coli and S. aureusafter 1 min and 24 h of contact, respectively [16,18]. The MBC val-ues of BC against clinical isolates of Streptococcus agalactiae after24 h of contact ranged between 0.70 and 12.50 mg L�1 [28]. TheAgNO3 at concentration of 2 g L�1 inhibited the growth of E. coliwithin 30 min [26]. The Cu, Ag and BC at concentration equivalentto the amounts loaded onto NZ which showed the MBC resulted in100% reduction of A. baumannii European clone I and II after 1 h ofcontact, where only 5 min was needed to obtain this value with BC(Fig. 4). The Cu, Ag or BC salts achieved the 100% reduction in amuch shorter time than CuNZ, AgNZ or BCNZ (Figs. 1–3). Althoughthe modified zeolites did not show the pronounced antimicrobialactivity when compared to the Cu, Ag or BC salts, the stabilityand slow release of cations from modified zeolites are the main

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15.6 31.2 62.5 125.0 250.0 500.0 1000.0

Concentration (mg/L)

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Fig. 3. Percent reduction in the number of A. baumannii isolates belonging toEuropean clone I and II during 24 h of contact with BCNZ as compared to thecorresponding control. c0 A. baumannii clone I (106 CFU mL�1) = 8.80 ± 0.17; c0 A.baumannii clone II (107 CFU mL�1) = 1.40 ± 0.17.

Table 1Leaching of cations (mg L�1/wt.% of the cation loaded onto NZ) from CuNZ, AgNZ andBCNZ in tubes showing the MBC and MIC in experiments with A. baumannii isolatesbelonging to European clone I and II.

Tube Cu Ag BC

Clone IMBC 0.403/7.9 0.090/0.7 10.28/48.4MIC 0.517/20.3 0.061/1.9 10.28/48.4

Clone IIMBC 0.103/4.1 0.130/8.2 22.49/52.9MIC 0.121/9.5 0.084/10.6 22.49/52.9

Cu Ag BC NZ-20

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clone 1

Cu Ag BC NZCompound

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clone 2

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Fig. 4. Percent reduction of Cu, Ag and BC at concentration loaded onto NZ showingthe MBC and no antibacterial activity of NZ against A. baumannii European clone Iand II during 24 h of contact as compared to the corresponding control. A.baumannii clone I: c0 CFU (106 mL�1) = 2.30 ± 0.08; c0 Cu (mg L�1) = 5.1; c0 Ag(mg L�1) = 12.7; c0 BC (mg L�1) = 21.3; c0 NZ (mg L�1) = 1000. A. baumannii clone II:c0 CFU (106 mL�1) = 6.95 ± 0.80; c0 Cu (mg L�1) = 2.5; c0 Ag (mg L�1) = 1.6; c0 BC(mg L�1) = 42.5; c0 NZ (mg L�1) = 1000.

J. Hrenovic et al. / Microporous and Mesoporous Materials 169 (2013) 148–152 151

characteristics which give them the advantage in use. NZ at thehighest tested concentration (1000 mg L�1) did not show any anti-bacterial activity against both clones of A. baumannii (Fig. 4). Inprevious studies it has been found that NZ did not exhibit the anti-bacterial activity against A. junii, E. coli and S. aureus [16,17].Accordingly, the antibacterial activity of CuNZ, AgNZ and BCNZagainst the A. baumannii European clone I and II could be ascribedto the activity of the cations loaded onto NZ but not to the NZ itself.

The alcian blue staining confirmed the presence of thick layer ofextracellular substances with embedded cells of A. baumannii Euro-pean clone I and II (Fig. 5). The bacterial extracellular substancesplay a role in detoxification of heavy metals through the putative

entrapment of metals [29]. It seems that the extracellular polymersare important virulence factor which protect A. baumannii from thebactericidal activity of the modified zeolites.

Infections with A. baumannii are associated with hospital instru-mentation, such as the use of mechanical ventilators, endotrachealand gastric tubes, vascular catherization, lumbar punctures, mye-lography, ventriculography, indwelling urinary catheterization, pe-netrating keratoplasty, and the fitting of contact lenses [25].Microbial resistance to antimicrobial agents causes serious prob-lems in the inactivation of microbes related to infectious diseases.Development of new antimicrobial agents is one of the priorities ofmodern community. Zeolites, well established porous aluminosili-cate materials, are good candidates for giving answer to this task.Their ion-exchange ability offers possibilities for hosting not onlydifferent metal ions with antimicrobial activity but also the bulkiercations such as benzalkonium ions which are usual additives inmany commercial products. The modified clinoptilolite can be con-sidered as a low cost material for bactericidal use having a pro-longed action. The obtained results clearly reveal theantibacterial properties of modified zeolites, which recommendthe natural clinoptilolite for the development of novel antibacterialmaterials-antimicrobial coatings for use in medical devices, mate-rials for filters in air or water purifiers in hospital facilities, etc.

Fig. 5. Thick layer of extracellular substances with embedded cells of A. baumanniiisolates belonging to European clone I and II.

152 J. Hrenovic et al. / Microporous and Mesoporous Materials 169 (2013) 148–152

4. Conclusions

Acinetobacter baumannii belonging to European clone I and IIshowed different sensitivity to the bactericidal action of the natu-ral clinoptilolite modified with Cu2+, Ag+ and benzalkonium ions.Clinical isolate from clone II was more sensitive to CuNZ and AgNZ,while the clone I was more sensitive to BCNZ. Modified zeolites asthe products other than antibiotics could find the application ascoating for different materials or as filters with antibacterial activ-ity. The antibacterial activity of modified zeolites opens a possibil-ity of clinical investigation on the higher number of multidrugresistant bacterial isolates on which the bactericidal effect can beexpected.

Acknowledgements

This work was supported by the Ministry of Science, Educationand Sports of the Republic of Croatia (Project No. 1191155-1203)

and Serbian Ministry of Science (Project No. 172018). The authorsare very grateful to Hemtek Company for HPLC/MS analysis.

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