Silver Nanoforms as a Therapeutic Agent for Killing Escherichia coli and Certain ESKAPE Pathogens

The scope of this study included the preparation of silver nanoforms with high antimicrobial efficacy, low cost, and ease of application. The term ‘silver nanoforms’ refers to silver located on the amorphous or crystalline titanium dioxide (TiO2). Silver nanoforms may be used as an alternative to antibiotics in killing bacteria. Pure and silver-incorporated titanium (used as a carrier) was prepared using the sol–gel-modified method. Physical and chemical properties of the samples were described, and the antibacterial activity was indicated using the following strains of bacteria: Staphylococcus aureus, Klebsiella pneumoniae (ESKAPE pathogens), and Escherichia coli. The results have shown that the antibacterial activity of silver nanoforms with amorphous TiO2 is much better than that in the samples based on anatase (crystalline TiO2). The sensitivity of the tested bacteria to silver nanoforms depends on physical and chemical properties of the nanoforms and individual characteristics of the bacteria. For the first time, significant participation of amorphous TiO2 in antibacterial compounds has been described through this study.

Abstract The scope of this study included the preparation of silver nanoforms with high antimicrobial efficacy, low cost, and ease of application. The term 'silver nanoforms' refers to silver located on the amorphous or crystalline titanium dioxide (TiO 2 ). Silver nanoforms may be used as an alternative to antibiotics in killing bacteria. Pure and silver-incorporated titanium (used as a carrier) was prepared using the sol-gel-modified method. Physical and chemical properties of the samples were described, and the antibacterial activity was indicated using the following strains of bacteria: Staphylococcus aureus, Klebsiella pneumoniae (ESKAPE pathogens), and Escherichia coli. The results have shown that the antibacterial activity of silver nanoforms with amorphous TiO 2 is much better than that in the samples based on anatase (crystalline TiO 2 ). The sensitivity of the tested bacteria to silver nanoforms depends on physical and chemical properties of the nanoforms and individual characteristics of the bacteria. For the first time, significant participation of amorphous TiO 2 in antibacterial compounds has been described through this study.

Introduction
Silver nanoforms are nanocompounds of silver located on the amorphous or crystalline titanium dioxide (TiO 2 ). TiO 2 is used as a carrier for biologically active silver (ions Ag ? and nanoparticles Ag 0 ), causing (1) prevention of silver ions inactivation by environmental factors (such as NaCl), (2) prevention of silver nanoparticles aggregating with each other, (3) increase in the surface area (S BET ) of silver nanoforms. The above points have one purpose: better (higher and faster) antibacterial activity of silver nanocompounds. TiO 2 appears in the amorphous form and the following polymorphic crystalline ones: anatase, rutile, and brookite. Anatase is known for its highest photocatalytic properties, and for this reason, it has a potential for various biomedical and environmental applications [15,20,30]. Photocatalysis is a process during which, in a water environment, radicals are generated under the influence of UV light [4]. Radicals change the metabolism of bacteria cells, which causes their death. Up to now (October 2015), over 20,000 results in the PubMed have focused on the crystalline TiO 2 (especially anatase) and its photocatalysis process. About 30 of them, concentrate on the comparison of their antibacterial efficacy, but only one refers to amorphous TiO 2 as a carrier for silver and describes its potential antibacterial applications [7]. Research on bacterial resistance to antibacterial agents is an important trend in contemporary microbiology and the medical area. During the last six decades, resistance to antibiotics has increased, and when antibiotics were introduced in the 1950s, researchers expected that they would eradicate all bacterial diseases. In the early 1960s, resistance to penicillin among Gram-positive bacteria was noted, and the same phenomenon was soon observed in Gram-negative bacteria [24]. Multidrug resistance (MDR) in both Grampositive and Gram-negative bacteria and extremely drugresistant bacteria (XDR) is an important issue in the treatment of infectious diseases [22]. Antibiotic resistance control is a long-term international program which surveys MDR bacteria. Many international reports indicated that the resistance of bacteria to antibiotic drugs is still high [6]. Klebsiella pneumoniae as a nosocomial pathogen that also causes septicemia in newborns is increasingly multidrugresistant [22]. MDR Staphylococcus aureus complicates the treatment of healthcare-associated infections. Also, carbapenems resistance in Gram-negative bacteria has been rapidly increasing [25]. Moreover, both K. pneumoniae and S. aureus belong to ESKAPE pathogens (together with Enterococcus faecium, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter spp.). The ESKAPE group describes pathogens responsible for hospital infections, which are most difficult to treat. However, E. coli causing bacteremia infections and sepsis showed increasing resistance to fluoroquinolones [24]. The spread of resistance among pathogens is becoming a serious problem [14], and therefore searching for alternative drugs, such as those containing nanocomposites doped with silver, is very desirable. Silver ions and silver nanoparticles show a broad spectrum of antibacterial activity and indicate an oligodynamic effect [11,18,19]. Silver has been known for its antibacterial activity since ancient times, but the development of bionanotechnology has given us possibilities for using it as an active biomedical factor [16]. The toxicity of silver compounds depends on the bioavailability of silver, and silver ions rapidly bind to factors in the environment (such as Cl -) or aggregate with each other and eventually become unable to kill bacteria. The presence of a carrier (such as titanium dioxide) acts as a ligand for silver ions or nanoparticles and preservatives before decreasing the availability of silver [16]. Kedziora et al. [12] proposed the synthesis of TiO 2 /Ag compounds, where anatase indicated very good antibacterial efficacy. We modified the previous method, and in this work, other nanoforms of TiO 2 were prepared-amorphous and crystalline (anatase) doped with silver-and their antibacterial activity was compared, with and without UV irradiation. This work proves excellent antibacterial activity of the nanoforms based on the amorphous TiO 2 -TiO 2 /Ag ? and TiO 2 /Ag 0 (even without UV), much better than that based on the anatases-TiO 2a /Ag ? and TiO 2a /Ag 0 (with UV irradiation). The amorphous nanoforms have a potential to be exploited in numerous biomedical, pharmaceutical, and industrial applications as an antimicrobial agent (e.g., for hard-to-heal wound infections as cream, ointment, bandages, disinfectants, etc., or as filters and preservatives in cosmetics). In order to maintain its excellent antibacterial activity, no additional factors such as UV irradiation are required.

Materials and Methods
Production of Pure Amorphous and Crystalline TiO 2 and TiO 2 Doped with Silver (TiO 2 /Ag 1 , TiO 2a / Ag 1 , TiO 2 /Ag 0 and TiO 2a /Ag 0 ) Nanocompounds All used reagents were described in a study by Kedziora et al. [12]. Titanium n-butoxide [Ti(O-Bu)], hydrofluoric acid, and ammonium hydroxide were added as described by Kedziora et al. [12]. The duration of stirring was established experimentally. In the previous study [12], diamminesilver(I) [Ag(NH 3 ) 2 ] ? was added during the above procedure to form nanosilver grain. In this work, we (1) finished the gel synthesis (by washing with methyl alcohol and water, drying at 80°C, and transforming to anatase, if necessary); and then (2) treated the powder of amorphous or crystalline TiO 2 with diamminesilver(I) and a reducing factor (glucose) during impregnation and reduction. After step 2, we received nanocompounds of amorphous and crystalline: TiO 2 -TiO 2 /Ag ? , TiO 2a /Ag ? (as an impregnation effect) and TiO 2 /Ag 0 and TiO 2a /Ag 0 (as a reduction effect). To prepare the anatase, amorphous TiO 2 was calcinated under the same conditions as described previously [12]. The content of silver (Ag ? or Ag 0 ) was measured by dissolving the composite in acetic acid or nitric acid accordingly, and then measured using atomic absorption spectroscopy (AAS, Perkin Elmer 1100).
Results of nanoforms characteristics are presented in Table 1.
Characteristics of Pure TiO 2 and TiO 2 Doped with Silver (TiO 2 /Ag 1 , TiO 2a /Ag 1 , TiO 2 /Ag 0 and TiO 2a /Ag 0 ) Nanocomposites The description of morphology is one of the basic features of materials in the nanoscale. The samples were characterized as follows: size and shape-with transmission electron microscopy (TEM, Philips CM20 Super Twin); crystalline structure-with X-ray diffraction (XRD, Stöe); porosity-with nitrogen adsorptions isotherms (Autosorb 1-C, Quantachrome Instruments); and content of silverwith atomic absorption spectroscopy (AAS, Perkin-Elmer). Diffraction patterns (degrees and intensity of peaks measured with XRD) were compared with the referenced patterns from the database of diffraction patterns: International Centre for Diffraction Data (PDF2 Database ICDD). Porosity measured with N 2 adsorption and desorption provides information about the pore size and the corresponding surface area. Porosity size is of fundamental importance in developing the surface area-a basic feature in nanotechnology. The smaller the pore sizes observed the larger the surface area described. A shape of the isotherm confirms a pore size. To measure the silver content in the nanocompounds with AAS, the solubility of silver ions in citric acid and that of silver nanoparticles in the nitric acid were used. The results obtained with the above techniques make the interpretation of antibacterial activity of silver nanomaterials easier. The photocatalytic activity of nanocompounds was measured using a model reaction of estimated decomposition of methyl blue (at room temperature, RT) [28].

Bacteria Strains
In all, 10 bacteria strains were used for antimicrobial study-three reference strains from American Type Culture Collection: Staphylococcus aureus ATCC 6538 (Gram-positive bacteria), Escherichia coli ATCC 11229 (Gram-negative bacteria), and Klebsiella pneumoniae ATCC 4352 (Gram-negative bacteria); and seven of the same genera strains, isolated from infected wounds purchased from the DiaLab Medical Laboratory. The antibiogram is presented in

Results and Discussion
Synthesis and Characterization of Silvered TiO 2 Sol-gel synthesis is one of the most popular methods used for nanocompounds production [2,3,23] and its modification leads to obtaining various products with differences in physicochemical and biological properties. In comparison with the study by Kedziora et al. [12], we showed that a source of silver [diamminesilver(I)] may be added during the synthesis of powder forming silver grain and to the ready gel (this work). The way of synthesizing has an impact on the surface area and antibacterial efficacy. In Fig. 1, the TEM images of the prepared basic samples are shown. Figure 1a presents amorphous TiO 2 , and anatase (TiO 2a ) is presented in Fig. 1b. The size of amorphous TiO 2 is less than or equal to 100 nm. Calcination of amorphous TiO 2 caused a decrease in grain size (from 100 to 40 nm), but silver grain embodied to TiO 2 structure [12] decreased the surface area (S BET ). The specific surface area of the powder decreased from 424.0 ± 8.0 m 2 /g (for amorphous TiO 2 ) to 52.8 ± 0.2 m 2 /g (for crystalline TiO 2a ) (Table 3). High reduction of surface area is probably related to the aggregation of TiO 2 during annealing. In a study by Kedziora et al. [12], adding diamminesilver(I) to the sol of TiO 2 was proposed. It caused a decrease in the size of silver nanoparticles to less than 5 nm [12]. Calcination of amorphous titanium (TiO 2 ) did not change the silver crystallite size. X-ray diffraction lines of amorphous and crystalline TiO 2 are shown in Fig. 2  of these are own results, and the obtained data were compared with the ICDD database. We confirmed that amorphous TiO 2 powder calcinated at 400°C is already well crystallized to anatase (Fig. 2b, d). All reflections in the diffraction patterns shown in Fig. 2b were identified as belonging to anatase (according to ICDD Database No. 99-200-3744, 38.78 2Theta). In the pattern observed in Fig. 2a Fig. 2d (diffraction lines of TiO 2a /Ag 0 ), peaks of anatase can be observed. However, no visible silver peak in Fig. 2d probably results from (1) a low content of silver (estimated at 1 wt% for TiO 2a /Ag 0 , see Table 1, proved with AAS) or (2) interference of the anatase and nanosilver peaks. N 2 adsorptiondesorption isotherms of the amorphous and crystalline TiO 2 are illustrated in Fig. 3. The isotherms of adsorption and desorption do not cover each other, which confirms that the prepared materials (amorphous TiO 2 and crystalline TiO 2a ) are porous. From the shape of hysteresis, it can be concluded that the material is mesoporous (the pore diameter of titanium dioxide is between 2 and 50 nm) [21]. Photocatalytic properties of the prepared anatase were experimentally confirmed by measuring the decomposition of methyl blue. Average time of decomposition of methyl blue by crystalline anatase (TiO 2a ) was estimated at 90 min. The knowledge of the nanocarrier morphology (size, shape, porosity, surface area, and crystal structures) is a fundamental issue and has an important impact on the following aspects: (1) silver content in the nanocompounds, (2) antibacterial efficacy of silver nanocompounds, (3) interaction of bacteria cells with silver nanocompounds, (4) antibacterial mechanism of action, and (5) mechanism of potential bacterial resistance to silver nanomaterials. Furthermore, this knowledge is fundamental for further research concerning the influence of physicochemical properties of the compounds on the answer of bacteria cells.

Antibacterial Activity
Results of the antibacterial efficacy of silver nanocomposites are summarized in Tables 4 and 5. We checked antimicrobial activity of temporary forms-TiO 2 impregnated with silver ions Ag ? (TiO 2 /Ag ? and TiO 2a /Ag ? ) and permanent forms-TiO 2 doped with silver nanoparticles Ag 0 (TiO 2 /Ag 0 and TiO 2a /Ag 0 ) against Gram-positive and Gram-negative bacteria. Pure TiO 2 and TiO 2a did not show antibacterial activity in the applied concentration (1024 lg/ ml). The best efficacy was indicated in the sample based on amorphous titanium dioxide used as a carrier for silver ions (TiO 2 /Ag ? ) where MIC was equal for ions with and without UV irradiation and reached 0.05 lg/ml. Excellent efficacy is indicated in the silver nanoparticles (Ag 0 ) also located on the amorphous TiO 2 (TiO 2 /Ag 0 ). Also, MIC of TiO 2 /Ag 0 comes to 0.4 lg/ml (the same values for UV irradiation and without it). Bionanotechnologists seem to overlook the amorphous TiO 2 , but we proved its excellent properties as a carrier for antibacterial agents such as silver (both silver ions and nanoparticles). It is very attractive for medical and biological applications-shows a very good antibacterial efficacy even without UV treatment. Annealing to the anatase structure may be limited and stopped after the amorphous TiO 2 phase. Silver nanoforms based on the crystalline TiO 2a indicated lower antibacterial activity than those based on an amorphous one even after UV treatment. MIC values for TiO 2a /Ag ? reached the level of 0.8-3.2 lg/ml without UV irradiation and 0.4-1.6 lg/ml for TiO 2a /Ag 0 after UV treatment. However, for TiO 2a /Ag 0 MIC values were established as 25.6-51.2 lg/ml without UV and 12.8-25.6 lg/ml after UV irradiation. It is worth underlining that the tested clinical isolates of E. coli (475, 555, 574) and K. pneumoniae 626 are resistant to penicillin and cefalosporins, and two of the tested S. aureus are MRSA bacteria strains (S. aureus 173, and 187, Table 2). When compared with the bacterial sensitivity and resistance to antibiotics, the obtained MIC values for silver nanoforms result from a different antibacterial mechanism of action. Yanez et al. [29] confirmed a good reduction of bacteria by TiO 2 nanotubes doped with polyethylene composites to over 40-99 % after UV light and about 28-43 % after white light irradiation. Results of our study confirm that the major reason for high antibacterial activity of silver nanoforms is the surface area, higher in amorphous than crystalline titanium dioxide. The increased S BET of anatase is due to lower bacterial susceptibility. Li et al. [15] also showed that a diameter of TiO 2 is one of the most important factors in the antibacterial efficacy. In contrast to our study, Li et al. [15] indicated that anatase has higher antibacterial activity than amorphous titania. In studies by Jasiorski et al. [10] and Wiglusz et al. [26], very good efficacy of silver nanoparticles deposited on other amorphous compounds such as silica surface [10] and hydroxyapatites [26] was proved. Amorphous TiO 2 has a potential to become a perfect carrier for antibacterial metals such as silver. In comparison with the previous research [12] the MIC values for the TiO 2 and TiO 2a impregnated with silver ions (TiO 2 /Ag ? , TiO 2a /Ag ? ) on S. aureus, E. coli, and K. pneumoniae were significantly lower than the MIC values obtained for Ag nanoparticles (Tables 4, 5), so antibacterial sensitivity depends on both the kinds of carrier and silver oxidation. Silver ions react quickly but, unlike silver nanoparticles, are rapidly inactivated by environment factors, e.g., Cl -. Gao et al. [7] also proposed the production of amorphous TiAg 2 O which revealed very effective antibacterial properties, cytocompatibility, and long lasting effectivity. Moreover, Zhao et al. [31] proved the long-term antibacterial ability of silver nanoparticles and biointegration. In contrast to Keleher et al. [13], we demonstrated less sensibility of Gram-negative bacteria to silver nanoparticles immobilized on the anatase (TiO 2a / Ag 0 ) than in the case of Gram-positive strains. However, Jamuna-Thevi et al. [9] and Mirzajani et al. [17] proved good activity of silver nanoparticles embedded on the anatase against S. aureus. They also proved that the minimum antibacterial inhibitory concentration of Ag 0 was less than the maximum cytotoxic concentration. Abdal-hay et al. [1] suggested that coating the Ti with different polymer layers causes more efficacy against bacteria.
b Fig. 2   In summary, we propose that silver nanoforms based on the amorphous TiO 2 have a potential to be used in the inhibition of growth or treatment of an infection caused by MDR E. coli and K. pneumoniae strains or MRSA S. aureus.

Conclusions
Various modifications of the sol-gel technology make it possible to synthesize different silver nanoforms with great possibilities to be used in the therapy of infections. Titanium dioxide with its physical and chemical properties may be used as a carrier of silver ions and nanoparticles. Amorphous TiO 2 is an ideal carrier for biologically active silver, both ions and nanoparticles, even without UV irradiation. The surface area of the carrier has an impact on the antibacterial efficacy. We have proven an alternative way to antibiotics for killing bacteria and have proposed amorphous TiO 2 as a carrier for biologically and medically active silver.
Acknowledgments This work was supported by the project ''Academy of Development as the key to strengthening human resources of the Polish economy'' co-financed by the European Union under the European Social Fund (beneficiary A. Kedziora, 08/12MDAR/2014). The authors thank Professor Leszek Kepinski for TEM images, Jerzy Kowalczyk PhD for research support, and DiaLab Medical Laboratory for bacteria strains.

Compliance with Ethical Standards
Conflict of interest The authors declare that there is no conflict of interest.
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