Enhanced Antimicrobial Efficacy of Bimetallic Porous CuO

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Enhanced Antimicrobial Efficacy of Bimetallic Porous CuO Microspheres Decorated with Ag Nanoparticles Xuemei Chen, Seockmo Ku, Justin A. Weibel, Eduardo Ximenes, Xingya Liu, Michael R Ladisch, and Suresh V. Garimella ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b11364 • Publication Date (Web): 23 Oct 2017 Downloaded from http://pubs.acs.org on October 24, 2017

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ACS Applied Materials & Interfaces

Enhanced Antimicrobial Efficacy of Bimetallic Porous CuO Microspheres Decorated with Ag Nanoparticles Xuemei Chen,† Seockmo Ku,‡+ Justin A. Weibel,† Eduardo Ximenes,‡ Xingya Liu,‡ Michael Ladisch,‡ Suresh V. Garimella†∗

†

School of Mechanical Engineering and Birck Nanotechnology Center, Purdue University, West

Lafayette, Indiana, USA ‡

Laboratory of Renewable Resources Engineering, and Department of Agricultural and

Biological Engineering, Purdue University, West Lafayette, Indiana, USA

Keywords: Antimicrobial efficacy; Antimicrobial mechanism; Bacteria; Bimetallic particles; Porous CuO Microspheres; Ag Nanoparticles

+

Current address: Fermentation Science Program, School of Agribusiness and Agriscience, College of Basic and Applied Sciences, Middle Tennessee State University ACS Paragon Plus Environment


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Abstract The antimicrobial action of porous CuO microspheres (µCuO), Ag nanoparticles (nAg), and bimetallic porous CuO microspheres decorated with Ag nanoparticles (µCuO/nAg) was evaluated against surrogate microorganisms representative of pathogens commonly implicated in foodborne and healthcare-associated human infections. This work addressed the gram-negative bacteria E. coli (Escherichia coli O157:H7-GFP B6-914), Salmonella (Salmonella enterica serovar enteritidis phage type PT21), and the gram-positive bacteria Listeria (Listeria innocua), as well as environmental microorganisms derived from local river water. Compared to particles composed only of CuO or Ag, the bimetallic porous µCuO/nAg particle exhibits enhanced antimicrobial efficacy. The antimicrobial action of bimetallic porous µCuO/nAg particles is dose-dependent, with 50 µg/mL particle concentration completely inhibiting the growth of both the gram-negative (Salmonella) and the gram-positive (Listeria) bacteria after 6 h. To assess the mechanism of antimicrobial action, the changes in surface morphologies of bacteria treated with the particles were observed using SEM. In the case of the gram-negative bacteria, the bacterial cell membrane is damaged, likely due to the release of metal ions from the particles; however, particle-induced cell membrane damage is not observed for gram-positive bacteria. Collectively, results from this work shed further light on possible mechanisms of antimicrobial action of micro/nanoparticles and highlight the potential for bimetallic particle-based inhibition of microbial infections.

2 Environment ACS Paragon Plus

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INTRODUCTION The attachment and aggregation of microorganisms (such as bacteria, fungi, viruses, and parasites) to a surface leads to colonization and subsequent biofilm formation. Biofilm contamination and the associated risk of infection pose serious problems in a wide range of fields including medical devices, healthcare products, food packaging, water purification systems, and household sanitation.1-6 In order to prevent infections, antimicrobial agents in the form of antiseptics, disinfectants, and antibiotics are used to inhibit the growth of microorganisms.7-8 However, the widespread overuse of antimicrobial agents has resulted in increasingly drugresistant microorganisms.9-10 There is a growing effort to replace existing antimicrobial agents with more effective, alternative therapies to mitigate this problem. Nanotechnology has the potential to develop nanomaterials to fight against the ever-growing number of antimicrobial-resistant microorganisms. A large number of studies have been conducted to explore metallic or metal oxide nanoparticles (such as Ag, Cu, CuO, TiO2, SiO2, MgO, and ZnO) as antimicrobial agents.11-24 Their high surface-to-volume ratio and small size allows such particles to closely interact with microbial cells, which trigger different biological responses compared to bulk metals, and thus exhibit promising antimicrobial efficacy (the ability to inhibit the growth of microorganism). Among the candidate particles, Ag nanoparticles (nAg) and Cu nanoparticles (nCu) exhibit the greatest antimicrobial properties against different species of microorganisms, including fungi and gram-positive and gram-negative bacteria.11-21 Although the antibacterial activities of nAg and nCu particles have been widely studied, the antibacterial properties of these nanoparticles vary among the different studies, depending on the nanoparticle size, shape, concentration, and type of microorganism.19-21 Ruparelia et al.11 evaluated the antimicrobial characteristics of nAg and nCu particles, and found that the antimicrobial efficacy



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of nAg was superior against Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus), while nCu showed better antimicrobial efficacy against Bacillus subtilis (B. subtilis). Zain et al.12 also confirmed that nAg displayed better antibacterial efficacy than nCu against E. coli and S. aureus. However, contrary observations by Valodkar et al.13 claimed greater biocidal efficacy for nCu compared to nAg against E. coli and S. aureus. Because both nAg and nCu particles have broad-spectrum antimicrobial properties, researchers have recently investigated the antimicrobial properties of bimetallic nanoparticles of Cu/Ag, either in the form of mixed, alloyed, or core-shell structures.12, 25-28 Compared to singlecomponent particles, enhanced antimicrobial efficacy was observed for bimetallic nanoparticles, which was associated with the synergetic antimicrobial efficacy of the two metals. Zain et al.12 tested the antimicrobial activities of nAg and nCu particles in different combinations, and the antimicrobial efficacy was found to increase in the following order: nCu < mixed nCu/nAg < nAg < alloyed nCu/nAg. Chen et al.25 studied the antibacterial properties of core-shell Cu(core)/Ag(shell) microparticles (1~1.5 µm), and demonstrated that their antibacterial efficacy against E. coli and S. aureus was related to the percentage of silver in the particles. Later, Rousse et al.26 evaluated the antibacterial action of core-shell nCu(core)/nAg(shell) particles (~7 nm), and showed that the bimetallic nanoparticles exhibited an enhanced bactericidal efficacy against both E. coli and S. aureus when the silver atomic percentage was greater than 40%. The antimicrobial mechanism of the metallic particles is still not clearly known and is debated in the literature. There are several theories for their biocidal action on microorganisms, including metal ion release,14-16, 28-30 generation of reactive oxygen species,31-33 and nanoparticle adhesion/penetration onto/into the surface of the cell membrane.34-36 Among all of these potential mechanisms, the release of metal ions by particles (rather than physical interaction of the


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particles themselves) is most commonly proposed as the main toxicity mechanism.37-38 It has been reported that the generation and release of ions from smaller particles into the biological system is faster than from larger ones.39-41 This is because the ion release is governed by diffusion and the high surface-to-volume ratio of small particles enables ions to release from the interior volume to the outer surfaces of the nanoparticles more rapidly. Antimicrobial micro/nanoparticles can be employed in various medical and industrial applications. For example, physicians administer topical silver agents on dermal wounds for their antimicrobial effects; these have traditionally consisted of ointments of silver nitrite, but clinicians are also relying on wound dressings loaded with nanocrystalline silver due to its enhanced (and extended) antimicrobial efficacy that results in improved clinical outcomes.42 Another application of metallic micro/nanoparticles is found in the food industry, where they can be used to create active packaging materials which are impregnated with antibacterial properties to prolong the shelf life of food and improve quality control.43 Because many of these applications feature substrates with a porous or mesh-like structure, manufacturers of such products would benefit from simple, scalable fabrication processes that allow in situ synthesis of emerging micro/nanoparticles with enhanced antimicrobial activity. In this work we develop a facile route to the synthesis of porous CuO microspheres (µCuO) with high surface areas decorated with Ag nanoparticle (nAg) to form bimetallic porous µCuO/nAg particles. The antimicrobial action of the bimetallic porous µCuO/nAg particles is evaluated against three microorganisms representative of major pathogens: the gram-negative bacteria E. coli (Escherichia coli O157:H7-GFP B6-914, with gene-coding for Shiga-toxins deleted, and expressing a green fluorescent protein44), Salmonella (Salmonella enterica serovar enteritidis phage type PT21), and the gram-positive bacteria Listeria (Listeria innocua), as well as



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environmental microorganisms derived from local river water. These microorganisms are clinically relevant due to the number of diseases caused by pathogenic forms of these bacteria including food poisoning, diarrhea, blood infection, and nosocomial infections after major surgeries.6, 45-47 The results are compared to the antimicrobial efficacy of two baseline samples, viz., porous µCuO and nAg particles. EXPERIMENTAL PROCEDURES Preparation of Porous CuO Microspheres (µ µCuO). Porous CuO microspheres were prepared by a hydrothermal method.48 First, 2 g of copper nitrate Cu(NO3)2 was mixed in 80 mL of ethanol C2H6O, followed by the addition of 60 mL of ammonia water NH4OH (25%) and 20 mL of sodium hydroxide NaOH (1 mM). After stirring for 20 min to form a clear solution, the solution was sealed and heated on a hot plate at 130 °C for 12 h and then cooled to room temperature. The resulting solid precipitate was collected by centrifugation, washed three times with deionized (DI) water, and dried on hot plate at 90 °C for 8 h (see Fig. 1). Preparation of Bimetallic Porous CuO Microspheres Decorated with Ag Nanoparticles (µ µCuO/nAg). For synthesis of the bimetallic porous µCuO/nAg particles, freshly prepared porous µCuO particles (1 g) were dispersed in 30 mL AgNO3 solution (1.2 mM) and stirred for 5 h. The nAg particles were decorated on the porous CuO microspheres via a galvanic replacement reaction (see Fig.1). The as-synthesized porous bimetallic µCuO/nAg particles were washed three times with DI water and dried on the hot plate at 90 °C for 8 h. Silver Nanoparticles (nAg). Silver nanoparticles with the size of 20-50 nm were obtained from Inframat Advanced Materials. Microbial Culture Conditions. Gram-negative Salmonella (Salmonella enterica serovar enteritidis phage type PT21) was obtained from the Bhunia Lab in the Department of Food 6 Environment ACS Paragon Plus

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Science at Purdue University, while gram-negative E. coli (Escherichia coli O157:H7-GFP B6914) and gram-positive Listeria (Listeria innocua) were obtained from the Deering Lab also in the Department of Food Science at Purdue University. All microorganisms were cultured in Lysogeny broth (LB) at 37 °C with shaking at 250 rpm; the cells were harvested after 24 h, at which point the concentration was ~108 colony forming units per milliliter (CFU/mL). Besides these common bacterial strains, naturally occurring microorganisms from a 10 L sample collected from a local river (Wabash river, West Lafayette, IN) were concentrated and recovered using a custom-built automated continuous cell concentration device (C3D), which utilizes crossflow microfiltration to rapidly separate and concentrate microorganisms from the liquid sample.49-52 This river sample was initially pre-filtered to remove large particles (cellulose filter, 40 µm pore size), followed by filtration using a hollow fiber (HF) membrane module (polysulfone membrane, 10 KDa pore size, catalog 500-017, Rancho Dominguez, CA, USA) in the C3D to reduce the volume of the river sample to ~100 mL. Further concentration and recovery of microorganisms is then accomplished in another HF membrane (0.2 µm pore size, Spectrum Laboratories, Inc., catalog D02-P20U-05-N, Rancho Dominguez, CA, USA) to recover a 4 mL final sample. Antimicrobial Action of Porous µCuO, nAg, and Bimetallic Porous µCuO/nAg Particles. All glassware and samples were sterilized at 120 °C for 30 min. The antimicrobial action of µCuO, nAg, and µCuO/nAg particles was tested against each different microorganism considered. The antibacterial action was first evaluated using the disk diffusion agar method. Particles of each type having a weight of 0.015 g were coated on a glass cover slip (1943-10012 German Glass Round Cover Slip, Bellco Glass; 0.13 mm thick; 12 mm diameter) with the help of spray adhesive (Scotch, Super 77). The microbial suspensions were diluted from ~108



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CFU/mL to ~105 CFU/mL with sterile DI water, and a 100 µL layer of the diluted microbial suspension was spread uniformly over the surface of the LB agar plate. Then, the glass cover slips coated with particles were placed face down on the agar plate with the microorganisms. The plates spread with microorganisms only were used as control. After incubation at 37 °C for 24 h, we compared the antimicrobial efficacy of particles against different microorganisms by measuring the zone of inhibition surrounding the particle samples in the test units. To detect how the particles inhibit the growth of the microorganisms, a growth inhibition assay was conducted. In each experiment, the microbial cell culture with an approximate concentration of 104 CFU/mL was injected into LB medium (5 mL) containing particles at different concentrations (25 µg/mL, 50 µg/mL, and 100 µg/mL). In order to ensure sufficient contact between particles and microbial cells, all experiments were performed in an incubator shaker at 37 °C and 250 rpm. Microbial suspensions were sampled (100 µL) from the medium after different durations of particle interactions with the cells at 0 min, 20 min, 1 h, 2 h, 3 h, 4 h, 6 h, and 8 h after initial introduction, and then spread on agar plates. After incubating the plates at 37 °C for 24 h, the number of colonies was counted in each test unit. The number of colonies on the agar plates without particles was counted as the control. The effect of particles on the growth of the microorganisms was analyzed. All assays were performed in duplicate plates and repeated three times with independent cell plating, to ensure repeatability of the results, and the standard error was calculated. Scanning Electron Microscopy (SEM) Characterization. SEM images of the microorganisms were taken after different lengths of interaction with particles in order to observe any changes in their morphologies. For this evaluation, gram-negative Salmonella and gram-positive Listeria were tested using bimetallic porous µCuO/nAg particles with a


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concentration of 100 µg/mL. After interacting with particles for 0 min, 1 h, 3 h and 6 h, the bacterial suspensions (100 µL) were added into 1 mL of sterile phosphate buffer saline (PBS). The cells were harvested by centrifugation at 14,000 rpm for 2 min, followed by removal of the supernatant with a pipette, leaving the microbial cell pellets at the bottom. Then 1 mL glutaraldehyde (2.5%) was added to the tube to fix the cells overnight at 4 °C; after fixing, the cells were washed twice with PBS. Thereafter, the cell pellets were dehydrated in a graded series of ethanol solutions (50, 70, 85, 95, and 100%), for 10 min each. The drying step was completed by drying pelletized cells at the critical point with a critical point dryer (Tousimis 931). After coating samples with gold via sputtering, SEM images were taken (S-4800, Hitachi).

Figure 1. Schematic drawing of the synthesis process for porous CuO microspheres (µCuO) and decoration with Ag nanoparticles to form the bimetallic porous µCuO/nAg particles.



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RESULTS AND DISCUSSION An SEM image of the synthesized µCuO particles is shown in Fig. 2a. It can be seen that the µCuO particles exhibit a dandelion-head-like hollow porous structure with an average diameter of ~ 3 µm. The zoomed-in image in the right column of the figure shows that the porous µCuO particles are composed of thin nanosheets protruding from the core; the spacing and thickness of the nanosheets are both on the order of ~20-100 nm (Fig. 2a). After the galvanic replacement reaction in silver nitrate solution, the interstices of the porous µCuO particles are decorated with platelet-shaped Ag nanoparticles, as indicated by red arrows in the higher magnification image of Fig. 2b. The as-purchased nAg particles are of similar size (~20-50 nm, Fig. 2c) to the plateletshaped Ag nanoparticles formed on the µCuO particles.

Figure 2. SEM images of (a) porous µCuO, (b) bimetallic porous µCuO/nAg, and (c) nAg particles; the images on the right show zoomed-in views of areas in the corresponding images on the left.


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The chemical compositions of the as-synthesized particles were analyzed by X-ray photoelectron spectroscopy (XPS). The XPS results were obtained at the Surface Analysis Facility in the Birck Nanotechnology Center at Purdue University. Figure 3a shows the full survey scan spectrum for all particle types (namely, porous µCuO, nAg, and bimetallic porous µCuO/nAg particles). Photoelectron lines for Cu and O elements are revealed for the porous µCuO and bimetallic porous µCuO/nAg particles; Ag lines appear for the nAg and µCuO/nAg particles. To obtain more detailed composition information, high-resolution XPS data were collected for Cu and Ag elements, as respectively shown in Fig. 3b and 3c. From assessing the high-resolution scan spectrums of Cu2p for µCuO and µCuO/nAg particles (Fig. 3b), there is no apparent change before and after decorating with Ag nanoparticles. The peaks at 950 and 933 eV are attributed to the bonding energies of Cu 2p1/2 and Cu 2p3/2 of Cu(II) oxide.53 Because each peak can be fitted to only one peak shape, the Cu 2p line corresponds to only one oxidation state, indicating that the synthesized product is pure CuO.54 From Fig. 3c, we can see Ag 3d5/2 and Ag 3d3/2 peaks located at 367 and 373 eV for µCuO/nAg particles of comparable intensity to the nAg particles, indicating that Ag nanoparticles have been successfully decorated onto the surface of the porous CuO microspheres.

Figure 3. XPS spectra of µCuO, nAg, and µCuO/nAg particles: (a) full spectrum and (b, c) corelevel spectra of Cu 2p and Ag 3d, respectively.



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The antimicrobial action of the particles was tested against gram-negative bacteria (E.coli and Salmonella), gram-positive bacteria (Listeria), and environmental microorganisms derived from river water, by using the disk diffusion method. Figure 4a shows a schematic diagram of the zone of inhibition for the antimicrobial action test. The zone of inhibition is a region around the particles where there is no growth of bacteria colonies during the test. A larger zone is considered to indicate a more successful antimicrobial agent for inhibiting bacterial growth. The photographs of the zone of inhibition (indicated with a solid white line) against the different microorganisms for each particle type are shown in Fig. 4b. It can be seen from Fig. 4b that all the particles exhibit some level of antimicrobial efficacy and develop an inhibition zone around the glass cover slips. In contrast, the control plates without particles did not show any inhibitionzone formation (see the left images of each microbial type in Fig. 4b). The corresponding ring width of the annular inhibition zone is shown for each case in Fig. 5. The size of the inhibition zone is strongly dependent on the type of particle and microorganism. The porous µCuO particles and nAg particles have different antimicrobial efficacy against grampositive and gram-negative bacteria; nAg particles exhibit higher antimicrobial efficacy toward E. coli and Salmonella (gram-negative bacteria), whereas porous µCuO particles have stronger antimicrobial efficacy against Listeria (gram-positive bacteria). For the river water microorganisms, the zone of inhibition for µCuO particles is larger than that with nAg particles. From Fig. 5, it is clear that bimetallic porous µCuO/nAg particles exhibit a more pronounced antimicrobial efficacy against all of the tested microorganisms compared to the particles of only CuO or nAg, which can be attributed to the cumulative antimicrobial efficacy of the two metals. The zone of inhibition with the bimetallic porous µCuO/nAg particles against gram-positive bacteria Listeria is larger (~8 mm) than against gram-negative bacteria E.coli and Salmonella


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(~3.5-6.5 mm), which could be attributed to the structural and compositional differences of the cell membranes and cell wall structure between these bacteria types.

Figure 4. (a) Schematic diagram representative of the observed inhibition zone, and (b) photographs of the zone of inhibition with each particle type against gram-negative bacteria E.coli and Salmonella, gram-positive bacteria Listeria, and environmental microorganisms derived from river water. Note that the white lines mark the boundary of the inhibition zones; the control is a negative control without particles.



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Figure 5. Comparisons of the inhibition zone size for different particles acting against each microorganism evaluated. For the bimetallic porous µCuO/nAg particles, which have the most pronounced antimicrobial efficacy, the effect of particle concentration on the growth of the microorganisms is assessed by observing the microorganism growth behavior after different lengths of interaction with the particles. Here, bimetallic porous µCuO/nAg particles with concentrations of 25 µg/mL, 50 µg/mL, and 100 µg/mL were evaluated; gram-negative bacteria Salmonella and gram-positive bacteria Listeria were used as representative microorganisms. Figure S1 (Supporting Information) shows photographs of the colony forming units of Salmonella and Listeria on the plates after being in contact with each particle concentration for different time periods. Table 1 gives the corresponding number of colony forming units per milliliter (CFU/mL). For the control samples without particles (0 µg/mL, top left panels of Fig. S1a and S1b), the number of colonies increases with incubation time; when particles are present, the number of colonies generally decreases with incubation time for the concentrations evaluated, except for the particle


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concentration of 25 µg/mL against Listeria, where the number of colonies increases after 3 h. The growth curves of bacteria as a function of interaction time in the presence/absence of bimetallic porous µCuO/nAg particles are shown in Fig. 6. Note that the results are presented as number of colony forming units per milliliter (CFU/mL), normalized to the number of CFU/mL at 0 min, on a log scale. It can be seen from Fig. 6 that the antimicrobial efficacy improves with the increase of particle concentration for both bacteria types, as indicated by a faster rate of decline in colonies. The growth curves of Salmonella (Fig. 6a) show that the 25 µg/mL particle concentration can inhibit bacteria growth up through 8 h (complete inhibition was observed after 24 h), while at higher concentrations of 50 µg/mL and 100 µg/mL, the time required for complete growth inhibition was reduced to ~6 h and ~3 h, respectively. In the case of Listeria (Fig. 6b), the particle concentrations of 50 µg/mL and 100 µg/mL completely inhibited the growth of bacteria after approximately the same durations (6 h and 4 h, respectively) as for Salmonella; however, in contrast to the behavior observed with Salmonella, the particle concentration of 25 µg/mL cannot completely prevent the growth of Listeria, as indicated by the increasing number of colonies after 3 h. From these tests, it can be deduced that in order to completely inhibit the growth of Salmonella and Listeria, the concentration of porous bimetallic µCuO/nAg particles should be at least ~50 µg/mL, which is comparable or even lower than previously reported metal-based particle concentrations used to inhibit the growth of various bacteria including E. coli, Staphylococcus aureus, Bacillus anthracis, Bacillus circulans BP2, and Pseudomonas aeruginosa BS3.55-57



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Table 1. The number of colony forming units per milliliter (CFU/mL, mean value) of Salmonella and Listeria after being in contact with bimetallic porous µCuO/nAg particles of different concentrations (0, 25, 50, and 100 µg/mL) for different lengths of time (0 min, 20 min, 1 h, 2 h, 3 h, 4 h, 6 h, and 8 h).

Microorganis m type

(µg/mL)

Particle/microorganism interaction time 0 min

20 min

1h

2h

3h

4h

6h

8h

0

9.3×103

8.9×103

8.8×103

6.8×103

1.5×104

4.9×104

\

\

25

8.1×103

9.6×103

5.8×103

7.6×103

6.3×103

4.1×103

8.9×102

1.8×103

50

5.3×103

5.0×103

4.0×103

3.5×103

9.7×102

4.5×102

1.9×102

\

100

2.9×103

4.6×103

5.3×103

8.0×102

2.5×102

\

\

\

0

9.3×103

9.4×103

1.2×104

2.6×104

4.6×104

\

\

\

25

7.1×103

4.7×103

1.9×103

1.2×103

1.1×103

3.7×103

4.6×103

1.9×104

50

4.7×103

4.5×103

3.2×103

2.6×103

1.1×103

6.1×102

2.2×102

\

100

3.1×103

3.2×103

2.5×103

1.2×103

2.7×102

1.0×102

\

\

Salmonella

Listeria

Number of colony forming units per milliliter (CFU/mL)

Particle concentration


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Figure 6. The growth and inhibition profiles for (a) Salmonella and (b) Listeria up to 8 h in the presence of µCuO/nAg particles at indicated concentrations; the control sample has no particles. The club symbol ♣ demonstrates that the bacteria are completely inhibited after the time point labelled, and the spade symbol ♠ indicates there too many colonies to count after the time point marked. Thus, no data are shown for times after these symbols. N is the number of colony forming units per milliliter (CFU/mL) and N0 is the initial number of CFU/mL (at time 0 min). One possible reason for the different bacterial inhibition rates of bimetallic porous µCuO/nAg particles against Salmonella and Listeria could be due to the differences in their cell membrane and cell wall structure, which is the basis for their classification as being gramnegative and gram-positive, respectively. The Cu and Ag ions released by the µCuO/nAg particles bind to the thiol groups of many vital enzymes present in the cell membrane, which may inactivate and damage them, ultimately leading to cell death.30 In order to observe if the cell membranes were damaged, a series of SEM images of Salmonella and Listeria were taken after different lengths of interaction with the bimetallic porous µCuO/nAg particles. Figure 7 shows representative SEM images of the Salmonella (a) and Listeria (b) bacteria morphology after interacting with bimetallic porous CuO/nAg particles for different time periods. As shown in Fig.



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7a, the untreated Salmonella shows an intact rod-like morphology. After the treatment with bimetallic porous µCuO/nAg particles, significant changes in morphology and cytoplasmic membrane integrity can be observed. As the interaction time increases, the Salmonella gradually lost its cellular integrity and shriveled from regular rod-like structures to irregular rod shapes with pits and cusps on the surface. The change in bacterial morphology is attributed to the interaction between Cu/Ag ions and the cellular membrane, which would cause desiccation and eventually cell death.58 However, noticeably different from Salmonella, the morphology of Listeria does not change even when the interaction time with particles increases to 6 h (see Fig. 7b), indicating that the antimicrobial mechanism of action of the particles is different for this gram-positive bacteria compared to Salmonella, a gram-negative bacteria. This may be because gram-positive bacteria have much thicker peptidoglycan cell wall compared to gram-negative bacteria, which makes Listeria less susceptible to particle-induced membrane damage.59 Other antimicrobial mechanisms, such as generation of reactive oxygen species31-33 and nanoparticles adhesion/penetration onto/into the surface of cell membrane,34-36 may be responsible for the antimicrobial action of bimetallic porous µCuO/nAg particles against Listeria. Further studies are needed to investigate the exact mechanism of antimicrobial action of the particles against gram-positive bacteria. Moreover, examination of the cytotoxic effect of particles toward human cells is required before considering their therapeutic use.


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Figure 7. SEM images of (a) Salmonella and (b) Listeria after interacting with bimetallic porous µCuO/nAg particles for different time periods of 0 min, 1 h, 3 h and 6 h. The images on the left and right of each panel show low- and high-magnification images, respectively. The scale bars are 2 µm.

CONCLUSIONS In summary, we have developed a simple technique to synthesize bimetallic porous µCuO/nAg particles via a hydrothermal method followed by galvanic replacement reaction. The antimicrobial efficacy of these bimetallic particles against gram-negative and gram-positive bacteria is stronger as compared to particles with just the CuO or Ag component alone. The antimicrobial action is dose-dependent and increases with particle concentration. The minimum bimetallic porous µCuO/nAg particle concentration to inhibit the growth of gram-positive and gram-negative bacteria considered in this study is recommended to be ~50 µg/mL. The



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antimicrobial mechanism of bimetallic porous µCuO/nAg particle is revealed to differ between the gram-positive and gram-negative bacteria, as supported by scanning electron microcopy examination. The µCuO/nAg particles tend to disrupt the cell membrane for gram-negative bacteria, while the cell membrane integrity is preserved for gram-positive bacteria. The cumulative antimicrobial efficacy of bimetallic porous µCuO/nAg particles makes them a potential alternative to other metal-based particles for prevention of hospital-acquired infections and for use as antimicrobial coatings for medical devices, wound dressings, and other applications.

AUTHOR INFORMATION Corresponding Author * E-mail: [email protected] ACKNOWLEDGEMENTS The authors gratefully acknowledge Professors Arun K. Bhunia and Amanda J. Deering of the Department of Food Science at Purdue University for providing the bacterial strains, and the staff at the Life Sciences Microscopy Facility of Purdue University for preparing the bacterial samples for SEM imaging. SUPPORTING INFORMATION Photographs of colony forming units of Salmonella and Listeria


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Enhanced Antimicrobial Efficacy of Bimetallic Porous CuO

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