Bactericidal efficiency of Au and Ag nanoparticles (NPs) is reported with and without photoactivation by white light. Au and Ag NPs were synthesized with an average size of and of , respectively. The size distribution of the Ag colloid was relatively wide. Less than 4 of these NPs were largely decahedral, which, based on numerical calculations, determined the position of the optical band. In contrast, the Au colloid had a narrow optical band; a concentration of was determined by theoretical and experimental spectra. Ag and Au NPs showed a superficial charge of and due to the presence of the citrate ions and cetyltrimethylammonium bromide on their surface, respectively. The effect of the NPs concentration on the viability of Escherichia coli and Staphylococcus aureus strains was investigated. It was found that Ag NPs were more effective against E. coli than Au NPs, whereas Au NPs were more effective against S. aureus than Ag NPs. The induced damage to the bacteria by the NPs was evaluated by AFM. The images show that the bacterial cell wall was changed in shape and in surface roughness, being more noticeable in S. aureus than in E. coli. The bactericidal activity of the photoactivated Ag NPs was almost doubled for both bacteria, whereas for the Au NPs, no bactericidal enhancement was observed for either strain. This can be explained by the high efficiency of Ag NPs to absorb white light and the consequent creation of hot spots that contribute to kill the bacteria.
The discovery of penicillin and its use around 1940 made a positive change in the treatment of infectious diseases. However, due to the ability of adaptation of the bacteria and the indiscriminate use of antimicrobials, there are numerous resistant microorganisms and diseases caused by bacteria that do not respond to the use of the most common antibiotics, becoming a problem worldwide1 especially of nosocomial infections. Staphylococcus aureus is one of the main causes of hospital-acquired infections with high antibiotic resistance, and one out of three people carry S. aureus.2 This microorganism usually causes pneumonia,3 endocarditis,4 and skin and soft tissue infections such as abscesses and cellulitis.5 Other common bacteria is Escherichia coli, which is mainly a commensal bacterium that colonizes the intestine of most mammals, although some strains under certain circumstances can be pathogenic producing gastrointestinal diseases such as diarrhea. E. coli has evolved through horizontal transfer of virulence genes that resulted into various pathovars that provide it with the ability to colonize and infect other regions of the human body such as urinary and circulatory systems.6,7 Therefore, there exists the necessity for treatment options for infections, stimulating research and discovery of new alternatives against these microorganisms. An available possibility is offered by using light, alone or combined with other elements. Recently, the photosensitization of bacteria has been reported, finding that its deactivation is not a consequence of heat, but rather a light-activated effect.8 Even more, to elucidate the role of light as an antibacterial agent, Lipovsky and colleagues used light of different wavelengths and intensities over E. coli and S. aureus concluding that visible light (400–800 nm) at high intensity kills bacteria, while low-power white light enhances bacterial proliferation.9 This finding proves the complex bactericidal activity of light. Moreover, light can excite molecules (photosensitizer molecules) that trigger a bactericidal effect through reactive oxygen species (ROS) production; until now, bacteria have not developed resistance to photoantimicrobials.10
A recent alternative is given by gold, silver, zinc oxide, titanium dioxide, or copper nanoparticles (NPs), among other nanomaterials, that have shown bactericidal activity against Gram negative and Gram positive bacteria. Ag nanoparticles (NPs) have a large bactericidal effectiveness that has been demonstrated against different bacterial species of clinical importance such as S. aureus, E. coli, and Pseudomonas aeruginosa, as well as fungus such as Candida.11,12 Additionally, bacteria are less likely to develop resistance against metallic NPs than to the conventional antibiotics, due to the numerous mechanisms of action of NPs.13 Unfortunately, the bactericidal efficiency of the NPs is not the same for Gram positive than Gram negative bacteria and, moreover, can be larger or lower from one kind of strain to another.14 NP shape and size have also been parameters to take into account in bacterial assays.15 Small Ag NPs have shown better antimicrobial effects,16 an outcome explained by the assumption that smaller NPs produce more Ag ions than large NPs.
Besides their bactericidal response, Ag and Au NPs are very well-known for their plasmonic properties. Their conduction electrons can be excited with light of a specific wavelength (usually in the visible spectrum) which corresponds to the resonance wavelength of the collective oscillation of conduction electrons at or near the surface (localized surface plasmon resonance, LSPR). Once the application of the external electromagnetic field ends, the electrons return to their unexcited state, releasing energy that is mostly transferred as heat. This phenomenon has been widely exploited by photothermal therapy to treat cancer tumors,17 using wavelengths close to the red and near infrared light.
In this work, we synthesize and characterize Ag and Au NPs and subsequently evaluate their antimicrobial effects against drug resistant S. aureus (Gram positive) and E. coli (Gram negative). As a hypothesis, we establish that the bactericidal efficiency of the NPs is enhanced when the bacteria are exposed to the NPs and at the same time are irradiated with light. To verify our hypothesis, bactericidal assays with NPs irradiated and unirradiated with white light are presented. These results can address the use of metal NPs for medical applications or within the agrofood industry.18,19
II. MATERIALS AND METHODS
A. Synthesis and characterization of silver and gold nanoparticles
Turkevich’s method was used to synthesize the metal NPs with sizes smaller than 50 nm.20,21 The size distribution of the particles was controlled by adjusting the temperature, pH, and the precursor concentration.22 Here, Ag NPs were synthesized as follows. 20 ml of 1 mM silver nitrate () Meyer 99.9% were heated to and vigorously stirred, next 2 ml of 1% sodium citrate () J. T. Baker 100% were rapidly added. The stirring was stopped when a yellow color was observed.
Independently, Au NPs were prepared following a seeded mediated synthesis method previously described.23 Sodium borohydride (), chloroauric acid trihydrated (), cetyltrimethylammonium bromide or CTAB (), ascorbic acid (), and sodium citrate () were supplied from Sigma-Aldrich. All reactants were used as received without further purification. The seeded mediated synthesis method started by first synthesizing the small Au NPs (the “seeds”) which were grown into bigger sizes by adding them into a “growth solution” containing Au precursor. We started by synthesizing the Au seeds (Au NPs of in diameter), which were obtained by abruptly adding of a freshly prepared 10 mM solution into a 30 ml solution containing 0.125 mM of and 0.250 mM of under stirring at room temperature. Afterward, the obtained solution is moved into a water bath at and kept under gently stirring for 15 min to ensure activity minimization of . Finally, the seeds solution was taken out of the water bath and the Au seeds were obtained. The second step was to prepare a 40 ml growth solution by mixing CTAB and in water (0.125 mM and 40 mM CTAB) at room temperature. Following the addition of of a 100 mM ascorbic acid solution, a color change resulted from pale yellow to transparent. Finally, 1.8 ml of Au seeds, previously synthesized, were added into the growth solution under stirring.
All the samples were characterized by transmission electron microscopy (TEM), UV-Vis spectroscopy, and Zeta potential experimental techniques. A Jeol-Jem 2010 transmission electron microscope was employed to determine the size distribution and shape of NPs. Prior to TEM characterization, the nanoparticles’ dispersion was washed/redispersed in de-ionized water to remove the surfactant excess. A couple of drops were placed on a holey carbon coated Cu grid. No staining was needed. The mean size was estimated from a statistical analysis over 100 Au NPs and 300 Ag NPs taken from representative TEM micrographs for each sample. The measurements were performed by using the “Image J” software. A Varian UV-Vis Cary 100 scan spectrophotometer was employed to obtain the UV-Vis spectra. Information of the electrophoretic mobility of NPs in colloidal solution was obtained using a Malvern Zetasizer Nano S90. The Zeta potential values reported correspond to an average of three measurements in water at pH 7 and .24
The concentration of NPs in the colloid was estimated using the well-known relation
where is the absorbance of the colloid, is the wavelength, is the length of the sample, is the concentration of NPs, and is the optical extinction cross section of a single NP. Introducing the extinction efficiency as ( is the radius of the NP) and considering values of and at the wavelength of the LSPR, then
of a single spherical, icosahedral, and decahedral NP with various sizes was calculated using the Discrete Dipole Approximation and DDSCAT code 7.3. Information about the model and computational code can be found elsewhere.25,26 The refractive index of Ag and Au in bulk are the reported by Johnson and Christy,27 properly modified to take into account of the finite size of the NP.28 The fact that the NPs are in an aqueous solution was also considered (refractive index 1.33). and were obtained from UV-Vis spectroscopy.
B. Determination of the bactericidal effect of NPs
The strains of E. coli MC 4100 and S. aureus L 27 used in this study were kindly provided by the Microbiological Sciences Research Center, BUAP, Mexico.
As a generality, all the experiments were done using strains taken from a cryovial at . The strains were incubated approximately 18–24 h in Luria-Bertani (LB) media at .
The determination of minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) values was done following the microdilution method of the Clinical and Laboratory Standard Institute (CLSI).29 First, the synthesized NPs were washed twice with triply distilled water to eliminate secondary products of the reduction reaction and then resuspended in LB media. Afterward, of different concentrations of NPs (see Table I) were put into the wells of a 96 well plate. Next, of a bacterial culture at a concentration of were added to each well. Finally, the strains and NPs within the wells were incubated at for 12 h (total incubation time).
|Ag NPs ) .||Au NPs ) .|
|Ag NPs ) .||Au NPs ) .|
The growth and viability curves were obtained from the absorbance measurements and the counting of colony forming units (CFU), respectively. During the incubation time, the absorbance was measured each hour with a Bio-Tek ELx800 microplate reader at 620 nm. Simultaneously, every 3 h, a sample was obtained, diluted (in LB 10, 100, and ), and plated in LB agar by the drop plate method, and then the CFUs were counted manually.
The whole procedure was repeated by photoactivating the NPs during the incubation time. A lamp with an array of 30 white light LEDs with a wavelength range of 400–700 nm and 150 lumens was used as the light source. The LED lamp was located under and at a distance of 3 cm from the 96 well plate, see Fig. 1. The distance from the source to the plate was kept to avoid any damage to the bacteria due to the heat radiated by the lamp. For completeness, incubated bacteria were also illuminated with the lamp and without NPs.
The MIC and MBC values were determined from the growth curves and viability assays for both strains, E. coli and S. aureus, with Au or Ag NPs.
To establish the shape, dimensions, and cell membrane damage of the bacteria, an atomic force microscope (AFM) XE-7 from Park Systems in the noncontact mode was used. The AFM topography and phase images were taken with PPP-NCHR tips at 1 Hz of scanning rate. Furthermore, the root mean square (rms) roughness of the specimens was determined using Gwyddion Software.30
III. RESULTS AND DISCUSSION
A. Size and shape of the synthesized NPs
A representative TEM micrograph of Au NPs is shown in Fig. 2. Rounded NPs are observed with probable spherical, icosahedral, and decahedral morphologies.31 From the statistical analysis performed to several TEM images, the diameter distribution, average size, and standard deviation were estimated, determining an average diameter of .
The measured optical absorbance () of Au NPs is shown in Fig. 3(a). The spectrum has a Full Width at the Half Maximum (FWHM) of about 88 nm, and its maximum is located at 525 nm. As the extinguished light is the absorbed plus the scattered light () and small metal NPs are well-known to be very good absorbers,32 then . Therefore, the maximum of is assigned to the characteristic LSPR; its position and intensity give account of the size and shape of the NPs in the colloid.33 To precise the representative morphology of the Au NPs, of some geometries was calculated. Panel (b) of Fig. 3 shows of a single spherical Au NP and a single icosahedral Au NP with the average size of the sample (). As we can see, the LSPR of a spherical NP (at 523 nm) is closer to the experimental LSPR than that of an icosahedral NP (at 531 nm). Moreover, the shape of the three spectral lines is very similar. Hence, we infer that in Au NP sample, NPs with spherical and icosahedral morphologies do exist. Then, the NPs’ concentration was estimated using the measured value of , calculated value of of a sphere, and Eq. (2), obtaining spherical Au ().
A similar procedure was intended for Ag NP sample. In Fig. 4, representative TEM images and the corresponding size distribution are shown. The sample contains mainly rounded small NPs with an average diameter of ; also, some large decahedral NPs are present but in a very small percentage (less than 4%). A feature of colloids with a wide size or shape distribution is a wide spectrum, and this sample is not an exception as it has a [see Fig. 5(a)], larger than that of the Au colloid. We did calculations exploring various sizes and morphologies. In Fig. 5(b), the optical efficiency of small and big Ag NPs is shown; surprisingly, although the amount of big NPs is not representative, its presence determines the position of the optical band.
On the other hand, Zeta potential was measured to have information of the stability and surface charge of the metal NPs. The zeta potential of Au NPs is of which indicates good stability. The positive sign suggests that the Au NPs are strongly cationic,34 and we attribute this to the hydrophilic group of CTAB stabilizer over the surface of the micelle.35–37 Ag NPs also have good stability but an anionic character (negative zeta potential );34 this fact is explained by the citrate anions adsorption given by two linked negative oxygen atoms and a third one free.38
B. E. coli and S. aureus in the presence of Ag and Au NPs
Both species of bacteria were treated using various concentrations of NPs (see Table I) and following the process described previously.
Bacterial growth and viability curves in the presence of Ag NPs are shown in Fig. 6. For E. coli, a complete inhibition was appreciated at a concentration of and higher [Fig. 6(a)], while lower concentrations than slightly inhibited its growth. On the other hand, S. aureus presented complete inhibition at and larger values; on the contrary, at lower concentrations growth rates kept similar to that of the control sample. However, at the particular concentration of , a bacteriostatic effect was promoted [Fig. 6(b)]. These data correlate well with the cell viability quantification (CFU/ml) shown in Figs. 6(c) and 6(d).
In panel (a) of Fig. 7, it is appreciated that a concentration of of Au NPs completely inhibits the growth of E. coli, while significant difference in the growth rate is not observed for the other concentrations and the control. These data agree with the respective viability curves [panel (c)]. In panel (b) of Fig. 7, it is noticed that concentrations of and higher have a good bactericidal action against S. aureus. In contrast, at lower concentrations, the culture is not affected, and a conclusion is drawn from the growth curve of the control sample and the viability curves [panel (d)]. Nevertheless, it is noteworthy that there is a retardation time of 3 and 6 h to reach a complete inhibition when the bacteria are exposed to a concentration of and , respectively.
The absorbance curves contain information of live and dead bacteria, while the CFU curves provide information only of live bacteria; therefore, both are complementary to specify the MIC and MBC values. The consequent MIC and MBC values are shown in Table II. The MIC value corresponds to the lowest concentration that inhibited the growth of the bacteria; meanwhile, MBC value is determined by the lowest level of antimicrobial agent that kills the bacteria. We found that the MBC/MIC ratio is equal to 1 for Ag and Au NPs against both species, which indicates their good role as antibacterial agents.39 However, the antimicrobial effect of Ag and Au NPs on each genera is different. We infer that Ag NPs have a better antimicrobial effect on E. coli than Au NPs do; this agrees well with the results reported by J. S. Kim and colleagues, notwithstanding the fact that they used different strains than the ones studied here.40 Inversely, Au NPs have a better efficacy on S. aureus than Ag NPs.
|.||MIC–MBC values .|
|Strain .||Ag NPs .||Au NPs .|
|E. coli MC4100||5.59–5.59||10.51–10.51|
|S. aureus L 27||11.19–11.19||1.31–1.31|
|.||MIC–MBC values .|
|Strain .||Ag NPs .||Au NPs .|
|E. coli MC4100||5.59–5.59||10.51–10.51|
|S. aureus L 27||11.19–11.19||1.31–1.31|
The MBC or MIC value reported here for Au NPs against E. coli is equal to . Bactericidal activity of Au NPs against other E. coli strains has also been detected, revealing a MBC/MIC ratio larger than 1 but smaller than 4.41 On strain , lower values of MIC than the ones established here were determined by the use of Au NPs with a size of 20–30 nm.42 Also, for the particular case of Au NPs against S. aureus, a low MIC value was settled but with the help of smaller NPs than those employed by us.43 Then, the efficacy of Au NPs as bactericidal agents does not obey a simple inverse relation with its size.
1. Visualization of the cell damage
To exhibit the damage to the bacteria, AFM images are presented. In Fig. 8, the differences in morphology, volume, and surface of the E. coli without and with Au or Ag NPs are evident. Images in panels (a), (b), and (c) correspond to the bacteria before being treated with NPs, where its characteristic shape, a rod with spherical caps, is recognized. From the 2D and 3D topography, the bacteria length is estimated to be approximately and its width around , typical values of this genus.44,45 Additionally, the surface of the cell is uniform with a root mean square (rms) roughness of 19.7 nm. Besides, the external layer of the bacteria can be clearly identified when the noncontact mode is selected in the AFM [phase of the cantilever vibration, panel (c)].45 The damage induced to the bacteria due the Au or Ag NPs [panels (d)–(f) and (g)–(i) of Fig. 8, respectively] is revealed through the changes suffered in the cell membrane, a complete loss of its integrity and an irregular shape is acquired. Similar harm has been produced by Ag NPs to E. coli and S. aureus but with the need of large concentrations ().46 From the color contrast in phase images [panels (f) and (i)], it is possible to distinguish at least two different materials surrounding the cell, indicative of intracellular material leakage. Also, the surface changed dramatically, and rms roughness of 36 nm and 55 nm were estimated after Au NPs and Ag NPs treatment, respectively. Therefore, the rms roughness value is a quantitative indicative of the damage induced to the bacteria.
S. aureus without and with NPs is shown in Fig. 9. Panels (a) and (b) show the 2D and 3D topography of the bacteria without NPs. The typical spherical shape of this bacteria is clearly observed, its diameter is approximately , and the surface rms roughness of 30 nm. Panel (c) reveals a uniform surface of the bacteria; besides, the color contrast observed on the zone surrounding the cell defines the region occupied by the capsule produced by S. aureus. Figures 9(d) and 9(e) exhibit the 2D and 3D topography of the S. aureus treated with Au NPs, clearly revealing that the shape of the bacteria is elongated compared to the spherical shape of the control [panel (a)]. A considerable decrease in its dimensions is evident because now the measured length is and the width is . However, the surface roughness is unchanged as the rms value is 31 nm. Additionally, in the phase image [panel (f)], brown and white zones are observed in the bacteria indicating depressions over the surface due to the interaction with Au NPs. Also, the contrast zone surrounding the cell could be the capsule but thinner. The 2D and 3D topography of S. aureus treated with Ag NPs is observed in panels (g) and (h), respectively; the shape of the bacteria has been deformed due to its interaction with Ag NPs, and consequently its roughness changed to a rms surface value of 37.3 nm. In the phase image [panel (i)], some bright spots are noticed, which are probably internal components of the bacteria indicating that the integrity of the cell has been completely lost.
Several experimental reports indicate that Ag and Au NPs show a better effect against Gram negative than Gram positive bacteria, this being attributed to the fact that nanoparticles can penetrate easier the Gram negative cell membrane. However, we found that Au NPs have a better effect against S. aureus than E. coli, which indicates there are other mechanisms of action besides the penetration of the nanoparticle that determine its bactericidal action. As they have a positive superficial charge, then its interaction is poor with the phospholipids of the membrane, inducing a lower damage than the one induced by the Ag NPs. Some other factors can be considered in order to explain the harm produced to the bacteria: (i) damage to the membrane (depolarization or collapse membrane potential), (ii) decrease in the bacterial metabolism (inhibition of the ATP synthase), and (iii) cellular breakdown.41,42
The damage due to the Ag NPs can be explained as follows: when they are adsorbed onto the cellular wall, injure the bacteria due to a reaction with the phosphate groups of the phospholipids, and promoting the formation of Ag ions. These ions and NPs can stay on the cellular wall or enter the cytoplasm. Ag NPs in the range of –100 nm show no penetration and also the smaller particles release much more ions than the bigger ones, due to an increased volume/superficial area ratio.47 When the ions enter the cell, they interact with the proteins, ribosomes, and DNA or induce the formation of reactive oxygen species (ROS), resulting in a dead cell or lysis.48
C. Bacterial assays with photoactivated NPs
All the previous results were obtained without direct application of light. In this section, we present bacterial assays done using white light of a LED lamp during the incubation time. White light was chosen to take advantage of the width of the Ag and Au NPs optical spectra; as it was observed, they cover a good interval of the visible spectrum. We found that the efficacy of the antimicrobial effect of Ag NPs is enhanced, but the efficacy of Au NPs is the same with and without light.
When E. coli is exposed to photoactivated Ag NPs, a growth inhibition is noticed at a concentration of (Fig. 10), while at the same concentration, a bacteriostatic effect was observed when light was absent [Fig. 6(a)]. A similar behavior is appreciated for S. aureus but with a Ag NPs concentration of [comparison between Figs. 10 and 6(b)].
Using Au NPs against E. coli, only the highest concentration () is the one that promotes a complete growth inhibition, with and without light. Meanwhile, concentrations of and higher induce cell death of S. aureus with and without illumination. We did not succeed with the enhancement of Au NPs’ bactericidal efficiency. However, an increase in the effectiveness of Au NPs against S. aureus was previously reported when a laser is used49 or Methylene Blue coated Au NPs are photoactivated.50
For completeness, we measured growth curves of E. coli and S. aureus without nanoparticles and with light (see Fig. 11). As the curves of the control culture and culture with light are very alike, it shows that no damage by heat is induced and the bacteria are not photosensitive to white light.
The improvement seen in the bactericidal effectiveness of Ag NPs when excited by light is associated with the width of their optical spectrum characterized by a (covering from violet to green), making that almost one third of the incident wavelengths that compose the white light be absorbed and transformed into the conduction electrons excitation energy. Meanwhile, for Au NPs, no improvement was observed because its spectrum has a and barely covers the green range. It is worth to continue investigating the effect of light on plasmonic NPs and their antibacterial action, specifically using laser light with wavelength close to the LSPR of the NPs.
In this work, we synthesized Au NPs of a mean size of (mostly spherical) and Ag NPs of . The FWHM of the Ag NPs spectrum is 120 nm and that of the Au NPs spectrum is 88 nm, revealing that the size distribution of Ag NPs is wider than that of Au NPs. In the colloid of Ag NPs, although there is a very small quantity of big decahedral NPs, their presence determines the position of the optical band. By zeta potential measurements, a negative superficial charge was assigned to Ag NPs () and a positive charge to Au NPs (), indicating that the nanoparticles are stable in water. We infer that the negative sign is due to the adsorption of layers of citrate ions on the surface of Ag NPs during the synthesis; meanwhile, the positive sign is conferred to the CTAB used in the synthesis of Au NPs.
Antimicrobial activity of the NPs was tested against Gram positive and Gram negative genera, specifically, S. aureus and E. coli, respectively. We found a MIC/MBC ratio equal to one, indicating the good role of the NPs as antibacterial agents. We also found that Ag NPs are more effective against E. coli than Au NPs; on the contrary, Au NPs are more effective against S. aureus than Ag NPs. By AFM images, evident damage was observed in the cellular wall of both genera, losing their shape due to the leaking of intracellular material. At least for E. coli, the damage can be estimated quantitatively through the rms roughness values, associating larger values to the complete rupture of the membrane. We assume that, due to the nature of the surface negative charge of Ag NPs, these can interact easily with the phosphate groups of the phospholipids of the membrane and enter the bacteria promoting its rupture releasing the cellular content. In the case of Au NPs, these have a surface positive charge allowing poor interaction with the phospholipids of the membrane, resulting in lower damage compared to the one induced by Ag NPs.
Furthermore, we repeated the assays using a white light LED lamp for activating the LSPR of the NPs and to take advantage of the width of their optical spectrum. The bactericidal action of Ag NPs was enhanced about twice, which means that to inhibit the bacterial growth with light, only half of Ag NPs concentration is required. The bactericidal action of Au NPs with light was not improved. We assume that because the optical spectrum of Ag NPs is wider than that of Au NPs, the first ones absorb the incident white light more efficiently and therefore excite the conduction electrons more efficiently producing hot spots that burn the bacteria locally. These results show that metallic nanoparticles can be used as a therapeutic approach against multidrug resistant bacteria and the antimicrobial efficiency may be enhanced via LSPR activation.
We conclude that photoactivating the metal NPs to enhance their bactericidal efficiency is possible; however, the enhancement factor is metal dependent and different for Gram positive than for Gram negative bacteria.
The authors gratefully acknowledge financial support from the UNAM Mexico funding through DGAPA-PAPIIT IA103117 (J.M.R.-H.) project and VIEP-BUAP projects: MAML-NAT17-1, SOUL-NAT17-1, 100504244-VIEP2018, 100409188-VIEP2018. P. Pfeiffer acknowledges CONACyT Fellowship No. 581420. The authors also thank Dr. Margarita Arenas and Dr. Rosa del Carmen Rocha for providing E. coli and S. aureus strains, respectively.