Carbon nanomaterials such as nanodiamond (ND) and graphene oxide (GO) are considered highly promising for diverse biomedical applications such as long-lasting medical implants, bone tissue engineering, biosensors or drug delivery [1
]. This is especially due to their beneficial chemical and physical properties such as general biocompatibility and non-toxicity, stable yet widely adjustable surface chemistry as well as suitable optical and electronic properties.
Even though these materials exhibit good biocompatibility, recent studies pointed out bactericidal properties of nanodiamonds and graphene oxide. It was shown that medical steel coated with a ND layer significantly suppresses the growth of the E. coli
cells compared to pure medical steel or titanium surface without coating [7
]. Another work stated that the bactericidal effect of NDs is most likely due to their partially oxidized surfaces where reactive oxygen-containing surface groups foster interactions of NDs with cellular components while the anisotropic distribution of charges on the ND surface facilitates alterations in bacterial surfaces [8
]. A similar mechanism of antibacterial interaction between NDs and bacteria was also deduced in [9
]. Our prior studies showed that NDs can modify different bacteria in a different manner. While Escherichia coli
indicated a drop in the number of colonies compared to the reference due to the presence of NDs, Bacillus subtilis
indicated a similar number of colonies but smaller colony sizes [10
GO is also extensively proposed as an effective antibacterial agent in commercial product packaging and for various biomedical applications. Various works confirmed bactericidal properties of graphene-based materials, namely pristine graphene (G), graphene oxide (GO) and reduced graphene oxide (rGO) [11
]. The suggested mechanism was different for various types of graphene-based materials. It was argued that superoxide anion generated by GO can disrupt the membrane of bacteria and that disruption of the cell membrane can also appear when it comes in direct contact with the sharp edges of GO nanowalls. Graphene nanosheets were also found able to be able to extract large amounts of phospholipids from the cell membranes due to strong interactions between graphene and lipid molecules [13
On the other hand, our recent study of interaction of various NDs, GO or rGO with E. coli
in Mueller Hinton (MH) broth and on MH agars showed that after 24 h the nanomaterials had no statistically significant antibacterial effect except for hydrogenated NDs that decreased the number of colony forming unit (CFU) by 50% [14
]. Thus, there are pronounced differences in the reported degree of antibacterial effects as well as the proposed mechanisms. This is not surprising as various inorganic nanoparticles have been found to exhibit bactericidal properties and cause growth inhibition but the mechanisms of toxicity are generally not yet fully understood [15
]. Most likely, it is due to different conditions in so far reported studies, such as specifically employed nanomaterial, a method of application (in the volume, on the surface) and also type of examined microorganism and culture medium.
In order to unambiguously elucidate the mechanism(s) of the antibacterial properties of ND or GO, one possible approach is to perform a comparison of the materials (with well-characterized properties) under the otherwise same conditions, such as in the case of cell interaction with graphene and nanocrystalline diamond thin films [3
]. Therefore, in the present study, we compare bactericidal properties of well-characterized NDs, GO and rGO under the same conditions including nanomaterial and bacterial concentrations. We perform the comprehensive study in two different culture media, Luria-Bertani (LB) and Mueller-Hinton (MH) broths. Thereby we disclose the specific effect of cultivation media as well as we elaborate on the origin of antibacterial properties of the aforementioned nanomaterials. The results may be useful for prosthetics and implants as well as for water purification and preservation of its quality.
2. Materials and Methods
Four types of nanomaterials were employed in our study and mentioned in the further text. As-received NDs produced by detonation process are labelled as HND due to their numerous C-H bonds and positive zeta potential [16
]. NDs that were annealed in air at 450 °C for 30 min to oxidize their structure [17
], are labelled as OND. The nominal diameter of both types of NDs is 5 nm.
The third nanomaterial was graphene oxide (GO), which was produced by oxidation from graphite powder according to Bangal method [18
], filtered through a nylon membrane and finally sonicated in an ultrasonic bath for 3 h. Estimated sizes of GO flakes is 0.5 to 2 μm. The fourth material, reduced graphene oxide (rGO) was produced by oxidation from graphite powder according to Brodie method [19
], filtered through a nylon membrane, dried and thermally reduced in an argon atmosphere at 750 °C to obtain well-exfoliated rGO flakes. Estimated size of rGO flakes is 0.1 to 1 μm. More details about the employed GO and rGO materials can be found in [14
All nanomaterials used in this work were dispersed in distilled water to achieve a concentration of 2 mg/mL. Figure 1
a shows a photograph illustrating appearance of such dispersed suspensions. The suspensions were homogenized in an ultrasonic bath for 30 min and sterilized by autoclave at 120 °C. We have confirmed that the sterilization in autoclave did not affect surface chemistries of NDs and GO/rGO sheets [14
]. Figure 1
b illustrates decreasing turbidity of suspension with E. coli
due to increasing dilution in the MH broth (samples in LB broth look similarly).
2.2. Microbiological Studies
The microbiological study was performed in two different media—Mueller-Hinton (ready made powder purchased from the Oxoid Ltd., Basingstoke, UK) and Luria-Bertani (powder components purchased from the Oxoid Ltd., Basingstoke, UK) in both liquid and solid phase: Mueller-Hinton broth (MHB) and agar (MHA), Luria-Bertani broth (LBB) and agar (LBA). The MH broth was prepared according to the instructions of the supplier by mixing of 21 g of ready-made MH powder with 1 L of distilled water. MH broth prepared by this way contains beef infusion, casein hydrolysate and starch [20
]. The LB broth was prepared according to the recipe [21
] by mixing of distilled water (1 L) with tryptone (10 g), yeast extract (5 g) and sodium chloride (10 g). The pH was balanced to 7.2 ± 0.2 in both cases. The agars were prepared from the broth liquids by the addition of 20 g of agar powder per 1 L of the broth.
The sample of bacterial suspension was prepared as follows: We spread 1 mL of concentrated E. coli suspension (taken from the freezer and melted for 15 min) on 9 cm Petri dishes with the agar (either MHA or LBA) and let it grow overnight in the thermostat at the temperature of 37 °C. Then all the bacteria from the Petri dish were wiped off and put into 5 mL of a broth (either MHB or LBB). This concentration of the bacteria was unity and marked as 100.
The initial bacterial suspension was diluted by the broth in several steps with dilution ratio 1:10 in each step. Figure 1
b shows a photograph illustrating the appearance of the suspensions with different bacterial concentrations. We stopped diluting of the suspension when its turbidity, around 0.5 MFU (MacFarland Units), was achieved. The closest value to this turbidity had a sample with relative concentration of 10−3
(turbidity of 0.4 MFU in case of both MHB and LBB). This bacterial concentration was used in all the performed experiments.
Then we prepared five test tubes for each broth type. Four of them were filled with 3 mL of bacterial suspension in appropriate broth and 3 mL of the suspension with the examined material. Thereby the concentration of examined nanomaterials was 1 mg/mL. The fifth test tube was a reference one, in which 3 mL of bacterial suspension were diluted by 3 mL of distilled water.
All test tubes were put into the shaker inside of the thermostat set at 37 °C. The first set of samples was taken from each tube after 5 h to examine the exponential growth phase of bacteria. Each sample was then gradually diluted to the relative concentration 10−10 while the original concentration taken from each test tube was 10−3. We used concentrations 10−9 and 10−10 for the further cultivation on Petri dishes.
These concentrations of each material were spread on three 9 cm Petri dishes in a triplet with the agar which represented a set of 15 Petri dishes for each type of media. The amount of the spread suspension was always 1 mL. Then we put all Petri dishes into the thermostat set at 37 °C for 24 h.
Another set of samples was taken from test tubes with broth after 24-h of incubation to examine the stationary phase performing the same dilution, spreading and cultivation procedure like we did with the 5 h set. The original concentration taken from each tube was still considered 10−3. The bacterial cultivation on agar plates (MHA or LBA), either original or enhanced by the addition of 40 g/L NaCl, was also performed for 24 h at 37 °C.
The bacterial colonies on Petri dishes were then counted and the average number of colonies was compared with the negative control sample (100% = the average for the control sample). The two-sample t-test for unequal variance with six participants in each group was used for the evaluation and comparison of colony unit counts against reference. There were two various bacteria concentrations in triplicates for each sample, which were recalculated to unity concentration.
We also performed an experiment with salty agars. To obtain salty agars we prepared MH and LB agars in the usual way and finally, we added sodium chloride into the agar liquid in a concentration of 40 g/L. Then we compared the growth of bacteria on normal and salty agars to find whether the combination of salt and nanomaterials would result in a synergic stress effect on the bacteria.
2.3. Material Analytical Techniques
Surface chemistry of the LB and MH broths was characterized by attenuated total reflectance Fourier transform infrared (ATR-FTIR) spectroscopy. Details about the spectrometer, its accessories and evaluation method can be found in [14
]. In all cases, the spectra represent an average of 128 scans recorded with a resuspension of 4 cm−1
. Spectra were normalized at 1632 cm−1
resp., AMID I band). Advanced ATR correction was applied on all measured spectra.
X-ray photo electron spectroscopy (XPS) analysis of the broth samples of MH and LB was performed on AXIS Supra (Kratos Analytical Ltd., Manchester, UK) using monochromated Al Kα X-ray source (1486.6 eV) and a hemispherical energy analyser (analysed area—0.7 × 0.3 mm2). XPS photoelectron survey spectra were acquired at a constant take-off angle of 90° using 80 eV pass energy. Samples were prepared by deposition of 100 µL broth suspension on an Au/Si substrate. After drying in a stream of nitrogen, the samples were further dried in a vacuum of 10−5 mbar for 2 days.
The size distribution and ζ-potential of the NDs, GO, rGO and E. coli colloidal suspensions were measured by dynamic light scattering (DLS) at 25 °C using a Nano-ZS (Malvern Instruments Ltd., Malvern, UK) equipped with HeNe laser. The disposable folded capillary cell was used to eliminate sample cross-contamination. The samples were not filtered nor centrifuged prior to the DLS measurements.
Scanning electron microscopy of the bacteria was performed by FE-SEM Mira 3 microscope (Tescan Brno s.r.o., Brno, Czech Republic) at an electron beam energy of 30 keV. The top-view micrographs were acquired using an in-beam detector in the secondary electrons mode at a working distance of about 5 mm. The bacteria were sampled directly from LB suspension with nanomaterials, diluted 100× in water and drop casted on a rough side of Si wafer substrate. The samples were consequently dried in air for 5 min.
Antibacterial effects of the employed carbon nanomaterials are significantly different in the specific media. While only HND exhibited significant reduction ratio of bacterial CFU in the MH medium, there was uniform reduction ratio in the LB medium across all the materials. The difference is probably caused by the presence of more stressors for the bacteria in case of the LB medium supported by the presence of nanomaterials and their antibacterial activity.
Since we tested a set of microorganisms, we had to keep in mind that negative influence on individual bacterium could be compensated by reaction of other bacteria. This surviving strategy is limited by stress factors. That is why the testing of biological properties of nanomaterials should include also analysis of stressors. In real conditions, we can expect the presence of several stressors (physical, chemical, nutritional).
There are two basic modes of action of nanomaterials affecting the E. coli
growth: direct mechanical interaction and/or oxidative stress [25
]. Accumulation of nanoparticles in close vicinity of bacteria can be caused by the electrostatic interaction of nanoparticles with (typically negatively) charged cellular surface. Inducted membrane stress can result in lethal changes of the cell structure [26
]. Negatively charged domains of bacterial flagella proteins can attract positively charged nanoparticles [27
]. Accumulated nanoparticles can form either thin layers around cells (in case of GO) or large aggregated particle clusters (rGO and NDs) [10
]. Consequential intimate contact with nanoparticles can cause modification of cell vital structures by local chemical or electrostatic interaction [24
]. Spectroscopic signatures obtained from biomolecules such as adenine and proteins from bacterial cultures with different concentrations of GO, were used to probe the antibacterial activity of GO at the molecular level. The observation of higher intensity Raman peaks from adenine and proteins in GO treated E. coli
correlated with induced death. The antibacterial action of GO was thus related to disruption of the cell membrane by GO [13
However, in our case, the values of ζ-potential of carbon nanomaterials and bacteria in cell culture media are all negative. Thus, direct electrostatic attraction of nanomaterials to bacteria can be excluded. In the LB, ζ-potential values of nanomaterials are quite comparable (considering also the statistical error), between −15 mV to −28 mV, not correlated with the bacterial growth inhibition trend. There are more pronounced ζ-potential differences of nanomaterials in MH, between −7 mV to −31 mV, yet again, there is no correlation with the inhibition trend. The antibacterial effect cannot be explained just in regard to the change of ζ-potential. The electrokinetic surface properties of nanomaterials or the electric interaction between the bacterial membrane and nanomaterial is therefore probably not the sole negative action of nanomaterial responsible for bacterial growth inhibition.
The nanomaterials may not attach and interact with the bacteria surface only electrostatically though. It can occur on the mechanical basis or by other chemical interactions. During the exposure to nanomaterials in the media, the bacterial culture is continuously agitated on a shaker plate. Thereby the mutual interaction is promoted, with or even without permanent nanomaterial attachment to the bacteria surface. To what degree are the bacteria coated or not by the nanomaterials is in our case still not clear though. Unfortunately, it was technically impossible to image unambiguously nanomaterial coverage on bacteria by optical or electron microscopy as we could not remove all residual materials in the culture medium.
Nevertheless, the results of E. coli cultivation on salty agars showed no further enhancement of antibacterial effect. Thus, disruption of bacterial membrane or difference in ionic concentration are not key factors behind the antibacterial effect and observed differences for specific nanomaterials and media.
Oxidative stress is an additional basic mechanism of nanomaterial toxicity. Superoxide anion (O2−
) is produced as a by-product of oxygen metabolism and, if not regulated, causes many types of cell damage. The exposure to nanomaterial is connected with the generation of reactive oxygen species (ROS), which are thought to be responsible for the bactericidal effects of many inorganic nanoparticles (e.g., Ag, Cu, MgO, ZnO, CeO2
demonstrated on B. subtilis
, E. coli
, P. aeruginosa
, E. faecalis
and S. aureus
, to name just a few) [15
]. However, the quantitative relationship between ROS activity and antibacterial activity have not been established so far. The factors for nanomaterial-induced oxidative stress in bacteria are many as it is a complex system in the oxidative metabolizing organism and therefore is influenced not only by the size of nanomaterial but also chemical composition of nanoparticle and its purity, surface charge, coatings and functionalization as well as band gap energy and illumination.
The role of hydroxyl radical abundance was excluded in pure rGO suspensions [28
] and superoxide anion abundance [24
] was excluded in GOs suspensions. In vitro glutathione (GSH) oxidation induced by the presence of graphite, graphite oxide, GO and rGO in suspensions showed that the GSH can be used as a perspective marker of general oxidative stress independent on particular reactive oxygen species detection [24
Previous study of the antibacterial activity of GO, rGO and other graphite materials towards E. coli
under similar concentrations and incubation conditions showed that GO dispersion exhibits the highest antibacterial activity, followed by rGO [24
]. No reactive oxygen species production was detected though. However, it was argued that GO and rGO materials can oxidize glutathione, which serves as redox state mediator in bacteria.
NDs were reported to increase superoxide dismutase (SOD) activity and at the same time decreased the activity of glutathione reductase (GR) and glutathione peroxidase (GPx) within erythrocytes [29
]. NDs did not significantly affect either the total antioxidative state (TAS) nor the thiobarbituric acid reactive substances (TBARS) in blood plasma.
Thus, oxidative stress seems to be the dominating factor behind the antibacterial effect of nanodiamonds (where it may be further enhanced by pronounced internalization) and graphene oxide. On the other hand, there is still remaining question is: Why does the LB broth enhance the effect of GO and rGO? The principal difference between the MH and LB cultivation media is in the concentration of the Mg2+
ions. The concentration of these ions is several times higher in case of the MH media compared to the LB ones [30
]. These ions are needed to bridge the highly negatively charged lipopolysaccharide molecules forming the outer membrane of the E. coli
. The absence of essential cations necessary for enzymatic biochemical reactions is an effective stressor for augmentation of nanomaterial effect on the microorganism growth. In addition, deficiency of Mg2+
ions has been correlated with increased oxidative stress signalled by increased superoxide anions in blood plasma [31
]. Another limiting factor for the LB media in comparison with MH media is consumption of utilizable nutrients. Unlike MH media where starch is a key component, LB media provides only a scant amount of carbohydrates. The dominant LB media nutrients source is tryptone and yeast extract peptides. However, utilization of these peptides is limited by a molecule size of approximately 650 daltons (peptides must be transferred into cells through porins). Consequently, the metabolism of cells is limited by consumption of easily utilizable amino acids—E. coli
growth behaviour goes through diauxie-like behaviour—and then available but hardly utilizable amino acids are used. This fact changes both growth rate and vulnerability of cells [21
A recent study suggested that the antibacterial activity of nanodiamond is linked to the presence of partially oxidized and negatively charged surfaces, specifically those containing acid anhydride groups [8
]. Furthermore, proteins were found to reduce the bactericidal properties of nanodiamonds by their covering with such surface groups. Our data do not confirm these assertions. Hydrogenated nanodiamonds acted here as the universal material, the most pronounced antibacterial agent in both LB and MH media, in spite of obvious encapsulation by proteins and a related in its ζ-potential to negative. Air annealed (oxidized) nanodiamonds with similar negative ζ-potential had only limited effect, even less than GO or rGO in the MH broth.