Study of Bacillus subtilis response to different forms of silver

Science of the Total Environment 661 (2019) 120–129

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Science of the Total Environment journal homepage: www.elsevier.com/locate/scitotenv

Study of Bacillus subtilis response to different forms of silver K. Rafińska a,b, P. Pomastowski b, B. Buszewski a,b,⁎ a b

Department of Environmental Chemistry and Bioanalytics, Faculty of Chemistry, Nicolaus Copernicus University, Gagarina 7, 87-100 Torun, Poland Interdisciplinary Centre of Modern Technology, Nicolaus Copernicus University, Wileńska 4, 87-100 Torun, Poland

H I G H L I G H T S

G R A P H I C A L

A B S T R A C T

• Bacillus subtilis is a bacteria with high resistance to AgNPs. • The mechanism of B. subtilis resistance to AgNPs has been explained. • AgNPs are modified in the environment of bacteria growth. • The mechanism of action of tetracycline and AgNPs functionalized with tetracycline is similar.

a r t i c l e

i n f o

Article history: Received 31 October 2018 Received in revised form 2 December 2018 Accepted 9 December 2018 Available online 04 January 2019 Editor: Henner Hollert Keywords: Bacillus subtilis Silver nanoparticles Silver ion Functionalization Antibiotic

a b s t r a c t Although silver nanoparticles are the most widespread product of nanotechnology, the mechanisms underlying AgNP microbial toxicity remain the subject of intense debate. In this study, Bacillus subtilis has been used as model organism to elucidate the molecular interactions between this class of bacteria and different forms of silver such as nanoparticles, nanoparticles functionalized with tetracycline and silver ions. For this purpose, we carried out transmission electron microscopy and MALDI-TOF MS analysis of cells treated with silver nanoparticles (AgNPs, AgNPs functionalized with tetracycline, combination of AgNPs with tetracycline) and silver ions as well as we measured the level of reactive oxygen species. The data demonstrate that B. subtilis exhibits high resistance to silver nanoparticles and this phenomenon is associated with following processes: (I) initiation of endospore formation, (II) reduction of free Ag+ released from nanoparticles and (III) modification of the AgNPs surface. However, high silver ions concentration appeared to be very toxic to studied strain of bacterium. MALDI-TOF MS analysis revealed that the spectra of B. subtilis cells treated with silver ions are significantly different from spectra of control cells and cell treated with AgNPs and antibiotic which can suggest that silver ions in the highest degree modify bacterial components. © 2018 Published by Elsevier B.V.

1. Introduction

⁎ Corresponding author at: Department of Environmental Chemistry and Bioanalytics, Faculty of Chemistry, Nicolaus Copernicus University, Gagarina 7, 87-100 Torun, Poland E-mail addresses: [email protected] (K. Rafińska), [email protected] (B. Buszewski).

https://doi.org/10.1016/j.scitotenv.2018.12.139 0048-9697/© 2018 Published by Elsevier B.V.

Silver nanoparticles are the most widespread product of nanotechnology. Their antibacterial activity against broad range of Grampositive and Gram-negative bacteria, fungi and viruses has been confirmed in many studies (Chen and Schluesener, 2008; Railean-Plugaru et al., 2016b, 2016a; Singh et al., 2015). The rapid development of nanotechnology has provided different types of materials for biomedical and

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cosmetic applications of silver nanoparticles. They are used to coat medical instruments and products, as well as in pharmacy for the production of cosmetics. It is suggested that silver nanoparticles can be a good alternative to antibiotics in fight with microorganisms which developed multi-drug resistance. Some authors suggest that bacteria do not develop resistance to silver nanoparticles and this phenomenon can be explained by the fact that silver nanoparticles work simultaneously at different targets (Rai et al., 2012). Despite the widespread use and growing level in the environment, the mechanism of antibacterial activity of silver nanoparticles is still topic of discussion. Some evidences indicate that silver nanoparticles release silver ions which can interact with thiol and amino groups of proteins, with nucleic acids, and with cell membranes (Chernousova and Epple, 2013; Hsueh et al., 2015; Kittler et al., 2010). Exposure of bacteria to silver nanoparticles can lead to membrane damage and oxidative stress, via AgNPs induced reactive oxygen species (Durán et al., 2010; Fabrega et al., 2009). A recent study has shown that surface-charge of silver nanoparticles is associated with their toxicity and has indicated that positive surface of Bacillus cells protects them against silver nanoparticles toxicity (El Badawy et al., 2011). These observations suggest that not only AgNPs but also Ag ions released from them could be responsible for antimicrobial activity of nanoparticles (Hsueh et al., 2015). Sublethal doses of AgNPs have been found to induce production of proteins related to quorum sensing, involved in stress responses and redox sensing. It was shown that in response to presence of silver nanoparticle, extracellular polysaccharides production and inorganic phosphate solubilization increases (Gambino et al., 2015). Little information is available regarding mechanism activated by bacteria to face AgNPs. The good model for such studies is strain which exhibits a certain degree of resistance to silver nanoparticles. Despite the common opinion about antibacterial activity of silver nanoparticles, in recent years reports about bacteria which are resistant to this factor appeared. Gunawan et al. reported the natural ability of environmental Bacillus subtilis (GenBank accession JX157667, JX157668) to adapt to silver nanoparticles (Gunawan et al., 2013). Some other studies also confirmed that Bacillus species isolated from soil are especially resistant to this antibacterial agent (Gambino et al., 2015). Last years brought new antibacterial formulation based on silver nanoparticles and antibiotics. Some studies indicate the synergistic effect of both agents. It was shown that this effect can be triggered by adding AgNPs and antibiotic in defined concentrations or by silver nanoparticles functionalized with antibiotic (Buszewski et al., 2016; Fayaz et al., 2010; Railean-Plugaru et al., 2016b, 2016a). Wherein, functionalization is considered as a process that involves covering the surface of nanoparticles with an antibiotic layer. Our previous research revealed that silver nanoparticles biologically synthesized by Actinomycetes called CGG 11n are very effective against Staphylococcus aureus, Klebsiella pneumoniae, Proteus mirabilis and Salmonella infantis (Railean-Plugaru et al., 2016a, 2016b) but they do not exhibit significant antibacterial activity against Bacillus subtilis PCM 2021. Therefore, we assumed that this strain will be a good model to compare the mechanism of antibacterial activity of different formulation based on silver. For this purpose, we studied growth curves, ultrastructure of Bacillus subtilis cells and level of oxidative stress. As it is postulated, silver ions released from nanoparticles are partly responsible for antibacterial properties, therefore during the analysis, we have monitored also the amount of silver reduced by B. subtilis. Moreover, we used MALDI-TOF MS to observe modifications in metabolism of bacterial cells. To explain the mechanism of action of silver nanoparticles as well their combination with antibiotic, all experiments have been carried out on cells treated with (I) silver nanoparticles, (II) combination of silver nanoparticles and tetracycline, (III) silver nanoparticles functionalized with tetracycline, (IV) tetracycline and (V) silver ions. Obtained results were compared with control.

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2. Materials and methods 2.1. Materials All solvents and materials i.e. Mueller-Hinton broth (MHB), silver nitrate, tetracycline, glutaraldehyde and contrast reagents were purchased from Sigma-Aldrich (Germany). Ultra-pure water was obtained from Milli-Q RG system by Millipore (Millipore Intertech,Bedford, MA, USA). Silver nanoparticles and silver nanoparticles functionalized with tetracycline were synthesized by Actinomycete strain called CGG 11n. The procedure of their synthesis and physicochemical characteristics (TEM, EDX, SAED) are detailed described by Railean-Plugaru et al. (2016a, 2016b) and Buszewski et al. (2016) (Fig. 1S). 2.2. Determination of minimum inhibitory concentration (MIC) The susceptibility of Bacillus subtilis PCM 2021 to CGG 11n silver nanoparticles, tetracycline, functionalized silver nanoparticles and Ag ions was evaluated by determination of minimum inhibitory concentration by microtiter broth dilution method. Mueller-Hinton broth (MHB) (Sigma-Aldrich) was used as solvent. The final concentration of bacteria in each well was 1 × 106 CFU/mL, and concentrations of AgNPs as well as functionalized AgNPs were 200, 100, 50, 25, 12.5, 6.25 μg/mL. The concentration of tetracycline and AgNO3 was 128, 64, 32, 16, 8, 4, 2, 1, 0.5 and 0.25 μg/mL. Prior and after 24 h incubation at 37 °C the microtitrate plates were read at 600 nm using Multiscan FC Microplate Photometer (Thermo Fisher, Finland). 2.3. Growth kinetics of Bacillus subtilis To examine the bacterial growth in the presence of tested antibacterial factors B. subtilis cells were grown in MHB. To achieve the same initial inoculation, the overnight culture was diluted 1:100 with fresh bacterial medium and subjected to growth curve determination. Flasks with samples (OD600 0.3) were supplemented with AgNPs at concentration 12.5 μg/mL and 100 μg/mL, functionalized silver nanoparticles at concentration of 12.5 μg/mL, combination of tetracycline (15 μg/mL) and silver nanoparticles (6.125 μg/mL) and AgNO3 at concentration 12.5, 1 and 0.1 μg/mL, respectively. Applied concentrations of antibacterial agents were based on our previous research. (Buszewski et al., 2017; Railean-Plugaru et al., 2016a, 2016b) A flask without antibacterial agent was kept as control to track the normal growth of the bacterial cells. All samples were incubated at 37 °C with continuous agitation. Growth of the culture was determined by measuring McFarland density with DEN-1B (Biosan, Riga, Latvia). 2.4. Preparation of material for transmission electron microscopy, MALDITOF MS analysis and detection of reactive oxygen species B. subtilis cells were cultivated at 37 °C in MHB at 200 rpm in a rotary shaker to McFarland density 0.3. Then samples were treated with AgNPs at concentration 12.5 μg/mL, functionalized silver nanoparticles at concentration of 12.5 μg/mL, combination of tetracycline (15 μg/mL) with silver nanoparticles (6.125 μg/mL) and AgNO3 at concentration 12.5 μg/mL. Culture without antibacterial agent served as control. Bacteria were incubated with above-mentioned agents for 2 and 6 h. 2.5. Transmission electron microscopy assay 10 mL culture broth was centrifuged and washed twice with sterilized phosphate buffered saline, and the biomass was subjected to transmission electron microscopy. Bacterial cells were fixed with 2% glutaraldehyde for 4 h. After eliminating the remaining glutaraldehyde, the dehydration process was conducted with 20, 30, 40, 50, 60, 70, 80, 90, and 100% of ethyl alcohol. The fixed cells were then infiltrated and embedded in LR Gold resin (Sigma-Aldrich). The embedded material

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was cut on Leica UCT ultramicrotome into ultra-thin sections (50 nm) and placed on grids coated with formvar. Ultrathin sections were stained with 2.5% uranyl acetate and 0.4% lead citrate solutions, and examined using a Tecnai F20 X-Twin transmission EM (FEI company, Hillsboro, USA).

2.9. Statistical analysis Results are expressed as the means of three replicates and standard error. Analysis of variance (ANOVA) was used to determine the significance of the difference between groups (p b 0.001). 3. Results and discussion

2.6. MALDI-TOF MS analysis 3.1. Bacillus subtilis viability All chemicals for the MALDI-MS analyses were purchased at the highest commercially available purity by Fluka Feinchemikalien (NeuUlm, Germany; a subsidiary of SigmaAldrich). Ground steel targets (Bruker Daltonik, Bremen, Germany) were used for sample deposition. Bacteria were grown and treated with antibacterial agent as for transmission electron microscopy analysis. Obtained bacterial suspension was centrifuged and washed with distilled water. Next, the ethanol/formic acid extraction, as found in the Biotyper manufacturers (Bruker) was carried out. In brief; 300 μL of water and 900 μL of ethanol was added to the pellet, mixed and centrifuged (13,000 rpm; 2 min). Then the supernatant was discarded and the pellet was allowed to air dry, resuspended in 50 μL of 70% formic acid and mixed. 50 μL of 100% acetonitrile was then added, the suspension was mixed thoroughly, and centrifuged (13,000 rpm; 2 min). 1 μL of supernatant was pipetted onto ground steel plate. As soon as the sample spot has dried, 1 μL HCCA matrix solution (in 50% acetonitrile, 47.5% water and 2.5% trifluoroacetic acid) was overlaid. Analysis was performed with the use of ultrafleXtreme MALDI-TOF/ TOF mass spectrometer (Bruker Daltonik, Bremen, Germany) equipped with a modified Nd:YAG laser (smartbeam II™) operating at the wavelength of 355 nm and the frequency of 2 kHz. ICM MS spectra of were recorded in linear positive mode within m/z range of 100–36,000 and 3000–55,000. All mass spectra were acquired and processed with the dedicated software: flexControl and flexAnalysis, respectively (both from Bruker). Peptide Calibration Standard II and Bacterial Test Standards (Bruker Daltoniks, Bremen) were used for external calibration for 100–3600 and 3000–55,000 m/z range, respectively. Obtained mass spectra were compared with using ClinProTools software.

2.7. Detection of reactive oxygen species The detection of ROS generation was carried out by staining cells with 2′7′-dichlorofluorescein diacetate (H2DCF-DA) (Sigma). A stock solution of 10 mM of H2DCF-DA in DMSO (Sigma) was stored at −20 °C. 2 mL of cell suspension treated with each of studied agents was incubated with 100 mM of H2DCF-DA in DMSO for 30 min. After this time, suspension was centrifuged and washed in sterile water. Next, cells were broken by shaking the sediment with glass beads (4 °C, 15 min). The homogenates were clarified by centrifugation (7000 rpm, 5 min) and fluorescence of the supernatant was measured in JASCO FP 8300 spectrofluorometer (excitation and emission wavelengths of 470 and 529 nm, respectively). Results were obtained by calculating the mean fluorescence of 3 independent triplicates.

2.8. ICP-MS determination of reduced silver To study the possibility of reduction silver ions by B. subtilis cells, the content of silver in precipitate was measured. In brief, silver nanoparticles were added to the culture of B. subtilis cells to a final concentration 12.5 μg/mL and incubated in 37 °C for 6 and 24 h. Then samples were centrifuged for 60 min, 14,000. The precipitate was mineralized with aqua regia for 30 min at 90 °C and dissolved with water. The concentration of silver was measured by ICP MS using the CX 7500 Spectrometer ICP-MS. As a control sample, alone medium MHB with silver nanoparticles was prepared and incubated for 6 h.

The antibacterial activity of (1) biologically synthesized silver nanoparticles (CGG 11n), (2) combination of nanoparticles with tetracycline, (3) silver nanoparticles functionalized with tetracycline, (4) tetracycline and (5) silver ions was compared by determination of MIC values and growth kinetics. Studies showed that MIC value obtained for AgNPs was above tested concentrations of silver nanoparticles i.e. 200 μg/mL. However, MIC values for nanoparticles functionalized with tetracycline were 50 μg/mL. Measurements of growth rates confirmed that B. subtilis is especially susceptible to tetracycline and combination of nanoparticles with tetracycline (Fig. 1). However, functionalized silver nanoparticles did not inhibit the growth completely and after 7 h of incubation the colony density was 60% lower compared to the control. Silver ions at concentration of 1 and 0.1 μg/mL decreased the growth rates by about half. However, concentration of 12.5 μg/mL completely inhibited B. subtilis growth (Fig. 1). The presence of 12.5 μg/mL of silver nanoparticles did not significantly affect the B. subtilis cells growth relative to control cells. Even concentration of 100 μg/mL inhibited the growth only by about 13% (Fig. 1). Similar phenomenon was observed for environmental (airborne) strain of Bacillus which developed the resistance to silver (I) oxide particles on crystalline TiO2 synthesized by the flame pyrolysis (FSP) technique (Gunawan et al., 2013). Studies of Gambino et al. (2015) also indicate that wild strain Cu1065 especially growing in colony biofilm is characterized by high level of resistance to AgNPs chemically synthesized and stabilized by PVP, in concentration even to 10 μg/mL. In turn, Yoon et al. reported that commercially available AgNPs at concentration of 30 μg/mL are required to inactivate 90% of B. subtilis culture (Yoon et al., 2007). Therefore, we can conclude that B. subtilis cells can develop the certain level of resistance to different types of silver nanoparticles both chemically as well as biologically synthesized. 3.2. Morphological changes of Bacillus subtilis after treatment with different silver forms Fig. 2A shows the internal structure of the control B. subtilis cells. Their shape is regular and their cytoplasm is unanimous electron density. Ultrastructure suggests that observed cells are in physiological condition without any environment disturbance. After 2 h of incubation with silver nanoparticles in samples of B. subtilis sporulation started (Fig. 2B, C, D, E). In some cells process of DNA coiling indicating the first stage of sporulation was visible (Fig. 2C). In other cells, engulfment of protoplast and core wall synthesis were noticed (Fig. 2D, E). It is well known that endospores are forms which exhibit a very high degree of resistance to inactivation by various physical and chemical insults, including heat, UV and gamma radiation, extreme desiccation, chemical damage and destruction. Similar, in Bacillus cereus after treatment with silver nanoparticle at a concentration of 0.5 μg/mL, formation of asymmetric septum indicating spores formation was also visible (Fajardo et al., 2014). After 6 h of incubation with silver nanoparticles, two populations of cells were present in the sample (Fig. 2F, G). Some of the cells had ultrastructure similar to control cells, they had unanimous cytoplasm and regular shape which indicates that they are live and metabolically active (Fig. 2G). In other cells electron-light cytoplasm was shrunk and detached from the cell wall. Cell wall was discontinuous and in the area of the cytoplasm, different membrane structures

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Fig. 1. Growth kinetics of Bacillus subtilis PCM 2021 strain in the presence of AgNPs, combination of tetracycline with AgNPs, silver ions, tetracycline and AgNPs functionalized with tetracycline.

were visible (Fig. 2F). Such morphology clearly indicate that these cells are dead. After 2 h of incubation with combination of silver nanoparticles and tetracycline, ultrastructure was similar as after incubation with alone silver nanoparticles. The process of DNA coiling - first stage of endopsore formation was visible (Fig. 2H, I). In few cells process of sporulation was noticed (Fig. 2J). However, another 4 h of incubation led to complete cell damage (Fig. 2 K, L). Cell cytoplasm was detached from the thickened cell wall and in its area many membrane structures were present (Fig. 2K). In some places naked protoplasts without cell walls or cell wall fragments were present (Fig. 2L). It seems that silver nanoparticles could induce process of sporulation in B. subtilis cells, but simultaneous activity of tetracycline prevents its completion. The spore development depends on the expression and phosphorylation level of transcription factor and regulator Spo0A~P which is responsible for the activation of subsequent sporulation genes (Eymard-Vernain et al., 2018). We suggest that tetracycline by inhibiting protein synthesis stops sporulation process. Therefore, the presence of AgNPs can play only an auxiliary role, for example through damaging of the cell walls and membranes which can facilitate penetration of the antibiotic into the cell. Treatment with tetracycline did not induce sporulation in B. subtilis cells (Fig. 2M, N). After 2 h of incubation with this antibiotic, cells had electrone-light and disintegrated cytoplasm. Their walls were very thick - even few times thicker than in control and interrupted. Such ultrastructure suggests that they are not metabolically active. Cells after incubation with silver nanoparticles functionalized with tetracycline looked similar to those after treatment with alone tetracycline, their cytoplasm was electron-light and their walls were much thicker (Fig. 3A, B, C). The thickening of the cell walls was previously observed in response of Gram-positive bacteria to antibiotics. Some strains of Staphylococcus aureus developed adaptative resistance to amikacin and vancomycin based on the presence of thick wall which is impermeable to antibiotics (Yuan et al., 2013). But in the case of B. subtilis thickening of the cell walls was not associated with acquisition of resistance. As growth curve indicates this strain was completely susceptible to used antibiotic. The reason may be discontinuity of walls surrounding protoplast, which was not observed in S. aureus cells. B. subtilis cells after two hours of incubation with silver ions at concentration of 12.5 μg/mL appeared to undergo lysis, resulting in the release of their cellular contents into the surrounding environment, and finally became disrupted (Fig. 3D, E). Similar effects were observed by Jung et al. in Staphylococcus aureus cells treated with 0.2 μg/mL of Ag+ (Jung et al., 2008). Although the mechanisms underlying the antibacterial actions of silver are still not fully understood, several previous

reports showed that the interaction between silver and the constituents of the bacterial membrane causes structural changes and damage to the membranes and intracellular metabolic activity (Mcdonnell and Russell, 1999; Pal et al., 2015; Sondi and Salopek-Sondi, 2004). Electron transmission microscopy pictures indicate that both silver nanoparticles and combination of silver nanoparticles and tetracycline initiate sporulation in B. subtilis cells. We cannot exclude that physical contact of AgNPs with bacterial cell wall activate the program of sporulation. However, results showed that in the case of alone silver nanoparticles after first defensive response consisting of i.a. sporulation, probably the neutralization of antibacterial agent occurs. After 6 h of incubation endospores were not visible which suggests that process of sporulation was not completed or formed spores earlier germinated. Soufo (2016) for the first time showed similar phenomenon of abortion of endospore formation in different Bacillus strains. His studies evidenced that under unfavorable conditions, Bacillus cells are able to initiate but after that fail to complete spore development and started normal growth (Soufo, 2016). During the preparation of samples for TEM analysis we have noticed that silver nanoparticles during incubation with B. subtilis aggregate. Observed aggregates were composed of many small nanoparticles (Fig. 4A, B). The stability of colloids depends on zeta potential value greater than ±20 mV indicates the stability of dispersion. One of the factors influencing the value of the zeta potential is the particle surface. The zeta potential of the applied nanoparticles was about 19.6 mV. However, during incubation of B. subtilis culture with AgNPs, it is possible that compounds secreted by cells to growth environment are adsorbed on the surface of nanoparticles and change their zeta potential so that they lose stability. This is consistent with the observation that polygamma-glutamate (PGA) secreted by B. subtilis can adsorb on AgNPs surface which results in loss of their biocidal effect (Eymard-Vernain et al., 2018). Recently studies have also evidenced that Gram-negative bacteria Escherichia coli and Pseudomonas aeruginosa can develop resistance to silver nanoparticles through the production of adhesive flagellum protein flagellin, which triggers the aggregation of the nanoparticles (Panáček et al., 2018). It is worth noting that functionalized with tetracycline silver nanoparticles did not induce process of sporulation. The surface of these silver nanoparticles is covered with thick layer of tetracycline and as our previous studies showed during functionalization, size of CGG 11n AgNPs measured by dynamic light scattering (DLS) methods grows from 86.36 ± 0.22 to 151 ± 11.47 nm (Buszewski et al., 2016). Probably this thick layer is a barrier that impedes the interaction of the silver core with the cell surface. Based on the observations made, it can be hypothesized that direct physical contact of silver nanoparticles with the cell surface is necessary to initiate sporulation.

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Fig. 2. Internal structure of Bacillus subtilis cells; (A) control cells; (B, C, D, E) cells incubated with AgNPs for 2 h; (F, G) cells incubated with AgNPs for 6 h; (H, I, J) cells incubated with combination of AgNPs and tetracycline for 2 h; (K, L) cells incubated with combination of AgNPs and tetracycline for 6 h; (M, N) cells incubated with tetracycline.

3.3. Reactive oxygen species generation Reactive oxygen species (ROS) are thought to have essential role in the mechanism by which both silver nanoparticles as well as antibiotics destroy cells. Studies on E. coli O157:H7 showed that ROS are responsible for the antibacterial activity of AgNPs, and the presence of ROS

scavenger significantly reduces antibacterial activity of silver nanoparticles (Xu et al., 2012). However, the level of ROS in B. subtilis cells after 2 and 6 h of incubation with AgNPs was lower than the level of ROS in control sample (Fig. 5). It is evidenced that biologically synthesized silver nanoparticles can act as free radical scavengers and we suggest that the antioxidant efficacy of used AgNPs may result from adsorption of

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Fig. 3. Internal structure of Bacillus subtilis cells; (A, B, C) cells treated with functionalized AgNPs; (D, E) cells treated with Ag ions.

antioxidant compounds produced by Actinomycete. This group of organisms has the ability to synthesize antioxidants which was confirmed by many studies (Dholakiya et al., 2017; Janardhan et al., 2014; Poongodi et al., 2012). Moreover, the silver nanoparticles synthesized by Actinomycete are also effective free radical scavengers (Shanmugasundaram et al., 2013). Therefore, the increase in the level of ROS in B. subtilis cells in response to AgNPs synthesized by Actinomycete CGG 11n can be compensated by their inherent antioxidant activity. A similar phenomenon was

observed in the case of Pseudomonas aeruginosa and Staphylococcus aureus, where the addition of a strong antioxidant was found to suppress AgNPs induced ROS generation (Yuan et al., 2017). The most pronounced increase of ROS was observed for silver nanoparticles functionalized with tetracycline after 2 h of incubation (about 2 times higher compared to the control). It was even higher than for tetracycline and silver nanoparticles combined with tetracycline. The surface of AgNPs functionalized with tetracycline is covered with tetracycline molecules and the effect of antioxidant compounds from

Fig. 4. Aggregation of silver after incubation of CGG 11n AgNPs with Bacillus subtilis culture; (A) optical microscopy, cells are labelled with methylene blue, silver aggregate is black colour (B, C) TEM, aggregation is composed of many small silver nanoparticles. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

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X

* #

X *X

# XX

XX

Fig. 5. Intracellular reactive oxygen species concentrations in Bacillus subtilis cells after 2 and 6 h of incubation with AgNPs, tetracycline, combination of AgNPs with tetracycline and AgNPs functionalized with tetracycline. Identical symbols correspond to no significant difference: *, X, XX, # (p N 0.001).

Actinomycete is probably inhibited (Buszewski et al., 2016). We cannot also exclude that AgNPs functionalized with tetracycline can exert synergistic effect, microdamage of cell walls and membranes together with releasing of tetracycline in these sites can cause significant oxidative stress. After 6 h of incubation level of ROS decreased but was still higher than in control. In samples treated with tetracycline and combination of silver nanoparticles with tetracycline the level of ROS was higher compared to control (about 2 times and 1.3, respectively) but after 6 h of incubation decreased. Significant drop in the level of ROS within 6 h can be probably related with the process of dying. Both growth kinetics as well as transmission electron analysis confirms that these two agents led to the cell death. Previous studies on B. subtilis showed that initiation of sporulation can be associated with the level of ROS. It has been evidenced that increase in the level of intracellular ROS concentration results in decrease of sporulation. Further, elevated level of intracellular ROS is probably responsible for the inhibition of sporulation (Sahoo et al., 2004). This is in accordance with the results obtained during this study where incubation with agents inducing significant amounts of reactive oxygen species such as tetracycline and functionalized silver nanoparticles, did not initiate sporulation. However, incubation with studied silver nanoparticles did not lead to the increase in ROS but induced sporulation in B. subtilis cells. Silver ions did not change significantly the level of ROS and did not induce process of sporulation. It can be the result of a rapid interaction of silver ions simultaneously with thiol and amino groups of proteins, with nucleic acids, and with cell membranes which can prevent and inhibit sporulation (Rai et al., 2012). On the other hand, on the basis of transmission electron microscopy analysis we cannot exclude that direct physical contact of silver nanoparticles with the surface of bacteria is necessary for sporulation and alone silver ions do not have the ability to initiate sporulation. Likewise, studies on Bacillus cereus showed that AgNPs do not change significantly the level of catalase expression, an enzyme involved in oxidative stress detoxification which can indicate that these nanoparticles also did not induce oxidative stress (Fajardo et al., 2014). Further Gambino et al. showed that silver nanoparticles at concentration 1 and 8 μg/mL do not induce oxidative stress in biofilm B. subtilis cells (Gambino et al., 2015).

free silver ions. ICP-MS measurements showed that concentration of free silver ions in medium released from silver nanoparticles is about 0.35 ± 0.146 μg/mL. Relatively high standard deviation can be the result of incomplete separation silver nanoparticles from the medium. Therefore, in a further stage of the research we have focused on monitoring the silver content in the pellets. Microscopic observations indicated that toxic properties of silver ions can be abolished by interaction with compounds secreted by B. subtilis cells. We applied ICP-MS to monitor changes in amount of silver in the pellet after 6 and 24 h of incubation with B. subtilis cells. Our experiment showed that during incubation of silver nanoparticles with B. subtilis cells, the amount of silver in the pellet increases which suggests that reduction of free Ag+ occurs (Fig. 6). It is well known that different B. subtilis strains are capable of biological synthesis of silver nanoparticles. Reddy et al. showed that B. subtilis reduces the silver ions extracellularly and that proteins of molecular weights between 66 and 116 kDa are probably responsible for this process (Reddy et al., 2010). Therefore, we put the hypothesis that addition of silver nanoparticles solution initiate sporulation of B. subtilis cells as a result of physical contact between nanoparticles and bacterial surface. At this time, free Ag+ ions present in AgNPs solution in very low concentration are reduced by proteins secreted by Bacillus cells to the medium. As the consequence the sporulation is aborted and cells resume divisions. Thus, we propose two mechanisms for neutralizing the toxic effects of nanoparticles by Bacillus subtilis. The first of these involves the

3.4. ICP-MS determination of reduced silver One of the postulated mechanism of the silver nanoparticles toxicity is releasing silver ions (Brunner et al., 2006; Kittler et al., 2010). We have also assumed that applied solution of silver nanoparticles contains

Fig. 6. Content of reduced silver in the pellet after 6 and 24 h incubation with Bacillus subtilis culture. Identical symbols correspond to significant difference: *, # (p b 0.001).

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precipitation of silver ions present in nanoparticle solution by the compounds secreted by Bacillus. The second one is adsorption of these compounds on the surface of nanoparticles and thus the formation of a barrier to their adverse effect. 3.5. MALDI-TOF-MS analysis Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) is a technology for routine identification of bacteria in clinical microbiology laboratories. This method detects a large spectrum of proteins and peptides (Biswas and Rolain, 2013; Gopal et al., 2011). The aim of the MALDI-TOF-MS analysis was to disclose changes in B. subtilis cells at the molecular level after treatment with tested antibacterial agents. Each MALDI spectrum of bacterial cells contains many signals of proteins and peptides, therefore their comparison can be quite tedious. Therefore, we applied new approach based on classification by hierarchical clustering by similarity of spectra with using ClinProTools (Bruker Daltonic) software. The hierarchical clustering allowed a quick and intuitive comparison of changes occurring in bacterial cells treated with various factors. As our results showed, spectrum of proteins isolated from cells treated with silver ions was simplicifolius clad which means that is substantially different from spectra of proteins isolated from both control

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cells and cells treated with tetracycline and different forms of silver nanoparticles (Fig. 7). Also, the view of the spectra in the form of a gel reveals that silver ions significantly modify bacterial proteins. It is well known that a silver cation is a soft Lewis acid that has an affinity to sulfur and nitrogen - elements commonly found in proteins. Thereby has many possibilities to distrupt cell function (Chernousova and Epple, 2013). Silver atoms can bind to thiol groups (-SH) in proteins i.a. enzymes and cause their deactivation. They especially bind with thiolcontaining compounds in the cell membrane that are involved in transmembrane energy generation and ion transport. It is also believed that silver can be involved in catalytic oxidation reactions that result in the formation of disulfide bonds. Silver can catalyze the reaction between oxygen molecules in bacterial cell and hydrogen atoms of thiol groups. As a result, two thiol groups are covalently bonded to one another. The adverse effect of silver ions can be due also to interactions with amino groups of proteins, nucleic acids and with cell membranes (Choi et al., 2008; Feng et al., 2000; Lansdown, 2006; Silver, 2003). Hence, disappearance and appearance of new peaks in the spectrum of the bacteria treated with silver ions may reflect the processes of structural modifications and inactivation of bacterial proteins. Moreover, in the spectrum of B. subtilis cells treated with silver ions were observed peaks (m/z about 1546) (Fig. 7C). They can indicate the presence of silver clusters because they correspond to silver isotopic

Fig. 7. (A) Cluster analysis based on comparison of MALDI-TOF MS spectra of Bacillus subtilis, (B) Gel-view of MALDI-TOF MS spectra of B. subtilis, (C) Signal present in B. subtilis after treatment with Ag ions and AgNPs. Its pattern resembles the isotopic pattern of silver ions and can indicate the process of Ag+ reduction.

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Fig. 8. Proposed mechanism of the interactions between silver nanoparticles and Bacillus subtilis cells.

patterns which were observed during biological synthesis of AgNPs (Viorica et al., 2017). Obtained results demonstrate that silver ions are probably reduced during incubation with B. subtilis, although transmission electron micrography did not show the presence of silver nanoparticles inside and outside the cells. We cannot exclude that created clusters are too small to see them in conditions used for cells preservation. The spectra obtained from cells treated with pure silver nanoparticles and combination of silver nanoparticles with tetracycline are similar (Fig. 7A, B). Only these two factors initiate sporulation process and similarity between spectra can reflect the changes occurring during sporulation. In gel view obtained from cells treated with AgNPs signals at 1304, 1560 and 3340 m/z were especially pronounced. An especially high degree of similarity occurs between the protein profile of control and tetracycline treated cells. Mechanism of tetracycline action is based on inhibition of protein synthesis by blocking the attachment of aminoacyl-tRNA to the ribosome (Nelson and Levy, 2011). Hence, this antibiotic does not change the structure of isolated proteins, only blocks translation which leads to cell death. This antibiotic also did not initiate sporulation. Therefore, differences in spectra may result only from changes in the expression of individual proteins. Spectrum of proteins isolated from cells treated with silver nanoparticles functionalized with tetracycline is in one clad together with spectra of proteins from cells after incubation with tetracycline and control cells. It indicates that tetracycline probably plays a key role as antibacterial agent in this composition. Silver nanoparticles after functionalization are primarily an antibiotic carrier and antibacterial activity of the silver core is probably abolished by a thick layer of antibiotic. MALDI-TOF-MS results confirmed TEM analysis which indicates that morphology of cells treated with functionalized silver nanoparticles is similar to morphology of cells treated with tetracycline. In summary, we present important observations which can partly explain the phenomenon of Bacillus subtilis resistant. We report the process of endospore creation in response to the presence of silver nanoparticles in the environment. Fig. 8 presents the possible mechanism of interaction silver nanoparticles with B. subtilis cell culture. We present two mechanisms that help eliminate the toxic effects of silver nanoparticles. They include the precipitation of silver ions present in nanoparticle solution by the compounds secreted by Bacillus and adsorption of these compounds on the surface of nanoparticles and thus aggregation. 4. Conclusion Our studies showed that Bacillus subtilis PCM 2021 is a bacterial strain which is very susceptible to silver ions but exhibits a high degree of resistance to biologically synthesized silver nanoparticles. It seems

that resistance of Bacillus cells was associated with following processes: reduction of free Ag ions released from silver nanoparticles and modification of silver nanoparticles surface. Moreover, we postulate that physical contact with silver nanoparticles initiate process of sporulation. Some symptoms of sporulation were visible also in cells incubated with combination of silver nanoparticles and tetracycline. Silver ions appeared to be very toxic, concentration of 0.1 μg/mL inhibited culture growth. MALDI TOF-MS spectra of cells treated with Ag ions were significantly different than spectra of control cells and cells treated with AgNPs which suggests that silver ions significantly modify structure of molecules of bacterial cells. However, further research on the resistance of B. subtilis to chemically synthesized AgNPs could give insight into the general mechanisms of interaction between nanoparticles and bacterial cells. Supplementary data to this article can be found online at https://doi. org/10.1016/j.scitotenv.2018.12.139. Acknowledgments The work was financially supported by the National Science Centre in the frame of the project Symfonia 1 no. 2013/08/W/NZ8/701 (2013–2016). We would like to thank to prof. H. Dahm and her team from Department of Microbiology, Nicolaus Copernicus University, Toruń, Poland for delivering of silver nanoparticles obtained in the framework of Symfonia 1 project and used in this study. References Biswas, S., Rolain, J.M., 2013. Use of MALDI-TOF mass spectrometry for identification of bacteria that are difficult to culture. J. Microbiol. Methods https://doi.org/10.1016/j. mimet.2012.10.014. Brunner, T.J., Wick, P., Manser, P., Spohn, P., Grass, R.N., Limbach, L.K., Bruinink, A., Stark, W.J., 2006. In vitro cytotoxicity of oxide nanoparticles: comparison to asbestos, silica, and the effect of particle solubility. Environ. Sci. Technol. https://doi.org/10.1021/ es052069i. Buszewski, B., Rafińska, K., Pomastowski, P., Walczak, J., Rogowska, A., 2016. Novel aspects of silver nanoparticles functionalization. Colloids Surf. A Physicochem. Eng. Asp. 506. https://doi.org/10.1016/j.colsurfa.2016.05.058. Buszewski, B., Railean-Plugaru, V., Pomastowski, P., Rafińska, K., Szultka-Mlynska, M., Kowalkowski, T., 2017. Antimicrobial effectiveness of bioactive silver nanoparticles synthesized by actinomycetes HGG16N strain. Curr. Pharm. Biotechnol. 18. https:// doi.org/10.2174/1389201018666170104112434. Chen, X., Schluesener, H.J., 2008. Nanosilver: A Nanoproduct in Medical Application. 176, pp. 1–12. https://doi.org/10.1016/j.toxlet.2007.10.004. Chernousova, S., Epple, M., 2013. Silver as antibacterial agent: ion, nanoparticle, and metal. Angew. Chem. Int. Ed. https://doi.org/10.1002/anie.201205923. Choi, O., Deng, K.K., Kim, N.-J., Ross, L., Surampalli, R.Y., Hu, Z., Ross Louis, J., Surampalli, R.Y., Hu, Z., 2008. The inhibitory effects of silver nanoparticles, silver ions, and silver chloride colloids on microbial growth. Water Res. https://doi.org/10.1016/j. watres.2008.02.021. Dholakiya, R.N., Kumar, R., Mishra, A., Mody, K.H., Jha, B., 2017. Antibacterial and antioxidant activities of novel actinobacteria strain isolated from Gulf of Khambhat, Gujarat. Front. Microbiol. 8, 1–16. https://doi.org/10.3389/fmicb.2017.02420.

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