Keywords: Pseudomonas aeruginosa, TzAgNPs, antimicrobial, antibiofilm, virulence factor.
Microorganisms live in nature in two forms; one is planktonic where the microorganism lives distantly free from each other while the other is known as biofilm where the microorganism lives in clustered colony form (Garrett et al. 2008). Biofilm can develop on both biotic and abiotic substances and are found in almost every environment (Cortes et. al, 2011). Biofilm are the accumulation of microbial cells that are irreversibly adhered on a surface and confined within a layer of self-secreted extracellular polymeric substances (EPS) containing proteins, polysaccharides and DNA etc. (Sharma et al. 2015). The EPS forms an interconnecting three-dimensional polymer network of cohesive nature which helps the microorganism to adhere to the surface as well as imbomilize biofilms cells transiently. (Gupta et al. 2016). Microorganism adapts to the harsh condition by forming biofilm. It was reported that microbial biofilm is often involved in microbial pathogenesis (Vasudevan 2014). Biofilm promotes gene transfer in the horizontal direction among bacterial population that steers an expansion in the population of virulent strains (Lewis 2001). According to an estimation by the centre for disease control (CDC) and National Institutes of Health (Joo and Otto, 2012), bacterial biofilm are involved in a multitude of serious, chronic infections, 65 and 80 percent of all infections, respectively. Moreover, cells of the biofilm can not only escape the immunity of the host but can also stay unaffected by wide range of antibiotics (Crossley et al. 2009). The infectious diseases caused by microbial biofilm include Endocarditis, Periodontitis, Osteomyelitis, Rhinosinusitis, Cystic fibrosis, etc (Gupta et al. 2016). Biofilm are also seen in medical implants, Joint prostheses, heart valves, Urinary catheters etc. Microbial biofilm often exhibits increased resistance to anti microbial agents. A plausible explanation concerning the biofilm-refereed resistance to drugs could be accredited to the varied gene expression of the cells in the biofilm in comparison to the planktonic form of the same organism (Whiteley et al. 2001). Thus, biofilm cells can exhibit antibiotic resistance 1000 fold higher than the antibiotic resistance properties exhibited by planktonic cells (Ceri et al. 1999). Among the various microbial pathogens capable of forming a biofilm, Pseudomonas aeruginosa can cause a biofilm on human host and is hence considered the most deadly. Pseudomonas aeruginosa is responsible for several diseases including urinary tract and kidney infections (Shirtliff and Mader 2000; Wagner and Iglewski 2008). The defence mechanism of microbial biofilm was found to be effective against a variety of antibiotics and disinfectants as illustrated in multiple publications. (Jabra-Rizk et al. 2006). In the light of the emergence of resistance to drugs, dissertations on new synthetic compounds having both antimicrobial and antibiofilm activity have attracted interest as an alternative potential drug against microbial pathogenesis (Kumar et al. 2013). Recent studies have showed that nano biotechnology have led to the development of newly creative therapeutic strategies and drug delivery alternatives particularly customized to deliver drugs to a selected site in order to enhance the efficacy of drug against microbial pathogenesis (Conde et al. 2012; Conde et al. 2012a; Martins et al. 2013). Therefore, in the current study, we had synthesized 3,6-di(pyridin-2-yl)-1,2,4,5-tetrazine (pytz) capped silver nanoparticles (TzAgNPs) in order to inhibit the formation of biofilm by Pseudomonas aeruginosa.
Materials and methods
Synthesis of pytz and TzAgNPs
Synthesis of 3,6-Di(pyridin-2-yl)-1,4-dihydro-1,2,4,5-tetrazine (H2pytz) and 3,6-di(pyridin-2-yl)-1,2,4,5-s-tetrazine (pytz) had been performed by following the protocol as described previously (Audebert et al. 2004; Clavier et al. 2010; Karver et al. 2011). 3, 6-Di (pyridin-2-yl)-1,2,4,5-s-tetrazine (pytz) capped-silver nanoparticles (TzAgNPs) were synthesized in accordance to the method reported previously (Samanta et al. 2014). In brief, the synthesis of 3,6-di(pyridin-2-yl)-1,2,4,5-s-tetrazine (pytz) capped-silver nanoparticles (TzAgNPs) was achieved by the reduction of silver nitrate (AgNO3) with 3,6-di(pyridin-2-yl)-1,4-dihydro-1,2,4,5-tetrazine (H2pytz). A 100 ml round bottom flask containing solution of 3,6-di(pyridin-2-yl)-1,4-dihydro-1,2,4,5-tetrazine (0.24 g, 1 mmol) in ethanol (20 ml) was placed in an ultrasonicator bath and silver nitrate solution (0.34 g, 2 mmol) in water was added to it drop wise. After five minutes, a grey precipitate was slowly formed indicating the formation of silver nanoparticles (TzAgNPs). The resulting particles were isolated by centrifugation (10 min, 12000 rpm) and washed subsequently with water, ethanol and dichloromethane. The product was then dried in vacuum oven at 50ºC for 4 hours and kept for further use.
Microbial strain, growth media and culture conditions
For the current study, Pseudomonas aeruginosa MTCC (424) was used as a test organism. This organism was a kind gift to us from Dr. Surajit Bhattacharjee, Tripura University, India. The organism was grown in Luria Bertani broth (LB) at 37ºC for varied lengths of time as per the necessity of experiment. LB medium was prepared by adding 10 g Sodium Chloride, 5 g Yeast Extract and 10 g Casein in 1 liter Milli-Q water. The pH of the growth media was set at 7.0 before autoclaving.
Determination of Minimum inhibitory concentration (MIC) by broth dilution assay
The Minimum inhibitory concentration (MIC) of TzAgNPs against Pseudomonas aeruginosa was determined by using broth dilution method (Clinical and Laboratory Standards Institute, 2005). To find out the value of MIC against Pseudomonas aeruginosa, an aliquot (10 μl) of an overnight saturated culture of Pseudomonas aeruginosa was independently added to different sterile test tubes having 5 ml of sterile LB media. Thereafter, different concentrations of TzAgNPs (10, 20, 30, 40, 50, 60 μg/ml) were independently added to the different test tubes containing 5 ml of sterile LB media. In the control set, the organism was grown devoid of any TzAgNPs. Afterwards, all the tubes including control were incubated at 37ºC for 48 hours. After the incubation, the turbidity was examined by quantifying OD at 600 nm and MIC was determined at the lowest concentration of TzAgNPs that does not allow any visible microbial growth in the test tubes.
Biofilm formation capability assessment
The extent of the microbial population that adhered to the glass surface was determined by performing Crystal Violet (CV) assay as described previously (Mukherjee et al. 2013; Sharma et al. 2015). Briefly, 100 µl of saturated culture (1X108 CFU/ml) of Pseudomonas aeruginosa was added independently into different test tubes containing 5 ml of sterile LB. Afterwards, various sub-MIC doses of TzAgNPs (10, 20 μg/ml) were independently added in each test tubes excluding control set. In the control set, the organism was grown in absence of TzAgNPs. All the test tubes were then incubated at 37ºC for 48 h. After the desired incubation time get over, planktonic cells were removed from each test tube and tubes were then gently washed with sterile Milli Q water. Afterwards, test tubes were air dried and stained with 0.4% of CV solution for 15 mins at room temperature to stain adhered microbial population if any on the glass surface. Thereafter, CV stain had been taken out and tubes were then subsequently gently washed with sterile Milli Q water. Then, 5 ml of 33% glacial acetic acid was added in each tube to dissolve the adsorbed CV if any. The intensity of color was then quantified by recording absorbance at 630 nm.
Microbial adherence on glass surface analysis by microscopy
To examine the extent of adherence of bacteria to the glass surface under the exposure of different concentration of TzAgNPs, 100 µl of saturated culture of Pseudomonas aeruginosa (1X108 CFU/ml) was independently inoculated into different test tubes having 5 ml of sterile LB. Different concentrations of sub-MIC doses TzAgNPs (10, 20 μg/ml) were separately added to each tube. In the control set, the organism was grown devoid of TzAgNPs. Sterile glass cover slips were added to all test tubes including control set. All test tubes were incubated at 37ºC for 48 h. After the incubation, cover slips were aseptically recovered from each conditioned media and subsequently stained with acridine orange (4 µg/ml) in order to examine the extent of microbial biofilm formation on the glass surface by fluorescence microscopy.
Estimation of biofilm total protein
The extent of adhered microbial population on glass surface was an indirect determination by measuring the extent of extractable protein from the glass surface as it is related to the extent of adhered microbial population (Das et al. 2016; Das et al. 2016a). In order to measure the amount of extractable protein from the glass surface, culture broths were removed from both TzAgNPs treated and untreated growth media after 2 days of incubation at 37ºC. All the tubes were then gently washed with sterile Milli Q water twice, air-dried and then boiled for 30 min in 5 ml of 0.3 (M) NaOH. The suspension was then centrifuged for 10 minutes at 8000 rpm. The supernatant was collected and the protein concentration of the supernatant was determined by the Lowry method (Lowry et al. 1951).
Evaluation of growth curve
To evaluate the effect of sub-MIC doses of TzAgNPs on the growth kinetics of Pseudomonas aeruginosa, 100 µl of saturated culture (1X108 CFU/ml) of Pseudomonas aeruginosa was aseptically inoculated into different conical flasks containing 100 ml of sterile LB media. Then, different concentrations (10 and 20 µg/ml) of TzAgNPs were added into each conical flask except control set. All the conical flasks were then incubated at 37ºC for 48 hours. At regular time gap, culture broth was collected from each conditioned media and absorbance was successively measured at 600 nm.
Microbial cell viability analysis
To inspect the cell viability of Pseudomonas aeruginosa under different sub-MIC doses of TzAgNPs, we inoculated identical numbers of microorganisms in different sterile test tubes containing 5 ml of sterile LB media and treated with different sub-MIC doses of TzAgNPs (10 and 20 µg/ml). In the control set, TzAgNPs was not added to the microorganisms. All the test tubes were then incubated at 37ºC for 48 hours. In order to determine the colony forming unit (CFU), 1 ml of culture media was collected from each conditioned media including control. Thereafter, 9 ml of sterile saline was added to 1 ml of culture media and after that different dilutions were prepared accordingly. Thereafter, 100 μl of these diluted samples were plated onto solid LA agar plates to get discrete pure single colony. All the plates were incubated at 37ºC for 2 days and CFU were counted accordingly (Bhattacharyya et al. 2017).
Intracellular measurement of Reactive Oxygen Species (ROS)
Microbial ROS was measured by using 2ꞌ, 7ꞌ – dichlorofluorescin diacetate (DCFDA) cellular ROS detection assay kit (ab113851). The kit was purchased from abcam and it uses the cell permeant reagent DCFDA, a fluorogenic dye that measures hydroxyl, peroxyl and other ROS activity within the cell. Once DCFDA gets inside the cell, it gets de-acetylated by cellular esterase to a non-fluorescent compound, which is thereafter oxidized by ROS into 2’, 7’- dichlorofluorescin (DCF). DCF is a highly fluorescent compound and it can be detected by fluorescence spectroscopy with excitation and emission spectra at 495nm and 529nm respectively.
For this study, Pseudomonas aeruginosa was cultured to 1X108 CFU/ml, and cells were washed three times with 1X Buffer. DCFDA was then mixed with the cultures at a ratio of 1:2000 and the mixture was shaken at 37ºC for 30 minutes. After the incubation get over, the bacteria were precipitated by centrifugation at 7000 rpm for 10 minutes. The collected bacterial cells were washed two times to remove the DCFDA outside the cell. Then, the cleaned bacterial cells were exposed to TzAgNPs at increasing concentration from 10 to 20 µg/ml. In the control set, the same organism was treated with DCFDA but TzAgNPs was not added to that culture. The fluorescence intensity of generated DCF was then measured by fluorescence spectrophotometer at an excitation wavelength of 488 nm and at an emission wavelength of 535 nm (Dwivedi et al. 2014).
Metabolic activity measurement
To determine the metabolic activity, Azocasein degrading proteolytic activity was measured in the cell free culture supernatant, according to the method of Kessler et al. 1993 with minor modifications (Das et al. 2016a). In order to do that, Pseudomonas aeruginosa was grown in different test tubes containing 5 ml of sterile LB. After that, sub-MIC doses of TzAgNPs (10 and 20 µg/ml) were added in different test tubes. In the control set, the organism was grown in absence of TzAgNPs. All the test tubes were then incubated at 37°C for 48 h. After the incubation get over, the culture broths either from TzAgNPs treated and untreated set were separately centrifuged at 8000 rpm for 10 minutes to collect the cell free culture supernatant. Two (2) ml of cell-free supernatant taken from either TzAgNPs treated or untreated sample was added with 500 µl of 0.3% azocasein and the reaction mixture was subsequently incubated at 37°C for 1 h. After the incubation, the reactions were then stopped by the addition of l0% Trichloroacetic acid. Thereafter, the reaction mixture were centrifuged at 10,000 rpm for 5 min and the absorbance of clear supernatant were recorded at 400 nm by a UV-Visible spectrophotometer.
Pyocyanin quantification assay
The pyocyanin quantification assay was performed according to the method reported previously (Essar et al. 1990; Das et al. 2017). Pseudomonas aeruginosa was incubated in presence and absence of sub-MIC doses (10 and 20 µg/ml) of TzAgNPs in LB growth media at 37°C for 48 h. After the incubation get over, each culture broth was centrifuged at 6000 rpm for 10 minutes to get the cell-free supernatant. A 5-ml cell free supernatant was extracted with 3 ml of chloroform and then re-extracted with 1 ml of 0.2 (N) HCl to produce an orange yellow to pink coloured solution. The absorbance of the coloured solution was measured at 520 nm.
Experimental results were statistically analyzed by performing one-way analysis of variance (ANOVA). The relationship between TzAgNPs, ROS and the formation of microbial biofilm was examined by the construction of a contour plot using Minitab 16 software.
Results and Discussion
Antimicrobial activity measurement of TzAgNPs against Pseudomonas aeruginosa
The broth dilution system (BDS) test was followed to ascertain the antimicrobial activity of TzAgNPs against Pseudomonas aeruginosa wherein TzAgNPs was solubilized in DMSO and subjected to determination of MIC. This BDS test was preferred to overcome the solubility and diffusion problems of TzAgNPs in agar diffusion method. MIC has been defined as the minimum concentration of an antimicrobial drug that inhibits any visible microbial growth after desired time of incubation of organism with the antimicrobial agent (Bhattacharyya et al. 2017). The result revealed that TzAgNPs showed considerable antimicrobial activity against Pseudomonas aeruginosa wherein the MIC of TzAgNPs was found at 40 µg/ml against Pseudomonas aeruginosa (Figure 1). DMSO was also tested for antimicrobial activity and it was observed that DMSO did not exhibit any antimicrobial activity against Pseudomonas aeruginosa (data not shown). It was thus observed from the results that TzAgNPs possess considerable antimicrobial property against Pseudomonas aeruginosa.
Antibiofilm activity measurement of TzAgNPs against Pseudomonas aeruginosa
Bacteria associated with biofilm often possess antibiotic resistance properties (Vasudevan 2014), virulence factors, secretion of different surface molecules and reduced growth rate that play an important role in the progression of the microbial pathogenicity (Hall-Stoodley and Stoodley, 2009). Attenuation in biofilm formation makes the microbial population more sensitive towards antimicrobial agents that lead to the removal of microbial pathogens from the target site comfortably (Bhattacharyya et al. 2017). Efficient strategies are therefore urgently required for preventing the development of biofilm to combat with the biofilm mediated microbial pathogenesis. After determining the antimicrobial effectiveness of TzAgNPs against Pseudomonas aeruginosa, we examined their possible antibiofilm activity if any on Pseudomonas aeruginosa. To determine the antibiofilm activity of TzAgNPs against Pseudomonas aeruginosa, 100 µl of saturated cultures of Pseudomonas aeruginosa were separately inoculated into different sterile test tubes containing LB. To that, sub-MIC concentrations (10, 20 µg/ml) of TzAgNPs were added and subsequently incubated at 37ºC for 48 h so that the TzAgNPs can only affect the biofilm formation ability of the bacteria instead of damaging the microbial cells. In the control set, organisms were grown in absence of TzAgNPs. After the similar period of incubation, planktonic cells were discarded from each experimental growth media including the control where the microorganism was not exposed to TzAgNPs. The biofilm microbial population on the glass surface if any was then stained with CV solution. Thereafter, 33% glacial acetic acid was added to dissolve the CV stained adhered microbial population if any. The result showed that TzAgNPs exhibited variable degree of antibiofilm activity against Pseudomonas aeruginosa (Figure 2). TzAgNPs showed ~30 % and ~52 % reduction in biofilm formation at 10 µg/ml and 20 µg/ml respectively from the level of biofilm formation by organisms that were not treated with TzAgNPs (Figure 2). The result reveals that TzAgNPs at a concentration of 20 µg ml-1 appears as a promising antibiofilm agent against Pseudomonas aeruginosa (Figure 2). The extent to which microbial biofilm was formed on test tubes in presence and absence of TzAgNPs was also measured indirectly by determining the amount of total extractable protein (Bhattacharyya et al; 2017; Das et al. 2017). Since it is very difficult to measure the extent of microbial association on glass surface by traditional colony counting assay, total protein extraction could be an important tool to count the extent of adhered microbial population on the glass surface as protein can only be collected from the bacteria not from abiotic test tubes (Bhattacharyya et al; 2017). More protein recovery reveals higher degree of microbial association on the test tube surface and vice versa. The result showed that less protein was extracted from those test tubes where microorganisms were treated with the sub-MIC doses of TzAgNPs (10, 20 µg/ml) compared to the extracted protein from the control tube where microorganisms were grown in absence of TzAgNPs (Figure 3). Thus, the results indicated that when microorganisms were treated with the sub-MIC doses of TzAgNPs, then microorganisms lost their ability to adhere on the glass surface that might inhibit the development of microbial biofilm.
TzAgNPs inhibits microbial attachment
Microbial colonization happens to be the most important stage of microbial biofilm development over the surface as it is the first stage of biofilm formation on a surface (Gupta et al. 2016). Thus, the compound interrupting the microbial adherence on the glass surface could be considered as a promising agent against microbial biofilm development. In order to test the effect of TzAgNPs against microbial colonization, equal numbers of bacteria (1X108 CFU/ml) were separately grown in sterile LB media containing sterile glass cover slips. Thereafter, sub- MIC doses of TzAgNPs (10, 20 µg/ml) were separately added to the organism. In the control test tube, organisms were allowed to grow in LB and allowed to colonize on the glass cover slip surface in absence of TzAgNPs. All test tubes were incubated under similar condition for same length of time. After the incubation got over, cover slips were recovered, stained with acridine orange and observed under fluorescence microscope. The microscopy results showed that microbial colonization varies considerably between TzAgNPs treated and untreated condition (Figure 4). The result showed that microorganisms colonize substantially on cover slip surface when they were not treated with TzAgNPs (Figure 4). Microbial colonization was gradually decreasing when the organisms were treated with increasing doses of TzAgNPs (Figure 4). Thus, the result explains that microorganisms lost its capacity to colonize when they were exposed to TzAgNPs. The result reveals that TzAgNPs inhibits the microbial colonization property of Pseudomonas aeruginosa considerably that might not allow organisms to develop biofilm over the surface.
Sub-MIC doses of TzAgNPs do not alter microbial cell viability
To examine the effect of the tested sub-MIC doses (10, 20 µg/ml) of TzAgNPs on the growth and viability of Pseudomonas aeruginosa, organisms were incubated with different sub-MIC doses (10, 20 µg/ml) of TzAgNPs and compared the growth kinetics of the bacteria with the control set where organisms were grown devoid of TzAgNPs. During the regime of incubation, we aseptically collected same volumes of culture broth from TzAgNPs treated and untreated set at regular time gap and recorded OD at 600 nm. The result showed that both TzAgNPs treated and untreated organisms showed the same growth pattern (Figure 5A) suggesting that TzAgNPs at its sub-MIC zone do not interfere with microbial growth machinery. To examine cell viability, we compared the viable microbial cell count between TzAgNPs treated and untreated condition. For this purpose, we counted the colony forming unit (CFU) of growth media by plating 50 μl of bacterial cultures collected separately from both TzAgNPs treated and untreated growth media. After the incubation got over, we observed no significant differences in CFU count between TzAgNPs treated and untreated culture media (Figure 5B). Thus, the results showed that the tested sub-MIC doses (10, 20 µg/ml) of TzAgNPs neither affect growth kinetics of organisms nor exhibit cell killing properties. Thus, the tested concentration (10, 20 µg/ml) of TzAgNPs inhibits microbial biofilm development without showing microbial growth arresting properties.
TzAgNPs generates Reactive oxygen species (ROS) in Pseudomonas aeruginosa
Reactive oxygen species (ROS) includes reactive chemical species harboring oxygen. Examples include peroxides, superoxide, hydroxyl radical, and singlet oxygen (Imlay 2003). Superoxide O2•− inadvertently produced by nearly all organisms by 1-electron transfer to oxygen during respiration. O2•− is subsequently converted to hydrogen peroxide (H2O2), which through the Fenton reaction generates the highly reactive •OH. Macromolecular damage (by reacting with DNA, proteins and lipids) is thus induced when cellular oxidant capacity is beaten by an increase in ROS production which is also responsible for disrupting thiol redox circuits in the cell (Briviba et al. 1997). It was reported that the increase in ROS level in bacteria decreases the microbial biofilm formation considerably (Dwivedi et al. 2014). In order to examine the role of TzAgNPs in ROS generation, microorganisms were grown in presence and absence of TzAgNPs. ROS level was measured and compared between TzAgNPs treated and untreated microbial cells. The result showed that there is a significant difference in ROS level between TzAgNPs treated and untreated cell (Figure 6). The result indicates that TzAgNPs treatment increases the ROS level considerably in microorganisms (Figure 6). Thus, the microbial biofilm inhibition could be attributed due to the accumulation of ROS. To obtain validation of the correlation between the sub-MIC doses of TzAgNPs, ROS and biofilm formation, we had constructed a contour plot (Figure 7). A contour plot is a two dimensional graphical representation of the relationships among three variables. The result indicated that the increase in sub-MIC doses of TzAgNPs accumulates ROS in microorganisms that lead to the inhibition of microbial development.
TzAgNPs reduces the secretion of virulence factor from Pseudomonas aeruginosa
Existing literature showed that microbe mediated pathogenesis is mostly associated with biofilm formation as microorganisms in biofilm secrete several virulence factors that could help the microorganism to spread the disease (Hall-Stoodley and Stoodley, 2009). An important role is played by Virulence Factors when microorganisms invade into host cells (Das et al. 2017). Pseudomonas aeruginosa, a gram negative bacterium often secrete diverse virulence factors including different kinds of proteases and pyocyanin for disease progression (Das et al. 2017). Thus, in the current study, we had examined the role of TzAgNPs on the secretion of protease enzyme and pyocyanin from the bacteria. Hydrolytic enzymes such as Proteases act by targeting the proteins of the host cells (infected tissue), thereby promoting bacterial invasion of the host by the bacteria. Azocasein happens to be a very sensitive substrate for proteases where the Azo dye gets released into the media after the enzymatic hydrolysis of the Casein protein. This released Azo dye can then be detected and quantified by measuring the absorbance at 440 nm using a spectrophotometer (Das et al. 2017). The result showed that microorganisms treated with TzAgNPs showed decreased level of proteases production compared to the amount of proteases secreted from the microorganisms that were not treated with TzAgNPs (Figure 8A). The increased concentration of TzAgNPs decreases the protease production from the bacteria considerably. Pyocyanin, a small organic molecule belong to the siderophore class gets expressed by several Gram-negative bacteria including Pseudomonas aeruginosa (Essar et al. 1990). Pyocyanin is a blue-green secondary pigment produced by Pseudomonas aeruginosa is capable of killing mammalian cells by targeting a wide range of cellular components and pathways including vesicular transport,electron transport chain and cell growth (Essar et al. 1990). The result showed reduced level of pyocyanin production from microorganisms that were treated with TzAgNPs than the control set where microorganisms were grown in absence of TzAgNPs (Figure 8B). Thus, all the results demonstrated that microorganisms showed reduced secretion of virulence factors under the exposure of TzAgNPs. Therefore, this TzAgNPs not only inhibits the microbial biofilm formation but also reduces the secretion of virulence factors from the microorganism that lead to compromise the spreading of pathogenicity.
Introduction of novel agents attenuating microbial biofilm open a new therapeutic approach to combat with biofilm associated human infections. Our current study demonstrates that sub-MIC doses (10, 20 µg/ml) of TzAgNPs exhibit efficient antibiofilm activity against Pseudomonas aeruginosa. Thus, TzAgNPs nanoparticles may work alone or in combination against drug resistant Pseudomonas aeruginosa infections, where biofilm formation plays a crucial role in disease progression.