Currently there is an urgent need to develop a new strategy for the treatment of antibiotic resistance bacterial pathogen and to reduce the virulent quorum-sensing ability of a microorganism. The development of eco-friendly process for the green synthesis of nanoparticles is an important aspect of modern nanotechnology. The purpose of this study was to evaluate the antiquorum sensing and antibacterial activity of silver nanoparticles synthesized by the mutant Klebsiella pneumoniae MTCC 3354. The synthesized silver nanoparticles were assessed by UV Visible Spectroscopy and Scanning Electron Microscope (SEM). The analysis of results confirms the formation of silver nanoparticles which was predominately spherical shape and polydispersed. The SEM study showed the formation of silver nanoparticles with particle size in the range of 2-20 nm. Furthermore, silver nanoparticles exhibit enhanced anti-quorum sensing and antibacterial activity on selected pathogens. The silver nanoparticles would be alternative strategies for treating the bacterial infection caused by antibiotic resistance bacteria in upcoming era.
INTRODUCTION
Nanotechnology has dynamically developed as an important field of modern research with potential effects in electronics and medicine (Glomm 2005; Chan 2006; Boisselier 2006 and Astruc 2009; Sadowski 2010). Production of metallic nanoparticles can be achieved through chemical, physical or biological methods. Chemical approaches are the popular mode of synthesis of nanoparticles. However; these methods cannot avoid the use of toxic chemicals. Now a day's biological synthesis of metallic nanoparticles is gaining importance as it is reliable and ecofriendly (Rajesh et al., 2012). The development of green process for the synthesis of nanoparticles is evolving into an important branch of nanotechnology (Ponaruselvam et al., 2012; Armendariz et al., 2002 Raveendran et al., 2002). Biological methods are regarded as safe, cost-effective, sustainable and environment friendly processes for the synthesis of nanoparticles(Schmid,1992).Silver nanoparticles (AgNPs) have been successfully synthesized using various bacteria(Kalimuthu et al., 2008 ; Chaudhari et al., 2012)., fungi (Ahmad et al.,2003 ; Kathiresan et al., 2009 )and plants (Raut et al.,2010; Masurkar et al., 2011). The biological methods of AgNPs synthesis using microorganism is an ecofriendly alternative method when compared to chemical and physical ones. These methods can be divided into two categories depending on the place where nanoparticles are created as many microorganism can provide inorganic materials either intra or extra cellularly (Mann 1996 and Ragunathan et al., 2009). In recent years, resistance to commercially available antimicrobial agents by pathogenic bacteria and fungi has been increasing at an alarming rate and has become a serious problem. Microorganisms, such as bacteria, molds, yeasts and viruses, in the living environment are often pathogenic and cause severe infections in human beings (Wright et al., 2000; Wright et al., 2009; Monali Gajbhiye et al., 2009). Quorum sensing is a cell communication mechanism through which signal molecules called autoinducers activate specific receptors associated with transcription signals for controlling various biochemical processes. Some of these processes are biofilm formation, expression of virulence factors, luminescence, pigment production and mechanisms of resistance to stress conditions (Gobbetti et al., 2007; Olivero et al.,2011), which are of major importance in bacterial pathogenesis (Vattem et al., 2007 and Bai et al., 2008). Furthermore, in most of the bacteria the quorum sensing signal molecules are important for the establishment of infection and also can serve as a switch to pathogenic state. Since the production of violacein is quorum sensing (QS)-driven, it has become an important tool for bacterial QS signal bioassays, especially for the N-acylhomoserine lactone autoinducers (AHLs). A C. violaceum mini-Tn5 mutant, CV026, is used as an indicator organism (Arunkumar et al., 2012; Mc Clean et al.,1997). There is a pressing need to search for new antimicrobial agents from natural and inorganic substances. Among inorganic antimicrobial agents, silver has been employed most widely since ancient times to fight infections. The aim of present study was to evaluate the antiquorum sensing and antibacterial activity of silver nanoparticles synthesized from the mutant Klebsiella pneumoniae.
MATERIALS AND METHODOLOGY
Chemicals
All chemicals used were of analytical grade. Muller-Hinton agar media was obtained from Hi-Media, India. Silver nitrate (purity>99%) was used as a precursor in the preparation of silver nanoparticles and AHL (Acyl Homoserine Lactone) were obtained from Sigma Aldrich, India.
Microorganisms
Klebsiella pneumoniae MTCC 3354, Staphylococcus aureus MTCC 7443, Escherichia coli MTCC 739 and Pseudomonas aeruginosa MTCC 2297 were procured from Microbial Type Culture Collection, Institute of Microbial Technology (IMTECH), and Chandigarh, India. The procured bacterial cultures were propagated in Nutrient Broth and incubated at 37°C for 24 hrs. The grown cultures were sub cultured at a regular interval of 15 days and stored at 4°C for further use.
RANDOM MUTAGENESIS AND MUTANT SELECTION
In the present investigation, two steps of mutagenesis were followed for the development of a highly mutated and stable bacterial strain. The first step of mutagenesis was carried out by exposing the wild strain Klebsiella pneumoniae MTCC 3354 to various mutagens like UV and Ethidium bromide. The mutated strains possessing higher rate of extracellular metabolite for biosynthesis of silver nanoparticles was screened out. During the second step, all the best mutants of first step mutation were again exposed to the same mutagens and double mutated strains were obtained. These strains further screened for the hyper production potential of extracellular metabolite for biosynthesis of silver nanoparticles and among these, best one was isolated and further work was continued on that mutant.
Synthesis of silver nanoparticles by Mutant Klebsiella pneumoniae MTCC 3354
The mutant Klebsiella pneumoniae MTCC 3354 were grown aerobically in nutrient broth amended with 1% glucose for the biosynthesis studies. The cultured flasks were incubated in orbital shaker at 37°C and agitated at 100 rpm for 24 hrs. After incubation the culture was centrifuged at 12,000 rpm for 20 minutes and the supernatant was collected, filtered (Whatman filter paper No. 1) and used for further experiments. For the synthesis of Silver nanoparticles, 100 ml of clean cell free filtrate was bought in contact with AgNO3 (1mM) final concentration in 250 ml Erlenmeyer flasks and agitated at 28°C under pH-7 in dark. Simultaneously control without silver ions was also run along with experimental flasks. Periodically, aliquots of the reaction solution were removed and absorptions were measured using a UV-Vis Spectrophotometer (PerkinElmer's LAMBDA 45).
UV-Vis Spectrophotometer analysis
After the period of incubation, the preliminary detection of silver nanoparticles produced by the mutant strain was carried out by visual observation of colour change of cell filtrate. These samples were later subjected to optical measurements, which were carried out by using a UV- visible spectrophotometer and the spectrum was measured between 200 and 700nm. Periodically aliquots of the reaction solution were removed and the absorptions were measured at multiple time intervals from 0 hr to 24 hrs.
Scanning Electron Microscopy
The silver nanoparticles were characterized by scanning electron microscopy (Hitachi S3000H) at Central Electro Chemical Research Institute (CECRI), Karaikudi, and Tamilnadu. The filtrate embedded with silver nanoparticles was subjected to freeze drying under high vacuum tube. After freeze-drying of the purified silver nanoparticles, the structure, composition, and average size of the synthesized silver nanoparticles were analyzed by Scanning Electron Microscopy. Atleast, three images of the sample were taken to have a clear representation of its morphology.
In vitro antibacterial activity of silver nanoparticles by well diffusion method
The in vitro antibacterial activity of silver nanoparticles was investigated against various bacterial pathogenic microorganisms such as S. aureus, E. coli and P. aeruginosa using well- diffusion method. The pure culture of organisms was sub cultured on Muller Hinton Broth at 37°C. Two wells of 6mm diameter were made on Muller Hinton Agar plates using gel puncture. The selected bacterial pathogenic suspension (100 µl of 104 -105 CFU) was applied uniformly on the surface of Muller Hinton Agar plates before adding a nanocolloids suspension to the well. Using sterile micropipette, 50 µl (5 mg/ml) of the sample of nanoparticles solution was loaded along with positive control (5 mg/ml Ciprofloxacin). After incubation at 37°C for 24 hrs, the different levels of zone of inhibition were measured using high antibiotic zone scale.
Synergistic effect of silver nanoparticles
A disc diffusion method was widely employed to detect the synergistic effects of silver nanocolloids with the combinations of commonly used antibiotic (Amoxicillin, Methicillin and Ampicillin) for the bactericidal activity against test bacterial strains on Muller-Hinton Agar plates. The standard antibiotics discs were purchased from Hi-media (Mumbai, India). To determine the synergistic effects, each standard antibiotic disc was impregnated with 10 µl of the freshly prepared AgNPs at a final content of 10 µg /disc. A single colony of bacterial pathogenic strain was grown overnight in Muller-Hinton liquid medium on a rotary shaker (100 rpm) at 37°C. The inoculum were applied to the plates along with the standard and prepared discs containing differing amounts of AgNPs. Cultures of S. aureus, E. coli and P. aeruginosa were used as test strains. Similar experiments were carried out with AgNPs alone. Standard antibiotic discs were used as positive control. Bacterial cell-free filtrate was used as negative control. After incubation at 37°C for 18 hours the zones of inhibition were measured. All the assays were performed in duplicate.
Antiquorum sensing activity of silver nanoparticles by disc diffusion assay
Antiquorum sensing activity of silver nanoparticles produced by mutant Klebsiella pneumoniae was assayed by disc diffusion method by using Chromobacterium violaceum as a bioreporter. Luria Bertani agar plates were seeded with 0.1 ml of approximately diluted (C.2.5Ã-106CFU ml-1) freshly grown cultures along with AHL as exogenous source of quorum sensing molecules. Sterile discs (6mm diameter) impregnated with different amounts (5, 10, 15 µl) of silver nanocolloids solutions. Solvent and sterile LB broth was used as control. These discs were placed on agar plates overlaid with the indicator strain. Plates were incubated for 18 - 24 hours at 28°C to check the inhibition of pigment violacein production around the well.
Result & Discussion:
The present study demonstrates the formation of silver nanoparticles by the reduction of silver metal ions during exposure to mutant Klebsiella pneumoniae. After 24 hrs of incubation, the intense colour change from colourless to yellowish brown was observed in mutant strain when compared to wild strain, which suggests that the synthesis of silver nanoparticles by the mutant strain is greater than that produced by the wild strain of K. pneumoniae (Fig-1). The appearance of brown colour was due to the excitation of surface plasmon vibrations, typical of silver nanoparticles (Ahmad et al. 2003; Shankar et al. 2004; Jae and Beom, 2009).
Similarly, the formation of silver nanoparticles was also confirmed by UV-visible spectrophotometer. The UV-visible spectra showed a strong plasma resonance which was centered approximately at 424nm of AgNPs, produced by the mutant strain. It was observed that the maximum absorption occurs at 424nm (Fig-2), broadening of peak indicated that the particles are poly-dispersed. Furthermore, AgNPs have free electrons, which give surface plasmon resonance (SPR) absorption band, due to the combined vibration of electrons of AgNPs in resonance with light wave (Nath et al. 2012). The exact mechanism for the synthesis of nanoparticles has not been clearly established but an enzyme NADH-dependent nitrate reductase is known to be involved in the process [Labrenz et al., 2000; Roh et al., 2011].
Scanning electron microscopy provided further insight into the morphology and size details of the silver nanoparticles. The SEM micrographs of nanoparticles obtained in the filtrate showed that the diameter of the nanoparticles in the solution was above 2-20nm. The silver nanoparticles are spherical in shape and well distributed without aggregations (Figure: 3-4).
The in vitro antibacterial activity of AgNPs was examined against three selected pathogenic bacteria, using ciprofloxacin as positive control, an antibacterial agent that is widely used against many bacterial infections. Figure 5 - represents the zone of inhibition for 50 µl of AgNps solution against test pathogens such as S. aureus (28mm), E. coli (32mm) and P. aeruginosa (35mm). The bactericidal effect of silver nanoparticles is dependent on the concentration of silver nanoparticles, size and nature of the microbial metabolites (Karthickraja et al., 2011; Raja et al 2012). This study clearly demonstrates that the zone of clearance increased in concentration dependent manner of silver nanoparticles against test pathogenic bacteria. The mechanism of the bactericidal effect of silver colloid particles against bacteria is not very well known (Panacek et al., 2006). AgNPs able to pass through the cell membrane of bacteria and thereby it may interact with DNA molecules, thus it arrest the replication process which may lead to the cell death. Synergistic effect of silver nanocolloids in combination with different antibiotics such as Methicillin, Amoxycillin, and Ampicillin was investigated against three pathogenic bacteria by using disc diffusion method. The zone of inhibition of silver nanocolloids was examined along with different antibiotic discs with and without AgNPs against selected organisms (Table-1) Activity of Amoxycillin, Ampicillin, and Methicillin increased in presence of Nanoparticles against P. aeruginosa, E. coli, and S. aureus. Methicillin was found to possess highest percentage fold increase, followed by Amoxycillin and Ampicillin. The maximum antibacterial activity for Methicillin in combination with silver nanocolloids was observed against S. aureus followed by P. aeruginosa, and E. coli with a percentage fold increase of 100, 30, and 57% respectively. In case of Ampicillin combination with silver nanocolloids the maximum activity was observed against P. aeruginosa with 40% fold increase in antibacterial activity and E. coli for 35% and followed by S. aureus with 33%. The use of Amoxycillin with silver nanoparticles showed fold increase in P. aeruginosa, E. coli, and S. aureus with fold ranges from 64, 58 and 54% respectively.
The enhanced synergetic effects of silver nanocolloids are due the bonding reaction between antibiotic and silver nanoparticles. Moreover, the nanoparticles have large surface area which allows them to closely interact with antibiotics. The antibiotic molecules contain active groups like hydroxyl and amino groups, which can easily react with AgNPs by chelation (Bataresh et al., 2004). Since the nanoparticles are too small in size, they can come in contact with antibiotics, thereby either it can inhibit peptidoglycan synthesis or AgNPs-antibiotics complex can react with DNA leading to the damage of the bacterial cells (Sindhu et al., 2013).
Chromobacterium violaceum assay was performed with different concentrations (15, 10, 5µl) of silver nanoparticles synthesized by mutant K. pneumoniae by disc diffusion assay using the bioreporter strain CV026. Loss of purple pigment in CV026 cultured with exogenous AHL is indicative of quorum sensing inhibition by the silver nanoparticles synthesized by mutant K. pneumoniae. A clear halo zone of inhibition around the wells of varying diameter indicates that quorum sensing inhibition effect was relative to the amount of silver nanocolloids added (Fig-6). Finding in this study, confirms for the first time quorum quenching activity of silver nanoparticles produced by the mutant strain. Furthermore, in the present study the silver nanocolloids exhibit varying degree of antibacterial activity and synergistic effect against selected pathogens.
Conclusions
We have demonstrated a simple biotechnological process for the hyper-intracellular synthesis of silver nanoparticles using this mutant bacterial strain. Furthermore, this research article aims at reviewing the literature and helping us to understand the ways of communication among bacteria, which further open up the prospects in the treatment of diseases caused by antibiotic resistance bacteria.
