|Year : 2016 | Volume
| Issue : 2 | Page : 89-101
Green-synthesized silver nanoparticles kill virulent multidrug-resistant Pseudomonas aeruginosa strains: A mechanistic study
Balaram Das1, Sandeep Kumar Dash2, Debasis Mandal1, Jaydeep Adhikary3, Sourav Chattopadhyay1, Satyajit Tripathy1, Aditi Dey1, Subhankar Manna1, Sankar Kumar Dey4, Debasis Das5, Somenath Roy1
1 Department of Human Physiology with Community Health, Immunology and Microbiology Laboratory, Vidyasagar University, Midnapore, West Bengal, India
2 Department of Human Physiology with Community Health, Immunology and Microbiology Laboratory, Vidyasagar University, Midnapore, West Bengal; Department of Physiology, University of Gour Banga, Malda, West Bengal, India
3 Department of Chemical Sciences, Ariel University, Ariel, Israel
4 Department of Physiology, Santal Bidroha Sardha Satabarshiki Mahavidyalaya, Midnapore, West Bengal, India
5 Department of Chemistry, University of Calcutta, Kolkata, West Bengal, India
|Date of Submission||10-Nov-2016|
|Date of Acceptance||10-Nov-2016|
|Date of Web Publication||19-Dec-2016|
Department of Human Physiology with Community Health, Immunology and Microbiology Laboratory, Vidyasagar University, Midnapore - 721 102, West Bengal
Source of Support: None, Conflict of Interest: None
Background: Due to abuse and improper prescribing policy of antibiotics, the antibiotics resistance were remarkably increased in Pseudomonas aeruginosa, so there are urgently need to develop a new kind of antimicrobial to treat Pseudomonas aeruginosa infection. Biosynthesized silver nanoparticles (Ag NPs) have showed its interesting impact against bacterial infection drawn researchers to green nanotechnology.
Aims: The study was conducted to evaluate the antibacterial activity of AgNPs against multi drug resistant P. aeruginosa isolated from urine sample of UTI patients.
Methods: 126 UTI patent's urine samples were included in the study. P. aeruginosa strains were isolated, identify, antimicrobial susceptibility, drug resistance mechanisms were done as per routine laboratory protocol. The antimicrobial activity and mechanisms of the killing of Ag NPs were studied.
Results: From this study, it was revealed that 25 (19.84%) isolates were multi drug resistant Pseudomonas aeruginosa. Green synthesized Ag NPs successfully destroyed the multi drug resistant strains via ROS generation and membrane damage. The prevalence of multidrug resistance is increased worldwide and there are urgently need another option to control the multidrug resistant strains.
Conclusion: The findings of the study suggested that Ag NPs might be used to treat the multi drug resistant Pseudomonas aeruginosa.
Keywords: Bacterial membrane damage, multidrug resistance, Pseudomonas aeruginosa, reactive oxygen species generation, silver nanoparticles
|How to cite this article:|
Das B, Dash SK, Mandal D, Adhikary J, Chattopadhyay S, Tripathy S, Dey A, Manna S, Dey SK, Das D, Roy S. Green-synthesized silver nanoparticles kill virulent multidrug-resistant Pseudomonas aeruginosa strains: A mechanistic study. BLDE Univ J Health Sci 2016;1:89-101
|How to cite this URL:|
Das B, Dash SK, Mandal D, Adhikary J, Chattopadhyay S, Tripathy S, Dey A, Manna S, Dey SK, Das D, Roy S. Green-synthesized silver nanoparticles kill virulent multidrug-resistant Pseudomonas aeruginosa strains: A mechanistic study. BLDE Univ J Health Sci [serial online] 2016 [cited 2017 Jul 24];1:89-101. Available from: http://www.bldeujournalhs.in/text.asp?2016/1/2/89/196087
Urinary tract infections (UTIs) are most important in humans that account for more than 300,000 hospital admissions in the United States annually. UTI is the most common infection of urological diseases., UTIs are common in females and caused by Escherichia More Details coli, Proteus mirabilis, Pseudomonas aeruginosa, Klebsiella pneumoniae, and Streptococcus faecalis. UTIs caused by P. aeruginosa are common in India. P. aeruginosa is the leading cause (12%) of hospital-acquired UTIs. P. aeruginosa is Gram-negative, asporogenous, obligate aerobic, motile bacilli generally found in soil, water, plants, and animals, including humans. It is occasionally pathogenic for plants as well as animals. Due to alteration of normal host defenses, P. aeruginosa begins disease process in humans. It rarely infects the healthy tissues; however, when defense system is compromised, it can infect virtually all tissues. P. aeruginosa exerts its pathogenesis by several virulence factors. These virulence factors and biofilm formation are regulated by quorum sensing (QS) (cell-to-cell communication mechanism by which bacteria can coordinate).
P. aeruginosa was less sensitive to the common antibiotics but highly sensitive to amikacin, piperacillin, gentamicin, and ciprofloxacin. The therapy of infections caused by P. aeruginosa presents a daunting problem to medical practitioners because of its multidrug resistance (MDR) property. A major reason for its importance as a pathogen is its high intrinsic resistance to antibiotics. This intrinsic antibiotic resistance of P. aeruginosa is attributed to factors such as active drug efflux and beta-lactamase production. For microbiologists, P. aeruginosa is one of the most challenging pathogens because of its constant evolution of resistance (continuing appearance of new resistance mechanisms) and the complexity of multi-resistant phenotypes; have forced to develop a new kind of diagnostic tool. Hence, there is urgently need to develop a new therapeutic strategy such as drugs acting on new targets as well as need to search out the prevalence of antimicrobial resistance on a national and international basis.
Silver and its products are used in biomedical purpose, water and air purification, food production, cosmetics, clothing, and numerous household products due to its broad-spectrum antimicrobial properties. With the rapid progress of nanotechnology, applications of nanomaterials have been extended further, and now, silver is the most commonly used engineered nanomaterial. Already, silver nanoparticles (Ag NPs) have been shown to be effective biocides against different bacteria such as E. coli, Staphylococcus aureus, Staphylococcus epidermidis, Bacillus subtilis, Klebsiella mobilis, K. pneumoniae, and P. aeruginosa.,,,,,,,, Biological methods involve in the synthesis of Ag NPs utilizing extracts from biological sources as reductant, capping agents, or both., Plant extracts such as apiin, leaf extract from magnolia, persimmon, geranium, and pine leaf have also been used as reducing agents in Ag NPs synthesis.,
The mechanisms of the activity of Ag NPs on bacteria are not yet fully understood; the most common mechanisms of activity are: (1) Ag NPs releases free silver ions that uptake cells, followed by disruption of adenosine triphosphate production and DNA replication, (2) Ag NP and silver ion augment the reactive oxygen species (ROS) generation, and (3) Ag NPs direct damage to cell membranes. Commonly proposed mechanisms in the background study begin with the release of silver ions, followed by generation of ROS, and cell membrane damage, but there are many contradictory findings reported.
Metals can act as catalysts and generate ROS in the presence of dissolved oxygen. In this context, Ag NPs may catalyze reactions with oxygen leading to excess free radical production. In bacterial cells, silver ions would likely induce the generation of ROS by impairing the respiratory chain enzymes through direct interactions with thiol groups in these enzymes or the superoxide radical scavenging enzymes such as superoxide dismutases. Excess ROS production may produce oxidative stress. Free radicals can attack membrane lipids and lead to a breakdown of membrane and mitochondrial function or cause DNA damage. Ag NPs interact with the bacterial membrane and are able to penetrate inside the cell. Ag NPs adhere to and penetrate into cells and also are able to induce the formation of pits in the cell membrane.,,
| Materials and Methods|| |
Culture media, chemicals, and quality control strains
Bacterial culture media, silver nitrate solution (AgNO3 ), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reagent, glutaraldehyde, fluorescence stains, and different chemicals and reagents were procured from Merck Ltd., SRL Pvt. Ltd., Mumbai, India, with highest grade available. Different antibiotics and antibiotics discs were purchased from HiMedia, India.
For quality control strains, different ATCC strains were used. These strains were obtained from Microbiology Laboratory, University of Calcutta, and Microbiology Laboratory, Midnapore Medical College and Hospital. These strains were stored in agar slants at 4°C for further studies and used as reference strain.
Collection, transport of sample, and culture of microorganisms
After proper inquiry of infection history and treatment summary of the patient in nearby Medical College and Hospital, a total of 126 UTI patent's urine samples were collected from catheter specimen between June 2013 and November 2014. Then, it transported to the laboratory in autoclaved Luria-Bertani (LB) broth within 2 h of collection. The urine samples kept in LB medium were incubated in a shaking incubator at 37°C for 24 h. Bacterial cultures were then growing on nutrient agar (NA) media; then, cultures were purified by single colony isolation technique. Isolates were subcultured on fresh NA plates for characterization studies.,
Morphological, biochemical, and physiological characterization
Colony characteristics, including pigment production, were determined on Pseudomonas P agar and NA. All strains were biochemically characterized by the following classical tests according to Bergey's Manual of Systematic Bacteriology: Gram staining, catalase production, oxidase reaction, indole test, urease test, gelatin hydrolysis, citrate utilization, esterase, lipase, methyl red-Voges-Proskauer (MR-VP) test, triple sugar iron (TSI) test, motility test, carbohydrate fermentation test, production of fluorescent pigment and growth on cetrimide agar, isolation agar, and 42°C.,, All the results were obtained in triplicate and then analyzed the results.
Resistance profile testing by disc agar diffusion
Resistance profile in terms of antimicrobial susceptibility testing was performed by Kirby-Bauer disk diffusion method. The tested bacterium was taken from an overnight culture and freshly grown for 4 h at approximately 106 CFU/ml. With this culture, a bacterial lawn was prepared on NA. Antibiotic discs were placed to observe antibiotic susceptibility patterns against different antibiotics. The diameter of zone of bacterial growth inhibition surrounding the disc (including the disc) was measured and compared with the standard for each antibiotic. This gave a profile of drug susceptibility vis-à-vis antibiotic resistance. Quality control was performed using P. aeruginosa 27,853.
Multiple antibiotic resistance index
The multiple antibiotic resistance (MAR) index was determined for each isolate by dividing the number of antibiotics to which the isolate is resistant by the total number of antibiotics tested.
Study of resistant mechanism and quorum-sensing-dependent virulence factors
The tested isolated strains were tooth-picked onto the surface of NA plates and incubated it for overnight at 37°C. In the next day, the plates were overlaid with 1% molten agarose containing 1% penicillin and 0.2% soluble starch. The plates were allowed to solidify at room temperature, and iodine solution was poured onto the agar plates. After 10 s, the residual iodine solution was damped out and the plates were incubated at room temperature until a discoloration zone appeared around the colonies. Thus, the presence of a clear zone around bacterial growth is indicative of β-lactamase production.
Study of efflux system
The efflux system of resistance of P. aeruginosa isolates was investigated according to the method of Lomovskaya et al., 2001. The minimum inhibitory concentrations (MICs) of antibiotics (ciprofloxacin) for 25 MDR P. aeruginosa isolates were examined in the presence and absence of 10 mM of the efflux pump inhibitor carbonyl cyanide m-chlorophenylhydrazone (CCCP) (Sigma). The reduction in MIC of a certain antibiotic with CCCP is an indication of resistance to this antibiotic mediated by an efflux system.
Elastase activity was measured using the elastin Congo red (electron cyclotron resonance [ECR], Sigma) assay. Cells were grown in LB broth at 37°C for 14 h. A 100 μl aliquot of bacterial supernatant was added to 900 μl of ECR buffer (100 mM Tris, 1 mM CaCl2 , pH 7.5) containing 20 mg of ECR and incubated with shaking at 37°C for 3 h. Insoluble ECR was removed by centrifugation, and the absorption of the supernatant was measured at 495 nm. LB medium was used as a negative control.
Production of rhamnolipids
LB agar plates containing 0.2 g cetyltrimethylammonium bromide and 5 mg methylene blue l-1 were inoculated with 2 μl of an overnight LB culture of P. aeruginosa strains. After an overnight incubation at 37°C, the diameter of the clearing zone around the bacterial spots was measured as evidence of rhamnolipid production.
Protease production and proteolytic activity were detected on 1.2% agar plates supplemented with 10% (v/v) sterile skimmed milk (105°C for 30 min). The cultures were streaked on the skim milk agar plates and incubated at 27°C for 24-36 h. Proteolytic strains caused a clearing zone around the colonies.
Quantitative assay for pyocyanin production
Pyocyanin was extracted from culture supernatants and measured by the method of Krishnan et al., 2012. A 3 ml volume of chloroform was added to 5 ml of culture supernatant and mixed. The chloroform layer was transferred to a fresh tube and mixed with 1 ml of 0.2 M HCl. After centrifugation, the top layer (0.2 M HCl) was removed. The amount of pyocyanin within the extracted layer was determined by measuring A520.
The amount of biofilm formation was measured according to Stepanovic et al., 2004 with minor modification. Bacteria were grown on LB agar, and several colonies were gently resuspended in LB (with or without the appropriate antibiotic); 100 μl aliquots were placed in a microtiter plate (polystyrene) and incubated 48 h at 37°C without shaking. After the bacterial cultures had been poured out, the plate was washed extensively with water, fixed with 2.5% glutaraldehyde, washed once with water, and stained with a 0.4% crystal violet solution. After solubilization of the crystal violet with ethanol-acetone (80:20, vol/vol), the absorbance at 590 nm was determined using a microplate reader (Bio-Rad, Hercules, Calif).
Synthesis and purification of silver nanoparticles
Ocimum gratissimum leaves were collected from Vidyasagar University campus and washed gently with distilled water to remove dust particles. The leaves were dry under sunlight. These leaves were then grinding to get dust in a grinder. The dust materials were dissolving in distilled water (10 g dust/100 ml double distilled water) and filtered with Whatman filter paper No. 1. The filtrate was collected, freeze-dried, and stored at 4°C until use.
Synthesis of Ag NPs using water extract of O. gratissimum leaf was done according to the method of Das et al., 2015. Ag NPs were synthesized by dissolving 0.001 M of AgNO3 with 100 mg freeze-dried plant leaf extract in 100 ml of deionized water in a 250 mL reaction vessel at room temperature for the bioreduction process. Immediate after the mixing of leaf extract, the pH of the solution was adjusted to 10.0 using a 7.7 M solution of NaOH. The entire system was thereafter shaken at a rotation rate of 150 rpm in the dark condition at 37°C for 48 h. The solution containing Ag NPs was then collected and centrifuged at 3000 rpm for 10 min for removal of excess extract components. To get fine nanoparticles, the solution then centrifuges at 13000 rpm for 20 min. Purification of the nanoparticles was performed by sucrose density gradient centrifugation.
Characterization of silver nanoparticles
Fourier transform infrared spectroscopy
Ag NPs were characterized by Fourier transform infrared (IR) spectroscopy with a PerkinElmer Spectrum RX I Fourier transform IR system, with a frequency ranging from 500 to 4000/cm and a resolution of 4/cm. The KBr was used to prepare the samples.
Dynamic light scattering and zeta potential
Dynamic light scattering (DLS) analysis was done with a Zetasizer Nano ZS (Malvern Instruments) according to standard method. The 100 μg/ml Ag NPs solution was prepared and sonicated for 10 min, and hydrodynamic particle sizes were measured by suspending few drops of an sonicated suspension of NPs in 10 ml of Millipore water. The experiments were repeated several times to obtain the average size of the NPs. The zeta potential of the Ag NPs was measured using a Zetasizer-Nano ZS (Malvern, Malvern Hills, UK). 1 mg/ml Ag NPs suspension was prepared in Milli-Q water. Then this suspension was used to experiment.
Scanning electron microscopy
The dry particle size, morphology, and microstructure of the nanoparticles were investigated by high-resolution scanning electron microscopy (SEM) (instrument from Nikon, Japan). In brief, Ag NPs were suspended in deionized Milli-Q water at a concentration of 1 mg/ml and then sonicated to get homogenous suspension. For size measurement, the sonicated stock solution of silver was diluted twenty times. Then, one drop of solution was taken on a glass plate and dried it. Then, the sample was gold coated and images were taken.
Transmission electron micrograph
The particle size, shape, morphology, and microstructure were studied by high-resolution transmission electron microscopy (TEM) in a JEOL 3010, Japan, operating at 200 kV according to the method of Chattopadhyay et al., 2012. In brief, 1 mg/ml Ag NPs solution was prepared in deionized water and then sonicated to form a homogenous suspension. For measurement, stock solution of Ag NPs was diluted twenty times and a drop of the aqueous Ag NPs suspension was taken onto carbon-coated copper grid and was dried and images were taken.
Antibacterial activity determination
Minimum inhibitory concentration and minimum bactericidal concentration determination
The MIC and minimum bactericidal concentration (MBC) values were determined by microdilution method in LB. In brief, 10 μl of bacterial strain containing 2.5 × 105 CFU/mL bacteria was added individually to 1 mL of LB. Pure suspension of Ag NPs was prepared by sonication. Different concentrations of test particles were added to the test tubes containing the bacterial strains and incubated for 24 h. After incubation, the MIC values were obtained by checking the turbidity of the bacterial growth. The MIC value corresponded to the concentration that inhibited 99% of bacterial growth.
The MBC values of the Ag NPs were determined according to the standard method. The MBC values were obtained by subculturing the MIC dilutions onto the sterile Mueller-Hinton agar plates and incubated at 37°C for 24 h. The lowest concentration of the Ag NPs where the tested bacteria were completely killed was tabulated as MBC level. The MBC value indicates the concentration where 100% bacterial growth was arrested.
The tolerance levels of P. aeruginosa against Ag NPs were determined according to May et al., 2006 using the following formula:
Tolerance = MBC/MIC.
Bacterial cell viability assay
Bacterial cell viability was performed after 24 h of treatment with Ag NPs by MTT method according to the standard method. After the treatment of Ag NPs, the bacterial cells were centrifuged at 1400 rpm for 10 min at 4°C, followed by repeated washing with sterile phosphate-buffered saline (PBS) (pH 7.4). Thereafter, the medium was replaced with fresh culture media containing 0.5 mg/ml of MTT and incubated for 3 h at 37°C. Then HCl-isopropanol solution was added, and after 15 min of incubation at room temperature, absorbance of solubilized MTT formazan product was measured in Shimadzu ultraviolet/visible 1800 spectrophotometer at 570 nm.
Cell viability count by fluorescence-activated cell sorter
Rh123 is a dye that stains the mitochondria of living cells; here, we used Rh123 as a viability stain. Bacterial intact membrane prohibits the dye to enter into the cell, but nonviable bacteria with depolarized membranes allow it to enter into the cell. Treated bacterial cells were centrifuged at 1800 rpm for 10 min at 4°C and washed with PBS (pH 7.4) and charged Rh123-labeled Ag NPs as required concentration (respective MBC concentration) and placed it at 37°C for 12 h in dark condition. Cells without Rh123 served as negative control. After incubation, cells were washed twice with PBS, and analyzed by flow cytometry (Model: FACSCalibur flow cytometer, Becton Dickinson).
Intracellular uptake study
Rh-B is a voltage sensitive cationic dye that is electrophoretically taken up by bacteria due to the transmembrane electrochemical potential of the plasma membrane. Ag NPs were labeled with rhodamine B (Rh-B) according to Mason et al., 1993. For this labeling, Rh-B dye (20 mg/ml) was added to Ag NPs to give a concentration of 0.2 mg/ml stain and it was kept at 37°C in dark for 6 h. Fresh bacterial cultures were centrifuged at 1400 rpm in 4°C for 10 min and then washed with PBS (pH 7.4), charged Rh-B-labeled Ag NPs as MBC concentration and placed it at 37°C in dark condition for 12 h. Cells without Rh-B served as negative control. After incubation, cells were washed and re-suspended in PBS, and a drop of the suspension was taken in a glass slide and examined with an Olympus research phase contrast with a fluorescence microscope (Model: CX41; Olympus Singapore Pvt. Ltd., Valley Point Office Tower, Singapore). Fluorescence images were acquired with 540 nm laser for differential interference contrast microscopy and 625 nm lasers for Rh-B excitation and emission.
Intracellular reactive oxygen species generation
The intracellular ROS generation of the bacterial cells was measured using 2,7-dichlorofluorescein diacetate (DCFH2 -DA). The oxidation of nonfluorescent DCFH to highly fluorescent 20, 70-dichlorofluorescein (DCF) provides a quantitative assay of ROS formation. The DCFH2 -DA passively enters the cell and reacts with ROS to form the highly fluorescent compound 2,7-dichlorofluorescein. Briefly, 10 mM DCFH2 -DA stock solution (in methanol) was diluted in culture medium to get 100 μM working solution. At the end of exposure with NPs, bacterial cells were cultured and washed with PBS. The cells were collected, and a homogeneous suspension was made by PBS. Then, cells were incubated with required amount of working solution of DCFH2 -DA at 37°C for 30 min. The cells were visualized under fluorescence microscope (Model: CX41; Olympus Singapore Pvt. Ltd., Valley Point Office Tower, Singapore). Another set was prepared in the same way for flow cytometry analysis. This intracellular ROS generation was confirmed by fluorescence-activated cell sorter analysis. The values were expressed as percent fluorescence intensity relative to control wells.
Pretreatment with N-acetyl-L-cysteine
To understand the involvement of ROS in Ag NP-induced bacterial cell death, P. aeruginosa cells were cultured overnight at a concentration of 2.5 × 105 CFU/mL. A stock solution of N-acetyl-L-cysteine (NAC) (Sigma-Aldrich) was made with sterile water and added to cells at 5 and 10 mM for 1 h. After NAC pretreatment, cells were treated with Ag NPs (16 μg/ml) for 48 h. Viability was determined by the MTT method. All measurements were done in triplicate.
Morphology of bacterial cells
Fresh overnight bacterial cultures were centrifuged at 1400 rpm in 4°C for 10 min; then, the bacterial cells were treated with required concentration of Ag NPs (MBC concentration) in 5 ml culture media and placed it at 37°C for 12 h with shaking at 198 rpm. Cells without Ag NPs served as negative control. After incubation, cells were washed and the bacterial palette was fixed with 50 μL of 2.5% glutaraldehyde for 5 min in 37°C and washed three times with 1X PBS. Fifty microliters PBS was added to this pallet to form a suspension. One drop of fixed pallet was taken on a glass plate and dried it. Then, the sample was gold coated used for observation in a SEM (Hitachi S-3000N).
In vivo antimicrobial efficacy study
For in vivo study, 6-8-week-old Swiss albino mice weighing 25-30 g were used. The animals were maintained in accordance with the guidelines of the National Institute of Nutrition, Indian Council of Medical Research, Hyderabad, India. The whole study was approved by the Ethical Committee of Vidyasagar University. The animals were housed for a week for acclimatization in standard polypropylene cages (Tarson) in the departmental animal house with stainless steel top grill. Clean paddy husk was used as a bedding material. The animals were fed on commercial pellet diet and water ad libitum in polypropylene bottles with stainless steel sipper tubes. The animals were maintained under standard conditions (humidity: 55%-65%, temperature: 22°C ± 3°C, and light: 12 h light/12 h dark cycles). The animals used in this study did not show any pathological process.
In vivo antibacterial activity using mice infection model
To evaluate the in vivo antibacterial effect of Ag NPs, the P. aeruginosa infection model was built. After 1 week acclimation, the mice were randomly divided into five groups (six mice in each group), comprising one normal control group, one P. aeruginosa infection control group, and three experimental groups. Before the experiment, Ag NPs were suspended in PBS (pH 7. 4) separately and ultrasonicated for 15 min. Mice were injected by subcutaneous injection with 100 μl of each NPs dose containing 500, 1000, and 2000 μg/kg body weight (B.W.) in PBS. PBS injections were used for control. Mice were injected by 3 days interval for 15 days. The duration was selected according to the previous report. In P. aeruginosa infection, the bacterial cell (50 μl of bacterial suspension containing 5 × 106 CFU/ml) was introduced into the bladder of the mice using a soft intramediac polyethylene tube. The doses were selected according to the previous report., Over the course of infection, the activity and B.W. of animals were recorded. The experiment was terminated at the end of 15 days and all animals were sacrificed and the blood and tissue samples were collected. Kidneys tissues were separated and homogenized in sterile PBS (5 mL) containing 1% Triton X-100. Aliquots of diluted homogenized tissues were plated on agar, on which the grown colonies were counted for analysis and the tissue samples were used in histopathological study.
The data were expressed as the mean ± the standard error of the mean (n = 6). Comparisons between the means of control and treated groups were made by one-way analysis of variance (using the statistical package Origin 6.1; Origin Laboratory, Northampton, MA, USA) with multiple comparison t-tests and P < 0.05 as the limit of significance.
| Results and Discussion|| |
Biochemical tests of isolates
The results of the phenotypic characterization based on physiological, morphological, and biochemical tests. P. aeruginosa was identified biochemically by means of several standard biochemical reactions. Single colony isolation technique was used to purify the bacterial cultures using NA. From the study, it was observed that 45.23% (57) isolates were Gram-negative and 54.76% (69) isolates were Gram-positive; 43.85% (25) of Gram-negative isolates are oxidase positive. One hundred percent of oxidase positive isolates were catalase positive, urease positive, gelatinase positive, motile, positive in pigment production (pyocyanin and pyoverdine) and also grow in 42°C. All oxidase positive isolates were negative for indole test, MR test, VP test and did not ferment glucose. More than 90% isolates were citrate test positive. It was also revealed that 100% of oxidase positive isolates were not fermented any substrate in TSI test. One hundred percent of oxidase positive isolates were not lactose fermenter. All they have not any DNase activity (results not shown). From the results of the biochemical tests, it was also revealed that they were release pigments. On cetrimide agar and Pseudomonas isolation agar, 100% of oxidase positive, indole negative isolates gave pigmented color colonies. Thus, among 126 clinical isolates, 25 (19.84%) isolates were confirmed to be P. aeruginosa.
The development and spread of antibiotic-resistant bacteria have been regarded as an unavoidable genetic response to the strong selective pressure imposed by antimicrobial chemotherapy, which plays a vital role in the evolution of antibiotic-resistant bacteria. These bacteria then pass the antibiotic resistance to other bacterial cells and species. The isolates obtained were identified by morphological and biochemical characteristics. Here, 45.23% clinical isolates were Gram-negative and 54.76% isolates were Gram-positive. As we know P. aeruginosa is Gram-negative bacteria, Gram-positive clinical isolates were discarded. Our results showed that 43.85% of Gram-negative isolates are oxidase positive that may be due to the presence of N, N, N', N'-tetramethyl-phenylenediamine dihydrochloride as artificial electron acceptor which takes the electron from cytochrome oxidase in the electron transport chain and changes color to a dark blue. Oxidase positivity supports that isolates were in Pseudomonas family. One hundred percent of oxidase positive isolates were catalase positive, urease positive, gelatinase positive, motile, positive in pigment production (pyocyanin and pyoverdine) and also grow in 42°C. It is evident from our study that all isolates were motile due to the presence of flagellum. The isolates release the urease and gelatinase enzyme which may degrade the urea and gelatin, respectively, in the medium. In our study, all oxidase positive isolates gave 92% positive and 8% negative in citrate utilization test. This is due to metabolism of citrate compound as a only source of carbon in the media. Hence, under basic condition, bromothymol blue changes the media color from green to blue. These isolates were also negative for MR-VP test. Reactions in TSI agar slant revealed that all isolates showed alkaline slant and butt without gas production. This indicates the glucose, lactose, and sucrose nonfermentation ability of those clinical isolates. P. aeruginosa strains produce two types of soluble pigments, the fluorescent pigment pyoverdin and the blue pigment pyocyanin. The latter is produced abundantly in media of low-iron content and functions in iron metabolism in the bacterium. Here, all the isolates release the pyocyanin the plate. In the selective media, 25 P. aeruginosa isolates successfully grow oxidase positive, catalase positive, motile, positive in pigment production (pyocyanin and pyoverdine) and also grow in 42°C of clinical isolates, which suggests that these are the P. aeruginosa strains. Hence, it is clear that the all 25 isolates were P. aeruginosa strains. Satisfactory results on all biochemical tests and colony characteristic on differential agar confirmed that all 25 isolates were P. aeruginosa. The clinically isolated P. aeruginosa strains were newly named as PA, from PA 1 to PA 25.
Resistance profile testing by disc agar diffusion
Antibiotic resistance pattern and profile of MDR was revealed by disc agar diffusion test [Table 1]. The results shows that out of 25 P. aeruginosa isolates 100% resistant to oxacillin, 96% resistance property were found to penicillin G, ampicillin; 88% resistant to cefotaxime and kanamycin, 84% resistant to erythromycin, 72% resistant to tetracycline, 60% resistant to chloramphenicol, 40% resistant to ciprofloxacin, 36% resistant to gentamycin and streptomycin, 32% resistant to norfloxacin, 20% resistant to imipenem, and 12% resistant to amikacin [Table 1]. It was found that 100% P. aeruginosa isolates were multidrug resistant as three or more antibiotic were resistant. Different types of resistance pattern were recognized. Among the 25 MDR P. aeruginosa strains, 8% were resistant to three antibiotics, 4% to four antibiotics, 16% to six antibiotics, 4% to seven antibiotics, 20% to eight antibiotics, 12% to nine antibiotics, and 36% to 11 or more antibiotics. Antibiotic susceptibility profile of P. aeruginosa isolates clearly shows that penicillin, ampicillin, tetracycline, erythromycin, cefotaxime, and oxacillin show more resistance to P. aeruginosa isolates and amikacin, norfloxacin, and imipenem were more sensitive than all other drugs used. It has been found that 0.4 and above MAR index from 86.96% isolated P. aeruginosa.
|Table 1: Antimicrobial susceptibility testing of 25 isolates of Pseudomonas aeruginosa |
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P. aeruginosa was less sensitive to commonly used antibiotics, but it highly was sensitive to the amikacin, piperacillin, ciprofloxacin, and gentamicin. Pseudomonas was susceptible to the second-line drugs, and most of these drugs are associated with high resistance to the first-line antibiotics (ampicillin and ciprofloxacin) used. It may be due to the widespread use of common antibiotics in the hospital and cross-resistance existing among various classes of antibiotics. P. aeruginosa is known to be resistant to penicillin, ampicillin, and first- and second-generation cephalosporins. In this study, the result of disc agar diffusion revealed that 20% isolates (PA-7, PA-10, PA-11, PA-18, and PA-23) were resistant to imipenem. Twelve percent isolates (PA-10, PA-11, and PA-13) were resistant and 8% isolates (PA-7, PA-18) were intermediate resistant to amikacin. From this study, it was revealed that all the isolates were multidrug resistant (MDR). The MDR phenotypic rate was high. The high level of antibiotic resistance among P. aeruginosa isolates may be due to self-prescription policy, comparatively cheaper antibiotics intake, lack of dependency on laboratory guidance, and inadequate doses of antibiotics intake. There is urgently needed to take a new antibiotic intake policy. If so, this may be helpful for the effective control of antibiotic resistance. Analysis of the MAR index of the isolated P. aeruginosa strains showed that 86.96% had MAR index of 0.4 and above. MAR index more than 0.2 indicates that the isolates were originating from other sources where antibiotics were often used., The MAR values can however be viewed as an indication of the extent of microbial exposure to antibiotics used within the community.
Study of resistant mechanism
All isolated P. aeruginosa strains were subjected to β-lactamase detection. The presence of a clear zone around bacterial growth was an indicative sign of β-lactamase production as described in the Materials and Methods section. Our results showed that 19 (76%) of the clinical P. aeruginosa isolates were β-lactamase producers. The evolution of MDR strains can be caused by an active efflux system that expels antibiotics from the cell. Results show the MICs of the antibiotic (ciprofloxacin) for 25 different MDR P. aeruginosa isolates in the presence and absence of the efflux inhibitor (CCCP). The addition of CCCP enhanced the activities of ciprofloxacin. This result emphasized the presence of an efflux-mediated resistance in the tested strains to ciprofloxacin.
We try to understand the main mechanisms of antibiotics resistance; we investigate β-lactamase production and efflux pump activity. P. aeruginosa was previously shown to use β-lactamase-mediated resistance to antibiotics., We observed high levels of β-lactamase production in P. aeruginosa isolates (76% in clinical isolates) in clinical samples. High levels of positive efflux pump activity were seen in our study. Efflux-mediated fluoroquinolone resistance of P. aeruginosa was reported previously.,, Another report also mentioned that overexpression of the efflux system was associated with antibiotics resistance in P. aeruginosa. Therefore, mechanisms of resistance used by P. aeruginosa isolates included β-lactamase production and the use of multiple drug resistance efflux pumps.
Prevalence of quorum-sensing-dependent virulence factors
In the present study, QS-dependent virulence factors (elastase, protease, pyocyanin, rhamnolipids and biofilm formation) were tested in the clinical isolates of P. aeruginosa (n = 25). Nearly 85% of the isolates were found to show the QS-dependent phenotypes. In this study, we found 80% isolates can form biofilm leads to more tolerant to antibiotics. Eighty-eight percent strains were produced elastase, where 100% strains were capable of releasing pyocyanin. Eighty-four percent and 72% strains were shows protease activity and rhamnolipid production respectively.
On the basis of the type of the infected tissue, lots of virulence factors play crucial roles in the pathogenesis of P. aeruginosa infections. In natural infections, the contribution of individual virulence factors to P. aeruginosa has been investigated in several studies. In the present study, QS-dependent virulence factors such as elastase, protease, pyocyanin, rhamnolipids, and biofilm formation were assayed in the clinical isolates of P. aeruginosa (n = 25). More than 85% isolates were found to show evidence of the QS-dependent phenotypes. Elastase, protease, rhamnolipid, and pyocyanin are important virulent factors in P. aeruginosa pathogenesis and these factors are totally regulated by QS. One of the virulence attributes of P. aeruginosa is its resistance to different antibiotics. Bacteria imbedded in biofilms are more tolerant to several antibiotics than their planktonic bacteria counterparts. In this study, we found that 89% isolates can form biofilm which causes tolerant to antibiotics. The QS-deficient biofilm was almost entirely eradicated in contrast with the wild-type biofilm, in which only cells in the top layer were killed. Hence, from our results and others, QS plays an important role in the pathogenicity in P. aeruginosa. Inhibition of the QS activity can decreased the secretion of virulent factors, and it will be a promising pathway to control bacterial infection.
Synthesis of silver nanoparticles
The appearance of a darkish-brown color in reaction mixture was the indication of Ag NPs formation. The intensity of color was increased as a function of time due to the reduction of Ag +. The reduction of silver ions was visibly evident from the color changes associated with it. During synthesis due to presence of biomass in the AgNO3 solution, the color of the solution was changed from colorless to darkish-brown. Here, biomasses play an important role in the synthesis of Ag NPs. It is known that color of the solutions was change due to excitation of surface plasmon vibrations with the Ag NPs.
Characterization of nanoparticles
Fourier transform infrared spectroscopy
The Fourier transform IR spectroscopy measurements of green-synthesized Ag NPs were carried out to identify the possible interaction between phytochemicals and proteins of the biomass with Ag NPs. Results showed sharp absorption peaks at about 1631 and 3435/cm [Figure 1]a. Absorption peak at 1631/cm and 3435/cm may be assigned to the amide bond of proteins arising due to the carbonyl stretch in proteins and OH stretching in alcohols and phenolic compounds, respectively. The absorption peak at 1631/cm was close to that reported for native proteins that means proteins were interacted with biosynthesized nanoparticles and also their secondary structure were not affected during reaction with Ag + ions or after binding with Ag NPs. Other bands were observed at 1588/cm due to the presence of aromatic ring and at 1452/cm, 1383/cm, 1349/cm due to skeletal vibration of the organic substances. These results prove that a carbonyl group of amino acid residue was strong binding ability with metal. It suggested that the formation of layer covering metal nanoparticles and acting as capping agent to prevent agglomeration and providing stability in the medium. This result confirms the presence of proteins acting as reducing and stabilizing agents.
|Figure 1: Characterization of synthesized silver nanoparticles. (a) Infrared spectroscopy (Fourier transform infrared spectroscopy). (b) The hydrodynamic size silver nanoparticles by dynamic light scattering. (c) Scanning electron microscopy. (d) Transmission electron microscopy of synthesized silver nanoparticles|
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Dynamic light scattering
DLS measures the hydrodynamic diameter of the particles which is much greater than the diameter obtained from SEM and TEM images. The average particle size and polydispersity index (PDI) of Ag NPs were evaluated by DLS technique. The DLS study shows that Ag NPs synthesized by green method had a Z average diameter of 35.69 nm with PDI of 0.569, suggesting that the nanoparticles were highly dispersive in aqueous medium [Figure 1]b.
The zeta potential of green-synthesized Ag NPs was −16.8 mV. Hence, Ag NPs showed good stability in water due to the electrostatic repulsion. The observed stability in combination with the measured value for the zeta potential hints the electrostatic mechanism due to adsorption of components of the biomass to the particles. These organic compounds present in biomass acts as spacers which prevent close contact between Ag NPs.
Scanning electron microscopy
The dry size, shape, and morphology of the Ag NPs were further characterized by electron microscopic analysis. Electron microscopic analysis revealed that the mean size of the Ag NPs was 16 ± 4 nm. The SEM morphology of individual Ag NPs showed that they had nearly triangular geometry with well-defined morphology [Figure 1]c. The TEM images support the steric stabilization mechanism.
Transmission electron micrography
TEM images of the Ag NPs were revealed that they were predominantly triangular in shape with maximum particles in size range with mean diameter of 14 ± 4 nm and were not in physical contact with each other. It was also observed that Ag NPs were evenly distributed in the sample [Figure 1]d.
Antibacterial activity of silver nanoparticles
Minimum inhibitory concentration and minimum bactericidal concentration determination
Antimicrobial activity of green-synthesized Ag NPs against Gram-negative P. aeruginosa at different concentrations showed that they revealed a strong dose-dependent antimicrobial activity. It was found that as the concentration of biosynthesized nanoparticles was increased, microbial growth decreases. The Gram-negative P. aeruginosa strain shows the MIC value of 16 μg/ml, whereas the MBC values were 32 μg/ml [Figure 2]a and b. It is well known that Gram-negative bacteria possess an outer membrane outside the peptidoglycan layer lacking in Gram-positive organisms. The important role of the outer membrane is to serve as a selective permeability barrier to protect bacteria from harmful agents such as detergents, drugs, toxins, and degradative enzymes and penetrating nutrients to sustain bacterial growth. These results suggest that growths were inhibited due to the penetration of Ag NPs into the bacterial cell that inhibits the bacterial growth and acts as a bactericidal agent followed by bacteriostatic activity.
|Figure 2: Antimicrobial activity of green synthesized silver nanoparticles. (a) Minimum inhibitory concentration of silver nanoparticles for multidrug-resistant Pseudomonas aeruginosa r PA-7. (b) Minimum bactericidal concentration of silver nanoparticles for multidrug-resistant Pseudomonas aeruginosa for PA-7. (c) Flow cytometric cell viability test of Pseudomonas aeruginosa strains against silver nanoparticles. (i) Control - Pseudomonas aeruginosa without silver nanoparticles treatment; (ii) treated - Pseudomonas aeruginosa with silver nanoparticles|
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The tolerance level of P. aeruginosa strain against Ag NPs was calculated from the respective MIC and MBC value. In P. aeruginosa strain, the tolerance level was 2. The MBC/MIC ratio is a parameter that reflects the bactericidal capacity of the analyzed compound. The bactericidal agents kill microbes, whereas bacteriostatic agents inhibit the bacterial growth. When MBC/MIC ratio is more than or equal to 16, then the antimicrobial agent is considered bacteriostatic whereas the ratio ≤4 indicates that the agent is considered bactericidal. In our study, Ag NPs exerted a bactericidal effect against P. aeruginosa because the MBC/MIC ratio values were 2.
Cell viability by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and by fluorescence-activated cell sorter
Ag NPs significantly decreased (P < 0.05) the cell viability of P. aeruginosa by 97.137% at 16 µg/ml. Flow cytometry and the MTT results were quite similar. Flow cytometric susceptibility test showed that Ag NPs slaughtered P. aeruginosa cells by 94.68% [Figure 2]c]. This type of results may be due to the uptake of Ag NPs into the bacterial cells that inhibits the bacterial growth and acts as a bactericidal agent, followed by bacteriostatic activity.
Intracellular uptake study
Nanoparticles binding to the plasma membrane and cellular uptake are probably a necessary condition for its exertion of activity. For assessment of the Ag NPs uptake by P. aeruginosa, fluorescence images showed that Ag NP-treated P. aeruginosa cells successfully uptake the nanoparticles [Figure 3]. Physical characteristics of nanoparticles such as size, shape, surface, and charge play a vital role in the uptake of nanomaterials. The uptake of nanoparticles is a two-step process: In first step, it binds to the cell membrane and second is the internalization step. The attachment of nanoparticles to cell membrane seems to be most affected by the surface charge of the particles. Variation of the particle surface charge could potentially control binding to the cell and direct NPs to cellular compartments. Bacterial cell membrane was highly electronegative in nature whereas Ag NPs were comparatively less negatively charge (nearly neutral charges). As the both bacterial cells had highly negative surface charge, the Ag NPs showed maximum uptake owing to have smaller size with nearly neutral surface charge; here, stability of the nanoparticles played important role in the uptake process. In the second step, the internalization of nanoparticles performed by endocytosis pathway, by which cells readily uptake nanomaterials and form a new intracellular vesicle around the substance to transport inside the cells.,
|Figure 3: Intracellular uptake of silver nanoparticles in multidrug resistant Pseudomonas aeruginosa strains. Intracellular uptake was examined using a fluorescence microscopy. Here, (a) Control cells: Pseudomonas aeruginosa cells without silver nanoparticles. (b) Pseudomonas aeruginosa treated with silver nanoparticles|
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Intracellular reactive oxygen species generation
The ROS generation has been shown to contribute to Ag NP-triggered cytotoxicity in bacteria. We found that the Ag NP-treated P. aeruginosa bacteria became DCF+, indicating that ROS were generated and participated in the Ag NP-mediated cell death [Figure 4]a. In contrast, after the same procedure without Ag NPs, no fluorescent cells were found, indicating no ROS generation. Flow cytometric analysis of ROS generation showed that Ag NPs enhance ROS generation in P. aeruginosa cells by 88.28% [Figure 4]b. From this result, it has been estimated that the antimicrobial activity of the Ag NPs involves the generation of intracellular ROS generation. Elevation of ROS levels are the main candidate mediators for the cell death. The production of ROS could be caused by the damage of plasma membrane.
|Figure 4: Intracellular reactive oxygen species generation of silver nanoparticles in multidrug-resistant Pseudomonas aeruginosa strains. (a) Fluorescence micrographic image. (b) Reactive oxygen species generation detected by flow cytometry. (i) Pseudomonas aeruginosa control. (ii) Pseudomonas aeruginosa treated|
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To find out whether ROS played a crucial role in Ag NPs persuaded bacterial cell death, P. aeruginosa cells were pretreated by 2 and 5 mM of a potent ROS inhibitor (NAC) for 4 h before Ag NPs exposure and cell viability was estimated by MTT method after 48 h of incubation. It was observed that pretreatment with 5 mM NAC effectively protected the cells from Ag NP-induced cytotoxicity. Cell viability of P. aeruginosa cells was reached to 81.56% cells compared with control group. It was found that pretreatment with NAC significantly protected the bacterial cells from Ag NP-induced toxicity. Restoration of cell viability >80% suggested that ROS is the main agent which played a crucial antibacterial potential caused by Ag NPs.
Action of silver nanoparticles on the structures of bacterial cells
The electron micrographs by SEM of P. aeruginosa cells treated and untreated with Ag NPs are displayed in [Figure 5]. Ag NPs present in the bacterial membrane as well as interior part of the bacteria was observed by electron microscopy. Electron microscopy determined the distribution and location of the Ag NPs, as well as the morphology of the bacteria after treatment with Ag NPs. Results showed that the bacterial cell surface of control group (untreated) was smooth, typical characters, intact, and some filaments around cells were obvious, while cells treated with Ag NPs were damaged severely. Some cells showed large leakage, others misshapen and fragmentary. Many pits and gaps appeared in the micrograph, and their membrane was fragmentary in the treated cells. The bacteria were almost disorganized to several parts and show many fragmentary bacteria. Ag NPs treatment results changes in its membrane morphology that produced a significant increase in its permeability affecting proper transport through the plasma membrane, leaving the bacterial cells incapable of properly regulating transport through the plasma membrane, resulting in cell death. From the previous study, it was observed that Ag NPs have penetrated inside the bacteria and have caused damage by interacting with phosphorus and sulfur containing compounds such as DNA, regulating enzymes. From this study, it has been suggest that the possible antibacterial mechanisms by which Ag NPs inhibit bacterial growth, as well as cellular responses to the Ag NPs treatment. Based on the present research, the mechanism of action of Ag NPs may be described as it make change the permeability of outer membrane first, resulting in the leakage of cellular materials, and Ag NPs enter the cells and produced ROS causing inhibiting bacterial growth. Simultaneously, Ag NPs may affect some cellular component to induce collapse of membrane, resulting in cell decomposition and death eventually.
|Figure 5: Action of silver nanoparticles on Pseudomonas aeruginosa cells observed by scanning electron microscopy (a) Pseudomonas aeruginosa control. (b) Pseudomonas aeruginosa treated with silver nanoparticles|
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In vivo antibacterial activity using mice infection model
Fifty microliters of P. aeruginosa suspension containing 5 × 106 CFU/ml elicited UTI after 5 days of its treatment as compared to PBS control group. During the entire study period, no mortality was observed in the treated group after 0.5 mg/kg B.W., 1 mg/kg B.W. and 2 mg/kg B.W. Ag NPs. In the case of intraperitoneal administration of Ag NPs in low dose, the B.W. was not changed significantly; however, in high dose, the B.W. was decreased. In infection control group, the B.W. was decreased slowly, and in normal control group mice, the B.W. increased slowly. The low dose 0.5 mg/kg B.W. Ag NPS showed the reduction percent reached 51.50%; moderate dose 1 mg/kg B.W. showed the reduction percent reached 68.75%; while the high dose 2 mg/kg B.W. Ag NPs showed reduction percent 98.50%. Hence, these results correlate between the inhibition percent of bacterial growth and the concentration of Ag NPs. MIC dose for P. aeruginosa inhibition and its ability to application onto animal were also studied. Results confirming the number of colony of the P. aeruginosa survival in kidney for the PBS control nearly zero because there is no P. aeruginosa administration while other infected mice get positive growth for the P. aeruginosa bacteria. P. aeruginosa infection control and untreated with any antimicrobial agent showed the highest rate of the bacterial growth nearly 360 × 106 CFU; however, on the other hand, the rates administrated with the same dose 50 μl of P. aeruginosa and treated with antimicrobial agent showed significant decrease in colony forming unit at the dose of 0.5 and 1 mg/kg BW, but high dose 2 mg/kg BW shows the number of colony was nearly zero, indicating clearance of infection by day 15 persistently infected of infection due to Ag NPs treatment. Inhibition of the P. aeruginosa in the presence of Ag NPs could have led to clearance of the organisms from the kidney tissue of infected mice.
On histopathological examination, the renal tissue of uninfected mice without Ag NPs treatment did not show any histopathological changes. P. aeruginosa-infected mice that did not receive the Ag NPs were lethargic and showed signs of histological damage in the kidney. The renal tissue of uninfected mice with Ag NPs treatment shows mild histopathological changes, suggesting mild effect of Ag NPs on renal tissue. Infected mice without Ag NPs treatment showed severe inflammation with infiltration of polymorphonuclear leukocytes and plasma cells in the renal pelvis [Figure 6] while infected mice treated with Ag NPs showed mild inflammation. In accordance with the bacteriological findings, the histopathological results showed significant differences between the treated and control groups. In the treated group, renal tissue destruction was reduced in comparison with the control groups. The N-acyl homoserine lactones play an important role in tissue inflammation.,, Reduced tissue inflammation in the treatment groups indicated that Ag NPs also play an anti-inflammatory role during UTIs and hence protects tissue from inflammation.
|Figure 6: Histopathological changes of the renal tissue (a) Renal tissue of normal uninfected mice. (b) Pseudomonas aeruginosa infection control. (c) Low-dose treatment group. (d) Moderate-dose treatment group. (e) High-dose group. All the sections were stained with hematoxylin and eosin. The images were taken at ×400 magnification|
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| Conclusion|| |
This study clearly shows that the Ag NPs have excellent antibacterial activity against P. aeruginosa both in vitro and in vivo. The study also suggests that the possible antibacterial mechanisms by which Ag NPs inhibit bacterial growth, as well as cellular responses to the Ag NPs treatment, make change the permeability of outer membrane, resulting in the leakage of cellular materials and Ag NPs enter the cells and produced ROS causing inhibiting bacterial growth. Simultaneously, Ag NPs may affect some cellular component to induce collapse of membrane, resulting in cell decomposition and death eventually. This study integrates bacteriology and nanotechnology, leading to possible breakthrough in the formulation of new kind of antibacterials. However, this study helps in the formulation of the biocidal agent in future on other Gram-positive and Gram-negative bacteria.
We express gratefulness to Vidyasagar University, Midnapore, and CRNN, University of Calcutta, for providing the facilities to execute these studies.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6]