Synergistic Effect of Biogenic Silver Nanoparticles and Antibiotics Against Multidrug-Resistant Pseudomonas aeruginosa

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INTRODUCTION
Infections from bacteria are one of the major public health concerns nowadays because of the increase in resistance of bacteria because they can survive under the influence of antimicrobial agents. Bacteria do this by reducing the permeability of cells, inactivation of antimicrobial agents by enzymes, production of flow pumps, and altering binding sites, which offer safety and stability to microorganisms (de Lacerda Coriolano et al., 2021).
These resistant bacterial strains are present all over the world. P. aeruginosa belongs to the class Gamma proteobacterium, is gram-negative, non-spore forming and aerobic. It is a rod-shaped organism responsible for causing infection in humans, plants, and animals (de Lacerda Coriolano et al., 2021). It is an opportunistic pathogen that produces indophenol oxidase enzyme, which means it is oxidase positive.
P. aeruginosa can also synthesize pyoverdine and pyocyanin. It can be found in soil, water, fresh vegetables, and fruits. It produces a sweet grape-like odor in the laboratory. P. aeruginosa gets energy by oxidizing sugar. It causes both communityacquired and nosocomial (like urinary tract infections, bacteremia, pneumonia, and ulcerative) infections. P. aeruginosa resists antiseptic solutions used for disinfecting surgical instruments (Salman et al., 2019). It causes hospital-acquired wound infections and bloodstream infections. It is also responsible for opportunistic infection in immunocompromised patients (Bayroodi & Jalal, 2016). The release of intracellular and extracellular virulence factors (Hydrogen cyanide, alkaline protease, elastase, phospholipases, and zinc metalloprotease) increase the pathogenesis of P. aeruginosa after colonization and results in high tissue damage due to toxA gene that encodes tissue damage enzyme and dissemination into the blood. Patients with persistent lung infections, pyocyanin may harm lung tissue (Haghi et al., 2018). P. aeruginosa now become able to overcome antibiotic treatment and has become resistant to various antibiotics. The misuse of antibiotics is a contributing factor, as is the organism's ability to thrive in new habitats and devise novel defensive mechanisms, such as biofilms (Hemmati et al., 2020;Hassani Sangani et al., 2015). In order to improve patient health, it is crucial to find an effective treatment for any barriers to treatment. P. aeruginosa has two primary antibiotic resistance types: intrinsic and acquired resistance (Fadwa et al., 2021). The pace of vaccine and antibiotic development has struggled to keep up with the emergence and spread of multidrug-resistant strains, highlighting the urgent need for more robust efforts in this area. That is why antibiotic resistance is a serious and major world problem. In the past few years, nanoparticles become popular and have been proven useful in various fields of diagnosis, medicine, and therapeutics. Nanoparticles have great antibacterial activity against all microorganisms, including bacteria and fungi. Nanotechnology in drug delivery systems consists of design, production, synthesis, characterization, device, and systems of nanoparticles with sizes ranging between 1-100 nm. Effective combinations of nanoparticles with antibiotics are among those techniques that are used nowadays to avoid bacterial growth (Abo-Shama et al., 2020;Sharma et al., 2016). Nanoparticles have garnered significant attention due to their unique properties and potential applications in various fields (Mba et al., 2021). Bose and Chatterjee (2016) conducted an environmentfriendly, cheap, and simple method to synthesize silver nanoparticles from the leaf extract of guava (Psidium guava) as a capping and reducing agent. This plant was used because of its medicinal properties and is available in every season. TEM and UV analysis verified the synthesis of AgNPs. Typically, nanoparticles were 40 nm in size. Disc diffusion, serial dilution turbidity assay, and agar cup assay were used to calculate the relative activity of AgNPs against the test organism. The findings show that guava leaf extract used in the green synthesis of AgNPs may reduce bacterial growth. The antibacterial activity of biosynthesized AgNPs against E. coli and P. aeruginosa was estimated by Ramalingam et al. (2016). The final findings show that AgNPs exhibit antibacterial action that is concentration-dependent and that these nanoparticles result in the complete eradication of both bacteria. By employing Atomic Force Microscopy (AFM), researchers have successfully visualized and characterized changes occurring at the nanoscale level, shedding light on the intricate modifications within cellular components and their mechanical behavior. Ramachandran and Sangeetha (2017) evaluate the activity of AgNPs against the biofilm production ability of MDR gram-negative bacteria. They studied the antibiofilm activity of AgNPs against five biofilms-producing MDR bacteria, P. aeruginosa, K. pneumoniae, E. coli, Acinetobacter baumannii, and Proteus mirabilis.
Jaiswal and Mishra (2018) studied formation of AgNPs from curcumin. These curcumin AgNPs were 25-35 nm in size, highly stable with low silver content, and had a bactericidal effect on gram-negative and gram-positive bacteria. In addition to their antibiofilm activity, silver nanoparticles (AgNPs) exhibited minimal cytotoxicity towards human cells, specifically keratinocytes. The assessment of bactericidal activity of AgNPs involved multiple methods, including determining the minimum inhibitory concentration (MIC), conducting post-agent effect studies, and performing time-kill assays. To further evaluate the antibacterial effect of curcumin-loaded AgNPs, scanning electron microscopy (SEM) and live-dead imaging techniques were employed. Cytotoxic results showed that AgNPs were cytotoxic at 156 mg/l, which is a very high concentration compared to its bacterial MIC value. The skin-keratinocytes test also revealed that AgNPs had antiinflammatory activity on human macrophages.
MATERIALS AND METHODS Study Design: The research study was carried out within the postgraduate research laboratories of the Department of Microbiology. Samples were obtained from patients having burns or surgical wounds from a tertiary care hospital. Sample Collection:A total collection of 120 samples was obtained using sterile swabs from patients' burn or surgical wounds at a tertiary care hospital (Omer et al., 2020). Samples were then transported to the postgraduate research lab in the Department of Microbiology.

Isolation
and Identification of Bacteria:Each sample was separately grown on selective media. Petri plates were incubated at 37ᵒC for 24 hours (Ijaz et al., 2019). After 24 hours, plates were taken, and Gram's staining was performed by picking isolated colonies from each plate. Bacteria were identified by observing morphological and cultural characteristics and biochemical tests (Omer et al., 2020). Preparation of Pseudomonas Cetrimide Agar: Cetrimide agar is commercially available in powder form that is used to culture Pseudomonas bacteria. It is selective media for the growth of P. aeruginosa. Composition:Pancreatic digest of gelatin (Peptone): 20.0g, Potassium sulfate:10.0g, Magnesium chloride: 1.4g, Cetyl-trimethylammonium bromide: 0.3g, Glycerin: 10ml, Agar: 13.6g and Distilled water: 1000ml.Final pH: 7.2 ± 0.2 at 25 ᵒC. . Smear was made by adding one drop of water and then mixing with a colony of bacteria from cetrimide agar. Smear was also fixed by heat on the slide. Heat fixed slide was then allowed to cool before stains were applied. Biochemical Tests: Biochemical tests (catalase , oxidase, urease, citrate utilization test, triple sugar iron test, methyl red, Voges Proskauer, and indole) were performed for all the 120 isolates to confirm bacterium (P. aeruginosa) (Quinn et al., 2002). Molecular Identification:All the positive isolates were further confirmed through Polymerase Chain Reaction (PCR) by amplification of bacterial DNA using OprL Gene-specific primer (Table 1).. DNA-Extraction: DNA is extracted from bacterial cells for use in molecular analysis. The lysis of bacterial cells is accomplished via chemical or physical means. Proteinase K and lysozyme are utilized for gram-negative bacterial lysis. In this study, DNA extraction was done by boiling method according to the protocol of (Ahmed & Dablool, 2017). Isolated DNA is then directly used for PCR. The broth culture of bacteria was transferred into Eppendorf tube and centrifuged for 10 minutes at 13000 rpm. The supernatant was discarded. The pallet was resuspended with normal saline or PBS buffer and centrifuged again at 13000 rpm for 10 minutes. The supernatant was discarded. The pallet was resuspended again in normal saline or PBS and boiled for about 10 min at 95 C. Immediately, Eppendorf tubes were transferred into an ice box and centrifuged for 10 minutes at 13000 rpm. The supernatant was collected and transferred to a fresh Eppendorf tube. Eppendorf tubes then stored at -20 C. Polymerase Chain Reaction: Table1: List of Primer Sequences.

Protocol:
The amplification was carried out in a total volume of 20 µl containing template DNA, forward/reverse primers, and Master Mix. The composition of the reaction reagent is described in Table 2. The conditions used in the thermo-cycler for PCR were as follows. In the first step, initial denaturation was carried out at 95ᵒC for 4-5 minutes. Then in the second step, denaturation was again accomplished at 95ᵒC for 45 seconds. In the third step, annealing was done at 57 C for 1 minute. In the fourth step, the extension of DNA was done at 72 C for 1 minute. Thirty cycles were repeated from the second to the fourth step and in the last step, the final polymerization of DNA was completed at 72 C for 5 minutes. After completing all steps, the reaction mixture was further analyzed by Agarose Gel (Table 3).

Agarose
Gel Electrophoresis: Gel electrophoresis facilitates the separation of DNA molecules based on their size. Under the influence of an electric current, negatively charged DNA migrates towards the positive electrode, allowing for distinct banding patterns to emerge. Genomic DNA was visualized to check the quality and purity by gel electrophoresis. Agarose gel was prepared to undergo electrophoresis (Lee et al., 2012). Preparation of Agarose Gel:Weigh 0.8 grams of agarose on the scale. Using a sterile measuring cylinder, 100ml of 0.5X TBE buffer was measured and deposited into the sterile beaker. After adding weighed agarose to 100ml of 0.5X TBE buffer, the beaker was sealed with aluminum foil. Microwave it for 1 to 2 minutes to dissolve agarose completely, but do not boil it. It was cooled to 50 degrees Celsius, and after 4-5 minutes, 6l of ethidium bromide was added. It was thoroughly combined to ensure that all ingredients were thoroughly mixed. The tray's rubber bands were affixed, and a 20-well comb was placed in its position. Agarose gel was steadily poured into a clean tray to prevent bubble formation. Allow the gel to solidify at an ambient temperature of 20 to 30 degrees Celsius. After solidification, rubber bands and a comb were removed with caution so as not to rupture the substance. The gel tray was deposited within the electrophoresis tank, and the gel was covered with 0.5X TBE buffer. Protocol of Sample Loading:In the initial well, a 6µl molecular weight ladder of P. aeruginosa was introduced. For each well, 1 µl of stain/dye was combined with 5 µl of DNA sample, using a micropipette for precise measurements. The dye-infused DNA samples were then meticulously loaded into their respective wells. The electrophoresis tank was securely covered, and the electrodes were appropriately connected, supplying a voltage of 120-135 for 45 minutes. To visualize the results, the spectrophotometer was utilized to observe the distinct bands obtained. Antimicrobial Susceptibility Testing (AST): Kirby Bauer disc diffusion technique using several antibiotic discs (meropenem 10 g, imipenem 10 g, colistin 10 g, Amikacin 30 g, ciprofloxacin 5 g, and ceftazidime 30 g) was used to test for antimicrobial resistance. Clinical and Laboratory Standards Institute (CLSI) 2021 criteria were used to quantify and interpret the zone of inhibition.

McFarland Standard Preparation:The
McFarland standard plays a crucial role in estimating bacterial density as it consists of a suspension containing either barium sulfate or latex particles. By comparing the turbidity of a test suspension with the standardized McFarland standards (Hudzicki, 2009), bacterial density can be accurately assessed. To establish a 0.5 McFarland standard, a solution was created by combining 0.05 ml of a 1.175% barium chloride solution with 9.95 ml of a 1% sulfuric acid solution. Following thorough mixing, the turbidity of the solution was carefully adjusted to an absorbance range of 0.08-0.1 OD by measuring the absorbance at 625nm (Table 4). This specific absorbance range ensured the accuracy of the McFarland standard for subsequent bacterial density estimations.

Bacterial Inoculum Preparation:
The inoculum was prepared using an 18-24 hours old culture of P. aeruginosa grown on cetrimide agar. To achieve a uniform bacterial suspension, 4-5 isolated bacterial colonies were carefully selected and placed into small test tubes containing normal saline. The mixture was vortexed for 5 minutes to ensure thorough dispersion of the bacteria ( Figure 1).
The turbidity of the bacterial suspension was then visually compared to the 0.5 McFarland standard. This assessment was done with the unaided eye against a blacklined background. If the bacterial suspension appeared more turbid than the standard, it was diluted by adding normal saline. Conversely, if the suspension appeared less turbid, 1-2 additional colonies of bacteria were added to enhance turbidity.
It's important to note that the prepared suspension was utilized within 30 minutes after its preparation to ensure accurate and reliable results. This time frame helped maintain the viability and consistency of the bacterial suspension for subsequent experimental procedures (Momenah et al., 2023). For disc diffusion assay, it has been the standard medium. Composition: Beef (dehydrated infusion from): 150g, Casein hydrolysate: 8.75g, starch: 1.5g, Agar 8.5g, Deionized water: 500ml.Final pH: 7.3 ± 0.1 at 25 ᵒC Testing of Antibiotic Resistance: Taken first were Muller Hinton agar plates. Create a lawn of the bacterial suspension with the use of a sterile swab, ensuring that the turbidity meets 0.5 McFarland criteria before disc displacement. The inoculum was swabbed onto the agar plate in a three-dimensional pattern to ensure uniform distribution. Within 15 minutes after inoculating the plates, place antibiotic discs on top of them. The plates should be inverted and incubated at 35-37 degrees Celsius for 16-18 hours after the discs have been added. Antibiotic discs' diameters inside their inhibition zones are measured using CLSI interpretation criteria. Placing the plate against a dark background and reflecting light enabled the results to be measured. A ruler was utilized to measure the diameter of each antimicrobial agent's zone. The results were analyzed in accordance with CLSI guidelines 2022.

Determination of Minimum Inhibitory Concentration (MIC) of Antibiotics:
The MIC value of the antibiotics (Colistin, Gentamicin, ciprofloxacin, Amikacin, ceftazidime, and cefepime) were determined using two folds broth microdilution method (Parvekar et al., 2020) in 96-well plates. A 1mg/ml stock of antibiotics (prepared in DMSO) was used to determine the MIC value against P. aeruginosa. Twenty-four hours old bacterial culture was used to prepare 0.5 McFarland. Next, on a sterile microtiter plate, 100 µl of MHB was placed into each well, and an equivalent volume of antibiotics stock solution was poured into well one and diluted by twofold serial dilution up to well 10. To ensure that each well had the same volume, 100 µl from well 10 was thrown away. Each well had 100 µl of bacterial inoculum put to it, except well 12, which served as a control. MHB and the positive-control bacterial inoculum are found in well 11. The plates were hermetically wrapped with parafilm to avoid evaporation, and then they were placed in a 37C incubator for 18 to 24 hours. After incubation, the optical density (OD) value was observed at 620 nm. Finally, 30 µl of Nitro-blue tetrazolium (NBT) redox dye was dispensed in each well and incubated at 37 ᵒC for 4 hours. Color change from Yellow to blue was observed to detect bacterial growth. Wells with blue color indicate growth, while wells with yellow color indicate no growth. Determination of Minimum Bacterial Concentration (MBC):The MBC was performed as described previously (Parvekar et al., 2020). Briefly, 50 l from each well exhibiting MIC and values above MIC were subcultured with the aid of a glass spreader on sterile petri plates containing MHA. Incubate these plates at 37 ᵒC for 24 to 48 hours and observe for bacterial growth. Plates with the lowest antibiotic concentration that showed no visible growth were determined as MBC. Evaluation of Antibacterial Properties of AgNPs: Preparation of 0.5 McFarland Standard:A 0.5 McFarland standard containing 1.5 108 CFU was created by combining 0.05 ml of a 1.175% barium chloride solution and 9.95 ml of a 1% sulfuric acid solution. After combining, the solution's turbidity was set between 0.08 and 0.1 O.D. by measuring absorbance at 625 nm (Table 5). Preparation of Bacterial Inoculum:The inoculum was prepared using an 18-24 hours old culture of P. aeruginosa grown on cetrimide agar. To ensure a uniform bacterial suspension, 4-5 isolated bacterial colonies were carefully selected and transferred into small test tubes containing normal saline. The mixture was then vortexed for 5 minutes to achieve thorough dispersion of the bacteria. The turbidity of the bacterial suspension was then compared to the 0.5 McFarland standard using the unaided eye against a black-lined background. If the bacterial suspension appeared more turbid than the standard, it was appropriately diluted by adding normal saline. Conversely, if the suspension showed less turbidity, 1-2 additional colonies of bacteria were added to increase the turbidity. It isimportant to note that the prepared suspension was used within 30 minutes after its preparation to maintain the viability and consistency of the bacterial suspension. This time frame ensured reliable and accurate results in subsequent experimental procedures.

Preparation of AgNPs Concentrations:
Three different concentrations of AgNPs, including 20 mg, 15 mg, 10 mg, and 5 mg, were weighted and mixed in 5 ml DMSO (dimethyl sulfoxide) to make 4 mg/ml, 3 mg/ml, 2 mg/ml, and 1 mg/ml solutions as shown in Figure 2. The solution of AgNPs was then sonicated for 2 hours to completely dissolve nanoparticles. These concentrations were then used for different antimicrobial tests. To perform this procedure, 24 hours old bacterial cultures were used to prepare an inoculum of 0.5 McFarland. A sterile cotton swab was used to spread 100 µl of bacterial culture on the surface of Muller Hinton agar, then left for 5 minutes to settle down. After swabbing, four wells of 5 mm diameter were punctured at appropriate positions with the help of a steel well borer. Each time a well was drilled, the good borer was heated to redhot temperatures and then permitted to cool. Then 100 µl of prepared concentrations of AgNPs were added in all wells except the control well. The negative control well was punctured in the middle of the plate and filled with DMSO only. All the plates were left at 4 ᵒC for 15-30 minutes to provide proper diffusion of AgNPs into media and then placed in the incubator for 24 hours at 37ᵒC. After incubation zone of inhibition was measured.

Preparation of Muller Hinton Broth (MHB):Muller
Hinton broth was used as a general-purpose medium for the growth of microorganisms. The media was prepared by dissolving 10.5 grams of MHB in 500 milliliters of distilled water. The media was then autoclaved at 121°C for 30 minutes to sterilize it. After autoclaving, allow the substance to cool and preserve it at 4 o C for future use (Table 6).

Determination of Minimum Inhibitory Concentration (MIC) of AgNPs:
The smallest amount of an antibiotic or other substance needed to stop the development of a particular strain of bacteria is known as its minimum inhibitory concentration (MIC). The antimicrobial activity of AgNPs was determined by measuring the MIC value using 2 folds broth microdilution method (Parvekar et al., 2020) in 96-well plates. A stock solution of 1mg/ml of AgNPs (prepared in DMSO) was used to determine the MIC value against P. aeruginosa. Twenty-four hours old bacterial culture was used to prepare 0.5 McFarland. Next, on a clean microtiter plate, 100 µl of MHB was put into each well, followed by 100 µl of AgNPs stock solution in well 1, serially diluted by 2 up to well 10. To ensure that each well had the same volume, 100 µl from well 10 was thrown away. Each well had 100 µl of bacterial inoculum put to it, with the exception of well 12, which served as a control. MHB and the positive-control bacterial inoculum are all that can be found in well 11. Parafilm was used to prevent evaporation while the plates were incubated at 37 C for 18 to 24 hours. An OD value of 620 nm was measured both before and after incubation.

Determination of Minimum Bacterial Concentration (MBC):
Minimum bacterial concentration (MBC) determines the lowest concentration of material that kills the bacteria. The MBC was performed by the method of (Parvekar et al., 2020). Pick up 50 µl from all wells that show MIC and above MIC value showing no visible growth and subculture on sterile petri plates containing MHA with the help of a glass spreader. Incubate these plates at 37 ᵒC for 24 to 48 hours and observe for bacterial growth. Plates that contained no visible growth were determined as MBC.

Checkerboard Assay To Estimate The Synergistic Effect of AgNPs and
Antibiotics:The combined effect of AgNPs and different antibiotics was determined by the calculation of fractional inhibitory concentration (FIC) in the two-dimensional checkerboard method (Fadwa et al., 2021). In the current research, we checked the activity of different antibiotics combined with Moringa oleifera synthesized silver nanoparticles against P. aeruginosa. The checkerboard method was used to perform FIC studies in a 96-well plate. A proper amount of Muller Hinton broth (MHB) was poured into all wells, then a two-fold serial dilution of antibiotics was poured into all the wells (in combination with AgNPs) from A to G (mentioned on 96 well plates). Similarly, a two-fold serial dilution of AgNPs, combined with antibiotics, was meticulously dispensed into all wells, numbered 1 to 11, on the 96well plates. Column 1 contains a two-fold serial dilution of antibiotics alone to determine the MIC of antibiotics, while row A contains a two-fold serial dilution of AgNPs alone to determine the MIC of AgNPs. Initial OD600 was determined just before completing the experiment, and then incubate the plates at 37C for 18-24 hours. The plates were parafilm to prevent evaporation. After 18-24 hours, the final OD600 was recorded again.

Gram Stain:
Smear was prepared on a clean glass slide by picking characteristics growth from cetrimide agar plates, and staining was performed using standard Gram's staining procedure as explained by (Boyanova, 2018). All the samples that showed growth on cetrimide agar were found pink with a rodshaped appearance at 100X under the microscope (Fig. 5).

Biochemical Characterization:
All the isolates were subjected to biochemical testing. The following biochemical tests were performed i.e., catalase, oxidase ( Figure 6), urease (Figure 7), citrate utilization test (Figure 8), triple sugar iron test, methyl red, Voges Proskauer, and indole. Results are demonstrated in Table 7.

Molecular Identification:
Microscopically and biochemically identified P. aeruginosa were confirmed at the genetic level as well. By using primers of gene OprL, P. aeruginosa was confirmed ( Fig.9). OprL is peptidoglycan-associated lipoprotein (PAL) of about 20kDa. By using ethidium bromide-stained agarose gel, the OprL amplicon was visualized.

Minimum Inhibitory Concentration (MIC) of antibiotics:
The minimum amount of antibiotic that inhibits the growth of microorganisms is called MIC. MIC of different antibiotics was evaluated in a 96-well plate; columns 11 and 12 act as a positive and negative control, and results were evaluated via optical density measured at 600nm (Fig. 13). The interpretation of each antibiotic's MIC was also performed (Table 9).

Minimum bactericidal concentration (MBC):
The minimum bactericidal concentration (MBC) represents the lowest concentration of antibiotics required to effectively eliminate bacteria. To determine the MBC, 50 µl of the content from each well of the microtitration plate was carefully spread onto autoclaved Muller Hinton Agar plates. These plates were then incubated for one day. Following the incubation period, the plates were thoroughly examined. Any plates showing no visible growth were identified as indicating the minimum bactericidal concentrations. This observation signifies the concentration at which the antibiotics successfully eradicated the bacteria, resulting in no detectable growth on the agar plates (Fig.  14).
The Minimum Bactericidal Concentration (MBC) values of antibiotics tested against Pseudomonas aeruginosa were also provided (Table 10). These values indicated the lowest concentration of each antibiotic that effectively kills the P. aeruginosa bacteria.

Antibacterial Activity of AgNPs: Agar Well Diffusion Method:
The antibacterial activity of biologically produced silver nanoparticles against multidrug-resistant P. aeruginosa was shown using the agar well diffusion technique. To estimate antibacterial activity, the zone of inhibition was measured. Different concentration of AgNPs, i.e., 1 mg/ml, 2 mg/ml, 3 mg/ml, and 4 mg/ml (prepared in DMSO), was used against MDR P. aeruginosa (Fig. 15). The Maximum zone of inhibition was measured at 62.5 µg/ml, while the minimum zone of inhibition was measured at 7.8 µg/ml (Table 11). No zone of inhibition was observed against DMSO. This indicates that the antibacterial activity of AgNPs is concentration dependent. As concentration increases zone of inhibition also increases.  Table 11: Zone of inhibition of AgNPs against P. aeruginosa by agar well diffusion method

Minimum Inhibitory Concentration:
The minimum inhibitory concentration that inhibits the growth of microorganisms is called MIC. MIC of AgNPs was evaluated in a microtitration (96 well) plate, and results were evaluated by visible eye or via optical density at 600nm. MIC of P. aeruginosa was found at 250 µg/ml of AgNPs. Results from the OD value are shown in Table 12. The results revealed a distinct pattern in the optical density readings of the wells. From well 4 onwards, an increase in optical density was observed, indicative of bacterial growth and turbidity. In contrast, wells 1 to 3 exhibited a decrease in optical density, suggesting that the presence of AgNPs effectively inhibited bacterial growth. Furthermore, a notable difference was observed within the first three wells. Well, 1 exhibited a higher optical density compared to wells 2 and 3. This discrepancy can be attributed to the higher concentration of AgNPs present in well 1, resulting in a stronger inhibitory effect on bacterial growth. These findings demonstrate the effectiveness of AgNPs in impeding bacterial growth, as evidenced by the decrease in optical density in wells 1 to 3. The subsequent increase in optical density from well 4 onwards indicates the absence of inhibitory effects, allowing for bacterial growth and turbidity to occur.

Minimum Bactericidal Concentration:
The minimum bactericidal concentration was the amount of AgNPs that kill bacteria. MBC was found by spreading 50µl of solution from MIC and below MIC well on MH agar plates. Plates that exhibited no visible growth after 24 hours of incubation were identified as representing the minimum bactericidal concentration (MBC). In our study, the results indicated that the Muller Hinton Agar (MHA) plate containing 250 µg/ml of the test substance effectively inhibited the growth of P. aeruginosa, as no visible growth was observed on this particular plate (Fig. 16).

Synergistic Effect of AgNPs with Drugs:
The checkerboard method was utilized to evaluate the combined effect of AgNPs and antibiotics. In this assay, only nanoparticles are serially diluted in column 1, while only antibiotics (ciprofloxacin, gentamicin, and colistin) are serially diluted in row 1 (Table 13-15). The rest of the wells have a serial dilution of both AgNPs and antibiotics in which antibiotic concentration decreases horizontally, and the dilution of nanoparticles decreases vertically. The MIC alone was used to calculate the FIC index to find the synergistic effect of synthesized AgNPs when combined with the drugs. The FIC index was calculated for P. aeruginosa, demonstrating a strong synergistic effect between antibiotics and AgNPs (Fig. 17). This synergy indicates that the combined application of these agents results in a highly effective inhibition of bacterial growth for P. aeruginosa.

DISCUSSION
One of the leading pathogens that can grow on any surface with very low growth requirements is P. aeruginosa. As an opportunistic pathogen it can grow in different types of wounds, ICU, and immunocompromised patients (Ijaz et al., 2019). Harsh or misuse of antibiotics administration leads to the occurrence of MDR P. aeruginosa, which is regarded as a red alert (Mohamed, 2019). It is a prevalent pathogen, accounting for a substantial proportion (38%) of such infections (Ijaz et al., 2019). More than 7 million people are annually affected by P. aeruginosa (Haghi et al., 2018). Biofilm producing ability of P. aeruginosa make its association more strong with chronicity (Farva & Bhutta, 2021). Out of a total of 120 clinical samples, P. aeruginosa was found in 46 of them (33.33%). Our study's findings were similar to those of Farva and Bhutta (2021), who reported a prevalence of 30%, and Ijaz et al. (2019), who reported a prevalence of 38%, but they were lower than those of Nasser et al. (2018), who reported a prevalence of 46%. Several classes of antibiotics have been described as being utilized as antipseudomonas medications in various research (Nasser et al., 2018;Bazghandi et al., 2020;Abdulhaq et al., 2020). We used the following antibiotics in this study: imipenem (10 g), meropenem (10 g), cefepime (30 g), ciprofloxacin (5 g), amikacin (30 g), ceftriaxone (30 g), ceftazidime (30 g), and gentamycin (10 g). The most often prescribed antibiotics for P. aeruginosa therapy were amikacin (30 mg), colistin (10 mg), and piperacillin (30 mg). The disc diffusion technique was used to determine the antibiotic susceptibility pattern of the aforementioned medications. Gentamycin (83%), meropenem (85%), and ceftriaxone (87%) showed the highest levels of resistance among P. aeruginosa isolates in the antimicrobial susceptibility pattern, followed by ciprofloxacin (65%), cefepime (70%), imipenem (76%), cefoxitin (65%), ampicillin (67%), amikacin (61%), colistin (33%), and piperacillin (59%). Overuse of antimicrobials may contribute to the alarming rise of antibiotic-resistant infections. Furthermore, mobile genetic elements play a crucial role in the acquisition and loss of resistance genes, which is a key factor in the emergence of MDR isolates. Results are consistent with those found by Nasser et al. (2018), who found that 65.2% of isolates were resistant to ciprofloxacin, 85.0% to Gentamycin, 76% to ceftazidime, and 71.0% to cefepime. The results are lower than the observation of Abdulhaq et al. (2020) with imipenem (90.38%), meropenem (92.3%), and ceftriaxone (94.3%).In our study, 70% of isolates showed resistance to one or more antimicrobial agents, and all were multi-drug resistant. As compared to other studies, MDR isolates were (16.5%) by Mirzaei et al. (2020), (38.46%) by Abdulhaq et al. (2020), (65.2%) by Nasser et al. (2018),(52%) by Bazghandi et al. (2020) and (100%) by Akinloye et al. (2021).To overcome these complications and antibiotic resistance, scientists have focused on nanomaterials to solve this challenge for their use in the medical industry for the last 30 years. Nanoparticles have an important role in different fields of biology and medicine (D'Lima et al., 2020). According to this data, the present study shows better performance than the study conducted by Abdullah et al. (2021), where they reported a mean zone of 8 mm against P. aeruginosa by agar well diffusion method. Another study by Ulagesan et al. (2021) determined the antibacterial activity of AgNPs against P. aeruginosa. They recorded a maximum zone of 24 mm at 50 mg/ml and a minimum zone of 12 mm at 4 mg/ml. Their results were consistent with the present study findings as they also reported a mean zone of 12 mm at 4 mg/ml concentration. In addition, Lefta et al. (2016) investigated the activity of AgNPs against P. aeruginosa using different concentrations (1,2,4,8 and 16) mg/ml. They recorded (7, 9, 11, 7, and 22) mm, which indicates an increase in the zone of inhibition with the increase of AgNPs concentration. As agar well diffusion method is a qualitative assay and do not tell the exact value of inhibition of microorganism. Thus, MIC and MBC were conducted because they are quantitative techniques and give exact values that inhibit and kill the microorganisms. Broth microdilution assay carried in 96 well plates was performed to calculate MIC and MBC. For MIC, 1 mg/ml concentration of AgNPs was used as starting concentration to make two-fold serial dilution, and MBC was determined by sub-culturing the solution from MIC well and below MIC wells on Muller Hinton agar plates. Results of MIC and MBC showed 250 µg and 500 µg for P. aeruginosa. MIC points were also seen visually, with bacterial growth showing turbidity, while wells with no visible growth showed no turbidity. Absorbance value (OD600) was also recorded, which indicates an increase in absorbance below 250 µg concentration. As compared to the study conducted by Ulagesan et al. (2021) recorded MIC and MBC of AgNPs 200 µg and 400 µg, respectively, for P. aeruginosa. The results of our study were less than the results recorded by Abdolhosseini et al. (2019). Danjuma and Abdullah (2021) recorded MIC of AgNPs 500 µg/ml against both gram-positive and gram-negative bacteria. The aim of the study was to check the synergistic effect of AgNPs with various antibiotics for different strains of P. aeruginosa isolated from surgical or burn wounds. P. aeruginosa strains that were used in our study showed resistance to multiple classes of drugs; however, these antibiotics may show synergistic effects when combined with nanoparticles. Results showed that drugs combined with silver nanoparticles have a good synergistic effect. The synergistic effect between AgNPs and various antibiotics was determined using the FIC (Fractional Inhibitory Concentration) index. The findings of the present study revealed notable synergism between AgNPs and colistin (mean FIC index: 0.49) as well as gentamicin (mean FIC index: 0.470). Additionally, a partial synergistic effect was observed with the combination of AgNPs and ciprofloxacin (mean FIC index: 0.70).
However, it is important to note that the results of the ciprofloxacin and AgNPs combination showed a slight antagonistic effect, indicating reduced effectiveness when used together, as opposed to the synergistic effects observed with the other antibiotic combinations.
These results provide valuable insights into the interactions between AgNPs and different antibiotics, highlighting the potential for enhanced efficacy and therapeutic benefits through synergistic combinations. The slight antagonistic effect observed in the ciprofloxacin and AgNPs combination warrants further investigation and consideration when developing treatment strategies (Mohamed, 2019). They recorded the FIC index of AgNPs with ciprofloxacin (0.5), which shows complete synergism. Likewise, another research conducted by Abdolhosseini et al. (2019) showed complete synergism of ciprofloxacin with AgNPs. The study conducted by Fadwa et al. (2021) showed that colistin showed a complete synergistic effect against P. aeruginosa, while ciprofloxacin showed partial synergism against clinical strains of P. aeruginosa. Esmaili and Hosseini Doust (2019) concluded in their research that AgNPs increased the antibacterial activity of gentamicin when both are used in combination. The best synergistic effect was observed when both were mixed equally at their MIC level. Colistin, as described (Dupuy et al., 2018), is a cationic polypeptide antibiotic that disrupts the bacterial outer membrane. The findings of the present research work showed that a combination of AgNPs and drugs (colistin, ciprofloxacin, and gentamicin) can be used as a strong antimicrobial agent against MDR P. aeruginosa (Tasleem et al., 2022). No resistance, even by a single isolate of P. aeruginosa, against AgNPs alone or in combinations of AgNPs with drugs was observed. Thus these nanoparticles could be used in combination with diverse antimicrobial agents for enhanced and significant antimicrobial effects against MDR bacteria. Moreover, to use these nanoparticles as a safe medicine, possibly for the treatment of human bacterial infection, there is a need to perform cytotoxic assays on numerous human cell lines (Jalal et al., 2023). CONCLUSION P. aeruginosa is among the most critical pathogens declared by WHO. Due to the increase in antibiotic resistance, now the world is moving toward using nanotechnology to overcome all these issues. It is shown by this research that infections caused by MDR P. aeruginosa can be overcome by using nanoparticles in combination with drugs. In the current study, a total of 120 samples were collected from surgical or burn wounds of patients 46 (38.3%) were positive, and 74 (61.7%) were negative for P. aeruginosa. Antimicrobial susceptibility test results showed that out of 46 positive isolates, 33 isolates of P. aeruginosa were multi-drug resistant (MDR). These isolates were further used in this study to check the antibacterial action of AgNPs individually or along three drugs. Results indicate that activity was higher when a higher concentration was used, as the highest zone at 4 mg/ml concentration was 12 mm. As the concentration decreased, activity was also decreased, such as at 1 mg/ml concentration zone size was only 2.5 mm. In addition, MIC and MBC of AgNPs were recorded at 250 µg/ml and 500 µg/ml. All tested isolates had MIC at 250 µg/ml and MBC at 500 µg/ml. 3 Isolates out of all 27 MDR P. aeruginosa isolates were processed via checkerboard assay to check the combined effect of AgNPs with 3 drugs (colistin, ciprofloxacin, and gentamicin). According to current study results, all 3 isolates showed synergistic and additive.