Transmission electron microscopy (TEM)

The morphology and size of AGNPs were researched using TEM. This was achieved using JEOL, JEM-2100 electron microscope, Tokyo, Japan. The liquid sample was loaded into a carbon grid before being scanned by an electron microscope.

Determination of cytotoxicity of silver nanoparticles

The cytotoxicity of AGNPs was determined using MTT assay. A series of concentrations of AGNPs (170, 85, 42.5, 21.25, 10.625, 5.312, 2.65, 1.32, 0.664, 0.332 µg/ml) were prepared. Cytotoxicity was tested on mononuclear cells grown at varying densities (1-5×10⁶ cells per ml) in a clear 96-well plate. The tested concentrations of AGNPs were separately added to the wells before 72 h of incubation at 37 ^oC. The liquid was then carefully aspirated, and 50 µl of serum-free media and 50 µl of MTT reagent were added into each well. The plate was incubated at 37 ^oC for 3 h. The developed dye was solubilized using 150 µl of MTT solvent. This was achieved by continuous shaking on the orbital shaker for 15 min. The optical density (OD) was measured at 590 nm. The recorded OD was corrected by subtracting the culture medium background and the cytotoxicity was computed using the following equation:

Specimen collection and bacterial strains

Clinical samples (280) were collected from patients admitted to the ENT and surgery departments at Tanta University Hospitals. This involved culturing the samples on cetrimide agar plates; separated bacterial colonies were exposed to conventional identification steps, including Gram staining techniques and biochemical identification, which include indole test negative, KIA test red, and citrate test blue. These investigations confirmed that 107 out of the 280 (61.7%) specimens were P. aeruginosa. These isolates were preserved in nutrient broth containing 10% v/v glycerol at – 80 ^οC for further studies.

Antimicrobial susceptibility testing

Antimicrobial susceptibility of all the P. aeruginosa isolates was conducted using the Kirby Bauer disc diffusion method. P. aeruginosa (ATCC 27,829) was used as reference strain. This was done on Muller Hinton agar (MHA) according to the guidelines of the Clinical Laboratory Standards Institute (CLSI, 2018). The following antibiotics were tested gentamycin (10 µg), tobramycin (10 µg), amikacin (30 µg), imipenem (10 µg), meropenem (10 µg), doripenem (10 µg) ceftazidime (30 µg), cefepime (30 µg), ciprofloxacin (5 µg), levofloxacin (5 µg), piperacillin (100 µg), piperacillin tazobactam (10 µg), aztreonam (30 µg). After 24 h of incubation at 37 ^oC, the plates were examined and inhibition zone diameters around antibiotic disks were measured. The isolates were categorized as resistant (R), intermediate resistant (I) and sensitive (S) based on the recorded diameter of the inhibition zone according to CLSI, 2018 specifications. The results of this study were employed to compute the multiple antibiotic resistance (MAR) index according to the following equation: [21]

Determination of multi-drug resistance isolates was done according to Magiorakos et al., (2012) [22], who considered any isolate showing resistance to at least one agent in three or more classes of antimicrobials as multidrug-resistant isolate (MDR). Those showing susceptibility for all agents from one or two antimicrobial classes and recording resistance to at least one agent from other classes is classified as extensively drug resistance (XDR) profile. Pan drug resistance (PDR) is indicated for isolates resistant to all agents in all antimicrobial categories.

Determining the minimum inhibitory concentration (MIC)

The MIC was determined for gentamicin, ciprofloxacin, ceftazidime, and AGNPs against the 25 resistant Pseudomonas isolates. The MIC was also determined for combinations of AGNPs with each tested antibiotic. Determination of MIC employed broth microdilution technique based on the CLSI standard methodology. Different concentrations of AGNPs were prepared, starting from a stock solution containing 170 µg/ml using a serial two-fold dilution method to reach a concentration containing 0.664 µg/ml. A two-fold dilution was conducted for the tested antibiotic, starting with stock solutions containing 1024 µg/ml.

The bacterial suspension was prepared by colony dilution method. Briefly, 10 ml of MHB was inoculated with a loopful of test organisms. The turbidity was diluted to a concentration equivalent to 0.5 McFarland Standard using sterile saline before further dilution 1:100 with fresh (MHB) medium. Aliquots of the final dilution (100 µl) were loaded into the wells of 96 well plates followed by the addition of 100 µL samples of the prepared concentrations of AGNPs or the tested antibiotics. A well containing 100 µL of bacterial suspension in the absence of silver or antibiotics was employed as a positive control. A well containing sterile MHB was used as negative control. The plates were occluded with their lids and incubated at 37 ^oC for 24 h at the end, MIC was determined as the lowest concentration showing no visible growth [23].

The minimum bactericidal concentration (MBC) was determined in the case of AGNPs. This involved subculturing 10 µL samples from each well on sterile nutrient agar plates. These were incubated aerobically at 37 ^oC for 24 h. The MBC was recorded as the lowest concentration showing no colony on the agar plate.

The synergistic potential of AGNPs with antibiotics was monitored by determination of the MIC of each antibiotic in the presence of AGNPs which were included in each well at a concentration equivalent to 0.5 MIC of silver on the corresponding bacteria.

Calculation of fractional inhibitory concentration (FIC) index

For calculating the index of the fractional inhibitory concentration (FICI), MIC values obtained for a given combination were used to evaluate the effects of the combination between each antimicrobial agent and AGNPs according to the formulas defined by Davidson (1989) [24]:

The MIC of compound A in the presence of compound B is represented by the MIC A+B value, and vice versa for MIC B+A. Determination of the MIC for the individual components is required for calculating the FIC value for either substance A or B. The obtained FICI values were used to classify the nature of the interaction as: synergistic (FICI≤0.5), additive (0.5<FICI≤1, indifferent (1<FICI<2), and antagonistic (FICI≥2) [25].

Detection of biofilm formation and effect of AGNPS on established biofilm

The isolates were tested for biofilm formation using the crystal violet assay described by Christensen et al. (1985) [26]. A loopful of the test bacteria was incubated at 37 °C for 24 h in 10 mL of LB broth. The culture was adjusted to 0.5 McFarland, and 200 µL of this bacterial suspension was transferred to the wells of 96 wells with flat-bottom TCPs. A well-containing sterile medium served as a negative control. The plates were incubated aerobically for 48 h at 37 °C. After incubation, the content of each well was removed, and wells were washed three times with phosphate buffer saline (PBS) to remove planktonic cells. Adherent biofilm was fixed with 95% ethanol for 15 min, followed by staining with 100 µL of 1% crystal violet for 10 min. The unbound stain was removed, and the wells were washed with 200 µl of 33% glacial acetic acid, and the plate was air dried. The stained adherent biofilm’s optical densities (OD) were obtained with an ELISA reader at wave-length 570 nm. Each bacterial isolate was tested in triplicates, and the average OD values were calculated for each isolate and negative control. The cut-off value (OD_C) was established according to be equal to three standard deviations (SD) above the mean OD of the negative control: OD_C = average OD of negative control + (3×SD of negative control), and the tested isolates were categorized according to biofilm formation into: [27]

1. Non biofilm producer OD≤OD_C

2. Weak biofilm producer OD_C < OD≤2×OD_C

3. Moderate biofilm producer 2×OD_C < OD≤4×OD_C

4. Strong biofilm producer 4× OD_C < OD

The antibiofilm activity of AGNPs was then researched. Biofilm were allowed to form as described previously in the presence of 0.5 MIC of AGNPs [6]. The percentage of biofilm inhibition was computed using the following equation:

Effect of silver NPs on the motility of Pseudomonas isolates

The effect of AGNPs on motility was determined using plate assay. Motility assay was conducted by using motility plates containing glucose (1%), bactoagar (0.5%), bactopeptone (0.6%), and yeast extract (0.2%) in the absence and in the presence of 0.5 MIC of AGNPs. The plates were inoculated with a sterile toothpick and incubated at 37 °C for 24 h. Motility was assessed by measuring the zone formed by the colonies migration array from the point of inoculation. The diameter of the swarming zone was then measured in millimeters (mm) (Fig. 4) [28].

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Fig. 4

Pseudomonas motility in the absence (A) and presence (B) of AGNPs

Effect of silver NPs on total proteases and gelatinase production

This was investigated according to the well-established method described by (Gupta et al., 2011; Vijayaraghavan and Vincent, 2013) [29, 30]. Briefly, 0.5 mL of overnight cultures (adjusted to 0.5 MacFarland) were inoculated in 5 mL LB broth both in the presence and absence of 0.5 MIC of AGNPs. These were incubated at 37 ^oC for 18 h at the end of which the cultures were centrifuged for 15 min at 8500 g and then filtered using a 0.45 μm filter. The supernatant was used for testing the protease and gelatinase activities.

Protease activity was tested using skimmed milk agar. The prepared supernatant (100 µl) was added to the wells made in the skimmed milk agar plates. The plates were incubated overnight for 24 h at 37 ^oC. The clear zones around the wells were measured and were taken as a measure for protease activity.

Gelatinase activity was similarly monitored using (1.5% LB agar supplemented with 3% gelatin). The existence and diameter of the clear zone were taken as a reflection for gelatinase activity.

Effect of AGNPs on pyocyanin production by P. Aeruginosa

Pyocyanin production was quantified as described by Das and Manefield (2012) [31]. Shortly, Pseudomonas suspension obtained after overnight culture in LB broth was diluted to 0.5 McFarland. This bacterial suspension (10 µL) was used to inoculate 1 mL of LB broth in the absence and presence of sub-MIC (0.5 MIC) concentration of AGNPs followed by 48 h incubation at 37 ^oC. Bacterial cells were separated by centrifugation at 10,000 rpm for 10 min to remove cells. The supernatant was used to quantify of pyocyanin by measuring the absorbance at 691 nm by spectrofluorometer [32].

Real-time PCR

The RT-PCR was performed in the presence of 0.5 MICs of AGNPs. Total RNA was extracted using Purelink^® RNA Mini Kit (Thermo SCIENTIFIC, USA) according to the manufacturer’s instructions. A single colony of each tested isolate was grown in LB broth adjusted to 0.5 MacFarland and incubated at 37 ^oC for 18 h in the presence and absence of 0.5 MIC of AGNPs. Reverse transcription followed by qRT-PCR of QS-regulatory genes lasR, rhlR, and pqsR was carried out using QuantiTects Reverse Transcription Kit (Qiagen, USA). Thermo Scientific Maxima SYBR Green/Fluoresce in qPCR Master Mix (2X) is a ready-to-use solution optimized for quantitative real-time PCR and two-step real-time RT-PCR. It contains SYBR^® Green I dye and is supplemented with fluorescein passive reference dye. Only templates and primers need to be added. The SYBR Green I intercalating dye allows DNA detection and analysis without sequence-specific probes. dUTP is included in the mix for optional carryover contamination control using uracil-DNA glycosylase (UDG). Each sample was subjected to real-time PCR in duplicate, and the mean values of the duplicates were used for subsequent analysis. Using 2^−ct [33] relative expression of the target gene was estimated as follows:

Control group was applied as calibrator. On the other hand, other dietaries groups were represented as tested groups for both target and reference genes.

Threshold cycler numbers (Ct) of target gene were normalized to reference genes, for tested and control groups according to following equations:

Ct of tested genes were normalized to the Ct of the calibrator as follows:

Relative gene expression fold change was estimated as follows:

Fold changes = (2^−ct).

Antimicrobial susceptibility testing

All P. aeruginosa isolates (107) were subjected to susceptibility testing against different antimicrobials using the agar dilution. The results were interpreted based on the inhibition zone diameter into resistant (R), intermediate (I) or sensitive (s) isolates according to the clinical breakpoints provided by the Clinical Laboratory Standards Institute CLSI (2018). The results of this interpretation are shown in Fig. 3. The incidence of resistance depended on the type of antibacterial agent tested, with the incidence ranging from 0.93 to 43% of the tested isolates. Ciprofloxacin exhibited the most significant resistance among the tested agents, with 43% of the bacterial isolates categorized as resistant. Levofloxacin was ranked second, with 37.38% of isolates being resistant to it. Ceftazidime was the third in this category, with the incidence of resistance being 35.51%. Imipenem showed the most minor incidence of resistance, with only 0.93% of the isolates showing resistance.

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Fig. 3

Incidence of resistance of P. aeruginosa isolates to different antimicrobials Meropenem 10 µg (MRP), Imipenem 10 µg (IPM), Doripenem 10 µg (DOR), Aztreonam 30 µg (AT), Cefepime 30 µg (CPM), Ceftazidime 30 µg (CAZ), Gentamycin 10 µg (GN), Amikacin 30 µg (AK), Tobramycin 10 µg (TOB), Piperacillin tazobactam 10 µg (PIT), Piperacillin 100 µg (PRL), Ciprofloxacin 5 µg (CIP) and Levofloxacin 5 µg (LEV)

Antimicrobial resistance patterns of the tested isolates

Resistance patterns and MAR indices of all tested isolates against the studied antimicrobial drugs are presented in Table 2. The results indicated the existence of heterogonous resistance patterns in 63 isolates out of the tested 107. The magnitude of resistance of bacterial isolates was further reflected from the calculated MAR index values, with high values indicating resistance to larger number of antimicrobial agents. The MAR indices ranged from 0.077 to 0.84, with 4 (6%) isolates having a MAR index of 0.84 (Table 2). Based on the recorded resistance pattern, the isolates were classified as MDR, XDR, and PDR. Fortunately, none of the tested isolates exhibited a PDR profile. However, out of 63 P. aeruginosa isolates, 12 (19%) and 13 (20.6%) showed MDR and XDR profiles (Table 2). Based on the resistance pattern of the MDR and XDR isolates, ciprofloxacin, ceftazidime, and gentamycin were selected for further investigations.

Minimum inhibitory concentration (MIC)

Table 3 shows the minimum inhibitory concentration (MIC) of AGNPs alone and in combination with gentamicin, ceftazidime, and ciprofloxacin against P. aeruginosa selected clinical isolates (25). It was found that the MIC values of AGNPs against tested isolates ranged from 2.65 to 21.25 µg/mL, and the MBC values ranged from 10.625 to 42.5 µg/mL (Table 3). The MIC values of the three tested antibiotics were determined in the absence and presence of 0.5 MIC of AGNPs against the bacterial isolates. In the case of gentamicin, MIC values decreased markedly in the presence of AGNPs, with a 4-9-fold reduction in the MIC being recorded. This reduction was manifested by recording MIC values of 1 µg/mL in 92% of the isolates. For ceftazidime, combination with AGNPs resulted in 1-9-fold reduction in the MIC values. The MIC values were reduced to reach as low as 1 µg/mL in 13 (52%) isolates. The combination of AGNPs with ciprofloxacin reduced its MIC by 3-10-fold, with the lowest MIC value (0.5 µg/mL) being recorded in 19 (76%) isolates (Table 3). The reduction in the MIC was further evaluated by calculation of the Fractional Inhibitory Concentration Index (FICI), which was employed to assess the existence of synergistic (FICI≤0.5), additive (0.5<FICI≤1), indifferent (1<FICI<2), or antagonistic (FICI≥2) as represented in Table 4. The results revealed that AGNPs highly synergized the action of aminoglycosides, cephems and fluoroquinolones against P. aeruginosa; specifically, gentamicin combined with 0.5 MIC of AGNPs showed synergism against approximately 96% of tested isolates of P. aeruginosa. The results also indicated that the combination of ciprofloxacin with 0.5 MIC of AGNPs had a synergistic mode of interaction against 84% of tested isolates. AGNPs provided a synergistic effect for ceftazidime in case of 76% of tested isolates (Table 4).

Table 3

Minimum inhibitory concentration (MIC) (µg/ml) of silver nanoparticles (SN), gentamicin (GN), Ceftazidime (CAZ), ciprofloxacin (CIP) and their combination

Isolate No	SN	GN	GN+SN	CAZ	CAZ+SN	CIP	CIP+SN
9	21.25(42.5)	32	1	512	1	32	0.5
15	21.25(42.5)	16	1	128	1	64	0.5
16	21.25(42.5)	512	1	256	1	64	0.5
17	21.25(42.5)	512	1	512	1	64	0.5
22	21.25(42.5)	512	1	128	4	64	0.5
25	21.25(42.5)	16	1	32	4	512	0.5
33	21.25(21.25)	16	1	128	1	256	0.5
34	21.25(42.5)	512	1	512	256	64	0.5
42	10.625(42.5)	512	1	32	16	64	0.5
49	2.656(10.625)	512	1	256	8	512	16
53	5.312(10.625)	16	1	256	128	512	1
55	5.312(21.25)	64	1	128	64	256	0.5
57	10.625(42.5)	256	1	512	256	512	0.5
64	10.625(10.625)	512	1	256	2	128	16
66	2.656(10.625)	512	1	256	1	512	64
68	5.312(10.625)	512	4	512	2	512	64
72	21.25(42.5)	512	2	128	1	256	0.5
73	21.25(42.5)	512	1	256	2	256	0.5
76	21.25(21.25)	512	1	128	2	512	0.5
79	21.25(21.25)	512	1	256	1	512	0.5
84	21.25(21.25)	512	1	256	1	32	0.5
85	10.625(10.625)	512	1	128	1	512	0.5
86	21.25(21.25)	512	1	256	1	128	0.5
94	5.312(21.25)	512	1	64	1	512	0.5
102	10.625(21.25)	64	1	64	1	64	1

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*Values between brackets are MBC of silver nanoparticles

Table 4

Fractional inhibitory concentration index (FICI) and the effect of combining silver nanoparticles (SN) with three antibiotics (gentamicin (GN), ceftazidime (CAZ) and ciprofloxacin (CIP))

Sample	FICI GN+SN	Effect	FICI CAZ+SN	Effect	FICI CIP+SN	Effect
9	0.078	Synergy	0.049	Synergy	0.038	Synergy
15	0.109	Synergy	0.054	Synergy	0.31	Synergy
16	0.049	Synergy	0.051	Synergy	0.31	Synergy
17	0.049	Synergy	0.049	Synergy	0.31	Synergy
22	0.049	Synergy	0.219	Synergy	0.31	Synergy
25	0.109	Synergy	0.313	Synergy	0.024	Synergy
33	0.109	Synergy	0.054	Synergy	0.025	Synergy
34	0.049	Synergy	12.5	Antagonism	0.030	Synergy
42	0.096	Synergy	2	Additive or indifference	0.054	Synergy
49	0.378	Synergy	3	Additive or indifference	6	Antagonism
53	0.25	Synergy	24.5	Antagonism	0.19	Synergy
55	0.20	Synergy	12.5	Antagonism	0.096	Synergy
57	0.098	Synergy	24.5	Antagonism	0.048	Synergy
64	0.096	Synergy	0.19	Synergy	1.625	Additive or indifference
66	0.378	Synergy	0.37	Synergy	24.125	Antagonism
68	0.760	Additive or indifference	0.37	Synergy	12.125	Antagonism
72	0.098	Synergy	0.054	Synergy	0.025	Synergy
73	0.049	Synergy	0.10	Synergy	0.025	Synergy
76	0.049	Synergy	0.11	Synergy	0.024	Synergy
79	0.049	Synergy	0.051	Synergy	0.024	Synergy
84	0.049	Synergy	0.051	Synergy	0.038	Synergy
85	0.096	Synergy	0.10	Synergy	0.048	Synergy
86	0.049	Synergy	0.051	Synergy	0.027	Synergy
94	0.190	Synergy	0.20	Synergy	0.095	Synergy
102	0.109	synergy	0.11	Synergy	0.11	Synergy

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Antibiofilm activity of silver nanoparticles

The tested isolates were screened for biofilm production using a crystal violet assay. The results of this screening are presented in Table 5. It was found that samples no. 25, 42, 64, and 85 (16%) are strong biofilm producers, isolates no. 9, 15, 16, 17, 22, 33, 34, 49, 57, 72, 79, 84, and 102 (52%) are moderate biofilm producers and isolates no. 53, 55 ,66, 68, 73, 76, 86, and 94 (32%) are weak biofilm producers. The results of 0.5 MIC of AGNPs against Pseudomonas biofilm are represented in Table 5. The percentage of biofilm inhibition ranged from 2 to 89% and it was found that AGNPs decreased biofilm formation by more than 50% in about 40% of the tested isolates. Statistical analysis studies revealed that the effect of AGNPs on Pseudomonas isolates biofilm is statistically significant (P<0.05) in most of the isolates except isolates no. 33 and 55 only (Table 5).

Table 5

Percentage of biofilm inhibition of AGNPs against P. Aeruginosa isolates

Sample	Biofilm forming ability	% inhibition
9	Moderate	17±1.026
15	Moderate	35.7±3.30
16	Moderate	24.25±2.10
17	Moderate	48±4.430
22	Moderate	53.37±10.5
25	Strong	58.68±18.9
33	Moderate	2±0.50
34	Moderate	41.6±6.38
42	Strong	42±10.97
49	Moderate	65±7.09
53	Weak	89±29.6
55	Weak	10±1.40
57	Moderate	32±5.63
64	Strong	62±12.45
66	Weak	74.2±2.968
68	Weak	54±2.4
72	Moderate	51.5±14.12
73	Weak	54.4±2.47
76	Weak	47±3.25
79	Moderate	36.8±3.80
84	Moderate	48.4±5.71
85	Strong	76.4±23.96
86	Weak	27.3±2.32
94	Weak	40.6±7.28
102	Moderate	26±2.32

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Effect of silver nanoparticles on the motility of pseudomonas isolates

The effect of AGNPs on the motility of Pseudomonas isolates was investigated by monitoring the swarming of the organism. The diameter of the swarming motility zone was assessed. This is exemplified in Fig. 4. The recorded zone diameters of the tested isolates are presented in Table 6 in the absence and presence of 0.5 MIC of AGNPs. In the absence of AGNPs, bacterial isolates were able to move within the agar plate with the swarming zone diameter ranging from 18 to 37 mm. In the presence of 0.5 MIC of AGNPs, the motility of the organism was reduced, with the swarming zone diameter values being in the range of 10 to 29 mm depending on the tested isolate. The diameter of the zone after treatment of the given isolate with AGNPs was correlated with the corresponding zone diameter in the absence of silver to compute the percentage inhibition of motility. This computation reflected a variable % reduction in motility, with the maximum reduction being 66.6% in isolates 49 and 66 (Table 6).

Effect of silver nanoparticles on the protease and gelatinase production

Protease and gelatinase assay were conducted in the absence and presence of 0.5 MIC of AGNPs. Proteolytic activity and gelatinase activity was detected on the skimmed milk agar plates and 1.5% LB agar supplemented with 3% gelatin, respectively. The proteolytic and gelatinase activities were shown as clear zones, as exemplified in Fig. 5. The clear zone diameter was used to estimate the effect of AGNPs, as presented in Table 7. The proteolytic activity data reflected the ability of 0.5 MIC concentration of AGNPs to reduce the ability of the tested isolates to secrete protease enzyme with the magnitude of reduction ranging between 5.88 and 52.38%. It should be noted that AGNPs failed to reduce the proteolytic activity in the case of one isolate out of the tested 25 isolates (Table 7). concerning gelatinase activity, a concentration equivalent to 0.5 MIC of AGNPs showed a significant reduction in gelatinase activity with a degree of reduction ranging from 13 to 100% (Table 7).

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Fig. 5

Examples of protease (left) and gelatinase (right) activities of Pseudomonas isolate in absence and presence of 0.5 MIC of AGNPs

Effect of silver nanoparticles on the pyocyanin production

The effect 0.5 MIC of AGNPs on the ability of P. aeruginosa to produce pyocyanin was investigated. The existence of pyocyanin is indicated by the green coloration of the solution with the color fading by inhibition of pyocyanin production (Fig. 6). The OD691 of P. aeruginosa pyocyanin after extraction in the absence and presence of 0.5 MIC of AGNPs is represented in Table 8 and was used for quantitative determination. The results showed that pyocyanin concentration decreased after adding AGNPs, with the magnitude of reduction ranging from 9 to 79%, depending on the tested isolate (Table 8).

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Fig. 6

Reduced pyocyanin color after treatment with 0.5 MIC of AGNPs

Not applicable.