Introduction

Multidrug-resistant Pseudomonas aeruginosa (MDR-PA) has emerged as a life-threatening opportunistic pathogen globally in the last two decades particularly in Pakistan, where it has become a severe concern of the health sector becoming a leading cause of nosocomial infections, particularly in patients with postoperative surgical injuries, intensive care units, burn and trauma units, and in those with a pre-existing pulmonary disease such as cystic fibrosis1,2. Long-term hospitalization frequently leads to infections by Pseudomonas aeruginosa namely malignant external otitis, endocarditis, ophthalmitis, meningitis, septicemia, and pneumonia1,2. Multidrug-resistant Pseudomonas aeruginosa is by nature resistant to various classes of antimicrobial drugs because of the presence of efflux pumps plus allowing low permeability of the microbial membranes2. Centers for Disease Control and Prevention (CDC) reports that more than 32,600 clinical infections in the US are caused by Pseudomonas aeruginosa, causing 2700 deaths annually3. Metallo-β-lactamase (MBL) enzymes break down a wide variety of β-lactam drugs, counting carbapenems too4. According to the Ambler classification of β-lactamase enzymes, they are divided into four classes (A, B, C, D); MBLs belong to class B enzymes, including VIM, IMP, NDM-1, and GIM4. Multidrug-resistant Pseudomonas aeruginosa-producing Metallo-β-lactamases are causative for various diseases, posing a severe health issue, as resistance against multiple antibiotics is on the rise, especially in Asian countries, including India, Pakistan, and Bangladesh therefore widespread use of antibiotics should be discouraged5. Recently, various investigations have reported the emergence of multidrug-resistant bacterial pathogens originating from humans, cattle, birds, and fish increasing the necessity of discovering novel, potent and safe antimicrobials calling for vigorous routine antibiotic susceptibility testing along with the screening of the emerging MDR strains6,7. In recent times, the slower improvement of antimicrobial agents has worsened the situation increasing the need of searching for alternative treatment regimes as a substitute for antibiotics6,7. A pioneering study reported the presence of 42% blaVIM gene multidrug-resistant gram-negative rods8. Yet another study from Pakistan reported the presence of blaVIM in 12% of multidrug isolates of Pseudomonas aeruginosa9,10. Algammal et al. report that on the basis of antibiogram as well as molecular analysis of the resistance genes, a majority of tested isolates prove to be multi-drug resistant to six major classes of antimicrobials (penicillins, tetracyclines, aminoglycosides, sulfonamides, fluoroquinolones, and lincosamides)11. It has been proposed that with an increasing incidence of MDR Pseudomonas aeruginosa, newer treatment options such as nanoparticles and other natural products should be explored12. Metallic nanoparticles were studied to act as promising alternatives to routine antibiotics for combating and defeating common resistance in pathogens that include target site modification, promoted efflux of drugs via membranes, and enhanced expression of the efflux pumps along with inactivation of enzyme and reduced permeability of the membranes13,14. Silver nanoparticles (AgNPs) have been stated as potential agents that have efficacy as antibacterial agents and can help combat nosocomial infections15. AgNPs adsorb and penetrate the bacterial cell walls, ultimately leading to the destruction of bacterial cells through the formation of free radicals13,14,15. Additionally, silver nanoparticles can give rise to silver ions14. These silver ions can form bonds with crucial organelles and lead to their dysfunction14,15,16. According to WHO, the point prevalence of nosocomial infections ranges from 5.7% in Low-Income and 19.1% in Middle-Income Countries (LMICs) to 3.5–12% in developed nations. The last few years have witnessed an increased interest in the application of Silver Nanoparticles as therapeutic regimens due to low toxicity in the environment, an increased rate of surface capacity, and the ability to inhibit the formation of biofilm that is required for the evasion of pathogens17.

The current study is a front-runner report from Pakistan, reporting the simultaneous existence of Metallo-β-lactamase (MBL), extended-spectrum β-lactamase (ESBL), and AmpC drug resistance genes. The current study is also the first to report efficient effects of multiple antibiotic-AgNPs combinations against multidrug-resistant isolates of Pseudomonas aeruginosa from Punjab, Pakistan.

Methodology

Ethical approval statement

The study was undertaken following ethical approval from the Research Ethics Committee, IMBB The University of Lahore viz Ref # IMBB/UOL/20/138.

Ethical guidelines and consent to participate

The current study was carried out following appropriate guidelines and regulations followed by informed written consent from all participants.

Sampling procedures

In total, 255 isolates of Pseudomonas aeruginosa obtained from multiple clinical specimens from different departments of a tertiary care hospital were obtained including urine (27.8%), wound swabs (34.9%), sputum (13.7%), pus (7.05%), blood (11.7%), and tissue (4.7%) were processed further. The prevalence of Pseudomonas aeruginosa was 22.0% (255/1159). 145 isolates that tested positive for Pseudomonas aeruginosa were from females, while 110 were from males.

Bacterial isolation and identification

The clinical isolates were confirmed by culturing on Pseudomonas cetrimide agar. The morphological characteristics of colonies were used for the identification of the isolates. Identification of isolates for Pseudomonas aeruginosa was based on bacterial culture and staining characteristics. Isolates were characterized biochemically using catalase, urease, indole, citrate utilization, lactose, lysine decarboxylation, and glucose fermentation tests18. Identification of Pseudomonas aeruginosa was by growth on Pseudomonas cetrimide agar plus API20NE identification strips (bioMerieux, France). Identified strains were stored in 30% glycerol broth at − 70 °C. The largest number of isolates were obtained from patients falling in the age group of 40–49 years. Percentage of Pseudomonas aeruginosa isolated from departments counted to surgery (36.8%), orthopedics (13.3%), gynecology (7.0%), ICU (11.4%), medicine (25.9%), and ENT (5.5%) (p ≤ 0.001). Specimen-wise isolation was performed with wound swabs (34.9%), tissue (4.7%), blood (11.7%), sputum (13.7%), urine (27.8%), and pus (7.05%). Resistance to imipenem was exhibited by 135 isolates while resistance to ceftazidime was shown by 153 isolates. These isolates were further evaluated employing molecular methodologies.

Determination of antibiotic susceptibility

Antibiotic sensitivity was studied using the Kirby-Bauer technique to evaluate antibiotic sensitivity of Pseudomonas aeruginosa isolates19. Following the recommendations of CLSI 2019 (Clinical & Laboratory Standards Institute), sensitivity testing was conducted on Mueller–Hinton agar20. The antibiotic discs (bioMérieux, France) specific for studying gram-negative bacteria, including Aminoglycosides (amikacin/AMK 30 µg, gentamicin/GEN 10 µg), Carbapenems (imipenem/IMP 10 µg, meropenem/MEM 10 µg), Monobactams (aztreonam/AZT 10 µg), Penicillins (piperacillin/tazobactam/TZP 100 µg), Gyrase inhibitors (ciprofloxacin/CIP 5 µg), Cephalosporin (ceftazidime/CAZ 30 µg, cefoxitin/CFX 30 µg, cefoperazone/sulbactam/SCF 75–10 µg) were used. The potency of the antibiotic discs was studied using the American Type Culture Collection (ATCC) standard reference strains (P. aeruginosa ATCC27853). For studying antimicrobial susceptibility, a 0.5% McFarland turbidity standard was employed for standardizing the bacterial inoculum suspension21. The sensitivity test results were utilized to determine the multiple antibiotic resistance index (MAR) of Pseudomonas aeruginosa isolates. MAR helps estimate resistance trends against multiple antimicrobial drugs and indicates the emergence of novel resistant bacterial strains. MICs (µg/mL) of meropenem and imipenem plus cefoxitin against multidrug-resistant Pseudomonas aeruginosa were determined by E-strip (Thermo Fisher Scientific, UK). A summarization of MIC of used antibiotics has been tabulated (Table 1). In accordance with Magiorakos et al., the tested isolates would fall in the category of MDR if the isolates expressed resistance to at least one antimicrobial in three used or more antibiotics22.

Table 1 Summarization of E-test® interpretive criteria of MIC for Pseudomonas aeruginosa in accordance with CLSI 201923.
Full size table

Phenotypic detection of carbapenemase and metallo-β-lactamases

Carbapenemases were studied following the combined disc synergy test (CDST) and Modified Hodge test (MHT)4. Whereas, the production of Metallo-β-lactamases was observed by the combined disc synergy test (CDST) as per the guidelines of CLSI 2019. This test is based on using a disc of imipenem alone and a disc of IMP/EDTA disc (Oxoid, Inc. Canada) according to methodology4. A combination disc synergy test (CDST) using a solitary imipenem disc along with an IMP/EDTA disc (Oxoid, Inc. Canada) was performed following the suggested method by Wadekar et al.24. The modified Hodge test (MHT) was performed following the methods of Kumar et al.25. Criteria by CLSI 2019 were used to analyze the results. IMP/EDTA E-Strips and IMP alone were used for detecting MBLs as per the manufacturer’s instructions (Liofilchem®, Italy)26.

Metallo-β-lactamase: molecular characterization

Molecular assays

In accordance with the previously stated method, extraction of the template DNA was performed from isolates27. The PCR mixture included 200 µM dNTPs, 50 ng DNA templates, 0.5 U Taq Polymerase, 10 pM primers, 1.5 mM MgCl2, and giving an eventual volume of 25 µL. Products of PCR were evaluated and construed for 30 min at 70 V. A 1.5 w/v agarose gel plus 500 µg/100 mL ethidium bromide was used to analyze the products.

Detection of MBL and ESBL genes

Confirmation of Pseudomonas aeruginosa isolates was done by singleplex PCR. Primer sequences selected for detecting the blaIMP-1, blaTEM, blaSHV, blaOXA, and blaVIM genes have been reported in previous studies28. Pseudomonas aeruginosa isolates for the blaVIM plus blaIMP-1 genes were screened with the help of singleplex PCR with reported primers29,30. The existence of gene blaAmpC in Pseudomonas aeruginosa was evaluated through PCR amplification of 1063 bp (Table 2). The DNA amplicons were placed in a 1.5% agarose gel for 60 min at 120 V. A UV light trans-illuminator gel documentation system helped visualize the amplified products (Thermo Fisher Scientific, US). Conditions for PCR have been enlisted in Supplementary Table 1 (ST_1).

Table 2 Sequence of primers used for detecting MBLa, ESBLb-type variants and blaAmpC.
Full size table

Fungal biomass cultivation

AgNPs myogenesis was carried out employing Aspergillus flavus. Aerobic cultivation of fungal biomass was carried out using a liquid medium that contained malt extract in a concentration of 0.3 g/100 mL, glucose 1.0 g/100 mL, yeast extract 0.3 g/100 mL, and peptone 0.5 g/100 mL. Medium’s pH was adjusted initially to be 5.8. Growth of fungal culture was carried out at 28 °C at 150 rpm using an orbital shaker. Extraction of the fungal mass was done through filtration employing Whatman filter paper no 1. Later, this culture helped synthesize nanoparticles.

Assay for synthesizing nanoparticles

Almost 200 mL of fungal culture without mycelia that contained 0.1 M AgNO3 as precursor salt was collected in an Erlenmeyer flask having a volume of 500 mL. This flask was incubated on a shaker at 150 rpm in an ill-lit condition at 28 °C for a duration of 96 h. In accordance with the methods described by Bhainsa et al. both positive control i.e. culture filtrate with the exception of silver salt along with negative control, i.e. AgNO3 solution was run along with experimental flasks33.

Characterization of AgNPs by transmission electron microscopy and X-ray diffraction

Characterization of myco-genized AgNPs was done by visual observations, Ultraviolet Visible (UV-Vis) Spectrophotometry, XRD, TEM, SEM and DLS. During the assay, 1 mL of sample volume was obtained from the reaction mixture at time intervals of 0, 2, 4, 6, 24, 48, 72 and finally 96 h. Absorbance of sample was recorded at wavelengths 200–800 nm employing a UV–visible spectrophotometer (Agilent 8453 UV–Vis, Agilent Technologies, USA). Colloid suspension from silver containing reaction mixture was eventually concentrated by centrifugation for 20 min at 12,000 rpm (centrifuge Model H-251, Kokusan Co, Ltd, Tokyo, Japan). AgNPs were obtained by washing the silver powder thrice with sterile deionized water and pure ethanol. Thereafter, nanoparticles had to pass the process of microcentrifugation (Microfuge® 18 Centrifuge, Beckman Coulter, USA). Eventually, the removal of the supernatant was performed followed by overnight drying in the oven. AgNPs were finally prepared in powdered form. Nanoparticle comprising dried sample drop-coated films on silica were eventually exposed to analysis by X-ray diffraction (XRD) working in transmission mode at 20 mA, 30 kV with Cu Kα radiation (X’pert PRO XRD, PANalytical BV, The Netherlands). The formation of AgNPs film took place on carbon-coated copper transmission electron microscopy (TEM) grids that were examined by TEM at an 80 kV accelerating voltage (JEM-1010, JEOL Ltd, Tokyo, Japan). Characterization of silver nanoparticles by UV–Vis Spectra, XRD analysis, Size distribution profile of AgNPs, and TEM Micrograph analysis has been discussed in detail by Naqvi et al.30,34.

AgNPs antibacterial activity against multidrug-resistant Pseudomonas aeruginosa.

AgNPs were prepared and procured from CRIMM (Centre for Research in Molecular Medicine) at the University of Lahore. To evaluate the antibacterial action of AgNPs, the "disc diffusion method" was employed35. Individual and combined antibacterial activities of common antibiotics plus AgNPs were explored against multidrug-resistant isolates of Pseudomonas aeruginosa by the Kirby–Bauer disk-diffusion method13,14.

Preparation of bacterial suspension and silver nanoparticles

LB broth (Oxoid, UK) was used to prepare a 0.5 MacFarland suspension of MBL-producing isolates of Pseudomonas aeruginosa. A uniform suspension of AgNPs concentrated at 1000 μg/10 mL (stock solution equal to 100 μg/mL) was prepared by dissolving AgNP powder (1 mg) in 10 mL normal saline36,37

Assays for antibacterial activity

The method of disc diffusion was employed for the analysis of the antibacterial efficacy of AgNPs against multiple drug-resistant strains of Pseudomonas aeruginosa that were identified from obtained specimens. MIC and MBC values for AgNPs were also evaluated38. Nanoparticles of silver nitrate derived from Aspergillus flavus were prepared and obtained by the microbiology lab (T-3 and 4), The University of Lahore. Measured weight One milligram of silver nanoparticles was mixed with 10 mL of normal saline for 15 min using a sonicator, and the prepared solution was 100 ppm (1000 µg/10 mL = 100 ppm). The prepared silver NP solution was dropped in amounts of 10, 20, 30, 40, and 50 µL with the help of a pipette on homemade 6 mm discs prepared from plain blotting paper and air-dried for a few seconds. Methodology of disc diffusion method was employed to assess the antibacterial action of AgNPs13,39. Pseudomonas aeruginosa was streaked on Mueller-Hinton agar for assessment of the antibacterial activity. The Petri plates were incubated at 37 °C for 24 h. Fresh cultures were then used to make the suspension. To maintain the turbidity of the bacterial culture, 0.5% McFarland solution was employed for comparison. One millilitre of this suspension (inoculum) was then added and spread on Mueller-Hinton Agar medium plates. Next, nanoparticles containing air-dried sterile filter paper discs were positioned at suitable distances. Following labelling, the plates were kept at 37 °C for 24 h.

MIC and MBC of AgNPs (µg/mL)

A volume of 200 µL of 0.5 McFarland bacterial suspension was prepared and added to LB broth from the 1st to the 10th well. Serial dilutions from stock solutions of AgNPs (1 mg/10 mL) were added to 96-well round-bottom microtiter plates (Thermo Fischer Scientific, UK) to finalize a 2 µg/mL concentration. Petri plates were incubated overnight at 37 °C following which the plates were observed to determine the absence of growth comparing each well with positive and negative controls40. MBC (minimum bactericidal concentration) measures the first dilution showing no growth on the agar. Wells showing no visible growth in the microtiter plate were further inoculated on nutrient agar (Oxoid, UK). A schematic illustration is shown (Fig. 1).

Figure 1
figure 1

Biosynthesis of AgNPs from Aspergillus flavus having antibacterial activity. (4a) Culture filtrate of A. flavus with silver nitrate solution (0.1 M) at 0 h. (4b) Culture filtrate of A. flavus with silver nitrate solution (0.1 M) at 96 h. (4cL) Size distribution profile of AgNPs synthesized by cultural filtrates of A. flavus {100 particles were analyzed}. (4cR) TEM micrographs of AgNPs produced by cultural filtrate of A. flavus. (4d) UV–Vis spectra recorded for the biosynthesis of AgNPs in response to 0.1 M. silver nitrate inoculated media of A. flavus. (4e) XRD patterns of AgNPs biosynthesized by cultural filtrates of A. flavus. (4f) Zones of inhibition with antibiotics alone and in combination with AgNPs*.Antibiotics in combination with AgNPs (A and B) have a wider zone of inhibition as compared to antibiotic alone showing thereby that AgNPs conjugated to antibiotics have a stronger antibacterial potency. (4 g) In vitro activity of AgNPs* at varying volumes (10 µL, 20 µL, 30 µL, 40 µL, 50 µL). AgNPs silver nanoparticles.

Full size image

Statistical analysis

Statistical analysis was performed for evaluating the percentage of resistance in relation to the ward and type of sample. Association between the type of sample [urine, sputum, wound, blood, pus, and tissue] and antibiotic resistance gene were evaluated by Chi-Square test. A p-value ≤ 0.05 was considered statistically significant. As per Pearson-correlation coefficient (r), the association between the phenotypic and genotypic variables was determined as follows: weak correlation: |r|< 0.3, moderate correlation: 0.3 <|r|< 0.5, strong correlation: 0.5 <|r|< 0.8541. Statistical analyses were carried out using SPSS software version 20 (IBM Corp., Armonk, NY, USA).

Results

Phenotypic characteristics of the recovered Pseudomonas aeruginosa isolates

Pseudomonas aeruginosa counts as a heterotrophic and motile, Gram-negative bacterium that is rod-shaped measuring 1–5 µm and 0.5–1.0 µm. It has been documented as a facultative aerobe that has the ability to grow through both aerobic and anaerobic respiration with nitrate studied to be the final electron acceptor. In accordance with Magiorakos et al., the tested isolates would fall in the category of MDR since the isolates were resistant to a minimum of one antimicrobial drug in three or exceeding antibiotic categories31. Pseudomonas aeruginosa has the ability to produce elastase and protease enzymes as well as hemolysins.

Antibiotic susceptibility testing

Sensitivity testing was accomplished on MH agar (Oxoid) as per recommendations of CLSI 2019 (Clinical and Laboratory Standards Institute 2019)20. This has been illustrated in Fig. 2. The pattern of antimicrobial resistance in Pseudomonas aeruginosa in association with departments has been tabulated in Table 3.

Figure 2
figure 2

Percentage antibiotic resistance and sensitivity Pseudomonas aeruginosa isolated from patients in Punjab, Pakistan (n = 255).

Full size image
Table 3 Pattern of antimicrobial resistance in Pseudomonas aeruginosa in association with departments.
Full size table

Phenotype detection of carbapenemase activity

Of 255 total isolates, 61.5% were carbapenemase producers, including 32 samples from urine (45%), wound swabs n = 25 (28%), sputum n = 28 (80%), blood n = 21 (70%), tissue n = 5 (41%) and pus n = 4 (22%). Phenotypic detection of Metallo-β-lactamases was observed by CDST as per guidelines of CLSI 2019 (Fig. 3). Antibiotic susceptibility of MBL producers is illustrated in Fig. 4. Prevalence of MBL and ESBL producers in correlation with the department is shown in Table 4.

Figure 3
figure 3

CDSTa for detection of MBL producers of Pseudomonas aeruginosa isolated from Punjab, Pakistan. This test shows using a disc of IMPb alone and IMP/EDTA disc as per methodology of Wadekar et al.24. A wider zone of inhibition is measured with IMP/EDTA disc as compared to IMP disc alone. This identifies metallo β-lactamase producers. aCDST Combined disc synergy disc; bImipenem.

Full size image
Figure 4
figure 4

Antibiotic susceptibility testing of MBL producing Pseudomonas aeruginosa (n = 83). MBL metallo β-lactamase.

Full size image
Table 4 Prevalence of MBL- and ESBL-producing Pseudomonas aeruginosa in correlation with department.
Full size table

Multiplex PCR for AMR genes (bla OXA, bla IMP, bla TEM, bla SHV, bla VIM)

Multiplex PCR detected the existence of resistance genes in 52.5% (n = 80) of the ESBL producers. Expression of blaTEM was of the order 43% of ESBL producers (n = 34) whereby blaSHV was detected in 32% of isolates. Likewise, the expression of blaOXA was 21%. blaVIM along with blaIMP-1 co-existed in 11.5% of MBL producers. 57.5% ESBL-positive strains exhibited simultaneous existence of blaTEM, blaSHV plus blaOXA, blaTEM was concomitantly expressed with blaOXA types in 19.5% of ESBL producers whereas blaTEM simultaneously showed presence with blaSHV in 22.5% isolates. Co-existence of blaOXA plus blaSHV was found in 9.5% of isolates while blaSHV, blaTEM, as well as blaOXA subsisted in 7.5% of ESBL-producers. Expression of blaAmpC was positive in 15% of isolates that were cefoxitin resistant. The correlation between phenotypic and genotypic MDR has been tabulated in Table 5. The co-expression of various genes is depicted in Fig. 5.

Table 5 Correlation matrix of phenotypic and genotypic MDR isolates of Pseudomonas aeruginosa by Pearson correlation co-efficient.
Full size table
Figure 5
figure 5

Co-expression of gene variants (MBL and ESBL) in isolates of Pseudomonas aeruginosa.

Full size image

MIC of MBL producing Pseudomonas aeruginosa

The MIC of imipenem against 10 (13%) blaIMP-producing Pseudomonas aeruginosa isolates inhibited five isolates at 16 μg/mL and five other isolates at 8 μg/mL. The MIC of imipenem for 12 (15%) blaVIM-producing Pseudomonas aeruginosa was 16 μg/mL, for three isolates was 8 μg/mL, and for the remaining three isolates measured 32 μg/mL. MIC of Imipenem for 17 (21%) blaOXA producers was 16 μg/mL for 12 isolates plus 8 μg/mL for five isolates.

Characteristics of AgNPs

Visual observations

Changes in the color of the reaction mixture were observed and images were recorded as the initial and ending stages of the experiment (Figs. 1–4a,b).

UV-spectroscopy

At the time of incubation, the reaction mixture’s UV–visible spectroscopy recorded spectra of increased intensity having a range of 350–600 nm; crucial peaks occurred at approximately 400–470 nm (Fig. 6). Moderately increasing peak absorbance accompanied with time was most notably connected with a change in the reaction mixture’s color exhibiting AgNPs synthesis positively (Fig. 6A). Particle size histogram has been depicted (Fig. 6B).

Figure 6
figure 6

Characterization of AgNPs biosynthesized using Aspergillus flavus. (A) UV–Vis spectra recorded for the biosynthesis of AgNPs in response to 0.1 M silver nitrate inoculated media of A. flavus. (B) Size distribution profile of AgNPs synthesized prepared by cultural filtrates of A. flavus {A total number of 100 particles were analyzed}. (C) XRD patterns of AgNPs biosynthesized from cultural filtrates of A. flavus. (D) TEM micrographs of AgNPs produced by cultural filtrates of A. flavus.

Full size image

XRD analysis

The crystalline nature of the AgNPs was depicted using the Debye–Scherrer analysis along with XRD of the dried sample’s drop-coated film. XRD analysis revealed four vital peaks in the total spectrum of 2θ value that extended in the range of 20°–80°. The crystallites averaged in size from 13 to 26 nm. Debye–Scherrer formulae were used to analyze the silver nanocrystallites ranging from 21 nm from total breadth at peak’s half maximum. The Debye–Scherrer equation is derived from Bragg’s law that determines the diameter of crystal samples according to the formula given below;

$${\text{D }} = k.\lambda ,$$
$$\beta \cos \theta ,$$

where D is the mean diameter, λ is the wavelength, k is the shape factor (0.9), θ is the Bragg angle for studied diffraction, β is the the full width at half maximum. XRD diractograms demonstrated four vital peaks around 2θ angles at 38, 44, 64, and 77. Crystallites estimated via XRD technique revealed size in the nanometer range (Fig. 6C).

TEM of AgNPs

Bright-field image mode was employed for the analysis of samples. Ultra-sonication produced pure ethanol based dilute suspensions of AgNPs. The suspension was spread drop-wise on 300-mesh lacy copper grids that were coated with carbon followed by drying for scanning under JEOL-1010 TEM. Accelerating high voltage was kept at 80 kV. Required adjustments and alignments were done following the selection of apertures and sample images were scanned on screen. Micrographs were recorded at definite magnifications on the photographic plates by focusing on the sample grids at the correct places. Standard developing procedures were adopted for processing the exposed photographic plates. These were scanned (flatbed high-resolution scanner) to finally achieve the image positives. TEM micrographs showed nanoparticles of different shapes where predominantly the shape was spherical. AgNPs acquired a size range of 5–30 nm30,34. The majority of the AgNPs were spread out in the micrographs with only a few places revealing larger aggregates of differing sizes (Fig. 6D).

Scanning electron microscopy (SEM)

Scanning electron micrographs were acquired with the JEOL 5600 following filtration of the samples through Millipore filters of 0.2 µm pore size to remove any contaminants that could possibly interfere with the SEM images. Samples for analysis were prepared by overnight fixation with 2.5% glutaraldehyde at room temperature. Subsequently, dehydration of the sample was carried out with gradient alcohol (10–95%) followed by incubation for 20 min in every gradient and soaked in absolute alcohol for about 2–5 min. Approximately 25 µL of the sample was pipetted out and loaded onto a ‘stub’ supplied for SEM analysis. The stub is approximately 1 cm in diameter has a cylindrical shape and is made of copper. Scanning of SEM was performed at the accelerating voltage of 25 kV from a distance of 5 mm to 5 cm (Fig. 7D).

Figure 7
figure 7

Myco-genized AgNPs (A) Zeta potential measurements for mycogenized AgNPs. (B) SAED analysis (C) Dynamic Light Scattering (DLS) measurements (D) SEM micrographs of AgNPs.

Full size image

Scattered area electron diffraction (SAED)

The crystalline character of NPs was determined by employing the SAED analysis supplementarily with TEM. Diffractions were specifically acquired at a distance of 80 cm. Myco-genized AgNPs were majorly crystalline in nature, which could be perfectly listed to the Bragg reflections of the face-centered cubic (fcc) nature of the crystalline silver (Fig. 7B).

Nano-suspensions of these myco-genized metallic NPs

Approximately 100 ppm of NP was diffused in test tubes that contained 100 mL of sterile deionized water to obtain nano-suspensions.

Dynamic light scattering (DLS)

Determination of the AgNPs size distribution was done by dynamic light scattering measurements on Malvern Zeta Sizer Nano ZS (Malvern Instruments Ltd., UK) using disposable clear zeta cells (DTS 1060C). The instrument allowed for an average diameter along with polydispersity index (PDI). Recordings for zeta average diameter plus PDI reported herewith were obtained by calculating an average of three separate measurements where each measurement was recorded after ten repetitions on each sample (Fig. 7C).

Zeta potential measurement

Zeta potential analysis is for the determination of the surface charge of AgNPs in solution form. The magnitude of zeta potential predicts the stability of the colloids. AgNPs having Zeta potential value exceeding + 25 mV or lesser than a value of − 25 mV possess a higher probability of stability. Dispersions having lower zeta potential value clump because of inter-particle Van Der Waal attractions.

Zeta potential measurements were executed with the AgNPs obtained from stock solution along with the resuspended employing Malvern Instruments Zeta-sizer Nano (Malvern Instruments Ltd., UK) that operated with a variable power of (5–50 mW) together with He–Ne laser at 632 nm. Measurements were obtained in zeta cells (DTS 1060C) at a temperature of 25 °C that were independently recorded thrice. Flow through the cell underwent washing three times before and during measurements with ultrahigh pure water prior to the addition of the subsequent sample. AgNPs zeta potential was evaluated at pH 7.4. Zeta potential measurements for AgNPs revealed that the maximum value was around − 19 mV (Fig. 7A).

MIC and MBC of AgNPs (µg/mL)

The MIC value for AgNPs was 1 µg/mL, and MBC was 2 µg/mL against MBL-producing Pseudomonas aeruginosa. The results depict that AgNPs conjugated with antibiotics are effective in the case of multidrug-resistant Pseudomonas aeruginosa and can be employed as an alternate treatment option following various clinical studies. The antibacterial potency of AgNPs was analyzed by recording the diameter of zones of inhibition in millimetres. This is illustrated in Fig. 8a,b.

Figure 8
figure 8

Antimicrobial effects: (a,b) AgNPs and antibiotic-AgNPs combinations, (c) AgNPs activity at varying volumes.

Full size image

Effect of AgNP solution volume on antibacterial action

Different volumes (10–50 μL) of 100 ppm AgNP solution were used to assess the impact of suspension volume on inhibition zone size. In the case of each test isolate, an increase in the inhibition zone was monitored with the corresponding rise in AgNP solution volume as shown in Fig. 8c.

Synergistic antibacterial effect of antibiotics in conjugation with AgNPs

The combined effect of AgNPs in conjugation with multiple antibiotics was construed against multidrug-resistant Pseudomonas aeruginosa using the disk diffusion method. It was observed that the antibacterial potency of the antibiotics was upgraded in the presence of AgNPs. The reducing order of antimicrobial activity of antibiotics and AgNPs plus their combinations was piperacillin/tazobactam + AgNPs (31 ± 1.4 mm), cefoxitin + AgNPs (30 ± 1.0 mm) > amikacin + AgNPs (25 ± 1.3 mm) > aztreonam + AgNPs (23 ± 1.5 mm) > meropenem + AgNPs (22 ± 1 mm) > imipenem + AgNPs (20 ± 1.5 mm) > gentamicin + AgNPs (17 ± 0.5 mm) > ciprofloxacin + AgNPs (16 ± 0.1 mm) > cefoperazone/sulbactam + AgNPs (14 ± 0.4 mm) ≥ ceftazidime + AgNPs (14 ± 1.2 mm). The combined effect of AgNPs plus antibiotics showed a 0.15–3.51 (average, 2.09) fold-area augmentation of the antibacterial activity. The potency of AgNPs alone and in combination with antibiotics was ascertained by measuring the inhibition zones. The inhibition zones were measured in a range of 14–31 mm. AgNPs used alone led to weaker bactericidal activities. Combinations of standard antibiotics with AgNPs ensued in a 0.15–3.51 (average 2.09) fold-area augmentation of the antibacterial activity. Typically, the highest zone of inhibition(s) was recorded by piperacillin/tazobactam in combination with AgNPs. Isolates resistant to ceftazidime showed a zone of inhibition measuring 14 ± 1.2 mm in combination with AgNPs (Table 6).

Table 6 Single and combined efficacy of AgNPs and antibiotics against MDR Pseudomonas aeruginosa.
Full size table

Discussion

The ability to produce Metallo-β-lactamases is the most vital mechanism by which Pseudomonas aeruginosa gains antimicrobial resistance against several drugs. Rapidly emerging strains of Pseudomonas aeruginosa that can produce MBLs are an urgent concern for hospitals and healthcare centers42. Hospital environment in lower middle economic countries has certain factors that contribute to the spreading of infections by MDR bacteria. These are improper handling of medical instruments, contaminated hands of medical personnel, and various unhygienic surfaces like floors and doorknobs42.

Multidrug-resistant phenotypes in Pseudomonas aeruginosa result from different mechanisms that are found to interact with one another thus providing antimicrobial resistance43. Increased expression of the efflux pump, reduction in the porins present in the external membrane, and modified geometry of the penicillin-binding proteins contribute to acquired resistance43. Acquisition of dual resistance to more than a single class of antimicrobials calls for reasoned treatment of infections owing to Pseudomonas aeruginosa39,43. Isolates of Pseudomonas aeruginosa resistant to ceftazidime and meropenem exhibit MBL and ESBL activity confirming that MBL-producing genes are considered crucial to resistance against these antimicrobials25,29.

In this study, more isolates were obtained from samples collected from female patients (145/523) as compared to male patients (110/636). These findings differ from a study that was carried out in Germany whereby out of a total of 168 patients, 67.3% of males, and 32.7% of females tested positive44. The highest isolates of Pseudomonas aeruginosa were from patients aged 40–49 years (25.4%). These results differ from another study that reported patients aged more than 55 years having a higher prevalence of Pseudomonas aeruginosa45. This study shows a much-increased prevalence of infections caused by Pseudomonas aeruginosa owing to poor health and sanitary condition of patients in hospital environments, improper prescription of antimicrobial drugs, and the presence of antimicrobial resistance in bacteria46. Almost 54.5% of the MDR strains of Pseudomonas aeruginosa were observed by Saderi et al.47. This increase in resistance against antibiotics may be due to activated efflux pumps in bacteria, modification of the target site of drugs, the presence of reducing enzymes, or the loss of membrane proteins48. Strains of Pseudomonas aeruginosa isolated in this study showed higher resistance against carbapenem drugs, i.e., imipenem (53%) and meropenem (51%). These findings are synonymous with a study conducted in the United States that mentions 65% resistance to carbapenem drugs49. The MIC values of meropenem and imipenem were in the range of 8 μg/mL to ≥ 32 μg/mL. These results agree with a previous study whereby the MIC of imipenem was > 32 μg/mL50. Results of the current study have intimated a prevalence of MBL producers as 61.5% of which 11.5% expressed blaIMP-1/blaVIM while blaTEM/blaOXA were expressed in 19.5% and blaOXA/blaSHV were concomitantly expressed in 9.5%. Tahmasebi et al. have reported the expression of 12 phenotypically MBL-producing isolates of Pseudomonas aeruginosa, of which PCR amplification confirmed blaVIM in 33.3%, and blaIMP in 25% of isolates41.

The antibacterial activities of silver have been discussed globally. Recently, thiol-dependent enzymes including thioredoxin (Trx) plus glutathione (GSH) systems have been discovered as potential bactericidal targets in MDR bacteria16,51. Reports have concluded that silver acts in conjugation with ebselen inhibiting the Trx system resulting in quick depletion of GSH in Gram-negative bacteria16,51. Additionally, silver augments the bacterial sensitivity towards antibiotics by blocking the Trx system16. Concomitantly, reactive oxygen species (ROS) generate helping bacteria become sensitive to conventional antibiotics16,51. Furthermore, the antibiotic- AgNPs combination strongly binds to the bacterial cells promoting the release of Ag(+) and resulting in an increased concentration of Ag(+) in the vicinity of bacteria. These findings are in support of the theory that Ag(+) release from AgNPs is the potential agent that causes toxicity52,53.

Minuscule AgNPs as found in the current study having a spherical shape possessing a microscale diameter are more sensitive to release silver because of enhanced surface area. Furthermore, AgNPs have the ability to penetrate the cell walls of bacteria by modifying their structure due to their nanoscale size14,52. Disruption of the cellular membranes can lead to rupture of the organelles resulting in cellular lysis13,14,39. The smaller size of nanoparticles in this study (5–30 nm) is attributed to their high efficiency in penetrating bacterial cells as compared to previous studies where the size of nanoparticles was 65–90 nm in diameter14. The collaborative effect of AgNPs plus antibiotics showed a 0.15–3.51 (average, 2.09) fold-area augmentation of the antibacterial activity. Several studies have recognized the antimicrobial potency of silver nanoparticles in combating bacteria. Unfortunately, the precise mechanism involved has not yet been ascertained44.

AgNPs can be toxic due to the release of ionic Ag in combination with surface properties, shape, and size. Lesser toxicity is associated with prismatic and cubic geometries of AgNPs at a concentration of ˂ 100 µg/L54. Auclair et al. have determined that sublethal toxicity ascertained at 96 h on the basis of salient characteristic morphological changes exhibits the following toxicity: ionic (2.6 µg/L), spherical (22 µg/L), and prismatic (32.5 µg/L) AgNPs54. Auclair et al. have also concluded that the structure of nanocube was not toxic at this concentration while nanoparticles possessing a low aspect ratio combined with high circularity as well as elongation properties exhibit high toxicity at both sublethal and lethal levels. AgNPs shape has been studied to influence the toxicity demanding further research in the field for understanding the mechanisms playing part in making AgNPs toxic54. Furthermore, varying neurobehavioral effects have been reported by Vogt et al. for various coatings and sizes of AgNPs while studying exposure of larvae to Ag + which suggests that AgNPs potentially act as a neurobehavioral disruptor55. Fu et al. have reported that exposure to AgNPs impairs social behavior and learning in the subjects studied which indicates a strong neurotoxic effect56. Bhalodia et al. reported an MIC ranging from 1.406 to 5.625 µg/mL plus an MBC ranging from 2.813 to 5.625 µg/mL for Metallo-β-lactamase producers in Pseudomonas aeruginosa35.

Uncoated AgNPs promote significant cytotoxic effects on PBMCs at proportionately lesser concentrations (< 5 μg/mL) and shorter exposure times (3–12 h). PBMCs are cells of the immune system that constitute lymphocytes (T cells, B cells, and NK cells) plus monocytes. This suggests that the coating of AgNPs decreases the interacting active surface area sites with the cellular components57,58. It has been reported by Kim et al. that AgNPs exhibit genotoxic effects in BEAS-2B cells that are bronchial epithelial cell lines59. The oxidative stress promoted by AgNPs might be a pivotal element in the genotoxic effects caused by AgNPs59. AgNPs have been studied to cause damage in DNA while creating formation of the micronucleus in a dose-dependent approach. The specific AgNPs dose-dependent activity due to the formation of reactive oxygen radicals has been studied to be reduced by superoxide dismutase most importantly as shown in the cytokinesis-block MN assay as well as the comet assay. The present study demonstrates that AgNPs conjugated with antibiotics inhibited Pseudomonas aeruginosa as the zone of inhibition significantly increased with the use of combined discs.

Conclusions

The current study concludes the high prevalence of multidrug-resistant Pseudomonas aeruginosa whereby a positive correlation has been observed between phenotypic and genotypic variants of Pseudomonas aeruginosa. Nevertheless, the antibiotic-silver nanoparticles combination has shown antibacterial potency against MDR Pseudomonas aeruginosa. Combinations of AgNPs with conventional antibiotics can possibly be researched as alternatives to antimicrobial agents for curing infections caused by MDR Pseudomonas aeruginosa following multiple clinical trials.