Antibacterial Potential of Biosynthesized Silver Nanoparticles Using Berberine Extract Against Multidrug-resistant Acinetobacter baumannii and Pseudomonas aeruginosa

Abstract

The emergence of multidrug resistance in bacterial infections has limited the use of antibiotics. Helping the action of antibiotics is one of the needs of the day. Today, the biosynthesis of nanoparticles (NPs) is considered due to its safety and cost-effectiveness. In this study, we investigated the effect of biosynthesized silver nanoparticles (AgNPs) by Berberine plant extract against standard strains of multidrug-resistant (MDR) Acinetobacter baumannii and Pseudomonas aeruginosa. Utilized UV–Vis, FTIR, FESEM/EDX, XRD, DLS, and Zeta potential techniques to confirm the biosynthesis of NPs. Then, disk diffusion agar (DDA) and minimum inhibitory concentration (MIC) tests were performed using common classes of standard antibiotics and AgNPs on the mentioned bacteria. The synergistic action between AgNPs and antibiotics was evaluated by the checkerboard method. First, we obtained the confirmation results of the biosynthesis of AgNPs. According to the DDA test, both standard bacterial strains were sensitive to NPs and had an inhibition zone. Also, the MIC values showed that AgNPs inhibit the growth of bacteria at lower concentrations than antibiotics. On the other hand, the results obtained from checkerboard monitoring showed that AgNPs, in combination with conventional antibiotics, have a synergistic effect. The advantage of this study was comparing the antibacterial effect of AgNPs alone and mixed with antibiotics. The antibacterial sensitivity tests indicated that the desired bacterial strains could not grow even in low concentrations of AgNPs. This property can be applied in future programs to solve the drug resistance of microorganisms in bacterial diseases.

Supplementary Information

The online version contains supplementary material available at 10.1007/s12088-023-01136-y.

Introduction

Recently, it has been observed that drug resistance in bacteria has become one of the therapeutic problems due to the widespread and improper use of antibiotics [1]. Also, outbreaks of rapid resistance to new antibiotics are not far away due to their indiscriminate use [2]. Bacteria resistant to more than one antibiotic are called multidrug-resistant (MDRs) bacteria. Among the features that cause antibiotic resistance in these bacteria, it can be attributed to the receipt of antibiotic resistance genes, the ability to form a strong biofilm, having efflux pumps, and numerous beta-lactamase enzymes [3]. Bacteria commonly exhibit social activities through acyl-homoserine lactones (AHLs)-based quorum sensing (QS) systems to form their unique social network [4]. AHL molecules can diffuse freely through the bacterial membrane and their concentration concomitantly accumulates with growth [5]. Treatment of infections caused by MDRs is challenging due to the limited choice of current antibiotics and the lengthy hospitalization that leads to elevated healthcare costs. Antibiotic resistance significantly threatens human health, causing approximately 0.7 million annual deaths worldwide [6].

According to the World Health Organization (WHO), Multi-drug resistant gram-negative bacilli (MDR-GNBs) like P. aeruginosa and A. baumannii are on the list of dangerous pathogens for humans, so the development of new therapies for them is essential [7]. A. baumannii and P. aeruginosa are the most common bacteria in nosocomial infections, especially burns. Patients with these infections are reservoirs of these microorganisms, ordinarily resistant to most antibiotics [8]. Therefore, there is an urgent need to discover new ways to fight multidrug-resistant bacteria and reduce the side effects of chemical drugs. To minimize the use of antibiotics, it is necessary to consider appropriate alternatives for them without side effects [9]. For example, the use of therapeutic agents (antimicrobials) of natural origin such as Polyhydroxyalkanoates (PHAs) [10] and cationic chitosan derivatives have been suggested as potential antimicrobial agents [11]; or NPs have displayed sustainable antimicrobial activity in surgical and food processing cases compared to their ions [12].

NPs in the size of 1 to 100 nm, due to their quantum properties and unique physical and chemical properties, can be applied to many fields in medical sciences [13]. The use of NPs for industrial biotechnological applications has been possible due to their high biocompatibility and biocatalytic activities [14]. Next to improving the feature of bioavailability, NPs contain a more vital ability for carrying capacity, mobility, cellular uptake, anti-inflammatory, and lower toxicity compounds compared to the cases of microparticles [15]. NPs have enormous potential in the delivery of drugs, proteins, and genes [16]. In addition, they can control and maintain the release profile of the drug that is given to the target site, as well as tailor the appointed particle to withstand detrimental pH, processing, and enzymatic conditions [17]. Scientists worldwide have focused on NPs because of these properties in several applications, one of which is the antibacterial potential and combating the problem of bacterial drug resistance [18]. Although some of the specific antimicrobial mechanisms of NPs are ambiguous and have not been well defined yet [19], these NPs can damage cellular structures by binding to the walls and membranes of cells and penetrating them, inducing the production of reactive oxygen species (ROS) and change the signal transmission in the cell [20]. According to past research, the AgNPs showed excellent antimicrobial activity against various pathogens, including bacteria and fungi [6, 21]. Several studies have suggested using AgNPs to prevent the spread of antibiotic-resistant [22]. AgNPs are possessed very low toxicity to human cells, high stability, and low volatility. Due to these properties, AgNPs have a variety of applications in medicine, including use against various infections, nanomedicine imaging, pharmacy, cosmetics, veterinary medicine, and bioassays [23]. Also, AgNPs can inhibit the growth of bacteria without any side effects or insignificance compared to antibiotics [24].

There are physical, chemical, and biological methods for synthesizing NPs [21]. The physical approaches such as photocatalytic synthesis, laser ablation, ultra-violet (UV) radiation, and hydrothermal technique are not economical due to the cost of consumable equipment [25]. The available methods for the chemical synthesis of NPs are fast but risky. Because chemical processes mediated by precipitation, acid decomposition, etc., are unsafe for biomedical applications, it utilizes extreme temperature, expensive methodology, acidic pH, and harsh chemicals that can cause toxicity to normal cells and hinder biomedical processes [26]. Green synthesis or biosynthesis, a new NPs synthesis method with a simple, biocompatible, safe, and economical approach, can replace chemical and physical processes [24, 27]. For the biosynthesis of NPs, natural products such as extracts of various plants or parts of plants (leaves, fruits, flowers, bark, seeds, roots, latex) have attracted the most attention. As a result, using NPS biosynthesized by plant extracts has replaced physicochemical methods and is increasing due to its advantages, such as high stability, non-toxicity, cheapness, simplicity, and fast [28].

Berberine plant grows in Asia and Europe and is very well known in Iran, and is widely used as a medicinal plant in traditional medicine [29]. The fruits of this plant are also used as a food additive with anti-oxidant and anti-inflammatory properties [30].

In the present study, AgNPs were biosynthesized with the help of Berberine plant extract. In the following, we compared the MIC values of AgNPs and antibiotics meropenem (MEN), ceftazidime (CAZ), ciprofloxacin (CIP), and penicillin (P) on the standard strains of MDR A. baumannii and P. aeruginosa. By the checkerboard assay, we also investigated the effect of co-synthesis of AgNPs with the desired antibiotics against the mentioned bacterial strains.

Materials and Methods

Antibiotics and Bacterial Samples

In this study, we used the standard strains of MDR A. baumannii: ATCC 19609 and MDR P. aeruginosa: ATCC 27853. They have been standardized in the Department of Microbiology of Pasteur Institute of Iran.

Preparation of AgNPs

The Berberine plant extract was used for the biosynthesis of AgNPs. First, for the biosynthesis of AgNPs, we combined 5.0 g of Berberine plant powder with 100 ml of H₂O. This mixture was stirred for 2 h at 60 °C. Added 30 ml of the plant extract dropwise to 100 ml of 1.0 mM silver nitrate (AgNO₃) (Sigma Aldrich, USA) previously prepared and stirred for 30 min and shaken at 25 °C for 48 h. The color of the reaction mixture changed from yellow to brown after the combination of AgNO₃ and reduction of silver (Ag) ions, which changed to dark silver over time and the complete reduction of Ag ions [13].

Instrumental Analysis

Confirmed the biosynthesis of AgNPs by various laboratory methods such as UV–Vis spectrophotometer with a Shimadzu (Japan) model 2550, Fourier transform infrared (FTIR) analysis (Thermo Nicolet model 370 Spectrum), X-Ray diffractometer (XRD) pattern (GNR Co., Model EXPLORER, Italy), the field emission scanning electron microscopy (FESEM) (TESCAN, Model MIRA3, Czech Republic), Energy-dispersive X-ray (EDX) spectroscopy, dynamic light scattering (DLS) device (Cordouan, model Vasco3, French) and zeta potential (CAD Co., Model Zeta Compact, France).

Cytotoxicity of AgNPs

MTT Assay

MTT [3-(4, 5-dimethylthiazol 2-yl)-2, 5-diphenyltetrazolium bromide] assay was performed to determine the cytotoxicity of the AgNPs against fibroblast cells of mouse connective tissue (3T3 cell line) according to the standard protocol [31].

Antibacterial Activity Tests

Disk Diffusion Agar (DDA)

First, to test the susceptibility of bacteria to antibiotics and AgNPs, the disk diffusion method was performed according to M51-A Clinical and Laboratory Standards Institute (CLSI) guideline [32]. By culturing the bacteria in the Muller Hinton Agar (MHA) plate. Then the antibiotic discs were placed next to the disc impregnated with a colloidal solution of AgNPs with a specific and same concentration, with a standard longitudinal distance. The plate was incubated at 37 °C for 24 h, and then we measured the aura of lack of growth with a ruler [23].

Minimum Inhibitory Concentration (MIC)

The reference gold standard CLSI (M07) broth micro-dilution was done on the MDR standard strains of P. aeruginosa and A. baumannii [33]. The MICs of AgNPs and four antibiotics, including ceftazidime (CAZ), penicillin (P), meropenem (MEN), and ciprofloxacin (CIP), against the mentioned bacteria were obtained. Poured sterile BHI alone into 12 wells of a column as a negative control, and the bacterial suspension in BHI was poured into 11 wells of a column as a positive control. In this method, the red color indicates the growth of bacteria, and no change of color indicates inhibition of its development [7].

Synergistic Effect

A checkerboard assay was performed to determine the antimicrobial effects of AgNPs combined with four antibiotics of meropenem, ceftazidime, ciprofloxacin, and penicillin against P. aeruginosa and A. baumannii in 96-well microplates (Supplementary file 1). The fractional inhibitory concentration index (FICI) was calculated for each combination separately. The FICI was expressed as FICI = FICS + FICL, where FICS was the MIC of S in combination/MIC of S alone, and FICL was the MIC of L in combination/MIC of L alone. FICI ≤ 0.5, 0.5 < FICI ≤ 1.0, 1.0 < FICI ≤ 4.0, and FICI > 4.0 were interpreted as synergy, additive, indifferent, and antagonism, respectively. Should note that performed all bacterial sensitivity tests in duplicate.

Results and Discussion

UV–Vis Spectrum

We performed UV–Vis spectroscopy for berberine extract alone and before the biosynthesis of NPs. After preparing the AgNPs, confirmed the existence of these NPs by using the UV–Vis spectrum recorded with a spectrophotometer in the absorption range of 410–430 nm. The UV–Vis spectrum of synthesized AgNPs using Bebeerine plant extract is presented in Fig. 1a, which exhibited the typical plasmon resonance bands of AgNPs at 424 nm (λmax).

Fig. 1.

UV–Vis (a) and FTIR spectra (b) recorded from Berberine extract and biosynthesized AgNPs; XRD pattern (c) and Zeta potential (d) of biosynthesized AgNPs

FTIR Analysis

Infrared spectroscopy was based on radiation absorption and provides data for studying the vibrational diffraction of molecules and polyatomic ions. To confirm the biosynthesized NPs, we investigated the FTIR spectra of the extract and the biosynthesized NPs. The FTIR spectra of AgNPs and Berberine plant extract were provided in Fig. 1b. The proper stability and dispersion of NPs were caused by inductive interactions with proteins, enzymes, and other organic substances in the plant extract. The role of the covering agent was filled by the proteins that were secreted through this plant. During the production of NPs, it appeared that the NPs were coated by the proteins, leading to their stabilization as any condensation was prevented. We performed the FTIR analysis in the wavelength range of 400–4000 cm⁻¹. According to the provided graphs of our report, we observed peaks throughout the spectra of 1078.97, 1357.23, 1565.61, 1729.69, and 3360.84 cm⁻¹ for AgNPs. In conformity to the FTIR outcomes, the existence and participation of proteins were confirmed through the stabilization of NPs, which was caused by the coating occurrence. To some extent, the number of peaks in plant extract before NPs biosynthesis are more and sharper, whereas after performing this process, the number and sharpness of peaks seemed to be decreased. The functionality of this procedure was to configure the interaction of NPs with plant extract proteins and determined the role of these proteins that surround NPs as stabilizing agents [13].

XRD Pattern

The XRD pattern obtained from the studied AgNPs confirmed that NPs were formed and found that the biosynthesized NPs were crystalline. Also, it investigated the average size of NPs through the XRD pattern of AgNPs. In Fig. 1c, the intensity of XRD with 2θ angles and in the range of 20° to 80° was observed. The average size of the NPs using the Debye–Scherrer equation [D = 0.9λ/ (β cos θ)] was about 36 nm [34].

Zeta Potential Analysis

After placing the colloidal solution in an ultrasonic bath for half an hour (at pH = 7.4 and a temperature of 30 °C), the average zeta potential for biosynthesized AgNPs was around − 30 mV, which indicated the stability of the NPs (Fig. 1d). Since similar charges repel each other, higher stability was provided for NPs, and their accumulation was prevented. Zeta potential analysis showed the surface charge of particles and was considered an essential factor in inspecting the stability of NPs. If this value exceeds ± 30 mV, the particles were more stable in solutions [24].

FESEM/EDX/DLS

As it could be observed in the EDX diagram, the existence of Ag element throughout the existing structure was quite evident in Fig. 2b. Furthermore, the presence AgNPs with an approximately spherical construction and size throughout the range of 35 nm, was proved by a TESCAN field emission scanning electron microscope (Fig. 2a).

Fig. 2.

FESEM image (a), EDX (b), DLS (c), and PSA (d) of biosynthesized AgNPs by Berberine extract

The average size of AgNPs based on DLS and particle size analysis (PSA) was about 51 nm (Fig. 2c, d), which was more significant than that obtained by XRD. This phenomenon may be attributed to materials that cover the surfaces of NPs, such as capping and stabilizing agents, which interfere with DLS measurements. Also, size-based DLS depends on the homogeneity of the solution and hydrodynamic NPs residues.

The In Vitro Cytotoxicity of AgNPs

Evaluated cellular responses of the AgNPs against 3T3 cell line by MTT assay. The IC₅₀ value was obtained by plotting cell viability against different concentrations (0.125 to 64 µg/ml) of the AgNPs. The in vitro cytotoxicity investigations of AgNPs have revealed non-toxic even in concentrations that enclosed up to 64 μg/ml of NPs (Supplementary file 2).

Antibacterial Activity/DDA Test

Using the DDA test, we investigated the antibacterial efficacy of AgNPs against MDR P. aeruginosa and MDR A. baumannii strains. As you can see in Supplementary file 3, MDR P. aeruginosa was resistant to ceftazidime, penicillin and meropenem. MDR A. baumannii was resistant to ceftazidime, penicillin, and ciprofloxacin. However, both MDR bacterial strains were sensitive to AgNPs and had an inhibitory zone.

Antibacterial Activity/MIC Test

The results of MIC of AgNPs and antibiotics for standard MDR strains P. aeruginosa, and A. baumannii were prepared in Table 1 (Supplementary file 4). According to these results, AgNPs had an antimicrobial effect against MDR P. aeruginosa and A. baumannii. Also, the combined impact of NPs with conventional antibiotics in treating A. baumannii and P. aeruginosa infections (meropenem, ceftazidime, ciprofloxacin, and penicillin) showed that they are synergistic (Supplementary file 5).

Also, we evaluated the effect of an aqueous extract of Berberine on the growth of MDR A. baumannii and P. aeruginosa standard strains as duplicated in serial dilutions. It was observed that the plates contain grown bacteria, which indicates the resistance of the mentioned strains to the aqueous extract of the plant and its ineffectiveness.

Antibacterial Activity/Checkerboard Assay

According to the results obtained from the checkerboard assay and investigating the combined effects of AgNPs with conventional antibiotics against the standard MDR strains of A. baumannii and P. aeruginosa infections, it was found that AgNPs had a synergistic effect with conventional antibiotics (ceftazidime, ciprofloxacin, penicillin, and meropenem). These results were shown in Table 2 (Supplementary file 6) and Supplementary file 7.

Considering the increasing trend of drug-resistant bacterial strains, as well as the increase in the use of antibacterial drugs to treat these infections and the side effects of these drugs, the importance of finding an alternative or complementary method with the property of inhibiting the growth of these microorganisms is revealed [7]. Based on this issue and looking at the upcoming problems, it is suggested to investigate other options instead of antibiotics. For example, these alternatives can be probiotic products [35], herbal extracts [36], cationic peptides [37], and NPs [38]. Among these, the use of biological NPs with broad applications and unique properties has attracted the attention of researchers [13]. As Wang et al. stated in their study in 2017, due to drug resistance to antibiotics in the future, the treatment of infections will be complicated, and NPs can be a suitable alternative for them [39]. A large number of living organisms, such as bacteria, fungi, algae, and plant extracts, are selected as mediators for synthesizing various NPs [40]. So far, several studies have reported the production of biological NPs from microorganisms such as Aspergillus flavus, Aspergillus niger, Escherichia coli, Bacillus subtilis, etc. [41], which, due to these items can be part of pathogenic microorganisms, they are limited for mass production and entering the industry [42]. But the use of plant extracts in the process of green synthesis of NPs is much more practical and is safe and available to a large extent. In this study, we have biosynthesized AgNPs using Berberine plant extract, which is completely non-toxic and clean, and then investigated the antimicrobial activity of the obtained product. In the research conducted by Rafique et al., it has been shown that the green synthesis of NPs and their use instead of physicochemical methods are faster and cheaper and has fewer risks for humans and the environment [43]. It should be noted that based on past studies, no cytotoxic effects have been reported for AgNPs. In addition, previous research has shown that green synthesized AgNPs are toxic only in cancer and non-toxic in standard cell lines, which is an excellent point about them [23, 24]. We confirmed the biosynthesized NPs with the help of different techniques. Salayová et al. synthesized AgNPs using medicinal plants widely used in folk medicine and characterized by several ways such as transmission electron microscopy (TEM), UV–Vis, FTIR, and zeta potential, which is somewhat similar to our work; still, our described verification techniques were more. Previous studies, like ours, found that the absorption peak of AgNPs is approximately in the range of 410–420 nm, which varies depending on the conditions and the size of the particles [23]. Also, knowing the content of the absorption peak provides us with information about the size of the particles, which Calvillo et al. concluded in their study that particles of different sizes produce different absorption peaks [44]. In 2014, Khodadadi et al. performed the biosynthesis of AgNPs with the help of oak fruit extract and reported its relatively good antimicrobial activity against hospital infections [45]. Also, the antibacterial activity of green synthesized AgNPs on Klebsiella pneumonia, E. coli, Staphylococcus aureus, and S. haemolyticus as bacteria have been investigated by Mekky et al. [23]. But what has been important in our research is working on the MDR strains of A. baumannii and P. aeruginosa, which are considered therapeutic problems.

A comparison of our results revealed that AgNPs could prevent the growth of bacteria even at low concentrations and have a better effect than the group of antibacterial drugs. Cittrarasu et al. showed in their studies that AgNPs could be an efficient antibacterial agent against human pathogens, and this effect is enhanced by increasing the concentration of AgNPs [46]. According to our collected data, the observed inhibitory effect on the growth of bacteria was entirely satisfactory since the applied AgNPs could inhibit the growth of bacteria in several wells. On the contrary, the individual application of the aqueous extract of this plant was not practical for the growth of bacteria. It can be said that the inhibitory effect of NPs is due to the easy penetration and transfer of these particles to the interior of the microbial cell and inactivating or reducing the expression of the drug-resistance genes of microorganisms [9, 47]. These particular types of substances can also disrupt cellular structure, production of ROS, and consequently alter the pathways of signal transduction [13]. Besides, in the synergistic phenomenon, the desired NPs can bind to the drugs and deliver them into the bacterial cell [48].

Compared with most of the reviewed studies, the advantage of our work is that we also investigated the synergistic effect between AgNPs and antibiotics using the checkerboard method, in which biosynthesized AgNPs had a synergistic effect with the intended antibiotics. It can be concluded that based on the past research and also the positive results obtained from the current study, synthesized NPs by biological methods have a better effect and more suitable efficiency compared to chemical NPs (commercial) and with more complete research on them, they can be employed in various medical fields. We recommend that other researchers investigate impact of AgNPs on other pathogenic microorganisms such as gram-negative and positive bacteria, fungi, parasites, and viruses in future studies. Although we used the high antimicrobial properties of AgNPs in this study, we suggest more comprehensive studies to evaluate the antimicrobial properties further. These NPs are considered a supplement or good alternative to solve the problems of drug resistance in pathogenic microorganisms. On the other hand, the application of these NPs is without unwanted side effects and is entirely affordable and economical in today's world. Due to this issue, the use of NPs in the diagnosis and treatment of diseases related to microorganisms such as bacteria, fungi, parasites, and viruses can be considered in the future. Meanwhile, it is expected that the antimicrobial activity of different types of NPs, including metallic, metal oxide, and nanocomposites, will be further investigated in future studies.

Conclusion

Based on past research and the positive results from the present study, biosynthesized AgNPs have favorable antibacterial properties, a better effect, and a more suitable efficiency than chemical antibacterial drugs such as common standard antibiotics. These NPs are considered an appropriate complement or alternative to solve drug resistance problems in pathogenic microorganisms and can significantly help various medical fields in the future. On the other hand, their application is without unwanted side effects and is quite affordable and economical today.

Supplementary Information

Below is the link to the electronic supplementary material.

Author Contributions

MT and SZ: Investigation and perform experiments, Writing an original draft, Formal analysis; AN: Writing-review & Editing; PM: Data curation; MB: Data curation; SJA: Formal analysis; SHHS: Software; MHB: Supervision, Project administration, Validation, Data curation, Writing-review & Editing. All authors have read and approved the manuscript.

Funding

No funding had been received.

Data Availability

All data is made available and presented in the manuscript.

Declarations

Competing interest

The authors declare no conflict of interest.

Ethical Approval

No experiments were conducted on animals or humans.