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 Table of Contents  
ORIGINAL ARTICLE
Year : 2022  |  Volume : 6  |  Issue : 3  |  Page : 400-409

Synthesis of silver nanoparticles using Nigella sativa seed extract and its efficacy against some multidrug-resistant uropathogens


1 Department of Microbiology, University of Nigeria, Nsukka, Enugu State, Nigeria
2 Department of Pharmaceutical Microbiology and Biotechnology, University of Nigeria, Nsukka, Enugu State, Nigeria

Date of Submission04-May-2022
Date of Decision28-Jun-2022
Date of Acceptance11-Jul-2022
Date of Web Publication17-Sep-2022

Correspondence Address:
Christian Kelechi Ezeh
Department of Microbiology, University of Nigeria, Nsukka, Enugu State
Nigeria
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/bbrj.bbrj_104_22

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  Abstract 


Background: Urinary tract infection (UTI) is one of the most chronic infections in humans, as well as the most common cause of multidrug-resistant (MDR) pathogen emergence, necessitating the quest for stringent antibiotic treatment. In the imperative search for effective drugs to combat MDR, silver nanoparticles (AgNPs) are given priority. The objective of this study was to synthesize, characterize, and evaluate the antimicrobial activity of AgNPs synthesized using Nigella sativa on MDR uropathogens. Methods: Urine samples of suspected UTI patients were collected using sterile sample bottles and cultured on different agar media to isolate and identify uropathogens using conventional method. All isolates were screened for multidrug resistance by disk diffusion method following the Clinical and Laboratory Standards Institute guideline with slight modification. N. sativa seed extract was used to synthesize AgNPs from aqueous solution of silver nitrate (AgNO3). AgNPs formed were characterized using ultraviolet-visible (UV-Vis) spectroscopy, scanning electron microscope (SEM), dynamic light scattering, Fourier transform infrared spectroscopy, and X-ray diffraction spectroscopy (XRD). Antibacterial activities of synthesized AgNPs were assessed (in vitro) by disc diffusion method against MDR isolates, and cytotoxicity was evaluated using brine shrimp lethality assay. The formed AgNPs were characterized using UV-Vis, and antibacterial activity of synthesized AgNPs was assessed (in vitro) by impregnated disc diffusion method against MDR isolates. Results: Escherichia coli, Klebsiella sp., and Staphylococcus aureus were isolated. Multiple antibiotic-resistant indexes of the isolates ranged between 0.4 and 0.75 against the different standard antibiotics tested. The color change from pale yellow to dark brown was indicative of AgNP formation. UV-Vis spectrum of solution containing AgNPs exhibited peak wavelengths of 378 nm. Fourier transform infrared spectroscopic analysis showed that terpenoids, flavonoid, and phenols played an important role in the biosynthesis. Irregular shapes within nanoscale range were detected using SEM. XRD showed that the particles were crystalline in structure with an average size of 32 nm. The mean diameter zone of inhibition (in mm) for the different isolates at the dose of 100 μg/mL concentration showed maximum for E. coli (18 mm) followed by both Klebsiella sp. and S. aureus at 17 mm. Conclusion: The study underscores the efficacy of the plant-mediated nanoparticles as well as their potential for use as broad-spectrum antimicrobial agents for the management of MDR uropathogens.

Keywords: Antibacterial activity, multidrug resistant, Nigella sativa, silver nanoparticles, urinary tract infection, uropathogens


How to cite this article:
Ezeh CK, Eze CN, Dibua ME, Emencheta SC, Ezeh CC. Synthesis of silver nanoparticles using Nigella sativa seed extract and its efficacy against some multidrug-resistant uropathogens. Biomed Biotechnol Res J 2022;6:400-9

How to cite this URL:
Ezeh CK, Eze CN, Dibua ME, Emencheta SC, Ezeh CC. Synthesis of silver nanoparticles using Nigella sativa seed extract and its efficacy against some multidrug-resistant uropathogens. Biomed Biotechnol Res J [serial online] 2022 [cited 2022 Oct 5];6:400-9. Available from: https://www.bmbtrj.org/text.asp?2022/6/3/400/356138




  Introduction Top


The human urinary tract consists of organs such as urethra, bladder, ureters, kidneys, and other ancillary parts responsible for collecting, storing, and release of urine. Inflammatory responses in any of these organs induced by the colonization and multiplication of microorganisms (mostly bacteria) result in urinary tract infection (UTI).[1] UTI is a community as well as a nosocomial infection and is one of the most common bacterial infections in humans,[2] affecting about 150 million people yearly around the world, with over 5 billion US dollars spent on its management.[3] Hence, it remains a major burden to the public health sector globally.

UTI is a common reason for antibiotic treatment.[4] Inappropriate usage of antibiotics has seen a shift in the antimicrobial susceptibility pattern of the causative pathogens; consequently, resistant strains have emerged.[5] Currently, UTI-causing pathogenic bacteria are resistant to numerous antibiotics and these constitute a serious health threat due to treatment failures in patients.[6],[7] Hence, it is imperative to develop an alternative source of antibacterial agents to combat such resistant strains[8] and nanoparticle technology is a very viable and promising approach.

Advances in nanoparticle technology come with the fabrication of nanosized objects with effective antimicrobial properties. The antimicrobial potential of metallic nanoparticles is extensively studied and has created an alternative platform to tackle the challenges created by multidrug-resistant (MDR) bacteria.[9] Nanoparticles as therapeutic agents have been shown to overcome the various mechanisms used by bacteria to resist antibiotics due to their multiple modes of action.[10] At nano-range, metals such as gold, zinc, silver, copper, and cadmium have shown antimicrobial activity. However, silver nanoparticles (AgNPs) are of high interest to researchers and have a wide range of applications, especially in the biomedical field owing to their unique properties such as chemical stability, catalytic, optical, and antimicrobial properties. Chemically, physically and biologically synthesized AgNPs have been shown to impede the growth of bacteria and yeasts.[11]

There are well-established methods of synthesizing AgNPs (chemical, physical, and biological approaches). The chemical and physical processes often involve high temperature/pressure for the reaction and utilization of noxious chemicals which are toxic to the biological system. Therefore, research in green synthesis is gaining importance. The biological method can either be microbial or plant-mediated synthesis. However, microbial facilitated synthesis is less appealing due to high maintenance of hygienic condition, intricate process of maintaining microbial culture, and longer incubation time. Hence, plant-based synthesis has gained much attention because it is a cost-effective, environmental friendly, single-step process, and efficient alternative for large-scale production of nanoparticles.[12],[13],[14],[15]

Phytochemicals including flavonoids, alkaloids, saponins, tannins, phenols, ketones, and aldehydes associated with plant extracts are responsible for the reduction of silver ions to AgNPs and subsequent capping of the formed particles with less toxicity.[16]

The plant selected for this synthesis is Nigella sativa seeds (black cumin seeds). N. sativa in the Ranunculaceae family is an annual plant growing mostly in arid regions. It bears terminal white flowers, alternate leaves, and capsule which contain numerous seeds of which seeds appear to show the most antimicrobial activities.[17],[18] Hence, in this study, AgNPs were synthesized using N. sativa seeds. The formed particles were characterized, and the antibacterial activity against selected multidrug uropathogens was evaluated.


  Methods Top


Plant collection

N. sativa seeds from Saudi Arabia were purchased from Middle East Merchants in Kano, Nigeria. The plant materials were packaged in sterile cellophane and transported promptly to laboratory for analyses and extraction. The plant was identified by the Department of Plant Science and Biotechnology, University of Nigeria, Nsukka, Nigeria.

Preparation of extracts

N. sativa seeds collected were washed and rinsed with de-ionized water to get rid of dust and other debris. The washed seeds were air-dried indoors for 15 days and ground into powder. Approximately, 100 ml of distilled water and 20 g of the seed powder were dispensed into a 500-ml conical flask. The solution was shaken, boiled for 10–15 min, and allowed to cool. The cooled infusion was filtered using Whatman No. 1 filter paper, and the filtrate was stored in a refrigerator for further use.

Isolation and identification of bacteria strains

Informed consent to collect clinical sample (urine) was sort for and approved by the ethical committee of Bishop Shanahan Hospital Nsukka, Enugu state, Nigeria. Midstream urine samples were collected aseptically using sterile universal sample containers from patients of all ages presenting UTI signs and symptoms in selected hospitals within the locality in accordance with standard operating procedures. Urine samples collected were observed macroscopically for the presence of pus. Urine culture was carried out according to the method described by Chessbrough.[19] The sample was inoculated onto sterile MacConkey, Eosin methylene blue, and Mannitol salt agar plates and incubated at 37°C for 24 h. Distinct colonies were subcultured on the appropriate media, and incubated as described above. The purified colonies were preserved in nutrient agar slants after incubation, and stored in the refrigerator for further use.

Antibiotic susceptibility test and multiple antibiotic resistance index screening

The bacterial isolates were screened for resistance to antibiotics using Kirby–Bauer disk diffusion method with slight modification, and the zone of inhibition (ZOI) of each antibiotic was interpreted using the criteria published by the Clinical and Laboratory Standards Institute (2012).[20] A 24-h culture of the test isolates (approximately 107 cfu/ml) was used for the sensitivity test. This was seeded onto sterile Mueller–Hinton plate and allowed to prediffuse. Commercially available discs containing the following antibiotics manufactured by Rapid Laboratories, UK, namely ceftazidime (30 μg), ciprofloxacin (5 μg), cefuroxime (30 μ), Augmentin (30 μg), gentamicin (10 μg), cefixime (10 μg), ampicillin (10 μg), nitrofurantoin (300 μg), and ofloxacin (5 μg), were aseptically placed on the surface of the sensitivity agar plates with a sterile forceps and were incubated at 37°C for 18 h. Zones of inhibition after incubation were observed and their diameters were measured in millimeters. Multiple antibiotic-resistant (MAR) index of recovered uropathogens was evaluated as the number of antibiotics the organisms were resistant to, divided by the total number of antibiotics the organisms were exposed to.[21] Isolates with MAR index >0.2 were tagged resistant to multiple antibiotics. These organisms were selected for further studies.

Preparation of 1 mM concentration of silver nitrate solution

About 1 mM concentration of silver nitrate solution was prepared by adding 0.17 g of silver nitrate salt into a 1000-ml conical flask containing 1000 ml of distilled water.

Nigella sativa seed-mediated synthesis of silver nanoparticles

Exactly 80 ml of aqueous silver nitrate was added to a flask holding 20 ml of the seed extract. This was shaken vigorously and incubated in a dark cupboard. The solution was observed for color change from pale yellow to dark brown which is indicative of AgNP formation.

Confirmation of nanoparticles synthesis by ultraviolet-visible spectrophotometry

The synthesized AgNPs were subjected to ultraviolet (UV)-spectrophotometer within the wavelength range of 300–800 nm for confirmation of synthesis.[22]

Harvesting and purification of silver nanoparticles

The AgNP solution synthesized was centrifuged at 4000 rpm for 15 min and the supernatant was discarded. The pellets were air-dried using a hand drier and the AgNPs were stored in a clean air-tight sterile container for further characterization.

Characterization of silver nanoparticles

Fourier transform infrared spectroscopy analyses

This technique was employed to check the possible functional groups representing the biomolecules associated with the seed extract responsible for the reduction of silver ions to AgNPs and the subsequent capping of the formed particles. The formed AgNPs and the seed extract each were mixed with potassium bromide (KBr) salt, and cast to form different pellets. The casted sample pellet was inserted into the Fourier transform infrared spectroscopy (FTIR) accessory sample holder, and the analyses were performed. To identify the functional groups present in each sample, the spectra obtained from both the plant extract and the formed nanoparticles were compared to a reference chart.

Scanning electron microscopy and energy-dispersive X-ray spectroscopy analyses

Scanning electron microscope (SEM) is a surface imaging tool utilized to illustrate the morphology and distribution of synthesized AgNPs. During SEM analysis, a thin smear of AgNPs was made on a copper lattice coated with carbon and allowed to dry. The dried smear was fixed in a vacuum chamber of the SEM in the sample holder and observed.[23] Meanwhile, electrodynamic suspension (EDS) was carried out using the fundamental principle that every element has a unique atomic structure which creates sets of distinctive peaks on X-ray emission spectrum to discover the surface element present in the sample. Electron beam was passed through the AgNPs, and an image of the elements' composition was formed.

X-ray diffraction analysis

X-ray diffraction (XRD) is a technique employed to determine the crystalline structure of a sample. For the determination of silver nanoparticle formation, XRD-7000s/7000 L Shimadzu was used. Dried powder of AgNPs obtained was washed three times in de-ionized water and then a pellet was formed. An Ultima IV X-ray powder diffractometer was used to measure X-ray powder diffraction (XRD) using Cu K radiation (=1.5418) (Rigaku, Tokyo, Japan).[23] Debye–Scherrer's equation was used to calculate the particle average size:



where D = crystalline size (nm)

K = Scherrer constant (0.9)

ʍ = wave length of the X-ray source (0.15406)

β = FWHM (radians)

θ = peak position (radians).

Antimicrobial activity of silver nanoparticles by disc diffusion method

The antibacterial activity of the synthesized AgNPs was evaluated against the selected MDR uropathogens using agar disc diffusion method. The National Committee for Clinical Laboratory Standards (2003)[24] breakpoint guidelines for susceptibility testing were used. The test suspension was prepared by obtaining a fresh culture of the test organisms inoculated into a nutrient broth and incubated at 37°C for 18–24 h. The bacteria broth was matched to 0.5 McFarland turbidity standard and streaked evenly with a sterile cotton swab stick on Mueller–Hinton agar plate and allowed to diffuse for 5 min. Using a sterile forceps, appropriate AgNP-impregnated discs of various concentrations (100 μg/ml, 50 μg/ml, and 25 μg/ml) were placed aseptically on the agar and gently pressed down to achieve contact. Plates were maintained at room temperature for 3–4 h for improved absorption and thereafter incubated for 24 h at 37°C. Antibacterial activity was determined by measuring the ZOI in millimeters and results were recorded.


  Results Top


Isolation and identification of uropathogens

The results of the morphological and biochemical tests performed on the isolates are presented in [Table 1]. Three different isolates were obtained and were presumptively identified as belonging to the genera Escherichia, Klebsiella, and Staphylococcus.
Table 1: Identification of uropathogenic isolates

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Antibiotic susceptibility test

[Figure 1] shows the antimicrobial sensitivity pattern of Gram-negative isolates (Escherichia coli and Klebsiella spp.). The figure revealed that 98% of the isolates were resistant to ampicillin and Augmentin while 2% were susceptible to each. It was shown that 100% of the isolates were resistant to cefuroxime while 91% showed resistance to ceftazidime with only 9% intermediate. Isolates that showed resistance to gentamicin and ciprofloxacin constitute 32% and 36%, respectively, with 40.9% susceptible to gentamicin and 36% to ciprofloxacin. Meanwhile, 93% and 67% of the isolates were susceptible to nitrofurantoin and ofloxacin, respectively.
Figure 1: Antimicrobial susceptibility pattern of Gram-negative isolates Key: AMP = Ampicillin, AUG = Augmentin, CAZ = Ceftazidime, CPR = Ciprofloxacin, CRX = Cefuroxime, GEN = Gentamicin, NIT = Nitrofurantoin, OFL = Ofloxacin

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[Figure 2] shows the antimicrobial sensitivity pattern of Gram-positive isolates (Staphylococcus aureus). The figure revealed that 90% of the isolates demonstrated resistance to ceftazidime while 10% were susceptible. It is also shown that 85%, 75%, and 56% of the isolates were resistant to Augmentin, cloxacillin, and erythromycin, respectively. Meanwhile, 67% and 60% of the isolates were susceptible to gentamicin and ofloxacin, respectively. However, 27% were resistant to gentamicin and 31% to ofloxacin. Isolates that were resistant to ceftriaxone and cefuroxime constituted 19% and 22% of the proportion while 46% of the isolates were susceptible to ceftriaxone and 27% to cefuroxime.
Figure 2: Antimicrobial susceptibility pattern of the Gram-positive isolates Key: AUG = Augmentin, CAZ = Ceftazidime; CRX = Cefuroxime, CTR = Ceftriaxone, CXC = Cloxacillin, ERY = Erythromycin, GEN = Gentamicin, OFL = Ofloxacin

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Multiple antibiotic resistance index

The MAR index of bacterial isolates is elucidated in [Table 2]. MAR index for Escherichia spp., Staphylococcus species, and Klebsiella bacterial isolates was within the range of 0.4–0.7. A total of 10 isolates, each that exhibited resistance to ceftazidime, cefuroxime, gentamicin, erythromycin, Augmentin, ampicillin, and ofloxacin, were selected.
Table 2: Multiple antibiotic resistance index of selected isolates

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Synthesis of silver nanoparticles

Treatment of silver nitrate solution with N. sativa seed extract resulted in color from pale yellow to dark brown, as illustrated in [Figure 3]. This visual identification is the first preliminary confirmation indicating the formation of AgNPs from the resulting solution of silver nitrate and seed extract. Particles began to settle at the bottom of the flask after some hours, indicating nanoparticle aggregation and the conclusion of the nanoparticle production process.
Figure 3: Silver nitrate solution and seed extract at A before incubation and the color change at B after incubation

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Characterization of silver nanoparticles

Ultraviolet-visible spectral analysis

The formation of AgNPs by reduction of silver ions due to the addition of N. sativa seed extract was followed by UV-visible spectroscopy within the wavelength 300 nm to 800 nm, as shown in [Figure 4]. Peak wavelength was observed at 378 nm which falls to 420 nm.
Figure 4: UV–visible spectrum of synthesized AgNPs using Nigella sativa seed extract, AgNPs: Silver nanoparticles, UV: Ultraviolet

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Fourier transform infrared spectroscopy analyses

The result of FITR assay carried out on both the aqueous N. sativa seed extract and the synthesized AgNPs and compared to a reference chart to determine the functional group present is highlighted in [Figure 5] and [Figure 6]. FTIR spectra [Figure 5] of seed extract of N. sativa showed peaks at 3283.8 representing O-H functional group of phenol; 2922.2 and 2855.1 indicate the presence of methyl group; 1744.4 indicates C = O stretch of carboxylate groups, while 1032.5 shows ether linkages. The observed functional groups are indicative of the presence of flavonoid, phenols, and terpenoids in the seed extracts.
Figure 5: FTIR spectrum of N. sativa seed extract, FTIR: Fourier transform infrared spectroscopy

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Figure 6: FTIR spectrum of silver nanoparticles synthesized using N. sativa seed extract, FTIR: Fourier transform infrared spectroscopy

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Similarly, the FTIR spectra of AgNPs shown in [Figure 6] had a peak at 3272.6, indicating the presence of OH groups; 2922.2 and 2855.1 show asymmetric stretching of methylene group; peak at 1632.8 indicates the presence of C = O stretch of carboxylate groups, while 1,038.2 cm−1 shows ether linkages, suggesting the presence of flavanones or terpenoids adsorbed on the surface of AgNPs.

Scanning electron microscopy and energy-dispersive X-ray spectroscopy analyses

SEM was used to gain a better understanding of the morphological characteristics of N. sativa-mediated AgNPs. [Figure 7] shows the SEM micrograph of the formed AgNPs; when a voltage of 15 kV was applied to the AgNPs, the morphology observed showed uneven sizes and shapes. [Figure 8] gives the result of energy-dispersive spectroscopy (EDS) analysis of the formed particles. The presence of metallic AgNPs in the synthesized particles was confirmed. It can be seen that Ag is the dominant element in the sample with traces of other elements confirming the formation AgNPs. Furthermore, elements such as carbon, oxygen, and nitrogen were observed.
Figure 7: SEM micrograph of silver nanoparticles synthesized using Nigella sativa seed extract

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Figure 8: EDS analysis of silver nanoparticles synthesized using N. sativa seed extract was found to be 32 nm, EDS: Energy-dispersive spectroscopy

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X-ray diffraction analysis

The XRD spectrum was recorded to confirm the crystalline structure of the formed particles and average particle size. The XRD spectra of AgNPs spanned from 10° to 80°. In the 2Θ range, major diffraction peaks were recorded at 38.1°, 44.2°, 64.4°, and 77.4°. The peaks falls within the hkl crystal planes (111), (200), (220), and (311) indicating the face-centered cubic structure of the formed particles, as shown in [Figure 9]. Using Scherrer's equation, the average particle size (D) was found to be 32 nm.
Figure 9: XRD analysis of AgNP synthesized using N. sativa seed extract, XRD: X-ray diffraction, AgNP: Silver nanoparticles

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Antimicrobial activity

The antimicrobial activity of AgNPs was evaluated against three MDR bacteria: Gram-positive S. aureus and Gram-negative Escherichia spp. and Klebsiella sp. [Figure 10]a, [Figure 10]b, [Figure 10]c clearly shows the ZOI for Staphylococcus sp., Klebsiella spp., and Escherichia spp., respectively, for AgNPs each in duplicate either side of the plates. Meanwhile, [Figure 11] shows that the MDR bacteria were susceptible to the AgNPs at different concentrations. At 100 μg/ml, Escherichia spp. had the highest ZOI of 18 mm while both Staphylococcus sp. and Klebsiella spp. were 17 mm.
Figure 10: (a-c) Represent Staphylococcus aureus, Klebsiella sp and Escherichia coli respectively

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Figure 11: Representation of inhibitory concentration of AgNPs at different concentrations against multidrug-resistant bacteria, AgNPs: Silver nanoparticles

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  Discussion Top


The macroscopic examination of the urine samples revealed the presence of pus in varying amounts in 68% of the samples which also showed significant growth on culture. Pus cells are white blood cells that have been obliterated by invading pathogens; hence, their presence in urine (pyuria) is an indicator of microbial infection. There are varying amounts in which pus cells occur; however, urine samples with a moderate-to-high amount of pus cells which yield significant microbial growth in culture (≥105 orgs/ml) are good indicators of UTI.[25] Urine samples screened without pus cells that yield significant microbial growth can be attributed to other underlying diseases such as enteric fever, bacteria endocarditis, diabetes, and so on. Identification tests of the isolates show that they belong to the genera Staphylococcus, Klebsiella, and Escherichia species and selective tests identified two isolates as S. aureus and E. coli. These three organisms have been known to be common and popular bacteria implicated in UTI. This is similar to the findings in Nigeria,[25],[26],[27],[28] and additionally, the occurrence of these organisms in this study is in conformity with the findings in Libya, Iraq, India, and Ethiopia.[29],[30],[31],[32] The differences between results from this study and those of other studies may be due to either variation in culture method, culture media used, sample size, region, or other environmental factors.

The antibiotic susceptibility pattern of Gram-negative isolates (E. coli and Klebsiella spp.) depicts a prevalence of antibacterial resistance by these strains to cephalosporin class of antibiotics (cefuroxime [100%] and ceftazidime [91%]), ampicillin (98%), and Augmentin (98%). However, all the strains had a varying percentage of susceptibility to ofloxacin (67%) and nitrofurantoin (93%). The results were similar to the work of Onyebueke et al.[33] in which E. coli and Klebsiella sp. showed resistance to cefuroxime (86.3%) and ceftazidime (71.1%). However, both organisms exhibited high resistance to nitrofurantoin (85%) when compared to the present study in which recovered isolates were highly susceptible. The work of Anejo-Okopi et al.[34] reported the pattern of resistance of UTI organisms in symptomatic patients in Maiduguri, Nigeria, and revealed the low percentage of resistance of Gram negative (E. coli and Klebsiella sp.) to ciprofloxacin (31.6%) and ofloxacin (15.8%) with high resistance to gentamicin (84.2%). The difference in resistance could be attributed to the type of antibiotics used in the locality of study and other environmental factors such as hygiene, medical facilities, and rate of antibiotic usage.

The Gram-positive isolates (S. aureus) in this study exhibited resistance to Augmentin (85%), cloxacillin (75%), and erythromycin (56%). However, the strains were susceptible to gentamicin (67%) and ofloxacin (60%). The result was similar to the work reported by Onyebueke et al.[33] which revealed that S. aureus showed resistance to erythromycin and cloxacillin at 64.3% apiece. However, they reported higher resistance to ofloxacin (64.3%) as compared to the current study which depicted that S. aureus were susceptible to ofloxacin. Lower resistance of S. aureus to ofloxacin (25%) and gentamicin (8, 3%) was also reported by Anejo-Okopi et al.[34] The resistance of UTI bacteria to commonly prescribed antibiotics is increasing in developing and developed countries. Resistance to multiple drugs has become a common feature, and most of the organisms associated with UTI have shown to be resistant to multiple antibiotics. From this study, some of the isolates were resistant to more than half of the antibiotics they were exposed to, hence they are MDR.

MAR indexing is considered a good tool for risk assessment because it gives an idea of the number of bacteria showing antibiotic resistance in the risk zone in any routine susceptibility testing. MAR index values <0.2 were considered to have originated from high-risk sources where antibiotics are often used.[35] In this study, MAR index of the isolates was found to be within the range of 0.4–0.7 depicting resistance to ceftazidime, cefuroxime, gentamicin, erythromycin, Augmentin, ampicillin, and ofloxacin antibiotics. The result showed that the isolates have MAR values <0.2, indicating that they organisms originated from areas with increased multidrug resistant organisms as previously reported by Akinleye et al.[26] This high resistance index may be attributed to the prolonged and undetected presence of pathogens in the urinary tract which were able to develop resistance to conventional antibiotics due to inappropriate use of antibiotics. Inappropriate use of antibiotics and blind diagnosis has been a consistent theme within the locality of study as individuals resort to self-medication without proper test. In addition, high prevalence of MDR in this study might be a result of the genetic features of the organisms used for the study.

The formation of AgNPs was caused by reduction of silver ions by N. sativa seed extract. Free electrons in metal nanoparticles produce surface plasmon resonance absorption band, which is caused by the combined vibrations of metal nanoparticles in resonance with light waves. Because the AgNPs were disseminated in the solution due to Brownian motion with no chance of aggregation, the crisp bands of AgNPs were observed to be around 400 nm throughout the observation time. The peak wavelength was found to be 378 nm which fell to 420 nm suggesting a high conversion of Ag+ to Ag0 as nanoparticles. Different parameters such as pH, temperature, optical charge, concentration of extract, and silver nitrate affect AgNP formation. Increase in concentration of plant extract tends to affect the size and shape of formed particles. Consequently, absorbance intensity increases resulting in the slow shift of surface plasmon peaks toward lower wavelength[36] as observed in the present study in which peak wavelength was 378 nm. This is normally rare; however, variation in absorbance peak toward lower wavelength of 360 nm was reported in a study using Catharanthus roseus leaf extract as a reducing agent in the formation of AgNPs.[37]

The dual role of N. sativa seed extract as a bioreductant and capping agent was confirmed through FTIR analysis. FITR analysis showed the different functional groups which were depicted as spectral lines. Each functional group represents the presence of a particular phytochemical. The identified functional groups representing terpenoids, phenols, and flavonoids were suggested to be responsible for the reduction of silver ions to AgNPs and subsequent capping of the formed particles. Carboxyl groups and aromatic rings were also implicated in the formation of AgNPs. According to some researchers, OH group present in flavonoid and phenols may be responsible for the reduction of silver ions to AgNPs.[38] It might also be argued that the tautomeric transformation from enol form to keto form in flavonoids leads to the release of reactive hydrogen atom that can reduce silver ions to AgNPs. In addition, concentrated terpenoids adsorbed on the surface of metal nanoparticles were due to interaction with carbonyl groups in the absence of other strong ligating agents.

SEM analysis revealed the irregular shape of the formed AgNPs. The existence of large-sized nanoparticles might be a result of protracted incubation period which resulted in aggregation. This is similar to the work reported by Widdatallah et al.[39] Irregular morphology was observed in the work using N. sativa seed to form AgNPs. The presence of metallic AgNPs in the synthesized AgNPs was further confirmed by EDS analysis. It was observed that Ag was the dominant element in the sample with traces of other elements confirming the presence AgNPs. Furthermore, elements such as carbon, oxygen, and nitrogen were observed. The presence of these biomaterials gives plant-facilitated synthesized AgNP advantage over the chemical and physically synthesized nanoparticles.

XRD analysis showed that the formed AgNPs were crystalline. Similar XRD result was reported for N. sativa-mediated AgNPs by Widdatallah et al.[39] There are identified peaks at 38.1°, 44.2°, 64.4°, and 77.4° corresponding to crystal planes (111), (200), (220), and (311) indicating the face-centered cubic structure of the formed particles. Furthermore, two unidentified intense peaks were observed occurring at 2 θ values of 28.1° and 38.4°. This indicates advanced degree of crystallinity as a result of the presence of some organic compounds incorporated on the surface of the formed particles from the seed extract. Using Scherer's equation, the average particle size (D) was found to be 32 nm.

The study proved that plant-mediated synthesized Ag-NPs were able to inhibit the growth of the uropathogens effectively. The mean diameter of ZOI (in mm) for the different isolates at the dose of 100 mg/mL concentration showed a maximum for Escherichia spp.(18 mm) followed by both Klebsiella sp., and S. aureus at (17 mm). The Gram-negative uropathogens, E. coli, showed the highest ZOI at 18 mm which can be attributed to the presence of thin peptidoglycan layer associated with Gram-negative bacteria. Gram-positive bacteria have thicker peptidoglycan which can limit the penetration of AgNPs. In a similar work on the antibacterial activity of AgNPs synthesized using N. sativa seeds, it was reported that the formed particles were effective against E. coli and S. aureus.[39] The ZOI decreases as the concentration decreases.

The phytochemicals which were implicated to be associated with the formed particles as revealed in the FTIR analysis include flavonoids, terpenoids, and phenols. These phytochemicals have been shown to possess antimicrobial activities as reported in other studies.[40] Plant mediated silver nanoparticles which are capped with flavonoids and terpenoids tend to have increased antimicrobial activity against multidrug resistant bacteria pathogens. This is because terpenoids have antimicrobial activity potential while flavonoids have been reported to possess the potential to reverse multidrug resistance mechanisms of bacteria. Thus, this combined quality potentiate the antimicrobial activity of plant mediated AgNPs. Hence, the antimicrobial activity of the formed particles was enhanced by the embedded phytochemicals pair wise. There are various proposed mechanisms of antibacterial activity of AgNPs. AgNPs are positively charged and when exposed to negatively charge bacterial cell, causes adhesion of nanoparticles onto the cell wall and the membrane. This adhesion leads to complex formation which results in membrane rupture.[41] AgNPs penetrate the cell and damage intracellular structures (mitochondria, vacuoles, and ribosomes) and biomolecules (proteins, lipids, and DNA). Interactions between the cellular structures and the biomolecules with AgNPs have a respective damaging effect on the microbes such as attaching to the ribosomes, thereby halting protein synthesis.[41],[42] AgNPs can induce cellular toxicity and oxidative stress caused by generation of reactive oxygen species and free radicals. These radicals have an adverse effect on bacteria leading to inactivation of the microbial cell.[43] AgNPs can also modulate the signal transduction pathway by dephosphorylation; thereby impacting negatively on the bacteria.[44]


  Conclusion Top


The biological synthesis of AgNPs using seeds of N. sativa as a reducing and capping agent was successful. Visual observation of color change from yellow to dark brown was indicative of AgNP formation. FTIR analysis indicated that flavonoids, terpenoids, and phenols were responsible for the synthesis of AgNPs. XRD analysis indicated the crystalline nature of the particles while SEM showed the irregular shape of the nanoparticles. Findings from this study show that AgNPs generated via biological technique utilizing N. sativa seed extract can suppress the growth of MDR bacteria pathogens that cause UTI. Thus, AgNPs synthesized from seeds of N. sativa can be used in the management of bacterial UTIs which have seen treatment failures in the past years due to the emergence of antibiotic-resistant bacteria.

Acknowledgments

The authors greatly acknowledge the staff of the Department of Microbiology, University of Nigeria, Nsukka, Nigeria, for their support toward the progress of this research. No funding was received for this research.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
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