Green Synthesis of AgNPs and Antibacterial Activity against MDR Pathogens Vol. 12 (2), Dec 2021 ISSN (Print): 2305 – 8722 ISSN (Online): 2521 – 8573 R A D S J . B i o l . R e s . A p p l . S c i . 98 Op e n Ac c e s s F u l l L e n g t h A r t i c l e Green Synthesis of Silver Nanoparticles from Flowers of Helianthus annus and Tagetes erecta and their Antibacterial Activity against MDR Pathogens Sadaf Shaheen1, Zelle Humma2, Iqra Arooj1, ⃰, Sana Batool1 1Department of Microbiology & Molecular Genetics, The Women University, Multan, Pakistan. 2Department of Biochemistry & Biotechnology, The Women University, Multan, Pakistan. A B S T R A C T Background: Multidrug Resistant (MDR) organisms are of major concern to healthcare sector as increasing antimicrobial resistance has made it obligatory to focus on alternative options. Investigating the medicinal benefits of nanoparticles synthesized from biological sources is relevant in this regard. Objectives: Main purpose of the current study was to explore antibacterial potential of silver nanoparticles (AgNPs) synthesized from flower extracts of Helianthus annus and Tagetes erecta against MDR bacteria. Methodology: MDR bacteria were isolated from sewage water of hospitals and characterized by Gram staining and biochemical analysis. From aqueous and methanolic extracts of H. annus and T. erecta flowers, silver nanoparticles were prepared and then characterized and confirmed by visual analysis, FTIR spectroscopy, and UV-Visible spectroscopy. Antibacterial activity of crude extracts and their nanoparticles was evaluated by well diffusion and disk diffusion methods, respectively. Results: Bacterial isolates included MDR Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumoniae, Salmonella spp., Shigella spp. and Proteus spp. as well as Methicillin-Resistant Staphylococcus aureus were identified. Instant color change in extract solutions upon addition of silver nitrate confirmed the formation of nanoparticles. FTIR and UV-Visible spectroscopy showed response of biomolecules present in flower extracts during formation of silver nanoparticles, indicating the presence of different phytochemicals. Antibacterial activity of crude extracts increased greatly after synthesis of nanoparticles and they produced zones of inhibition against all of the MDR bacteria included. Methanolic extracts and their nanoparticles possessed more antibacterial power compared to aqueous extracts. Conclusion: Our findings suggest the presence of potent antibacterial components in flowers of H. annus and T. erecta which necessitates further evaluation. Keywords Antibiotic resistance, Helianthus annus, Multidrug Resistance, Nanoparticles, MDR, Tagetes erecta. *Address of Correspondence iqra.6051@wum.edu.pk Article info. Received: May 09, 2021 Accepted: July 06, 2021 Cite this article Shaheen S, Humma Z, Arooj I, Batool S. Green Synthesis of Silver Nanoparticles from Flowers of Helianthus annus and Tagetes erecta and their Antibacterial Activity against MDR Pathogens. 2021; 12(2):98-107. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium provided the original work is properly cited. I N T R O D U C T I O N Multidrug Resistant (MDR) organisms are the principal cause for the morbidity and mortality worldwide. In terms of contagious diseases, MDR pathogens are the obligatory reason for annual mortality1. Due to acquired chromosomal mutations or transposon-mediated transfer, microbes incorporate genes that make them intrinsically resistant to O R I G I N A L A R T I C L E Green Synthesis of AgNPs and Antibacterial Activity against MDR Pathogens Vol. 12 (2), Dec 2021 ISSN (Print): 2305 – 8722 ISSN (Online): 2521 – 8573 R A D S J . B i o l . R e s . A p p l . S c i . 99 antibiotics2. Methicillin-Resistant Staphylococcus aureus (MRSA) and Extensive-Spectrum Beta-Lactamase (ESBL) producers have attained the ability to dissolve penicillins and cephalosporins. Members of several bacterial genera have acquired ESBL genes and produce enzymes that enable them to demonstrate resistance against multiple antimicrobial agents3. The mechanism of resistance in microbes may involve metabolic rearrangements in some pathogens4. Bacteria acquire antibiotic resistance owing to the modification of extracellular proteins and DNA, as well as formation of thick biofilms5. Rapidly increasing resistance against known antibiotics necessitates the exploration of alternative drug sources to combat microbial diseases. Phytochemicals present in plants such as vitamins (A, C, E, and K), flavonoids, terpenoids, saponins, enzymes, pigments, and minerals have been reported to possess antimicrobial, antioxidant and anti-cancerous activities6. According to the World Health Organization (WHO), approximately 80% of people rely on medicinal plants for treatment of ailments7. Recently, nanotechnology has extended biological systems for the production of nanoscale materials by using superficial techniques8. Regarding biomedical approaches, nano techniques are being used in tissue engineering, drug delivery, and biosensor production5. In phyto-nanotherapy, the effect of plants is combined with metal nanoparticles which are equivalent to synthetic drugs, with the advantage of being associated with less side effects9. Nanoparticles clasp more surface atoms which enhances their functional aptitude. Among metals employed for nanoparticle synthesis, silver holds great promise owing towards exhibition of high level of toxicity against microorganisms and representation of the minimal harmful effects on mammals10. Silver has been incorporated in many ointments and creams where it acts as an antimicrobial agent to prevent infections from burns and wounds11. Silver Nanoparticles (AgNPs) synthesized from flower extracts have exhibited antimicrobial activity against several pathogens12. They were detected as a reservoir of Ag+ ions, which lead to deprivation of cell membrane by incorporating themselves into bacterial proteins13. This biosynthesis-based approach is attractive because AgNPs synthesis could be done extra-cellularly by using cell-free extracts of biomolecules loaded in free amino acids and organic acids. AgNPs suspension was shown to demonstrate intrinsic antibacterial activity against MDR pathogens as well, and it has been established that the colloidal AgNPs could eradicate growth of pathogenic bacteria14. With reference to Helianthus annus, ozonated vegetable oils like canola, olive, and sunflower are used against bacterial infections15. Tagetes erecta flower possesses many pharmacological properties and is also used as food colorant as it possesses many bioactive compounds which are the key determinants for its antioxidant and antibacterial activity16. In view of these observations, the present study aimed at exploring the antibacterial potential of AgNPs synthesized from Helianthus annus and Tagetes erecta flower extracts against MDR pathogens. M A T E R I A L S A N D M E T H O D S Isolation and Characterization of Bacteria Sewage water was collected from Nishtar Hospital and Family Hospital, Multan, Pakistan, in air-tight containers. Serial dilutions of sewage water were plated onto nutrient agar to obtain pure isolates, which were then characterized based on Gram staining, growth on McConkey, EMB and mannitol salt agar, as well as a series of biochemical tests including MR-VP, TSI, citrate utilization, starch hydrolysis, gelatin hydrolysis, and indole production. Using Kirby- Bauer disk diffusion method, MDR status of isolates was ascertained. For this purpose, isolated organisms were cultured on Mueller-Hinton agar plates and incubated at 37ºC for 18-24hrs after placement of antibiotic disks. Antibiotics used for this purpose included Vancomycin (30µg), Imidazole (20µg), Imipenem (30µg), Augmentin (10µg), Ceftazidime (30µg) and Methicillin (30µg). Subsequently, zones of inhibition were measured using CLSI guidelines so as to establish status of bacteria as sensitive (S), intermediate (I) or resistant (R) to the antibiotics used. Preparation of Flower Extracts Floral part of two plants Helianthus annus and Tagetes erecta, were collected from field area of Multan in sterile plastic bags and transported to the laboratory of Department of Microbiology and Molecular Genetics, The Women University, Multan. The collected floral parts were washed several times with running water followed by distilled water in last step to remove dust particles. For Green Synthesis of AgNPs and Antibacterial Activity against MDR Pathogens Vol. 12 (2), Dec 2021 ISSN (Print): 2305 – 8722 ISSN (Online): 2521 – 8573 R A D S J . B i o l . R e s . A p p l . S c i . 100 aqueous extraction, 20g of fresh petals of each flower were added to 60ml of distilled water and boiled on slow heat for 1hr with continuous stirring. Then the extract was filtered, allowed to cool down at room temperature and stored at 4ºC for future use. For the preparation of methanolic extracts, petals were oven dried at 45ºC for 3-5 days and grinded into fine powder later on. Oven-dried powder (20g) of each flower was added separately to 60ml of methanol and mixed with continuous shaking in shaking incubator at 15,000rpm for 72hrs. Then, it was evaporated to obtain concentrated solution and stored at 4ºC for further use. Synthesis and Characterization of Silver Nanoparticles Stock solutions of flower extracts were added to 5mM colorless silver nitrate solution separately in dark room to minimize photoactivation of silver. Color change observed after 5min and 10min served as an indication for successful synthesis of nanoparticles. These solutions were centrifuged 2-3 times at 12,000rpm for 15min, supernatant was discarded every time to obtain purified particles and pellet was oven-dried at 45-50°C12. Characterization of these Silver Nanoparticles (AgNPs) was done by UV- Visible spectroscopy and Fourier Transform Infrared (FTIR) spectroscopy. FTIR spectrum was recorded in the range of 500-4000cm-1. Various modes of vibration were observed in transmittance mode. The reduction of Ag+ to Ag° was monitored by measuring the UV-Visible spectrum within the range of 400-800nm at a resolution of 10nm in the UV-Vis spectrophotometer including methanol as control. Analysis of Antibacterial Activity of Flower Extracts and AgNPs For the assay of antibacterial potential of crude floral extracts as well as synthesized AgNPs, two alternative approaches were employed: well diffusion and disk diffusion. All the analyses were performed in triplicates so as to minimize chances of error. For well diffusion analysis to check antibacterial activity of crude extracts, holes of 6mm were punched on Mueller Hinton agar plates after swabbing isolates on them. Extract solution (20μl) was added to each well and plates were left at room temperature for 1hour to allow diffusion of extracts followed by incubation at 37ºC for 24hrs. Afterwards, zones of inhibition were measured and recorded in mm. For AgNPs activity analysis, filter paper disks were made by soaking those in AgNPs solution for 30min and subsequently drying for 5-10min. These discs were placed onto Mueller Hinton agar plates after swabbing of isolates and incubated at 37ºC for 24hrs, subsequent to which, zones of inhibition were measured in mm. R E S U L T S Bacterial Isolates and their MDR Status Isolated organisms included Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, Klebsiella pneumoniae, Salmonella spp., Shigella spp. and Proteus spp. whose identity was ascertained following the scheme provided by Bergey’s Manual of Systematic Bacteriology. MDR status of these isolates was established based on resistance to more than one of the antibiotic drugs tested as documented in Table 1. With the exception of S. aureus, all pathogens were MDR. S. aureus was however, resistant to methicillin. Majority of the isolates demonstrated resistance against vancomycin and augmentin whereas, the most effective drug against majority of the pathogens was ceftazidime. Table 1. Antibiotic Sensitivity Profiles of Bacteria Isolated from Sewage Water. Test Organism Vancomycin Imidazole Imipenem Ceftazidime Augmentin Methicillin E. coli S R S R R I S. aureus S I I S S R P. aeruginosa R I R S R I K. pneumoniae R I R S R S Salmonella spp. R S S I I R Shigella spp. S I R S R I Proteus spp. R I S R R I Green Synthesis of AgNPs and Antibacterial Activity against MDR Pathogens Vol. 12 (2), Dec 2021 ISSN (Print): 2305 – 8722 ISSN (Online): 2521 – 8573 R A D S J . B i o l . R e s . A p p l . S c i . 101 Characterization of AgNPs Synthesized from Floral Extracts Silver Nanoparticles (AgNPs) synthesized from H. annus and T. erecta floral extracts were characterized based on visual analysis, FTIR spectroscopy and UV-Visible spectroscopy. Visual analysis on the basis of color change is used as a convenient indication for successful synthesis of AgNPs and the reason for the observation of this color change is surface plasmon resonance. For H. annus, the color of solution changed from colorless to dark yellow 5min after the addition of silver nitrate solution while that of T. erecta changed to dark green (Fig. 1). After 10min, the color became dark as resonance increases with the passage of time. FTIR analysis was performed to evaluate the presence of characteristic phytochemicals in floral extracts, which act as reducing and capping agents. FTIR bands for H. annus were observed in different regions with the broadest absorbance band observed at 3404.30cm-1 (Fig. 2). FTIR bands for T. erecta were also observed at different values but the broadest band was observed at 3401cm-1 (Fig. 3). UV-Visible spectroscopy analysis was performed to establish reduction of Ag+ to Agº which is characteristic feature of silver demonstrating maximum absorbance in the range of 425nm. Absorbance for both flowers was between 1.0 and 1.5, in the range of 400- 450nm which confirmed the presence of AgNPs (Fig. 4). Figure 1. Visual analysis of AgNPs synthesized using (A) H. annus and (B) T. erecta floral extracts. Figure 2. FTIR analysis of AgNPs synthesized from H. annus flowers. Green Synthesis of AgNPs and Antibacterial Activity against MDR Pathogens Vol. 12 (2), Dec 2021 ISSN (Print): 2305 – 8722 ISSN (Online): 2521 – 8573 R A D S J . B i o l . R e s . A p p l . S c i . 102 Figure 3. FTIR analysis of AgNPs synthesized from T. erecta flowers. Figure 4. UV-Visible spectrum of AgNPs synthesized from (A) H. annus and (B) T. erecta flowers. Antibacterial Activity of Crude Floral Extracts and their AgNPs In general, the antibacterial activity of crude floral extracts was enhanced after synthesis of their AgNPs, although exceptions existed (Table 2). This observation is reinforced by the fact that aqueous floral extract of H. annus was completely ineffective against E. coli and Salmonella spp. while that of T. erecta was not efficacious against P. aeruginosa. However, these demonstrated antibacterial effect against aforementioned pathogens subsequent to the synthesis of AgNPs. It was further documented that crude extract as well as AgNPs of H. annus flowers exhibited stronger bactericidal effect against most of the MDR isolates (Fig. 5). However, T. erecta flowers were much more effective against E. coli as compared to H. annus flowers. Likewise, in comparison with corresponding extract or AgNPs of H. annus flowers, crude aqueous extract of T. erecta flowers possessed stronger Green Synthesis of AgNPs and Antibacterial Activity against MDR Pathogens Vol. 12 (2), Dec 2021 ISSN (Print): 2305 – 8722 ISSN (Online): 2521 – 8573 R A D S J . B i o l . R e s . A p p l . S c i . 103 bactericidal activity against Salmonella spp. and AgNPs synthesized from its methanolic extract displayed more potency against Shigella spp. On the whole, methanolic extracts of both flowers as well as AgNPs synthesized from it were more effective against MDR isolates in comparison with aqueous extract and AgNPs synthesized from it, and the difference was noteworthy in some cases. The strong antibacterial potential of both flowers was revealed by the observation that AgNPs synthesized from methanolic extract of H. annus completely cleared the growth of S. aureus, and K. pneumoniae and those of T. erecta cleared growth of Shigella spp. Table 2. Antibacterial Activity Assay of H. annus and T. erecta Floral Extracts and their Synthesized AgNPs. Test organism / Type of extract AgNPs of T. erecta (Zone of inhibition in mm) AgNPs of H. annus (Zone of inhibition in mm) Crude extract of T. erecta (Zone of inhibition in mm) Crude extract of H. annus (Zone of inhibition in mm) E. coli Aqueous extract 17 12 08 00 Methanol extract 13 15 19 06 S. aureus Aqueous extract 14 18 12 17 Methanol extract 14 Clear 16 16 P. aeruginosa Aqueous extract 10 16 00 14 Methanol extract 11 16 04 14 K. pneumoniae Aqueous extract 13 17 06 15 Methanol extract 12 Clear 12 16 Salmonella spp. Aqueous extract 12 15 07 00 Methanol extract 14 19 06 08 Shigella spp. Aqueous extract 14 15 09 10 Methanol extract Clear 19 09 12 Proteus spp. Aqueous extract 08 10 15 16 Methanol extract 12 12 14 19 Green Synthesis of AgNPs and Antibacterial Activity against MDR Pathogens Vol. 12 (2), Dec 2021 ISSN (Print): 2305 – 8722 ISSN (Online): 2521 – 8573 R A D S J . B i o l . R e s . A p p l . S c i . 104 Figure 5. Evaluation of antibacterial activity of extracts by well diffusion and disk diffusion methods: A) Crude extracts against Methicillin-resistant S. aureus, T= T. erecta, H= H. annus; B) Crude extracts against Proteus spp., T= T. erecta, H= H. annus; C) AgNPs against E. coli, T= T. erecta, H= H. annus, Ag=AgNO3 solution, C=Control; C) AgNPs against P. aeruginosa, T= T. erecta, H= H. annus, Ag=AgNO3 solution, C=Control. D I S C U S S I O N Due to its multi-targeted action, silver has already been established as a promising agent in overcoming antibiotic resistance because bacterial cell membranes have been documented to be harmed by AgNPs owing to structural changes, and this property can be used to enhance susceptibility of bacteria towards antibiotics17. Silver Nanoparticles (AgNPs) have specific optical, electrical, and thermal properties, as well as a large surface area to volume ratio, allowing them to associate with bacterial surfaces optimally, resulting in superior antimicrobial activity18. Binding of silver ions to bacterial cell macromolecules causes structural alterations and deformations as well as degradation of essential enzymes by interaction with thiol groups, cumulating in the form of oxidative stress19. Plant-based compounds possess pharmacologically significant attributes as their secondary metabolites act as anti-oxidant, anti-microbial, anti-cancer, anti-inflammatory agent etc20. Furthermore, various parts of plants are non-toxic, economic, eco-friendly and safe for the synthesis of nanoparticles with an added advantage of being environment friendly21. In the present study, nanoparticles were synthesized using T. erecta and H. annus whose flowers are used for ornamental, cosmetic and medicinal purposes. Characterization of AgNPs synthesized from T. erecta and H. annus flower extracts relied upon spectroscopic analysis. Visual analysis in terms of color change serves as a simple spectroscopic signature marking the successful synthesis of nanoparticles12. Color change observed after addition of silver nitrate solution to crude flower extracts was rapid and instantaneous and the intensity of color increased with the passage of time due to shift in surface plasmon resonance. Several previous studies have documented change in the color of solution as an indication for formation of nanoparticles from various plant sources and it occurs due to reduction of silver ions and excitation of surface plasmon vibrations which are characteristic attributes of silver22-24. To identify the functional groups responsible for reduction of silver ions to AgNPs, FTIR spectrum was generated in the range of 500- Green Synthesis of AgNPs and Antibacterial Activity against MDR Pathogens Vol. 12 (2), Dec 2021 ISSN (Print): 2305 – 8722 ISSN (Online): 2521 – 8573 R A D S J . B i o l . R e s . A p p l . S c i . 105 4000cm-1. FTIR spectrum for T. erecta produced peaks in the range of 557.45-3862.58cm-1 and H. annus produced the broadest band at 3404.30cm-1. These narrow peaks correspond to functional groups like flavonoids, saponins, terpenoids etc. These observations were in accordance to those reported earlier for some other plant sources25. The presence of narrow peaks implies that nanoparticles synthesized were of small size within the range of 1-100nm. Presence of AgNPs was further confirmed by UV-Visible spectroscopy and maximum absorbance was observed at 425nm. This is characteristic attribute of silver which is used to confirm the presence of AgNPs, and has been reported previously by other researchers as well26. Two different types of extracts were prepared from flowers of T. erecta and H. annus i.e. aqueous and methanolic. Methanolic extract was more effective against MDR organisms possibly due to the presence of hydrophobic phytochemicals in the solution. Effectiveness of methanolic extract compared to other solvents has been reported for Aegle marmelos fruit as well27. Silver Nanoparticles (AgNPs) of H. annus possessed greater antibacterial activity as compared to T. erecta particularly against gram- positive bacteria. This was probably due to difference in cell wall composition as thin wall of gram-negative bacteria leads to poor penetration of silver nanoparticles28, 29. Additional factors determining antibacterial activity of AgNPs include availability of particles in the medium, their electrostatic interactions and bacterial sensitivity to them30, 31. We observed a general increase in the antibacterial potential of both floral extracts subsequent to the synthesis of AgNPs which is in line with previous reports32. Plant-AgNPs exhibited antibacterial activity against all the pathogens tested but particularly against S. aureus, Shigella spp. Similar to these findings, zones of inhibition have been shown by synthesized nanoparticles against pathogens like S. aureus, B. cereus, and P. aeruginosa33. Additionally, antibacterial potential of plant-AgNPs has been reported against MDR pathogens including E. coli, K. pneumoniae and A. baumannii34. The exact mechanism underlying antibacterial potential of AgNPs has not been deciphered yet. Theories proposed in this regard implicate altered membrane permeability, generation of reactive oxygen species, release of membrane lipopolysaccharides as well as disintegration of membrane potential35-38. C O N C L U S I O N Inappropriate use of antibiotics has become significant and predominant problem as it is leading towards rapid increase in resistance among pathogens. The combined effect of plant and metal nanoparticles is equivalent to synthetic drugs with lower side effects. Biosynthesis-based approach is attractive and effective as demonstrated by antibacterial potential of H. annus and T. erecta flowers against MDR organisms in this study which must be explored by further research. C O N F L I C T S O F I N T E R E S T None. F U N D I N G S O U R C E None. A C K N O W L E D G M E N T S None. 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