Antibacterial Activity of Silver Nanoparticles Synthesized by Aqueous Extract of Carthamus oxycantha M.Bieb. Against Antibiotics Resistant Bacteria

: Antibiotics resistant bacteria have become a global problem as a result of the unprogrammed use of antibiotics, resulting in bacterial strains resistant to many antibiotics, or to all available antibiotics. Plants are a good source of primary and secondary metabolites that have a major role in reducing silver nitrate to silver nanoparticles (AgNPs). The production of these nanoparticles were carried out by using aqueous extract of Carthamus oxycantha M.Bieb. This can be verified by color change of the reaction solution from yellow to dark brown because of the excitation of the surface plasmon resonance. AgNPs were characterized by UV-Vis spectroscopy, where they recorded the peak at 420 nm. Fourier Transformation-infrared (FTIR) was conducted to identify the effective plant group that contributes to the formation of AgNPs and it was found that proteins and phenols have the major role in the formation of those nanoparticles. Shapes and sizes of the synthesized AgNPs were characterized by Scanning Electron Microscope (SEM) with a range of 50-80nm in size and spherical in shapes. Antibacterial activity of AgNPs were tested against Multi-Drug Resistant bacteria (MDR), Extremely antibiotics Resistant (XDR), and Pan drug-resistant (PAN) bacteria, was done in concentrations ranging from 1000-63 µg/ml. The results showed that there were significant variations between the concentrations, the tested bacteria also showed significant differences in its sensitivity to AgNPs. The results recorded a proportional relation between the type of bacterial resistance to antibiotics and it's resistant to AgNPs, therefore the most resistant bacteria to AgNPs in this study Enterobacter cloacae EN2 was resistant to all antibiotics (PAN), while Escherichia coli E11 recorded was the most sensitive bacteria to AgNPs and its resistant only to 3 antibiotics


Introduction:
There is a competition between human and pathogenic bacteria and as described by Baptista 2018 1 "A battle of the Titans", when human discovered new antibiotics, the bacteria developed new mechanisms to fight these antibiotics until the limit is reached to obtain bacteria resistant to more than one antibiotics multidrug-resistant bacteria (MDR), and then resistant to most of the antibiotics extremely antibiotic resistant (XDR) bacteria, finally resistant to all types of antibiotics pan-drugresistant bacteria (PAN). And if the situation remains as it is, it is expected that in 2050 there will be no antibiotic capable of eliminating these bacteria expect that humans discover new antimicrobial agents 2 .
Green synthesis of metal nanoparticles using plant extracts had a universal interest due to their physiochemical and its implementation in different fields of biotechnology. These methods had attention in the last decade because these metal nanoparticles are mediated by eco-friendly plant extracts with low toxicity to human. Metal nanoparticles mediated by plant extracts are characterized by high productivity in addition to its stability in size and shape and as well as having a good antimicrobial activity 3 . Plant extracts have an important role in reducing and stabilizing metal nanoparticles as they reduce the toxicity compared with using other methods in synthesizing nanoparticles. Plant extracts have secondary metabolites that play an important role in the manufacturing of metal nanoparticles such as polyphenols 4 . Silver nanoparticles show great attention because of their special characters like shapes and sizes. In the current years, green synthesis of silver nanoparticles by using plant extracts have been studied and invested for a wide range. Silver nanoparticles manufactured using plant extracts have numerous implantations as a result of their unique characters higher than in their bulk form. Silver nanoparticles have been used nearly 120 years and called colloidal silver 5 .
Carthamus oxycantha M.Bieb.belonging to the family Compositae, spread as a wild plant in Iraq. According to 6,7 , it is present in dry and open places. It is distributed in Iraq and central Asia. The plant is used in the treatment of rheumatism, male infertility, chest disease, and high blood pressure 8 .
The emergence of antibiotic-resistant bacteria is one of the biggest problems facing humanity. Antibiotic resistance bacteria first arise in hospitals and then spread everywhere, and this is because of the uncontrolled use of antibiotics, and this will lead to fewer antibiotics available to treat diseases 2. The current study aims to produce AgNPs composed by aqueous extract of C. oxycantha as antimicrobial agents since that is the first time that aqueous extract of this plant was tested as an antimicrobial agent to inhibit MDR,XDR, and PAN human pathogenic bacteria.

Materials and Methods: Preparation of plant extracts
Carthamus oxycantha plant shoot was collected from Basrah University campus in flowering period and brought to a laboratory, washed with a much amount of tap water, then washed again with DW to remove dust, left to dry at room temperature, shoot was finely grounded to a fine powder using an electrical grinder. Refluxing extraction protocol was used to extract plant materials according to 9 with some modifications.10 g of plant powdered was added to 100 ml of DW supplemented to around, mixed well, refluxed for 5 hours. It was left to cool down filtered with no.1 Whatman filter paper. Filtrate was concentrated at room temperature and kept at 4 °C until used.

Synthesis of silver nanoparticles
Green synthesis of silver nanoparticles was done according to 10 . Formation of silver nanoparticles was indicated by color change from pale green to dark brown. The formed AgNPs were centrifuged at 3000 rpm followed by washing with double distilled water.

Characterization of AgNPs
Synthesized AgNPs manufactured by using aqueous extract of C.oxycantha were characterized by using UV-Vis spectrophotometer, the range of absorbance was recorded from 350-700 nm. The dried AgNPs were documented by FTIR-84005-Shimadzu machine, Germany and in the region (500-4000cm-1) at room temperature; spectra were recorded at the University of Basrah / College of Education for Pure Sciences. SEM (Scanning Electron Microscope) analysis was done using Leo 1455vp (Germany) machine. AgNPs were coated with golds to conduct the samples.

In vitro antibacterial activity of silver nanoparticles
The antibacterial activity of AgNPs mediated by using aqueous extract of C.oxyacantha shoot was estimated against 9 isolates of MDR, XDR, and PAN Gram negative and Gram positive human pathogenic bacteria isolated from urine, stool and blood sample from out visit Al-Sadr Teaching Hospital, Central Laboratory, by agar well diffusion method according to Elbeshehy et al.2015 11 with some modifications. Briefly, tested bacterial suspension was swabbed with approximately 1X10 6 CFU/ml on Muller-Hinton agar medium wells filled with synthesized AgNPs in concentrations ranging from 1000-63 µg/ml. Antibacterial activity was recorded by the diameter of inhibition zone in mm. Statical analysis was done by Chi-Square -Friedman test with probability value P≤0.05.

Results and Discussion: Characterization of AgNPs
The color changing the reaction mixture from yellow to dark brown indicated the formation of AgNPs due to the reduction of silver ions to AgNPs, mediated by biomolecule founded in plant extracts 12 as shown in Fig. 1. The color changed due to the plasmon resonance surface of sedimented silver nanoparticles because of coherent and collective surface electron oscillation 13 . Color changed of solution due to the reduction of silver nitrate to silver nanoparticles as a result of the plant component extract, which acts as reducing, capping, and stabilizing of silver nanoparticles 14 . Uv-Vis spectrophotometer analysis technique widely used in the characterization of AgNPs formation with range of absorbance ranging from 350-700nm AgNPs recorded absorbance peak at (420) nm showed in Fig. 2. The color changing the reaction mixture from yellow to dark brown indicated formation of AgNPs due to excitation of the surface Plasmon resonance 15 . This finding is also recorded by Aboutorabi et al.2018 16 when they studied flower extract at Carthamus tinctorius to synthesized AgNPs, and they reported that Uv-Vis peak at (420)nm. that the compounds responsible for reducing silver nitrate to silver nanoparticles included free and bonded amide group of protein and polyphenols. The results above showed that protein and polyphenols may act as reducing, capping, and stabilizers of AgNPs.

. FTIR analysis of AgNPs composed by aqueous extract of Carthamus oxycantha.
Scanning Electron Microscope (SEM) images of AgNPs composed by aqueous extract of C. oxycantha are explained in Fig. 4A and B. These results characterized the shapes and sizes of Ag NPs synthesized by this method. SEM pictures showed the formation of AgNPs with range of 50-80 nm in size and spherical in shape. Aggregation of AgNPs resulted in agglomeration of these particles, which consist of large aggregates leading to the formation of large particles of AgNPs. This finding was also recorded by former researchers such as Kumar et al. 2013 20 who founded that AgNPs size was 270nm, Shah et al.2015 21 showed that AgNPS size ranges from 20-150nm, whereas Kumar et al.2017 22 reported their size at 85-120nm. Formation of these aggregations can be attributed to the aggregation of nanoparticles during sample preparation 23,19 . The formation of large-sized AgNPs was common when plant extracts were used in the green synthesis of these nanoparticles. It was found that the frequency of these large-sized nanoparticles was promoted by directly proportional to reaction temperature according to Ostwald ripening theory 24,16 .  The antibacterial activity of AgNPs synthesized by aqueous extracts of C .oxycantha. has been investigated against MDR, XDR, and PAN pathogenic bacteria showed the zone of inhibition produced. The results of the antibacterial activity of AgNPs showed that there were significant differences between the concentrations with Chi-Square 35.435, also the tested bacteria varied significantly in their sensitivity to AgNPs (Chi-Square 28.707) where Escherichia coli E1 was the most sensitive with mean rank value of 8.8, while the bacteria Enterobacter cloacae EN2 was the most resistant to AgNPs and the value of mean rank was 2 as shown in Table 1 and Fig. 5. The resistance of the bacteria AgNPs was directly proportional to its resistance to antibiotics, as the most resistant bacteria Enterobacter cloacae EN2, was recorded as resistant to all antibiotics, while Escherichia coli E11 bacteria was the most sensitive to AgNPs and was resistant to 3 antibiotics only Table 2. This can be explained as the targeted bacteria treated with AgNPs might change the permeability of the outer membrane and increased efflux pumps 25 . Here it can be concluded that the greater the resistance of bacteria to antibiotics, the greater their resistance to silver nanoparticles becomes. This can be explained by the fact that antibiotic-resistant bacteria have many mechanisms to resist anti-bacterial agents, including silver nanoparticles, and from those mechanisms is that bacteria can minimize the toxic impact of AgNPs because of reduced uptake of the plasma membrane and cell wall or augmented of nanoparticles 26 . Also, resistance to AgNPs can be assigned to the genetic changed in bacterial cells resulting in the quick emergence of resistance to AgNPs and induced transfers of managers antibiotics resistance genes 27,28 .E. coli treated with AgNPs, bacterial cells evolution of phenotypic changes may lead to the production of flagellin which weakened the antibacterial effect of AgNPs 25,29. Whereas Enterobacter cloacae EN2 in this study resistant to all antibiotics (PAN) was recorded sensitive to AgNPs, although it is the most resistant species studied. Here it can be concluded that the most resistant bacteria to antibiotics, can be inhibited with AgNPs.
Furthermore, silver nanoparticles have an effect on the growth of the MDR mechanisms involved in the resistance to antimicrobial agents 30, and these AgNPs can beat the bacterial resistant mechanisms in several ways such as Cracking the cell wall, production of reactive oxygen species (ROS) which results in inhibition of ATP production, DNA replication, cell wall shatter, increase of cell membranes permeability, inhibit RNA and proteins production, attack bacteria immune system, disarray of electron transport chain 1.

Conclusions:
Aqueous extract of Carthamus oxycantha M.Bieb was a good source to synthesize silver nanoparticles (AgNPs). These nanoparticles were characterized by UV-Vis (FTIR) and (SEM) spectroscopy, Antibacterial activity of AgNPs was tested against MDR,XDR, and PAN bacteria, in concentrations ranging from 1000-63 µg/ml. This study recorded a proportional relation between the type of bacterial resistance to antibiotics and it's resistance to AgNPs, The most resistant bacteria to AgNPs was Enterobacter cloacae EN2 was resistant to all antibiotics, while Escherichia coli E11was recorded the most sensitive bacteria to AgNPs and its resistant only to 3 antibiotics.