Evaluation of the Antibacterial Inhibitory Activity of Chitosan Nanoparticles Biosynthesized by Streptococcus thermophilus

INTRODUCTION
Chitosan is a natural cationic nontoxic biopolymer made from the deacetylation of chitin, which consists of N-acetyl-D-glucosamine and D-glucosamine components crosslinked by -1,4-glycosidic links [1,2]. Many studies have investigated the antibacterial action of Chitosan [1–4], and until now various forms of Chitosan derivates have been fabricated to improve its antibacterial action [5–8]. Chitosan demonstrates further unique biological properties, for instance, biodegradability and biocompatibility that assist in its use in numerous medical applications [2,3,9]. As nanotechnology has recently played a progressively more significant role because it increases the capability to expand antibacterial research to the molecular and atomic levels [2]. Chitosan nanoparticles NPs have exceptional biochemical characterization [10–14] and have been synthesized by several methods [12]. 
There is strong evidence that the bacterial cell wall or membrane is involved in Chitosan nanoparticles’ antibacterial action. Many other ideas have been proposed to try and make sense of this behavior. Electrostatic repulsion between the glucosamine’s positively charged amino groups and the bacteria’s negatively charged cell membranes is one of them [15]. By causing a shift in membrane permeability, this interaction ultimately causes an osmotic imbalance, followed by the outflow of intracellular chemicals and cytoplasm, and finally cell death [16-18]. Additionally, chitosan nanoparticles can modify the electron transport chain of bacteria [19]. 
It is generally known that bacteria may fuse and gather molten ions of metals and metalloids. Toxic metal ions can be converted by individual bacteria into safe nanoparticles [20,21]. In this case, some bacteria are used as nano-bioreactors, synthesizing materials with unique properties [22]. since it has low energy consumption and process controllability, the bacterial synthesis of nanoparticles is an extremely promising field [20].  At present, several studies have revealed synthesizing of metal nanoparticles such as zinc oxide ZnO, silver, copper oxide CuO and iron oxide nanoparticles by various types of bacteria [23]. The antimicrobial characteristics of nanoparticles have been known for the most recent periods, which paved the route to be exploited in various contemporary bio-medical applications to terminate or control some microbial infection diseases [22]. Streptococcus thermophilus (S. thermophilus), one of the most important lactic acid bacteria (LAB), has been extensively applied in combination with other bacteria species in the dairy industry [21]. S. thermophilus have antibacterial and antioxidative features and the current study indicated its ability to synthesize the chitosan nanoparticles, therefore the main aim of the current experiment is to produce the chitosan nanoparticles by the S. thermophilus and apply these nanoparticles to verify its antimicrobial abilities. To the best of our knowledge, there are no reports about the antibacterial activities of the chitosan nanoparticles from S. thermophilus especially the effect on the bacteria isolated from the diabetic feet named Aeromonas hydrophila and Citrobacter freundii.

MATERIALS AND METHODS
Identification of the efficient isolate and large-scale production of chitosan NPs
The identification of species sequence was revealed previously via PCR with universal primers [24] for the S. thermophilus. The S. thermophilus isolate was screened against E. coli using the Muller Hinton agar well diffusion assay and incubated for 24 hours at 37ºC. An efficient isolate was selected based on antimicrobial activity, color change, and absorption spectrum efficiency. Preparation of cell-free supernatant of Streptococcus thermophilus was inoculated in ready and autoclaved (sterile) broth. After that, bacteria were grown in brain heart infusion broth at 37ºC for 24h. After 15 minutes of incubation, the colloidal suspension was centrifuged at 10,000 rpm to separate the precipitate. The supernatant was then mixed with a solution of distilled water, (chitosan, 2 mg), and (acetic acid, 2ml). After the inoculum was activated, the BHI broth was inoculated (incubation time: 24 hours at 37°C), the culture was incubated for biomass production, and the cell-free supernatant was prepared by centrifugation at 4200xg for 15 minutes at 4°C, after which the pellets of cells of microorganism were discarded and the cell-free supernatants were collected for use in the biosynthesis of chitosan nanoparticles. 
The supernatant was collected by centrifuging the broth culture at 4200xg in a large-scale, refrigerated centrifuge that could accommodate 50 ml tubes, thereby pelleting the cells at the bottom of the tubes. To make chitosan nanoparticles, the supernatant was collected in a clean flask. The strains were inoculated, and then incubated for 24 hours at 37 °C with a shaking incubator at 150 rpm and 400 ml of BHI broth with 4 gm of chitosan as a substrate and 4 ml of acetic acid in each autoclaved flask. This was repeated at different times.

Characterization of biosynthesized chitosan NPs
Physical properties of biosynthetic NPs were studied using XRD, UV visible, and AFM. Detection of the chitosan nanoparticle formation was estimated with UV-Visible spectroscopy (Shimadzu UV-visible1800 spectrophotometer) in the research laboratory of Imam Ali Holy Shrine. While Scanning Electron Microscope (SEM) Analysis, Energy Dispersive X-Ray Spectroscopy (EDS) Analysis, Atomic Force Microscope (AFM), and X-ray Diffraction (XRD) Analysis were examined at the University of Tehran’s electron microscopy unit 

Antibacterial activity of chitosan NPs
The antibacterial activity of chitosan NPs were examined at various concentrations against diabetic feet bacteria Aeromonas hydrophila and Citrobacter freundii using the agar diffusion method. With 100 μl of bacterial suspension, the agar plate was inoculated. Bores (6 mm in diameter) were then produced using a sterile borer and filled with 150 μL of chitosan NPs. Subsequently, the Petri dishes were kept at 4 ° C for 2 h, then again incubated at 37 ° C for 24. Antibacterial activities were calculated, and the values were provided as means of triplicate analysis by measuring the growth inhibition zone diameter in millimeters.

Antioxidant Activity of Chitosan NPs
One hundred pure chemical compounds were evaluated using a modified DPPH radical cation technique. DPPH (8 mg) dissolved in MeOH (100 mL) for 80 µl /mL solution. 100 µl DPPH reagent was combined with 100 µl of sample in a 96-well microplate and incubated at room temperature for 30min. After incubation, absorbance was measured at 514 nm using an ELISA reader (TECAN, Gröding, Austria), and 100% methanol was used as a control. Formula for DPPH scavenging effect:

DPPH scavenging impact (%) = (Ao –A1) / Ao × 100 

where Ao was the absorbance of the control and A1 was the absorbance in the presence of the chitosan. The real absorption decrease caused by test compounds was compared with the positive controls [25]. We extrapolated the results of regression analysis to determine the IC50 DPPH values (the concentration of sample necessary to block 50% of DPPH radicals). Based on this IC50 value, the antioxidant was rated.

RESULTS AND DISCUSSION
Selection of the efficient isolate that produces chitosan NPs
Twenty-five isolates of various Streptococcus thermophilus strains were screened based on the ability of bacteria to synthesize the chitosan NPs depending on the color change from light to dark, UV-Visible Spectroscopy, and the biological activities of the chitosan NPs against E. coli to select the efficient strains. The efficient isolate was identified as Streptococcus thermophilus based on morphological, biochemical identification, and VITEK-2 compact system. 
Diverse microorganisms can synthesize inorganic materials either intracellularly or extracellularly or both ways. The microbial cell has an exceptional ion transfer mechanism in an intracellular way. The negatively charged bacterial cell wall absorbs positively charged metal ions due to electrostatic activity [19]. In addition, enzymes in the bacterial cell wall break down the metal ions into their component nanoparticles. The extracellular technique, on the other hand, involves the bioreduction of metal ions into the corresponding NPs by the secretion of reductases from the microbial cell [22].

Large scale production of chitosan nanoparticles using Streptococcus thermophilus
Streptococcus thermophilus bacteria were subjected to use in the biosynthesis of chitosan NPs, demonstrating the competence in extracellular biosynthesis by using cell-free supernatant after the addition of chitosan as a substrate under previously optimized conditions. The reaction mixture turns from light to dark. The production of chitosan NPs by Streptococcus thermophilus was monitored by observing the color change and antibacterial activity after incubation in shaking at 150 rpm. Many techniques exist for the production of chitosan nanoparticles, including ionic gelation, complex coacervation, emulsion cross-linking, spray drying, and a biological method [26]. Manufacturing through biological systems is a relatively new field, and microorganisms are being eyed by scientists as a possible environmentally benign nano-factory [12]. Because of its repeatability and adaptability, the biological method was used. The size and surface charge of the resulting particles may be changed to some extent using this technology, making it a highly manipulable process [26].

Characterization of biosynthesized chitosan NPs
UV-visible Spectroscopy
Nanoparticle biogenesis may be confirmed by seeing their formation and by measuring their absorbance band with UV-visible spectroscopy. Nanoparticles formed from the reaction mixture have a 280 nm peak in their absorption (Fig. 1). This demonstrates the existence of nanoparticle surface plasmon resonance (SPR), and a single SPR band shows that the nanoparticles are spherical in form. The production of nanoparticles was investigated using a UV-Visible spectrophotometer, which indicated a single peak at 280 nm, suggesting the presence of chitosan nanoparticles in the reaction solution. According to a report performed by Gabriel et al., 2017 [27].

Scanning Electron Microscopy (SEM) Analysis
Biogenic chitosan nanoparticles were approved for morphology and size by scanning electron microscopy (SEM). Chitosan nanoparticles generated by Streptococcus thermophilus were well-distributed and spherical in form, with sizes ranging from 29.9 nm to 51.8 nm (Fig. 2).
Nanoparticles (NPs) varied in size from 10 nm to 60 nm. The majority of NPs were the same size, and just a tiny fraction was larger than 60 nm. While prior research has employed various biological chemicals as reducing and stabilizing agents, the approach adopted in the current work allowed for the generation of chitosan NPs of lower size.

EDS analysis of biogenic chitosan NPs 
Energy Dispersive Spectroscopy (point and mapping analysis) was used to quantify the presence of chitosan nanoparticles by observing the optical absorption peaks of elements. Elemental analysis exhibited the percentage of elements constituent of CHNPs synthesis by Streptococcus thermophilus was 33.31% carbon, 10.24 % nitrogen, 50.55% oxygen, 5.34% phosphate, and 0.55% chloride (Fig. 3). The energy dispersive spectroscopy analysis recognized components, allowing for quantitative and qualitative analysis of the carbon, oxygen, nitrogen, phosphate, and chloride constituents in the formulations produced.

Atomic Force Microscope (AFM)
 AFM image of chitosan nanoparticle synthesis by Streptococcus thermophilus, gave information on the morphology of biogenic chitosan NPs. The average diameter of chitosan NPs biosynthesis from Streptococcus thermophilus was 47.18 (Fig. 4) which showed a three-dimensional image. 
AFM’s extraordinary resolution allows for precise three-dimensional visualization of molecular structures, as well as atomic-scale strategies. The procedure for preparing samples for AFM is straightforward. Because samples may be seen under circumstances that are almost identical to those found in living organisms, AFM can capture the dynamic processes that occur within molecules, organelles, and other structures in real-time [28].

X-ray Diffraction (XRD) Analysis
The crystalline structure of CHNPs was studied using XRD. The diffraction pattern of the polymorph was assigned to the characteristic peaks of chitosan polymer found at 2Ɵ=21o. The XRD spectrum of biogenic chitosan nanoparticles seemed at 20o -26o. The sharp peak indicated the presence of bioorganic coupons/proteins in the nanoparticles during the synthesis process (Fig. 5). 
X-ray diffraction was used to calculate the typical size of a chitosan nanoparticle. X-ray diffraction (XRD) was used to look into the crystalline structure of CHNPs, and the peak at 2Ɵ=21o was assigned to the polymorph of chitosan. When X-ray diffraction was applied to CHNPs, the spectra occurred at 20o -26o. Sharp peaking suggested the incorporation of bioorganic couponed/proteins into the nanoparticles.

Antibacterial activity of chitosan NPS 
Biogenic chitosan nanoparticles synthesized by Streptococcus thermophilus have been evaluated for their antibacterial activity against some multi-drug resistance bacterial pathogens (MDR). The agar well diffusion method was used for detecting the antibacterial activity of biogenic chitosan nanoparticles. Chitosan NPS with different concentrations (100, 200, 400 ug/ml) showed inhibition activity against all tested bacteria and the highest inhibition zone of Chitosan NPs observed in Gram-Positive bacteria was (26mm) in Aeromonas hydrophila and Citrobacter freundii with concentration (400 ug/ml), while the lower inhibition zone in Gram-Negative bacteria was (18mm) in E. coli with the same concentration. This inhibitory effect increased with increasing concentrations of chitosan NPS as in Fig. 6. Consistent with previous studies [16], we found that high quantities of Chitosan NPs inhibited bacterial growth. Based on the findings, it is clear that nanoparticles may effectively halt the proliferation of both gram-positive and gram-negative bacteria. When exposed to biogenic chitosan NPs, gram-positive bacteria were more sensitive than gram-negative bacteria. This agrees with [29] who reported GP bacteria are more susceptible to chitosan’s antibacterial effect than GN bacteria. As a result, interpreting bacterial exposure to chitosan is difficult. Since the rising threat of multidrug resistance (MDR), the scientific community has made it their mission to find an effective alternative to replace existing antibiotics that have become resistant. Areas an effective new entry-drug supplement to antibiotics Nanoparticles are now seen as a promising solution to antibiotics and seem to have a high potential to overcome the bacterial MDR problem [21].

Antioxidant Activity of Biogenic Chitosan Nanoparticles
 The 1-Diphenyl-2-picrylhydrazyl (DPPH) free radical scavenging experiment was performed to assess the antioxidant activity of Streptococcus thermophilus NPs in vitro by decreasing DPPH free radicals. After adding 100, 200, 300, and 400 ug/ml nanoparticles to DPPH solution, absorbance was measured at 517 nm. Color change showed nanoparticles’ capacity to scavenge DPPH free radicals. DPPH reduces the activity of biogenic chitosan NPs as their concentration increases. 28% in 100ug/ml, 30% in 200ug/ml, 42% in 300ug/ml, and 54% in 400ug/ml (Fig. 7).
 The DPPH scavenging activity of NPs increased as their concentration increased, suggesting that the percentage of inhibition of DPPH increased as the concentration of chitosan NPs increased, indicating that DPPH showed more inhibition due to increased electron donation and agrees. Antioxidants work not only by scavenging free radicals but also by inhibiting free radical formation [30].

Anti-biofilm activity of chitosan
The most crucial characteristic of bacteria is a biofilm, which increases bacterial adherence to surfaces like those found on surgical tools and prostheses. Changes in colony color on congo red agar were recorded as a qualitative measure of biofilm development; Figs (3-8). Chitosan NPs were tested with three different concertation (100,200,400) for antibiofilm activity against two strains of bacteria involving (Aeromonas hydrophila and Citrobacter freundii). The formation of biofilm in both isolates was prevented by chitosan NPs. The anti-biofilm activity increased with increasing the concentration of biogenic Chitosan NPs (Figs 8 and 9).
Biofilms are multicellular bacterial communities that may evade antibiotics and the body’s immune system. Because there are currently no antibiotics on the market that are capable of killing off biofilms. This issue was fixed by using nanoparticles. The antibiofilm activity shown in NPs suggests that they might be a promising candidate therapy. The concentration of antibiotics needed to destroy bacteria in biofilms is substantially greater than the MIC of planktonic cells, hence antibiotics typically fail to remove these cells [31].

Determination of minimal inhibitory concentration (MIC)
The MIC values were defined as the lowest concentration of the chitosan NPs able to inhibit visible growth of bacteria, while the MBC values are defined as the lowest concentration of the chitosan NPs at which 99.9 % of the inoculated microorganisms are killed (32).
A broth microdilution protocol was performed according to CLSI guidelines in flat bottom 96-well microtiter plates. Cultures of each pathogen were taken with an inoculating needle after 7 days and suspended in sterile saline containing 0.1% Tween 80. Bacteria were put to 5 ml of RPMI1640 once settling time had passed for the heavier particles. First, 100 μl of the medium was poured into each well, then 90 l in Column 3 wells, and 80 l in Column 4 wells. Then, a chitosan NPs suspension, in volumes of 10 μl and 20 μl, respectively, was added to each well in Columns 3 and 4. In order to get the final NPs concentrations, which ranged from 2.5 mg/ml (4th well) to 0.020 mg/ml (11th well), we did serial dilutions from Column 4 to Column 11. (11th well). Each well had 100 μl of bacterial inoculum put to it, with the exception of all wells in Columns 2 and 3, for a total of 200 μl. As a growth control, we used a well (Column 12) without any NPs that contained media and inoculum.
The microtiter plates were inoculated, then placed in a moist chamber and incubated at 37°C in a B.O.D. incubator to see whether any infections will thrive. We used spectrophotometry to take readings of the optical density (OD530 nm) every 24 hours, 48 hours, and 72 hours (3 times), and the findings are depicted in Figs 10 and 11. no visible signs of growth or a reduction in the growth of 80% compared to the drug-free well. The IC50 value was calculated as the concentration at which growth was inhibited by 50% relative to that in the drug-free well.

CONCLUSION
Streptococcus thermophilus has the capability to synthesize Chitosan NPs through biological techniques and the nanoparticle’s characterizations showed optimal properties by conducting AFM, XRD, SEM, and EDS examinations. It showed a great inhibition activity against pathogenic bacteria related to diabetic feet (Aeromonas hydrophila and Citrobacter freundii) at a concentration of 400 ug/ml. Current biosynthesized Chitosan NPs exhibited antioxidant activity and antibiofilm inhibition activity which made it suitable for various medical applications in regards of fighting diseases that emerge by microorganisms. 

CONFLICT OF INTEREST
The authors declare that there is no conflict of interests regarding the publication of this manuscript.