A comparison of the Effects of Lemon Peel -Silver Nanoparticles Versus Brand Toothpastes and Mouthwashes on Staphylococcus Spp. Isolated From Teeth Caries

Thirty one samples of gum swabs were collected from patients with tooth caries (5-30 years old) from the College of Science (Biology department )University of BaghdadIraq for the period from October 2018 to December 2018. , The samples were transported, after inoculation in a transport media (nutrient broth), to the laboratory of the College of Science and then cultured on mannitol salt agar and blood agar). The isolates belonging to Staphylococcus spp. were identified by biochemical tests and Vitek 2 compact system, while the more antibiotic resistant isolates were identified by using Polymerase Chain Reaction(﴾PCR) and sequencing of 16SrRNA . The results showed sharp UV absorption peaks at 303 303nm and AFM at 56 nm. Transmission Electron Microscope (TEM) results showed that lemon silver nanoparticles (NPs) are spherical in shape. The results also demonstrated that the silver NPs have a higher antibacterial activity with a diameter of inhibition zone that reached 16mm as compared with the common three mouthwash and toothpaste brands (Colgate, Crest and Close up) that had inhibition zone values of 5-8mm.

All specimens were streaked on mannitol salt agar plates, then incubated at 37°C under aerobic conditions for 18-24 hrs. After incubation, the pure colonies were selected to be diagnosed.

Biochemical Analysis
The physical and biochemical tests were conducted according to the methods described by Bergey's manual of determinative bacteriology.

Antimicrobial susceptibility testing
Kirby-Bauer's disc diffusion method for testing antibiotic sensitivity was performed for each isolate The test was performed on Mueller-Hinton agar with the antibiotic discs shown in Table-1 . After incubation (24 hrs at 35ºC), sensitivity was read. The bacteria isolates were regarded as sensitive or resistant according to CLSI criteria [9]. Molecular assay DNA extraction Genomic DNA was extracted from the detected bacterial isolate no. according to the protocol of Wizard Genomic DNA Purification Kit, Promega. Quantus Florometer was used to detect the concentration of the extracted DNA [10] .

Primers Selection
The set of primers 27F (AGAGTTTGATCTTGGCTCAG) and 1492R (TACGGTTACCTTGTTACGACTT) was used for the amplification of the 16s rRNA of the identified bacteria [11]. Toothpastes Three types of the toothpastes (Colgate, Crest , and Close up) were used in this study (Table1)., as listed on the (package and the manufacturer's name). Toothpastes without antimicrobial agents and water were used as controls [12].

Preparation of Bio Extract
Fresh peels of lemon (20 gm) were washed with tap water and air dried for a short time. They were finely cut and soaked in 100 ml D.W for 10 min and filtered through a Whatman filter paper (no.0.5 ). This bio extract was always used as fresh .

Preparation of 1mM Silver Nitrate
Silver nitrate was brought from Lobachemie. A weigh of 0.0169 gm was dissolved in 100mL of distilled water in an amber colored bottle.

Synthesis of Silver Nanoparticles
Three milliliters of the prepared peel extract was added to 40mL of silver nitrate solution in a 100mL conical flask and incubated at room temperature for 2-3 hours. The control sample contained only 40 mL of silver nitrate solution.

Well Diffusion Method
(WDA)Well diffusion method then of the suspension was spread on the test plate. 6 mm diameter were impregnated with 10 μL of the citrus juices and placed on the surface of the test plate was used. The plates were incubated at 37 0 C for 24 hrs and the inhibition zone was measured [14] .
Physio-chemical characterization of silver nanoparticles Force Field Microscopy (AFM) UV-Vis spectrophotometer was used at a resolution of 1 nm and a range from 250 nm to 800 nm. The size of nanoparticles was performed with Atomic Force Field Microscopy (AFM)

Scanning Electron Microscope (SEM) Analysis
The morphological features of the synthesized silver nanoparticles prepared with the plant extract were studied by Scanning Electron Microscope (JSM-6480 LV).

Characterization of Silver Nanoparticles
Then synthesis of silver nanoparticles was analyzed using UV-Visible spectroscopy at the wavelength of 300-700nm.

Staphylococcus. aureus)
The bacterial cultures were examined and showed that gram positive cocci appeared as single cells, pairs, tetrads and chains. The macroscopic examination of the isolates on mannitol salt agar demonstrated the ability to ferment mannitol and turn the color of the medium from red to yellow. The bacteria were classified as presumptive S. aureus and S. epidermidis (Figure-1).  The thirty one samples were taken from patient from both sex groups with an age range of 5-30 years. All the swab samples were positive for microorganisms (Table-2). The results agree with those previously reported [16]. These bacteria were characterized by using biochemical tests as described. All these isolates were obtained from amalgam tooth filling We show that the percentage of the bacterial isolates was higher in males (24%) than in females (16%), as demonstrated in Table-2. The percentage increased with the increase in the age of the donors, which may be due to the decrease in immunity and resistance to infection by bacteria. . The result agrees with those reported by other authors [17].

Antibiotic susceptibility test (AST)
The antibiotic resistance patterns of the isolated gram positive bacteria included resistance to Erythromycin and Azithromycin as well as sensitivity to Climdamycin , Levofloxacin, and Amoxicillin. These results are similar to those obtained by a previous investigation [18] which showed that most of the isolates of Staphylococcus aureus were multi-resistant for antibiotics, with a high level of resistance against Methicillin, Penicillin G, Methicillin, Chloramphenicol, whereas they demonstrated sensitivity to Vancomycin.
In this study, the DNA of the bacterial isolates was successfully extracted and showed an appropriate quality to perform PCR (10 ng/µl). 16S rDNA was amplified by PCR using specific primers that give a distinct with a size of 1500 bp when analyzed with gel electrophoresis (Figure-3) [19].

Atomic Force Electron Microscopy (AFM)
The AFM micrograph obtained for the -lemon-silver nanoparticles (Figure-5) indicates the surface roughness changes values as identified by root mean square (Rp). The sample's roughness value was 56 nm, while the section analysis of the sample's grain size showed a value of 34 nm.

Emad and Salama
Iraqi Journal of Science, 2020, Vol. 61, No. 8, pp: 1894-1901 1899 We then compared the antimicrobial activities of silver NPs versus those of toothpastes. The NPs synthesized using the natural plant extracts represented the only formulation that had activity against Gram positive bacteria. The diffusion method was used as a preliminary test for detecting the antimicrobial activity. The results showed that the NPs synthesized using lemon had a higher activity with a larger diameter of inhibition zone as compared to the different brands of toothpastes and mouth washes (Table -2). The result agree with those of an earlier study [20] which showed that the mouthwash Colgate was the most active solution which caused an inhibitory zone of about 4 mm in diameter, followed by ZAK (3.7 mm.) and Listerine (0.5mm.).
The conventional antimicrobial toothpastes increase the effectiveness in the control involved in a wide variety of the primary etiological agents of dental caries the antimicrobial activity of commercial toothpaste [20] The inhibitory effects of a 10 % concentration of six different mouthwashes against six oral bacteria was previously measured. The inhibition effect of the six mouthwashes were tested at different dilutions 25%, 50% and 75% using agar well diffusion method against Streptococcus mutants, Streptococcus sanguinis, Staphylococcus aureus, Klebsiella pneumoniae , Heamophilus influenza and Streptococcus pyogenes [21].

Conclusions
Because of the increased bacterial resistance to antibiotics, nanoparticle silver particles were produced with lemon peel and their effectiveness against bacteria isolated from the teeth caries was compared to that of toothpastes and mouthwashes. A high efficacy was observed for the nanoparticles as compared to the toothpastes and mouthwashes.