MACRO AND NANO ECBALLIUM ELATERIUM IN VITRO ACTIVITY AGAINST MULTIDRUG-RESISTANT SALMONELLA TYPHI WITH GENETIC DETECTION AND SEQUENCING

. S. typhi was discovered in 76/113 (67.25%) of the collected specimens. 41/76 samples were multidrug-resistant. However, men were likely more prone to infection than women. The findings showed that 95.12% of S. typhi isolates harbored the blaCMY-2 gene, 92.68% harbored the DHFR gene, 48.78% harbored the acrB gene, and 85.36% harbored the rpID gene while 31.70% of them harbored the four genes. Agreement of the four genes sequences showed mutations ranging between deletion, insertion, transversion, and inversion mutations. GC-MS illustrated that the Biochanin B compound was the most popular component, with a percentage of 19.31% in E. elaterium essential oil. 10mg/ml of E. elaterium nanoparticles were found to be the minimal inhibitory concentration (MIC). E. elaterium nanoparticles damaged the cell wall, nuclear material, and cytoplasmic structures of MDR S. typhi . Moreover, they had a more significant cell viability effect on human gastric epithelial cell line (GESI) than chloramphenicol drug. The IC50 of E. elaterium nanoparticles was 1230.05±72.9ug/ml, and the chloramphenicol drug was 73.6924±4.05ug/ml, showing that chloramphenicol was more cytotoxic on GES1 normal cells than E. elaterium nanoparticles. E. elaterium nanoparticles have high effect against MDR S. typhi and more safe than using chloramphenicol drug.


INTRODUCTION
Salmonella serovar Typhi (S. typhi), a part of the Family enterobacteriaceae among the most major Gram-negative motile bacterium that causes typhoid, is the main cause of viral gastroenteritis (Elumalai et al., 2014).There are no cuttingedge ways to avoid typhoid or enteric fever other than the use of antibiotics, making it one of the most frequent health issues globally.In places with poor sanitation and hygiene, just one enteric illness for which fatality levels has not been reduced is typhoid dropping (Hardjo Lugito and Cucunawangsih, 2017).S. typhi has recently developed greater renitence to many antimicrobials, including chloramphenicol, ampicillin, sulfonamides, and tetracycline.As a result, the bacteria are now considered multidrug-renitent (Das et al., 2017).Multi-drug resistant S. typhi is classified as S. typhi insulate, renitent to three separate types of antibiotics (Aljanaby, 2013).Ceftriaxone is one of the most recent antibiotics introduced for medical use; Caution should be expressed about the 2 gene of blaCMY, which confers Salmonella renitence to cephalosporins (Mthembu et al., 2019).The resistance to trimethoprim is due to interfering with folate synthesis in Gram-negative bacteria.Being competitive and dihydrofolate reductase binding (DHFR), which catalyzes the production of dihydrofolate to tetrahydrofolate, it exhibits bacteriostatic behavior.Although trimethoprim can also attach to DHFRs from eukaryotic cells, the medication has a higher affinity for bacterial enzymes (Chiu et al., 2004).Ecballium elaterium fruits' diluted aqueous extract has long been used as an analgesic and anti-inflammatory for chronic sinusitis.It has various applications, such as treating rheumatic diseases, dropsy, hypertension, cancer, fever, and liver disorders (Chan et al., 2010).The plant has been prepared for the production of nanoparticles using the literature review as a research method (El Moussaoui et al., 2019).In order to stop and manage MDR S. typhi strains, this research seeks to isolate, identify multi-drug resistant S. typhi isolates in clinical stool separates from Egypt, detect special genes involved in various medicines defenses additionally their sequences, and assess the impact of various E. elaterium nanoparticle concentrations.Moreover, the study has conducted the cytotoxicity effect of E. elaterium nanoparticles compared with the drug of choice on the human gastric epithelial cell line (GES1).

Bacterial isolation & identification from collected samples
Distinct clinical stool specimens (113) were collected during June 2019 to January 2021.From various hospitals in Egypt: 84 samples from Abbassia Fever Hospital and 29 samples from Abo El-Reesh Al Mounira Hospital with research ethics approval (no.30-2020/10) by the Egyptian National Center for Research and Health Development.Patients' ages ranged from 18 to 45 years old.This study included male and female patients to evaluate the connection between gender, age, and the frequency of S. typhi infection Stool tests were obtained and handled utilizing sterilized, established procedures in a moisture container (Inusa et al., 2018).Salmonella Shigella agar and MacConkey media (Oxoid, England) were used to culture the samples, incubated at 37°C for a day (Amsalu et al., 2021) and purified in the same medium.First, the system of VITEK 2, diversion 9.02, from BioMerieux in the United States, verified the bacterial identity (Henning et al., 2015).sérotyping technique (SSI Diagnostica, Denmark) was used to identify S. typhi (Diep et al., 2019).
Multidrug-resistant (MDR) Salmonella typhi is involved in a number of illnesses such as typhoid fever.This research sought to recognize and quantify Salmonella typhi in clinical stool samples by serotyping, with detection and sequencing of blaCMY-2 gene in charge of cephalosporin resistance, DHFR gene responsible for sulfonamide resistance and acrB and rplD genes responsible for macrolide resistance and use of Ecballium elaterium nanoparticles as an all-natural remedy for MDR S. typhi strains.S. typhi was discovered in 76/113 (67.25%) of the collected specimens.41/76 samples were multidrug-resistant.However, men were likely more prone to infection than women.The findings showed that 95.12% of S. typhi isolates harbored the blaCMY-2 gene, 92.68% harbored the DHFR gene, 48.78% harbored the acrB gene, and 85.36% harbored the rpID gene while 31.70% of them harbored the four genes.Agreement of the four genes sequences showed mutations ranging between deletion, insertion, transversion, and inversion mutations.GC-MS illustrated that the Biochanin B compound was the most popular component, with a percentage of 19.31% in E. elaterium essential oil.10mg/ml of E. elaterium nanoparticles were found to be the minimal inhibitory concentration (MIC).E. elaterium nanoparticles damaged the cell wall, nuclear material, and cytoplasmic structures of MDR S. typhi.Moreover, they had a more significant cell viability effect on human gastric epithelial cell line (GESI) than chloramphenicol drug.The IC50 of E. elaterium nanoparticles was 1230.05±72.9ug/ml,and the chloramphenicol drug was 73.6924±4.05ug/ml,showing that chloramphenicol was more cytotoxic on GES1 normal cells than E. elaterium nanoparticles.E. elaterium nanoparticles have high effect against MDR S. typhi and more safe than using chloramphenicol drug.

Characterization of synthesized nanoparticles
TEM (Transmission Electron Microscope) at 80.0 Kv of accelerating voltage was used (JEOL JEM-1400 series TEM, Japan) to study the morphological size of E. elaterium nanoparticles.First, 1mg of E. elaterium nanoparticles was suspended in 10ml of distilled water, and then 2 μl drops of nanoparticles were placed onto a parafilm and directly put on electron microscope (E.M.) grids.Finally, the filter paper was used to wick away specimen drop and placed in a Petri dish.

Antibacterial screening of E. elaterium extract and nanoparticles on MDR S. typhi
To test the antibacterial effects of various solvent extracts and nanoparticles, agar well diffusion method has been used.Filter paper discs (about 6 mm in diameter), each consisting 50 μl of the extract solution, were placed on the agar surface.Agar plates are then kept the following day at 37°C under the proper conditions, depending on the test microorganism.

Detection of the effect of E. elaterium nanoparticles on MDR S. typhi by using transmission electron microscopy (TEM) and protein profile SDS-PAGE
The effect of E. elaterium nanoparticles on MDR S. typhi were specified by TEM images (JEOL JEM-1400 series TEM, Japan) to study the morphology of bacteria as a control sample compared to S. typhi treatment with (E.elaterium nanoparticles).First, the bacterial was inoculated onto 2 ml brain-heart broth, then 1mg E. elaterium nanoparticles were added and incubated for 24 hrs at 37 o C.
Centrifuging the mixture at 7500 rpm for 10 min., the palette was preserved in glutaraldehyde and osmium tetroxide after the excess was eliminated, and dehydration in alcohol.Next, the test had an epoxy resin coating.The thickness of the microtome sections was set between 500 and 1000 m (Leica Ultracut UCT Ultramicrotome).Toluidine blue (1X) staining was applied to small fragments and examined by Camera Leica ICC50 HD.
The effect of E. elaterium nanoparticles on MDR S. typhi was selected by protein profile (lane 2) compared to S. typhi as a control (lane 1) using sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE).The bacteria were inoculated into 2 ml brain-heart broth, then 1mg E. elaterium nanoparticles added, then incubated for a day at 37°C.After centrifuging the mixture for 10 minutes at 7500 rpm, the supernatant was removed.SDS-PAGE was done by Biometra 010-130 minigel-twn, Germany.

Examination assay of cytotoxicity of E. elaterium nanoparticles compared with chloramphenicol drug
The American Type Culture Collection provided the human gastric epithelial cell line (GESI), and the cells were increased in Dulbecco's Modified Eagle Medium (DMEM) with 10% Fetal Bovine Serum (FBS) (Grand Island, NY, USA), 10 ug/ml of insulin (Sigma), and 1% penicillin-streptomycin (Sigma).Cell plates (well plates at a density of 1x10 3 cells/well) were placed in a 96-well plate with 100 ul of the tested substance per well and 100 l of full growth media for a day (Chen et al.,

2018).
Cytotoxicity was calculated using the original enzymatic reduction modification of viability assay 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) to generate blue crystals named formazan (Elshal et al., 2022).First, the culture medium was deleted from a centrifuge tube, and the cell layer was rinsed with 0.25% (w/v) Trypsin 0.53 mM EDTA solution to remove all traces of serum that contained trypsin inhibitor.Next, 2.0ml of trypsin EDTA solution was added to the flask, and cells were observed under an inverted microscope until the cell layer was dispersed.Subsequently, 6.0ml of complete growth medium was added, and cells were aspirated by pipetting gently, then centrifuged at 125 xg for 5 min.The supernatant was discarded, and the cell pellet was in a new growing media suspended; finally, For 24 hours, cultures were kept at 37°C.Following an elevated microscope inspection of the plates, the MTT assay was conducted.
Multiwell plates worked well for the MTT in vitro cytotoxicity measurement technique.Each vial of MTT [M-5655] to be used was reconstitute with 3 ml of medium and reconstituted MTT were added in an amount equal to 10% of the culture medium volume.Cultures were returned to the incubator for 2hrs based on the maximal cell density and the type of cell, and then The formed formazan crystals were eliminated by adding MTT solubilization solution [M-8910] in an amount equal to the volume of the original culture medium.Different concentrations of cells were incubated with E. elaterium nanoparticles (4.0, 16.0, 63.0, 250.0, 1000.0 ug/l) and Chloramphenicol drug (0.4, 1.6, 6.3, 25.0, 100.0 ug/l), dissolved in 10% FBS for 24 hrs.Spectrophotometrically (BioTek Instruments, Inc.Winooski, VT, USA) measured absorbance at wavelength 450 nm, and the plates were measured.

Statistical analysis
The experimental data were expressed using the mean ± standard error of the mean (SEM).Additionally, one-way ANOVA was performed to analyze the data gathered, and a significant difference was defined as one with a p-value of 0.05 or lower (Elshal et al., 2022).

RESULTS AND DISCUSSION
Salmonella typhi causes an intestinal disorder called typhoid fever.Fever, malaise, stomach pain, and constipation are common symptoms in patients (Umair and Siddiqui, 2020).Typhoid fever is estimated to cause between 11 and 21 million cases worldwide and 128,000 to 161,000 fatalities per year (WHO, 2019).

Collection data with co-relation to patient gender and age
The current study showed that seventy-six isolates out of 113 (67.25%) were identified as S. typhi.Males were more influenced by S. typhi than females with a rate of 62 and 38% respectively and the age rate was categorized as 18-25 (male 90% & female 10%), >25-35 (male 65% & female 35%), and >35-45 (male 54% & female 46%).It is obvious that males were infected by S. typhi more than females in each category (Fig. 1).Anook and Farooq conducted a similar investigation (2017).They found that S. typhi grew 57.9% in 119 clinical stool samples from Al-Samawah general hospital in Iraq from March 2017 to February 2018, and illustrated that the frequent pathogens isolates from the patients admitted to hospitals in Iraq were higher in males (55.0%) than in females (45.0%).This result was in contradiction with Inusa et al. (2018), who found that females (55.5%) had a statistically significant higher frequency of S. typhi than males (44.5%).Series alignment was performed for each detected gene and compared to the World Wide Web's gene bank database, revealing the presence of several types of mutation (Fig. 3, 4, 5 and 6).Table 2 illustrated different types and numbers of mutations found in the 4 detected genes.In the case of the blaCMY-2 gene, the deletion mutations were recorded at nine query positions, while the inversion mutations were detected at thirty-one query positions.At the same time, transversion mutations were recorded at sixty query positions.The insertion mutations were recorded at eleven query positions.Nevertheless, in the case of the DHFR gene, one type of mutation was substitution (inversion and transversion mutations).
Figure 3 Amplified 648 bp blaCMY-2 gene of MDR S. typhi and its sequence alignment compared to GenBank.M as Marker & 1, 2, 3, ….to 41 as no. of sample.Deletion mutations were recorded at nine query positions, while the inversion mutations were detected at thirty-one query positions.At the same time, transversion mutations were recorded at sixty query positions.The insertion mutations were recorded at eleven query positions There were three types of mutations in the acrB gene were deleted at three query positions, substitution (inversion recorded at twelve query positions and transversion were recorded at thirteen query positions) and insertion mutations were recorded at one query position.One type of deletion mutation was recorded at three query positions in the rplD gene.It's possible that Egypt's excessive or incorrect usage of antibiotics is to blame for the abnormalities in both genes.Ecballium elaterium extract and nanoparticles were tested for their antibacterial activity on MDR S. typhi strains contained four resistance genes (blaCMY-2, DHFR, rplD and acrB) by filter paper disc method.Figure 7 illustrated that the diameter of the growth inhibition zone of E. elaterium nanoparticles (22mm) on MDR S. typhi was larger than that of E. elaterium extract (16mm).Characterization of E. elaterium nanoparticles carried out using a transmission electron microscope (TEM) showed that their size was 16.In the current study, for the bacterial strains carrying the blaCMY-2, DHFR, rplD and acrB genes, the minimal bactericidal concentration (MBC) was 20 µg/ml Nano E. elaterium.The strain with four genes has a minimum inhibitory concentration (MIC) was 10 µg/ml shown in Table 3.

The effect of E. elaterium nanoparticles on MDR S. typhi by using transmission electron microscopy (TEM) and protein profile SDS-PAGE
The effect of E. elaterium nanoparticles on MDR S. typhi strains harboring four genes was determined by TEM and Protein profile SDS-PAGE.After being treated with E. elaterium nanoparticles, the cell wall was damaged (the nuclear materials and cytoplasmic structures were not seen) (Fig. 9).The molecular weight of each band in the protein profile of MDR S. typhi (lane 1) compared to the treated bacteria (lane 2) was determined and molecular weights are indicated (Table 4).

Results of cytotoxicity of E. elaterium nanoparticles compared with chloramphenicol drug
The viability of cells was detected by a reduction assay of MTT.These results showed that chloramphenicol 30μg (the drug of choice that treat MDR S. typhi) had a more potent inhibitory activity towards GES1 normal cells than E. elaterium nanoparticles.
The inhibitory concentration that is half as large (IC50)was used to measure the cytotoxicity of E. elaterium nanoparticles compared to chloramphenicol.The results showed that E. elaterium nanoparticles were 1230.05±72.9ug/ml, and the chloramphenicol drug was 73.6924±4.05ug/ml.Consequently, chloramphenicol was more cytotoxic on GES1 normal cells than E. elaterium nanoparticles (Table 5).

CONCLUSION
Using natural compounds is safer for humans than using chemical compounds, E. elaterium nanoparticles have a higher infectivity against MDR S. typhi that contain (blaCMY-2, DHFR, rplD, and acrB) resistance genes and have lower cytotoxicity on GES1 normal cells than chloramphenicol (the most effective drug on MDR S. typhi), so we are recommended to use natural products in the treatment of multidrug-resistant bacteria.

Figure 1
Figure 1 Percentage of S. typhi in clinical samples with co-relation to patient gender Detection of MDR S. typhi Despite the development of improved antibacterial medications, enteric fever remained a serious health issue (Zaki and Karande, 2011).In addition to gaining resistance to effective medications like ciprofloxacin, S. typhi also became resistant in comparison to antibiotics like co-trimoxazole, ceftriaxone, and ampicillin (Inusa et al., 2018)..In the current study, S. typhi isolates were evaluated for antibiotic susceptibility (Fig.2).Fifty-four percent (41 out of 76) of them were found to be multidrug-resistant strains.S. typhi strains were resistant to macrolides, sulfonamides, penicillins, and cephalosporins.Ghurnee et al. (2021), found that S. typhi most renitent to the antibiotics ciprofloxacin (99.52%) and nalidixic acid

Figure 2
Figure 2 Percentage of susceptible, intermediate and resistant S. typhi isolates to different antibiotic groups (AM (ampicillin), AZM (azithromycin), CEC (cefaclor), CEP (cefoperazone), FEP (cefepime), CIP (ciprofloxacin), NA (nalidixic acid), CN (gentamycin), SXT (trimethoprim/sulphamathoxazole), C (chloramphenicol) and DO (doxycycline))Results of genes in charge of multidrug-resistance and mutations in DNA gene fragmentsIn the current study, the existence of the blaCMY-2, DHFR, acrB, and rplD genes responsible for multidrug-resistance in S. typhi strains was determined by PCR in this work.The PCR product revealed that 39 strains out of 41 (95.12%) harbored the blaCMY-2 gene, 38 isolates (92.68%) harbored the DHFR gene, 20 isolates (48.78%) harbored the acrB gene, and 35 isolates (85.36%) harbored the rplD gene, whereas thirteen isolates (31.70%) harbored the four resistance genes (Fig.3, 4, 5 and 6).S. typhi strains resistant to cephalosporins are common worldwide, and this is probably related to the synthesis of the CMY enzyme (s) (Li et al., 2007).S. typhi strains harboring blaCMY-2 were associated with community-acquired infections (Liebana et al., 2012).Previous research showed that CMY-2-carrying plasmids may around self-transfer several strains alone but infrequently co-transfer numerous genes (Li et al., 2008).The discovery of the plasmid-encoded DHFR gene in S. typhi provided proof that this specific DHFR is widely distributed.According to the study, there are many S. typhi strains in Solan, Himachal Pradesh, that are resistant to trimethoprim, co-trimoxazole, and sulfanilamide.India.Therefore consideration must be given to controlling and preventing the MDR strains that emerged in this region and became a severe hazard to public health (Neha et al., 2016).The overexpression of the acrB gene resulted in the AcrAB-TolC efflux pump turning on.The establishment of a multi-resistant phenotype in Salmonella was caused by the transporter protein acrB, which may extrude a variety of unrelated chemicals from bacterial cells (Giraud et al., 2006).Series alignment was performed for each detected gene and compared to the World Wide Web's gene bank database, revealing the presence of several types of mutation(Fig.3, 4, 5 and 6).Table2illustrated different types and numbers of mutations found in the 4 detected genes.In the case of the blaCMY-2 gene, the deletion mutations were recorded at nine query positions, while the inversion mutations were detected at thirty-one query positions.At the same time, transversion mutations were recorded at sixty query positions.The insertion mutations were recorded at eleven query positions.Nevertheless, in the case of the DHFR gene, one type of mutation was substitution (inversion and transversion mutations).

Figure 4 Figure 5
Figure 4 Amplified 413 bp DHFR gene of MDR S. typhi and its sequence alignment compared to GenBank.M as Marker & 1, 2, 3, ….to 41 as no. of sample.One type of mutation was substitution (inversion and transversion mutations) Figure 5 Amplified 930 bp acrB gene of MDR S. typhi and its sequence alignment compared to GenBank.M as Marker & 1, 2, 3, ….to 41 as no. of sample.Deletion mutations were recorded at three query positions, substitution (inversion recorded at twelve query positions and transversion recorded at thirteen query positions) and insertion mutations recorded at one query position 3 nm.Adwan et al. (2011), cleared that the educator of E. elaterium exhibited antibiotic efficacy against multidrug-resistant bacteria (15mm).

Figure 6 Figure 7
Figure 6 Amplified 509 bp rplD gene of MDR S. typhi and its sequence alignment compared to GenBank.M as Marker & 1, 2, 3, ….to 41 as no. of sample.One type of deletion mutation was recorded at three query positions

Figure 8
Figure 8 Diagrams showing GC-MS for chemical composition, structure, and concentrations of E. elaterium essential oil

Figure 9
Figure 9 Images of TEM shows MDR S. typhi strain compared with after adding E. elaterium nanoparticles

Detection of blaCMY-2, DHFR, acrB, and rplD genes obtaining S. typhi DNA To
extract the bacterial DNA fragment, a QIAamp DNA Mini Kit (Cat.No. 51304 and 51306, QIAGEN, USA).On a 5 ml brain-heart broth medium, S. typhi was freshly cultured for 24 hours at 37°C (

Oates et al., 2012).
The use of PCR analysis to find the blaCMY-2 gene is in charge of cephalosporin resistance, DHFR gene is in charge of sulfonamide resistance and acrB & rplD

Table 1
The sequences of the specific primers used to detect blaCMY-2, DHFR, acrB and rplD

Su et al., 2021).
system (MultiDoc-It™ system) was used to do data analysis (ww.totallab.com,Ver.1.0.1).Purified PCR results were measured spectrophotometrically at 312 nm and with micro spin filtering (SYNGENE Model 680XHR, U.K) (Hall and Beiko, 2018).Sequencing of DNA gene fragmentsABI 3730xl DNA sequencer" and analysis software v3.1 were used for the in comparison to arrangement.The four genes' sequences and homology were compared to the GenBank database applying the Basic Local Alignment Search Tool (BLAST) and BLAST nucleotide (BLASTN 2.2.13) programs (GATC Company, Germany) (Ibrahim et al., 2016).Preparation of Ecballium elaterium extract and nanoparticlesMature E. elaterium fruits were purchased from El-sheikh Zowaid, North Sinai, then it was identified in the National Research Center, Egypt.Fresh plant samples were dried by air in a dim area at room temperature (28°C) for 7 days (Mahmoud M. Saker, 2012).The dried herbs were pulverized into a powder by grinding device (Ahmadi et al., 2017).30-40 g of dried components was added 200 ml of sterile distilled water at room temperature also the mixing was left for 24 h.The extract was evaporated till it became dry at 37°C (Adwan et al., 2011).E. elaterium was collected, rinsed with deionized water multiple times, and room temperature dried afterward.They were processed into a powdery form by a ball milling instrument (TENCAN, Changsha Tianchuang Powder Technology Company, China).In the grinding method, 5 g of E. elaterium was placed in nanotubes and ground into extremely fine powders for four hours.The ball milling process's collision of the tiny stiff balls in a covert container process would generate localized high pressure (Rabiee et al., 2020).

Mutlu-Ingok et al., 2021). Chemical characterization of E. elaterium nanoparticles in conjunction with gas chromatography-mass spectrometry
Tubes contained 95 μl of Nutrient broth, 100 μl serial E. elaterium nanoparticles dilutions were inoculated with five μl of microbial inoculum.Negative control tubes contain bacterial suspension without E. elaterium nanoparticles, while positive control tubes only contain E. elaterium nanoparticle suspension.For 24 hours, every tube was incubated at 37°C.MIC and MBC values were calculated spectrophotometrically using optical density as a gauge at 600 nm on a spectrophotometer (T80 UV/VIS Spectrometer, PG instrument, United Kingdom).Experiments were conducted in triplicate ( (Daoud et al., 2019)de on the diameter of growth inhibitory zones(Daoud et al., 2019).Serial dilutions of E. elaterium nanoparticles were prepared to the MDR S. typhi that was subcultured overnight on Mueller-Hinton agar to examine their impact.The minimal inhibitory concentration (MIC) and the minimal bactericidal (GC-MS)Following the steps outlined in the European Pharmacopoeia, the dried E. elaterium plant sample (500g) was hydro-distilled for 4 hours using a Clevengerstyle apparatus.The tests of essential oils were kept at 4 o C in the darkness (Mahdavi et al., 2017).An Agilent 7000 series Triple Quad Gas Chromatograph linked up to a Mass Spectrometer (GC-MS fitted with an Elite-5MS (5% diphenyl / 95% dimethyl polysiloxane)) was applied to perform gas chromatography-mass spectrometry (GC-MS) to chemically analyze the specific oil.The National Bureau of Standards and Technology (NIST) database, which contains over than 62,000 patterns, was used for mass spectrum interpretation.The molecular weight and structure of the test material's constituent parts were determined (Neelamegam and Ezhilan, 2012).

Table 3
Effect of different concentrations of E. elaterium nanoparticles on MDR S.

Table 4
Molecular weight of each band in SDS-PAGE

Table 5
Cytotoxicity to GES1 normal cells with E. elaterium nanoparticles and chloramphenicol drug