Evaluation and comparison of antimicrobial e�cacy of snail mucus of Egyptian Eremina desertorum and Helix aspersa with novel approach of their anti-inammatory and wound healing potencies

Snail mucus is composed of bioactive compounds thought to have different biological properties for the treatment of some skin problems. Although Helix aspersa mucus is used in several cosmetic products, a detailed characterization of Eremina desertorum mucus composition and its biological activities is still missing. Mucus extracts (MEs) from H. aspersa and E. desertorum were prepared and tested for their antimicrobial, anti-inammatory activities with their potencies in wound healing. Also, chemical characterization is done by GC-MS analysis. Results showed that ME of E. desertorum gave higher inhibitory activity against resistant strains related to burn wound infections compared to ME of H. aspersa. Also, it revealed a signi�cant anti-inammatory activity. Moreover, we found that ME of E. desertorum lacked cytotoxicity and was able to signi�cantly induce cell proliferation and migration through up-regulation expression of TGFβ1 and VEGF genes. Our results suggested that MEs of E. desertorum have higher biological effects compared to H. aspersa, which are attributable to antimicrobial, anti-inammatory activities, cell proliferation and pave the way for further investigating its potential effect as a human therapeutic agent.


Introduction
Snails have a thick mucus coating that may aid in minimizing moisture loss, reducing friction which helps them to glide smoothly across dry surfaces, as well as protecting their bodies from physical harm 1 .Mucus secretions have a wide range of functions and biological activity 2 .Trail mucus is mostly composed of big, carbohydrate-rich polymers with a few tiny proteins 3 , which can relieve heartburn as mucus neutralizes stomach acidity and gastro esophageal re ux based on the role of snail mucus in mending ulcers and the role of human mucus in preventing or ghting acidity 4 .Also, snails may produce a lot of mucin in their mucus secretion, which contains antibacterial proteins and gives them some resistance to infection by pathogens 5 .Moreover, several scienti c studies have shown that bioactive compounds-derived from different mucus snails can be utilized in a wide range of therapies, such as creams to treat skin abrasions and scars, respiratory disorders, and heartburn 6 .
Eremina is very con ned genus to many countries of North Africa region 7 and considered as part of the natural ecosystem of Egypt 8 .Eremina desertorum is one of the common desert species occurred in many different locations along the Mediterranean region, between Alexandria till the border of Egypt with Libya [9][10][11] .Despite the spread of this species in Egypt, so far there is no study explaining the chemical composition or even proving the medical importance of the mucus extracted from it.
Burn wounds are one of the most important health issues worldwide, especially in the developing countries 12 .Microbial infections for burn wound patients are considered a huge problem as about 50-75% of mortality in hospitalized burn patients is due to microbial infections 13 .Moreover, lack of researches in Egypt on pathogenicity, resistance of microorganisms on burn wounds and statistical information makes the problem more complicated.Also, many studies on burn wound infections ignored host microbiota-associated pathogens 14 .Recently, Kopeck 15 reported that the presence of some resistant microbial strains in burns could lower the e ciency of burn wound healing.Wound healing process is controlled by different cytokines and growth factors, such as transforming growth factorbeta 1 (TGFβ1), and vascular endothelial growth factor (VEGF) 16 .TGFβ1 is created by cells such as T cells, platelets and macrophages, which releases neutrophils and broblasts to the site of damage at the in ammatory phase of wound healing 17 .Also, TGFβ1 helps in migration, growth, and motivation of broblasts 18 .Moreover, VEGF is created by several cells as well as endothelial cells, broblasts, platelets and neutrophils 19 .TGFβ1 and VEGF can suppress severe in ammation as in ammation is the response of living tissues to infected wound.The mechanism of anti-in ammatory agents depends on inhibiting the release of lysosomal constituents of activated neutrophils which can cause tissue damage and in ammation 20 .
Although the huge commercial diffusion of products from garden snail Helix aspersa mucus [21][22][23] , there have been no reports to discuss antimicrobial and anti-in ammatory activities of E. desertorum mucus.According to our knowledge, there are no studies on chemical composition of E. desertorum mucus related to its biological activities and its mechanisms in wound healing activity.Therefore, the aim of the present study is the rst to identify the mucus chemical composition of the desert snail E. desertorum comparing to garden snail H. aspersa under Egyptian condition and explore it as a new antimicrobial, and anti-in ammatory approach against resistant pathogens of burn wound infections and its wound healing potency on human skin broblasts through expression of some growth factors genes.

Antimicrobial activities and MIC
The present study is might be the rst to investigate effect of MEs of H. aspersa and E. desertorum against MDR or PDR pathogenic microorganisms isolated from burn wound infections.Antimicrobial activities of both snails were tested against eight resistant pathogens as in Fig. 1.ME of E. desertorum showed higher signi cant inhibitory activity against tested strains with differences in their susceptibility than H. aspersa.While, both snails didn't show any inhibitory activity against KP-1 (Table 1).Fungal strains were found to be more susceptible strains to MCE of E. desertorum.The highest mean zones of inhibition ranged from 3 ± 0.0 to 55.2 ± 0.1mm and from 9.5 ± 0.0 to 30.5 ± 0.06 mm against fungal and bacterial strains, respectively compared to DMSO (1%) which didn't show any inhibition zone (Fig. 1).The values of minimum inhibitory concentrations (MIC) for each organism were shown in Table 1.MIC ranged between (5 and 20µg/ml) against bacterial strains, while MIC for fungal strains ranged between 7 and 32 µg/ml.

Anti-in ammatory activities of MEs of H. aspersa and E. desertorum
The anti-in ammatory activities of MEs of both snails were determined through membrane stabilization, albumin denaturation, and proteinase inhibitory activity compared with aspirin as a refrence drug (Fig. 2).Both snails showed anti-in ammatory activities, while E. desertorum showed higher activity.E. desertorum showed highly signi cant stabilization toward human red blood cells membrane.Also, the percentage inhibition of albumin denaturation for E. desertorum at concentration 2000 µg/ml was higher than that of aspirin at the same concentration with inhibition 92.8% and 85.3%, respectively.Moreover, a signi cant increase in inhibiting proteinase activity was highly similar to that of aspirin with inhibition of 89.9% and 89.2%, respectively at concentration of 2000 µg/ml.

Lack of cytotoxicity of MEs of H. aspersa and E. desertorum
To evaluate the biological effects of MEs of both snails, human skin broblasts (HSF) cells were treated in vitro with different concentrations (0.03-300µg/ml) of both snails, to show their effect on normal cell viability and morphology.
Figure (3 A, B) showed lack of cytotoxicity of both snails as the percentage viability of HSF cells at the highest treated concentration of MEs of H. aspersa and E. desertorum were observed to be 93% and 75.8%, respectively compared to untreated samples and DMSO(1%) and (10%) as different controls.The concentrations of MEs of both snails used for treatment and their corresponding percentage cell viability showed IC 50 > 300 µg/ml in both snails which con rmed the disappearance of any toxic effect of treated concentration.

Induction of MEs of both snails to cell migration and wound repair
Beside the cell viability, cell migration and proliferation properties of both snails were determined by the scratch wound assay.As shown in Fig. 4A, both snails improved wound healing process compared to untreated cells as ME of E. desertorum induced the migration of HSF cells resulting in complete wound closure after 48 hrs.faster than ME of H. aspersa.Figure 4B indicated that E. desertorum, at 300µg/ml, closed the gap created by the scratch by 99.2% after 48 hrs.While, in untreated cells, 55.1% of the gap was closed at 48 hrs.

Upregulation of TGFβ1 and VEGF genes expression
The present investigation determined changes in the expression of TGFβ1 and VEGF genes by realtimePCR in HSF cells with MEs of both snails at 48hrs after treatment.To determine possible molecular mechanism of the induction of MEs of both snails to wound repair and healing, we tested the expression level of TGFβ1 and VEGF genes.Expression of TGFβ1 gene treated by MEs of H. aspersa and E. desertorum was signi cantly upregulated by 5-fold, and 7.5-fold, respectively, when compared to the control (Fig. 5).Also, expression of VEGF gene was signi cantly upregulated by 2fold, and 3.5-fold when treated with MEs of H. aspersa and E. desertorum, respectively.
Chemical analysis of MEs of both snails using GC-MS Chemical constituents, molecular weight and peak area of each component for MEs of both snails were listed in Tables (2, 3).Our results indicated that the major compounds in ME of E. desertorum were 3H-1,2,4-triazole-3thione,4,5-dihydro-4,5-diphenyl followed by phthalic acid, 7-bromoheptyl ethyl ester and methyl 1,2-benzisothiazole-3acetate.While in ME of H. aspersa, the major compounds were Thiophene, 3-(decyloxy)tetrahydro-, 1,1-dioxide followed by 4-(Nona uoro-tert-butyl) nitrobenzene.So, further study will be done for the isolation and the puri cation of these active compounds with a comprehensive toxicological analysis to determine its safety as it is beyond the scope of this paper.

Discussion
Based on previous investigations, antimicrobial activities of mucus from mollusks including snails and slugs have never been suggested extensively 24 .According to several reports, antimicrobial activity depends on snail species, extraction method, and the resistance of the tested organism 25 .In the present study, ME of E. desertorum was the most effective snail against the most selected resistant strains with a strong inhibitory activity.These results were similar to Lopez 26 who evaluated the antimicrobial activity of the crude extract of marine snail C. muricatus.Although, there are few reports on potent antimicrobial activities of extracts from H. aspersa, our study is considered the rst to explore antimicrobial activities of E. desertorum compared to H. aspersa against resistant pathogens related to burn wound infection.
ME of E. desertorum showed a signi cant anti-in ammatory activity through membrane stabilization, albumin denaturation, and proteinase inhibitory activity compared with commercially aspirin.It might be the rst study to discuss the in vitro anti-in ammatory activity of this snail.Therefore, we suggest it as a new alternative agent with a potent anti-in ammatory activity in the treatment of burn wounds infections.Hence, ME of E. desertorum treatment was further conducted in order to evaluate the e cacy of this snail in curing the burn wound infections.
Moreover, ME of E. desertorum accelerates wound healing by inducing the migration of broblasts with enhancing the expression of wound healing related gene (TGFβ1 and VEGF).This is in agreement with Coppe 27 who demonstrated that methanolic extract of C. molmol and ethanolic extract of henna signi cantly improved the expression of TGFβ1 and VEGF genes at 48 hrs.after treatment of normal mouse broblast cells.However, there are some reports on wound healing activity of mucus of different snails 28 , there are no reports on effect of this snail on expression of wound healing related genes.
It was necessary as a next step to check the chemical composition of bioactive compounds in both snails.The differences in their biological activities may be due to differences in the active compounds that present in both snails.
In addition, the third one showed a strong antimicrobial activity 31 .While in Egyptian H. aspersa, there were another two major compounds; Thiophene, 3-(decyloxy)tetrahydro-, 1,1-dioxide followed by 4-(Nona uoro-tert-butyl) nitrobenzene which had different biological activities 32,33 .This variation in mucus composition could be attributed to species differences, as well as mechanical factors such as temperature, humidity, light intensity, soil conditions, and food supply.These data agreements with Meikle 34 who found substantial differences between the mucus of six coral species.Also, Sallam 35 observed several chemical variations in the composition of three common Egyptian land snails, Eobania vermiculata, Theba pisana and Monacha obstructa mucus.Between the two species in this study, it should not be surprising that different forms of mucus have different compositions and different mechanical properties according to their environmental living condition.These environmental conditions also affect the physical properties of the two snail species in terms of color and viscosity.The garden snail Helix aspersa was colorless and less viscosity comparing with mucus dessert snail Eremina desertorum with slightly cloudy-white with high viscous.Dessert snail's high viscosity acted as a barrier, preventing moisture loss and safeguarding the snail from bacterial infection 1,36 .Finally, these results suggest that E. desertorum snail is a mixture of several compounds, and each component might contribute to its biological activity than if they acted alone.Therefore, the current study suggested that ME of E. desertorum snail is a potential source of natural components that possessed antimicrobial and antiin ammatory properties that may be used for the treatment of burn wound infections.Also, it can induce wound healing by improving expression of growth factors genes.However, so far, there is no available toxicological data on human regarding the E. desertorum snail; therefore, further assessment should be performed to de ne the safety doses of this novel snail for human use.

Conclusion
This study has evidenced the e cacy of ME of E. desertorum snail as a new antimicrobial and anti-in ammatory agent in burn wound infections, highlighting its e ciency in wound healing for future usage in topical technology.
Moreover, in vivo and human studies need to be performed further to con rm the snail biological properties.

Snail collection and mucus extraction:
Thirty adult of garden snail Helix aspersa and desert snail Eremina desertorum were collected from Foah region, Kafr El-sheikh, Egypt (31°06′42″N 30°56′45″E) and El Alamein, Western Coast, Egypt (30°50′N 28°57′E), respectively.The samples were identi ed according to Schileyko 37 as reported in supplementary data (Fig S1).Each species of snail was housed in two separately plastic boxes, each with 15 snails.To keep the plastic boxes damp, they were sprayed with water every day.Then snails were transferred individually packaged in plastic containers and stored.To avoid infection, go 3 days without eating.
Snails were manually stimulated at the pedal glands in their foot.Each individual's mucus sample is collected and then pooled for each species.About 100ml of crude extract from 25 snails of each species was collected.The harvested mucus was ltered).Mucus was than sterilized by ltering through 0.45-µm membrane then stored at − 80°C.In order to obtain only the dry part, Mucus samples were lyophilized overnight to obtain a solid powder that was used for the biological characterization.
Microbiological characterization.Bacterial contamination was tested by plating 100 µl of mucus extracts (MEs) of both snails on Tryptic Soy agar (TSA) medium (Biomerieux, Italy).Colonies number were counted after incubation for 24-48 hrs. at 37°C and expressed as colony forming unit (CFU).Also, fungal and yeast contamination was evaluated by plating 100 µl of MEs of both specimens on Sabouraud medium plates (Biomerieux, Italy).Fungal growth was noticed after incubation for 5-7 days at 30°C 23 .Microbiological evaluation of MEs of selected strains is reported in supplementary data (Table S1), which con rmed the sterilization of MEs of both snails by the absence of fungal and bacterial contaminations without addition of any preservative.
and C. albicans (CA-11) 38,39 .All isolates were identi ed as MDR or PDR strains as described previously in our studies 12,38,39 and stored at − 70°C.Active cultures for further experiments were prepared by transferring a loop full of culture from frozen glycerol stock cultures of each strain to test tubes of Mueller-Hinton broth (MHB) (Merck, Darmstadt, Germany) for bacteria, and Sabouraud Dextrose (SD) broth for fungi, and were incubated for 24-48 hrs. at 37°C.

Antimicrobial activity assay and Minimum Inhibitory Concentration (MIC)
Antimicrobial activities of MEs from both snails were assessed against the eight selected strains by agar well diffusion method as detailed in El-Zawawy 40 .The agar plates were swabbed with 100 µl broth culture of selected strains.Wells were made in agar plates using a sterile cork borer of 5 mm.MEs were dissolved in 1% pure dimethyl sulfoxide (DMSO; Sigma-Aldrich, St. Louis, Missouri, USA) to a nal concentration of 100 µg/ml.Twenty microliters of various concentrations (10, 20, 30, 40, 50 µg/ml) were added to each well.DMSO (1%) was used as a negative control.
The minimum inhibitory concentration (MIC) was determined by micro dilution method 41 .The growth was observed and the optical density was read at 595 nm spectrophotometrically.MIC was determined by the lowest concentration of sample that inhibited the development of turbidity.

Anti-in ammatory activity
The anti-in ammatory activities of MEs from both snails were determined in vitro by three experiments as described in our previous studies 12,42 in details; membrane stabilization of human red blood cells, albumin denaturation and proteinase inhibitory activity.Different concentrations (100, 200, 300, 400, 500, 1000 and 2000 µg/ml) of both snails were prepared and compared with DMSO (1%) as a negative control and aspirin (Bayer, Leverkusen, Germany) as a reference drug.

Cell culture
Human Skin Fibroblast (HSF) cell line employed in this study was obtained from Nawah Scienti c Inc., (Mokatam, Cairo, Egypt).Cells were maintained in DMEM media supplemented with 100 mg/ml of streptomycin, 100 units/ml of penicillin and 10% of heat-inactivated fetal bovine serum in humidi ed, 5% (v/v) CO 2 atmosphere at 37°C.Cells were counted by hemocytometer and viability was calculated to seed the cells at appropriate densities, to perform the assays.

Cell viability and Cytotoxicity studies
The cytotoxicity of MEs of both snails on HSF cells was evaluated by SRB assay 43 .Brie y, HSF cells with initial density (5x10 3 cells) were seeded in 96-well plate and incubated with 100 µl of DMEM media for 24 h.Cells were then treated with another aliquot of 100 µl media containing MEs of both snails separately at various concentrations (0.03, 0.3 ,3, 30, 300 µg/ml).After 72 h of treatment exposure, cells were xed by replacing media with 150 µl of 10% TCA and incubated at 4°C for 1 h.The TCA solution was removed, and the cells were washed 5 times with distilled water.Aliquots of 70 µl SRB solution (0.4% w/v) were added and incubated in a dark place at room temperature for 10 min.
Plates were washed 3 times with 1% acetic acid and allowed to air-dry overnight.Then, 150 µl of Tris (10 mM) was added to dissolve protein-bound SRB stain; the absorbance was measured at 540 nm using a BMG LABTECH®-FLUOstar Omega microplate reader (Ortenberg, Germany).The cells treated with DMEM alone, 1% DMSO and 10% DMSO were considered as negative, vehicle and positive controls, respectively 23 .

Scratch assay and assessment of cell migration
The healing properties of MEs of both snails were tested on HSF cells scratch assay 44 .Brie y, cells were seeded at density of 3x10 5 cells/well in 6-well plate and were cultured overnight.After 24 hrs.medium was removed and a linear scratch in the middle of the well was done using a p200 tip.Then 400µl of selected snails with a concentration of 300 µg/ml or media (control) were added to each well.Scratch repair and cell migration were observed in the images taken by inverted microscope, equipped with digital camera.The experiments were performed in triplicate.The width of the scratch and wound closure at different time intervals (0, 24 and 48hrs.)was analyzed by MII Image View software version 3.7.
Effect of MEs of selected snails on the expression of transforming growth factorbeta 1 gene TGFβ1 and vascular endothelial growth factor gene (VEGF), was evaluated by qRT-PCR.Hot phenol/chloroform extraction method 45 was used in extraction of total RNA.The obtained cDNA was then used for realtime polymerase chain reaction (PCR) using master SYBR Green I (Takara Bio, Japan) on ABI 7900HT.Realtime PCR was executed at 95°C for 10 s, 62°C for 15 s, and 72°C for 8 s using the primers for the normalizing GAPDH gene against the Tgfβ1 and VegfA target genes.Primers were designed by Gen-Script according to the cDNA sequences of mouse TGFβ1 and VEGF and GAPDH in Gene Bank as shown in S2.Table 2. Realtime PCR was performed in triplicate for every cDNA.Expression in broblast cells was treated with each extract at 24 and 48 hrs after treatment were compared with the control (nontreated cells) after normalization with GAPDH.We used relative gene expression, to identifying the increase or decrease of a transcript of target gene in treated sample versus control sample via normalizing with a housekeeping gene.To determine the difference of the gene expression between groups, the data were analyzed using the Relative Expression Software Tool (REST; version 2009).Gas chromatography-mass spectrometer (GC-MS) analysis MCEs of both snails were investigated for their phytoconstituents using GC-MS (Trace GC Ultra, USA), at the National Research Centre (NRC), El Dokky, Giza Governorate.Identi cation of unknown compounds was based on comparing their retention time relative to those of the known compounds by matching spectral peaks available with Wiley 9 Mass Spectral Library 46 .

Statistical analysis
All data were expressed as mean ± standard deviation of three replicates and submitted to variance analysis using SPSS-20.

Declarations Figures
Page 17/   Anti-in ammatory activity of MEs of both snails compared to aspirin.
Cytotoxicity evaluation of MEs of both selected snails.HSF cells were exposed to 300µg/ml of MEs of both selected snails and cell viability was examined by SRB assay.A) Representative images with magni cation of (10X) taken by light microscopy of HSF cells untreated and treated with 300µg/ml of both selected strains at 48 hrs.B) Cell viability was calculated at 24, 48 and 72 hrs compared to untreated cells (control), DMSO (10%) and (1%) were used as positive and vehicle controls of cell death, respectively.
Scratch-wound healing assay.A) Percentage of wound closure at 0, 24, 48 hrs. in the absence and presence of MEs of both selected snails (300µg/ml).B) Microscopical representative images for wound healing of MEs of both selected snails.

Table 1
Antimicrobial activity of MEs of selected snails Values are the mean of three replicates ± SD.Means with the same letters in the same column showed the insigni cant difference (P ≤ 0.05).Data obtained from our previous studies[12, 20, 25, 26].

Table 2
Chemical Constituents ME of E. desertorum using GC-MS