In vitro evaluation of pomegranate ( Punica granatum L.) peels antibacterial activity against bacteria responsible of diabetic foot infections

Diabetic foot infections are common complications of diabetes. Antibioresistant bacteria are associated with a higher incidence of lower-limb amputation. Punica granatum L. is one of the plants used by many civilisations in the treatment of infectious maladies. Our work consists of evaluating in vitro the antibacterial activity of three extracts of pomegranate peels on Gram positive and Gram negative bacterial strains isolated from diabetic foots pus. The active molecules were obtained by maceration of pomegranate peel powder in ethanol/water (30/70) and fractionation using three solvents: dichloromethane (EDCM), ethyl acetate (EEA) and the n-Butanol (En-BuOH). The bacterial susceptibility to the extracts was determined using the disks diffusion test and the Minimal Inhibitory Concentration (MIC) by dilution in liquid medium using sterile microplates. The three extracts were active against the tested strains with maximum activity of the extract (En-BuOH) against Staphylococcus epidermidis (35.3 ±0.3 mm). Enterobacter aerogenes was less sensitive to the extracts (EDCM) and (EEA) (9±0.9 and 9.6±0.6 mm respectively). The weakest MIC (0.12 mg/ml) was recorded within Morganilla morganii with the three fractions and within Proteus mirabilis with the fraction ethyl acetate. Furthermore, the highest MIC (> 250 mg/ml) was fond within Staphylococcus aureus and Enterobacter aerugenes with dichloromethane, Escherichia coli and Pseudomonas aeruginosa with different fractions. More of that, the extracts were characterised by an antibacterial activity better than some used antibiotics to whom the strains were multiresistant.


Introduction
Diabetes has become an increasingly prevalent and severe public health issue in Algeria.The national evidence suggests that the prevalence of diabetes in Algeria has increased from 6.8% in 1990 to 12.29% in 2005 (Lamri et al., 2014).Algeria is particularly impacted by the epidemic to the point of appearing in the "top 10" 2016 of countries with very high incidence of T1D (Touhami et al., 2019).Foot problems in diabetes are common and costly, and people with diabetes make up about half of all hospital admissions for amputations (Boulton et al., 2018).Among patients with diabetes presenting with a foot wound, about half have clinical evidence of infection (Lipsky et al., 2016).For people with diabetes, DFIs are the most common diabetes-related reason for hospitalizations and for lower extremity amputations (Boulton et al., 2018).This is due to the disruption of the protective skin.The deformed foot becomes colonised with infectious bacteria (Fisher et al., 2010).Gram cocci bacteria and specially Staphylococcus aureus are the most isolated germs from DFI infections (Lipsky et al., 2012;Citron et al., 2007;Roberts and Simon, 2012).Antibiotics are one of the mainstays of treating diabetic foot infections.Drug-resistant organisms are over-represented in samples obtained from diabetic foot ulcers (Powlson and Coll , 2010) and are associated with a higher incidence of lower-limb amputation (Richard, 2008).Plants are prospective source of antimicrobial agents in different countries (Alviano and Alviano, 2009).Punica granatum L. (pomegranate) is one of the plants arousing interest for its virtues medicinal.Their Peel have been commonly employed as a crude drug in traditional medicine for the treatment of diarrhea as well as for use as an astringent, antihelminthic, asphrodisacs, laxative, diuretic, stomachic, cardiotonic and refrigerant (Pradeep et al., 2008).Therefore, our objective is firstly to confirm In Vitro the antibacterial activity of the pomegranate peel extracts (En-BuOH, EDCM and EEA) against multiresistant bacteria isolated from diabetic foot ulcers and secondly to ameliorate the costs of diabetic foot infections treatment by substituting the classical antibiotics by the tested extracts or conjugating the two treatments and this to limit the rate of lower-limb amputation.

Material and Methods
Our work consists of three parts; extraction of active molecules from grenade peel, isolation of bacterial strains from infected diabetic foot and testing the effect of the extracted active molecules on the isolated bacterial strains.

Sampling and macroscopic exams
The method of sampling depends on the infection level, superficial or deep.When samples are quite abundant, macroscopic exams may provide interesting information: a bad smell pus for the anaerobic bacteria, granular aspect for streptococci and creamy aspect for staphylococci or pneumococci.

Cytologic exam
It consists of counting the number of polynuclear and neutrophil cells as well as the presence and absence of germs by the methods of Hanging-drop.The methods of simple coloration using methylene blue and Gram coloration determine the form, the size and the regrouping mode of the bacterial cells.They are indicative exams for the choice of the culture media.

Bacteriological exams
The isolation of bacterial strains was done by culturing pus directly in three media: Chapman, Hektoen and Blood agar.In case of negative culture the previously enriched pus in brain heart infusion broth ( BHIB) or in glucose buffered broth medium was used.Enterobacteria were identified by the biochemical miniaturised kits (API 20E).Staphylococci strains were identified according to the presence and absence of the catalase and coagulase enzymes.Two reference strains were also used, Escherichia coli ATCC25922 and Staphylococcus aureus ATCC29213.

Preparation of the three extracts
The pomegranate fruit (Punica granatum L.) was obtained from Ramdan Djamel town, the state of Skikda during the month of October 2015.The fruit peel was then isolated and dried at the temperature room, in the darkness and grinded.The obtained powder was used to prepare the different extracts.

Maceration
The method of Merghem et al. (1995) was used with modification of the used solvents.The pomegranate peel powder (200g) was added to ethanol/water (30/70) v/v.The mixture was well shacked during 72h with renewal of the mixture each 24h.

Extraction liquid/liquid
The macerates were reunited and filtered by filter paper.The recovered filtrate was then evaporated using a rotary evaporator.The dry residue was recuperated in 200ml boiling distilled water.The extract was then decanted during a night and the limpid phase underwent fractionation using three solvents with increasing polarity: dichloromethane (EDCM), ethyl acetate (EAA) and the n-butanol (En-BuOH).The aqueous phase and the solvent were well agitated and left to rest for 30 minutes.The aqueous phase in the bottom of the separating funnel and the phase charged with active molecules were recovered separately.The active phase charged with polyphenols was dried and the following equation was used for the determination of the extraction yield (Harborne, 1998): Yield= (weight of the extract / weight of the vegetal material) *100.

Determination of antibacterial activity of pomegranate peel extracts
Antibacterial susceptibility to pomegranate peel extracts was tested using agar disks diffusion technique.Stock solutions of the three tested extracts were prepare by diluting each dried extract in dimethyl sulfoxide (DMSO) to obtain a final concentration of 250mg/ml.Muller Hinton medium was aseptically poured into sterile Petri plates.The bacterial inoculums (300Ul) adjusted to 0.5 Mac Farland (10 8 Cells/ml) (CA-SFM, 2012) and diluted to 1/10 (10 7 cells/ml) (CA-SFM, 2010) was swabbed on Muller-Hinton medium.6 mm filter paper disks loaded into 10ul of the tested extract were placed on the plates at equivalent interval.A pure DMSO loaded disk was added as a negative control.The cultures were left 15 minutes for a pre-diffusion before they were incubated at 37°C for 24 hours.The experiment was realised in triplicate and mean value of zone inhibitions was calculated.The bacterial sensitivity toward the extracts was classified according to Moreira et al. (2005): 8≤mm: Not sensitive (-), 9-14 mm: Sensitive (+), 15-19 mm: Very sensitive (++), ≥20 mm: Extremely sensitive (+++)

Evaluation of minimal inhibitory concentration (MIC) in liquid medium:
The minimal inhibitory concentration was determined using sterile microplates (8x12 wells).0.1 ml of nutrient broth were added to the wells of the same line, then 0.1 of the tested extract (250mg/ml) were added to the first well and well homogenized with nutrient broth.0.1 ml of the mixture were transferred from well to well to obtain dilutions at a factor of 1/2.Finally, 0.1 ml of the inoculums previously diluted to 1/100 (10 6 cells/ml) were added to each well (EUCAST, 2003).Thereby, the concentration 250 mg/ml was diluted successively to 1/2, 1/4, 1/8, 1/16, 1/32, 1/64, 1/128, 1/156, 1/512, 1/1024 and 1/2048.The plates were incubated at 37°C for 24 hours.The MIC was determined by the concentration where no trouble was observed.

Antibiogram
Antibacterial susceptibility to the tested antibiotics was determined based on the directives of CA-SFM (2010).A bacterial suspension of 0.5 Mac Farland was previously prepared.Muller Hinton medium poured into Petri plates was seeded by swabbing the swab onto its surface to achieve tight steaks.The antibiotics disks were placed on the surface of the medium and the plates were incubated at 37°C for 16-24 hours (CA-SFM, 2010).The obtained inhibition zones around the antibiotics were measured and the bacterial strains were classified as sensitive (S) or resistant (R) (CA-SFM, 2012).

Statistical analysis
Inhibition zones were done in triplicate sets and the results were expressed in mean value± SD (Steel et al., 1995)

Bacterial strains
Eight bacterial strains were isolated from diabetic foots: Proteus mirabilis, Pseudomonas aeruginosa, Morganella morganii, Enterobacter aerogenes, Enterobacter cloacae, Escherichia coli, Staphylococcus epidermidis and Staphylococcus aureus.Two reference strains Escherichia coli ATCC25922 and Staphylococcus aureus ATCC29213 provided by Pasteur institute of Algeria were also tested.Enterobacteria strains were identified using their macroscopic, microscopic and biochemical characters (Table1).Staphylococcus aureus was Catalase and Coagulase positive however Staphylococcus epidermidis was Catalase positive and Coagulase negative.

Antibacterial activity of the three extracts
The obtained results (Table 2 and Figure 1) showed that the three fractions of pomegranate peel were active against the eight strains.This activity indicates the presence of toxic secondary metabolites towards the tested strains (Voravuthikunchai et al., 2004).Pomegranate is known as a rich source of pharmacological properties which have been evaluated due to antiparasitic, antibacterial, antifungal antiproliferative, apoptotic and anti-cancer effects (Reddy et al., 2007; Kim et al., 2002; Naz et al., 2007 ) .The inhibition zones vary according to the used extract and the tested strain.The comparison of the average of three inhibition zones replicates revealed that the highest antibacterial activity was recorded against Staphylococcus aureus and Staphylococcus epidermidis as well as Staphylococcus aureus ATCC29213 with the three extracts (29.3±0.3-35.3±0.3 mm) and against Morganella morganii and Proteus mirabilis with En-BuOH and EEA (21±0.6-22.6±0.6 mm).Moderate inhibition zones were observed against Escherichia coli ATCC 25922 within the three extracts (14.6±0.3-17±07mm) as well as Pseudomonas aeuginosa, Enterobacter cloacae and Morganella morganii within En-BuOH, EEA and EDCM respectively (14.6±0.2-18.3±0.3mm).Escherichia coli and Enterobacter aerugenes were less sensitive to EEA and EDCM fractions (9±0.9-12±0.2mm) as well as Proteus mirabilis and Pseudomonas aeruginosa with EDCM (11.6±0.6-13.3±0.3 mm).Gram negative bacteria were less sensitive in comparison with Gram positive bacteria.The outer membrane of Gram negative bacteria high in polysaccharides prevents certain antibacterial biomolecules from entering into the cell (Bagamboula et al., 2004).Our results corroborate those of Reddy et al. (2007) and those of Celia and Jenifer (2018) who demonstrated that the pomegranate peel extracts constitute a very strong inhibitor of Staphylococcus aureus growth (Inhibition zones> 20mm).Hence, the antibacterial activity of Punica granatum L. may be related to polyphenol structures (Rathia et al., 2014;Barathikannan et al., 2016) because polyphenols may affect the bacterial cell wall, inhibit enzymes by oxidized agents, interact with proteins and disturb co-aggregation of microorganisms (Naz et al., 2007;Vasconcelos et al., 2003).3).They were resistant to three antibiotics families (B-Lactamines, Polymixines and Aminoglygosides).They were on the other hand sensitive to the three tested extracts with inhibition zones varying from 9±0.9-21.6±0.0mm(Table2).Pradeep et al. (2008) and Khan and Hanee (2011) demonstrated that the tested E.coli was more sensitive to Tetracyclin, Viprofloxacin and Oflaxacin in comparison with the tested extracts.Proteus mirabilis and Escherichia coli ATCC259 were however sensitive to the tested antibiotics as well as the tested extracts (13.3±0.3-21.3±0.3).Pseudomonas aeruginosa which is a multi resistant strain to more than three antibiotics families (B-Lactamines, Quinolones, Aminosides, Tetracyclines and Fosfomycines) (Table 4) was very sensitive to the extract En-BuOH (17±0.7mm)and sensitive to EEA (14.6±0.2 mm) and EDCM (11±0.7mm).These values are superior of that obtained by Belaidi (2012) and Pradeep et al. (2008) as well as those of Sajjad et al. (2015) with ethyl acetate extract.They were on the other hand inferior of that obtained by Khan and Hanee (2011).This is due to the differences in the solubility of active molecules in each used solvent.The tested Gram positive bacteria, Staphylococcus aureus, Staphylococcus epidermidis and Staphylococcus aureus ATCC29213 were highly sensitive to the tested extracts (29.3±0.3-35.3±0.3mm)(Table 2).

Conclusion
It was concluded from the results of current study that n-Butanol extract of Punica granatum L. peel demonstrated high activity against Gram positive bacteria in comparison with Gram negative bacteria.
Our in vitro research confirms the possibility of exploiting pomegranate peels as an alternative or a conjugating treatment of diabetic foot infections, but more experiments are necessary to determine the concentration and the nature of the active molecules in the pomegranate peel.

8≤mm:Figure
Figure-1.Inhibition zones of the three extracts En-BuOH, EAA and EDCM against the tested bacterial strains