EVALUATION OF ANTIMICROBIAL POTENTIAL OF DIFFERENT COMMERCIAL GRAPEFRUIT SEED EXTRACTS

(MDR) bacteria. The highest inhibitory potential is conducted for Citrofit®, while CitroBiotic® has shown the weakest effect. These results are in concordance with the obtained MIC and MBC values. It could be concluded that frequent use of commercial GSEs is justified due to their powerful antimicrobial activity. These findings are promising in terms of utilization of natural products as novel antimicrobial agents.

were triple tested.Antimicrobial activity of tested GSEs was evaluated based on diameter of inhibition zones, after the extract diffusion in the medium.Four different antibiotics: Colistin (10 µg), Streptomycin (10 µg), Ampicillin (10 µg) and Amoxicillin (25 µg), as well as standard antifungal drug Nystatin (100 IU), all made by Oxoid™, Great Britain, were used as positive control.

Broth microdilution method
Broth microdilution method (CLSI, 2018) was applied in order to determine the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of tested grapefruit seed extracts.Considering the antifungal activity, we used the term MFC (minimum fungicidal concentration).Serial dilutions were made in sterile 96 well microtiter plate.Amoxicillin (Sigma-Aldrich, USA) and Nystatin (Sigma-Aldrich, USA) were used as reference, prepared in initial concentration of 1024 µg/ml and 1000 µg/ml (5916 IU/ml), respectively.Dimethyl Sulfoxide, DMSO (Sigma-Aldrich, USA) was used as the solvent.At first, 100 µL of liquid MH medium was added to each well in a microtiter plate, except in the wells of the first column.Amount of 200 µl of tested GSEs at initial concentration of 330 µg/ml was added in the first microtiter column.Then, 100 µl of extract was transferred from the first column to the second, and after mixing 100 µl of content, transferred from the second to the third column.This procedure was repeated to the tenth column, from which 100 µl of content was finally removed.In this way, in each row of the microtiter plate, from position 2 to position 10, a serial two-fold dilution of the test substance in the range of 165 to 0.64 µg/ml was obtained.The last row in the microtiter plate contained double serial dilution of antibiotic, starting from 512 to 2 μg/ml.Wells from the second to the eleventh column were then inoculated with the 5 µl of test microorganisms' suspension, at the concentration of 1-2 x 10 8 CFU/ml, and final concentration of 5 x 10 5 CFU/ml was obtained.Column 11 containing 100 μl of standard inoculum without tested GSEs served as the positive control, while column 12 containing only the sterile liquid medium and DMSO was used as the negative control.Each test was performed twice.Microtiter plates were incubated for 16-18 hours at 37 °C.

Resazurin assay
Reading of MIC is done manually using an inverted mirror and after that 30 μl of 0.015% Resazurin sodium salt solution (Sigma-Aldrich, USA) was added to all wells.Resazurin dye allows easier interpretation of the results (Elshikh et al., 2016).The wells containing the living microorganisms were coloured pink, while those with death microorganisms, destroyed by the test substance, were stained blue.All columns with no visible colour change represent the concentration of the extract above the MIC value.The last position in the row where the blue colour remained, while there is a presence of pink colour in the next position, represents MIC.

Minimum bactericidal concentration (MBC) and minimum antifungal concentration (MFC) assays
After the determining of minimum inhibitory concentration (MIC), the determination of the minimum bactericidal concentration (MBC) and minimum fungicidal concentration (MFC) was proceeded (CLSI, 2018).The volume of 10 µl of content from the well whose position was marked as MIC, as well as from the previous three wells in the same row with no observed microbial growth, was transferred on agar plates with MH/SGA medium.Inoculated plates were incubated at 37 °C for 18-24 hours.The MBC/MFC was recorded as the value closest to the determined MIC value, at which there was no growth of microorganisms after incubation.All experiments were performed in triplicate.

Statistical analysis
Performed tests were done in three replications, therefore descriptive statistical parameters: mean values ± standard deviation (SD) were calculated, using the Microsoft Office 2019 Excel (Microsoft Corporation, USA).In order to determine the presence of statistically significant differences between collected data, One-way ANOVA and post hoc Fisher's Least Significant Difference (LSD) test (STATISTICA 10; StatSoft.Inc.) were applied, at the significance level of p<0.05.

Antibacterial and antifungal assays
All three investigated GSEs showed inhibitory effects against reference microorganisms, but with different intensity.Overall results suggest Citrofit® as the strongest GSE, with widest inhibition zones observed in the case of B. subtilis subsp.spizizenii (31.00±0.00)and MRSA (30.00±0.00).The most sensitive Gram-negative strain to this extract was S. enterica subsp.enterica serovar Enteritidis (24.30±1.15).Citrofit® caused the greatest inhibition of C. albicans (26.30±0.57)as well, which is significantly different in comparison to the standard antimycotic (Tab 1).These results are in accordance with the investigation of Krajewska-Kulak et al. (2003), where 33% grapefruit-water glycerol solution also exhibited strong activity against the yeast-like fungi.NutriBiotic® performed balanced antibacterial activity, with the greatest zones on P. aeruginosa (25.30±1.52),followed by slightly different inhibition of multidrug-resistant ESBL producing E. coli (25.00±1.00),MRSA, E. faecalis, S. enterica subsp.enterica serovar Enteritidis (24.60±0.57),and B. subtilis subsp.spizizenii (22.60±1.52).Both Citrofit® and NutriBiotic® have shown significantly higher antibacterial and antifungal activity compared to the CitroBiotic® (Tab 1).A possible explanation for such results could be the fact that CitroBiotic® preparation is completely free of benzethonium chloride, benzalkonium chloride and triclosan, as well as of organochlorine-and organophosphorus-pesticides (Manufacturer Report No. K 2465 FN -III and K 2465 FN -I).There is a chance that some of the listed substances may act as antimicrobial agents.Statistical analysis has shown that antimicrobial activity of tested GSEs was significantly higher from antibacterial and antifungal effects of tested antimicrobials (Tab 1).None of four used antibiotics didn't cause inhibition of MRSA, in contrast to the effects of GSEs.The obtained results coincide with the data that GSE has a pronounced effect on methicillin-resistant Staphylococcus aureus (Su et al., 2012), which indicates that investigated extract could be used in the treatment of infections caused by this multiresistant bacterial strain.In addition, ESBL producing E. coli was inhibited by Colistin and Streptomycin, but with lower rate in comparison to the GSEs (Tab 1).In most cases, Gram-positive bacteria have shown greater sensitivity to the investigated extracts compared with Gram-negative bacteria, which is in line with previous findings (Reagor et al., 2002;Heggers et al., 2004;Xu et al., 2007;Bangwoo et al., 2015).This could be explained by the fact that Gram-positive bacteria have only a thick layer of peptidoglycan in their cell wall, which makes them susceptible to inhibitory substances, while Gram-negative bacteria have an outer lipid membrane in their cell wall, as a kind of barrier, making them less sensitive.GSE probably acts by destroying the cell membrane and ultimately leading to lysis and death of the bacterium (Heggers et al., 2004;Bhatia et al., 2015).

Determination of minimum inhibitory concentration (MIC)
In terms of antibacterial and antifungal activity, tested GSEs have showed MIC values of <0.64-82.50µg/ml (Tab 2).Comparing the three GSEs used in this investigation, Citrofit® has showed a higher antimicrobial activity than CitroBiotic® and NutriBiotic®.Furthermore, antimicrobial activity of this extract almost completely surpassed the activity of Amoxicillin and Nystatin.Tested antibiotic didn't cause the growth inhibition of MRSA, E. faecalis and ESBL producing E. coli, while all tested extracts were successful in this sense (Tab 2).

Determination of MBC and MFC
The microdilution method of testing the minimum inhibitory concentration of Citrofit® has showed the most effective action on four microorganisms (B.subtilis, MRSA, P. aeruginosa, and C. albicans) with the MIC less than 0.64 µg/ml, whereby Gram-positive bacteria were more susceptible than Gramnegative.The same results were observed in the study of the minimum bactericidal and minimum fungicidal concentration following the results of the MIC, where Citrofit® had the strongest effect (Tab 2, Tab 3).

CONCLUSION
Commercial GSEs investigated in this study have shown antimicrobial activity against all tested Gram-positive and Gram-negative bacteria, as well as the strain of pathogenic fungus C. albicans.The strongest inhibitory effects were observed for Citrofit®, especially against multidrug-resistant pathogens (MRSA and ESBL E. coli).Tested antimicrobials proved to be less effective against bacteria and C. albicans strain in comparison to the GSEs.There are differences between the antimicrobial activity of each extract, which could be attributed to the chemical content variation.It is known that phytochemical profile, as well as biological activity of plant products, could be related to the specific abiotic factors in the plant habitat, but also the conditions of mass production process.Also, there is a possibility that artificial additives, frequently present in such extracts, could be related to overall antimicrobial potential.It is promising that commercially available products of natural origin, possess such remarkable antimicrobial activity, especially in times where most synthetic antibiotics are less and less effective.However, it must be noted that investigated extracts could represent a dietary supplement and not an official treatment against infectious diseases.Wider medicinal use of GSEs should be supported with exact evidence regarding its phytochemicals, pharmacological and biological properties.

Table 1
Diameter of inhibition zones* (mm) obtained through the agar well diffusion method 't share the same letters, differ significantly at p<0.05 after post hoc LSD test.

Table 2
Minimum inhibitory concentration (MIC) of tested extracts and controls