Antimicrobial Potential of Genes from Garlic (Allium sativum L.)

2.1 Antibacterial potential

Allium extracts containing thiosulfinates have the potential to retard the growth of Gram-positive and Gram-negative bacteria. It is, however, reported that garlic can inhibit the Gram-negative bacteria more than Gram-positive bacteria [26]. The permeability of inhibitory compounds from Allium might be affected by the cell wall and cell membrane structure. However, the results were quite opposite with diallyl trisulfide and dimethyl trisulfide and with garlic extracts to conclude that the Gram-positive bacteria were more sensitive than Gram-negative bacteria [27, 28].

Extracts from the garlic are reported to exhibit the effective results against saprobic and pathogenic bacteria, which are resistant to various drugs [29]. Garlic along with ciprofloxacin exhibits the pronounced inhibition of E. coli Z17, O2:K1:H- and Helicobacter pylori, but no significant evidence was found in the case of H. pylori infection in human [30, 31]. It has previously been proved that allicin is the main compound in garlic responsible for the antimicrobial activity, as garlic oil and extracts deficient in allicin do not exhibit any kind of antimicrobial activity [32]. It was later found that garlic oil and its constituting sulfides exhibit the more and significant inhibition of microbes and work as strong antifungal than the antibacterial agent [28].

Studies have reported that oils and sulfides from elephant (A. ampeloprasum) and shallot (A. ascalonicum L.) garlic have the potential to inhibit the food-borne pathogenic bacteria [33, 34]. Ajoene, an unsaturated disulfide, has been reported for its broad-spectrum antibacterial activities, which can be reduced by cysteine, a sulfhydryl compound [35]. Later, it was proved that disulfide in ajoene is a necessary component for the inhibition of bacteria as reduction by sulfhydryl compounds reduces the antibacterial activity. Gram-positive bacteria and yeast are more sensitive to ajoene than Gram-negative bacteria.

2.2 Antifungal potential

It is reported in different studies that oils and sulfides from the Allium have the more potential to inhibit the fungi than bacteria [28, 36]. Antifungal activity of sulfide molecules is directly proportional to increase in the number of sulfur atoms up to sulfur number three or four in sulfide molecules [28, 37].

Another study has also reported that sterilized/autoclaved garlic and its active compounds exhibit significant antifungal activities than that of antibacterial. Further analysis of garlic antimicrobial products revealed that these products are the heterocyclic sulfides [38], allyl alcohol [39], and 3-(allyltrisulfanyl)-2-aminopropanoic acid [40]. For bacteria and yeasts, minimum inhibitory concentrations (MICs) of heterocyclic sulfides are more than 100 and 1–6 ppm [38], respectively, while for the allyl alcohol, 4% and 55–140 ppm MICs are recorded for bacteria and yeasts [39], respectively. In the case of 3-(allyltrisulfanyl)-2-aminopropanoic acid, 100 ppm and less than 50 ppm MICs are observed for bacteria and yeasts [40], respectively. In previous studies, it was mistakenly stated that autoclaved garlic exhibit less antimicrobial activities than fresh garlic. For this statement, the only reason was that they tested autoclaved garlic against bacteria, which was already very less sensitive than yeasts against garlic [41]. Recent studies have explored the germicidal potential of sterilized/autoclaved garlic.

2.3 Antiviral activity

Diallyl polysulfides, as transformation product of allicin, and ajoene exhibit the antiviral activities. From all the reported Allium products, it is observed that ajoene exhibits more inhibition than other compounds like allicin and thiosulfinates, but on the other hand, allicin is considered as strong antimicrobial agent [42, 43]. It is thought that antimicrobial compounds from garlic react with viral envelope and inhibit the penetration and exponentiation of influenza virus in animal kidney cells [44]. Garlic aqueous extracts have also been studied to observe the inhibition against potato virus Y under in vivo and in vitro conditions [45].

2.4 Antiparasitic potential

A number of parasites, including Leishmania donovani [46], Spironucleus vortens [47], and Eimeria papillata [48], are sensitive to garlic extracts. The MIC values of allicin, dithiins, and ajoene for the inhibition of S. vortens growth are higher than the MICs reported for the inhibition of bacteria and fungi, indicating the high tolerance of S. vortens for Allium extracts [46].

From the above discussed literature, it is clear that garlic has a certain pool of antimicrobial genes which can be isolated and studied further to explore their mechanisms. It will provide some new directions for antimicrobial research. Now we will discuss some techniques to isolate and study the antimicrobial genes from garlic.

3.1 Bacillus subtilis and Escherichia coli expression systems

An experiment was designed to study the antimicrobial genes from the garlic. For this purpose, cDNA libraries from garlic were constructed by using two different vectors, pBE-s and pET22 (b), and then transformed into expression systems, B. subtilis and E. coli, respectively. For the library quality analysis, two parameters were considered, recombination rate and library titer [49]. For the E. coli expression system, 96.7% and 4.6 × 10⁶ pfu/ml, recombination rate and library titer were observed, respectively. On the other hand, recombination rate and library titer for B. subtilis expression system were 91.7% and 7.8 × 10⁶ pfu/ml, respectively. Quality analysis revealed gene library in E. coli expression system was marginally better than that of the B. subtilis expression system.

For the screening of libraries, it was considered that because of the toxicity of protein products of cDNA libraries, B. subtilis and E. coli cells would be showing autolysis to indicate the antimicrobial potential of these libraries’ inserts. For more confirmation, trypan blue dye was also used to indicate the viability of E. coli cells [50]. By using this strategy, a number of antimicrobial genes were screened from garlic to reveal its further potentials. For example, in case of B. subtilis expression system, a total of 48 antimicrobial genes were screened, including AsR 416, while AsRE 67 was identified by using E. coli expression system [51].

3.2 Antimicrobial potential of genes from A. sativum

Antimicrobial potential of A. sativum genes was studied against fungi and Gram-positive and Gram-negative bacteria [50], and the results were observed as follows (Tables 1–3).

3.3 Action mechanism of antimicrobial proteins

A study was designed to explore the action mechanism of antimicrobial peptides. In this study, B. subtilis cells were treated with antimicrobial peptide, AsR 416, and then PI (propidium iodide) staining was performed [51]. PI is fluorescent agent that has the ability to bind with DNA through broken cell membrane. Red fluorescence in all bacterial cells treated with antimicrobial peptide was observed under confocal laser microscope [52], while the flow cytometry analysis revealed that cell membrane damages increase with increase in the protein concentration [53]. All findings collectively support that the target of antimicrobial peptide is to destroy the cell membrane of target bacteria.

3.4 Thermal stability and safety analysis of antimicrobial proteins

Proteins from AsR 117, AsR 416, and AsR 498 were heated at different temperatures for 15 min, and it was found that AsR 117 and AsR 416 proteins were thermally stable at all temperature ranges, while AsR 498 became thermally unstable after 50°C, as it exhibited the reduced antimicrobial activity. For the safety analysis, these proteins were analyzed against sheep red blood cells [54, 55]. This analysis revealed these antimicrobial proteins as nontoxic to mammalian cells with maximum 1000 μg/ml concentration [51]. From the thermal and safety analyses, it is also obvious that antimicrobial genes from garlic can also be used in human medicines in the future, which needs further investigations.