Garlic is one of the most studied medicinal plants in the world and has been used in traditional medicine for many centuries for the treatment of many diseases [34, 35]. According to Majewski [16], the properties of garlic are due to the combination of several biologically active substances with antimicrobial properties, which are responsible for its curative effect, which aroused our interest in studying its antimicrobial activities. To this end, we evaluated its susceptibility and drug interaction when combined with the antibiotic ampicillin and penicillin G, both belonging to the class of β-lactams, against clinical isolates of S. agalactiae.
Ampicillin and penicillin G were used because they are first-line antibiotics for IAP in pregnant women [36]. In addition, no studies were found that evaluated the interaction of these antibiotics with garlic or the prospect of finding ways to enhance the action of these antibiotics, which could help in the prophylaxis and/or treatment of this infection. It is worth noting that our study is the first of its kind to be published in the literature to date.
The complete phytochemical analyses of the SP80 fraction obtained from the raw garlic extract (RGE) were performed by Torres et al [27] to isolate and identify the bioactive compounds present in garlic using reverse phase ultrafast liquid chromatography with UV spectroscopy (RP-UFLC-UV), mass spectrometry (LC-MS) and nuclear magnetic resonance (NMR). Some of the compounds identified in their study were y-glutamyl-S-allylcysteine, y-glutamyl-phenylalanine, and (E) and (Z)-ajoenes, and the yields of these compounds were 8.48%, 2.77%, and 0.31%, respectively. Therefore, the present study aimed to evaluate the synergistic activity of the association of the SP80 fraction with the antibiotic’s ampicillin and penicillin G.
The SP80 fraction was used instead of the isolated fractions mentioned above because, according to Majewski [16], the properties of garlic result from the combination of several biologically active substances with antimicrobial properties, and since the yield of each isolated compound was very low, the ideal would be to use synthetic compounds purchased commercially, but this will be the objective of future work.
The methods used to evaluate antimicrobial and synergistic activity were disk diffusion and broth microdilution [37–41]. The disk diffusion method was used because it is the most used method in clinical analysis laboratories and hospitals, in Brazil, to verify antimicrobial susceptibility, and broth microdilution is used by the Microbiology Society.
The MIC of the SP80 fraction obtained by purification of the RGE and the antibiotics were determined by broth microdilution, in triplicate. The MICs used in our tests were the lowest that completely inhibited microbial growth and showed bacteriostatic activity [35]. As our work is pioneering, different extraction methods may lead to different biological responses.
When the MIC value was determined using the microdilution method, this garlic fraction was shown to have antimicrobial activity against S. agalactiae. This is an important finding that confirms all other studies described in the literature on the antimicrobial activity of this plant [37, 42–45].
The association of the SP80 fraction with ampicillin was more efficient than the association of the SP80 fraction with penicillin G compared to the antibiotics alone. The combination of SP80 + ampicillin indicates an additive effect, but we cannot consider a synergistic effect.
According to CLSI [31], inhibition halos ≥ 24 mm formed by ampicillin and penicillin G indicate that the strains tested are sensitive to these antibiotics. We compared the antimicrobial effect of ampicillin and, penicillin G, respectively, isolated against S. agalactiae strains, and of the 56 strains tested, 25 strains were resistant to penicillin G and another 2 were resistant to ampicillin, thus demonstrating that the strains tested are more resistant to penicillin G than they are to ampicillin.
There is little information on the mechanism behind reduced penicillin G susceptibility in GBS. According to Hayes et al [46], β-lactams exert their antibacterial effect by blocking the action of enzymes involved in cell wall synthesis. Penicillin G inhibits bacterial peptidoglycan formation by binding the β-lactam ring to enzymes known as penicillin-binding proteins (PBPs) at DD-transpeptidase. Therefore, Chu et al [47] believe that modification of penicillin binding proteins is a possible explanation for the reduced penicillin G susceptibility observed in their GBS isolates.
According to Van der Linden et al [48], the acquisition of point mutations in the PBPs genes, leading to the production of proteins with low affinity for β-lactam binding, could explain why some S. agalactiae strains may be non-susceptible to penicillin. However, further molecular work is needed for confirmation.
Hayes et al [45] performed checkerboard and time-kill assays in vitro to determine the synergistic activity of erythromycin and nisin against clinical isolates of Group B Streptococcus against invasive and colonizing GBS strains. Their results suggest that erythromycin and nisin may act synergistically to inhibit the growth of GBS. This study differs from ours in the antibiotics used, and our results showed an additive effect of combining the SP80 fraction with antibiotics, respectively.
Garlic has a variety of bioactive compounds, including organosulfur compounds, saponins, phenolic compounds, and polysaccharides. The most important are organosulfur compounds, especially allicin [25, 49]. According to Choo et al [50], the antibiotic activity of garlic is mainly due to allicin, which acts by destroying and inhibiting gram-positive and gram-negative bacteria.
In the work of Torres et al [27], the authors showed that other compounds present in garlic, in addition to allicin, have antimicrobial activity and can be used against strains of S. agalactiae, it is enough to know how they act to inhibit these bacteria.
From this research, the SP80 fraction from A. sativum L. has in vitro antimicrobial activity against the bacteria S. agalactiae; its association with the antibiotics showed an additive effect.
Based on our results, we believe that further studies should be conducted to pave the way for the development of translational research, as this drug-drug interaction may prove beneficial in the therapy of S. agalactiae infections in humans.