In Vitro Antimicrobial Effectiveness Tests Using Garlic (Allium sativum) against Salmonella enterica Subspecies enterica Serovar Enteritidis

In recent years, there has been a more prudent use of drugs on livestock farms and alternative products have been considered, with a view of reducing the risk of the onset of antibiotic resistance. Salmonella enterica subsp. enterica serovar Enteritidis (S. Enteritidis) may cause disease in poultry, and it is also responsible for human food poisoning. The aim of this study was to evaluate the efficacy of garlic against S. Enteritidis and to define its Minimal Inhibitory Concentration (MIC)90 and MIC50 values. The study was carried out in vitro, testing 26 S. Enteritidis strains identified in laying hens from various farms in Southern Italy. A preliminary efficacy trial was carried out on two strains, S. Enteritidis and Escherichia coli, using a garlic concentration of 10 mg/mL (1%). Later, 26 strains of S. Enteritidis at 106 Colony Forming Unit (CFU) and 104 CFU were tested with different concentrations of garlic, ranging from 10 mg/mL to 1 mg/mL. Based on the results, intermediate concentrations of garlic, from 5 mg/mL to 4 mg/mL and 4 mg/mL to 3 mg/mL, were used to test 106 CFU and 104 CFU, respectively. The data were statistically analyzed. The MIC90 was 4.75 mg/mL for strains tested at 106 CFU and 4 mg/mL for strains tested at 104 CFU. The results highlight garlic’s potential to inhibit the growth of Salmonella enterica ser. Enteritidis in vitro. Efficacy was dependent on the microbial concentration used. In vivo efficacy trials will be crucial to confirm the efficacy of garlic against S. Enteritidis and to assess whether garlic can be used in poultry flocks to prevent the spread of the bacterium in the field.


Introduction
In recent years, due to fears surrounding the risk of antibiotic resistance, there has been growing interest in alternative methods to pharmaceutical approaches for controlling potential pathogens on livestock farms. Antimicrobials are used in herds both as a treatment and for prophylactic purposes. The misuse of drugs has contributed to the onset of antimicrobial resistance (AMR), especially in the past. AMR is the consequence of the evolutionary response and evasive strategies that bacteria exhibit in response to a selection pressure caused by an external insult, such as exposure to antibiotics. This leads to the expression of mutations or the transmission of mobile genetic elements through horizontal gene transfer (HGT) among bacterial populations [1]. Currently, the greatest risk is the spread of so-called 'superbugs', i.e., multidrug-resistant bacteria (MDR).
On the basis of Regulation (EU) 2016/429 of the European Parliament and of the Council of 9 March 2016 on transmissible animal diseases and amending and repealing certain acts in the area of animal health (Animal Health Law) [2], biosecurity measures have been increased, health and hygiene standards within farms have been raised, animal densities have been regulated, and there has been a greater use of vaccine prophylaxis even for the prevention of some bacterial forms. The possibility of using natural substances, or their derivatives, as an alternative to the use of antibiotics has also prompted the scientific community to investigate their potential effectiveness.
The health benefits associated with the use of garlic are attributed to its anticancer, antiinflammatory, antifungal, antiviral and antibacterial properties. Several in vitro, in vivo and epidemiological studies indicate that garlic exhibits anticancer activity, and its likely mechanism of action is the activation of metabolizing enzymes, inhibiting reactive oxygen species and radical scavenging, preventing DNA damage and inhibiting tumors [19]. Its immunomodulatory effects are mediated through its ability to modulate cytokine production as well as to activate immune responses by stimulating antibody secretion and immune cells [20].
Garlic contains different bioactive complexes, and organosulfur compounds are its main bioactive components, with allicin, which is responsible for garlic's strong odor, being the most commonly described substance with medicinal activity in the literature [23,24]. Allicin was recognized by Cavallito and Bailey [25] and has been shown to have antimicrobial activities against Gram-positive and Gram-negative bacteria [26]. It is transformed from its precursor alliin by the enzyme alliinase. Being rather unstable, it soon participates in a cascade of non-enzymatic reactions, producing such compounds as vinyl dithiins, ajoene and polysulphides, which have been reported to exhibit antimicrobial activity [23,27].
Taking into account such potential, the aim of our study was to evaluate how effective garlic might be against strains of Salmonella enterica ser. Enteritidis. This bacterium is a pathogen for poultry, in which it can induce morbid conditions characterized by enteritis, production losses and mortality while, via contamination of meat and eggs, it is also responsible for food poisoning in humans [31].

Strains Used for Bacterial Suspensions
The study was carried out in vitro on 26 Salmonella enterica subsp. enterica ser. Enteritidis previously isolated in laying hen farms for the production of eggs for consumption, following the methodology proposed by Mooijman et al. [32]. These strains were stored at −20 • C in Brucella broth and glycerol (10%) at the Department of the Veterinary Medicine, University of Bari (Valenzano, Italy). All strains were grown on Tryptic Soy Agar (TSA) (OXOID, Basingstoke, UK) at 37 • C overnight before testing. Starting from each strain, two bacterial suspensions were prepared with respective concentrations of 1 × 10 6 CFU/mL and 1 × 10 4 CFU/mL. To obtain the predefined concentrations, according to CLSI standards [33], bacterial suspensions equal to the 0.5 McFarland standard were prepared, corresponding to 1-2 × 10 8 CFU/mL. The concentrations obtained were confirmed by serial dilutions and plate bacterial counts. Ten microliters of the suspensions with a concentration of 10 8 CFU/mL was inoculated in spots, in order to analyze in the tests a charge equal to 10 6 CFU. Meanwhile, for the tests using 10 4 CFU, suspensions at 10 8 CFU/mL were diluted stepwise in sterile 0.9% saline solution to obtain 10 6 CFU/mL, and 10 µL was then inoculated in each spot.

Preparation of Aqueous Garlic Extract (AGE)
For the efficacy trials, commercial freeze-dried garlic was used. The lyophilized powder was weighed and placed in 100 mL of sterile distilled water in different quantities (10 g, 9 g, 8 g, etc.) based on the different concentrations of garlic that we intended to test. The efficacy tests were performed on Mueller-Hinton agar (OXOID), reconstituted using 900 mL of water, sterilized in an autoclave at 121 • C and subsequently brought to 50 • C before adding garlic suspensions at the different concentrations to the medium.

Preliminary Test
A 1% solution, with a garlic concentration of 10 mg/mL, was used for a first preliminary efficacy test on a strain of Escherichia coli (ATCC 25922) and a strain of Salmonella enterica subsp. enterica ser. Enteritidis. From the bacteria, 10 8 CFU/mL suspensions were prepared and inoculated in a plate containing simple Mueller-Hinton agar to which garlic was added, using common inoculation methods: with a bacteriological loop, with a sterile swab and by spot.
The presence of garlic in the added medium resulted in total inhibition of growth of both E. coli and Salmonella, regardless of the method of inoculation (Figures 1 and 2).

Preparation of Efficacy Tests
Several series of Mueller-Hinton agar plates were prepared, containing concentrations of garlic from 10 mg/mL to 1 mg/mL (10 mg/mL; 9 mg/mL; 8 mg/mL; 7 mg/mL; 6 mg/mL; 5 mg/mL; 4 mg/mL; 3 mg/mL; 2 mg/mL; 1 mg/mL), which were used to test both bacterial concentrations (10 6 CFU and 10 4 CFU) for each strain. The plates were incubated at 37 • C, and the results were read after 24 h of incubation, evaluating the efficacy of AGE based on growth/no growth in the spot (Figure 3). Each strain was identified using a numbered grid placed under the plate (Figure 4).

Preparation of Efficacy Tests
Several series of Mueller-Hinton agar plates were prepared, containing concentrations of garlic from 10 mg/mL to 1 mg/mL (10 mg/mL; 9 mg/mL; 8 mg/mL; 7 mg/mL; 6 mg/mL; 5 mg/mL; 4 mg/mL; 3 mg/mL; 2 mg/mL; 1 mg/mL), which were used to test both bacterial concentrations (10 6 CFU and 10 4 CFU) for each strain. The plates were incubated at 37 °C, and the results were read after 24 h of incubation, evaluating the efficacy of AGE based on growth/no growth in the spot (Figure 3). Each strain was identified using a numbered grid placed under the plate (Figure 4).    Each experiment was carried out twice on two different days. Based on the results obtained, the bacterial loads at 10 6 CFU were evaluated with a range of garlic concentrations from 5 mg/mL to 3.5 mg/mL (5 mg/mL; 4.75 mg/mL; 4.5 mg/mL; 4.25 mg/mL; 4 mg/mL; 3.75 mg; 3.5 mg) and at 10 4 CFU with concentrations from 4 mg/mL to 3.25 mg/mL (4 mg/mL; 3.75 mg/mL; 3.25 mg/mL). Each experiment was carried out twice on two different days. Based on the results obtained, the bacterial loads at 10 6 CFU were evaluated with a range of garlic concentrations from 5 mg/mL to 3.5 mg/mL (5 mg/mL; 4.75 mg/mL; 4.5 mg/mL; 4.25 mg/mL; 4 mg/mL; 3.75 mg; 3.5 mg) and at 10 4 CFU with concentrations from 4 mg/mL to 3.25 mg/mL (4 mg/mL; 3.75 mg/mL; 3.25 mg/mL).

Statistical Data Evaluation
The data obtained from the different efficacy tests were compared to evaluate their statistical significance using the two-sided Fisher test. Statistical analysis was carried out in R v. 4.2.1 [34].

Results
Garlic concentrations greater than or equal to 5 mg/mL were found to be effective, inhibiting the growth of all strains regardless of the bacterial concentration tested. MIC 90 and MIC 50 values were found to be 5 mg/mL at 10 6 CFU, while only 6 of 26 strains (23.07%) were completely inhibited by a concentration of 4 mg/mL (Table 1). At 10 4 CFU, the corresponding MIC 90 and MIC 50 values were 4 mg/mL, with the growth of 24 out of 26 strains (92.3%) being inhibited.
A concentration of 3 mg/mL proved to be ineffective for all the strains analyzed at a concentration of 10 6 CFU, while at 10 4 CFU, the growth of just one bacterial strain was inhibited.
Analyzing the effectiveness of the intermediate concentrations of garlic, from 5 mg/mL to 3.5 mg/mL for the 10 6 CFU inoculations (Table 2), the minimum concentration inhibiting the growth of the strains in 90% of cases (MIC 90 ) was found to be 4.75 mg/mL, while a concentration of 4.5 mg/mL was not as effective (p < 0.001), as less than 50% of the strains were inhibited at this concentration.
At 10 4 CFU, MIC 90 stood at 4 mg/mL, while the concentration of 3.75 mg/mL inhibited the growth of 22 out of 26 strains, i.e., well above 50% (MIC 50 ) and significantly higher than at 3.5 mg/mL (p = 0.034). Comparing the two different bacterial concentrations used, an inhibitory effect at 4 mg/mL was observed in one strain equal to 10 4 CFU. However, the same concentration did not have the same efficacy on suspensions at 10 6 CFU (p < 0.001).

Discussion
The results show that garlic possesses a good inhibitory capacity in vitro against Salmonella enterica sub. enterica ser. Enteritidis. This capacity was variable and dependent on the bacterial load used. Given that the rate of bacteria inhibition is not necessarily proportional to the concentration of AGE in a linear way, the same decrease in the rates is not expected to be observed when concentrations are decreased from 5 to 4.75 mg/mL vs. when they are decreased from 4.75 mg/mL to 4.5 mg/mL, etc., with 0.25 mg/mL intervals.
The inhibitory capacity of garlic is predominantly due to its bacteriostatic rather than bactericidal properties, as MIC values are generally below the Minimum Bactericidal Concentration (MBC) [35].
Studies on the antimicrobial efficacy of garlic, conducted on individual strains by bacterial species, have shown in vitro MIC values of 6.25 mg/mL for Streptococcus mutans [36] and for Salmonella Tiphi, Escherichia coli, Staphylococcus aureus and Pseudomonas [37]. These are all higher than the MIC 90 values obtained in this study for S. e. sub e. ser. Enteritidis.
In another experiment [38], also including only one strain of Salmonella Tiphi, the MIC value obtained was much lower, at 0.02 mg/mL. Such divergences could be attributable to the individual sensitivity of a particular strain, since in these studies there was no comparison with other strains and the bacterial load adopted.
When evaluating the effectiveness of natural substances, unlike in the pharmacological field, any divergence among the values obtained may be linked to a lack of standardization of the methods adopted in the different laboratories. For example, in the two studies cited on Salmonella Tiphi, in one case [37], the evaluation was carried out through serial dilutions in broth, while in the other case [38], the MIC was determined in a plate in solid medium according to the Kirby-Bauer method, soaking discs with garlic extracts specially constructed for the experiment. Furthermore, the different concentration panels adopted in the two studies led-in the research conducted by Andualem [37]-to MIC values of 6.25 mg/mL, corresponding to the lowest concentration of garlic evaluated in the trial, while, on the other hand, Gull et al. [38], who evaluated even lower concentrations, obtained MIC concentrations ranging from 0.02 to 0.2 mg/mL for the bacteria tested.
In a larger study, involving 25 strains of E. coli and S. aureus analyzed in bacterial concentrations of 10 7 CFU and by serial dilutions in broth, the MIC values were between 4 mg/mL and 8 mg/mL for both bacteria [39].
Other factors that can influence the discrepancies found in different studies may include the instability of allicin; the active ingredient responsible for its antimicrobial activity against Gram-positive and Gram-negative bacteria; or else the physical form of garlic used, for example, fresh or dried, and, when using an extract, the type and method of preparation [26,40]. Allicin is transformed from its precursor alliin by the enzyme alliinase. Being rather unstable, it soon participates in a cascade of non-enzymatic reactions, producing such compounds as vinyl dithiins, ajoene and polysulphides, which have been reported to exhibit antimicrobial activity [23,27].
Comparative tests with garlic extracts, i.e., aqueous, alcoholic in ethanol and alcoholic in methanol, showed better efficacy of the aqueous extract compared to the alcoholic ones, whose efficacy can be superimposed [38], but these results were variable depending on the germ tested [35]. AGE exhibited in vitro antibacterial activity against various pathogenic bacteria including Bacillus cereus, Shigella and Salmonella species and enterotoxigenic E. coli [41].
The antimicrobial efficacy of natural substances could be enhanced by their combination [26,42]. For example, thyme, peppermint, sage, black pepper and garlic appear to have a greater in vitro antimicrobial effect against Bacillus subtilis and Salmonella Enteritidis if tested in synergy rather than individually [26,43], although Andualem [37] showed no real enhancement of garlic's efficacy when it was combined with honey.
Field tests carried out on groups of turkeys have shown the effectiveness of a mixture composed of organic acids (acetic, formic and propionic acid), and cinnamic aldehyde, a cinnamon extract, added to the feed at a concentration of 0.2% in reducing the intestinal concentration of E. coli and in limiting the lesions induced by a 078 antibiotic-resistant strain [44].

Conclusions
The results obtained in this preliminary study are quite encouraging. However, further evaluations on a larger number of strains and on possible combinations of garlic with other natural substances are desirable in order to assess its applicability. Indeed, its combination with other natural substances could help to limit the amount of garlic administered to animals, thus reducing the risk of transmitting unpleasant odors to feed and animal-derived products, such as eggs or meat.