Antibiofilm and Antibacterial Activities of Garlic Extract Against Clinical Isolates of Pseudomonas Aeruginosa

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1. Introduction

Plant derived antimicrobials have extremely large therapeutic effects. They are considered as an efficient tool in treating infectious diseases though reducing the complications that connected with synthetic antimicrobials (Saranraj & Sivasakthi, 2014) [23].The changed pharmacological properties of plant products are generally the result of secondary product combinations resulting from plants. Such plant compounds are often secondary metabolites such as steroids, alkaloids, and fatty acid secretions that have a certain functional influence on the body (Shang et al., 2019 and Josling, 2005) [24, 13].There are many chemicals and natural elements are found in garlic, in addition to numerous enzymes, sulfur compounds, different vitamins, minerals, and amino acids. Allicin is among garlic’s greatest crucial biologically dynamic substances where contain the main antioxidant composite, polar compounds of phenolic and steroidal origin. It is displayed diverse pharmacological countenance without odor and are heat stable (Mohammed et al., 2021) [18].The antibacterial activity of garlic has been basically attributed to the entity of allicin generated by the enzymatic activity of allinase on allicin (Alam et al., 2018) [1]. Garlic is also effective to antibiotic resistant microbes, and combining garlic extracts with antimicrobial agents results in partial and total synergism (Yadav et al., 2015) [26].Pseudomonas aeruginosa is gram negative, rod shape, a aerobic or facultative anaerobic and monoflagellated bacteria. P. aeruginosa is a very invasive microbe that has been discovered in environments, animals, plants, people, and hospital (International Journal of Health & Medical Research, 2025).Furthermore, P. aeruginosa is considered as a globally foodborne pathogens due to its great metabolic adaptability, (Fabre et al., 2019) [10] quick reproducibility, and capability to grow at lower temperature.P. aeruginosa is capable to cause disease include keratitis, otitis, burn, wound, and endocarditis, urinary tract infections (Montero et al., 2020 and Hilliam et al., 2020) [19, 11].The increasing multidrug resistance of P. aeruginosa characterizes a main risk to community health and wellbeing. Alteration enzymes can change antibiotics and render them ineffective. For example, this bacterium produces aminoglycoside-modifying enzymes, also carbapenem antibiotics P. aeruginosa resistance it by carbapenemases that can break down (Anju et al., 2021) [4]. These genes are often located on mobile genetic elements such as plasmids, transposons.Biofilms are assembly of bacterial cells, a single or mixed species enclosed in a polysaccharide matrix secreted by the bacteria (Rajput & Kumar, 2018) [22] leading to the production and specific secretion of exopolysaccharides, DNA, and protein by the bacterium itself (Anju et al., 2021) [4]. The extracellular polysaccharide matrix EPS matrix provides protection to biofilm cells from several negative environmental stresses, like UV radiation, sudden changes in pH values (Zhou et al., 2013 and Dubois-Brissonnet et al., 2016)) [28, 8] Biofilm formation is the source of a major problem for the host, as these biofilms are often resistant to antibiotics, phagocytosis, and surfactants, making them difficult to remove once established (Olivares et al., 2019) [20].Biofilm formation is the source of a major problem for the host, as these biofilms are often resistant to antibiotics, phagocytosis, and surfactants, making them difficult to remove once established (de Sousa et al., 2023).The biofilm formation process in P. aeruginosa is regulated by a number of genetic and environmental factors. One of the key systems involved in biofilm formation is the quorum sensing system. Based on cell density, this system is allowed bacteria to communicate with one another and coordinate their behavior, including acyl-homoserine lactones (AHLs) and 2-heptyl-3-hydroxy-4- quinolone (PQS). Another important factor in biofilm formation is production of extracellular polymeric substances (EPS), which are secreted by the bacteria and form a matrix helps them to anchor to surfaces and protect from environmental stresses (de Sousa et al., 2023).This study is aimed to evaluate the antibacterial activity of garlic extract against P. aeruginosa and examine its effect on biofilm formation.

2. Methodology

2.1. Collection Bacteria Isolation

Forty samples of Pseudomonas aeruginosa were isolated from burn where all isolates are identified and cultured on nutrient agar medium, MacConkey agar and Cetrimide agar, and diagnosed by microscopic examination, biochemical tests, and the VITEK2device.

2.2. Antibiotic Susceptibility test

Antibiotic susceptibility testing was completed on twenty of the forty bacterial isolates using the VITEK® 2 Compact system conferring to the manufacturer's instructions, and results were interpreted by depending on Clinical and Laboratory Standards Institute (CLSI) (Yang et al., 2009) [25] The antibiotics test was included Cefixime(CFM), Erythromycin (E), Chloramphenicol(C), Trimethoprim (TR), Piperacillin (PC), Amikacin (AK)

2.3. Detection Biofilm Formation

The biofilm formation of P. aeruginosa was determined by spectrophotometric and using microtiter plate assay (Li et al., 2019 and Borlinghaus et al., 2021) [5].Bacterial colonies were inoculated into (10 mL) of Trypticase soy broth supplemented with 1% glucose and incubated at 37C° for 24 h. Following incubation, the bacterial cultures were diluted at a ratio of 1:100 using TSB medium. 200 µL of the dilute bacterial suspension was transferred into individual wells of a sterile 96 well flat bottom. Negative control wells containing only sterile broth were included to verify sterility and exclude non-specific binding. After incubation, the contents of the wells were discarded, and the wells were washed 4 times with phosphate buffered saline to remove non-adherent cells (Lee & Zhang, 2021) [15] Then, the attached biofilms were fixed with acetation of 2% sodium for 30 min. The fixed biofilms were stained with (0.1% ) crystal violet for 30 min. Throughout the deionized water, the walls was washed and the excess stain was removed and finally the plates were allowed to air dry at room temperature. By measuring the optical density (OD) of stained adherent cells, biofilm formation was counted by using a microplate ELISA reader at a wavelength of 600 nm. All assesses were performed in triplicate, and the mean OD value was deliberate for each isolate. Based on the obtained OD values, isolates were categorized as, non-weak, moderate, or strong biofilm producers.Table as described in previous studies (Panda et al., 2016) [21]

Table 1: Classification of biofilm formation by tissue culture plate method Optical densities values Adherence Biofilm formation > 0.240 Strong Strong 0.120- 0.240 Moderate Moderate < 0.120 Non/weak Non/weak

2.4. Preparation of crude extracts

Garlic extract was performed by using maceration technique. 100-gram sample of fresh garlic cloves was peeled and ground. Then, it is divided to prepare both aqueous and 70% ethanolic extracts (Borlinghaus et al., 2021) [5].Whilst aqueous extract is prepared with 100 grams of ground garlic by soaking it in 100 mL of hot distilled water for four hours with continuous stirring. Then the mixture was cool and filtered to remove impurities. consequently, by using a freeze dryer, the water was removed, to obtain the dry extract. For ethanolic extract, 100 grams of ground garlic were soaked in 70% ethanol for five days with room temperature and with daily stirring to ensure extraction efficiency (Mohammed et al., 2021) [18] Yield percentage for both extracts was calculated by using formula underneath:

Yield (%) = (Weight of dry extract ÷ Initial weight of plant material) × 100 Finally, these extracts were stored in tightly sealed and sterilized glass vials at 4°C until that is used in subsequent experiments (Fadwa et al., 2021) [9]

2.5. Determination of Minimum Inhibitory

Minimum inhibitory concentration firmed by using broth microdilution method in a 96 well microtiter plate. A serial dilution of substance was prepared in Mueller-Hinton Broth (MHB), where 100 µL of each concentration was added to the wells, then followed with 100 µL of a standardized bacterial suspension ( ≈1.5 × 10⁸ CFU/mL).The plates were hatched at 37°C for 24 hours, after that the wells were checked to see bacterial growth (De Marco et al., 2017)

2.6. Effect Garlic Extract on Biofilm Formation

In vitro, biofilm assay was completed using a 96 well microtiter plate as formerly described with some modification (Al-Fridawy et al., 2020) [29].Overnight cultures of P. aeruginosa were diluted (1:100) into fifteen mL of Tryptic Soy Broth (TSB) added with2%sucrose, in presence or in absence of different extracts tested at the concentrations shown below. At 37∘C for 24h, cultures were incubated under. After incubation, the biofilm developed in each well was washed twice with 200??????L of distilled water and then dried for 45min. In each well, 100??????L of 0.4% crystal violet was added for 30–45min. After that, four times the wells were washed with distilled water and directly discolored with 200??????L of 95% ethanol, after 45 minutes, 100??????L of discolored solution was transferred to a well of a new plate and the crystal violet was measured at 570nm in a microplate reader. The amount of biofilm formed was measured comparation the absorbance values of the compounds treated wells against untreated control wells. Biofilm formation bioassays were performed in triplicates in at least three individual experiments for each concentration (Abdel-Moneim et al., 2021) [2].

2.7. Antibacterial Testing for Garlic Extract

The paper disc diffusion method was being used to evaluate the antimicrobial property of garlic extracts using MHA. The dilution factor of Bacterial suspension was done with a sterile physiological solution to 108 CFU/ ml (turbidity = McFarland standard 0.5). After that 100 μl of bacterial suspension moistened frequently on MHA and was permitted to dry for 5 minutes. Then filter paper discs (6 mm in diameter) was sterilized in an oven at 80oC for 30 minutes, and placed on MHA and soaked with 20 μl of a solution of each garlic extract with diluted different serial dilution as (1mg/ml, 2mg/ml, 5mg/ml, ) respectively, next suspended by methanol for 70% ethanolic extract and the same concentration used after resuspended by sterile distilled water for aqueous extract. Then inoculated plates were incubated at 37°C for 24h (IBM Corp., 2019) [12].

2.8. Statistical analysis

The Statistical Packages of Social Sciences -SPSS (2019) program was used to detect the effect of difference groups parameters. Least significant difference (LSD) was used to significant compare between means in this study(Khadim & Al Marjani, 2019) [14].

3. Result and Discussion

3.1. Collection Bacteria Isolation

Pseudomonas aeruginosa isolates were diagnosed based on microscopic characteristics and biochemical tests and their growth on Cetrimide agar, MacConkey agar and nutrient agar medium.The diagnosis was also confirmed by using VITEC device, as shown in figure 1:

Fig 1: Pseudomonas aeruginosa on Cetrimide agar

3.2. Antimicrobial Susceptibility Test

In this study, a total of forty P. aeruginosa clinical isolates collected from burn samples. Seven types of antibiotic were tested against twenty bacterial isolates, as shown in table 2 and figure 2 Table 2: Antimicrobial susceptibility testing in clinical isolates of Pseudomonas aeruginosa Antibiotics NO. of isolate Resistance (%) NO. of isolate Intermediate (%) NO. of isolate Sensitive Cefixime(CFM) 13(65%) 7(35%) 0(0%)

All isolates were revealed resistance to Erythromycin and Trimethoprim. 70% of isolates are resisted ciprofloxacin, these results are algin with those obtained by Mansouri et al., (2013) [17] which explain that 55.55% of isolated P. aeruginosa were resisted to ciprofloxacin and 87.3 % for cefotaxime where the isolates of P. aeruginosa are taken from different infection sites. This high multi resistance due to the production of hydrolytic enzymes and the acquisition of resistance mechanisms by P. aeruginosa strains. In the current study, isolates exhibited resistances for β-lactams in a varied levels especially for the third-generation cephalosporin, but at the same time several isolates were ESBLs producers. Clinically, antibiotics-resistant bacteria are responsible for the increased length of hospitalization and mortality (Zhang et al., 2024) [27].

Fig 2: Antimicrobial susceptibility testing of Pseudomonas aeruginosa

3.2. Biofilm Formation

Twenty isolate were conferring to biofilm analysis. then they were divided into three categories depend on it production for biofilm, which are : 25 % (n=5) formed strong biofilm, 75 % (n=15) produced moderate biofilm, and 0 % (n=0) of isolates were considered as weak biofilm producers. Isolates from burns had shown a greater ability to form a strong and moderate biofilm compared with the isolates that obtained from other sources, as illustrated in table 3, 4 Table 3: Optical densities values of biofilm formation in Pseudomonas aeruginosa NO.OF Isolates Optical densities values Biofilm formation Ps1 0.339 Strong Table 4: Classification of biofilm formation by tissue culture plate method in P. aeruginosa Biofilm formation NO. of isolate (Percentage) Strong 5 (25 %)

The results of the current study showed all isolates had ability to form biofilm. Biofilm formation is an important mechanism in resistance of bacteria. Biofilm formation protects bacterial cells from host immune defenses and decreases the penetration and efficacy of antimicrobial agents, thereby contributing to chronic and recurrent infections. The absence of weak or non-biofilm-forming isolates stresses that biofilm production is a preserved characteristic in these isolates and may play a vital role in their pathogenicity and antimicrobic resistance.

3.3. Determination of Minimum Inhibitory

MIC is defined as lowest concentration, which show no visible bacterial growth was recorded as 2mg/mL, while sub-inhibitory concentration was determined to be 1m g/mL. In subsequent experiments, the minimum bactericidal concentration is determined by taking samples from wells showing no bacterial growth and cultured on Mueller Hinton Agar (MHA) plates, which were then incubated at 37°C for 24 hours. The lowest concentration of crude garlic extract that produced no bacterial growth on the solid medium was recorded as the MBC, with a value of 5 mg/mL.

3.4. Effect of Garlic Extract on Biofilm Formation

The results show that garlic extract caused a significant decrease (P ≤ 0.05) in biofilm formation across all clinical isolates (Ps1, Ps10, Ps12, Ps13, and Ps14) of P. aeruginosa, as illustrated in table 5 and figure 3:

Table 5: Biofilm formation before and after treatment with garlic extract in difference isolates of pseudomonas aeruginosa No. of Isolates Biofilm formation before treatment with garlic extract Biofilm formation after treatment with garlic extract Sub-MIC =1mg/ml MIC=2mg/ml MBC=5mg/ml L.S.D. 12 0.352 ±0.04 a 0.266 ±0.05 ab 0.168 ±0.03 b 0.092 ±0.02 b 0.156 * Means having with the different letters in same row differed significantly. * (P≤0.05).

Fig 3: Biofilm formation before and after treatment with garlic extract in difference isolates of pseudomonas aeruginosa Before treatment, biofilm formation values were high and it was 0.315–0.352. Then they decreased in a concentration dependent. Specifically, values dropped gradually from the initial range of 0.315–0.352 to 0.236–0.272 at the sub-MIC concentration of 1 (mg/ml), then to 0.133–0.195 at the MIC 2 (mg/ml), reaching a minimum of 0.072–0.095 at the MBC 5 (mg/ml). This progressive decline indicates that the efficacy of the garlic extract increases with concentration, demonstrating a clear inhibitory effect on biofilm formation. These results are dealt with the findings of Alvi et al., (2021) [3] which demonstrated that sulfur compounds which derived from garlic such as diallyl disulfide act as inhibitors of the quorum sensing system in P. aeruginosa. This inhibition is reduced biofilm formation and attenuation of bacterial virulence factors. which leads to inhibite the cell communication system and diminishes the bacteria's ability to adhere and produce extracellular polymeric substances (EPS) that is required for biofilm maturation, thereby explaining the markable decreasing in optical absorbance values which observed in the present study. These findings also support recent studies that are indicating: compounds such as allicin and its sulfur derivatives that that derived from garlic are considered as inhibit the expression of genes regulating the quorum sensing system. This leads to a dose-dependent reduction in the production of virulence factors and biofilm formation, consistent with the progressive decline observed in this study when transitioning from sub-MIC to MIC and subsequently to MBC levels.

3.5. Antibacterial effects of garlic extract

The antibacterial activity of garlic extract for three isolates of bacteria P. aeruginosa has been tested by using the agar well diffusion (34). At different concentration (1, 2 and 5 mg /ml) of garlic extract, as indicated by the formation of inhibition zones, as shown in table (6) and figure (4 ):

Table 6: Antibacterial activity of garlic extract against Pseudomonas aeruginosa at different concentrations No. of isolates MIC=1 mg /ml Sub-MIC=2 mg /ml MBC=5 mg /ml At 1 mg/mL, the inhibition zones ranged from 8 to 9 mm, whereas increasing the concentration to 2 mg/mL resulted in inhibition zones ranging from 10 to 12 mm. At 5 mg/mL, the inhibition zones were 9–15 mm, with the largest inhibition zone observed against isolate Ps1 (15 mm), followed by Ps2 (13 mm) and Ps3 (9 mm). Overall, the inhibitory effect was more pronounced at the higher concentrations, although the response varied among the isolates. Isolate Ps1 showed the greatest susceptibility to the garlic extract, while Ps3 exhibited the lowest response at 5 mg/mL.

Fig 5: Antibacterial Effects of Garlic Extract

4. Conclusion

This study is determined the ability of P. aeruginosa isolates for biofilm formation and pyocyanin production where biofilm formation is associated with the increased resistance to the antibiotic and bacterial colonization within burning infections. The isolates have high levels of biofilm specific resistance despite its moderate or weak biofilms production which means that biofilm formation and pyocyanin could increase the toxicity and pathogenicity of this bacterium. The results is indicated that garlic extract inhibited biofilm formation by P. aeruginosa in a dose-dependent manner; mean optical density (OD) values decreased progressively as the extract concentration increased from Sub-MIC to MIC and then to MBC.

Acknowledgement

The authors would like to thank Medical Technical Institute/Baghdad, Middle Technical University, Baghdad – Iraq for its support in the present work Funding Information None