Abstract

Staphylococcus aureus is a major opportunistic pathogen responsible for wound and burn infections, and its increasing resistance to conventional antibiotics, especially methicillin-resistant strains (MRSA), represents a serious public health problem. The urgent need for affordable and effective alternatives has directed attention toward plant-based therapies. This study aimed to evaluate the antibacterial activity of Aloe vera and clove (Syzygium aromaticum) extracts against clinical isolates of S. aureus. Fifty swab samples were collected from patients with wound and burn infections at Fallujah Teaching Hospital between October and December 2024. Bacterial isolates were identified using morphological and biochemical tests, with confirmation through the VITEK 2 system. Twenty isolates (26%) were confirmed as S. aureus, with a higher prevalence in burn infections (57.14%) compared to wound infections (42.86%).The antibacterial effects of both extracts were tested using the disc diffusion method. Clove extract showed stronger inhibitory activity, producing a mean inhibition zone of 20.21 ± 2.72 mm, while Aloe vera exhibited moderate inhibition with a mean zone of 16.43 ± 2.38 mm. Importantly, some isolates resistant to standard antibiotics were inhibited by the plant extracts, suggesting their potential use against resistant strains. The pronounced activity of clove is attributed to eugenol and related phenolic compounds that damage bacterial membranes and disrupt essential cellular processes. Aloe vera, while less potent in terms of inhibition zones, contains bioactive compounds such as aloin and anthraquinones that not only exhibit antibacterial action but also promote wound healing and tissue regeneration.The results highlight the therapeutic promise of Aloe vera and clove as complementary agents for managing S. aureus infections. Clove demonstrated superior antibacterial potency, whereas Aloe vera provided additional healing benefits. Their effectiveness, affordability, and accessibility support their potential role in reducing reliance on synthetic antibiotics. Further in vivo and clinical studies are recommended to validate these findings and to explore synergistic applications.

Keywords: Staphylococcus aureus, Aloe vera, Clove extracts

Introduction

Under a microscope, the Gram-positive, facultative anaerobic coccus Staphylococcus aureus usually appears in clusters resembling grapes. Because of the pigment staphyloxanthin, which also helps it withstand oxidative stress, colonies of this non-motile, non-spore-forming organism frequently have a golden-yellow appearance. The organism can survive as a component of the regular flora and frequently colonises human skin, mucous membranes, and anterior nares (nostrils). It has a survival advantage on the skin’s surface and in hospital settings due to its resistance to desiccation and high salt concentrations. The ability of S. aureus to manufacture coagulase, an enzyme that allows it to coagulate blood plasma and aid in immune evasion, makes it the most pathogenic of the more than 25 species and subspecies of staphylococci to humans (Foster et al., 2009).

1. aureus is an extremely versatile and opportunistic organism that can cause a wide range of illnesses, from minor skin infections like boils, abscesses, and impetigo to more serious invasive diseases including pneumonia, septic arthritis, endocarditis, osteomyelitis, and sepsis. Numerous virulence factors, including surface proteins that promote adherence, enzymes that break down host tissues, toxins (such as haemolysins and leukocidins), and immune evasion strategies like protein A, are responsible for the pathogen’s success. These elements improve its capacity to infiltrate host tissues, evade immunological reactions, and endure in a variety of settings (Chambers et al., 2009).

The rise of methicillin-resistant Staphylococcus aureus (MRSA), a strain that has developed resistance to beta-lactam antibiotics, such as cephalosporins and penicillin, is a cause for increasing concern. This resistance is mainly caused by the mecA gene, which produces a modified penicillin-binding protein (PBP2a) with decreased affinity for beta-lactams. Hospital-acquired MRSA (HA-MRSA) and community-acquired MRSA (CA-MRSA) are the two primary types of MRSA strains. The former is more resistant and linked to nosocomial infections, while the latter frequently affects otherwise healthy people in community settings. MRSA infections have grown to be a major public health concern, increasing morbidity, death, and medical expenses worldwide (Garoy et al., 2019).

The term “multidrug-resistant S. aureus” refers to infections that are more challenging to treat since there are fewer antibiotic alternatives available. In many cases, resistance extends beyond methicillin to multiple antibiotic classes. The persistence of MRSA in both community and healthcare settings emphasises the significance of infection control, surveillance, and the creation of innovative treatment approaches to fight this dangerous disease.

Methods

Sample collection

This study was conducted on 50 patients suffering from burns and wounds by taking a swab from the burn and wound site during the period from October 2024 to December 2024from Fallujah Teaching Hospital, and each patient’s information was recorded in terms of gender and age.

Ethical approval

The ethical approval was obtained from the ethics committee for Fallujah Teaching Hospital/Ministry of Health.

Bacteria isolated and diagnosis

The medium was prepared according to the instructions fixed on their containers as indicated by the manufacturing company. It was sterilized by autoclaving at 121ᵒC for 15 minutes under 1.5 bars.It was used nutrient agar, nutrient broth, mannitol salts agar, Mueller-Hinton II Agar, and blood agar (Baird, 1996; Forbes et al.,2002). It was cultured bacteria on the media to isolate it.

Further, the bacteria were characterized by their growth colonies on the Mannitol salt agar and Blood agar and then studied the growth colony forms and identified by their color, shape, and size. In addition to other general characteristics, such as lactose fermentation or not, and hemolysis on the blood agar media or not.

Diagnose bacteria by biochemical tests and Vitek 2 System

After the completion of microscopic examination and cultured the following biochemical diagnostic tests were used catalase test, oxidase test, motility test,Coagulase test )Tiwari et al ,2009; Kloos and Bannerman, 1994; Freney et al., 1999).

Preparation extract

Aloe vera leaves are soaked in water for two hours so that all the toxic yellow substance comes out from under the aloe vera leaves, then extracted  the aloe vera gel from the leaves.

In a clean and sterile container, we put 100 ml of sterile water with 20 ml of white vinegar and 20 ml of lemon juice, then the aloe vera gel weighing 35 g put the aloe vera gel in the solution and soaked it for 5 hours. To keep the aloe vera gel from oxidizing. After 5 hours, we filter the aloe vera gel and rinse it well with distilled water. Finally aloe vera gel put in a clean and sterile glass container, and 65 ml of vegetable glycerin was added on top of it. Then, it is kept in a cool, dark place for a week, after which the extract is ready to use

Preparing clove extract

50 ml of distilled water added to 4 gm clove powder put it on medium heat until it starts to boil, then leave it on the fire for 5 minutes. Filter the solution and place it in a sterile glass container(Danthu et al. ,2020).

Antibiotic susceptibility test by the disc diffusion method

This method is one of the best standardized tests, and its performance is continually updated by the CLSI consensus effort (CLSI 2012), Table 1(Wayne, 2002). The inoculum bacterial was prepared by transferring a single colony to a test tube containing a nutrient broth, then incubating at 37ᵒC for 24 hrs. After preparing the incubation and inoculum. Sterile swabs were taken and put in a test tube containing inoculum then swabbed evenly across the surface of a Muller- Hinton agar plate, after inoculation, the antibiotics were applied by forceps with hard pressure, where developed 3discs in each dish. The plate was inverted and incubated at 37ᵒC for 18 hrs. (Mahon and Manuselis, 2000). Then, after incubation, the zone of inhibition for each antibiotic was compared with standard diameters of inhibition.

Table 1: Antibiotic concentrations

Statistical Analysis:

A statistical package (SPSS 26) was used for data input and analysis. All results were presented as mean ± standard deviation (M±SD). T-test for two independent samples and Anova test. P- Values (P < 0.05) were measured statistically significantly. Findings with P value equal or less than 0.05 were considered significant.

Results and Discussion

Bacterial Isolate

Fifty samples were collected from burns and wounds October 2024 to December 2024. After morphological, microscopic and biochemical tests, 20 isolates were Staphylococcus aureus, which represents about 26% of the total samples.

Morphological identification

Isolates of S. aureus bacteria were initially identified by relying on phenotypic characteristics, after their development on each Mannitol salt agar. Orientation The results indicated that the bacteria were fermented the mannitol and gave gold colonies show in figure 1.

Figure 1: The S. aureus fermented the mannitol and gave gold colonies

Microscopic examination

A smear of the developing colonies was made on the Mannitol salt agar medium, and the dye was applied to it using Gram stain and examined under the microscope Staphylococcus aureus appeared as typical Gram-positive cocci in grape-like groups clusters figure 4.2 (Mohamed et al ,2018).

Figure 2: Sow the Staphylococcus aureus shows as Gram- stain bacteria under the microscope

Biochemical tests

Biochemical tests were performed on all isolates, and all of them were positive to coagulase test.  The bacteria were negative for oxidase test, with no color change, and positive for the urease test(Table 2).

Table2: Biochemical tests used for identification S. aureus

A total of 7 clinical samples were collected during this study; 4 (57.14%) S. aureus isolates were obtained from burn samples, and 3 isolates (42.86%) from wounds. In the present study, the majority of S. aureus isolates were detected in burn clinical samples (57.14%), as shown in (Table 3), Figure 3).

Table3 : S. aureus isolates counts and prevalence rates from various sources

Figure 3: Percentages of S. aureus isolates from various sources

Evaluation of plant extracts bioactivity

The effect of two different plant extracts, Clove and Aloe Vera were examined against Staphylococcus aureus isolates were chosen by using the disk diffusion technique. The effects of the plant extracts against the tested S. aureus are shown in the (Table 4, Figure 4).

The Clove extract showed a significant increase in activity against S. aureus 5 and S. aureus 7 isolates with Mean ±SD (22.50±2.121 and 22.50±0.707 mm), p<0.0001) respectively, and the lowest Mean ±SD inhibition diameter was 18.00±4.243 mm for S. aureus 6 isolate.

While Aloe Vera extract showed a significant increased activity against S. aureus 5 with Mean ±SD (33.00±17.66 mm), and the lowest Mean ±SD inhibition diameter was 28.00±12.91mm for S. aureus 1 isolate.The result shows a most significant increase of Aloe Vera extract against S. aureus (42.93±6.844) mm by comported with Clove extract (20.21±2.723) mm. (Figure 5 and 6).

Figure 4: Determination zone the effective aloe vera and clove

Table 4: Antimicrobial activity of plant extracts on S. aureus by well diffusion method.

*Significant

Figure 5: Mean of antimicrobial activity of plant extracts on S. aureus isolates.

Figure 6: Mean of antimicrobial activity of plant extracts on S. aureus isolates.

The results clearly show that Aloe Vera extract is more effective than Clove extract in inhibiting S. aureus. This is consistent with previous studies that have found Aloe Vera contains active compounds, such as aloin and anthraquinones, which exhibit antimicrobial activity, while Clove contains eugenol, a compound also known for its antibacterial properties (Boudreau & Beland, et al 2006).Clove contains eugenol, which is effective against a wide range of bacteria, including S. aureus. However, its efficacy can vary depending on the bacterial strain and extraction method. )Boudreau and Beland, 2006).Aloe Vera contains compounds such as alone and anthraquinones, which have shown antimicrobial effects in numerous studies (Gupta & Jain, et al 2010).

Antibiotics Sensitivity testing:

The effect of different antibiotics on the isolated bacteria was studied by using disc diffusion method (known as Kirby- Bauer method). Antibiotics that were used for this study were: Ciprofloxacin (CIP) (10 μg), Norfloxacin (NOR) (30μg), Trimethoprim (TMP) (10μg), Gentamicin (CN) (10μg), Levofloxacin (LEV) (5μg), Tetracycline (TE) (10μg), Azithromycin (AZM) (15μg), Ceftriaxone (CRO) (10μg), Cefixime (CFM) (5μg), Carbenicillins (PY) (25 μg). S. aureus isolate No 1 was cultured on Muller-Hinton agar. The zone of inhibition was determined after 24 h of incubation at 37. This test was used as a screening test (CLSI, 2011). As shown in Table 5..Ciprofloxacin (CIP), Norfloxacin (NOR), and Gentamicin (CN) had a clear zone of inhibition, indicating bacterial sensitivity. Resistant antibiotics: Trimethoprim (TMP), Levofloxacin (LEV), Tetracycline (TE), Azithromycin (AZM), Ceftriaxone (CRO), Cefixime (CFM), and Carbenicillin (PY) showed no significant inhibition, suggesting bacterial resistance.This highlights the growing issue of antibiotic resistance, where common antibiotics no longer effectively control bacterial growth, especially in clinical settings (Van Boeckel et al., 2014).

Table5: Evaluation of antibiotics activity against S. aureus .

S= Sensitive, R= Resistant.

Comparison of the activity of plant extracts with antibiotics

The results are shown in Table 6. That the highest inhibition diameter of the plant extracts was the Aloe Vera extract, and it was 52. mm compared to the antibiotics Norfloxacin and Ciprofloxacin, which were 33 and 29, respectively)Figure 6).

Table 6: Comparison of the high inhibition diameters of plant extracts with the highest diameter of antibiotic inhibition on S. aureus .

Figure7 :Analysis results by the Vitek

The Kirby-Bauer disk diffusion method is widely used for determining antibiotic susceptibility to bacteria. S. aureus is known for developing resistance to many antibiotics, including those tested in this study. The resistance to drugs like Trimethoprim, Tetracycline, and Ceftriaxone is concerning, as these are commonly prescribed antibiotics for treating S. aureus infections (Chambers & Deleo, 2009).

Aloe Vera has long been recognized for its antimicrobial properties, which may be due to compounds such as alone, anthraquinones, and other bioactive compounds (Boudreau & Beland, 2006). Conclusions

The study’s results support the idea that Aloe Vera extract could be a promising alternative or adjunct to traditional antibiotics, especially in cases of antibiotic-resistant infections.

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