Antibacterial Activity of Rose Geranium (Pelargonium graveolens) Essential Oil Against Clinical Isolates of Staphylococcus aureus and Pseudomonas aeruginosa with Different Antimicrobial Resistance Profiles

2.1 Study population

A group of clinical bacterial isolates of patients who visited Al-Imamain Al-Kadhimain Medical City, Baghdad, Iraq, was employed as the study population. There were 60 non-duplicate isolates comprising 30 Staphylococcus aureus and 30 Pseudomonas aeruginosa. The isolates were obtained from various clinical specimens in the hospital microbiology laboratory and were reflective of both male and female infections in diverse age groups. The study included only confirmed, pure, and viable cultures as determined by standard microbiological and biochemical techniques. Duplicates of an isolate of a single patient were eliminated to prevent sampling bias. The organisms collected were maintained in the right laboratory conditions and utilized in antimicrobial susceptibility tests and assessment of the antimicrobial influence of Pelargonium graveolens EO.

2.2 Ethical approval and sample source description

This investigation received ethical approval from the Health Research Ethics Committee of Al-Nahrain University, Baghdad, Iraq (approval number: 20241141). The management of Al-Imamain Al-Kadhimain Medical City was also contacted to give consent to the collection and use of the clinical bacterial isolates. All the experiments with the clinical samples were carried out according to the ethical principles of the institutional and national research committees and the principles of the Declaration of Helsinki. There was no access or recording of patient identifiers or other isolates. The specimens were collected from wound isolates, blood isolates, throat swabs, urine, burn swab isolates, and fluid isolates. All isolates were treated anonymously to guarantee confidentiality and biosafety regulations.

2.3 Source of plant materials

Pelargonium graveolens plants were collected from the AL-Razi Center for Medicinal Herbs, Baghdad, Iraq. The plants were identified and verified by Prof. Ibrahim Saleh AI-Jubouri in the Department of Pharmacy, University of Al-Mustansiriya, Baghdad, Iraq. Plant identification was confirmed by an official certificate (Letter No. 437, dated June 3, 2025).

The new aerial sections, such as leaves and stems of Pelargonium graveolens, were cut and cleansed thoroughly with distilled water to remove any surface contaminants.

2.4 Source of microbial isolates

The microbiology laboratories of Al-Imamain Al-Kadhimain Medical City were sources of the clinical isolates of Staphylococcus aureus and Pseudomonas aeruginosa. Species identification was confirmed using VITEK 2 automated identification systems following preliminary biochemical characterization [11]. Upon identification, pure isolates were cultured and kept in proper media at 4 ℃ until the time they were needed to undergo further analyses.

2.5 Extraction of essential oil

Hydrodistillation was employed to extract EOs by means of a Clevenger-type apparatus. The method was applied to the plant materials (approximately 100 g) in distilled water (500 mL) for 3 hours. The acquired EO was then distilled off the aqueous layer and dried with anhydrous sodium sulphate to remove the remaining moisture. The oil was then put in sterile amber glass vials and kept at 4 ℃ until further examination. Hydrodistillation is also a common standard method of extracting EOs of aromatic plant materials [12]. The EO yield was calculated as the percentage volume per dry weight of plant material employing the following equation:

Oil yield (%) = (volume of extracted oil/weight of dried plant material) × 100

2.6 Gas chromatography-mass spectrometry

The chemical characterization of Pelargonium graveolens EO was done through the gas chromatograph method coupled with a mass selective detector by the use of GC–MS. The separation was done in a capillary column (30 m × 0.25 mm × 0.25 μm film thickness) in the presence of helium as the carrier gas at a flow rate of 1 mL/min. One microliter was injected in split mode (1:20). The temperature of the oven was kept at 50 ℃ at the beginning, with the increase of the temperature to 200 ℃ at 5 ℃/min, then to 250 ℃ at 10 ℃/min, and sustained at 250 ℃ at 10 ℃/min. The injector and detectors were heated to 250 ℃ and 280 ℃, respectively. Mass spectrometric analysis was performed in electron ionization (EI) at 70 eV with a scan range of 40-500 m/z and a scan rate of 1 scan/sec. Compounds were identified by comparing mass spectra with the NIST library and other literature. Normalization of peak areas was employed to calculate relative percentages of identified constituents. The GC–MS is a well-known method of identifying volatile compounds in EOs [13].

2.7 Culture media

The culture media employed in the study were mannitol. Salt agar to selectively isolate Staphylococcus aureus, Cetrimide agar to grow Pseudomonas aeruginosa, and Mueller-Hinton agar (MHA) to test antimicrobial susceptibility. MHA plates were inoculated according to the instructions provided on the plate and the Clinical and Laboratory Standards Institute (CLSI) guidelines. To achieve uniformity of test conditions, the agar depth was kept at about 4 mm, following the CLSI protocols [14].

2.8 Preparation of inoculum

Colonies that were freshly grown were harvested and suspended in sterile normal saline and taken 18-24 hours later. The resulting suspension was then diluted to correspond to the turbidity of a 0.5 McFarland standard. This standardization was in line with the approximate concentration of 1 × 10⁸ CFU/mL of bacterial isolates. The inoculum density was critically adjusted in line with the CLSI to maintain uniformity and reproducibility of the antimicrobial susceptibility testing [14].

2.9 Antimicrobial susceptibility testing

The agar disc diffusion method was employed to conduct antimicrobial susceptibility testing following the recommendations of the CLSI [14]. Pelargonium graveolens EO antimicrobial activity was assessed on sterile MHA plates in which evenly spread, standardized suspensions of microbes were inoculated using sterile cotton swabs. The plates that were inoculated were then left to stand for about 10 minutes to allow the absorption and drying of the surfaces before the disc was applied. EO solutions were prepared in dimethyl sulfoxide (DMSO) at concentrations of 50, 100, and 200 mg/mL. A volume of 20 µL of each solution was applied to each disc, providing final doses of 1, 2, and 4 mg/disc, respectively. A fixed volume of 20 µL was applied to each disc. Therefore, the tested concentrations of 50, 100, and 200 mg/mL corresponded to EO loadings of 1, 2, and 4 mg/disc, respectively.

The discs of 6 mm diameter filter paper were impregnated with the EO to prepare the discs. The discs were then dried in sterile conditions to make sure that the test substance adhered properly. The impregnated discs were then placed on the agar surface using sterile forceps and pressed to make contact with the media. Gentamicin (10 µg/disc) was used as the positive control for 30 clinical isolates. Discs containing solvent only served as negative controls. The bacterial cultures were incubated at 37 ℃ for 18-24 hours under the recommended conditions, as indicated by CLSI [14]. After incubation, the areas of inhibition were measured in millimeters with a calibrated ruler. Each assay was repeated three times, and the average values were obtained. Diameters of the inhibition zone were interpreted according to CLSI standards [14]. Multidrug resistance was defined as resistance to at least one agent in three or more antimicrobial categories according to internationally accepted criteria.

2.10 Statistical analysis

Statistical Package for the Social Sciences (SPSS; version 24) software was employed to analyze data statistically. The mean and the standard deviation were employed to present the results. One-way analysis of variance (ANOVA) was employed to determine differences between the experimental groups. A p-value of more than 0.05 was deemed not to be statistically significant. Appropriate statistical analysis is a key to reliable interpretation and validation of antimicrobial activity results [15]. All 60 clinical isolates were included in the essential-oil antibacterial assay (30 Staphylococcus aureus and 30 Pseudomonas aeruginosa isolates). For each concentration (50, 100, and 200 mg/mL), the inhibition-zone diameter is presented as mean ± Standard Deviation (SD) calculated from the 30 clinical isolates of each bacterial species.

The current study demonstrated that Pelargonium graveolens EO has a significant antibacterial influence on clinically isolated, antibiotic-resistant Staphylococcus aureus and Pseudomonas aeruginosa, with an apparent concentration-dependent response observed in the tested organisms. The findings are consistent with the emerging literature that shows that plant-based EOs are promising alternative antimicrobial agents in the age of increasing rates of AMR [16, 17]. The growing clinical burden of multidrug-resistant pathogens, especially S. aureus and P. aeruginosa, has been extensively reported as a global public health issue with limited treatment options and high adaptability of the organisms [3, 5, 18].

The GC–MS analysis showed that the major component of the oil was phenylethyl alcohol, and the others were geraniol, citronellol, and eugenol. Through this chemical profile, it is possible to agree with the past literature on the compositions of P. graveolens EO, but differences in ratios of the constituents are usually dependent on geographical location, climate, and method of extraction. Phenylethyl alcohol was not separately evaluated because the study focused on commercially available major bioactive standards. The strong antimicrobial potential of oxygenated monoterpenes like geraniol and citronellol is well documented, especially because of the lipophilic property that enables them to interact with the cell membranes of the microbes. These compounds can disrupt lipid bilayers, raise membrane permeability, and eventually cause leakage of intracellular contents and loss of membrane integrity [19, 20]. The high activity of the EO toward S. aureus can be explained by its higher susceptibility to the EO since it is a Gram-positive bacterium. Gram-positive bacteria have a thick peptidoglycan layer but no outer membrane, which means that the outer membrane is permeable to hydrophobic compounds in EOs. Geraniol was found to exert the greatest inhibitory activity on S. aureus, then citronellol and eugenol, which suggests that monoterpene alcohols significantly contribute to ABA.

The findings align with previous studies that reported that P. graveolens oil exhibits strong inhibitory influences against S. aureus [21]. Conversely, P. aeruginosa was more resistant to each of the oil components, whereas the crude EO showed medium but stable activity. The intrinsic resistance mechanisms are also known to be intrinsic to this organism, such as low outer membrane permeability, overexpression of efflux pumps, and biofilm formation, which all restrict the efficacy and penetration of antimicrobial agents [3]. The comparatively low activity of the isolated components toward P. aeruginosa in this research supports the idea that Gram-negative bacteria demand increased concentrations or a combination of bioactive compounds to be inhibited. Nevertheless, the crude oil was measurably active, indicating that mixture effects between its compounds contribute to antimicrobial influences in the crude oil.

The hypothesis that the concentration of EOs affects the availability of active phytochemicals to promote membrane disruption and metabolic interference is further supported by the concentration-dependent antibacterial influence in this study. More concentration-dependent trends were noted in recent research on the assessment of geranium oil encapsulation and antimicrobial delivery systems, with higher dose levels significantly enhancing the zones of bacterial inhibition [19, 22-24]. Moreover, the moderate-to-high inhibition of S. aureus over P. aeruginosa is also a characteristic trend in EO research since Gram-positive organisms are generally more sensitive than Gram-negative organisms because of the structural variation of cell envelopes. The antimicrobial influence of P. graveolens EO is thought to act on a variety of targets, mechanistically. Phenolic and monoterpene components have the potential to cause disruption of cytoplasmic membranes, a block on adenosine triphosphate (ATP) synthesis, and enzyme activity. In addition, phenylethyl alcohol, which was found to be the dominant compound in this study, has been noted to display antimicrobial influences by destabilising membranes and disrupting quorum-sensing signalling pathways and thus may potentially reduce bacterial virulence and biofilm formation. The broad-spectrum activity is probably due to the mixture effects of geraniol, citronellol, eugenol, and phenylethyl alcohol [25, 26].

The findings of the present study are consistent with the recent reviews, which highlighted the renewed interest in plant EOs as alternative antimicrobial agents. Schardong et al. [27] emphasized that mathematical and biological modelling of AMR is more and more contributing to the incorporation of phytochemicals into antimicrobial development pipelines because of their multi-target and reduced resistance development potential. On the same note, the WHO [1] has stressed the importance of using alternative and adjunct antimicrobial interventions, especially in the treatment of multidrug-resistant infections in clinical practice. Though the crude EO usually had a lower antibacterial efficacy than some of the individual compounds (e.g., geraniol) against S. aureus, the overall findings indicate that whole EO preparations could possess greater efficacy owing to the mixture effects of multiple bioactive compounds.

The greater activity observed with crude EO compared with individual tested constituents may suggest a possible mixture effect; however, this requires confirmation using dedicated synergy assays. This mixture effect could be the reason why crude P. graveolens oil showed significant inhibitory activity against both the pathogens tested, even though the activity of individual compounds varied.

The present study has its limitations, though, such as the use of in vitro disc diffusion only, which might not entirely recapitulate in vivo pharmacokinetics, host immune interactions, and bioavailability. Also, inconsistency in the EO composition and the absence of the minimum inhibitory concentration (MIC) can restrict the direct clinical translation. In spite of these shortcomings, the results give good preliminary data on the ABA of P. graveolens EO against resistant clinical isolates. MRSA classification in this study was based on the phenotypic antimicrobial susceptibility results obtained using the VITEK 2 AST-GP67 system. Molecular confirmation by mecA gene or PBP2a detection was not performed, which represents a limitation of this study.