The In Vitro Antimicrobial Efficacy of Lemon Oil (Citrus limon) Against Clinically Relevant Pathogens

The In Vitro Antimicrobial Efficacy of Lemon Oil (Citrus limon) Against Clinically Relevant Pathogens

R. K. Vijayraj, Pavan R

Department of Microbiology, Sri Siddhartha Medical College and Hospital, Sri Siddhartha Academy of Higher Education, Agalakote, B H Road, Tumkur-572107, India.

Abstract – The global rise in antimicrobial resistance has intensified the search for natural antimicrobial agents. This study evaluated the in vitro efficacy of lemon oil (Citrus limon) against Escherichia coli, Staphylococcus aureus, and Candida albicans using quantitative suspension tests. A two-way ANOVA demonstrated significant concentration- and time-dependent effects (p < 0.001). A 1 Log10 reduction was achieved at 5% concentration within 3040 minutes. These findings support lemon oil as a potential natural disinfectant. Keywords – Lemon oil, Citrus limon, antimicrobial activity, essential oils, suspension test, ANOVA, disinfectant.

1. INTRODUCTION

   Antimicrobial resistance (AMR) represents a major global health crisis, threatening the effectiveness of antibiotics and disinfectants [1], [2]. Healthcare-associated infections caused by resistant pathogens remain a significant burden [3]. Essential oils have emerged as promising alternatives due to their multi-target mechanisms [4], [5]. Lemon oil, rich in limonene and citral, exhibits antimicrobial potential [6][8]. However, standardized quantitative data remain limited [9].

2. MATERIALS AND METHODS

   1. Microorganisms

      Standard ATCC strains were used: E. coli ATCC 25922, S. aureus ATCC 29213, and C. albicans ATCC 10231.

   2. Preparation of Inoculum

      Cultures were grown in Tryptic Soy Broth and adjusted to 0.5 McFarland standard (~10 CFU/mL).

   3. Lemon Oil Preparation

      Water-soluble lemon oil was prepared at 3%, 4%, 5%, 6%, and 7% (v/v) concentrations.

   4. Quantitative Suspension Test

      Testing was conducted according to a modified EN 13697 protocol [10]. Equal volumes of inoculum and test solution were incubated for 10, 20, 30, and 40 minutes at 20°C.

   5. Neutralization and Enumeration

      Dey-Engley broth was used for neutralization [11]. Viable counts were determined by the pour plate method.

   6. Statistical Analysis

      Data were analyzed using two-way ANOVA to assess the effects of concentration and time. Tukeys HSD test was applied for

      post hoc analysis. Significance was set at p < 0.05.

3. Results

   Lemon oil exhibited concentration- and time-dependent antimicrobial activity against all test organisms. A 1 Log10 reduction was

   consistently achieved at 5% concentration within 3040 minutes.

   1. Statistical Analysis

Two-way ANOVA revealed significant effects of concentration, time, and their interaction for all organisms (p < 0.001).

Figure 1 Figure 2

Figure 3

Figure 1. Time-kill kinetics of 7% lemon oil against E. coli. Data represent mean ± SD (n = 3). ***p < 0.001 versus 10 min. Figure 2. Time-kill kinetics of 7% lemon oil against S. aureus. Data represent mean ± SD (n = 3). ***p < 0.001 versus 10 min. Figure 3. Time-kill kinetics of 7% lemon oil against C. albicans. Data represent mean ± SD (n = 3). ***p < 0.001 versus 10 min. Effect of Concentration

For E. coli, S. aureus, and C. albicans, increasing lemon oil concentration produced a significant increase in Log reduction (p < 0.001). Post hoc analysis revealed that concentrations 5% resulted in significantly higher microbial reduction compared with 3% and 4% (p < 0.01).

Effect of Contact Time

Contact time also significantly influenced antimicrobial activity (p < 0.001). Reductions observed at 30 and 40 minutes were significantly greater than those at 10 and 20 minutes for all organisms (p < 0.01).

Interaction Between Concentration and Time

A significant interaction between concentration and contact time was observed (p < 0.001), indicating that the antimicrobial effect of lemon oil increased synergistically with prolonged exposure and higher concentrations.

Inter-Organism Comparison

One-way ANOVA comparing mean Log reductions among organisms at 7% concentration and 40 minutes revealed significant

differences (p = 0.012). E. coli showed significantly greater susceptibility than S. aureus and C. albicans (Tukey test, p < 0.05).

E. coli

S. aureus

C. albicans

Tukey HSD Test Concentration Comparison

Tukey HSD Test Time Comparison

One-Way ANOVA Inter-Organism Comparison (7%, 40 min)

1. DISCUSSION

   The results confirm strong antimicrobial activity of lemon oil. E. coli exhibited greater susceptibility, possibly due to membrane destabilization by terpenes [12]. The antifungal activity may be attributed to citral-mediated membrane disruption [13]. The significant interaction between concentration and time indicates synergistic effects.

2. LIMITATIONS AND FUTURE WORK

   The suspension model does not reflect biofilm conditions. Future studies should evaluate biofilm efficacy, organic load interference, formulation development, and cytotoxicity [14], [15].

3. CONCLUSION

Lemon oil demonstrated significant antimicrobial activity against clinically relevant pathogens. Concentrations of 57% achieved meaningful microbial reductions, supporting its potential as a natural disinfectant.

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