Evaluation of the antimicrobial effect of 14 essential oils against bacterial pathogens from Meat Processing SurfacesAbstract
This study aimed to evaluate the antimicrobial activity of 14 essential oils against bacterial isolates recovered from meat processing surfaces in selected supermarkets. A total of 23 isolates were identified using morphological, biochemical, and molecular techniques targeting the uidA and Ddl genes for Escherichia coli and Enterococcus spp., respectively. The antimicrobial activity of the essential oils was assessed using the disc diffusion method. The results indicated that E. coli strains were generally more susceptible to essential oils compared to Enterococcus spp. Thyme oil exhibited the highest antibacterial effect (up to 34 mm), followed by lemongrass and clove oils. Conversely, eucalyptus, rosemary, and sweet basil oils showed minimal to no activity. These findings confirm the antimicrobial efficacy of certain essential oils, particularly thyme oil, under the specified laboratory conditions. However, further research is required to evaluate their effectiveness and stability in the complex, real-world conditions of a meat processing environment before they can be considered a viable alternative to conventional chemical disinfectants.
Keywords:
Essential oils; Antibacterial activity; Meat processing; Natural disinfectants; Escherichia coli; Enterococcus spp
HIGHLIGHTS
Thyme oil showed strong antibacterial activity with a 34 mm inhibition zone
Enterococcus spp. were more resistant to essential oils than Escherichia coli
Essential oils are promising alternatives to control multidrug-resistant foodborne bacteria
1 Introduction
In recent years, the rising consumption of ready-to-eat and ready-to-cook food products has been associated with a notable increase in foodborne disease outbreaks and growing concerns about food safety. This trend, driven by rapid urbanization and changing lifestyles, has introduced new challenges in maintaining hygiene across food production and distribution systems (Purice et al., 2024). High-income countries such as Canada have reported numerous incidents linked to contaminated processed foods, resulting in severe public health outcomes and considerable economic losses (Farber and Pagotto, 2023; Canadian Food Inspection Agency, 2022). The complexity of modern food supply chains has further exacerbated the difficulty in tracing sources of contamination, highlighting the critical need for robust safety monitoring and natural antimicrobial alternatives. Recently, South Africa has experienced one of the biggest foodborne outbreaks caused by the bacterium Listeria that was responsible for economic loss and high rate of mortality (Kaptchouang Tchatchouang et al., 2020). In addition, this was the first time for such tremedous outbreak in South Africa.
Historically, the control and prevention of foodborne pathogens have relied predominantly on chemical agents employed for cleaning and disinfection. Prolonged use of chemical disinfectants poses risks to human health and the environment due to their limited biodegradability and associated toxic effects, including acute toxicity, carcinogenicity, and teratogenicity (Richardson & Plewa, 2020). Despite improvements in hygiene and food production, food safety remains a major public health concern (World Health Organization, 2022). Food-contact surfaces act as reservoirs for pathogenic bacteria, which can contaminate food directly or via airborne particles (El Ayeb Naceur et al., 2021). Microorganisms naturally adhere to these surfaces as a survival strategy, often enhancing their resistance to chemical treatments (Mazaheri et al., 2021a).
According to the World Health Organization (2020a, 2020b, 2022), nearly one in ten people globally fall ill each year due to consuming contaminated food, resulting in approximately 420,000 deaths annually. Moreover, about a quarter of the world’s population is considered at higher risk of foodborne diseases due to factors such as age, immune status, and underlying health conditions. Therefore, there is an ongoing need to identify novel alternatives to eliminate or reduce the spread of foodborne pathogens.
In recent years, attention has shifted toward naturally derived products as potential solutions (Sishuba et al., 2021). Among these, essential oils (EOs) have been widely incorporated into products such as creams, soaps, food flavorings, dentistry applications, agricultural preservatives, fragrances, and massage products (Sharma et al., 2023). Essential oils are complex blends of volatile compounds biosynthesized by aromatic plants and extracted using techniques such as steam distillation, cold pressing, or maceration (Sharma et al., 2023). These bioactive metabolites, particularly monoterpenes like thymol, carvacrol, eugenol, and citral, exhibit broad-spectrum antimicrobial activity by disrupting cell membranes, altering ion transport, and interfering with enzymatic systems in microbial cells (Perricone et al., 2020; Rehman et al., 2021).
The antimicrobial potential of EOs has been documented against key foodborne pathogens, including Escherichia coli, Listeria monocytogenes, and Salmonella spp., highlighting their promise for applications in food safety and surface sanitation (Nazzaro et al., 2023; Kačániová et al., 2021). They have been applied not only as disinfectants for medical equipment and surfaces but also in aerosolized forms to reduce contamination in food processing environments (Yu et al., 2020). This study aimed to assess the antibacterial properties of selected essential oils against bacterial strains isolated from food processing surfaces, with the broader goal of exploring natural antimicrobials as alternatives or complements to synthetic disinfectants in industrial settings.
Recent technological advancements have enabled the formulation of EOs in nanoemulsions and polymeric matrices, enhancing antimicrobial efficiency, reducing volatility, and improving compatibility with food-grade materials (Moghimi et al., 2022; Huang et al., 2023). Regulatory agencies and consumer preferences increasingly favor eco-friendly interventions, creating an ideal context for integrating essential oils into hygiene protocols. Research demonstrates that EOs from Thymus vulgaris L., Cymbopogon citratus (DC.) Stapf, and Syzygium aromaticum (L.) Merr. & L.M. Perry exhibit both bactericidal and antibiofilm effects, crucial for addressing persistent microbial contamination on meat processing surfaces (Wang et al., 2022).
2 Materials and methods
2.1 Selection and sourcing of essential oils
Fourteen (14) essential oils (EOs) were selected for this study based on their documented biological activities, including antimicrobial properties, as supported by existing literature. The EOs were commercially procured from a reputable supplier in Pretoria, South Africa, ensuring quality and authenticity. Details of each essential oil, including their botanical source, extraction method, and reported biological activities, are summarized in Table 1. All oils were stored at 4 °C in airtight containers until further use to preserve their bioactive compounds.
2.2 Isolation of bacterial isolates from meat processing surfaces
Surface swab samples were collected aseptically from four meat processing supermarkets located in Mafikeng, North-West Province. Sampling focused on high-contact food processing surfaces, including cutting boards, knives, workbenches, and conveyor belts. A total of 16 swabs were obtained using sterile cotton swabs pre-moistened with sterile peptone water (PW; Merck Chemicals, Gauteng, South Africa). Swabs were collected immediately after routine surface sanitization to assess the efficacy of cleaning procedures in removing bacterial contaminants. Each swab was placed into a sterile Eppendorf tube containing 5 mL of PW and transported on ice to the laboratory for immediate processing. From each sample, 100 µL was inoculated into 900 µL Nutrient Broth (NB; Merck Chemicals, Gauteng, South Africa) and incubated at 37 °C for 48 hours to enrich bacterial populations. Post-incubation, serial ten-fold dilutions ranging from 101 to 106 were prepared in sterile saline. Subsequently, 100 µL aliquots from 104 to 106 dilutions were spread plated onto selective media targeting specific bacterial genera:
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Staphylococcus aureus: Mannitol Salt Agar (MSA; Merck, Cape Town, South Africa)
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Salmonella spp.: Salmonella-Shigella Agar (SS; Merck, Cape Town, South Africa)
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Escherichia coli: Eosin Methylene Blue Agar (EMB; Merck, Cape Town, South Africa)
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Enterococcus spp.: Bile Esculin Agar (BE; Merck, Cape Town, South Africa)
Plates were incubated at 37 °C for 24 hours. Presumptive colonies characteristic of each target organism were selected for further morphological, biochemical, and molecular identification.
2.3 Morphological and biochemical characterization
Pure bacterial colonies were obtained by streaking individual colonies onto fresh nutrient agar plates using the quadrant streak method. Plates were incubated at 37 °C for 24–48 hours, and distinct colonies exhibiting uniform morphology were repeatedly sub-cultured until a single bacterial type was confirmed. Pure bacterial colonies obtained from selective media were subjected to Gram staining and examined microscopically to confirm morphological characteristics. Subsequently, isolates were screened via standard biochemical tests, including oxidase, catalase, citrate utilization, and triple sugar iron (TSI) assays, to aid in preliminary species identification.
2.4 DNA extraction
Genomic DNA was extracted from all presumptive bacterial isolates using a modified boiling method based on Kao & Alocilja (2025). Briefly, bacterial cultures were centrifuged at 15,000 × g for 15 minutes, supernatant discarded, and pellets resuspended in sterile distilled water. After a second centrifugation at 15,000 × g for 10 minutes, pellets were resuspended in nuclease-free water and boiled at 100 °C for 10 minutes in a water bath (Memmert, Gauteng, South Africa). The concentration and purity of the extracted bacterial DNA were assessed using a NanoDrop spectrophotometer (Thermo Fisher Scientific, USA). DNA purity was evaluated by measuring the absorbance ratios at 260/280 nm and 260/230 nm, with ratios of ~1.8 and 2.0–2.2, respectively, indicating high-quality DNA suitable for downstream molecular analyses. Samples were then rapidly cooled on ice, centrifuged briefly at 15,000 × g for 10 seconds, and the supernatant containing DNA was stored at −20 °C until use.
2.5 Polymerase Chain Reaction (PCR) amplification and sequencing
PCR amplification was performed targeting the 16S rRNA gene for broad bacterial identification across all isolates. The PCR reaction mixture (25 µL total volume) consisted of 12 µL master mix, 0.25 µL each of forward (27F) and reverse (1492R) primers (Table 2), 1 µL DNA template, and 11.5 µL nuclease-free water. The cycling conditions for 16S rRNA gene amplification were as follows: initial denaturation at 94 °C for 3 minutes; 25 cycles of denaturation at 94°C for 1 minute, annealing at 55 °C for 1 minute, and extension at 72 °C for 2 minutes; followed by a final extension at 72 °C for 10 minutes.
Species-specific PCRs were performed to confirm E. coli and E. faecalis identities, targeting the uidA and ddl genes, respectively (Table 2). Genomic DNA for these reactions was extracted directly from purified bacterial colonies. PCR amplification for E. coli uidA used an initial denaturation at 95 °C for 10 min, followed by 35 cycles of 95 °C for 45 s, 59 °C for 30 s, and 72 °C for 1 min 30 s, with a final extension at 72 °C for 10 min. For E. faecalis ddl, amplification consisted of an initial denaturation at 95 °C for 4 min, 30 cycles of 95 °C for 30 s, 55 °C for 60 s, 72 °C for 60 s, and a final extension at 72 °C for 7 min (Table 2).
PCR products were electrophoresed on 1.5% agarose gel in 1X TAE buffer at 80 V for 45 minutes (Smith et al., 2023). Gels were stained with ethidium bromide (Et Br) and visualized under Ultraviolet (UV) light using a UV transilluminator (Spectroline, Lasec, Gauteng, South Africa). Representative 16S rRNA gene amplicons were purified and subjected to Sanger sequencing (performed by [specify sequencing facility]) to confirm species identification by Basic Local Alignment Search Tool (BLAST) analysis against the National Center for Biotechnology Information (NCBI) database.
2.6 Antimicrobial assay of essential oils
The antibacterial activities of the selected essential oils were evaluated using the Kirby-Bauer disk diffusion method. Sterile 6 mm diameter filter paper disks were aseptically impregnated with 10 µL of each essential oil and placed on Mueller-Hinton agar (MHA; Merck Chemicals) plates previously inoculated with 100 µL of standardized bacterial suspensions (0.5 McFarland standard). Plates were incubated at 37 °C for 24 hours, after which zones of inhibition around the disks were measured in millimeters using a digital calliper. All assays were performed in triplicate.
3 Results and discussion
The findings showed that a total of 23 strains of potential pathogens were successfully isolated from the selective media used. Based on preliminary results, the bacterial isolates were identified as E. coli and Enterococcus spp. Identification was initially performed using standard biochemical tests, including Gram staining, catalase and oxidase reactions, sugar fermentation profiles, and motility assessments. Species confirmation was further achieved using species-specific Polymerase Chain Reaction (PCR) targeting the uidA gene for E. coli and the ddl gene for E. faecalis (Table 2). Notably, the occurrence of these bacteria varied across the four meat processing establishments. While E. coli and Enterococcus spp. were detected in all locations, their prevalence and isolation patterns differed, reflecting variability in hygiene practices, surface contamination levels, and environmental conditions. Figure 1 shows amplification of the 16S rRNA gene with a 1420bp size, which was displayed in all 23 tested isolates. Ddl gene region for Enterococcus spp. had a band size of 475bp. These primers were to identify the Enterococcus spp. A total of twelve (n = 12) strains were confirmed to be E. faecelis using the Ddl primers. Amplification of the uidA gene region for E. coli with a band size of 556bp was also performed. These primers were to identify the E. coli. A total of ten (n = 10) strains were confirmed to be E. coli using uidA specific primers.
Gel electrophoresis (1.5%) for 16S rRNA gene (a); (b) uidA specific primers (Escherichia coli); (c) Ddl specific primers (Enterococcus spp.) Lane M 1 kb marker, other lane test samples.
Antimicrobial assay was determined using the disk diffusion assay and analysed by measuring the zone of inhibition. Table 3 shows the inhibitory effects of the selected oils against the bacterial isolates. Thyme, lemongrass, and clove oil showed the highest effectiveness, displayed by zone of inhibition measuring 34 mm, 30 mm, and 18 mm, respectively. While eucalyptus, sweet basil, and rosemary showed the least activity, with no activity in most isolates. The trend of activity towards the Gram-positive isolates against thyme, lemongrass, and clove oil was similar, as they all had relatively large zones of inhibition but very low zones of inhibition against the rest of the selected oils used. However, Gram negative bacteria were more sensitive towards all the oils studied as compared to Gram positive bacteria. Enterococcus spp. was more resistant than E. coli. Enterococcus (Sample ID: EnC3) was the most resistant isolate, and E. coli (Sample: EcP1) was the most susceptible.
Inhibitory effects of selected essential oils against Escherichia coli and Enterococcus spp. Isolates.
The present study provides comprehensive insights into the microbiological quality of meat processing surfaces and the potential of essential oils as natural antimicrobial agents. A total of 23 bacterial isolates were recovered from various supermarket meat processing surfaces, highlighting the persistence of microbial contamination despite routine cleaning. Molecular analysis confirmed the identities of the isolates as E. coli and E. faecalis, two well-known foodborne pathogens of public health concern. The detection of E. coli and E. faecalis from surfaces commonly involved in food contact emphasizes the role of environmental surfaces as reservoirs for cross-contamination. These findings are consistent with earlier reports indicating that food processing environments can harbour pathogens even under good manufacturing practices (Tshabalala et al., 2021b; Mazaheri et al., 2021b). The amplification of the 16S rRNA gene in all isolates validated their bacterial origin, while species-specific PCR targeting the uidA and ddl genes enabled accurate confirmation of E. coli and E. faecalis, respectively.
Antimicrobial assays using the disk diffusion method demonstrated significant inhibitory activity of certain essential oils. Thyme, lemongrass, and clove oils exhibited pronounced antibacterial effects, with inhibition zones of up to 34 mm, 30 mm, and 18 mm, respectively. These results align with those of Thyme oil, which has been widely recognized for its bioactive constituents, primarily thymol and carvacrol, which are known to disrupt bacterial membranes and interfere with vital metabolic processes (Kosakowska et al., 2021). Clove oil, rich in eugenol, also demonstrated notable efficacy. Eugenol exerts its antibacterial effect by damaging cell walls, inhibiting enzymes, and disrupting genetic material (Kaur et al., 2022). Lemongrass oil contains citral and geraniol, both of which have strong bactericidal effects by increasing membrane permeability and inducing cell leakage (Almasi et al., 2021; Oliveira et al., 2021). The effectiveness of these oils against both Gram-negative E. coli and Gram-positive E. faecalis suggests broad-spectrum applicability, although E. coli isolates in this study exhibited greater susceptibility. Interestingly, while Gram-negative bacteria are typically more resistant due to their outer membrane barrier, essential oils used in this study were able to permeate and exert inhibitory effects. This could be attributed to the small, lipophilic nature of key essential oil compounds that allow for membrane penetration, a phenomenon supported by other studies (Vasilatis & Gianfagna, 2021).
Among the Enterococcus isolates, one strain (EnC3) displayed marked resistance to all tested oils, indicating possible intrinsic resistance mechanisms such as efflux pumps or biofilm formation, which are known to confer protection against antimicrobials (Garcia-Contreras et al., 2023). Conversely, E. coli strain EcP1 exhibited the highest level of susceptibility, suggesting strain-dependent variability.
The global burden of foodborne illnesses remains substantial, with E. coli and E. faecalis among the most commonly identified pathogens in outbreaks linked to contaminated food and contact surfaces (Nyenje & Ndip, 2022; Tian et al., 2023). The increasing resistance of these pathogens to conventional disinfectants and antibiotics underscores the urgent need for safer and more sustainable alternatives (Singh et al., 2021; Bakour et al., 2022). Essential oils, owing to their natural origin, complex phytochemical profiles, low toxicity, and broad-spectrum antimicrobial activity, have emerged as viable options for enhancing hygiene in food handling and processing environments (Chouhan et al., 2023; Mahdavi et al., 2022). Studies have shown that oils such as thyme, clove, and lemongrass are particularly effective in inhibiting key foodborne pathogens, offering promising avenues for non-toxic disinfection strategies (Bakour et al., 2022; Chouhan et al., 2023).
This study supports the incorporation of essential oils into sanitation strategies, either as direct surface disinfectants or as components in antimicrobial packaging. However, further research is needed to optimize application methods, assess stability, and evaluate potential sensory impacts on food products.
4 Conclusion
Foodborne illnesses resulting from pathogenic bacteria on contaminated food remain a significant public health concern. Contamination of meat processing surfaces is a key contributor to the spread of these pathogens. While conventional chemical disinfectants are commonly used to control surface contamination, their potential negative impact on human health and the environment has raised concerns. This study assessed the antibacterial properties of selected essential oils against bacterial strains isolated from meat processing surfaces. The results indicate that certain essential oils exhibit inhibitory effects against these bacteria, suggesting their potential as natural, eco-friendly agents to complement existing sanitation strategies. However, further research is needed to optimize their application and to fully evaluate their efficacy under industrial conditions.
Acknowledgements
The authors extend their appreciation to their respective universities, institutes and research niche areas.
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Cite as:
Kabelinde, A., Foka, F. E. T., Ateba, C. N., & Manganyi, M. C. (2026). Evaluation of the antimicrobial effect of 14 essential oils against bacterial pathogens from Meat Processing Surfaces. Brazilian Journal of Food Technology, 29, e2025072. https://doi.org/10.1590/1981-6723.07225
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Data Availability Statement
All data generated or analyzed in this study are included in this published article.
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Funding:
None.
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Edited by
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Associate Editor:
Felipe Alves de Almeida.
All data generated or analyzed in this study are included in this published article.
