The antimicrobial activity of tee tree oil nanoliposomes against Escherichia coli

Even though antibiotics are used as feed additives to manage various animal bacterial infectious diseases, the continuous emergence of drug-resistant bacterial strains has limited their efficacy. Since plant essential oils have shown a significant antibacterial effect, it has been considered a potential candidate to alleviate the problem of antibiotic resistance. 

Study: Tea tree oil nanoliposomes: optimization, characterization, and antibacterial activity against Escherichia coli in vitro and in vivo. Image Credit: ronstik/Shutterstock

In a recent Poultry Science study, researchers have formulated tee tree oil nanoliposomes (TTONL) and analyzed their antibacterial efficacy against pathogenic Escherichia coli (E. coli) that significantly affects the poultry industry. The main aim of the current study is to develop a new drug to promote sustainable and healthy animal husbandry in China.

E.coli infection in poultry

Although most E. coli strains are non-pathogenic and are present naturally in the intestine, some virulent strains can cause Crohn’s disease, gastroenteritis, and hemorrhagic colitis. Additionally, pathogenic E. coli cause colibacillosis, which is a systemic or localized infection commonly found in poultry. Colibacillosis has also been associated with multiorgan lesions, such as air sac inflammation, peri-hepatitis, and peritonitis, causing significant mortality in poultry.

E. coli cell wall is mainly composed of lipopolysaccharide (LPS), and cellular lysis of this bacteria causes a massive release of LPS which triggers an inflammatory response. In this context, NLRP3 has been found to play an important role in inducing inflammatory response during E. coli infection. Another factor that mediates late-stage inflammation is HMGB1.

Tee tree oil (TTO) and nanoliposomes

TTO is an essential oil that is extracted from the leaves of the tea tree. This essential oil is light yellow to clear in color and contains a fresh camphor odor. Typically, tea tree is found on the coast of southern Queensland to northern New South Wales, Australia.

TTO has many medicinal applications, including the treatment of herpes, acne, scabies, insect bites, and microbial skin infection. Importantly, this essential oil has shown a minimal inhibitory concentration (MIC) of less than 1% against most bacteria and fungi. Hence, TTO is considered a promising antimicrobial agent.

Terpinen-4-ol and α-terpineol are the main components of TTO that promote antibacterial activity. Mechanistically, the bactericidal effect of these components has been associated with microbial cell membrane disruption, causing cell lysis.

Besides its advantages, some of the limitations that restrict the application of TTO include insolubility in water, its unstable nature, and the strong inclination of its active ingredients to change when exposed to air. These shortcomings could be overcome by using nanoliposomes, a bilayer vesicle carrier system formed by self-assembly in aqueous media.

Nanoliposomes carrying essential oil reaches the target site through cellular interactions (e.g., phagocytosis, adsorption, and fusion). Importantly, these have significantly improved the poor stability of essential oils during storage and application. In this study, TTONL was optimally produced to inhibit E.coli disease in poultry.

Synthesis and characterization of TTONL

TTONL was synthesized using thin film hydration and sonication technique. The developmental process was optimized using the Box-Behnken response surface method. The optimal conditions determined for TTONL synthesis were lecithin to cholesterol mass ratio of 3.7:1, pH of the hydration medium of 7.4, and TTO concentration of 0.5. These conditions lead to a TTONL encapsulation rate of 80.31 ± 0.56%.

Transmission electron microscope (TEM) analysis revealed that TTONL was nearly spherical shaped and uniform in size. The average particle size of this bilayer structure containing TTO was 227.8 ± 25.3 nm with a negative charge. The characteristic absorption peak of TTONL revealed insignificant modification of the basic skeleton of a liposome. Importantly, experimental results indicated that TTONL was more stable at 4oC, compared to room temperature, for 35 days.

Antibacterial efficacy of TTONL

The antibacterial efficacy of TTONL was assessed against E. coli through in vitro and in vivo experiments. For the in vivo study, the efficacy of TTONL was investigated using chickens infected with colibacillosis.

The MIC test’s findings indicated that the nanoliposomes improved the antibacterial efficacy of TTO against various E. coli strains. After 8 hours of treatment with 75 mg/mL of TTONL, a complete bactericidal effect was observed against the test strains.

In vitro experiments showed that TTONL exposure caused varying degrees of structural damage to the E. coli strains. An in vivo study revealed that oral administration of TTONL significantly reduced the clinical symptoms and intestinal lesions of infected chickens. Importantly, TTONL treatment considerably decreased mRNA expression of NLRP3 and NF-κB in the cecum and duodenum of E. coli-infected chickens.


The newly synthesized TTONL exhibited a greater encapsulation rate, slow release, and improved stability with promising antibacterial activity against the tested pathogens. Considering all the experimental results, the current study strongly recommended the prophylactic application of TTONL to manage avian bacterial diseases.

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