Bacterial resistance in diarrhea and tea tree oil as a potential alternative treament: a review

Resistencia bacteriana en diarrea y aceite esencial de arbol de té como potencial tratamiento: revisión

Review



Javier Nicolás González-González1 , Ildefonso Guerrero-Encinas1 , Gloria Guadalupe Morales-Figueroa1 , Gustavo A. González-Aguilar1 , Jesus F. Ayala-Zavala1 , Humberto F. Astiazarán-García1 , Marco A. López- Mata2 , Raymundo R. Rivas-Cáceres3 & Luis Quihui-Cota1*

1 Centro de Investigación en Alimentación y Desarrollo, A.C. (CIAD, A.C.) Carretera Gustavo Enrique Astiazarán Rosas No. 46. Col. La Victoria 83304. Hermosillo, Sonora, México.

2 Departamento de Ciencias de la Salud, Universidad de Sonora, Campus Cajeme, Blvd. Bordo Nuevo S/N, A.P. 85040, Antiguo Providencia, Cd. Obregón, Sonora, México.

3 Universidad Autónoma de Ciudad Juárez, Ave. Plutarco Elías Calles No. 1210, FOVISSSTE Chamizal Cd, Juarez C.P. 32310, México.

ABSTRACT

Bacterial diarrhea is a global health concern, particularly in developing countries like Mexico, where high morbidity and mortality rates persist, especially in children under five years of age. While antibiotics like ciprofloxacin, ceftriaxone, and azithromycin are effective, increasing bacterial resistance has led to the search for alternatives. Tea tree essential oil (TTEO) has been proposed as a potential treatment, but research, especially in vivo, remains limited due to oil composition variability and a lack of standardized protocols. This review compiles current data (2000-2024) on the epidemiology, diagnosis, treatment, and antibiotic resistance of critical dia- rrhea-causing bacteria (E. coli, Shigella spp., Campylobacter spp., and Salmonella spp.) and evaluates TTEO’s antibacterial potential. In vitro studies show its bactericidal and bacte- riostatic effects, while in vivo studies assess its therapeutic impact on animal models. In conclusion, TTEO holds promise as an alternative or adjuvant to antibiotics for treating bacte- rial diarrhea. However, further in vivo studies are required to confirm its efficacy and optimize its clinical application.

Keywords: Diarrhea, Antibiotics, Antibiotic-resistant, Essen- tial oils, Tea tree.


RESUMEN

La diarrea bacteriana es un problema de salud pública mun- dial, especialmente en países en desarrollo como México, donde persisten altas tasas de morbilidad y mortalidad, sobre todo en niños menores de cinco años. Aunque los an- tibióticos como la ciprofloxacina, ceftriaxona y azitromicina son efectivos, el aumento de la resistencia bacteriana nos ha forzado a buscar alternativas. El aceite esencial de árbol de té (TTEO) ha sido propuesto como tratamiento potencial, pero la investigación, especialmente in vivo, es limitada de- bido a la variabilidad en la composición del aceite y la falta de protocolos estandarizados. Esta revisión recopila datos actuales (2000-2024) sobre la epidemiología, diagnóstico,

tratamiento y resistencia a antibióticos de bacterias clave que causan diarrea (E. coli, Shigella spp., Campylobacter spp. y Salmonella spp.), y evalúa el potencial antibacteriano del TTEO. Los estudios in vitro muestran sus efectos bactericidas y bacteriostáticos, mientras que los estudios in vivo evalúan su impacto terapéutico en modelos animales. En conclusión, el TTEO tiene potencial como una alternativa o complemen- to a los antibióticos para tratar la diarrea bacteriana, pero se necesitan más estudios in vivo para confirmar su eficacia y optimizar su aplicación en la práctica clínica.

Palabras clave: Diarrea, Antibióticos, Resistente a los anti-

bióticos, Aceites esenciales, Árbol de té.


INTRODUCTION

One of the leading health problems worldwide is malnutri- tion and infection. Both conditions have been associated with high morbidity and mortality rates in children and adults, resulting in intestinal alterations and a negative impact on nutrient absorption (FDA, 2023). In addition, the indiscri- minate or inappropriate use of antibiotics in animals and humans has contributed to the increased bacterial resistance to multiple drugs (van den Bogaard, 2000; Bouarab-Chibane, 2019). Pathogen bacteria such as Escherichia coli, Shigella spp., Campylobacter spp., and Salmonella spp. (Hirose and Sato, 2011) are on the list of the highest epidemiological surveillance worldwide, causing mild symptoms to severe diseases (WHO, 2017). Thus, they negatively impact on the host’s nutritional status and are associated with alterations in the intestinal mucosa, resulting in a low absorption capacity of nutrients (Fagundes-Neto and Affonso-Scaletsky, 2000).

Natural alternatives with antibacterial properties have been sought, and they may be an appropriate adjuvant for conventional antibiotics (Calo et al., 2015). Essential oils (EOs) of some plants have shown antibacterial properties in vitro, producing inhibition percentages ranging from deficient (13

%) to very satisfactory (96 %) against some pathogenic bac-


*Author for correspondence: Luis Quihui-Cota e-mail: lquihui@ciad.mx

Received: February 27, 2023

Accepted: October 8, 2024

Published: November 7, 2024


Volume XXVI


1

DOI: 10.18633/biotecnia.v26.2270

Journal of biological and health sciences http://biotecnia.unison.mx

Universidad de Sonora

ISSN: 1665-1456

teria (Kon and Rai, 2012; Chouhan et al., 2017). In addition, EO’s components can exert antioxidant effects (Miguel, 2010; Yang et al., 2019), which may positively impact the host’s nutritional status since they act as functional foods for both animals and humans (Firmino et al., 2020).

Search strategy

This review was conducted by retrieving data from reputable scholarly databases, including Google Scholar, PubMed, Springer, Science Direct, and Wiley Online Library. The search used keywords pertinent to the efficacy of tree tea essential oil against Shigella, Salmonella, Campylobacter, and Escheri- chia coli, encompassing both in vitro and in vivo evaluations at the gastrointestinal level. The inclusion criteria for selected studies encompassed publications from 2000 to 2023. Fur- thermore, an investigation into plant extracts’ safety profile and compositional characteristics demonstrating efficacy was undertaken to provide a holistic perspective on their potential applications.


Shigella spp.

Shigella is a Gram-negative bacterium from the Enterobacte- riaceae family and causes approximately 125 million diarrhea cases and 160,000 deaths annually, and a third of this figure are children. One of the important clinical aspects of Shigella is that even a low infectious dose is associated with aggressi- ve watery diarrhea or bloody diarrhea (Baker and Chung-The, 2018). In addition, this bacterium is the third most isolated pathogen by clinical laboratories in the United States. Shige- lla is subdivided into four major species: Shigella dysenteriae, Shigella boydii, Shigella flexneri, and Shigella sonnei (Bowen, 2018).

Shigellosis is a disease that occurs once the bacteria settle in the intestine. Shigella spp. can tolerate the acidic pH of the stomach, colonizing the digestive tract, generating pores in the membrane of epithelial cells, and invading their cytoplasm. Shigella spp. can then multiply in adjacent cells without moving through the extracellular medium (Barran- tes-Jiménez and Achí-Araya, 2009). Although all Shigella spp. can cause bloody diarrhea, S. dysenteriae can produce Shiga toxin, which causes hemolytic uremic syndrome, associated with blood in the urine and, occasionally, clots in blood ves- sels of the kidneys (Lampel, 2012). To prevent this infection, hand washing, food care, precautions in drinking water (especially in developing countries), use of alcohol-based sanitizers, and avoiding fecal exposure in the sexual act are more often recommended (Bowen, 2018). On the other hand, special attention must be paid to fulfill those measures, particularly to children under five years old. In addition, when an infant is infected, special care must be taken so that there is no person-to-person transmission, especially if the child’s diaper must be changed (CDC, 2023c).

To diagnose properly, a doctor must suspect shigellosis based on its usual symptoms such as pain, fever, and watery or bloody diarrhea. Subsequently, confirmation is carried out with serological or molecular methods (Lampel, 2012).

Salmonella spp.

Salmonella is a Gram-negative bacterium belonging to the Enterobacteriaceae family. Salmonella strains can cause two types of illnesses: typhoid fever and non-typhoid salmone- llosis. Although both cause fever, the former is more severe and has a higher mortality rate (Hammack, 2012). However, globally, non-typhoid salmonellosis is one of the four leading causes of diarrheal illness, with around 153 million cases of gastroenteritis and 57,000 deaths (Hunter and Francois- Watkins, 2018). There are 2,500 serotypes of the two main species, Salmonella bongori, and Salmonella enterica, the second being the most relevant clinically and in public health (WHO, 2019).

Most Salmonella strains are pathogenic due to their ability to invade and survive in host cells. When Salmonella enters the digestive tract, it can penetrate epithelial cells by injecting effectors through the membrane into the cyto- plasm, or some adhesion systems, detected in strains such as S. enterica (Shu-Kee et al., 2015). Within these systems, adhesins, invasins, exotoxins, and endotoxins have been identified to ensure its survival in low pH. These properties give specificity to the different serotypes to adapt to the host, promoting the advancement of the disease (Jajere, 2019).

Therefore, since it has been reported that salmonellosis is the most significant foodborne infection worldwide, its prevention is a concern for public health (Jajere, 2019), so risk prevention is focused on primary production to food transportation (WHO, 2019). In addition, the usual safety measures, such as hand washing and precautions when in contact with farm or domestic animals, are recommended. On the other hand, greater caution is recommended in chil- dren under five years old, who are the most susceptible to salmonellosis (Hunter and Francois-Watkins, 2018).

Regarding diagnosis, coprological analysis is the selec- tion technique for timely detection. Similarly, this can be detected in cultures from samples of blood, urine, abscesses, and cerebrospinal fluid (Hunter and Francois-Watkins, 2018; CDC, 2022b). In addition, in countries such as the United States, it is important that the laboratories responsible for clinical diagnosis report to the Center for Disease Control and Prevention (CDC) for proper epidemiological surveillance (CDC, 2019).

Campylobacter spp.

Campylobacter is a Gram-negative, spiral S-shaped bacteria belonging to the family Campylobacteriaceae. It causes diarrhea and is one of the most prevalent enteric pathogens in developing and developed countries. In the United States,

1.3 million are infected every year (CDC, 2023a). Although the disease by this bacterium is considered mild, in populations under five years of age, it may be fatal (WHO, 2020), where Campylobacter jejuni is the most common agent found asso- ciated with diarrheal disease, in Argentina (30.1 %), Peru (23

%) and Colombia (14.4 %) (Fernández, 2011).

C. jejuni is competitive in the intestine of the host, taking DNA from the environment, allowing recombination bet- ween strains, and favoring its genetic diversity (Young et al., 2007), which allows it to adapt to adverse acidic and aerobic environments. Through flagella and union factors, C. jejuni can attach to epithelial cells of the intestine, colonizing the host’s intestine and causing severe diarrhea, fever, and blood in the stool (Dasti et al., 2010).

There are no vaccines to prevent infection, but preven- tion is achieved by applying basic hygiene recommendations, such as hand washing, correct cooking of food, and proper treatment of contaminated water (Geissler et al., 2018). In addition, some organizations emphasize disease control with timely hygiene measures at all stages of the food chain, from farms to homes (WHO, 2020).

The diagnosis of C. jejuni is made with unique isolation and growth conditions due to its microaerophilic characteris- tics. The stool or rectal samples are inoculated in a selective medium, and the culture is kept at an ideal temperature (42

°C) for 72 h, with 5 % oxygen (Foley, 2012). On the other hand, microscopic visualization has been recommended, where the spiral S shape of the bacterium can be observed to establish an opportune diagnosis. A relevant issue is that laboratories analyze the sample in no more than 2 hours, taking advan- tage of the unique bacterial characteristics (Geissler et al., 2018).


Escherichia coli

E. coli is a group of Gram-negative bacilli and facultative anaerobes belonging to the Enterobacteriaceae family (Vila et al., 2016). E. coli strains are predominant in the intestinal microbiota of mammals and, specifically, in humans, showing a prevalence higher than 90 % (Denamur et al., 2021). At the epidemiological level, the diarrheagenic E. coli (DEC) sub- group is the leading cause of diarrhea, a condition that kills infants in developing countries (Hebblestrup, 2014). Around 6 DECs have been identified, including Enteropathogenic E. coli (EPEC), Enterotoxigenic E. coli (ETEC), Shiga-toxigenic E. coli (STEC), Enteroinvasive E. coli (EIEC), Enteroaggregative E. coli (EAEC) and diffusely adherent E. coli (DAEC) (O’Reilly et al., 2018).

Diseases associated with DEC had a 30–40 % preva- lence in Asia, the Middle East Africa, Central America, South America, and Mexico, especially within children populations (Gomes et al., 2016; O’Reilly et al., 2018). In Mexico, these bacteria represent the top pathogens that cause diarrhea in children, with 30.9 %, followed by S. enterica (11.4 %), Shigella spp. (10.8 %), and Campylobacter spp. (5.6 %) (Rios-Muñiz et al., 2019). For example, EPEC has been known to cause 17 to 19% of childhood diarrhea (Vidal et al., 2007). On the other hand, ETEC and EAEC, which produce “traveler’s diarrhea,” affect both children and adults, with prevalences ranging from 5.1 to 12.2 % in Northwest Mexico. The rest of the DEC seems less frequent, with prevalences ranging from 0.2% to

1.4 % (Rios-Muñiz et al., 2019).

The pathogenesis of DEC depends on the pathotype and the specific strains. However, most of them mainly affect the small or large intestine, with an incubation period ran- ging from 8 hours to 10 days, mainly causing watery diarrhea (Gomes et al., 2016; O’Reilly et al., 2018; Rios-Muñiz et al., 2019). Specifically, ETEC, EIEC, EAEC, and STEC adhere to the intestinal mucosa, releasing enterotoxins and cytotoxins that promote inflammation, triggering diarrhea (Vidal et al., 2007; O’Reilly et al., 2018). In addition, EPEC adheres to the intes- tinal mucosa, causing a flattening of villi and inflammatory changes, leading to conformational changes and reduction of hydrolysis and absorption of nutrients (Morales-Cruz and Huerta-Romano, 2010). Finally, although little is known about its pathogenicity, DAEC is believed to form a diffuse adheren- ce that induces protruding structures protecting its colonies and allowing disease development (O’Reilly et al., 2018).

Some strategies have been proposed to prevent infec- tion by DEC, among which general hygiene in food prepara- tion, hand washing, and food cooking (62.6 °C) stand out. In addition, it is recommended to avoid consuming foods such as raw milk and dairy products and unpasteurized juices (CDC, 2023b). This type of prevention may be impractical for humans since there are different pathogenic strains with di- fferent transmission routes. Therefore, the most widely used strategy worldwide is the epidemiological surveillance of specific strains that can contaminate food and infect humans. This strategy includes the detection of potential hazards of specific groups such as STEC, estimation of risk of food con- tamination, counts of viable organisms to trigger the disease, and timely diagnosis (Newell and La Ragione, 2017).

A diagnosis of EPEC can be carried out using a copro- logical analysis to confirm the presence of these bacteria. However, diagnostic tests that only detect a subset of STEC, known as enterohemorrhagic E. coli (EHEC), and recently for ETEC, are usually performed in most hospitals due to its pre- valence and clinical importance (O´Reilly et al., 2018). In the case of EHEC, detection of specific physiological markers pro- duced by the EHEC O157:H7 strain are performed since this is the primary bacterium of this pathotype. Similarly, some tests to detect ETEC are performed by culturing the sample in selective media, such as eosin agar and methylene blue, fo- llowed by serotyping or the use of molecular techniques for serotype identification (Morales-Cruz and Huerta-Romano, 2010; Newell and La Ragione, 2017).


Conventional treatment

The usual treatments for gastrointestinal diseases caused by these bacteria, especially diarrhea, are based on fluid and electrolyte replacement (WHO, 2020b). However, in some cases of immunocompetent patients with bloody diarrhea, patients who are immunocompromised, or in contact with another infected patient, antibiotic treatment is recommen- ded (Shane et al., 2017). The prescription of these drugs va- ries depending on different factors, such as the patient’s age or the pathogenic bacteria that causes the disease (Cohen et

al., 2017). Among the most important and the first choices to treat infections by E. coli are azithromycin, ceftriaxone, and ciprofloxacin.

Azithromycin is a second-generation broad-spectrum macrolide antibiotic. Guidelines for treating gastrointestinal diseases, especially diarrhea, recommend this antibiotic to treat Campylobacter, Shigella, and DEC infections (Shane et al., 2017). Its primary mechanism of action is based on bacte- rial protein synthesis by interfering with their 50S ribosomal subunits (Parnham et al., 2014). On the other hand, effective doses vary depending on age, reaching doses of 500 mg (Co- hen et al., 2017). However, this antibiotic is not recommen- ded in patients with STEC, especially children, since it can cause hemolytic uremia syndrome, characterized by kidney damage, hemolytic anemia, and thrombocytopenia (Tarr et al., 2018).

Ceftriaxone is a third-generation cephalosporin prescri- bed for treating Salmonella and Shigella infections (Lamb et al., 2002). The main action of this antibiotic lies in its ability to inhibit the synthesis of mucopeptides in the bacterial cell wall. In addition, it has been shown that ceftriaxone establishes bonds with the enzymes responsible for cell wall synthesis and cell division, such as carboxypeptidases and endopeptidases, which cause bacterial cell death. Doses range from 20-50 mg/kg for standard treatment, and up to 80 mg/kg in the case of severe infections (Schleibinger et al., 2015).

Finally, ciprofloxacin is a fluoroquinolone effective aga- inst Salmonella, Shigella, and E. coli (Shane et al., 2017; Hunter and Francois-Watkins, 2018). The antimicrobial mechanism is based on inhibiting the DNA gyrase enzyme, a DNA to- poisomerase involved in bacterial cell replication. It causes bacterial DNA breaks and suppresses the division of the target cell (Campoli-Richards et al., 1988; Ojkicet al., 2020). Doses range from 20 mg/kg/day in children to a maximum of 500-1000 mg in adults for 3-5 days (Shane et al., 2017; Cohen et al., 2017). However, in recent years, the loss of effectiveness of antibiotics against bacteria was reported.

Antibiotic resistance

Drug-resistant diseases currently cause 700,000 deaths per year, and estimations are that by 2050, there will be around 10 million deaths per year (WHO, 2022b). Although antibiotics are the drugs of choice for treating gastrointes- tinal illnesses caused by bacteria, antibiotic resistance is an increasing problem. This natural phenomenon is observed in some medications, but their indiscriminate use in humans and animals has aggravated this problem (CDC, 2023c).

It has been reported that Shigella spp. strains have acquired resistance to antibiotics, including ciprofloxacin (8.9 %) and ceftriaxone (9.3 %) (Puzari et al., 2018; Hussen et al., 2019). On the other hand, Campylobacter spp. strains have developed resistance with prevalences of 45% and 89.9 % to azithromycin and ciprofloxacin, respectively, at their usual doses (Yang et al., 2019). In the case of non-typhoidal Salmo- nella, a resistance increase from 12.3 % to 19.2 % in children

has been reported in China within a 4-year period (Wu et al., 2021). Finally, different E. coli strains have presented resis- tance of up to 14.2, 9, and 20 % to ciprofloxacin, ceftriaxone, and azithromycin, respectively (Eltai et al., 2018; Xiang et al., 2020).

The WHO and CDC have developed several recommen- dations and strategies, among which, are the prescription of antibiotics only when necessary, and investing in the deve- lopment of new antibiotics and vaccines, which are the ones that stand out (WHO, 2022e; CDC, 2023c). Recently, work has been done on developing these new strategies, among which the tea tree essential oil (TTEO) can be found. This essential oil has been proven effective in vitro and in vivo to inhibit the growth of some bacteria. It may be an alternative or natural adjuvant in treating human and animal bacterial infections (Chouhan et al., 2017).

Tea Tree essential oil as alternative treatment Antimicrobial compounds and preservatives have delayed food spoilage (Perricone et al., 2015). EOs have been an alter- native since ancient times, gaining relevance in recent years for their antimicrobial properties. In addition, studies have confirmed their different antioxidant and anti-inflammatory properties (Dagli et al., 2015). EOs are volatile secondary metabolites produced by plants and are responsible for their aromatic properties (Bakkali et al., 2008). In general, EOs are liquid, volatile, and soluble compounds in lipids and organic solvents, which are obtained from different parts of plants (e.g., flowers, seeds, leaves, grouts, and bark) through different extraction techniques (Aziz et al., 2018). Among their components stand out the terpenoid and non-terpenic compounds raised by the phenylpropanoid pathway of eugenol, cinnamaldehyde, and safrole (Dhifi et al., 2016). The concentration of these components depends on different factors, such as the source of extraction, geographical loca- tion, season, and maturity of the plant from which they are extracted (Dagli et al., 2015).

TTEO, obtained from Melaleuca alternifolia, has been used for almost 100 years in countries such as Australia, due to its properties as a complementary and alternative medi- cine (Carson et al., 2006). This EO contains oxygenated cyclic monoterpenes and hydrocarbons, such as terpinen-4-ol, ƴ-terpinene, α-terpinene, and 1,8-cineol (Dagli et al., 2015). Among them, the predominant is terpinen-4-ol (30-40%), to which most of its bioactivities are attributed (Groot and Schmidt, 2006).

TTEO has shown some in vitro antimicrobial properties, for example, as antifungal, especially against Candida albi- cans the leading cause of vaginal infections, with minimum inhibitory concentrations (MIC) ranging from 0.06 to 8.0 %. TTEO seems to alter the properties of the membrane and inhibit the respiration of the fungus at MICs from 0.25 % to

1.0 % (v/v) and reaching minimal bactericidal concentration (MBC) (Carson et al., 2006). Also, TTEO has shown some effect against viruses that cause herpes, which is better when com- bined with eucalyptus EO (Gavanji et al., 2016). On the other

hand, in vitro studies show that TTEO reduce by 50 % of the growth of protozoa such as Leishmania major and Trypano- soma brucei, and it has inhibited all Trichomonas vaginalis at 300 mg/mL (Carson et al., 2006).

In summary, TTEO exhibits various antimicrobial proper- ties in vitro, supporting its potential application in treating infections caused by various microorganisms. However, it is essential to consider that results from in vitro studies may not always translate directly to clinical efficacy, highlighting the need to explore these effects in animal models.

Tea tree oil against bacteria: in vitro and in vivo assays Regarding antibacterial properties, concentrations from 0.78 to 50 mg/mL have been tested against bacteria that cause urinary tract infections. This demonstrates the TTEO in vitro and in vivo ability to reduce bacterial load (Loose et al., 2020). Table 1 summarizes some studies reporting their MBC and MIC in vitro against E. coli, Shigella spp., Campylobacter spp., and Salmonella spp. On the other hand, in vivo assays with TTEO (1000 mg/kg), resulted in an increased count of Lacto- bacillus colonies within the cecal contents in Partridge Shank chickens (n = 144) at 50 days of supplementation. Terpinen- 4-ol, the primary constituent of TTEO, has been identified to possess selective antimicrobial properties against intestinal pathogens in vitro, which results in the modulation of the cecal microbiota composition through the enhanced Lacto- bacillus population because of TTEO supplementation (Qu et al., 2019).

Additionally, TTEO as a dietary supplement, at concen-

trations ranging from 50 to 150 mg/kg in broiler chicken diets, exhibited notable effects. A significant enhancement in daily weight gain (7% approximately) was observed as a reduction in both morbidity and mortality rates (Bakkali et al., 2008). Also, the same concentrations described above (50 mg/kg to 150 mg/kg) of TTEO supplementation resulted in an increa- sed average daily feed intake and a tendency of daily gain in weanling piglets (n = 120) after 21 days (Aziz et al., 2018).

Table 1. In vitro effect of Tea Tree essential oil on different bacteria associa- ted with diarrhea.

Tabla 1. Efecto in vitro del aceite esencial de Árbol de Té sobre diferentes bacterias asociadas a la diarrea.


Bacteria

Inhibition zone (mm)

MBC

(μL/ mL)


MIC (%)


Reference


Shigella spp


-


-


0.25

Harkenthal et al.,





1999

Campylobacter spp

26.7-30

-

0.001

Kureckci et al.,





2013

Salmonella spp

37.4

4

-

Swamy et al.,





2013




0.03-

Bučková et al.,

E. coli

17.0-35.03

4

0.5

2018; Kureckci et





al., 2019

MBC: Minimum Bactericidal Concentration; MIC: Minimum Inhibitory Concentration.

This study suggested that the passage of these compounds through the gastrointestinal tract might not affect the native microbiota of an organism; however, information is limited, and additional studies exploring its effects in different living organisms may provide more precise information.

On the other hand, the consumption of TTEO encapsu- lated with n-hexane lactulose, gum Arabic, and maltodextrin for 28 days seems to modulate the microbiota in weanling pigs (n = 144) and reduced the E. coli in the intestine and diarrhea episodes (Wang et al., 2021). However, despite the evidence about its effective activity against Shigella, Salmo- nella, Campylobacter, and E. coli, additional in vivo studies are still required to test the efficacy of a TTEO oral administration against these bacteria in infected animal models.

Some studies administer TTEO orally for other purposes in different animal models. It was observed that the con- sumption of 0.2 mg/kg of TTEO (60 days) added to the diet of goats (n = 24) improved intestinal immune function (Lv et al., 2022). Likewise, using TTEO integrated into the chickens’ diet also improved immune function (Abo Ghanima et al., 2021). Therefore, these findings indicate that the treatment’s potential effects go beyond its antimicrobial activity.

While existing studies have investigated the antibac- terial properties of TTEO, a critical research gap exists in understanding its efficacy through oral administration against specific gastrointestinal pathogens in infected animal models. In vitro studies have demonstrated TTEO’s ability to reduce bacterial load. However, a comprehensive exploration of its effectiveness in vivo against critical bacteria such as Shigella, Salmonella, Campylobacter, and E. coli must be explored. Current research indicates positive outcomes, such as increased Lactobacillus colonies in Partridge Shank chickens and notable effects on growth rates and morbidity in broiler chickens and weanling piglets when supplemented with TTEO in their diets. Nevertheless, the passage of TTEO through the gastrointestinal tract and its impact on native microbiota remains misunderstood. Additionally, despite encapsulation studies showing promise in modulating mi- crobiota and reducing E. coli in weanling pigs, more research needs to be done on the oral administration of TTEO, spe- cifically targeting other bacteria in infected animal models. Bridging this gap will provide valuable insights into the practical efficacy and safety of TTEO as an oral therapeutic intervention against gastrointestinal pathogens, informing potential applications and advancing our understanding of microbial communities’ impact on different organisms.


Mechanisms to support the TTE biological activities

The mechanisms proposed to explain the TTEO effects are diverse and will depend on the doses used and the analyzed pathogen characteristics. It is suggested that modifying the cell membrane structure is most important, reducing the membrane potential (Swamy et al., 2016). In addition, it has been reported that TTEO can affect potassium ion channel homeostasis and interfere with glucose-dependent respiration, affecting bacterial membrane integrity, e.g., E.

coli (Yadav et al., 2016). Also, TTEO can improve intestinal development, cytokine secretion, gene expression of tight junction proteins, and Notch2 signaling to some extent, surpassing the effect of some antibiotics. The integrity of tight junctions is a crucial indicator of intestinal well-being, and any disturbance in their structure and function is often linked to intestinal stress injury. Perturbations in critical tight junction proteins, including occlusions, claudins, ZOs, and MUC2, can result in augmented intestinal permeability and compromised nutrient transport. In addition, the disrup- tion of tight junctions has been implicated in developing inflammatory bowel disease, irritable bowel syndrome, and infectious diarrhea (Dong et al., 2019).

On the other hand, previous works have report the toxicity of TTEO in epithelial-like cells (Hela), at IC50 of 2.7 ±

0.07 g/L (Carson and Riley, 1995). Likewise, toxicity tests were also reported in ICR male mice using nano TTEO emulsions in agreement with the guidelines drawn up by the Organi- zation for Economic Co-operation and Development. In this context, it was shown that TTEO nanoemulsions showed lower toxicity (oral LD50 1656 mg/kg) compared to TTEO alo- ne (oral LD50 854 mg/kg). Similarly, the antibacterial activity against Salmonella typhimurium and E. coli has been tested in vitro, concluding that TTEO can be used as a potential oral antimicrobial agent (Wei et al., 2021). Emulsions have been proposed as a strategy to make efficient use of TTEO. This sys- tem allows for the encapsulation and protection of bioactive compounds in droplets, with sizes ranging from micrometers to nanometers (Singh and Pulikkal, 2022).

The mechanisms underlying the effects of TTEO are multifaceted and contingent, on both the administered do- ses and the specific characteristics of the studied pathogen. These findings endorse TTEO’s suitability as a potential oral antimicrobial agent. Moreover, the adoption of emulsions as a delivery strategy for TTEO is proposed, presenting an efficient means to encapsulate and safeguard bioactive compounds, offering potential applications in antimicrobial interventions.


Challenges in the development of treatments based on essential oils

Effective implementation of essential oil-based antibacterial treatments faces multifaceted challenges that require careful attention in development and clinical application. The varia- bility in the composition of these oils raises questions about the identification of essential compounds, and determining optimal concentrations for consistent antibacterial activity. Furthermore, addressing technical challenges in formulation and administration, along with the need to achieve clinical acceptance, completes the picture of challenges that must be overcome to fully exploit EO’s therapeutic potential in treating bacterial infections.

On the other hand, regulation of the consumption of essential oil-based products varies significantly depending on the jurisdiction and the specific purpose of the product. In general, EOs are commonly used in dietary supplements and

cosmetic products, and regulations cover aspects such as labeling, allowable concentrations, and safety requirements. When used for medicinal purposes, some countries may sub- ject these products to more rigorous regulations. Regulation on alternative therapies can be diverse, from limited to more detailed, depending on local laws. Safety and toxicity are key considerations, with regulatory agencies evaluating the safe- ty of EO and setting limits to protect consumers. Additionally, in the marketing of dietary supplements, specific regulations regarding content and health claims may apply. Given the constant evolution of the EO industry, consumers and ma- nufacturers should stay informed about local regulations and consult with relevant authorities to ensure regulatory compliance, empowering them with the knowledge to make informed decisions.

The acceptance of products based on EO is diverse and depends on several factors. In recent years, there has been a rise in the popularity of these products, driven by increasing attention towards natural and alternative approaches to wellness. Acceptance may be influenced by factors such as knowledge and education about the associated benefits and risks, personal experience with positive results, cultural and traditional attitudes towards natural medicine, and the ge- neral perception of effectiveness. However, acceptance may also vary depending on individual perceptions of alternative medicine and preference for more conventional approaches. Considering EO as an oral treatment for gastrointestinal bacterial infections involves several key factors. Although in vitro studies suggest antimicrobial properties, the transition to clinical efficacy requires further investigation. Local regu- lations and safety should be considered, as some EOs can be toxic in large quantities in clinical practice. The diversity of gastrointestinal infections and the variability in efficacy aga- inst different pathogens are important to evaluate. Finally, using EOs in this context requires not just consideration but also careful evaluation and medical supervision to ensure safety, efficacy, effectiveness, and consideration of individual circumstances, thereby reassuring patients and healthcare professionals about the thoroughness of the treatment pro-

cess.


CONCLUSIONS

As described, public health concerns about antibiotic- resistant bacteria are growing worldwide. So, essential oils may offer an alternative or adjuvant solution to establish new treatment strategies. Essential oils such as TTEO can po- tentially be used to treat gastrointestinal diseases associated with bacteria, because of their excellent antimicrobiological properties. However, the effects reported in the literature are primarily in vitro studies. So, further studies in animal models are required to investigate the TTEO properties.


ACKNOWLEDGMENTS

The author thanks CIAD and CONAHCYT for supporting this review.

CONFLICTS OF INTEREST

The authors declare no conflict of interest.


REFERENCES

Abo Ghanima, M.M., Swelum, A.A., Shukry, M., Ibrahim, S.A., Abd El-Hack, M.E., Khafaga, A.F., Alhimaidi, A.R., Ammari, A.A., El-Tarabily, K.A. and Younis, M.E.M. 2021. Impacts of tea tree or lemongrass essential oils supplementation on growth, immunity, carcass traits, and blood biochemical parameters of broilers reared under different stocking densities. Poultry Science, 100(11):101443.

Aghraz, A., Benameur, Q., Gervasi, T., Ait Dra, L., Ben-Mahdi, M., Larhsini, M., Markouk, M. and Cicero, N. 2018. Antibacterial activity of Cladanthus arabicus and Bubonium imbricatum essential oils alone and in combination with conventional antibiotics against Enterobacteriaceae isolates. Letters in Applied Microbiology. 67: 175-182.

Aziz, Z.A.A., Ahmad, A., Setapar, S.H.M., Karakucuk, A., Azim,

M.M., Lokhat, D., Rafatullah, M., Ganash, M., Kamal, M.A. and Ashraf, G. 2018. Essential oils: Extraction techniques, pharmaceutical and therapeutic potential. Current Drug Metabolism. 19: 1100-1110.

Baker, S. and Chung-The, H. 2018. Recent insights into Shigella.

Current Opinion in Infectious Diseases, 31, 449-454.

Bakkali, F., Averbeck, S., Averbeck, D. and Idaomar, M. 2008. Biological effects of essential oils: A review. Food and Chemical Toxicology. 46, 446-475.

Barrantes-Jiménez, K. and Achí-Araya, R. 2009. Interacciones celulares en el proceso de invasión de Shigella sp. Revista Panamericana de Infectología. 11: 56-61.

Bouarab-Chibane, L., Degraeve, P., Ferhout, H., Bouajila, J. and Oulahal, N. 2019. Plant antimicrobial polyphenols as potential natural food preservatives. Journal of the Science of Food and Agriculture. 99: 1457-1474.

Bowen, A. 2018. Shigellosis: En: In Yellow Book 2018: Health Information for International Travel. G. Brunette (Ed.), pp. 319-320. Oxford University Press, U.K.

Bučková, M., Puškárová, A., Kalászová, V., Kisová, Z. and Pangallo,

D. 2018. Essential oils against multidrug resistant gram- negative bacteria. Biologia. 73: 803-808.

Calo, J.R., Crandall, P.G., O’Bryan, C.A. and Ricke, S.C. 2015. Essential oils as antimicrobials in food systems—A review. Food Control. 54: 111-119.

Campoli-Richards, D.M., Monk, J.P., Price, A., Benfield, P., Todd,

P.A. and Ward, A. 1988. Ciprofloxacin. Drugs. 35: 373-447. Carson, C.F. and Riley, T.V. 1995. Toxicity of the essential oil of

Melaleuca alternifolia or tea tree oil. Journal of Toxicology and Clinical Toxicology. 33(2): 193-194.

Carson, C.F., Hammer, K.A. and Riley, T.V. 2006. Melaleuca alternifolia (tea tree) oil: a Review of antimicrobial and other medicinal properties. Clinical Microbiology Reviews. 19: 50- 62.

CDC. Campylobacter (Campylobacteriosis). [Consultado 13 mayo 2023] 2023a. Disponible en: https://www.cdc.gov/ campylobacter/faq.html

CDC. E. coli (Escherichia coli). [Consultado 13 mayo 2023] 2023b.

Disponible en: https://www.cdc.gov/ecoli/index.html

CDC. La resistencia a los antibióticos, los alimentos y los animales de producción. [Consultado 13 de mayo 2023] 2023c. Disponible en: https://www.cdc.gov/foodsafety/es/ challenges/antibiotic-resistance.html

CDC. Salmonella: Diagnostic and Public Health Testing. [Consultado 13 de mayo de 2023] 2019. Disponible en: https://www.cdc.gov/salmonella/general/diagnosis- treatment.html?CDC_AA_refVal=https%3A%2F%2Fwww. cdc.gov%2Fsalmonella%2Fgeneral%2Fdiagnosis.html

CDC. Shigella-Shigellosis. [Consultado 13 de mayo de 2023] 2023c. Disponible en: https://www.cdc.gov/shigella/ prevention-control.html.

Chouhan, S., Sharma, K. and Guleria, S. 2017. Antimicrobial activity of some essential oils—Present status and future perspectives. Medicines. 4(3): 58.

Cohen, R., Raymond, J. and Gendrel, D. 2017. Antimicrobial treatment of diarrhea/acute gastroenteritis in children. Archives de Pédiatrie. 24: 26-29.

Dagli, N., Dagli, R., Mahmoud, R.S. and Baroudi, K. 2015. Essential oils, their therapeutic properties, and implication in dentistry: A review. Journal of International Society of Preventive & Community Dentistry. 5: 335-340.

Dasti, J.I., Tareen, A.M., Lugert, R., Zautner, A.E. and Groß, U. 2010. Campylobacter jejuni: A brief overview on pathogenicity- associated factors and disease-mediating mechanisms. International Journal of Medical Microbiology. 300: 205-211. Denamur, E., Clermont, O., Bonacorsi, S. and Gordon, D. 2021. The population genetics of pathogenic Escherichia coli.

Nature Reviews Microbiology. 19: 37-54.

Dhifi, W., Bellili, S., Jazi, S., Bahloul, N. and Mnif, W. 2016. Essential oils’ chemical characterization and investigation of some biological activities: A critical review. Medicines. 3: 1-16.

Eltai, N.O., Yassune, H.M., Al-Thani, M.A., Ismail, A., Ibrahim, E. and Alali, W.Q. 2018. Prevalence of antibiotic resistant Escherichia coli isolates from fecal samples of food handlers in Qatar. Antimicrobial Resistance and Infection Control. 7: 1-7.

Fagundes-Neto, U. and Affonso-Scaletsky, I. 2000. Escherichia coli infections and malnutrition. The Lancet. 356(27).

FDA. Nutrición e infección, salud y enfermedad. [Consultado 13 de mayo de 2023] 2023. Disponible en: http://www.fao. org/3/w0073s/w0073s07.htm

Fernández, H. 2011. Campylobacter y Campylobacteriosis: Una mirada desde América del Sur. Revista Peruana de Medicina Experimental y Salud Pública. 28: 121-127.

Firmino, J.P., Vallejos-Vidal, E., Sarasquete, Ortiz-Delgado, J.B., Balasch, J.C., Tort, L., Estevez, A., Reyez-López, F.E. and Gisbert, E. 2020. Unveiling the effect of dietary essential oils supplementation in Sparus aurata gills and its efficiency against the infestation by Sparicotyle chrysophrii. Scientific reports. 10(1):17764.

Foley, S. 2012. Campylobacter jejuni. En: Bad Bug Book: Foodborne Pathogenic Microorganisms and Natural Toxins Handbook. K.A. Lampel (Ed.), pp. 14-17. FDA, Atlanta.

Gavanji, S., Sayedipour, S.S., Larki, B. and Bakhtari, A. 2015. Antiviral activity of some plant oils against herpes simplex virus type 1 in Vero cell culture. Journal of Acute Medicine. 5: 62-68.

Geissler, A.L., Mahon, B.E. and Fitzgerald, C. 2018. Campylobacteriosis. En: Yellow Book 2018: Health Information for International Travel. G. Brunette (Ed.), pp. 149-150. Oxford University, New York.

Gomes, T.A., Elias, W.P., Scaletsky, I.C., Guth, B.E., Rodrigues, J.F., Piazza, R.M., Ferreira, L.C. and Martinez, M.B. 2016. Diarrheagenic Escherichia coli. Brazilian Journal of Microbiology. 47: 3-30.


Groot, A.C. and Schmidt, E. 2006. Tea tree oil: Contact allergy and chemical composition. Contact Dermatitis. 75: 128-143.

Hammack, T. 2012. Salmonella species. En: Bad Bug Book: Foodborne Pathogenic Microorganisms and Natural Toxins Handbook. K.A. Lampel (Ed.) pp. 9-13. FDA, Atlanta.

Harkenthal, M., Reichling, J., Geiss, H.K. and Saller, R. 1999. Comparative study on the in vitro antibacterial activity of Australian tea tree oil, cajuput oil, niaouli oil, manuka oil, kanuka oil, and eucalyptus oil. Pharmazie. 54: 462-463.

Hebblestrup-Jensen, B., Olsen, K.E., Struve, C. and Krogfelt,

K.A. 2014. Epidemiology and clinical manifestations of enteroaggregative Escherichia coli. Clinical Microbiology Reviews. 27: 614-630.

Hirose, K. and Sato, E. 2011. Comparative analysis of combination kanamycin-furosemide versus kanamycin alone in the mouse cochlea. Hearing Research. 272: 108-116.

Hunter, J.C. and Francois-Watkins, L.K. 2018. Salmonella (Nontyphoidal). En: Yellow Book 2018: Health Information for International Travel. G. Brunette (Ed.), pp. 304-306. Oxford University Press, New York.

Hussen, S., Mulatu, G. and Kassa, Z.Y. 2019. Prevalence of Shigella species and its drug resistance pattern in Ethiopia: a systematic review and meta-analysis. Annals of Clinical Microbiology and Antimicrobials. 18: 1-11.

Jajere, S.M. 2019. A review of Salmonella enterica with particular focus on the pathogenicity and virulence factors, host specificity and antimicrobial resistance including multidrug resistance. Veterinary World. 12: 504-521.

Kabir-Mumu, S. and Mahboob-Hossain, M. 2018. Antimicrobial activity of tea tree oil against pathogenic bacteria and comparison of its effectiveness with eucalyptus oil, lemongrass oil and conventional antibiotics. American Journal of Microbiology Research. 6: 73-78.

Kon, K.V. and Rai, M.K. 2012. Plant essential oils and their constituents in coping with multi-drug-resistant bacteria. Expert Review of Anti-infective Therapy. 10: 775-790.

Kurekci, C., Padmanabha, J., Bishop-Hurley, S.L., Hassan, E., Al-Jassim, R.A. and McSweeney, C.S. 2013. Antimicrobial activity of essential oils and five terpenoid compounds against Campylobacter jejuni in pure and mixed culture experiments. International Journal of Food Microbiology. 166: 450-457.

Lamb, H.M., Ormrod, D., Scott, L.J. and Figgitt, D.P. 2002. Ceftriaxone: An update of its use in the management of community-acquired and nosocomial infections. Drugs. 62: 1041-1089.

Lampel, K.A. 2012. Shigella Species. En: Bad Bug Book: Foodborne Pathogenic Microorganisms and Natural Toxins Handbook.

K.A. Lampel (Ed.), pp. 22-25. FDA, Atlanta.

Dong, L., Liu, J., Zhong, Z., Wang, S., Wang, H., Huo, Y., Wei, Z. and Yu L. 2019. Dietary tea tree oil supplementation improves the intestinal mucosal immunity of weanling piglets. Animal Feed Science and Technology. 255: 114209.

Loose, M., Pilger, E. and Wagenlehner, F. 2020. Anti-bacterial effects of essential oils against uropathogenic bacteria. Antibiotics. 9: 1-8.

Lv, X., Chen, L., Zhou, C., Guo, Y., Zhang, G., Kang, J. and Liu, Z. 2022. Dietary tea tree (Melaleuca alternifolia) oil supplementation enhances the expressions of amino acid transporters in goat ileal mucosa and improves intestinal immunity. Food Science & Nutrition. 10(11): 3749-3758.

Miguel, M.G. 2010. Antioxidant and anti-inflammatory activities of essential oils: A short review. Molecules. 15: 9252-9287.

Morales-Cruz, V.G. and Huerta-Romano, J.F. 2010. Escherichia coli diarreogénica. Conocimientos vigentes. Revista Mexicana de Pediatría. 77: 271-276.

Newell, D.G. and La Ragione, R.M. 2017. Enterohaemorrhagic and other Shiga toxin-producing Escherichia coli (STEC): Where are we now regarding diagnostics and control strategies? Transboundary and Emerging Diseases. 65: 49-71.

O´Reilly, C.E., Iwamoto, M. and Griffin, P.M. 2018. Escherichia coli, Diarrheagenic. En: Yellow Book 2018: Health Information for International Travel. G. Brunette (Ed.), pp. 174-177. Oxford University Press, New York.

Ojkic, N., Lilja, E., Direito, S., Dawson, A., Allen, R.J. and Waclaw, B. 2020. A roadblock-and-kill mechanism of action model for the DNA-targeting antibiotic ciprofloxacin. Antimicrobial Agents and Chemotherapy. 64: 1-17.

Parnham, M.J., Haber, V.E., Giamerellos-Bourboulis, E.J., Perletti, G., Verleden, G.M. and Vos, R. 2014. Azithromycin: Mechanisms of action and their relevance for clinical applications. Pharmacology Therapeutics. 143: 225-245.

Perricone, M., Arace, E., Corbo, M.R., Sinigaglia, M. and Bevilacqua, A. 2015. Bioactivity of essential oils: a review on their interaction with food components. Frontiers in Microbiology. 6: 1-7.

Puzari, M., Sharma, M. and Chetia, P. 2018. Emergence of antibiotic resistant Shigella species: A matter of concern. Journal of Infection and Public Health. 11: 451-454.

Qu, H., Cheng, Y., Chen, Y., Zhao, Y., Li, J., Wen, C. and Zhou, Y. 2019. Dietary tea tree (Melaleuca alternifolia) oil supplementation improves growth performance, cecal microflora, immunity, and antioxidant capacity of partridge shank chickens. Journal of Poultry Science. 56(3): 212-219.

Rios-Muñiz, D., Cerna-Cortés, J.F., Morán-García, N., Meza- Segura, M. and Estrada-García, T. 2019. Escherichia coli enterotoxigénica y enteroagregativa: prevalencia, patogénesis y modelos muridos. Gaceta Médica de México. 155: 410-416.

Schleibinger, M., Steinbach, C.L., Topper, C., Kratzer, A., Liebchen,

U. and Kees, F. 2015. Protein binding characteristics and pharmacokinetics of ceftriaxone in intensive care unit patients. British Journal of Clinical Pharmacology. 80: 525- 533.

Shane, A.L., Mody, R.K., Crump, J.A., Tarr, P.I., Steiner, T.S., Kotloff,

K. and Pickering, L.K. 2017. 2017 Infectious Diseases Society of America Clinical Practice Guidelines for the Diagnosis and Management of Infectious Diarrhea. Clinical Infectious Diseases. 65: 45-80.

Shu-Kee, E., Pusparajah, P., Nurul-Syakima, A.M., Hooi-Leng, S., Kok-Gan, C. and Learn-Han, L. 2015. Salmonella: A review on pathogenesis, epidemiology and antibiotic resistance. Frontiers in Life Science. 8: 284-293.

Singh, I.R. and Pulikkal, A.K. 2022. Preparation, stability and biological activity of essential oil-based nanoemulsions: A comprehensive review. OpenNano. 8: 100066.

Swamy, M.K., Akhtar, M.S. and Sinniah, U.R. 2016. Antimicrobial properties of plant essential oils against human pathogens and their mode of action: An updated review. Evidence- Based Complementary and Alternative Medicine. 2016: 1-21.

Tarr, G.A., Oltean, H.N., Phipps, A.I., Rabinowitz, P. and Tarr, P.I. 2018. Strength of the association between antibiotic use and hemolytic uremic syndrome following Escherichia coli O157:H7 infection varies with case definition. International Journal of Medical Biology. 308: 921-926.

van den Bogaard, A.E. and Stobberingh, E.E. 2000. Epidemiology of resistance to antibiotics: Links between animals and humans. International Journal of Antimicrobial Agents. 14: 327-335.

Vidal, J., Canizález-Román, A., Futiérrez-Jiménez, J. and Navarro- García, F. 2007. Patogénesis molecular, epidemiología y diagnóstico de Escherichia coli enteropatógena. Salud Pública de México. 49: 376-386.

Wang, L., Zhang, Y., Liu, L., Huang, F. and Dong, B. 2021. Effects of three-layer encapsulated tea tree oil on growth performance, antioxidant capacity, and intestinal microbiota of weaned pigs. Frontiers in Veterinary Science. 8: 789225.

Wei, S., Zhao, X., Yu, J., Yin, S., Liu, M., Bo, R. and Li, J. 2021.

Characterization of tea tree oil nanoemulsion and its acute and subchronic toxicity. Regulatory Toxicology and Pharmacology, 124: 104999.

WHO. Enfermedades diarreicas. [Consultado 14 de mayo de 2023] 2017. Disponible en: https://www.who.int/es/news- room/fact-sheets/detail/diarrhoeal-disease.

WHO. Salmonella (no tifoidea). [Consultado 14 de mayo de 2023] 2018. Disponible en: https://www.who.int/es/news-room/ fact-sheets/detail/salmonella-(non-typhoidal).

WHO. Campylobacter. [Consultado 14 de mayo de 2023] 2020. Disponible en: https://www.who.int/es/news-room/fact- sheets/detail/campylobacter.

WHO. Resistencia a los antibióticos. [Consultado 14 de mayo de 2023]2020b. Disponible en: https://www.who. int/es/news-room/fact-sheets/detail/resistencia-a-los- antibi%C3%B3ticos.

Wu, L.J., Luo, Y., Shi, G.I. and Li, Z.Y. 2021. Prevalence, clinical characteristics and changes of antibiotic resistance in children with nontyphoidal Salmonella infections from 2009–2018 in Chongqing, China. Infectious Drug Resistance. 14: 1403-1413.

Xiang, Y., Wu, F., Chai, Y., Xu, X., Yang, L., Tian, S., Zhang, H., Li,

Y., Yang, C., Liu, H., Qiu, S., Song, H. and Sun, Y. 2020. A new plasmid carrying mphA causes prevalence of azithromycin resistance in enterotoxigenic Escherichia coli serogroup O6. BMC Microbiology. 20: 1-9.

Yadav, E., Kumar, S., Mahant, S., Khatkar, S. and Rao, R. 2016. Tea tree oil: a promising essential oil. Journal of Essential Oil Research. 29: 1-13.

Yang, C., Zhang, L., Cao, G., Feng, J., Yue, M., Xu, Y., Dai, B., Han, Q. and Guo, X. 2019. Effects of dietary supplementation with essential oils and organic acids on the growth performance, immune system, fecal volatile fatty acids, and microflora community in weaned piglets. Journal of Animal Science. 97: 133-143.

Young, K.T., Davis, L.M., and DiRita, V.J. 2007. Campylobacter jejuni: Molecular biology and pathogenesis. Nature Reviews Microbiology. 5: 665-679.