**5. Use of essential oils against multidrug-resistant bacteria**

Antibiotics are effective drugs that play an important role in treating infections and decreasing morbidity and mortality rates [59, 60]. In general, antibiotics kill multidrug-resistant (MDR) bacteria through various mechanisms. Examples include the β-lactam antibiotics that inhibit the bacterial cell wall synthesis, fluoroquinolones that inhibit DNA synthesis, tetracycline which is an inhibitor of protein synthesis, sulfonamides as a metabolic pathway or folic acid synthesis inhibitor, and polymyxin B which interferes with cell membrane integrity [60–63]. Antibiotic resistance develops naturally but is accelerated when the antibiotics are misused in both human and animals; the bacteria will evolve and develop resistance toward antibiotics, preventing the antibiotic from killing the bacteria [59, 64]. The bacteria subsequently become resistant by many mechanisms depending on the selective pressure incurred by the antibiotic used; for example, if the penicillin is used, the bacteria will become resistant to it by producing enzymes that will act against the antibiotic which is in this situation penicillinase enzyme [39]. For instance, a study conducted in 173 hospitals in Europe showed that high antibiotic consumption hospitals have a higher number of methicillin-resistant *Staphylococcus aureus* (MRSA) [65].

Antibiotic resistance in microorganisms is increasing at a worrisome rate [66]. Hence, over the years, researchers are exploring possible alternative sources that will be helpful to mitigate MDR bacteria. Of all the potential sources, EO was identified as one of the good alternative sources, because of their effectiveness in folk medicine [67]. Bacteria can be divided into two main types: the gram-positive and the gram-negative. The gram-positive have a thicker peptidoglycan layer than the gram-negative bacteria [68]. Besides that, the gram-negative bacteria also have an outer membrane that is absent in the gram-positive bacteria (**Figure 1**).

Generally, the gram-positive bacteria are less resistant to EOs than gram-negative bacteria [69, 70]. In gram-positive bacteria, hydrophobic molecules are able to penetrate the cell and act on the cell wall and cytoplasm. This is exemplified by the phenolic compounds in EOs against gram-positive bacteria [66]. In the gram-negative bacteria, a thin layer of peptidoglycans is present with an outer membrane that contains LPS. LPS consists of lipid A, core LPS, and O-side chain, which makes the gramnegative bacteria more resistant to EOs than gram-positive bacteria [66, 71]. Small hydrophilic solutes will make use of the porin proteins in the gram-negative bacteria to pass through the outer membrane; it is this porin selectivity that also makes the gram-negative bacteria less susceptible to hydrophobic antibiotics [66, 72, 73].

EOs via their different components have different targets against microorganisms such as the membrane and the cytoplasm [8]. Scientists have also found that the solubility of EO in water allowed them to decipher how EOs penetrated the cell wall of microbes; in other words EOs, being soluble in the cell membrane phospholipid bilayer, diffuse through the membrane [74]. A study done using the EO of *Melaleuca alternifolia* (tea tree) against MDR gram-negative bacteria (e.g., *Escherichia coli* and carbapenem-resistant *K. pneumoniae*) and methicillinresistant *S. aureus* (MRSA) showed that there is a bactericidal effect of tea tree EO on these microorganisms [75]. This indicated that the EO can be used to kill

#### **Figure 1.**

*Schematic of different gram-positive (at the top) and gram-negative (at the bottom) cell walls.*

resistant bacteria [74]. Moreover, EO phenolic compounds' effect is concentrationdependent, whereby at low concentrations the phenolic compound will work with enzymes to produce energy, while at high concentration it will denature proteins [66, 76].
