**5.1 Determination of MIC of EOs for bacteria**

Minimal inhibitory concentration (MIC) is the lowest concentration of a specific drug to inhibit the growth of microorganisms such as bacteria [1, 77]. After knowing that a particular EO has bactericidal, viricidal, and antiparasitic effects, the lowest concentration of EO to inhibit microbial growth should be measured [57, 78]. There are many assays to evaluate and screen for antimicrobial activity such as the disk diffusion test, microdilution (resazurin) or broth method, and agar dilution method [79, 80]. The agar disk diffusion test is commonly used to determine the antibacterial activity of the EO, but this method works only for EO with known components. This is because, for the EOs with unknown components, the antimicrobial effect may give rise to false or negative result caused by the unknown components [81]. Previously, in a study performed using the disk diffusion test to examine the antimicrobial activity of *Eucalyptus globulus* leaves, EO showed that there was a bacterial inhibitory effect on *E. coli* and *S. aureus* [82].

The commonly used alternative method to determine antimicrobial activity is the dilution method through a serial dilution of the EO in several tubes, and then determining the MIC after adding the test microorganism, turbidity is measured as a signal for growth [81]. In this method, the EO is first diluted; then it will be added to the medium that contains the broth culture, followed by incubation for 18 h in 37°C [69]. After the incubation period, the tube with the lowest concentration that showed no sign of growth is the MIC of the EO [69, 83]. However, this method

**57**

ability properties [39, 96].

*Essential Oils: Partnering with Antibiotics DOI: http://dx.doi.org/10.5772/intechopen.86575*

**6. Mechanisms of actions with antibiotic**

**7. Application of essential oils in therapy**

agar dilution assay [80].

therapy [39].

requires a large quantity of the plant extract [81]. A study using the redox dye resazurin for the new modified microdilution method has been carried out to determine the MIC for tea tree EO (*Melaleuca alternifolia*) against the gram-positive and gram-negative bacteria. The results showed that the resazurin method is accurate to determine the MIC and is higher in sensitivity than the results obtained from the

EOs' mechanism of action is poorly understood, but in general it depends on their chemical composition [8, 66, 84]. As antimicrobial resistance to antibiotics is increasing, scientists are currently exploring the ability of the plant extract to modify bacterial resistance against drugs [39]. The three main types of interactions that occur between the combination of antibiotic and EO are synergism, additivity, and antagonism [85]. Synergistic interaction is when the effect of the combined chemicals is greater than the effect of a chemical alone; additive interaction is when the sum of two chemicals is equal to the sum of chemical effect alone, while antagonism is when the whole effect of the two chemicals is less than the sum of effect of a single chemical alone [86]. In a study performed using the tea tree EO against the MDR bacteria, when a combination of tea tree EO with antibiotic (e.g., oxacillin) was tested on the bacteria, in particular the MRSA, a high synergistic index in the sub-inhibitory concentration was recorded [75]. This indicates that the EO can be used to overcome bacterial resistance to antibiotic. The synergism level increases when the combined effect is higher than the individual effect in the combination

Combination therapy is a new method that combines antibiotics and EO to kill resistant bacteria, via enhancement of the antimicrobial activity [39, 87]. Moreover, EOs have more components possessing different mechanisms of actions for many targets than antibiotics that have only one target. Combination therapy would be useful and able to provide a new treatment option for resistance bacteria [39].

Daily, the human body comes into contact with EOs through various sources such as herbs, spices, orange, spearmint, lemongrass, etc., but only limited information about the amount of EO uptake is known [4, 88]. Effects of EO begin to appear after it penetrates the human body in several ways such as by ingestion, by absorbing the EO or diffusion, and by inhalation [4, 89]. EOs can be taken by inhalation through the lungs and distributed into the blood because of their volatility [90–92]. Moreover, consumption of EO by ingestion should be taken with care because EOs may cause probable toxicity [4]. EOs are used in folk medicine to treat many health problems and can also be used as food preservatives by giving antimi-

Many studies investigated the efficiency of EOs in combination with antibiotics to combat bacterial resistance; EOs with its compounds and secondary metabolites have shown promising synergistic interaction as an indication that they would be helpful to treat and decrease bacterial resistance to antibiotics [39, 95]. The advantages that make the EOs preferable are that they will decrease adverse reactions, besides being comparatively more cost-effective, with more public acceptance due to traditional usage, and being renewable with better biodegrad-

crobial, antioxidant, and anti-inflammation properties [93, 94].

*Essential Oils - Oils of Nature*

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

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

Minimal inhibitory concentration (MIC) is the lowest concentration of a specific drug to inhibit the growth of microorganisms such as bacteria [1, 77]. After knowing that a particular EO has bactericidal, viricidal, and antiparasitic effects, the lowest concentration of EO to inhibit microbial growth should be measured [57, 78]. There are many assays to evaluate and screen for antimicrobial activity such as the disk diffusion test, microdilution (resazurin) or broth method, and agar dilution method [79, 80]. The agar disk diffusion test is commonly used to determine the antibacterial activity of the EO, but this method works only for EO with known components. This is because, for the EOs with unknown components, the antimicrobial effect may give rise to false or negative result caused by the unknown components [81]. Previously, in a study performed using the disk diffusion test to examine the antimicrobial activity of *Eucalyptus globulus* leaves, EO showed that

The commonly used alternative method to determine antimicrobial activity is the dilution method through a serial dilution of the EO in several tubes, and then determining the MIC after adding the test microorganism, turbidity is measured as a signal for growth [81]. In this method, the EO is first diluted; then it will be added to the medium that contains the broth culture, followed by incubation for 18 h in 37°C [69]. After the incubation period, the tube with the lowest concentration that showed no sign of growth is the MIC of the EO [69, 83]. However, this method

there was a bacterial inhibitory effect on *E. coli* and *S. aureus* [82].

**56**

[66, 76].

**Figure 1.**

**5.1 Determination of MIC of EOs for bacteria**

requires a large quantity of the plant extract [81]. A study using the redox dye resazurin for the new modified microdilution method has been carried out to determine the MIC for tea tree EO (*Melaleuca alternifolia*) against the gram-positive and gram-negative bacteria. The results showed that the resazurin method is accurate to determine the MIC and is higher in sensitivity than the results obtained from the agar dilution assay [80].
