**4.5 Antibacterial activity**

#### *4.5.1 Bacterial strains, culture media and drugs*

For the antibacterial tests, standard strains were used to determine minimum inhibitory concentration (MIC), being *Escherichia coli* ATCC 25922, *Pseudomonas aeruginosa* ATCC 25853 and *Staphylococcus aureus* ATCC 25923. While for the modulation and also MIC tests, strains resistant cells (**Table 3**), being *Escherichia coli* 06, *Pseudomonas aeruginosa* 03 and *Staphylococcus aureus* 10.

As for the culture medium for the antibacterial assays, Brain Heart Infusion (BHI) was prepared according to the measures recommended by the manufacturer. While for *in vitro* modulation assays, the drugs used were Gentamicin from class aminoglycoside, Norfloxacin, belonging to the classes of fluoroquinolones and Imipenem of the carbapenem class.

#### *4.5.2 Minimal inhibitory concentration (MIC)*

It was followed the methodology employed in the work Bezerra et al. [3] for the determination of the Minimum Inhibitory Concentration (MIC). In this study, concentrations ranging from 1 to 1024 μg/mL of the essential oil of *M. suaveolens* against pathogenic bacteria were evaluated. For that, the inoculants of the strains were mixed with BHI (10%), being distributed in microdilution plates with the natural product. After 24 hours of microbial growth at a temperature of 37°C, the MIC was evaluated with the addition of resazurin.

#### *4.5.3 Effect modulator of antibiotics*

To assess the modulating effect of essential oil, sub-inhibitory concentrations (MIC/8) of the product against multidrug-resistant bacteria were used. For that, concentrations of standard antibiotics (1–1024 μg/mL) were added to microdilution plates containing BHI (10%) and bacteria inoculum, as well as volatile *M. suaveolens* terpenes in sub-inhibitory concentrations. After 24 hours in a bacteriological oven (37°C), a resazurin solution was added to determine the MIC [7].

#### **4.6 Statistical analysis**

The results were analyzed in the GraphPad Prism program, version 6, in which the data were analyzed by Anova One-way and followed by post hoc Tukey test and were considered significant when *p* < 0.05.
