**2. Results**

#### *2.1. Susceptibility of Planktonic and Biofilm E. coli O157:H7 Cells to C. citratus EO, Citral, and Geraniol*

Citral was effective inhibiting the growth of planktonic cells (minimal inhibitory concentration or MIC = 1.0 mg/mL) compared to biofilm (minimal biofilm inhibitory concentration or MBIC = 2.0 mg/mL), followed by *C. citratus* EO [MIC = 2.2 mg/mL and MBIC = 2.0 mg/mL] and geraniol (MIC = 3.0 mg/mL and MBIC = 4.0 mg/mL). In general, higher concentrations were needed to inhibit the cell adhesion and the biofilm formation compared to those needed to inhibit the growth of planktonic cells. Lower concentrations than the MIC and the MBIC values of the treatments (*C. citratus* EO = 0.5 mg/mL, citral = 0.5 mg/mL, geraniol = 0.25 mg/mL) were selected to avoid interference of the loss of viability on the biofilm formation and glucans production responses (Figure 1).

Figure 2A shows the *E. coli* O157:H7 biofilm cells on stainless steel coupons in the presence of the treatments. It can be observed that the viable cell in the control bacteria increased steadily as a function of the incubation time, reaching a maximum growth at 12 h at 37 ◦C. On the other hand, *C. citratus* EO, citral, and geraniol significantly reduced biofilm cells on stainless steel surfaces. *C. citratus* EO reduced 1.64 log CFU/cm<sup>2</sup> the cell adhesion at 12 h compared to the control bacteria, whereas citral and geraniol completely inhibited cell adhesion at the end of incubation time. Figure 2B shows the microphotographs of *E. coli* biofilm development at different incubation times in the absence and presence of the compounds. A significant increase in bacterial aggregation was observed in the control (a) as the incubation time increased, being at 10 h a complete surface colonization. In the case of *C. citratus* EO (b), we observed a significant reduction in aggregation compared to the control, keeping constant at 10 and 12 h, whereas for citral (c), a significant reduction was observed after 8 h. Geraniol

(d) completely inhibited *E. coli* biofilm formation, since no bacterial aggregation was observed after 2 h of incubation at 37 ◦C.

**Figure 1.** Viability changes of planktonic *E. coli* O157:H7 exposed to non-lethal concentrations of *C. citratus* essential oil (EO), citral, and geraniol. Different letters among treatments indicated significant differences among them (*p* < 0.05). The values are means ± SD, *n* = 3.

#### *2.2. E*ff*ect of C. citratus EO, Citral, and Geraniol on the Glucans Content in E. coli O157:H7 Biofilms*

Figure 3 shows the glucan content in *E. coli* biofilms exposed to *C. citratus* EO (0.5 mg/mL), citral (0.5 mg/mL), and geraniol (0.25 mg/mL). It can be observed that the glucan content of control increased exponentially during the incubation time, whereas in those treated with *C. citratus* EO, citral, and geraniol, the glucans production during the biofilm formation was significantly reduced. The stainless steel coupons exposed to citral and geraniol had a lower glucan content compared to the control and the *C. citratus* EO treated bacteria. The relationship between the secreted glucans and the biofilm cells on stainless steel surfaces showed a Pearson correlation coefficient of 0.768 with a probability of 0.0000119.

#### *2.3. Inhibition of Glucosyltransferase Activity by Citral and Geraniol*

The activity of pure glucosyltransferase was affected by the presence of citral and geraniol showing IC50 values of 8.5 and 6.5 μM, respectively (Figure 4). The reaction pattern of the tested glucosyltransferase showed a Michaelis–Menten kinetic (Figure 5A,B). Table 1 shows the calculated kinetic constants, where both Km and Vmax decreased with increasing citral and geraniol concentrations (Figure 5C,D). On the other hand, low Ki values indicated that both inhibitors showed affinity towards the enzyme–substrate complex, this being higher in the case of geraniol. The steric arrangements that could explain the interference of terpenes were proposed by the computational docking analysis. Docking analysis showed that the most probable interactions among citral or geraniol and the enzyme occurred within the hydrophobic pocket located below the gating loop and next to the helix finger of the glucosyltransferase enzyme (Figure 6). The affinity energy obtained for the citral–enzyme-substrate complex was -5.8 kcal/mol with a root-mean-square deviation of atomic positions or RMSD 1.382 Å (Figure 6B), while for the geraniol–enzyme-substrate complex, it was -6.1 kcal/mol with RMSD 1.649 Å (Figure 6C).

**Figure 2.** (**A**) Viability changes of biofilm embedded *E. coli* O157:H7 cells exposed to non-lethal concentrations of *C. citratus* EO, citral, and geraniol; different letters indicate significant differences among average of treatments (*p* < 0.05). The values are means ± SD, *n*= 3. (**B**) Light microscopy analysis of *E. coli* O157:H7 biofilms: (a) control, (b) *C. citratus* EO, (c) citral, (d) geraniol. Microphotographs were captured at 600x magnification in an Axio-Vert Microscope.

**Figure 3.** Glucans content on stainless steel coupons produced by *E. coli* O157:H7 biofilms exposed to non-lethal concentrations of *C. citratus* EO, citral, and geraniol; different letters indicate significant differences among treatments (*p* < 0.05). The values are means ± SD, *n* = 3.

**Figure 4.** Glucosyltransferase inhibition by the presence of citral and geraniol at different. concentrations (*p* < 0.05). The values are means ± SD, *n* = 3.


**Table 1.** Kinetic parameters of glucosyltransferase exposed to citral and geraniol.

\* Values are means of three replicated experiments. \*\* UDP: Uridine diphosphate.

**Figure 5.** *Cont.*

**Figure 5.** Reaction velocity of glucosyltransferase as a function of substrate concentration in the presence of citral (**A**) and geraniol (**B**). Lineweaver–Burk double reciprocal plot of the glucosyltransferase activity in the presence of citral (**C**) and geraniol (**D**). The double reciprocal plot of the glucosyltransferase activity as a function of citral (**E**) and geraniol (**F**) as a graphical method to calculate Ki. Every point is a mean of three replicated experiments.

**Figure 6.** Glucosyltransferase (**a**) interactions with citral (**b**) and geraniol (**c**) blocking the gating loop (green) and the helix finger (yellow) movements during cellulose processing.
