**3. Discussion**

The contamination of food contact surfaces and the resistance of *E. coli* O157:H7 to disinfection processes are associated with its ability to form biofilms. The important role of glucosyltransferase producing glucans to strengthen the *E. coli* O157:H7 biofilms makes its inhibition an attractive target to reduce the biofilm formation. In this regard, *C. citratus* EO, citral, and geraniol have shown antimicrobial activities against many Gram-positive and Gram-negative bacteria, including *E. coli*. However, their e ffect on glucosyltransferase activity in relation with the biofilm formation has not been previously evaluated.

*C. citratus* EO, citral, and geraniol inhibited the planktonic growth of *E. coli* O157:H7, and this effect could be attributed to their abilities to degrade membrane proteins and cell permeability. The higher antibacterial activity of citral and *C. citratus* EO compared with geraniol could be related to their hydrophobic characteristics, since they have partition coe fficients (Log P) of 3 and 3.5, respectively [16,17], and these values could reflect a higher rate of interaction with the bacterial membrane. On the other hand, geraniol showed the lowest antibacterial activity against *E. coli* O157:H7, which may be explained considering its relatively lower lipophilic character (Log P = 2.9) [16] given by its hydroxyl group, which makes it more di fficult to pass through non-polar environments such as the cell membrane [18] compared with citral and *C. citratus* EO. A similar situation was described for thymol (possess a hydroxyl group), which showed a lower e fficacy against *E. coli* (MIC = 5 mg/mL) compared to p-cymene (absence of hydroxyl groups), which showed higher antibacterial activity (MIC = 2.5 mg/mL) [18].

Previously, Ortega-Ramirez et al. [10] reported the inhibitory e ffect of *C. citratus* EO against planktonic *E. coli* at 2.21 mg/mL. On the other hand, other EOs also showed e fficacy to inhibit *E. coli* O157:H7; for example, Kim et al. [19] reported that concentrations of 0.001 to 0.01mg/mL of bay, clove, and pimento berry EO significantly inhibited the biofilm formation of *E. coli* O157:H7. Bazargani and Rohlo ff [20] reported an inhibition of *E. coli* O157:H7 adhesion of 72.3, 56.2, and 98.4% by coriander (1.6 mg/mL), anise (12.5 mg/mL), and peppermint EO (6.3 mg/mL), respectively. These results showed that *C. citratus* EO and its terpenes, citral and geraniol, showed e fficacy as antibacterial agents inhibiting planktonic growth of *E. coli* O157:H7 even at low doses. It is important to mention that no previous reports of MBICs of these treatments were found in the revised literature; however, few mechanistic studies have been proposed. It is possible that the lower adhesion of the treated bacteria could be related to the interference in the adhesion process. Therefore, it has to be highlighted that the interest of this study was to evaluate the e ffect of *C. citratus* EO, citral, and geraniol on glucosyltransferase activity, glucan production, and biofilm development to propose a more complete mechanisms against cell communities that are the natural way of bacterial organization instead individual planktonic cells. For this reason, lower doses than MICs and MBICs were used to only a ffect the production of glucans without a ffecting cell viability.

Glucan production during *E. coli* O157:H7 biofilm formation was significantly reduced by citral and geraniol. Among the factors regulating the production of glucans in biofilms are the intercellular communication and the biosynthetic pathways [21]. Intercellular communication in *E. coli* occurs throughout the detection of acyl-homoserine lactones [22]; this process triggers the expression of virulence genes and the enzymatic production of glucans [23]. Thus, within the potential mechanisms of action of terpenes inhibiting glucans production are: (i) down-regulation of glucans synthase genes or a (ii) direct e ffect on the activity of such system [24]. Both approaches have been tested in other bacterial systems; however, most of the evidence has been directed to a possible e ffect on the enzymatic production of this polymer, as was done in the present study [24,25].

The ability of bacteria to adhere and form biofilms on di fferent surfaces has substantial implications in the food industry due to safety, quality, and economic issues [26]. As mentioned above, the presence of glucans protects cells from the action of disinfectants and physical cleaning processes. In this sense, it is possible to use *C. citratus* EO, citral, and geraniol as alternative disinfectants to inhibit biofilm formation as well as to help enhance the e ffect of other cleaning methods. These data can be

compared with previous studies that showed the efficacy of plant extracts and their active constituents to inhibit the production of water-insoluble glucans and biofilms of plaque-forming bacteria. Extracts of *Plectranthus barbatus*, *Plectranthus ecklonii*, and *Rheum undulatum* were effective in inhibiting the production of glucans in crude extracts of *Streptococcus sobrinus* and *S. mutans* [24].

For the same bacteria, Koo et al. [25] reported IC50 of 0.35 and 0.28 mg/mL for apigenin and farnesol, respectively. Also, epigallocatechin gallate, epigallocatechin, tannic acid, and catechol at 0.1 mg/mL inhibited the production of water-insoluble glucans of 73.1, 68.5, 68, and 67.6%, respectively [27]. In these studies, the reduction of glucans production was related with biofilm inhibition; however, most of them were done on dental plaque and tooth decay bacteria, not in a foodborne pathogen such as *E. coli* O157:H7. From the obtained results, it was observed that *C. citratus* EO, citral, and geraniol were effective in inhibiting the glucans production at non-lethal concentrations, maintaining their effect during the biofilm formation process.

*C. citratus* terpenes affected glucosyltransferase activity and, based on the obtained kinetic constants, this suggested an uncompetitive inhibition mechanism of glucosyltransferase by citral and geraniol, indicating that both terpenes bound reversibly to the enzyme–substrate complex, forming a ternary complex catalytically inactive. Citral and geraniol are molecules capable of accepting and donating hidrogens atoms and possess non-polar properties to establish hydrophobic interactions [16]. The interaction of terpenes within the hydrophobic pocket below the gating loop and the helix finger could affect the consequent UDP-glucose binding and glucan synthesis [6]. Cellulose synthase is activated by the presence of c-di-GMP, specifically by conformational changes caused by binding c-di-GMP, leading to an open state of the gating loop away from the active site cleft and near the water–lipid interface, where the loop is stabilized by the hydrophobic interactions with the BcsA's amphipathic interface helices forming a transmembrane channel [6]. In this sense, the interruption of the helix finger movement by the presence of citral or geraniol affected the glucan polymerization by influencing the retraction and the insertion of the gating loop [6].

Although there is no evidence of the effect of plant extracts on the glucosyltransferase activity of *E. coli*, there are studies with the dental bacteria *Streptococcus* [24,25]. Plant extracts of *P. barbatus*, *P. ecklonii*, and *R. undulatum* inhibited the activity of glucosyltransferase in crude extracts of *S. sobrinus* (IC50 = 1.0, 1.2 and 0.142 mg/mL, respectively) and *S. mutans* (IC50 = 3.1, 1.6 and 0.079 mg/mL, respectively) [24]. Within the same study, rosmarinic acid, one of the main components of these plants, showed IC50 of 2.1 and 3.9 mg/mL for *S. sobrinus* and *S. mutans* enzyme extracts, respectively. However, these studies did not propose any inhibition mechanism. On the other hand, oleic and linoleic acids showed to be uncompetitive inhibitors of glucosyltransferase; these fatty acids interacted with the substrate–enzyme complex, decreasing the velocity reaction in a similar way to that observed with *C. citratus* EO terpenes [28].

*C. citratus* EO and its components also inhibited the activity of other enzymes; for example, *C. citratus* EO inhibited MARK4, a kinase enzyme involved in apoptosis, inflammation, and many other regulatory pathways [14]. In another study, seven monoterpenes of *C. citratus* EO were evaluated on pentoxyresorufin activity, obtaining IC50 of 0.087 mM for (-)-α-pinene, 0.089 mM for (+)-α-pinene, 0.76 mM for α-terpinene, and 1.19 mM for citral [29]. For this reason, it is important to consider the effect of the rest of the EO components against glucosyltransferase activity, glucan production, and biofilm inhibition of *E. coli* O157:H7. As shown in previous studies, there is evidence that *C. citratus* EO and its compounds were capable of inhibiting different enzymes, but there was no evidence of the effect of this EO against *E. coli* O157:H7 biofilm-glucans-glucosyltransferase, which is the contribution of this study.

#### **4. Material and Methods**

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

The antibacterial e fficacies of *C. citratus* EO (W523100), citral (W230316), and geraniol (W250716) (Sigma-Aldrich, St. Louis, MO, USA) were evaluated against the growth of planktonic and biofilm *E. coli* O157:H7 (ATCC 43890). MIC experiments were performed by the broth microdilution method reported by the Clinical and Laboratory Standards Institute or CLSI [30] with some modifications. Briefly, 5 μL of an overnight inoculum of *E. coli* O157:H7 (1 × 10<sup>6</sup> CFU/mL) diluted in sterile saline solution were added to a sterile 96-well microtitre plate (Costar 96, Sigma-Aldrich, St. Louis, MO, USA), followed by 295 μL of EO, citral, and geraniol diluted in Luria Bertani o MH (LB) broth at concentrations from 1 to 20 mg/mL, obtaining 2-fold dilutions, respectively. The microplate was incubated at 37 ◦C for 24 h, and the MICs were determined as the lowest concentrations of each agen<sup>t</sup> that completely inhibited the visible growth of planktonic cells.

For inhibiting biofilm bacteria, MBICs were determined as the lowest dose of each compound inhibiting the bacterial adhesion on stainless steel coupons (1 × 1 × 0.1 cm, grade 304) during 24 h of incubation at 37 ◦C [31]. Di fferent concentrations of natural compounds (0–20 mg/mL) were added into test tubes with 10 mL of MH broth containing stainless steel coupons. Then, the tubes were inoculated with *E. coli* O157:H7 (1 × 10<sup>6</sup> CFU/mL, diluted in sterile saline solution) and incubated at 37 ◦C for 24 h under static conditions; then, the coupons were removed from the culture medium and washed with sterile distilled water to remove weakly adhered cells. Afterward, the coupons were placed in 5 mL of sterile peptone water and subjected to an ultrasonic bath (40 kHz) for 5 min to release the strongly adhered cells and were counted by plating on MH agar after 24 h of incubation at 37 ◦C (log CFU/cm2). Both inhibitory concentrations were obtained by triplicate from three independent experiments, and the obtained results were expressed as mg/mL [31].

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

Lower doses than MICs and MBICs were used to only a ffect the production of glucans without affecting cell viability. The conditions used for biofilm formation were as described above; applying *C. citratus* EO (0.5 mg/mL), citral (0.5 mg/mL), and geraniol (0.25 mg/mL), viable cells were counted at di fferent times (0, 2, 4, 8, 10, 12 h) at 37 ◦C. Biofilm cells adhered to stainless steel coupons as well as planktonic cells in the culture medium were determined as described above, expressing results as log CFU/cm<sup>2</sup> and log CFU/mL, respectively. Also, biofilms were stained with 0.1% crystal violet solution for 10 min and fixed with Lugol to observe morphological changes during the exposure to the treatments using an inverted microscope (Zeiss Axio Vert A1 Inverted, Carl Zeiss, NY, USA), viewing with phase contrast at 600× [32].

The glucans production by treated bacteria was expressed as glucose equivalents (GE) per area of stainless steel (cm2) [32]. Coupons were removed from the culture medium after incubation and then washed with water to remove weakly adhered cells. Subsequently, they were placed into tubes containing 5 mL of water and 30 μL of formaldehyde (33%) (Sigma Aldrich, St. Louis, MO, USA) and left at 4 ◦C for 1 h. Subsequently, 2 mL of NaOH (1 M) (Sigma Aldrich, St. Louis, MO, USA) were added to the tubes, sonicated for 5 min, and stored for 3 h at 4 ◦C. The final volume (7 mL) was filtered (millipore 0.22 μm) and dialyzed with Milli-Q water using a dialysis membrane (3500 Da) (Sigma Aldrich, St. Louis, MO, USA) at 4 ◦C for 24 h, and the > 3500 Da fraction was lyophilized. The lyophilized sample was diluted in 300 μL of Milli-Q water for the subsequent quantification of glucans adhered to the stainless steel coupons. The glucans were determined with the phenol/sulfuric acid method [33] using glucose as standard and expressing results as mg of glucose equivalents per area, GE/cm2.

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

Glucosyltransferase (SRP0416, Sigma Aldrich, St. Louis, MO, USA) activity was measured in the presence of citral and geraniol at 0, 8, and 10 μM; lemongrass EO was excluded from this assay considering the variety of chemical structures in its content, making it difficult to establish a molar relation. This was measured in 300 μL of buffer solution (40 mM Tris-HCl, pH 8, 15 mM MgCl2, 1 mM CaCl2, and 5 mM UDP-glucose) containing each concentration of terpenes; this mixture was pre-incubated at 30 ◦C for 10 min, and the reaction was initiated by adding the glucosyltransferase (EC 2.4.1.). The enzyme activity was measured using the fluorometric assay [34] that monitored the release of UDP-fluorescein (λex 490 nm; λem 514 nm) as a product of the UDP-glucose hydrolysis (the absence of terpenes in the reaction was taken as 100% activity).

The initial reaction velocities (Vo) were obtained using 2 mM of glucosyltransferase, substrate at 2, 4, 8, 10, and 20 μM, and the individual terpenes at 8 and 10 μM, respectively. The experimental data were fitted to a non-linear model, applying the equation of Michaelis–Menten for Km and Vmax calculation, and then these values were fitted to the Lineweaver–Burk equation. The type of inhibition was determined analyzing the Lineweaver–Burk graph, and the Ki values of the individual terpenes were taken from the x-intercepts of 1/Vmax versus the terpene concentration [35]; this assay was performed three times to assure reproducibility.

#### *4.4. Molecular Docking of Glucosyltransferase with Citral and Geraniol*

Molecular docking was used to identify possible interactions between the individual terpenes (citral and geraniol, respectively) with the glucosyltransferase crystallographic model (PDB 5EIY) [6]; the used citral and geraniol models were PubChem 638011 and PubChem 637566. This analysis was done using the AutoDoc Vina application in the UCSF Chimera version 1.13 software (Resource for Biocomputing, Visualization, and Informatics, University of California, San Francisco, CA, USA) to obtain affinity energies (kcal/mol) with the lowest root-mean-square deviation (RMSD, Å) between glucosyltransferase and each terpene. Ten binding modes with a 3 level of exhaustiveness search and a 3 kcal/mol level of maximum energy difference were set as basic parameters during the analysis.

#### *4.5. Statistical Analysis*

A completely randomized experimental design was done for all assays. The effect of *C. citratus* EO, citral, and geraniol, as well as the exposure time (0, 2, 4, 8, 10, and 12 h) were evaluated on the count of viable planktonic and biofilm cells and the glucans production. In addition, a Pearson correlation was done between the secreted glucans and the biofilm formation. All experiments were done by triplicate, expressing the results as means ± standard deviation. An analysis of variance (ANOVA) was done for all the assays to estimate significant differences among treatments, and the means were compared by the Tukey–Kramer test. All experiments were performed at *p* ≤ 0.05 using the statistical software NCSS 2007 (NCSS, LLC, Utah, USA).
