Salpianthus macrodontus Extracts, a Novel Source of Phenolic Compounds with Antibacterial Activity against Potentially Pathogenic Bacteria Isolated from White Shrimp

This study aimed to evaluate the antibacterial activity in vitro of Salpianthus macrodontus and Azadirachta indica extracts against potentially pathogenic bacteria for Pacific white shrimp. Furthermore, the extracts with higher inhibitory activity were analyzed to identify compounds responsible for bacterial inhibition and evaluate their effect on motility and biofilm formation. S. macrodontus and A. indica extracts were prepared using methanol, acetone, and hexane by ultrasound. The minimum inhibitory concentration (MIC) of the extracts was determined against Vibrio parahaemolyticus, V. harveyi, Photobacterium damselae and P. leiognathi. The polyphenol profile of those extracts showing the highest bacterial inhibition were determined. Besides, the bacterial swimming and swarming motility and biofilm formation were determined. The highest inhibitory activity against the four pathogens was found with the acetonic extract of S. macrodontus leaf (MIC of 50 mg/mL for Vibrio spp. and 25 mg/mL for Photobacterium spp.) and the methanol extract of S. macrodontus flower (MIC of 50 mg/mL for all pathogens tested). Both extracts affected the swarming and swimming motility and the biofilm formation of the tested bacteria. The main phenolic compounds related to Vibrio bacteria inhibition were naringin, vanillic acid, and rosmarinic acid, whilst hesperidin, kaempferol pentosyl-rutinoside, and rhamnetin were related to Photobacterium bacteria inhibition.


Introduction
The Pacific white shrimp (Litopenaeus vannamei) is globally one of the most important aquaculture species [1]. The shrimp aquaculture industry was intensified between 2002 and 2012; however, the intensification caused different sanitary problems related to mortality caused by bacteria, such as Vibrio spp., challenging productivity, and survival intensive farms [2][3][4]. Among the main causative agents of bacterial diseases in shrimp are Vibrio angullarum, V. ordalii, V. salmonicida, V. vulnificus, V. alginolyticus, V. harveyi, V. ponticus, V. parahaemolyticus, V. mimicus, Photobacterium damselae, and P. leiognathi [5][6][7][8]. Table 1 shows the maximum inhibition percentage and the MIC at the evaluated conditions. The extracts obtained from the leaves and flowers of S. macrodontus showed higher antibacterial activity against the bacteria tested (p < 0.05) than the A. indica extracts. Figure 1 shows that at higher extract concentrations, the inhibition rate is greater in most cases. However, the highest concentration was not always the most effective to inhibit bacteria.  bacteria.    The MICs of ALE (acetone leaves extract) were 50 mg/mL against V. parahaemolyticus and V. harveyi, 25 mg/mL against P. damselae, and P. leiognathi. The MIC to MFE (methanol flower extract) was 50 mg/mL for all bacteria evaluated (Table 1). From the A. indica extracts, only the MNE (methanol leaves of A. indica extract) was effective against the four bacteria evaluated in 50 and 100 mg/mL concentrations. However, several extracts obtained from the leaves and flowers of S. macrodontus were better (p < 0.05) than the MNE (Figure 1). In this regard, at a 100 mg/mL concentration, HLE (hexane leaves extract) was better against P. damselae, and HFE (hexane flowers extract) was better against V. parahaemolyticus and P. damselae. Due to the above, ALE and MFE were chosen for the following tests. Therefore, ALE and MFE extracts can be used to treat infectious diseases caused by resistant pathogens. This is the first report of the antibacterial activity of S. macrodontus.

Analysis of Polyphenolic Compounds of Extracts
The polyphenolic profiles of the S. macrodontus flowers and leaves extracts are shown in Table 2. A total of 45 compounds were identified, of which 27 were flavonoids, and 18 were phenolic acids.
The MLE (methanol leaves extract) showed the highest concentration of the phenolic compounds (1017.34 mg/mL), followed by MFE (253.72 mg/mL), and then ALE (215.35 mg/mL). Quercetin hexoside stands out as the phenolic compound with the highest concentration in the MLE, followed by quercetin hexoside-rhamnoside, kaempferol dihexoside, kaempferol rutinoside, and kaempferol hexosyl-rhamnosyl-hexoside. The main compounds found in ALE were kaempferol dihexoside, quercetin hexoside, eriocitrin, and kaempferol rutinoside. On the other hand, the main compounds in MFE were kaempferol hexosyl-rhamnosyl-hexoside, followed by quercetin hexoside-rhamnoside, quercetin dihexoside, kaempferol trihexoside, and kaempferol dihexoside.  Figure 2 shows PLS-DA plots constructed with polyphenol profile of extracts and inhibition percentage. The preceding is in order to identify the bioactive compounds associated with the inhibition of the different bacteria evaluated. Those compounds with VIP > 0.8 and coefficient values > 0 can be considered responsible for the extracts' inhibitory activity. Likewise, compounds with VIP > 0.8 and coefficient values < 0 can be regarded as growth stimulators.  Figure 2 shows PLS-DA plots constructed with polyphenol profile of extracts and inhibition percentage. The preceding is in order to identify the bioactive compounds associated with the inhibition of the different bacteria evaluated. Those compounds with VIP > 0.8 and coefficient values > 0 can be considered responsible for the extracts' inhibitory activity. Likewise, compounds with VIP > 0.8 and coefficient values < 0 can be regarded as growth stimulators. The effects of the compounds on the growth of V. parahaemolyticus and V. harveyi are shown in Figure 2A,B, respectively. Both bacteria had a similar response to phenolic compounds. Naringin (F_8), vanillic acid (PA_3), and rosmarinic acid (PA_18) were the compounds mainly related to the inhibition of Vibrio bacteria since they had higher VIP and coefficient values. Naringin was found in the methanolic and acetone extracts of S. macrodontus flowers and leaves. Vanillic acid was found in MFE, AFE (acetone flower extract), and ALE. Rosmarinic acid was found in all the evaluated extracts, except HLE.

Effect of Polyphenols on the Bacterial Inhibition
Moreover, Figure 2C,D show the compounds related to inhibition of P. damselae and P. leiognathi, respectively. Hesperidin (F_6), kaempferol pentosyl-rutinoside (F_16), and rhamnetin (F_27) are mainly related to inhibition of Photobacterium bacteria since they had higher VIP and coefficient values. Hesperidin was found in all evaluated extracts. kaempferol pentosyl-rutinoside was found in all evaluated extracts except HFE. Rhamnetin was presented in the acetone and hexene extracts of flower and leaf.
It is important to note that the three main compounds that inhibit the growth of Photobacterium bacteria (hesperidin (F_6), kaempferol pentosyl-rutinoside (F_16), and rhamnetin (F_27)), enhanced the growth of Vibrio bacteria ( Figure 2). The effects of the compounds on the growth of V. parahaemolyticus and V. harveyi are shown in Figure 2A,B, respectively. Both bacteria had a similar response to phenolic compounds. Naringin (F_8), vanillic acid (PA_3), and rosmarinic acid (PA_18) were the compounds mainly related to the inhibition of Vibrio bacteria since they had higher VIP and coefficient values. Naringin was found in the methanolic and acetone extracts of S. macrodontus flowers and leaves. Vanillic acid was found in MFE, AFE (acetone flower extract), and ALE. Rosmarinic acid was found in all the evaluated extracts, except HLE.
Moreover, Figure 2C,D show the compounds related to inhibition of P. damselae and P. leiognathi, respectively. Hesperidin (F_6), kaempferol pentosyl-rutinoside (F_16), and rhamnetin (F_27) are mainly related to inhibition of Photobacterium bacteria since they had higher VIP and coefficient values. Hesperidin was found in all evaluated extracts. kaempferol pentosyl-rutinoside was found in all evaluated extracts except HFE. Rhamnetin was presented in the acetone and hexene extracts of flower and leaf.

Motility Assays
The result obtained from the motility assays of ALE and MFE extracts ( Figure 3) showed significant differences among four bacteria and control (without extract) (p < 0.05). The MFE extract had a higher effect against the four bacterial strains tested for both types of motilities.

Motility Assays
The result obtained from the motility assays of ALE and MFE extracts ( Figure 3) showed significant differences among four bacteria and control (without extract) (p < 0.05). The MFE extract had a higher effect against the four bacterial strains tested for both types of motilities. The ALE increased the swarming motility of V. parahaemolyticus, V. harveyi, and P. damselae on 254.17, 232.43, and 650%, respectively. In contrast, P. leiognathi had a reduction of 25%. On the other hand, MFE increased the swarming motility of V. parahaemolyticus, V. harveyi, and P. damselae by 87.5, 18.92, and 21.42%, respectively. However, P. leiognathi decreased its swarming motility by 70% in the presence of MFE ( Figure 3A).

Microplate Assay for Biofilm Quantification
In this study, statistical analysis indicated a significant difference in ALE and MFE on the biofilm formation of these bacteria (p < 0.05). ALE reduced biofilm formation on 69.25, 100, and 61.13% in V. parahaemolyticus, V. harveyi, and P. leiognathi. However, P. damselae did not form biofilm. Furthermore, the MFE did not affect significantly the biofilm formation in V. parahaemolyticus and P. leiognathi (p > 0.05); however, V. harveyi increased the biofilm formation 372.4% (p < 0.05).

Discussion
Several plant extracts and essential oils have been used to control pathogenic bacteria in aquaculture (Vibrio and Photobacterium bacteria), such as boiled water extract of Psidium guava leaf, green tea leaf, and water and oil extracts of Calendula officinalis [19,20], Piper betle ethyl acetate [24], and Scutellaria baicalensis water extract [24]. This tendency has responded for the need of new antimicrobials that can be obtained from a low-impact technology (reduction in solvents, industrial process, residuals) and that contain different active ingredients to reduce the development of resistance.
In the case of A. indica, there are several studies where extracts of this plant have been effective in the control of genus Vibrio, including V. parahaemolyticus at concentrations of 0.1 to 100 mg/mL [17][18][19][20][21][22], V. alginolyticus at concentrations of 0.075 mg/mL to 250 mg/mL The ALE increased the swarming motility of V. parahaemolyticus, V. harveyi, and P. damselae on 254.17, 232.43, and 650%, respectively. In contrast, P. leiognathi had a reduction of 25%. On the other hand, MFE increased the swarming motility of V. parahaemolyticus, V. harveyi, and P. damselae by 87.5, 18.92, and 21.42%, respectively. However, P. leiognathi decreased its swarming motility by 70% in the presence of MFE ( Figure 3A).

Microplate Assay for Biofilm Quantification
In this study, statistical analysis indicated a significant difference in ALE and MFE on the biofilm formation of these bacteria (p < 0.05). ALE reduced biofilm formation on 69.25, 100, and 61.13% in V. parahaemolyticus, V. harveyi, and P. leiognathi. However, P. damselae did not form biofilm. Furthermore, the MFE did not affect significantly the biofilm formation in V. parahaemolyticus and P. leiognathi (p > 0.05); however, V. harveyi increased the biofilm formation 372.4% (p < 0.05).

Discussion
Several plant extracts and essential oils have been used to control pathogenic bacteria in aquaculture (Vibrio and Photobacterium bacteria), such as boiled water extract of Psidium guava leaf, green tea leaf, and water and oil extracts of Calendula officinalis [19,20], Piper betle ethyl acetate [24], and Scutellaria baicalensis water extract [24]. This tendency has responded for the need of new antimicrobials that can be obtained from a low-impact technology (reduction in solvents, industrial process, residuals) and that contain different active ingredients to reduce the development of resistance.
In the case of A. indica, there are several studies where extracts of this plant have been effective in the control of genus Vibrio, including V. parahaemolyticus at concentrations of 0.1 to 100 mg/mL [17][18][19][20][21][22], V. alginolyticus at concentrations of 0.075 mg/mL to 250 mg/mL [17,18], and V. cholerae at concentrations of 0.1 mg/mL to 15 mg/mL [22]. Banerjee [17] found an MIC of 3.13% (equivalent to 31.3 mg/mL) of A. indica juice, which is lower than that shown in the present study. Moreover, the aqueous extract of leaves from A. indica (MIC of 10 mg/mL) reported by Sharma and Patel [22], besides the ethanol, methanol, chloroform, and acetone extracts from the leaves of A. indica (MIC of 0.1, 0.25, 0.075, and 0.25 mg/mL, respectively) reported by Dhayanithi et al. [18]. However, in the present study, A. indica extracts did not completely inhibit the four bacteria tested. The bacteria probably developed a differential resistance against this plant extract in a similar manner as some Vibrio strains have shown resistance to antibiotics [9,[12][13][14]. Pathogenic bacteria can become resistant to antibacterial agents through mutation and selection or by acquiring genetic information that encodes other bacteria [25].
In this study, we established the novel use of S. macrodontus extracts' efficacy against Vibrionaceae family bacteria tested, indicating that the plant produces compounds that affect the bacterial defense mechanisms. The antimicrobial activity of both ALE and MFE showed inhibition of Vibrio species was similar (MIC 50 mg/mL). However, ALE demonstrated a higher capacity to inhibit Photobacterium species (MIC of 25 mg/mL) than MFE (MIC of 50 mg/mL). In both extracts, MIC does not suggest a dose-response relationship, however, a high dose of the extract can stimulate the growth of bacteria causing a hormesis effect. Therefore, in both extracts, phytochemical compounds probably exert a differential effect based on their active compounds as we can observe in Table 2.
We found flavonoids, such as flavanols, flavanones and flavonols, as well as phenolic acids, such as hydroxybenzoic acids and hydroxycinnamic acids, as the main components of S. macrodontus extracts. Daglia [26] mentions that flavanols and flavonols have a wide spectrum and higher antimicrobial activity than other polyphenols since they can suppress virulence factors, such as biofilm formation inhibition and the reduction in host ligands adhesion, and the neutralization of bacterial toxins.
Vanillic acid has been proven to have antibacterial effects against E. coli, Pasteurella multocida, Neisseria gonorrhoeae, Klebsiella pneumoniae, and Staphylococcus aureus, possibly to increased membrane permeability and antibiotic accumulation in pathogens [28][29][30]. Liu et al. [31] proved that vanillic acid presents an antibacterial and an antivirulence effect on Vibrio alginolyticus, with a MIC of 1 mg mL −1 . The vanillic acid effect on V. alginolyticus causes cell membrane damage and increasing membrane permeability and affects biofilmforming capability, mobility and exotoxin production.
Rosmarinic acid has shown antibacterial activity against Pseudomona aureaginosa and E. coli due to their strong cytotoxic potency and genotoxic effects [32][33][34][35]. This activity is related to enzyme inhibition by oxidized compounds due to reactions with sulfhydryl groups of non-specific interactions with proteins [36]. Corrales et al. [37] and Khalid et al. [27] reported that hesperidin shows inhibitory action against a wide-spectrum of Grampositive and Gram-negative bacteria, which is related to bacterial membrane disruption and interference with microbial enzymes [36]. Biharee et al. [38] and Daglia [26] reported that rhamnetin has antimicrobial activity against Gram-positive, Candida albicans, and Chlamidia pneumoniae. Rhamnetin can cause membrane disruption [38] and decrease the infective yields and the compounds related to pathogenesis [26]. Furthermore, Cid-Ortega and Monroy-Rivera [39] and Sati et al. [40] mention that kaempferol glycosides, such as kaempferol pentosyl-rutinoside, have antibacterial activities against Gram-positive and Gram-negative bacteria.
Also, we observed that hesperidin (F_6), kaempferol pentosyl-rutinoside (F_16), and rhamnetin (F_27)) inhibited Photobacterium bacteria but enhanced the growth of Vibrio bacteria. This can be related to the hormesis phenomenon, where stimulatory responses (bacterial growth) occur at low doses of antibacterial compounds. In contrast, inhibitory responses (antibacterial activity) appear at higher doses, which form a dose-response relationship [41]. These three compounds could be in enough doses to inhibit Photobacterium bacteria but not enough to inhibit Vibrio bacteria.
On the other hand, ALE and MFE showed antipathogenic activities since they affected the virulence factors, such as motility (swarming and swimming) and the biofilm formation capacity of bacteria.
Both extracts (ALE and MFE) increased the swarming motility of V. parahaemolyticus, V. harveyi, and P. damselae, and swimming motility to V. harveyi. Increased motility in bacteria (chemotaxis) could respond to avoiding contact with the antimicrobial compounds present in the extracts [42]. On the other hand, P. leignathi decreased its swarming motility in the presence of ALE and MFE, as well as V. parahaemolyticus, P. damselae, and P. laiognathi decreasing their swimming motility in the presence of MFE at sublethal doses. One of the responsible compounds for this phenomenon could be naringin, which was reported as an inhibitor of swimming and swarming motility in Chromobacterium violaceum and Yersinia enterocolitica [32,34]. This could be due to the inhibition of the microorganisms in question or directly affected by the bacteria flagella [42].
Regarding the virulence factor biofilm production, ALE reduced biofilm production, even using 50% of the minimum inhibitory concentration. This reduction can be because of the naringin, rosmarinic acid, and hesperidin, which have been reported as inhibitors in biofilm production [32,34,35,[43][44][45]. Santhakumari and Ravi [44] mention that naringin interferes with the acyl homoserine lactone-based QS of a wide range of Gram-negative bacteria, which is related to biofilm production.
However, MFE had low control of biofilm formation capacity or even increased its production. This may be because the dose was inadequate for the bacteria tested since MFE showed a lower concentration of the main antibacterial compounds than ALE. The low concentration of antibacterial compounds with reports of antibiofilm activity, such as naringin, rosmarinic acid, and hesperidin, can cause stress and induce biofilm production to protect themselves from toxic substances [26].

Plant Material
Leaves and flowers of S. macrodontus were collected near Tuxpan, Nayarit, Mexico

Preparation of the Vegetable Extracts
For the preparation of the extracts, a solvent was added in a 1:10 ratio (dry sample: solvent) and sonicated in an ultrasonic bath Branson ® 5510 (47 kHz at 130 W) for 30 min at ≤40 • C [46]. In this way, the methanol, acetone, and hexane extracts were obtained from A. indica leaves and S. macrodontus leaves and flowers. Then, the supernatant was recovered by vacuum filtration (40 Torr) through Whatman paper No. 1, and the solvent was eliminated in a rotary evaporator (Büchi R-3) at no more than 40 • C under vacuum conditions (40 Torr). Afterward, the extracts were resuspended in glycerol at 20% until reaching a concentration of 1 g/mL. Then, the extracts were dissolved in the same manner in tryptic soy broth at 50% (TSB 50%) supplemented with 20 g/L of sodium chloride (TSB20 50%) in order to get a final concentration of 100 mg/mL (stock solution). Finally, all extracts were sterilized using filters with a pore size of 0.22 µm and stored at −20 • C until later analysis to avoid denaturation. The extracts were named by three characters. The first one means the solvent used (M for methanol, A for acetone, and H for Hexane). The second character is according to the vegetable source from which it was obtained (L for S. macrodontus leaves, F for S. macrodontus flowers, and N for A. indica leaves). The last character means extract.

Bacterial Strains
Four pathogenic strains, previously isolated from white shrimp showing signs of AHPND in Mexico shrimp farms (2013), were used for the susceptibility analysis. These bacteria were kindly provided from the CIBNOR collection (Environmental Microbiology Group, CIBNOR, La Paz, México). The strains used for this study were: Vibrio parahaemolyticus 2, Vibrio harveyi 6F, Photobacterium damselae 7F, and Photobacterium leiognathi 8F.

Antibacterial Assay and Minimum Inhibitory Concentration (MIC)
Antibacterial activity and MIC were determined based on the broth microdilution technique described by the Clinical and Laboral Standards Institute (CLSI) [47], with minor modifications. In order to do this, serial dilutions of the extracts were made with TSB20 50% to obtain a concentration range of 12.5-100 mg/mL from the stock solutions. Later, 150 µL of each culture medium was added to the microplate wells, followed by 10 µL of a bacterial suspension (0.4 optical density at 620 nm, which corresponds to 1 × 10 8 cells/mL) of the strain to be evaluated. Afterward, the plates were incubated for 20 h at 35 • C, and the optical density at 620 nm was recorded with a microplate reader (Thermo Scientific). Finally, the inhibition percentage was calculated of the extracts tested concerning the control (no extract). In the present study, the MIC was taken as the extract concentration that reduced the bacteria growth between 95 and 100%, according to CLSI (Clinical and Laboratory Standards Institute) [47].

Polyphenol Profile by UPLC-ESI-Q-ToF MS
Samples of the concentrated plant extract (200 mg) were dissolved in 8 mL of methanol: water (50:50 v/v) acidity with HCl (pH 2); next, it was thoroughly shaken at room temperature for 1 h, it was centrifugated at 16,000× g for 10 min at 4 • C. The supernatant was recovered. A total of 20 mL of acetone/water (70:30 v/v) was added to the residue. The shaking and centrifugation were repeated. The methanol and acetone extracts were mixed and filtered through PVDF syringe filters (13 mm, 0.45 µm).
The elution gradient was performed with a binary system consisting of (A) 0.1% formic acid in water and (B) 0.1% formic acid in acetonitrile. The following gradient was applied at a flow rate of 0.4 mL/min: 0 min at 0% B, 2.5 min at 15% B, 10 min at 21% B, 12 min at 90% B, 13 min at 95% B; 15 min at 0% B, and 17 min 0% B. The injection volume was 2 µL, and the sample temperature was set at 10 • C.
The Q-ToF MSE conditions were as follows: data were acquired at negative ionization (ESI-) within a mass range of 100 to 1200 Da; capillary voltage, 2.5 kV (ESI-) and 3.5 kV (ESI+); cone voltage, 40 eV; low collision energy, 6V. The conditions of the mass spectrometer were as follows: the temperature of the source was adjusted to 120 • C and nitrogen was used as the desolvation gas (800 L/h) at a temperature of 450 • C. The sampling cone was 40 eV, and capillary voltages were 2.0 kV (ESI-) and 3.5 kV (ESI+). Data acquisition was performed using the high definition MSE negative ionization mode with a 50-2000 Da mass range. Leucine-enkephalin (50 pg/mL) at 10 mL/min was used for mass correction. Peak identification was carried out by identifying the exact mass of the pseudo-molecular ion (mass error < 5 ppm), isotope distribution, and fragmentation pattern. Calibration curves were constructed with ellagic acid (hydroxycinnamic acids), gallic acid (hydroxybenzoic acids), (-)-epicatechin (flavanols), naringenin (flavanones), and quercetin (flavonols). Data acquisition was performed with the UNIFI Scientific Information System (Waters Co., MA, USA). The extracts were analyzed in triplicate.

Motility Assays
Swimming (flagella-directed movement in aqueous environments) and swarming (flagella-directed rapid movement onto solid surfaces) assays were performed as described by de la Fuente-Núñez et al. [48] with some modifications. Briefly, individual colonies were transferred from TSB20 agar to the surface of swimming agar (0.3% Difco Bacto Agar) and swarming agar (0.5% Difco Bacto Agar) using a sterile sharp toothpick. After incubation at 35 • C for 20 h, the motility was assessed by measuring the distance the bacteria had moved off the inoculation point, expressed as diameter (mm).

Microtiter Plate Assay for Biofilm Quantification
Biofilm formation assays were performed according to Naves et al. [49] with some modifications. A volume of 10 µL of inoculum with 0.4 OD620 was inoculated in 200 µL of tryptic soy broth (TSB 20%) containing 20 g/L of sodium chloride was added in peripheral wells. Then, the microplate was incubated for 20 h at 35 • C (without agitation). After, the biofilms were fixed with a crystal violet solution (1%) for 15 min. Then, the excess crystal violet dye was removed with water, plates were washed twice, and air-dried. At that point, 200 µL of 95% ethanol was added to all well and kept in orbital shaking (130 rpm) for 18 h. Finally, biofilm measurements were determined using Equation (1).
where SBF is the specific biofilm formation, AB is the OD540 of the attached and stained bacteria, CW is the OD540 of the control medium (no bacteria), and G is the microbial growth before crystal violet staining (OD620). The spectrophotometric measures were obtained using a microplate reader (Thermo Fisher Scientific Mutiskan Go, Vantaa, Finland).
The SBF values were classified into two categories: strong biofilm producers (SBF index 1.00) and weak biofilm producers (SBF index 1.00).

Statistical Analysis
The results were expressed as means ± SD. Each extract was tested in triplicate in three independent experiments. Statistical significance of the differences between means was established by testing homogeneity of variance and normality of distribution followed by ANOVA with Tukey test (analysis of classes of phytochemical compounds). The non-parametric methods (Kruskal-Wallis test) were used for the antibacterial activity of extracts. The p values below 0.05 were considered statistically significant. All analyses were performed using SAS software version 9.4 for Windows.
Associations between the polyphenolic compounds and inhibition (%) were assessed with the Variable Importance in the Projection (VIP) vs. coefficient score plots constructed from the supervised Partial Least Squares-Discriminant Analysis (PLS-DA) with centered and scale data. A non-linear iterative partial least squares (NIPALS) was used. This analysis was carried out with JMP software (v10) (Sytat Software, Inc., San José, CA, USA).

Conclusions
The present study shows the potential antibacterial activity of S. macrodontus ALE and MFE against shrimp pathogens. The ALE and MFE antimicrobial potential against evaluated Vibrio bacteria is mainly due to naringin, vanillic acid, and rosmarinic acid, while against photobacterium bacteria is mainly due to hesperidin, kaempferol pentosylrutinoside, and rhamnetin. The ALE and MFE showed antipathogenic activity modifying the speed of motility (swarming and swimming) and biofilm formation, which could be related to compounds present in extracts, mainly naringin, rosmarinic acid, and hesperidin.

Patents
The patent MX 391053 B resulted from the work reported in this manuscript.