Antibacterial Ingredients and Modes of the Methanol-Phase Extract from the Fruit of Amomum villosum Lour.

Epidemics of infectious diseases threaten human health and society stability. Pharmacophagous plants are rich in bioactive compounds that constitute a safe drug library for antimicrobial agents. In this study, we have deciphered for the first time antibacterial ingredients and modes of the methanol-phase extract (MPE) from the fruit of Amomum villosum Lour. The results have revealed that the antibacterial rate of the MPE was 63.64%, targeting 22 species of common pathogenic bacteria. The MPE was further purified by high performance liquid chromatography (Prep-HPLC), and three different constituents (Fractions 1–3) were obtained. Of these, the Fraction 2 treatment significantly increased the cell membrane fluidity and permeability, reduced the cell surface hydrophobicity, and damaged the integrity of the cell structure, leading to the leakage of cellular macromolecules of Gram-positive and Gram-negative pathogens (p < 0.05). Eighty-nine compounds in Fraction 2 were identified by ultra HPLC-mass spectrometry (UHPLC-MS) analysis, among which 4-hydroxyphenylacetylglutamic acid accounted for the highest 30.89%, followed by lubiprostone (11.86%), miltirone (10.68%), and oleic acid (10.58%). Comparative transcriptomics analysis revealed significantly altered metabolic pathways in the representative pathogens treated by Fraction 2 (p < 0.05), indicating multiple antibacterial modes. Overall, this study first demonstrates the antibacterial activity of the MPE from the fruit of A. villosum Lour., and should be useful for its application in the medicinal and food preservative industries against common pathogens.


Introduction
Epidemics of infectious diseases threaten human health, cause loss of life, and seriously impact the economy [1,2].In recent decades, due to the inappropriate use of antibiotics, clinical antibiotic therapy has become increasingly ineffective in preventing outbreaks and spreading of infectious diseases [3].Therefore, it is imperative to search for safe and effective antimicrobial alternatives.Pharmacophagous plants with safety and low toxicity properties are traditionally used to treat many diseases.These plant extracts constitute an ideal drug library for antimicrobial agents [4].
Amomum villosum Lour. is a comestible medicinal plant that belongs to the Zingiberaceae family.This plant is mainly distributed in the tropical regions of Asia and Oceania.Its dry fruits and seeds are often used as cooking condiments, with a unique and rich aroma, and also used as valuable traditional medicines, such as for the obstruction of body dampness and turbidity, stomach deficiency and cold, and vomiting and diarrhea [5,6].Recent studies have provided experimental evidence for the pharmacological activities of A. villosum Lour., such as anti-ulcer, anti-diarrhea, and anti-inflammation [7].For example, Yin et al. [8] reported that the extracted labdane and norlabdane diterpenoids from the Based on our recent studies [11][12][13][14], the M-CE method was employed to extract antibacterial substances from the fruit of A. villosum Lour.Its water loss rate was 73.68% after freeze-drying at −80 • C for 48 h.The extraction yields of the MPE and chloroformphase extract (CPE) of A. villosum Lour.were 12.00% and 2.80%, respectively.
The antibacterial activities of the MPE and CPE of A. villosum Lour.were determined, targeting 22 species of common pathogenic bacteria.As shown in Table 1, the MPE showed an inhibition rate of 63.64% and repressed the growth of 14 species of bacteria, including the following: 2 species of Gram-positive bacteria: S. aureus and Bacillus cereus; and 12 species of Gram-negative bacteria: Aeromonas hydrophila, Pseudomonas aeruginosa, Shigella dysenteriae, Shigella flexneri, Shigella sonnei, Salmonella enterica subsp.enterica (ex Kauffmann and Edwards), Vibrio alginolyticus, Vibrio cholerae, Vibrio harveyi, Vibrio metschnikovi, Vibrio mimicus, and Vibrio parahaemolyticus.The CPE showed an inhibition rate of 54.55% and inhibited 1 species of Gram-positive and 11 species of Gram-negative bacteria (Table 1).Note: CPE: chloroform-phase extract.MPE: methanol-phase extract.-: no antibacterial activity.DIZ: diameters of inhibitory zone, including the diameter of the disc (6 mm).MIC: minimum inhibitory concentration.The values are expressed as the mean ± standard deviation (S.D.) of three parallel measurements.
Tang et al. [10] used water as a solvent to extract the EO of A. villosum Lour.and found its inhibitory effect on S. aureus ATCC43.In this study, our results indicated that the M-CE method was more effective in extracting antibacterial substances in A. villosum Lour.than water.
Given the higher antibacterial rate (63.64%), the MPE of A. villosum Lour.was subjected to further analysis in this study.The minimum inhibitory concentrations (MICs) of the MPE were determined, targeting the 14 species of pathogenic bacteria.As shown in Table 1, the MICs of the MPE ranged from 128 to 1024 µg/mL.The most effective inhibition was observed against the Gram-positive bacterium S. aureus GIM1.441 and the Gram-negative bacterium V. parahaemolyticus B2-28, with MICs of 128 µg/mL and 256 µg/mL, respectively (Table 1).

Purification of the MPE of A. villosum Lour.
The MPE of A. villosum Lour.was prepared in large quantities using the Prep-highperformance-liquid-chromatography (Prep-HPLC) technique.As shown in Figure 1, three distinct constituents (designated as Fractions 1-3) were observed at 1.9-4.2min when scanning at OD 211 for 15 min.
The antibacterial activity of the three different Fractions was further determined, and the results are presented in Table 2 and Figure 2. Fraction 2 displayed inhibitory effects on the two species of Gram-positive and eight species of Gram-negative bacteria.The diameters of the inhibitory zone (DIZ) ranged between 7 and 11.5 mm.In contrast, Fraction 1 and Fraction 3 had weak and no inhibitory effects, respectively (Table 2).The antibacterial activity of the three different Fractions was further determined, and the results are presented in Table 2 and Figure 2. Fraction 2 displayed inhibitory effects on the two species of Gram-positive and eight species of Gram-negative bacteria.The diameters of the inhibitory zone (DIZ) ranged between 7 and 11.5 mm.In contrast, Fraction 1 and Fraction 3 had weak and no inhibitory effects, respectively (Table 2).
We also determined the MICs of Fraction 2. As shown in Table 2, the strongest antibacterial efficacy of Fraction 2 was observed against S. aureus GIM1.441,B. cereus Y1, and V. parahemolyticus B2-28, with MICs of 256 µg/mL, 512 µg/mL, and 512 µg/mL, respectively, consistent with the results yielded from the MPE of A. villosum Lour.We also determined the MICs of Fraction 2. As shown in Table 2, the strongest antibacterial efficacy of Fraction 2 was observed against S. aureus GIM1.441,B. cereus Y1, and V. parahemolyticus B2-28, with MICs of 256 µg/mL, 512 µg/mL, and 512 µg/mL, respectively, consistent with the results yielded from the MPE of A. villosum Lour.
The Gram-positive bacterium S. aureus can cause human skin and tissue infections and sepsis in severe cases [15], while B. cereus can cause self-limiting emetic and diarrhea diseases [16].The Gram-negative bacterium V. parahemolyticus is a leading sea-food-borne pathogen worldwide, and common clinical symptoms include headache, nausea, vomiting, and diarrhea.Severe infections caused by V. parahemolyticus can develop into sepsis or even death [17].
In order to decipher the antibacterial mechanisms of Fraction 2 of A. villosum Lour., based on the above results, the Gram-positive bacteria S. aureus GIM1.441 and B. cereus Y1 and the Gram-negative bacterium V. parahemolyticus B2-28 were chosen as target strains for further analyses in this study.The Gram-positive bacterium S. aureus can cause human skin and tissue infections and sepsis in severe cases [15], while B. cereus can cause self-limiting emetic and diarrhea diseases [16].The Gram-negative bacterium V. parahemolyticus is a leading sea-food-borne pathogen worldwide, and common clinical symptoms include headache, nausea, vomiting, and diarrhea.Severe infections caused by V. parahemolyticus can develop into sepsis or even death [17].
In order to decipher the antibacterial mechanisms of Fraction 2 of A. villosum Lour., based on the above results, the Gram-positive bacteria S. aureus GIM1.441 and B. cereus Y1 and the Gram-negative bacterium V. parahemolyticus B2-28 were chosen as target strains for further analyses in this study.

Inhibited Growth of the Target Strains Treated with Fraction 2 of A. villosum Lour.
We determined growth curves of the three target strains treated with Fraction 2 of A. villosum Lour.As shown in Figure S1, S. aureus GIM1.441 was strongly inhibited when incubated in tryptone soybean broth (TSB) medium supplemented with 1 x MIC of Fraction 2. The inhibition showed a concentration-dependent mode, as S. aureus GIM1.441 was found to grow at 1/2 x MIC of Fraction 2, but showed lower biomass (maximum OD600 = 0.9065), as compared to the control group (maximum OD 600 = 1.205) (Figure S1A).
Similarly, the inhibition by the 1 x MIC of Fraction 2 was also stronger on B. cereus Y1 than the 1/2 x MIC (Figure S1B).The same case was apparent for the Gram-negative bacterium V. parahemolyticus B2-28, but this bacterium appeared to be the most sensitive to the Fraction 2 treatment among the test strains (Figure S1C).

Changed Cell Surface Hydrophobicity (CSH), Cell Membrane Fluidity (CMF), and Cell Membrane Permeability (CMP) of the Target Strains Treated with Fraction 2 of A. villosum Lour.
The interaction between microbial cells and the host is influenced by the biophysical properties of the cell membrane, such as the CSH [18].In this study, we observed that the CSH of the target strains was remarkably reduced in all of the treatment groups after 4 h-  We determined growth curves of the three target strains treated with Fraction 2 of A. villosum Lour.As shown in Figure S1, S. aureus GIM1.441 was strongly inhibited when incubated in tryptone soybean broth (TSB) medium supplemented with 1 x MIC of Fraction 2. The inhibition showed a concentration-dependent mode, as S. aureus GIM1.441 was found to grow at 1/2 x MIC of Fraction 2, but showed lower biomass (maximum OD 600 = 0.9065), as compared to the control group (maximum OD 600 = 1.205) (Figure S1A).
Similarly, the inhibition by the 1 x MIC of Fraction 2 was also stronger on B. cereus Y1 than the 1/2 x MIC (Figure S1B).The same case was apparent for the Gram-negative bacterium V. parahemolyticus B2-28, but this bacterium appeared to be the most sensitive to the Fraction 2 treatment among the test strains (Figure S1C).
Taken together, the 1 x MIC (256 µg/mL, 512 µg/mL, 512 µg/mL) of Fraction 2 was chosen as the treatment conditions for S. aureus GIM1.441,B. cereus Y1, and V. parahemolyticus B2-28, respectively, in the further analyses in this study.The interaction between microbial cells and the host is influenced by the biophysical properties of the cell membrane, such as the CSH [18].In this study, we observed that the CSH of the target strains was remarkably reduced in all of the treatment groups after 4 h-and 6 h-treatment of Fraction 2 of A. villosum Lour., as compared to the control groups (p < 0.05).Moreover, the decreased CSH of the strains was closely related to prolonged treatment time (Figure 3A).For example, after being treated with Fraction 2 for 2 h, the CSH of S. aureus GIM1.441 did not significantly change (p > 0.05).However, the decreased CSH was observed to be 1.23-fold and 2.05-fold after treatment for 4 h and 6 h, respectively (p < 0.001).Similarly, the CSH of B. cereus Y1 decreased by 1.07-fold to 2.56-fold after the treatment for 2 h to 6 h (p < 0.05).The strongest decrease (2.12-fold) in the CSH was found in V. parahaemolyticus B2-28 after being treated with Fraction 2 for 2 h (p < 0.001).
The CMF is also a key parameter of the bacterial cell membrane.In this study, 1,6diphenyl-1,3,5-hexatriene (DPH) was used as a probe to detect the changes in CMF of the target strains.Higher DPH values indicated weaker CMF [19].As shown in Figure 3B, as compared to the control groups, the CMF of the three target strains increased significantly (1.09-fold, 1.15-fold, and 1.63-fold) after being treated with Fraction 2 for 2 h (p < 0.05).Among the three strains, the CMF of V. parahaemolyticus B2-28 and B. cereus Y1 increased the most (3.06-fold and 9.35-fold) after the 4-h-and 6-h-treatment, respectively (p < 0.001).The bacterial cell membrane is a permeable barrier against external harmful substances; therefore, it is the therapeutic target of antimicrobial agents [20].In this study, the o-nitrophenyl-β-D-galactopyranoside (ONPG) was used as a probe to detect the changes in the CMP of the target strains.As shown in Figure 4A, as compared to the control group, there were no significant changes in the CMP of S. aureus GIM1.441 after being treated with Fraction 2 of A. villosum Lour.for 2 h and 4 h (p > 0.05).However, a significant increase in the CMP was observed after the 6 h-treatment (p < 0.05).B. cereus Y1 showed a 1.08-fold increase in the CMP after the treatment for 2 h (p < 0.05, Figure 4B).Similarly, the increased CMP of V. parahemolyticus B2-28 was also observed to be 1.14-fold to 1.25-fold after being treated with Fraction 2 for 2 h to 6 h (p < 0.05, Figure 4C).
Taken together, the Fraction 2 treatment can significantly reduce the CSH but increase the CMP and CMF of Gram-positive S. aureus GIM1.441 and B. cereus Y1 and Gramnegative V. parahaemolyticus B2-28.The antibacterial effects are exerted with the treatmenttime-dependent mode.The CMF is also a key parameter of the bacterial cell membrane.In this study, 1,6diphenyl-1,3,5-hexatriene (DPH) was used as a probe to detect the changes in CMF of the target strains.Higher DPH values indicated weaker CMF [19].As shown in Figure 3B, as compared to the control groups, the CMF of the three target strains increased significantly (1.09-fold, 1.15-fold, and 1.63-fold) after being treated with Fraction 2 for 2 h (p < 0.05).Among the three strains, the CMF of V. parahaemolyticus B2-28 and B. cereus Y1 increased the most (3.06-fold and 9.35-fold) after the 4-h-and 6-h-treatment, respectively (p < 0.001).
The bacterial cell membrane is a permeable barrier against external harmful substances; therefore, it is the therapeutic target of antimicrobial agents [20].In this study, the onitrophenyl-β-D-galactopyranoside (ONPG) was used as a probe to detect the changes in the CMP of the target strains.As shown in Figure 4A, as compared to the control group, there were no significant changes in the CMP of S. aureus GIM1.441 after being treated with Fraction 2 of A. villosum Lour.for 2 h and 4 h (p > 0.05).However, a significant increase in the CMP was observed after the 6 h-treatment (p < 0.05).B. cereus Y1 showed a 1.08-fold increase in the CMP after the treatment for 2 h (p < 0.05, Figure 4B).Similarly, the increased CMP of V. parahemolyticus B2-28 was also observed to be 1.14-fold to 1.25-fold after being treated with Fraction 2 for 2 h to 6 h (p < 0.05, Figure 4C).Based on the above results, we wondered whether the cell structure of the target strains was damaged by the Fraction 2 treatment.Therefore, we observed the cell structure Based on the above results, we wondered whether the cell structure of the target strains was damaged by the Fraction 2 treatment.Therefore, we observed the cell structure changes in S. aureus GIM1.441,B. cereus Y1, and V. parahaemolyticus B2-28 using a scanning electron microscope (SEM).As shown in Figure 5, the bacterial cells in the control groups were intact, with a full shape and clear structure.However, in the treatment groups, the cells showed varying degrees of folds, breaks, and pores after being treated with Fraction 2 for 2 h to 6 h.Based on the above results, we wondered whether the cell structure of the target strains was damaged by the Fraction 2 treatment.Therefore, we observed the cell structure changes in S. aureus GIM1.441,B. cereus Y1, and V. parahaemolyticus B2-28 using a scanning electron microscope (SEM).As shown in Figure 5, the bacterial cells in the control groups were intact, with a full shape and clear structure.However, in the treatment groups, the cells showed varying degrees of folds, breaks, and pores after being treated with Fraction 2 for 2 h to 6 h.For example, for the Gram-positive bacterium S. aureus GIM1.441, no significant change in the bacterial cell surface was observed after the 2 h-treatment with Fraction 2. However, the wrinkled cell surface occurred after the 4 h-treatment, and they even burst after the 6 h-treatment (Figure 5A).A similar case was found for B. cereus Y1 (Figure 5B).
For the Gram-negative bacterium V. parahemolyticus B2-28, severe ruffling on the cell surface, and even a wrinkled cell structure, were observed after the treatment for 2 h.Remarkably, the bacterial cells were fully ruptured after the treatment for 6 h (Figure 5C).
Tang et al. [10] reported that the surface of S. aureus ATCC43 appeared irregular, wrinkled, and uneven, but did not burst after being treated with the EO of A. villosum Lour.for 6 h.These results have provided additional evidence to validate that the MPE of A. villosum Lour.has a stronger antibacterial efficacy on the target strains than the EO.
Taken together, Fraction 2 of A. villosum Lour.can destroy the cell structure of Grampositive and Gram-negative bacteria to varying degrees.Moreover, the treatment is the most effective against the Gram-negative bacterium V. parahemolyticus B2-28.Damage to the cell structure may lead to the leakage of macromolecules.Therefore, we examined the nucleotide acid and protein exudation of the target strains treated with Fraction 2 of A. villosum Lour.As shown in Figure 6A, as compared to the control group, the amount of nucleotide acids exuded from S. aureus GIM1.441 was significantly increased by 1.55-fold after being treated with Fraction 2 for 2 h (p < 0.001).More extracellular nucleotide acids (2.22-fold and 3.18-fold) were detected with the longer treatment time (4 h and 6 h) (p < 0.001).A similar case was observed in B. cereus Y1 and V. parahaemolyticus B2-28.
markably, the bacterial cells were fully ruptured after the treatment for 6 h (Figure 5C).
Tang et al. [10] reported that the surface of S. aureus ATCC43 appeared irregular, wrinkled, and uneven, but did not burst after being treated with the EO of A. villosum Lour.for 6 h.These results have provided additional evidence to validate that the MPE of A. villosum Lour.has a stronger antibacterial efficacy on the target strains than the EO.
Taken together, Fraction 2 of A. villosum Lour.can destroy the cell structure of Grampositive and Gram-negative bacteria to varying degrees.Moreover, the treatment is the most effective against the Gram-negative bacterium V. parahemolyticus B2-28.

Nucleotide Acid and Protein Exudation of the Target Strains Treated with Fraction 2 of A. villosum Lour.
Damage to the cell structure may lead to the leakage of macromolecules.Therefore, we examined the nucleotide acid and protein exudation of the target strains treated with Fraction 2 of A. villosum Lour.As shown in Figure 6A, as compared to the control group, the amount of nucleotide acids exuded from S. aureus GIM1.441 was significantly increased by 1.55-fold after being treated with Fraction 2 for 2 h (p < 0.001).More extracellular nucleotide acids (2.22-fold and 3.18-fold) were detected with the longer treatment time (4 h and 6 h) (p < 0.001).A similar case was observed in B. cereus Y1 and V. parahaemolyticus B2-28.
As shown in Figure 6B, the number of extracellular proteins of S. aureus GIM1.441 was also significantly increased by 1.97-fold after being treated with Fraction 2 for 24 h (p < 0.001).Likewise, the proteins were exuded by 1.82-fold and 1.80-fold from B. cereus Y1 and V. parahemolyticus B2-28, respectively, after the Fraction 2 treatment (p < 0.001).As shown in Figure 6B, the number of extracellular proteins of S. aureus GIM1.441 was also significantly increased by 1.97-fold after being treated with Fraction 2 for 24 h (p < 0.001).Likewise, the proteins were exuded by 1.82-fold and 1.80-fold from B. cereus Y1 and V. parahemolyticus B2-28, respectively, after the Fraction 2 treatment (p < 0.001).
Bouyahya et al. [21] reported that the EO of Origanum compactum was involved in the changed membrane permeability and leakage of macromolecules.In this study, it can be concluded that the treatment with Fraction 2 of A. villosum Lour.results in the leakage of nucleotide acids and proteins from the Gram-positive and Gram-negative strains, consistent with the damaged bacterial cell structure observed with the SEM.In order to obtain insights into the changes in gene expression at the whole-genome level, we determined the transcriptomes of S. aureus GIM1.441,B. cereus Y1, and V. parahaemolyticus B2-28 treated with Fraction 2 (1 x MIC) for 6 h.The lists of the differential expressed genes (DEGs) in the three strains were deposited in the NCBI SRA database (https://sub-mit.ncbi.nlm.nih.gov/subs/bioproject/,accessed on 25 September 2023) under the accession number PRJNA1020669.The DEGs in the treatment group accounted for 11.12% (291/2617) of the S. aureus GIM1.441 genes, as compared to the control group.Of these, 91 DEGs showed lower transcription levels (fold change (FC) ≤ 0.5), whereas 200 DEGs were up-regulated (FC ≥ 2.0).Eight metabolic pathways were significantly altered in S. aureus GIM1.441, including valine, leucine, and isoleucine biosynthesis; the biosynthesis of various other secondary metabolites; ascorbate and aldarate metabolism; C5-branched dibasic acid metabolism; alanine, aspartate, and glutamate metabolism; o-antigen nucleotide sugar biosynthesis; ribosome; and the biosynthesis of various antibiotics (Figure 7, Table S1).
In alanine, aspartate, and glutamate metabolism, the expression of two DEGs was also significantly down-regulated (0.413-to 0.472-fold) in S. aureus GIM1.441 (p < 0.05).For example, the adenylosuccinate synthase (B4602_RS00095) was significantly inhibited (0.413-fold), which catalyzes the first committed step in the synthesis of adenosine [26].Conversely, four DEGs were significantly up-regulated (2.014-to 2.141-fold), e.g., the glutamate synthase large subunit (B4602_RS02195) (2.141-fold).Li et al. reported that this enzyme may play a key role in the antimicrobial resistance in cocci through its involvement in folate metabolism or cell membrane integrity [27].
Taken together, Fraction 2 of A. villosum Lour.altered the eight metabolic pathways in S. aureus GIM1.441, thereby likely inhibited the bacterial amino acid and secondary metabolite metabolisms, cell membrane biosynthesis, and resistance.The up-regulated expression of ribosome-related proteins may serve as a self-saving strategy for the bacterium to survive under the unfavorable circumstance elicited by the Fraction 2 treatment.The DEGs in the treatment group accounted for 44.45% (2363/5316) of the B. cereus Y1 genes.Of these, notably, 2173 DEGs were down-regulated (FC ≤ 0.5), whereas 190 DEGs were up-regulated (FC ≥ 2.0).Seven metabolic pathways were significantly altered in B. cereus Y1, including bacterial chemotaxis; nucleotide excision repair; histidine metabolism; mismatch repair; valine, leucine, and isoleucine degradation; the biosynthesis of siderophore group nonribosomal peptides; and o-antigen nucleotide sugar biosynthesis (Figure 8, Table S2).For example, in the bacterial chemotaxis, the expression of 24 DEGs was significantly down-regulated (0.11-to 0.479-fold) at the transcriptional level in B. cereus Y1 after being treated with Fraction 2 (p < 0.05), e.g., the methyl-accepting chemotaxis protein (MCP) (EJ379_25705), chemotaxis signal transduction protein CheV (EJ379_08390), flagellar motor protein MotB (EJ379_23055), and glutamate O-methyltransferase CheR (EJ379_05155).For instance, the MCP plays an important role in cell survival and biodegradation [30], and can also control the direction of flagella motors, promoting cell rolling and smooth For example, in the bacterial chemotaxis, the expression of 24 DEGs was significantly down-regulated (0.11-to 0.479-fold) at the transcriptional level in B. cereus Y1 after being treated with Fraction 2 (p < 0.05), e.g., the methyl-accepting chemotaxis protein (MCP) (EJ379_25705), chemotaxis signal transduction protein CheV (EJ379_08390), flagellar mo-tor protein MotB (EJ379_23055), and glutamate O-methyltransferase CheR (EJ379_05155).For instance, the MCP plays an important role in cell survival and biodegradation [30], and can also control the direction of flagella motors, promoting cell rolling and smooth swimming [31].
In mismatch repair, the expression of 13 DEGs was significantly down-regulated (0.242to 0.500-fold) in B. cereus Y1 (p < 0.05).For example, the DNA mismatch repair endonuclease MutL (EJ379_19260) was significantly repressed (0.332-fold).The MutL family of DNA mismatch repair proteins plays a key role in the cleavage and repair of mismatch errors during DNA replication [33].
For example, in glyoxylate and dicarboxylate metabolism, the expression of eight DEGs was significantly inhibited (0.126-fold to 0.499-fold) in V. parahemolyticus B2-28 after being treated with Fraction 2 of A. villosum Lour., whereas two DEGs were significantly up-regulated (2.081-to 4.678-fold) (p < 0.05).For instance, the multidrug transporter AcrB (Vp_B2_28_4316) was highly repressed (0.126-fold), which is a component of the multidrug efflux pumps that play a key role in the process of bacterial resistance [34].In contrast, the catalase (Vp_B2_28_0438) was significantly up-regulated (2.081-fold), which has been reported to prevent cellular oxidative damage by decomposing hydrogen peroxide into water and oxygen [35].
In propanoate metabolism, the expression of nine DEGs was also significantly inhibited (0.199-fold to 0.432-fold) (p < 0.05), e.g., the DNA mismatch repair protein MutT, histone acetyltransferase, and phosphoenolpyruvate protein phosphotransferase (PtsA).The latter was significantly down-regulated (0.264-fold), which is involved in the monosaccharide phosphotransferase system.Nebenzahl et al. [36] reported that the cell-wall-localized PtsA can function as an adhesin, and anti-PtsA antisera were shown to inhibit the adhesion of S. pneumoniae to cultured human lung adenocarcinoma cells A549.
being treated with Fraction 2 of A. villosum Lour., whereas two DEGs were significantly up-regulated (2.081-to 4.678-fold) (p < 0.05).For instance, the multidrug transporter AcrB (Vp_B2_28_4316) was highly repressed (0.126-fold), which is a component of the multidrug efflux pumps that play a key role in the process of bacterial resistance [34].In contrast, the catalase (Vp_B2_28_0438) was significantly up-regulated (2.081-fold), which has been reported to prevent cellular oxidative damage by decomposing hydrogen peroxide into water and oxygen [35].In propanoate metabolism, the expression of nine DEGs was also significantly inhibited (0.199-fold to 0.432-fold) (p < 0.05), e.g., the DNA mismatch repair protein MutT, histone acetyltransferase, and phosphoenolpyruvate protein phosphotransferase (PtsA).The latter was significantly down-regulated (0.264-fold), which is involved in the monosaccharide phosphotransferase system.Nebenzahl et al. [36] reported that the cell-wall-localized PtsA can function as an adhesin, and anti-PtsA antisera were shown to inhibit the adhesion of S. pneumoniae to cultured human lung adenocarcinoma cells A549.
In the QS, the expression of 20 DEGs was significantly inhibited (0.116-fold to 0.495-fold) in V. parahemolyticus B2-28 (p < 0.05).Many previous studies have indicated that the formation of QS and biofilm promotes the development of antibiotic resistance in microorganisms [41].In this study, for example, the DEG encoding a ketol-acid reductoisomerase (KARI) (Vp_B2_28_4615) was significantly down-regulated (0.434-fold), which has been reported to be a potential drug target against pathogenic bacteria [42].
In arginine and proline metabolism, the expression of 13 DEGs was significantly inhibited (0.071-fold to 0.284-fold) (p < 0.05).In contrast, remarkably, the expression of endonuclease I (Vp_B2_28_2907) was strongly enhanced (70.055-fold), suggesting that DNA breakage may have occurred in V. parahaemolyticus B2-28 after the Fraction 2 treatment.
In taurine and hypotaurine metabolism, the expression of four DEGs was significantly down-regulated (0.353-to 0.488-fold) in V. parahaemolyticus B2-28 (p < 0.05).For instance, the RNA chaperone ProQ (Vp_B2_28_2494) and tail-specific protease (Vp_B2_28_2495) were repressed (0.353-fold and 0.42-fold).The former mediates sRNA-directed gene regulation in Gram-negative bacteria, while the latter is involved in tolerance to heat stress and virulence [43,44].In addition, the DEG encoding a phage shock protein (Psp) G was greatly down-regulated (0.098-fold).The Psp stress response system can sense and respond to cell membrane damage [45].
Taken together, Fraction 2 of A. villosum Lour.significantly altered sixteen metabolic pathways in V. parahemolyticus B2-28, and thus hindered the amino acid metabolism, cell membrane biosynthesis, and substance transportation; and repressed the intercellular communication, stress regulation, and virulence, leading to cellular oxidative damage, DNA breakage, and cell death.
Additionally, to validate the transcriptome data, we performed real-time reverse transcription quantitative PCR (RT-qPCR) analysis on fifteen representative DEGs, and the obtained results were generally consistent with the transcriptome data (Tables S4 and S5).As shown in Table S6, Fraction 2 of A. villosum Lour.displayed different antibacterial modes against the Gram-positive and Gram-negative bacteria and hindered a series of metabolic pathways, leading to varying levels of cell damage and even death.On the other hand, the same metabolic pathways, such as o-antigen nucleotide sugar biosynthesis and valine, leucine, and isoleucine metabolisms, were all inhibited in the Gram-positive bacteria S. aureus GIM1.441 and B. cereus Y1 by Fraction 2. Additionally, some metabolic pathways were only hindered in the Gram-negative bacterium.parahemolyticus B2-28 by Fraction 2, such as the repressed substance transporting, intercellular communication, stress regulation, and virulence.
Overall, the results of this study demonstrate that Fraction 2 of A. villosum Lour.exerts the strongest inhibitory efficacy on the Gram-negative bacterium V. parahemolyticus B2-28, followed by the Gram-positive bacteria B. cereus Y1 and S. aureus GIM1.441 through multiple antibacterial modes.

Identification of Potential Antibacterial Compounds in Fraction 2 of A. villosum Lour.
Based on the above results, we wondered what compounds functioned in Fraction 2 of A. villosum Lour.Therefore, the antibacterial components of Fraction 2 were further identified using the ultra-HPLC and mass spectrometry (UHPLC-MS) technique.As shown in Table 3, eighty-nine compounds were identified.The most abundant compound in Fraction 2 was 4-hydroxyphenylacetylglutamic acid (30.89%), followed by lubiprostone (11.86%), miltirone (10.68%), oleic acid (10.58%), and oxymorphone (5.69%).The other compounds (4.81-0.10%),such as alkaloids, flavonoids, phenols, and coumarins, were also identified (Table 3).The 4-hydroxyphenylacetylglutamic identified in this study is an acetyl compound of glutamate.It has been reported that this compound can pass through the bloodbrain barrier, improve nerve cell metabolism, maintain nervous stress, and reduce blood ammonia [46].The lubiprostone identified in this study can safely and effectively treat chronic idiopathic constipation and irritable bowel syndrome with constipation [47].Terpenoids, such as the miltirone identified in this study, have enormous inhibitory potential against microorganisms through different mechanisms such as membrane disruption, anti-QS, and protein and ATP synthesis inhibition [48].The 8-Geranyloxypsoralen, bergamotine, and 3,4-Dihydrocoumarin identified in this study are coumarins, with pharmacological activities such as antibacterial, anti-inflammatory, and anti-cancer [49].The piperlonguminine identified in this study is a compound of the alkaloid class that has been proved to have anti-inflammatory activity [50].The isoquercitrin identified in this study has a variety of chemical protection effects in vitro and in vivo against oxidative stress, cancer, cardiovascular disease, diabetes, and allergic reaction [51].
Taken together, these results have revealed potential antibacterial compounds in Fraction 2 of A. villosum Lour., a promising antibacterial source of natural products.
The major limitation of this study was that the top compounds identified in Fraction 2 were not available commercially, therefore, the antibacterial activity of each compound could not be analyzed.It would be interesting to continue with the proposed analysis through a chemical synthesis method to obtain single compounds in future research.

Bacterial Strains and Culture Conditions
The bacterial strains and media used in this study are shown in Table S7.The incubation conditions of the bacterial strains were the same as those described in our recent reports [11][12][13][14].

Extraction of Bacteriostatic Substances from A. villosum Lour.
The fresh fruit samples were purchased from the production base of A. villosum Lour. in Yangchun City (22 • 41 01 N, 111 • 16 27 E), Guangdong Province, China (Figure S2).The fruit is oval, purplish red when ripe, and brown after dry, with a unique and rich aroma.The antibacterial components in the samples were extracted using the M-CE method, as reported in our recent studies [11][12][13][14].Briefly, the fresh samples were washed with water, cut into small pieces of about 1/4 size, and pre-frozen at −80 • C for 8 h.Thereafter, the samples were further freeze-dried, crushed, and extracted with the methanol-chloroform (2:1, v/v, analytical grade, Merck KGaA, Darmstadt, Germany) at a solid-to-liquid ratio of 1:10 (m/v) for 5 h.A certain amount of H 2 O (analytical grade) was added, sonicated, and filtered, and then the MPE and CPE were separated using the same equipment and parameters described in our recent reports [11][12][13][14].

Antibacterial Activity Assay
The sensitivity of the bacterial strains to the MPE and CPE from A. villosum Lour.was measured according to the standard method approved by the Clinical and Laboratory Standards Institute, Malvern, PA, USA (CLSI, M100-S23, 2018).The MICs of the extracts from A. villosum Lour.were determined against the target strains.The definition of antibacterial activity and MICs were described in our recent reports [11][12][13][14].

Prep-HPLC Analysis
The MPE of A. villosum Lour.was isolated using a Waters 2707 autosampler (Waters, Milford, MA, USA) linked with a UPLC Sunfifire C 18 column (Waters, Milford, MA, USA).The parameters of the Prep-HPLC analysis were the same as those described in our recent reports [11][12][13][14].

Growth Curve Assay
The 1 x MIC and 1/2 x MIC of Fraction 2 of A. villosum Lour.were individually added into the bacterial cell culture of the target strains at the mid-logarithmic growth phase (mid-LGP) and then incubated at 37 • C for 24 h.The growth curves of the target strains were determined using the automatic growth curve analyzer (Synergy, BioTekInstruments, Winooski, VT, USA).

The CSH, CMF, and CMP Assays
The CSH of the target strains was determined according to the method of Cui et al. [52].Briefly, the target strains at the mid-LGP were treated with Fraction 2 of A. villosum Lour for 2 h, 4 h, and 6 h, respectively.A total of 1 mL of hexadecane (National Pharmaceutical Group Corporation Co., Ltd., Shanghai, China) was added to an equal volume of the treated bacterial suspension, mixed for 1 min, and set at 37 • C for 30 min.Thereafter, the absorbance values of the mixture were measured at 600 nm.The CMF of the target strains was determined using the DPH (Sangon, Shanghai, China) as a probe, according to the method described in our recent reports [11][12][13][14].The CMP of the target strains was measured using the OPNG (Beijing Solarbio Science & Technology Co., Ltd., Beijing, China) as a probe [11][12][13][14].

Bacterial Nucleotide Acid and Protein Exudation Assays
The nucleotide acid exudation of the target strains was measured according to the method of Lin et al. [53], with minor modifications.Briefly, the bacterial cell culture at the mid-LGP was treated with Fraction 2 (1 x MIC) of A. villosum Lour for 2 h, 4 h, and 6 h, respectively.The bacterial cell suspension was centrifuged at 4 • C at 3500 rpm for 5 min.Thereafter, the absorbance values of the supernatant were measured at 260 nm.
The bacterial protein exudation was determined according to the method of Atta et al. [54].Briefly, Fraction 2 (1 x MIC) of A. villosum Lour was added into the bacterial cell culture at the mid-LGP and then incubated at a stationary condition at 37 • C for 24 h.The extracellular protein concentrations were determined using the Bradford method protein concentration determination kit (Sangong, Shanghai, China) according to the manufacturer's instructions.

Illumina RNA Sequencing
The bacterial cell culture at the mid-LGP of the target strains was individually treated with Fraction 2 (1 x MIC) of A. villosum Lour for 6 h.The total RNA of the harvested bacterial cells was extracted, purified, and analyzed, as described in our recent reports [11][12][13][14].Three independently prepared RNA samples were used for each Illumina RNA-sequencing analysis, which was conducted by Shanghai Majorbio Bio-pharm Technology Co., Ltd.(Shanghai, China) using Illumina HiSeq 2500 platform (Illumina, Santiago, CA, USA) [11][12][13][14].

Figure 2 .
Figure 2. The DIZs of the MPE and Fraction 2 of MPE from A. villosum Lour.(A-C): the MPE, Fraction 2 of MPE, and negative control, respectively.

Figure 2 .
Figure 2. The DIZs of the MPE and Fraction 2 of MPE from A. villosum Lour.(A-C): the MPE, Fraction 2 of MPE, and negative control, respectively.

2. 3 .
Inhibited Growth of the Target Strains Treated with Fraction 2 of A. villosum Lour.

2. 5 .
Changed Cell Morphological Structure of the Target Strains Treated with Fraction 2 of A. villosum Lour.

2. 6 .
Nucleotide Acid and Protein Exudation of the Target Strains Treated with Fraction 2 of A. villosum Lour.

2. 7 .
The Altered Metabolic Pathways in the Target Strains Treated with Fraction 2 of A. villosum Lour.

Figure 7 .
Figure 7.The eight significantly altered metabolic pathways in S. aureus GIM1.441 treated with Fraction 2 of A. villosum Lour.(A,B): Volcano plot of the DGEs and the changed metabolic pathways, respectively.

Figure 7 .
Figure 7.The eight significantly altered metabolic pathways in S. aureus GIM1.441 treated with Fraction 2 of A. villosum Lour.(A,B): Volcano plot of the DGEs and the changed metabolic pathways, respectively.

Figure 8 .
Figure 8.The significantly altered metabolic pathways in B. cereus Y1 treated with Fraction 2 of A. villosum Lour.(A,B): Volcano plot of the DGEs and the changed metabolic pathways, respectively.

Figure 8 .
Figure 8.The significantly altered metabolic pathways in B. cereus Y1 treated with Fraction 2 of A. villosum Lour.(A,B): Volcano plot of the DGEs and the changed metabolic pathways, respectively.

Figure 9 .
Figure 9.The significantly altered metabolic pathways in V. parahemolyticus B2-28 treated with Fraction 2 of A. villosum Lour.(A,B): Volcano plot of the DGEs and the changed metabolic pathways, respectively.

Figure 9 .
Figure 9.The significantly altered metabolic pathways in V. parahemolyticus B2-28 treated with Fraction 2 of A. villosum Lour.(A,B): Volcano plot of the DGEs and the changed metabolic pathways, respectively.

Table 1 .
Antibacterial activities of the MPE and CPE from the fruit of A. villosum Lour.

Table 2 .
Antibacterial activities of Fraction 2 of the MPE of A. villosum Lour.

Table 3 .
Potential antibacterial compounds identified in Fraction 2 of A. villosum Lour.by the UHPLC-MS analysis.