Identification of Antibacterial Components in the Methanol-Phase Extract from Edible Herbaceous Plant Rumex madaio Makino and Their Antibacterial Action Modes

Outbreaks and prevalence of infectious diseases worldwide are some of the major contributors to morbidity and morbidity in humans. Pharmacophageous plants are the best source for searching antibacterial compounds with low toxicity to humans. In this study, we identified, for the first time, antibacterial components and action modes of methanol-phase extract from such one edible herbaceous plant Rumex madaio Makino. The bacteriostatic rate of the extract was 75% against 23 species of common pathogenic bacteria. The extract was further purified using the preparative high-performance liquid chromatography (Prep-HPLC) technique, and five separated componential complexes (CC) were obtained. Among these, the CC 1 significantly increased cell surface hydrophobicity and membrane permeability and decreased membrane fluidity, which damaged cell structure integrity of Gram-positive and -negative pathogens tested. A total of 58 different compounds in the extract were identified using ultra-HPLC and mass spectrometry (UHPLC-MS) techniques. Comparative transcriptomic analyses revealed a number of differentially expressed genes and various changed metabolic pathways mediated by the CC1 action, such as down-regulated carbohydrate transport and/or utilization and energy metabolism in four pathogenic strains tested. Overall, the results in this study demonstrated that the CC1 from R. madaio Makino are promising candidates for antibacterial medicine and human health care products.


Introduction
China is one of the richest countries in biodiversity, with very high levels of plant endemism [1]. Pharmacopoeia of the Peoples' Republic of China (2020 Edition) contains 2711 species of Chinese herbal plants, which constitute a gold mine for exploiting medicine candidates and health care products [2]. For instance, R. madaio Makino is an edible, perennial and herbaceous plant that belongs to the Dicotyledoneae class, Polygonaceae family, and Rumex genus. According to the National Compilation of Chinese Herbal Medicine (1996 Edition), leaf and root tissues of R. madaio Makino can be used as medicine such as clearing heat and detoxification, removing blood stasis, and defecating and killing insects. Nevertheless, current studies on the antibacterial activity of R. madaio Makino are rare.
In this study, antibacterial components and action modes of methanol-phase extract from R. madaio Makino were for the first time identified. The objectives of this study were: (1) to extract bioactive substances from R. madaio Makino using the methanol and chloroform extraction (MCE) method, and determine their inhibition activity against 23 species In this study, antibacterial components and action modes of methanol-phase extract from R. madaio Makino were for the first time identified. The objectives of this study were: (1) to extract bioactive substances from R. madaio Makino using the methanol and chloroform extraction (MCE) method, and determine their inhibition activity against 23 species of pathogenic bacteria; (2) to purify the methanol-phase extract from R. madaio Makino by preparation high-performance liquid chromatography (Prep-HPLC) analysis, and identify bioactive compounds in componential complex 1 (CC 1) using an ultra-HPLC and mass spectrometry (UHPLC-MS) technique; (3) to determine cell surface hydrophobicity, cell membrane permeability, fluidity, and the damage of four representative pathogenic strains treated with the CC 1; (4) to decipher possible molecular mechanisms underlying antibacterial activity by comparative transcriptomic analysis. The results of this study meet the increasing need for novel antibacterial agent candidates against common pathogenic bacteria.

Antibacterial Activity of Crude Extracts from R. madaio Makino
Antibacterial substances in fresh leaf and stem tissues of R. madaio Makino were extracted using the MCE method. The results showed that the water loss rate of the plant material was 93.32%, and extraction rates of the methanol phase and chloroform phase were 32.10% and 29.60%, respectively. Antibacterial activity of the crude extracts against 23 species of pathogenic bacteria was determined, most of which are common foodborne pathogens, and the results are presented in Table 1. The chloroform-phase crude extract from R. madaio Makino showed a bacteriostatic rate of 39%, inhibiting 2 species of Gram-positive and 11 species of Gram-negative pathogens (Table 1, Figure 1). Remarkably, the methanol-phase crude extract from R. madaio Makino inhibited the growth of 33 bacteria strains tested with a bacteriostatic rate of 75%, including 2 species of Gram-positive and 18 species of Gram-negative pathogens (Table 1). Based on the higher bacteriostatic rate (75%), the methanol-phase crude extract from R. madaio Makino was chosen for further analysis in this study.

Purification of the Methanol-Phase Crude Extract from R. madaio Makino
Large amounts of the methanol-phase crude extract from R. madaio Makino were further purified by the Prep-HPLC analysis. As shown in Figure 2, five obviously separated peaks (designated as componential complex, CCs 1 to 5) were observed by scanning at OD 280 nm for 15 min. These five single peaks were individually collected for antibacterial activity analysis. The results revealed that the CC 1 had strong inhibitory effects on Vibrio parahaemolyticus ATCC17802, Vibrio alginolyticus ATCC17749, Bacillus cereus A1-1, and V. parahaemolyticus B4-10. Moreover, the growth of the other four strains was also depressed, including V. parahaemolyticus ATCC33847, V. parahaemolyticus B3-13, V. parahaemolyticus B5-29, and Staphylococcus aureus ATCC6538 (Table 2). Among these, V. alginolyticus is an opportunistic pathogenic bacterium that can infect a broad range of marine host animals, including fish, crab and pearl oysters, and can also infect the human ear, soft tissue and wounded sites [3,4], while V. parahaemolyticus is a leading seafood-borne pathogen worldwide and can cause acute gastroenteritis and septicemia in humans [5]. B. cereus is a Gram-positive bacterium for food poisoning. This bacterium has been incriminated in clinical conditions such as anthrax-like progressive pneumonia, fulminant sepsis, and devastating central nervous system infections, particularly in immunosuppressed individuals, intravenous drug abusers, and neonates [6].
Conversely, the other four peaks (CCs 2 to 4) showed weak or no antibacterial activity, indicating that bioactive compounds in the methanol-phase extract from R. madaio Makino existed in the CC 1.
MIC values of the CC 1 were also determined, which was 64 μg/mL against V. alginolyticus ATCC17749 and V. parahaemolyticus ATCC17802; 128 μg/mL against B. cereus A1-1; and 256 μg/mL against V. parahaemolyticus B4-10.  These five single peaks were individually collected for antibacterial activity analysis. The results revealed that the CC 1 had strong inhibitory effects on Vibrio parahaemolyticus ATCC17802, Vibrio alginolyticus ATCC17749, Bacillus cereus A1-1, and V. parahaemolyticus B4-10. Moreover, the growth of the other four strains was also depressed, including V. parahaemolyticus ATCC33847, V. parahaemolyticus B3-13, V. parahaemolyticus B5-29, and Staphylococcus aureus ATCC6538 (Table 2). Among these, V. alginolyticus is an opportunistic pathogenic bacterium that can infect a broad range of marine host animals, including fish, crab and pearl oysters, and can also infect the human ear, soft tissue and wounded sites [3,4], while V. parahaemolyticus is a leading seafood-borne pathogen worldwide and can cause acute gastroenteritis and septicemia in humans [5]. B. cereus is a Gram-positive bacterium for food poisoning. This bacterium has been incriminated in clinical conditions such as anthrax-like progressive pneumonia, fulminant sepsis, and devastating central nervous system infections, particularly in immunosuppressed individuals, intravenous drug abusers, and neonates [6]. Conversely, the other four peaks (CCs 2 to 4) showed weak or no antibacterial activity, indicating that bioactive compounds in the methanol-phase extract from R. madaio Makino existed in the CC 1.

Changed Bacterial Cell Surface Structure by the CC 1 Extract
To decipher possible mechanisms underlying bacteriostatic activity of the CC 1, the cell structure of the four highly inhibited strains were observed by the transmission electron microscope (TEM) analysis. As shown in Figure 3, in remarkable contrast to control groups whose cell surface structure was intact, showing rod cells, a flat surface, and a clear structure, bacterial cells in the treatment groups showed different degrees of contraction and rupture, some of which were deformed with obvious depressions, folds or cavities on the surface. For example, for the Gram-positive B. cereus A1-1, the 2 h treatment by the CC 1 resulted in the bacterial cell surface shrinking seriously, the flagella breaking, and some contents leaking. After being treated for 4 h, cell surface shrinkage was intensified, and more cells were ruptured. After being treated for 6 h, the cell structure was seriously damaged, a large number of contents exuded, and only a few cells still maintained rod shape ( Figure 3A). For the Gram-negative V. parahaemolyticus ATCC17802, after being treated with the CC 1 for 2 h, its cell surface shrunk slightly, and pili structure was still visible. However, after being treated for 4 h, the cell surface shrinkage increased and the cell membrane folded. V. parahaemolyticus ATCC17802 cells were destroyed, seriously shrunk and deformed after being treated for 6 h ( Figure 3C). These results indicated that the CC 1 from R. madaio Makino damaged the cell surface structure of the Gram-negative and Gram-positive pathogens. To decipher possible mechanisms underlying bacteriostatic activity of the CC 1, the cell structure of the four highly inhibited strains were observed by the transmission electron microscope (TEM) analysis. As shown in Figure 3, in remarkable contrast to control groups whose cell surface structure was intact, showing rod cells, a flat surface, and a clear structure, bacterial cells in the treatment groups showed different degrees of contraction and rupture, some of which were deformed with obvious depressions, folds or cavities on the surface. For example, for the Gram-positive B. cereus A1-1, the 2 h treatment by the CC 1 resulted in the bacterial cell surface shrinking seriously, the flagella breaking, and some contents leaking. After being treated for 4 h, cell surface shrinkage was intensified, and more cells were ruptured. After being treated for 6 h, the cell structure was seriously damaged, a large number of contents exuded, and only a few cells still maintained rod shape ( Figure 3A). For the Gram-negative V. parahaemolyticus ATCC17802, after being treated with the CC 1 for 2 h, its cell surface shrunk slightly, and pili structure was still visible. However, after being treated for 4 h, the cell surface shrinkage increased and the cell membrane folded. V. parahaemolyticus ATCC17802 cells were destroyed, seriously shrunk and deformed after being treated for 6 h ( Figure 3C). These results indicated that the CC 1 from R. madaio Makino damaged the cell surface structure of the Gram-negative and Gram-positive pathogens.

Changed Bacterial Cell Surface Hydrophobicity, Cell Membrane Fluidity, Permeability, and Damage by the CC 1 from R. madaio Makino
Cell surface hydrophobicity plays an important role in the adhesion to abiotic and biological surfaces and infiltration of host tissue [7]. In this study, bacterial cell surface hydrophobicity of all four experimental groups was significantly increased (p < 0.05) when compared with the control groups ( Figure 4A). The effect was highly enhanced with the increase in treatment time. For example, cell surface hydrophobicity was significantly increased in V. parahaemolyticus ATCC17802 (1.47-fold), V. parahaemolyticus B4-10 (1.62-fold) and B. cereus A1-1 (1.42-fold) after being treated with the CC1 for 2 h (p < 0.05), whereas a similar change was observed in the treatment group of V. alginolyticus ATCC17749 (1.48-fold) after being treated for 4 h. Moreover, the highest increase in cell surface hydrophobicity was observed in B. cereus A1-1 (3.75-fold) after being treated with the CC1 for 6 h ( Figure 4A).
Membrane fluidity is also a key parameter of the bacterial cell membrane that undergoes quick adaptation in response to environmental challenges [8]. It has recently been regarded as an important factor in the antibacterial mechanism of membrane-targeting antibiotics [9]. In this study, compared with the control groups, there was no significant difference in cell membrane fluidity of V. parahaemolyticus ATCC17802 and  Cell surface hydrophobicity plays an important role in the adhesion to abiotic and biological surfaces and infiltration of host tissue [7]. In this study, bacterial cell surface hydrophobicity of all four experimental groups was significantly increased (p < 0.05) when compared with the control groups ( Figure 4A). The effect was highly enhanced with the increase in treatment time. For example, cell surface hydrophobicity was significantly increased in V. parahaemolyticus ATCC17802 (1.47-fold), V. parahaemolyticus B4-10 (1.62-fold) and B. cereus A1-1 (1.42-fold) after being treated with the CC1 for 2 h (p < 0.05), whereas a similar change was observed in the treatment group of V. alginolyticus ATCC17749 (1.48-fold) after being treated for 4 h. Moreover, the highest increase in cell surface hydrophobicity was observed in B. cereus A1-1 (3.75-fold) after being treated with the CC1 for 6 h ( Figure 4A).
Membrane fluidity is also a key parameter of the bacterial cell membrane that undergoes quick adaptation in response to environmental challenges [8]. It has recently been regarded as an important factor in the antibacterial mechanism of membrane-targeting antibiotics [9]. In this study, compared with the control groups, there was no significant difference in cell membrane fluidity of V. parahaemolyticus ATCC17802 and B4-10, as well as V. alginolyticus ATCC17749 after being treated with the CC 1 for 2 h (p > 0.05). However, a significant decrease in membrane fluidity of these three strains was observed after the treatment for 4 h. Additionally, cell membrane fluidity significantly declined in B. cereus A1-1 (1.20-fold) treated with the CC 1 for 2 h, and sharply lost for 6 h (8.11-fold) ( Figure 4B). The change of membrane lipid composition likely contributed to the observed membrane fluidity change to resist the lipid disorder effect by therapeutic agents [10].
Molecules 2022, 26, x 7 of 30 B4-10, as well as V. alginolyticus ATCC17749 after being treated with the CC 1 for 2 h (p > 0.05). However, a significant decrease in membrane fluidity of these three strains was observed after the treatment for 4 h. Additionally, cell membrane fluidity significantly declined in B. cereus A1-1 (1.20-fold) treated with the CC 1 for 2 h, and sharply lost for 6 h (8.11-fold) ( Figure 4B). The change of membrane lipid composition likely contributed to the observed membrane fluidity change to resist the lipid disorder effect by therapeutic agents [10]. The o-nitrophenyl-β-D-galactopyranoside (o-nitrophenyl)-β-D-galactopyranoside (ONPG) was used as a probe to monitor the inner cell membrane permeability of the four bacterial strains, and the results were illustrated in Figure 5. Different influence of the CC 1 from R. madaio Makino on inner cell membrane permeability was observed among the four treatment groups. For example, V. alginolyticus ATCC17749 did not change significantly in the inner cell membrane permeability after the treatment for 2 h (p > 0.05), whereas a significant increase was observed after being treated for 4 h (1.15-fold) and 6 h (1.18-fold), respectively (p < 0.05) ( Figure 5). The o-nitrophenyl-β-D-galactopyranoside (o-nitrophenyl)-β-D-galactopyranoside (ONPG) was used as a probe to monitor the inner cell membrane permeability of the four bacterial strains, and the results were illustrated in Figure 5. Different influence of the CC 1 from R. madaio Makino on inner cell membrane permeability was observed among the four treatment groups. For example, V. alginolyticus ATCC17749 did not change significantly in the inner cell membrane permeability after the treatment for 2 h (p > 0.05), whereas a significant increase was observed after being treated for 4 h (1.15-fold) and 6 h (1.18-fold), respectively (p < 0.05) ( Figure 5).
N-Phenyl-1-naphthylamine (NPN) was used as a probe to monitor the bacterial outer membrane permeability. As shown in Figure 6, the outer membrane permeability in the four experimental groups were all highly increased after the treatment with the CC 1 for 2 h (p < 0.01). The highest increase was found in B. cereus A1-1 (6.06-fold) after being treated for 6 h, whereas an opposite pattern was observed in V. parahaemolyticus ATCC17802 (1.77-fold).
As shown in Figure 4C, when compared with the control groups, cell membrane damage rates of all four experimental groups significantly increased (p < 0.05), which raised with the increase in treatment time. Significant damage was observed in B. cereus A1-1 (2.95-fold) and V. parahaemolyticus B4-10 (2.21-fold) after being treated for 2 h, whereas a similar change was found in the other two strains treated for 4 h. Moreover, cell membrane damage of B. cereus A1-1 was the most severe among the four strains after being treated for 6 h (8.54-fold).
Taken together, these results demonstrated that the CC 1 from R. madaio Makino significantly increased bacterial cell surface hydrophobicity and membrane permeability and decreased membrane fluidity of V. parahaemolyticus ATCC17802, V. parahaemolyticus B4-10, V. alginolyticus ATCC17749, and B. cereus A1-1, consistent with the observed bacterial surface structure by the TEM analysis. The damaged cell surface and membrane structure integrity were beneficial for the CC1 to penetrate bacterial cell envelope to target intracellular processes. N-Phenyl-1-naphthylamine (NPN) was used as a probe to monitor the bacterial outer membrane permeability. As shown in Figure 6, the outer membrane permeability in the four experimental groups were all highly increased after the treatment with the CC 1 for 2 h (p < 0.01). The highest increase was found in B. cereus A1-1 (6.06-fold) after being treated for 6 h, whereas an opposite pattern was observed in V. parahaemolyticus ATCC17802 (1.77-fold).
As shown in Figure 4C, when compared with the control groups, cell membrane damage rates of all four experimental groups significantly increased (p < 0.05), which raised with the increase in treatment time. Significant damage was observed in B. cereus A1-1 (2.95-fold) and V. parahaemolyticus B4-10 (2.21-fold) after being treated for 2 h, whereas a similar change was found in the other two strains treated for 4 h. Moreover, cell membrane damage of B. cereus A1-1 was the most severe among the four strains after being treated for 6 h (8.54-fold).
Taken together, these results demonstrated that the CC 1 from R. madaio Makino significantly increased bacterial cell surface hydrophobicity and membrane permeability and decreased membrane fluidity of V. parahaemolyticus ATCC17802, V. parahaemolyticus B4-10, V. alginolyticus ATCC17749, and B. cereus A1-1, consistent with the observed bacterial surface structure by the TEM analysis. The damaged cell surface and membrane

Identification of Potential Antibacterial Compounds in the CC 1 from R. madaio Makino
The obtained CC 1 resolved in H 2 O was subjected to UHPLC-MS analysis. As shown in Table 3, a total of 58 different compounds were identified. The highest percentage of these compounds in the CC 1 was p-phenol ethanolamine (18.62%), followed by D-2aminobutyric acid (9.46%), sucrose (7.01%), turanose (7.01%), and lactulose (7.01%). Some compounds with lower concentrations were also identified from the extract (0.83-0.07%), including a galactose 1-phosphate, L-glutamic acid, and kojibiose (Table 3). Phenols and organic acids have good antioxidant and antibacterial activities [11], while alkaloids can inhibit the formation of and/or disperse bacterial biofilms [12]. For example, the indole of alkaloids is a versatile heterocyclic compound with various pharmacological activities such as anticancer, anticonvulsant, antimicrobial, antitubercular, antimalarial, antiviral, antidiabetic and other miscellaneous activities. Indole also regulates various aspects of bacterial physiology, including spore formation, plasmid stability, resistance to drugs, biofilm formation and virulence [13]. Saccharides have been used to preserve foods for a long history by changing cell osmolarity to inhibit harmful bacterial growth. Kojibiose is a natural disaccharide comprising two glucose moieties linked by an α-1,2 glycosidic bond. It has been reported that Kojibiose can inhibit bacterial proliferation and have anti-inflammatory and antiviral activities [14,15]. In contrast, the certain content of the identified amino acids may not contribute to the observed antibacterial activity by the CC 1 from R. madaio Makino.
Molecules 2022, 26, x 9 of 30 structure integrity were beneficial for the CC1 to penetrate bacterial cell envelope to target intracellular processes. Figure 6. Effects of the CC 1 from R. madaio Makino on outer cell membrane permeability of the four bacterial strains. The results were represented as the mean ± standard deviation of three repetitions. **: p < 0.01; ***: p < 0.001.

Identification of Potential Antibacterial Compounds in the CC 1 from R. madaio Makino
The obtained CC 1 resolved in H2O was subjected to UHPLC-MS analysis. As shown in Table 3, a total of 58 different compounds were identified. The highest percentage of these compounds in the CC 1 was p-phenol ethanolamine (18.62%), followed by D-2-aminobutyric acid (9.46%), sucrose (7.01%), turanose (7.01%), and lactulose (7.01%). Some compounds with lower concentrations were also identified from the extract (0.83-0.07%), including a galactose 1-phosphate, L-glutamic acid, and kojibiose (Table 3). Phenols and organic acids have good antioxidant and antibacterial activities [11], while alkaloids can inhibit the formation of and/or disperse bacterial biofilms [12]. For example, the indole of alkaloids is a versatile heterocyclic compound with various pharmacological activities such as anticancer, anticonvulsant, antimicrobial, antitubercular, antimalarial, antiviral, antidiabetic and other miscellaneous activities. Indole also regulates various aspects of bacterial physiology, including spore formation, plasmid stability, resistance to drugs, biofilm formation and virulence [13]. Saccharides have been used to    (Table S2) by RT-qPCR analysis, and the resulting data were correlated with those yielded from the transcriptome analysis (Table S2).
2.6.1. The Major Altered Metabolic Pathways in V. alginolyticus ATCC17749 Approximately 6.73% (316/4698) of V. alginolyticus ATCC17749 genes were expressed differently in the experimental group compared with the control group. Among these, 238 genes showed higher transcription levels (FC ≥ 2.0), and 78 genes were down-regulated (FC ≤ 0.5). Based on the comparative transcriptomic analyses, 11 significantly changed metabolic pathways were identified, including valine, leucine and isoleucine degradation; nitrogen, histidine, tryptophan, glyoxylate and dicarboxylate metabolisms; quorum sensing (QS); lysine degradation; fatty acid degradation; amino sugar and nucleotide sugar metabolism; ABC transporters; and mitogen-activated protein kinase (MAPK) signal pathway (Figure 7). ABC transporters are ATP-dependent efflux transporters to transport lipids, metabolites, exogenous substances and other small molecules out of the cell [16]. They are also the main type of transporters associated with bacterial multidrug resistance [17]. In this study, comparative transcriptome analysis revealed 23 DEGs in ABC transporters and QS that were significantly up-regulated in V. alginolyticus ATCC17749 (2.104-to 7.585-fold) (p < 0.05) ( Table 4). ABC transporter can also catalyze the turnover of lipids in the lipid bilayer that play a critical role in the occurrence and functional maintenance of the cell membrane [18]. In this study, the up-regulated expression of these DEGs suggested that the treatment with the CC 1 from R. madaio Makino enhanced the bacterial pumping of exogenous and endogenous metabolites to eliminate cell damage.  Remarkably, approximately 60 DEGs involved in 10 changed metabolic pathways were significantly up-regulated in V. alginolyticus ATCC17749 (2.002-to 87.807-fold) (p < 0.05) ( Table 4). For example, in the valine, leucine and isoleucine degradation, expression of nine DEGs were significantly up-regulated at the transcription level (2.117-to 4.619-fold) (p < 0.05); six DEGs encoding key enzymes in the histidine metabolism were also significantly upregulated (2.001-to 3.187-fold) (p < 0.05); similarly, in the tryptophan metabolism, expression of three DEGs were significantly enhanced (2.123-to 5.154-fold) (p < 0.05); additionally, in the lysine degradation, expression of a transcriptional regulator (N646_3623) and an arginine/lysine/ornithine decarboxylase (N646_1979) were significantly up-regulated (2.972-to 3.332-fold) (p < 0.05). These four pathways are related to amino acid degradation metabolisms.
ABC transporters are ATP-dependent efflux transporters to transport lipids, metabolites, exogenous substances and other small molecules out of the cell [16]. They are also the main type of transporters associated with bacterial multidrug resistance [17]. In this study, comparative transcriptome analysis revealed 23 DEGs in ABC transporters and QS that were significantly up-regulated in V. alginolyticus ATCC17749 (2.104-to 7.585-fold) (p < 0.05) ( Table 4). ABC transporter can also catalyze the turnover of lipids in the lipid bilayer that play a critical role in the occurrence and functional maintenance of the cell membrane [18]. In this study, the up-regulated expression of these DEGs suggested that the treatment with the CC 1 from R. madaio Makino enhanced the bacterial pumping of exogenous and endogenous metabolites to eliminate cell damage.  In contrast, all DEGs in the MAPK signaling pathway were significantly inhibited (0.123-to 0.369-fold) (p < 0.05) ( Table 4), which likely led to a highly toxic reactive oxygen species (ROS) accumulation and cell damage.
Approximately 44 DEGs involved in six energy metabolism pathways in V. parahaemolyticus ATCC17802 were also significantly inhibited (p < 0.05). For example, the DEG encoding a pyruvate dehydrogenase complex dihydrolipoyllysine-residue acetyltransferase (VP_RS12210) was significantly down-regulated (0.331-fold), which connects glycolysis with tricarboxylic acid cycle (TCA) and plays a key role in glucose metabolism [19]. The down-regulation of this enzyme led to a decrease in ATP production and insufficient energy supply [20], which consequently affected bacterial growth and mobility.     Approximately 44 DEGs involved in six energy metabolism pathways in V. parahaemolyticus ATCC17802 were also significantly inhibited (p < 0.05). For example, the DEG encoding a pyruvate dehydrogenase complex dihydrolipoyllysine-residue acetyltransferase (VP_RS12210) was significantly down-regulated (0.331-fold), which connects glycolysis with tricarboxylic acid cycle (TCA) and plays a key role in glucose metabolism [19]. The down-regulation of this enzyme led to a decrease in ATP production and insufficient energy supply [20], which consequently affected bacterial growth and mobility.
The bacterial flagellum is a complex mobility machine with a diversity of roles in pathogenesis, including attachment, colonization, invasion, maintenance and post-infection dispersal in the host [21,22]. In this study, expression of 23 DEGs involved in three substructures of the flagellum, including the filament, hook and basal body [23], were significantly downregulated at the transcriptional level in V. parahaemolyticus ATCC17802 (0.055-to 0.49-fold) (p < 0.05), which indicated the depressed flagellum assembly that led to inactive motility of V. parahaemolyticus ATCC17802. The 17 down-regulated DEGs in the bacterial chemotaxis [24] (0.101-to 0.491-fold) (p < 0.05) provided indirect evidence for this result.
Interestingly, 23 DEGs encoding type III secretory system (T3SS) components were also significantly down-regulated (0.055-to 0.490 -fold) (p < 0.05). T3SS enables pathogenic bacteria to directly inject effector proteins into host cells, facilitating bacterial colonization in the host [25]. This result suggested that the cytotoxicity of V. parahaemolyticus ATCC17802 was significantly reduced after being treated with the CC 1 from R. madaio Makino.
Approximately eight DEGs in the TCSs were significantly down-regulated. TCSs are widespread regulatory systems that can help bacteria to control their cellular functions and respond to a diverse range of stimuli [27]. In this study, in the HIF-1 signaling pathway, the expression of a L-lactate dehydrogenase (BCN_RS24725) was also significantly downregulated (0.191-fold). These results indicated the inhibited signal transduction systems in B. cereus A1-1.
Additionally, 17 DEGs in the arginine biosynthesis, thiamine metabolism, and alanine, aspartate and glutamate metabolism were all significantly down-regulated (0.031-to 0.498-fold) (p < 0.05) ( Table 7), which suggested the inhibited energy metabolism in B. cereus A1-1 after being treated by the CC 1 from R. madaio Makino.

Bacterial Strains and Culture Conditions
Bacterial strains and culture media used in this study are listed in Table S1. Bacterial culture media were purchased as described previously [28]. Vibrio strains were inoculated in media (pH 8.4-8.5) with 3.0% NaCl, while non-Vibrios in media (pH 7.0-7.2) with 1% NaCl [28].

Extraction of Bioactive Substances from R. madaio Makino
R. madaio Makino was collected in Lishui City (27 • 25 37 N, 118 • 41 28 E), Zhejiang Province, China in September of 2020. A 500 g of fresh leaf and stem tissues of R. madaio Makino was washed clean, dried at room temperature, and then freeze-dried using ALPHA 2-4 LD Plus Freeze Dryer (Martin Christ, Osterode, Germany) at −80 • C for 48 h. The freezedried material was crushed using FW-135 High-Speed Crusher (Beijing Kangtuo Medical Instruments Co., Ltd., Beijing, China) and passed through 300 mesh screen. Then, 10.0 g of the powder was mixed with 99-mL chloroform: methanol (2:1, v/v, analytical grade, Merck KGaA, Darmstadt, Germany) at a solid to liquid ratio of 1.10 (m/v) for 5 h [29]. A 60 mL of H 2 O (Analytical grade, Merck KGaA, Darmstadt"Germany) was then added, fully mixed, and then sonicated using Scientz IID ULtrasonic Cell Crusher (SCIENT Z, Ningbo, China) at the following parameters: power: 300 W; ultrasonic on time: 1 s; ultrasonic off time: 1 s; working time: 20 min; and probe size: 6 mm. The sonicated mixture was filtered through 20-25 µm membrane (Shanghai Sangon Biological Engineeing Technology and Service Co., Ltd., Shanghai, China), and the filtration was collected for the secondary extraction. The methanol phase was separated from the chloroform phase and then individually evaporated, concentrated on pasting using Rotary Evaporator (IKA, Staufen, Germany).

Antimicrobial Susceptibility Assay
Susceptibility of bacterial strains (Table S1) to the extracts from R. madaio Makino was determined according to the method issued by Clinical and Laboratory Standards data were analyzed using SPSS statistical analysis software version 17.0 (SPSS Inc., Armonk, NY, USA).

Conclusions
In this study, we identified, for the first time, antibacterial components and action modes of methanol-phase extract from one edible herbaceous plant R. madaio Makino. The bacteriostatic rate of the extract was 75% against 23 species of common pathogenic bacteria, which was higher than that of the chloroform-phase extract (39%). The methanol-phase extract was further purified using the Prep-HPLC technique, and five separated CCs were obtained. Among these, the CC 1 from R. madaio Makino significantly increased bacterial cell surface hydrophobicity and membrane permeability and decreased membrane fluidity of Gram-positive and Gram-negative pathogens, such as V. parahaemolyticus ATCC17802, V. parahaemolyticus B4-10, V. alginolyticus ATCC17749, and B. cereus A1-1. The damaged cell surface and membrane structure integrity facilitated the CC1 to penetrate bacterial cell envelope to target intracellular processes. A total of 58 different compounds in the extract were identified using UHPLC-MS technique. Comparative transcriptomic analyses revealed a number of differentially expressed genes (DGEs) and various changed metabolic pathways mediated by the CC1 action, such as down-regulation of carbohydrate transport and/or utilization, and energy metabolism; upward regulation of amino acid and fatty acid degradation, and nitrogen metabolism; and inactive flagellar assembly and mobility in the four bacterial strains. Taken, the results in this study demonstrated that the CC1 from R. madaio Makino are promising candidates for antibacterial medicine and human health care products.
Supplementary Materials: The following supporting information can be downloaded at. Table S1: Bacterial strains and media used in this study; Table S2: Expression of representative DEGs by RT-qPCR assay.