Identification of Antibacterial Components and Modes in the Methanol-Phase Extract from a Herbal Plant Potentilla kleiniana Wight et Arn

Abstract

The increase in bacterial resistance and the decline in the effectiveness of antimicrobial agents are challenging issues for the control of infectious diseases. Traditional Chinese herbal plants are potential sources of new or alternative medicine. Here, we identified antimicrobial components and action modes of the methanol-phase extract from an edible herb Potentilla kleiniana Wight et Arn, which had a 68.18% inhibition rate against 22 species of common pathogenic bacteria. The extract was purified using preparative high-performance liquid chromatography (Prep-HPLC), and three separated fragments (Fragments 1–3) were obtained. Fragment 1 significantly elevated cell surface hydrophobicity and membrane permeability but reduced membrane fluidity, disrupting the cell integrity of the Gram-negative and Gram-positive pathogens tested (p < 0.05). Sixty-six compounds in Fragment 1 were identified using Ultra-HPLC and mass spectrometry (UHPLC-MS). The identified oxymorphone (6.29%) and rutin (6.29%) were predominant in Fragment 1. Multiple cellular metabolic pathways were altered by Fragment 1, such as the repressed ABC transporters, protein translation, and energy supply in two representative Gram-negative and Gram-positive strains (p < 0.05). Overall, this study demonstrates that Fragment 1 from P. kleiniana Wight et Arn is a promising candidate for antibacterial medicine and food preservatives.

1. Introduction

Infectious diseases caused by pathogenic bacteria continue to be a global concern for public health, causing millions of deaths worldwide per year [1]. Since the introduction of sulfonamides in 1933, a large number of antibiotics have been applied in clinics [2]. Nevertheless, in recent decades, the overuse and/or misuse of antibiotics have accelerated the spread of antibiotic-resistant bacteria, leading to ineffective drug treatment [3]. It was estimated that at least 700,000 people worldwide die each year due to antimicrobial resistance [4].

Pharmacophagous plants are recognized as a rich source of phytochemicals with antimicrobial potential [5]. Phytocompounds extracted from such plants are long known for their therapeutic uses, and characterized by safety and low toxicity [6]. The application of herbal products may be a better choice for the extensive and imprudent use of synthetic antibiotics [7]. For example, In China, approximately 34,984 native higher plant species have been recorded [8]. Of these, the herbal plant Potentilla kleiniana Wight et Arn was first recorded in the earliest pharmaceutical book “Divine Farmer’s Classic of Materia Medica” during the Warring States period (475–221 B.C.) in China. It belongs to the phylum of Angiospermae, the class of Dicotyledoneae, the order of Rosales Bercht. and J. Presl, and the family of Rosaceae Juss. P. kleiniana Wight et Arn is widely distributed in China, and many Asian countries such as Japan, India, Malaysia, Indonesia, and North Korea. Its whole plant has been used as a traditional Chinese medicine to treat fever, arthritis, malaria, insect and snake bites, hepatitis, and traumatic injury [9]. Recently, Zhou et al. identified bioactive components from P. kleiniana Wight et Arn with anti-human immunodeficiency virus-1 (HIV-1) protease activity [10]. Liu et al. developed an efficient method for the rapid screening and separation of α-glucosidase inhibitors from P. kleiniana Wight et Arn [11]. Li et al. [12] found antihyperglycemic and antioxidant effect of the total flavones of P. kleiniana Wight et Arn in streptozotocin induced diabetic rats, which may be helpful in the prevention of diabetic complications associated with oxidative stress [12]. However, to the best of our knowledge, there are few studies so far in the current literature on antibacterial activity of P. kleiniana Wight et Arn. Tao et al. [9] reported that total flavonoids from P. kleiniana Wight et Arn (TFP) inhibited biofilm formation and virulence factor production in methicillin-resistant Staphylococcus aureus (MRSA). The TFP also damaged cell membrane integrity of Pseudomonas aeruginosa. These results supported potential application of the TFP as a novel natural bioactive preservative in food processing [13]. Song et al. also reported that bioactive components extracted from P. kleiniana Wight et Arn showed antibacterial effects against S. aureus, Candida albicans, P. aeruginosa, and Escherichia coli, but not against the mold Aspergillus niger [14].

To further exploit bioactive nature products in P. kleiniana Wight et Arn, in the present study, we extracted bacteriostatic components in P. kleiniana Wight et Arn using the methanol and chloroform method [15,16]. Antimicrobial action modes of the methanol-phase extract were further investigated. The results of this study provide useful data for potential pharmaceutical application of P. kleiniana Wight et Arn against the common pathogenic bacteria.

2. Results and Discussion

2.1. Antibacterial Activity of Crude Extracts from P. kleiniana Wight et Arn

Antibacterial substances in the fresh P. kleiniana Wight et Arn were extracted using the methanol and chloroform method [15,16]. The water loss rate of the fresh plant sample was 94.12% after freeze-drying treatment of the sample. The extraction rates of the methanol-phase and chloroform-phase crude extracts were 31.13% and 25.43%, respectively. As shown in

Table 1. Antibacterial activity of crude extracts from P. kleiniana Wight et Arn.

Strain	Inhibition Zone (Diameter, mm)		MIC (mg/mL)
	CPE	MPE	CPE	MPE
Aeromonas hydrophila ATCC35654	-	-	-	-
Bacillus cereus A1-1	7.03 ± 0.01	10.54 ± 0.48	50	6.25
Bacillus cereus A2-2	7.11 ± 0.02	10.54 ± 0.75	50	1.56
Enterobacter cloacae ATCC13047	7.00 ± 0.11	7.11 ± 0.26	50	50
Enterobacter cloacae C1-1	-	-	-	-
Escherichia coli ATCC8739	-	7.62 ± 0.37	-	25
Escherichia coli ATCC25922	-	-	-	-
Escherichia coli K12	-	7.51 ± 0.29	-	25
Enterobacter sakazakii CMCC45401	-	-	-	-
Klebsiella pneumoniae 8-2-10-8	-	-	-	-
Klebsiella pneumoniae 8-2-1-14	-	-	-	-
Pseudomonas aeruginosa ATCC9027	-	10.51 ± 0.41	-	6.25
Pseudomonas aeruginosa ATCC27853	-	8.14 ± 0.32	-	25
Salmonella enterica subsp. enterica (ex Kauffmann and Edwards) Le Minor and Popoff serovar Choleraesuis ATCC13312	-	-	-	-
Salmonella paratyphi-A CMCC50093	-	-	-	-
Salmonellaenterica subsp. enterica (ex Kauffmann and Edwards) Le Minor and Popoff serovar Vellore ATCC15611	7.09 ± 0.09	10.11 ± 0.61	50	6.25
Salmonella E1-1	-	-	-	-
Shigella dysenteriae CMCC51252	7.02 ± 0.11	9.29 ± 0.51	50	12.5
Shigella flexneri CMCC51572	7.82 ± 0.20	10.17 ± 0.20	25	6.25
Shigella flexneri ATCC12022	-	-	-	-
Shigella flexneri CMCC51574	-	9.17 ± 0.21	-	12.5
Shigella sonnei ATCC25931	7.00 ± 0.11	8.19 ± 0.51	50	25
Shigella sonnet CMCC51592	-	-	-	-
Staphylococcus aureus ATCC25923	7.03 ± 0.14	9.41 ± 0.27	50	12.5
Staphylococcus aureus ATCC8095	7.07 ± 0.15	10.15 ± 0.24	50	6.25
Staphylococcus aureus ATCC29213	7.78 ± 0.10	9.21 ± 0.01	25	12.5
Staphylococcus aureus ATCC6538	7.62 ± 0.61	9.55 ± 0.37	25	12.5
Staphylococcus aureus D1-1	7.11 ± 0.25	7.00 ± 0.51	50	50
Vibrio alginolyticus ATCC17749	-	10.11 ± 0.24	-	3.13
Vibrio alginolyticus ATCC33787	-	-	-	-
Vibrio cholerae GIM1.449	-	7.00 ± 0.14	-	50
Vibrio cholerae Q10-54	7.22 ± 0.10	7.02 ± 0.21	50	50
Vibrio fluvialis ATCC33809	-	7.12 ± 0.03	-	50
Vibrio harvey ATCC BAA-1117	-	-	-	-
Vibrio harveyi ATCC33842	-	-	-	-
Vibrio mimicus bio-56759	7.21 ± 0.41	11.00 ± 0.32	25	3.13
Vibrio parahemolyticus ATCC17802	-	10.67 ± 1.21	-	1.56
Vibrio parahemolyticus ATCC33847	8.63 ± 0.24	7.14 ± 0.12	12.5	50
Vibrio parahemolyticus B3-13	7.17 ± 0.29	12.33 ± 0.65	50	3.13
Vibrio parahemolyticus B4-10	-	11.26 ± 0.34	-	6.25
Vibrio parahemolyticus B5-29	7.17 ± 0.04	13.77 ± 0.85	50	3.13
Vibrio parahemolyticus B9-35	7.20 ± 0.09	13.15 ± 0.44	25	3.13
Vibrio parahemolyticus A1-1	7.13 ± 0.15	10.35 ± 0.58	50	3.13
Vibrio vulnificus ATCC27562	7.65 ± 0.44	7.01 ± 0.23	25	50

Note: CPE: chloroform-phase extract. MPE: methanol-phase extract. -: no bacteriostasis activity. Inhibition zone: diameter includes the disk diameter (6 mm). MIC: minimum inhibitory concentration. Values were means ± standard deviation (S.D.) of three parallel measurements.

, the chloroform-phase extract from P. kleiniana Wight et Arn had a 50.00% inhibition rate, which inhibited one species of Gram-positive bacterium S. aureus, and 10 species of Gram-negative bacteria, including Bacillus cereus A1-1, B. cereus A2-2, Enterobacter cloacae ATCC13047, Salmonella typhimurium ATCC15611, Shigella dysenteriae CMCC51252, Shigella flexneri CMCC51572, Shigella sonnei ATCC25931, Vibrio cholerae Q10-54, Vibrio mimicus bio-56759, Vibrio parahemolyticus ATCC33847, V. parahemolyticus B3-13, V. parahemolyticus B5-29, V. parahemolyticus B9-35, V. parahemolyticus A1-1, and Vibrio vulnificus ATCC27562 (Table 1).

Of note, the methanol-phase crude extract from P. kleiniana Wight et Arn inhibited the growth of 15 bacterial species, including one species of Gram-positive S. aureus, and 14 species of Gram-negative bacteria, P. aeruginosa ATCC9027, S. typhimurium ATCC15611, S. dysenteriae CMCC51252, S. flexneri CMCC51572, S. flexneri CMCC51574, S. sonnei ATCC25931, V. alginolyticus ATCC17749, V. cholerae Q10-54, V. fluvialis ATCC33809, V. mimicus bio-56759, V. parahemolyticus ATCC17802, and V. vulnificus ATCC27562, which showed a 68.18% inhibition rate (Table 1, Figure 1).

In this study, the methanol and chloroform extract method exhibited a broader antibacterial spectrum, consistent with our previous reports [15,16]. Previous studies also reported effective extraction of bioactive compounds from P. kleiniana Wight et Arn. For example, Tao et al. [13] extracted TFP in P. kleiniana Wight et Arn using an ethanol-water solution, and the obtained extract was further partitioned using petroleum ethers, chloroform and ethyl acetate. The extracted TFP inhibited survival and virulence of P. aeruginosa, and MRSA. Song et al. [14] extracted bioactive compounds from P. kleiniana Wight et Arn using ethanol and ethyl acetate, and the obtained extract showed antibacterial activity against P. aeruginosa, S. aureus, C. albicans, and E. coli. The difference in bioactive compounds extracted from P. kleiniana Wight et Arn using the different methods may explain the distinct antibacterial profiles between this study and the previous reports [13,14].

We further determined minimum inhibitory concentrations (MICs) of the crude extracts from P. kleiniana Wight et Arn, and the results are shown in Table 1. The MICs of the chloroform-phase extract ranged from 12.5 mg/mL to 50 mg/mL against the eleven species of the bacteria. Notably, for the methanol-phase extract, the MICs were between 1.56 mg/mL and 50 mg/mL against the fifteen bacterial species. Of these, the growth of B. cereus A2-2 and V. parahemolyticus ATCC17802 was the most strongly repressed by the methanol-phase extract with the MICs of 1.56 mg/mL, followed by V. alginolyticus ATCC17749, V. mimicus bio-56759, V. parahemolyticus B3-13, V. parahemolyticus B5-29, V. parahemolyticus B9-35, and V. parahemolyticus A1-1 with MICs of 3.13 mg/mL. In addition, the growth of B. cereus A1-1, P. aeruginosa ATCC9027, S. typhimurium ATCC15611, S. flexneri CMCC51572, S. aureus ATCC8095, and V. parahemolyticus B4-10 was also inhibited by the methanol-phase extract with lower MICs (6.25 mg/mL). Of these pathogens, for example, V. alginolyticus is a foodborne marine Vibrio that can cause gastroenteritis, otitis media, otitis externa, and septicemia in humans [17]. V. mimicus can also cause gastroenteritis in humans due to contaminated fish consumption and seafood [18]. P. aeruginosa is an opportunistic pathogen and can cause serious infections, especially in patients with compromised immune systems [19].

Recently, Song et al. [14] reported that the ethyl acetate extract of P. kleiniana Wight et Arn inhibited E. coli, P. aeruginosa, and C. albicans, with MICs of 5 mg/mL, 2.5 mg/mL, and 5 mg/mL, respectively. Tao et al. reported the MIC value of the TFP against MRSA was 20 μg/mL [9].

These results indicated that the methanol-phase crude extract had a higher inhibition rate (68.18%), showing a more broad inhibitory profile with much lower MICs (1.56–50 mg/mL) against the pathogens tested, as compared to the chloroform-phase crude extract (50.00%; 12.5–50 mg/mL). Thus, the methanol-phase crude extract was chosen for further analysis in this study.

2.2. Purification of the Methanol-Phase Crude Extract from P. kleiniana Wight et Arn

Based on the obtained results, a large amount of the methanol-phase crude from P. kleiniana Wight et Arn was prepared and further purified using Prep-HPLC analysis. As shown in Figure S1, three separated fragments (designated Fragments 1–3) were observed via scanning at OD₂₁₁ for 12 min, including Fragment 1 (2.45 min), Fragment 2 (6.75 min), and Fragment 3 (9.83 min). The main peak of the methanol-phase crude was observed to occur at 2.45 min, wherein the absorption peak of Fragment 1 reached its maximum.

The three single fragments were subjected for antibacterial activity analysis. Fragment 1 had strong inhibitory effects on V. parahemolyticus ATCC17802, V. parahemolyticus B5-29, V. parahemolyticus B9-35, V. parahemolyticus B3-13, and V. parahemolyticus B4-10. In addition, the growth of the other six strains was also effectively repressed, including B. cereus A2-2, V. parahemolyticus A1-1, S. flexneri CMCC51572, S. aureus ATCC25923, S. aureus ATCC8095, and S. aureus ATCC6538 (

Table 2. Antibacterial activity of Fragment 1 of the methanol-phase extract from P. kleiniana Wight et Arn.

Strain	Inhibition Zone (Diameter, mm)	MIC (mg/mL)
B. cereus A2-2	8.03 ± 0.45	6.25
S. flexneri CMCC51572	7.50 ± 0.50	6.25
S. aureus ATCC25923	8.03 ± 0.40	12.5
S. aureus ATCC8095	9.53 ± 0.35	6.25
S. aureus ATCC6538	7.10 ± 0.36	50.0
V. parahemolyticus ATCC17802	10.31 ± 0.62	6.25
V. parahemolyticus A1-1	8.57 ± 0.60	25.0
V. parahemolyticus B3-13	10.37 ± 0.32	6.25
V. parahemolyticus B4-10	10.30 ± 0.50	12.5
V. parahemolyticus B5-29	11.30 ± 0.26	6.25
V. parahemolyticus B9-35	11.27 ± 0.40	12.5

). Of these, V. parahaemolyticus is a Gram-negative halophilic bacterium that can cause diseases in marine animals, leading to huge economic losses to the aquaculture. V. parahaemolyticus can also cause gastrointestinal infections and other health complications in humans [20]. B. cereus is a Gram-positive foodborne pathogen that can cause diarrhea and emesis [21]. S. flexneri is a Gram-negative intracellular pathogen that invades colonic cells and causes bloody diarrhea in humans [22]. S. aureus is a Gram-positive opportunistic pathogen leading to food poisoning as well as human and animal infectious diseases [23,24].

We also determined MICs of Fragment 1 against the four species of pathogenic bacteria (Table 2). The synergistic effect may explain the observed MICs of Fragment 1 (6.25–50 mg/mL), as compared to the methanol-phase extract from P. kleiniana Wight et Arn. Among the Gram-negative pathogens, V. parahemolyticus ATCC17802 and V. parahemolyticus B5-29 were the most sensitive strains to Fragment 1, with MICs of 6.25 mg/mL. For the Gram-positive pathogen, the growth of S. aureus ATCC8095 and S. aureus ATCC25923 was also effectively repressed, with MICs of 6.25 mg/mL and 12.5 mg/mL, respectively.

Conversely, the other two peaks (Fragments 2 and 3) showed weak or no antibacterial activity. To further investigate possible antibacterial modes of Fragment 1, the two Gram-negative strains V. parahemolyticus ATCC17802 and V. parahemolyticus B5-29, and two Gram-positive stains S. aureus ATCC8095 and S. aureus ATCC25923 were chosen for the further analysis in this study.

2.3. Bacterial Cell Surface Hydrophobicity, Membrane Fluidity and Permeability Changes Triggered by Fragment 1 from P. kleiniana Wight et Arn

2.3.1. Cell Surface Hydrophobicity

Cell surface hydrophobicity is an important cellular biophysical parameter that affects cell surface interactions and cell–cell communication [25]. In this study, the hexadecane was used as a probe to assess cell surface hydrophobicity change. The difference between before and after the absorbance value of bacterial fluid can indicate the change of hydrophobicity, and the larger the difference, the more hydrophobicity of the surface [26]. The cell surface hydrophobicity of the four experimental groups (1× MIC of Fragment 1) was significantly increased, as compared to the control groups (p < 0.05) (Figure 2A). For instance, after being treated with Fragment 1 for 2 h, bacterial cell surface hydrophobicity was significantly increased, including V. parahaemolyticus B5-29 (8.62%, 1.42-fold), V. parahaemolyticus ATCC17802 (8.27%, 1.50-fold), S. aureus ATCC25923 (10.34%, 1.24-fold), and S. aureus ATCC8095 (12.20%, 1.19-fold) (p < 0.05). Increasing treatment time, the cell surface hydrophobicity was further increased. After the 4 h treatment, the cell surface hydrophobicity was the most significantly increased (11.97%, 1.97-fold) in the V. parahaemolyticus B5-29 treatment group. The highest increase (15.96%, 2.63-fold) was also observed in V. parahaemolyticus B5-29, after treatment for 6 h. The results indicated that Fragment 1 from P. kleiniana Wight et Arn can significantly increase the cell surface hydrophobicity of both Gram-negative V. parahemolyticus and Gram-positive S. aureus pathogens.

2.3.2. Cell Membrane Fluidity

Cell membrane is a natural barrier to prevent extracellular substances from freely entering the cell [27]. In this study, as shown in Figure 2B, when compared to the control groups, the membrane fluidity of V. parahaemolyticus B5-29, S. aureus ATCC25923, and S. aureus ATCC8095 did not change significantly after treatment with Fragment 1 (1× MIC) for 2 h and 4 h. However, a significant decrease (1.16-fold, 1.25-fold, and 1.24-fold) was observed in these three treatment groups after treatment for 6 h, respectively (p < 0.05). In addition, a significant decrease in cell membrane fluidity was only found in V. parahaemolyticus ATCC17802 after treatment for 4 h (1.16-fold) and 6 h (1.24-fold), respectively (p < 0.05). These results indicated that Fragment 1 from P. kleiniana Wight et Arn can significantly reduce the cell membrane fluidity of both Gram-negative V. parahemolyticus and Gram-positive S. aureus pathogens.

2.3.3. Cell Membrane Permeability

β-galactosidase is a macromolecular protein naturally found in the interior of cells that can hydrolyze the substrate o-nitrophenyl-β-D-galactopyranosi (ONPG) to galactose and o-nitrophenol in yellow. If the inner membrane of bacterial cells is damaged, ONPG will quickly enter the cell [28]. In this study, the ONPG was used as a probe to assess whether the bacterial inner membrane is damaged. As illustrated in Figure 3D, the inner cell membrane permeability of S. aureus ATCC8095 did not change significantly after treatment with Fragment 1 (1× MIC) from P. kleiniana Wight et Arn for 2 h (p > 0.05); conversely, significant increases were observed in V. parahaemolyticus B5-29, V. parahaemolyticus ATCC17802, and S. aureus ATCC25923 treatment groups (1.15-fold, 1.18-fold, and 1.04-fold), respectively (p < 0.05). After being treated for 4 h, the highest increase was found in V. parahaemolyticus B5-29 (1.22-fold). After treatment for 6 h, significant increases were also observed in V. parahaemolyticus B5-29, V. parahaemolyticus ATCC17802, S. aureus ATCC25923, and S. aureus ATCC8095 (1.20-fold, 1.17-fold, 1.07-fold, and 1.08-fold), respectively (p < 0.05). These results indicated that Fragment 1 from P. kleiniana Wight et Arn can significantly increase the inner cell membrane permeability of both Gram-negative V. parahemolyticus and Gram-positive S. aureus pathogens.

Outer membrane permeability was assessed by measuring the uptake of a hydrophobic fluorescent probe N-phenyl-1-naphthylamine (NPN) [29]. The outer membrane permeability increased significantly in the four treatment groups, after being treated with Fragment 1 for 2 h (1.38-fold to 1.66-fold) (p < 0.01), and 4 h (1.77-fold to 2.72-fold), respectively (p < 0.001) (Figure 2C). The highest increase was found in V. parahaemolyticus ATCC17802 (2.70-fold), after treatment for 6 h. These results indicated that Fragment 1 from P. kleiniana Wight et Arn can significantly increase the outer cell membrane permeability of the Gram-negative V. parahemolyticus and Gram-positive S. aureus pathogens. Recently, Tao et al. also reported that the TFP from P. kleiniana Wight et Arn increased cell membrane permeability of MRSA [13].

Taken together, the results of this study demonstrated that Fragment 1 (1× MIC) from P. kleiniana Wight et Arn can significantly increase the cell surface hydrophobicity and membrane permeability, but decreases the cell membrane fluidity of both Gram-negative V. parahemolyticus and Gram-positive S. aureus pathogens. Antibacterial compounds (e.g., flavonoids) in Fragment 1 from P. kleiniana Wight et Arn may have interacted with lipid components of the bacterial cell membrane. The disorder in lipid chains resulted in changed permeability and fluidity of the bacterial cell membrane [30]. The compounds may also have interacted with the bacterial cell surface proteins, leading to the altered nanomechanical properties, which consequently changed cell surface hydrophobicity and fluidity [31]. The two common pathogens V. parahemolyticus and S. aureus were chosen for further analysis in this study. The former is the leading sea foodborne pathogen worldwide [20], while the latter leads to food poisoning, as well as human and animal infections [23].

2.4. Bacterial Cell Surface Structure Changes Triggered by Fragment 1 from P. kleiniana Wight et Arn

Based on the obtained results in this study, the representative Gram-negative V. parahaemolyticus ATCC17802 and Gram-positive S. aureus ATCC25923 strains were chosen for further scanning electron microscope (SEM) analysis. As shown in Figure 4, the cells of V. parahaemolyticus ATCC17802 were intact in shape with a flat surface, showing a typical rod-like structure, while those of S. aureus ATCC25923 were also intact and clear, showing a typical spherical structure. In remarkable contrast to the control groups, the bacterial morphological structures were altered to varying degrees in the treatment groups triggered by Fragment 1 (1× MIC) for different times.

For the Gram-negative V. parahaemolyticus ATCC17802, its cell surface was slightly shrunken after being treated with Fragment 1 for 2 h. After 4 h of treatment, the cell surface was more wrinkled and was slightly depressed, the cell membrane was folded and some contents were exuded. After 6 h of the treatment, the cells were severely deformed and crumpled, with a large amount of content leaked.

For the Gram-positive S. aureus ATCC25923, its cell surface was rough and slightly wrinkled, but certain cells were depressed, with a small amount of content leaked after the treatment for 2 h. Upon the increased treatment time (4 h), more cells were obviously wrinkled and deformed with the irregularly spherical, and more content leaked out. The cell morphological structure was seriously damaged after being treated for 6 h.

These results demonstrated that Fragment 1 (1× MIC) from P. kleiniana Wight et Arn can severely damage the cell surface structure of both Gram-negative V. parahaemolyticus and Gram-positive S. aureus after treatment for 6 h.

2.5. Identification of Potential Antibacterial Compounds in Fragment 1 from P. kleiniana Wight et Arn

Potential antibacterial components in Fragment 1 from P. kleiniana Wight et Arn were further identified using UHPLC-MS analysis. As shown in

Table 3. Compounds identified in Fragment 1 from P. kleiniana Wight et Arn via UHPLC–MS analysis.

Peak No.	Identified Compound	Compound Nature	Rt (min)	Formula	Exact Mass	Peak Area (%)
1	D-Maltose	Carbohydrates	0.76	C12H22O11	342.1162	6.77%
2	Oxymorphone	Phenanthrenes and derivatives	11.18	C17H19NO4	301.1314	6.29%
3	Rutin	Flavonoids	12.99	C27H30O16	281.0899	6.29%
4	D-Proline	Amino acid and derivatives	0.76	C5H9NO2	115.0633	5.41%
5	L-Proline	Amino acid and derivatives	0.73	C5H9NO2	115.0633	5.41%
6	L-Glutamic acid	Amino acid and derivatives	0.66	C5H9NO4	147.0532	5.20%
7	Sucrose	Carbohydrates	0.89	C12H22O11	342.1162	3.62%
8	Cynaroside	Flavonoids	12.98	C21H20O11	282.162	3.37%
9	Piperlonguminine	Alkaloids	10.57	C16H19NO3	273.1365	3.21%
10	5-Aminovaleric acid	Amino acid and derivatives	1.11	C5H11NO2	117.079	3.12%
11	D-Glutamine	Carboxylic acids and derivatives	0.66	C5H10N2O3	146.0691	2.99%
12	L-Lysine	Amino acid and derivatives	0.64	C6H14N2O2	146.1055	2.99%
13	p-Octopamine	Phenols	3.84	C8H11NO2	153.079	2.96%
14	Oleic acid	Fatty acyls	13.03	C18H34O2	282.2559	2.91%
15	Isoquercitrin	Flavonoids	10.58	C21H20O12	274.1933	2.44%
16	L-Pipecolic acid	Amino acid and derivatives	0.69	C6H11NO2	129.079	2.31%
17	Moracin C	Phenols	0.67	C19H18O4	129.0426	2.31%
18	Kojibiose	Fatty acyls	0.72	C12H22O11	342.1162	2.22%
19	Gluconic acid	Carbohydrates	0.69	C6H12O7	196.0583	1.97%
20	Betaine	Alkaloids	1.06	C5H11NO2	117.079	1.51%
21	L-Valine	Amino acid and derivatives	0.93	C5H11NO2	117.079	1.49%
22	D-alpha-Aminobutyric acid	Carboxylic acids and derivatives	0.65	C4H9NO2	103.0633	1.46%
23	cis-Aconitic acid	Organic acids and derivatives	1.46	C6H6O6	174.0164	1.34%
24	Lactulose	Organooxygen compounds	0.77	C12H22O11	342.1162	1.33%
25	Turanose	Fatty acyls	0.79	C12H22O11	342.1162	1.33%
26	L-Pipecolic acid	Amino acid and derivatives	1.47	C6H11NO2	129.079	1.15%
27	DL-Norvaline	Amino acid and derivatives	1.05	C5H11NO2	117.079	1.11%
28	L-Asparagine	Amino acid and derivatives	0.64	C4H8N2O3	132.0535	1.11%
29	Malic acid	Hydroxy acids and derivatives	0.8	C4H6O5	134.0215	0.90%
30	Trigonelline	Alkaloids	0.82	C7H7NO2	137.0477	0.90%
31	Acetamide	Alkaloids	13.95	C2H5NO	59.03711	0.88%
32	Beta-D-fructose 2-phosphate	Organooxygen compounds	0.75	C6H13O9P	260.0297	0.77%
33	22-Dehydroclerosterol	Steroids	12.59	C29H46O	410.3549	0.76%
34	Artemisinin	Sesquiterpenoids	13.02	C15H22O5	282.1467	0.72%
35	Kaempferol-3-O-rutinoside	flavonoids	6.29	C27H30O15	594.1585	0.54%
36	L-Homoserine	Amino acid and derivatives	0.67	C4H9NO3	119.0582	0.52%
37	L-Threonine	Amino acid and derivatives	0.64	C4H9NO3	119.0582	0.50%
38	Palmitic acid	Lipids	12.92	C16H32O2	256.2402	0.49%
39	O-Acetylethanolamine	Alkaloids	0.67	C4H9NO2	103.0633	0.46%
40	Galactose 1-phosphate	Organooxygen compounds	0.65	C6H13O9P	260.0297	0.46%
41	Glucose 1-phosphate	Organooxygen compounds	13	C6H13O9P	260.0297	0.45%
42	Adenosine 5′-monophosphate	Nucleotide and its derivates	1.38	C10H14N5O7P	347.0631	0.43%
43	L-Arginine	Amino acid and derivatives	0.6	C6H14N4O2	174.1117	0.43%
44	Maltotriose	Organooxygen compounds	1.23	C18H32O16	504.169	0.40%
45	Indole	Alkaloids	3.82	C8H7N	117.0578	0.38%
46	D-Glucose 6-phosphate	Carbohydrates	0.65	C6H13O9P	260.0297	0.37%
47	D-Aspartic acid	Alkaloids	0.76	C4H7NO4	133.0375	0.36%
48	Vitexin rhamnoside	Flavonoids	6.78	C27H30O14	578.1636	0.35%
49	L-Aspartic acid	Amino acid and derivatives	0.63	C4H7NO4	133.0375	0.33%
50	Maltol	Flavonoids	0.9	C6H6O3	126.0317	0.33%
51	Astragalin	Flavonoids	6.52	C21H20O11	448.1006	0.32%
52	3-Hydroxy-3-methylpentane-1,5-dioic acid	Amino acid and derivatives	2.32	C6H10O5	162.0528	0.31%
53	Campesterol	Steroids and steroid derivatives	12.18	C28H48O	400.3705	0.30%
54	L-Ornithine	Amino acid and derivatives	0.55	C5H12N2O2	132.0899	0.30%
55	Adenosine	Nucleotide and its derivates	2.58	C10H13N5O4	267.0968	0.29%
56	Vidarabine	Purine nucleosides	2.28	C10H13N5O4	267.0968	0.27%
57	Nicotinic acid	Nicotinic acid derivatives	0.73	C6H5NO2	123.032	0.27%
58	Pelargonidin-3-O-glucoside	Flavonoids	1.11	C21H20O10	100.0524	0.26%
59	L-Citruline	Amino acid and derivatives	0.66	C6H13N3O3	175.0957	0.26%
60	Diallyl disulfide	Miscellaneous	0.68	C6H10S2	146.0224	0.26%
61	Sarracine	Alkaloids	13.14	C18H27NO5	337.1889	0.22%
62	N-Acetylputrescine	Phenolamides	1.79	C6H14N2O	130.1106	0.22%
63	Salicylic acid	Organic acid	7.06	C7H6O3	138.0317	0.22%
64	5-Methylcytosine	Nucleotide and its derivates	2.26	C5H7N3O	125.0589	0.21%
65	Ellagic acid	Phenols	6.12	C14H6O8	302.0063	0.21%
66	Isodiospyrin	Quinones	11.28	C22H14O6	374.079	0.21%

, a total of 66 different compounds were identified. The highest relative percentage of the compounds was D-maltose (6.77%), followed by oxymorphone (6.29%), rutin (6.29%), D-proline (5.41%), and L-proline (5.41%). In addition, alkaloids, flavonoids, phenols, sesquiterpenoids, fatty acyls, and organic acids were also detected (Table 3).

Highly concentrated sugar solutions, such as the D-maltose identified in this study, are known to be effective antimicrobial agents [32]. Previous research has indicated that the antibacterial activity of phenanthrenes and derivatives, such as the oxymorphone identified in this study, was primarily related to the destruction of the bacterial cell wall structure [33]. Plant extracts contain a large number of bioactive compounds, mainly polyphenols including flavonoids and phenolic compounds. Flavonoids, such as the rutin identified in this study, could exert antibacterial activity via damaging the cytoplasmic membrane, inhibiting energy metabolism and synthesis of nucleic acids [34]. Tao et al. also reported the major compounds of the TFP were 3-O-methylducheside A, naringenin, rutin and quercetin [9,13]. Phenols, such as the p-octopamine identified in this study, are potent antibacterial agents against both Gram-positive and Gram-negative bacteria via the disruption of the bacterial membrane, leading to bacterial lysis and leakage of intracellular contents [35]. Indole alkaloids, such as the indole identified in this study, possess not only intriguing structural features but also biological/pharmacological activities e.g., antimicrobial activity [36]. Additionally, amino acids and its derivatives, such as the D-proline, L-proline, glutamic acid, 5-aminovaleric acid, lysine, pipecolic acid, and L-valine identified in this study, are a kind of antibacterial agent with the advantages of being not easily drug-resistant, and having low toxicity or harmless metabolites [37].

2.6. Differential Transcriptomes Triggered by Fragment 1 from P. kleiniana Wight et Arn

To obtain the genome-wide gene expression changes triggered by Fragment 1 from P. kleiniana Wight et Arn, we determined transcriptomes of the Gram-negative V. parahaemolyticus ATCC17802 and the Gram-positive S. aureus ATCC25923 pathogens treated with Fragment 1 (1× MIC) for 6 h using the Illumina RNA sequencing technology. A complete list of differently expressed genes (DEGs) in the two strains are available in the National Center for Biotechnology Information (NCBI) SRA database under the accession number PRJNA906658.

2.6.1. The Major Changed Metabolic Pathways in V. parahaemolyticus ATCC17802

Approximately 13.07% (580 of 4436 genes) of V. parahaemolyticus ATCC17802 genes were differentially expressed in the treatment group, as compared to the control group. Of these, 238 DEGs showed higher transcriptional levels (fold change ≥ 2.0), whereas 342 DEGs were significantly down-regulated (fold change ≤ 0.5) (p < 0.05). Sixteen significantly altered metabolic pathways were identified in V. parahaemolyticus ATCC 17802, including the citrate cycle; glyoxylate and dicarboxylate metabolism; fatty acid degradation; glycine, serine, and threonine metabolism; oxidative phosphorylation; pyruvate metabolism; propanoate metabolism; beta-Lactam resistance; ABC transporters; two-component system; alanine, aspartate, and glutamate metabolism; phosphotransferase system (PTS); butanoate metabolism; lysine degradation; quorum sensing (QS); and nitrogen metabolism (Figure 5,

Table 4. The major altered metabolic pathways in V. parahaemolyticus ATCC17802.

Metabolic Pathway	Gene ID	Gene Name	Fold Change	Gene Description
Citrate cycle	WU75_19785	sucA	0.146	2-oxoglutarate dehydrogenase
	WU75_07425	pckA	0.465	Phosphoenolpyruvate carboxykinase
	WU75_19790	sucB	0.133	Dihydrolipoamide succinyltransferase
	WU75_11550	acnB	0.143	Bifunctional aconitate hydratase 2/2-methylisocitrate dehydratase
	WU75_19795	sucC	0.134	Succinyl-CoA synthetase subunit beta
	WU75_19800	sucD	0.16	Succinyl-CoA synthetase subunit alpha
	WU75_19770	sdhD	0.199	Succinate dehydrogenase
	WU75_19780	sdhB	0.157	Succinate dehydrogenase
	WU75_19765	sdhC	0.182	Succinate dehydrogenase
	WU75_13785	fumA	0.497	Fumarate hydratase
	WU75_09605	icd	0.179	Isocitrate dehydrogenase
	WU75_19775	sdhA	0.144	Succinate dehydrogenase
	WU75_06430	mdh	0.177	Malate dehydrogenase
	WU75_16530	lpd	0.35	Dihydrolipoamide dehydrogenase
Glyoxylate and dicarboxylate metabolism	WU75_19760	gltA	0.129	Type II citrate synthase
	WU75_19150	aceA	0.37	Isocitrate lyase
	WU75_19145	aceB	0.352	Malate synthase
	WU75_00290	aceB	0.315	Malate synthase
	WU75_10840	phbB	0.277	3-ketoacyl-ACP reductase
	WU75_03265	katE	2.389	Catalase
Fatty acid degradation	WU75_22235	fadB	0.151	Multifunctional fatty acid oxidation complex subunit alpha
	WU75_08655	fadE	0.184	Acyl-CoA dehydrogenase
	WU75_20175	fadJ	0.204	Multifunctional fatty acid oxidation complex subunit alpha
	WU75_22230	fadA	0.208	3-ketoacyl-CoA thiolase
	WU75_20180	fadA	0.305	3-ketoacyl-CoA thiolase
	WU75_10835	atoB	0.433	Acetyl-CoA acetyltransferase
	WU75_10445	atoB	0.445	Acetyl-CoA acetyltransferase
	WU75_12560	fadE	0.452	Acyl-CoA dehydrogenase
	WU75_19885	fadD	0.493	Long-chain fatty acid—CoA ligase
Glycine, serine and threonine metabolism	WU75_14910	gcvP	0.113	Glycine dehydrogenase
	WU75_14915	gcvH	0.127	Glycine cleavage system protein H
	WU75_10395	betA	0.162	Choline dehydrogenase
	WU75_14930	gcvT	0.184	Glycine cleavage system protein T
	WU75_16130	lysC	0.187	Aspartate kinase
	WU75_14920	glyA	0.203	Serine hydroxymethyltransferase
	WU75_16140	ectB	0.222	Diaminobutyrate-2-oxoglutarate aminotransferase
	WU75_16145	ectA	0.246	L-2,4-diaminobutyric acid acetyltransferase
	WU75_10400	betB	0.259	Betaine-aldehyde dehydrogenase
	WU75_00565	sdaA	0.264	Serine dehydratase
	WU75_16135	ectC	0.27	Ectoine synthase
	WU75_02030	trpB	0.397	Tryptophan synthase subunit beta
	WU75_05755	thrC	0.429	Threonine synthase
	WU75_05760	thrB	0.47	Serine kinase
	WU75_05330	glxK	0.495	Glycerate kinase
Oxidative phosphorylation	WU75_06010	petC	0.195	Cytochrome C
	WU75_06015	petB	0.209	Cytochrome B
	WU75_14570	ccoO	0.228	Peptidase S41
	WU75_14575	ccoN	0.272	Cbb3-type cytochrome c oxidase subunit I
	WU75_14560	ccoP	0.301	Cytochrome Cbb3
	WU75_06485	ppa	0.339	Inorganic pyrophosphatase
	WU75_06020	petA	0.442	Ubiquinol-cytochrome C reductase
	WU75_14565	ccoQ	0.475	Cytochrome C oxidase
	WU75_02240	cyoC	0.478	Cytochrome o ubiquinol oxidase subunit III
	WU75_19125	ppk2	2.159	Polyphosphate kinase
	WU75_09420	cydA	3.637	Cytochrome d terminal oxidase subunit 1
	WU75_09415	cydB	4.11	Cytochrome d ubiquinol oxidase subunit 2
	WU75_09410	cydX	5.362	Membrane protein
Pyruvate metabolism	WU75_01940	yiaY	0.171	Alcohol dehydrogenase
	WU75_03655	lldD	0.276	Lactate dehydrogenase
	WU75_22155	dld	0.322	Lactate dehydrogenase
	WU75_16665	oadA	0.324	Oxaloacetate decarboxylase
	WU75_16060	aldB	0.397	Aldehyde dehydrogenase
	WU75_20855	gloA	2.451	Lactoylglutathione lyase
	WU75_12805	pta	8.464	Phosphate acetyltransferase
	WU75_02150	ackA	8.851	Acetate kinase
	WU75_12810	ackA	10.365	Acetate kinase
	WU75_09685	pflD	12.853	Pyruvate formate-lyase
	WU75_00810	gloA	13.536	Glyoxalase
Propanoate metabolism	WU75_15760	prpF	0.402	3-methylitaconate isomerase
	WU75_15770	prpC	0.435	Methylcitrate synthase
beta-Lactam resistance	WU75_09315	acrA	6.699	Hemolysin D
	WU75_09310	acrB	8.911	Multidrug transporter
	WU75_09925	acrA	40.366	Hemolysin D
ABC transporters	WU75_10385	proW	0.106	ABC transporter permease
	WU75_16175	proX	0.116	Glycine/betaine ABC transporter substrate-binding protein
	WU75_10390	proX	0.122	Glycine/betaine ABC transporter substrate-binding protein
	WU75_12775	oppC	0.133	Peptide ABC transporter permease
	WU75_10380	proV	0.138	ABC transporter ATP-binding protein
	WU75_09655	aotM	0.143	Amino acid ABC transporter permease
	WU75_09665	aotJ	0.144	Nickel transporter
	WU75_13090	yejA	0.151	Diguanylate cyclase
	WU75_12770	oppB	0.164	Oligopeptide transporter permease
	WU75_12780	oppD	0.172	Oligopeptide transporter ATP-binding component
	WU75_09660	aotQ	0.176	ABC transporter
	WU75_16170	proW	0.199	Glycine/betaine ABC transporter permease
	WU75_08085	oppA	0.201	Peptide ABC transporter substrate-binding protein
	WU75_07210	yejA	0.204	Diguanylate cyclase
	WU75_12765	oppA	0.214	Peptide ABC transporter substrate-binding protein
	WU75_07220	yejB	0.22	Hypothetical protein
	WU75_07215	yejE	0.221	Peptide ABC transporter permease
	WU75_09670	aotP	0.228	Amino acid transporter
	WU75_12785	oppF	0.228	Peptide ABC transporter ATP-binding protein
	WU75_04720	oppA	0.341	Peptide ABC transporter substrate-binding protein
	WU75_16165	proV	0.343	Glycine/betaine ABC transporter ATP-binding protein
	WU75_14765	aapQ	0.377	Amino acid ABC transporter permease
	WU75_03180	malE	0.4	Sugar ABC transporter substrate-binding protein
	WU75_14775	aapP	0.405	ABC transporter ATP-binding protein
	WU75_04605	vcaM	0.406	Multidrug ABC transporter ATP-binding protein
	WU75_14055	mdlB	0.411	Multidrug ABC transporter ATP-binding protein
	WU75_10275	rbsD	0.438	D-ribose pyranase
	WU75_05845	btuF	0.487	Vitamin B12-binding protein
	WU75_14760	aapJ	0.491	Amino acid ABC transporter substrate-binding protein
	WU75_03185	malK	2.175	Maltose/maltodextrin transporter ATP-binding protein
	WU75_19815	znuA	2.204	Zinc ABC transporter substrate-binding protein
	WU75_19810	znuC	2.491	Zinc ABC transporter ATPase
	WU75_02265	artP	2.617	Arginine ABC transporter ATP-binding protein
	WU75_19805	znuB	2.666	Membrane protein
	WU75_00425	macB	14.353	Macrolide transporter
Two-component system	WU75_07480	glnG	0.186	Nitrogen regulation protein NR(I)
	WU75_13735	mcp	0.218	Chemotaxis protein
	WU75_15795	tctB	0.237	TctB
	WU75_21750	dctD	0.288	C4-dicarboxylate ABC transporter
	WU75_13155	ttrB	0.31	4Fe-4S ferredoxin
	WU75_21770	dctP	0.31	C4-dicarboxylate ABC transporter
	WU75_01920	mcp	0.32	Chemotaxis protein
	WU75_21745	dctB	0.352	ATPase
	WU75_10200	phoA	0.353	Alkaline phosphatase
	WU75_21765	dctQ	0.368	C4-dicarboxylate ABC transporter permease
	WU75_00210	dctD	0.406	C4-dicarboxylate ABC transporter
	WU75_16210	qseC	0.423	Histidine kinase
	WU75_23015	fliC	0.435	Flagellin
	WU75_07100	mcp	0.453	Chemotaxis protein
	WU75_13380	crp	0.457	Transcriptional regulator
	WU75_09825	mcp	0.471	Chemotaxis protein
	WU75_16525	hapR	0.477	LuxR family transcriptional regulator
	WU75_15800	tctA	0.485	Tripartite tricarboxylate transporter TctA
	WU75_14800	mcp	0.491	Chemotaxis protein
	WU75_06085	tolC	2.068	Outer membrane channel protein
	WU75_15630	dcuB	2.125	C4-dicarboxylate transporter
	WU75_06045	degP	2.148	Serine endoprotease DegQ
	WU75_04355	mcp	2.163	Chemotaxis protein
	WU75_10915	luxQ	3.377	ATPase
	WU75_22175	mcp	4.001	Chemotaxis protein
	WU75_02450	pfeR	4.828	Transcriptional regulator
	WU75_18570	cpxA	10.981	Two-component sensor protein
	WU75_18575	cpxR	26.5	Transcriptional regulator
Alanine, aspartate and glutamate metabolism	WU75_06265	glmS	0.037	Glucosamine-fructose-6-phosphate Aminotransferase
	WU75_07465	glnA	0.123	Glutamine synthetase
	WU75_04655	putA	0.145	Pyrroline-5-carboxylate dehydrogenase
	WU75_14680	-	0.286	NAD-glutamate dehydrogenase
	WU75_05875	carB	0.343	Carbamoyl phosphate synthase large subunit
	WU75_05820	gltB	0.414	Glutamate synthase
	WU75_05825	gltD	0.44	Glutamate synthase
	WU75_05880	carA	0.46	Carbamoyl phosphate synthase small subunit
	WU75_18095	pyrI	0.462	Aspartate carbamoyltransferase regulatory subunit
	WU75_18090	pyrB	0.466	Aspartate carbamoyltransferase catalytic subunit
	WU75_20915	ansA	2.141	Cytoplasmic asparaginase I
	WU75_01110	ansB	2.718	L-asparaginase II
	WU75_18550	aspA	7.015	Aspartate ammonia-lyase
PTS	WU75_03285	ptsN	0.462	PTS fructose transporter subunit IIA
	WU75_12990	ptsG	0.5	PTS glucose transporter subunit IIBC
	WU75_17910	celC	2.36	Molecular chaperone TorD
	WU75_14970	fruB	2.451	Bifunctional PTS system fructose-Specific transporter subunit IIA/HPr protein
	WU75_19555	ptsH	3.973	PTS sugar transporter
	WU75_00455	ulaB	3.977	PTS ascorbate transporter subunit IIB
	WU75_19550	ptsI	4.075	Phosphoenolpyruvate-protein Phosphotransferase
	WU75_00460	cmtB	4.118	PTS system mannitol-specific Transporter subunit IIA
	WU75_01640	cmtB	4.539	PTS mannitol transporter subunit IIA
	WU75_14960	fruA	5.096	PTS fructose transporter subunit IIBC
	WU75_00450	ulaA	6.946	PTS beta-glucoside transporter subunit IIBC
Butanoate metabolism	WU75_01985	acsA	0.334	Acetoacetyl-CoA synthetase
	WU75_10825	phaC	0.336	Poly(3-hydroxyalkanoate) synthetase
Lysine degradation	WU75_21960	ldcC	7.207	Lysine decarboxylase LdcC
QS	WU75_07805	-	0.109	Cytochrome C
	WU75_07800	-	0.181	ABC transporter permease
	WU75_07795	-	0.202	ABC transporter permease
	WU75_07810	ddpD	0.216	ABC transporter ATP-binding protein
	WU75_11620	-	0.218	Peptide ABC transporter permease
	WU75_11630	-	0.233	Peptide ABC transporter substrate-binding protein
	WU75_11625	-	0.261	Peptide ABC transporter permease
	WU75_11610	ddpF	0.358	Chemotaxis protein
	WU75_11615	ddpD	0.484	Sugar ABC transporter ATP-binding protein
	WU75_21410	aphA	2.288	Transcriptional regulator
Nitrogen metabolism	WU75_00760	ncd2	0.276	2-nitropropane dioxygenase
	WU75_10810	napA	2.286	Nitrate reductase
	WU75_15655	nirD	3.934	Nitrite reductase
	WU75_10815	napB	6.27	Nitrate reductase
	WU75_08850	hcp	63.107	Hydroxylamine reductase

).

In the citrate cycle, all the DEGs (n = 14) were significantly repressed (0.146-fold to 0.35-fold) (p < 0.05) in V. parahaemolyticus ATCC17802 after treatment by Fragment 1 from P. kleiniana Wight et Arn. For instance, the DEGs (sucABCD, WU75_19785 and WU75_19790, WU75_19795, and WU75_19800), encoding a 2-oxoglutarate dehydrogenase, a dihydrolipoamide succinyltransferase, and succinyl-CoA synthetase subunits alpha and beta, respectively, were highly inhibited (0.146-fold, 0.133-fold, 0.134-fold, and 0.16-fold) (p < 0.05). Moreover, the DEGs (sdhABCD, WU75_19775, WU75_19780, WU75_19765, and WU75_19770) encoding a succinate dehydrogenase were also highly repressed (0.144-fold to 0.199-fold) (p < 0.05), which links two essential energy-producing processes, the citrate cycle and oxidative phosphorylation [38]. The inhibited key enzymes in the citrate cycle highlighted inactive energy production in V. parahaemolyticus ATCC17802 triggered by Fragment 1.

In the propanoate metabolism, all the DEGs (n = 2) were significantly inhibited (0.402-fold to 0.435-fold) in the V. parahaemolyticus ATCC17802 treatment group (p < 0.05). For example, the DEG (prpC, WU75_15770) encoding a 2-methylcitrate synthase was significantly inhibited (0.435-fold) (p < 0.05). It has been reported that the strategic inhibition of organic acid catabolism in P. aeruginosa through inhibition of PrpC activity may be a potent mechanism to halt the growth of this pathogen [39].

In the glyoxylate and dicarboxylate metabolism, five of the six DEGs were significantly repressed (0.129-fold to 0.277-fold) (p < 0.05). For instance, the DEGs (aceAB, WU75_19150, WU75_19145, and WU75_00290), encoding an isocitrate lyase and a malate synthase of the glyoxylate shunt (GS) carbon cycle, were significantly inhibited (0.315-fold to 0.370-fold) (p < 0.05). The GS could avoid unnecessary reactive oxygen species (ROS) generation by bypassing nicotinamide adenine dinucleotide (NADH) production, and respiration, eventually helping cells to survive in harsh conditions [40,41].

In the glycine, serine, and threonine metabolism, all the DEGs (n = 15) were significantly inhibited (0.113-fold to 0.495-fold) in V. parahaemolyticus ATCC17802 (p < 0.05). For example, the DEGs (ectBAC, WU75_16140, WU75_16145, and WU75_16135), encoding a diaminobutyrate-2-oxoglutarate aminotransferase, a 2% 2C4-diaminobutyric acid acetyltransferase, and an ectoine synthase, which are involved in the synthesis of ectoine that is commonly found in halophilic and halotolerant microorganisms to maintain cell osmotic balance [42]. Additionally, in the alanine, aspartate, and glutamate metabolism, ten of the thirteen DEGs were significantly down-regulated (0.037-fold to 0.466-fold) in V. parahaemolyticus ATCC17802 as well (p < 0.05). Conversely, the DEGs (ansAB, WU75_20915, and WU75_01110) were up-regulated (2.141-fold and 2.718-fold) (p < 0.05), which encoded a cytoplasmic asparaginase I and a L-asparaginase II. The asparaginase I is required for bacterial growth on asparagine as the sole nitrogen source [43], while asparaginases are important in maintaining nitrogen balance and the levels of amino acids within cells [43]. These results indicated that the amino acid synthesis was inhibited in V. parahaemolyticus ATCC17802 mediated by Fragment 1.

For the ABC transporters, 29 of the 35 DEGs were significantly down-regulated (0.106-fold to 0.491-fold) in V. parahaemolyticus ATCC17802 (p < 0.05). Of these, the DEGs (proVXW, WU75_10380, WU75_10390, and WU75_10385), encoding a choline ABC transporter ATP-binding protein, a choline ABC transporter substrate-binding protein, and a choline ABC transporter permease subunit that are responsible for the choline transport, were all significantly repressed (0.106-fold to 0.138-fold). The DEGs (oppABCDF, WU75_12765, WU75_12770, WU75_12775, WU75_12780, and WU75_12785) encoding a peptide ABC transporter substrate-binding protein, an oligopeptide transporter permease, a peptide ABC transporter permease, an oligopeptide transporter ATP-binding component, and a peptide ABC transporter ATP-binding protein, respectively, were all highly repressed (0.172-fold and 0.214-fold). Additionally, the DEGs (yejABE, WU75_13090, WU75_07210, WU75_07220, and WU75_07215) encoding a diguanylate cyclase, an ABC transporter permease subunit, and a peptide ABC transporter permease, respectively, were highly repressed as well (0.151-fold and 0.220-fold). The ABC transporter YejABEF is required for resistance to antimicrobial peptides and virulence of Brucella melitensis [44]. These results indicated that the inhibited ABC transporters likely led to the repressed substance transport and harmful substances discharged in V. parahaemolyticus ATCC17802.

In the oxidative phosphorylation, nine of the thirteen DEGs were significantly down-regulated in V. parahaemolyticus ATCC17802 (0.195-fold to 0.478-fold) (p < 0.05). Oxidative phosphorylation is a major metabolic pathway to obtain energy required for cell growth and proliferation [45] (Huang et al., 2019). For instance, the DEGs (ccoNOQ, WU75_14575, WU75_14570, and WU75_14565) were significantly inhibited (0.228-fold to 0.475-fold) (p < 0.05), which regulated the bacterial adhesion in environmental stresses in V. alginolyticus [45].

In the QS, most DEGs (n = 9) were significantly inhibited (0.109-fold to 0.484-fold) (p < 0.05), e.g., cytochrome c (WU75_06010), cytochrome B (WU75_06015), and peptidase S41 (WU75_14570). For instance, the cytochrome c mediates electron-transfer in the respiratory chain and acts as a detoxifying agent to dispose of reactive oxygen species (ROS) [46].

In contrast, in the PTS, nine of the eleven DEGs were significantly up-regulated (2.36-fold to 6.946-fold) in the V. parahaemolyticus ATCC17802 treatment group (p < 0.05). Of these, the DEGs (fruA, WU75_14960; ulaA, WU75_00450), encoding a PTS fructose transporter subunit IIBC and a PTS beta-glucoside transporter subunit IIBC, respectively, were highly up-regulated (5.096-fold and 6.946-fold) (p < 0.05).

In the nitrogen metabolism, most of the DEGs (n = 4) were significantly up-regulated (2.286-fold to 63.107-fold) (p < 0.05). Remarkably, the DEG (hcp, WU75_08850) encoding a hydroxylamine reductase was strongly up-regulated (63.107-fold) (p < 0.05), and is involved in the processes of scavenging hydroxylamine with NO detoxification [47].

In the two-component system, 19 DEGs were significantly inhibited (0.186-fold to 0.491-fold), whereas 9 DEGs were significantly enhanced (2.068-fold to 26.5-fold) (p < 0.05). The two-component system is one of the primary pathways by which bacteria adapt to environmental stresses [48]. For instance, the DEGs (cpxAR, WU75_18570, and WU75_18575) encoding a two-component sensor protein and a transcriptional regulator were strongly up-regulated (10.981-fold and 26.500-fold) (p < 0.05). The CpxAR is a key modulator of capsule export that facilitates Actinobacillus pleuropneumoniae survival in the host [49]. It also regulates cell membrane permeability and efflux pump activity and induces multidrug resistance (MDR) in Salmonella enteritidis [50].

Additionally, in the beta-lactam resistance, all the DEGs (acrAB, WU75_09925, WU75_09315, and WU75_09310) were strongly up-regulated (6.699-fold to 40.366-fold) in the V. parahaemolyticus ATCC17802 treatment group (p < 0.05), which encoded a multidrug efflux resistance nodulation division (RND) transporter periplasmic adaptor subunit and a multidrug transporter. The RND family efflux pumps, including the major pump AcrAB-TolC, are important mediators of intrinsic and evolved antibiotic resistance [51].

Taken together, these results indicated that Fragment 1 from P. kleiniana Wight et Arn can significantly change sixteen metabolic pathways in the Gram-negative V. parahaemolyticus ATCC17802, which consequently led to repressed substance transporting, energy production, and protein translation, but enhanced stringent response, and harmful substance discharging, and thereby cell death.

2.6.2. The Major Changed Metabolic Pathways in S. aureus ATCC25923

Approximately 7.3% (196 of 2672 genes) of S. aureus ATCC25923 genes were differentially expressed in the treatment group, as compared to the control group. Of these, 156 DEGs showed higher transcriptional levels (fold changes ≥ 2.0), whereas 40 DEGs were significantly down-regulated (fold changes ≤ 0.5) (p < 0.05). Based on the comparative transcriptomic analysis, seven significantly altered metabolic pathways were identified in S. aureus ATCC25923, including the two-component system; nitrogen metabolism; riboflavin metabolism; arginine and proline metabolism; atrazine degradation; alanine, aspartate and glutamate metabolism; and pyrimidine metabolism (Figure 6,

Table 5. The major altered metabolic pathways in S. aureus ATCC25923.

Metabolic Pathway	Gene ID	Gene Name	Fold Change	Gene Description
Two-component system	KQ76_00500	-	0.373	Capsular biosynthesis protein
	KQ76_00560	wecC	0.490	UDP-N-acetyl-D-mannosamine dehydrogenase
	KQ76_12475	nreC	2.117	Nitrate respiration regulation response regulator NreC
	KQ76_12480	nreB	2.276	Nitrate respiration regulation sensor histidine kinase NreB
	KQ76_12485	nreA	2.433	Nitrate respiration regulation accessory nitrate sensor NreA
	KQ76_10520	agrB	2.565	Histidine kinase
	KQ76_03245	graS	2.989	Histidine kinase
	KQ76_10785	kdpF	5.371	ATPase
	KQ76_04230	dltC	28.924	Alanine-phosphoribitol ligase
Nitrogen metabolism	KQ76_12490	narI	3.529	Nitrate reductase
	KQ76_12515	nirD	4.199	Nitrite reductase
	KQ76_12520	nirB	5.060	Nitrite reductase
	KQ76_12460	narT	6.376	Nitrate transporter NarT
	KQ76_12500	narH	5.799	Nitrate reductase
	KQ76_12505	narZ	8.442	Nitrate reductase
	KQ76_12495	narJ	10.404	Nitrate reductase
Riboflavin metabolism	KQ76_09200	ribE	0.373	Riboflavin synthase subunit alpha
	KQ76_09195	ribBA	0.413	GTP cyclohydrolase
	KQ76_09205	ribD	0.430	Diaminohydroxyphosphoribosylaminopyrimidine deaminase
	KQ76_09190	ribH	0.480	6,7-dimethyl-8-ribityllumazine synthase
Arginine and proline metabolism	KQ76_09185	fadM	0.109	Proline dehydrogenase
	KQ76_00580	-	0.218	Aldehyde dehydrogenase
	KQ76_13360	-	0.303	1-pyrroline-5-carboxylate dehydrogenase
	KQ76_11235	rocF	0.461	Arginase
Atrazine degradation	KQ76_11915	ureC	0.406	Urease subunit alpha
	KQ76_11910	ureB	0.412	Urease subunit beta
Alanine, aspartate and glutamate metabolism	KQ76_13360	-	0.303	1-pyrroline-5-carboxylate dehydrogenase
	KQ76_05770	carB	2.158	Carbamoyl phosphate synthase large subunit
	KQ76_05765	carA	3.084	Carbamoyl phosphate synthase small subunit
Pyrimidine metabolism	KQ76_05745	pyrR	2.968	Phosphoribosyl transferase
	KQ76_05760	pyrC	3.115	Dihydroorotase
	KQ76_05755	pyrB	3.213	Aspartate carbamoyltransferase

).

In the arginine and proline metabolism, all the DEGs (n = 4) were significantly down-regulated at the transcription levels (0.109-fold to 0.461-fold) in S. aureus ATCC25923 (p < 0.05). The arginine metabolism converts L-arginine to urea and L-ornithine, which are further metabolized into proline and polyamides that drive collagen synthesis and bioenergetic pathways critical for cell proliferation, respectively [52]. For instance, the DEG (rocF, KQ76_11235) encoding an arginase was significantly down-regulated (0.461-fold) (p < 0.05), and was associated with the ability of Helicobacter pylori to establish chronic infections [53].

All the DEGs (n = 4) in the riboflavin metabolism were also significantly inhibited (ribBADEH, 0.3734-fold to 0.480-fold) (p < 0.05). In this pathway, the redox cofactors flavin mononucleotide and flavin adenine dinucleotide and their precursor riboflavin play important roles in many cellular processes, such as respiration, DNA repair, biosyntheses of heme groups, cofactors and nucleotides, fatty acid beta-oxidation, and bioluminescence [54].

Bacteria use two-component signal transduction systems to elicit adaptive responses to environmental changes [55]. In this study, seven DEGs in the two-component system were significantly up-regulated (2.117-fold to 28.924-fold) in S. aureus ATCC25923 (p < 0.05). For instance, the DEGs (agrB, KQ76_10520; and graS, KQ76_03245) encoding histidine kinases were significantly up-regulated by 2.565-fold and 2.989-fold, respectively (p < 0.05). The accessory gene regulator (agr) quorum-sensing system contributes to its pathogenicity of S. aureus [56]. GraS, the sensor histidine kinase of the GraXRS system, has been suggested to directly activate the response regulator ArlR [53]. Loss of the ArlR alone impairs the ability of S. aureus to respond to host-imposed manganese starvation and glucose limitation [57].

Interestingly, expression of all the DEGs (n = 7) in the nitrogen metabolism was significantly increased at the transcription level (3.529-fold to 10.404-fold) in S. aureus ATCC25923 (p < 0.05). The seven DEGs (nirBD, narHIJZT) were all involved in nitrate reduction [58,59,60]. Of these, the NirD (KQ76_12515) was a small subunit of cytoplasmic NADH-dependent nitrite reductase complex NirBD [61,62]. Over-expression of nirD limits RelA-dependent accumulation of guanosine 5′-triphosphate 3′-diphosphate ((p)ppGpp) in vivo and can prevent activation of the stringent response during amino acid starvation in E. coli [62].

In the alanine, aspartate, and glutamate metabolism, two DEGs (carBA, KQ76_05770 and KQ76_05765) encoding carbamoyl phosphate synthase were significantly up-regulated (2.154-fold and 3.084-fold) in S. aureus ATCC25923 (p < 0.05). The interface residues located near the CarB region of carboxy phosphate synthetic domain plays a key role in carbamoyl phosphate synthetase, aspartate transcarbamoylase, and dihydroorotase (CAD) complex regulation in the pyrimidine biosynthesis [63]. Correspondingly, in the pyrimidine metabolism, four DEGs (pyrBCR, KQ76_05755, KQ76_05760, and KQ76_05745) were also significantly up-regulated (2.968-fold to 3.213-fold) (p < 0.05), and encoded an aspartate carbamoyltransferase, a dihydroorotase, and a phosphoribosyl transferase, respectively. The pyrimidines are involved in the synthesis of DNA, RNA, lipids, and carbohydrates. The pyrimidine metabolism is involved in the synthesis, degradation, salvage, interconversion, and transport of these compounds [64].

Taken together, these results indicate that Fragment 1 from P. kleiniana Wight et Arn can significantly influence seven metabolic pathways in the Gran-positive S. aureus ATCC25923. Of these, the two-component system, alanine, aspartate and glutamate metabolism, and nitrogen metabolism were also changed in the Gram-negative V. parahaemolyticus ATCC17802, which led to the enhanced regulation of stringent response in the two pathogens. On the other hand, we also found distinct transcriptomic profiles between the Gram-positive and Gram-negative pathogens triggered by Fragment 1. For example, consistent with the results obtained from the cell structure analysis, V. parahaemolyticus ATCC17802 was more sensitive to Fragment 1 treatment, as more metabolic pathways were altered, such as the citrate cycle, glyoxylate and dicarboxylate metabolism, fatty acid degradation, glycine, serine and threonine metabolism, oxidative phosphorylation, pyruvate metabolism, propanoate metabolism, beta-lactam resistance, ABC transporters, PTS, butanoate metabolism, lysine degradation, and QS, which resulted in cell destruction and even death.

In addition, to validate the transcriptome data, we tested 16 representative DEGs (Table S1) via reverse transcription real time-quantitative PCR (RT-qPCR) analysis, and the resulting data were generally correlated with those yielded from the transcriptome analysis (Table S2).

3. Materials and Methods

3.1. Bacterial Strains and Culture Conditions

The bacterial strains and culture media used in this study are listed in Table S3. Vibrio strains and non-Vibrio strains were incubated as described in our recent studies [15,16,65].

3.2. Extraction of Bioactive Substances from P. kleiniana Wight et Arn

Fresh P. kleiniana Wight et Arn was purchased from the Qian Shan Zhen Pin shop in Guiyang City (26°36′5.01″ N, 106°41′19.90″ E), Guizhou Province, China, in October of 2021. Bioactive substances were extracted from the samples using the methanol and chloroform method described in our recent reports [15,16,66]. Briefly, aliquot of a 500 g of the whole plant sample was lyophilized, pulverised, powded, sonicated, and then filtered and collected for the secondary extraction. The methanol and chloroform phases were separated and then concentrated using the Rotary Evaporator (IKA, Staufen, Germany) [15,16].

3.3. Antimicrobial Susceptibility Assay

The susceptibility of the bacterial strains (Table S3) to the extracts from P. kleiniana Wight et Arn were determined according to the standard method issued by the Clinical and Laboratory Standards Institute, USA (CLSI, M100-S23, 2018). The antibacterial activity was defined as described previously [15,16]. Broth dilution testing (microdilution) (CLSI, M100-S18, 2018) was used to determine MICs of the extracts. The MIC was defined as described previously [15,16].

3.4. Prep-HPLC Analysis

Aliquots of the extracted samples (10 mg/mL) were resolved, centrifuged, filtered, and subjected for the Prep-HPLC Analysis, using Waters 2707 (Waters, Milford, MA, USA) linked with UPLC Sunfire C18 column (5 µm, 10 × 250 mm) (Waters, Milford, MA, USA) with the same parameters and elution conditions described in our recent reports [15,16].

3.5. UHPLC–MS Analysis

The UHPLC–MS analysis was conducted using the EXIONLC System (Sciex, Framingham, MA, USA) by Shanghai Hoogen Biotech, Shanghai, China [67].

3.6. Bacterial Cell Surface Hydrophobicity and Membrane Fluidity Assays

The cell surface hydrophobicity was measured according to the method of Cui et al. [68]. The cell membrane fluidity was measured according to the method of Kuhry et al. [69], using the 1,6-diphenyl-1,3,5-hexatriene (DPH, Sangon, Shanghai, China).

3.7. Cell Membrane Permeability Analysis

Cell outer membrane permeability was measured according to the method of Wang et al. [70], with the NPN solution (Sangon, Shanghai, China). The inner membrane permeability was measured according to the method of Huang et al. [71], with the ONPG solution (Sangon, Shanghai, China).

3.8. Scanning Electron Microscope (SEM) Assay

The preparation of the samples for the SEM analysis was performed using the method described in our recent reports [15,16,72]. The samples were observed using the Scanning Electron Microscope (Tescan Mira 3 XH, Tescan, Brno, Czech Republic, 5.0 kV, 30,000×).

3.9. Illumina RNA Sequencing

The bacterial cell culture at the mid-LGP was treated with Fragment 1 (1× MIC) from P. kleiniana Wight et Arn for 6 h, and then collected via centrifugation for the total RNA preparation [15,16,72]. Three independently prepared RNA samples for each strain were subjected for the Illumina RNA sequencing analysis, using Illumina HiSeq 2500 platform (Illumina, Santiago, CA, USA) [72].

3.10. RT-qPCR Assay

The RT-qPCR assay was performed according to the method described in our recent reports [15,16,72]. The oligonucleotide primers were designed (Table S1), and synthesized via Sangon (Shanghai, China).

3.11. Data Analysis

The DEGs were analyzed as described in our recent reports [15,16,72]. All tests were carried out in triplicate. The data were analyzed using the SPSS statistical analysis software version 17.0 (SPSS Inc., Armonk, NY, USA). One-way analysis of variance (ANOVA) was performed using the least-significant difference (LSD) method and homogeneity of variance test. There was no significant difference between the control and the treatment groups if the generalized p-values were more than 0.05; conversely, there was significant difference if p-values were less than 0.05.

4. Conclusions

In this study, the methanol-phase extract from P. kleiniana Wight et Arn showed an inhibition rate of 68.18% against 22 species of common pathogenic bacteria. The methanol-phase extraction inhibited the growth of one species of Gram-positive S. aureus, and 14 species of Gram-negative bacteria, including B. cereus, E. cloacae, E. coli, P. aeruginosa, S. typhimurium 1, S. dysenteriae, S. flexneri, S. flexneri, S. sonnei, V. alginolyticus, V. cholerae, V. fluvialis, V. mimicus, V. parahemolyticus, and V. vulnificus strains. This extract was further purified using the Prep-HPLC, and three separated fragments were obtained. Fragment 1 significantly increased bacterial cell surface hydrophobicity and membrane permeability and decreased membrane fluidity, disrupting the cell integrity of the Gram-positive and Gram-negative bacteria such as S. aureus ATCC25923, S. aureus ATCC8095, V. parahaemolyticus ATCC17802, and V. parahaemolyticus B5-29. The MIC values of Fragment 1 ranged from 6.25 mg/mL to 50 mg/mL. A total of 66 different compounds in Fragment 1 were identified. The highest relative percentage of the compounds was D-maltose (6.77%), followed by oxymorphone (6.29%), rutin (6.29%), D-proline (5.41%), and L-proline (5.41%). Highly concentrated sugar solutions, such as the D-maltose identified in Fragment 1, are known to be effective antimicrobial agents. The identified oxymorphone and rutin could exert antibacterial activity via damaging the bacterial cell wall and cytoplasmic membrane, respectively. Multiple cellular metabolic pathways altered by Fragment 1 in the representative Gram-negative V. parahaemolyticus ATCC17802 and Gram-positive S. aureus ATCC25923 pathogens after treatment with Fragment 1 (1× MIC) for 6 h (p < 0.05). These results indicated that the energy supply and protein translation of the tested strains was inhibited, the signal transduction was blocked, and the ability to pump foreign harmful substances was reduced, leading to cell death. Overall, the results of this study demonstrate that Fragment 1 from P. kleiniana Wight et Arn is a promising candidate for antibacterial medicine and food preservatives.