Antibiofilm Efficacy and Mechanism of the Marine Chlorinated Indole Sesquiterpene Against Methicillin-Resistant Staphylococcus aureus

Abstract

Methicillin-resistant Staphylococcus aureus (MRSA) can easily form biofilms on food surfaces, thus leading to cross-contamination, which is difficult to remove. Therefore, there is an urgent need to find alternatives with good antibacterial and antibiofilm effects. In this study, two indole sesquiterpene compounds, xiamycin (1) and chlorinated metabolite chloroxiamycin (2), were isolated from the fermentation liquid of marine Streptomyces sp. NBU3429 for the first time. The chemical structures of the two compounds were characterized by spectroscopic data interpretation, including 1D NMR and HRESIMS analysis. Antimicrobial test showed that chloroxiamycin (2) (minimum inhibitory concentration, MIC = 16 μg/mL) exhibited superior antibacterial activity than xiamycin (1) (MIC = 32 μg/mL) against MRSA ATCC43300. Moreover, compound (2) decreased the biofilm formation rate of MRSA ATCC43300 by 12.7%−84.6% in the concentration range of 32–512 μg/mL, which is relatively stronger than xiamycin (1) (4.1%−49.9%) as well. Antibacterial/antibiofilm mechanism investigation indicated that chloroxiamycin (2) could disrupt the cell wall and membrane of MRSA, inhibiting the production of biofilm extracellular polysaccharides. All these results illustrated that chloroxiamycin (2) is an effective antibacterial/antibiofilm agent, which makes it an attractive candidate for food preservatives.

Introduction

In recent years, food safety problems have emerged endlessly, among which microbial contamination is an important triggering factor (Ripolles-Avila et al., 2020). Staphylococcus aureus (S. aureus) is one of the common foodborne pathogens, after massive propagation, the toxic substances of the bacteria often exist in starchy foods and milk products, resulting in food poisoning-related diseases (Bravo-Santano et al., 2019; Bravo-Santano et al., 2018; Gao et al., 2018; Thwaites and Gant 2011).

Bacterial biofilm is an aggregate formed by bacteria that actively adhere to the surface of animate or inanimate objects and encapsulate themselves in the extracellular matrix, to protect themselves during environmental changes (Colagiorgi et al., 2016). Extracellular matrix of biofilm mainly consists of extracellular polysaccharide (EPS), extracellular protein, and extracellular DNA, among which EPS plays an important role in maintaining the structure and function of biofilm, enhancing the resistance of the bacterium in nature (Flemming et al., 2016; Xie et al., 2017). Therefore, it is one of the main concerns of health problems and economic losses in the food industry. As a strain with biofilm-forming ability, S. aureus can continue to exist on various food contact surfaces, including food processing equipment or packaging materials, which is difficult to remove. Hence, there is an urgent need for food preservative candidates to control the bacterium and its biofilm contamination.

The natural product indole alkaloids have attracted the continuous attention of pharmacologists due to their complicated structure and promising bioactivities. Among them, the indole sesquiterpenes represent a little group of secondary metabolites mainly originating from fungi, plants, and actinomycetes as exemplified by the family of xiamycin. They comprise a six-membered benzene ring fused to a five-membered nitrogen-containing pyrrole ring and three isoprene units. Until 2012, some indole sesquiterpenoids derived from marine actinomycetes were found (Zhang et al., 2012). Many of them were reported to have antibacterial, anticancer, and anti-HIV activities (Marcos et al., 2013).

In this article, two indole sesquiterpenes, xiamycin (1) and chloroxiamycin (2) were purified and identified from marine sponge-associated Streptomyces sp. NBU3429 for the first time. Phylogenetic analysis by 16S rRNA gene sequencing identified the actinomycete as Streptomyces sp. In addition, the isolated compounds were evaluated for antibacterial and antibiofilm activities, respectively. Herein, we report the isolation, structure elucidation, and biological evaluation of xiamycin (1) and chloroxiamycin (2).

Materials and Methods

The preparation of compounds

General experimental procedures

The ¹H and ¹³C NMR chemical shifts were recorded relative to the solvent signal Dimethyl sulfoxide-d ₆ (DMSO-d ₆) (¹H 2.5 ppm; ¹³C 39.5 ppm) on a Bruker AVANCE NEO 600 MHz instrument (Bruker Biospin AG, Fällanden, Germany) using Tetramethylsilane (TMS) as an internal standard. Medium-pressure liquid chromatography (MPLC) was performed using a Bonna-Agela FLEXA purification instrument. Silica gel (200–300 mesh, Qingdao), C18 reversed-phase silica gel (50 μm, YMC Co., Ltd.), was used for column chromatography. HRESIMS were recorded on a Waters (Milford, MA, United States) G2-XS Q-TOF Liquid Chromatography Mass Spectrometry (LC/MS) system. Compound purification was carried out by Agilent Technologies 1260 High Performance Liquid Chromatography (HPLC) equipped with a semipreparative C18 column (YMC Pack ODS-A, 250 × 10 mm, 5 μm, Tokyo, Japan).

Bacterial strains

The actinomycete used in this experiment was Streptomyces sp. NBU3429, which was isolated from marine sponge Spongia sp. collected from Paracel Islands, China (16°02'24.60''N 111°48'35.49''E) in May 2020. The BLAST analysis showed that the rDNA sequence was most similar (99.8%) to the sequence of Streptomyces olivaceus. The sequence data for this actinomycete strain have been submitted to NCBI (Accession No. OP055907). A voucher specimen (No. NBU3429) was preserved at the Ningbo University, Ningbo (deposited in the bacterial collection of China General Microbiological Culture Collection Center, CGMCC, accession number 24623). Methicillin-resistant S. aureus (MRSA) ATCC 43300 was purchased from the American Type Culture Collection (ATCC) (VA, USA).

Fermentation, extraction, and isolation

The endophytic actinomycete Streptomyces sp. NBU3429 was inoculated into a 1000 mL Erlenmeyer flask containing 400 mL of A medium (soluble starch 2%, peptone 0.5%, yeast extract 0.2%, KBr 0.01%, CaCO₃ 0.1%, Fe₂(SO₄) H₂O 0.004%, NaCl 3%). The flask was shaken on a rotary shaker (180 rpm) at 28°C for 3 days. The seed culture (3 mL) was transferred into 300 1000-mL Erlenmeyer flasks, each containing 400 mL of YMG liquid medium (soluble starch 2%, glucose 1%, yeast extract 0.5%, malt extract 0.5%, CaCO₃ 0.05%, NaCl 3%). The inoculated flasks were shaken on a rotary shaker (180 rpm) at 28°C for 10 days.

The fermented broth (120 L) was repeatedly and batchically extracted with ethyl acetate (EtOAc) (3 × 120 L, for 1 h each) to afford 25.14 g of organic extract by evaporating under reduced pressure. The EtOAc extract (25.14 g) was subjected to a silica gel column (200–300 mesh) using gradient mixtures of petroleum ether (PE) and EtOAc (from 1:0 to 0:1, v/v, flow rate 80 mL/min, 150 min) to afford 5 fractions (Fr.1−Fr.5). Fr.3 (6.5 g) was further separated using a reversed-phase silica gel (ODS) column to afford 12 fractions (Fr.3.1−Fr.3.12), eluting with a step gradient of MeOH/H₂O (from 1:9 to 1:0, v/v, flow rate 20 mL/min, 100 min). Fr.3.7 was further separated using semipreparative HPLC (YMC Pack ODS-A, 250 × 10 mm, 5 μm; 35% CH₃CN−H₂O; containing 0.1% FA; 2 mL/min) to give compounds 1 (t _R 36 min; 3.5 mg) and 2 (t _R 42 min; 5.5 mg).

Antimicrobial activity

Minimum inhibitory concentration

The minimum inhibitory concentration (MIC) values of xiamycin (1) and chloroxiamycin (2) were determined by the microtiter broth dilution method as reported previously (Wiegand et al., 2008). Briefly, MRSA ATCC43300 of midlogarithmic phase was diluted to 1 × 10⁵ CFU/mL in fresh MH media. Serial twofold dilutions were prepared in DMSO (256−0.5 μg/mL). Then 2 μL of compounds and 98 μL of bacterial suspension were added to a 96-well plate. The plate was incubated in an incubator at 37°C for 16–18 h. The MIC was determined as the lowest drug concentration at which no significant bacterial growth was observed. Vancomycin was used as a positive control, and all experiments were repeated three times (Faqueti et al., 2015).

Effects of chloroxiamycin (2) on cell wall and membrane

Observation of bacterial morphology by scanning electron microscope

The bacteria of midlogarithmic phase were diluted to 1 × 10⁸ CFU/mL with PBS and treated with 4×MIC of chloroxiamycin (2) at 37°C for 2 h. At the end of the incubation, the bacteria were washed twice with PBS and fixed with 2.5% glutaraldehyde at 4°C for 12 h. Next, bacterial dehydration was performed with different concentrations of ethanol (20%−50%−70%−85%−95%×2–100%×2) for 5 min each time and overnight drying. Gold-palladium was sputtered on samples and the images were observed by an S4800 scanning electron microscope (SEM) (Yang et al., 2019).

Membrane permeabilization analysis

The effect of chloroxiamycin (2) on bacterial cell membrane permeability was evaluated with propidium iodide dye (PI) (Li et al., 2022). Midlog-phase MRSA ATCC43300 was diluted to 1 × 10⁸ CFU/mL and then incubated with 1×MIC, 2×MIC, and 4×MIC of chloroxiamycin (2) solution for 30 min and 120 min at 37°C, respectively. At the end of the incubation, the bacteria were washed twice with PBS and then incubated with 50 μg/mL of PI for 15 min. Finally, the fluorescence was analyzed by FACSCalibur Flow Cytometer.

Ability of chloroxiamycin (2) to inhibit MRSA biofilm

Effect of chloroxiamycin (2) on MRSA biofilm formation

To test the effect of chloroxiamycin (2) on the biofilm formation process, log-medium MRSA 43300 was diluted to 1 × 10⁸ CFU/mL with tryptic soy broth (TSB) medium and then added to 96-well plates along with different concentrations of chloroxiamycin (2) (32–512 μg/mL) and incubated at 37°C for 24 h. Fresh TSB medium was used as negative control. After the incubation, the medium was discarded and washed twice with PBS to remove the planktonic bacteria, then the biofilm was fixed with 2.5% glutaraldehyde solution for 15 min, washed twice more with PBS, and dried on an ultraclean table for 1 h. Next, 0.1% crystal violet solution was added for 30 min, washed twice more with PBS to remove the excess stain, and dried overnight. Finally, 95% ethanol was added to dissolve the crystalline violet for 30 min, and the absorbance at 570 nm was measured using microplate reader (Martinez et al., 2019).

Biofilms observed by SEM

To evaluate the effect of chloroxiamycin (2) on the biofilm formation process, midlogarithmic phase MRSA ATCC43300 (1 × 10⁸ CFU/mL) was grown in TSB medium on 24-well plates at 37°C for 24 h in the presence (128 μg/mL) or absence of chloroxiamycin (2). At the end of incubation, PBS was used twice to remove planktonic bacteria, and then, 2.5% glutaraldehyde was added for a 15-min fixation, followed by gradient dehydration using different concentrations of ethanol (20%−50%−70%−85%−95%×2–100%×2) for 5 min each time and overnight drying. Gold-palladium was sputtered on samples and observed on an SEM.

Effect of chloroxiamycin (2) on EPS in biofilms

To determine the effect of chloroxiamycin (2) on the production of EPS during biofilm formation of MRSA ATCC43300, the polysaccharide content was determined by ruthenium red staining. First, the overnight culture of MRSA ATCC43300 was diluted to 1 × 10⁸ CFU/mL with fresh MH medium, and then, chloroxiamycin (2) was added to the suspension to final concentrations of 8, 16, 32, 64, 128, and 256 μg/mL. The samples were added to 96-well plates and incubated at 37°C for 24 h. After incubation, wash twice with PBS buffer solution to remove suspended bacteria, then add 200 μL ruthenium red dye solution and incubate at 37°C for 60 min. Finally, a microplate reader was used to measure the absorbance at 450 nm. The treatment group without the addition of chloroxiamycin (2) was used as the control (Upadhyay et al., 2013).

Results

Structural identification and MICs of xiamycin (1) and chloroxiamycin (2)

The molecular formula of xiamycin (1) (Fig. 1) was established as C₂₃H₂₅NO₃ (m/z 364.1935 [M + H]⁺) by HRESIMS (Supplementary Fig. S8). The ¹H NMR spectrum (600 MHz, Table 1) displayed resonances for six signals of aromatic protons at δ_H 8.04 (1H, d, 7.7), 7.08 (1H, t, 7.4), 7.3 (1H, t, 7.6), 7.38 (1H, d, 8.0), 7.99 (1H, s), and 7.06 (1H, s,), one signal of oxymethine at δ_H 3.87 (1H, dd, 10.6, 5.5), two signals of aliphatic methyl groups at δ_H 1.2 (3H, s) and 1.1 (3H, s). The ¹³C NMR, together with DEPT NMR and HSQC NMR data of xiamycin (1) (150 MHz, Table 1) revealed the presence of an oxymethine carbon at δ_C 74.0 (C-15), an aliphatic methine carbon at δ_C 52.7 (C-16), four methylene carbons at δ_C 37.4 (C-13), 27.7 (C-14), 20.9 (C-18), and 30.4 (C-19), and nine quaternary carbons [one carbonyl at δ_C 180.1 (C-24), six aromatic carbons at δ_C 138.3 (C-2), 121.1 (C-3), 128.8 (C-4), 140.2 (C-9), 140.8 (C-11), 132.7 (C-20), and two aliphatic carbons at δ_C 36.7 (C-12) and 52.7 (C-16)]. Then the above data were compared with the NMR data of the reference (Takada et al., 2010), and it was finally determined that it was xiamycin (1) (Supplementary Figs. S1-S7).

FIG. 1.

Structures of xiamycin (1) and chloroxiamycin (2) compounds.

Table 1.

The ¹H and ¹³C NMR Data of Compounds Xiamycin (1) and Chloroxiamycin (2) in DMSO-d ₆ (δ in ppm)

	1		2
Position	δ_C, Mult	δ_H (J in Hz)	δ_C, Mult	δ_H (J in Hz)
1-N
2	138.3 C		135.6 C
3	121.1 C		122.4 C
4	128.8 C		123.1 C
5	119.9 CH	8.04 (d, 7.7)	120.6 CH	8.09 (d, 7.8)
6	118.1 CH	7.08 (t, 7.4)	119.2 CH	7.14 (t, 7.5)
7	125.0 CH	7.3 (t, 7.6)	126.0 CH	7.37 (t, 7.6)
8	110.7 CH	7.38 (d, 8.0)	111.6 CH	7.48 (d, 8.1)
9	140.2 C		140.4 C
10	115.5 CH	7.99 (s)	114.9 CH	8.04 (s)
11	140.8 C		142.4 C
12	36.7 C		37.1 C
13	37.4 CH₂	1.58 (dt, 12.5, 5.5); 2.55 (dt, 13.1, 3.4)	37.7 CH₂	1.56 (dt, 12.5, 5.4); 2.57 (m)
14	27.7 CH₂	1.73 (m)	27.8 CH₂	1.73 (m)
15	74.0 CH	3.87 (dd, 10.6, 5.5)	74.0 CH	3.87 (dd, 10.5, 5.6)
16	52.7 C		52.9 C
17	45.9 C	2 (dd, 12.5, 2.4)	45.5 C	1.97 (d, 13.0)
18	20.9 CH₂	1.44 (m); 1.89 (dt, 12.;4, 6.9)	20.5 CH₂	1.48 (dd, 12.7, 8.1); 1.91 (ddd, 11.9, 6.2)
19	30.4 CH₂	2.95 (m); 3.07 (dd, 16.5, 7.1)	28.4 CH₂	3.09 (dd, 17.7, 6.5); 2.77 (ddd, 18.0, 10.7, 8.1)
20	132.7 C		129.5 C
21	109.7 CH	7.06 (s)	114.6 C
22	25.6 CH₃	1.2 (s)	25.7 CH₃	1.21 (s)
23	11.1 CH₃	1.1 (s)	11.1 CH₃	1.09 (s)
24	180.1 C		178.8 C

The molecular formula of chloroxiamycin (2) (Fig. 1) was established as C₂₃H₂₄ClNO₃ (m/z 396.1363 [M-H]⁻) by HRESIMS (Supplementary Fig. S16). Comparison of 1D and 2D spectroscopic data (Table 1) exhibited high structurally similarity between xiamycin (1) and chloroxiamycin (2). The most apparent difference was that the chemical shift of C-21 was shifted from δ_C = 109.7 ppm in 1 to δ_C = 114.6 ppm in chloroxiamycin (2) and the tertiary carbon was replaced by the quaternary carbon, indicating the location of the chlorine atom at C-21 in chloroxiamycin (2) (Supplementary Figs. S9-S15).

Notably, it has long been shown that halogen-containing compounds possess excellent antimicrobial activity. The inhibition activity of xiamycin (1) (MIC = 32 μg/mL) was about a half of that of chloroxiamycin (2) (MIC = 16 μg/mL). The MIC value of vancomycin against Staphylococcus aureus is 1 μg/mL. Therefore, chloroxiamycin (2) was selected for the following experiments related to antibacterial and antibiofilm.

Effects of chloroxiamycin on cell wall and membrane

SEM observations

SEM was used to observe the morphology and cell membrane integrity of MRSA ATCC 43300. In the untreated control group, normal bacterial morphology was observed and the cell membrane remained relatively intact, however, after 2 h of treatment with chloroxiamycin (2), wrinkling and bulging of the bacterial surface were observed, indicating some degree of damage to the bacteria (Fig. 2).

FIG. 2.

Scanning electron micrographs of randomly chosen areas of MRSA ATCC 43300 biofilm at 2 h. (A) and (B) untreated control. (C and D) Treated with chloroxiamycin (2). The magnifications were: (A) and (C) ×20,000; (B) and (D) ×40,000.

Membrane permeabilization analysis

PI is a nucleic acid fluorescent dye that penetrates damaged bacterial cell membranes, and therefore, it was used to assess the effect of chloroxiamycin (2) on bacterial cell walls and cell membranes. The fluorescence rate in the untreated group was only 3.56%, which indicated that the vast majority of bacterial cell membranes remained well-integrated when untreated. The percentage of bacterial fluorescence rates ranged from 9.63 to 39.6% after 30 min and a 120-min incubation with 1×, 2×, and 4× MICs of chloroxiamycin (2). The results indicate that compound chloroxiamycin (2) has high cell membrane permeability (Fig. 3).

FIG. 3.

Membrane permeability of MRSA ATCC 43300 treated by chloroxiamycin (2).

Ability of chloroxiamycin (2) to inhibit MRSA biofilm

Effect of chloroxiamycin (2) on MRSA biofilm formation

Crystalline violet staining was used to test the inhibitory effect of chloroxiamycin (2) compounds on the biofilm formation of MRSA ATCC43300. The results showed that chloroxiamycin (2) inhibited biofilm formation in a concentration-dependent manner (Fig. 4), and when treated with chloroxiamycin (2) at concentrations of 512, 256, 128, and 64 μg/mL for 24 h, the percentages of biofilm formed decreased by 78.9%, 84.6%, 78.3%, and 12.7%, respectively, which indicated that chloroxiamycin (2) could effectively inhibit the formation of MRSA ATCC43300 biofilm.

FIG. 4.

The effect of chloroxiamycin (2) on biofilm formation of MRSA ATCC43300.

Biofilms observed by SEM

To further test the effect of compound chloroxiamycin (2) on the biofilm formation process, the morphology was observed by scanning electron microscopy after treatment with 128 μg/mL of chloroxiamycin (2). As shown in the Figure 5, MRSA ATCC43300 in the control group formed a biofilm on the surface of the wafer, while the amount of biofilm formation was greatly reduced and some of the bacteria showed surface crumpling after treatment with chloroxiamycin (2). These results indicate that chloroxiamycin (2) has a good ability to inhibit biofilm formation.

FIG. 5.

Scanning electron micrographs of randomly chosen areas of MRSA ATCC43300 biofilm formation. (A and B) Untreated control. (C and D) Treated with chloroxiamycin (2) (128 μg/mL). The magnifications were: (A) and (C) ×5000; (B) and (D) ×15,000.

Effect of chloroxiamycin (2) on EPS in biofilms

The effect of chloroxiamycin (2) on the secretion of EPS in biofilms is shown in Figure 6. The amount of polysaccharide formation was reduced by 61.0% in the chloroxiamycin (2)-treated group with 256 μg/mL compared with the control group. These results indicated that chloroxiamycin (2) treatment effectively inhibited the secretion of EPS in MRSA ATCC43300.

FIG. 6.

The effect of chloroxiamycin (2) on the secretion of extracellular polysaccharides in biofilms.

Discussion

S. aureus can continue to exist on various food contact surfaces, including food processing equipment or food packaging materials, through its biofilm-forming ability. Bacterial biofilm is one of the main concerns of health problems and economic losses in the food industry. Bacterial biofilm can form an extracellular matrix to wrap the bacterial cell within a biofilm to be protected from an antibacterial agent, which means the difficulty of cleaning is increased (Avila-Novoa et al., 2018).

In this study, xiamycin (1) and chloroxiamycin (2) were isolated from a marine Streptomyces strain and chloroxiamycin (2) has been proven to possess significant antimicrobial and antibiofilm activities against MRSA ATCC43300. In addition, chloroxiamycin (2) (MIC = 16 μg/mL) showed better antimicrobial activity against MRSA than the common food preservative sodium benzoate (MIC = 25,000 μg/mL). The subsequent antimicrobial and antibiofilm mechanism investigation indicated that compound chloroxiamycin (2) can kill bacteria by disrupting bacterial cell wall and membranes, thereby increasing bacterial membrane permeability. Furthermore, as shown in Figure 2, the MRSA ATCC43300 of the control group (CK) exhibited normal morphology and smooth surface. Shrinkage and hollow appeared on the bacterial surface in chloroxiamycin (2) groups, which indicated the destructive effect of chloroxiamycin (2) on the bacterial cell wall. In addition, the antibacterial mechanism of chloroxiamycin (2) against MRSA was similar to that of peptides, so chloroxiamycin (2) may also have the advantage of being less susceptible to drug resistance.

In addition, chloroxiamycin (2) also showed low cytotoxicity, suggesting a high safety profile and potential application in food preservation. The selectivity indices (SIs) were calculated for the two metabolites by measuring the 50% cytotoxic concentration (CC₅₀), 50% effective concentration (EC₅₀) (Khodabandehloo and Maghsoudlou 2023). In prior studies, xiamycin (1) and chloroxiamycin (2) have exhibited no cytotoxicity up to 138 µM and 162 µM, which demonstrated promising results in terms of safety, same as some other metabolites with antibacterial activity (Kim et al., 2016). Reciprocally, whereas xiamycin (1) and chloroxiamycin (2) showed leak inhibitory activity with EC₅₀ values of more than 10 μM, resulting in lower selectivity toward PEDV (SI values of 20 and 13, respectively). They possessed similar antibacterial activities to galangin but a little bit inferior to the metabolite (Khodabandehloo and Maghsoudlou 2023).

In addition, S. aureus is prone to form biofilms on the food surface, and due to the protection of the biofilm matrix, the tolerance of the bacteria inside the film to the bacteriostatic and bactericidal substances in the external environment is tens to thousands of times higher than that of the free bacteria (De la Fuente-Núñez et al., 2014), which makes it difficult to remove the pathogenic bacteria from the food. Currently, the traditional methods applied for biofilm control in the food industry include physical and chemical methods, but they are prone to problems such as bacterial resistance. Natural antibiofilm active substances have the advantage of being green and safe compared with traditional methods and have received wide attention both at home and abroad (Mishra et al., 2020). In addition, extracellular polymers are mainly composed of EPS, proteins, and DNA, and are essential for biofilm formation and maintenance of the three-dimensional structure of biofilms (Di Martino 2018). The secretion of extracellular polymers is reflected by measuring the amount of polysaccharide production. In this study, we discussed the effect of chloroxiamycin (2) on MRSA ATCC43300 biofilm and found that chloroxiamycin (2) inhibited biofilm formation in a concentration-dependent manner. EPS play an important role in biofilm formation, and this study found that chloroxiamycin (2) interfered with the synthesis of EPS in the extracellular matrix of MRSA ATCC43300 biofilm.

Based on the quoted content (Dakhili et al., 2019), it has been observed that compounds containing halogens consistently show higher antibacterial activities compared with compounds that are halogen-free. This indicates that the presence of halogens in the molecular structure plays a crucial role in enhancing antibacterial properties. Furthermore, researchers have conducted structure–activity relationship (Abd Halim and Ngaini 2017) studies to understand the relationship between the position of halogens in aromatic groups and their impact on antibacterial activities. These studies have revealed that halogens situated at different positions within the aromatic groups contribute significantly to the differing activities against microorganisms. For example, thiourea derivatives with fluorine atoms attached to the ortho position of an aromatic group have been found to exhibit excellent activities against various fungal strains such as Rhizopus oryzae, Fusarium oxysporum, Aspergillus niger, and A. fumigatus. These findings suggested that the presence of halogens at specific positions in the molecular structure can enhance the efficacy of compounds against fungal infections. Moreover, the concept of halogen-bonding has been identified as a key factor in guiding antibacterial activities. Maja Kokot’s (Kokot et al., 2021) research further supported the significance of halogen bonding in influencing the effectiveness of antibacterial compounds. Halogen bonding refers to the interaction between halogen atoms and other atoms or molecules, and it is believed to contribute to the enhanced antibacterial properties of halogen-containing compounds. In addition, investigations into chitosan quaternary ammonium salts (Zhang et al., 2018) have shown that the antibacterial performance of these compounds was influenced by the electron-withdrawing ability of different halogenated chitosan quaternary ammonium salts. Chitosan quaternary ammonium salts with stronger electron-withdrawing ability display relatively higher antibacterial activities. In summary, various research studies have highlighted the importance of halogens in enhancing the antibacterial activities of compounds. The position of halogens within aromatic groups, the concept of halogen bonding, and the electron-withdrawing ability of halogenated compounds all play crucial roles in determining the effectiveness of these compounds against microorganisms.

In conclusion, indole sesquiterpene compound chloroxiamycin (2) was isolated from the fermentation liquid of marine Streptomyces sp. NBU3429 for the first time. Chloroxiamycin (2) exhibited potent antibacterial and antibiofilm activity against MRSA ATCC43300. Mechanism investigation indicated that chloroxiamycin (2) could disrupt the cell wall and membrane of MRSA, inhibiting the production of biofilm EPS. All these results illustrated that chloroxiamycin (2) is an attractive candidate for a food preservative. However, the application effect of chloroxiamycin (2) in the food field needs to be further explored.

Footnotes

Data Availability

All data analyzed in this study are included in this published article and its supplementary files.

Ethics Approval

This article does not contain any studies with human participants or animals performed by the authors.

Consent to Participate

The participant has consented to the participants of the article.

Disclosure Statement

The authors declare no competing interests.

Author Contribution

L.D. and X.W. designed this project. Q.W., J.L., and H.W. conducted the experiments. L.D., Q.W., G.H., and S.H. analyzed the data. G.H., Q.W., and H.W. wrote the article. All authors discussed the results and approved the article.

Funding Information

This study was supported by the Ningbo Key Science and Technology Development Program (2021Z046), the National 111 Project of China (D16013), and the Li Dak Sum Yip Yio Chin Kenneth Li Marine Biopharmaceutical Development Fund.

Supplementary Material

Supplementary Figure S1

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Supplementary Figure S15

Supplementary Figure S16

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