# Brown and Red Seaweeds Serve as Potential Efflux Pump Inhibitors for Drug-ResistantEscherichia coli

Authors: Wen-Jung Lu; Hsuan-Ju Lin; Pang-Hung Hsu; Margaret Lai; Jen-Yu Chiu; Hong-Ting Victor Lin
Journal: Evidence-Based Complementary and Alternative Medicine
 (2019)

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## Abstract

Multidrug-resistant pathogens are a significant clinical problem. Efflux pump inhibitors (EPIs) can restore the activities of existing antibiotics by interfering with drug efflux pumps located in bacterial cell membranes. Seaweeds are important sources of biologically active metabolites of natural origin; however, their potential as EPIs remains uninvestigated. Here, functional extracts from the brown seaweedsLaminaria japonica andSargassum horneri and the red seaweedsGracilariasp. andPorphyra dentata were evaluated as potential EPIs against drug-resistantEscherichia coli. All these extracts were found to potentiate the activities of drugs in modulation tests, although not to the same extent. Synergistic effects of the extracts and the drug clarithromycin were observed from the onset of Time-kill assays, with no evidence of bacterial regrowth. Ethidium bromide accumulation studies revealed that the efflux decreased in the presence of each extract, as indicated by the presence of EPIs. Most identified EPIs that have been discovered to date have aromatic structures, and the seaweed extracts were found to contain various terpenes, terpenoids, phenolic compounds, indoles, pyrrole derivatives, alkaloids, and halogenated aromatic compounds. Our study highlights the potential of these compounds of the seaweeds as drug EPIs.


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## Body

## 1. Introduction

Multidrug-resistant (MDR) pathogens are a significant clinical problem. O’Neill [1], commissioned by the British government, estimated that, by 2050, global deaths due to drug-resistant infections will increase from 700,000 to 10 million annually and that the global economic loss may reach US$100 trillion. Attempts by the pharmaceutical industry to reverse this trend have achieved limited success, and the development and release of novel antimicrobial agents have faltered [2, 3]. Efflux pumps in bacteria are major contributors to drug resistance; they extrude a broad spectrum of antibiotics to the exterior of the organism. Hence, infections caused by these pathogens can be difficult to treat [4]. For example, the inner membrane transporter AcrB (resistance-nodulation-division family) is the major MDR efflux pump inEscherichia coli and can assemble with a periplasmic adaptor protein AcrA and an outer membrane factor TolC. This assembly confers drug resistance by translocating various types of antibiotics, such as macrolides [5], tetracyclines [6, 7], fluoroquinolones [8], and β-lactams [9], across the inner and outer membranes [10–12]. The use of EPIs has been rapidly gaining attention as a novel approach to treat infections caused by pathogens expressing MDR efflux pumps [13, 14]. Their use in adjunctive therapy may restore the activities of existing antibiotics by interfering with efflux pumps, thereby allowing therapeutically ineffective antibiotics to be reintroduced into clinical practice. EPIs may affect the function of efflux pumps by (i) regulating the expression of the pump, (ii) inhibiting the functional assembly of the membrane transporter complex involved in drug efflux, (iii) interfering with the energy required for active drug transport, and (iv) inhibiting drug transport via competitive/noncompetitive binding or by physically blocking the efflux channel [15]. Several classes of EPIs, including antibiotic (tetracyclines, aminoglycosides, and fluoroquinolones) analogs [16–18], amide derivatives (aromatic nitrogen-containing compounds) [19, 20], indoles [21], alkaloids [22–24]), flavonoids [25, 26], aromatic ketones [27], terpenes [28], and oligosaccharides [29], have been reported to date. Several well-known EPIs, such as phenylalanine-arginine β-naphthylamide (PAβN) [30], carbonyl cyanidem-chlorophenylhydrazone (CCCP) [31], verapamil [32], and reserpine [33], have seen limited clinical use and development owing to their toxicity [14]. Plants are a promising source of novel EPIs owing to their low toxicity and compound diversity. In addition, plants are natural, sustainable, and largely unexplored [34]. Capsaicin (8-methyl-N-vanillyl-6-nonenamide), extracted from hot chilies (genusCapsicum), has been shown to significantly reduce the minimum inhibitory concentration of ciprofloxacin by targeting the transporter NorA ofStaphylococcus aureus strains [35]. Piperine, a plant alkaloid found in black pepper (Piper nigrum) and long pepper (Piper longum) and belonging to the familyPiperaceae, potentiates the activity of ciprofloxacin againstS. aureus strains expressing drug efflux pumps [22, 36]. Lanatoside C and daidzein were chosen from a phytochemical database viain silico screening. Both compounds were shown to potentiate the activities of levofloxacin and carbenicillin and increase the accumulation of ethidium bromide (EB) inE. coli[37]. Seaweeds have been used as food and medicine since centuries. They have a short generation cycle and can be easily cultivated in various aquatic environments [38, 39]. Seaweeds are an important source of biologically active metabolites owing to their availability, diversity, and productivity [40]. They are rich in phenolics [41], terpenoids [42], alkaloids [43], flavonoids [44], and polysaccharides [45], some of which possess specific characteristics that are rare or absent in terrestrial plants. Seaweeds are thought to possess antioxidant [46], antimicrobial [47], anti-inflammation [48, 49], antidiabetic [50, 51], and anticancer activities [52]. Despite these factors, their potential as EPIs has not yet been evaluated. Gram-negative pathogenE. coli is one of the most troublesome clinical bacterial species. Moreover, to date, very few EPIs for Gram-negative bacteria have been reported [53]. To our knowledge, this study was the first to evaluate the potential of ethanolic extracts of the brown seaweedsLaminaria japonica andSargassum horneri and red seaweedsGracilariasp. andPorphyra dentata as EPIs against drug-resistantE. coli.


## 2. Materials and Methods

### 2.1. Preparation of Ethanolic Extracts from Seaweeds

L. japonica,Gracilariasp., andP. dentatawere purchased from a local market in Chao-Ching Park.S. horneriwas harvested in May 2017 along the north east coast of Taiwan. The seaweeds were washed, air-dried (50°C), ground, and sieved through 0.25 mm pores and stored in a freezer until use. The ethanolic seaweed extracts were obtained by maceration. In brief, the seaweed powders were soaked in 95% ethanol (solid/solvent ratio = 1/10) with slow stirring at room temperature for 24 h for extraction. The mixture was filtered through, and the filtered algal residue was extracted once again using the previous procedure. The ethanolic extracts were obtained by rotary evaporation and lyophilization and stored in dark at -20°C until use.


### 2.2. Bacterial Strains, Media, and Chemicals

E.coliKam3 (DE3) which is theacrB deletion strain was used in drug susceptibility, modulation and drug accumulation assays [54]. The bacteria were grown in Luria-Bertani broth (LB) and Mueller-Hinton broth (MH broth) for cultivation and broth microdilution experiments.


### 2.3. Cloning of E. coli Efflux Pump AcrB

acrB gene was cloned from theE. coliK12 chromosome by using PCR method. TheacrB gene amplified by using primers 5′-AAAACCCATATG1CCTAATTTCTTTATCGATCGCC-3′ and 5′-AAAACCGTCGAC2TCAATGATGATCGACAGTAT-3′ which was digested with NdeI and XhoI restriction enzymes and inserted onto pSYC vector (pQE100 derivative, T5 promoter) at the NdeI-XhoI site. The pSYC plasmid encodingacrB was transform intoE. coli Kam3 (DE3) in drug susceptibility, modulation, and drug accumulation assays. The ampicillin (100 μg/mL) was used in the experiments.


### 2.4. IC50 and Modulation Tests

The IC50 experiments and modulation tests were carried out as previously described with some modifications [55]. The IC50 of the antibiotic erythromycin, clarithromycin, and tetracycline, and the ethanolic seaweed extracts against drug-sensitive and drug-resistantE. colistrains were determined by using microdilution methods (MH broth), individually, with an inoculum of logarithmic-phase cells (cell density of an OD600 of 0.05 to 0.1). For the modulation tests, the IC50 of the antibiotic erythromycin, clarithromycin, and tetracycline were determined in the presence of 1/2 or 1/4 IC50 of the seaweed extracts, individually. 500 μg/mL was chosen as the IC50 concentration of the seaweed extracts in the modulation assays when the IC50 > 500 μg/mL.


### 2.5. Time-Kill Assays

The Time-kill experiments were carried out according to a previous study [27], with some modifications. Time-kill study of clarithromycin (1/2 IC50) alone or in the presence of seaweed extract (IC50, 1/2 IC50 or 1/4 IC50) was performed in 50 ml volume conical flasks containing 20 mlE. coli cells (7 Log CFU/mL). 500 μg/mL were chosen as the IC50 concentration for the seaweed extracts in the Time-kill assays when the IC50 > 500 μg/mL. Each analysis was done in triplicate with a control without seaweed extract.


### 2.6. EB Accumulation Assay

The EB accumulation assay was performed according to previous studies [56, 57], with the following modifications. TheE. coli cells were grown to mid-log phase in MH broth and collected by centrifugation (5000 ×g, 5 min and 4°C). The cells were resuspended twice in phosphate buffered saline (PBS) (10 mM Na2HPO4, 1.8 mM KH2PO4, 137 mM NaCl, 2.7 mM KCl, 1 mM CaCl2, and 0.5 mM MgCl2 at pH 7.4) and diluted in PBS in a final OD600 of 0.5. The cell suspension was incubated in 96 well plate with the filter-sterilized glucose to a final concentration of 25 mM at room temperature for 3 min. The EB was added to a final concentration of 25 μM and the fluorescence was measured over 38 min using at excitation and emission wavelengths of 520 nm and 600 nm. The effects of CCCP (final concentration of 20 μg/mL) and algal ethanolic extracts (1/2 IC50), individually, were added to bacterial suspension before the fluorescence was measured.


### 2.7. Gas-Chromatography-Mass Spectrometry

Gas Chromatography tandem Mass Spectrometry (GC-MS/MS) was performed by a PolarisQ Ion Trap GC-MS/MS system with a split/splitless injector and Equity®-5 capillary GC column (L × I.D. 30 m × 0.25 mm,d
f 0.25 μm). The carrier gas used was Helium (He) at a flow rate of 1 ml/min, and the injection port was maintained at 280°C. The temperature programming at 50°C for 2 min and increased at a rate of 15°C/min until 250°C and held at 250°C for 2 min. Electron impact ionization (EI) of the GC column eluents screening ranges from 100 to 1000 m/z, and the mass spectrometry data was analyzed by database provided by National Institute of Standards and Technology.


### 2.8. Statistical Analysis

Data are analyzed statistically by using SPSS version 12 (Chicago, IL, USA) and presented as means ± standard deviation. One-way analysis of variance (ANOVA) was used to determine statistical differences between samples means, with the level of significance set atp < 0.05, and multiple comparisons of means were accomplished by Tukey test.


## 2.1. Preparation of Ethanolic Extracts from Seaweeds

L. japonica,Gracilariasp., andP. dentatawere purchased from a local market in Chao-Ching Park.S. horneriwas harvested in May 2017 along the north east coast of Taiwan. The seaweeds were washed, air-dried (50°C), ground, and sieved through 0.25 mm pores and stored in a freezer until use. The ethanolic seaweed extracts were obtained by maceration. In brief, the seaweed powders were soaked in 95% ethanol (solid/solvent ratio = 1/10) with slow stirring at room temperature for 24 h for extraction. The mixture was filtered through, and the filtered algal residue was extracted once again using the previous procedure. The ethanolic extracts were obtained by rotary evaporation and lyophilization and stored in dark at -20°C until use.


## 2.2. Bacterial Strains, Media, and Chemicals

E.coliKam3 (DE3) which is theacrB deletion strain was used in drug susceptibility, modulation and drug accumulation assays [54]. The bacteria were grown in Luria-Bertani broth (LB) and Mueller-Hinton broth (MH broth) for cultivation and broth microdilution experiments.


## 2.3. Cloning of E. coli Efflux Pump AcrB

acrB gene was cloned from theE. coliK12 chromosome by using PCR method. TheacrB gene amplified by using primers 5′-AAAACCCATATG1CCTAATTTCTTTATCGATCGCC-3′ and 5′-AAAACCGTCGAC2TCAATGATGATCGACAGTAT-3′ which was digested with NdeI and XhoI restriction enzymes and inserted onto pSYC vector (pQE100 derivative, T5 promoter) at the NdeI-XhoI site. The pSYC plasmid encodingacrB was transform intoE. coli Kam3 (DE3) in drug susceptibility, modulation, and drug accumulation assays. The ampicillin (100 μg/mL) was used in the experiments.


## 2.4. IC50 and Modulation Tests

The IC50 experiments and modulation tests were carried out as previously described with some modifications [55]. The IC50 of the antibiotic erythromycin, clarithromycin, and tetracycline, and the ethanolic seaweed extracts against drug-sensitive and drug-resistantE. colistrains were determined by using microdilution methods (MH broth), individually, with an inoculum of logarithmic-phase cells (cell density of an OD600 of 0.05 to 0.1). For the modulation tests, the IC50 of the antibiotic erythromycin, clarithromycin, and tetracycline were determined in the presence of 1/2 or 1/4 IC50 of the seaweed extracts, individually. 500 μg/mL was chosen as the IC50 concentration of the seaweed extracts in the modulation assays when the IC50 > 500 μg/mL.


## 2.5. Time-Kill Assays

The Time-kill experiments were carried out according to a previous study [27], with some modifications. Time-kill study of clarithromycin (1/2 IC50) alone or in the presence of seaweed extract (IC50, 1/2 IC50 or 1/4 IC50) was performed in 50 ml volume conical flasks containing 20 mlE. coli cells (7 Log CFU/mL). 500 μg/mL were chosen as the IC50 concentration for the seaweed extracts in the Time-kill assays when the IC50 > 500 μg/mL. Each analysis was done in triplicate with a control without seaweed extract.


## 2.6. EB Accumulation Assay

The EB accumulation assay was performed according to previous studies [56, 57], with the following modifications. TheE. coli cells were grown to mid-log phase in MH broth and collected by centrifugation (5000 ×g, 5 min and 4°C). The cells were resuspended twice in phosphate buffered saline (PBS) (10 mM Na2HPO4, 1.8 mM KH2PO4, 137 mM NaCl, 2.7 mM KCl, 1 mM CaCl2, and 0.5 mM MgCl2 at pH 7.4) and diluted in PBS in a final OD600 of 0.5. The cell suspension was incubated in 96 well plate with the filter-sterilized glucose to a final concentration of 25 mM at room temperature for 3 min. The EB was added to a final concentration of 25 μM and the fluorescence was measured over 38 min using at excitation and emission wavelengths of 520 nm and 600 nm. The effects of CCCP (final concentration of 20 μg/mL) and algal ethanolic extracts (1/2 IC50), individually, were added to bacterial suspension before the fluorescence was measured.


## 2.7. Gas-Chromatography-Mass Spectrometry

Gas Chromatography tandem Mass Spectrometry (GC-MS/MS) was performed by a PolarisQ Ion Trap GC-MS/MS system with a split/splitless injector and Equity®-5 capillary GC column (L × I.D. 30 m × 0.25 mm,d
f 0.25 μm). The carrier gas used was Helium (He) at a flow rate of 1 ml/min, and the injection port was maintained at 280°C. The temperature programming at 50°C for 2 min and increased at a rate of 15°C/min until 250°C and held at 250°C for 2 min. Electron impact ionization (EI) of the GC column eluents screening ranges from 100 to 1000 m/z, and the mass spectrometry data was analyzed by database provided by National Institute of Standards and Technology.


## 2.8. Statistical Analysis

Data are analyzed statistically by using SPSS version 12 (Chicago, IL, USA) and presented as means ± standard deviation. One-way analysis of variance (ANOVA) was used to determine statistical differences between samples means, with the level of significance set atp < 0.05, and multiple comparisons of means were accomplished by Tukey test.


## 3. Results and Discussion

### 3.1. Ethanol Extraction of the Four Seaweeds and Their IC50 against Drug-Susceptible and Drug-Resistant E. coli

The chemical structures of several EPIs derived from natural sources have been identified. These include the alkaloids reserpine [58] and piperine [22], the flavonolignans 5′-methoxyhydnocarpin [59] and silybin [60], the flavones baicalein [61] and chrysosplenol-D [62], and the diterpene carnosol [63]. These compounds were initially extracted using organic solvents owing to their lipophilic nature. We used ethanol to extract potential EPIs fromL.japonica,S. horneri, Gracilariasp., andP. dentata and obtained yields of 5.2%, 4.3%, 6.3%, and 3.9%, respectively (Table 1). This is in agreement with the findings of Dickson et al. [64], who used ethanol to extract drug-potentiating substances from the terrestrial plantsMicroglossa pyrifolia,Mezoneuron benthamianum, andSecurinega virosa; they obtained yields of 5.9%, 4.3%, and 3.6%, respectively.Table 1
Ethanol extraction yield of the seaweed extracts and their IC50 against drug-susceptible and drug-resistant E. coli.
Macroalgae
Extraction yield (%)
IC50 (μg/mL)
Kam3
Kam3-AcrB
Brown
Laminaria japonica
5.2
125
>500
Sargassum horneri
4.3
250
>500
Red
Gracilariasp.
6.3
62.5
>500
Porphyra dentata
3.9
250
>500The ethanolic extracts were tested using the microdilution method against drug-susceptible (Kam3) and drug-resistant (Kam-AcrB)E. colistrains based on their IC50 measurements. As shown in Table 1, the IC50 values of theL.japonica,S. horneri,Gracilariasp., andP. dentataextracts against Kam3 were 125, 250, 62.5, and 250 μg/mL, respectively, thereby indicating that the extracts might contain some antibacterial compounds. The naturally occurring aromatic organic compound p-cymene found in some algal extracts [65] has been shown to exhibit antibacterial activity againstE. coliO157: H7 [66].Interestingly, the ethanolic extracts at the tested concentrations showed no inhibitory effects against the drug-resistantE. colistrain. The IC50 values of all the four extracts against Kam3-AcrB were >500 μg/mL, indicating that the antibacterial substances in the extracts are expelled by the multidrug transporter AcrB. Lomovskaya et al. [30] reported that Mex pumps inPseudomonas aeruginosa confer resistance to EPI PAβN, thereby indicating that PAβN is efficiently extruded.


### 3.2. Seaweed Extracts Potentiate the Activities of Macrolides against Drug-Susceptible and Drug-Resistant E. coli

AcrAB-TolC inE. coliis the most studied tripartite pump to date; it is linked to a wide range of drugs, including macrolides [5], tetracyclines [6, 7], fluoroquinolones [8], and β-lactams [9], and dyes, such as EB [67]. The four ethanolic extracts were added at subinhibitory concentrations of 1/2 and 1/4 IC50 in the modulation assays with the macrolides erythromycin and tetracycline, both of which are known to be substrates of the RND drug transporter AcrB.As shown in Table2, the extracts from brown seaweedsL. japonica andS. horneri and red seaweedsGracilariasp. at a concentration of 1/2 IC50 were able to potentiate the activity of erythromycin against the Kam3 strain, with a modulation factor of 4, and theS. horneriandGracilaria sp. extracts were even found to have potentiating activities at 1/4 IC50. In addition, theS. horneriandGracilaria sp. extracts were able to potentiate the activity of erythromycin against the Kam3-AcrB strain, with a modulation factor of 8 and 2 at 1/2 IC50, respectively, and 2 at 1/4 IC50. Intriguingly, the potentiating activities of the extracts were not observed in the modulation assays using tetracycline (data not shown); this antibiotic shares its mechanism of action (i.e., protein synthesis inhibition) with erythromycin. Lomovskaya et al. [30] demonstrated that PAβN did not potentiate the activities of all antibiotic substrates of the MexAB-OprM efflux pump to the same extent. They speculated that different antibiotics may have different binding sites on the pump and that PAβN-induced inhibition is binding site specific.Table 2
Erythromycin-modulation activity of the seaweed extracts for Kam3 and Kam3-AcrB.
E. coli
Macroalgalextracts
Extractsconc.
IC50 of Erythromycin (μg/mL)
Modulationfactor
No extract
With extract
Kam3
Laminaria japonica
IC50/2
15.63
3.90
4
IC50/4
15.63
15.63
1
Sargassum horneri
IC50/2
15.63
3.90
4
IC50/4
15.63
7.81
2
Gracilaria sp.
IC50/2
15.63
3.90
4
IC50/4
15.63
3.90
4
Porphyra dentata
IC50/2
15.63
15.63
1
IC50/4
15.63
NA
NA
Kam3-AcrB
Laminaria japonica
IC50/2
62.5
62.5
1
IC50/4
62.5
62.5
1
Sargassum horneri
IC50/2
62.5
7.81
8
IC50/4
62.5
31.25
2
Gracilariasp.
IC50/2
62.5
31.25
2
IC50/4
62.5
31.25
2
Porphyra dentata
IC50/2
62.5
125
0.5
IC50/4
62.5
62.5
1
NA, not applicable.We further investigated the potentiating activities of the extracts with another macrolide clarithromycin in the modulation assay (Table3). The extracts fromL. japonica,S. horneri, andGracilariasp. at 1/2 IC50 exhibited potentiating activities with clarithromycin against Kam3 (modulation factor, 8). In addition, all the extracts were found to potentiate the activity of clarithromycin against Kam3-AcrB at 1/2 and 1/4 IC50. The extract fromS. horneri appeared to possess the greatest potentiating activity (modulation factor, 16 at 1/2 IC50).Table 3
Clarithromycin-modulation activity of the seaweed extracts for Kam3 and Kam3-AcrB.
E. coli
Macroalgalextracts
Extractsconc.
IC50 of Clarithromycin
Modulationfactor
No extract
With extract
Kam3
Laminaria japonica
IC50/2
21.88
2.73
8
IC50/4
21.88
21.88
1
Sargassum horneri
IC50/2
21.88
2.73
8
IC50/4
21.88
21.88
1
Gracilaria sp.
IC50/2
21.88
2.73
8
IC50/4
21.88
5.47
4
Porphyra dentata
IC50/2
21.88
21.88
1
IC50/4
21.88
NA
NA
Kam3-AcrB
Laminaria japonica
IC50/2
175
43.75
4
IC50/4
175
87.5
2
Sargassum horneri
IC50/2
175
10.94
16
IC50/4
175
43.75
4
Gracilariasp.
IC50/2
175
87.5
2
IC50/4
175
87.5
2
Porphyra dentata
IC50/2
175
87.5
2
IC50/4
175
87.5
2
NA, not applicable.


### 3.3. Effect of Seaweed Extracts on Time-Kill Curves

To determine the synergistic effect of the seaweed extracts with macrolide over time, the growth of Kam3-AcrB was monitored in the presence of clarithromycin and clarithromycin + a seaweed extract. Figure1(a) indicates that Kam-AcrB was allowed to grow to log phase and then exposed to clarithromycin, theL. japonica extract, or the clarithromycin +L.japonicaextract. The bacteria exposed to the extract alone showed a growth pattern similar to the control group (no addition), whereas the cells exposed to clarithromycin gradually decreased in number.Figure 1
Time-kill curves of clarithromycin alone and combined with brown seaweed (a)L. japonica extract and (b)S. horneriextract against drug-resistantE. coli. The Kam3-AcrBE. coli cells at a cell density of 7 Log CFU/mL were added with clarithromycin alone or combined with seaweed extracts, and the cell numbers were monitored every 3 h for 12 h.
(a)
(b)Furthermore, the number of viable cells exposed to clarithromycin +L. japonica extract (1/2 and 1/4 IC50) sharply decreased from 7 to 4.4 log CFU/mL after 12 h of incubation. Similar results were observed in the assays withS. horneri (Figure 1(b)). Inhibitory effects were not observed when testing the bacteria withS. horneri alone. Moreover, the addition of the clarithromycin +S. horneri extract (1/2 and 1/4 IC50) resulted in a greater inhibitory effect than that of clarithromycin alone. The approximate reduction in cell number was 7 to 4 log CFU/mL after 12 h (1/2 IC50).We also investigated the synergistic inhibitory effects of clarithromycin and red seaweed extracts against Kam3-AcrB over time. Figure2 shows that the addition ofGracilaria sp. orP. dentata extracts to the cells in the presence of clarithromycin resulted in greater inhibition than when clarithromycin was used alone. Our data suggest that the extracts had synergistic effects on clarithromycin from the onset of incubation, with no bacterial regrowth occurring. Zhou et al. [68] reported similar results of Time-kill assays, thus demonstrating the synergistic antibacterial effects of the alkaloid EPI berberine and the antibiotic ciprofloxacin in a MDR clinical isolate ofKlebsiella pneumoniae.Figure 2
Time-kill curves of clarithromycin alone and combined with red seaweed (a)Gracilariasp. extract and (b)P. dentataextract against drug-resistantE. coli. The Kam3-AcrBE. coli cells at a cell density of 7 Log CFU/mL were added with clarithromycin alone or combined with seaweed extracts, and the cell numbers were monitored every 3 h for 12 h.
(a)
(b)


### 3.4. Efflux-Mediated Properties of the Seaweed Extracts

To analyze the potential efflux-mediated properties of the seaweed extracts, we monitored the accumulation of EB into Kam3-AcrB in their presence. EB is a substrate of the multidrug transporter AcrB inE. coli [67] and intensely fluoresces when bound to DNA. Figure 3(a) shows that EB accumulation was greater in cells exposed to the extract and positive control CCCP (20 μg/mL; a proton uncoupler) rather than the control (no addition). This indicates that theL. japonicaextract possesses an efflux pump inhibitor that interferes with EB efflux from Kam3-AcrB. Similar results were observed in cells exposed toS. horneri(Figure 3(b)),Gracilaria sp. (Figure 4(a)), andP. dentataextracts (Figure 4(b)). The EB accumulation assays were also performed in Kam3 in the presences of the seaweed extracts and observed no obvious increase of EB accumulation (data not shown). This might suggest that the seaweed extracts increase the EB accumulation possibly by mediating efflux pump rather than by increasing membrane permeability. Our data indicate that all the extracts contain functional compounds that increase EB accumulation in Kam-AcrB. This highlights their potential as EPIs of AcrB, which is known to be overexpressed with a strong promoter in Kam3-AcrB strains. However, we were unable to rule out the possibility that the extracts interfere with other efflux pumps that translocate EB.Figure 3
Effects of brown seaweeds (a)L. japonica extract and (b)S. horneriextract on EB accumulation in drug-resistantE. coli. TheE. coli Kam3-AcrB cells were added with glucose (25 mM) and EB (25 μM) in presence or absence of CCCP (20 μg/mL) or seaweed extracts (1/2 IC50). The fluorescence was monitored at Ex 520 nm and Em 600 nm.
(a)
(b)Figure 4
Effects of red seaweeds (a)Gracilaria sp. extract and (b)P. dentata extract on EB accumulation in drug-resistantE. coli. TheE. coli Kam3-AcrB cells were added with glucose (25 mM) and EB (25 μM) in presence or absence of CCCP (20 μg/mL) or seaweed extracts (1/2 IC50). The fluorescence was monitored at Ex 520 nm and Em 600 nm.
(a)
(b)


### 3.5. Chemical Compositions of the Seaweed Extracts

Table4 shows the chemical composition of the seaweed extracts as determined using gas chromatography-mass spectrometry (GC-MS). The classes with the symbol +
+ have more chemicals than the classes with the symbol + in each seaweed extract. All the extracts contained terpenes, terpenoids, and phenolic compounds. TheS. horneriandP. dentata extracts contained indoles, whereas theGracilariasp. extract contained pyrrole derivatives. In addition, halogenated aromatic compounds and alkaloids were identified in the red seaweeds.Table 4
Chemical composition of the ethanol extracts from the seaweeds.
Classifications
Brown seaweeds
Red seaweeds
L. japnoica
S. horneri
Gracilaria sp.
P. dentata
Terpenes
+
++
++
+
Terpenoids
+
+
+
+
Phenolic compounds
++
++
++
++
Indoles
–
+
–
+
Pyrrole derivatives
–
–
+
–
Halogenated aromatic compounds
–
–
+
+
Alkaloids
–
–
+
+
++, dominantly present in the extract; +, present in the extract; –, absent in the extract.The chemical structure of several plant-derived and synthetic EPIs has been characterized. The diterpenes carnosol and carnosic acid obtained from the herbRosmarinus officinalis are known to potentiate erythromycin and tetracycline againstS. aureus strains expressing Tet(K) and Msr (A) pumps [63]. Lorenzi et al. [69] indicated that the monoterpene geraniol obtained fromHelichrysum italicum increases the efficacy of quinolones and chloramphenicol inE. coli. Synthetic indole derivatives, such as [4-benzyloxy-2-(5-nitro-1H-2-yl)-phenyl]-methanol, have been reported as EPIs for the efflux pump NorA inS. aureus [70].Furthermore, synthetic halogenated phenothiazine derivatives, such as chlorpromazine, are thought to inhibit drug efflux pumps via various mechanisms [71]. Dwivedi et al. [23] indicated that alkaloid chanoclavine isolated fromIpomoea muricata potentiates the activity of tetracycline against MDR clinicalE. coli isolate, possibly by inhibiting drug efflux and downregulating the expression of drug transporters. In addition, catharanthine isolated from the leaves of flowering plantCatharanthus roseus has been shown to potentiate the activity of tetracycline, possibly due to the inhibition of the efflux pumps inP. aeruginosa [24]. 3,4-Dibromopyrrole-2,5-dione, a bacterial halogenated metabolite, is an effective EPI against bacterial strains that overexpress AcrB-TolC, MexAB-OprM, and MexXY-OprM [72]. The plant-derived alkaloids reserpine and piperine are also effective EPIs for fluoroquinolones inS. aureus [22, 33]. EPIs hinder the functions of efflux pumps via several mechanisms.Seaweeds are rich in bioactive terpenes and aromatic compounds, some of which possess specific characteristics that are absent or rarely found in terrestrial plants. For instance, approximately 25% of all halogenated natural products are alkaloids, and most of them are found in marine organisms [73]. Halogenated compounds obtained from seaweeds are varied and range from indoles, terpenes, and phenols to halogenated hydrocarbons; investigations into their potential use as EPIs would be beneficial [74]. Our IC50 data in Table 1 indicated that some antibacterial substances in the extracts are expelled by the multidrug transporter AcrB, which might suggest that the inhibition of drug transport was a result of competitive binding to AcrB. Further chromatographic purification of the ethanolic extracts obtained from seaweeds is required to identify and produce pure, active compounds. The findings of this study should encourage further research into seaweeds and their extracts with a view to identifying new EPIs with potential clinical applications.


## 3.1. Ethanol Extraction of the Four Seaweeds and Their IC50 against Drug-Susceptible and Drug-Resistant E. coli

The chemical structures of several EPIs derived from natural sources have been identified. These include the alkaloids reserpine [58] and piperine [22], the flavonolignans 5′-methoxyhydnocarpin [59] and silybin [60], the flavones baicalein [61] and chrysosplenol-D [62], and the diterpene carnosol [63]. These compounds were initially extracted using organic solvents owing to their lipophilic nature. We used ethanol to extract potential EPIs fromL.japonica,S. horneri, Gracilariasp., andP. dentata and obtained yields of 5.2%, 4.3%, 6.3%, and 3.9%, respectively (Table 1). This is in agreement with the findings of Dickson et al. [64], who used ethanol to extract drug-potentiating substances from the terrestrial plantsMicroglossa pyrifolia,Mezoneuron benthamianum, andSecurinega virosa; they obtained yields of 5.9%, 4.3%, and 3.6%, respectively.Table 1
Ethanol extraction yield of the seaweed extracts and their IC50 against drug-susceptible and drug-resistant E. coli.
Macroalgae
Extraction yield (%)
IC50 (μg/mL)
Kam3
Kam3-AcrB
Brown
Laminaria japonica
5.2
125
>500
Sargassum horneri
4.3
250
>500
Red
Gracilariasp.
6.3
62.5
>500
Porphyra dentata
3.9
250
>500The ethanolic extracts were tested using the microdilution method against drug-susceptible (Kam3) and drug-resistant (Kam-AcrB)E. colistrains based on their IC50 measurements. As shown in Table 1, the IC50 values of theL.japonica,S. horneri,Gracilariasp., andP. dentataextracts against Kam3 were 125, 250, 62.5, and 250 μg/mL, respectively, thereby indicating that the extracts might contain some antibacterial compounds. The naturally occurring aromatic organic compound p-cymene found in some algal extracts [65] has been shown to exhibit antibacterial activity againstE. coliO157: H7 [66].Interestingly, the ethanolic extracts at the tested concentrations showed no inhibitory effects against the drug-resistantE. colistrain. The IC50 values of all the four extracts against Kam3-AcrB were >500 μg/mL, indicating that the antibacterial substances in the extracts are expelled by the multidrug transporter AcrB. Lomovskaya et al. [30] reported that Mex pumps inPseudomonas aeruginosa confer resistance to EPI PAβN, thereby indicating that PAβN is efficiently extruded.


## 3.2. Seaweed Extracts Potentiate the Activities of Macrolides against Drug-Susceptible and Drug-Resistant E. coli

AcrAB-TolC inE. coliis the most studied tripartite pump to date; it is linked to a wide range of drugs, including macrolides [5], tetracyclines [6, 7], fluoroquinolones [8], and β-lactams [9], and dyes, such as EB [67]. The four ethanolic extracts were added at subinhibitory concentrations of 1/2 and 1/4 IC50 in the modulation assays with the macrolides erythromycin and tetracycline, both of which are known to be substrates of the RND drug transporter AcrB.As shown in Table2, the extracts from brown seaweedsL. japonica andS. horneri and red seaweedsGracilariasp. at a concentration of 1/2 IC50 were able to potentiate the activity of erythromycin against the Kam3 strain, with a modulation factor of 4, and theS. horneriandGracilaria sp. extracts were even found to have potentiating activities at 1/4 IC50. In addition, theS. horneriandGracilaria sp. extracts were able to potentiate the activity of erythromycin against the Kam3-AcrB strain, with a modulation factor of 8 and 2 at 1/2 IC50, respectively, and 2 at 1/4 IC50. Intriguingly, the potentiating activities of the extracts were not observed in the modulation assays using tetracycline (data not shown); this antibiotic shares its mechanism of action (i.e., protein synthesis inhibition) with erythromycin. Lomovskaya et al. [30] demonstrated that PAβN did not potentiate the activities of all antibiotic substrates of the MexAB-OprM efflux pump to the same extent. They speculated that different antibiotics may have different binding sites on the pump and that PAβN-induced inhibition is binding site specific.Table 2
Erythromycin-modulation activity of the seaweed extracts for Kam3 and Kam3-AcrB.
E. coli
Macroalgalextracts
Extractsconc.
IC50 of Erythromycin (μg/mL)
Modulationfactor
No extract
With extract
Kam3
Laminaria japonica
IC50/2
15.63
3.90
4
IC50/4
15.63
15.63
1
Sargassum horneri
IC50/2
15.63
3.90
4
IC50/4
15.63
7.81
2
Gracilaria sp.
IC50/2
15.63
3.90
4
IC50/4
15.63
3.90
4
Porphyra dentata
IC50/2
15.63
15.63
1
IC50/4
15.63
NA
NA
Kam3-AcrB
Laminaria japonica
IC50/2
62.5
62.5
1
IC50/4
62.5
62.5
1
Sargassum horneri
IC50/2
62.5
7.81
8
IC50/4
62.5
31.25
2
Gracilariasp.
IC50/2
62.5
31.25
2
IC50/4
62.5
31.25
2
Porphyra dentata
IC50/2
62.5
125
0.5
IC50/4
62.5
62.5
1
NA, not applicable.We further investigated the potentiating activities of the extracts with another macrolide clarithromycin in the modulation assay (Table3). The extracts fromL. japonica,S. horneri, andGracilariasp. at 1/2 IC50 exhibited potentiating activities with clarithromycin against Kam3 (modulation factor, 8). In addition, all the extracts were found to potentiate the activity of clarithromycin against Kam3-AcrB at 1/2 and 1/4 IC50. The extract fromS. horneri appeared to possess the greatest potentiating activity (modulation factor, 16 at 1/2 IC50).Table 3
Clarithromycin-modulation activity of the seaweed extracts for Kam3 and Kam3-AcrB.
E. coli
Macroalgalextracts
Extractsconc.
IC50 of Clarithromycin
Modulationfactor
No extract
With extract
Kam3
Laminaria japonica
IC50/2
21.88
2.73
8
IC50/4
21.88
21.88
1
Sargassum horneri
IC50/2
21.88
2.73
8
IC50/4
21.88
21.88
1
Gracilaria sp.
IC50/2
21.88
2.73
8
IC50/4
21.88
5.47
4
Porphyra dentata
IC50/2
21.88
21.88
1
IC50/4
21.88
NA
NA
Kam3-AcrB
Laminaria japonica
IC50/2
175
43.75
4
IC50/4
175
87.5
2
Sargassum horneri
IC50/2
175
10.94
16
IC50/4
175
43.75
4
Gracilariasp.
IC50/2
175
87.5
2
IC50/4
175
87.5
2
Porphyra dentata
IC50/2
175
87.5
2
IC50/4
175
87.5
2
NA, not applicable.


## 3.3. Effect of Seaweed Extracts on Time-Kill Curves

To determine the synergistic effect of the seaweed extracts with macrolide over time, the growth of Kam3-AcrB was monitored in the presence of clarithromycin and clarithromycin + a seaweed extract. Figure1(a) indicates that Kam-AcrB was allowed to grow to log phase and then exposed to clarithromycin, theL. japonica extract, or the clarithromycin +L.japonicaextract. The bacteria exposed to the extract alone showed a growth pattern similar to the control group (no addition), whereas the cells exposed to clarithromycin gradually decreased in number.Figure 1
Time-kill curves of clarithromycin alone and combined with brown seaweed (a)L. japonica extract and (b)S. horneriextract against drug-resistantE. coli. The Kam3-AcrBE. coli cells at a cell density of 7 Log CFU/mL were added with clarithromycin alone or combined with seaweed extracts, and the cell numbers were monitored every 3 h for 12 h.
(a)
(b)Furthermore, the number of viable cells exposed to clarithromycin +L. japonica extract (1/2 and 1/4 IC50) sharply decreased from 7 to 4.4 log CFU/mL after 12 h of incubation. Similar results were observed in the assays withS. horneri (Figure 1(b)). Inhibitory effects were not observed when testing the bacteria withS. horneri alone. Moreover, the addition of the clarithromycin +S. horneri extract (1/2 and 1/4 IC50) resulted in a greater inhibitory effect than that of clarithromycin alone. The approximate reduction in cell number was 7 to 4 log CFU/mL after 12 h (1/2 IC50).We also investigated the synergistic inhibitory effects of clarithromycin and red seaweed extracts against Kam3-AcrB over time. Figure2 shows that the addition ofGracilaria sp. orP. dentata extracts to the cells in the presence of clarithromycin resulted in greater inhibition than when clarithromycin was used alone. Our data suggest that the extracts had synergistic effects on clarithromycin from the onset of incubation, with no bacterial regrowth occurring. Zhou et al. [68] reported similar results of Time-kill assays, thus demonstrating the synergistic antibacterial effects of the alkaloid EPI berberine and the antibiotic ciprofloxacin in a MDR clinical isolate ofKlebsiella pneumoniae.Figure 2
Time-kill curves of clarithromycin alone and combined with red seaweed (a)Gracilariasp. extract and (b)P. dentataextract against drug-resistantE. coli. The Kam3-AcrBE. coli cells at a cell density of 7 Log CFU/mL were added with clarithromycin alone or combined with seaweed extracts, and the cell numbers were monitored every 3 h for 12 h.
(a)
(b)


## 3.4. Efflux-Mediated Properties of the Seaweed Extracts

To analyze the potential efflux-mediated properties of the seaweed extracts, we monitored the accumulation of EB into Kam3-AcrB in their presence. EB is a substrate of the multidrug transporter AcrB inE. coli [67] and intensely fluoresces when bound to DNA. Figure 3(a) shows that EB accumulation was greater in cells exposed to the extract and positive control CCCP (20 μg/mL; a proton uncoupler) rather than the control (no addition). This indicates that theL. japonicaextract possesses an efflux pump inhibitor that interferes with EB efflux from Kam3-AcrB. Similar results were observed in cells exposed toS. horneri(Figure 3(b)),Gracilaria sp. (Figure 4(a)), andP. dentataextracts (Figure 4(b)). The EB accumulation assays were also performed in Kam3 in the presences of the seaweed extracts and observed no obvious increase of EB accumulation (data not shown). This might suggest that the seaweed extracts increase the EB accumulation possibly by mediating efflux pump rather than by increasing membrane permeability. Our data indicate that all the extracts contain functional compounds that increase EB accumulation in Kam-AcrB. This highlights their potential as EPIs of AcrB, which is known to be overexpressed with a strong promoter in Kam3-AcrB strains. However, we were unable to rule out the possibility that the extracts interfere with other efflux pumps that translocate EB.Figure 3
Effects of brown seaweeds (a)L. japonica extract and (b)S. horneriextract on EB accumulation in drug-resistantE. coli. TheE. coli Kam3-AcrB cells were added with glucose (25 mM) and EB (25 μM) in presence or absence of CCCP (20 μg/mL) or seaweed extracts (1/2 IC50). The fluorescence was monitored at Ex 520 nm and Em 600 nm.
(a)
(b)Figure 4
Effects of red seaweeds (a)Gracilaria sp. extract and (b)P. dentata extract on EB accumulation in drug-resistantE. coli. TheE. coli Kam3-AcrB cells were added with glucose (25 mM) and EB (25 μM) in presence or absence of CCCP (20 μg/mL) or seaweed extracts (1/2 IC50). The fluorescence was monitored at Ex 520 nm and Em 600 nm.
(a)
(b)


## 3.5. Chemical Compositions of the Seaweed Extracts

Table4 shows the chemical composition of the seaweed extracts as determined using gas chromatography-mass spectrometry (GC-MS). The classes with the symbol +
+ have more chemicals than the classes with the symbol + in each seaweed extract. All the extracts contained terpenes, terpenoids, and phenolic compounds. TheS. horneriandP. dentata extracts contained indoles, whereas theGracilariasp. extract contained pyrrole derivatives. In addition, halogenated aromatic compounds and alkaloids were identified in the red seaweeds.Table 4
Chemical composition of the ethanol extracts from the seaweeds.
Classifications
Brown seaweeds
Red seaweeds
L. japnoica
S. horneri
Gracilaria sp.
P. dentata
Terpenes
+
++
++
+
Terpenoids
+
+
+
+
Phenolic compounds
++
++
++
++
Indoles
–
+
–
+
Pyrrole derivatives
–
–
+
–
Halogenated aromatic compounds
–
–
+
+
Alkaloids
–
–
+
+
++, dominantly present in the extract; +, present in the extract; –, absent in the extract.The chemical structure of several plant-derived and synthetic EPIs has been characterized. The diterpenes carnosol and carnosic acid obtained from the herbRosmarinus officinalis are known to potentiate erythromycin and tetracycline againstS. aureus strains expressing Tet(K) and Msr (A) pumps [63]. Lorenzi et al. [69] indicated that the monoterpene geraniol obtained fromHelichrysum italicum increases the efficacy of quinolones and chloramphenicol inE. coli. Synthetic indole derivatives, such as [4-benzyloxy-2-(5-nitro-1H-2-yl)-phenyl]-methanol, have been reported as EPIs for the efflux pump NorA inS. aureus [70].Furthermore, synthetic halogenated phenothiazine derivatives, such as chlorpromazine, are thought to inhibit drug efflux pumps via various mechanisms [71]. Dwivedi et al. [23] indicated that alkaloid chanoclavine isolated fromIpomoea muricata potentiates the activity of tetracycline against MDR clinicalE. coli isolate, possibly by inhibiting drug efflux and downregulating the expression of drug transporters. In addition, catharanthine isolated from the leaves of flowering plantCatharanthus roseus has been shown to potentiate the activity of tetracycline, possibly due to the inhibition of the efflux pumps inP. aeruginosa [24]. 3,4-Dibromopyrrole-2,5-dione, a bacterial halogenated metabolite, is an effective EPI against bacterial strains that overexpress AcrB-TolC, MexAB-OprM, and MexXY-OprM [72]. The plant-derived alkaloids reserpine and piperine are also effective EPIs for fluoroquinolones inS. aureus [22, 33]. EPIs hinder the functions of efflux pumps via several mechanisms.Seaweeds are rich in bioactive terpenes and aromatic compounds, some of which possess specific characteristics that are absent or rarely found in terrestrial plants. For instance, approximately 25% of all halogenated natural products are alkaloids, and most of them are found in marine organisms [73]. Halogenated compounds obtained from seaweeds are varied and range from indoles, terpenes, and phenols to halogenated hydrocarbons; investigations into their potential use as EPIs would be beneficial [74]. Our IC50 data in Table 1 indicated that some antibacterial substances in the extracts are expelled by the multidrug transporter AcrB, which might suggest that the inhibition of drug transport was a result of competitive binding to AcrB. Further chromatographic purification of the ethanolic extracts obtained from seaweeds is required to identify and produce pure, active compounds. The findings of this study should encourage further research into seaweeds and their extracts with a view to identifying new EPIs with potential clinical applications.


## 4. Conclusions

The ethanolic seaweed extracts were able to potentiate the activities of macrolides againstE. coli and inhibit the action of efflux pumps in clinically important pathogens, thereby highlighting their potential as effective EPIs. We believe these extracts should be further investigated to exploit their ability to block drug efflux pumps, thereby facilitating the development of novel antimicrobial agents effective against clinically important MDR pathogens.



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*Source: 1836982-2019-01-01.xml*