Antibacterial and anti-virulence potential of plant phenolic compounds against <i>Vibrio parahaemolyticus</i>

F. Javier Vazquez-Armenta; M. Olivia Aros-Corrales; M. Lizeth Alvarez-Ainza; A. Thalia Bernal-Mercado; J. Fernando Ayala-Zavala; Adrian Ochoa-Leyva; A. Alexis Lopez-Zavala

doi:10.12688/f1000research.141268.2

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Research Article

Revised

Antibacterial and anti-virulence potential of plant phenolic compounds against Vibrio parahaemolyticus

[version 2; peer review: 1 approved, 2 approved with reservations]

F. Javier Vazquez-Armenta¹, M. Olivia Aros-Corrales¹, M. Lizeth Alvarez-Ainza¹, [...] A. Thalia Bernal-Mercado², J. Fernando Ayala-Zavala³, Adrian Ochoa-Leyva⁴, A. Alexis Lopez-Zavala¹

F. Javier Vazquez-Armenta¹, M. Olivia Aros-Corrales¹, [...] M. Lizeth Alvarez-Ainza¹, A. Thalia Bernal-Mercado², J. Fernando Ayala-Zavala³, Adrian Ochoa-Leyva⁴, A. Alexis Lopez-Zavala¹

PUBLISHED 03 Jul 2024

Author details Author details

¹ Departamento de Ciencias Quimico Biologicas, Universidad de Sonora, Hermosillo, Sonora, 83000, Mexico
² Departamento de Investigacion y Posgrado en Alimentos, Universidad de Sonora, Hermosillo, Sonora, 83000, Mexico
³ Coordinacion de Tecnologia de Alimentos de Origen Vegetal, Centro de Investigacion en Alimentacion y Desarrollo AC, Hermosillo, Sonora, 83304, Mexico
⁴ Departamento de Microbiologia Molecular, Instituto de Biotecnologia, Universidad Nacional Autonoma de Mexico, Mexico City, Mexico City, 62210, Mexico

F. Javier Vazquez-Armenta
Roles: Conceptualization, Formal Analysis, Investigation, Methodology, Project Administration, Visualization, Writing – Original Draft Preparation

M. Olivia Aros-Corrales
Roles: Formal Analysis, Methodology, Visualization

M. Lizeth Alvarez-Ainza
Roles: Formal Analysis, Writing – Review & Editing

A. Thalia Bernal-Mercado
Roles: Formal Analysis, Writing – Review & Editing

J. Fernando Ayala-Zavala
Roles: Formal Analysis, Writing – Original Draft Preparation, Writing – Review & Editing

Adrian Ochoa-Leyva
Roles: Funding Acquisition, Investigation, Project Administration, Resources, Writing – Review & Editing

A. Alexis Lopez-Zavala
Roles: Conceptualization, Funding Acquisition, Investigation, Methodology, Project Administration, Resources, Supervision, Visualization, Writing – Original Draft Preparation

OPEN PEER REVIEW

REVIEWER STATUS

This article is included in the Pathogens gateway.

Abstract

Background: Vibrio parahaemolyticus is a pathogenic bacterium that affects shrimp aquaculture; its infection can lead to severe production losses of up to 90%. On the other hand, plant phenolic compounds have emerged as a promising alternative to combat bacterial infections. The antibacterial and anti-virulence activity of the plant phenolic compounds quercetin, morin, vanillic acid, and protocatechuic acid against two strains of V. parahaemolyticus (Vp124 and Vp320) was evaluated.

Methods: The broth microdilution test was carried out to determine phenolic compounds' antibacterial activity. Moreover, the biofilm-forming ability of V. parahaemolyticus strains in the presence of phenolic compounds was determined by total biomass staining assay using the cationic dye crystal violet. The semisolid agar displacement technique was used to observe the effect of phenolic compounds on the swimming-like motility of V. parahaemolyticus.

Results: Results showed that phenolic compounds inhibited both strains effectively, with minimum inhibitory concentrations (MICs) ranging from 0.8 to 35.03 mM. Furthermore, at 0.125 – 0.5 × MIC of phenolic compounds, V. parahaemolyticus biofilms biomass was reduced by 63.22 – 92.68%. Also, quercetin and morin inhibited the motility of both strains by 15.86 – 23.64% (Vp124) and 24.28 – 40.71% (Vp320).

Conclusions: The results suggest that quercetin, morin, vanillic, and protocatechuic acids may be potential agents for controlling V. parahaemolyticus.

Keywords

anti-virulence, natural compounds, vibriosis, food safety

Corresponding author: A. Alexis Lopez-Zavala

Competing interests: No competing interests were disclosed.

Grant information: CONAHCYT grant Ciencia-Frontera-2019-263986 and UNISON-DCBS-2022-USO313007854 funded this research. The authors acknowledge to UNAM-CIC-UNISON-2019 and − 2023 grants for the academic exchange program and to CONAHCYT for the postdoctoral scholarship grant ESTANCIAS POSDOCTORALES POR MÉXICO 2022.
The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Copyright: © 2024 Vazquez-Armenta FJ et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

How to cite: Vazquez-Armenta FJ, Aros-Corrales MO, Alvarez-Ainza ML et al. Antibacterial and anti-virulence potential of plant phenolic compounds against Vibrio parahaemolyticus [version 2; peer review: 1 approved, 2 approved with reservations]. F1000Research 2024, 12:1256 (https://doi.org/10.12688/f1000research.141268.2) First published: 02 Oct 2023, 12:1256 (https://doi.org/10.12688/f1000research.141268.1) Latest published: 03 Jul 2024, 12:1256 (https://doi.org/10.12688/f1000research.141268.2)

Revised Amendments from Version 1

A statement on the importance of Vibrio parahaemolyticus infection in shrimp was added to the introduction section.
Information about the manufacturers of reagents and equipment was included in the materials and methods section.
A comparison between effective doses of antibiotics and phenolic compounds, as well as the challenges of using phenolic compounds in aquaculture, were included in the discussion section.

See the authors' detailed response to the review by Chumporn Soowannayan
See the authors' detailed response to the review by Saharuetai Jeamsripong

Introduction

According to the Food and Agriculture Organization of the United Nations (FAO), shrimp farming represents 53% of world aquaculture production of crustaceans.¹ In recent years, stocking densities have increased, which has led to the proliferation of diseases and the appearance of new pathologies that directly affect the profitability of shrimp production systems. Twenty percent of the diseases that appear during shrimp farming are related to bacteria, including the genus Vibrio.² Vibrio parahaemolyticus is considered of veterinary importance in shrimp farming, as some strains can infect shrimp, causing various diseases such as vibriosis. Strains that have acquired the pVA-1 plasmid, encoding the PirAB toxin, are of special concern because they cause Acute Hepatopancreatic Necrosis Disease (AHPND), initially defined as Early Mortality Syndrome (EMS).³ This atypical vibriosis has been responsible for losses of about 90% of production and, in some cases, up to 100%.³^,⁴ The increasing prevalence of antibiotic-resistant strains has made the control of V. parahaemolyticus infections progressively challenging.

Once effective in controlling these infections, traditional antibiotics are now facing limitations due to the emergence of resistant strains and the negative environmental impacts resulting from their extensive use in aquaculture.⁵ Moreover, the complex and dynamic nature of aquatic environments makes preventing the spread of bacterial infections in these systems difficult. Waterborne pathogens, such as V. parahaemolyticus, can quickly disseminate throughout aquaculture facilities, contaminating water sources and other organisms.

In addition, the biofilm-forming capacity of V. parahaemolyticus further complicates the control measures, as biofilms provide protection against antimicrobial agents and enable the bacteria to persist in the environment.⁶ Biofilms are communities of microorganisms attached to biotic or abiotic surfaces embedded in a self-produced matrix consisting of polysaccharides, proteins, and nucleic acids.⁷ In the inner part of the biofilms, the oxygen concentration is lower. Hence, an anaerobic atmosphere with a low pH develops, which causes a decrease in the activity of some antibiotics.⁸ Other phenomena caused by these altered microenvironment conditions are decreased bacterial metabolism and dropped replication times.⁹ Therefore, once the biofilm is formed, its eradication becomes complicated. This challenge requires the development of novel strategies and compounds capable of effectively inhibiting bacterial growth, biofilm formation, and motility, without causing significant environmental harm or promoting antibiotic resistance.

The use of natural compounds, such as phenolic compounds, offers a promising alternative due to their antimicrobial properties and potential to address the limitations of traditional antibiotics.¹⁰ Phenolic compounds, which are secondary metabolites produced by a wide variety of plants, have shown antibacterial activity against Gram-negative (Escherichia coli, Salmonella spp., Vibrio spp.) and Gram-positive (Listeria monocytogenes and Staphylococcus aureus) pathogenic bacteria.¹¹^–¹³ They are widely distributed in plants as defense mechanisms against microbial pathogens. Owing to their structural diversity and biological activity, phenolic compounds have gained increasing interest as potential antibacterial agents in various applications, including food preservation, agriculture, and human health.¹⁴^,¹⁵ For example, the flavonoids quercetin and myricetin showed a minimum inhibitory concentration (MIC) against V. parahaemolyticus of 0.125 and 0.25 mg/mL, respectively.¹⁶ In comparison, catechin and isorhamnetin showed MIC values of 0.05 and 0.025 mg/mL, respectively, against V. cholerae.¹⁷ Moreover, some of them, such as quercetin, catechin, ferulic acid, vanillic acid, and protocatechuic acid, have been shown to disrupt biofilm formation of pathogenic bacteria through different mechanisms of action, including inhibition of motility and EPS synthesis or/and disruption of intercellular communication.¹⁸^–²¹ However, the effect of these phenolic compounds on V. parahaemolyticus virulence factors, such as motility and biofilm formation capacity is unknown. Therefore, the present study aimed to evaluate (quercetin, morin, vanillic acid, and protocatechuic acid) phenolic compounds’ antibacterial and anti-virulence activity against a V. parahaemolyticus reference strain (CAIM 320; Vp320), and a strain isolated from white shrimp samples (Vp124). It is hypothesized that the selected phenolic compounds will significantly inhibit the growth, biofilm formation, and motility of V. parahaemolyticus strains, thus highlighting their potential as alternative agents for controlling infections and the persistence of this pathogen. Furthermore, the flavonoids (quercetin and morin) are expected to exhibit different mechanisms of action compared to the phenolic acids (vanillic and protocatechuic acids), which may offer unique advantages in tackling V. parahaemolyticus.

Methods

Strains and culture conditions

The human pathogenic reference strain CAIM 320 (Vp320) harboring the virulence genes tlh (thermolabile haemolysin), tdh (thermostable direct haemolysin), trh (TDH-related haemolysin), T3SS1: VP1680 (type III secretion system 1) and VPI and a strain isolated from shrimp, designated as Vp124²² were donated by the Laboratory of Clinical Microbiology of the University of Sonora. The studied strains were seeded on Thiosulfate Citrate Bile Sucrose agar (TCBS; BD Difco, Sparks, MD, USA), reseeded in Trypticase Soy Broth (TSB; BD Bacto, Sparks, MD, USA) with 3% NaCl, and incubated at 37°C during the assays.

Phenolic compounds

Standards (purity >95%) of gallic acid (G7384), vanillic acid (94770), protocatechuic acid (03930590), rutin (R5143), morin (M4008), and quercetin (Q4951) were purchased from Sigma-Aldrich (St Louis, MO, USA).

Antibacterial activity of phenolic compounds against Vibrio parahaemolyticus

The antibacterial activity of phenolic compounds against V. parahaemolyticus strains was determined as described by Alvarez et al.²³ An inoculum of each strain (Vp124 and Vp320) was prepared from exponential phase cultures (16 h in TSB + NaCl 3% at 37 °C), adjusting the optical density (600 nm) to 0.1 absorbance units, which is equivalent to 1 × 10⁸ CFU/mL. Concentrations of 0.8 mM - 40 mM of each compound (quercetin, morin, vanillic acid, protocatechuic acid, gallic acid, and rutin) were prepared in TSB + NaCl 3% at 37°C from dimethyl sulfoxide (DMSO; Sigma-Aldrich, St Louis, MO, USA) stocks. Subsequently, 5 μL of the adjusted inoculum and 295 μL of each concentration of the compounds were taken and placed in microplate wells (Costar 96; Corning, NY, USA) and incubated for 24 h at 37°C. The positive control was the same bacterial inoculum in TSB + NaCl 3% + DMSO 5%. As negative controls, each concentration of the tested compound and TSB + NaCl were placed in microplate wells without bacterial inoculum. The lowest compound concentration at which no visible growth was observed was considered the minimum inhibitory concentration (MIC). In addition, each compound's minimum bactericidal concentration (MBC) was determined by placing 50 μL aliquots from wells with three concentrations above the MIC on Mueller-Hinton agar (BD Difco, Sparks, MD, USA) + NaCl 3% plates. The MBC was considered the minimum concentration of each compound where no growth of V. parahaemolyticus was observed on the plate.²³

Effect of phenolic compounds on V. parahaemolyticus biofilm formation

The biofilm-forming ability of V. parahaemolyticus strains in the presence of phenolic compounds was determined by total biomass staining assay using the cationic dye crystal violet according to Beltran-Torres et al.²⁴ An inoculum of each strain at an optical density of 0.1 units at 600 nm was prepared from exponential phase cultures. Each compound was dissolved in DMSO (5% of final volume) and added to TSB at 0.125, 0.25, and 0.5 × MIC. Subsequently, 5 μL of the inoculum and 295 μL of each concentration were taken, placed in microplate wells, and incubated for 24 h at 37 °C.

At the end of the incubation, the culture medium was removed from the microplate wells by aspiration. Three consecutive washes were performed with distilled water to remove weakly adherent cells, and then the microplate was allowed to dry at 50°C. After that, 300 μL of a 0.1% crystal violet (Sigma-Aldrich; St Louis, MO, USA) solution was added to each well and allowed to stand for 45 min to stain the biomass of the formed biofilms. The crystal violet solution was then carefully removed, and three consecutive washes were carried out to remove the excess dye with distilled water, followed by drying at 50°C. Finally, 300 μL of 20% acetic acid was added to each well to solubilize the crystal violet for 15 min, and the absorbance of the solution was read at 594 nm. The absorbance of the solubilized violet crystal is proportional to the total biomass of the biofilms formed in each well. Each experiment was performed in triplicate, and the results were expressed as a percentage of the reduction of total biomass compared to the positive control (biofilms formed without the presence of phenolic compounds) according to the following equation:

Biomass reduction (%) = \frac{Abs {positive control}_{594} - Abs {treatment}_{594}}{Abs {positive control}_{594}} \times 100

Effect of phenolic compounds on swimming motility of V. parahaemolyticus

The semisolid agar displacement technique was used to observe the effect of phenolic compounds on the swimming-like motility of V. parahaemolyticus following the protocol of Beltran-Torres et al.²⁴ Phenolic compounds were added to TSB + NaCl 3% medium at a final concentration of 0.5 × MIC. An inoculum of each V. parahaemolyticus strain from exponential phase cultures (18 h at 37°C in TSB + NaCl 3%) was prepared at a final 1 × 10⁸ CFU/mL concentration. Subsequently, 10 μL of bacterial suspension were taken and placed in the center of a Petri dish with semi-solid TSB + NaCl 3% agar (0.42% agar) and incubated for 16 h at 37°C. Finally, the diameter of motility halos was measured. The experiment was performed in triplicate, and cultures of each V. parahaemolyticus strain on TSB + NaCl 3% without phenolic compounds were used as positive controls.

Statistical analysis

A complete randomized experimental design was used for all experiments. In the biofilm formation assay, the factors were the type of compound (quercetin, morin, vanillic acid, and protocatechuic acid) and the tested concentration (0.125, 0.25, and 0.5 × MIC), and the response variable was biomass reduction (%). In the motility assay, the factors were also the type of compound and concentration, and the response variable was motility inhibition (%). An analysis of variance (ANOVA) was performed, and where differences were found, multiple comparisons of means were performed by the Tukey-Kramer method at 95% confidence using the statistical software NCSS 2007.

Results

Antibacterial activity of phenolic compounds against V. parahaemolyticus

The MICs of the assessed compounds are shown in Table 1, where four of the six compounds were able to inhibit the growth of both Vibrio strains. The flavonoid quercetin was the most effective, presenting the lowest MIC (0.8 mM for both strains). The flavonoid morin and protocatechuic acid showed the same effect on both Vibrio strains, with MICs of 1.6 and 28.43 mM, respectively. On the other hand, vanillic acid presented a MIC equal to 31.73 mM for the Vp124 strain and 35.03 mM for the Vp320 strain. In contrast, rutin and gallic acid did not inhibit the growth of V. parahaemolyticus at the evaluated concentrations, 0 - 4 mM and 0-41.12 mM, respectively. For this reason, they were discarded in the following determinations. The minimum bactericidal concentration was also evaluated; however, this effect was not observed at any tested concentrations. The observed MICs of quercetin, morin, protocatechuic acid, and vanillic acid against both Vibrio strains support our hypothesis that certain phenolic compounds can effectively inhibit the growth of V. parahaemolyticus.

Table 1. Minimum inhibitory concentrations (MIC) and minimum bactericidal concentrations (MBC) of phenolic compounds against V. parahaemolyticus.

Phenolic compound	Strain
	Vp124		Vp320
	MIC (mM)	MBC (mM)	MIC (mM)	MBC (mM)
Quercetin	0.8	>1.6	0.8	>1.6
Morin	1.6	>2.4	1.6	>2.4
Rutin	>4.0	>4.0	>4.0	>4.0
Protocatechuic acid	28.43	>35.03	28.43	>35.03
Vanillic acid	31.73	>38.33	35.03	>41.12
Gallic acid	>41.12	>41.12	>41.12	>41.12

Effect of phenolic compounds on V. parahaemolyticus biofilm formation

To determine the effect of phenolic compounds on V. parahaemolyticus biofilm formation, sub-inhibitory concentrations of each compound were selected based on the MIC (0.125, 0.25, and 0.5 × MIC). Figure 1 shows the percentage of total biomass of V. parahaemolyticus biofilms formed at different concentrations of quercetin and morin. Both compounds were able to interfere with the biofilm formation process. For quercetin at 0.125 × MIC, a 71.97% reduction (p<0.05) was observed on the Vp124 strain, while no significant differences were found between 0.25 and 0.5 × MIC, achieving a reduction of 77.97-80.85% (p<0.05) respect to control. A similar pattern was observed in strain Vp320, where quercetin at 0.125 × MIC reduced 48.44% of the total biomass of V. parahaemolyticus biofilms, and for 0.25-0.5 × MIC, total biomass reduction was 85.31 – 91.66%. Regarding the effectiveness of morin, a reduction (p<0.05) in the total biomass of biofilms of strain Vp124 of 73.39%, 84.08%, and 89.07% was observed at concentrations of 0.125, 0.25, and 0.5 × MIC, respectively. While in the Vp320 strain, no differences were observed in the percentages of reduction at the evaluated concentrations (p>0.05), showing a reduction of 88.84-89.93 % compared to the control. The significant decrease of V. parahaemolyticus biofilm formation observed at sub-inhibitory concentrations of quercetin and morin supports our hypothesis that certain phenolic compounds can effectively disrupt biofilm formation processes.

Figure 1. Effect of the flavonoids quercetin and morin on the total biomass of V. parahaemolyticus biofilms.

Values represent mean ± standard error (n = 3). Different literals represent differences (p<0.05) between the evaluated concentrations for each strain.

Figure 2 shows the total biomass of biofilms formed by V. parahaemolyticus in the presence of vanillic and protocatechuic acids. Both phenolic acids inhibited the biofilm formation capacity of both strains compared to the control. However, no differences were found (p>0.05) between the evaluated concentrations. Vanillic acid reduced the total biomass of Vp124 biofilms by 85.92% to 92.68% and 89.55% - 90.27%for Vp320 strain. In comparison, protocatechuic acid inhibited 91.61% - 93.12% and 91.04% - 92.82% of the biofilm formation of Vp124 and Vp320 strains, respectively.

Figure 2. Effect of vanillic and protocatechuic acids on the total biomass of V. parahaemolyticus biofilms.

Values represent mean ± standard error (n = 3). Different literals represent differences (p<0.05) between the concentrations evaluated for each strain.

Effect of phenolic compounds on the motility of V. parahaemolyticus

Table 2 shows the effect of quercetin, morin, vanillic and protocatechuic acids on the motility of Vp124 and Vp320 strains. At the tested concentrations, the flavonoids quercetin and morin inhibited the motility of both strains on soft agar (0.42% agar). In this assay, differences (p<0.05) were observed in the inhibition percentages of each strain. For the Vp124 strain, quercetin inhibited 15.86% of the displacement of the bacteria on soft agar; while an inhibition of 23.63% was observed for the Vp320 strain. In the presence of morin, a similar trend was observed, where Vp320 showed the highest (p<0.05) percentage of motility inhibition (40.71%), while for the Vp124 strain, the percentage of inhibition was 24.28%. However, phenolic acids at the evaluated concentration did not show the capacity to interfere with the motility of V. parahaemolyticus strains (p>0.05). These results demonstrated that the flavonoids quercetin and morin effectively inhibited the motility of V. parahaemolyticus strains Vp124 and Vp320, following the hypothesis that certain phenolic compounds can impact bacterial motility. On the other hand, phenolic acids, such as vanillic and protocatechuic acids, did not exhibit the same inhibitory effect on motility, highlighting the varying impacts of different phenolic compounds on V. parahaemolyticus motility.

Table 2. Effect of phenolic compounds at 0.5 × MIC on the swimming motility of V. parahaemolyticus.

Values are mean ± standard error (n = 3).

Compound	Motility inhibition (%)
Compound	Vp124	Vp320
Quercetin	15.86 ± 0.87	23.63 ± 4.13
Morin	24.28 ± 1.02	40.71 ± 3.53
Vanillic	0	0
Protocatechuic acid	0	0

Discussion

Phenolic compounds are known for their antimicrobial activity against various pathogenic bacteria, including Gram-negative and Gram-positive species.¹⁰^,¹¹ In this study, vanillic and protocatechuic acids, along with the flavonoids quercetin and morin, effectively inhibited the growth of V. parahaemolyticus. Notably, flavonoids exhibited 20-40 times stronger antibacterial activity than phenolic acids, as evidenced by their lower MICs, which aligns with findings from previous research. Abuga et al.¹⁶ demonstrated that quercetin and myricetin inhibited V. parahaemolyticus, with MICs of 0.4 and 0.8 mM, respectively. Similarly, Tinh et al.¹² evaluated the antibacterial activity of phenolic compounds against 96 V. parahaemolyticus isolates from Pacific white shrimp (L. vannamei) in Thailand. Among the tested compounds, vanillic acid displayed significant bactericidal activity, with MICs and MBCs ranging from 6.09 to 12.18 mM. Pyrogallol, however, exhibited the strongest antibacterial properties, with MICs and MBCs between 0.25 and 2.02 mM. Wu et al.²⁵ also investigated the MIC of 3-p-trans-coumaroyl-2-hydroxyquinic acid against various pathogenic bacteria, including V. parahaemolyticus. Their results showed MICs ranging from 7.06 to 28.24 mM for all evaluated pathogenic bacteria, with a MIC of 14.12 mM for V. parahaemolyticus. In conjunction with our current research findings, the outcomes of these earlier studies underscore the potential of phenolic compounds as effective antibacterial agents against V. parahaemolyticus. Our study contributes to the growing body of evidence by demonstrating the comparative effectiveness of flavonoids and phenolic acids, highlighting the promising role of these natural compounds in combating V. parahaemolyticus infections.

MIC values of the evaluated phenolic compounds were higher than MICs of commonly used antibiotics in aquaculture. For example, enrofloxacin, florfenicol, and oxytetracycline showed MIC values against V. parahaemolyticus strains isolated from diseased white shrimp ranging from 33 – 44.51 μM, 2.9 – 11.6 μM, and 0.55 – 17.4 μM, respectively.²⁶ However, MIC values of antibiotics in the millimolar range have also been reported, as in the case of oxytetracycline hydrochloride, which showed MICs against V. parahaemolyticus strains of 0.257 – 0.515 mM.²⁷ Despite the differences in the effective doses to inhibit planktonic growth, common antibiotics not only do not inhibit the formation of biofilms but, in certain bacterial species, promote their development.²⁸^–³⁰

One notable aspect of these results is the direct comparison between flavonoids and phenolic acids regarding their antibacterial activity against V. parahaemolyticus. While previous research has separately investigated various phenolic compounds for their antimicrobial properties, our study offers a unique contribution by evaluating and contrasting the effectiveness of both flavonoids and phenolic acids. The results of our study emphasize that flavonoids, such as quercetin and morin, exerted stronger antibacterial activity than phenolic acids, as evidenced by their lower MICs. This novel finding highlights the potential advantages of using flavonoids over phenolic acids in developing new strategies to control and prevent V. parahaemolyticus infections in aquaculture systems. Additionally, our study contributes to the growing body of evidence supporting using natural compounds as alternatives to traditional antibiotics, which is particularly important in increasing antibiotic resistance.

The antibacterial activity of phenolic compounds is closely related to their physicochemical properties and the ability to interact with the lipid membrane and intracellular targets. Phenolic acids can alter the morphology of treated cells and cause hyperpolarization of the lipid membrane with its consequent loss of integrity.²⁵^,³¹ While flavonoids can intercalate in different regions of lipid bilayers, causing changes in their fluidity, this effect depends on the number and position of hydroxyl groups.³² Wu et al.³³ found a strong and positive correlation (r = 0.921) between the antimicrobial activity of flavonoids against E. coli and their ability to reduce the fluidity of lipid membranes. Furthermore, flavonoids can inhibit essential enzymes for bacterial growth, e.g., quercetin, kaempferol, luteonin, galangin, and myricetin are inhibitors of DNA gyrase (IC₅₀ = 0.037-1.18 mg/mL).³⁴ Also, quercetin and apigenin can inhibit d-alanine:d-alanine ligase, the enzyme responsible for the binding of alanine residues during the assembly of peptidoglycan precursors.³⁵

Biofilms enable pathogenic bacteria to survive in adverse environments, such as nutrient scarcity and exposure to antimicrobial agents.⁷^,³⁶ V. parahaemolyticus can form biofilms on various surfaces in aquaculture-related settings, acting as a reservoir that compromises the safety of aquaculture products.⁶ Our study reveals that vanillic and protocatechuic acids and the flavonoids quercetin and morin can effectively interfere with V. parahaemolyticus biofilm formation (Figures 1 and 2), contributing new insights into the potential of these compounds. Liu et al.²¹ demonstrated that vanillic acid reduced the total biomass of V. alginolyticus biofilms by approximately 65% at a concentration of 2.9 mM. Additionally, 2,6-di-tert-butyl-4-methylphenol, found in garlic, green algae, and cyanobacteria, inhibited the biofilm formation of V. harveyi, V. parahaemolyticus, and V. vulnificus by 80%, 83%, and 80%, respectively.³⁷ Moreover, ethanolic extracts of ginger and its major compounds, 6-gingerol, 8-gingerol, and 6-shogaol, effectively inhibited V. parahaemolyticus biofilm formation, with 6-shogaol exhibiting the highest efficacy by reducing biofilm biomass by up to 80% at a concentration of 0.15 mM.³⁸ This study breaks new ground by assessing the anti-biofilm activity of selected flavonoids and phenolic acids against V. parahaemolyticus, demonstrating their potential as anti-biofilm agents in combating infections in aquaculture systems. This research significantly expands on prior findings, offering a deeper and more extensive understanding of phenolic compounds' capabilities in mitigating biofilm-associated risks within the aquaculture industry.

Motility is a key virulence factor in the Vibrio genus, essential in the initial adhesion to abiotic surfaces and subsequent biofilm formation.³⁹^,⁴⁰ Our study reveals that flavonoids quercetin and morin inhibited V. parahaemolyticus swimming motility, while vanillic and protocatechuic acids did not exert any inhibitory effects (Table 2). V. parahaemolyticus exhibits swimming motility in aquatic environments, driven by a single polar flagellum,⁴¹ which relies on the expression of approximately 60 genes organized in at least 11 operons.⁴²^,⁴³ Roy et al.⁴⁴ reported that quercetin (0.09-0.36 mM) repressed flaA and flgL genes encoding flagellin, inhibiting V. parahaemolyticus motility and biofilm formation on shrimp and crab tissues. Our results corroborated these findings and further suggested that quercetin and morin may impair biofilm formation due to their motility-inhibiting effects. Notably, Vp124 (shrimp isolate) and Vp320 (clinical isolate) exhibited differential responses to flavonoids, with Vp124 being more resistant. Previous studies have indicated that mutations in sodium-type flagellar motor genes (motX and motY) confer resistance to known bacterial motility inhibitors, such as phenamil and amiloride.⁴⁵ Investigating a potential similar resistance mechanism in these strains could be worthwhile. In contrast, the lack of inhibitory effects of vanillic and protocatechuic acids on V. parahaemolyticus motility suggests that they may interfere with biofilm formation through alternative mechanisms, such as modifying the physicochemical properties of the bacterial surface.¹⁸^,²⁰ This study provides insights into the potential role of phenolic compounds as motility inhibitors and enhances our understanding of biofilm disruption strategies in the aquaculture industry.

The different responses observed with flavonoid treatments between the Vp124 strain (isolated from shrimp), and the reference strain (clinical isolate) warrant further investigation to elucidate the underlying resistance mechanisms. It is plausible that genetic or phenotypic differences between the two strains may contribute to their varying susceptibilities to the tested compounds. The disparity in response could also be attributed to differences in gene expression profiles, metabolic pathways, or the presence of specific efflux pumps that confer resistance to these flavonoids.⁴⁶ Further research employing genomic, transcriptomic, and proteomic approaches could help to identify the key genetic determinants and molecular pathways responsible for these differential responses. By understanding the resistance mechanisms in play, it may be possible to tailor more targeted and effective interventions for controlling V. parahaemolyticus infections in different settings, such as aquaculture and clinical environments.

Despite the valuable insights gained from this study, some limitations should be acknowledged. Investigating a broader range of phenolic compounds and a more extensive and diverse set of V. parahaemolyticus strains, including environmental isolates, would help to create a more complex panorama. In addition, the specific mechanisms exerted by phenolic compounds in motility and biofilm formation could go deeper into exploring the molecular targets and pathways involved in the observed inhibitory effects. Additionally, the potential synergistic or antagonistic interactions between the tested compounds and their combination with other antimicrobial agents should be investigated to identify more effective biofilm disruption and motility inhibition strategies.

Finally, the challenge of using phenolic compounds in aquaculture includes obtaining enough quantities due to resource-intensive extraction processes, the variability in yield and purity based on plant species and extraction methods, and stability issues as these compounds can degrade under environmental conditions such as light, temperature, and pH changes. Effective delivery mechanisms are also needed to ensure the compounds reach their target sites in sufficient concentrations. Addressing these limitations involves developing enhanced extraction techniques to increase yield and purity, employing encapsulation methods to improve stability, and/or creating innovative delivery systems like controlled-release formulations. Exploring synergistic combinations with other antimicrobial agents can also enhance effectiveness and mitigate costs and side effects. Addressing these limitations will deepen our understanding of the potential applications of phenolic compounds in controlling V. parahaemolyticus infections and mitigating biofilm-associated risks in the aquaculture industry.

Conclusions

This study provides valuable insights into the potential of phenolic compounds, particularly quercetin and morin, in controlling V. parahaemolyticus infections by inhibiting biofilm formation and motility. Quercetin and morin exhibited strong antibacterial activity compared to vanillic and protocatechuic acids. Moreover, both flavonoids effectively reduced biofilm formation and motility, supporting the hypothesis that these compounds can interfere with key virulence factors in V. parahaemolyticus. Finally, further research should address the study's limitations and focus on the underlying molecular mechanisms, synergistic or antagonistic interactions between the tested compounds, and in vivo efficacy to fully harness the potential of these phenolic compounds in combating V. parahaemolyticus infections.

Data availability

Underlying data

Figshare: Underlying data supporting the results of the manuscript “Antimicrobial and Anti-Virulence Potential of Plant Phenolic Compounds Against Vibrio parahaemolyticus” https://doi.org/10.6084/m9.figshare.24123315.v4.⁴⁷

Data are available under the terms of the CC BY 4.0 Attribution International license (CC BY 4.0).

Acknowledgments

The authors thank Q.B. Cesar Otero-Leon for technical support.

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Version 2

VERSION 2 PUBLISHED 02 Oct 2023

Author details Author details

F. Javier Vazquez-Armenta
Roles: Conceptualization, Formal Analysis, Investigation, Methodology, Project Administration, Visualization, Writing – Original Draft Preparation

M. Olivia Aros-Corrales
Roles: Formal Analysis, Methodology, Visualization

M. Lizeth Alvarez-Ainza
Roles: Formal Analysis, Writing – Review & Editing

A. Thalia Bernal-Mercado
Roles: Formal Analysis, Writing – Review & Editing

J. Fernando Ayala-Zavala
Roles: Formal Analysis, Writing – Original Draft Preparation, Writing – Review & Editing

Adrian Ochoa-Leyva
Roles: Funding Acquisition, Investigation, Project Administration, Resources, Writing – Review & Editing

A. Alexis Lopez-Zavala
Roles: Conceptualization, Funding Acquisition, Investigation, Methodology, Project Administration, Resources, Supervision, Visualization, Writing – Original Draft Preparation

Competing interests

No competing interests were disclosed.

Grant information

CONAHCYT grant Ciencia-Frontera-2019-263986 and UNISON-DCBS-2022-USO313007854 funded this research. The authors acknowledge to UNAM-CIC-UNISON-2019 and − 2023 grants for the academic exchange program and to CONAHCYT for the postdoctoral scholarship grant ESTANCIAS POSDOCTORALES POR MÉXICO 2022.
The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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https://doi.org/10.12688/f1000research.141268.2

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https://doi.org/10.12688/f1000research.141268.1

© 2024 Vazquez-Armenta FJ et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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Vazquez-Armenta FJ, Aros-Corrales MO, Alvarez-Ainza ML et al. Antibacterial and anti-virulence potential of plant phenolic compounds against Vibrio parahaemolyticus [version 2; peer review: 1 approved, 2 approved with reservations]. F1000Research 2024, 12:1256 (https://doi.org/10.12688/f1000research.141268.2)

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ApprovedThe paper is scientifically sound in its current form and only minor, if any, improvements are suggested

Approved with reservations A number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.

Not approvedFundamental flaws in the paper seriously undermine the findings and conclusions

Version 2

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Reviewer Report 27 Sep 2024

Nguyen Ngoc Phuoc, University of Agriculture and Forestry, Hue University, Phung Hung, Vietnam

Approved

https://doi.org/10.5256/f1000research.168486.r316670

This study investigates the antibacterial and anti-virulence effects of the plant phenolic compounds quercetin, morin, vanillic acid, and protocatechuic acid on two strains of Vibrio parahaemolyticus (Vp124 and Vp320). As a proof-of-concept, it has the potential to support the hypothesis that these compounds significantly inhibit growth, biofilm formation, and motility of V. parahaemolyticus strains. The findings could inform larger studies involving additional V. parahaemolyticus strains. The experiments are well-designed and persuasive, providing a promising method for assessing the antibacterial and anti-virulence potential of these compounds. Results are clearly presented, and the discussion effectively highlights how this study contributes new knowledge to the existing literature in the field. However, the handling of individual details in the article is not perfect. There are some minor suggestions, which are specified below:

The introduction highlights the importance of investigating the antibacterial and anti-virulence effects of plant phenolic compounds. While this study focuses on two virulence factors of V. parahaemolyticus-biofilm formation and motility-there is an emphasis on biofilm formation, with insufficient discussion on motility. It would strengthen the introduction to include information on why studying bacterial motility is essential, as it plays a crucial role in the pathogen's ability to invade host tissues and contribute to infection dynamics. Providing this context will enhance the rationale for the study's
The method section for the antibacterial activity of phenolic compounds should include the preparation method for each compound in DMSO. This will help readers understand why the positive control was performed in TSB + NaCl 3% + DMSO 5%. Additionally, please specify the percentage of NaCl in the negative control treatment in the sentence: “As the negative controls, each concentration of the tested compound and TSB + NaCl were placed in microplate wells without bacterial inoculum.”
The limitation of small sample size should be mentioned in the discussion section to provide a more comprehensive assessment of the study's findings.

Is the work clearly and accurately presented and does it cite the current literature?

Yes
Is the study design appropriate and is the work technically sound?

Yes
Are sufficient details of methods and analysis provided to allow replication by others?

Yes
If applicable, is the statistical analysis and its interpretation appropriate?

Yes
Are all the source data underlying the results available to ensure full reproducibility?

Yes
Are the conclusions drawn adequately supported by the results?

Yes

Competing Interests: No competing interests were disclosed.

Reviewer Expertise: My research area focuses on the pathology of aquatic animals, particularly their immunology. I study the application of immunostimulants to combat diseases in fish and shrimp, exploring how these compounds can enhance immune responses and improve overall health in aquaculture.

I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.

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Reviewer Report 21 May 2024

Chumporn Soowannayan, Center of Excellence for Shrimp Molecular Biology and Biotechnology (CENTEX SHRIMP), Faculty of Science, Mahidol University, Salaya, Nakhon Pathom, Thailand

Approved with Reservations

https://doi.org/10.5256/f1000research.154697.r260789

The work is interesting and could be helpful to aquatic animal and human health research or bacteria disease ... Continue reading

The work is interesting and could be helpful to aquatic animal and human health research or bacteria disease research in general. The comments and questions are posted in the attached main manuscript file which can be viewed here.

Is the work clearly and accurately presented and does it cite the current literature?

Partly
Is the study design appropriate and is the work technically sound?

Partly
Are sufficient details of methods and analysis provided to allow replication by others?

Partly
If applicable, is the statistical analysis and its interpretation appropriate?

I cannot comment. A qualified statistician is required.
Are all the source data underlying the results available to ensure full reproducibility?

Yes
Are the conclusions drawn adequately supported by the results?

Yes

Competing Interests: No competing interests were disclosed.

Reviewer Expertise: Fish and shrimp diseases

I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above.

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Author Response 26 Jun 2024

Francisco Javier Vázquez Armenta, Departamento de Ciencias Quimico Biologicas, Universidad de Sonora, Hermosillo, 83000, Mexico

26 Jun 2024

Author Response

Dear reviewer,

We appreciate your valuable feedback, which has greatly enhanced the quality and clarity of our manuscript. Below, we address each of your major and minor concerns point-by-point.
... Continue reading Dear reviewer,

We appreciate your valuable feedback, which has greatly enhanced the quality and clarity of our manuscript. Below, we address each of your major and minor concerns point-by-point.

Please check if this sentence is correct. If I'm not wrong, the world cultured shrimp production in 2023 was around 6 million tons, but the total aquaculture production of that year was around 96 million tons.

Response: We appreciate your observations. The sentence was modified to indicate that this percentage is relative to crustacean production. Also, the reference was updated to the last published report.

Many Vibrio species can cause AHPND, not just V. parahaemolyticus.

Response: We appreciate your observations and agree that many AHPNDs are caused by vibrio and non-vibrio species carrying the pVA-1 plasmid, which expresses the PirAB toxin.
The paragraph was modified as follows:
“Vibrio parahaemolyticus is considered of veterinary importance in shrimp farming, as some strains can infect shrimp, causing various diseases such as vibriosis. Strains that have acquired the pVA-1 plasmid, encoding the PirAB toxin, are of special concern because they cause Acute Hepatopancreatic Necrosis Disease (AHPND), initially known as Early Mortality Syndrome (EMS)³. This atypical vibriosis has been responsible for losses of about 90% of production and, in some cases, up to 100%.”

Are the bacteria causing AHPND the same as those causing disease in humans? If I'm right, they are different.

Response: The bacteria causing Acute Hepatopancreatic Necrosis Disease (AHPND) in shrimp are typically strains of Vibrio parahaemolyticus, which are different from the strains that usually cause disease in humans. The paragraph was restructured to mention only the consequences of V. parahaemolyticus infections in shrimp farming.

What was the concentration of each compound in DMSO? What were the final concentrations of the DMSO left in the final cultures?

Response: The concentration of the DMSO stocks varies based on the evaluated compound, and the final cultures (both treatments and controls) had a 5% DMSO concentration.

How many replicates were performed for each compound and each concentration?

Response: MIC and MBC were made in triplicate.

Is this the correct reference?

Response: The correct reference was included.

How many replicates?

Response: Each experiment was performed in triplicate.

Why use standard error, not standard deviation?

Response: We have chosen to display the standard error on the graphs to show the accuracy of the estimate of the population mean, thereby providing a clear indication of the precision of our results. Additionally, using standard error helps readers visualize the differences among treatments, facilitating an intuitive comparison of the variability and significance between different groups.

How do we know if these two are the cause or the effect?

Response: Vibrio parahaemolyticus has two types of motilities: swimming and swarming. The former uses a polar flagellum in liquid media, and the latter uses lateral flagella in solid media or viscous environments. In our study, we focused on swimming motility since this kind of movement is related to the initial attachment of Vibrio to solid surfaces to start biofilm development (Zhang et al. 2023, Lu et al. 2021).

Thank you again for your comments.
Sincerely,
The authors

References:
Lu, Renfei, Junfang Sun, Yue Qiu, Miaomiao Zhang, Xingfan Xue, Xue Li, Wenhui Yang, Dongsheng Zhou, Lingfei Hu, and Yiquan Zhang. 2021. "The quorum sensing regulator OpaR is a repressor of polar flagellum genes in Vibrio parahaemolyticus." Journal of Microbiology 59 (7):651-657. doi: 10.1007/s12275-021-0629-3.
Zhang, Yiquan, Tingting Zhang, Yue Qiu, Miaomiao Zhang, Xiuhui Lu, Wenhui Yang, Lingfei Hu, Dongsheng Zhou, Bo Gao, and Renfei Lu. 2023. "Transcriptomic Profiles of Vibrio parahaemolyticus During Biofilm Formation." Current Microbiology 80 (12):371. doi: 10.1007/s00284-023-03425-7.
Dear reviewer,

We appreciate your valuable feedback, which has greatly enhanced the quality and clarity of our manuscript. Below, we address each of your major and minor concerns point-by-point.

Please check if this sentence is correct. If I'm not wrong, the world cultured shrimp production in 2023 was around 6 million tons, but the total aquaculture production of that year was around 96 million tons.

Response: We appreciate your observations. The sentence was modified to indicate that this percentage is relative to crustacean production. Also, the reference was updated to the last published report.

Many Vibrio species can cause AHPND, not just V. parahaemolyticus.

Response: We appreciate your observations and agree that many AHPNDs are caused by vibrio and non-vibrio species carrying the pVA-1 plasmid, which expresses the PirAB toxin.
The paragraph was modified as follows:
“Vibrio parahaemolyticus is considered of veterinary importance in shrimp farming, as some strains can infect shrimp, causing various diseases such as vibriosis. Strains that have acquired the pVA-1 plasmid, encoding the PirAB toxin, are of special concern because they cause Acute Hepatopancreatic Necrosis Disease (AHPND), initially known as Early Mortality Syndrome (EMS)³. This atypical vibriosis has been responsible for losses of about 90% of production and, in some cases, up to 100%.”

Are the bacteria causing AHPND the same as those causing disease in humans? If I'm right, they are different.

Response: The bacteria causing Acute Hepatopancreatic Necrosis Disease (AHPND) in shrimp are typically strains of Vibrio parahaemolyticus, which are different from the strains that usually cause disease in humans. The paragraph was restructured to mention only the consequences of V. parahaemolyticus infections in shrimp farming.

What was the concentration of each compound in DMSO? What were the final concentrations of the DMSO left in the final cultures?

Response: The concentration of the DMSO stocks varies based on the evaluated compound, and the final cultures (both treatments and controls) had a 5% DMSO concentration.

How many replicates were performed for each compound and each concentration?

Response: MIC and MBC were made in triplicate.

Is this the correct reference?

Response: The correct reference was included.

How many replicates?

Response: Each experiment was performed in triplicate.

Why use standard error, not standard deviation?

Response: We have chosen to display the standard error on the graphs to show the accuracy of the estimate of the population mean, thereby providing a clear indication of the precision of our results. Additionally, using standard error helps readers visualize the differences among treatments, facilitating an intuitive comparison of the variability and significance between different groups.

How do we know if these two are the cause or the effect?

Response: Vibrio parahaemolyticus has two types of motilities: swimming and swarming. The former uses a polar flagellum in liquid media, and the latter uses lateral flagella in solid media or viscous environments. In our study, we focused on swimming motility since this kind of movement is related to the initial attachment of Vibrio to solid surfaces to start biofilm development (Zhang et al. 2023, Lu et al. 2021).

Thank you again for your comments.
Sincerely,
The authors

References:
Lu, Renfei, Junfang Sun, Yue Qiu, Miaomiao Zhang, Xingfan Xue, Xue Li, Wenhui Yang, Dongsheng Zhou, Lingfei Hu, and Yiquan Zhang. 2021. "The quorum sensing regulator OpaR is a repressor of polar flagellum genes in Vibrio parahaemolyticus." Journal of Microbiology 59 (7):651-657. doi: 10.1007/s12275-021-0629-3.
Zhang, Yiquan, Tingting Zhang, Yue Qiu, Miaomiao Zhang, Xiuhui Lu, Wenhui Yang, Lingfei Hu, Dongsheng Zhou, Bo Gao, and Renfei Lu. 2023. "Transcriptomic Profiles of Vibrio parahaemolyticus During Biofilm Formation." Current Microbiology 80 (12):371. doi: 10.1007/s00284-023-03425-7.
Competing Interests: There is no conflict of interest. Close
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COMMENTS ON THIS REPORT

Author Response 26 Jun 2024

Francisco Javier Vázquez Armenta, Departamento de Ciencias Quimico Biologicas, Universidad de Sonora, Hermosillo, 83000, Mexico

26 Jun 2024

Author Response

Dear reviewer,

We appreciate your valuable feedback, which has greatly enhanced the quality and clarity of our manuscript. Below, we address each of your major and minor concerns point-by-point.
... Continue reading Dear reviewer,

We appreciate your valuable feedback, which has greatly enhanced the quality and clarity of our manuscript. Below, we address each of your major and minor concerns point-by-point.

Please check if this sentence is correct. If I'm not wrong, the world cultured shrimp production in 2023 was around 6 million tons, but the total aquaculture production of that year was around 96 million tons.

Response: We appreciate your observations. The sentence was modified to indicate that this percentage is relative to crustacean production. Also, the reference was updated to the last published report.

Many Vibrio species can cause AHPND, not just V. parahaemolyticus.

Response: We appreciate your observations and agree that many AHPNDs are caused by vibrio and non-vibrio species carrying the pVA-1 plasmid, which expresses the PirAB toxin.
The paragraph was modified as follows:
“Vibrio parahaemolyticus is considered of veterinary importance in shrimp farming, as some strains can infect shrimp, causing various diseases such as vibriosis. Strains that have acquired the pVA-1 plasmid, encoding the PirAB toxin, are of special concern because they cause Acute Hepatopancreatic Necrosis Disease (AHPND), initially known as Early Mortality Syndrome (EMS)³. This atypical vibriosis has been responsible for losses of about 90% of production and, in some cases, up to 100%.”

Are the bacteria causing AHPND the same as those causing disease in humans? If I'm right, they are different.

Response: The bacteria causing Acute Hepatopancreatic Necrosis Disease (AHPND) in shrimp are typically strains of Vibrio parahaemolyticus, which are different from the strains that usually cause disease in humans. The paragraph was restructured to mention only the consequences of V. parahaemolyticus infections in shrimp farming.

What was the concentration of each compound in DMSO? What were the final concentrations of the DMSO left in the final cultures?

Response: The concentration of the DMSO stocks varies based on the evaluated compound, and the final cultures (both treatments and controls) had a 5% DMSO concentration.

How many replicates were performed for each compound and each concentration?

Response: MIC and MBC were made in triplicate.

Is this the correct reference?

Response: The correct reference was included.

How many replicates?

Response: Each experiment was performed in triplicate.

Why use standard error, not standard deviation?

Response: We have chosen to display the standard error on the graphs to show the accuracy of the estimate of the population mean, thereby providing a clear indication of the precision of our results. Additionally, using standard error helps readers visualize the differences among treatments, facilitating an intuitive comparison of the variability and significance between different groups.

How do we know if these two are the cause or the effect?

Response: Vibrio parahaemolyticus has two types of motilities: swimming and swarming. The former uses a polar flagellum in liquid media, and the latter uses lateral flagella in solid media or viscous environments. In our study, we focused on swimming motility since this kind of movement is related to the initial attachment of Vibrio to solid surfaces to start biofilm development (Zhang et al. 2023, Lu et al. 2021).

Thank you again for your comments.
Sincerely,
The authors

References:
Lu, Renfei, Junfang Sun, Yue Qiu, Miaomiao Zhang, Xingfan Xue, Xue Li, Wenhui Yang, Dongsheng Zhou, Lingfei Hu, and Yiquan Zhang. 2021. "The quorum sensing regulator OpaR is a repressor of polar flagellum genes in Vibrio parahaemolyticus." Journal of Microbiology 59 (7):651-657. doi: 10.1007/s12275-021-0629-3.
Zhang, Yiquan, Tingting Zhang, Yue Qiu, Miaomiao Zhang, Xiuhui Lu, Wenhui Yang, Lingfei Hu, Dongsheng Zhou, Bo Gao, and Renfei Lu. 2023. "Transcriptomic Profiles of Vibrio parahaemolyticus During Biofilm Formation." Current Microbiology 80 (12):371. doi: 10.1007/s00284-023-03425-7.
Dear reviewer,

We appreciate your valuable feedback, which has greatly enhanced the quality and clarity of our manuscript. Below, we address each of your major and minor concerns point-by-point.

Please check if this sentence is correct. If I'm not wrong, the world cultured shrimp production in 2023 was around 6 million tons, but the total aquaculture production of that year was around 96 million tons.

Response: We appreciate your observations. The sentence was modified to indicate that this percentage is relative to crustacean production. Also, the reference was updated to the last published report.

Many Vibrio species can cause AHPND, not just V. parahaemolyticus.

Response: We appreciate your observations and agree that many AHPNDs are caused by vibrio and non-vibrio species carrying the pVA-1 plasmid, which expresses the PirAB toxin.
The paragraph was modified as follows:
“Vibrio parahaemolyticus is considered of veterinary importance in shrimp farming, as some strains can infect shrimp, causing various diseases such as vibriosis. Strains that have acquired the pVA-1 plasmid, encoding the PirAB toxin, are of special concern because they cause Acute Hepatopancreatic Necrosis Disease (AHPND), initially known as Early Mortality Syndrome (EMS)³. This atypical vibriosis has been responsible for losses of about 90% of production and, in some cases, up to 100%.”

Are the bacteria causing AHPND the same as those causing disease in humans? If I'm right, they are different.

Response: The bacteria causing Acute Hepatopancreatic Necrosis Disease (AHPND) in shrimp are typically strains of Vibrio parahaemolyticus, which are different from the strains that usually cause disease in humans. The paragraph was restructured to mention only the consequences of V. parahaemolyticus infections in shrimp farming.

What was the concentration of each compound in DMSO? What were the final concentrations of the DMSO left in the final cultures?

Response: The concentration of the DMSO stocks varies based on the evaluated compound, and the final cultures (both treatments and controls) had a 5% DMSO concentration.

How many replicates were performed for each compound and each concentration?

Response: MIC and MBC were made in triplicate.

Is this the correct reference?

Response: The correct reference was included.

How many replicates?

Response: Each experiment was performed in triplicate.

Why use standard error, not standard deviation?

Response: We have chosen to display the standard error on the graphs to show the accuracy of the estimate of the population mean, thereby providing a clear indication of the precision of our results. Additionally, using standard error helps readers visualize the differences among treatments, facilitating an intuitive comparison of the variability and significance between different groups.

How do we know if these two are the cause or the effect?

Response: Vibrio parahaemolyticus has two types of motilities: swimming and swarming. The former uses a polar flagellum in liquid media, and the latter uses lateral flagella in solid media or viscous environments. In our study, we focused on swimming motility since this kind of movement is related to the initial attachment of Vibrio to solid surfaces to start biofilm development (Zhang et al. 2023, Lu et al. 2021).

Thank you again for your comments.
Sincerely,
The authors

References:
Lu, Renfei, Junfang Sun, Yue Qiu, Miaomiao Zhang, Xingfan Xue, Xue Li, Wenhui Yang, Dongsheng Zhou, Lingfei Hu, and Yiquan Zhang. 2021. "The quorum sensing regulator OpaR is a repressor of polar flagellum genes in Vibrio parahaemolyticus." Journal of Microbiology 59 (7):651-657. doi: 10.1007/s12275-021-0629-3.
Zhang, Yiquan, Tingting Zhang, Yue Qiu, Miaomiao Zhang, Xiuhui Lu, Wenhui Yang, Lingfei Hu, Dongsheng Zhou, Bo Gao, and Renfei Lu. 2023. "Transcriptomic Profiles of Vibrio parahaemolyticus During Biofilm Formation." Current Microbiology 80 (12):371. doi: 10.1007/s00284-023-03425-7.
Competing Interests: There is no conflict of interest. Close
Report a concern

Views

Reviewer Report 15 May 2024

Saharuetai Jeamsripong, Department of Veterinary Public Health, Faculty of Veterinary Science, Chulalongkorn University, Bangkok, Bangkok, Thailand

Approved with Reservations

https://doi.org/10.5256/f1000research.154697.r272299

Antibacterial and anti-virulence potential of plant phenolic compounds against Vibrio parahaemolyticus
This study investigated antibacterial and anti-virulence effects of the plant phenolic compounds quercetin, morin, vanillic acid, and protocatechuic acid on two strains of V. parahaemolyticus (Vp124 and ... Continue reading

No reference protocols and equations are provided in the materials and methods section.
Details of media and reagents used, including company, city, and country should be included.
Why did the authors choose to test only two strains of V. parahaemolyticus? Testing only two isolates of V. parahaemolyticus is a major limitation due to the small sample size, which may affect the accuracy and generalizability of the study. This limitation should be addressed.
How many replicates were used in the experiments?
The rationale for selecting phenolic compounds over other chemicals should be explained.
The antibiotics and their concentrations used for treatment of Vibrio infection comparison with phenolic compounds should be specified. Additionally, the challenges of using plant phenolic compounds, including quantity of phenolic compounds and field application feasibility, should be discussed.

Minor concerns

Please verify the unit of MIC in the abstract.
There is an inconsistency in the results for vanillic acid in the table (31.73) and the text (32.73). This discrepancy needs to be corrected.
Data regarding the antibacterial activity of quercetin and morin, which is 20-40 times higher than that of phenolic acids, is redundant in discussion and should be revised.

Is the work clearly and accurately presented and does it cite the current literature?

Partly
Is the study design appropriate and is the work technically sound?

No
Are sufficient details of methods and analysis provided to allow replication by others?

Partly
If applicable, is the statistical analysis and its interpretation appropriate?

Yes
Are all the source data underlying the results available to ensure full reproducibility?

Partly
Are the conclusions drawn adequately supported by the results?

Yes

Competing Interests: No competing interests were disclosed.

Reviewer Expertise: antimicrobial resistance, aquaculture, epidemiology, food safety

CITE

Report a concern

Author Response 26 Jun 2024

Francisco Javier Vázquez Armenta, Departamento de Ciencias Quimico Biologicas, Universidad de Sonora, Hermosillo, 83000, Mexico

26 Jun 2024

Author Response
Dear reviewer,

We want to express our gratitude for your constructive comments, which have significantly helped us improve the quality and clarity of our manuscript. Here, we provide a ... Continue reading
Dear reviewer,

We want to express our gratitude for your constructive comments, which have significantly helped us improve the quality and clarity of our manuscript. Here, we provide a point-by-point response to major and minor concerns.

Major concerns

No reference protocols and equations are provided in the materials and methods section.

Response: we appreciate your observations. The materials and methods section was improved, including the reference in the first sentence of each section.

Details of media and reagents used, including company, city, and country should be included.

Response: Details of media and reagents were included in the materials and method section.

Why did the authors choose to test only two strains of V. parahaemolyticus? Testing only two isolates of V. parahaemolyticus is a major limitation due to the small sample size, which may affect the accuracy and generalizability of the study. This limitation should be addressed.

Response: we appreciate your feedback and acknowledge the limitation regarding the sample size of V. parahaemolyticus strains tested in our study. We selected two strains with distinct phenotypic and genotypic profiles to provide a starting point for understanding the potential variability within this species. However, we agree that the small sample size limits the generalizability of our findings. We plan to expand our study to include a larger and more diverse set of V. parahaemolyticus isolates to address this. This will allow us to validate our preliminary results and enhance the accuracy and applicability of our conclusions. We will also explicitly mention this limitation in the discussion section of our manuscript and outline our future research plans to address this issue. By doing so, we aim to provide a more comprehensive understanding of V. parahaemolyticus and contribute to the broader body of knowledge in this field.

How many replicates were used in the experiments?

Response: All experiments were done in triplicate.

The rationale for selecting phenolic compounds over other chemicals should be explained.

Response: thank you for your observation. We appreciate the opportunity to clarify our rationale for selecting phenolic compounds for this study.
The selection of phenolic compounds was driven by the proven antimicrobial properties, safety profile, natural origin, and the potential for synergistic effects. Phenolic compounds exert antimicrobial effects by disrupting cell membranes, inactivating essential enzymes, and interfering with microbial metabolism, enhancing their inhibitory potential against bacteria. Their natural occurrence in plants and presence in the human diet generally make them safer than synthetic chemicals, aligning with the trend toward plant-based antimicrobials. Previous research has demonstrated their efficacy against various Vibrio species, and the current study aims to confirm their effectiveness specifically against V. parahaemolyticus. Additionally, phenolic compounds can work synergistically with other antimicrobial agents, suggesting their potential in developing more effective treatment strategies.
We will incorporate this rationale into the manuscript to provide a clearer understanding of our choice and strengthen the context of our research. Thank you again for your insightful feedback.

The antibiotics and their concentrations used for treatment of Vibrio infection comparison with phenolic compounds should be specified. Additionally, the challenges of using plant phenolic compounds, including quantity of phenolic compounds and field application feasibility, should be discussed.

Response: we appreciate the opportunity to address these important points and provide additional clarity in our study. We recognize the significance of discussing the concentration of antibiotics used in aquiculture and the challenges of using plant-derived phenolic compounds to treat Vibrio infections. Accordingly, we have included a detailed paragraph in the discussion section to address these issues comprehensively.

Minor concerns

Please verify the unit of MIC in the abstract.

Response: the MIC unit in the abstract was verified.

There is an inconsistency in the results for vanillic acid in the table (31.73) and the text (32.73). This discrepancy needs to be corrected.

Response: we appreciate your observation; the correct value of MIC is 31.73 mM.

Data regarding the antibacterial activity of quercetin and morin, which is 20-40 times higher than that of phenolic acids, is redundant in discussion and should be revised.

Response: we appreciate your observation; this issue was addressed.

Thank you again for your comments.

Sincerely,

The authors
Dear reviewer,

We want to express our gratitude for your constructive comments, which have significantly helped us improve the quality and clarity of our manuscript. Here, we provide a point-by-point response to major and minor concerns.

Major concerns

No reference protocols and equations are provided in the materials and methods section.

Response: we appreciate your observations. The materials and methods section was improved, including the reference in the first sentence of each section.

Details of media and reagents used, including company, city, and country should be included.

Response: Details of media and reagents were included in the materials and method section.

Why did the authors choose to test only two strains of V. parahaemolyticus? Testing only two isolates of V. parahaemolyticus is a major limitation due to the small sample size, which may affect the accuracy and generalizability of the study. This limitation should be addressed.

Response: we appreciate your feedback and acknowledge the limitation regarding the sample size of V. parahaemolyticus strains tested in our study. We selected two strains with distinct phenotypic and genotypic profiles to provide a starting point for understanding the potential variability within this species. However, we agree that the small sample size limits the generalizability of our findings. We plan to expand our study to include a larger and more diverse set of V. parahaemolyticus isolates to address this. This will allow us to validate our preliminary results and enhance the accuracy and applicability of our conclusions. We will also explicitly mention this limitation in the discussion section of our manuscript and outline our future research plans to address this issue. By doing so, we aim to provide a more comprehensive understanding of V. parahaemolyticus and contribute to the broader body of knowledge in this field.

How many replicates were used in the experiments?

Response: All experiments were done in triplicate.

The rationale for selecting phenolic compounds over other chemicals should be explained.

Response: thank you for your observation. We appreciate the opportunity to clarify our rationale for selecting phenolic compounds for this study.
The selection of phenolic compounds was driven by the proven antimicrobial properties, safety profile, natural origin, and the potential for synergistic effects. Phenolic compounds exert antimicrobial effects by disrupting cell membranes, inactivating essential enzymes, and interfering with microbial metabolism, enhancing their inhibitory potential against bacteria. Their natural occurrence in plants and presence in the human diet generally make them safer than synthetic chemicals, aligning with the trend toward plant-based antimicrobials. Previous research has demonstrated their efficacy against various Vibrio species, and the current study aims to confirm their effectiveness specifically against V. parahaemolyticus. Additionally, phenolic compounds can work synergistically with other antimicrobial agents, suggesting their potential in developing more effective treatment strategies.
We will incorporate this rationale into the manuscript to provide a clearer understanding of our choice and strengthen the context of our research. Thank you again for your insightful feedback.

The antibiotics and their concentrations used for treatment of Vibrio infection comparison with phenolic compounds should be specified. Additionally, the challenges of using plant phenolic compounds, including quantity of phenolic compounds and field application feasibility, should be discussed.

Response: we appreciate the opportunity to address these important points and provide additional clarity in our study. We recognize the significance of discussing the concentration of antibiotics used in aquiculture and the challenges of using plant-derived phenolic compounds to treat Vibrio infections. Accordingly, we have included a detailed paragraph in the discussion section to address these issues comprehensively.

Minor concerns

Please verify the unit of MIC in the abstract.

Response: the MIC unit in the abstract was verified.

There is an inconsistency in the results for vanillic acid in the table (31.73) and the text (32.73). This discrepancy needs to be corrected.

Response: we appreciate your observation; the correct value of MIC is 31.73 mM.

Data regarding the antibacterial activity of quercetin and morin, which is 20-40 times higher than that of phenolic acids, is redundant in discussion and should be revised.

Response: we appreciate your observation; this issue was addressed.

Thank you again for your comments.

Sincerely,

The authors
Competing Interests: There is no conflict of interest. Close
Report a concern
Respond or Comment

COMMENTS ON THIS REPORT

Author Response 26 Jun 2024

Francisco Javier Vázquez Armenta, Departamento de Ciencias Quimico Biologicas, Universidad de Sonora, Hermosillo, 83000, Mexico

26 Jun 2024

Author Response
Dear reviewer,

We want to express our gratitude for your constructive comments, which have significantly helped us improve the quality and clarity of our manuscript. Here, we provide a ... Continue reading
Dear reviewer,

We want to express our gratitude for your constructive comments, which have significantly helped us improve the quality and clarity of our manuscript. Here, we provide a point-by-point response to major and minor concerns.

Major concerns

No reference protocols and equations are provided in the materials and methods section.

Response: we appreciate your observations. The materials and methods section was improved, including the reference in the first sentence of each section.

Details of media and reagents used, including company, city, and country should be included.

Response: Details of media and reagents were included in the materials and method section.

Why did the authors choose to test only two strains of V. parahaemolyticus? Testing only two isolates of V. parahaemolyticus is a major limitation due to the small sample size, which may affect the accuracy and generalizability of the study. This limitation should be addressed.

Response: we appreciate your feedback and acknowledge the limitation regarding the sample size of V. parahaemolyticus strains tested in our study. We selected two strains with distinct phenotypic and genotypic profiles to provide a starting point for understanding the potential variability within this species. However, we agree that the small sample size limits the generalizability of our findings. We plan to expand our study to include a larger and more diverse set of V. parahaemolyticus isolates to address this. This will allow us to validate our preliminary results and enhance the accuracy and applicability of our conclusions. We will also explicitly mention this limitation in the discussion section of our manuscript and outline our future research plans to address this issue. By doing so, we aim to provide a more comprehensive understanding of V. parahaemolyticus and contribute to the broader body of knowledge in this field.

How many replicates were used in the experiments?

Response: All experiments were done in triplicate.

The rationale for selecting phenolic compounds over other chemicals should be explained.

Response: thank you for your observation. We appreciate the opportunity to clarify our rationale for selecting phenolic compounds for this study.
The selection of phenolic compounds was driven by the proven antimicrobial properties, safety profile, natural origin, and the potential for synergistic effects. Phenolic compounds exert antimicrobial effects by disrupting cell membranes, inactivating essential enzymes, and interfering with microbial metabolism, enhancing their inhibitory potential against bacteria. Their natural occurrence in plants and presence in the human diet generally make them safer than synthetic chemicals, aligning with the trend toward plant-based antimicrobials. Previous research has demonstrated their efficacy against various Vibrio species, and the current study aims to confirm their effectiveness specifically against V. parahaemolyticus. Additionally, phenolic compounds can work synergistically with other antimicrobial agents, suggesting their potential in developing more effective treatment strategies.
We will incorporate this rationale into the manuscript to provide a clearer understanding of our choice and strengthen the context of our research. Thank you again for your insightful feedback.

The antibiotics and their concentrations used for treatment of Vibrio infection comparison with phenolic compounds should be specified. Additionally, the challenges of using plant phenolic compounds, including quantity of phenolic compounds and field application feasibility, should be discussed.

Response: we appreciate the opportunity to address these important points and provide additional clarity in our study. We recognize the significance of discussing the concentration of antibiotics used in aquiculture and the challenges of using plant-derived phenolic compounds to treat Vibrio infections. Accordingly, we have included a detailed paragraph in the discussion section to address these issues comprehensively.

Minor concerns

Please verify the unit of MIC in the abstract.

Response: the MIC unit in the abstract was verified.

There is an inconsistency in the results for vanillic acid in the table (31.73) and the text (32.73). This discrepancy needs to be corrected.

Response: we appreciate your observation; the correct value of MIC is 31.73 mM.

Data regarding the antibacterial activity of quercetin and morin, which is 20-40 times higher than that of phenolic acids, is redundant in discussion and should be revised.

Response: we appreciate your observation; this issue was addressed.

Thank you again for your comments.

Sincerely,

The authors
Dear reviewer,

We want to express our gratitude for your constructive comments, which have significantly helped us improve the quality and clarity of our manuscript. Here, we provide a point-by-point response to major and minor concerns.

Major concerns

No reference protocols and equations are provided in the materials and methods section.

Response: we appreciate your observations. The materials and methods section was improved, including the reference in the first sentence of each section.

Details of media and reagents used, including company, city, and country should be included.

Response: Details of media and reagents were included in the materials and method section.

Why did the authors choose to test only two strains of V. parahaemolyticus? Testing only two isolates of V. parahaemolyticus is a major limitation due to the small sample size, which may affect the accuracy and generalizability of the study. This limitation should be addressed.

Response: we appreciate your feedback and acknowledge the limitation regarding the sample size of V. parahaemolyticus strains tested in our study. We selected two strains with distinct phenotypic and genotypic profiles to provide a starting point for understanding the potential variability within this species. However, we agree that the small sample size limits the generalizability of our findings. We plan to expand our study to include a larger and more diverse set of V. parahaemolyticus isolates to address this. This will allow us to validate our preliminary results and enhance the accuracy and applicability of our conclusions. We will also explicitly mention this limitation in the discussion section of our manuscript and outline our future research plans to address this issue. By doing so, we aim to provide a more comprehensive understanding of V. parahaemolyticus and contribute to the broader body of knowledge in this field.

How many replicates were used in the experiments?

Response: All experiments were done in triplicate.

The rationale for selecting phenolic compounds over other chemicals should be explained.

Response: thank you for your observation. We appreciate the opportunity to clarify our rationale for selecting phenolic compounds for this study.
The selection of phenolic compounds was driven by the proven antimicrobial properties, safety profile, natural origin, and the potential for synergistic effects. Phenolic compounds exert antimicrobial effects by disrupting cell membranes, inactivating essential enzymes, and interfering with microbial metabolism, enhancing their inhibitory potential against bacteria. Their natural occurrence in plants and presence in the human diet generally make them safer than synthetic chemicals, aligning with the trend toward plant-based antimicrobials. Previous research has demonstrated their efficacy against various Vibrio species, and the current study aims to confirm their effectiveness specifically against V. parahaemolyticus. Additionally, phenolic compounds can work synergistically with other antimicrobial agents, suggesting their potential in developing more effective treatment strategies.
We will incorporate this rationale into the manuscript to provide a clearer understanding of our choice and strengthen the context of our research. Thank you again for your insightful feedback.

The antibiotics and their concentrations used for treatment of Vibrio infection comparison with phenolic compounds should be specified. Additionally, the challenges of using plant phenolic compounds, including quantity of phenolic compounds and field application feasibility, should be discussed.

Response: we appreciate the opportunity to address these important points and provide additional clarity in our study. We recognize the significance of discussing the concentration of antibiotics used in aquiculture and the challenges of using plant-derived phenolic compounds to treat Vibrio infections. Accordingly, we have included a detailed paragraph in the discussion section to address these issues comprehensively.

Minor concerns

Please verify the unit of MIC in the abstract.

Response: the MIC unit in the abstract was verified.

There is an inconsistency in the results for vanillic acid in the table (31.73) and the text (32.73). This discrepancy needs to be corrected.

Response: we appreciate your observation; the correct value of MIC is 31.73 mM.

Data regarding the antibacterial activity of quercetin and morin, which is 20-40 times higher than that of phenolic acids, is redundant in discussion and should be revised.

Response: we appreciate your observation; this issue was addressed.

Thank you again for your comments.

Sincerely,

The authors
Competing Interests: There is no conflict of interest. Close
Report a concern

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Version 2

VERSION 2 PUBLISHED 02 Oct 2023

Open Peer Review

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Reviewer Reports

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	1	2	3
Version 2 (revision) 03 Jul 24			read
Version 1 02 Oct 23	read	read

Saharuetai Jeamsripong, Chulalongkorn University, Bangkok, Thailand
Chumporn Soowannayan, Mahidol University, Salaya, Thailand
Nguyen Ngoc Phuoc, University of Agriculture and Forestry, Hue University, Phung Hung, Vietnam

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Reviewer Report

3 Views

27 Sep 2024 | for Version 2

Nguyen Ngoc Phuoc, University of Agriculture and Forestry, Hue University, Phung Hung, Vietnam

3 Views Cite this report Responses(0)

Approved

The introduction highlights the importance of investigating the antibacterial and anti-virulence effects of plant phenolic compounds. While this study focuses on two virulence factors of V. parahaemolyticus-biofilm formation and motility-there is an emphasis on biofilm formation, with insufficient discussion on motility. It would strengthen the introduction to include information on why studying bacterial motility is essential, as it plays a crucial role in the pathogen's ability to invade host tissues and contribute to infection dynamics. Providing this context will enhance the rationale for the study's
The method section for the antibacterial activity of phenolic compounds should include the preparation method for each compound in DMSO. This will help readers understand why the positive control was performed in TSB + NaCl 3% + DMSO 5%. Additionally, please specify the percentage of NaCl in the negative control treatment in the sentence: “As the negative controls, each concentration of the tested compound and TSB + NaCl were placed in microplate wells without bacterial inoculum.”
The limitation of small sample size should be mentioned in the discussion section to provide a more comprehensive assessment of the study's findings.

Is the work clearly and accurately presented and does it cite the current literature?

Yes
Is the study design appropriate and is the work technically sound?

Yes
Are sufficient details of methods and analysis provided to allow replication by others?

Yes
If applicable, is the statistical analysis and its interpretation appropriate?

Yes
Are all the source data underlying the results available to ensure full reproducibility?

Yes
Are the conclusions drawn adequately supported by the results?

Yes

Competing Interests

No competing interests were disclosed.

Reviewer Expertise

My research area focuses on the pathology of aquatic animals, particularly their immunology. I study the application of immunostimulants to combat diseases in fish and shrimp, exploring how these compounds can enhance immune responses and improve overall health in aquaculture.

I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.

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Reviewer Report

12 Views

21 May 2024 | for Version 1

Chumporn Soowannayan, Center of Excellence for Shrimp Molecular Biology and Biotechnology (CENTEX SHRIMP), Faculty of Science, Mahidol University, Salaya, Nakhon Pathom, Thailand

12 Views Cite this report Responses(1)

Approved With Reservations

Is the work clearly and accurately presented and does it cite the current literature?

Partly
Is the study design appropriate and is the work technically sound?

Partly
Are sufficient details of methods and analysis provided to allow replication by others?

Partly
If applicable, is the statistical analysis and its interpretation appropriate?

I cannot comment. A qualified statistician is required.
Are all the source data underlying the results available to ensure full reproducibility?

Yes
Are the conclusions drawn adequately supported by the results?

Yes

Competing Interests

No competing interests were disclosed.

Reviewer Expertise

Fish and shrimp diseases

Respond to this report

Responses (1)

Author Response

26 Jun 2024

Francisco Javier Vázquez Armenta, Departamento de Ciencias Quimico Biologicas, Universidad de Sonora, Hermosillo, 83000, Mexico

Dear reviewer,

We appreciate your valuable feedback, which has greatly enhanced the quality and clarity of our manuscript. Below, we address each of your major and minor concerns point-by-point.

Please check if this sentence is correct. If I'm not wrong, the world cultured shrimp production in 2023 was around 6 million tons, but the total aquaculture production of that year was around 96 million tons.

Response: We appreciate your observations. The sentence was modified to indicate that this percentage is relative to crustacean production. Also, the reference was updated to the last published report.

Many Vibrio species can cause AHPND, not just V. parahaemolyticus.

Response: We appreciate your observations and agree that many AHPNDs are caused by vibrio and non-vibrio species carrying the pVA-1 plasmid, which expresses the PirAB toxin.
The paragraph was modified as follows:
“Vibrio parahaemolyticus is considered of veterinary importance in shrimp farming, as some strains can infect shrimp, causing various diseases such as vibriosis. Strains that have acquired the pVA-1 plasmid, encoding the PirAB toxin, are of special concern because they cause Acute Hepatopancreatic Necrosis Disease (AHPND), initially known as Early Mortality Syndrome (EMS)³. This atypical vibriosis has been responsible for losses of about 90% of production and, in some cases, up to 100%.”

Are the bacteria causing AHPND the same as those causing disease in humans? If I'm right, they are different.

Response: The bacteria causing Acute Hepatopancreatic Necrosis Disease (AHPND) in shrimp are typically strains of Vibrio parahaemolyticus, which are different from the strains that usually cause disease in humans. The paragraph was restructured to mention only the consequences of V. parahaemolyticus infections in shrimp farming.

What was the concentration of each compound in DMSO? What were the final concentrations of the DMSO left in the final cultures?

Response: The concentration of the DMSO stocks varies based on the evaluated compound, and the final cultures (both treatments and controls) had a 5% DMSO concentration.

How many replicates were performed for each compound and each concentration?

Response: MIC and MBC were made in triplicate.

Is this the correct reference?

Response: The correct reference was included.

How many replicates?

Response: Each experiment was performed in triplicate.

Why use standard error, not standard deviation?

Response: We have chosen to display the standard error on the graphs to show the accuracy of the estimate of the population mean, thereby providing a clear indication of the precision of our results. Additionally, using standard error helps readers visualize the differences among treatments, facilitating an intuitive comparison of the variability and significance between different groups.

How do we know if these two are the cause or the effect?

Response: Vibrio parahaemolyticus has two types of motilities: swimming and swarming. The former uses a polar flagellum in liquid media, and the latter uses lateral flagella in solid media or viscous environments. In our study, we focused on swimming motility since this kind of movement is related to the initial attachment of Vibrio to solid surfaces to start biofilm development (Zhang et al. 2023, Lu et al. 2021).

Thank you again for your comments.
Sincerely,
The authors

References:
Lu, Renfei, Junfang Sun, Yue Qiu, Miaomiao Zhang, Xingfan Xue, Xue Li, Wenhui Yang, Dongsheng Zhou, Lingfei Hu, and Yiquan Zhang. 2021. "The quorum sensing regulator OpaR is a repressor of polar flagellum genes in Vibrio parahaemolyticus." Journal of Microbiology 59 (7):651-657. doi: 10.1007/s12275-021-0629-3.
Zhang, Yiquan, Tingting Zhang, Yue Qiu, Miaomiao Zhang, Xiuhui Lu, Wenhui Yang, Lingfei Hu, Dongsheng Zhou, Bo Gao, and Renfei Lu. 2023. "Transcriptomic Profiles of Vibrio parahaemolyticus During Biofilm Formation." Current Microbiology 80 (12):371. doi: 10.1007/s00284-023-03425-7.

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Competing Interests

There is no conflict of interest.

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Reviewer Report

19 Views

15 May 2024 | for Version 1

Saharuetai Jeamsripong, Department of Veterinary Public Health, Faculty of Veterinary Science, Chulalongkorn University, Bangkok, Bangkok, Thailand

19 Views Cite this report Responses(1)

Approved With Reservations

No reference protocols and equations are provided in the materials and methods section.
Details of media and reagents used, including company, city, and country should be included.
Why did the authors choose to test only two strains of V. parahaemolyticus? Testing only two isolates of V. parahaemolyticus is a major limitation due to the small sample size, which may affect the accuracy and generalizability of the study. This limitation should be addressed.
How many replicates were used in the experiments?
The rationale for selecting phenolic compounds over other chemicals should be explained.
The antibiotics and their concentrations used for treatment of Vibrio infection comparison with phenolic compounds should be specified. Additionally, the challenges of using plant phenolic compounds, including quantity of phenolic compounds and field application feasibility, should be discussed.

Minor concerns

Please verify the unit of MIC in the abstract.
There is an inconsistency in the results for vanillic acid in the table (31.73) and the text (32.73). This discrepancy needs to be corrected.
Data regarding the antibacterial activity of quercetin and morin, which is 20-40 times higher than that of phenolic acids, is redundant in discussion and should be revised.

Is the work clearly and accurately presented and does it cite the current literature?

Partly
Is the study design appropriate and is the work technically sound?

No
Are sufficient details of methods and analysis provided to allow replication by others?

Partly
If applicable, is the statistical analysis and its interpretation appropriate?

Yes
Are all the source data underlying the results available to ensure full reproducibility?

Partly
Are the conclusions drawn adequately supported by the results?

Yes

Competing Interests

No competing interests were disclosed.

Reviewer Expertise

antimicrobial resistance, aquaculture, epidemiology, food safety

Respond to this report

Responses (1)

Author Response

26 Jun 2024

Francisco Javier Vázquez Armenta, Departamento de Ciencias Quimico Biologicas, Universidad de Sonora, Hermosillo, 83000, Mexico

Dear reviewer,

We want to express our gratitude for your constructive comments, which have significantly helped us improve the quality and clarity of our manuscript. Here, we provide a point-by-point response to major and minor concerns.

Major concerns

No reference protocols and equations are provided in the materials and methods section.

Response: we appreciate your observations. The materials and methods section was improved, including the reference in the first sentence of each section.

Details of media and reagents used, including company, city, and country should be included.

Response: Details of media and reagents were included in the materials and method section.

Why did the authors choose to test only two strains of V. parahaemolyticus? Testing only two isolates of V. parahaemolyticus is a major limitation due to the small sample size, which may affect the accuracy and generalizability of the study. This limitation should be addressed.

Response: we appreciate your feedback and acknowledge the limitation regarding the sample size of V. parahaemolyticus strains tested in our study. We selected two strains with distinct phenotypic and genotypic profiles to provide a starting point for understanding the potential variability within this species. However, we agree that the small sample size limits the generalizability of our findings. We plan to expand our study to include a larger and more diverse set of V. parahaemolyticus isolates to address this. This will allow us to validate our preliminary results and enhance the accuracy and applicability of our conclusions. We will also explicitly mention this limitation in the discussion section of our manuscript and outline our future research plans to address this issue. By doing so, we aim to provide a more comprehensive understanding of V. parahaemolyticus and contribute to the broader body of knowledge in this field.

How many replicates were used in the experiments?

Response: All experiments were done in triplicate.

The rationale for selecting phenolic compounds over other chemicals should be explained.

Response: thank you for your observation. We appreciate the opportunity to clarify our rationale for selecting phenolic compounds for this study.
The selection of phenolic compounds was driven by the proven antimicrobial properties, safety profile, natural origin, and the potential for synergistic effects. Phenolic compounds exert antimicrobial effects by disrupting cell membranes, inactivating essential enzymes, and interfering with microbial metabolism, enhancing their inhibitory potential against bacteria. Their natural occurrence in plants and presence in the human diet generally make them safer than synthetic chemicals, aligning with the trend toward plant-based antimicrobials. Previous research has demonstrated their efficacy against various Vibrio species, and the current study aims to confirm their effectiveness specifically against V. parahaemolyticus. Additionally, phenolic compounds can work synergistically with other antimicrobial agents, suggesting their potential in developing more effective treatment strategies.
We will incorporate this rationale into the manuscript to provide a clearer understanding of our choice and strengthen the context of our research. Thank you again for your insightful feedback.

The antibiotics and their concentrations used for treatment of Vibrio infection comparison with phenolic compounds should be specified. Additionally, the challenges of using plant phenolic compounds, including quantity of phenolic compounds and field application feasibility, should be discussed.

Response: we appreciate the opportunity to address these important points and provide additional clarity in our study. We recognize the significance of discussing the concentration of antibiotics used in aquiculture and the challenges of using plant-derived phenolic compounds to treat Vibrio infections. Accordingly, we have included a detailed paragraph in the discussion section to address these issues comprehensively.

Minor concerns

Please verify the unit of MIC in the abstract.

Response: the MIC unit in the abstract was verified.

There is an inconsistency in the results for vanillic acid in the table (31.73) and the text (32.73). This discrepancy needs to be corrected.

Response: we appreciate your observation; the correct value of MIC is 31.73 mM.

Data regarding the antibacterial activity of quercetin and morin, which is 20-40 times higher than that of phenolic acids, is redundant in discussion and should be revised.

Response: we appreciate your observation; this issue was addressed.

Thank you again for your comments.

Sincerely,

The authors

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Competing Interests

There is no conflict of interest.

Alongside their report, reviewers assign a status to the article:

Approved - the paper is scientifically sound in its current form and only minor, if any, improvements are suggested

Approved with reservations - A number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.

Not approved - fundamental flaws in the paper seriously undermine the findings and conclusions

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Antibacterial and anti-virulence potential of plant phenolic compounds against Vibrio parahaemolyticus

Abstract

Keywords

Revised Amendments from Version 1

Introduction

Methods

Strains and culture conditions

Phenolic compounds

Antibacterial activity of phenolic compounds against Vibrio parahaemolyticus

Effect of phenolic compounds on V. parahaemolyticus biofilm formation

Effect of phenolic compounds on swimming motility of V. parahaemolyticus

Statistical analysis

Results

Antibacterial activity of phenolic compounds against V. parahaemolyticus

Table 1. Minimum inhibitory concentrations (MIC) and minimum bactericidal concentrations (MBC) of phenolic compounds against V. parahaemolyticus.

Effect of phenolic compounds on V. parahaemolyticus biofilm formation

Figure 1. Effect of the flavonoids quercetin and morin on the total biomass of V. parahaemolyticus biofilms.

Figure 2. Effect of vanillic and protocatechuic acids on the total biomass of V. parahaemolyticus biofilms.

Effect of phenolic compounds on the motility of V. parahaemolyticus

Table 2. Effect of phenolic compounds at 0.5 × MIC on the swimming motility of V. parahaemolyticus.

Discussion

Conclusions

Data availability

Underlying data

Acknowledgments

References

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