Enhancement of pyocyanin production by subinhibitory concentration of royal jelly in <i>Pseudomonas aeruginosa</i>

Dina Auliya Amly; Puspita Hajardhini; Alma Linggar Jonarta; Heribertus Dedy Kusuma Yulianto; Heni Susilowati

doi:10.12688/f1000research.27915.1

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Research Article

Enhancement of pyocyanin production by subinhibitory concentration of royal jelly in Pseudomonas aeruginosa

[version 1; peer review: 1 approved with reservations, 1 not approved]

Dina Auliya Amly¹, Puspita Hajardhini¹, Alma Linggar Jonarta², Heribertus Dedy Kusuma Yulianto³, Heni Susilowati ²

Dina Auliya Amly¹, Puspita Hajardhini¹, [...] Alma Linggar Jonarta², Heribertus Dedy Kusuma Yulianto³, Heni Susilowati ²

PUBLISHED 11 Jan 2021

Author details Author details

¹ Master of Dental Sciences Program, Faculty of Dentistry, Universitas Gadjah Mada, Sleman, Yogyakarta, 55281, Indonesia
² Department of Oral Biology, Faculty of Dentistry, Universitas Gadjah Mada, Sleman, Yogyakarta, 55281, Indonesia
³ Department of Dental Biomedical Sciences, Faculty of Dentistry, Universitas Gadjah Mada, Sleman, Yogyakarta, 55281, Indonesia

Dina Auliya Amly
Roles: Data Curation, Investigation, Writing – Original Draft Preparation

Puspita Hajardhini
Roles: Data Curation, Investigation

Alma Linggar Jonarta
Roles: Formal Analysis, Supervision, Writing – Review & Editing

Heribertus Dedy Kusuma Yulianto
Roles: Supervision, Writing – Review & Editing

Heni Susilowati
Roles: Conceptualization, Funding Acquisition, Project Administration, Resources, Supervision, Writing – Review & Editing

OPEN PEER REVIEW

REVIEWER STATUS

This article is included in the Cell & Molecular Biology gateway.

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Abstract

Background: Pseudomonas aeruginosa, a multidrug resistant Gram-negative bacterium, produces pyocyanin, a virulence factor associated with antibiotic tolerance. High concentrations of royal jelly have an antibacterial effect, which may have the potential to overcome antibacterial resistance. However, in some cases, antibiotic tolerance can occur due to prolonged stress of low-dose antibacterial agents. This study aimed to investigate the effect of subinhibitory concentrations of royal jelly on bacterial growth and pyocyanin production of P. aeruginosa.
Methods: Pseudomonas aeruginosa ATCC^®10145™ and clinical isolates were cultured in BHI media for 18 hours followed by optical density measurements at 600 nm wavelength to determine minimum inhibitory concentration (MIC). After 36 hours of incubation, pyocyanin production was observed by measuring the absorbance at 690 nm. Pyocyanin concentrations were calculated using extinction coefficient 4310 M^-1cm^-1.
Results: Results of the MIC tests of both strains were 25%. The highest production of pyocyanin was observed in the subinhibitory concentration group 6.25%, which gradually decreased along with the decrease of royal jelly concentration. Results of one-way ANOVA tests differed significantly in pyocyanin production of the two strains between the royal jelly groups. Tukey HSD test showed concentrations of 12.5%, 6.25%, and 3.125% significantly increased pyocyanin production of ATCC^®10145™, and the concentrations of 12.5% and 6.25% significantly increased production of the clinical isolates.
Conclusions: This study concluded royal jelly concentrations of 25% or above could inhibit bacterial growth; however, only the concentrations of 12.5% and 6.25% could increase pyocyanin production in P. aeruginosa, both in ATCC^® 10145™ and clinical isolates. In conclusion, it is advisable to determine the appropriate concentration of royal jelly to obtain beneficial virulence inhibiting activity.

Keywords

royal jelly, antibacterial effect, Pseudomonas aeruginosa, pyocyanin

Corresponding author: Heni Susilowati

Competing interests: No competing interests were disclosed.

Grant information: Part of this research was supported by funding from the Ministry of Research, Technology and Higher Education, Republic of Indonesia, The Excellence Basic Research [Grant No. 26023/ UN1.DITLIT/DIT.LIT/LT/2019] to Heni Susilowati.
The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Copyright: © 2021 Amly DA 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: Amly DA, Hajardhini P, Jonarta AL et al. Enhancement of pyocyanin production by subinhibitory concentration of royal jelly in Pseudomonas aeruginosa [version 1; peer review: 1 approved with reservations, 1 not approved]. F1000Research 2021, 10:14 (https://doi.org/10.12688/f1000research.27915.1) First published: 11 Jan 2021, 10:14 (https://doi.org/10.12688/f1000research.27915.1) Latest published: 07 Dec 2021, 10:14 (https://doi.org/10.12688/f1000research.27915.4)

Introduction

Pseudomonas aeruginosa (P. aeruginosa) is one of the Gram-negative bacilli bacteria which causes nosocomial infections that can be fatal, especially in immunocompromised patients^1–3. These bacteria are often found in the dental unit waterlines which allows the transmission of these bacteria into the oral cavity⁴. As an opportunist pathogen, P. aeruginosa is also frequently involved in oral infections, such as necrotizing ulcerative gingivitis, periodontitis, and mandibular osteomyelitis^5–7. Although the mechanism is not clear yet, its presence in the oral cavity has been shown to result in systemic infections, such as nosocomial pneumonia⁸.

Based on the reports from several clinical cases, the infection caused by P. aeruginosa bacteria can be fatal. Treatment of P. aeruginosa infection is sometimes ineffective, which is closely related to the number of virulence factors possessed by the bacteria⁹. The bacterial cell surface components and some secretory products are important virulence factors of P. aeruginosa, one of which is pyocyanin¹⁰. Pyocyanin is a cytotoxic pigment from the Phenazine group of compounds that can facilitate biofilm development, cause pro-inflammatory effects, and result in host cell death¹¹.

The resistance of P. aeruginosa to various spectrums of antibiotics creates difficulties in handling the infection it causes¹². It has been reported recently that the administration of antibiotics below the minimum inhibitory concentration (MIC) can cause specific bacterial responses, such as an increase in pyocyanin production in P. aeruginosa. PAO1 and P14 are the attempts by the bacteria to survive under antibiotic stress¹³. This certainly motivates researchers to further analyze the infection they cause, and find the appropriate antibiotic concentration or dose to overcome the problem.

Royal jelly is a natural bee product that has the potential to be developed to overcome antibiotic resistance. Royal jelly has anti-inflammatory, antibacterial, and antioxidant effects¹⁴. Royal jelly proteins, such as Jelleine, major royal jelly protein-1 (MRJP1), and royalicin are known to have antibacterial effects against P. aeruginosa. Major royal jelly protein-1 and Jelleine can interfere with the permeability of the outer membrane of the cell, causing the loss of vital contents of bacterial cells, which in turn causes cell death. Cationic antimicrobial peptides, such as royalicin, are known to also interfere with cell membrane permeability in various Gram-positive and Gram-negative bacteria, such as P. aeruginosa^15–17. Results of previous studies have shown that royal jelly can inhibit the growth of P. aeruginosa. In this study, royal jelly showed inhibition of the growth of P. aeruginosa ATCC^® 27853™¹⁸. In addition, it has also been known that royal jelly in various concentrations can inhibit the nonspecific attachment of P. aeruginosa ATCC^® 27853™¹⁹, but so far, the effect of the subinhibitory concentration of royal jelly against these bacteria is unknown. Furthermore, as pyocyanin is an indicator of the pathogenicity of P. aeruginosa strains, the aim of this study was to determine the effect of subinhibitory royal jelly concentration on pyocyanin production in representative strains of a high level pyocyanin-producer P. aeruginosa ATCC^® 10145™ and clinical isolates.

Methods

This in vitro laboratory experimental research was done at the Integrated Research Laboratory of the Faculty of Dentistry, Universitas Gadjah Mada, Yogyakarta. All research procedures have been approved by the Ethics Committee of the Faculty of Dentistry, Universitas Gadjah Mada, Yogyakarta (No. 00393/KKEP/ FKG-UGM/EC/2020).

The royal jelly used in this study was obtained from Nusukan, Surakarta, Central Java, Indonesia. This product is produced from Apis mellifera bees that have been identified previously¹⁹. Royal jelly 5.5 grams was dissolved in 10 ml of cold phosphate buffered saline (PBS), then homogenized using a magnetic stirrer (24 hours, 4°C). The royal jelly solution was centrifuged (12,000 g, 45 minutes, 4°C), then the supernatant was filtered using 0.45 µm millipore to produce 55% royal jelly. Furthermore, royal jelly was stored at a temperature of 4–8°C²⁰.

Pseudomonas aeruginosa ATCC^® 10145™ (Thermo Scientific) was obtained from the Integrated Research Laboratory of the Faculty of Dentistry, Universitas Gadjah Mada. A clinical isolate of P. aeruginosa derived from patient sputum was obtained from the Laboratory of Microbiology, Faculty of Medicine, Public Health and Nursing, Universitas Gadjah Mada. Both of these strains were each inoculated in Luria Bertani broth and incubated at 37°C for 24 hours. After that, the culture was centrifuged at 3000 rpm for 15 minutes and then resuspended using 0.98% NaCl to obtain a bacterial concentration equivalent to 1.5 × 10⁵ CFU/ml.

Measurement of the effect of royal jelly on the viability of P. aeruginosa

A sterile 55% w/v royal jelly solution was diluted in brain heart infusion (BHI; Himedia Laboratories) broth to obtain a concentration of 50% and then serial dilution was performed in 96 well microplates. A total of 5 µl of the P. aeruginosa ATCC^® 10145™ suspension or clinical isolate bacteria (1.5 × 10⁵ CFU/ml) was inoculated in all groups, except the groups that had been determined as blanks (blanko). The culture was then incubated at 37°C for 18 hours. After that, the microplate was scanned using the Spark® Multimode Microplate Reader (Tecan trading AG) to measure optical density (OD) using a 600 nm wavelength. The percentage of bacterial viability inhibition was determined based on the OD value of the treatment group against the control.

Analysis of the effect of royal jelly on pyocyanin production

Royal jelly solution was diluted into sterile BHI broth to get the concentration of 12.5%, 6.25%, 3.125%, 1.56%, 0.78%, 0.39%, 0.19%, and 0.098% w/v. Both strains of P. aeruginosa were cultured on BHI broth containing various concentrations of royal jelly as treatment and BHI broth only as a blank (blanko). The cultures were incubated at 37°C for 36 hours, then the pyocyanin production of each strain was observed visually, which appeared green in the culture supernatant. The pyocyanin concentration was further quantified using previously published methods²¹. Briefly, after 36 hours of incubation, the culture supernatant was transferred to a sterile tube and centrifuged at a rate of 10,000 g for 30 minutes. The supernatant was filtered using a 0.45 µm Millipore filter and transferred to 96 new well microplates. The absorbance value of the supernatant containing pyocyanin was measured at a wavelength of 690 nm, then the pyocyanin concentration was calculated using the following equation²¹.

Concentration of pyocyanin = A₆₉₀ nm (A₆₉₀ nm of sample - A₆₉₀ nm of blank) / ε * d

ε = extinction coefficient (pyocyanin at A₆₉₀ nm = 4310 M^-1cm^-1)

d = path length (0.23 cm for 96 well microplate)

Statistical analysis

The data in this study were presented as the percentage of bacterial viability and pyocyanin concentration in the P. aeruginosa culture supernatant. All data were tested for normality using the Shapiro-Wilk and the Levene Test for homogeneity using SPSS Statistic v20. Furthermore, one-way ANOVA and Games-Howell parametric analysis were performed for bacterial cell viability data; and parametric one-way ANOVA followed by Tukey HSD on pyocyanin concentration data.

Results

Antibacterial activity of royal jelly against P. aeruginosa

The antibacterial activity of royal jelly against the two strains of P. aeruginosa is shown in Figure 1. Data on the percentage of bacterial growth inhibition shows normal distribution data (p>0.05), but has a non-homogeneous variant (p<0.05). One-way ANOVA showed a significant difference in the percentage of growth inhibition in P. aeruginosa ATCC^® 10145™ (p = 0.000) and P. aeruginosa clinical isolate (p = 0.000) between royal jelly treatment groups and negative control. In this study, it was proven that royal jelly can inhibit the viability of both P. aeruginosa strains starting from a concentration of 25%. The results of the multi-comparison analysis showed that there was no significant difference between the concentrations of 25% and 50% and significant differences were identified between the concentrations of 25% and 50% with 12.5% to 0.098% in both strains. It can be concluded that the MIC for both strains is 25%.

Figure 1. Percentage of inhibition of growth of P. aeruginosa bacteria (1.5 × 10⁵ CFU/well).

Bacterial cultures were incubated with varying concentrations of royal jelly for 18 hours at 37°C. Royal jelly 50% and 25% inhibit bacterial growth. The difference is based on the results of the Games-Howell analysis at the significance value (*) p <0.05. ATCC^® 10145™ (a) strain; clinical isolate (b).

Exposure to subinhibitory royal jelly concentrations induced increased pyocyanin production in P. aeruginosa

Pyocyanin was identified as green in culture supernatant P. aeruginosa ATCC^®10145™ and clinical isolate. After 36 hours of incubation, pyocyanin production was increased in the stimulated culture group with subinhibitory concentrations below 25%. The intensity of green color in the culture medium increased with the increase in the concentration of royal jelly (Figure 2). The change in the color intensity of the culture supernatant was consistent with the results of the pyocyanin concentration measurement.

Figure 2. Pyocyanin was identified from the green color of the P. aeruginosa culture supernatant after 36 h incubation at 37°C.

P. aeruginosa bacteria (1.5 × 10⁵ CFU/well) ATCC^® 10145™ (a); clinical isolate (b). Royal jelly 12.5% (A); 6.25% (B); 3,125% (C); 1.56% (D); 0.78% (E); 0.39% (F); 0.19% (G); 0.098% (H); 0% (I); No treatment (J).

Pyocyanin concentration data in each royal jelly treatment group and negative control were the results of experiments on triplicate cultures. Figure 3 shows the average pyocyanin concentration for each group. The highest average pyocyanin concentration was identified in P. aeruginosa ATCC^® 10145™ induced by royal jelly with a concentration of 6.25%, which was 23.59 μM, while the lowest mean was identified in clinical isolates of P. aeruginosa without exposure to royal jelly, which was 0.7 μM. The pyocyanin concentration of P. aeruginosa ATCC^® 10145™ was seen to be higher than clinical isolate in the same concentration in all treatment groups.

Figure 3. Pyocyanin production in P. aeruginosa bacteria (1.5 × 10⁵ CFU/well) after 36 hours of exposure to various concentrations of royal jelly.

Pyocyanin production increased due to exposure to sub-inhibitory royal jelly concentrations of 6.25% and decreased at lower royal jelly concentrations. The difference was based on the results of the Tukey-HSD analysis at the significance value (*) p <0.05. ATCC^® 10145™ (a) strain; (b) clinical isolate.

Pyocyanin concentration data both on ATCC^® 10145™ and clinical isolate in all groups were normally distributed (p>0.05) and homogeneous (p>0.05). There was a significant difference in the concentration of pyocyanin ATCC^® 10145™ (p = 0.000) and clinical isolate (p = 0.000) between the treatment groups. The results of multiple comparison analysis of Tukey-HSD on P. aeruginosa cultures of ATCC^® 10145™ showed a significant difference between the royal jelly groups with concentrations of 0% with 12.5%, 6.25%, and 3.125%. In addition, a significant difference in pyocyanin concentrations in clinical isolate was found between the 0% royal jelly group with 12.5% and 6.25%.

Discussion

The antibacterial effect of royal jelly has been widely reported by previous researchers^15–17. The ability of royal jelly to inhibit the growth of P. aeruginosa is thought to be related to the variety and concentration of its antibacterial protein. Royal jelly components that have been identified as having antibacterial activity are major royal jelly protein-1 (MRJP-1), Jelleine I–III, royalicin, and 10-hydroxy-2-decenoic (10-HDA)^15,17,22.

This study showed that royal jelly concentrations of 25% and 50% had antibacterial activity against P. aeruginosa ATCC^® 10145™ and clinical isolate. The results of this observation are different from previous studies that showed P. aeruginosa growth could be inhibited at concentrations >50%¹⁸. This difference is thought to be closely related to differences in geographical location, botanical origin, climate, and storage conditions of royal jelly, which affect the antibacterial component of royal jelly²³. Previous studies have shown that royal jelly originating from different geographic and botanical locations affects the quantity of 10-HDA. Royal jelly originating from tropical climates is reported to contain lower concentrations of 10-HDA than cold climates²³. The higher temperature and longer storage time also resulted in a significant reduction in the quantity of MRJP1²⁴. However, the bacterial strains studied probably also had an effect, as previously reported there was a variable response between clinical isolates and standard bacteria^18,19.

Pyocyanin is an indicator of the pathogenicity of P. aeruginosa. To our knowledge, this study report is the first to demonstrate a dualism effect of royal jelly on P. aeruginosa. The subinhibitory concentration of royal jelly amplify the effect of an autoinducer. It was able to increase the production of pyocyanin in ATCC^® 10145™ and clinical isolates to protect and maintain their survival¹³. The pyocyanin concentration in the ATCC^® 10145™ strain appeared to be significantly higher than the clinical isolates. This observation is in accordance with previous studies that found the ATCC^® 10145™ strain produced more pyocyanin than the clinical isolate strains from active ulcerative keratitis patients²⁵. The presence of phzM and phzS genes was thought to affect the concentration of pyocyanin produced²⁶. This was proven by previous studies that the phzM and phzS gene expression of multidrug resistance (MDR) clinical isolate P. aeruginosa was lower leading to less pyocyanin production than non-MDR isolates and PAO1 strains²⁷. Some clinical isolates were also reported not to have the genes so that these bacteria cannot produce pyocyanin²⁶. Other research results also showed that the pyocyanin concentration of ATCC^® 10145™ strains is higher than that of PAO1 and PA14 strains after incubation for 60 hours²⁸. It is estimated that ATCC^® 10145™ is one of the strong pyocyanin producing strains. However, other virulence factors possessed by this strain were lower than the clinical isolate strains so that they were considered less virulent²⁵.

Various virulence factors, including pyocyanin are generally associated with the quorum sensing mechanism²⁹. Quorum sensing refers to the communication process between microbial cells using autoinducer molecules³⁰. One of the autoinducer molecules that plays an important role in the regulation of pyocyanin production is the pseudomonas quinolone signal (PQS). Mutation of the PQS gene results in reduced pyocyanin production³¹. When bacterial cells are exposed to exogenic stress, such as an antibacterial agent that can threaten their survival, the bacteria immediately respond to the stimulus by inducing the production of PQS which is responsible for activating various genes involved in the production of virulence factors, including pyocyanin^29,32. Although the effect of royal jelly subinhibitor concentration on this autoinducer molecule is not yet known, several studies have reported that the increase in pyocyanin production is closely related to the effect of subinhibitor antibiotics that increase PQS gene expression³³. It is thought that this is the cause of increased pyocyanin production at subinhibitory concentrations.

The increase in pyocyanin production in P. aeruginosa bacteria will have implications for the mechanism of bacterial attachment and biofilm formation. Apart from its production, which is closely related to the quorum sensing mechanism, pyocyanin is also a signaling factor in the quorum sensing process itself. This was identified from the results of research on P. aeruginosa PAO1 and PA14³⁴. In addition, the increase in pyocyanin is likely to have an impact on the activity of bacteria to produce extracellular DNA (eDNA). Extracellular DNA is an important part of extracellular polymeric substance (EPS) which is the main component of the biofilm matrix. The increase in EPS production is very beneficial for the bacterial attachment process and subsequently the formation of biofilms. Pyocyanin can induce eDNA production in low level pyocyanin-producer strains, PAO1 and pyocyanin-deficient strains, PA14. In this study, it was proven that pyocyanin caused an increase in the production and release of eDNA, which is the main component in forming and stabilizing bacterial biofilms³⁵.

The increase in pyocyanin production induced by subinhibitory royal jelly concentrations in P. aeruginosa ATCC^® 10145™ and clinical isolates in this study is an interesting phenomenon. Although, subinhibitory royal jelly concentrations were not effective in inhibiting the growth of these bacteria, on the other hand, they increased production of pyocyanin virulence factors. This has inspired the alleged biphasic nature of royal jelly which has antibacterial potential, but at different exposure concentrations, it can induce the production of P. aeruginosa bacteria virulence factors. This phenomenon leads us to think that researchers, as well as medical practitioners, should be careful in determining the concentration of royal jelly for its antibacterial research purposes or its therapeutic potential. This of course requires further research on the mechanisms associated with bacterial response to subinhibitory concentrations of royal jelly.

Conclusions

Royal jelly at a concentration of 25% was only able to inhibit the growth of P. aeruginosa bacteria, but at subinhibitory concentrations it could increase pyocyanin production in P. aeruginosa strain ATCC^® 10145™ and clinical isolate. Based on the results of this study, we suggest selecting the appropriate dose or concentration for the purpose of inhibiting the growth and production of P. aeruginosa virulence factors.

Data availability

Underlying data

Figshare: Pseudomonas aeruginosa pyocyanin, https://doi.org/10.6084/m9.figshare.13247429.v1³⁶.

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

Acknowledgements

Researchers appreciate the help of Bunga Artika as a laboratory assistant who helped in this research.

Faculty Opinions recommended

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Version 4

VERSION 4 PUBLISHED 11 Jan 2021

Author details Author details

Dina Auliya Amly
Roles: Data Curation, Investigation, Writing – Original Draft Preparation

Puspita Hajardhini
Roles: Data Curation, Investigation

Alma Linggar Jonarta
Roles: Formal Analysis, Supervision, Writing – Review & Editing

Heribertus Dedy Kusuma Yulianto
Roles: Supervision, Writing – Review & Editing

Heni Susilowati
Roles: Conceptualization, Funding Acquisition, Project Administration, Resources, Supervision, Writing – Review & Editing

Competing interests

No competing interests were disclosed.

Grant information

Part of this research was supported by funding from the Ministry of Research, Technology and Higher Education, Republic of Indonesia, The Excellence Basic Research [Grant No. 26023/ UN1.DITLIT/DIT.LIT/LT/2019] to Heni Susilowati.
The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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© 2021 Amly DA 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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Amly DA, Hajardhini P, Jonarta AL et al. Enhancement of pyocyanin production by subinhibitory concentration of royal jelly in Pseudomonas aeruginosa [version 1; peer review: 1 approved with reservations, 1 not approved]. F1000Research 2021, 10:14 (https://doi.org/10.12688/f1000research.27915.1)

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Open Peer Review

Version 1

VERSION 1

PUBLISHED 11 Jan 2021

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Reviewer Report 04 Feb 2021

Theerthankar Das, Department of Infectious Diseases and Immunology, School of Medical Science, The University of Sydney, Camperdown, NSW, Australia

Not Approved

https://doi.org/10.5256/f1000research.30869.r77077

The intention of this study is good and to enlighten the antibacterial and anti-virulence effect of royal jelly in P. aeruginosa.

To be more conclusive this study needs more work:

Its better to

The intention of this study is good and to enlighten the antibacterial and anti-virulence effect of royal jelly in P. aeruginosa.

To be more conclusive this study needs more work:

Its better to use chloroform-HCl (standard and more popular) assay to quantify pyocyanin yield - its more rigorous than just taking absorbance at 690nm of pyocyanin (broad peak).
Biofilm (Crystal violet for biomass quantification) study needs to be done to ensure impact of royal jelly/pyocyanin on biofilm development.
Confocal microscopy will be of great help to show biofilm architecture.

Specific comments:

Why solubilized royal jelly is centrifuged and supernatant is only taken? What happens with pellets (are there any pellets found after centrifugation?). If yes - then how can we know that supernatant has all royal jelly?
At 25% royal jelly we see only 60% growth inhibition for both bacterial strains. Its not MIC - you can call it LC50 (lethal dose to reduce at least 50% growth). MIC is when there is zero or 1-2% growth only.

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?

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

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

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

Partly
Are the conclusions drawn adequately supported by the results?

Partly

Competing Interests: No competing interests were disclosed.

Reviewer Expertise: Biofilms, antimicrobial agents

I confirm that I have read this submission and believe that I have an appropriate level of expertise to state that I do not consider it to be of an acceptable scientific standard, for reasons outlined above.

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Report a concern

Author Response 13 Jul 2021

Heni Susilowati, Department of Oral Biology, Faculty of Dentistry, Universitas Gadjah Mada, Sleman, 55281, Indonesia

13 Jul 2021

Author Response

We thank you for the thorough and careful review of our manuscript. We apologize for our delay in revisions due to the various obstacles we face regarding the availability of ... Continue reading We thank you for the thorough and careful review of our manuscript. We apologize for our delay in revisions due to the various obstacles we face regarding the availability of laboratory facilities and access during the Covid-19 pandemic.
The following are our responses to the suggestions and specific comments.

The response to comment 1 (quantification of pyocyanin)
We measured the concentration of pyocyanin contained in the culture supernatant of P. aeruginosa. This condition is in accordance with the previous research conducted by Price-Whelan et al. (2007). Indeed, we only measured the absorbance value of the supernatant filtrate of the bacterial culture at a wavelength of 690 nm, but the pyocyanin concentration was then known by calculations using a formula published by previous researchers (Price-Whelan et al, 2007). The constant value of 4310 M -1cm -1 for the absorbance at 690 nm has been determined based on the research of Dietrich et al. 2006. Since our experimental conditions match the experimental conditions in both studies, both of which are published in outstanding journals, we feel it is sufficient to refer to their methods.

The response to comment 2 (Biofilm [Crystal violet for biomass quantification] study)
We have conducted additional experiments to detect the association between pyocyanin production in bacteria exposed to royal jelly and biofilm formation using the static microtiter plate biofilm assay (crystal violet staining) method. To analyze the relationship between pyocyanin production and biofilm mass production, we have used P. aeruginosa ATCC 10145 as this strain is more responsive in producing pyocyanin when compared to the clinical isolate strains we previously use. The results showed that the detectable biofilm mass significantly increased in bacterial cultures exposed to 12.5% royal jelly extract. Meanwhile, cultures treated with 25% royal jelly did not show any biofilm formation. The 6.25% royal jelly extract and the lower concentrations induced biofilm mass formation but at a lower quantity than 12.5% royal jelly. This evidence suggests a relationship between royal jelly concentration, pyocyanin production, and biofilm mass formation. The subinhibitory concentration (12.5%, 6.23%) of the royal jelly extract induced more pyocyanin production and biofilm mass formation rather than the higher concentrations.
Related changes were in the Methods, Results, and Conclusion of the Abstract, paragraph 6 of the Methods, paragraph 5 of Results, Figure 4, and line 5 of paragraph 6 on the Discussion section in the manuscript.

The response to comment 3 (microscopy of biofilm architecture)
In order to complement the data regarding the effect of royal jelly on the mass formation of biofilms, we have carried out observations of the microscopic architecture of P. aeruginosa ATCC 10145 biofilms using scanning electron microscopy, since we could not access the confocal microscopy method at our university. We chose secondary electron (SE) mode rather than a backscattered electron (BSE) to investigate biofilm in a three-dimensional perspective. The results of observations on representative samples showed that in the culture exposed to 25% royal jelly extract there was no biofilm mass deposition, while the group exposed to 12.5% royal jelly showed a larger density of biofilm than the 6.25% royal jelly group. Chlorhexidine 0.2% control showed total inhibition of biofilm mass formation.
Methods, results, discussion, and related references have been added to the manuscript on Methods, Results, and Conclusion in the Abstract, paragraph 7 of the Methods section, paragraph 6-7 of the Results section, and line 5 in paragraph 6 of the Discussion section.

Specific comments:

1. Why solubilized royal jelly is centrifuged and supernatant is only taken?
Answer: The extraction method using phosphate buffer saline (Hu et al, 2019) was chosen in order to dissolve major royal jelly protein 1 (MRJP1), the active components that are antibacterial or anti-adhesion. It is known that MRJP1 is a hydrosoluble protein dissolved in PBS, which is higher in supernatants than in pellets (Gismondi et al., 2017).

What happens with pellets (are there any pellets found after centrifugation?). If yes - then how can we know that supernatant has all royal jelly?

After centrifugation, there were indeed pellets, and it has been found that the pellets contain more liposoluble proteins than supernatants. To ensure that the compounds contained in the PBS royal jelly extract supernatant, further research is needed; However, from the research of Furusawa et al. (2016), it was found that MRJP1 was contained in the supernatant of PBS royal jelly extract in quite a large amount, reaching 27.6%.
The active components that are antibacterial or anti-adhesion in the supernatant of royal jelly extract in this study cannot be explained with certainty, but the royal jelly used in this study was thought to mainly contain MRJP1.
We add this explanation to paragraph 2 of the Discussion section. Additional sources of literature have been written in the reference list numbers 37, 38, and 39.

2. At 25% royal jelly, we see only 60% growth inhibition for both bacterial strains. It is not MIC - you can call it LC50 (lethal dose to reduce at least 50% growth). MIC is when there is zero or 1-2% growth only.
Answer: We agree with the reviewer's comments regarding the MIC score. In clinical isolates, 25% royal jelly extract caused 60% death in the bacterial population; meanwhile, in the standard strain (P. aeruginosa ATCC 10145), this concentration caused growth inhibition of 85%.
We have revised the first sentence in Methods of Abstract and two last sentences in the Results section.
We thank you for the thorough and careful review of our manuscript. We apologize for our delay in revisions due to the various obstacles we face regarding the availability of laboratory facilities and access during the Covid-19 pandemic.
The following are our responses to the suggestions and specific comments.

The response to comment 1 (quantification of pyocyanin)
We measured the concentration of pyocyanin contained in the culture supernatant of P. aeruginosa. This condition is in accordance with the previous research conducted by Price-Whelan et al. (2007). Indeed, we only measured the absorbance value of the supernatant filtrate of the bacterial culture at a wavelength of 690 nm, but the pyocyanin concentration was then known by calculations using a formula published by previous researchers (Price-Whelan et al, 2007). The constant value of 4310 M -1cm -1 for the absorbance at 690 nm has been determined based on the research of Dietrich et al. 2006. Since our experimental conditions match the experimental conditions in both studies, both of which are published in outstanding journals, we feel it is sufficient to refer to their methods.

The response to comment 2 (Biofilm [Crystal violet for biomass quantification] study)
We have conducted additional experiments to detect the association between pyocyanin production in bacteria exposed to royal jelly and biofilm formation using the static microtiter plate biofilm assay (crystal violet staining) method. To analyze the relationship between pyocyanin production and biofilm mass production, we have used P. aeruginosa ATCC 10145 as this strain is more responsive in producing pyocyanin when compared to the clinical isolate strains we previously use. The results showed that the detectable biofilm mass significantly increased in bacterial cultures exposed to 12.5% royal jelly extract. Meanwhile, cultures treated with 25% royal jelly did not show any biofilm formation. The 6.25% royal jelly extract and the lower concentrations induced biofilm mass formation but at a lower quantity than 12.5% royal jelly. This evidence suggests a relationship between royal jelly concentration, pyocyanin production, and biofilm mass formation. The subinhibitory concentration (12.5%, 6.23%) of the royal jelly extract induced more pyocyanin production and biofilm mass formation rather than the higher concentrations.
Related changes were in the Methods, Results, and Conclusion of the Abstract, paragraph 6 of the Methods, paragraph 5 of Results, Figure 4, and line 5 of paragraph 6 on the Discussion section in the manuscript.

The response to comment 3 (microscopy of biofilm architecture)
In order to complement the data regarding the effect of royal jelly on the mass formation of biofilms, we have carried out observations of the microscopic architecture of P. aeruginosa ATCC 10145 biofilms using scanning electron microscopy, since we could not access the confocal microscopy method at our university. We chose secondary electron (SE) mode rather than a backscattered electron (BSE) to investigate biofilm in a three-dimensional perspective. The results of observations on representative samples showed that in the culture exposed to 25% royal jelly extract there was no biofilm mass deposition, while the group exposed to 12.5% royal jelly showed a larger density of biofilm than the 6.25% royal jelly group. Chlorhexidine 0.2% control showed total inhibition of biofilm mass formation.
Methods, results, discussion, and related references have been added to the manuscript on Methods, Results, and Conclusion in the Abstract, paragraph 7 of the Methods section, paragraph 6-7 of the Results section, and line 5 in paragraph 6 of the Discussion section.

Specific comments:

1. Why solubilized royal jelly is centrifuged and supernatant is only taken?
Answer: The extraction method using phosphate buffer saline (Hu et al, 2019) was chosen in order to dissolve major royal jelly protein 1 (MRJP1), the active components that are antibacterial or anti-adhesion. It is known that MRJP1 is a hydrosoluble protein dissolved in PBS, which is higher in supernatants than in pellets (Gismondi et al., 2017).

What happens with pellets (are there any pellets found after centrifugation?). If yes - then how can we know that supernatant has all royal jelly?

After centrifugation, there were indeed pellets, and it has been found that the pellets contain more liposoluble proteins than supernatants. To ensure that the compounds contained in the PBS royal jelly extract supernatant, further research is needed; However, from the research of Furusawa et al. (2016), it was found that MRJP1 was contained in the supernatant of PBS royal jelly extract in quite a large amount, reaching 27.6%.
The active components that are antibacterial or anti-adhesion in the supernatant of royal jelly extract in this study cannot be explained with certainty, but the royal jelly used in this study was thought to mainly contain MRJP1.
We add this explanation to paragraph 2 of the Discussion section. Additional sources of literature have been written in the reference list numbers 37, 38, and 39.

2. At 25% royal jelly, we see only 60% growth inhibition for both bacterial strains. It is not MIC - you can call it LC50 (lethal dose to reduce at least 50% growth). MIC is when there is zero or 1-2% growth only.
Answer: We agree with the reviewer's comments regarding the MIC score. In clinical isolates, 25% royal jelly extract caused 60% death in the bacterial population; meanwhile, in the standard strain (P. aeruginosa ATCC 10145), this concentration caused growth inhibition of 85%.
We have revised the first sentence in Methods of Abstract and two last sentences in the Results section.
Competing Interests: I declare that the authors have no conflict of interest with the reviewer. Close
Report a concern
Author Response 09 Jul 2021

Heni Susilowati, Department of Oral Biology, Faculty of Dentistry, Universitas Gadjah Mada, Sleman, 55281, Indonesia

09 Jul 2021

Author Response

We thank you for the thorough and careful review of our manuscript.
The following are our responses to the suggestions and specific comments.

The response to comment 1 (quantification of ... Continue reading We thank you for the thorough and careful review of our manuscript.
The following are our responses to the suggestions and specific comments.

The response to comment 1 (quantification of pyocyanin)
We measured the concentration of pyocyanin contained in the culture supernatant of P. aeruginosa. This condition is in accordance with the previous research conducted by Price-Whelan et al. (2007). Indeed, we only measured the absorbance value of the supernatant filtrate of the bacterial culture at a wavelength of 690 nm, but the pyocyanin concentration was then known by calculations using a formula published by previous researchers (Price-Whelan et al, 2007). The constant value of 4310 M -1cm -1 for the absorbance at 690 nm has been determined based on the research of Dietrich et al. 2006. Since our experimental conditions match the experimental conditions in both studies, both of which are published in outstanding journals, we feel it is sufficient to refer to their methods.

The response to comment 2 (Biofilm [Crystal violet for biomass quantification] study)
We have conducted additional experiments to detect the association between pyocyanin production in bacteria exposed to royal jelly and biofilm formation using the static microtiter plate biofilm assay (crystal violet staining) method. To analyze the relationship between pyocyanin production and biofilm mass production, we have used P. aeruginosa ATCC 10145 as this strain is more responsive in producing pyocyanin when compared to the clinical isolate strains we previously use. The results showed that the detectable biofilm mass significantly increased in bacterial cultures exposed to 12.5% royal jelly extract. Meanwhile, cultures treated with 25% royal jelly did not show any biofilm formation. The 6.25% royal jelly extract and the lower concentrations induced biofilm mass formation but at a lower quantity than 12.5% royal jelly. This evidence suggests a relationship between royal jelly concentration, pyocyanin production, and biofilm mass formation. The subinhibitory concentration (12.5%, 6.23%) of the royal jelly extract induced more pyocyanin production and biofilm mass formation rather than the higher concentrations.
Related changes were in the Methods, Results, and Conclusion of the Abstract, paragraph 6 of the Methods, paragraph 5 of Results, Figure 4, and line 5 of paragraph 6 on the Discussion section in the manuscript.

The response to comment 3 (microscopy of biofilm architecture)
In order to complement the data regarding the effect of royal jelly on the mass formation of biofilms, we have carried out observations of the microscopic architecture of P. aeruginosa ATCC 10145 biofilms using scanning electron microscopy, since we could not access the confocal microscopy method at our university. We chose secondary electron (SE) mode rather than a backscattered electron (BSE) to investigate biofilm in a three-dimensional perspective. The results of observations on representative samples showed that in the culture exposed to 25% royal jelly extract there was no biofilm mass deposition, while the group exposed to 12.5% royal jelly showed a larger density of biofilm than the 6.25% royal jelly group. Chlorhexidine 0.2% control showed total inhibition of biofilm mass formation.

Methods, results, discussion, and related references have been added to the manuscript on Methods, Results, and Conclusion in the Abstract, paragraph 7 of the Methods section, paragraph 6-7 of the Results section, and line 5 in paragraph 6 of the Discussion section.

Specific comments
a. Why solubilized royal jelly is centrifuged and supernatant is only taken?
Answer: The extraction method using phosphate buffer saline (Hu et al, 2019) was chosen in order to dissolve major royal jelly protein 1 (MRJP1), the active components that are antibacterial or anti-adhesion. It is known that MRJP1 is a hydrosoluble protein dissolved in PBS, which is higher in supernatants than in pellets (Gismondi et al., 2017).

What happens with pellets (are there any pellets found after centrifugation?). If yes - then how can we know that supernatant has all royal jelly?
Answer: After centrifugation, there were indeed pellets, and it has been found that the pellets contain more liposoluble proteins than supernatants. To ensure that the compounds contained in the PBS royal jelly extract supernatant, further research is needed; However, from the research of Furusawa et al. (2016), it was found that MRJP1 was contained in the supernatant of PBS royal jelly extract in quite a large amount, reaching 27.6%.
The active components that are antibacterial or anti-adhesion in the supernatant of royal jelly extract in this study cannot be explained with certainty, but the royal jelly used in this study was thought to mainly contain MRJP1.

We add this explanation to paragraph 2 of the Discussion section. Additional sources of literature have been written in the reference list numbers 37, 38, and 39.

b. At 25% royal jelly, we see only 60% growth inhibition for both bacterial strains. It is not MIC - you can call it LC50 (lethal dose to reduce at least 50% growth). MIC is when there is zero or 1-2% growth only.
Answer: We agree with the reviewer's comments regarding the MIC score. In clinical isolates, 25% royal jelly extract caused 60% death in the bacterial population; meanwhile, in the standard strain (P. aeruginosa ATCC 10145), this concentration caused growth inhibition of 85%.
We have revised the first sentence in Methods of Abstract and two last sentences in the Results section.
We thank you for the thorough and careful review of our manuscript.
The following are our responses to the suggestions and specific comments.

The response to comment 1 (quantification of pyocyanin)
We measured the concentration of pyocyanin contained in the culture supernatant of P. aeruginosa. This condition is in accordance with the previous research conducted by Price-Whelan et al. (2007). Indeed, we only measured the absorbance value of the supernatant filtrate of the bacterial culture at a wavelength of 690 nm, but the pyocyanin concentration was then known by calculations using a formula published by previous researchers (Price-Whelan et al, 2007). The constant value of 4310 M -1cm -1 for the absorbance at 690 nm has been determined based on the research of Dietrich et al. 2006. Since our experimental conditions match the experimental conditions in both studies, both of which are published in outstanding journals, we feel it is sufficient to refer to their methods.

The response to comment 2 (Biofilm [Crystal violet for biomass quantification] study)
We have conducted additional experiments to detect the association between pyocyanin production in bacteria exposed to royal jelly and biofilm formation using the static microtiter plate biofilm assay (crystal violet staining) method. To analyze the relationship between pyocyanin production and biofilm mass production, we have used P. aeruginosa ATCC 10145 as this strain is more responsive in producing pyocyanin when compared to the clinical isolate strains we previously use. The results showed that the detectable biofilm mass significantly increased in bacterial cultures exposed to 12.5% royal jelly extract. Meanwhile, cultures treated with 25% royal jelly did not show any biofilm formation. The 6.25% royal jelly extract and the lower concentrations induced biofilm mass formation but at a lower quantity than 12.5% royal jelly. This evidence suggests a relationship between royal jelly concentration, pyocyanin production, and biofilm mass formation. The subinhibitory concentration (12.5%, 6.23%) of the royal jelly extract induced more pyocyanin production and biofilm mass formation rather than the higher concentrations.
Related changes were in the Methods, Results, and Conclusion of the Abstract, paragraph 6 of the Methods, paragraph 5 of Results, Figure 4, and line 5 of paragraph 6 on the Discussion section in the manuscript.

The response to comment 3 (microscopy of biofilm architecture)
In order to complement the data regarding the effect of royal jelly on the mass formation of biofilms, we have carried out observations of the microscopic architecture of P. aeruginosa ATCC 10145 biofilms using scanning electron microscopy, since we could not access the confocal microscopy method at our university. We chose secondary electron (SE) mode rather than a backscattered electron (BSE) to investigate biofilm in a three-dimensional perspective. The results of observations on representative samples showed that in the culture exposed to 25% royal jelly extract there was no biofilm mass deposition, while the group exposed to 12.5% royal jelly showed a larger density of biofilm than the 6.25% royal jelly group. Chlorhexidine 0.2% control showed total inhibition of biofilm mass formation.

Methods, results, discussion, and related references have been added to the manuscript on Methods, Results, and Conclusion in the Abstract, paragraph 7 of the Methods section, paragraph 6-7 of the Results section, and line 5 in paragraph 6 of the Discussion section.

Specific comments
a. Why solubilized royal jelly is centrifuged and supernatant is only taken?
Answer: The extraction method using phosphate buffer saline (Hu et al, 2019) was chosen in order to dissolve major royal jelly protein 1 (MRJP1), the active components that are antibacterial or anti-adhesion. It is known that MRJP1 is a hydrosoluble protein dissolved in PBS, which is higher in supernatants than in pellets (Gismondi et al., 2017).

What happens with pellets (are there any pellets found after centrifugation?). If yes - then how can we know that supernatant has all royal jelly?
Answer: After centrifugation, there were indeed pellets, and it has been found that the pellets contain more liposoluble proteins than supernatants. To ensure that the compounds contained in the PBS royal jelly extract supernatant, further research is needed; However, from the research of Furusawa et al. (2016), it was found that MRJP1 was contained in the supernatant of PBS royal jelly extract in quite a large amount, reaching 27.6%.
The active components that are antibacterial or anti-adhesion in the supernatant of royal jelly extract in this study cannot be explained with certainty, but the royal jelly used in this study was thought to mainly contain MRJP1.

We add this explanation to paragraph 2 of the Discussion section. Additional sources of literature have been written in the reference list numbers 37, 38, and 39.

b. At 25% royal jelly, we see only 60% growth inhibition for both bacterial strains. It is not MIC - you can call it LC50 (lethal dose to reduce at least 50% growth). MIC is when there is zero or 1-2% growth only.
Answer: We agree with the reviewer's comments regarding the MIC score. In clinical isolates, 25% royal jelly extract caused 60% death in the bacterial population; meanwhile, in the standard strain (P. aeruginosa ATCC 10145), this concentration caused growth inhibition of 85%.
We have revised the first sentence in Methods of Abstract and two last sentences in the Results section.
Competing Interests: We declare that we have no conflict of interest with this peer review. Close
Report a concern
Respond or Comment

COMMENTS ON THIS REPORT

Author Response 13 Jul 2021

Heni Susilowati, Department of Oral Biology, Faculty of Dentistry, Universitas Gadjah Mada, Sleman, 55281, Indonesia

13 Jul 2021

Author Response

We thank you for the thorough and careful review of our manuscript. We apologize for our delay in revisions due to the various obstacles we face regarding the availability of ... Continue reading We thank you for the thorough and careful review of our manuscript. We apologize for our delay in revisions due to the various obstacles we face regarding the availability of laboratory facilities and access during the Covid-19 pandemic.
The following are our responses to the suggestions and specific comments.

The response to comment 1 (quantification of pyocyanin)
We measured the concentration of pyocyanin contained in the culture supernatant of P. aeruginosa. This condition is in accordance with the previous research conducted by Price-Whelan et al. (2007). Indeed, we only measured the absorbance value of the supernatant filtrate of the bacterial culture at a wavelength of 690 nm, but the pyocyanin concentration was then known by calculations using a formula published by previous researchers (Price-Whelan et al, 2007). The constant value of 4310 M -1cm -1 for the absorbance at 690 nm has been determined based on the research of Dietrich et al. 2006. Since our experimental conditions match the experimental conditions in both studies, both of which are published in outstanding journals, we feel it is sufficient to refer to their methods.

The response to comment 2 (Biofilm [Crystal violet for biomass quantification] study)
We have conducted additional experiments to detect the association between pyocyanin production in bacteria exposed to royal jelly and biofilm formation using the static microtiter plate biofilm assay (crystal violet staining) method. To analyze the relationship between pyocyanin production and biofilm mass production, we have used P. aeruginosa ATCC 10145 as this strain is more responsive in producing pyocyanin when compared to the clinical isolate strains we previously use. The results showed that the detectable biofilm mass significantly increased in bacterial cultures exposed to 12.5% royal jelly extract. Meanwhile, cultures treated with 25% royal jelly did not show any biofilm formation. The 6.25% royal jelly extract and the lower concentrations induced biofilm mass formation but at a lower quantity than 12.5% royal jelly. This evidence suggests a relationship between royal jelly concentration, pyocyanin production, and biofilm mass formation. The subinhibitory concentration (12.5%, 6.23%) of the royal jelly extract induced more pyocyanin production and biofilm mass formation rather than the higher concentrations.
Related changes were in the Methods, Results, and Conclusion of the Abstract, paragraph 6 of the Methods, paragraph 5 of Results, Figure 4, and line 5 of paragraph 6 on the Discussion section in the manuscript.

The response to comment 3 (microscopy of biofilm architecture)
In order to complement the data regarding the effect of royal jelly on the mass formation of biofilms, we have carried out observations of the microscopic architecture of P. aeruginosa ATCC 10145 biofilms using scanning electron microscopy, since we could not access the confocal microscopy method at our university. We chose secondary electron (SE) mode rather than a backscattered electron (BSE) to investigate biofilm in a three-dimensional perspective. The results of observations on representative samples showed that in the culture exposed to 25% royal jelly extract there was no biofilm mass deposition, while the group exposed to 12.5% royal jelly showed a larger density of biofilm than the 6.25% royal jelly group. Chlorhexidine 0.2% control showed total inhibition of biofilm mass formation.
Methods, results, discussion, and related references have been added to the manuscript on Methods, Results, and Conclusion in the Abstract, paragraph 7 of the Methods section, paragraph 6-7 of the Results section, and line 5 in paragraph 6 of the Discussion section.

Specific comments:

1. Why solubilized royal jelly is centrifuged and supernatant is only taken?
Answer: The extraction method using phosphate buffer saline (Hu et al, 2019) was chosen in order to dissolve major royal jelly protein 1 (MRJP1), the active components that are antibacterial or anti-adhesion. It is known that MRJP1 is a hydrosoluble protein dissolved in PBS, which is higher in supernatants than in pellets (Gismondi et al., 2017).

What happens with pellets (are there any pellets found after centrifugation?). If yes - then how can we know that supernatant has all royal jelly?

After centrifugation, there were indeed pellets, and it has been found that the pellets contain more liposoluble proteins than supernatants. To ensure that the compounds contained in the PBS royal jelly extract supernatant, further research is needed; However, from the research of Furusawa et al. (2016), it was found that MRJP1 was contained in the supernatant of PBS royal jelly extract in quite a large amount, reaching 27.6%.
The active components that are antibacterial or anti-adhesion in the supernatant of royal jelly extract in this study cannot be explained with certainty, but the royal jelly used in this study was thought to mainly contain MRJP1.
We add this explanation to paragraph 2 of the Discussion section. Additional sources of literature have been written in the reference list numbers 37, 38, and 39.

2. At 25% royal jelly, we see only 60% growth inhibition for both bacterial strains. It is not MIC - you can call it LC50 (lethal dose to reduce at least 50% growth). MIC is when there is zero or 1-2% growth only.
Answer: We agree with the reviewer's comments regarding the MIC score. In clinical isolates, 25% royal jelly extract caused 60% death in the bacterial population; meanwhile, in the standard strain (P. aeruginosa ATCC 10145), this concentration caused growth inhibition of 85%.
We have revised the first sentence in Methods of Abstract and two last sentences in the Results section.
We thank you for the thorough and careful review of our manuscript. We apologize for our delay in revisions due to the various obstacles we face regarding the availability of laboratory facilities and access during the Covid-19 pandemic.
The following are our responses to the suggestions and specific comments.

The response to comment 1 (quantification of pyocyanin)
We measured the concentration of pyocyanin contained in the culture supernatant of P. aeruginosa. This condition is in accordance with the previous research conducted by Price-Whelan et al. (2007). Indeed, we only measured the absorbance value of the supernatant filtrate of the bacterial culture at a wavelength of 690 nm, but the pyocyanin concentration was then known by calculations using a formula published by previous researchers (Price-Whelan et al, 2007). The constant value of 4310 M -1cm -1 for the absorbance at 690 nm has been determined based on the research of Dietrich et al. 2006. Since our experimental conditions match the experimental conditions in both studies, both of which are published in outstanding journals, we feel it is sufficient to refer to their methods.

The response to comment 2 (Biofilm [Crystal violet for biomass quantification] study)
We have conducted additional experiments to detect the association between pyocyanin production in bacteria exposed to royal jelly and biofilm formation using the static microtiter plate biofilm assay (crystal violet staining) method. To analyze the relationship between pyocyanin production and biofilm mass production, we have used P. aeruginosa ATCC 10145 as this strain is more responsive in producing pyocyanin when compared to the clinical isolate strains we previously use. The results showed that the detectable biofilm mass significantly increased in bacterial cultures exposed to 12.5% royal jelly extract. Meanwhile, cultures treated with 25% royal jelly did not show any biofilm formation. The 6.25% royal jelly extract and the lower concentrations induced biofilm mass formation but at a lower quantity than 12.5% royal jelly. This evidence suggests a relationship between royal jelly concentration, pyocyanin production, and biofilm mass formation. The subinhibitory concentration (12.5%, 6.23%) of the royal jelly extract induced more pyocyanin production and biofilm mass formation rather than the higher concentrations.
Related changes were in the Methods, Results, and Conclusion of the Abstract, paragraph 6 of the Methods, paragraph 5 of Results, Figure 4, and line 5 of paragraph 6 on the Discussion section in the manuscript.

The response to comment 3 (microscopy of biofilm architecture)
In order to complement the data regarding the effect of royal jelly on the mass formation of biofilms, we have carried out observations of the microscopic architecture of P. aeruginosa ATCC 10145 biofilms using scanning electron microscopy, since we could not access the confocal microscopy method at our university. We chose secondary electron (SE) mode rather than a backscattered electron (BSE) to investigate biofilm in a three-dimensional perspective. The results of observations on representative samples showed that in the culture exposed to 25% royal jelly extract there was no biofilm mass deposition, while the group exposed to 12.5% royal jelly showed a larger density of biofilm than the 6.25% royal jelly group. Chlorhexidine 0.2% control showed total inhibition of biofilm mass formation.
Methods, results, discussion, and related references have been added to the manuscript on Methods, Results, and Conclusion in the Abstract, paragraph 7 of the Methods section, paragraph 6-7 of the Results section, and line 5 in paragraph 6 of the Discussion section.

Specific comments:

1. Why solubilized royal jelly is centrifuged and supernatant is only taken?
Answer: The extraction method using phosphate buffer saline (Hu et al, 2019) was chosen in order to dissolve major royal jelly protein 1 (MRJP1), the active components that are antibacterial or anti-adhesion. It is known that MRJP1 is a hydrosoluble protein dissolved in PBS, which is higher in supernatants than in pellets (Gismondi et al., 2017).

What happens with pellets (are there any pellets found after centrifugation?). If yes - then how can we know that supernatant has all royal jelly?

After centrifugation, there were indeed pellets, and it has been found that the pellets contain more liposoluble proteins than supernatants. To ensure that the compounds contained in the PBS royal jelly extract supernatant, further research is needed; However, from the research of Furusawa et al. (2016), it was found that MRJP1 was contained in the supernatant of PBS royal jelly extract in quite a large amount, reaching 27.6%.
The active components that are antibacterial or anti-adhesion in the supernatant of royal jelly extract in this study cannot be explained with certainty, but the royal jelly used in this study was thought to mainly contain MRJP1.
We add this explanation to paragraph 2 of the Discussion section. Additional sources of literature have been written in the reference list numbers 37, 38, and 39.

2. At 25% royal jelly, we see only 60% growth inhibition for both bacterial strains. It is not MIC - you can call it LC50 (lethal dose to reduce at least 50% growth). MIC is when there is zero or 1-2% growth only.
Answer: We agree with the reviewer's comments regarding the MIC score. In clinical isolates, 25% royal jelly extract caused 60% death in the bacterial population; meanwhile, in the standard strain (P. aeruginosa ATCC 10145), this concentration caused growth inhibition of 85%.
We have revised the first sentence in Methods of Abstract and two last sentences in the Results section.
Competing Interests: I declare that the authors have no conflict of interest with the reviewer. Close
Report a concern
Author Response 09 Jul 2021

Heni Susilowati, Department of Oral Biology, Faculty of Dentistry, Universitas Gadjah Mada, Sleman, 55281, Indonesia

09 Jul 2021

Author Response

We thank you for the thorough and careful review of our manuscript.
The following are our responses to the suggestions and specific comments.

The response to comment 1 (quantification of ... Continue reading We thank you for the thorough and careful review of our manuscript.
The following are our responses to the suggestions and specific comments.

The response to comment 1 (quantification of pyocyanin)
We measured the concentration of pyocyanin contained in the culture supernatant of P. aeruginosa. This condition is in accordance with the previous research conducted by Price-Whelan et al. (2007). Indeed, we only measured the absorbance value of the supernatant filtrate of the bacterial culture at a wavelength of 690 nm, but the pyocyanin concentration was then known by calculations using a formula published by previous researchers (Price-Whelan et al, 2007). The constant value of 4310 M -1cm -1 for the absorbance at 690 nm has been determined based on the research of Dietrich et al. 2006. Since our experimental conditions match the experimental conditions in both studies, both of which are published in outstanding journals, we feel it is sufficient to refer to their methods.

The response to comment 2 (Biofilm [Crystal violet for biomass quantification] study)
We have conducted additional experiments to detect the association between pyocyanin production in bacteria exposed to royal jelly and biofilm formation using the static microtiter plate biofilm assay (crystal violet staining) method. To analyze the relationship between pyocyanin production and biofilm mass production, we have used P. aeruginosa ATCC 10145 as this strain is more responsive in producing pyocyanin when compared to the clinical isolate strains we previously use. The results showed that the detectable biofilm mass significantly increased in bacterial cultures exposed to 12.5% royal jelly extract. Meanwhile, cultures treated with 25% royal jelly did not show any biofilm formation. The 6.25% royal jelly extract and the lower concentrations induced biofilm mass formation but at a lower quantity than 12.5% royal jelly. This evidence suggests a relationship between royal jelly concentration, pyocyanin production, and biofilm mass formation. The subinhibitory concentration (12.5%, 6.23%) of the royal jelly extract induced more pyocyanin production and biofilm mass formation rather than the higher concentrations.
Related changes were in the Methods, Results, and Conclusion of the Abstract, paragraph 6 of the Methods, paragraph 5 of Results, Figure 4, and line 5 of paragraph 6 on the Discussion section in the manuscript.

The response to comment 3 (microscopy of biofilm architecture)
In order to complement the data regarding the effect of royal jelly on the mass formation of biofilms, we have carried out observations of the microscopic architecture of P. aeruginosa ATCC 10145 biofilms using scanning electron microscopy, since we could not access the confocal microscopy method at our university. We chose secondary electron (SE) mode rather than a backscattered electron (BSE) to investigate biofilm in a three-dimensional perspective. The results of observations on representative samples showed that in the culture exposed to 25% royal jelly extract there was no biofilm mass deposition, while the group exposed to 12.5% royal jelly showed a larger density of biofilm than the 6.25% royal jelly group. Chlorhexidine 0.2% control showed total inhibition of biofilm mass formation.

Methods, results, discussion, and related references have been added to the manuscript on Methods, Results, and Conclusion in the Abstract, paragraph 7 of the Methods section, paragraph 6-7 of the Results section, and line 5 in paragraph 6 of the Discussion section.

Specific comments
a. Why solubilized royal jelly is centrifuged and supernatant is only taken?
Answer: The extraction method using phosphate buffer saline (Hu et al, 2019) was chosen in order to dissolve major royal jelly protein 1 (MRJP1), the active components that are antibacterial or anti-adhesion. It is known that MRJP1 is a hydrosoluble protein dissolved in PBS, which is higher in supernatants than in pellets (Gismondi et al., 2017).

What happens with pellets (are there any pellets found after centrifugation?). If yes - then how can we know that supernatant has all royal jelly?
Answer: After centrifugation, there were indeed pellets, and it has been found that the pellets contain more liposoluble proteins than supernatants. To ensure that the compounds contained in the PBS royal jelly extract supernatant, further research is needed; However, from the research of Furusawa et al. (2016), it was found that MRJP1 was contained in the supernatant of PBS royal jelly extract in quite a large amount, reaching 27.6%.
The active components that are antibacterial or anti-adhesion in the supernatant of royal jelly extract in this study cannot be explained with certainty, but the royal jelly used in this study was thought to mainly contain MRJP1.

We add this explanation to paragraph 2 of the Discussion section. Additional sources of literature have been written in the reference list numbers 37, 38, and 39.

b. At 25% royal jelly, we see only 60% growth inhibition for both bacterial strains. It is not MIC - you can call it LC50 (lethal dose to reduce at least 50% growth). MIC is when there is zero or 1-2% growth only.
Answer: We agree with the reviewer's comments regarding the MIC score. In clinical isolates, 25% royal jelly extract caused 60% death in the bacterial population; meanwhile, in the standard strain (P. aeruginosa ATCC 10145), this concentration caused growth inhibition of 85%.
We have revised the first sentence in Methods of Abstract and two last sentences in the Results section.
We thank you for the thorough and careful review of our manuscript.
The following are our responses to the suggestions and specific comments.

The response to comment 1 (quantification of pyocyanin)
We measured the concentration of pyocyanin contained in the culture supernatant of P. aeruginosa. This condition is in accordance with the previous research conducted by Price-Whelan et al. (2007). Indeed, we only measured the absorbance value of the supernatant filtrate of the bacterial culture at a wavelength of 690 nm, but the pyocyanin concentration was then known by calculations using a formula published by previous researchers (Price-Whelan et al, 2007). The constant value of 4310 M -1cm -1 for the absorbance at 690 nm has been determined based on the research of Dietrich et al. 2006. Since our experimental conditions match the experimental conditions in both studies, both of which are published in outstanding journals, we feel it is sufficient to refer to their methods.

The response to comment 2 (Biofilm [Crystal violet for biomass quantification] study)
We have conducted additional experiments to detect the association between pyocyanin production in bacteria exposed to royal jelly and biofilm formation using the static microtiter plate biofilm assay (crystal violet staining) method. To analyze the relationship between pyocyanin production and biofilm mass production, we have used P. aeruginosa ATCC 10145 as this strain is more responsive in producing pyocyanin when compared to the clinical isolate strains we previously use. The results showed that the detectable biofilm mass significantly increased in bacterial cultures exposed to 12.5% royal jelly extract. Meanwhile, cultures treated with 25% royal jelly did not show any biofilm formation. The 6.25% royal jelly extract and the lower concentrations induced biofilm mass formation but at a lower quantity than 12.5% royal jelly. This evidence suggests a relationship between royal jelly concentration, pyocyanin production, and biofilm mass formation. The subinhibitory concentration (12.5%, 6.23%) of the royal jelly extract induced more pyocyanin production and biofilm mass formation rather than the higher concentrations.
Related changes were in the Methods, Results, and Conclusion of the Abstract, paragraph 6 of the Methods, paragraph 5 of Results, Figure 4, and line 5 of paragraph 6 on the Discussion section in the manuscript.

The response to comment 3 (microscopy of biofilm architecture)
In order to complement the data regarding the effect of royal jelly on the mass formation of biofilms, we have carried out observations of the microscopic architecture of P. aeruginosa ATCC 10145 biofilms using scanning electron microscopy, since we could not access the confocal microscopy method at our university. We chose secondary electron (SE) mode rather than a backscattered electron (BSE) to investigate biofilm in a three-dimensional perspective. The results of observations on representative samples showed that in the culture exposed to 25% royal jelly extract there was no biofilm mass deposition, while the group exposed to 12.5% royal jelly showed a larger density of biofilm than the 6.25% royal jelly group. Chlorhexidine 0.2% control showed total inhibition of biofilm mass formation.

Methods, results, discussion, and related references have been added to the manuscript on Methods, Results, and Conclusion in the Abstract, paragraph 7 of the Methods section, paragraph 6-7 of the Results section, and line 5 in paragraph 6 of the Discussion section.

Specific comments
a. Why solubilized royal jelly is centrifuged and supernatant is only taken?
Answer: The extraction method using phosphate buffer saline (Hu et al, 2019) was chosen in order to dissolve major royal jelly protein 1 (MRJP1), the active components that are antibacterial or anti-adhesion. It is known that MRJP1 is a hydrosoluble protein dissolved in PBS, which is higher in supernatants than in pellets (Gismondi et al., 2017).

What happens with pellets (are there any pellets found after centrifugation?). If yes - then how can we know that supernatant has all royal jelly?
Answer: After centrifugation, there were indeed pellets, and it has been found that the pellets contain more liposoluble proteins than supernatants. To ensure that the compounds contained in the PBS royal jelly extract supernatant, further research is needed; However, from the research of Furusawa et al. (2016), it was found that MRJP1 was contained in the supernatant of PBS royal jelly extract in quite a large amount, reaching 27.6%.
The active components that are antibacterial or anti-adhesion in the supernatant of royal jelly extract in this study cannot be explained with certainty, but the royal jelly used in this study was thought to mainly contain MRJP1.

We add this explanation to paragraph 2 of the Discussion section. Additional sources of literature have been written in the reference list numbers 37, 38, and 39.

b. At 25% royal jelly, we see only 60% growth inhibition for both bacterial strains. It is not MIC - you can call it LC50 (lethal dose to reduce at least 50% growth). MIC is when there is zero or 1-2% growth only.
Answer: We agree with the reviewer's comments regarding the MIC score. In clinical isolates, 25% royal jelly extract caused 60% death in the bacterial population; meanwhile, in the standard strain (P. aeruginosa ATCC 10145), this concentration caused growth inhibition of 85%.
We have revised the first sentence in Methods of Abstract and two last sentences in the Results section.
Competing Interests: We declare that we have no conflict of interest with this peer review. Close
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Reviewer Report 01 Feb 2021

Ariadna A. Djais, Department of Oral Biology, Faculty of Dentistry, University of Indonesia, Jakarta, Indonesia

Approved with Reservations

https://doi.org/10.5256/f1000research.30869.r77076

This study examined royal jelly and Apis mellifera bees as a base ingredient. These ingredients are antibacterial with pycocyanin as a pigment by the bacterium Psedomonas aeroginosa. The P. aeroginosa was selected because these bacteria are known as multidrug-resistant bacterium. This research is interesting and specific. In this study, the data was analyzed as an indicator of the difference in the contrast of the green pigment color, which was carried out by measuring at a wavelength of 690 nm.

In my opinion, this research needs to add more information regarding the methods chapters:

It is necessary to add information about the stage of royal jelly making as the test material.
It is necessary to add information about the bacterium identification technique which used by the researcher when confirming clinical isolates as P. aeroginosa.
Number of replication of independent experiment conducted by the researcher.

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?

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?

Yes
Are the conclusions drawn adequately supported by the results?

Yes

Competing Interests: No competing interests were disclosed.

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.

CITE

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Author Response 13 Jul 2021

Heni Susilowati, Department of Oral Biology, Faculty of Dentistry, Universitas Gadjah Mada, Sleman, 55281, Indonesia

13 Jul 2021

Author Response

Thank you for providing a review and suggestions for improving our manuscript. Below are our revisions to the manuscript according to the review comments.

1. Response to comments 1:
... Continue reading Thank you for providing a review and suggestions for improving our manuscript. Below are our revisions to the manuscript according to the review comments.

1. Response to comments 1:
We explain the stages of preparing royal jelly on the manuscript, written in the Methods section (page 3) as follows:
Royal jelly used in this study was obtained from Nusukan, Surakarta, Central Java, Indonesia and harvested in May-October 2019. This product was obtained from the beekeeper Apis mellifera who lives in the randu trees (Ceiba pentandra) and sono wood (Dalbergia latifolia). Apis mellifera bee species have been identified through previous research19. Royal jelly 5.5 grams was dissolved in 10 ml of cold phosphate buffered saline (PBS), then homogenized using a magnetic stirrer (24 hours, 4^oC). The royal jelly solution was centrifuged (12,000 g, 45 minutes, 4°C), then the supernatant is taken and checked the pH. The results of pH measurements with a pH meter showed a pH of 3.79. Furthermore, the supernatant was filtered using 0.45 µm Millipore to produce 55% royal jelly.

2. Response to comment 2:
We write an explanation of how to identify bacteria in the Methods section (page 3), which is as follows:
Identification of the clinical isolate was carried out using API 2ONE (bioMérieux, Inc).

3. Response to comment 3:
Each experiment was carried out with 6 replications.
We write the revision on page 3 of the Methods section, on bacterial viability tests and pyocyanin detection.
Thank you for providing a review and suggestions for improving our manuscript. Below are our revisions to the manuscript according to the review comments.

1. Response to comments 1:
We explain the stages of preparing royal jelly on the manuscript, written in the Methods section (page 3) as follows:
Royal jelly used in this study was obtained from Nusukan, Surakarta, Central Java, Indonesia and harvested in May-October 2019. This product was obtained from the beekeeper Apis mellifera who lives in the randu trees (Ceiba pentandra) and sono wood (Dalbergia latifolia). Apis mellifera bee species have been identified through previous research19. Royal jelly 5.5 grams was dissolved in 10 ml of cold phosphate buffered saline (PBS), then homogenized using a magnetic stirrer (24 hours, 4^oC). The royal jelly solution was centrifuged (12,000 g, 45 minutes, 4°C), then the supernatant is taken and checked the pH. The results of pH measurements with a pH meter showed a pH of 3.79. Furthermore, the supernatant was filtered using 0.45 µm Millipore to produce 55% royal jelly.

2. Response to comment 2:
We write an explanation of how to identify bacteria in the Methods section (page 3), which is as follows:
Identification of the clinical isolate was carried out using API 2ONE (bioMérieux, Inc).

3. Response to comment 3:
Each experiment was carried out with 6 replications.
We write the revision on page 3 of the Methods section, on bacterial viability tests and pyocyanin detection.
Competing Interests: I hereby confirm that there I have no competing interest with the reviewer. Close
Report a concern
Author Response 09 Jul 2021

Heni Susilowati, Department of Oral Biology, Faculty of Dentistry, Universitas Gadjah Mada, Sleman, 55281, Indonesia

09 Jul 2021

Author Response

Thank you for providing a review and suggestions for improving our manuscript.
Below are our revisions to the manuscript according to the review comments.

1. The response to comment ... Continue reading Thank you for providing a review and suggestions for improving our manuscript.
Below are our revisions to the manuscript according to the review comments.

1. The response to comment 1
We explain the stages of preparing royal jelly on the manuscript, written in line 2-6 of paragraph 2 of the Methods section as follows:
Royal jelly used in this study was obtained from Nusukan, Surakarta, Central Java, Indonesia and harvested in May-October 2019. This product was obtained from the beekeeper Apis mellifera who lives in the randu trees (Ceiba pentandra) and sono wood (Dalbergia latifolia). Apis mellifera bee species have been identified through previous research (Hartono et al., 2019). Royal jelly 5.5 grams was dissolved in 10 ml of cold phosphate buffered saline (PBS), then homogenized using a magnetic stirrer (24 hours, 4^oC). The royal jelly solution was centrifuged (12,000 g, 45 minutes, 4°C), then the supernatant was taken and checked for the pH. The results of pH measurements using a pH meter showed a pH of 3.79. Furthermore, the supernatant was filtered using 0.45 µm Millipore to produce 55% royal jelly.

2. The response to comment 2
We have written an explanation of how to identify bacteria in line 5 of paragraph 3 the Methods section, which is as follows:
Identification of the clinical isolate was carried out using API 2ONE (BioMérieux, Inc).

3. Response to comment 3
The bacterial viability assay experiment was carried out in quadruplicate, meanwhile, the pyocyanin experiments were performed in triplicate.
We write the revision in the last line of related paragraphs in the Methods section.
Thank you for providing a review and suggestions for improving our manuscript.
Below are our revisions to the manuscript according to the review comments.

1. The response to comment 1
We explain the stages of preparing royal jelly on the manuscript, written in line 2-6 of paragraph 2 of the Methods section as follows:
Royal jelly used in this study was obtained from Nusukan, Surakarta, Central Java, Indonesia and harvested in May-October 2019. This product was obtained from the beekeeper Apis mellifera who lives in the randu trees (Ceiba pentandra) and sono wood (Dalbergia latifolia). Apis mellifera bee species have been identified through previous research (Hartono et al., 2019). Royal jelly 5.5 grams was dissolved in 10 ml of cold phosphate buffered saline (PBS), then homogenized using a magnetic stirrer (24 hours, 4^oC). The royal jelly solution was centrifuged (12,000 g, 45 minutes, 4°C), then the supernatant was taken and checked for the pH. The results of pH measurements using a pH meter showed a pH of 3.79. Furthermore, the supernatant was filtered using 0.45 µm Millipore to produce 55% royal jelly.

2. The response to comment 2
We have written an explanation of how to identify bacteria in line 5 of paragraph 3 the Methods section, which is as follows:
Identification of the clinical isolate was carried out using API 2ONE (BioMérieux, Inc).

3. Response to comment 3
The bacterial viability assay experiment was carried out in quadruplicate, meanwhile, the pyocyanin experiments were performed in triplicate.
We write the revision in the last line of related paragraphs in the Methods section.
Competing Interests: We declare that we have no conflict of interest with this peer review. Close
Report a concern
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COMMENTS ON THIS REPORT

Author Response 13 Jul 2021

Heni Susilowati, Department of Oral Biology, Faculty of Dentistry, Universitas Gadjah Mada, Sleman, 55281, Indonesia

13 Jul 2021

Author Response

Thank you for providing a review and suggestions for improving our manuscript. Below are our revisions to the manuscript according to the review comments.

1. Response to comments 1:
... Continue reading Thank you for providing a review and suggestions for improving our manuscript. Below are our revisions to the manuscript according to the review comments.

1. Response to comments 1:
We explain the stages of preparing royal jelly on the manuscript, written in the Methods section (page 3) as follows:
Royal jelly used in this study was obtained from Nusukan, Surakarta, Central Java, Indonesia and harvested in May-October 2019. This product was obtained from the beekeeper Apis mellifera who lives in the randu trees (Ceiba pentandra) and sono wood (Dalbergia latifolia). Apis mellifera bee species have been identified through previous research19. Royal jelly 5.5 grams was dissolved in 10 ml of cold phosphate buffered saline (PBS), then homogenized using a magnetic stirrer (24 hours, 4^oC). The royal jelly solution was centrifuged (12,000 g, 45 minutes, 4°C), then the supernatant is taken and checked the pH. The results of pH measurements with a pH meter showed a pH of 3.79. Furthermore, the supernatant was filtered using 0.45 µm Millipore to produce 55% royal jelly.

2. Response to comment 2:
We write an explanation of how to identify bacteria in the Methods section (page 3), which is as follows:
Identification of the clinical isolate was carried out using API 2ONE (bioMérieux, Inc).

3. Response to comment 3:
Each experiment was carried out with 6 replications.
We write the revision on page 3 of the Methods section, on bacterial viability tests and pyocyanin detection.
Thank you for providing a review and suggestions for improving our manuscript. Below are our revisions to the manuscript according to the review comments.

1. Response to comments 1:
We explain the stages of preparing royal jelly on the manuscript, written in the Methods section (page 3) as follows:
Royal jelly used in this study was obtained from Nusukan, Surakarta, Central Java, Indonesia and harvested in May-October 2019. This product was obtained from the beekeeper Apis mellifera who lives in the randu trees (Ceiba pentandra) and sono wood (Dalbergia latifolia). Apis mellifera bee species have been identified through previous research19. Royal jelly 5.5 grams was dissolved in 10 ml of cold phosphate buffered saline (PBS), then homogenized using a magnetic stirrer (24 hours, 4^oC). The royal jelly solution was centrifuged (12,000 g, 45 minutes, 4°C), then the supernatant is taken and checked the pH. The results of pH measurements with a pH meter showed a pH of 3.79. Furthermore, the supernatant was filtered using 0.45 µm Millipore to produce 55% royal jelly.

2. Response to comment 2:
We write an explanation of how to identify bacteria in the Methods section (page 3), which is as follows:
Identification of the clinical isolate was carried out using API 2ONE (bioMérieux, Inc).

3. Response to comment 3:
Each experiment was carried out with 6 replications.
We write the revision on page 3 of the Methods section, on bacterial viability tests and pyocyanin detection.
Competing Interests: I hereby confirm that there I have no competing interest with the reviewer. Close
Report a concern
Author Response 09 Jul 2021

Heni Susilowati, Department of Oral Biology, Faculty of Dentistry, Universitas Gadjah Mada, Sleman, 55281, Indonesia

09 Jul 2021

Author Response

Thank you for providing a review and suggestions for improving our manuscript.
Below are our revisions to the manuscript according to the review comments.

1. The response to comment ... Continue reading Thank you for providing a review and suggestions for improving our manuscript.
Below are our revisions to the manuscript according to the review comments.

1. The response to comment 1
We explain the stages of preparing royal jelly on the manuscript, written in line 2-6 of paragraph 2 of the Methods section as follows:
Royal jelly used in this study was obtained from Nusukan, Surakarta, Central Java, Indonesia and harvested in May-October 2019. This product was obtained from the beekeeper Apis mellifera who lives in the randu trees (Ceiba pentandra) and sono wood (Dalbergia latifolia). Apis mellifera bee species have been identified through previous research (Hartono et al., 2019). Royal jelly 5.5 grams was dissolved in 10 ml of cold phosphate buffered saline (PBS), then homogenized using a magnetic stirrer (24 hours, 4^oC). The royal jelly solution was centrifuged (12,000 g, 45 minutes, 4°C), then the supernatant was taken and checked for the pH. The results of pH measurements using a pH meter showed a pH of 3.79. Furthermore, the supernatant was filtered using 0.45 µm Millipore to produce 55% royal jelly.

2. The response to comment 2
We have written an explanation of how to identify bacteria in line 5 of paragraph 3 the Methods section, which is as follows:
Identification of the clinical isolate was carried out using API 2ONE (BioMérieux, Inc).

3. Response to comment 3
The bacterial viability assay experiment was carried out in quadruplicate, meanwhile, the pyocyanin experiments were performed in triplicate.
We write the revision in the last line of related paragraphs in the Methods section.
Thank you for providing a review and suggestions for improving our manuscript.
Below are our revisions to the manuscript according to the review comments.

1. The response to comment 1
We explain the stages of preparing royal jelly on the manuscript, written in line 2-6 of paragraph 2 of the Methods section as follows:
Royal jelly used in this study was obtained from Nusukan, Surakarta, Central Java, Indonesia and harvested in May-October 2019. This product was obtained from the beekeeper Apis mellifera who lives in the randu trees (Ceiba pentandra) and sono wood (Dalbergia latifolia). Apis mellifera bee species have been identified through previous research (Hartono et al., 2019). Royal jelly 5.5 grams was dissolved in 10 ml of cold phosphate buffered saline (PBS), then homogenized using a magnetic stirrer (24 hours, 4^oC). The royal jelly solution was centrifuged (12,000 g, 45 minutes, 4°C), then the supernatant was taken and checked for the pH. The results of pH measurements using a pH meter showed a pH of 3.79. Furthermore, the supernatant was filtered using 0.45 µm Millipore to produce 55% royal jelly.

2. The response to comment 2
We have written an explanation of how to identify bacteria in line 5 of paragraph 3 the Methods section, which is as follows:
Identification of the clinical isolate was carried out using API 2ONE (BioMérieux, Inc).

3. Response to comment 3
The bacterial viability assay experiment was carried out in quadruplicate, meanwhile, the pyocyanin experiments were performed in triplicate.
We write the revision in the last line of related paragraphs in the Methods section.
Competing Interests: We declare that we have no conflict of interest with this peer review. Close
Report a concern

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VERSION 4 PUBLISHED 11 Jan 2021

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Ariadna A. Djais, University of Indonesia, Jakarta, Indonesia
Theerthankar Das, The University of Sydney, Camperdown, Australia
Shizuo Kayama, National Institute of Infectious Diseases, Higashimurayama, Japan
Philip S. Bird, The University of Queensland, Gatton, Australia

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30 Views

20 Jan 2022 | for Version 4

Philip S. Bird, School of Veterinary Science, Faculty of Science, The University of Queensland, Gatton, Qld, Australia

30 Views Cite this report Responses(0)

Approved With Reservations

I still have a few issues with the latest revision.

First paragraph of Introduction:
"Pseudomonas aeruginosa (P. aeruginosa) is transitional pathogen. This Gram-negative bacillus can be involved in seriously opportunistic-systemic-infections, mainly in immunocompromised patients^1–3. These bacteria are often found in the dental unit waterlines which allows the transmission of these bacteria into the oral cavity⁴. As an opportunist pathogen, P. aeruginosa is also frequently involved in oral infections, such as necrotizing ulcerative gingivitis, periodontitis, and mandibular osteomyelitis^5–7. Although the mechanism is not clear yet, its presence in the oral cavity has been shown to result in systemic infections, such as nosocomial pneumonia⁸."

My comment was to illustrate that Pa is a transitional bacterium in the oral cavity. It is an opportunistic pathogen. It is not a frequent oral pathogen as the references refer to case studies. A better reference to cite to illustrate Pa infections is Klochgetter and Tümmler (2017¹), for example.

Methods: "Royal jelly used in this study was obtained from Nusukan, Surakarta, Central Java, Indonesia and harvested in May–October 2019. Royal jelly produced by Apis mellifera bees live in kapok tree (Ceiba pentandra) and sono wood (Dalbergia latifolia). Apis mellifera bee species have been identified through previous research²⁰. Royal jelly 5.5 grams was dissolved in 10 ml…"

I’m not sure what the tree species have to do with the production of RJ - maybe say that RJ was harvested from feral bee colonies found in kapok trees etc. - but usually to collect royal jelly hives as set up so that bees produce a number of queen cells are from those royal jelly is collected. Maybe the authors could look at how royal jelly is produced, cited in reference Fratini et al. (2016) #14.
I’m not sure what the sentence with cited reference #20 means.
Was the RJ freeze-dried or a raw fresh extracted solution?

My comments in the review:
Just as an addendum, while this paper is mainly a technical paper I thought it would be of interest to readers with a few sentences on bee products as well as royal jelly. Something on how royal jelly is produced by bees. How and what would royal jelly be used for in treatment and what infections?

It is good to see that some discussion on other bee products was included in the last paragraph of the discussion. Bee products appear to be used currently for treatment of wounds. How does this research lead to royal jelly treatments of Pa infections? Are there any current papers that can be cited?

References

1. Klockgether J, Tümmler B: Recent advances in understanding Pseudomonas aeruginosa as a pathogen.F1000Res. 2017; 6: 1261 PubMed Abstract | Publisher Full Text

Competing Interests

No competing interests were disclosed.

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Reviewer Report

20 Views

18 Oct 2021 | for Version 3

Philip S. Bird, School of Veterinary Science, Faculty of Science, The University of Queensland, Gatton, Qld, Australia

20 Views Cite this report Responses(1)

Approved With Reservations

Introduction - Pa is not frequently involved in oral infections. Pa is a transitional pathogen and can be involved in opportunistic infections. Please change to indicate that. Please change throughout the article 'isolates' to 'isolate' (delete "s"). There is nothing in the introduction or discussion on the other products from bees such as honey, propolis. Why only introduce royal jelly and not these products which have anti-bacterial properties? References Pasupuleti et al. (2017¹) and Camplin and Maddocks (2014²) would be useful additions. Was Royal jelly freeze-dried? The royal jelly came from bees, not beekeeper, and what race of bees?

Just as an addendum, while this paper is mainly a technical paper I thought it would be of interest to readers with a few sentences on bee products as well as royal jelly. Some thing on how royal jelly is produced by bees. How and what would royal jelly be used for in treatment and what infections?

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?

Yes
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

References

1. Pasupuleti VR, Sammugam L, Ramesh N, Gan SH: Honey, Propolis, and Royal Jelly: A Comprehensive Review of Their Biological Actions and Health Benefits.Oxid Med Cell Longev. 2017; 2017: 1259510 PubMed Abstract | Publisher Full Text
2. Camplin AL, Maddocks SE: Manuka honey treatment of biofilms of Pseudomonas aeruginosa results in the emergence of isolates with increased honey resistance.Ann Clin Microbiol Antimicrob. 2014; 13: 19 PubMed Abstract | Publisher Full Text

Competing Interests

No competing interests were disclosed.

Reviewer Expertise

Oral microbiologist and beekeeper

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Author Response

07 Dec 2021

Heni Susilowati, Department of Oral Biology, Faculty of Dentistry, Universitas Gadjah Mada, Sleman, 55281, Indonesia

Reviewer comment 1:
Introduction - Pa is not frequently involved in oral infections. Pa is a transitional pathogen and can be involved in opportunistic infections. Please change to indicate that.
Response:
We have changed the first sentence of the first paragraph in the introduction as follows:
Pseudomonas aeruginosa (P. aeruginosa) is transitional pathogen. This Gram-negative bacillus can be involved in seriously opportunistic-systemic-infections, mainly in immunocompromised patients ¹^–³.

Reviewer comment 2:
Please change throughout the article 'isolates' to 'isolate' (delete "s").
Response:
Correction was done on paragraph 4 of Introduction, paragraph 1 and 3 of Results, paragraph 4 and 8 of the Discussion section.

Reviewer comment 3:
There is nothing in the introduction or discussion on the other products from bees such as honey, propolis. Why only introduce royal jelly and not these products which have anti-bacterial properties? References Pasupuleti et al. (2017) and Camplin and Maddocks (2014) would be useful additions.
Response:
Additional information about honey products in the 6^th paragraph of the Discussion section, written as 4^th-7^th sentences.
The increase in pyocyanin production ……. The known bee products are honey, propolis, and royal jelly. All three have antibacterial and antibiofilm properties, in addition to other therapeutic potentials in the process of tissue healing, reproduction, and infectious diseases. (Pasupuleti et al, 2017) Manuka honey has been widely studied and proven to be antibacterial and inhibit the formation of biofilms in P. aeruginosa; however, bacteria derived from biofilms experienced a decrease in sensitivity to manuka honey due to the presence of variants in the bacterial population (Camplin and Maddocks, 2014) or due to subinhibitory concentrations of manuka honey. (Lu et al, 2019). Our results indicate that the sensitivity of P. aeruginosa bacteria to royal jelly may also vary, depending on the concentration of royal jelly exposed to the bacteria. At sub-inhibitory concentration, we found the increase of the pyocyanin production and biofilm growth. …….. of royal jelly.

Reviewer comment 4:
Was Royal jelly freeze-dried?
Response:
The royal jelly preparation used in this study was a fresh extract in PBS as described at the end of the first paragraph of the Methods section.

Reviewer comment 5:
The royal jelly came from bees, not beekeeper, and what race of bees?
Response:
The royal jelly we use is produced by Apis mellifera bees. We have revised it according to the reviewer's suggestion, which can be read in the 4^th sentence of the first paragraph of the Methods section.

Reviewer comment 6:
Just as an addendum, while this paper is mainly a technical paper, I thought it would be of interest to readers with a few sentences on bee products as well as royal jelly. Something on how royal jelly is produced by bees. How and what would royal jelly be used for in treatment and what infections?
Response:
Additional information regarding royal jelly is written at the beginning of paragraph 4 of the Introduction section as follows:
Royal jelly is produced by hypopharyngeal and mandibular glands of worker bees. This substance is a "superfood" for the queen bee. It is widely used as a nutrient-rich supplement to overcome various health conditions such as inflammation, bacterial infection, tumor, allergy, and antioxidant and immunomodulatory effects. [Fratini et al., 2016; Pasupuleti et al., 2017]

Response to the reviewer comments on statistical analysis:
We have consulted statistical analysis of the results of this study with statisticians and stated that no changes were needed.

Additional references:

41. Pasupuleti VR, Sammugam L, Ramesh N, et al: Honey, propolis, and royal jelly: A comprehensive review of their biological actions and health benefits. Oxid Med Cell Longev. 2017;2017:1259510. doi: 10.1155/2017/1259510

42. Camplin AL and Maddocks SE: Manuka honey treatment of biofilms of Pseudomonas aeruginosa results in the emergence of isolates with increased honey resistance. Ann Clin Microbiol Antimicrob. 2014;13:19

43. Lu J, Cokcetin NN, Burke CM, et al.: Honey can inhibit and eliminate biofilms produced by Pseudomonas aeruginosa. Sci Rep. 2019; 9(1):18160

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6 Views

06 Oct 2021 | for Version 3

Shizuo Kayama, Antimicrobial Resistance Research Center, National Institute of Infectious Diseases, Higashimurayama, Japan

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Approved

The revised manuscript has been improved compared to the previous version. I have no more comment.

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bacteriology

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10 Views

28 Sep 2021 | for Version 3

Theerthankar Das, Department of Infectious Diseases and Immunology, School of Medical Science, The University of Sydney, Camperdown, NSW, Australia

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Approved

I approve this manuscript.

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8 Views

07 Sep 2021 | for Version 2

Theerthankar Das, Department of Infectious Diseases and Immunology, School of Medical Science, The University of Sydney, Camperdown, NSW, Australia

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Approved

I approve this manuscript and recommend for indexing.

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17 Views

31 Aug 2021 | for Version 2

Shizuo Kayama, Antimicrobial Resistance Research Center, National Institute of Infectious Diseases, Higashimurayama, Japan

17 Views Cite this report Responses(1)

Approved With Reservations

Amly et al. investigated that 25% royal jelly was able to inhibit P. aeruginosa, but it could increase pyocyanin and biofilm production at subinhibitory concentrations. This bilateral reaction of P. aeruginosa against royal jelly is interesting. However, it seems that this manuscript is constructed according to mainly observation. Your estimation of the molecular mechanism brought from your observation is detail. But I am interested in the relationship between the expression of these genes and those phenotypes. I want to know the molecular mechanism more deeply. The reader of your manuscript will also want to know the gene expression related to pyocyanin, or quorum sensing, and so on. If possible, please measure the gene expression.

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

bacteriology

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Author Response

07 Oct 2021

Heni Susilowati, Department of Oral Biology, Faculty of Dentistry, Universitas Gadjah Mada, Sleman, 55281, Indonesia

We are very grateful for your invaluable input. We agree that an analysis of the effect of royal jelly on the expression of quorum sensing-related genes that play important roles in the regulation of biofilm formation and P. aeruginosa pyocyanin production is urgently needed. Accordingly, we add this discussion to the manuscript version 3, paragraph 7 of the discussion section. We have also added one related reference to the bibliography (no. 39).

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10 Views

22 Jul 2021 | for Version 2

Ariadna A. Djais, Department of Oral Biology, Faculty of Dentistry, University of Indonesia, Jakarta, Indonesia

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Approved

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

Competing Interests

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39 Views

04 Feb 2021 | for Version 1

Theerthankar Das, Department of Infectious Diseases and Immunology, School of Medical Science, The University of Sydney, Camperdown, NSW, Australia

39 Views Cite this report Responses(2)

Not Approved

The intention of this study is good and to enlighten the antibacterial and anti-virulence effect of royal jelly in P. aeruginosa.

To be more conclusive this study needs more work:

Its better to use chloroform-HCl (standard and more popular) assay to quantify pyocyanin yield - its more rigorous than just taking absorbance at 690nm of pyocyanin (broad peak).
Biofilm (Crystal violet for biomass quantification) study needs to be done to ensure impact of royal jelly/pyocyanin on biofilm development.
Confocal microscopy will be of great help to show biofilm architecture.

Specific comments:

Why solubilized royal jelly is centrifuged and supernatant is only taken? What happens with pellets (are there any pellets found after centrifugation?). If yes - then how can we know that supernatant has all royal jelly?
At 25% royal jelly we see only 60% growth inhibition for both bacterial strains. Its not MIC - you can call it LC50 (lethal dose to reduce at least 50% growth). MIC is when there is zero or 1-2% growth only.

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?

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

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

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

Partly
Are the conclusions drawn adequately supported by the results?

Partly

Competing Interests

No competing interests were disclosed.

Reviewer Expertise

Biofilms, antimicrobial agents

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Author Response

13 Jul 2021

Heni Susilowati, Department of Oral Biology, Faculty of Dentistry, Universitas Gadjah Mada, Sleman, 55281, Indonesia

We thank you for the thorough and careful review of our manuscript. We apologize for our delay in revisions due to the various obstacles we face regarding the availability of laboratory facilities and access during the Covid-19 pandemic.
The following are our responses to the suggestions and specific comments.

The response to comment 1 (quantification of pyocyanin)
We measured the concentration of pyocyanin contained in the culture supernatant of P. aeruginosa. This condition is in accordance with the previous research conducted by Price-Whelan et al. (2007). Indeed, we only measured the absorbance value of the supernatant filtrate of the bacterial culture at a wavelength of 690 nm, but the pyocyanin concentration was then known by calculations using a formula published by previous researchers (Price-Whelan et al, 2007). The constant value of 4310 M -1cm -1 for the absorbance at 690 nm has been determined based on the research of Dietrich et al. 2006. Since our experimental conditions match the experimental conditions in both studies, both of which are published in outstanding journals, we feel it is sufficient to refer to their methods.

The response to comment 2 (Biofilm [Crystal violet for biomass quantification] study)
We have conducted additional experiments to detect the association between pyocyanin production in bacteria exposed to royal jelly and biofilm formation using the static microtiter plate biofilm assay (crystal violet staining) method. To analyze the relationship between pyocyanin production and biofilm mass production, we have used P. aeruginosa ATCC 10145 as this strain is more responsive in producing pyocyanin when compared to the clinical isolate strains we previously use. The results showed that the detectable biofilm mass significantly increased in bacterial cultures exposed to 12.5% royal jelly extract. Meanwhile, cultures treated with 25% royal jelly did not show any biofilm formation. The 6.25% royal jelly extract and the lower concentrations induced biofilm mass formation but at a lower quantity than 12.5% royal jelly. This evidence suggests a relationship between royal jelly concentration, pyocyanin production, and biofilm mass formation. The subinhibitory concentration (12.5%, 6.23%) of the royal jelly extract induced more pyocyanin production and biofilm mass formation rather than the higher concentrations.
Related changes were in the Methods, Results, and Conclusion of the Abstract, paragraph 6 of the Methods, paragraph 5 of Results, Figure 4, and line 5 of paragraph 6 on the Discussion section in the manuscript.

The response to comment 3 (microscopy of biofilm architecture)
In order to complement the data regarding the effect of royal jelly on the mass formation of biofilms, we have carried out observations of the microscopic architecture of P. aeruginosa ATCC 10145 biofilms using scanning electron microscopy, since we could not access the confocal microscopy method at our university. We chose secondary electron (SE) mode rather than a backscattered electron (BSE) to investigate biofilm in a three-dimensional perspective. The results of observations on representative samples showed that in the culture exposed to 25% royal jelly extract there was no biofilm mass deposition, while the group exposed to 12.5% royal jelly showed a larger density of biofilm than the 6.25% royal jelly group. Chlorhexidine 0.2% control showed total inhibition of biofilm mass formation.
Methods, results, discussion, and related references have been added to the manuscript on Methods, Results, and Conclusion in the Abstract, paragraph 7 of the Methods section, paragraph 6-7 of the Results section, and line 5 in paragraph 6 of the Discussion section.

Specific comments:

1. Why solubilized royal jelly is centrifuged and supernatant is only taken?
Answer: The extraction method using phosphate buffer saline (Hu et al, 2019) was chosen in order to dissolve major royal jelly protein 1 (MRJP1), the active components that are antibacterial or anti-adhesion. It is known that MRJP1 is a hydrosoluble protein dissolved in PBS, which is higher in supernatants than in pellets (Gismondi et al., 2017).

What happens with pellets (are there any pellets found after centrifugation?). If yes - then how can we know that supernatant has all royal jelly?

After centrifugation, there were indeed pellets, and it has been found that the pellets contain more liposoluble proteins than supernatants. To ensure that the compounds contained in the PBS royal jelly extract supernatant, further research is needed; However, from the research of Furusawa et al. (2016), it was found that MRJP1 was contained in the supernatant of PBS royal jelly extract in quite a large amount, reaching 27.6%.
The active components that are antibacterial or anti-adhesion in the supernatant of royal jelly extract in this study cannot be explained with certainty, but the royal jelly used in this study was thought to mainly contain MRJP1.
We add this explanation to paragraph 2 of the Discussion section. Additional sources of literature have been written in the reference list numbers 37, 38, and 39.

2. At 25% royal jelly, we see only 60% growth inhibition for both bacterial strains. It is not MIC - you can call it LC50 (lethal dose to reduce at least 50% growth). MIC is when there is zero or 1-2% growth only.
Answer: We agree with the reviewer's comments regarding the MIC score. In clinical isolates, 25% royal jelly extract caused 60% death in the bacterial population; meanwhile, in the standard strain (P. aeruginosa ATCC 10145), this concentration caused growth inhibition of 85%.
We have revised the first sentence in Methods of Abstract and two last sentences in the Results section.

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Competing Interests

I declare that the authors have no conflict of interest with the reviewer.

Author Response

09 Jul 2021

Heni Susilowati, Department of Oral Biology, Faculty of Dentistry, Universitas Gadjah Mada, Sleman, 55281, Indonesia

We thank you for the thorough and careful review of our manuscript.
The following are our responses to the suggestions and specific comments.

The response to comment 1 (quantification of pyocyanin)
We measured the concentration of pyocyanin contained in the culture supernatant of P. aeruginosa. This condition is in accordance with the previous research conducted by Price-Whelan et al. (2007). Indeed, we only measured the absorbance value of the supernatant filtrate of the bacterial culture at a wavelength of 690 nm, but the pyocyanin concentration was then known by calculations using a formula published by previous researchers (Price-Whelan et al, 2007). The constant value of 4310 M -1cm -1 for the absorbance at 690 nm has been determined based on the research of Dietrich et al. 2006. Since our experimental conditions match the experimental conditions in both studies, both of which are published in outstanding journals, we feel it is sufficient to refer to their methods.

The response to comment 2 (Biofilm [Crystal violet for biomass quantification] study)
We have conducted additional experiments to detect the association between pyocyanin production in bacteria exposed to royal jelly and biofilm formation using the static microtiter plate biofilm assay (crystal violet staining) method. To analyze the relationship between pyocyanin production and biofilm mass production, we have used P. aeruginosa ATCC 10145 as this strain is more responsive in producing pyocyanin when compared to the clinical isolate strains we previously use. The results showed that the detectable biofilm mass significantly increased in bacterial cultures exposed to 12.5% royal jelly extract. Meanwhile, cultures treated with 25% royal jelly did not show any biofilm formation. The 6.25% royal jelly extract and the lower concentrations induced biofilm mass formation but at a lower quantity than 12.5% royal jelly. This evidence suggests a relationship between royal jelly concentration, pyocyanin production, and biofilm mass formation. The subinhibitory concentration (12.5%, 6.23%) of the royal jelly extract induced more pyocyanin production and biofilm mass formation rather than the higher concentrations.
Related changes were in the Methods, Results, and Conclusion of the Abstract, paragraph 6 of the Methods, paragraph 5 of Results, Figure 4, and line 5 of paragraph 6 on the Discussion section in the manuscript.

The response to comment 3 (microscopy of biofilm architecture)
In order to complement the data regarding the effect of royal jelly on the mass formation of biofilms, we have carried out observations of the microscopic architecture of P. aeruginosa ATCC 10145 biofilms using scanning electron microscopy, since we could not access the confocal microscopy method at our university. We chose secondary electron (SE) mode rather than a backscattered electron (BSE) to investigate biofilm in a three-dimensional perspective. The results of observations on representative samples showed that in the culture exposed to 25% royal jelly extract there was no biofilm mass deposition, while the group exposed to 12.5% royal jelly showed a larger density of biofilm than the 6.25% royal jelly group. Chlorhexidine 0.2% control showed total inhibition of biofilm mass formation.

Methods, results, discussion, and related references have been added to the manuscript on Methods, Results, and Conclusion in the Abstract, paragraph 7 of the Methods section, paragraph 6-7 of the Results section, and line 5 in paragraph 6 of the Discussion section.

Specific comments
a. Why solubilized royal jelly is centrifuged and supernatant is only taken?
Answer: The extraction method using phosphate buffer saline (Hu et al, 2019) was chosen in order to dissolve major royal jelly protein 1 (MRJP1), the active components that are antibacterial or anti-adhesion. It is known that MRJP1 is a hydrosoluble protein dissolved in PBS, which is higher in supernatants than in pellets (Gismondi et al., 2017).

What happens with pellets (are there any pellets found after centrifugation?). If yes - then how can we know that supernatant has all royal jelly?
Answer: After centrifugation, there were indeed pellets, and it has been found that the pellets contain more liposoluble proteins than supernatants. To ensure that the compounds contained in the PBS royal jelly extract supernatant, further research is needed; However, from the research of Furusawa et al. (2016), it was found that MRJP1 was contained in the supernatant of PBS royal jelly extract in quite a large amount, reaching 27.6%.
The active components that are antibacterial or anti-adhesion in the supernatant of royal jelly extract in this study cannot be explained with certainty, but the royal jelly used in this study was thought to mainly contain MRJP1.

We add this explanation to paragraph 2 of the Discussion section. Additional sources of literature have been written in the reference list numbers 37, 38, and 39.

b. At 25% royal jelly, we see only 60% growth inhibition for both bacterial strains. It is not MIC - you can call it LC50 (lethal dose to reduce at least 50% growth). MIC is when there is zero or 1-2% growth only.
Answer: We agree with the reviewer's comments regarding the MIC score. In clinical isolates, 25% royal jelly extract caused 60% death in the bacterial population; meanwhile, in the standard strain (P. aeruginosa ATCC 10145), this concentration caused growth inhibition of 85%.
We have revised the first sentence in Methods of Abstract and two last sentences in the Results section.

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We declare that we have no conflict of interest with this peer review.

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36 Views

01 Feb 2021 | for Version 1

Ariadna A. Djais, Department of Oral Biology, Faculty of Dentistry, University of Indonesia, Jakarta, Indonesia

36 Views Cite this report Responses(2)

Approved With Reservations

It is necessary to add information about the stage of royal jelly making as the test material.
It is necessary to add information about the bacterium identification technique which used by the researcher when confirming clinical isolates as P. aeroginosa.
Number of replication of independent experiment conducted by the researcher.

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?

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?

Yes
Are the conclusions drawn adequately supported by the results?

Yes

Competing Interests

No competing interests were disclosed.

Respond to this report

Responses (2)

Author Response

13 Jul 2021

Heni Susilowati, Department of Oral Biology, Faculty of Dentistry, Universitas Gadjah Mada, Sleman, 55281, Indonesia

Thank you for providing a review and suggestions for improving our manuscript. Below are our revisions to the manuscript according to the review comments.

1. Response to comments 1:
We explain the stages of preparing royal jelly on the manuscript, written in the Methods section (page 3) as follows:
Royal jelly used in this study was obtained from Nusukan, Surakarta, Central Java, Indonesia and harvested in May-October 2019. This product was obtained from the beekeeper Apis mellifera who lives in the randu trees (Ceiba pentandra) and sono wood (Dalbergia latifolia). Apis mellifera bee species have been identified through previous research19. Royal jelly 5.5 grams was dissolved in 10 ml of cold phosphate buffered saline (PBS), then homogenized using a magnetic stirrer (24 hours, 4^oC). The royal jelly solution was centrifuged (12,000 g, 45 minutes, 4°C), then the supernatant is taken and checked the pH. The results of pH measurements with a pH meter showed a pH of 3.79. Furthermore, the supernatant was filtered using 0.45 µm Millipore to produce 55% royal jelly.

2. Response to comment 2:
We write an explanation of how to identify bacteria in the Methods section (page 3), which is as follows:
Identification of the clinical isolate was carried out using API 2ONE (bioMérieux, Inc).

3. Response to comment 3:
Each experiment was carried out with 6 replications.
We write the revision on page 3 of the Methods section, on bacterial viability tests and pyocyanin detection.

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Competing Interests

I hereby confirm that there I have no competing interest with the reviewer.

Author Response

09 Jul 2021

Heni Susilowati, Department of Oral Biology, Faculty of Dentistry, Universitas Gadjah Mada, Sleman, 55281, Indonesia

Thank you for providing a review and suggestions for improving our manuscript.
Below are our revisions to the manuscript according to the review comments.

1. The response to comment 1
We explain the stages of preparing royal jelly on the manuscript, written in line 2-6 of paragraph 2 of the Methods section as follows:
Royal jelly used in this study was obtained from Nusukan, Surakarta, Central Java, Indonesia and harvested in May-October 2019. This product was obtained from the beekeeper Apis mellifera who lives in the randu trees (Ceiba pentandra) and sono wood (Dalbergia latifolia). Apis mellifera bee species have been identified through previous research (Hartono et al., 2019). Royal jelly 5.5 grams was dissolved in 10 ml of cold phosphate buffered saline (PBS), then homogenized using a magnetic stirrer (24 hours, 4^oC). The royal jelly solution was centrifuged (12,000 g, 45 minutes, 4°C), then the supernatant was taken and checked for the pH. The results of pH measurements using a pH meter showed a pH of 3.79. Furthermore, the supernatant was filtered using 0.45 µm Millipore to produce 55% royal jelly.

2. The response to comment 2
We have written an explanation of how to identify bacteria in line 5 of paragraph 3 the Methods section, which is as follows:
Identification of the clinical isolate was carried out using API 2ONE (BioMérieux, Inc).

3. Response to comment 3
The bacterial viability assay experiment was carried out in quadruplicate, meanwhile, the pyocyanin experiments were performed in triplicate.
We write the revision in the last line of related paragraphs in the Methods section.

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Competing Interests

We declare that we have no conflict of interest with this peer review.

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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Enhancement of pyocyanin production by subinhibitory concentration of royal jelly in Pseudomonas aeruginosa

Abstract

Keywords

Introduction

Methods

Measurement of the effect of royal jelly on the viability of P. aeruginosa

Analysis of the effect of royal jelly on pyocyanin production

Statistical analysis

Results

Antibacterial activity of royal jelly against P. aeruginosa

Figure 1. Percentage of inhibition of growth of P. aeruginosa bacteria (1.5 × 105 CFU/well).

Exposure to subinhibitory royal jelly concentrations induced increased pyocyanin production in P. aeruginosa

Figure 2. Pyocyanin was identified from the green color of the P. aeruginosa culture supernatant after 36 h incubation at 37°C.

Figure 3. Pyocyanin production in P. aeruginosa bacteria (1.5 × 105 CFU/well) after 36 hours of exposure to various concentrations of royal jelly.

Discussion

Conclusions

Data availability

Underlying data

Acknowledgements

References

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Figure 1. Percentage of inhibition of growth of P. aeruginosa bacteria (1.5 × 10⁵ CFU/well).

Figure 3. Pyocyanin production in P. aeruginosa bacteria (1.5 × 10⁵ CFU/well) after 36 hours of exposure to various concentrations of royal jelly.