Preliminary study on the inhibitory effect of seaweed <i>Gracilaria verrucosa</i> extract on biofilm formation of <i>Candida albican</i>s cultured from the saliva of a smoker

Zaki Mubarak; Adintya Humaira; Basri A. Gani; Zainal A. Muchlisin

doi:10.12688/f1000research.14879.2

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Preliminary study on the inhibitory effect of seaweed Gracilaria verrucosa extract on biofilm formation of Candida albicans cultured from the saliva of a smoker

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

Zaki Mubarak ¹, Adintya Humaira¹, Basri A. Gani¹, Zainal A. Muchlisin²

PUBLISHED 16 Aug 2018

Author details Author details

¹ Faculty of Dentistry, Syiah Kuala University, Banda Aceh, 23111, Indonesia
² Faculty of Marine and Fisheries, Syiah Kuala University, Banda Aceh, 23111, Indonesia

Zaki Mubarak
Roles: Conceptualization, Formal Analysis, Funding Acquisition, Investigation, Methodology, Supervision, Validation

Adintya Humaira
Roles: Data Curation, Investigation, Project Administration, Software, Visualization, Writing – Original Draft Preparation

Basri A. Gani
Roles: Conceptualization, Formal Analysis, Investigation, Methodology, Resources, Writing – Original Draft Preparation

Zainal A. Muchlisin
Roles: Software, Visualization, Writing – Review & Editing

OPEN PEER REVIEW

REVIEWER STATUS

Abstract

Background: Candida albicans is an opportunistic fungus that might infect the oral cavity. Increased colony numbers of C. albicans in the mouth can be caused by multiple factors, such as smoking, weakened immune system, antibiotics use and immune-compromised condition. Smoking can increase expression of virulence factors of C. albicans and make it stronger. One virulence factor of C. albicans is biofilm formation. The ability of creating biofilm makes C. albicans more tolerant to commercial antifungal agents. The objective of this preliminary study was to examine the ability of the seaweed G. verrucosa extracts to inhibit the formation of biofilm by C. albicans isolated from the saliva of a smoker.
Methods: The extract of G. verrucosa was prepared by maceration using 96% methanol and subjected for phytochemical analysis. C. albicans was isolated from the saliva of a smoker who voluntarily participated in the study after providing informed consent. In triplicate, the fungus was cultured in the growth medium containing increased concentrations of G. verrucosa (6.25, 12.5, 25, 50, 75 and 100% ).The same reaction using fluconazole 0.31 µg/ml C. albicans was prepared as positive control. Biofilm formation was accessed based on optical density of cell mixtures using an ELISA reader. The data obtained were subjected to Kruskal-Wallis test at a significance limit of 0.05.
Results: Methanol extract of seaweed G. verrucosa contained three bio-active compounds namely steroids, terpenoid, and tannins. Inhibitory activity of seaweed extracts on C. albicans biofilm formation increased as their concentration increased. The highest inhibitory effect was recorded at fungus culture treated with seaweed concentration of 25% at 24 hours of time exposure.
Conclusions: Seaweed G. verrucosa extract contained steroids, terpenoids and tannins that were able to effectively inhibit the formation of biofilm by C. albicans at the concentration of 25% after 24 hours of time exposure.

Keywords

Candida albicans, oral candidiasis, seaweed Gracilaria verrucosa

Corresponding author: Zaki Mubarak

Competing interests: No competing interests were disclosed.

Grant information: The author(s) declared that no grants were involved in supporting this work.

Copyright: © 2018 Mubarak Z 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. Data associated with the article are available under the terms of the Creative Commons Zero "No rights reserved" data waiver (CC0 1.0 Public domain dedication).

How to cite: Mubarak Z, Humaira A, Gani BA and Muchlisin ZA. Preliminary study on the inhibitory effect of seaweed Gracilaria verrucosa extract on biofilm formation of Candida albicans cultured from the saliva of a smoker [version 2; peer review: 1 approved, 2 approved with reservations]. F1000Research 2018, 7:684 (https://doi.org/10.12688/f1000research.14879.2) First published: 31 May 2018, 7:684 (https://doi.org/10.12688/f1000research.14879.1) Latest published: 18 Sep 2018, 7:684 (https://doi.org/10.12688/f1000research.14879.3)

Revised Amendments from Version 1

Some revisions and improvements have been done throughout the manuscript as responses to the good comments and suggestions from reviewers. Major revisions performed include grammar check and corrections; sentence improvements; and reorganization and adding appropriate details in each section of the Methods part. Grammar issues, a concern of all reviewers, have been addressed. The ways to present results were also changed as response to comment from Dr Heni Susilowati about the meaning of OD values in presenting the growth of C. albicans and how to interpret them. Restructure of Results and Discussion, therefore, was also performed. These changes became the basis for synthesizing more appropriate Conclusion sentences. One reference was also added to support discussion about the importance of extract concentration for resulting in expected medicinal effects, in this study was growth inhibition that reflected by biofilm formation. Since this study is still in preliminary step, no non-smoker participant was participated. As consequence, no comparison could be made between smoker and non-smoker groups. Yet, the study was still able to confirm potential of seaweed Gracia verrucose extract as anti candidiasis in smokers. Potential of doing further studies was mentioned in relation to this weakness of our study.

See the authors' detailed response to the review by Shahida Mohd-Said
See the authors' detailed response to the review by Heni Susilowati

Introduction

Smoking is a common problem in both developed and developing countries, including in Indonesia. Based on a survey by the Tobacco Atlas in 2015, Indonesia has the highest number of smokers in Asia, with 66% of men in Indonesia being active smokers¹. Smoking can lead to addiction owing to the nicotine contents, and harm due to the presence of toxic compounds such as carbon monoxide, ammonia and tar contents in tobacco¹. Substances in cigarettes can also contribute for the occurrence of oral candidiasis, infection of in the mouth cavity caused by the fungus Candida albicans². This fungus is part of the normal flora of the human mouth, but it can become pathogenic in certain conditions, for example, due to nicotine exposure³.

The C. albicans that infects human tissues generally form biofilm³, an extracellular matrix consisting of C. albicans colonies⁴. The size of the biofilm increases when the fungus was exposed to substances in cigarette smoke because cigarette contains chemicals that can initiate the growth of and nourish C. albicans^5,6.

Currently, fluconazole and nystatin are the most effective drugs for treating oral candidiasis. Unfortunately, these drugs could result in undesired side effects. Prolonged use of fluconazole, for example, can lead to resistance⁷ whereas high dosages of nystatin give gastrointestinal discomfort and increase plaque formation⁸. Therefore, plant-derived antifungals may be viable oral treatment options for candidiasis. One of these potential plants is Gracilaria verrucosa. This seaweed contains several bioactive compounds, including alkaloids, flavonoids, phenolics, saponins, steroids and terpenoids⁹. Aceh Province, Indonesia, has large G. verrucosa resources, although this aquatic plant has not been commonly used for medicinal purposes. Hence, the objective of the present study was to examine the ability of seaweed G. verruosa extract to inhibit the biofilm formation of C. albicans isolated from the saliva of smoker.

Methods

Time and site

The study was conducted in August 2017 at the Laboratory of Microbiology, Veterinary Faculty, Syiah Kuala University. The G. verrucosa seaweeds were collected from a farmer in Pulo Aceh, Aceh Province.

Ethics

All research protocols used in this study were approved by the Research Ethics Committee of the Dentistry Faculty of Syiah Kuala University No. 1741/UN11.1.21/TU/2017.

Saliva collection

The saliva was collected from an active smoker who worked as administrative staff at the Faculty of Dentistry Medicine of Syiah Kuala University and voluntarily participated in this study after providing written informed consent. Inclusion criterion of the volunteer was active smoker who smoked 20 cigarettes per day. Saliva was collected once by spitting into a glass jar (15 ml) right after the subject finished smoking, and added with 10 ml of PBS (0.01 M, pH 7.2). The jar was centrifuged at 10,000 rpm for 10 minutes. The precipitate was stored for the microbiological examination.

Candida albicans Isolation and preparation

Precipitate was cultured in ChromAgar Candida medium and incubated at 37 °C for 2 days. The C. albicans fungus grew as green colonies. One colony of C. albicans was mixed with 5 ml of peptone in a tube and incubated at 37°C for 24 hours. Turbidity of medium was compared to a 0.5 McFarland solution standard, which was equivalent to 1.5 × 10⁸ CFU/ml.

Seaweed extraction

Seaweed extraction was performed by maceration using 96% methanol¹⁰. In brief, a total of 500 g of fresh seaweeds were washed with tap water then with distilled water. Seaweed samples were dried at 25°C for 24 hours, chopped into small pieces (2 mm), and soaked in 1.5 liters of 96% methanol. Macerate was filtered using Whatman filter paper No. 42. Filtrate collected was concentrated using a vacuum rotary evaporator (Laborta 4003 control, Heildolph) 60°C and at a speed of 80-90 rpm for 3 days. Concentrated extract was put in a sealed dark bottle and stored at 4 °C.

Phytochemical tests

Flavonoid test. A 0.5 cm magnesium plate was rinsed in 5 ml of seaweed extract, mixed with two drops of HCl, and heated by passing it over a Bunsen flame. Red or purple coloration formed on the heating indicated the presence of flavonoids¹¹.

Alkaloid test. Seaweed extract (5 ml) was mixed with 8 ml of HCl and filtered. Filtrate was subjected to Mayer, Wagner and Dragendroff tests for alkaloids¹¹. This was done by mixing 2 ml of filtrate with 5 g potassium mercuric iodide (Mayer test), 2 ml of Wagner reagent, or 2 ml of bismuth potassium iodide solution (Dragendroff test). The formation of white or pale precipitates (Mayer test), brown or reddish-brown precipitates (Wagner test) and red precipitates (Dragendroff test) indicated the presence of alkaloids.

Tannin/phenolic test. Two drops of 1% FeCl₃ was added to 1 ml seaweed extract. The change in the color to a blackish green indicated the presence of tannin/phenolic¹².

Saponin test. Seaweed extract, 1 ml, was diluted in 20 ml of distilled water and shaken vertically for 15 seconds. Persistent foaming indicated the presence of saponin content.

Steroid test. Two milliliters of seaweed extract was added with 2 ml of CHCl₃, 2 drops of H₂S and 1 ml of CH₃COOH. The formation of green or blue precipitates indicated the presence of steroid¹¹.

Terpenoid test. Seaweed extracts (5 ml) was mixed with 2 ml of chloroform. Concentrated H₂SO₄, 3 ml, were carefully added. The formation of reddish brown layer at the interface of extract and chloroform solution indicated the presence of terpenoids¹³.

Examination of Biofilm Formation

Casein-peptone lecithin polysorbate broths (Merck-1117230500), 100 µl, were poured in each well of a 96-well plate, incubated at room temperature for 5 minutes and discarded. Fifty microliter C. albicans culture had turbidity was equal to 0.5 McFarland standard were added to each well and incubated for 5 minutes. In triplicate, decreased concentrations of seaweed extracts (100, 75, 50, 25, 12.5 and 6.25%) were added. The same reaction using fluconazole 0.31 µg/ml was prepared as positive control. The plate was then incubated at 37 °C for 24, 48 or 72 hours. Each well was added with 200 µl of 0.1% violet crystal, incubated at 25 °C for 15 min, and washed three times with 200 µl of 0.01 M PBS. The crystal violet in each well was then removed by adding 100 µl of 96% ethanol for 2 min. The biofilm formation was analyzed by reading optical density of mixture using an ELISA reader at 620 nm^14,15.

Data analysis

The data obtained were subjected to Kruskal-Wallis test using SPSS software v20.0 for windows.

Results

The results of phytochemical tests in Table 1 show that methanol extract of seaweed G. verrucosa contained bioactive compounds belonged to steroids, terpenoids, and tannins/polyphenols.

Table 1. Phytochemical contents of seaweed Gracilaria verrucosa extract.

Substance	Reagent	Result	Indication
Alkaloid	Mayer	-	White deposit
	Wagner	-	Brown deposit
	Dragendroff	-	Red deposit
Steroid	Uji Lieberman-Burchard	+	Green or blue colors
Terpenoid	Uji Lieberman-Burchard	+	Red or purple colors
Saponin	Shuffling method	-	Stable foams
Flavonoid	0.5 Mg and HCl	-	Red or purple colors
Tannin/Phenolic	MgCl₃	+	Dark green

In general, the inhibitory effect was reduced as seaweed concentration increased (Figure1). The highest inhibition after 24 and 48 hours time exposures was recorded from C. albicans culture treated with 25% seaweed extract, followed by those with 50% and 75%. The best inhibition after 75 hours time exposure, however, was found in C. albicans culture treated with 25% seaweed extract. In all time exposures, the inhibitory effect caused by fluconazole (control) was the worst, followed by those caused by 100% seaweed extracts, but there was no difference between these two groups. Results of Kruskal-Wallis analysis showed that seaweed extract significantly (p<0.05) inhibited the formation of biofilm by C. albicans, indicated potential of the extract to inhibit the growth of the fungus.

Figure 1. The formation of biofilm by Candida albicans exposed to seaweed Gracilaria verrucosa extract on different time exposures and concentrations.

Dataset 1.The raw data of the Triplo anti-Biofilm seaweed to C. albicans for 24, 48 and 72 h at a wavelength 620 nm.

Discussion

The study showed that 25–75% seaweed extracts are promising for inhibiting the growth of C. albicans as shown by much lower biofilm formation after 24, 48 and 72 hours exposure compared to those caused by positive control (fluconazole) and 100% seaweed extract. This indicated potential of seaweed extract as natural anti-fungus to treat oral candidiosis caused by against C. albicans in smokers.

C. albicans is a normal micro-organism in the human mouth. This fungus, however, can be pathogenic in certain circumstances³ such as in the mouth of smokers². Smoking can stimulate synthesis of HWP1, EAP1 and SAP2 proteins in C. albicans, causing higher virulence of the fungus. This can lead to increased formation of biofilm and finally cause oral candidiasis⁶. Smoking also cause a decreased immune function, making individuals more susceptible to oral infections, including candidiasis^4,6.

The ability of 25–75% seaweed extracts to inhibit the formation of C. albicans biofilm significantly stronger than those of fluconazole (positive control) was probably caused by bioactive compounds presence in the extracts. These included steroids, terpenoids, and tannins/polyphenols, all of which have been known their benefits for human health. According to Sampaio et al.¹⁶, antifungal activity of a substance strongly depends on the composition of its bioactive compounds. Steroids can kill C. albicans through their lypophilic properties, interfering with the formation of fungal spores and mycelium¹⁷. This activity weakens C. albicans, inhibiting the formation of the biofilm. To function optimally, steroids require oligosaccharides that are also present in the seaweed content¹⁸.

Terpenoids are derivatives of saponins that may act as an antifungals by damaging organelles of the fungus and by inhibiting secretion of enzymes, leading to the inhibition of C. albicans cell growth. Terpenoids can also damage the morphology of C. albicans¹⁹. Tannins may inhibit chitin synthesis in C. albicans cell walls, leading to lost of membrane cell protection and disrupted cellular metabolism. Tannins also can inhibit ergosteron activity of C. albicans²⁰.

The effectiveness of seaweed extracts in inhibiting fungal growth is influenced by at least three factors, namely concentration, exposure time, and contact surface media²¹. The present study showed that diluted concentration (25–75%) of extracts showed better inhibitory effect on the growth of C. albicans than the more concentrated 100% extract. Concentrated extract usually has lesser effectiveness in vitro due to solubility and import problems. In more aqueous condition plant extracts generally show better medicinal properties with increased concentration as shown by a study testing antifungal activity of ethanol extracts of Syzygium jombolanum, Cassia siamea, Ordina wodier, Momodica charantia and Melia azedarach as well as two algal species Sargassum wightii and Saulerpa scalpellformis against 25 C. albicans isolates in vitro²².

This study also showed the best inhibitory effect of seaweed extracts was recorded at 24 hour of exposure. This is probably because the farnesol, a quorum-sensing molecule that has the potency to inhibit C. albicans growth²³, works effectively after 48–72 hours of exposure.

This study, however, was still preliminary due to limited number of subject and unavailability of non-smoker, control. Virulence factor of C. albicans analyzed was also only biofilm formation. Since more virulence factors are possibly synthesized by the fungus under certain environmental condition, further studies are need to be conducted to investigate effect of individual bioactive compound contained in the seaweeds on the formation of biofilm and on the expression of other virulence factors of C. albicans isolated form both smoker and non-smoker individuals.

Conclusion

Extract of Gracilaria verrucosa seaweed could inhibit the growth of C. albicans isolated from the saliva of a smoker. The inhibitory effect decreased with the increase of concentration, and reached the highest at concentration of 25% and time exposure of 24 hours.

Data availability

Dataset 1. The raw data of the Triplo anti-Biofilm seaweed to C. albicans for 24, 48 and 72 h at a wavelength 620 nm. DOI: 10.5256/f1000research.14879.d204270²⁴.

Competing interests

No competing interests were disclosed.

Grant information

The authors declare that no funding was involved in supporting this work.

Faculty Opinions recommended

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Comments on this article Comments (1)

Version 3

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Version 1

VERSION 1 PUBLISHED 31 May 2018

Discussion is closed on this version, please comment on the latest version above.

Author Response 16 Aug 2018

Zaki Mubarak, Faculty of Dentistry, Syiah Kuala University, Banda Aceh, 23111, Indonesia

16 Aug 2018

Author Response

Some revisions and improvements have been done throughout the manuscript as responses to the good comments and suggestions from reviewers. Major revisions performed include grammar check and corrections; sentence improvements; ... Continue reading Some revisions and improvements have been done throughout the manuscript as responses to the good comments and suggestions from reviewers. Major revisions performed include grammar check and corrections; sentence improvements; and reorganization and adding appropriate details in each section of the material and methods part. Grammar issues, were concerned by all reviewers, had been addressed. The ways to present results were also changed as response to comment from Indonesia reviewer about the meaning of OD values in presenting the growth of C. albicans and how to interpret them. Restructure of result and discussion, therefore, was also performed. These changes became the basis for synthesizing more appropriate conclusion sentences. One reference was also added to support discussion about the importance of extract concentration for resulting in expected medicinal effects, in this study was growth inhibition that reflected by biofilm formation. Since this study is still in preliminary step, no non-smoker participant was participated. As consequence, no comparison could be made between smoker and non-smoker groups. Yet the study was still able to confirm potential of seaweed Gracia verrucose extract as anti candidiasis in smokers. Potential of doing further studies was mentioned in relation to this weakness of our study.
Some revisions and improvements have been done throughout the manuscript as responses to the good comments and suggestions from reviewers. Major revisions performed include grammar check and corrections; sentence improvements; and reorganization and adding appropriate details in each section of the material and methods part. Grammar issues, were concerned by all reviewers, had been addressed. The ways to present results were also changed as response to comment from Indonesia reviewer about the meaning of OD values in presenting the growth of C. albicans and how to interpret them. Restructure of result and discussion, therefore, was also performed. These changes became the basis for synthesizing more appropriate conclusion sentences. One reference was also added to support discussion about the importance of extract concentration for resulting in expected medicinal effects, in this study was growth inhibition that reflected by biofilm formation. Since this study is still in preliminary step, no non-smoker participant was participated. As consequence, no comparison could be made between smoker and non-smoker groups. Yet the study was still able to confirm potential of seaweed Gracia verrucose extract as anti candidiasis in smokers. Potential of doing further studies was mentioned in relation to this weakness of our study.
Competing Interests: No competing interests were disclosed. Close
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Author details Author details

¹ Faculty of Dentistry, Syiah Kuala University, Banda Aceh, 23111, Indonesia
² Faculty of Marine and Fisheries, Syiah Kuala University, Banda Aceh, 23111, Indonesia

Zaki Mubarak
Roles: Conceptualization, Formal Analysis, Funding Acquisition, Investigation, Methodology, Supervision, Validation

Adintya Humaira
Roles: Data Curation, Investigation, Project Administration, Software, Visualization, Writing – Original Draft Preparation

Basri A. Gani
Roles: Conceptualization, Formal Analysis, Investigation, Methodology, Resources, Writing – Original Draft Preparation

Zainal A. Muchlisin
Roles: Software, Visualization, Writing – Review & Editing

Competing interests

No competing interests were disclosed.

Grant information

The author(s) declared that no grants were involved in supporting this work.

Article Versions (3)

version 3

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Published: 18 Sep 2018, 7:684

https://doi.org/10.12688/f1000research.14879.3

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© 2018 Mubarak Z 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. Data associated with the article are available under the terms of the Creative Commons Zero "No rights reserved" data waiver (CC0 1.0 Public domain dedication).

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Mubarak Z, Humaira A, Gani BA and Muchlisin ZA. Preliminary study on the inhibitory effect of seaweed Gracilaria verrucosa extract on biofilm formation of Candida albicans cultured from the saliva of a smoker [version 2; peer review: 1 approved, 2 approved with reservations]. F1000Research 2018, 7:684 (https://doi.org/10.12688/f1000research.14879.2)

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Version 2

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Reviewer Report 23 Aug 2018

Shahida Mohd-Said, Periodontology Unit, Centre for Restorative Dentistry, Faculty of Dentistry, Universiti Kebangsaan Malaysia, Kuala Lumpur, Malaysia

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Heni Susilowati, Department of Oral Biology, Faculty of Dentistry, Gadjah Mada University, Yogyakarta, Indonesia

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I really appreciate the efforts of the authors who have revised the report, but I would like to add suggestions to the authors to explain why the optical density value of the treatment group treated with Fluconazole and extracts of ... Continue reading

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Reviewer Report 27 Jun 2018

Cristiane Y. Koga-Ito, Oral Biopathology Graduate Program, Institute of Science and Technology, São Paulo State University - UNESP, São José dos Campos, Brazil

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https://doi.org/10.5256/f1000research.16195.r34560

The manuscript by Mubarak et al. aimed to evaluate the effect of G. verrucosa extract on Candida albicans biofilm formation. The rationale of the study should be clearer. Also, there is lack of essential information throughout the text. The English should be revised.

Specific comments

Introduction
The period “Smoking is a common problem in most developing countries, including Indonesia.”. A reference should be added. Also, smoking is not a problem only or mainly in developing countries. Please, consider revising.
The authors stated that “This fungus is part of the normal flora of the human mouth, but it can become pathogenic in certain conditions, for example, due to nicotine exposure.”. The exposure to nicotine has been correlated with increasing in C. albicans virulence factor expression. The predisposing conditions for candidiasis are much more related to immunologic state of the host and imbalance in microflora. Please, consider revising.
Revise the period “Infection with C. albicans will increase the formation of a biofilm of the fungus”, it is confusing.
Revise the period “The biofilm is an extracellular matrix consisting of C. albicans colonies”, it is confusing.
The authors stated that “high dosages of nystatin give gastrointestinal discomfort and increase plaque formation”. What do authors mean by “increase plaque formation”?
The rationale of the study is not clear. Why did the authors select G. verrucosa? Is this plant commonly used? Why did the authors decide to use saliva from a smoker individual?

Methods
Revise the period “C. albicans was extracted from the saliva”, C. albicans was isolated from saliva.
The authors reported that “G. verrucosa seaweed was collected from a farmer in Pulo Aceh, Aceh Province.”. More information on the plant, the exact location it was collected from, the period of the year, identification procedure (how and who did the identification?), registration in herbarium, number of voucher should be included in the text.
Please, revise the period “Saliva was collected by spitting into a glass jar (15 ml), then 1 ml PBS (0.01 M, pH 7.2) was added to the jar.” Why did 1 ml of PBS added to the saliva? What was the final volume of saliva collected? Was saliva stimulated?
The authors stated that “If the colour of a colony was green, this indicated that the colony was C. albicans.”. However, the color of the colony in CHROMagar is only a presumptive test and phenotypic or genotypic definitive identification should be done.
The inclusion of a reference strain is highly needed.

Include the number of experiments/replicates performed.

The inclusion of more clinical isolates from non-smokers patients is needed.

The methodology of activity of extract on biofilm formation is not clear. Why and how peptone was removed from the wells? Why did the fungal suspension left in the wells for 5 min? How 100% extract concentration was obtained in the well (you already has the broth inside the well)? Why the concentration of 0.31 was chosen for fluconazole?

Figure 1 should be revised. Note that optical density at 620 nm is higher after treatment with 100% extract when compared to the other concentrations.

Discussion and Conclusion sections should be revised after the revision of the aforementioned points.

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

No
Is the study design appropriate and is the work technically sound?

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

No
If applicable, is the statistical analysis and its interpretation appropriate?

Not applicable
Are all the source data underlying the results available to ensure full reproducibility?

No
Are the conclusions drawn adequately supported by the results?

No

Competing Interests: No competing interests were disclosed.

Reviewer Expertise: Microbiology

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

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Reviewer Report 19 Jun 2018

Shahida Mohd-Said, Periodontology Unit, Centre for Restorative Dentistry, Faculty of Dentistry, Universiti Kebangsaan Malaysia, Kuala Lumpur, Malaysia

Approved with Reservations

https://doi.org/10.5256/f1000research.16195.r34561

Overall fair manuscript with sound finding and relevant area of study. Can much be improved with grammar check and essential scientific writing reorganisation especially in Introduction and Discussion section. Inclusion of results for negative control (untreated biofilm) would critically improve Results presentation and appreciation of findings. Lacks relevant information on findings between smoker and non-smoker in the study that may not strongly support the conclusion on the anti-fungal effect of agent on smoker.

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?

No
Are the conclusions drawn adequately supported by the results?

Yes

Competing Interests: No competing interests were disclosed.

Reviewer Expertise: Periodontology, natural product drug discovery

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Author Response 16 Aug 2018

Zaki Mubarak, Faculty of Dentistry, Syiah Kuala University, Banda Aceh, 23111, Indonesia

16 Aug 2018

Author Response

Some improvement in grammar, sentences and writing reorganization have been done as a response to reviewer Dr Shahida M. Said. Some details in methods and analysis have also been provided ... Continue reading Some improvement in grammar, sentences and writing reorganization have been done as a response to reviewer Dr Shahida M. Said. Some details in methods and analysis have also been provided to make better readibility and potential reproducibility of the work. As a result the work in this study is much better presented. These improvements can be found in track changes.

As this study is still preliminary comparison between smoker and non-smoker has not been performed yet, but the finding clearly show potential of use of seaweed extract to treat oral candidiasis.
Some improvement in grammar, sentences and writing reorganization have been done as a response to reviewer Dr Shahida M. Said. Some details in methods and analysis have also been provided to make better readibility and potential reproducibility of the work. As a result the work in this study is much better presented. These improvements can be found in track changes.

As this study is still preliminary comparison between smoker and non-smoker has not been performed yet, but the finding clearly show potential of use of seaweed extract to treat oral candidiasis.
Competing Interests: No competing interests were disclosed. Close
Report a concern
Respond or Comment

COMMENTS ON THIS REPORT

Author Response 16 Aug 2018

Zaki Mubarak, Faculty of Dentistry, Syiah Kuala University, Banda Aceh, 23111, Indonesia

16 Aug 2018

Author Response

Some improvement in grammar, sentences and writing reorganization have been done as a response to reviewer Dr Shahida M. Said. Some details in methods and analysis have also been provided ... Continue reading Some improvement in grammar, sentences and writing reorganization have been done as a response to reviewer Dr Shahida M. Said. Some details in methods and analysis have also been provided to make better readibility and potential reproducibility of the work. As a result the work in this study is much better presented. These improvements can be found in track changes.

As this study is still preliminary comparison between smoker and non-smoker has not been performed yet, but the finding clearly show potential of use of seaweed extract to treat oral candidiasis.
Some improvement in grammar, sentences and writing reorganization have been done as a response to reviewer Dr Shahida M. Said. Some details in methods and analysis have also been provided to make better readibility and potential reproducibility of the work. As a result the work in this study is much better presented. These improvements can be found in track changes.

As this study is still preliminary comparison between smoker and non-smoker has not been performed yet, but the finding clearly show potential of use of seaweed extract to treat oral candidiasis.
Competing Interests: No competing interests were disclosed. Close
Report a concern

Views

Reviewer Report 12 Jun 2018

Heni Susilowati, Department of Oral Biology, Faculty of Dentistry, Gadjah Mada University, Yogyakarta, Indonesia

Approved with Reservations

https://doi.org/10.5256/f1000research.16195.r34557

This preliminary research is interesting enough to be developed but there are some things that need to be reconsidered:

ABSTRACT

Research background that written on the abstract (lines

This preliminary research is interesting enough to be developed but there are some things that need to be reconsidered:

ABSTRACT

Research background that written on the abstract (lines 6 and 7) does not match the purpose of the study. The sentence can be interpreted in contrast to the antifungal potential possessed by Garcinia verrrucosa. It is dubious to investigate the potency of biofilm inhbition effect if Candida albicans more tolerant to Gracilaria extract.

2. METHODS

Methods need to explain the following:

Systemic conditions and state of teeth and oral soft tissue volunteers,
The predestined determination of the Gracilaria verrucosa plant should be mentioned.
Were the culture washed after the incubation period on treatment?
How many times an experiment produces a representative result?

3. RESULTS

Interpretation of the results is confusing; as far as I know the higher the optical density value the more biofilms are formed. The results in Fig. 1 show that 100% and Fluconazole extracts have higher optical densities rather than the lower concentration of extracts, as far as I know this shows that the mass of biofilms formed in the group is higher. Please observe the methods and results of research reported by Sebaa et al 2016.
The statistical method used was only Kruskal-Wallis, is there a multiple difference analysis? Researchers need to discuss the effect of antibiofilm extract at lower concentrations, because of course 100% extract is not a good recommendation for subsequent experimental use.

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?

No
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?

No

References

1. Sebaa S, Hizette N, Boucherit-Otmani Z, Courtois P: Dose‑dependent effect of lysozyme upon Candida albicans biofilm.Mol Med Rep. 2017; 15 (3): 1135-1142 PubMed Abstract | Publisher Full Text

Competing Interests: No competing interests were disclosed.

CITE

Report a concern

Author Response 16 Aug 2018

Zaki Mubarak, Faculty of Dentistry, Syiah Kuala University, Banda Aceh, 23111, Indonesia

16 Aug 2018

Author Response

Abstract
Comments from reviewer :
The objective has been clarified
Action done:
We think aims of study still need to be stated
Rearrangement and sentences improvement were done in some sections of abstract

Introduction
Comments from reviewer ... Continue reading Abstract
Comments from reviewer :
The objective has been clarified
Action done:
We think aims of study still need to be stated
Rearrangement and sentences improvement were done in some sections of abstract

Introduction
Comments from reviewer :
No comments
Action done:
Grammar check, revise and improvement were done to provide more concise and clear sentences/statements

Material and method
Comments from reviewer :
Need clarification on sentence:
“This volunteer has a bad OHIS4” (time and place)
“.Then microtiter wells plate wells were washed three times with 200µl of PBS buffer and dried for 15 minutes.”(biofilm examination)
“This examination was repeted three times with triplo method”( biofilm examination)
Action done:
Sentences improvement, grammar check and correction had been done throughout this section to provide better readability, clarity and possible reproducibility

Results
Comments from reviewer :
No comments
Action done:
Rewritten this part due to misinterpretations we did in the first previous manuscript about which treatment gave best inhibition effect on C. albicans

Discussion
Comments from reviewer :
No specific comments
Action done:
Rewritten has been done to this part in realtion to due to changes in results presentation
Sentences improvement, grammar check and reorganization had been done throughout this section to provide better readability and clarity

Conclusion
Comments from reviewer :
No specific comments
Action done:
This part had been rewritten in relation to the revision performed in result and discussion.
Abstract
Comments from reviewer :
The objective has been clarified
Action done:
We think aims of study still need to be stated
Rearrangement and sentences improvement were done in some sections of abstract

Introduction
Comments from reviewer :
No comments
Action done:
Grammar check, revise and improvement were done to provide more concise and clear sentences/statements

Material and method
Comments from reviewer :
Need clarification on sentence:
“This volunteer has a bad OHIS4” (time and place)
“.Then microtiter wells plate wells were washed three times with 200µl of PBS buffer and dried for 15 minutes.”(biofilm examination)
“This examination was repeted three times with triplo method”( biofilm examination)
Action done:
Sentences improvement, grammar check and correction had been done throughout this section to provide better readability, clarity and possible reproducibility

Results
Comments from reviewer :
No comments
Action done:
Rewritten this part due to misinterpretations we did in the first previous manuscript about which treatment gave best inhibition effect on C. albicans

Discussion
Comments from reviewer :
No specific comments
Action done:
Rewritten has been done to this part in realtion to due to changes in results presentation
Sentences improvement, grammar check and reorganization had been done throughout this section to provide better readability and clarity

Conclusion
Comments from reviewer :
No specific comments
Action done:
This part had been rewritten in relation to the revision performed in result and discussion.
Competing Interests: No competing interests were disclosed. Close
Report a concern
Respond or Comment

COMMENTS ON THIS REPORT

Author Response 16 Aug 2018

Zaki Mubarak, Faculty of Dentistry, Syiah Kuala University, Banda Aceh, 23111, Indonesia

16 Aug 2018

Author Response

Abstract
Comments from reviewer :
The objective has been clarified
Action done:
We think aims of study still need to be stated
Rearrangement and sentences improvement were done in some sections of abstract

Introduction
Comments from reviewer ... Continue reading Abstract
Comments from reviewer :
The objective has been clarified
Action done:
We think aims of study still need to be stated
Rearrangement and sentences improvement were done in some sections of abstract

Introduction
Comments from reviewer :
No comments
Action done:
Grammar check, revise and improvement were done to provide more concise and clear sentences/statements

Material and method
Comments from reviewer :
Need clarification on sentence:
“This volunteer has a bad OHIS4” (time and place)
“.Then microtiter wells plate wells were washed three times with 200µl of PBS buffer and dried for 15 minutes.”(biofilm examination)
“This examination was repeted three times with triplo method”( biofilm examination)
Action done:
Sentences improvement, grammar check and correction had been done throughout this section to provide better readability, clarity and possible reproducibility

Results
Comments from reviewer :
No comments
Action done:
Rewritten this part due to misinterpretations we did in the first previous manuscript about which treatment gave best inhibition effect on C. albicans

Discussion
Comments from reviewer :
No specific comments
Action done:
Rewritten has been done to this part in realtion to due to changes in results presentation
Sentences improvement, grammar check and reorganization had been done throughout this section to provide better readability and clarity

Conclusion
Comments from reviewer :
No specific comments
Action done:
This part had been rewritten in relation to the revision performed in result and discussion.
Abstract
Comments from reviewer :
The objective has been clarified
Action done:
We think aims of study still need to be stated
Rearrangement and sentences improvement were done in some sections of abstract

Introduction
Comments from reviewer :
No comments
Action done:
Grammar check, revise and improvement were done to provide more concise and clear sentences/statements

Material and method
Comments from reviewer :
Need clarification on sentence:
“This volunteer has a bad OHIS4” (time and place)
“.Then microtiter wells plate wells were washed three times with 200µl of PBS buffer and dried for 15 minutes.”(biofilm examination)
“This examination was repeted three times with triplo method”( biofilm examination)
Action done:
Sentences improvement, grammar check and correction had been done throughout this section to provide better readability, clarity and possible reproducibility

Results
Comments from reviewer :
No comments
Action done:
Rewritten this part due to misinterpretations we did in the first previous manuscript about which treatment gave best inhibition effect on C. albicans

Discussion
Comments from reviewer :
No specific comments
Action done:
Rewritten has been done to this part in realtion to due to changes in results presentation
Sentences improvement, grammar check and reorganization had been done throughout this section to provide better readability and clarity

Conclusion
Comments from reviewer :
No specific comments
Action done:
This part had been rewritten in relation to the revision performed in result and discussion.
Competing Interests: No competing interests were disclosed. Close
Report a concern

Comments on this article Comments (1)

Version 3

VERSION 3 PUBLISHED 18 Sep 2018

Revised

Comment

Version 1

VERSION 1 PUBLISHED 31 May 2018

Discussion is closed on this version, please comment on the latest version above.

Author Response 16 Aug 2018

Zaki Mubarak, Faculty of Dentistry, Syiah Kuala University, Banda Aceh, 23111, Indonesia

16 Aug 2018

Author Response

Some revisions and improvements have been done throughout the manuscript as responses to the good comments and suggestions from reviewers. Major revisions performed include grammar check and corrections; sentence improvements; ... Continue reading Some revisions and improvements have been done throughout the manuscript as responses to the good comments and suggestions from reviewers. Major revisions performed include grammar check and corrections; sentence improvements; and reorganization and adding appropriate details in each section of the material and methods part. Grammar issues, were concerned by all reviewers, had been addressed. The ways to present results were also changed as response to comment from Indonesia reviewer about the meaning of OD values in presenting the growth of C. albicans and how to interpret them. Restructure of result and discussion, therefore, was also performed. These changes became the basis for synthesizing more appropriate conclusion sentences. One reference was also added to support discussion about the importance of extract concentration for resulting in expected medicinal effects, in this study was growth inhibition that reflected by biofilm formation. Since this study is still in preliminary step, no non-smoker participant was participated. As consequence, no comparison could be made between smoker and non-smoker groups. Yet the study was still able to confirm potential of seaweed Gracia verrucose extract as anti candidiasis in smokers. Potential of doing further studies was mentioned in relation to this weakness of our study.
Some revisions and improvements have been done throughout the manuscript as responses to the good comments and suggestions from reviewers. Major revisions performed include grammar check and corrections; sentence improvements; and reorganization and adding appropriate details in each section of the material and methods part. Grammar issues, were concerned by all reviewers, had been addressed. The ways to present results were also changed as response to comment from Indonesia reviewer about the meaning of OD values in presenting the growth of C. albicans and how to interpret them. Restructure of result and discussion, therefore, was also performed. These changes became the basis for synthesizing more appropriate conclusion sentences. One reference was also added to support discussion about the importance of extract concentration for resulting in expected medicinal effects, in this study was growth inhibition that reflected by biofilm formation. Since this study is still in preliminary step, no non-smoker participant was participated. As consequence, no comparison could be made between smoker and non-smoker groups. Yet the study was still able to confirm potential of seaweed Gracia verrucose extract as anti candidiasis in smokers. Potential of doing further studies was mentioned in relation to this weakness of our study.
Competing Interests: No competing interests were disclosed. Close
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Discussion is closed on this version, please comment on the latest version above.

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Heni Susilowati, Gadjah Mada University, Yogyakarta, Indonesia
Shahida Mohd-Said, Universiti Kebangsaan Malaysia, Kuala Lumpur, Malaysia
Cristiane Y. Koga-Ito, São Paulo State University - UNESP, São José dos Campos, Brazil

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6 Views

26 Sep 2018 | for Version 3

Cristiane Y. Koga-Ito, Oral Biopathology Graduate Program, Institute of Science and Technology, São Paulo State University - UNESP, São José dos Campos, Brazil

6 Views Cite this report Responses(0)

Approved

The revised manuscript has improved. I recommend indexing of the revised manuscript as a Research Note.

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.

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7 Views

21 Sep 2018 | for Version 3

Heni Susilowati, Department of Oral Biology, Faculty of Dentistry, Gadjah Mada University, Yogyakarta, Indonesia

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Approved

I have read the revised version and can accept the revisions that have been made by the author insofar as this research is still an initial research.

Competing Interests

No competing interests were disclosed.

Reviewer Expertise

Molecular biology and oral microbiology

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

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8 Views

23 Aug 2018 | for Version 2

Shahida Mohd-Said, Periodontology Unit, Centre for Restorative Dentistry, Faculty of Dentistry, Universiti Kebangsaan Malaysia, Kuala Lumpur, Malaysia

8 Views Cite this report Responses(0)

Approved

Improvements made are acknowledged and approved.

Competing Interests

No competing interests were disclosed.

Reviewer Expertise

Periodontology, natural product drug discovery

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

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13 Views

20 Aug 2018 | for Version 2

Heni Susilowati, Department of Oral Biology, Faculty of Dentistry, Gadjah Mada University, Yogyakarta, Indonesia

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Approved With Reservations

Competing Interests

No competing interests were disclosed.

Reviewer Expertise

Molecular Biology and Oral Microbiology

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21 Views

27 Jun 2018 | for Version 1

Cristiane Y. Koga-Ito, Oral Biopathology Graduate Program, Institute of Science and Technology, São Paulo State University - UNESP, São José dos Campos, Brazil

21 Views Cite this report Responses(0)

Approved With Reservations

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

No
Is the study design appropriate and is the work technically sound?

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

No
If applicable, is the statistical analysis and its interpretation appropriate?

Not applicable
Are all the source data underlying the results available to ensure full reproducibility?

No
Are the conclusions drawn adequately supported by the results?

No

Competing Interests

No competing interests were disclosed.

Reviewer Expertise

Microbiology

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Reviewer Report

19 Views

19 Jun 2018 | for Version 1

Shahida Mohd-Said, Periodontology Unit, Centre for Restorative Dentistry, Faculty of Dentistry, Universiti Kebangsaan Malaysia, Kuala Lumpur, Malaysia

19 Views Cite this report Responses(1)

Approved With Reservations

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

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

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?

No
Are the conclusions drawn adequately supported by the results?

Yes

Competing Interests

No competing interests were disclosed.

Reviewer Expertise

Periodontology, natural product drug discovery

Respond to this report

Responses (1)

Author Response

16 Aug 2018

Zaki Mubarak, Faculty of Dentistry, Syiah Kuala University, Banda Aceh, 23111, Indonesia

Some improvement in grammar, sentences and writing reorganization have been done as a response to reviewer Dr Shahida M. Said. Some details in methods and analysis have also been provided to make better readibility and potential reproducibility of the work. As a result the work in this study is much better presented. These improvements can be found in track changes.

As this study is still preliminary comparison between smoker and non-smoker has not been performed yet, but the finding clearly show potential of use of seaweed extract to treat oral candidiasis.

View more View less

Competing Interests

No competing interests were disclosed.

Back to all reports

Reviewer Report

22 Views

12 Jun 2018 | for Version 1

Heni Susilowati, Department of Oral Biology, Faculty of Dentistry, Gadjah Mada University, Yogyakarta, Indonesia

22 Views Cite this report Responses(1)

Approved With Reservations

This preliminary research is interesting enough to be developed but there are some things that need to be reconsidered:

ABSTRACT

Research background that written on the abstract (lines 6 and 7) does not match the purpose of the study. The sentence can be interpreted in contrast to the antifungal potential possessed by Garcinia verrrucosa. It is dubious to investigate the potency of biofilm inhbition effect if Candida albicans more tolerant to Gracilaria extract.

2. METHODS

Methods need to explain the following:

Systemic conditions and state of teeth and oral soft tissue volunteers,
The predestined determination of the Gracilaria verrucosa plant should be mentioned.
Were the culture washed after the incubation period on treatment?
How many times an experiment produces a representative result?

3. RESULTS

Interpretation of the results is confusing; as far as I know the higher the optical density value the more biofilms are formed. The results in Fig. 1 show that 100% and Fluconazole extracts have higher optical densities rather than the lower concentration of extracts, as far as I know this shows that the mass of biofilms formed in the group is higher. Please observe the methods and results of research reported by Sebaa et al 2016.
The statistical method used was only Kruskal-Wallis, is there a multiple difference analysis? Researchers need to discuss the effect of antibiofilm extract at lower concentrations, because of course 100% extract is not a good recommendation for subsequent experimental use.

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?

No
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?

No

References

1. Sebaa S, Hizette N, Boucherit-Otmani Z, Courtois P: Dose‑dependent effect of lysozyme upon Candida albicans biofilm.Mol Med Rep. 2017; 15 (3): 1135-1142 PubMed Abstract | Publisher Full Text

Competing Interests

No competing interests were disclosed.

Respond to this report

Responses (1)

Author Response

16 Aug 2018

Zaki Mubarak, Faculty of Dentistry, Syiah Kuala University, Banda Aceh, 23111, Indonesia

Abstract
Comments from reviewer :
The objective has been clarified
Action done:
We think aims of study still need to be stated
Rearrangement and sentences improvement were done in some sections of abstract

Introduction
Comments from reviewer :
No comments
Action done:
Grammar check, revise and improvement were done to provide more concise and clear sentences/statements

Material and method
Comments from reviewer :
Need clarification on sentence:
“This volunteer has a bad OHIS4” (time and place)
“.Then microtiter wells plate wells were washed three times with 200µl of PBS buffer and dried for 15 minutes.”(biofilm examination)
“This examination was repeted three times with triplo method”( biofilm examination)
Action done:
Sentences improvement, grammar check and correction had been done throughout this section to provide better readability, clarity and possible reproducibility

Results
Comments from reviewer :
No comments
Action done:
Rewritten this part due to misinterpretations we did in the first previous manuscript about which treatment gave best inhibition effect on C. albicans

Discussion
Comments from reviewer :
No specific comments
Action done:
Rewritten has been done to this part in realtion to due to changes in results presentation
Sentences improvement, grammar check and reorganization had been done throughout this section to provide better readability and clarity

Conclusion
Comments from reviewer :
No specific comments
Action done:
This part had been rewritten in relation to the revision performed in result and discussion.

View more View less

Competing Interests

No competing interests were disclosed.

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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[24] 24. Mubarak Z, Humaira A, Gani BA, et al.: Dataset 1 in: Preliminary study on the inhibitory effect of seaweed Gracilaria verrucosa extract on biofilm formation of Candida albicans cultured from the saliva of a smoker. F1000Research. 2018. http://www.doi.org/10.5256/f1000research.14879.d204270

Preliminary study on the inhibitory effect of seaweed Gracilaria verrucosa extract on biofilm formation of Candida albicans cultured from the saliva of a smoker

Abstract

Keywords

Revised Amendments from Version 1

Introduction

Methods

Time and site

Ethics

Saliva collection

Candida albicans Isolation and preparation

Seaweed extraction

Phytochemical tests

Examination of Biofilm Formation

Data analysis

Results

Table 1. Phytochemical contents of seaweed Gracilaria verrucosa extract.

Figure 1. The formation of biofilm by Candida albicans exposed to seaweed Gracilaria verrucosa extract on different time exposures and concentrations.

Discussion

Conclusion

Data availability

Competing interests

Grant information

References

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