The acid tolerance response and pH adaptation of Enterococcus faecalis in extract of lime Citrus aurantiifolia from Aceh Indonesia.

Background: The objective of the present study was to evaluate the acid tolerance response and pH adaptation when Enterococcusfaecalis interacted with extract of lime ( Citrus aurantiifolia).
Methods: We used E. faecalis ATCC 29212 and lime extract from Aceh, Indonesia. Both materials were analyzed for their pH adaptation, acid tolerance response, adhesion assay, and mass profiles using a light microscope with a magnification of x1000. Further, statistical tests were performed to analyze both correlation and significance of the acid tolerance and pH adaptation also the interaction activity.
Results: E. faecalis was able to adapt to a very acidic environment (pH 2.9), which was characterized by an increase in its pH (reaching 4.2) at all concentrations of the lime extract (p < 0.05). E. faecalis was also able to provide acid tolerance response to lime extract based on spectrophotometric data (595 nm) (p < 0.05). Also, the interaction activity of E. faecalis and the lime extract was relatively stable within 6 up to 12 hours (p < 0.05), but it became unstable within 24-72 hours (p > 0.05) based on the mass profiles of its interaction activity. Conclusions:E. faecalis can adapt to acidic environments (pH 2.9-4.2); it is also able to tolerate acid generated by Citrus aurantiifolia extract, revealing a stable interaction in the first 6-12 hours.

Introduction

Enterococcus faecalis is a significant agent in the pathogenesis of root canal infections, especially in post-endodontic treatment, with a prevalence of 24–77% in these infections [1]. This bacterium is very difficult to eliminate because the pathogen can survive in poor nutrient conditions. It can adapt to acidic conditions, including living in the dentin tubule of a closed root canal with a smear layer. It can also express the dominant biofilm protein to maintain its attachment to host cells [2].

E. faecalis has been shown tolerate to acidic environments as well as to adapt to pH changes, which are the essential virulence factors in maintaining antibacterial balance [3]. Fisher reported that E. faecalis could survive in environments with high NaCl concentrations at extreme temperatures of 5–65°C with a pH of 4.5–10.0 [4]. Stuart et al. [1] reported that E. faecalis are less sensitive, with a pH of 5.0 at 25°C after it has been incubated for 10 h. The author also found that it has an excellent growth capability at pH 8.5 and low adhesion at pH 7.1 in a medium coated with bovine serum albumin (BSA).

E. faecalis is resistant to medication materials such as calcium hydroxide [5] and chlorhexidine (CHX) [6]. The long-term use of both medication materials can lead to parachloroaniline (PAC), causing blockage of the dentinal tubules and eventually becoming toxic [7]. Fosfomycin may also interfere with acid tolerance systems and pH changes of E. faecalis in tooth root canals by inhibiting phosphoenolpyruvate synthetase [8].

Indonesia, especially in Aceh has a tropical climate with a variety of plants that can be utilized in medical treatment, including lime ( Citrus aurantiifolia). Lime peel extract contains phenols, flavonoids, hydrogen peroxide, tannins, alkaloids, and saponins that have antibacterial, antioxidant, antifungal, analgesic, and anti-inflammatory properties [9]. Nwankwo [10] reported that lime extract helped to prevent Klebsiella pneumonia, Salmonella, and Escherichia coli. Here, the acid in lime extract influenced the bacterial development and cell metabolism. The present study evaluates the acid tolerance response and pH adaptation of E. faecalis when the bacterium grows as biofilm in the presence of lime extract with different concentration.

Methods

Materials

The lime extract and E. faecalis (ATCC-29212) were used in this study. The extractions were conducted at the Laboratory of Microbiology at the Faculty of Veterinary, University of Syiah Kuala, Darussalam, Banda Aceh, Indonesia. The material and bacterium were prepared in vitro to analyze the pH adaptation, acid tolerance response, and interaction activity of E. faecalis in different concentration of lime extracts.

Lime extraction

Lime peel was separated from the flesh then dried using dehydrator until the water content reduced to 10%. Dried lime peel was grinded into powders. The powder was put into a glass container and macerated with ethanol 70% for two days and then strained using a gauze. Filtrate was evaporated using a rotary evaporator at 80°C to obtain the pure lime extracts.

Culture of E. faecalis

One colony of E. faecalis bacteria was subsequently re-cultured in 5 ml of Mueller-Hinton Broth (MHB) medium (Thermo Fisher Scientific Inc, Paisley, UK) in anaerobic conditions at a temperature of 37°C for 48 hours. Afterward, the E. faecalis grown on the liquid medium was synchronized further with McFarland 0.5 (1 × 10 8 CFU/ml) (TM50, Dalynn Biological Inc., Calgary, Canada). The accurate density of McFarland standard was checked using a spectrophotometer with an absorbance reading of 0.08 to 0.1 at 625 nm [11].

Adaptation to pH assay

A total of 50 ml of lime extracts in several different concentrations (100% v/v, 75% v/v, 50% v/v, 25% v/v, 12.5% v/v, and 6.25% v/v) was put into different beaker glasses. Then, 5 ml of E. faecalis in MHB (1:10) were added to each of the beakers. The initial pH of each mixture prior to incubation (0 hours) was 2.89, 2.75, 2.91, 2.92, 2.95, 2.98 and 3.10, respectively. Next, bacterium containing beaker was put into incubator (37°C) for 6 hours, 12 hours, 24 hours, 48 hours, and 72 hours in an anaerobic atmosphere using Anaerogen TM GasPack (Oxoid, Basing stoke UK), at each of these times, the beakers’ pH was measured using a pH meter (Thermo Fisher Scientific Inc, Paisley, UK). Various changes in pH from 0 hours to the specified time can be used as an indicator of whether E. faecalis has a tolerance response to the acidic environment and can adapt to changing pH [12].

Acid tolerance assay

The cultures of the pH measurements were used to measure the acid tolerance response of E. faecalis to lime extract utilizing the principle of spectrophotometry [13]. The analysis was performed based on the incubation time that had been determined following the measurement of pH shaken at 500 rpm. Here, 50 ul of MHB was put into a microplate in triplicate (Thermo Fisher Scientific Inc., Paisley, UK). The microplate was incubated at room temperature for 15 minutes and vacuumed. The materials tested and E. faecalis derived from the incubation processes at 6 h, 12 h, 24 h, 48 h, and 72 hours were added to each well. The microplate was put into an incubator 37 °C for 15 minutes. Then bio-tolerant activity was measured by Elisa Reader (Bio-Rad Laboratories, Hercules, CA) at a wavelength of 595 nm.

Adhesion assay

Adhesion assay was conducted based on the principles of Gram-staining [14]. This incubation time-based interaction activity on the microplate 96 wells series was done following the Gamble’s working principle [15]. It was modified using violet crystalline and safranin staining to confirm the possible occurrence of bacterial contaminants. First, microplate in triplicate wells were coated with 50 μl of MHB (Thermo Fisher Scientific Inc., Paisley, UK) for 15 min, and aspirated. Second, 50 uL of E. faecalis in MHB were added and incubated for 15 min at room temperature. Third, 100 uL of different concentrations of LE were added and incubated for 6 h, 12 h, 24 h, 48 h, and 72 hours (as adapted from research conducted by Bachtiar) [16]. All residues of the test materials ( E. faecalis + lime extract) in the microplate were aspirated and the plate was settled for 10 min at room temperature. Then, 50 μL of 2% violet crystalline were added to each well for 5 minutes; the wells were washed with phosphate buffer saline (PBS) two times (Merck, Darmstadt, Germany). A total of 100 μL of Lugol solution was added for 1 minute and washed with PBS. Cell metabolites and dye were removed by the addition of 100 μL of 96% alcohol for 20 seconds. Safranin solution, 50 μL, were added for 2 minutes and washed with PBS [17, 18]. The microplate was measured using an Elisa reader using with optical density of 595 nm [13]. For each period, the turbidity of the medium was visually compared with a 0.5 McFarland standard

Statistical analysis

E. faecalis acid tolerance and adhesion to lime extract were calculated to determine average values and standard deviations for each concentration. Two-ways analysis of variance (ANOVA) was performed with significance set at p < 0.05. The analysis was performed using SPSS ver. 20.0 software.

Results

The experiment was performed in triplicate wells. This study showed that the presence of lime extracts decreased pH, but reduction of low pH did not have a significant effect on the ability of E. faecalis to adhere and form biofilm, compared to the control (fosfomycin). All results (adaptation to pH, acid tolerance assays and interaction activity are shown in Figure 1– Figure 3). Interestingly, the tolerance effect was not influenced by exposure time and the concentrations of lime peel extract set in this study ( Figure 3), and the correlation between time exposure and lime extract concentration was positive (r2 = 0.98).

Figure 1.

The pH adaptation response of E. faecalis to lime extract at different concentration and exposed time.

Figure 3.

Optical density of adhesion of E. faecalis at different concentration of lime extracts and exposed time.

E. faecalis did not express an ability to adapt to result in pH changes after interacting with fosfomycin (as a positive control) ( Figure 1), although it still expressed acid tolerance response ( Figure 2) and robust adhesion activity ( Figure 3).

Figure 2.

The optical density of the acid tolerance response of E. faecalis to lime extract at different concentration and exposed time.

Click here for additional data file.

Click here for additional data file.

Click here for additional data file.

Discussion

In searching for pH adaptation of E. faecalis to ethanolic extract of lime peel, the initial pH of concentrated extract (100% v/v) without E. faecalis was 2.89. This is in agreement with those reported by Sitanggang et al. [19] that the water extract of lime has highly acidic pH ranges (1.7–3.1). As illustrated in Figure 1, this highly acidic pH only slightly changed under serial dilution and after the addition of E. faecalis suspension prior to incubation (0 hour).

This relatively stable pH of lime extract after serial dilution in water and after the addition of E. faecalis might be related to the presence of buffering compounds in the extract that is able to maintain pH. According to Bolhari et al. the juice of lime ( C. aurantiifolia) contains 88% water, 6–8% citric acid, 2% potassium citrate and calcium, 0.4–0.6% and other substances [20]. The water extract of the fruit contains a number of bioactive compounds such as alkaloids, phenols, flavonoids, steroids, terpenoids, reducing sugar, saponins and cardiac glycosides [21]. Moreover, the peel of C. aurantiifolia contained 7 percent essential oil consisting of 46 compounds, most of which are terpenes [22]. The acidity is generated by citric acid and amino acids, while the essential oils contribute to maintaining its acidic pH [23]. Citric acid is reported to play a crucial role as a natural material to maintain pH balance and possesses antibacterial activity [24].

After incubation there was a significant increase in the acidic pH of lime extract containing E. faecalis compared to lime extract alone (as a negative control) (p < 0.05). The increased pH occurred in all different concentration of lime extracts, from 6.25% (v/v) to 100% (v/v) (see Figure 1) indicates that E. faecalis can adapt to environments with an acidic pH (2.9–4.2) at a temperature of 37°C. The ability of E. faecalis to adapt to situations with a low pH and temperature has been reported by Morandi et al. [25] in the experiment using pH adjusted to pH of 5.0 at 25°C within 10 hours.

Whilst better E. faecalis growth was observed in different lime extract concentrations with long exposure, increased acid tolerance response shown by the microbe as the concentration of the lime extract increased ( Figure 2) (p < 0.05). These phenomena probably relate to the ability of E. faecalis to produce a number of virulence factors in the extreme environment, such as the presence of potent antimicrobial agents or highly acidic condition [26]. Among them is lipoteichoic acids that contribute to biofilm formation, a bacterial community-based resistant mechanism developed by certain bacteria to survive in extremely disadvantages environment [27].

Molecularly, the acid tolerance response of E. faecalis is influenced by the EfCitH gene, which encodes the citrate transporter protein on the surface of the bacterial cell membrane that acts to maintain the balance of the effects of citric acid generated from the environment [28]. Sarantinopoulos found that enterococcal strains have metabolic potential against the citrate metabolism; this supports their acid tolerance response to environmental influences such as aroma and fermentation products [29]. In this research, in the presence of fosfomycin with a pH of 7.2 ( Figure 1) E. faecalis could still slightly show pH tolerance. The acid tolerance response is related to the ability of certain E. faecalis strains to grow in environments with an alkaline pH (9.5–12) within 48–72 hours [12].

In general, E. faecalis showed progressively decreased adhesion ability in the presence of different concentrations of lime extract within 6 – 24 hours ( Figure 3). The adhesion started to increase after the microbe was exposure to different concentration of lime extract for 24 hours. The relatively high error bar (standard deviation values) obtained indicates such variation might be existed among the E. faecalis isolates toward the pH and concentration of lime extracts.

Varoni et al. [30] reported that anti-adhesion activity between plant polyphenol-rich extract and Streptococcus mutans bacteria was at its maximum within 24 hours, while within 6, 7, and 8 hours, the activity was stable but not yet maximal. This is probably caused by the adaptation and tolerant mechanisms developed by E. faecalis against bioactive compounds presence in the extracts. Lime peel extract contains a number of metabolites (phenols, flavonoids, hydrogen peroxide, tannins, alkaloids, and saponins) have various therapeutic properties such as antibacterial, antioxidant, antifungal, analgesic, and anti-inflammatory [9].

The mechanism utilized by bacteria to survive heat and low-pH of the environment operate in many different ways. The most successful means of surviving low-pH stress is the complete avoidance of extremely acidic environments. However, none more critical than the sensing of mild acidification to prevent the potentially lethal consequences of the inappropriate production of potentially antigenic proteins. Bacteria that are forewarned by mild acidification can prepare through the induction of a wide range of protective measures. It can alter the composition of the cell membrane, extrude protons, protect macromolecules, alter metabolic pathways, and generate alkaline [31].

The lower adhesion activity observed in E. faecalis exposed to different concentrations of lime extract compared to that exposed to fosfomycin indicated better antibacterial activity of lime extract on the microbe than that of fosfomycin. Among active ingredients contained in the lime extract are flavonoids (polyethoxylated flavones and flavanones), coumarin, and terpenoids, all of which act as antibacterials [32]. Extract prepared from peel, fruit and leaves of lime show promising antibacterial activity against some microbes belong to both gram positive bacteria ( Staphylococcus aureus, S. epidermis and E. faecalis) and gram negative bacteria ( Klebsiella pneumonia and Proteus vulgaris) [33].

Conclusion

Enterococcus faecalis can adapt to environments with a pH of 2.9–4.3 generated by lime extracts. In addition E. faecalis also expressed a tolerance response to the acidic environment. The interaction activity of E. faecalis in different concentrations of lime extract become stable within 6–12 hours at a temperature of 37°C. Therefore, the lime extract can be used to inhibit the E. faecalis growth.

Data availability

Dataset 1: pH adaptation of E. Faecalis in lime extract based on replications 10.5256/f1000research.13990.d196643 [34].

Dataset 2: Optical density (OD) of acid tolerance respond of E. Faecalis in lime extract based on replications 10.5256/f1000research.13990.d196644 [35].

Dataset 3: The OD value of the interaction activity of E. Faecalis in lime extract based on replications 10.5256/f1000research.13990.d196646 [36].

I have read the revised manuscript, and the authors have made modifications to the manuscript, and have no further comments. Thus, I recommend this work for publication in F1000Research.

Thank you for considering me as a reviewer.

Kindly Regards

Boy M Bachtiar

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

In general, the paper would still benefit from a review by a native English speaker to correct the language.

Additional recommended actions to consider are the following:

Abstract

in Methods (2nd line): instead of “Both materials were analyzed for their adaptation, …” please use “The microbe was analyzed for its adaptation, …”

Methods

in Culture of E. faecalis bacteria: please rephrase the last sentence “The Accurate of the density ...” to make it more understandable.

in Adaptation to pH assay: first sentence: how do the concentration values, given here in %, compare with units of Figures 1 to 3 where concentration is given in micrograms per milliliter?

Results, Discussion

Figure 1: why is the initial pH value (at 0 hours) about the same at all lime extract concentrations? Are there any known inhibitor (buffering) substances in the lime extract (or in the bacterial culture) that could explain relatively constant initial pH at a range of concentrations?

Figure 2: why is the indicated acid tolerance apparently higher towards increasing lime extract concentration also at 0 hours of exposure?

Figure 3: why is the indicated adhesion level apparently higher towards increasing lime extract concentration also at 0 hours of exposure? Where are the error bars mentioned in the text?

The paper is referring in many places to “interaction activity” between lime extract and E. faecialis. However, could the results also involve a concentration-dependent interaction between e.g. citric acid and other antibacterial compounds of the extract?

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

We have made the revision based on suggestion and comment from reviewers

Introduction

The last paragraph: ………..acid tolerance response and pH adaptation of E. faecalis  when it interacts with lime extract.

Reviewer’s comment:  please change the bold word with: when the bacterium grows as biofilm in the presence of lime extract with difference concentration.

Method

-           “Both materials were made in vitro to analyze”.

Reviewer’s comment: please change with: The material and bacterium were prepared

- In lime extract, typo……………………………………into a glass

- Culture of E. faecalis bacteria; please discard; “bacteria”

- E. faecalis ATCC 29212 taken from glycerol ……………………………………………………………………………………………..and incubator (Memmert, Germany).

Reviewer’s suggestion: discard this part, and please replace with:

One colony of E. faecalis bacteria was subsequently re-cultured in 5 ml of Mueller-Hinton Broth (MHB) medium (Thermo Fisher Scientific Inc, Paisley, UK) in anaerobic conditions at a temperature of 37°C for 48 hours. Afterward, the E. faecalis grown on the liquid medium was synchronized further with McFarland 0.5 (1 x 108 CFU/ml) (TM50, Dalynn Biological Inc., Calgary, Canada). The Accurate of the density of McFarland standart (typo: it should be standard) can be checked using a spectrophotuometer with an absorbance reading of 0.08 to 0.1 at 625 nm11.

Adaptation to pH assay

A total of 50 ml of lime extracts in several different concentrations

(100%, 75%, 50%, 25%, 12.5%, and 6.25%) was placed into different beaker glasses. Then, 5 ml containing  1 X 108 CFU/mL of E. faecalis in MHB (1:10) were added to each of the beakers. The initial pH

Reviewer’s suggestion: please indicate the initial pH that was measured at the zero h.,  before incubation. Reviewer’s suggestion:  Next, bacterium-containing beaker was put into incubator (37°C)  for 6 hours, 12, hours, 24 hours, 48 hours, and 72 hours in an anaerobic atmosphere using Anaerogen TM GasPack (Oxoid, Basing stoke UK), at each of these times, the beakers’ pH was measured using a pH meter (Thermo Fisher Scientific Inc, Paisley, UK). Various changes in pH from 0 hours to the specified time can be used as an indicator of whether E. faecalis has a tolerance response to the acidic environment and can adapt to changing pH.

Acid tolerance assay

-           “Here, 96 wells of the triple microplate series were coated with 50 μl of MHB”.

Reviewer suggestion: Please change to: Here, 50 ul of MHB was put into microplate in triplicate.

-           “the test materials (E. faecalis + lime extract) derived…….

Reviewer suggestion: please change to: material tested  and E. faecalis……………………………………………”Each well” change to the microplate was put into incubator for 15 min.

-           “Then bio-tolerant activity was measured using the Elisa Reader (Bio-Rad Laboratories, Hercules, CA) at a wavelength of 595 nm”. I suggest to discard this part, as it does not has a clear meaning, ….activity was measure by ELISA reader……..

Adhesion assay

-           “The standard protein concentration of the E. faecalis and the active component concentration of the lime extract were measured via the Bradford method”

It is not clear, why the authors need to analyze (by measuring) the E. faecalis’s proteins (I assume the whole cell proteins). Thus, what does the authors would like to say with "the standard protein concentration of E. faecalis and the concentration of active component of the lime extract”?. My suggestion is please discard this part.

-           “Spectrophotometry detected the interaction of E. faecalis with lime extract at a wavelength of 595 nm”

It is rather confusing. Spectrophotometry was used to detect the interaction between the bacterium and the lime extract. Did  you use this method to determine the bacterium growth rate? I suggest to discard this spectrometric method. Otherwise, please focus the methods (adaptation to pH, acid tolerance, and adhesion assay) used only to address your hypothesis; "that biofilm formation or bacterial adherence ability contributes to E. faecalis survival in biofilm-related environment in the presence of lime extract."

-           The principle of incubation time-based interaction activity on the microplate 96 wells series used in this research based on Gamble’s working principle. I think you only tested the bacterial adherence capability, not interaction activity between E. faecalis and……….?

-           It was modified using violet crystalline and safranin staining. First, 96 wells of the triple microplate series were coated with………..

Suggestion: Please change this part: “First, microplate in triplicate wells. ...................Second, 50 uL of E. faecalis in ( ADD IN WHAT MEDIUM USED) ...........Third, 100 uL of different concentration of LE was added and incubated for..........

-           “A total of 100 μL of Lugol solution was added for 1 minute and then washed with PBS. The rest of the cell metabolism that was not bacterial cells was dissolved in 96% alcohol for 20 seconds until the dye completely removed. 50 μL of safranin solution was added for 2 minutes and then washed again with PBS18. The interaction activity between the lime extract and the E. faecalis bacteria in the microplate wells was assessed via an Elisa reader using a spectrophotometer (Bio-Rad Laboratories, Hercules, CA, USA) at a wavelength of 595 nm”

Suggestion:

As you only tested mono-species biofilm (E. faecalis is a Gram (+) ve bacterium), please add an explanation, why you used both crystal violet and safranin solution?, and you did it similar to the procedure for Gram staining method.

Please discard the word: “The interaction activity between the lime extract and the E. faecalis Bacteria”. It would better to say: the bacterial adherence was assessed using crystal violet and measured using ELISA reader with optical density of 595 nm.

Observation of adhesion mass. This method is not necessary for this study, as you have used crystal violet (CV) assay to measure biofilm mass. CV provides a good measure of biofilm mass, although it cannot measure biofilm viability.  This means, CV stains both  bacteria cells and the polymer substance (carbohydrate) as part of the extra cellular matrix.

Statistical analysis

………..standard deviations for each concentration.

Reviewer comment: why there are no SD displayed in each graph shown in the figures

Results

-The experiment was conducted in three replicates

Reviewer suggestion:

Please changed:  the experiment was performed in triplicate wells

This study showed that the presence of lime extracts decreased pH, but reduction of low pH did not have a significant effect on the ability of E. faecalis to adhere and form biofilm, compared to the control (fosfomycin). All result (crystal violet, adaptation to pH, and acid tolerance assays) are shown in Fig .1-3).

Interestingly, the tolerance effect was not influenced by exposure time and the concentration of LE set in this study (Fig. 3), and the correlation between time exposure and LE concentration was positive (However, the authors need to show the regression graph as they said r2 = 0.98). Otherwise, please exclude this part.

“the results showed that the interaction activities of E. faecalis in Lime extract will decrease from 6, 12, 24, and 72 hours. Reviewer‘s comment: I am wondering what the authors mean with the interaction activity. I suggest to discard this part.

The mass profile also indicated interaction activity between lime extract and E. faecalis. Antibiotics are capable of forming a covalent bond to activate the cysteine residue of a bacterial cell, triggering UDP-N-acetylglucosamine to form hydrogen bonds. It inhibits the synthesis of peptidoglycan as an antibacterial defense. Reviewer’s comment: Please discard this part, as 1. I cannot see the data (mass profile). 2. Antibiotic are capable........this is not the result of this study. When necessary please put the sentence in the discussion section.

Discussion

Reviewer’s comment: please focus the results you got. Especially, please do not repeat the result, and exclude: “The interaction activity between lime extract and E. faecalis can be assumed to be the antibacterial activity, because there was a decrease in the interaction activity between E. faecalis and the biological components of lime extract within 6–24 hours and again within 48–72 hours. The interaction activity is related to the activity of the active ingredients contained in the lime extract, such as flavonoids (polyethoxylated flavones and flavanones), coumarin, and terpenoids, all of which act as antibacterials”. I suggest to rewrite this part ……….The survival of E. faecalis in the presence of LE………..

Conclusion

E. faecalis can adapt to environments with a pH of 2.9–4.3 generated by lime extracts. In addition E. faecalis also expressed a tolerance response to the acidic environment. The interaction activity between E. faecalis and lime extract become stable within 6–12 hours at a temperature of 37°C. Therefore, the lime extract can be used to inhibit the E. faecalis growth.

Reviewer’s comment: Your conclusion should succinctly describe the overall result of the experiment. Please delete: 1. The interaction activity between E. faecalis and lime extract become stable within 6–12 hours at a temperature of 37°C. 2/.  Therefore, the lime extract can be used to inhibit the E. faecalis growth.

Please refer your conclusion to the title that reflects the result.

I have read this submission. I 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.

Revision has been made according to the comment of the reviewer