Exploration of the Bioactivity of Pigmented Extracts from Streptomyces Strains Isolated Along the Banks of the Guaviare and Arauca Rivers (Colombia)

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Peer Reviewer comment

Author response

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The manuscript entitled ‘Exploration of the Bioactivity of Pigmented Extracts from Streptomyces Strains Isolated along the Banks of the Guaviare and Arauca Rivers (Colombia)’, presents the exploration of the bioactive activities of pigmented extracts produced by Streptomyces isolates from riverbank sediment samples in Colombia. The exploration focuses on showing the bioactive potential of pigmented extracts of these bacteria. The search for new microbial pigments and their potential activity is an area of interest. It allows the discovery of new super-producing strains of pigments and metabolites of scientific interest. The manuscript presents the results of an extensive screening study of bacteria in different culture media and extracts evaluating their bioactive activities; generating a significant number of results that establish the microbial potential of specific sites.

Exploratory studies of new biological sources of pigment production should be carried out, not only to identify the strains but also to establish the initial color potential characteristics of the extracts, such as the level of pigmentation, the color produced (wavelength of maximum absorption in the visible), as well as the characterization of the behavior of the strain under different culture conditions (pH, temperature, nutrients, etc.). The authors address the isolation and identification of the strains, the determination of the behavior in different media, and the potential bioactivity evaluation of the pigmented extracts as a way to know the potential of the isolated strains. However, publication of the manuscript in its present form is not recommended. The manuscript should resolve some important issues related to the information presented in the methods, results, and discussion before publication is considered.

We sincerely thank the reviewer for the thoughtful and detailed evaluation of our study, as well as the positive and encouraging feedback on our manuscript. We have enthusiastically addressed the detailed and relevant suggestions provided by the reviewer, and made every effort to improve the manuscript, accordingly, resulting in an enhanced version of our study.

1

The manuscript does not present a hypothesis explicitly or it is not defined, what is presented is an exploratory study. The authors should describe the definition of their hypothesis concerning their research interest in the manuscript.

We appreciate too much your kind remark. Accordingly, in the introduction section, this passage was introduced under the developed context to explicitly define and inform about the hypothesis of our study: “Given this context, we hypothesized that the unique environmental conditions of these tropical rivers might induce the production of diverse pigment-related metabolites in Streptomyces strains growing along the riverbanks. We further posited that the pigmented extracts from these strains could exhibit multifaceted properties due to the presence of bioactivity specialized metabolites. Therefore, the aim of this study was to explore the biological activity of pigmented extracts from Streptomyces isolated from the Guaviare and Arauca rivers in Colombia, focusing on their antimicrobial, antioxidant, and anticancer properties, in support of the further hypothesis that these strains could lead to the discovery of novel bioactive compounds.”.

2

The authors indicate in some sections of the methods that they have carried out triplicate trials, however, they do not mention anywhere in the manuscript the statistical methods of these analyses. The authors should report these analyses in the manuscript, the methods of statistical evaluation, and the type of comparison performed, and then indicate this in the results that require it.

Thanks a lot for your important observation. We agree with it. Accordingly, we added a description of the statistical analysis performed on the bioactivity data (Section 2.6) and the groups derived from the post-hoc multiple comparisons according to the Tukey test to define significant differences between the means of all possible pairs per concentration along tested extracts were also added in Tables S1-S5.

3

In section 2.2, more detail needs to be given in the manuscript on the preparation methods of strains (20 isolates) for culture. Inoculum preparation conditions (type of culture, use of spores or mycelium, aliquot used, etc.), and type of medium (agar, liquid culture, semi-solid, etc.) should be reported.

Thank you for your kind comment. We agree with it, so this passage was then inserted in the section 2.2.: “A subset of 20 pigmented isolates was reactivated on semi-solid isolation medium across four different culture media (see Table 1). For this, 100 µL of spore suspension (pH 7) was massively seeded onto the media and incubated at 30°C for 7 days (Friocell, Querétaro, Mexico). Once the strain fully colonized the agar surface, 1-cm² sections were used to initiate liquid cultures”.

4

In the same section 2.2, it is necessary to report in the manuscript the culture conditions for the pathogenic bacteria used in the antimicrobial evaluation (type of seeding, amount of inoculum, etc.), so that reproducibility of the methods by other authors can be ensured.

We also appreciate this suggestion. Accordingly, this paragraph was insert in the section 2.2.: “For each pathogen, 100 µL of cryopreserved strains in TSA medium with 40% glycerol (at 1 x 10⁹ CFU/mL) were thawed and added to 10 mL of TSA medium, then incubated at 30°C for 24 hours. The bacterial concentration was adjusted to an optical density (OD) of 600 nm, with values between 0.8 and 1.0, equivalent to a 0.5 McFarland standard. Subsequently, 100 µL of the bacterial suspension was spread uniformly onto TSA agar plates. After incubation at 4°C for 10 minutes, sensi-discs containing different concentrations of the extracts were placed on the semi-solid medium for antibacterial assays (vide infra)”.

5 & 6

In section 2.3, it is suggested to change the term ‘Fermentation of isolates’ to ‘culture preparation’.

Sections 2.2. and 2.3, contain information showing continuity in the isolate preparation and culture procedures. It is therefore suggested to make a single section with subsections.

Thanks a lot for these kind observations. We agree with them, and, consequently, this section was removed, and the respective text was then merged with section 2.2. In addition, the subheading was changed and subheadings were also added as suggested.

7

In these sections (2.2 and 2.3) the authors should be more specific in establishing their cultivation procedures and the timeline for obtaining the pigmented extracts, i.e., they should describe in such detail the procedures from strain activation to cultivation for pigment production.

Thank you very much for your kind remark. We agree with this, and, in line with the previous comments, we re-organized this section 2.2.. In addition, this paragraph was inserted to improve the provided information: “This culture (1 mL) was subsequently mixed with fresh medium (9 mL) and incubated under the same conditions (30°C, 150 rpm) in 25-mL glass Erlenmeyer flasks for 7 days. Finally, the 10-mL culture was transferred to 90 mL of fresh medium in 250 mL Erlenmeyer flasks and incubated for an additional 7 days at 150 rpm and 30°C”.

8 & 9

In lines 112 and 113 (section 2.3), it is unclear whether the strains grown on activated ISP2 medium were grown on a new ISP2 medium in addition to the other three culture media. The authors should indicate in the manuscript these details to know how the cells were handled. Conventionally in pigment production, the first level of activation is not used as inoculum for pigment production.

In line 113, the level of spores inoculated from the ISP2 medium to the other three culture media is not indicated. The authors should report in the manuscript the details of the levels of inoculated spores and the method of obtaining them

We appreciate your kind remarks. Accordingly, this sentence was inserted to clarify the information: “A subset of 20 pigmented isolates was reactivated on semi-solid isolation medium across four different culture media (see Table 1). For this, 100 µL of spore suspension (pH 7) was massively seeded onto the media and incubated at 30°C for 7 days.”

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Authors should report in the manuscript (lines 118 - 120) the volumetric capacities of the flasks for the liquid culture and the amount of medium used. This parameter is very important to know the oxygenation state of the cultures because the agitations indicated in the manuscript are relatively low.

Thaks a lot for your kind comment. Consequently, this passage was added to complete this missing information: “a 1-cm² section was cut from each solid medium and transferred to 3 mL of the corresponding liquid medium in 15 x 1 cm glass test tubes. These cultures were then incubated at 30°C for 7 days with agitation at 150 rpm (New Brunswick Scientific, Connecticut, USA). This culture (1 mL) was subsequently mixed with fresh medium (9 mL) and incubated under the same conditions (30°C, 150 rpm) in 50-mL glass Erlenmeyer flasks for 7 days. Finally, the 10-mL culture was transferred to 90 mL of fresh medium in 500 mL Erlenmeyer flasks and incubated for an additional 7 days at 150 rpm and 30°C.”

11 & 12

In section 2.4, it is suggested that the term ‘preparation’ be changed to obtention, or some other term, to indicate that the extracts were recovered from the cultures and to avoid misinterpretation.

It is strongly suggested to replace the term fermentation with culture in the different sections of the manuscript. This term is commonly used when the cultivation process is by fermentation (from a metabolic perspective).

Thank you for your kind suggestions. We accepted them and, therefore, these terms were changed as suggested.

13

The authors indicate in lines 124 - 125 that two phases (supernatant and/or sediment) were obtained in the extracts. The authors should report in the manuscript the treatment of the pigment-containing phases for the isolates that produced intra- and extracellular pigments, as this is not reported in the manuscript, i.e., were the phases treated independently, or were they mixed before or after lyophilization?

We appreciate so much this remark. We agree to add information to avoid confusing passages since the relevant phase (i.e., supernatant, sediment, or both) was selected according to the pigmentation and extracted independently without mixing. In this regard, this sentence was added for clarity: “Depending on whether the pigment was intracellular or secreted into the medium, the relevant phase (supernatant and/or sediment) containing the pigment was independently lyophilized for 24 hours”.

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In section 2.5.1. Lines 134 - 135, it is stated that the extracts were evaluated at different concentrations (5 to 5000 ppm). To avoid confusion, the authors should indicate from which type of extract these concentrations were prepared, e.g., were they from the freeze-dried extracts or the ethanolic extract? The same observation is made for section 2.5.2, line 148 where it is indicated that a solution was prepared at 30 mg/mL

Thanks a lot for this observation related to a missing information. Accordingly, the word “ethanolic” was included in sections 2.5.1 y 2.5.2.

15

It is suggested to use the term OD (optical density) instead of absorbance, throughout the manuscript

Thank you for this relevant comment. Consequently, Absorbance was changed by “optical density” or “OD” throughout the manuscript.

16

In section 2.5.2, lines 150 - 151, indicate that discs were used to impregnate the pigmented extract solutions. Authors should report disc sizes and disc material in the manuscript to be able to properly interpret these assays when using different volumes of extracts (e.g., 5 microlitres).

Thank you for this kind observation. Accordingly, the sentence was changed as: “The 1-cm diameter cellulose discs were impregnated with 5, 15, and 30 µL of the pigment solutions to examine the variation of bioactivity depending on pigment amounts in the range of 150 µg to 900 µg”

17

In line 155, the respective antibiotics used for each microorganism as a positive control should be specified in the manuscript.

Thank you very much for your kind suggestion. This sentence was then inserted for clarity: “ciprofloxacin (5 µg) against K. pneumoniae and E. coli, gentamycin (10 µg) against B. subtilis, and vancomycin (30 µg) against S. aureus and S. epidermidis”.

18

The authors present the discussion of their results, as well as an overview of their findings from antecedent information; however, it is highly suggestive that in the different subsections of the results they present their research findings first (results) and then discuss their results, so that the main focus of their manuscript is their contributions.

Thank you very much for this comment, which allows us to improve our manuscript. Therefore, we agree to reorganize the R&D section by presenting the results first, followed by the respective discussion for each subsection.

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The focus of the manuscript is directed towards the exploration of biological activities of pigmented extracts of actinomycetes, and they employ colorimetric methods for these determinations; however, the authors do not present results of scanning scans in the visible spectrum. Such scans are commonly reported for microbial pigments. Thus, interference by the color of the extract in the colorimetric determinations can be accounted for, if any. It is strongly recommended that the authors carry out these scanning scans, analyze and report them in the manuscript, and if necessary, reinterpret their results.

Thanks a lot for this kind and valuable comment. Effectively, we recorded scanning scan results in the visible spectrum (400-800 nm) for the pigmented extracts and we found that none of the pigmented extracts, even at the highest concentrations, showed absorption at test wavelengths (i.e., 525 to 734 nm). This clarification about the non-absorption in the wavelength range of the colorimetric-based assays was added in the manuscript. Additionally, we also cordially clarify to the reviewer that we did not compile this information for this study since we are planning a future publication that will involve detailed pigment characterization, including scanning scans in the visible spectrum. Thus, considering that these scans are not necessary for the biological assays presented in this paper, with the pertinent clarification (vide supra), they will form a critical part of our ongoing research into the broader applications of these extracts, and we do not want to report previous information. We hope that the reviewer empathizes with our perspective as the authors.

20 & 21

The authors indicate in section 2.3, that some isolates did not grow on some culture media and were re-evaluated in liquid culture. It is recommended that these findings be reported to establish the relationship of the different isolates in each culture medium.

Line 246 presents Table 1. Pigmentation produced by the strain in the different culture media. The authors should indicate for the results in this table, whether the apparent colours presented correspond to the extracts obtained in agar or liquid culture media

Thank you for these comments. We agree with them, and, accordingly, the results were added to Table 2 and, therefore, its caption was modified: “Pigmentation produced by the strains in the different culture media ((B) = biomass, (L) = liquid media or (–) = no pigmentation)”

22 & 23

Section 3.1., lines 246, revise the numbering of the tables in the manuscript. Table 1 was previously presented (line 106).

In Fig. 2, lines 302 - 307, the results of antioxidant activity are presented, and in the figure legend reference is made to table 4; however, in the manuscript, no such table is presented. Review the information presented and correct it if necessary, or annex the table indicated. Also, indicate the type of analysis used to present the results (e.g. heat map).

Thanks a lot for your kind comment. We performed a detailed scrutiny of the table numbering. Hence, we found some discrepancies and they were accordingly revised. In addition, the sentence was changed “Table 4” by “Table 2”. In the title of Fig. 2 was added “Heat map of antioxidant activity…”.

24

In the results section, both for the pigment production in the different media and for the evaluation of the biological activities, the authors should expand the discussion of other investigations that have presented similar results, so that possible relationships with other studies can be established and their results can be compared.

Thanks a lot for your kind suggestion. We agree with it, and, consequently, we added several brief passages to expand discussion with previous results.

25 & 26

In lines 308 - 309, section 3.3.1, the authors indicate that the higher the concentration of the extract, the higher its antioxidant capacity. Is there a possibility of interference by the color of the extract in these colorimetric assays, and can the authors demonstrate that such results are due to the activity of the extract, and not interference by the color of the extract?

The comment immediately above applies to the evaluations of cytotoxic activity (section 3.3.3) lines 406 - 407. Did the authors try to measure the OD of the pigmented extracts in each treatment to establish the pigment concentration behaviour?

We appreciate this comment. These assays involve discoloration of radical solutions or , comparing a blank (without an antioxidant agent), where the color is maximally produced by the presence of the radical. The more active the extract or antioxidant agent, the greater the discoloration, as the radical is reduced or disappears, either prepared in situ or in advance. In the case of cytotoxic assay, the formation of formazan from MTT depends on the cell viability after incubation with pigmented extracts. Given the pigmentation of the extracts, solutions of each extract at the final concentration were scanned at 515, 570, 600 and 734 nm to assess potential interferences. None of the pigments, even at the highest concentrations, showed absorption at these wavelengths. This information was added in the manuscript.

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The authors indicate that they have used different concentrations of pigmented extracts to evaluate the antimicrobial activity; however, in the manuscript, they do not indicate in their results which of the tested concentrations show antimicrobial activity for the strain. This is of utmost importance to show the antimicrobial potential of the pigmented extracts. The authors should include this result in their manuscript

Thanks a lot for your kind comment. In agreement with it, in the R&D section, the minimum tested concentrations of extract, at which antibacterial activity was detected, were added in the respective passages.

28

In Fig. 3, lines 422 - 427, the results of the cytotoxicity evaluation are presented, and the % viability is presented; however, it is not indicated how this determination was carried out; the authors should present this data for a better understanding of their results.

We appreciate this kind remark. Accordingly, the following sentence in the cytotoxic activity procedure was inserted: “The cell viability percentage (CVP) was calculated by using the mean OD of four wells in the indicated groups, using the formula: CVP = (OD_treatment − DO_blank)/(DO_control − DO_blank) × 100%.”. In addition, the Figure 3 includes a modified caption: “For more information, the mean data with standard deviation (n = 3) are presented in supplementary material (Tables S3, S4, and S5)”.

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In section 3.4, line 430, the authors state that ‘the most promising pigmented extracts’, what do they mean by this term, i.e., on what basis do they establish a promising pigment? It is strongly suggested that the authors make this term clear to ensure the objectivity of their work

Thank you very much for your kind suggestion. We agree with it. Therefore, the sentence was modified: “The most promising pigmented extracts (i.e., those presented one or more bioactivities with high levels among the extracts evaluated) were analyzed by HPLC-MS to chemically characterize such extracts and annotate those detected compounds”.

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In section 3.4, the authors have identified the compounds present in the extracts. In this regard and because the focus of the work is on the pigments of their Streptomyces strains, it is suggested that the authors put a focus on the pigments found in each isolate, reporting their summary data to show the presence of known pigments in the extracts obtained

Thanks a lot for your kind suggestion. We totally agree with it. Accordingly, we added the Table S25, which gathers the pigment-related compounds among the entire set of the LC-MS-based annotated metabolites. In addition, we added a brief discussion, highlighting the presence of anthraquinones and phenazines in the bioactive extracts (lines 630-666).

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Check-in Fig.2, in the legend, the error is marked... (line 306 - 307).

Thanks for this remark. In the legend of Figure 2., the number of Table 4 were changed to Table 2.

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To give relevance to the isolation of actinobacteria from underexplored environments, it is recommended that the authors extend the information regarding the sites where the microorganisms were isolated in the introduction section. An attempt can be made to report on the characteristics of the isolation sites, characteristics of the region, environmental conditions, etc.

Thank you very much for this relevant observation. Thus, in the introduction section, this sentence was added: “In the present study, two rivers located in tropical zones were selected as sources of actinomycetes. These rivers have average temperatures ranging from 25 °C to 30 °C and receive substantial solar radiation throughout the year, supporting rich biodiversity and a variety of ecosystems. However, little is known about the biodiversity in these watersheds, particularly their microbiota”.

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In the introductory section, little is addressed about the bioactive activities of pigments or pigmented extracts from microorganisms, especially actinomycetes. Since this is the focus of the manuscript, the authors should delve deeper into this topic, to establish an overview of previous studies in this regard.

Thanks a lot for your kind comment. We agree with it. Consequently, this paragraph was inserted: “The bioactive properties of pigments derived from actinomycetes have attracted significant attention due to their potential applications in medicine and industry. These pigments exhibit a wide range of activities, including antimicrobial, antioxidant, and cytotoxic effects, making them valuable for various uses. For instance, pigments from actinomycetes like Gordonia terrae have demonstrated strong antimicrobial activity against Gram-positive bacteria and fungi, including Bacillus subtilis and Candida albicans [34]. Marine actinomycetes strains VES 01 and VES 04 also exhibited notable antibacterial effects against Escherichia coli and B. subtilis, with significant cytotoxicity observed in the Brine Shrimp Lethality Test [35]. Additionally, pigment extracts from G. terrae and marine actinomycetes have shown antioxidant activity, with IC₅₀ values supporting their potential as natural antioxidants [34]. Studies on Streptomyces strains further revealed their ability to produce bioactive pigments with anticancer and antioxidant properties, underscoring their biotechnological significance [36]. Beyond scientific applications, actinomycete pigments have also been explored in non-scientific fields, such as painting, highlighting their versatility [37]. Their potential for eco-friendly applications, including fabric dyeing, has also been demonstrated [38]. While the bioactive properties of actinomycete pigments are promising, further research is required to fully elucidate their mechanisms of action and to optimize their production for commercial use”

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La conclusión del manuscrito parece resumir los hallazgos y se presenta en una extensión relativamente grande que podría ajustarse para mayor claridad y presentación. Se recomienda que los autores modifiquen y acoten su conclusión centrándose en los hallazgos de su investigación y en la importancia de su contribución a la comunidad científica, específicamente en el campo de los pigmentos microbianos.

We agree with this comment, hence, in general, conclusion organization was modified to shorten and provide a more concise section.

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Las bibliografías podrían actualizarse para presentar un mayor porcentaje de citas de los últimos cinco años.

Thank you for this remark. We added several recent references from the last five years as suggested. However, the used references were retained since they are the proper sources to support our manuscript structure and findings.

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Peer Reviewer comment

Author response

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The study reveals important findings about the potential of Colombian Actinobacteria as a source of bioactive pigments. The authors used robust methodologies, such as 16S rRNA gene sequencing for isolate identification and LC-MS for chemical composition analysis. The pigmented extracts obtained from Streptomyces bacteria had antioxidant, antibacterial, and cytotoxic properties, making them significant for potential practical applications in industries like pharmaceuticals, cosmetics, and food as multitasking compounds.

The authors provided sufficient background in the introduction. The methodology was adequately described, which allows for the reproduction of research. The results and discussion were prepared in a clear and concise way, but below I provide my comments and suggestions on the submitted paper

We sincerely appreciate the reviewer’s thoughtful and detailed evaluation of our study, as well as the positive and encouraging feedback. We have carefully addressed all the relevant suggestions and made every effort to improve the manuscript, leading to a significantly enhanced version of our work.

1

There are two Tables 1, i.e., in the materials and methods section and in the results and discussion part.

Thanks a lot for your kind remark. This mistake was corrected in the manuscript.

2

How were the colors for the Table of produced dyes selected? Was it based on visual (human eye) evaluation or using the CIELab color measurements?

We appreciate so much this kind remark. We cordially clarify that pigmentation was assessed by measuring the color coordinates of each culture (solid or liquid) at three random points on the surface of the liquid media or biomass powder placed in a petri dish. This information was added in the M6M section (lines 149-156). The results were added in Table 2 to provide elements for reproducibility of the pigmented cultures.

3

I wonder why there are 20 isolates in Table 1, but only 18 strains are identified by molecular analysis for strains with pigmentation (Table 2; isolates 263 and 247 were omitted).

Thank you very much for this observation. You are correct; we have been unable to isolate high-quality DNA from strains 247 and 263 to successfully amplify the 16S rRNA gene. As a result, we have not yet obtained sequences that would allow us to accurately identify or closely approximate their identification. While we have classified these strains morphologically as Streptomyces, we still need to complete their molecular sequencing in future studies. This clarification was added in the manuscript.

4

Tables S1-S5 – units for antioxidant or cytotoxic properties should be provided.

Thank you very much for this remark. The units for antioxidant and cytotoxic properties were provided in the title of tables.

5

The conclusion should be rephrased. In my humble opinion, it is too long.

We agree with this comment, hence, in general, conclusion organization was modified to shorten and provide a more concise section.

6

Lines 306-307 – it should be corrected. Line 362 – the same

Thank you for your kind observation. Thus, the number of Table was corrected in these lines.