Evaluation of antimicrobial and antiproliferative activities of Actinobacteria isolated from the saline lagoons of northwestern Peru

Rene Flores Clavo; Nataly Ruiz Quiñones; Álvaro Jose Hernández-Tasco; Marcos José Salvador; Ana Lúcia Tasca Gois Ruiz; Lúcia Elaine de Oliveira Braga; Jonas Henrique Costa; Taícia Pacheco Fill; Zhandra Lizeth Arce Gil; Luis Miguel Serquen Lopez; Fabiana Fantinatti Garboggini

doi:10.1371/journal.pone.0240946

. 2021 Sep 8;16(9):e0240946. doi: 10.1371/journal.pone.0240946

Evaluation of antimicrobial and antiproliferative activities of Actinobacteria isolated from the saline lagoons of northwestern Peru

Rene Flores Clavo ^1,^2,^3,^*, Nataly Ruiz Quiñones ^2,³, Álvaro Jose Hernández-Tasco ⁴, Marcos José Salvador ⁴, Ana Lúcia Tasca Gois Ruiz ⁵, Lúcia Elaine de Oliveira Braga ⁶, Jonas Henrique Costa ⁷, Taícia Pacheco Fill ⁷, Zhandra Lizeth Arce Gil ⁸, Luis Miguel Serquen Lopez ^3,⁹, Fabiana Fantinatti Garboggini ²

Editor: Luis Angel Maldonado Manjarrez¹⁰

PMCID: PMC8425546 PMID: 34495972

Abstract

Extreme environments Morrope and Bayovar Salt lagoons, several ecosystems and microhabitats remain unexplored, and little is known about the diversity of Actinobacteria. We suggest that the endemic bacteria present in this extreme environment is a source of active molecules with anticancer, antimicrobial, and antiparasitic properties. Using phenotypic and genotypic characterization techniques, including 16S rRNA sequencing, we identified these bacteria as members of the genera Streptomyces, Pseudonocardia, Staphylococcus, Bacillus, and Pseudomonas. Actinobacteria strains were found predominantly. Phylogenetic analysis revealed 13 Actinobacteria clusters of Streptomyces, the main genus. Three Streptomycetes, strains MW562814, MW562805, and MW562807 showed antiproliferative activities against three tumor cell lines: U251 glioma, MCF7 breast, and NCI-H460 lung (non-small cell type); and antibacterial activity against Staphylococcus aureus ATCC 6538, Escherichia coli ATCC 10536, and the multidrug resistant Acinetobacter baumannii AC-972. The antiproliferative activities (measured as total growth inhibition [TGI]) of Streptomyces sp. MW562807 were 0.57 μg/mL, for 0.61 μg/mL, and 0.80 μg/mL for glioma, lung non-small cell type, and breast cancer cell lines, respectively; the methanolic fraction of the crude extract showed a better antiproliferative activity and could inhibit the growth of (U251 (TGI = 38.3 μg/mL), OVCAR-03 (TGI = 62.1 μg/mL), and K562 (TGI = 81.5 μg/mL)) of nine tumor cells types and one nontumor cell type. Extreme enviroments, such as the Morrope and Bayovar Salt saloons are promising sources of new bacteria, whose compounds may be useful for treating various infectious diseases or even some types of cancer.

Introduction

Several regions in Peru have extreme environments, such as the salt marshes located on the coast, center, and south of the country. These regions, have several unexplored ecosystems and microhabitats, and consequently limited reports on the diversity of bacteria and other organisms [1–3]. Among microorganisms, members of the phylum Actinobacteria can be found in all types of extreme environments. Micromonospora, Actinomadura, and Nocardiopsis have been reported from saline soils of ephemeral salty lakes in Buryatiya [4], whereas Streptomyces, Nocardiopsis, and Nocardioides have been isolated from the Western Ghats region of India [5]. Micromonospora, Streptomyces, Salinispora, and Dietzia have been isolated from the coastal zone in Chile [6]. Additionally, halophilic and halotolerant strains of Actinobacteria show heterogeneous physiological characteristics for different genera because these bacteria can synthesize secondary metabolites to cope with the high salinity and extreme temperature conditions of their environments [7–9]. These extreme conditions favor the development of metabolic competitiveness to the production enzyme, which can bacterial population adapt to high salinity [10]. Furthermore, these microorganisms can perform essential processes, such as carbon cycle, metal transfer, and the removal of organic pollutants at higher trophic levels [11, 12]. Interestingly, this microbial group presents a unique ability to produce new products, mainly antibiotics [13, 14]. However, information on substances isolated from microorganisms inhabiting saline environments is scarce.

The search for these microorganisms has been mainly associated with the production of antibiotics and antitumor substances [15]. Approximately 22,500 biologically active substances are obtained from microorganisms, 45% of which are represented by Actinomycetes, and 70% are Streptomyces-derived [16, 17]. According to Lam (2006), new Actinobacteria from these unexplored habitats could serve as the source of new bioactive secondary metabolites [18].

The bio-guided study strategy helped us identify several different microorganisms with biotechnological potential [19]. In this study, we performed a phylogenetic analysis of a collection of bacterial isolates from the saline lagoons of northwestern Peru. Furthermore, we explored their potential as producers of secondary metabolites with antimicrobial and antiproliferative activities. we have revealed that the Actinobacteria existing in these extreme environmental conditions have diverse characteristics and could comprise new species that produce novel and biologically active compounds.

Materials and methods

Site description and sampling

Samples were collected from zone “1”, zone “2”, and zone “3” of Morrope saline lagoons in December 2012, January 2013, December 2014, respectively; and zone “4”) at Bayovar saline lagoons in March 2015 (Fig 1). This study did not require permission from a competent authority since it excludes any protected species; besides, documentation is currently being processed to achieve its registration. The sampling areas were the lagoons, and sampling was performed based on the Nagoya Protocol.

Fig 1 — Sampling sites: a, b, c = lagoons 1, 2, and 3 (State Morrope); and d = lagoon 4 (State Bayovar).

Table 1 indicates the sources and locations from which samples were obtained. Samples were collected aseptically, placed in sterile plastic bags, and refrigerated at 4°C for further processing in the Bacteriology Laboratory, Regional Hospital of Lambayeque. A flowchart depicting the methodological strategy adopted in this study is shown (S1 Fig).

Table 1. Data related to saline lagoon samples.

Morrope lagoons
Collection zone	Sample type	Geographic location
Zone 1 (December 2012)	A; AES	6°10S;80°35W (3 samples × triplicate)	6°11S;80°37W (3 samples × triplicate)	6°9S;80°39W (3 samples × triplicate)
Zone 2 (January 2013)	AES; SE	6°19S;80°28W (3 samples × triplicate)	6°17S;80°26W (3 samples × triplicate)	6°18S;80°27W (3 samples × triplicate)
Zone 3 (December 2014)	A; SE	6°08S;80°50W (3 samples × triplicate)	6°08S;80°51W (3 samples × triplicate)	6°08S;80°40W (3 samples × triplicate)
Bayovar lagoons
Zone 4 (March 2015)	A; AES; SE	6°23S;80°66W (3 samples × triplicate)	6°25S;80°26W (3 samples × triplicate)	6°23S;80°78W (3 samples × triplicate)

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A = saline water, AES = water and sediment, SE = sediment.

Isolation of cultivable Actinobacteria

A 10.0 mL aliquot of saline lagoon sample was transferred to an Erlenmeyer flask containing 10.0 mL of salt broth (0.6% (w/v) yeast extract Difco, 2% (w/v) glucose Merck, 5% (w/v) peptone Merck, 3% (w/v) meat extract Difco supplemented with chloramphenicol Medrock, and 1% (w/v) fluconazole Genfar at pH 7.0). Erlenmeyer flasks were homogenized and maintained at 50°C in a water bath for 60 min to reduce the pollutant load [20, 21], as described in a previously reported protocol [22]. The isolates were reactivated and stored in a refrigerated chamber at -20°C for transporting to the Microbial Resources Division Laboratory of the Pluridisciplinary Center for Chemical, Biological and Agricultural Research (CPQBA) in August 2015.

Identification of bacterial isolates

Reactivation and morphological phenotypic characterization of bacterial strains

The reactivation of the isolates bacterial was performed as previously described [22], the isolates from the Culture Collection of Research Center and Innovation and Sciences Actives Multidisciplinary (CIICAM), Department of Biotechnology, Chiclayo, Lambayeque, Perú; these were labeled with location codes according to the salt saloons from which they were collected, for example (B-81), which refers to the Bayovar saline saloon sample 8 culture 1.

DNA extraction and 16S rRNA gene sequencing

An isolated colony was used for genomic DNA, according to the method described by Pospiech and Neuman [23], with some modifications according to a previously published protocol [24]. The Amplification of the 16S rRNA gene was performed via polymerase chain reaction (PCR). The specifications of this protocol have been previously described [25]. The samples were purified using minicolumns GFX PCR DNA & gel band purification kit (GE Healthcare Bio-Sciences AB Uppsala, Sweden), according to a previously described protocol [26], and sequenced using an ABI3500XL Series automatic sequencer (Applied Biosystems Foster City, California, USA) in the Laboratory of the Division of Microbial Resources, Chemical, Biological and Agricultural Pluridisciplinary Research Center (CPQBA), University of Campinas (UNICAMP), Paulínia, Sao Paulo, Brazil). Sequencing reactions were performed using the Big Dye Terminator Cycle Sequencing Ready Reaction Kit (Applied Biosystems) according to the manufacturer’s modified by the authors protocol [27]. Partial sequences of the 16S ribosomal RNA gene obtained from each isolate were assembled into a contig using BioEdit 7.0 [28]. The sequences of organisms were added to the EZBioCloud 16S Database (https://www.ezbiocloud.net/)) using the “Identify” service [29], and species assignment was based on the closest hits [30]. 16S rRNA gene sequences retrieved from the database and related to the unknown organism gene were selected for alignment in the Clustal X program [31], and phylogenetic analyses were performed using the MEGA version 7.0 program [32]. The evolutionary distance matrix was calculated using the Kimura-2 model parameters [33], and the phylogenetic tree was constructed from the evolutionary distances calculated by the neighbor-joining method [34], with bootstrap values based on 1000 resamples.

Therapeutic potential of bacterial isolates

Crude extract produced from extremophilic bacteria

The evaluation of the secondary metabolites of bacterial isolates extracted with ethyl acetate was performed in three culture media—R2A broth (Himedia ref. 1687), ISP2 broth (Difco ref 277010), and nutrient broth (Termofisher Scientific ref CM0001)—as described in a previously published protocol [35]. The crude extracts of three representative strains were used for antimicrobial and antiproliferative activity tests. Simultaneously, the crude extracts were analyzed by ultra-high pressure liquid chromatography-mass spectrometry (UHPLC-MS) in a Thermo Scientific QExactive® Hybrid Quadrupole-Orbitrap Mass Spectrometer.

Fractionation of the crude extract from extremophilic bacteria

The crude extracts from the isolates were fractionated using a previously described protocol [35], and the fractions were tested for their antibacterial and antiproliferative activities.

In vitro antibacterial activity assay

Three crude extracts from Streptomyces sp. MW562814, MW562807, and MW562805 were tested for antimicrobial activity using the minimum inhibitory concentration (MIC) assay, following the protocol reported by Siddharth and Vittal [36]. The crude extracts were partially diluted in 1% dimethyl sulfoxide (DMSO; 0,39mg/mL–1 mg/mL), and the microbial cultures were grown in sterile broth to obtain a total volume of 200 μL. The 96- well plate was incubated at 37°C (room temperature); the lowest concentration of the extract, that completely inhibited bacterial growth was considered the MIC. Each biological assay was performed in triplicate. The pathogenic bacteria used in this test were Escherichia coli ATCC 10536, Staphylococcus aureus ATCC 6538, and Acinetobacter baumannii AC-972. The source strain was obtained from the bank of multidrug-resistant (MDR) isolates from a patient with pneumonia from the Intensive Care Unit (ICU) of the Hospital Regional Lambayeque. Patient data linked to the samples were anonymized for access.

In vitro antiproliferative activity assay

This assay aimed detected anticancer activities by evaluating antiproliferative activity against human tumor cells [37]. In vitro tests assessing the effects of the crude extract of Streptomyces sp. MW562805, MW562807, and MW562814 on human tumor cell lines of different origins and a non-tumor cell line were performed. Human tumor cell lines [U251 (glioblastoma), UACC-62 (melanoma), MCF-7 (breast, adenocarcinoma), NCI-ADR/RES (multi-drug resistant ovarian adenocarcinoma), 786–0 (renal, adenocarcinoma), NCI-H460 (lung, non-small cell carcinoma), PC-3 (prostate, adenocarcinoma), OVCAR-03 (ovarian, adenocarcinoma), and K562 (chronic myeloid leukemia)] were kindly donated by Frederick Cancer Research & Development Center, National Cancer Institute, Frederick, MA, USA. One immortalized cell line (HaCaT, human keratinocyte) was kindly donated by Dr. Ricardo Della Coletta (University of Campinas). Stock cultures were grown in a medium containing 5 mL of RPMI 1640 (GIBCO BRL, Gaithers-Burg, MD, USA) supplemented with 5% (v/v) fetal bovine serum (GIBCO) at 37°C and 5% (v/v) CO₂. Penicillin:streptomycin (1000 μg·L⁻¹:1000 U·L⁻¹, 1 mL·L⁻¹, Vitrocell, Campinas, SP, Brazil) was added to the experimental cultures. Cells in 96-well plates (100 μL cells well⁻¹) were exposed to the extracts in DMSO (Sigma-Aldrich)/RPMI (0.25, 2.5, 25, and 250 μg·mL−1) at 37°C and 5% (v/v) CO₂ for 48 h. The DMSO final concentration of 0.2% (w/v) did not affect cell viability. Before (T₀) and after (T₁) sample application, cells were fixed with 50% (w/v) trichloroacetic acid (Merck), and cell proliferation was determined by the spectrophotometric quantification (540 nm) of cellular protein content using the sulforhodamine B assay. Verified in the concentration-response curve for each cell line, the values of the sample concentration required to produce total growth inhibition (TGI) or cytostatic effect through non-linear regression analysis using ORIGIN 8.6® (OriginLab Corporation, Northampton, MA, USA).

Mass spectrometry analysis

Streptomyces sp. MW562807 extract was resuspended in 1 mL of methanol (HPLC grade), and 100 μL of this suspension was diluted in 900 μL of methanol to a final concentration of 6.4 mg/mL. (UHPLC-MS) analyses were performed using a Thermo Scientific QExactive® Hybrid Quadrupole-Orbitrap Mass Spectrometer according to a previously published protocol [38].

Molecular MS/MS network

A molecular network for Streptomyces sp. MW562807 was constructed using the online workflow at Global Natural Products Social Molecular Networking (GNPS) (https://gnps.ucsd.edu/). The library spectra were filtered in the same manner as the input data. All matches maintained between network and library spectra were required to have a score above 0.5 and at least five matched peaks [39]. These procedures were performed using a previously described protocol [38].

Statistical analyses

The data are expressed as the mean ± standard error of the mean (SEM). Statistical comparisons were performed using a one-way ANOVA followed by the Student–Newman–Keuls test, and the differences were considered statistically significant when P < 0.05 and were determined through non-linear regression analysis using ORIGIN 8.6® (OriginLab Corporation, Northampton, MA, USA).

Results and discussion

Isolation, identification, and selection of bacterial species from the northern saline lagoons of Peru

In total, 50 pure cultures showing different colony morphologies were obtained, which were grown on R2A medium containing saline water (A) only, water and sediment (AES), and sediment (SE) only (Table 2). Isolates were clustered into 42 filamentous and 8 non-filamentous bacterial groups with similar characteristics based on morphological characteristics, including the aerial mycelium, morphological spore mass color, pigmentation of vegetative or substrate mycelium, and the production of diffusible pigment [40].

Table 2. 16S rRNA gene-based identification of bacteria isolated from lagoons Morrope and Bayovar.

Sample type	Number of isolates (%)	Phylum	Family	Genus
AES, SE	39 (78%)	Actinobacteria	Streptomycetaceae	Streptomyces
SE	3 (6%)	Actinobacteria	Pseudonocardiaceae	Pseudonocardia
A	2 (4%)	Firmicutes	Bacillaceae	Bacillus
A	4 (8%)	Firmicutes	Staphylococcaceae	Staphylococcus
AES	2 (4%)	Proteobacteria	Pseudomonadaceae	Pseudomonas

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The isolates were regrouped into their similar phenotypic characteristics and identified based on the sequencing and alignment from the 16S rRNA gene analyses. In total, 13 bacterial isolates from the saline lagoons of northwestern Peru (Morrope and Bayovar) cultivars were identified after the 16S rRNA gene sequencing (Fig 2).

Fig 2 — Numbers at branching points refer to percentages of bootstrap values from 1000 replicates. Bar, 0.02 substitutions per nucleotide position was used. *Bacillus cereus* ATCC 14579^T (AE016877) was the outgroup.

Therefore, these strains should be subjected to further taxonomic and analytical chemistry analyses to confirm their novelty at the species level and as a source of novel chemical entities.

According to EZBioCloud, 13 isolates were identified in the Morrope and Bayovar salt saloons, of wich eight and five groups of Streptomyces were identified in the Morrope salt flats only and the Bayovar salt flats, respectively. Three isolates were similar to S. olivaceus NRRL B-3009: MW562807 (99.93%), MW562805 (99.84%) (Fig 2), and MW562808 (97.98%) MW562806 was similar to S. hyderabadensis OU-40 (98.06). Addionally, MW562809 presented higher similarity with S. pactum NBRC 13433 (97.98%); MW562810 and MW562811 presented high similarity to S. griseorubens NBRC 12780 and S. labedae NBRC 15864 with 97.57% and 97.58% similarity, respectively. MW562812 and MW562813 were similar to S. griseoincarnatus LMG 19316 (97.58%), and S. variabilis NBRC 12825 (97.58%), respectively; the latter is another actinobacterium recovered from the Morrope salt saloons. The isolates MW562814, MW562815, MW562816, and MW562817 exhibited similarity with S. luteus TRM 45540 (99.16%) (Fig 2), S. violascens ISP 5183 (97.37%), S. hydrogenans NBRC 13475 (97.27%), and Pseudonocardia antimicrobica YIM 63235 (98.41%), respectively. This study is the first to report this genus of Streptomyces present in the Bayovar and Morrope saline lagoons. Due to the physicochemical characteristics of the environment, they are considered extremophilic bacteria; however, a different genus such as Pseudonocardia, has been reported by Zhang et al. (2016) as producers of γ-butyrolactone molecules present in Streptomyces genera [41]. Our culture-based approach using pre-enrichment to decrease the bacterial load corresponds to the reports described in literature, in which the isolation of halophilic bacteria reveals low species richness and dominance of the genus Streptomyces [42]. Ballav et al. (2015) reported Streptomyces especies as the most predominant group contributing to 46% of the total isolates in crystallizer pond sediments of Ribandar saltern in Goa, India [43]. In this study, isolation was performed using three types of culture media and four different concentrations of salt in the lagoons because the concentration of salts ranged from 0 to 300 psu. Cortés-Albayay et al. (2019) in Salar de Huasco reported similar results; they isolated Streptomyces (86%), Nocardiopsis, (9%), Micromonospora (3%), Bacillus (1%), and Pseudomonas (1%) [44].

Antibacterial activity screening

The MIC assay was used to screen antibacterial metabolite-producing strains against the indicator strains Escherichia coli ATCC 10536, Staphylococcus aureus ATCC 6538, and Acinetobacter baumannii AC-972 MDR. The crude extracts tested from the three Actinobacteria were effective against the pathogenic bacteria tested. The antimicrobial activity of the crude extract of Streptomyces sp. MW562807 crude extract exhibited the highest inhibitory activity with the lowest MIC (7.82 μg/mL) against the three pathogens, followed by Streptomyces sp. MW562814, which showed an MIC of 7.82 μg/mL against E. coli ATCC 10536 and S. aureus ATCC 6538 and MIC of 15.63 μg/mL against A. baumannii AC-972 MDR. MW562805 showed an MIC of 15.63 μg/mL against the pathogens tested. Notably, all crude extracts of the strains presented low MIC compared with the reference antibiotic (Table 3).

Table 3. Minimum inhibitory concentration of three Streptomyces sp. crude extracts, as determined by broth dilution method.

Isolates of Streptomyces sp.	Minimum inhibitory concentration (μg/mL)
	E. coli ATCC 10536	S. aureus ATCC 6538	A. baumannii AC-972 MDR	Antibiotic Chloramphenicol
MW562814	7.82	7.82	15.63	22.2
MW562805	15.63	15.63	15.63	23.1
MW562807	7.82	7.82	7.82	22.3

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Similar results were reported by Siddharth & Vidal (2018) [36] for a marine Streptomyces sp. S2A with an MIC of 31.25 μg/mL against Klebsiella pneumoniae, 15.62 μg/mL against Staphylococcus epidermidis, Staphylococcus aureus, Bacillus cereus, Escherichia coli, and Micrococcus luteus with 7.8 μg/mL of the Streptomyces sp. MW562807 showed an MIC of 7.82 μg/mL against Escherichia coli, Staphylococcus aureus and A. baumannii. The crude extract of Streptomyces sp. YBQ59 showed MICs ranging from 10.5 to 22.5 μg/mL, against nine pathogens, which were similar to those we obtained [45].

Antiproliferative activity screening

Among all isolated microorganisms, crude extracts could be prepared from 23 isolated microorganisms. These extracts were evaluated against a panel of three human tumor cell lines. Only the crude extracts of Streptomyces sp. MW562805, MW562807, and MW562814 showed moderate antiproliferative effects, indicating the concentration required to elicit a TGI (Table 4).

Table 4. Antiproliferative activity of three Streptomyces extracts against human tumor cell lines.

Crude extract and positive control	TGI (μg/mL) ^a
	U251 ^b	MCF-7 ^b	NCI-H460 ^b
Doxorubicin ^c	2.5 ± 2.2 (P)	6.11^* (P)	<0.025 (P)
Streptomyces sp. MW562814	5.1 ± 2.8 (P)	8.0 ± 3.6 (M)	9.3 ± 6.9 (M)
Streptomyces sp. MW562805	5.4 ± 4.0 (P)	8.0 ± 1.5 (M)	5.1 ± 4.0 (P)
Doxorubicin ^c	1.5 ± 1.2 (P)	>25 (W)	1.8 ± 1.1 (P)
Streptomyces sp. MW562807	0.57 ± 0.05 (P)	0.8 ± 0.2 (P)	0.6 ± 0.1 (P)

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a) Results expressed as concentration required to elicit total growth inhibition (TGI) in μg ml^-1 followed by standard error, calculated by sigmoidal regression using Origin 8.0 software; Results classified according to CSIR’s criteria: inactive (I, TGI ≥ 50 μg ml^-1), weak (W, 15 μg ml^-1 ≤ TGI < 50 μg ml^-1), moderate (M, 6.25 μg ml^-1 ≤ TGI < 15 μg ml^-1) or potent (P, TGI < 6.25 μg ml^-1) activity [46]

* Estimated TGI value = when experimental data did not converge (standard error higher than calculated effective concentration).

b) Human tumor cell lines: U251 (glioblastoma); MCF7 (breast, adenocarcinoma); NCI-H460 (lung, non-small cell carcinoma).

c) Doxorubicin (positive control, 0.025 to 25 μg ml^-1).

According to previous studies [46, 47], TGI values higher than 50 μg/mL were exhibited by inactive samples. The most active extract was Streptomyces sp. MW562807 with TGI ranging from 0.57 μg/mL (U251, glioblastoma) to 0.80 μg/mL (MCF-7, mammary adenocarcinoma) (Table 4).

Based on these results, Streptomyces sp. MW562807 strain was cultured in three media to evaluate the influence of nutrient conditions on the production of bioactive compounds. The nutrient broth growth medium (NA) yielded an less active extract, while ISP2 and R2A media yielded active extracts with different profiles (Table 5).

Table 5. Antiproliferative evaluation of isolate Streptomyces sp. MW562807 fermented in three different media and fractions of the R2A medium against nine tumors and one non-tumor cell line.

Cell lines ^b	TGI (μg/ml) ^a
	Dox ^c	MW562807 ^d			MW562807 R2A Fractions ^e
	Dox ^c	NB	ISP2	R2A	1	2	3	4	5	6
U251	0.04 ± 0.03 (P)	44.7 ± 29.7 (W)	14.1 ± 5.2 (M)	1.6 ± 1.5 (P)	^#	^#	38.3 ± 23.4 (W)	73.5 ± 36.6 (I)	^#	163.0 ± 109.6 (I)
UACC-62	0.8 ± 0.3 (P)	250 (I)	17.2 ± 5.1 (M)	21.3 ± 17.2 (W)	^#	^#	127.2 ± 21.5 (I)	89.4 ± 31.6 (I)	^#	^#
MCF-7	>25 (W)	^#	^#	8.0^* (M)	^#	^#	154.6 ± 53.4 (I)	184.5 ± 23.7 (I)	^#	^#
NCI-ADR/RES	>25 (W)	^#	155.9 ± 65.2 (I)	31.0 ± 10.6 (W)	^#	^#	189.8 ± 118.1 (I)	143.2 ± 43.1 (I)	^#	^#
786–0	0.2 ± 0.1 (P)	^#	^*	39.0 ± 35.1 (W)	^#	^#	172.6 ± 162.8 (I)	^#	^#	^#
NCI-H460	>25 (W)	^#	38.9 ± 11.0 (W)	11.2 ± 6.2 (M)	^#	^#	108.0 ± 16.0 (I)	235.1 ± 26.7 (I)	^#	^#
OVCAR-03	14.4 ± 6.8 (P)	^#	55.2 ± 18.0 (I)	19.4 ± 3.5 (W)	^#	^#	62.1 ± 30.0 (I)	81.6 ± 32.1 (I)	^#	^#
HT29	>25 (W)	^#	155.7 ± 147.4 (I)	89.7 ± 72.5 (I)	^#	^#	167.4 ± 33.5 (I)	195.2 ± 37.8 (I)	^#	^#
K562	6.3 ± 2.2 (P)	^#	^#	11.6 ± 7.3 (M)	^#	^#	81.5 ± 26.8 (I)	100.4 ± 78.3 (I)	^#	^#
HaCat	>25 (W)	^#	^#	>250 (I)	^#	^#	^#	^#	^#	^#

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* Estimated TGI value = when experimental data did not converge (standard error higher than calculated effective concentration)

# TGI > 250 μg ml^-1.

b) Human tumor cell lines: U251 (glioblastoma), UACC-62 (melanoma), MCF-7 (breast, adenocarcinoma), NCI-ADR/RES (multi-drug resistant ovarian adenocarcinoma); 786–0 (kidney, adenocarcinoma); NCI-H460 (lung, non-small cell carcinoma); OVCAR-3 (ovarian adenocarcinoma); HT29 (colon, adenocarcinoma); and K562 (chronic myeloid leukemia). Human non-tumor cell line: HaCaT (immortalized keratinocytes).

Samples

c) Dox = doxorubicin (positive control)

d) MW562807 = Streptomyces sp. crude extract in nutrient broth growth medium (NB), yeast extract-malt medium (ISP2), or R2A growth medium (R2A)

e) MW562807 R2A fractions: 1 = H20; 2 = MeOH-H₂O; 3 = MeOH; 4 = MeOH–EtOAc; 5 = 100% EtOAc; 6 = EtOAc-acid (MW562807 R2A crude extract submitted to vacuum chromatographic C18-column).

The crude extract of Streptomyces sp. MW562807 in the ISP2 medium moderately inhibited the proliferation of glioblastoma (U251, TGI = 14.1 μg/mL) and melanoma (UACC-62, TGI = 17.2 μg/mL) cell lines. In contrast, the crude extract of Streptomyces sp. MW562807 in the R2A medium potently inhibited glioblastoma cell growth (U251, TGI = 1.6 μg/mL) along with moderate anti-proliferative effects against lung carcinoma (NCI-H460, TGI = 11.2 μg/mL) and leukemia (K562, TGI = 11.6 μg/mL) cell lines. Despite the expectation of a fractionation-mediated increase in antiproliferative activity, fractions 1–6 showed lower antiproliferative activity. The best activity was observed for fraction 3, which weakly inhibited the proliferation of U251 (TGI = 38.3 μg/mL glioma) cells (Table 5). Besides synergism and antagonism, the combined effect of natural products has been extensively studied over the years. Many studies have demonstrated that the crude extract exhibits more (synergistic) or less (antagonistic) effects than less complex fractions or even the isolated compounds [48–50].

Due to its metabolic and genetic capacities, several secondary metabolites produced by Streptomyces species have shown anti-proliferative activity [51]. Furthermore, throughout the evolutionary history of aquatic organisms and as a response to the pressure of environmental selection, actinomycetes isolated from marine environments have a greater capacity to express secondary metabolites with unique chemical structures [18]. This pressure has generated high specificity and a complex three-dimensional conformation of the compounds to act in the marine environment [52]. In this particular case, the geographical isolation, geological conditions of the saline lagoon formation, and extreme environmental generate a greater environmental selection pressure, which enables the analysis of antibiotic and antiproliferative activity in the isolates.

The anticancer potential of halophilic actinomycetes has been extensively studied. Secondary metabolites Salternamides A-D isolated from halophilic actinomycetes of saline on the island of Shinui (Republic of Korea) have potential cytotoxicity against human colon cancer (HCT116) and gastric cancer (SNU638) cell lines [53]. Another moderately halophilic Streptomyces sp. Nov WH26 with the GenBank accession number JN187420.1, isolated from Weihai Solar Saltern in China, exhibits potent cytotoxicity against human cancer cell lines of the lung (A549, IC₅₀ 78.6 μM), cervical epithelium (HeLa, IC₅₀ 56.6 μM), liver cancer cell (BEL-7402, IC₅₀ 47.1 μM), and colon (HT-29, IC₅₀ 94.3 μM) [54]. Four compounds, shellmicin A-D isolated from the Streptomyces sp. shell-016 were described as cytotoxic agents against five human tumor cell lines: non-small cell lung cancer (H1299, ATCC CRL-5803), malignant melanoma (A375, ATCC CRL- 1619), hepatocellular carcinoma (HepG2, ATCC HB-8065), colorectal adenocarcinoma (HT29 ATCC HTB-38), and breast cancer (HCC1937, ATCC CRL-2336) [55]. Moreover, the compounds Shellmicin A, B, and D showed greater cytotoxicity (IC₅₀ ranging from 0.69 μM to 3.11 μM at 72 h) than Shellmicin C. Interestingly, shellmicin C and D are a pair of stereoisomers, and their biological activity differ considerably.

Two active substances Chromomycin SA and 1-(1H-indol-3-yl)-propane-1,2,3-triol, were isolated and identified from the actinomycete strain Streptomyces sp. KML-2 wich was isolated from a saline soil mine in Khewra, Pakistan, Both substances, could reduce Hela cell viability (human cervical cancer) in a similar magnitude (IC₅₀ 8.9 and 7.8 μg/mL, respectively) while MCF-7 cells (human breast cancer) were more sensitive to 1-(1H-indol-3-yl)-propane-1,2,3-triol (IC₅₀ 0.97 μg /mL [56]. This study also revealed that the Khewra salt mine from which the KML-2 strain was isolated is a powerful ecological niche with inimitable strain diversity that is yet to be discovered, a fact that reinforces our study approach in the search for new compounds with biotechnological activity.

Secondary metabolite analysis of Streptomyces sp. MW562807 isolate

The crude extract obtained from Streptomyces sp. MW562807 was analized by UHPLC-MS and demonstrated a broad biomolecule profile (S2 Fig). Furthermore, we screened the metabolites in the GNPS platform, and molecular networking revealed two clusters (A and B) that exhibited secondary metabolites produced by Streptomyces sp. MW562807 extract (pink) (Fig 3).

All compounds close to cholic acid are described in the pink circle (Fig 3), there are described as close compounds because they are grouped within the same cluster derived from the proximity of their specific masses and according to what it generates after comparing in the GNPS platform database. Thus, cluster B contains compounds called Loboforins with Loboforins A, B, E and K at number 2, 3, 4 and 5 with a specific masses of 1157.64, followed 1187.61, 1171.61, and 1173.63, respectively.

Metabolites were identified as a hit in the GNPS database or manually identified by accurate mass analyses, which showed mass errors below 5 ppm (Table 6).

Table 6. MS data obtained for secondary metabolites detected in Streptomyces sp. MW562807 extract.

Compound	Ion formula	Calculated m/z	Experimental m/z	Error (ppm)
Cholic Acid	C₂₄H₄₄NO₅	426.3219	426.3212	-1.6
Lobophorin A	C₆₁H₉₃N₂O₁₉	1157.6372	1157.6373	0.1
Lobophorin B	C₆₁H₉₁N₂O₂₁	1187.6114	1187.6108	-0.5
Lobophorin E	C₆₁H₉₁N₂O₂₀	1171.6165	1171.6160	-0.4
Lobophorin K	C₆₁H₉₃N₂O₂₀	1173.6322	1173.6321	-0.1
Compound 6	C₁₁H₁₇O₃	197.1177	197.1173	-2.0

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The observed signals corresponded to Cholic Acid (1), Lobophorin A (2), Lobophorin B (3), Lobophorin E (4), and Lobophorin K (5).

In cluster A, the GNPS database indicated the presence of cholic acid in Streptomyces sp. MW562807 extract (S3 and S4 Figs). In molecular networking, each MS/MS spectrum is represented by nodes, which are grouped into clusters based on their fragmentation pattern similarity. Thus, compounds of the same molecule class are grouped in the same cluster [39]. The Cholic Acid cluster is composed of various nodes, suggesting the presence of other analogs (Fig 4). Cholic acid and other bile acids have been reported as secondary metabolites of specific members of the genus Streptomyces and Myroides [57]. In the literature, Cholic Acid derivatives and bile acids are termed antimicrobial agents, displaying activity against Gram-positive and Gram-negative bacterial or improving the antimicrobial effect of antibiotics [58–61].

In cluster B, the GNPS database indicated the production of Lobophorin production by Streptomyces sp. MW562807 strain (S5 and S6 Figs), related to the loss of the sugar units in the structure, as revealed in literature [62]. Similar to cluster A, cluster B also revealed other Lobophorin compounds such as lobophorins B, E, and K, which were identified based on their accurate masses and fragmentation profiles with typical fragments at m/z 184.09, 157.09, 108.08, and 97.06 (S7–S9 Figs). Lobophorins were discovered by Jiang et al. (1999) in the expedition to Belize on board the Columbus research ship [63]. On the occasion, a new strain of actinomycete, named # CNC-837, was isolated from the surface of a brown algae from the Caribbean called Lobophora variegata, and was reported to produce lobophorins A and B, potent anti-inflammatory agents [63]. For the cytotoxic activity, Lobophorins A and E were inactive, while Lobophorins B and F were active against MCF-7 cells (human breast adenocarcinoma cell line), NCI-H460 (human non-small cell lung cancer cell line), and SF-268 (human glioma cell line) [64]. Further, Lobophorin C exhibited potent cytotoxic activity against human liver cancer cells, while Lobophorin D displayed a significant inhibitory effect on human breast cancer cells [65]. For the antibacterial activity, Lobophorins A, B, E, and F demonstrated activity against Bacillus thuringensis SCSIO BT01 [64], Lobophorin H exhibited antibacterial activity against Bacillus subtilis [66] and S. olivaceus SCSIO T05, a marine-derived strain, was previously reported to be isolated from the Indian Ocean deep-sea-derived sediment [67]. To explore the biosynthetic potential of this strain, metabolic engineering and genome mining were performed in S. olivaceus SCSIO T05, leading to the isolation and identification of one known compound, Lobophorin CR4 [68]. A compound of the Loboforin family, Lobophorin K promoted bacteriostatic effects against Staphylococcus aureus EPI1167 MSSA [69], Streptomyces sp. SCSIO 01127 was isolated from deep sea sediment in southern China and the phylogenetic tree generated by a neighbor-joining method clearly revealed that the evolutionary relationship had the greatest similarities to Streptomyces olivaceus NBRC 12805^T (AB249920) (100%) and Streptomyces pactum NBRC 13433^T (100%). Thus, this strain was designated as Streptomyces sp. SCSIO 01127 presented biological activities product of Lobophorins A, B, F and E against Bacillus thuringensis SCSIO BT01, S. aureus ATCC 29213, E. faecalis ATCC 29212 and three human tumor cell lines SF-268, MCF-7 and NCI—H460, thus the strain MW562807 that we present in this manuscript have active biological activities. [70].

Besides Cholic Acid and Lobophorins, a Furan-Type compound (6) was also detected in Streptomyces sp. MW562807 extract (S10 Fig). The structures of the metabolites 1–6 are shown in Fig 4.

Compound 6 exhibited fragments at m/z 168.07, 167.07, and 153.05, in agreement with the findings of a previous study [62]. Besides Lobophorin A, Compound 6 was reported as a secondary metabolite of the Streptomyces sp. VN1 strain, a microorganism isolated from sea sediment from the coastal region of Vietnam [62]. For the Compound 6 displayed by our data further investigation is needed to correlate the antimicrobial and cytotoxic activities, observed for Streptomyces sp. MW562807 extract, to Bile Acids, Lobophorins, and Furan Compounds. Additionally, extremophilic environments, such as Saloons of Bayovar, are prolific sources of microorganisms, including the identified unique Streptomyces sp. Due to the extreme conditions, these microorganisms produce different compounds to adapt to surviving in the environment, this makes Streptomyces sp. MW562807 an attractive source of bioactive compounds.

Conclusion

This study successfully determined the diversity and bioactive potential of the actinobacterial isolates sourced from locations with extreme environments (Morrope and Bayovar saloons) in northwestern Peru; these regions had not been previously explored for this type of study. Furthermore, the Actinobacteria isolates, namely, Streptomyces sp. MW562814, MW562805, and MW562807, could be new species; their importance lies in their antibacterial and antiproliferative potential, with Streptomyces sp. MW562807 demonstrating the most promising activity. Six biomolecules (Cholic Acid; Lobophorin A, B, E, and K; in addition to a Sixth Compound) were detected in this cultivable actinobacterial isolated from Bayovar’s saloons, but these have not yet been found in the GNPS curated database. In this study, we revealed that the Actinobacteria existing in these extreme environmental conditions have diverse characteristics, and could comprise new species that produce novel and biologically active compounds. The antibacterial and antiproliferative potential of these compounds renders the study of these saline lagoons of northwestern Peru valuable. In a Future work, the genome sequencing of these three Streptomyces species will be presented out to identify these molecules to purify and characterize them, as this can result in the economically beneficial production of bioactive compounds for future pharmaceutical applications.

Supporting information

S1 Fig. Flowchart depicting the methodological strategy adopted in this study.

(DOCX)

Click here for additional data file.^{(35.6KB, docx)}

S2 Fig

Total ion chromatogram (TIC) of UHPLC-MS analyses for (A) Streptomyces sp. MW562807 extract and (B) control.

(DOCX)

Click here for additional data file.^{(49KB, docx)}

S3 Fig

MS/MS match between GNPS database (green) and Cholic Acid (1) from Streptomyces sp. MW562807 extract (black).

(DOCX)

Click here for additional data file.^{(33KB, docx)}

S4 Fig

Extracted ion chromatograms of m/z 426.32 for (A) Streptomyces sp. MW562807 extract and (B) control. (C) Mass spectrum of ion [M+NH₄]⁺ m/z 426.3212 obtained for compound Cholic Acid (1) (error = -1.6 ppm) at 8.1 min.

(DOCX)

Click here for additional data file.^{(63.5KB, docx)}

S5 Fig

MS/MS match between GNPS database (green) and Lobophorin A (2) from Streptomyces sp. MW562807 extract (black).

(DOCX)

Click here for additional data file.^{(40.2KB, docx)}

S6 Fig

Extracted ion chromatograms of m/z 1157.63 for (A) Streptomyces sp. MW562807 extract and (B) control. (C) Mass spectrum of ion [M+H]⁺ m/z 1157.6373 obtained for Lobophorin A (2) (error = 0.1 ppm) at 7.9 min.

(DOCX)

Click here for additional data file.^{(58.8KB, docx)}

S7 Fig

Extracted ion chromatograms of m/z 1187.61 for (A) Streptomyces sp. MW562807 extract and (B) control. (C) Mass spectrum of ion [M+H]⁺ m/z 1187.6108 obtained for Lobophorin B (3) (error = -0.5 ppm) at 6.9 min. (D) MS/MS spectrum of Lobophorin B.

(DOCX)

Click here for additional data file.^{(81.6KB, docx)}

S8 Fig

Extracted ion chromatograms of m/z 1171.61 for (A) Streptomyces sp. MW562807 extract and (B) control. (C) Mass spectrum of ion [M+H]⁺ m/z 1171.6160 obtained for Lobophorin E (4) (error = -0.4 ppm) at 7.2 min. (D) MS/MS spectrum of Lobophorin E.

(DOCX)

Click here for additional data file.^{(83.3KB, docx)}

S9 Fig

Extracted ion chromatograms of m/z 1173.63 for (A) Streptomyces sp. MW562807 extract and (B) control. (C) Mass spectrum of ion [M+H]⁺ m/z 1173.6321 obtained for Lobophorin K (5) (error = -0.4 ppm) at 7.2 min. (D) MS/MS spectrum of Lobophorin K.

(DOCX)

Click here for additional data file.^{(84.1KB, docx)}

S10 Fig

Extracted ion chromatograms of m/z 197.11 for (A) Streptomyces sp. MW562807 extract and (B) control. (C) Mass spectrum of ion [M+H]⁺ m/z 197.1173 obtained for compound 6 (error = -2.0 ppm) at 7.2 min. (D) MS/MS spectrum of Compound 6.

(DOCX)

Click here for additional data file.^{(90.9KB, docx)}

Acknowledgments

We are grateful to the anonymous reviewers whose constructive criticism has significantly improved the quality of the manuscript, and we would like to thank Editage (www.editage.com) for English language editing.

Data Availability

Funding Statement

This study has been financed by the Concytec - World Bank Project "Improvement and Expansion of the Services of the National System of Science, Technology and Technological Innovation" 8682-PE, to the "World Bank", to "CONCYTEC" through its executing unit ProCiencia [contract number 190-2018]" and "The postgraduate programs in Genetics and Molecular Biology.

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PLoS One. doi: 10.1371/journal.pone.0240946.r001

Decision Letter 0

Luis Angel Maldonado Manjarrez

23 Nov 2020

PONE-D-20-29463

EVALUATION OF ANTIMICROBIAL AND ANTIPROLIFERATIVE ACTIVITIES OF ACTINOBACTERIA ISOLATED FROM THE SALINE LAGOONS OF NORTHWEST PERU

PLOS ONE

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"At the research agency of Peru (Concytec-Fondecyt) financially supported this work within the framework of the 041-01 call with the 190-2018 contract; as well as the National Council for Scientific and Technological Development of Brazil (CNPq), it was the authors are grateful to the cooperation of the postgraduate programs in Genetics and Molecular Biology and Biosciences of the Biology Institute of the State University of Campinas. Also, Coordination for the Improvement of Higher Education Personnel - Brazil (CAPES) - (Scholarship-code 001)."

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Reviewer #1: Yes

Reviewer #2: Partly

**********

2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: No

**********

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Reviewer #1: No

Reviewer #2: No

**********

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Reviewer #1: No

Reviewer #2: No

**********

5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: This manuscript describes the isolation of actinobacterial strains from a saline lagoon in Northwest Peru. The authors identified a selected group of actinobacteria by 16S rRNA gene sequencing and subjected the isolates to tests in order to determine whether they produce any bio-active compounds with activity against bacteria and various cancer cell lines. The extract from one of the most promising strains was evaluated via molecular networking, allowing for the prediction of compounds produced by this strain.

It is clear that the manuscript represents a large body of work, which also resulted in a rather lengthy manuscript. The authors would need to look at ways in which the manuscript can be shortened so that it is more streamlined. In addition, the authors also need to make sure that the way of citing references in the main text follow the journal’s requirements.

Here are some additional comments/suggestions to the authors:

1. The manuscript will require language editing before it can be considered for publication.

2. Figure 1: the caption for image ‘d’ is not included in the figure legend.

3. Figure 2 can probably be presented as supplementary material.

4. Page 8, section 2.2.: The authors described a very specific isolation protocol. This obviously would limit the amount and type of actinobacteria that would be isolated. What was the rationale behind using this method and were other isolation methods explored, but yielded no isolates of interest? In addition, many researchers often base their isolation protocol on the observed physicochemical characteristics of the environment the sample was collected from. Were any physicochemical analyses performed (e.g. sediment pH, level of salinity, presence of K, P, N, C, etc.)?

In addition, in this section and throughout the manuscript, when indicating a % solution, please indicate whether it is v/v or w/v, e.g. isolates were preserved in 20% (v/v) glycerol…

5. Page 9, section 2.3.1: What was the final pH of the R2A-ASW broth used?

6. Page 9-10, section 2.32: It is mentioned that modifications were made to the DNA extraction method reported by Pospiech and Neuman (1995), but no details are provided. Please expand on the modifications effected.

Line 161: The PCR protocol also calls for a large amount of DNA to be added. Is there any particular reason for that?

Line 166: What % agarose gel was used for the analysis of the amplicons?

Lines 171-172: Which program was used to assemble to DNA sequences?

7. Page 11, line 186: What was the rationale behind the use of R2A broth for the production of bio-active compounds? Actinobacteria can produce numerous different compounds, a process that is greatly affected by the type of growth media used. Were different types of media tested, or did you solely focus on this medium?

8. Page 11, lines 196-197: Here mention is made that the crude extracts were also analysed by LC-MS. Is this the same analyses reported later on in the methodology section (in which case the authors need to refer to the section) or was this a separate analysis (in which case more details are required)?

9. Page 12, line 208: The wording of this sentence does not make sense – the ‘three crude bacterial extracts’ can’t be ‘tested as antimicrobial producers’. Best to say that they were tested for antimicrobial activity.

10. Page 12, lines 211-212: What is meant by ‘pre-coated microbial cultures’?

11. Page 12, lines 217-218: Define the abbreviations ‘MDRs’ and ‘UCI’ – these have not been defined previously.

12. Throughout the manuscript, make sure that ‘sp.’ is not written in italics, e.g. page 13, line 244 – the ‘sp.’ should not be in italics.

13. Page 13, lines 244-245: It is indicated that the B-81 extract was diluted 1 in 10 in methanol, but there is no indication as to the final concentration of the extract. Please indicate, e.g. 1mg/ml, 0.1mg/ml, etc.

14. Page 15, section 3.1: The use of phenotypic characteristics to group actinobacteria is notoriously unreliable. Genera within a family may have very similar morphological features and you stand a chance of ‘missing out’ potentially unique/’rare’ actinobacteria, e.g. members of Kitasatospora and Streptomyces share phenotypic features, similarly members of Pseudonocardia and Nonomuraea. Why not identify all 166 strains?

15. Page 17: The text provided in this section is basically a repeat of what is presented in Table 3. Only highlight the most interesting rather than repeating what is already presented.

16. Page 18, line 317 and 324: ‘Streptomyces’ should be written in italics.

17. Page 18, lines 321-325: Here the authors highlight the types of actinobacteria isolated from the different environments. Care should be taken here especially since the authors only made use of a single isolation approach, which will result in isolation bias. They therefore can’t compare what they detected during their isolations to that of others – they can simply report it and mention that these were isolated under the experimental conditions applied.

18. Figure 3: A phylogenetic tree containing only the three selected strains (and related streptomycetes) is presented in this figure. It seems a bit out of place since the importance of these three strains only becomes clear once the results for antimicrobial and anticancer tests have been reported. This makes it seem out of order. I would recommend amending the tree to include all the streptomycete strains identified in the study and once the bio-activity has been reported, reference can be made to this figure. This also means that Table 3 can be shifted to supplementary material.

19. Page 20, line 364: No evidence has been provided to support the classification of the isolates as ‘extremophilic’.

20. Page 22, line 395: Note the spelling of ‘doxorubicin’ – please correct.

21. Page 22, line 398: It is mentioned that the extracts from ‘three different media’ were selected. This is not reported in the methodology section – please amend.

22. Page 30, line 544: Note the spelling of ‘cholic’ – please correct.

23. Page 18, line 333: incomplete surname provided of the reference Cortés-Albayay.

24. Page 36, reference 40: Not cited in the main text.

25. Page 37, reference 42: Not cited in main text or incorrect spelling of lead author.

26. Page 37, reference 48: Not cited in main text or incorrect spelling of l

Reviewer #2: The article “EVALUATION OF ANTIMICROBIAL AND ANTIPROLIFERATIVE ACTIVITIES OF ACTINOBACTERIA ISOLATED FROM THE SALINE LAGOONS OF NORTHWEST PERU” describes the isolation and antimicrobial and anticancer capacity of strains isolated from saline environments. Although some of the results are of interest the quality of the manuscript is not enough, and a careful revision of data presentation and English should be taken into account before publication.

It is not clear at any point, why the three strains of Streptomyces have been selected for further analysis, it is said that they have better inhibition results, but it has not been shown any experiment with the other strains.

The authors should clarify that the sampling was in agreement with Nagoya protocol (check comment from line 110).

Lines 30-31: Reconsider this sentence, it has no many sense as it is.

Line 35: add “and /or”

Line 36: add “these bacteria” before “were identified”

Lines 36-38: I imagine these numbers refer to abundance of strains but it is not clear here.

Line 38: Actinobacteria should be in italics

Line 60: Here should be a final dot instead of semicolon.

Line 69: Italics

Line 77: give a reference.

Line 110: The Nagoya protocol was signed by Peru in 2014 so, at least the sampling of 2015 should be authorised by the goverment.

Line 112: it should be b, c and d, isn't it?

Table 1: before was indicated that sampling was in July, clarified it. Indeed if it was in december the Nagoya protocol should be applied.

Figure 2: correct “bacterial” and “extremophilic bacteria”. But indeed this figure can be omitted.

Line 126: it should be actinomycetes, not actinomyces, but indeed better if you use "Actinobacteria"

Line 144: this amount means 2.43% not 5%, verified your data.

Lines 276-279: These characteristics do not allow the classification into genus, you can group them into groups with similar characteristics, but not genus. This classification could be obtained after 16S rRNA gene analysis.

Line 286: based on what? which strains were included in those groups? this should be further explained.

Line 288: What are the other 116 strains isolated?

Page 292: delete strain, and put "isolates" and “samples” in plural

Line 303: correct to “were similar” or “presented higher similarities with”

Lines 300-317: it has no sense to give all the data here again if you give them in table 3, keep just the text or the table but not both. I recommend to keep the table.

Line 317: Streptomyces should be in italics; delete “of the”

Line 318: change “to” by “from”

Line 323: add a space before “has”

Lines 324 and 388: Streptomyces should be in italics

Line 328: correct the reference

Figure 3: I do not understand why the authors has selected only three of their strains to construct the phylogenetic tree, a good analysis of their strains should include all the ones for which the 16s rRNA gene has been obtained, independently of their ability to produce secondary metabolites. The 16S rRNA gene sequences should be deposited in public databases, and their accession numbers included in the tree.

Line 345: genus names should be in italics.

Line 353-356: this is not true, that is only true for the strain M-92, but not for the other two strains represented in the tree.

Line 379: check the reference

Line 401: delete “the”

Lines 405-407: these values are higher than 50, Shouldn’t they be considered as inactive according to line 394 reference? This should be further discussed by the authors.

Line 474: give details about the molecules marked as 1, 2 ,3, 4, and 5 in the figure.

Line 541: it should be a comma after the first “study”. Indeed one “study” should be replaced.

Line 542: correct “actinobacteria”

Line 550: delete the final dot after “potential”

**********

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Reviewer #1: No

Reviewer #2: No

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PLoS One. 2021 Sep 8;16(9):e0240946. doi: 10.1371/journal.pone.0240946.r002

Author response to Decision Letter 0

3 Mar 2021

Response to the Editor comments

Comments from editor:

1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming.

Reply: We have corrected the tittle, headings, authors’ names, and affiliations according to PLOS ONE's style requirements.

2. We note that figure 1 in your submission contains map images which may be copyrighted. All PLOS content is published under the Creative Commons Attribution License (CC BY 4.0), which means that the manuscript, images, and Supporting Information files will be freely available online, and any third party is permitted to access, download, copy, distribute, and use these materials in any way, even commercially, with proper attribution. For these reasons, we cannot publish previously copyrighted maps or satellite images created using proprietary data, such as Google software (Google Maps, Street View, and Earth). For more information, see our copyright guidelines.

Reply: We have modified figure 1 in our manuscript. Now, the map has been removed and only the sampling sites photos remain.

3. Please ensure that reagents are described in sufficient detail for another researcher to reproduce the experiments described (source, product number, lot number).

Reply: We have made elaborated detailed protocols which are now published in protocols.io to make the requested information available.

4. Please provide additional information about each of the cell lines used in this work, including any quality control testing procedures (authentication, characterization, and mycoplasma testing).

Reply: We have added the requested information about the cell lines in the description to methods.

5. To comply with PLOS ONE submission guidelines, in your Methods section, please provide additional information regarding your statistical analyses.

Reply: We have added detailed information in the 2.7 Statistical analysis in the Methods section.

6. We suggest you thoroughly copyedit your manuscript for language usage, spelling, and grammar.

Reply: We have purchased the services of the suggested Editage editors for the language corrections.

7. We note that you have provided funding information that is not currently declared in your Funding Statement. However, funding information should not appear in the Acknowledgments section or other areas of your manuscript. We will only publish funding information present in the Funding Statement section of the online submission form.

Please remove any funding-related text from the manuscript and let us know how you would like to update your Funding Statement. Currently, your Funding Statement reads as follows:

"CONFLICT OF INTEREST STATEMENT

The authors declare no competing financial interests and the funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript."

Please include your amended statements within your cover letter; we will change the online submission form on your behalf.

Reply: We have removed the funding information from the acknowledgments section and put it on the Funding Statement in the Cover Letter.

8. Please include captions for your Supporting Information files at the end of your manuscript, and update any in-text citations to match accordingly.

Reply: We have added the captions information in the Supporting information section.

Comments from the nomenclature reviewers to be forwarded to the authors: The nomenclature reviewers agree with the proposed name and its etymology.

Reply: Thank you.

Response to the reviewer’s comments

Here are some additional comments/suggestions to the authors:

1. The manuscript will require language editing before it can be considered for publication.

REPLY: According to your suggestions, this manuscript has been sent to Editage Editors for language corrections.

2. Figure 1: the caption for image 'd' is not included in the figure legend.

REPLY: We have modified figure 1 in our manuscript. Now, the map has been removed and only the sampling sites photos remain. Now “d” is included in Figure 1.

3. Figure 2 can probably be presented as supplementary material.

REPLY: We have put Figure 2 to the Supplementary material section.

4. Page 8, section 2.2.: The authors described a very specific isolation protocol. This obviously would limit the amount and type of actinobacteria that would be isolated. What was the rationale behind using this method and were other isolation methods explored, but yielded no isolates of interest? In addition, many researchers often base their isolation protocol on the observed physicochemical characteristics of the environment the sample was collected from. Were any physicochemical analyses performed (e.g., sediment pH, level of salinity, presence of K, P, N, C, etc.)?

REPLY: We have developed and optimized a selective isolation protocol because the purpose of the research was to recover only actinobacteria, at the expense of Bacillus and Micrococcus, which has a faster growth. We have considered pH, solute concentrations, and other conditions from previous physicochemical analysis.

In addition, in this section and throughout the manuscript, when indicating a % solution, please indicate whether it is v/v or w/v, e.g., isolates were preserved in 20% (v/v) glycerol...

REPLY: Thank you. We have corrected those specifications about percentage.

5. Page 9, section 2.3.1: What was the final pH of the R2A-ASW broth used?

REPLY: We have corrected that specification o pH, which is 7.

Line 161: The PCR protocol also calls for a large amount of DNA to be added. Is there any reason for that?

Reply: We have made elaborated detailed protocols which are now published in protocols.io to make the requested information available. Link: https://dx.doi.org/10.17504/protocols.io.bpvjmn4n

Line 166: What % agarose gel was used for the analysis of the amplicons?

Reply: We have made elaborated detailed protocols which are now published in protocols.io to make the requested information available. Link: https://dx.doi.org/10.17504/protocols.io.brrmm546

Lines 171-172: Which program was used to assemble to DNA sequences?

REPLY: The BioEdit 7.0 software was used for DNA sequences assembly and it is now written in the Methods section.

REPLY: We have made elaborated detailed protocols which are now published in protocols.io to make the requested information available. R2A Broth, Nutrient Broth and SP2 Broth.

REPLY: Those analysis are the same. We have now corrected the reference in the Methods section.

9. Page 12, line 208: The wording of this sentence does not make sense – the 'three crude bacterial extracts' can't be 'tested as antimicrobial producers'. Best to say that they were tested for antimicrobial activity.

Reply: We have corrected that sentence and now it says “tested for antimicrobial activity.

10. Page 12, lines 211-212: What is meant by 'pre-coated microbial cultures'?

REPLY: We have corrected that sentence in the Methods section and now it just says that they were added.

11. Page 12, lines 217-218: Define the abbreviations 'MDRs' and 'UCI' – these have not been defined previously.

REPLY: We have detailed the definitions of those acronyms in the Methods section.

12. Throughout the manuscript, make sure that 'sp.' is not written in italics, e.g. page 13, line 244 – the 'sp.' should not be in italics.

REPLY: We have now corrected the italics in the “sp” throughout the manuscript.

13. Page 13, lines 244-245: It is indicated that the MW562807 extract was diluted 1 in 10 in methanol, but there is no indication as to the final concentration of the extract. Please indicate, e.g. 1mg/ml, 0.1mg/ml, etc.

REPLY: We have now specified the final concentration of 6.4 mg/mL of the MW562807 extract in the Methods section.

14. Page 15, section 3.1: The use of phenotypic characteristics to group actinobacteria is notoriously unreliable. Genera within a family may have very similar morphological features and you stand a chance of 'missing out' potentially unique/'rare' actinobacteria, e.g. members of Kitasatospora and Streptomyces share phenotypic features, similarly members of Pseudonocardia and Nonomuraea. Why not identify all 166 strains?

REPLY: We did not identify all 166 strains because we were identifying bacteria because the main objective was to know the bacteria with antimicrobial and antiproliferative activities.

15. Page 17: The text provided in this section is basically a repeat of what is presented in Table 3. Only highlight the most interesting rather than repeating what is already presented.

REPLY: We have now kept only the most interesting instead of than repeating the same as Table 3.

16. Page 18, line 317 and 324: 'Streptomyces' should be written in italics.

REPLY: We have now corrected that word.

17. Page 18, lines 321-325: Here the authors highlight the types of actinobacteria isolated from the different environments. Care should be taken here especially since the authors only made use of a single isolation approach, which will result in isolation bias. They therefore can't compare what they detected during their isolations to that of others – they can simply report it and mention that these were isolated under the experimental conditions applied.

REPLY: We have now specified that we just report those bacteria according to the conditions of the samples they were obtained.

18. Figure 3: A phylogenetic tree containing only the three selected strains (and related streptomycetes) is presented in this figure. It seems a bit out of place since the importance of these three strains only becomes clear once the results for antimicrobial and anticancer tests have been reported. This makes it seem out of order. I would recommend amending the tree to include all the Streptomycete strains identified in the study and once the bio-activity has been reported, reference can be made to this figure. This also means that Table 3 can be shifted to supplementary material.

REPLY: The phylogenetic tree is has been edited considering a representative strains for each of the 13 identified sub-groups.

19. Page 20, line 364: No evidence has been provided to support the classification of the isolates as 'extremophilic'.

REPLY: We have now specified that they were considered as extremophilic since they are able to grow in all media (Artificial Sea Water - ASW) supplemented with 5% of NaCl.

20. Page 22, line 395: Note the spelling of 'doxorubicin' – please correct.

REPLY: Thank you. We have now corrected that spelling.

21. Page 22, line 398: It is mentioned that the extracts from 'three different media' were selected. This is not reported in the methodology section – please amend.

REPLY: We have now specified that we just report those bacteria according to the conditions of the samples they were obtained.

22. Page 30, line 544: Note the spelling of 'cholic' – please correct.

REPLY: Thank you. We have now corrected that word.

23. Page 18, line 333: incomplete surname provided of the reference Cortés-Albayay.

REPLY: Thank you. We have now corrected that reference. Now we are using Mendeley to manage references.

24. Page 36, reference 40: Not cited in the main text.

REPLY: Thank you. We have now corrected that citation. Now we are using Mendeley to manage references.

25. Page 37, reference 42: Not cited in main text or incorrect spelling of lead author.

REPLY: Thank you. We have now corrected that citation. Now we are using Mendeley to manage references.

26. Page 37, reference 48: Not cited in main text or incorrect spelling

REPLY: Thank you. We have now corrected that citation. Now we are using Mendeley to manage references.

Reviewer #2: The article "EVALUATION OF ANTIMICROBIAL AND ANTIPROLIFERATIVE ACTIVITIES OF ACTINOBACTERIA ISOLATED FROM THE SALINE LAGOONS OF NORTHWEST PERU" describes the isolation and antimicrobial and anticancer capacity of strains isolated from saline environments. Although some of the results are of interest the quality of the manuscript is not enough, and a careful revision of data presentation and English should be taken into account before publication. It is not clear at any point, why the three strains of Streptomyces have been selected for further analysis, it is said that they have better inhibition results, but it has not been shown any experiment with the other strains. The authors should clarify that the sampling was in agreement with Nagoya protocol (check comment from line 110).

REPLY: Thank you. We have now specified that sampling was carried out according to The Nagoya protocol in the Methods section.

Lines 30-31: Reconsider this sentence, it has no many sense as it is.

REPLY: Thank you. We have now corrected those lines.

Line 35: add "and /or"

REPLY: Thank you. We have added “and/or”.

Line 36: add "these bacteria" before "were identified"

REPLY: Thank you. We have added “these bacteria”

Lines 36-38: I imagine these numbers refer to abundance of strains but it is not clear here.

REPLY: Thank you. We have now edited that line in the Methods section. We now just mention the Streptomyces genera without expressing quantities to avoid confusions.

Line 38: Actinobacteria should be in italics

REPLY: Thank you. We have now corrected that word.

Line 60: Here should be a final dot instead of semicolon.

REPLY: Thank you. We have now corrected the punctuation.

Line 69: Italics

REPLY: We have now corrected the italics.

Line 77: give a reference.

REPLY: We have now provided the corresponding reference.

Line 110: The Nagoya protocol was signed by Peru in 2014 so, at least the sampling of 2015 should be authorised by the goverment.

REPLY: We have initiated the corresponding procedures according to Peruvian laws we are currently obtaining the final documentation for the samplings from 2015 since the competent Governmental Entities and Normative Guidelines for the access to genetic resources specific to these sampling environments have been stablished recently in 2020, the print of the message is sent to the responsible entity with whom you are working to obtain the agreement for the use of the genetic resources.

Line 112: it should be b, c and d, isn't it?

REPLY: Yes, thank you. We have now modified Figure 1.

Table 1: before was indicated that sampling was in July, clarified it. Indeed if it was in december the Nagoya protocol should be applied.

REPLY: Thank you. The procedures according to the Nagoya protocol were stablished in 2020 for the specific conditions and samples sites. We are currently obtaining the final documentation for the access to genetic resources.

Figure 2: correct "bacterial" and "extremophilic bacteria". But indeed this figure can be omitted.

REPLY: Thank you. We have now corrected in supplementary Fig 1.”.

Line 126: it should be actinomycetes, not actinomyces, but indeed better if you use "Actinobacteria"

REPLY: We have now corrected that word.

Line 144: this amount means 2.43% not 5%, verified your data.

REPLY: We have now specified that ASW was supplemented with 5% of NaCl described protocol with reference 23.

REPLY: We have modified that part in the Methods section and we classify them grouped in bacterial with similar characteristics.

Line 286: based on what? which strains were included in those groups? this should be further explained.

REPLY: We based this grouping on their genotypic similarities after the 16s rDNA gene alignment as shown in Table 2, and figure 2.

Line 288: What are the other 116 strains isolated?

REPLY: We isolated a total amount of 166 strains and then we could re-activate only 50 strains that were grouped into 13 clusters of actinobacteria identified by 16S rRNA.

Page 292: delete strain, and put "isolates" and "samples" in plural

REPLY: Thank you. We have now corrected that in the Results section.

Line 303: correct to "were similar" or "presented higher similarities with"

REPLY: Thank you. We have corrected that line.

Lines 300-317: it has no sense to give all the data here again if you give them in table 3, keep just the text or the table but not both. I recommend to keep the table.

REPLY: Thank you. We have now deleted in the table.

Line 317: Streptomyces should be in italics; delete "of the"

REPLY: Thank you. We have edited that word and that line.

Line 318: change "to" by "from"

REPLY: Thank you. We have edited that word.

Line 323: add a space before "has"

REPLY: Thank you. We have edited that word.

Lines 324 and 388: Streptomyces should be in italics

REPLY: Thank you. We have edited that word.

Line 328: correct the reference

REPLY: Thank you. We have edited that reference.

REPLY: Thank you. We made the phylogenetic tree considering the three. Representative strains from each group from 13 sequenced strains. Also, we have now added information about de deposited sequences in NCBI database.

Line 345: genus names should be in italics.

REPLY: Thank you. We have edited that word.

Line 353-356: this is not true, that is only true for the strain M-92, but not for the other two strains represented in the tree.

REPLY: Thank you. We have now corrected that information in the Results section.

Line 379: check the reference

REPLY: Thank you. We have now corrected the reference.

Line 401: delete "the"

REPLY: Thank you. We have deleted the “the”.

Lines 405-407: these values are higher than 50, Shouldn't they be considered as inactive according to line 394 reference? This should be further discussed by the authors.

REPLY: Thank you. We have corrected and specified that information in the Results section.

Line 474: give details about the molecules marked as 1, 2 ,3, 4, and 5 in the figure.

REPLY: Thank you. We have now made a description specifying those molecules in the figure.

Line 541: it should be a comma after the first "study". Indeed one "study" should be replaced.

REPLY: Thank you. We have edited that line.

Line 542: correct "actinobacteria"

REPLY: Thank you. We have edited that word.

Line 550: delete the final dot after "potential"

REPLY: Thank you. We have now corrected the punctuation.

Attachment

Submitted filename: Response to Reviewers.pdf

Click here for additional data file.^{(291.2KB, pdf)}

PLoS One. doi: 10.1371/journal.pone.0240946.r003

Decision Letter 1

Luis Angel Maldonado Manjarrez

27 May 2021

PONE-D-20-29463R1

EVALUATION OF ANTIMICROBIAL AND ANTIPROLIFERATIVE ACTIVITIES OF ACTINOBACTERIA ISOLATED FROM THE SALINE LAGOONS OF NORTHWEST PERU

PLOS ONE

Dear Dr. Flores Clavo,

As you can notice from the comments of the two reviewers, one has suggested major and one has suggested minor revisions to your manuscript. However, I have decided to go for the "minor revisions" decision based on the first revision of your manuscript. Despite the latter, I would kindly appreciate if you would check on those comments of both reviewers and address all of their points accordingly as indicated in the previous paragraph.

Please submit your revised manuscript by Jul 11 2021 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

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Luis Angel Maldonado Manjarrez, Ph.D.

Academic Editor

PLOS ONE

Journal Requirements:

Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice.

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: (No Response)

Reviewer #3: (No Response)

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

Reviewer #1: Yes

Reviewer #3: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #3: N/A

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

Reviewer #1: Yes

Reviewer #3: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

Reviewer #1: Yes

Reviewer #3: Yes

**********

6. Review Comments to the Author

Reviewer #1: It is clear from the revised manuscript that the authors applied great effort to address the comments/recommendations/queries of the reviewers. However, with the editing and additions there are still some additional comments/recommendations for the revised manuscript:

1. Page 2-3, lines 54-55: ‘Three Streptomyces sp. strains’ sound cumbersome – consider changing the wording to ‘Three streptomycetes, strains MW562814,…’

2. Page 3, lines 59-60: To streamline the sentence, consider writing ‘… and the multi-drug resistant Acinetobacter baumannii AC-972.’

3. Page 3, line 61: ‘…in the R2A medium was determined using a…’

4. Page 4, lines 79-81: Consider merging the two sentences to provide more flow to the information provided, e.g., ‘…were reported from saline soils of the ephemeral salty lakes in Buryatiya [4], while Streptomyces, Nocardiopsis, and…’

5. Page 4, lines 81-83. The sentence on biological activities seems out of place – I would recommend removing it.

6. Page 4, line 89: ‘…to the production of enzymes, which can…’

7. Page 8, lines 147, 156, 159-160: It is conventional to refer to 16S rRNA (as per other sections in the manuscript).

8. Page 8, line 158: What is meant by ‘represented’? Did you mean that the sequences were submitted to the EZBioCloud 16S database ‘Identify’ service?

9. Page 9, line 162: ‘Mega’ should be written as ‘MEGA’

10. Throughout the manuscript: For any % solutions, please indicate whether they are v/v or w/v solutions.

11. Page 10, line 187: What is meant by ‘adjusted to a total volume of 200�l’? And is the ‘plate’ referred to a 96-well plate?

12. Page 13, lines 251-252: ‘…were used to group isolates into 42 filamentous and 8 non-filamentous bacterial groups’. Can’t use the information to ‘classify’, rather ‘group’, and since all the isolates were bacteria, best to keep it generic at this stage and use an inclusive term such as ‘bacteria’.

13. Table 2: The Family names should also be written in italics.

14. Page 14, line 283: Here is it indicated that MEGA7 was used, whereas in the methodology section it is indicated that MEGA6 was used – please correct.

15. Page 15, line 298: Remove ‘to’

16. Page 15, line 303: Replace ‘Actinobacteria’ with ‘actinobacterium’

17. Page 16, line 310: Replace ‘like’ (colloquial term) with ‘such as’ (more scientific).

18. Page 16, line 312: Did you mean to say that a member of the genus Pseudonocardia was also recovered during this study? In which case, the wording needs to change to reflect this: ‘…and plants, was also recovered in this study.’

19. Page 16, line 316 and throughout the manuscript: If there is no reference made to specific strains of a genus, then it is best to write out spp. as species. For example, here, it is best to write ‘…Streptomyces species as the most…’

20. Table 3: Replace ‘Isolates of Streptomyces sp.’ with ‘Streptomyces isolates’

21. Figure 3: It is recommended that the figure should be removed. The addition of all streptomycete isolates to Figure 2 negated the need for this figure.

22. Page 22: Be consistent in how the names of bioactive compounds are presented, e.g., shellmicin is written in both lower case and upper case in this section (see lines 430 and 436 vs 434 and 436).

23. Figure 4: How does the inset information (Streptomyces B-81 and Control) relate to the figure presented?

24. Page 25, line 463: What is meant by ‘rolling in the GNPS platform database’?

25. Page 25: Please see comment above about writing the names of bioactive compounds in lower case/upper case. Choose one and be consistent in its use.

26. Page 26, line 484: ‘gram-negative’ needs to be replaced with ‘Gram-negative’. ‘Gram’ refers to the surname of the scientist who designed the staining technique and should therefore always be written with a capital letter.

27. Page 28, line 519: Remove the additional full stop after ‘sp.’

28. Page 28, line 527: Remove ‘one’.

29. Even though it was attempted to highlight additional language editing required, it is recommended that the revised manuscript should be evaluated once again before resubmission.

Reviewer #3: The manuscript by Flores and colleagues deals with the isolation and application of actinobacteria recovered from lagoons in Peru. No doubt the MS is interesting but I feel some sections should be improved.

1. It does not seem to be fully details on the culture conditions, time, and morphology of the colonies as these were recovered from the isolation plates and subcultured for further analyses. I think this is important should other authors who read the MS may also be encouraged to perform or follow the protocols indicated on their MS. If this information is missing, then I do not see how other authors may follow or identify their cultures.

2. After sequencing the 16S of the isolates, the authors indicate a size of 1,206 nt but the 16S rDNA gene is nearly 1,500 nt. Is there any particular reason why the gene was only partially sequenced and not completed? I am not saying that the full gene should be sequenced to accept the ms but an explanation within the MS would be useful on why 300nt were missing (technical problems? primers used? sequencing reactions?).

3. Figure 3 indicates: Type strains within the genus Streptomyces and Pseudonocardia but the figure is not related nor include any Pseudonocardia, only Streptomyces!

4. In Figure 2 Streptomyces strain MW562807 falls in a subclade together with many other isolates. However, in that same tree MW562807 is also related to Streptomyces olivaceus and S. pactum. Have the authors checked if those two streptomycetes also show the same or similar biological activities to their isolate? Also since MW562807 is the most interesting strain, why the authors did not include on their study all related isolates to that strain acording to Figure 2? It would definitively be interesting to know if all the isolates within that subclade in Figure 2 do also show similar activities. Again, this is not meant to hold the MS any longer but a few lines within the conclusions or future work could be mention.

5. Conclusion. It is clear that the authors have a good set of interesting strains which have been analysed in the MS. There is the MW562805 and related isolates but, in my view, MS562814 also deserves more comments. That isolate is not related to any type strain and it seems to be a putative novel species as it is not related to any other streptomycete in their phylogenetic trees. If the authors are to continue with this kind of studies, which one they think deserve more attention? The MSW562805 subclade or the MS562814?

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #3: No

PLoS One. 2021 Sep 8;16(9):e0240946. doi: 10.1371/journal.pone.0240946.r004

Author response to Decision Letter 1

14 Jun 2021

Response to the Editor comments

Comments from editor:

1. Journal Requirements:

Reply: We have corrected the references according to PLOS ONE's requirements.

Response to the reviewer’s comments

Reviewer #1:

1. Page 2-3, lines 54-55 before, now 47-48 lines: ‘Three Streptomyces sp. strains’ sound cumbersome – consider changing the wording to ‘Three streptomycetes, strains MW562814,…’

REPLY: Thank you. According to your suggestions, this manuscript we have corrected.

2. Page 3, lines 59-60 before, now 51-52 lines: To streamline the sentence, consider writing ‘… and the multi-drug resistant Acinetobacter baumannii AC-972.’

REPLY: Thank you. According to your suggestions, this manuscript we have corrected.

3. Page 3, line 61: ‘…in the R2A medium was determined using a…’

REPLY: Thank you. According to your suggestions, the manuscript has been corrected, and the abstract has been reduced for clarity, and this sentence in the manuscript text has been deleted.

4. Page 4, lines 79-81 before, now 67-68 lines: Consider merging the two sentences to provide more flow to the information provided, e.g., ‘…were reported from saline soils of the ephemeral salty lakes in Buryatiya [4], while Streptomyces, Nocardiopsis, and…’

REPLY: Thank you. According to your suggestions, this manuscript we have corrected.

5. Page 4, lines 81-83. The sentence on biological activities seems out of place – I would recommend removing it.

REPLY: Thank you. According to your suggestions, this manuscript we have removing it.

6. Page 4, line 89 before, now 74: ‘…to the production of enzymes, which can…’

REPLY: Thank you. According to your suggestions, this manuscript we have corrected.

7. Page 8, lines 147, 156, 159-160 before, now 137,147, 150: It is conventional to refer to 16S rRNA (as per other sections in the manuscript).

REPLY: Thank you. According to your suggestions, this manuscript we have corrected.

8. Page 8, line 158 before, now 148,149: What is meant by ‘represented’? Did you mean that the sequences were submitted to the EZBioCloud 16S database ‘Identify’ service?

REPLY: Thank you. According to your suggestions, this manuscript we have corrected for “were added”.

9. Page 9, line 162 before, now 152: ‘Mega’ should be written as ‘MEGA’

REPLY: Thank you. According to your suggestions, this manuscript we have corrected.

10. Throughout the manuscript: For any % solutions, please indicate whether they are v/v or w/v solutions.

REPLY: Thank you. According to your suggestions, this manuscript we have corrected.

11. Page 10, line 187 before, now 177,178: What is meant by ‘adjusted to a total volume of 200�l’? And is the ‘plate’ referred to a 96-well plate?

REPLY: Thank you. According to your suggestions, this manuscript we have corrected “total volume of 200�l” and add 96-well plate.

12. Page 13, lines 251-252 before, now 239,240: ‘…were used to group isolates into 42 filamentous and 8 non-filamentous bacterial groups’. Can’t use the information to ‘classify’, rather ‘group’, and since all the isolates were bacteria, best to keep it generic at this stage and use an inclusive term such as ‘bacteria’.

REPLY: Thank you. According to your suggestions, this manuscript we have corrected.

13. Table 2: The Family names should also be written in italics.

REPLY: Thank you. According to your suggestions, this manuscript we have corrected.

14. Page 14, line 283: Here is it indicated that MEGA7 was used, whereas in the methodology section it is indicated that MEGA6 was used – please correct.

REPLY: Thank you. According to your suggestions, this manuscript we have corrected.

15. Page 15, line 298 before, now 269 line: Remove ‘to’

REPLY: Thank you. According to your suggestions, this manuscript we have corrected.

16. Page 15, line 303 before, now 274: Replace ‘Actinobacteria’ with ‘actinobacterium’

REPLY: Thank you. According to your suggestions, this manuscript we have corrected.

17. Page 16, line 310 before, now 280 line: Replace ‘like’ (colloquial term) with ‘such as’ (more scientific).

REPLY: Thank you. According to your suggestions, this manuscript we have corrected.

18. Page 16, line 312 before, now 281 line: Did you mean to say that a member of the genus Pseudonocardia was also recovered during this study? In which case, the wording needs to change to reflect this: ‘…and plants, was also recovered in this study.’

REPLY: Thank you. According to your suggestions, this manuscript we have corrected.

19. Page 16, line 316 before, now 285 line (and throughout the manuscript): If there is no reference made to specific strains of a genus, then it is best to write out spp. as species. For example, here, it is best to write ‘…Streptomyces species as the most…’

REPLY: Thank you. According to your suggestions, this manuscript we have corrected.

20. Table 3: Replace ‘Isolates of Streptomyces sp.’ with ‘Streptomyces isolates’

REPLY: Thank you. According to your suggestions, this manuscript we have corrected.

21. Figure 3: It is recommended that the figure should be removed. The addition of all streptomycete isolates to Figure 2 negated the need for this figure.

REPLY: Thank you. According to your suggestions, this manuscript we have removing it.

22. Page 22: Be consistent in how the names of bioactive compounds are presented, e.g., shellmicin is written in both lower case and upper case in this section (see lines 430 and 436 vs 434 and 436) before, now (384,391,395,396,etc)

REPLY: Thank you. According to your suggestions, this manuscript we have corrected Shellmicin A, …

23. Figure 4: How does the inset information (Streptomyces B-81 and Control) relate to the figure presented?

REPLY: Thank you. According to your suggestions, it was corrected for better understanding, than is indicated in the description in the figure 3.

24. Page 25, line 463 before, now 423,424 lines: What is meant by ‘rolling in the GNPS platform database’?

REPLY: Thank you. According to your suggestions, this manuscript we have added term “comparing in the GNPS platform database”.

25. Page 25: Please see comment above about writing the names of bioactive compounds in lower case/upper case. Choose one and be consistent in its use.

REPLY: Thank you. According to your suggestions, this manuscript we have corrected.

26. Page 26, line 484: ‘gram-negative’ before, now 443 line needs to be replaced with ‘Gram-negative’. ‘Gram’ refers to the surname of the scientist who designed the staining technique and should therefore always be written with a capital letter.

REPLY: Thank you. According to your suggestions, this manuscript we have corrected.

27. Page 28, line 519 before, now 492 line: Remove the additional full stop after ‘sp.’

REPLY: Thank you. According to your suggestions, this manuscript we have corrected.

28. Page 28, line 527 before, now 498 line: Remove ‘one’.

REPLY: Thank you. According to your suggestions, this manuscript we have corrected.

29. Even though it was attempted to highlight additional language editing required, it is recommended that the revised manuscript should be evaluated once again before resubmission.

REPLY: Thank you, if there has been a resignation of the major revision of the language in this new version committed.

Reviewer #3:

REPLY: Thank you for your comment, we appreciate your evaluation very much. This information was described in the protocol on open access data base which was mentioned in the methodology section 132-133 (as described in a previously reported protocol https://www.protocols.io/view/actinobacteria-collection-enrichment-and-isolation-brztm76n). We describe the culture conditions, growth time and type of colonies morphology by microscopic observation.

2. After sequencing the 16S of the isolates, the authors indicate a size of 1,206 nt but the 16S rDNA gene is nearly 1,500 nt. Is there any particular reason why the gene was only partially sequenced and not completed? I am not saying that the full gene should be sequenced to accept the MS but an explanation within the MS would be useful on why 300nt were missing (technical problems? primers used? sequencing reactions?).

REPLY: Thank you. The primers used amplify about 1000 bp covering regions V1 to V8, whose variability for genera level distinction is commonly used in diversity studies. It was added in the description of the phylogenetic tree in the specification if sequenced with primer 10F to 1492R, which resulted in cured sequences between 1206-1500 bp.

3. Figure 3 indicates: Type strains within the genus Streptomyces and Pseudonocardia but the figure is not related nor include any Pseudonocardia, only Streptomyces!

REPLY: Thank you. This figure has been replaced to include the genera Streptomyces and Pseudonocardia.

REPLY: Thank you. According to your suggestions, this manuscript, were added three new references of Lobophorins compounds isolated from Streptomyces olivaceus strains (on lines 461-475), and it is presented that the strains that are reported for their biological activity from different lobophorins are also close S. olivaceus and S. pactum. In this study, a representative clade isolate was chosen to assess biological activities, however, we do not rule out this potential in future studies.

REPLY: Thank you. According to your suggestions, this manuscript we could not only discriminate a single strain, because all three have bioactive activities that is why the three will be taken to future work as we have mentioned in the conclusion within the lines (508-512). Taxonomic studies to characterize the strain MS562814 have been conducted by the group to describe a possible new species of the genus Streptomyces.

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PLoS One. doi: 10.1371/journal.pone.0240946.r005

Decision Letter 2

Luis Angel Maldonado Manjarrez

16 Jul 2021

EVALUATION OF ANTIMICROBIAL AND ANTIPROLIFERATIVE ACTIVITIES OF ACTINOBACTERIA ISOLATED FROM THE SALINE LAGOONS OF NORTHWEST PERU

PONE-D-20-29463R2

Dear Dr. Flores Clavo,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.

If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

Despite the "acceptance" of your Manuscript, there are a few extra comments that should be addressed together with the technical requirements. A list of such minor comments which are merely mine follows:

1. The abstract should be shortened 3-5 lines. The chemical details can be easily avoided in the abstract.

2. Line 158, it says "on closest hits" and should be "on the closest hits"

3. Lines 277, 278, 281, 282, 284 285 and 286. All of these lines use the word "Streptomyces" but this paragraph is mainly devoted to Streptomyces hence it is redundant to re-write Streptomyces for each species and the corresponding comparisons. I suggest you simply use "S." which it is understood, due to the context of the paragraph, referring to Streptomyces.

4. Line 292. It currently says "has recovered" but I think it should be "was recovered".

5. Line 354. It says "Kitasospora" but it should be "Kitasatospora". Despite this typographical mistake I'm not quite sure if this outgroup would be the best because Kitasatospora belongs to the same family as Streptomyces. Perhaps a total different actinobacteria such as Micromonospora which is not related neither to Pseudonocardia nor Streptomyces would be better.

I strongly believe that these minor corrections should contribute to the improvement of the manuscript hence I kindly ask you to follow them together with the technical requirements of the editing process.

Kind regards,

Luis Angel Maldonado Manjarrez, Ph.D.

Academic Editor

PLOS ONE

PLoS One. doi: 10.1371/journal.pone.0240946.r006

Acceptance letter

Luis Angel Maldonado Manjarrez

27 Aug 2021

PONE-D-20-29463R2

Evaluation of antimicrobial and antiproliferative activities of actinobacteria isolated from the saline lagoons of northwestern Peru

Dear Dr. Flores Clavo:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

If we can help with anything else, please email us at plosone@plos.org.

Thank you for submitting your work to PLOS ONE and supporting open access.

Kind regards,

PLOS ONE Editorial Office Staff

on behalf of

Dr. Luis Angel Maldonado Manjarrez

Academic Editor

PLOS ONE

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

S1 Fig. Flowchart depicting the methodological strategy adopted in this study.

(DOCX)

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S2 Fig

Total ion chromatogram (TIC) of UHPLC-MS analyses for (A) Streptomyces sp. MW562807 extract and (B) control.

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S3 Fig

MS/MS match between GNPS database (green) and Cholic Acid (1) from Streptomyces sp. MW562807 extract (black).

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S4 Fig

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S5 Fig

MS/MS match between GNPS database (green) and Lobophorin A (2) from Streptomyces sp. MW562807 extract (black).

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S6 Fig

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S7 Fig

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S8 Fig

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S9 Fig

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S10 Fig

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Attachment

Submitted filename: Response to Reviewers.pdf

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Attachment

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Data Availability Statement

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PERMALINK

Evaluation of antimicrobial and antiproliferative activities of Actinobacteria isolated from the saline lagoons of northwestern Peru

Rene Flores Clavo

Nataly Ruiz Quiñones

Álvaro Jose Hernández-Tasco

Marcos José Salvador

Ana Lúcia Tasca Gois Ruiz

Lúcia Elaine de Oliveira Braga

Jonas Henrique Costa

Taícia Pacheco Fill

Zhandra Lizeth Arce Gil

Luis Miguel Serquen Lopez

Fabiana Fantinatti Garboggini

Roles

Abstract

Introduction

Materials and methods

Site description and sampling

Fig 1.

Table 1. Data related to saline lagoon samples.

Isolation of cultivable Actinobacteria

Identification of bacterial isolates

Reactivation and morphological phenotypic characterization of bacterial strains

DNA extraction and 16S rRNA gene sequencing

Therapeutic potential of bacterial isolates

Crude extract produced from extremophilic bacteria

Fractionation of the crude extract from extremophilic bacteria

In vitro antibacterial activity assay

In vitro antiproliferative activity assay

Mass spectrometry analysis

Molecular MS/MS network

Statistical analyses

Results and discussion

Isolation, identification, and selection of bacterial species from the northern saline lagoons of Peru

Table 2. 16S rRNA gene-based identification of bacteria isolated from lagoons Morrope and Bayovar.

Antibacterial activity screening

Table 3. Minimum inhibitory concentration of three Streptomyces sp. crude extracts, as determined by broth dilution method.

Antiproliferative activity screening

Table 4. Antiproliferative activity of three Streptomyces extracts against human tumor cell lines.

Table 5. Antiproliferative evaluation of isolate Streptomyces sp. MW562807 fermented in three different media and fractions of the R2A medium against nine tumors and one non-tumor cell line.

Secondary metabolite analysis of Streptomyces sp. MW562807 isolate

Fig 3. Molecular networking obtained for Streptomyces sp. MW562807 extract Cholic Acid and its derivatives were grouped in cluster A, and Lobophorins were grouped in cluster B.

Table 6. MS data obtained for secondary metabolites detected in Streptomyces sp. MW562807 extract.

Fig 4. Structures of secondary metabolites identified in Streptomyces sp. MW562807 extract.

Conclusion

Supporting information

Acknowledgments

Data Availability

Funding Statement

References

Decision Letter 0

Luis Angel Maldonado Manjarrez

Roles

Author response to Decision Letter 0

Decision Letter 1

Luis Angel Maldonado Manjarrez

Roles

Author response to Decision Letter 1

Decision Letter 2

Luis Angel Maldonado Manjarrez

Roles

Acceptance letter

Luis Angel Maldonado Manjarrez

Roles

Associated Data

Supplementary Materials

Data Availability Statement

ACTIONS

PERMALINK

RESOURCES

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