# Broad-spectrum antimicrobial activity of wetland-derived streptomyces sp. actif450

Mabrouka Benhadj, Metrouh, Roumaisa, Taha Menasria, Djamila Gacemi-Kirane, Slim Fatma Zohra, Stephane Ranque

► To cite this version:

Mabrouka Benhadj, Metrouh, Roumaisa, Taha Menasria, Djamila Gacemi-Kirane, Slim Fatma Zohra, et al.. Broad-spectrum antimicrobial activity of wetland-derived streptomyces sp. actif450. EXCLI Journal, 2020, 19, pp.360-371. 10.17179/excli2020-1124. hal-02529318

HAL Id: hal-02529318

https://amu.hal.science/hal-02529318

Submitted on 15 Mar 2021

HAL is a multi-disciplinary open access
archive for the deposit and dissemination of sci-
entific research documents, whether they are pub-
lished or not. The documents may come from
teaching and research institutions in France or
abroad, or from public or private research centers.

L'archive ouverte pluridisciplinaire HAL, est
destinée au dépôt et à la diffusion de documents
scientifiques de niveau recherche, publiés ou non,
émanant des établissements d'enseignement et de
recherche français ou étrangers, des laboratoires
publics ou privés.

<figure><img src="image_1.png" /></figure>

Distributed under a Creative Commons Attribution 4.0 International License
Original article:

BROAD-SPECTRUM ANTIMICROBIAL ACTIVITY OF
WETLAND-DERIVED *STREPTOMYCES* SP. ACTIF450

Mabrouka Benhadj¹,², Roumaisa Metrouh¹, Taha Menasria¹*, Djamila Gacemi-Kirane³,
Fatma Zohra Slim¹, Stephane Ranque⁴

¹ Department of Applied Biology, Faculty of Exact Sciences and Natural and Life Sciences, Larbi Tebessi University, 12002 Tebessa, Algeria
E-mail: mabrouka.benahdj@univ-tebessa.dz

² Biomolecules and Application Laboratory, Faculty of Exact Sciences and Natural and Life Sciences, Larbi Tebessi University, 12002 Tebessa, Algeria

³ Department of Biochemistry, Faculty of Science, University Badji Mokhtar Annaba, Annaba, 23000, Algeria

⁴ Aix Marseille University, IRD, APHM, SSA, VITROME, IHU-Méditerranée Infection, 19-21 Boulevard Jean Moulin, 13005 Marseille, France

* Corresponding author: T. Menasria, Department of Applied Biology, Faculty of Exact Sciences and Natural and Life Sciences, Larbi Tebessi University, 12002 Tebessa, Algeria. E-mail: tahamenasria@hotmail.com; ORCID: https://orcid.org/0000-0003-4925-6165

http://dx.doi.org/10.17179/excli2020-1124

This is an Open Access article distributed under the terms of the Creative Commons Attribution License
(http://creativecommons.org/licenses/by/4.0/).

ABSTRACT

The increased incidence of invasive infections and the emerging problem of drug resistance particularly for commonly used molecules have prompted investigations for new, safe and more effective microbial agents. Actinomycetes from unexplored habitats appear as a promising source for novel bioactive compounds with a broad range of biological activities. Thus, the present study aimed to isolate effective wetland-derived actinomycetes against major pathogenic fungi and bacteria. Water samples were collected from various locations of Fetzara Lake, Algeria. Thereafter, an actinomycete designated ActiF450 was isolated using starch-casein-agar medium. The antimicrobial potential of the newly isolated actinomycete was screened using the conventional agar cylinders method on Potato Dextrose Agar (PDA) against various fungal and bacterial pathogens. A wetland-derived *Streptomyces* sp. Actif450 was identified as *Streptomyces malaysiensis* based on its physiological properties, morphological characteristics, and 16S rDNA gene sequence analysis. The antimicrobial activity of *Streptomyces* sp. ActiF450 showed a potent and broad spectrum activity against a range of human fungal pathogens including moulds and yeasts, such as *Arthroderma vanbreuseghemii*, *Aspergillus fumigatus*, *A. niger*, *Candida albicans*, *C. glabarta*, *C. krusei*, *C. parapsilosis*, *Fusarium oxysporum*, *F. solani*, *Microsporum canis*, *Rhodotorula mucilaginous* and *Scodapulariopsis candida*. In addition, high antibacterial activity was recorded against pathogenic staphylococci. The novel *Streptomyces* sp. ActiF450 may present a promising candidate for the production of new bioactive compounds with broad-spectrum antimicrobial activity.

**Keywords:** Coastal wetland, *Streptomyces*, antifungal activity, *Candida* spp.

INTRODUCTION

The last two decades have seen unprece-
dented changes in the pattern of fungal dis-

eases (FDs) in humans. The rising prevalence of FDs, such as candidiasis, aspergillosis, pneumocytosis and cryptococcosis, have
become a major health problem worldwide (Casadevall, 2018; Richardson and Warnock, 2012).

Fungal diseases are difficult to manage because they tend to be chronic, hard to diagnose, and more recalcitrant to therapy such that most mycoses require treatment courses lasting months or longer (Liu et al., 2018). These diseases have gained a much greater importance, largely because of their increasing incidence among transplant recipients and immune compromised patients, including those with acquired immunodeficiency syndrome (AIDS) (Richardson and Warnock 2012). In fact, the more widespread use of immunosuppressive therapies and increased movements of patients at risk are among the main acquired risk factors contributing to FDs (Liu et al., 2018).

The world is facing an ever-increasing problem of antimicrobial resistance (Menasria et al., 2015). In fact, new resistance mechanisms emerge and spread globally threatening our ability to treat common infectious diseases, resulting in death and disability of individuals (Boukoucha et al., 2018). Different saprophyte and commensal fungi from both yeast and mold forms are recognized among agents that cause human mycosis (Benammar et al., 2017), including species of Candida spp., Aspergillus spp., Pneumocysti spp., dimorphic (Coccidioides and Paracoccidioides), dermatophytes (Trichophyton spp.), and encapsulated yeast Cryptococcus spp., which are present in the localized and disseminated forms of the disease (Liu et al., 2018; Richardson and Warnock, 2012). Fungal infections including superficial and invasive fungal infections 'IFIs' have increased significantly during the past last decades, which can be an economical burden and a substantial medical concern, particularly in immunocompromised population (Antinori et al., 2018). Also, the effectiveness of current antifungal therapies in the management of these infections is under discussion, due to several limitations, such as off-target toxicity and drug-resistant emergence (Liu et al., 2018).

The increased incidence of invasive mycoses and the emerging problem of drug resistance (Casadevall, 2018), particularly for the azole and polyene compounds or commonly used molecules, have prompted investigations for new, safe and more effective antifungal agents (Liu et al., 2018). In addition, the incidence of *S. aureus*-linked infections has increased, with highly virulent strains being encountered (Menasria et al., 2015). The aforementioned pathogens can be acquired from hospitals and increasingly from non-clinical environments (i.e., community-associated infections) (Antinori et al., 2018; Merradi et al., 2019).

Microorganisms are known to produce various bioactive compounds with great potential to be developed as therapeutic drugs for humans and animal uses (Chaudhary et al., 2013). In fact, many of these compounds were derived from the genus *Streptomyces* (Benhadj and Gacemi-Kirane, 2016). So far, over 850 species of *Streptomyces* have been isolated and validly published (LPSN, 2019), and more than 600 species have been recorded to be excellent sources of bioactive molecules (Supong et al., 2017).

*Streptomyces* are generally prevalent in soils and diverse natural habitats (Seipke et al., 2012). It is known to be the largest anti-biotic-producing genus in the microbial world discovered so far (Benhadj et al., 2019), also having the ability to produce other important bioactive secondary metabolites, such as antifungals, antivirals, antitumorals, anti-hypertensives, and especially immuno-suppressants (de Lima Procópio et al., 2012). Accumulated evidence indicates that the production of bioactive compounds from actinomycetes is associated with nonribosomal peptide synthetase (NRPS) and polyketide synthase (PKS) pathways (Albright et al., 2014), suggesting that nonribosomal peptides, polyketides and their hybrid compounds are the major secondary metabolites of actinomycetes (Komaki et al., 2018).

Sequencing of *Streptomyces* genomes has shown treasure troves of unsuspected and uncharacterized biosynthetic gene clus-
ters, referred as silent or cryptic pathways,
for secondary metabolites and antibiotic-like
substances than originally anticipated (Niu et
al., 2016). These cryptic gene clusters are
substantially tied to the environmental condi-
tions in which secondary metabolites pro-
duction may evolve (Onaka, 2017). General-
ly, studies on actinobacteria are confined to
the terrestrial ecosystems and less signifi-
cance has given to marine or fresh water sys-
tems. It has been shown that marine actino-
mycetes and strains from unexplored habitats
were found to represent a rich source for di-
verse bioactive metabolites with potential
applications (Benhadj et al., 2018).

Algeria harbors several wetlands of
which fifty are classified as Ramsar sites of
international importance in terms of biodi-
versity and functional role (Menasria et al.,
2019). However, aspects related to microbio-
ta remain little known. It is suggested that
the changes in salinity, light, temperature,
nutrient availability and other physicochemi-
cal and microbiological processes in such
ecosystem turned out to be the driving forces
for metabolic pathway adaptations that could
result in the production of valuable metabo-
lites (Menasria et al., 2018). In this study, a
potent antimicrobial actinobacteria was iso-
lated from a coastal wetland (Ramsar wet-
land) within the West Mediterranean Basin
(Fetzara Lake, northeastern Algeria)
(www.ramsar.org). This strain was identified
as Streptomyces sp. ActiF450 and exhibits
broad antifungal spectrum activities against
different medically important bacteria
(Satphylococcus aureus), yeast and moulds
species like Candida spp., Kluveromyces
spp., Rhodotorula spp., Aspergillus spp.,
Fusarium spp., Microsporum spp., and oth-
ers. In addition, to evaluate the antimicrobial
potency, various extractions of solid cultures
using different organic solvents were per-
formed.

MATERIAL AND METHODS

Sample collection and isolation

Water samples from Fetzara Lake
(36°43' and 36°50'N, 7°24' and 7°39'E)

were collected during winter (January 2017). The samples were heat treated at 50 °C for 30 min and 10-fold serial dilutions were prepared. Aliquots of 0.1 ml were spread plated onto Casein Starch agar (CSA) (g/l) (soluble starch, 10; casein, 0.3; KNO3, 2.0; MgSO4·7H2O, 0.05; K2HPO4, 2.0; NaCl, 2; CaCO3, 0.02; FeSO4·7H2O, 0.01 agar 20) supplemented with (2.5 µg/ml of rifampicin, 10 µg/ml of amphotericin B and 75 µg/ml of fuconazol). Plates were incubated at 30 °C for 7 days up to 4 weeks, and actinobacteria-like strain designated ActiF450 was isolated, subcultured and maintained on CSA slants at 4 °C. Spore suspensions were prepared in glycerol 20 % and stored at -80 °C for further use.

Morphological characteristics of the isolate

Morphological characteristics such as
aerial mass color and substrate mycelium
were observed on soya flour mannitol (SFM)
plates. The aerial mass and color of the sub-
strate mycelium were recorded and classified
according to Bergey's Manual of Systematic
Bacteriology and ISCC-NBS Color Charts
standard (Kelly and Judd, 1964; Vos et al.
2009). The tolerance of NaCl, pH, and the
effect of temperature was determined by cul-
tivating on ISP2 medium. Nitrate reduction,
hemolytic activity and production of amyl-
ase, gelatinase, protease and lipase were de-
termined by cultivation on various media as
described by Benhadj et al. (2019) using
starch, gelatin, casein and Tween 80 as sub-
strates, respectively.

DNA extraction, molecular identification
and phylogenetic analyses

The genomic DNA of the strain
ActiF450 was extracted as described by Kie-
ser et al. (2000). A loopful of mycelium was
scraped from colonies grown on CSA and
suspended in 5 ml of saline-EDTA (5M
NaCl, 0.5 M EDTA [pH 8.0], Tris-HCL [pH
7.5]) by vortexing. Lysozyme was added to a
final concentration of 1 mg/ml, followed by
incubation at 37 °C for 60 min. Later, 10 µl
of 1 % (wt/vol) proteinase K and 200 µl of
10% sodium dodecyl sulfate were added, and the mixed solution was incubated at 50 °C for 2 hours. Subsequently, 500 µl of 5M NaCL was added and the lysate was centrifuged (15,000 g, 5 min) before sequential extractions with phenol, followed by chloroform. The aqueous phase was precipitated using 0.6 ml isopropanol and two volumes of 70% ethanol. The DNA was suspended in 100 µl of TE 1X (10 mM Tris-HCl at pH 8, 1 mM EDTA) and stored at -20 °C for further use.

The 16S rRNA gene was amplified by PCR using the universal primers Fd1 (5' AGAGTTTGATCCTGGCTCAG) and rP2 (5'-AAGGAGGTGATCCAGCC) (Weisburg et al., 1991). Sequence similarity calculations were carried out using an alignment search program with the EzTaxon-server (Chun et al., 2007). Evolutionary trees based on the aligned sequences were inferred using the neighbor-joining method (Saitou and Nei, 1987) and topologies were evaluated by bootstrap sampling expressed as percentage of 1000 replicates. Phylogenetic analyses were conducted using MEGA software version 6 (Tamura et al., 2013).

### Test microorganisms

For antifungal activity investigation, the following pathogenic yeasts and filamentous fungi were used: *Candida krusei* ATCC6258, *Candida parapsilosis* ATCC22019, clinical *Candida* isolates (ICF18, ICF19, ICF20, ICF21, ICF22, ICF23, ICF24, ICF35, ICF37 and ICF38), *Saccharomyces* sp. ICF43, *Kluveromyces* sp. ICF44, *Rhodotorula mucilaginosa* YA1, *Aspergillus fumigatus* MA1, *A. calidoustus* A5, *A. niger* (IAF27 and MA2), *Aspergillus* sp. ICF58V, *Arthroderma vanbreuseghemii* ICF62B, *Scopulariopsis brevicaulis* ICF57, *Fusarium oxysporum*, *F. solani*, *Scedosporium apiospermum*, *Lichtheimia corymbifera* ST87, *Lomentospora prolificans* ST67, *Microsporum canis* ICF58B, *Pecilomyces variotii*, *Penicillium chrysogenum* ICF 59, *Rhinopus oryzae* and *Scodapulariopsis candida* ICF53. For antibacterial activity, seven clini-

cal *Staphylococcus aureus* isolates were used including two reference strains *Staphylococcus aureus* ATCC25293 and *Staphylococcus aureus* ATCC43300. All fungal and bacterial cultures were maintained on Potato Dextrose Agar (PDA) and Luria Bertani slants respectively at 4 °C.

### Antimicrobial activity screening

Antimicrobial activity was first screened using the conventional agar cylinders method on Potato Dextrose Agar (PDA) and Luria Bertani (LB) plates for fungi and bacteria respectively (Benhadj et al., 2019). Mycelium plugs (7 mm diameter) of ActiF450 inoculated at different time (3, 7, 10 and 14 days) were inoculated onto PDA and LB plates previously inoculated with target pathogens. Secondly, a double layer method was used for confirmation. The active strain ActiF450 was inoculated as a spot in the center of ISP2 plates at 30 °C for 7 days. After incubation, the plates were then covered by 10 ml of PDA and LB previously inoculated with target fungi and bacteria respectively (Badji et al., 2005). The inhibition zones around each spot were measured (mm) after 24 h at 37 °C for bacteria, 48 h and 7 days at 30 °C for yeast and moulds respectively. Two replicates were prepared for each test and plates with indicator strain were used as control.

### Extraction of bioactive molecules

To extract antimicrobial compounds, strain ActiF450 was inoculated onto ISP2 plates at 30 °C for 10 days. After growth, the cultures were fragmented and extracted with an equal volume of different solvents (n-butanol, dichloromethane, ethanol, ethyl acetate, methanol and hexane). The mixtures were filtered through a Whatman No. 1 filter after a vigorous agitation for one hour, and the collected organic extracts were concentrated using a rotary vacuum evaporator. The crude extracts were separately dissolved in 10 % dimethyl sulfoxide (DMSO) and absolute methanol with a final concentration of 20.0 mg/ml. After filtering through a
0.22 µm Millipore filter, different crude ex-
tract solutions were used to analyze the inhi-
bition ability using the disc diffusion method
(Mehalaine et al., 2017).

RESULTS AND DISCUSSION

Phenotypic and molecular characterization
of ActiF450

In the present study, an active actinobac-
terium ActiF450 strain was isolated from a
natural and unexplored wetland ecosystem
(Fetzara Lake) located in Northeastern Alge-
ria. The vegetative and aerial mycelia as well
as soluble pigments of the strain ActiF450
were evaluated after cultures on different
media for 10 days at 30 °C. Colors of myce-
lia were determined using the ISCC-NBS
centroid color chart (Kelly and Judd, 1964).
ActiF450 can grow well on ISP1, ISP2,
Bennett and Glucose Yeast Extract Agar
(GYEA). The strain produced white grayish
aerial and substrate mycelia on ISP2 agar.
However, no diffusible pigments were pro-
duced, after 10 days of incubation. The mi-
croscopic analysis revealed that the strain
ActiF450 produced well developed and
branched aerial mycelium with spore chains
in the top ends (Figure 1). The strain was
moderately halo tolerant up to 5 % of NaCl
concentration. Growth occurs at 25 to 40 °C
/optimum, 37 °C) and pH 6.0–9.0 (optimum,
pH 7.0). ActiF450 could degrade casein,
starch, Tween 80 and liquefy gelatin indicat-
ing the variety for their complex metabolites
and genomic organization (Bentley et al.,
2002). However, the strain was unable to
produce urease and H2S.

The 16S rRNA partial sequence (1,417
bp) of the strain ActiF450 was amplified and
sequenced. The data indicated that strain
ActiF450 belongs to the genus *Streptomyces*
and was referred to as *Streptomyces* sp.
ActiF450. The sequence was subjected to
alignment and the BLAST search showed
high level of similarity values (99.65 %,
99.22 % and 99.15 %) with *Streptomyces*
*malaysiensis* NBRC16446 T (Al-Tai et al.,

1999), *S. samsunensis* M1463T (Sazak et al., 2011) and *S. solisilvae* HNM0141T (Zhou et al., 2017), respectively. The result was supported by the phylogenetic tree constructed using the neighbor-joining method, where *Streptomyces* sp. ActiF450 formed a subclade and clustered with both *S. malaysiensis* and *S. samsunensis* (Figure 1).

Antimicrobial activity of ActiF450

The results presented in Table 1 showed
that the strain *Streptomyces* sp. ActiF450 ex-
hibited a broad-spectrum antimicrobial activ-
ity against both indicator organisms (bacteria
and fungi), from which highly activity were
recorded. High inhibitory activities were
found against the yeast *Rhodotorula muci-
laginosa* YA1 (inhibition zone diameter of
67.6±2.7 mm), and filamentous fungi *Asper-
gillus niger* MA2 (54.0 ± 6.0 mm), followed
by *Microsporum canis* ICF58B (55.0 mm),
*Arthroderma vanbreuseghemii* ICF62B
(49.2±2.8 mm), *Fusarium oxysporum* F15
(41.2±7.4 mm), *Penicillium chrysogenum*
ICF59 (41.1±5.9 mm) and *Scodapulariopsis
candida* ICF53 (35.0±2.5 mm). Data clearly
indicate that strain ActiF450 exhibited a sig-
nificant antimicrobial activity against *Can-
dida* spp. (15 to 27 mm), *Candida*-like spe-
cies *Kluveromyces* sp. ICF44
(25.6±6.2 mm), *Saccharomyces* sp. ICF43
(17.0±0.0 mm)as well as Gram-positive bac-
teria (*Staphylococcus aureus* clinical iso-
lates) (26.5-35.5 mm).

As shown in Figure 2a, the antimicrobial
activity of the strain ActiF450 against select-
ed *S. aureus* isolates started after two days of
incubation and reached a maximum after
seven days of culture in ISP2. These activi-
ties were persistent until the end of the incu-
bation. Furthermore, the anticandidal activity
was observed at the first four days of incuba-
tion. However, the highest activity was rec-
orded at the third day of incubation period.
Thereafter, the activity dramatically declined
after one week (Figure 2b).
<figure><img src="image_2.png" /><figcaption>**Figure 1:** Neighbor-joining tree based on 16S RNA gene sequences of *Streptomyces* sp. ActiF450 and closely related species. Numbers at the nodes indicate levels of bootstrap support based on an analysis of 1000 re-sampled datasets. The scale bar corresponds to 0.01 substitutions per nucleotide position (A). Morphological characters of colony (B), and cell morphology (C) of the strain ActiF450. The strain ActiF450 was grown on SFM medium and the aerial mycelia morphology was viewed using optical microscopy (x100).</figcaption></figure>
<table><caption>**Table 1:** Antimicrobial activity of *Streptomyces* sp. ActiF450</caption><thead><tr><th>Test microorganism</th><th>Activity mm (Day)</th></tr></thead><tbody><tr><td>Yeast</td><td></td></tr><tr><td>Candida krusei ATCC6258</td><td>25.0±0.0 (D7)</td></tr><tr><td>Candida parapsilosis ATCC22019</td><td>20.0±0.0 (D7)</td></tr><tr><td>Candida albicans sp. ICF19</td><td>22.0±0.5 (D3)</td></tr><tr><td>Candida albicans sp. ICF22</td><td>25.0±0.0 (D3)</td></tr><tr><td>Candida albicans sp. ICF23</td><td>27.0±1.0 (D3)</td></tr><tr><td>Candida albicans sp. ICF24</td><td>15.0±0.0 (D7)</td></tr><tr><td>Candida albicans sp. ICF35</td><td>19.3±1.5 (D3)</td></tr><tr><td>Candida albicans sp. ICF37</td><td>18.0±2.6 (D3)</td></tr><tr><td>Candida albicans sp. ICF38</td><td>15.6±1.1 (D7)</td></tr><tr><td>Candida glabarta sp. ICF18</td><td>25.0±0.0 (D3)</td></tr><tr><td>Candida glabarta sp. ICF20</td><td>25.0±0.0 (D3)</td></tr><tr><td>Candida glabarta sp. ICF21</td><td>21.0±1.0 (D3)</td></tr><tr><td>Saccharomyces sp. ICF43</td><td>17.0±0.0 (D7)</td></tr><tr><td>Kluveromyces sp. ICF44</td><td>25.6±6.2 (D3)</td></tr><tr><td>Rhodotorula mucilaginosa YA1</td><td>67.6±2.7 (D7)</td></tr><tr><td>Arthroderma vanbreuseghemii ICF62B</td><td>49.2±2.8 (D7)</td></tr><tr><td>Aspergillus fumigatus MA1</td><td>14.9±2.0 (D7)</td></tr><tr><td>A. calidoustus A5</td><td>20.9±2.0 (D7)</td></tr><tr><td>A. niger IAF27</td><td>45.0±6.0 (D7)</td></tr><tr><td>A. niger MA2</td><td>54.0±6.0 (D7)</td></tr><tr><td>Aspergillus sp. ICF58V</td><td>35.0±0.0 (D7)</td></tr><tr><td>Fusarium oxysporum F15</td><td>41.2±7.4 (D7)</td></tr><tr><td>F. solani</td><td>19.9±4.0 (D7)</td></tr><tr><td>Lichtheimia corymbifera ST87</td><td>Not detected</td></tr><tr><td>Lomentospora prolificans ST67</td><td>Not detected</td></tr><tr><td>Microsporum canis ICF58B</td><td>55.0±0.0 (D7)</td></tr><tr><td>Pecilomyces variotii</td><td>30.0±6.0 (D7)</td></tr><tr><td>Penicillium chrysogenum ICF 59</td><td>41.1±5.9 (D7)</td></tr><tr><td>Scedosporium apiospermum</td><td>28.0±0.0 (D7)</td></tr><tr><td>Scopulariopsis brevicaulis ICF57</td><td>15.3±1.1 (D7)</td></tr><tr><td>Scodapulariopsis candida ICF53</td><td>35.0±2.5 (D7)</td></tr><tr><td>Staphylococcus aureus ATCC25293</td><td>35.5±3.5 (D10)</td></tr><tr><td>S. aureus ATCC43300</td><td>26.5±0.5 (D10)</td></tr><tr><td>S. aureus IC13</td><td>30.0±0.0 (D7)</td></tr><tr><td>S. aureus IC29</td><td>30.0±0.5 (D10)</td></tr><tr><td>S. aureus IC30</td><td>32.5±0.5 (D10)</td></tr><tr><td>S. aureus IC31</td><td>33.0±1.0 (D7)</td></tr><tr><td>S. aureus IC32</td><td>31.0±0.0 (D14)</td></tr><tr><td>S. aureus IC33</td><td>21.0±0.5 (D14)</td></tr><tr><td>S. aureus IC34</td><td>31.5±4.0 (D10)</td></tr></tbody></table>

Several reports highlighted the production of various antimicrobial compounds by *Streptomyces* strains with different and variable activities depending on culture conditions including; media nature and/or composition (nitrogen and carbon sources), time of incubation even the indicator organisms used in the confrontation test (Vijayakumar et al., 2012).

The differences in susceptibility among tested organisms are in concordance with previous studies by the fact that fungal organisms are eukaryotic, and their structures are different from bacteria (Benhadj et al., 2019). The absence of acidic phospholipids and presence of sterols may reduce susceptibility of eukaryotic cells to lytic molecules (Alan and Earle, 2002). The maximum antibacterial activity after seven to ten days of
incubation may be attributed to the fact that ActiF450 reached the stationary phase of growth. It has been reported that the bioactive metabolites production by *Streptomyces* takes place in the stationary phase of the growth (Augustine et al., 2004) and the decrease in the anticandidal activity after three days can be attributed to the decrease in the supply of nutrients or the accumulation of toxic substances.

Members of the genus *Streptomyces* have attracted extensive attention due to the tremendous success of their natural products in practical application. With their complex life cycle, *Streptomyces* are renowned for the

production of an outstandingly large number of bioactive metabolites and accounts for 80 % of the currently available antibiotic-like substances including antifungals and antibacterials (Niu et al., 2016). However, few studies related to the antimicrobial activities of *Streptomyces malaysiensis*, or the closest strains (*S. samsunensis* and *S. solisilvae*) were attempted so far. Our results indicate that ActiF450 could be a potential strain for the development of antimicrobial drugs against a wide range of human pathogenic bacteria and fungi including *Candida*-like species and dermatophytes (Figure 3).

<figure><img src="image_3.png" /><figcaption>**Figure 2:** Effect of incubation period on antimicrobial activity of *Streptomyces* sp. ActiF450 against *Staphylococcus* spp. (A) and *Candida* spp. strains (B).</figcaption></figure>
<figure><img src="image_4.png" /><figcaption>**Figure 3:** Antifungal activity of *Streptomyces* sp. ActiF450 on the growth of different test organisms. The active strain ActiF450 was inoculated as a spot in the center of ISP2 plates at 30 °C for 7 days. After, the plates were then covered by 10 ml of PDA previously inoculated with target fungi.</figcaption></figure>
Extraction of bioactive molecules

As an effort to study the antimicrobial potential of the strain, assays were conducted on the different extracts of ActiF450 against Candida albicans sp. ICF23 and S. aureus IC30 (Table 2). Traditionally, the discovery of bioactive molecules from microbial sources such as actinomycetes has generally involved, cultivation under different growth conditions, screening of biological activities, extraction of the metabolites, and analysis of the extract for bioactivity. The results reflected that the ethyl acetate, hexane and methanol extracts dissolved in DMSO at 10 % exhibited potent activity against both C. albicans ICF23 and S. aureus IC30. Other extracts showed moderate activity. In addition, significant differences in susceptibility were noted; where S. aureus was high susceptible to ActiF450 extracts compared to C. albicans.

This fact might indicate the existence of a synergistic effect among different metabolites produced by ActiF450 which is lost when they are separated during the extraction (Genilloud, 2017). Otherwise, the antifungal activity might be related to the presence of proteins or enzymes labile to hydrophilic solvents such as chitinases and glucanases capable of degrading the cell wall of the fungi (Benhadj et al., 2019). Studies re-

viewed by Genilloud (2017) show that actinomycetes continue to be a valuable source of antibiotic activity as they produce a broad spectrum of different antifungal and antibacterial compounds. Moreover, study results indicate a marked effect of the extraction solvents on the isolation of bioactive compounds. It has also been reported that organic solvents provides a higher efficiency in extracting compounds for antimicrobial activities (Lima-Filho et al., 2002).

Previous study on *Streptomyces malaysiensis* MJM1968 reported a strong inhibitory activity *in vitro* by dual-culture *in vitro* assay on several phytopathogenic fungi such as *Alternaria mali*, *Cladosporium cladosporioides*, *Colletotrichum gloeosporioides*, *Fusarium chlamydosporum*, *Fusarium oxysporum*, *Rhizoctonia solani* (Cheng et al., 2010). In addition, the compound, which exhibited antifungal activity, was identified as Azalomycin F complex.

Recently, Zhang et al. (2019) have reported the effect of different fermentation factors for Azalomycin F production from S. malaysiensis strain ECO00002. Similarly, Li et al. (2008) have also reported another promising antifungal activity of perhydrofuropyran C-nucleoside named Malayamycin isolated from *Streptomyces malaysiensis* ATB-11 (Al-Tai et al., 1999).

<table><caption>**Table 2:** Effect of extraction solvent on antimicrobial activity of ActiF450</caption><thead><tr><th rowspan="3">Extraction solvent</th><th colspan="4">Antimicrobial activity (mm)</th></tr><tr><th>Candida albicans sp. ICF38</th><th>S. aureus IC30</th><th>Candida albicans sp. ICF38</th><th>S. aureus IC30</th></tr><tr><th colspan="2">Dissolved in methanol</th><th colspan="2">Dissolved in DMSO 10 %</th></tr></thead><tbody><tr><td>Dichloromethane</td><td>15.5±1.5</td><td>-</td><td>19.0±1.0</td><td>-</td></tr><tr><td>Ethyl acetate</td><td>13.5±1.5</td><td>18.0±1.0</td><td>-</td><td>47.5±2.5</td></tr><tr><td>Hexane</td><td>15.0±0.0</td><td>26.5±1.5</td><td>15.5±0.5</td><td>38.5±2.5</td></tr><tr><td>Methanol</td><td>10.0±1.0</td><td>17.5±0.5</td><td>27.0±3.0</td><td>19.5±0.5</td></tr><tr><td>Ethanol</td><td>11.5±0.5</td><td>-</td><td>15.5±1.5</td><td>-</td></tr><tr><td>Butanol</td><td>15.0±1.0</td><td>16.0±1.0</td><td>09.5±0.5</td><td>-</td></tr></tbody></table>
CONCLUSION

The present study provides evidence that Algerian wetlands are a rich and valuable resource of potentially active actinomycetes. Concisely, this study highlighted for the first time the antimicrobial potentials of wetland-associated actinobacteria with the ability to suppress major human fungal and bacterial pathogens *in vitro*. Further studies are needed to enhance isolation and selection of actinomycetes from unexplored ecosystems for antibiotic discovery. Hopefully, these new agents will meet the challenges as we attempt to manage serious underlying infection diseases. In addition, knowledge of the actinobacteria gene clusters may provide important answers toward understanding the metabolites biosynthetic pathway.

Disclosure of interest

The authors declare no conflicts of interest regarding the publication of this study.

Funding sources

This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

REFERENCES

Alan AR, Earle ED. Sensitivity of bacterial and fungal plant pathogens to the lytic peptides, MSI-99, magainin II, and cecropin B. Mol Plant Microbe Interact. 2002;15:701-8.

Albright JC, Goering AW, Doroghazi JR, Metcalf WW, Kelleher NL. Strain-specific proteogenomics accelerates the discovery of natural products via their biosynthetic pathways. J Ind Microbiol Biotechnol. 2014;41:451-9.

Al-Tai A, Kim B, Kim SB, Manfio GP, Goodfellow M. *Streptomyces malaysiensis* sp. nov., a new streptomycete species with rugose, ornamented spores. Int J Syst Evol Microbiol. 1999;49:1395-402.

Antinori S, Corbellino M, Parravicini C. Challenges in the diagnosis of invasive fungal infections in immunocompromised hosts. Curr Fung Infect Rep. 2018;12: 12-22.

Augustine SK, Bhavsar SP, Baserisalehi M, Kapadnis BP. Isolation, characterization and optimization of antifungal activity of an actinomycete of soil origin. Indian J Exp Biol. 2004;42:928-31.

Badji B, Riba A, Mathieu F, Lebrihi A, Sabaou N. Activité antifongique d'une souche d'Actinomadura d'origine saharienne sur divers champignons pathogènes et toxinogènes. J Mycol Méd. 2005;15:211-9.

Benammar L, Menasria T, Chergui A, Benfiala S, Ayachi A. Indoor fungal contamination of traditional public baths (Hammams). Int Biodeter Biodegrad. 2017;117C:115-22.

Benhadj M, Gacemi-Kirane D. Les actinomycètes: source de biomolécules d'intéret. Sarrebruck: Éditions universitaires européennes, 2016.

Benhadj M, Gacemi-Kirane D, Toussaint M, Hotel L, Bontemps C, Duval RE, et al. Diversity and antimicrobial activities of *Streptomyces* isolates from Fetzara Lake, north eastern Algeria. Ann Biol Clin. 2018; 76: 81-95.

Benhadj M, Gacemi-Kirane D, Menasria T, Guebla K, Ahmane Z. Screening of rare actinomycetes isolated from natural wetland ecosystem (Fetzara Lake, north-eastern Algeria) for hydrolytic enzymes and antimicrobial activities. J King Saud Univ Sci. 2019;31:706-12.

Bentley SD, Chater KF, Cerdeño-Tárraga AM, Chalis GL, Thomson NR, James KD, et al. Complete genome sequence of the model actinomycete *Streptomyces coelicolor* A3(2). Nature. 2002;417(6885):141-7.

Boukoucha M, Menasria T, Bouguerra N. Phenotypic characterization and genotypic subtyping of *Salmonella enterica* Serovars enteritidis and Gallinarum isolated from human and poultry-related samples. Food Biotechnol. 2018;32:206-21.

Casadevall A. Fungal diseases in the 21st century: the near and far horizons. Pathog Immun. 2018;3:183-96.

Chaudhary HS, Yadav J, Shrivastava AR, Singh S, Singh AK, Gopalan N. Antibacterial activity of actinomycetes isolated from different soil samples of Sheopur (A city of central India). J Adv Pharm Technol Res. 2013;4:118-23.

Cheng J, Yang SH, Palaniyandi SA, Han JS, Yoon TM, Kim TJ, et al. Azalomycin F complex is an antifungal substance produced by *Streptomyces malaysiensis* MJM1968 isolated from agricultural soil. J Korean Soc Appl Biol Chem. 2010;53:545-52.

Chun J, Lee JH, Jung Y, Kim M, Kim S, Kim BK, et al. EzTaxon: a web-based tool for the identification of prokaryotes based on 16S ribosomal RNA gene sequences. Int J Syst Evol Microbiol. 2007;57:2259-61.

de Lima Procópio RE, da Silva IR, Martins MK, de Azevedo JL, de Araújo JM. Antibiotics produced by *Streptomyces*. Braz J Infect Dis. 2012;16:466-71.

Genilloud O. Actinomycetes: still a source of novel antibiotics. Nat Prod Rep. 2017;34:1203-32.
Kelly KL, Judd DB. ISCC-NBS color-name charts illustrated with centroid colors. Washington, DC: U.S. National Bureau of Standards, 1964 (Supplement to National Bureau of Standards circular, Vol. 553).

Kieser T, Bibb MJ, Buttner MJ, Chater KF, Hopwood DA. Practical *Streptomyces* genetics. Norwich: John Innes Foundation, 2000.

Komaki H, Sakurai K, Hosoyama A, Kimura A, Igarashi Y, Tamura T. Diversity of nonribosomal peptide synthetase and polyketide synthase gene clusters among taxonomically close *Streptomyces* strains. Sci Rep. 2018;8(1):1-11.

Li W, Csukai M, Corran A, Crowley P, Solomon PS, Oliver RP. Malayamycin, a new streptomycete antifungal compound, specifically inhibits sporulation of *Stagonospora nodorum* (Berk) Castell and Germano, the cause of wheat glume blotch disease. Pest Manag Sci. 2008;64:1294-302.

Lima-Filho JVM, Carvalho AF, Freitas SM, Melo VM. Antibacterial activity of extracts of six macroalgae from the northeastern Brazilian coast. Braz J Microbiol. 2002;33:311-4.

Liu N, Tu J, Dong G, Wang Y, Sheng C. Emerging new targets for the treatment of resistant fungal infections. J Med Chem. 2018;61:5484-511.

LPSN. List of Prokaryotic names with Standing in Nomenclature. Founded in 1997 by Jean P. Euzéby. 2019. http://www.bacterio.net/.

Mehalaine S, Belfadel O, Menasria T, Messaili A. Chemical composition and antibacterial activity of essential oils of three medicinal plants from Algerian semi-arid climatic zone. Phytothérapie. 2017;15:1-9.

Menasria T, Samir TINE, Mahcene D, Benammar L, Megri R, Boukoucha M, et al. External bacterial flora and antimicrobial susceptibility patterns of *Staphylococcus* spp. and *Pseudomonas* spp. isolated from two household cockroaches, *Blattella germanica* and *Blatta orientalis*. Biomed Environ Sci. 2015;28:316-20.

Menasria T, Aguilera M, Hocine H, Benammar L, Ayachi A, Si Bachir A, et al. Diversity and bioprospecting of extremely halophilic archaea isolated from Algerian arid and semi-arid wetland ecosystems for halophilic-active hydrolytic enzymes. Microbiol Res. 2018;207:289-98.

Menasria T, Monteoliva-Sánchez M, Benammar L, Benhadj M, Ayachi A, Hacène H, et al. Culturable halophilic bacteria inhabiting Algerian saline ecosystems: a source of promising features and potentialities. World J Microbiol Biotechnol. 2019;35(9):132.

Merradi M, Kassah-Laouar A, Ayachi A, Heleili N, Menasria T, Hocquet D, et al. Occurrence of VIM-4 metallo-β-lactamase-producing *Pseudomonas aeru-

ginosa* in an Algerian hospital. J Infect Dev Ctries. 2019;13:284-90.

Niu G, Chater KF, Tian Y, Zhang J, Tan H. Specialised metabolites regulating antibiotic biosynthesis in *Streptomyces* spp. FEMS Microbiol Rev. 2016;40:554-73.

Onaka H. Novel antibiotic screening methods to awaken silent or cryptic secondary metabolic pathways in actinomycetes. J Antibiot. 2017;70:865-70.

Richardson MD, Warnock DW. Fungal infection: diagnosis and management. 4th ed. Hoboken, NJ: Wiley-Blackwell, 2012.

Saitou N, Nei M. The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol. 1987;4:406-25.

Sazak A, Şahin N, Güven K, Işık K, Goodfellow M. *Streptomyces samsunensis* sp. nov., a member of the *Streptomyces violaceusniger* clade isolated from the rhizosphere of *Robinia pseudoacacia*. Int J Syst Evol Microbiol. 2011;61:1309-14.

Seipke RF, Kaltenpoth M, Hutchings MI. *Streptomyces* as symbionts: an emerging and widespread theme? FEMS Microbiol Rev. 2012;36:862-76.

Supong K, Sripreechasak P, Tanasupawat S, Danwisetkanjana K, Rachtawee P, Pittayakhajonwut P. Investigation on antimicrobial agents of the terrestrial *Streptomyces* sp. BCC71188. Appl Microbiol Biotechnol. 2017;101:533-43.

Tamura K, Stecher G, Peterson D, Filipski A, Kumar S. MEGA6: molecular evolutionary genetics analysis version 6.0. Mol Biol Evol. 2013;30:2725-9.

Vijayakumar R, Panneerselvam K, Muthukumar C, Thajuddin N, Panneerselvam A, Saravanamuthu R. Optimization of antimicrobial production by a marine actinomycete *Streptomyces afghaniensis* VPTS3-1 isolated from Palk Strait, East Coast of India. Indian J Microbiol. 2012;52:230-9.

Vos P, Garrity G, Jones D, Krieg NR, Ludwig W, Rainey FA, et al. Bergey's manual of systematic bacteriology. New York, NY: Springer-Verlag, 2009.

Weisburg WG, Barns SM, Pelletier DA, Lane DJ. 16S ribosomal DNA amplification for phylogenetic study. J Bacteriol. 1991;173:697-703.

Zhang Q, Bai T, Li M, Shi Z, Yang Q, Peng R, et al. Optimization of seed culture conditions of S. malaysiensis for producing azalomycin F. Southwest China J Agric Sci. 2019;32:316-21.

Zhou S, Yang X, Huang D, Huang X. *Streptomyces solisilvae* sp. nov., isolated from tropical forest soil. Int J Syst Evol Microbiol. 2017;67:3553-8.