Diversity and Antimicrobial Activities of Actinobacteria Isolated from Mining Soils in Midelt Region, Morocco

Multidrug-resistant bacteria have emerged as a serious global health threat that requires, more than ever before, an urgent need for novel and more effective drugs. In this regard, the present study sheds light on the diversity and antimicrobial potential of Actinobacteria isolates in mining ecosystems. We have indeed investigated the production of bioactive molecules by the Actinobacteria isolated from abandoned mining areas in Midelt, Morocco, where average contents of lead (Pb) and cadmium (Cd) are higher than normal world levels. One hundred and forty-five Actinobacteria isolates were isolated and characterized based on morphological, chemotaxonomical, biochemical, and molecular data. Most of the 145 isolates were identified as Streptomyces. Isolates affiliated to the genera Amycolatopsis, Lentzea, Actinopolymorpha, and Pseudonocardia were also found. Antimicrobial producing potentials of Actinobacteria isolates were assessed against eight test microorganisms Gram+ and Gram− bacteria and yeast. Out of 145 isolates, 51 showed antimicrobial activities against at least one test microorganism. 31 isolates inhibited only bacteria, 7 showed activity against bacteria and Candida albicans, and 13 displayed activity against C. albicans solely. Our findings suggest that Actinobacteria isolated from natural heavy metal ecosystems may be a valuable source of novel secondary metabolites and therefore of new biotechnologically promising antimicrobial compounds.


Introduction
Actinobacteria are a group of Gram-positive flamentous bacteria with high G+C DNA content and are ubiquitously found in aquatic and terrestrial environments [1]. Tese bacteria represent the most renowned group of microorganisms for the production of bioactive compounds with diversifed chemical structures [2,3]. Tey are well known for antibiotics and antitumor agents production and are considered the source of about 45% of the obtained molecules from the microbial origin [4][5][6]. Among Actinobacteria, Streptomyces is still considered the best important source of bioactive secondary metabolites [6].
Since the discovery of streptomycin from Streptomyces griseus in 1944, immense and various antimicrobial and antitumor products produced by Streptomyces have gained worldwide scientifc consideration [7]. Generally, 80% of discovered antibiotics are produced by the genus Streptomyces and rare Actinobacteria, such as Actinomadura, and only 20% are synthesized by fungal species [8]. Actually, genomes of Streptomyces species can possess a panel of biosynthetic gene clusters (BGCs) responsible for the production of various secondary metabolites with antimicrobial activities [9][10][11].
Resistance to antibiotics remains a serious global health issue, and there is an urgent need for novel and efective antibiotics. By the end of the last century, despite the extensive eforts in screening soil-dwelling Actinobacteria for new bioactive compounds for pharmaceutical use, most attempts went in vain [12]. As a result, investigating Actinobacteria living in unexplored habitats such as desert soils, marine biotopes, saline environments, and other extreme environments, in which certain physical and/or chemical factors difer signifcantly from those in natural and wellstudied habitats, has shown great importance because of the novelty of their antimicrobial compounds [13,14].
Lead mines (Ahouli-Zaida-Mibladen), located in Midelt, Morocco, were considered as the biggest Moroccan Pb mining sites of the last century. Currently abandoned, they remain largely unexplored and unexploited. To the best of our knowledge, no prior study has reported the diversity and antimicrobial efects of Actinobacteria living in these mining ecosystems. Tus, the present work aims frstly to isolate and characterize Actinobacteria from extreme mines and secondly to analyze the distribution and the antimicrobial potential of those isolates. Four distinct locations within an area of 20 m 2 were determined for sampling in each site. Each soil sample was collected at 5 to 10 cm depth in sterilized plastic bags and stored at 4°C for further analyses. For chemical analyses, samples were mixed to generate a composite soil and sieved until a fne powder was obtained.

Materials and Methods
One gram of each soil sample was used to determine organic matter content. Soil samples were dried in an oven at 500°C for 4 h. After drying, each sample was weighed to determine the lost weight [24]. Soil pH was measured with a calibrated pH 210 Microprocessor pH meter (Hanna Instruments, Romania). Five grams of soil sample were mixed with 50 mL of distilled water. After shaking for 20 min, the suspension was left to settle for 2 hours. Te concentrations of 10 metals, Cd 2+ , Co 2+ , Cr 2+ , Cu 2+ , Fe 2+ , Mg 2+ , Na + , Ni 2+ , Pb 2+ , and Zn 2+ , were determined by inductively coupled plasma atomic emission spectroscopy (ICP-AES) using a spectrometer (Activa Horiba Jobin Yvon-Ovou 1048, France) after mineralization according to the method recommended by Alsac [25]. An amount of 0.5 g of the soil powder was mixed with 6 mL of hydrochloric acid and 2 mL of nitric acid. Te mixture obtained was incubated at 95°C for 75 min, and the volume of the obtained fltrate was adjusted to 50 mL prior to ICP-AES analysis. Te concentration values were expressed in mg/kg (metal/sol).

Isolation of Actinobacteria.
Te isolation of Actinobacteria was carried out using the spread plate method by serially diluting 1 g of each sample in 9 mL of distilled water using SCA as a culture medium. Prior to isolation, suspended soil samples were incubated in a shaking incubator at 30°C for 30 min and 250 rpm, and then the suspension was sonicated 3 times for 30 s ON and 30 s OFF at 60 kHz. Dilutions up to 10 -6 were prepared from each soil sample and 100 μL of each dilution was plated onto the SCA medium. Te agar plates were incubated at 30°C and examined every 2 days for microbial growth for up to 15 days. Morphologically distinct colonies with a special earthy odor were purifed through repeated subculturing, and then sporulating and non-sporulating pure isolates were stored in sterile glycerol (20%, v/v) at −80°C.  [26]. Micromorphology and sporulation of isolates were examined under the light microscope (Olympus, Japan).

Chemotaxonomical Study.
For the chemotaxonomical study, the isolates were cultured in shake fasks (150 rpm, 30°C, 7 days) using modifed Bennett's medium broth (glucose 10.0 g, yeast extract 1.0 g, meat extract 1.0 g, and distilled water 1000 mL; pH 7.0 ± 0.2). Te dried biomass of each isolate was obtained by centrifugation at 9000 g. It was washed 3 to 5 times until complete elimination of any trace of culture medium before being well dried overnight at 45°C.
Regarding the diagnostic isomers of diaminopimelic acid (DAP), the dried biomass (10 mg) was hydrolyzed for 18 h with 1 mL of 6 N HCI in a sealed tube by incubation at 100°C in a sand bath. After cooling, the mixture was fltered through paper flter. Te liquid hydrolysate was dried at 40°C, washed with distilled water to remove most of the HCl, and then dried again at 40°C. Te remaining residue was solubilized in 0.3 mL of distilled water, and 20 µL of the mixture was spotted on Whatman no. 1 paper with the amino acid standards [27]. Tin-layer chromatography (TLC) was performed for 4 hours by elution with methanolwater-10 N HCl-pyridine (80 : 17.5 : 2.5 : 10). Amino acids were detected by spraying the paper with acetone ninhydrin solution (0.1%, w/v), followed by heating for 2 min at 100°C. Diaminopimelic acid spots were green-olive fading to yellow, and thus the LL form of DAP acid migrates faster than the DL form. Other amino acids, such as glycine, in the mixture gave purple-pink spots and moved faster than the diaminopimelic acid [27].
As for the whole-cell sugar pattern, the dried biomass (25 mg) was hydrolyzed for 2 h with 1.5 mL of 1 N sulfuric acid in a sealed tube and incubated in a boiling water bath. After cooling, the obtained hydrolysate was poured into a conical centrifuge tube, and saturated barium hydroxide solution was added by drops until the pH reached about 5.2-5.5. Te supernatant, obtained after centrifugation at 9000 g, was evaporated to dryness, and the residue was redissolved in 0.3 mL of distilled water. A volume of 1 µL of the mixture was deposited on the base line of a cellulose plate (DC-Fertigfolien, F 1440, Cellulose) with 1 µL of two solutions containing standard sugars. Te frst solution contains Lrhamnose, L-mannose, D-glucose, and D-ribose at 1%, and the second one consists of D-galactose, L-arabinose, and Dxylose, each at 1% as well [28]. TLC was performed with an elution system composed of n-butanol-distilled water-pyridine-toluene (10 : 6:6 : 1) for approximately 4 h. Te spots were developed by spraying the plate with acid aniline phthalate solution (3.35 g phthalic acid solubilized in 100 mL of watersaturated butanol plus 2 mL of aniline) and dried at 100°C for 5 min. Te hexoses (glucose, galactose, and mannose) appeared with a yellow color and the pentoses (ribose, xylose, and arabinose) developed a pink color after drying [28].
Te degradation of casein (1%, w/v, skimmed milk), tyrosine (0.5%, w/v), and starch (1%, w/v) was carried out in agar nutrient medium. Nitrate reduction was studied by the addition of 0.2 mL of Griess reagent to the culture carried out on a nitrate medium composed of nutrient broth supplemented with 0.1% (w/v) of KNO 3 . Te appearance of red color indicates the reduction of nitrates. For the decomposition of urea, 10 mL of 15% of urea solution sterilized by fltration was added to 75 mL of sterile urea broth (KH 2 PO 4 10.0 g, Na 2 HPO 4 9.5 g, yeast extract 1.0 g, 0.04% of phenol red solution 20 mL, and distilled water 1000 mL, pH 6.7). Te mixture was transferred aseptically into 2.5 mL volume sterile tubes and inoculated with the tested isolate. An alkaline reaction demonstrated the presence of urease. Catalase production was carried out by adding a few drops of H 2 O 2 onto old colonies cultivated on Bennett's agar medium.
Analysis of the ability of isolates to grow on Bennett's agar medium at diferent temperatures (15,37, and 42°C) was performed. All results of biochemical and physiological studies were recorded after incubation for 7 days at 30°C.

Molecular Identifcation.
For DNA extraction, a bacterial pellet was prepared from a pure colony culture. Te pellet was resuspended in 500 µL of TE bufer (10 mmol/L Tris-HCl and 1 mmol/L EDTA; pH 8), and then 25 µL of SDS (20%) was added. Te mixture was incubated under stirring at 50°C for 10 min. After the incubation period, the mixture was centrifuged at 12000 g for 10 min. A 0.8 × V volume of isopropanol was added to the supernatant, and the mixture was centrifuged at 15000 g for 10 min at room temperature. Te obtained pellet was washed twice with 75% ethanol, dried, and then resuspended in 50 µL of distilled water.
Amplifcation of the 16S rDNA was carried out in a Mastercycler personal thermocycler (Germany) using the universal primers: 27F (AGAGTTTGATCCTGGCTCAG) and 1492r (GGTTACCTTGTTACGACTT). Te reaction mix was prepared at a fnal volume of 20 µL containing 2 µL of Taq bufer (5x), 1. Sanger sequencing was carried out using an ABI PRISM 3130XL Genetic Analyzer (Applied Biosystems) at the Innovation Center (USMBA, Fez, Morocco). Te 16S rRNA gene sequences were compared with the GenBank database using BLASTN. Multiple sequence alignments were performed using ClustalW [30]. Te neighbor-joining method was applied for the construction of the phylogenetic tree, and the analysis was conducted in MEGA11 [31,32].
Each isolate was spot deposited on culture media, and the agar plates were incubated at 30°C for 7 days. Afterward, these plates were covered with a 5 mL soft agar (0.5% agar) of either LB (Luria-Bertani) medium for bacteria or YPG (yeast extract-peptone-glucose) medium for Candida growth, preinoculated with 100 µL from an overnight culture of the test microorganism (OD600 nm ≈ 0.1). Tese cultures were poured carefully on the surface of the plates and incubated during 24 h at 30°C for C. albicans and 37°C for the tested bacteria. Antimicrobial activity was assessed by measuring the diameter of the clear zone of growth inhibition (DI = mm). Each experiment was conducted in

Soil Sample Characterization and Isolation of
Actinobacteria. pH, organic matter, and trace metal elements were measured for each soil sample collected from the mining sites. Te soil's pH average values varied from 7.08 (Zaida site) to 8.79 (Ahouli site), characterizing the soil as slightly alkaline (Table 1). Trace metal element concentrations varied considerably between samples from diferent sites but also between samples within the same site. Tis was mainly observed for Cr, Cu, Fe, Na, Pb, and Zn. For all soil samples, except S1, S2, and S4 taken from the Midelt site, the content of Pb was greater than the normal world averages (35 mg/kg) for uncontaminated soils given by Bown [33] (P < 0.05). Tis content ranged from 47.97 to 113.86 mg/kg, 495.28 to 55623 mg/kg, and 131.80 to 634.69 mg/kg in Ahouli, Mibladen, and Zaida sites, respectively, compared to 35 mg/kg measured in the normal world averages (Table 1). Cd content was also higher than the normal world average (0.35 mg/kg) (P < 0.01) with average values ranging from 3.58 mg/kg in the Ahouli site to 5.15 mg/kg in the Zaida site. Te average values of Pb and Cd exceeded the normal world averages (these values are 2.08, 11.10, and 69.01 folds increase in Ahouli, Zaida, and Mibladen sites, respectively, for Pb, and 10 to 14.73 folds increase for Cd in the four sites), revealing that the study areas, mainly Mibladen and Zaida sites, are highly rich in Pb and Cd metals (Table 1).
Te assessment of organic matter revealed that the Zaida site exhibits a low content with an average of 1.66%. Te maximum content of organic matter reached an average of 4.3% in the Ahouli site, followed by an average of 3.05 and 3.56% in the Mibladen and Midelt sites, respectively. Te bacterial concentration determined in all sites varied considerably between 9.2 × 10 4 and 2.28 × 10 8 CFU/g of soil. One hundred and forty-fve isolates were distributed over the isolation sites as follows: 41 in the Ahouli site, 19 in the Mibladen site, 25 in the Midelt site, and 60 isolates in the Zaida site (Table 1).
A positive correlation was observed between the bacterial load and both Mg and Cd metals at the four studied sites. A negative correlation was observed between Pb and Zn metals and both bacterial load and number of Actinobacteria isolates in Ahouli and Midelt sites. However, in the Zaida site, a positive correlation was observed between Pb and the bacterial load. No infuence of the Pb concentration was detected on the bacterial load and the Actinobacteria isolates in Mibladen site ( Figure 2).
Te infuence of the other metal ions on the two parameters (bacterial load and Actinobacteria isolates) was variable in all sites. For organic matter, the infuence of this parameter on both bacterial load and Actinobacteria isolates was variable in all samples with a preference for a high percentage of organic matter ( Figure 2).

Phenotypic Analysis of Actinobacteria Isolates.
Te phenotypic characterization of the 145 actinobacterial isolates was carried out through the observation of Actinobacteria's distinctive characteristics, such as colony pigmentation, difusible and melanoid pigments, the color of aerial and substrate mycelia, spore chains, and aerial hyphae arrangement. Chemotaxonomical and classical biochemical tests were performed to better characterize the isolates.
Based on the above mentioned tests, the 145 isolates were grouped into several clusters, each containing many subclusters, suggesting a potential diversity within the isolated Actinobacteria. Figures 3(a) and 4(a) illustrate the clustering according to the morphological and biochemical studies, classifying the isolates into more than 12 groups.
According to Bergey's Manual of Systematic Bacteriology [34], phenotypic characteristics positioned the isolates at the genus level. Most isolates grew well on Bennett's and ISP2 agar plates and showed well-developed aerial hyphae and a good sporulation on both media. Tey also produced a wide range of pigments (responsible for the color of substrate and aerial mycelia) and can produce difusible pigments ranging from yellow to brown-red (Figure 3(b)). Te aerial mycelium, which rarely fragments, forms short, medium, or long spore chains. Some isolates were able to hydrolyze starch, produce nitrate reductase, and show good growth on specifc carbohydrates as the only source of carbon.
Te chemotaxonomical study of isolates showed that their peptidoglycan cell contained LL-isomer of DAP in addition to the glycine, glutamic acid, and alanine for most isolates. Characteristic sugars in the whole-cell hydrolysates were rarely present and even absent for most isolates, and these properties are characteristic of Streptomyces, which represent 87.59% of all isolated Actinobacteria. Te other isolates (no Streptomyces) contained in their peptidoglycan the DL isomer of DAP with or without glycine. In addition, they contained some characteristic sugars such as the combination of the three sugars: galactose, arabinose, and mannose or the combination of the two sugars: galactose and arabinose.
All the gathered data showed that the 145 actinobacterial isolates belong to fve bacterial genera, which are Streptomyces, Amycolatopsis, Pseudonocardia, Lentzea, and Actinopolymorpha. All the studied sites contain Streptomyces as an abundant genus (Figure 4(b)).
Te analysis of the results illustrated in Figure 4(b) showed that the Ahouli and Mibladen sites contain, in addition to Streptomyces, other bacterial genera: Lentzea and Actinopolymorpha, with a percentage of 9.76% and 7.32% in Ahouli site and 5.26% for each genus in Mibladen site, respectively. Midelt site contains only one isolate afliated to the genus Actinopolymorpha (4%), and Zaida site contains 3 genera: Amycolatopsis (3%), Pseudonocardia (5%), and Lentzea (5%) (Figure 4(c)). It should be noted that genera Amycolatopsis and Pseudonocardia were present only in Zaida site, characterized by its high content mainly in sodium (Na), which indicates the halophilic trait of these genera (Figure 4(c)).

Phylogenetic Analysis.
Twenty-fve strains with important antagonistic activities and fve strains (AS20, BS56, CS86, DS182, and DS189) without antimicrobial activity were identifed up to the species level using the 16S rRNA gene fragment and considered for carrying out the phylogenetic analysis. To determine the relatedness among the selected strains, 16S rRNA gene sequences were aligned with the related sequences from the GenBank database.
Te results showed that strains belong to two families Streptomycetaceae and Pseudonocardiaceae. With regard to the sampling site, the majority of strains were identifed as  Table 2).
Te neighbor-joining method was applied for the construction of the phylogenetic tree. Based on the results obtained, the sequences of Actinobacteria strains were divided into two major clades (clade I and clade II) ( Figure 5). Clade I was formed by the majority of strains belonging to the genus Streptomyces and diverged into fve subclades with a certain geographical link. Subclades Ia and Id were the largest and were clustered from sequences of strains obtained from Ahouli, Mibladen, and Zaida sites ( Figure 5). Subclade Ia is composed of sequences from strains of Mibladen and Zaida sites, very close to each other with an identical bootstrap value, and only two sequences from      Te Scientifc World Journal   from Mibladen site. Furthermore, it clustered two strains with the same bootstrap value: AS20 without activity and AS34 with activity ( Figure 5). Clades Ic and Ie were the smallest, with only one sequence for each clade: DS169 in clade Ic and CS86 in clade Ie ( Figure 5). Strains in the genera Lentzea and Amycolatopsis were clustered to subclades IIa, IIb, and IIc to create clade II. Te clade sequence IIa of Amycolatopsis DS182 was detected exclusively in Zaida site. Similarly and fnally, the clades IIb and IIc only appeared in Ahouli and Zaida sites ( Figure 5).

Antimicrobial Activities.
Te potential of each isolate to produce molecules with antimicrobial activity against both Gram-positive and Gram-negative bacteria and yeast (C. albicans) was assessed via double layer assay using three diferent culture media (Bennett's, GYM, and CSA) (Figure 6). Results indicate that among the 145 tested isolates, 51 (35.17%) exhibited antimicrobial activities against at least one test microorganism. Out of these 51 isolates, 13 (25.49%) displayed a positive activity against C. albicans, 31 (60.78%) inhibited Gram+ and/or Gram − bacteria, and 7 (13. 72%) isolates displayed a positive activity against both bacteria and C. albicans. Te strongest inhibitory activities were noted against Gram-positive bacteria (for 23 and 22 active isolates against B. subtilis and S. aureus, respectively) and the yeast C. albicans (for 20 active isolates) as compared to those tested against Gram-negative bacteria (Figure 6(a)).
As shown in the Venn diagram ( Figure 6(b)), most of the strains produced a narrow spectrum of antimicrobial compounds. Of 51 active strains, 31 (60.78%) antimicrobial producing strains seemed to be active against a specifc category of microorganisms: Gram-positive bacteria (13 strains), Gram-negative bacteria (5 strains), or C. albicans (13 strains) (Figure 6(b)). Eighteen strains (35.29%) could be characterized as producers of a middle spectrum of activity for both, Gram-positive and Gram-negative bacteria inhibition (13 strains) or Gram-positive bacteria and C. albicans inhibition (5 isolates) (Figure 6(b)). Only two (3.92%) strains, Streptomyces sp. AS22 and Streptomyces sp. DS169 could be characterized as producers of a large spectrum of activity against some of the tested Grampositive and Gram-negative bacteria, as well as the yeast C. albicans (Figure 6(b)).
Tree Streptomyces strains (AS3, AS34, and BS61) were found to be remarkably active against all tested Grampositive bacteria (S. aureus, B. subtilis, and L. innocua). Strain AS34 had a high inhibitory efect on the growth of three tested Gram-positive bacteria with inhibition zones ranging from 36.50 ± 09.26 to 80.25 ± 01.36 mm in all tested media (Figure 6(c)).
It is worth mentioning that BS61 lost its activity against L. innocua when cultivated on GYM and Bennett's media. In addition, strains AS28, AS45, BS57, and BS63 showed a high production of antimicrobial compounds against Gramnegative and Gram-positive bacteria in both Bennett's and GYM media as compared to the CSA medium (Figure 6(c)   Te strain BS57 displayed a medium-dependent inhibition specifcity. It was active against B. subtilis solely in Bennett's medium, exhibited activity against E. coli and D. solani in Bennett's and GYM media and inhibited L. innocua in all tested media. Te strain BS63 displayed a middle spectrum of activity against both Gram-positive and Gram-negative bacteria. It was more active against the phytopathogenic bacteria (P. brasiliensis and D. solani) in the GYM medium with 42.38 ± 00.65 and 29.83 ± 04.65 mm inhibition zones towards P. brasiliensis and D. solani, respectively. However, the strain was inactive against E. coli and D. solani when cultivated on the CSA medium ( Figure 6(c)).
Te strain DS169 inhibited the growth of C. albicans when grown on Bennett's and CSA media and was able to inhibit the growth of S. aureus only on Bennett's medium. Four isolates (BS68, BS69, DS106, and DS107) were similarly active only against C. albicans on the three used culture media (Figure 6(c)).
Representative agar plate pictures illustrating selected examples of the antagonistic activities are shown in Figure 7

Discussion
Te Ahouli-Mibladen-Zaida mines were the largest Moroccan Pb-Zn mining districts of the last century. Tey had been discovered in 1916 and were closed back in 1986. A hundred-year period could have been sufcient for the development and the adaptation of microorganisms to severe living conditions.
In line with prior research on the assessment of trace metal contamination levels in sediments and water from these mining sites [16,35,36], we have been exploring extreme environments (the Dead Sea in Jordan and hypersaline sites in the Pre-Rif region in Morocco) [22,37] for the isolation of bacteria of biotechnological potential. We were the frst group searching for Actinobacteria from Ahouli-Mibladen-Zaida mining areas and determining their potential to produce antimicrobial agents.
From the data analysis, we noted that the bacterial load and the number of Actinobacteria isolates inside samples decreased along with the increase of Pb concentration in the Ahouli and Midelt sites. In the Zaida site, the bacterial load is proportional to Pb concentration. We speculate that this increase or decrease might be due to the ability of bacteria to adapt (Mibladen and Zaida sites) or not (case of Ahouli and Midelt sites) to heavy metals in the diferent mining sites. In accordance with our fndings, prior studies have shown that heavy metals in lead-zinc mines decrease microbial diversity by increasing Proteobacteria and by decreasing Actinobacteria and other phyla [38][39][40][41]. Other studies demonstrated that Actinobacteria and Proteobacteria were predominant in heavy metal contaminated soils [42][43][44][45]. Terefore, the present study on actinobacterial isolates from mining sites in the region of Midelt, Morocco, will be considered as an adding value to the previous researches on Actinobacteria.
Phenotypic analysis based on morphological, chemotaxonomical, and biochemical characteristics afliated the Actinobacteria isolates to fve genera, including Streptomyces, Pseudonocardia, Lentzea, Amycolatopsis, and Actinopolymorpha. We have also observed some diferences at the genus level between sites: isolates of the genus Actinopolymorpha were found only in soil samples of Ahouli and Mibladen, while Amycolatopsis and Pseudonocardia were identifed only within the Zaida mining site. Isolates afliated to these latest genera or to the genus Lentzea were found in the soil samples of Ahouli, Mibladen, and Zaida sites, which present higher concentrations of Pb when compared to the Midelt site where Streptomyces was predominant, with only one isolate belonging to the genus Actinopolymorpha. Tis indicates that the chemical characteristics, mainly the high Pb concentration, might be favorable to these bacterial genera. However, the Amycolatopsis and Pseudonocardia genera were identifed as habitat-specifc genera since they were specifcally found only in the Zaida site. Tese diferences could also be attributed to the specifc physicochemical and biological characteristics of each mining site.
Te most abundant genus from all sites was Streptomyces (87.59%) followed by Lentzea (5.51%). Tis fnding goes in line with previous studies showing that Streptomyces is the most abundant microorganism in heavy metal natural soils and other heavy metal contaminated and uncontaminated soils [46][47][48][49]. It has also been reported that several Streptomyces strains were resistant to heavy metal elements [47][48][49][50][51][52][53].    DI  80  70  60  50  40  30  20  10  0   AS22  AS25  AS28  AS30  AS34  AS37  AS45  AS52  BS54  BS57  BS58  BS59  BS60  BS61  BS63  BS68  BS69  CS79  CS88  CS89  DS93  DS102  DS103  DS104  DS105  DS106  DS107  DS109  DS120  BS132  BS134  BS136  BS137  CS138  CS143  CS145  CS147  DS149  DS151  DS155  DS156  DS158  DS159  DS161  DS167  DS169  DS174   Te rare genera belonging to the Pseudonocardiaceae family (Amycolatopsis and Pseudonocardia) found in our studied areas have been previously reported from other heavy metal soils and were reported to be resistant to certain metals [46,47,54,55]. However, little is known about the Lentzea genus living in heavy metal ecosystems [56,57], and no prior data have been reported on the isolation of Actinopolymorpha from heavy metal ecosystems. Twenty-nine strains with high antimicrobial potential and other fve inactive strains were characterized at the species level. All strains were classifed within two families and three genera, indicating a partial diversity among mining Actinobacteria. Te composition of Actinobacteria as obtained by the phylogenetic tree was more diverse as compared to Actinobacteria isolated from abandoned mining areas of Marrakech, Morocco [47]. Streptomyces strains fall under one major clade which is divided into fve subclades (Ia-Ie), and rare Actinobacteria Amycolatopsis and Lentzea were assembled together to form a second major cluster. More than half of the strains displayed diferent phenotypic characteristics and bioactivity.
Regarding bioactivity, strains with closely similar 16S rDNA sequences showed diferent bioactivity profles. For example, the strain AS20 (OP122985) from site Ahouli is without antimicrobial activity and is clustered with the strain AS34 (OP125838), from the same site, which is potentially active against Gram-positive bacteria. Te same observation is noted for the inactive strain BS56 (OP131863) which is closer to DS102 strain (OP164544) which was active against C. albicans suggesting a potential diversity among strains from the same site.
In general, the phylogeny of Streptomyces strains showed a geographical-antimicrobial relationship between the subclades of clade I. Subclade Ia contained the majority of strains from Zaida and Mibladen sites displaying activity against only C. albicans, while subclade Id was composed of strains with diferent antimicrobial activities and isolated from three diferent sites. It is worth mentioning that the strain CS86 (OP164536), without antimicrobial activity,  from the Midelt site, forms another subclade which difers from the major subclades. Tis suggests a potential diversity among Streptomyces strains. Rare Actinobacteria DS189 (OP141210) and AS16 (OP117486) of the genus Lentzea isolated from two diferent sites were clustered in two different subclades, and only AS16 displayed antimicrobial activity. Our results show that Actinobacteria strains display certain selectiveness towards tested pathogens, and that there is certainly a relationship between the antimicrobial capacities of these strains and their geographical site. Tis result is in line with the fndings of previous studies [58]. Soil Actinobacteria have been largely studied, worldwide, for their production of antimicrobial agents. In Morocco, several studies have reported the isolation of Actinobacteria from many biotopes and demonstrated their antimicrobial potential. Recently, Rammali et al. highlighted the isolation of Streptomyces species from cold biotopes showing antimicrobial and antioxidant potentials [23]. Nafs et al. reported two new non-polyenic macrolide derivatives from Streptomyces Z26 isolated from a rhizospheric soil showing antifungal activity [59]. From the unexplored hot Merzouga desert, one hundred and sixty-three Actinobacteria isolates were obtained, of which 59% showed a high biological diversity with the greatest growth inhibition of pathogens tested [21]. Te study reported by Oubaha et al. shows that Streptomyces species isolated from rhizospheric soils displayed a potent inhibitory efect against Aphanomyces euteiches causing the damping-of disease of pea (Pisum sativum L.) [60]. However, Actinobacteria from Moroccan mining habitats have not been investigated for their biocontrol potential. Tis shows the interest of our study for the valorisation of Actinobacteria living in these biotopes by researching their antimicrobial potential.
From the obtained data, it was established that the majority of the 51 bioactive strains in all sites are potent antimicrobial strains and belonged to Streptomyces sp. For example, Streptomyces sp. AS34, from the Ahouli site, showed strong and specifc antibacterial activity against Grampositive bacteria. Streptomyces sp. AS22, from the same site, produces a large spectrum of activities with a mix of Gram-positive and Gram-negative bacteria and the yeast C. albicans. Similarly, previous reports have indeed shown that species of Streptomyces produce a variety of bioactive metabolites with both antibacterial and antifungal activities [23,61,62]. Moreover, other Streptomyces sp. strains (AS28, AS45, BS57, and BS58) and Amycolatopsis sp. DS158 exhibited relatively important activities against both Grampositive and Gram-negative bacteria in line with previous studies that reported strains belonging to the Amycolatopsis genus exhibiting diferent antimicrobial compounds [63,64].
Some strains afliated to the Lentzea genus in the current study have antimicrobial activities that corroborate previous reports; this includes, for example, the recently reported strain Lentzea tibetensis sp. nov. that exhibits antimicrobial activity against Gram-positive bacteria and Fusarium oxysporum [65]. Antimicrobial activities in Lentzea indicate that rare Actinobacteria strains are appropriate sources of bioactive compounds. Te recent analysis of the Lentzea genome reveals a repertoire of biosynthetic gene clusters (BGCs) among which more than 90% code for antimicrobial molecules including mainly terpenoids, lipopeptides, and thiopeptides [66].

Conclusion
Te present study is the frst of its kind to explore the diversity of actinobacterial isolates thriving in soils of mining areas with high Pb and Cd contents in the region of Midelt, Morocco. Data showed that the obtained Actinobacteria isolates were mainly assigned to fve major genera (Streptomyces, Amycolatopsis, Pseudonocardia, Lentzea, and Actinopolymorpha). Te genus Actinopolymorpha was, for the frst time, isolated from a mining ecosystem. Actinobacteria isolates afliated to Streptomyces genus exhibited a broad antimicrobial efect spectrum, indicating that mining biotopes are valuable sources of discovery for potential biocontrol Actinobacteria against human pathogen microorganisms.

Data Availability
Te data used to support the fndings of this study are included within this article. Any required further information can be provided by the corresponding author upon request.