Isolation and screening of actinomycetes producing antimicrobial substances from an extreme Moroccan biotope

Introduction This study was carried out to isolate and screen actinomycetes from soil of two salterns in Taza-Morocco, for the production of antimicrobial compounds against a set of target bacteria. Also, it aims to highlight some practices in order to isolates actinomycetes and screen for their ability to produce antibacterial compounds. Methods Soil samples were analyzed for physical and chemical parameters including pH, electrical conductivity, and salinity. The actinomycetes were isolated on Casein Starch Agar (CSA) medium and purified on International Streptomyces Project 2 (ISP-2) medium. Antimicrobial activity of actinomycete isolates was evaluated by measuring the inhibition zone. These activities were tested against Dickeya solani IP2222, Pectobacterium brasiliensis 13471a, Escherichia coli K12, Proteus mirabilis, Pseudomonas aeruginosa CECT118, Listeria innocua CECT4030, Staphylococcus aureus CECT976, Bacillus subtilis DSM 347 and Candida alibicans, using three different culture media (CSA, Bennett and Mueller Hinton) and at two temperatures of incubation (30°C and 37°C). Results Physical and chemical analysis of soil samples showed that both sites are alkaline. Also, with regards to salinity, the second site showed to contain high salt concentration compared the first site. The abundance of bacteria isolated on CSA medium from both sites showed correlation with the physical-chemical properties of the sampling soils. Incubation temperature of 30°C resulted in a high number of actinomycetes (18/22) isolates with antimicrobial effect relative to the temperature of 37°C (4/22). Some actinomycetes isolates show antimicrobial effect on only one culture medium, which shows a special nutritional requirement to express their antimicrobial effect. On the other hand, some isolates, they express their antimicrobial effect on the three media at the same time. Additionally, some isolates of actinomycetes inhibit the growth of several microorganisms at once. While others inhibit the growth of only one microorganism tested which reflects a possible specificity of antimicrobial substances. Conclusion Growth conditions including, media composition, temperature of incubation and the spectrum of test strain tailors the behavior of the antimicrobial screening.

Microorganisms found in extreme environments have attracted a great deal of attention, due to the production by such microorganisms of various natural compounds and their specialized mechanisms for adaption to extreme environments [4]. Among the various extremophiles, halophilic microorganisms have developed several strategies to survive and to function in hypersaline ecosystems, such as salterns, salt mines and other hypersaline environments [5]. The cultivation of actinomycetes from extreme environments including saline habitats is very difficult than common environments, because of their slow growth rate. In the conventional isolation techniques, several factors must be considered, namely, the choice of screening source, the selective medium, culture conditions and the recognition of candidate colonies in the primary isolation. Furthermore, choosing appropriate media and growth conditions is important and published media are typically associated with a particular microbial genus or species. As with other microbial discovery research, when working with environmental samples harboring communities of novel microbial populations, the media and growth conditions chosen will enrich for certain populations and not others [6]. Actinomycetes are known as the most biotechnologically valuable prokaryotic microorganisms.
They are well known as a source of antibiotics and bioactive molecules. Most of their bioactive molecules have been shown to have antibacterial (streptomycin, tetracycline and chloramphenicol), antifungal (nystatin), antiviral (tunicamycin) and antiparasitic (avermectin) properties [7]. Indeed, most of the antimicrobials used today in remedying diseases caused by pathogens have been developed from actinomycetes. Currently, over 5000 antibiotics have been screened from Gram positive, Gram negative bacteria as well as fungi. However, only 100 of these antibiotics have been developed to clinical applications [8]. Nowadays, the control of pathogenic microorganisms by synthetic products is becoming less attracting due to the emergence of resistant strains and because of the undesirable effects of these products on the environment. Therefore, it's absolutely necessary to find antagonistic microorganisms that are used as a means of bio-control. In this study we were interested in isolating actinomycetes from two Moroccan saline soils and screening of isolates for the production of antimicrobial substances in three Media: the Casein Starch Agar (CSA) medium used for actinomycetes isolation contained (soluble starch 10g, casein 0.3 g, KNO 2 g, NaCl 2 g, K2HPO4 2 g, MgSO4-7H2O 0.05 g, CaCO3 0.02 g, FeSO4-7H2O 0.01 g, agar 18g and distilled water to 1 l (pH 7.2) was used for isolation of actinomycetes [11]. This medium was supplemented with Ampicillin (20 μg/ml) and Fluconazol (25 μg/ml) to inhibit the growth of bacterial and fungal contaminants respectively. The International Streptomyces Project (ISP-2) medium (malt extract 10 g, yeast extract 4 g, glucose 4 g, agar 20 g and distilled water to 1 l (pH 7.3)) was used for purification of actinomycetes isolates [12]. The antimicrobial activity of the isolated actinomycetes was determined in three media: CSA, Bennett's agar medium (yeast extract 1 g, beef extract 1 g, casamino acids 2 g, glucose 10 g, agar 15 g and distilled water to 1 l (pH 7.3) [13] and Mueller-Hinton (MH) agar medium containing beef extract 2 g, acid hydrolysate of casein 17.5 g, soluble starch 1.5 g, agar 17 g and distilled water to 1 l (pH 7.4).
Strains: the strains tested (Table 1) were cultivated on Luria Bertani (LB) medium (peptone 10 g, yeast extract 5 g, sodium chloride 10 g and distilled water to 1 l) for bacteria and Sabouraud medium (Casein 5 g, meat extract 5 g, glucose 40 g, agar 15 g and distilled water to 1 l) for yeast at 30°C. Isolation of actinomycetes: 10 g of each soil sample were suspended in 100 ml of sterile physiologic water (0.9% NaCl in distilled water). Heat treatment was performed in a water bath at 50°C for 60 min under agitation [14]. Serial 10-fold dilutions of the samples were prepared with sterile 0.9% saline. Dilutions were then plated in triplicate on CSA and incubated for 4 to 6 weeks at 28°C.

Purification
and conservation of actinomycetes isolates: actinomycetes isolates were recognized by their morphological aspects and by microscopic observation. Isolates were purified by successive streaking on ISP2 medium. They were stored in 20% glycerol at -80°C and/or maintained at 4°C for less than 1 month.
Antimicrobial activity of actinomycetes isolates: antimicrobial activities of pure isolates were determined using the double layer agar method test according to the protocol described by Ouhdouch [15].
Pure actinomycetes isolates were spotted on each one of the three tested culture media: CSA, Bennett and Mueller Hinton. After incubation at 30°C or at 37°C for 7 days, the plates were exposed to chloroform vapor for 40 min. Emergent colonies were then covered with a 0.6% agar layer of Bennett's medium, previously seeded with one of the test strains (3.10 +8 -4.10 +8 CFU/ml) and incubated for 48 h at 30°C or at 37°C. Antimicrobial activity was evaluated by measuring the inhibition zone. Each test was carried out in triplicate.
For the negative control, the tested strains were grown in the same culture medium without inoculation with actinomycetes.

Results
Soil chemical analysis: pH values obtained for two sites are greater than 7, which qualifies our biotope in the category of calcareous soils (alkaline). Also, the results obtained show that the second site is very salty compared to the first site, which is qualified salty ( Table 2).
Isolation of actinomycetes: a total of 22 isolates (S1-S22) were obtained on CSA medium from the two sites, 13 isolates from the first site and 9 from the second site. In site 1, green isolates are dominant.
However, in site 2, white isolates are dominant (Table 3). Figure   2 shows the aspect of actinomycetes isolates developed on ISP-2 medium after 4 weeks at 30°C.
Antimicrobial activity of actinomycetes isolates: antimicrobial activity was evaluated by measuring the inhibition zone around the actinomycetes colonies. An example of an inhibition zone is shown in Figure 3. Screening results of actinomycetes isolates for the production of antimicrobial substances, on three culture media and at two incubation temperatures, are shown in Table 4 and Table 5.

Effect of incubation temperature on antimicrobial substances
production: among 22 actinomycetes isolated from both sites and grown at 30°C, 18 isolates seemed to inhibit the growth of at least one microorganism (≈ 82%) ( Table 4). On the other hand, when grown at 37°C, most of them aren't even capable to grow on all threeculture media and only 4 isolates (≈ 18%) showed the ability of producing antimicrobial substances ( Table 5).

Effect of culture medium on antimicrobial substances
production: screening results of actinomycetes isolates incubated at 30°C producing antimicrobial substances showed that from the 22 isolates, 11 (50%) are capable of producing antimicrobial substances on Bennett medium, 10 (> 45%) on CSA medium and only 3 on MH medium (> 13%) ( Table 4, Table 5).

Discussion
Actinomycetes are known as versatile producers of antimicrobials active metabolites [16]. Screening has always been the essential method for isolation of new antimicrobial molecules. Several parameters can condition the synthesis and the production of these antimicrobial molecules by the actinomycetes such as the composition of the culture medium, the temperature and the incubation time [17]. Other studies have reported that the Gelatin Broth (GB) medium (glycerol 20 g, soluble starch 20 g, peptone 10 g, meat extract 5 g, CaCO3 3 g, distilled water 1 000 ml, Agar 15 g (pH 7.0)) is favorable to the production of antimicrobial agents in contrary to ISP2 medium [18]. Screening on Bennett medium at 30°C showed that show that the temperature of 30°C allows a better production of antifungal substances [15].  Table 1: strains used in this study         11.6±1.5 against C.a 0 0 S10 0 13±1 against E.c 0 S11