Inducing and Enhancing Antimicrobial Activity of Mining-Soil-Derived Actinomycetes Through Component Modification of Bennett’s Culture Medium

Abstract

This study investigated the effect of different culture agar media, derived from Bennett’s medium, on the antimicrobial activity of 15 Streptomyces sp. and 1 Lentzea sp. strains isolated from mining environments. The media were prepared from the standard Bennett’s medium by suppressing one, two, or three ingredients—yeast extract (YE), beef extract (BE), or casein (Cas)—while maintaining glucose (Gluc) or by substituting it with fructose (Fruc) or galactose (Gal) and keeping the same suppressions. The antimicrobial activity was investigated against Candida albicans ATCC 10231, Staphylococcus aureus ATCC 29213, Bacillus subtilis ATCC 6633, and Escherichia coli K12. The antimicrobial activity of actinomycete strains was positively influenced by media modifications, though the response was actinomycete strain and target pathogen-dependent. Unexpectedly, thirteen strains exhibited poor growth on a pure agar-agar medium, including six Streptomyces strains (AS34, AS3, BS59, BS68, BS69, and DAS104) that showed notable antimicrobial activity, with inhibition zone diameters ranging from 10.75 ± 1.06 to 18.00 ± 0.00 mm. Modifications of Bennett’s medium, including replacing glucose with fructose or galactose and maintaining yeast extract or both yeast extract and beef extract, induced and enhanced the antimicrobial activity of several actinomycete strains. Notably, the new media induced antimicrobial activity in strains that showed no activity in Bennett’s medium. They led, compared to Bennett’s medium, to the detection of eight additional active strains against S. aureus, eight against B. subtilis, six against E. coli, and four against C. albicans. This study is the first to explore the modification of Bennett’s medium, either by subtraction or substitution, in order to investigate the effect on antimicrobial activity of actinomycete strains. These results highlight the importance of the composition of culture media on inducing or boosting antimicrobial activity in Streptomyces and Lentzea.

1. Introduction

In the early 1900s, around 80% of all medicines were derived from plants. However, the discovery of penicillin from Penicillium notatum and its widespread therapeutic use in the 1940s marked an important shift, with plants being replaced by microorganisms as a source of natural medicines [1]. Since then, bioactive compounds from microorganisms have been extensively used in agriculture, the food industry, scientific research, and medicine. Among these microorganisms, actinomycetes represent a key source of commercially valuable products [2]. These filamentous bacteria, especially the Streptomyces genus, are recognized as one of the richest sources of many bioactive molecules, including antibiotics, with 12,000 of all bioactive metabolites described [3].

The potential of actinomycetes to produce bioactive compounds is not an unchanging trait but can be significantly increased, decreased, or completely lost under different environmental conditions and nutritional variations [4,5,6]. Temperature, pH, incubation time, and nutrient availability (mainly carbon, nitrogen, and phosphate) significantly affect the production of these bioactive compounds by microorganisms [7]. However, the production of specialized metabolites, like antibiotics, and their concentrations are specifically connected to primary metabolism (as precursors or cofactors); in particular, nutrient (primary metabolites) limitation strongly induces secondary metabolism in the Streptomyces genus [8]. Hence, media composition plays a crucial role in the efficiency and cost-effectiveness of the final process. Therefore, developing a suitable fermentation medium is of critical importance in the production of specialized compounds [6].

In our previous study, 145 actinomycete strains were tested for their potential to inhibit pathogenic microorganisms [9]. In three distinct culture agar media, 51 strains exhibited antimicrobial activity against at least one pathogenic microorganism. We found that a strain’s ability to produce antimicrobial compounds could differ depending on the medium used; some strains can produce antimicrobial compounds in one medium but not in another. In particular, Bennett’s agar medium was able to detect almost all strains showing antimicrobial activity. As confirmed by other studies, it is the medium of choice for evaluating the inhibitory properties of actinomycetes. Indeed, its nutrient composition promotes growth and enhances the antimicrobial potential of bacteria, such as actinomycetes [9,10,11,12]. However, the antimicrobial activity assay on other media allowed the detection of other activities, not detected on Bennett’s medium. Then, some questions that arise are whether and how the composition of the medium affects the production of antimicrobial metabolites; which components of the medium have an inductive effect or not on such production? The objective of the present study is to evaluate the influence of various culture media, prepared by removing or substituting some components of the standard Bennett’s medium, on the antimicrobial activity of selected actinomycete strains.

2. Materials and Methods

2.1. Actinomycete Strains

Sixteen actinomycete strains formed the basis for the present study. They were previously isolated from soil samples, with high levels of lead and cadmium, at different mining sites in Morocco’s Midelt region [9]. The isolation, morphological, biochemical, and molecular identifications, as well as the phylogenetic analysis of these strains, were conducted following the methodology detailed in our previous study [9]. Based on 16 rRNA gene sequence analyses, fifteen strains were identified as Streptomyces species and one as a Lentzea species. Their sequences were deposited in the GenBank database under the accession numbers summarized in

Table 1. Actinomycete strains and their corresponding accession numbers in GenBank.

Actinomycete Strain	Accession Number
Lentzea sp. AS16	OP117486
Streptomyces sp. AS2	OP122091
Streptomyces sp. AS3	OP122569
Streptomyces sp. AS22	OP124048
Streptomyces sp. AS28	OP125353
Streptomyces sp. AS30	OP125546
Streptomyces sp. AS34	OP125838
Streptomyces sp. AS45	OP164531
Streptomyces sp. AS52	OP142749
Streptomyces sp. BS59	OP132389
Streptomyces sp. BS68	OP133137
Streptomyces sp. BS69	OP133217
Streptomyces sp. DS104	OP133558
Streptomyces sp. DS106	OP164552
Streptomyces sp. CS143	OP135549
Streptomyces sp. DS167	OP137212

. Additionally, their phylogenetic relationships were analyzed, and the corresponding phylogenetic tree was constructed in our previous study [9].

2.2. Preparation of Culture Media

Bennett’s agar medium was used as the standard culture medium: 10 g/L of glucose (Solvapur, Casablanca, Morocco), 2 g/L of casein hydrolysate (Sigma-aldrich, St. Louis, MO, USA), 1 g/L of yeast extract (Oxford, Maharashtra, India), 1 g/L of beef extract (Biokar diagnostics, Paris, France), and 18 g/L of agar (Biokar diagnostics, Paris, France).

The new media were prepared by removing one, two, or three components (yeast extract, beef extract, or casein) from the basal medium in the presence of glucose, fructose, or galactose. Consequently, 25 different media were prepared and are listed in

Table 2. Media composition (in %).

Medium N°	Gluc	Fruc	Gal	YE	BE	Cas	Agar-Agar
1							1.8
2	1			0.1	0.1	0.2	1.8
3	1			0.1	0.1		1.8
4	1			0.1		0.2	1.8
5	1				0.1	0.2	1.8
6	1			0.1			1.8
7	1				0.1		1.8
8	1					0.2	1.8
9	1						1.8
10		1		0.1	0.1	0.2	1.8
11		1		0.1	0.1		1.8
12		1		0.1		0.2	1.8
13		1			0.1	0.2	1.8
14		1		0.1			1.8
15		1			0.1		1.8
16		1				0.2	1.8
17		1					1.8
18			1	0.1	0.1	0.2	1.8
19			1	0.1	0.1		1.8
20			1	0.1		0.2	1.8
21			1		0.1	0.2	1.8
22			1	0.1			1.8
23			1		0.1		1.8
24			1			0.2	1.8
25			1				1.8

, with glucose-based media numbered from 3 to 9, fructose-modified media from 10 to 17, and galactose-modified media from 18 to 25. Bennett’s agar medium (N°2) and agar-agar medium (N°1) were used as positive and negative controls, respectively.

2.3. Cultivation of Actinomycete Strains

Actinomycete strains were cultivated on previously prepared culture media using the spot inoculation method. A 5 µL aliquot of spore suspension (≈10⁷ spores/mL), harvested from cultures grown on Bennett’s medium, was aseptically deposited onto the surface of each medium. The plates were incubated at 30 °C for four days. Following incubation, colony development was evaluated based on aerial mycelium formation, sporulation abundance, and overall colony morphology. These features were examined under a magnifying glass and recorded according to growth levels classified as good (+++), moderate (++), weak (+), or absent (−) (refer to Figure 1).

2.4. In Vitro Antimicrobial Assay

The antimicrobial activity of actinomycete strains was performed using a qualitative double-layer microbial method [9]. The target pathogens used for antimicrobial activity detection were Staphylococcus aureus ATCC 29213, Bacillus subtilis ATCC 6633, Escherichia coli K12, and fluconazole-resistant Candida albicans strain ATCC 10231. The plates, containing cultivated actinomycete strains, were covered with a 5 mL soft agar (0.5% w/v agar-agar) of either LB (Luria–Bertani) medium (10 g/L of tryptone (Oxford, Maharashtra, India), 5 g/L of yeast extract, 10 g/L of NaCl (Emsure, Søborg, Denmark), and 5 g/L of agar) for bacteria or YPG (yeast extract-peptone-glucose) medium (20 g/L of glucose, 10 g/L of peptone (Biokar diagnostics, Paris, France), 10 g/L of yeast extract, and 5 g/L of agar) for Candida growth, pre-inoculated with 100 μL from a culture of the test microorganism (OD_600nm = 0.1). These cultures were carefully poured over the plates and incubated for 24 h at 30 °C for C. albicans and 37 °C for bacteria. Antimicrobial activity was determined by measuring the growth inhibition zone diameter (IZD = mm) using a graduated ruler. The IZD was measured according to the following formula:

$$Inhibition zone diameter \left(mm\right)=Total inhibition diameter-Colony diameter$$

Each experiment was performed in duplicate, and the mean IZD was calculated.

Any mean IZD value below 5 mm indicates no activity, and the IZD was noted as zero. Consequently, a strain was considered active when the IZD was ≥5 mm and inactive when the IZD was <5 mm.

2.5. Data Analysis

For data analysis, each target test strain was considered separately, and the activity of the 16 actinomycete strains was assessed collectively in each culture medium. IZDs were presented as mean± standard deviation. The significance of differences was analyzed using Student’s t-test, and the heatmaps were performed under GraphPad Prism version 9.3.1 software (GraphPad Software, LLC, San Diego, CA, USA).

3. Results

3.1. Quantification of Active or Inactive Actinomycete Strains Depending on the Growth Conditions

The growth of sixteen actinomycete strains was examined to determine how the culture medium affected the production of antimicrobial compounds. These strains were cultivated in 25 agar media formulated by subtracting or substituting ingredients from the standard Bennett’s medium. Following four days of incubation, growth was noted, and antimicrobial activity was evaluated against the four indicator strains using the double-layer microbial method. A total of 25 different culture media were tested, including Bennett’s medium and a pure agar-agar medium, which served as positive and negative controls, respectively. Each indicator strain evaluated in this experiment was tested in duplicate, yielding 200 observations (3200 total).

The data collected are summarized in

Table 3. Growth of strains in modified media prepared from standard Bennett’s medium (refer to Table 2 for media components).

	1	2	3	4	5	6	7	8	9	10	11	12	13	14	15	16	17	18	19	20	21	22	23	24	25
AS2	-	++	+++	+++	+++	+++	+++	+++	-	+++	+++	+++	+++	+++	+++	+++	++	++	++	++	++	++	++	++	+
AS3	+	++	+++	+++	++	+++	+++	++	+	+++	+++	+++	+++	+++	+++	+++	++	++	++	++	++	++	++	++	+
AS16	-	++	++	++	++	++	++	++	-	++	++	++	++	++	++	+++	++	++	++	++	++	++	++	++	++
AS22	+	++	++	++	++	++	++	++	-	+++	+++	+++	+++	+++	+++	+++	+++	+++	+++	+++	+++	+++	+++	+++	+++
AS28	+	++	++	+++	++	++	++	++	-	++	+++	+++	++	+++	+++	++	+++	+++	++	+++	+++	+++	++	++	+++
AS30	+	+++	+++	+++	+++	+++	+++	+++	-	+++	+++	+++	+++	+++	+++	+++	++	+++	++	+++	++	+++	++	+++	+
AS34	+	+++	+++	+++	+++	+++	+++	+++	+	+++	+++	+++	+++	+++	+++	+++	++	+++	+++	+++	+++	+++	+++	+++	+
AS45	+	++	++	++	++	++	++	++	-	++	+++	+++	++	+++	+++	++	++	+++	++	+++	+++	+++	++	++	+++
AS52	-	+++	+++	+++	+++	+++	+++	+++	-	+++	+++	+++	+++	+++	+++	+++	++	+++	++	+++	++	+++	++	+++	+
BS59	+	++	++	++	++	++	++	++	+	+++	+++	+++	+++	+++	+++	++	+	++	++	++	++	++	++	++	+
BS68	+	++	++	++	++	++	++	++	-	++	++	+++	++	++	+++	++	+	++	++	++	++	++	++	++	++
BS69	+	++	++	++	++	++	++	++	-	++	++	++	++	++	+++	++	+	++	++	++	++	++	++	++	++
DS104	+	++	++	++	++	++	++	++	-	+++	++	+++	+++	+++	+	++	+	+++	++	++	+++	++	++	++	+
DS106	+	++	++	++	++	++	++	++	-	+	++	++	++	++	+	++	++	++	++	++	++	++	++	++	+
CS143	+	++	++	++	++	++	++	++	-	+	+++	++	++	++	++	++	++	++	++	++	++	++	++	++	+
DS167	+	+++	++	++	++	++	++	++	-	+++	+++	++	+++	+++	+++	+++	+	+++	+++	++	+++	+++	+++	+++	+

-: No growth, +: weak growth, ++: moderate growth, and +++: good growth.

. The classification criteria for growth levels are illustrated in Figure 1, showing examples of good (+++), moderate (++), and weak (+) growths, based on aerial mycelium abundance and colony appearance.

The result showed that the growth of the different strains on the various agar media varied from no growth (-) to good growth (+++). Some strains, such as Streptomyces sp. AS30 and AS34, consistently showed a similar growth profile in almost all culture media, suggesting their ability to develop under a wide range of nutrient conditions. In contrast, strains BS59 to CS143 showed moderate growth in almost all media (Table 3).

Unexpectedly, despite containing only agar-agar and lacking any additional nutrient sources, medium N°1 supported weak growth for all strains, except for Streptomyces sp. AS2, AS16, and AS52, which failed to grow entirely. Moreover, the addition of fructose or galactose to agar-agar led to weak or even moderate growth in most strains. However, medium N°9, consisting of only glucose, proved unfavorable to growth, as the majority of strains failed to develop except Streptomyces sp. AS3, AS34, and BS59 (Table 3).

The capacity to develop on pure agar-agar medium (N°1) suggests that many actinomycete strains can grow weakly on agar-agar, probably producing hydrolytic enzymes such as agarase, which hydrolyzes agar to enable its assimilation. It has already been reported that a strain of Streptomyces (Streptomyces coelicolor A 3(2)) has the unusual ability to use agar as its sole carbon source [13,14], thanks to the production of an extracellular agarase.

However, when glucose was added to the agar-agar and used as the sole carbon source, strain growth was mostly inhibited, possibly due to either an inability to metabolize glucose or the glucose’s inhibitory effect on agarase production. This aligns with the previous study, which found that a glucose presence caused the intracellular degradation of pre-agarase in S. coelicolor and S. lividans TK21 and reduced the secretion of proteases [15]. These observations illustrate the complexity of bacteria’s nutrient requirements and their ability to adapt to diverse environmental conditions.

An overview of the actinomycete strains’ antimicrobial activities evaluated from different growth media, derived from the standard Bennett’s medium, is shown in Figure 2. The left side displays the number of inactive strains (IZD < 5 mm), while the right side presents the number of active strains (IZD ≥ 5 mm). The different tested culture media are illustrated in the center, including from which one, two, or three components of Bennett’s medium were removed (N°3 to N°9) and those from which glucose was substituted with fructose (N°10 to N°17) or galactose (N°18 to N°23). Bennett’s agar medium (N°2) and the agar-agar medium (N°1) are also included. The detailed data are presented in supplementary tables (Tables S1–S12).

The results show that, of the 13 strains showing weak growth on the negative control (agar-agar medium (N°1)), 6 unexpectedly demonstrated the capacity to produce antimicrobial compounds (Figure 2). Similarly surprising, most strains failed to grow in the glucose-only medium (N°9), with the exception of three strains (AS3, AS34, and BS59) (Table 3). Among these, Streptomyces sp. AS34 exhibited antimicrobial activity against B. subtilis. In contrast, when glucose was replaced by fructose (N°17) or galactose (N°25), antimicrobial compound production was observed in 14 and 12 strains, respectively, against at least 1 of the tested target strains (Figure 2).

Overall, media supplemented with fructose or galactose (whether combined with one, two, or three additional ingredients) appear to stimulate more effectively the production of antimicrobial compounds compared to glucose-containing media. However, the number of active strains, against at last one test pathogen, in Fruc-Bennett (N°10) and Gal-Bennett (N°18) media remains comparable to that observed in the standard Bennett’s medium, which detects 13 active strains. Specifically, 12 active strains were detected in the Gal-Bennett medium, while 14 active strains were observed in the Fruc-Bennett medium (Figure 2).

3.2. Assessment of the Effect of Various Culture Media Derived from Bennett’s Medium on the Antimicrobial Activity

Figure 3 presents heat maps illustrating the antimicrobial activity of actinomycete strains against S. aureus, B. subtilis, E. coli, and C. albicans in different culture media derived from Bennett’s medium. These maps provide a clear and comparative visualization of the effect of medium modifications on the production of antimicrobial compounds. Figure 4, on the other hand, showcases photos that further illustrate examples of antimicrobial activity.

• Activity against S. aureus

The results show that strain DS106 remained inactive across all tested media, whereas strain AS34 consistently exhibited the highest activity across all media (Figure 3a).

Compared to Bennett’s medium, the modified media supplemented with fructose and galactose displayed a positive effect on stimulating active strains, exceeding the effect observed in Bennett’s medium. In particular, Fruc (N°17), Fruc-YE-BE (N°11), Fruc-YE (N°14), and Gal-YE (N°2) exhibited superior stimulation, inducing 11, 10, 10, and 9 strains, respectively, compared to only 7 strains in Bennett’s medium (Figure 3a).

Overall, fructose and galactose media outperformed glucose-based media in inducing anti-S. aureus activity. Notably, the rare Lentzea AS16 displayed enhanced activity in Fruc-YE (N°14) and Gal-YE (N°22) (IZD: 37.00 ± 01.41 mm) compared to Gluc-YE (N°6) (IZD: 20.00 ± 01.41 mm) and the absence of activity in Bennett’s medium (p < 0.05) (Tables S1–S3). In contrast, strains AS3 and AS28 exhibited reduced activity in fructose and galactose-based media (Figure 3a).

• Activity against B. subtilis

The results show that strains DS106 and BS69 showed no anti-B. subtilis activity across all tested media. In contrast, strain CS143 displayed activity exclusively in the Glu-YE medium (N°6), with an IZD of 38.00 ± 01.41 mm, and strain AS34 exhibited the highest anti-B. subtilis activity in all media tested (Figure 3b; Tables S4–S6).

When the medium composed of agar-agar solely (N°1) was tested, thirteen strains exhibited growth. Among them, two strains (AS3 and AS34) displayed IZDs ranging from 10.75 ± 01.06 to 18.00 ± 00.00 mm against B. subtilis. The addition of glucose (N°9) supported weak growth in only three strains, including the AS34, which retained anti-B. subtilis activity with an IZD of 15.00 ± 00.00 mm. Supplementing glucose with yeast extract Gluc-YE (N°6) or beef extract Gluc-BE (N°7) induced anti-B. subtilis activity in three strains (Streptomyces sp. AS28, AS45, and CS143) not detected in Bennett’s medium (Figure 3b).

Replacing glucose with fructose or galactose in Bennett’s medium induced anti-Bacillus activity in four strains (AS2, AS16, AS45, and BS59) not detected in Bennett’s medium. This activity seems to be specifically linked to the carbon source. Additionally, the Fruc-YE-BE medium (N°11) induces anti-B. subtilis activity in the same three strains (AS45, BS59, and AS2). Notably, this medium enables the detection of anti-B. subtilis activity in a total of eight strains, in contrast to the Bennett’s medium, which reveals activity in only five Streptomyces strains (Figure 3b).

• Activity against E. coli

Analysis of Figure 3c reveals that, out of 16 Streptomyces strains, seven (AS2, AS3, AS16, BS68, BS69, DS104, and DS106) showed no anti-E. coli activity in the 25 tested media. The Bennett’s medium enabled the detection of activity in only three strains, while Glc-YE-BE (N°3) and Fruc-YE-BE (N°11) enabled the detection of eight active strains in total, suggesting that these two media allow the induction of five additional strains not detectable in Bennett ‘s medium.

Media containing glucose, particularly Glc-YE-BE (N°3) and Glc-BE-Cas (N°5), were the 2 media out of the 25 tested, which successfully induced activity in strains CS143 and DS167 (Figure 3c).

When glucose was replaced with fructose, Fruc-YE-BE medium (N°11) allowed the detection of five positive strains, including three strains not induced in Bennett’s medium (Streptomyces sp. AS34, AS52, and BS59). However, strain AS28 was no longer detected in medium N°11, but it was detected in Fruc-YE-Cas (N°12) and Fruc-BE-Cas (N°14). It was also detected in medium N°3 (Glu-YE-BE), with an IZD = 38.50 ± 00.70 mm similar to that observed in Bennett’s medium (Figure 3c; Tables S7 and S8).

Additionally, AS22 and AS45 exhibited increased activity in fructose and galactose media. For example, Fruc-BE (N°15), and even media containing only fructose (N°17) or galactose (N°25), enhanced activity in AS22, with an IZD ranging from 37.50 ± 02.12 to 43.50 ± 02.12 mm, compared to an IZD of only 15.50 ± 00.70 mm observed in Bennett’s medium (p < 0.001) (Tables S8 and S9). Gal-BE (N°23) exhibited a strong inhibitory effect on AS34, which was absent in fructose or glucose-based media. However, both fructose and galactose repressed anti-E. coli activity in CS143 and DS167 (Figure 3c).

• Activity against C. albicans

Figure 3d illustrates that 5 out of 16 strains (AS16, AS28, AS34, BS59, and CS143) exhibited no anti-C. albicans activity in the 25 different media tested. In contrast, strain AS45 was exclusively detected in the Gal-Cas medium (N°24).

The media Fruc-YE (N°14), Fruc-Cas (N°16), Gal-YE (N°22), and Gal-YE-BE (N°19) detected anti-C.  albicans activity in 10, 10, 9, and 9 strains, respectively, outperforming Bennett’s medium, which detected activity in only 7 strains (Figure 3d).

Media derived from Bennett’s medium by component subtraction generally failed to detect all strains exhibiting anti-C. albicans activity, with the exception of Glu-BE-Cas (N°5). This medium detected activity in seven strains, comparable to Bennett’s medium, but uniquely induced activity in two strains (AS30 and AS52), not detected in Bennett’s. Oppositely, the medium repressed the anti-C. albicans activity in two strains (AS22 and DS167), which were yet detected in Bennett’s medium (Figure 3d).

Replacing glucose with fructose or galactose significantly enhanced anti-C. albicans activity. Fructose-based media notably increased activity in strains AS2, AS22, BS69, and DS104 compared to Bennett’s medium. Specifically, strain AS2 displayed an IZD of 38.50 ± 02.12 mm in Fru-YE-Cas (N°12), compared to 14.50 ± 00.70, 16.00 ± 01.41, and 20.50 ± 00.70 mm in Gal-YE-Cas (N°20), Gluc-YE-Cas (N°4), and Bennett’s medium, respectively (p < 0.001) (Tables S10–S12). Additionally, galactose-based media strongly enhanced anti-C. albicans activity in strains BS68, BS69, DS104, and DS106 (Figure 3d; Table S12).

Based on the obtained results, the Fruc-YE-BE (N°11) and Fruc-YE (N°14) media are recommended as the first choice for exploring anti-S. aureus (strain ATCC 29213) activity. When used separately, these two media induced anti-S. aureus activity in 13 actinomycete strains, compared to only 7 strains detected using Bennett’s medium.

For anti-B. subtilis activity, Fruc-YE-BE (N°11) and Glu-YE (N°6)-amended agar media are the preferred choices and are recommended for investigating anti-B. subtilis activity (strain ATCC 6633). When these two media were used separately, 10 actinomycete strains were induced for anti-B. subtilis activity, compared to only 5 strains detected in Bennett’s medium.

Regarding anti-E. coli activity, Fruc-YE-BE (N°11) and Glu-YE-BE (N°3) are recommended for searching anti-E. coli activity (strain K12). When used separately, these media induced anti-E. coli activity in five additional actinomycete strains.

For anti-C. albicans (strain ATCC 10231), Fruc-YE (N°14) is the most preferred medium, detecting activity in the majority of Streptomyces strains (nbr = 10), surpassing Bennett’s medium (nbr = 7).

These findings highlight the importance of selecting an appropriate culture medium to maximize the detection of antimicrobial-producing actinomycete strains, especially those thriving in mining biotopes.

3.3. Induction of Antimicrobial Activity of Actinomycete Strains Against Target Strains

We refer to the different culture media formulated from Bennett’s medium as “new media”, and we determine the effect of these media on the antimicrobial activity of actinomycete strains on the target strain, compared to Bennett’s medium. Figure 5 illustrates that the “new media” significantly induced antimicrobial activity compared to Bennett’s medium. For S. aureus, the new media induced the production of antibacterial compounds in 8 additional strains, resulting in a total of 15 active strains, compared to only 7 strains observed in Bennett’s medium. Similarly, for B. subtilis, 8 additional strains were induced for antibacterial activity in the new media, enabling the detection of a total of 13 active strains, whereas only 5 strains were detected in Bennett’s medium.

Regarding E. coli, the new media induced antibacterial activity in six additional strains, bringing the total to nine strains, while only three were activated in Bennett’s medium. This highlights the capacity of the actinomycete strains to produce anti-E. coli compounds in the newly formulated media. As for C. albicans, the new media induced antifungal activity in 4 additional strains and retained the activity of 7 strains also induced in Bennett’s medium, totaling 11 active strains.

Overall, no actinomycete strain was found to be active exclusively in Bennett’s medium without also being induced in the new media (Figure 5).

4. Discussion

For decades, optimizing the production of secondary metabolites linked to biological activities has been a strategy in both the pharmaceutical industry and the research institutions. This optimization mainly involves modifying physicochemical parameters, such as temperature, pH, and minerals, but more rarely the composition of the culture medium. Given this, the main objective of the present study was to investigate the effect of different culture media on the antimicrobial activity of mining-derived actinomycetes.

These strains were isolated from mining biotopes in a previous work. These ecosystems have been poorly investigated for the search of actinomycetes able to produce antimicrobial compounds [9]. While studies demonstrated that the antimicrobial potential of actinomycetes is influenced by the culture medium and the environmental conditions, mining-derived strains might possess unique adaptations to heavy metals that could enhance their ability to produce novel bioactive compounds. Furthermore, given that, the relationship between the heavy metal resistance and the ability of microorganisms to synthesize metabolites with antimicrobial activity is still poorly understood [16]. There is a strong possibility that our actinomycetes harbor novel antimicrobial metabolites that are yet to be discovered.

In this context, we modified the composition of the standard Bennett’s medium to assess how these changes might influence the antimicrobial potential of the mining-derived actinomycetes. The culture media were modified, either by suppressing one, two, or three ingredients (yeast extract, beef extract, and casein) while retaining glucose or by combining these subtractions with the substitution of glucose by other carbon sources, such as fructose or galactose.

The results obtained with the four microbial strains tested (B. subtilis, S. aureus, E. coli, and C. albicans) highlight distinct effects of the culture media modifications on the antimicrobial activity of actinomycete strains. These observations provide valuable insights on how to optimize the composition of the culture medium to enhance the production of antimicrobial compounds against specific pathogens.

Depending on the target pathogen, modifications by component subtraction from Bennett’s medium showed contrasting results. For B. subtilis, the media often maintained or even induced antimicrobial activity in certain actinomycete strains (37.5%), particularly with combinations like Gluc-YE and Gluc-BE. However, for C. albicans, subtraction-derived media resulted in reducing antimicrobial activity, especially for the strain Streptomyces sp. BS68, where the IZD dropped significantly to 10.50 ± 00.70 mm compared to 38.50 ± 02.12 mm in Bennett’s medium. Regarding S. aureus, certain modified media, such as Gluc-YE, boosted antimicrobial activity in strains like Streptomyces sp. AS3 and AS34, compared to Bennett’s medium. However, other strains showed reduced or suppressed activity under the same conditions. Similarly, for E. coli, media composed of three ingredients such as Gluc-YE-BE and Gluc-BE-Cas induced antibacterial activity in some strains (12.5%) but led to suppressing it in others (12.5%).

It is well known that various sugars serve as carbon sources for both growth and secondary metabolite production. At the molecular level, the preferential use of one carbon source over another, as well as the secondary metabolite production, is modulated by transcriptional activations or repression mechanisms [17]. These forms of regulation, referred to as carbon catabolite repression (CCR), mainly involve glucose as a key regulator in Streptomyces, where it inhibits or reduces secondary metabolite production by regulating dedicated precursors [18]. It has been reported that the production of over 20 examples of antibiotics has been suppressed by changing the carbon source. Sugars such as glucose, glycerol, maltose, mannose, sucrose, and xylose have been found to interfere with antibiotic production [17,19]. Glucose, which is generally an excellent carbon source for growth, when used at high concentrations, interferes with the formation of many other secondary metabolites and delays the morphological differentiation [20,21].

In our study, most strains (13 out of 16) were able to grow in the pure agar-agar negative control medium. However, the addition of 1% glucose to the agar-agar inhibited the growth of most strains (13 out of 16). This result is consistent with previous research, showing that glucose interferes with the production of glycoside hydrolases, such as agarase, an enzyme responsible for agar-agar degradation [22]. Notably, this inhibitory effect was alleviated upon the addition of yeast extract, leading to the restoration of growth. Furthermore, when either fructose or galactose was individually supplemented to the agar-agar medium, all the Streptomyces strains exhibited growth.

The use of glucose as a carbon source has been shown to negatively influence the production of antimicrobial compounds. This effect was particularly evident when we substituted glucose with fructose or galactose. This substitution generally induced or enhanced antimicrobial activity against almost tested pathogens, though the extent of this effect varied depending on the actinomycete strain and the target species. In the presence of glucose alone, no antimicrobial activity was detected. However, fructose alone induced anti-S. aureus activity in 11 strains, anti-B. subtilis activity in 8 strains, anti-E. coli activity in 1 strain, and anti-C. albicans activity in 8 strains, while galactose alone triggered these activities in 8, 7, 3, and 4 strains, respectively. On the other hand, fructose- or galactose-modified media, particularly those combined with yeast extract (Fruc-YE-BE, Fruc-YE, and Gal-YE), showed a significant enhancement of antimicrobial activity in many strains. However, in strains such as Streptomyces sp. CS143 and DS167, the anti- E. coli activity detected in Glu-BE-Cas medium was repressed when cultivated in fructose or galactose-based media.

This is consistent with other studies showing that fructose and galactose can either inhibit or induce antibiotic production, depending on the microbial strain. Fructose has been reported to interfere with penicillin production in Penicillium chrysogenum by repressing it [23]. This same sugar can stimulate the production of actinomycin by Streptomyces antibioticus [24] and gentamicin by Micromonospora purpurea [23]. Galactose has also been reported to inhibit penicillin production in P. chrysogenum [23] while having no effect on actinomycin production by S. antibioticus or cephalosporin production by Cephalosporium acremonium [23].

Similarly, to the carbon source, the nitrogen source significantly influenced the antimicrobial activity of the tested actinomycetal strains. Yeast extract proved to be the most effective, and its combination with beef extract further boosted antimicrobial activity. The use of 0.1% yeast extract and beef extract provided the most favorable conditions for antimicrobial activity development. This relatively low concentration might have supplied sufficient organic nitrogen compounds, such as peptides, amino acids, and vitamins, essential for antibiotic production. In contrast, casein (0.2%) combined with a carbon source suppressed or reduced antimicrobial activity in some strains, such as BS59 and AS45 against E. coli, AS52 against B. subtilis and S. aureus, or AS28 against S. aureus. Our results align with those reported by Tan et al. [25] in their critical review, which reported that yeast extract enhances anti-Vibrio activity of Streptomyces metabolites compared to casein [25]. Combining casein, yeast extract, and beef extract with a carbon source (Bennett, Fruc-Bennett and Gal-Bennett media) negatively influenced antimicrobial activity, possibly due to nitrogen overload, which is known to inhibit specialized metabolism production in Streptomyces [26]. Nitrogen limitation is recognized as a key signal for initiating specialized metabolites like antibiotics [27]. For instance, high ammonium concentrations have been demonstrated to inhibit actinorhodin production in S. coelicolor [28].

Among all the culture media tested, the bioactivity of the actinomycete strains varied depending on the target pathogen. Against S. aureus, only one strain (DS106) was found to be inactive, whereas, in Bennett’s medium, nine strains exhibited no activity. In the case of B. subtilis, only two strains (DS106 and BS69) showed no inhibitory effect across all media tested, while, on Bennett’s medium, eleven strains displayed no activity. For E. coli, only 7 strains showed no activity across the evaluated culture media, but, in Bennett’s medium, this number increased to 13. Similarly, for C. albicans, five strains were inactive across all the tested media, while nine strains failed to show any activity in Bennett’s medium.

Media containing fewer ingredients are more effective in stimulating or enhancing antimicrobial activity compared to nutrient-rich media. The results allow us to propose recommendations for studying the antimicrobial activity of actinomycete strains. To search anti-S. aureus (strain ATCC 29213) activity, the use of Fruc-YE-BE (N°11) and Fruc-YE (N°14) media is recommended. To detect anti-B. subtilis (strain ATCC 6633) activity, Fruc-YE-BE (N°12) and Fruc-YE-Cas (N°11) media are preferred. Anti-E. coli (strain K12) activity is best observed using Fruc-YE-BE (N°11) and Glu-YE-BE (N°3) media. Finally, for anti-C. albicans (strain ATCC 10231) activity, Fruc-YE (N°14) medium is the most suitable.

In this study, we investigated how different culture media influence antimicrobial activity in agar media. However, further research is needed to analyze the specific secondary metabolites produced in each culture medium to optimize antimicrobial metabolite production.

5. Conclusions

This study demonstrates the crucial role that culture media composition plays in modulating the antimicrobial activity of actinomycetes. Our findings show that minimal media, often limited in energy sources, enhanced antimicrobial activity. Oppositely, complex media often interfere with secondary metabolite production. The substitution of glucose by fructose or galactose was shown to be particularly useful, enhancing and even inducing the production of antimicrobial compounds in most actinomycetal strains. This result highlights a crucial observation: during the screening of antimicrobial activities of microorganisms, it is essential to diversify culture media, particularly carbon sources, rather than relying solely on standard media. By strategically varying and combining nutrient components within a single medium or across different media, it is possible to maximize the detection of microorganisms with significant antimicrobial potential. This approach reduces the risk of overlooking strains with promising antimicrobial activities that do not express themselves under overly standard culture conditions. Moreover, the strains isolated from mining biotopes, which have not been extensively explored for antimicrobial properties, present a promising area for further research, especially regarding their potential to produce novel antimicrobial metabolites. Strains AS22 and AS34, which demonstrated significant antimicrobial activities, particularly merit further studies.

Overall, this study suggests that culture medium composition is a crucial factor for modulating antimicrobial agent production, and the optimization of its composition is a critical factor in (i) detecting the majority of antimicrobial-producing strains and (ii) maximizing the production of antimicrobial compounds, potentially resulting in more effective antibiotics to fight against multi-drug-resistant pathogens.