Introduction

Antimicrobial is used to spare people from contamination. It is a substance or a synthetic item with an inhibitory or destructive impact on the microorganisms (Piddock, 2012). The advancement of safe strains is a characteristic wonder that happens through choice weight on the antimicrobial microorganism populace (Chellat et al., 2016). Nonetheless, some of these antimicrobials have been rendered dormant by resistant systems (Nikolaidis et al., 2014). Several natural antimicrobials were extracted from soil microbes (Jain & Pundir, 2011; Qinn et al. 2020).

Actinomycetes are a group of prokaryotic microorganisms, gram-positive bacteria with high guanine⁺cytosine in their DNA (Adegboye & Babalola, 2012). They are considered biotechnologically important organisms since they are responsible for producing about half of the bioactive secondary metabolites, including antibiotics. They are filamentous bacteria that produce two types of branching mycelium, namely aerial and substrate mycelium (Yuan et al., 2010).

Streptomyces (family Streptomycetaceae) are the most significant Actinobacteria (Rong et al., 2009). It is disseminated in soil, water, and other indigenous habitats (Singh et al., 2006). In all years, Streptomyces strains as a wellspring of various bioactive mixes, for example, anthelminthic chemicals, herbicides, hostile to malignancy drugs, development components, and insusceptible modulators (Kumar et al., 2020; Adeyemo et al., 2021).

Factors influencing the number and types of actinomycetes present in a particular soil are geographical location, such as soil type, temperature, organic matter content, moisture content, cultivation, and aeration. Actinomycetes act as a major component of the microbial population in most soil. About 90% of the total actinomycetes population consists of Streptomyces species (Harir et al., 2018). Almost 80% of the world's antibiotics are known to come from actinomycetes, mostly from the genera Streptomyces and Micromonospora (Pandey et al., 2004).

A major global healthcare problem has occurred in the 21^st century because of the increasing resistance of microorganisms against commonly used antibiotics. A renewed search for new antimicrobial agents from Streptomyces has occurred because of this rapid emergence of antimicrobial resistance among pathogenic microorganisms (Alanis, 2005; Sebak et al. 2021).

Microscopic organisms are profoundly versatile pathogens that create protection from antimicrobials through a few mechanisms. The creation of β-lactamases that hydrolyze the β-lactam ring is the most widely recognized resistant system for these microscopic organisms against b-lactam antimicrobials (Pfeifer et al., 2010; Tooke et al., 2019). Creating board range β-lactamases (ESBLs) is a noteworthy opposition instrument that obstructs the antimicrobial treatment of contaminations brought about by Enterobacteriaceae and is a genuine risk to the presently accessible antimicrobial arsenal (Shaikh et al., 2015). Bacterial efflux pumps effectively transport numerous antimicrobials out of the cell. They are significant contributors to the characteristic resistance of gram-negative microscopic organisms to many medications that can be utilized to treat gram-positive bacterial contaminations (Blair et al., 2015).

This study aimed to optimize the growth conditions required for the maximum antibacterial efficacy of Streptomyces globosus (S. globosus) separated from soil samples in Saudi Arabia.

Materials and Methods

Effect of different pH values

The 100 ml Calibration of sterile starch-nitrate broth medium for different pH levels; 6, 6.5, 7, 7.5, 8, 8.5, 9, and 9.5 using Na OH for alkaline and HCl for acidic before sterilizing medium was performed. The cultures were incubated at 30C^o. The culture filtrates were centrifuged (4000 rpm in 30 m). The antibacterial efficacy of CFF was investigated using the agar well diffusion technique. Twenty-four hours post-incubation (35 ± 2^°C), the antibacterial efficacy was calculated by observing the zone of inhibition radius in millimeters.

Effects of the light factor

Nine Petri plates by the selected streptomyces isolate were incubated (three in light, dark, or 12 h light/ 12 h dark) at 28^°C for 7 days. Two agar discs (6 diameters) of Streptomyces isolate were placed over the surface of the tested bacteria's cultures with equal distance and incubated at 35 ± 2 ^°C for one day. After incubation, the antibacterial activity was estimated by measuring the inhibition zones' diameter in (mm).

Effect of different incubation periods at different incubation temperatures

The chosen Streptomyces sp. was cultured on sterile starch-nitrate broth (100 ml) medium at different temperatures (25^°, 30^°, 35^° and 40^° C) for various periods (1, 2, 3, 4, 5, 6,7, 8 and 9). At each incubation period and different temperatures, the culture filtrates were centrifuged (4000 rpm in 30 m). The antibacterial efficacy of cell-free filtrate (CFF) was investigated using the agar well diffusion technique. Twenty-four hours post-incubation (35 ± 2 ^° C), the antibacterial efficacy was calculated by observing the zone of inhibition radius in millimeters.

Effect of shaking incubator

The chosen Streptomyces sp. was cultured on sterile starch-nitrate broth (100 ml) medium in optimum conditions. Half of the cultures were incubated on a shaking incubator, and others were incubated in a constant incubator. The culture filtrates were centrifuged (4000 rpm in 30 m). The antibacterial efficacy of CFF was investigated using the agar well diffusion technique. Twenty-four hours post-incubation (35 ± 2 ^° C), the antibacterial efficacy was calculated by observing the zone of inhibition radius in millimeters.

Extraction of an antibacterial compound with organic solvents

Four different solvents (ethyl-alcohol, methanol, chloroform, ethyl-acetate, and acetone) were used to extract the antibacterial activity agent that was produced by the selected Streptomyces sp. The chosen Streptomyces sp. was cultured on sterile starch-nitrate broth (100 ml) medium in optimum conditions. The culture filtrates were centrifuged (4000 rpm in 30 m). The CFF was transferred aseptically into the conical flasks, and an equal volume of solvents was separately added to the CFF and shaken for 20 min. After gathering the organic phase, the solvent was evaporated under a vacuum. 50 µl of the extract was used to investigate antibacterial efficacy using the agar well diffusion technique. Twenty-four hours post-incubation (37 ^° C), the antibacterial efficacy was calculated by observing the zone of inhibition radius in millimeters.

MIC values against nosocomial infection bacteria

The process includes the preparation of a double dilution of the antimicrobial agents in a liquid medium. Upon diluting the standardized bacterial culture, each tube with a bacterial suspension was packed in the same medium. The inoculated tubes were incubated (mostly without agitation) under suitable conditions depending upon the test microorganism.

Statistics

The data presented as a mean of 3 replicates ± SD. The difference between the means was statistically compared using Microsoft Excel software.

rESULTS AND DISCUSSION

Effect of the light factor on antibacterial activity of S. globosus

Table 1 revealed no significant difference in the antibacterial activity when S. globosus was grown in different lighting. It had antibacterial activity against all the test bacteria except  P.earuginosa ATCC27853, but no differences in the antibacterial activity against the other test bacterial isolates when S. globosus was grown in light, dark, or 12h light / 12h dark, it showed antibacterial activity against E.coli ATCC35218 only when it was grown in dark.

Table 1. Effect of the light factor on antibacterial activity of S. globosus

Effect of different incubation periods and different incubation temperatures on antibacterial activity of S. globosus

Incubation of S. globosus at 25^oC showed antibacterial activity against S. aureus ATCCBAA977 only after five days of incubation and increased by increasing the incubation period. The most excellent antibacterial activity was after seven days of incubation, and the inhibition zone was 14.3 mm. Still, there was no antibacterial activity against other Gram-positive and Gram-negative bacteria (Figure 1).

Results in Figures 1a-1c revealed that antibacterial activity against most test bacteria was noticed and increased with increasing the incubation period at 30^oC. The optimum antibacterial activity was after seven days of incubation. The diameters of inhibition zones were (13.3 mm, 18.0 mm, 12.0 mm, 19.3 mm, and 16.3 mm) against E. coli ATCC35218, E. coli ATCC25922, Salmonella sp., S. aureus ATCCBAA977 and St. pneumonia ATCC49619, respectively.

From the results in Figure 1, when S. globosus was incubated at 35^oC, the antibacterial efficacy was versus E. coli ATCC25922, Salmonella sp., and S. aureus ATCCBAA977. The antibacterial activity against Salmonella sp. and S. aureus ATCCBAA977 began from the third day but after five days produced antibacterial activity against E. coli ATCC25922. The optimum antibacterial activity was on the fifth day, and the diameters of inhibition zones were (12.6 mm, 15.0 mm, and 16.3 mm) versus E. coli ATCC25922, Salmonella sp, and S. aureus ATCCBAA977. S. globosus at 40^oC did not show antibacterial activity against all test bacteria.

Impact of various pH conditions on antibacterial efficacy of S. globosus

S. globosus produce antibacterial activity against S. aureus ATCCBAA977 at pH 6, against E. coli ATCC25922, and S. aureus ATCCBAA977 at pH 6.5, and against all test bacterial isolate except P. earuginosa ATCC27853 at pH 7. Figure 2 shows that the antibacterial activity of S. globosus against all test bacteria increased by increasing the fermentation medium's pH values. The highest antibacterial activity was at pH 8.5, which was decreased above. It was noticed that S. globosus produced antibacterial activity against all test isolates and the highest was against S. aureus ATCCBAA977. The inhibition zone diameter was (20.3 mm) and decreased slowly above.

Effect of shaking incubator on antibacterial activity of S. globosus

Results in Figure 3 show the difference between the antibacterial activity produced by S. globosus when the cultures of this isolate were incubated in a constant and shaking incubator. When incubated in a shaking incubator, S. globosus can't have antibacterial activity agents against all the test bacteria. Different antibacterial activities were produced when cultures of S. globosus were incubated in a constant incubator.

The diameters of inhibition zones were (15.3 mm, 14.0 mm, 13.3 mm, 14.3 mm, 24.0 mm, and 15.3 mm) against E. coli ATCC35218, E. coli ATCC25922, Salmonella sp., P. earuginosa ATCC27853, S. aureus ATCCBAA977 and St. pneumonia ATCC49619, respectively.

Effect of different organic solvents used in the extraction of the antibacterial compound from S. globosus on its antibacterial activity.

Figure 4 revealed that all organic solvents succeeded in extracting the antibacterial compound. Better extraction was obtained using chloroform. The diameters of inhibition zones were (17.6 mm, 16.3 mm, 17.3 mm, 15.6 mm, 27.6 mm, and 17.3 mm) against E. coli ATCC35218, E. coli ATCC25922, Salmonella sp., P. earuginosa ATCC27853, S. aureus ATCCBAA977 and St. pneumonia ATCC49619, respectively.

MIC values of the chloroform extract against nosocomial infection bacteria

The MIC values against all the tested bacteria showed different results. S. aureus ATCCBAA977 has been inhibited at (16 µg/m), which was the lowest concentration for inhibition. E. coli ATCC35218, Salmonella sp., and St. Pneumonia ATCC49619 bacteria have been inhibited at (32 µg/ml). E. coli ATCC25922 inhibited at (64 µg/ml). The highest concentration for inhibition was at (125 µg/ml) against P. earuginosa ATCC27853 (Table 2).

Table 2. MIC values of the chloroform extract against nosocomial infection bacteria.