# Evaluation of SoilStreptomyces Isolates from North-Western Ethiopia as Potential Inhibitors against Spoilage and Foodborne Bacterial Pathogens

Authors: Abayeneh Girma; Aleka Aemiro
Journal: Journal of Chemistry
 (2022)

---

## Abstract

The outbreak and spread of foodborne diseases is a serious concern for global healthcare and security. Finding novel antimicrobial agents with diverse mechanisms of action against the current spoilage and foodborne bacterial pathogens is a central strategy to overcome the problems of antibiotic resistance. Soil actinomycetes are the major antimicrobial producers with great biopreservative and medical value. This study was aimed at isolatingStreptomyces from soil samples of northwestern Ethiopia against spoilage and foodborne bacterial pathogens. Thirty-six soil samples were collected at a depth of 5–10 cm in the rhizosphere and agricultural soils of soybean. A total of 118 actinomycete strains were isolated and screened primarily using the perpendicular streak plate method against 3 Gram-positive and 3 Gram-negative bacterial strains. Out of 118 isolates, 36/118 (30.50%) were active against at least two of the tested bacteria, of which 8 isolates were selected for their wide-spectrum antibacterial activities. During the disc diffusion assay, the eight in vitro ethyl acetate extract antibacterial activities range from 7 to 24 mm. The minimum inhibitory concentration and minimum bactericidal concentration values range from 0.10 to 0.25 μg/mL and 0.15 to 0.40 μg/mL, respectively. Following the morphological, physiological, biochemical, and molecular characteristics, eight potent isolates were identified as follows: Streptomyces fasciculus, Streptomyces roseochromogenes, Streptomyces ruber, Streptomyces glaucus, Streptomyces griseus, Streptomyces cellulosae, Streptomyces griseoflavus, and Streptomyces xanthophaeus. After the treatment of potent Streptomyces cell-free culture supernatant with proteinase K, papain, α-amylase, and lysozyme enzymes, their antagonistic effects were also observed. Most Streptomyces cell-free culture supernatant antibacterial activity was highly resistant to heat, acidity, organic solvents, and additives. Thus, the results of this investigation revealed that soil actinomycetes could be a valuable source for novel antibacterial agents applicable in food biopreservation and the treatment of spoilage and foodborne bacterial pathogens.


---

## Body

## 1. Introduction

Foodborne diseases are illnesses caused by the consumption of foods contaminated with pathogenic microbes (bacteria, fungi, viruses, and parasites) or their toxins (in case of bacteria and fungi). Of those, bacteria are the most causative agents, contributing to two-thirds of foodborne disease outbreaks. Unhygienic practices during the food supply chain (from production to consumption) are the causes of severe foodborne diseases such as food intoxication, toxicoinfection, and infection. War, famine, crowding, poverty, competition for food, and natural disasters contribute to the foodborne pathogens’ ability to survive and spread easily [1].Globally, foodborne diseases are still not under control, and outbreaks can cause health and economic loss. Morbidity and mortality are serious consequences of outbreaks caused by foodborne pathogens in both developed and developing countries. Annually, the burdens of foodborne diseases in low-income countries, including Ethiopia, are responsible for two million deaths. Foodborne diseases can also cause both short-term symptoms (nausea, vomiting, and diarrhea) and longer-term illnesses (tissue damage, cancer, kidney or liver failure, brain disorders, and neural disorders). These symptoms and illnesses cause serious public health problems, especially in children, pregnant women, elderly people, and immunocompromised individuals [1–3].Actinomycetes have an ability to synthesize and provide a wide array of bioactive substances, which have been confirmed in numerous institutional and industrial laboratories for commercial and medical value. This has resulted in the isolation of metabolites from actinomycetes of different sources that have been found applicable in treating a variety of human infections [4]. More than half of the naturally occurring antibiotics have been isolated from different genera of actinomycetes such as Streptomyces, Micromonospora, and Nocardia [5]. Of them, the Streptomyces genus occupies the most prominent place for the production of bioactive secondary metabolites in terms of versatility, diversity, medical value, and structural complexity of the produced compounds.The improper use of antimicrobial drugs for therapeutic purposes in human, veterinary, and agriculture is favoring the survival and spread of resistant bacterial pathogens due to the acquisition of novel resistant genes from different sources, such as the environment. Some antibiotics, for example, penicillin, erythromycin, and methicillin, which were active against bacterial infectious diseases, are now less effective due to the acquisition of resistance genes [6]. For instance, methicillin and vancomycin-resistant strains of Staphylococcus aureus cause an enormous threat to the treatment of serious infections [7].Besides,Salmonella spp., Shigella spp., Vibrio spp., Clostridium spp., Campylobacter spp., Escherichia coli, Bacillus cereus, Listeria monocytogenes, and Yersinia enterocolitica are some of the drug-resistant bacterial pathogens that cause foodborne diseases and death across the world [1–3]. Because of this, treatment is becoming more complex and options for therapy are often limited. To overcome this urgent problem, there is a need to screen new strains of actinomycetes capable of extending the shelf life of food and producing novel classes of antibiotics with unique mechanisms of action against the current food-origin multidrug-resistant bacterial pathogens. Thus, the present study was undertaken to isolate actinomycetes from the rhizosphere and agricultural soil samples of Pawe Agricultural Research Center (PARC) against spoilage and foodborne bacterial pathogens.


## 2. Materials and Methods

### 2.1. Description of the Study Area

The study was conducted at PARC, Metekel Zone of Benishangul Gumuz region, North-West Ethiopia, at a distance of 575 km from Addis Ababa. Its geographical coordinates are located at a latitude and longitude of 11°19′N and 36°24′E with an elevation range of 980-1200 meters above sea level. Its annual minimum and maximum temperatures are 16.3 and 32.6 degrees Celsius, respectively, and its annual rainfall ranges from 900 to 1587 millimeters. It is situated about 3 km outskirts of the town, with an area of about 294.18 hectares. Nitosols, vertisols, and luvisols are the major soil types of PARC. The agricultural center is characterized by its hot to warm, moist agroecological zone and is well-known for the production of different varieties of crops like soybean [8].


### 2.2. Soil Sample Collection

A total of 30 soil samples were collected from the rhizosphere and agricultural soils of the soybean crop. The soils were excavated from a depth of 5-10 cm by using a sterile spatula and collected in clean, dry, and sterile polyethylene bags [9]. All samples were well-labeled and transported to PARC, Microbiology Laboratory with the help of an icebox. The rhizosphere and agricultural soil samples were air-dried for 7 days at room temperature, crushed, and sieved through a 250 μm pore size sieve.


### 2.3. Isolation and Maintenance of Actinomycetes

For both rhizosphere and agricultural soil samples, stock solutions were prepared by diluting a 1 g sieved soil sample in 10 mL of physiological saline (NaCl, 0.9 g/L) and shaking well using a vortex mixer [10]. From the stock solution, 1 mL was used to prepare the final volumes of 10-1up to 10-7 using a serial dilution technique. From 10-4 to 10-7 dilutions, 0.1 mL was taken and spread on the prepared Starch Casein Agar (SCA) medium (HiMedia, India) using a bent glass spreader. The medium was supplemented with 25 μg mL−1 cycloheximide and 20 μg mL−1 nalidixic acid to minimize the contaminant fungi and bacteria, respectively. Plates were incubated at 30 °C for 7 to 14 days. Then, the colonies with a tough or powdery texture, a dry or folded appearance, and branching filaments with or without aerial mycelia were subcultured on similar agar plates. The purified isolates were maintained in starch casein broth (SCB) (HiMedia, India) and kept at 4 °C for further screening and characterization tests [11].


### 2.4. Collection of Spoilage and Foodborne Bacterial Pathogens

Staphylococcus aureus (ATCC® 29213), Bacillus cereus (ATCC® 11778), Salmonella enterica serovar Typhimurium (ATCC® 14028), Listeria monocytogenes (ATCC® 7644), Pseudomonas aeruginosa (ATCC® 27853), and Escherichia coli (ATCC® 8739) were obtained from Amhara Public Health Institute (APHI) and used as test microorganisms. The strains were selected as a major foodborne illness causing bacterial pathogens.


### 2.5. Standardization and Inoculum Preparation for Antibacterial Production Test

0.5 McFarland standard was prepared by mixing 0.50 mL of (1.175% w/v) dehydrated barium chloride (BaCl2.2H2O) solution with 99.50 mL of (1% v/v) sulfuric acid (H2SO4). The turbidity standard solution was aliquoted into identical test tubes that were used to prepare the inoculum suspension. The prepared 0.5 McFarland standard solution was also further confirmed spectrophotometrically to have an absorbance reading of 0.08 to 0.12 at 625 nm. To prevent evaporation and light, the standard solution tube was tightly sealed and stored in a dark area at room temperature. Before being compared with the bacterial suspension, the turbidity standard tube was mixed using a vortex mixer to get a uniform turbid appearance [12].From a 24-h old pure agar culture, 4-5 morphologically similar bacterial colonies were suspended in 5 mL of sterile nutrient broth (HiMedia, India) and compared to that of the 0.5 McFarland standards, which is approximately equivalent to that of1.5×108 CFU/mL. After adjusting the turbidity, a sterile cotton swab was dipped into the suspension and streaked over the entire surface of the prepared Mueller-Hinton Agar (MHA) medium (HiMedia, India) by rotating the plate at 60° to ensure even distribution of the inoculum [13].


### 2.6. Primary Screening

The antibacterial activities of the actinomycetes isolated from the rhizosphere and agricultural soil samples were primarily screened against bacterial strains using the perpendicular streak plate method. The isolates to be screened for antibiotic production were streaked horizontally at the diameter of the MHA medium and incubated at 30 °C for 7 days [14]. After incubation, 0.5 McFarland standard adjusted bacterial strains (Viz., S. aureus, B. cereus, E. coli, L. monocytogenes, S. enterica, and P. aeruginosa) were streaked vertically at 90° angle very close to the screened one from left to right, respectively. The plates were then further incubated at 37 °C for 24 h, and zones of inhibition were indicated between the antibiotic-producing isolates and the test organisms that were considered positive for antibiotic production. The positive isolates were selected based on a wide spectrum of activity against tested bacterial strains. Then, the isolates were maintained in SCB and kept at 4 °C for further antibacterial compound production, secondary screening, and characterization tests.


### 2.7. Antibacterial Compound Production

Antagonistically active broad-spectrum actinomycete isolates were subjected to the submerged state fermentation method to produce crude extracts. 10% of fresh cultures of each antimicrobial-producing isolate were inoculated separately in a 500 mL Erlenmeyer flask containing 200 mL of SCB. For the production of the antibacterial compound, each flask was incubated at 30 °C for 7 days with constant shaking in a rotary shaker at 190 rpm. After incubation, each sample was centrifuged at 6,000 rpm for 10 minutes at 4°C to obtain cell-free culture supernatant (CFS). The supernatant obtained from each potential isolate was mixed with equal volume of ethyl acetate (1 : 1 v/v) and subjected to shaking on a rotary shaker at 250 rpm for 4 h. The organic phase was separated from the aqueous phase by using a separating funnel and concentrated to dryness under vacuum using a rotary evaporator at a temperature of 50 °C [15].


### 2.8. Antibacterial Activity Screening

The antibacterial activities of the CFS were evaluated against bacterial strains using the disc diffusion assay as described by Kirby-Bauer with some modifications [16]. After adjusting the turbidity to that of 1.5×108 CFU/mL, the bacterial strains, viz., S. aureus, B. cereus, E. coli, L. monocytogenes, S. enterica, and P. aeruginosa, were swabbed uniformly on sterile MHA medium using a sterile cotton swab. Sterile 6 mm diameter paper discs were impregnated with 10 μl of the CFS. The impregnated discs were introduced to the upper layer of the seeded agar plate and incubated at 37 °C for 24 h. The antibacterial activities of the CFS were compared with those of the known antibiotic gentamicin (10 μg/disc) as a positive control and ethyl acetate (10 μl/disc) as a negative control. Antibacterial activity was evaluated by measuring the diameter of the inhibition zone (mm) on the surface of the plates, and the results were reported as mean±SE after three repeats of the experiment [17].


### 2.9. Characterization of Selected Strains and Their Antimicrobial Compounds

Colony characteristics (aerial and substrate mycelium and consistency) and standard biochemical and physiological (Gram staining; starch, casein, gelatin, and urea hydrolysis; nitrate reduction, citrate utilization; indole and H2S production; motility test; catalase reaction; and lysine decarboxylase test) tests were conducted. The potent broad-spectrum antimicrobial compound-producing actinomycetes were identified following morphological, physiological, biochemical, and molecular characterizations [18].A 16S rRNA gene was used for molecular characterization of selected strains based on DNA sequencing. Based on the manufacturer’s instructions, genomic DNA was extracted from pure cultures using the genomic DNA extraction kit for Gram positive bacteria (QIAGEN). 16S rRNA gene sequences in target-specific screening strains were amplified with the universal primers RP1 (5′-AGAGTTT-GATCATGGCTCAG-3′) and FD2 (5′-ACGGTTACCTT- GTTACGACTT-3′) [18]. PCR was carried out at a total volume of 50 μl. The reaction mixture contained 25 μl of Taq master mix (Genet Bio, Seoul, South Korea), 2 μl of each of forward and reverse primers (10 μM stock), 2 μl of template DNA (50 ng μl–1), and 19 μl of PCR grade pure water. The reaction mixture was contained for 4 min at 95 °C, and then, in 35 cycles, amplification was performed. For each cycle, 94 °C for 1 min, 51 °C for 1 min, and 72 °C for 2 min were the denatured, annealed, and extended temperatures and times, respectively. The final extension was performed at 72 °C for 10 min. The PCR products from potent strains were purified using the QIAGEN PCR purification kit. PCR-purified 16S rRNA genes of the potent strains were sequenced partially in both directions using the same two primers. The results obtained via sequencing were compared with other related taxa in the GenBank databases using the NCBI BLAST (http://www.ncbi.nlm.nih.gov/). [18].Various enzymes, namely, proteinase K, pepsin,α-amylase, and lysozyme (BIO BASIC Canada INC) were added to the CFS of the potent strains to evaluate their effect on antibacterial activity. The potent CFS obtained were treated with catalase in a water bath at 25 °C for 1 h, filtered, and stored at 4 °C. Calcium chloride (CaCl2) buffer was added at 1 mg/mL of proteinase K, papain, α-amylase, and lysozyme enzymes. Then, the solutions were filter-sterilized and set at 37 °C for 3 h, and the mixture was boiled for 3 min to inactivate the enzymes. The inhibitory effects were analyzed using the disc diffusion assay. By observing the presence of an inhibition zone, the activity of the residual was finally determined [2, 3].The antibacterial activity of the inhibitory substances was also evaluated after treatment under various conditions. The effects of temperature on CFS activity were estimated by heating for 30 minutes at 45, 65, 85, 100, and 121°C. The effect of pH on the CFS antibacterial activity was determined by adjusting the pH of the CFS to 2, 4, 6, 8, 10, and 12, using 1 N hydrochloric acid (HCl), 1 N sodium hydroxide (NaOH), and a pH meter. The CFS was exposed to 30 °C for 1 h, and the samples were readjusted to pH 6.0, and the activity was determined. To observe the effect of an organic solvent on the bioactivity of CFS, methanol (CH3OH) and ethanol (CH3CH2OH) solvents were added to the CFS at 1 : 9 (v/v) and placed at 30 °C for 30 min. To observe the effect of the additives on the antibacterial activity, 1% (w/t) sodium citrate (Na3C6H5O7) and potassium chloride (KCl2) were added and mixed into the CFS. All of the treated solutions in the antibacterial experiments were incubated for 24 h at 37 °C using the disc diffusion method. The inhibitory activity of the residual was determined by observing their zone of inhibition. Sterile water and untreated CFS were used as negative and positive controls, respectively [2–4].


### 2.10. Determination of the Minimum Inhibitory and Bactericidal Concentrations

The minimum inhibitory concentration and minimum bactericidal concentration were determined by the broth twofold serial dilution method as described by Andrews [19]. Twelve screw-capped test tubes were picked and a volume of 1000 μL of nutrient broth (HiMedia, India) was dispensed into tubes 1-10 and 2 mL into tube 11 (negative control). One milliliter of CFS was added to test tube 1, mixed, and transferred to test tube 2, and the process was repeated serially up to test tube 10 by mixing and changing the micropipette tips at each dilution, with 2000 μL added to test tube 12 (positive control). 100 μL of standardized inoculum was added to test tubes 1–10 and incubated for 24 hours at 37 °C. After incubation, the MIC value was determined spectrophotometrically by absorbance reading. From the above test tubes with no growth (no turbidity), 0.1 mL was spread on the surface of MHA plates to note the survival of the pathogens. The concentration of extract resulting in no growth of bacterial colonies on the plate after incubation at 37 °C for 24 h was considered the MBC value.


### 2.11. Statistical Analysis

Triplicate independent experiments were performed and recorded from screening. The secondary screening data were analyzed by one-way analysis of variance (ANOVA) using the statistical package for social science (SPSS) version, 23. Differences among means were tested for significance (P<0.05) by Duncan’s multiple range test.


## 2.1. Description of the Study Area

The study was conducted at PARC, Metekel Zone of Benishangul Gumuz region, North-West Ethiopia, at a distance of 575 km from Addis Ababa. Its geographical coordinates are located at a latitude and longitude of 11°19′N and 36°24′E with an elevation range of 980-1200 meters above sea level. Its annual minimum and maximum temperatures are 16.3 and 32.6 degrees Celsius, respectively, and its annual rainfall ranges from 900 to 1587 millimeters. It is situated about 3 km outskirts of the town, with an area of about 294.18 hectares. Nitosols, vertisols, and luvisols are the major soil types of PARC. The agricultural center is characterized by its hot to warm, moist agroecological zone and is well-known for the production of different varieties of crops like soybean [8].


## 2.2. Soil Sample Collection

A total of 30 soil samples were collected from the rhizosphere and agricultural soils of the soybean crop. The soils were excavated from a depth of 5-10 cm by using a sterile spatula and collected in clean, dry, and sterile polyethylene bags [9]. All samples were well-labeled and transported to PARC, Microbiology Laboratory with the help of an icebox. The rhizosphere and agricultural soil samples were air-dried for 7 days at room temperature, crushed, and sieved through a 250 μm pore size sieve.


## 2.3. Isolation and Maintenance of Actinomycetes

For both rhizosphere and agricultural soil samples, stock solutions were prepared by diluting a 1 g sieved soil sample in 10 mL of physiological saline (NaCl, 0.9 g/L) and shaking well using a vortex mixer [10]. From the stock solution, 1 mL was used to prepare the final volumes of 10-1up to 10-7 using a serial dilution technique. From 10-4 to 10-7 dilutions, 0.1 mL was taken and spread on the prepared Starch Casein Agar (SCA) medium (HiMedia, India) using a bent glass spreader. The medium was supplemented with 25 μg mL−1 cycloheximide and 20 μg mL−1 nalidixic acid to minimize the contaminant fungi and bacteria, respectively. Plates were incubated at 30 °C for 7 to 14 days. Then, the colonies with a tough or powdery texture, a dry or folded appearance, and branching filaments with or without aerial mycelia were subcultured on similar agar plates. The purified isolates were maintained in starch casein broth (SCB) (HiMedia, India) and kept at 4 °C for further screening and characterization tests [11].


## 2.4. Collection of Spoilage and Foodborne Bacterial Pathogens

Staphylococcus aureus (ATCC® 29213), Bacillus cereus (ATCC® 11778), Salmonella enterica serovar Typhimurium (ATCC® 14028), Listeria monocytogenes (ATCC® 7644), Pseudomonas aeruginosa (ATCC® 27853), and Escherichia coli (ATCC® 8739) were obtained from Amhara Public Health Institute (APHI) and used as test microorganisms. The strains were selected as a major foodborne illness causing bacterial pathogens.


## 2.5. Standardization and Inoculum Preparation for Antibacterial Production Test

0.5 McFarland standard was prepared by mixing 0.50 mL of (1.175% w/v) dehydrated barium chloride (BaCl2.2H2O) solution with 99.50 mL of (1% v/v) sulfuric acid (H2SO4). The turbidity standard solution was aliquoted into identical test tubes that were used to prepare the inoculum suspension. The prepared 0.5 McFarland standard solution was also further confirmed spectrophotometrically to have an absorbance reading of 0.08 to 0.12 at 625 nm. To prevent evaporation and light, the standard solution tube was tightly sealed and stored in a dark area at room temperature. Before being compared with the bacterial suspension, the turbidity standard tube was mixed using a vortex mixer to get a uniform turbid appearance [12].From a 24-h old pure agar culture, 4-5 morphologically similar bacterial colonies were suspended in 5 mL of sterile nutrient broth (HiMedia, India) and compared to that of the 0.5 McFarland standards, which is approximately equivalent to that of1.5×108 CFU/mL. After adjusting the turbidity, a sterile cotton swab was dipped into the suspension and streaked over the entire surface of the prepared Mueller-Hinton Agar (MHA) medium (HiMedia, India) by rotating the plate at 60° to ensure even distribution of the inoculum [13].


## 2.6. Primary Screening

The antibacterial activities of the actinomycetes isolated from the rhizosphere and agricultural soil samples were primarily screened against bacterial strains using the perpendicular streak plate method. The isolates to be screened for antibiotic production were streaked horizontally at the diameter of the MHA medium and incubated at 30 °C for 7 days [14]. After incubation, 0.5 McFarland standard adjusted bacterial strains (Viz., S. aureus, B. cereus, E. coli, L. monocytogenes, S. enterica, and P. aeruginosa) were streaked vertically at 90° angle very close to the screened one from left to right, respectively. The plates were then further incubated at 37 °C for 24 h, and zones of inhibition were indicated between the antibiotic-producing isolates and the test organisms that were considered positive for antibiotic production. The positive isolates were selected based on a wide spectrum of activity against tested bacterial strains. Then, the isolates were maintained in SCB and kept at 4 °C for further antibacterial compound production, secondary screening, and characterization tests.


## 2.7. Antibacterial Compound Production

Antagonistically active broad-spectrum actinomycete isolates were subjected to the submerged state fermentation method to produce crude extracts. 10% of fresh cultures of each antimicrobial-producing isolate were inoculated separately in a 500 mL Erlenmeyer flask containing 200 mL of SCB. For the production of the antibacterial compound, each flask was incubated at 30 °C for 7 days with constant shaking in a rotary shaker at 190 rpm. After incubation, each sample was centrifuged at 6,000 rpm for 10 minutes at 4°C to obtain cell-free culture supernatant (CFS). The supernatant obtained from each potential isolate was mixed with equal volume of ethyl acetate (1 : 1 v/v) and subjected to shaking on a rotary shaker at 250 rpm for 4 h. The organic phase was separated from the aqueous phase by using a separating funnel and concentrated to dryness under vacuum using a rotary evaporator at a temperature of 50 °C [15].


## 2.8. Antibacterial Activity Screening

The antibacterial activities of the CFS were evaluated against bacterial strains using the disc diffusion assay as described by Kirby-Bauer with some modifications [16]. After adjusting the turbidity to that of 1.5×108 CFU/mL, the bacterial strains, viz., S. aureus, B. cereus, E. coli, L. monocytogenes, S. enterica, and P. aeruginosa, were swabbed uniformly on sterile MHA medium using a sterile cotton swab. Sterile 6 mm diameter paper discs were impregnated with 10 μl of the CFS. The impregnated discs were introduced to the upper layer of the seeded agar plate and incubated at 37 °C for 24 h. The antibacterial activities of the CFS were compared with those of the known antibiotic gentamicin (10 μg/disc) as a positive control and ethyl acetate (10 μl/disc) as a negative control. Antibacterial activity was evaluated by measuring the diameter of the inhibition zone (mm) on the surface of the plates, and the results were reported as mean±SE after three repeats of the experiment [17].


## 2.9. Characterization of Selected Strains and Their Antimicrobial Compounds

Colony characteristics (aerial and substrate mycelium and consistency) and standard biochemical and physiological (Gram staining; starch, casein, gelatin, and urea hydrolysis; nitrate reduction, citrate utilization; indole and H2S production; motility test; catalase reaction; and lysine decarboxylase test) tests were conducted. The potent broad-spectrum antimicrobial compound-producing actinomycetes were identified following morphological, physiological, biochemical, and molecular characterizations [18].A 16S rRNA gene was used for molecular characterization of selected strains based on DNA sequencing. Based on the manufacturer’s instructions, genomic DNA was extracted from pure cultures using the genomic DNA extraction kit for Gram positive bacteria (QIAGEN). 16S rRNA gene sequences in target-specific screening strains were amplified with the universal primers RP1 (5′-AGAGTTT-GATCATGGCTCAG-3′) and FD2 (5′-ACGGTTACCTT- GTTACGACTT-3′) [18]. PCR was carried out at a total volume of 50 μl. The reaction mixture contained 25 μl of Taq master mix (Genet Bio, Seoul, South Korea), 2 μl of each of forward and reverse primers (10 μM stock), 2 μl of template DNA (50 ng μl–1), and 19 μl of PCR grade pure water. The reaction mixture was contained for 4 min at 95 °C, and then, in 35 cycles, amplification was performed. For each cycle, 94 °C for 1 min, 51 °C for 1 min, and 72 °C for 2 min were the denatured, annealed, and extended temperatures and times, respectively. The final extension was performed at 72 °C for 10 min. The PCR products from potent strains were purified using the QIAGEN PCR purification kit. PCR-purified 16S rRNA genes of the potent strains were sequenced partially in both directions using the same two primers. The results obtained via sequencing were compared with other related taxa in the GenBank databases using the NCBI BLAST (http://www.ncbi.nlm.nih.gov/). [18].Various enzymes, namely, proteinase K, pepsin,α-amylase, and lysozyme (BIO BASIC Canada INC) were added to the CFS of the potent strains to evaluate their effect on antibacterial activity. The potent CFS obtained were treated with catalase in a water bath at 25 °C for 1 h, filtered, and stored at 4 °C. Calcium chloride (CaCl2) buffer was added at 1 mg/mL of proteinase K, papain, α-amylase, and lysozyme enzymes. Then, the solutions were filter-sterilized and set at 37 °C for 3 h, and the mixture was boiled for 3 min to inactivate the enzymes. The inhibitory effects were analyzed using the disc diffusion assay. By observing the presence of an inhibition zone, the activity of the residual was finally determined [2, 3].The antibacterial activity of the inhibitory substances was also evaluated after treatment under various conditions. The effects of temperature on CFS activity were estimated by heating for 30 minutes at 45, 65, 85, 100, and 121°C. The effect of pH on the CFS antibacterial activity was determined by adjusting the pH of the CFS to 2, 4, 6, 8, 10, and 12, using 1 N hydrochloric acid (HCl), 1 N sodium hydroxide (NaOH), and a pH meter. The CFS was exposed to 30 °C for 1 h, and the samples were readjusted to pH 6.0, and the activity was determined. To observe the effect of an organic solvent on the bioactivity of CFS, methanol (CH3OH) and ethanol (CH3CH2OH) solvents were added to the CFS at 1 : 9 (v/v) and placed at 30 °C for 30 min. To observe the effect of the additives on the antibacterial activity, 1% (w/t) sodium citrate (Na3C6H5O7) and potassium chloride (KCl2) were added and mixed into the CFS. All of the treated solutions in the antibacterial experiments were incubated for 24 h at 37 °C using the disc diffusion method. The inhibitory activity of the residual was determined by observing their zone of inhibition. Sterile water and untreated CFS were used as negative and positive controls, respectively [2–4].


## 2.10. Determination of the Minimum Inhibitory and Bactericidal Concentrations

The minimum inhibitory concentration and minimum bactericidal concentration were determined by the broth twofold serial dilution method as described by Andrews [19]. Twelve screw-capped test tubes were picked and a volume of 1000 μL of nutrient broth (HiMedia, India) was dispensed into tubes 1-10 and 2 mL into tube 11 (negative control). One milliliter of CFS was added to test tube 1, mixed, and transferred to test tube 2, and the process was repeated serially up to test tube 10 by mixing and changing the micropipette tips at each dilution, with 2000 μL added to test tube 12 (positive control). 100 μL of standardized inoculum was added to test tubes 1–10 and incubated for 24 hours at 37 °C. After incubation, the MIC value was determined spectrophotometrically by absorbance reading. From the above test tubes with no growth (no turbidity), 0.1 mL was spread on the surface of MHA plates to note the survival of the pathogens. The concentration of extract resulting in no growth of bacterial colonies on the plate after incubation at 37 °C for 24 h was considered the MBC value.


## 2.11. Statistical Analysis

Triplicate independent experiments were performed and recorded from screening. The secondary screening data were analyzed by one-way analysis of variance (ANOVA) using the statistical package for social science (SPSS) version, 23. Differences among means were tested for significance (P<0.05) by Duncan’s multiple range test.


## 3. Results

### 3.1. Isolation and Characterization of Potent Isolates

A total of 118 different actinomycete isolates were recovered from the rhizosphere 83 (70.30%) and agricultural 35 (29.70%) soil samples of PARC using starch casein agar growth medium. Of those, 36 (30.50%) isolates showed antibacterial activity against at least two of the test bacterial strains (Table1). Out of the 36 bioactive isolates, only eight strains (five from the rhizosphere and three from agricultural soil samples) demonstrated broad-spectrum antibacterial activity (Table 2 and Table 3). These potential isolates (RS18, RS25, RS37, RS48, RS72, AS89, AS97, and AS115) were characterized following morphological, physiological, biochemical, and molecular tests. The isolates showed red, gray, and white aerial mycelium, while the substrate mycelium showed yellow, gray, green, orange, and brown colors (Figure 1). All isolates showed positive Gram and catalase reactions, nitrate reduction, starch, casein, and urea hydrolysis. Negative indole and H2S production, motility, and lysine decarboxylase tests were observed (Table 2). According to molecular and a series of morphological, physiological, and biochemical characterizations, eight highly active and wide-spectrum actinomycete isolates were included under the genus Streptomyces (Table 4).Table 1
Primary screening of actinomycete isolates for antimicrobial compound production against spoilage and foodborne bacterial pathogens.
Gram-positive test bacterial strainsGram-negative test bacterial strainsIsolatesS. aureusB. cereusL. MonocytogenesE. coliS. EntericaP. aeruginosaRS1—++——+RS3+———+—RS7+———+—RS9—+++——+RS12+—+———RS14++—++—RS18++++++++++++RS19+++———RS21—+++———RS23——+—+—RS25++++++++++++RS26+——+——RS28—++——+RS29—++————RS30—++———RS31+——+——RS33++++———RS35+++——+—RS37++++++++++++RS40—++———RS42+——++—RS45+———+—RS46—+——+—RS48++++++++++++RS49+———+—RS72++++++++++++AS78+————+AS84+———+—AS89++++++++++++AS93+——++—AS97++++++++++++AS100+——+——AS104++————AS107—++—+——AS111—+++——AS115++++++++++++Keys: RS = rhizosphere soil; AS = agricultural soil. ̶ denotes no clear zone + inhibitionzone<8mm; ++ have inhibition zone>10mm.Table 2
The morphological, physiological, and biochemical characteristics of the isolates with broad-spectrum antagonistic activity.
CharacteristicsRS18RS25RS37RS48RS72AS89AS97AS115Aerial myceliumWhiteWhiteWhiteRedWhiteGrayWhiteWhiteSubstrate myceliumGrayBrownGrayOrangeGreenYellowYellowBrownConsistencyDryDryDryMoistDryMoistDryMoistGram reaction++++++++Starch hydrolysis++++++++Casein hydrolysis++++++++Nitrate reduction++++++++Gelatin hydrolysis+—+—++—+Urea hydrolysis++++++++Citrate utilization+—++—+—+Indole production————————H2S production————————Motility test————————Catalase reaction+++++—++Lysine decarboxylase test————————Presumptive identificationStreptomycesStreptomycesStreptomycesStreptomycesStreptomycesStreptomycesStreptomycesStreptomycesKey: + = positive for test; ̶ = test negative.Table 3
Secondary screening ofStreptomyces isolates in triplicate mean±SE of the clear zone in mm against spoilage and foodborne bacterial pathogens using the disc diffusion assay.
Test strainsStreptomyces isolates inhibition zone in millimeter (mm)S18±SERS25±SERS37±SERS48±SERS72±SEAS89±SEAS97±SEAS115±SECN±SEEASa14±0.7c16±0.1b15±0.2c23±1.0c14±0.4b22±0.6c15±0.3c24±1.4c19±1.4aNDBc13±0.4a11±0.8b10±0.1a20±1.4b12±0.6b22±0.5b9.5±1.4b21±0.5b24±0.5cNDEc12±0.6c7.0±0.5a11±1.4b20±0.8b10±0.5a18±1.4a7.6±0.6a19±1.4c23±1.4cNDLm12±0.3a8.5±0.5c10±0.4a21±0.5c14±1.4c22±0.3b7.8±0.4a21±0.3c23±0.8cNDSe11±0.7b12±0.6b13±0.8b23±0.3c10±0.5a22±1.4c12±0.7b21±0.7b22±1.4bNDPa10±0.5c7.5±0.5a9.0±0.3a17±0.6b11±0.8a18±0.8b12±0.8b19±0.1a21±0.0aNDKeys: Sa =Staphylococcus aureus; Bc = Bacillus cereus; Ec = Escherichia coli; Lm = Listeria monocytogenes; Se = Salmonella enterica; Pa = Pseudomonas aeruginosa; CN = gentamicin (10 μg/disc) as a positive control; EA = ethylene acetate (10 μl/disc) as a negative control; ND = not detected. Data are average of triplicates; numbers followed by the same letter superscripts in the same row do not vary significantly by Duncan’s multiple range test at P<0.05; SE: standard error.Figure 1
Colony characteristics of broad-spectrum bioactive compound producingStreptomyces isolates.Table 4
Identification ofStreptomyces strains through 16S rRNA gene analysis.
Isolates codeIdentifiedStreptomyces strainsPercent (%) of similarityRS18Streptomyces fasciculus85RS25Streptomyces roseochromogenes80RS37Streptomyces ruber80RS48Streptomyces glaucus98RS72Streptomyces griseus98AS89Streptomyces cellulosae98AS97Streptomyces griseoflavus85AS115Streptomyces xanthophaeus80The sequences of 16S rRNA gene products of the eightStreptomyces strains with antibacterial activities result were observed at 1500 bp of ladder sequence and compared with the GenBank databases using the NCBI BLAST and gave 98% similarity to Streptomyces glaucus, Streptomyces griseus, and Streptomyces cellulosae, 85% identical to Streptomyces fasciculus, and Streptomyces griseoflavus were observed, while 80% homology was observed for Streptomyces roseochromogenes, Streptomyces ruber, and Streptomyces xanthophaeus (Table 4 and Figure 2).Figure 2
PCR amplification of the 16S rRNA from the isolatedStreptomyces on a 0.8% agarose gel. M: marker. RS18, RS25, RS37, RS48, RS72, AS89, AS97, and AS115 refer to the isolates code denoted in the tables.The CFS was treated with different enzymes (Proteinase K, Papain,α-Amylase, and Lysozyme) and their antagonistic effects were examined after 24 h of incubation at 37 °C. The antibacterial effect of the CFS of strains was reduced after treatment with proteinase K and papain, while the lysozyme and α-amylase enzymes had no effect on the antagonistic activity of the CFS of the Streptomyces strains (Table 5).Table 5
Effects of different enzymes on the antibacterial activities of metabolites of the stains.
EnzymesRS18RS25RS37RS48RS72AS89AS97AS115Proteinase K++++++++Papain++++++++α-Amylase++++++++++++++++Lysozyme+++++++++++++++++ Inhibitionzone<8 mm; ++ have inhibition zone>10mm.Low and medium temperatures had no effect on the antibacterial activity of theStreptomyces CFS. However, high temperatures (100 and 121°C) had a high effect. pH of 4 and 6 is a preferable environment for the inhibitory effect of the CFS. Ethanol was a good solvent that enhanced the antibacterial effects of the CFS as compared to methanol. The eight Streptomyces strain CFS were treated with additives (sodium citrate and potassium chloride) to verify the effect of food additives and other chemicals on the antibacterial substances of Streptomyces. Sodium citrate was an effective additive with Streptomyces strain CFS as compared to potassium chloride (Table 6).Table 6
The antagonistic effects of temperature, pH, organic solvents, and additives on the CFS.
Concentrations used for treatmentRS18RS25RS37RS48RS72RS89RS97RS115Temperature/time45°C/30 min++++++++++++++++65°C/30 min++++++++++++++++85°C/30 min+++++++++++100°C/30 min̶̶—++—++̶++121°C/30 min̶̶—̶̶—̶̶pH/time2/30 min++++++++++++4/30 min++++++++++++++++6/30 min++++++++++++++++8/30 min+++++++++++10/30 min̶̶̶̶̶̶̶̶12/30 min̶̶—̶̶—̶̶Organic solvent/timeMethanol 10% (vol/vol)/30 min+++++++++++Ethanol 10% (vol/vol)/30 min++++++++++++++++Additive/timePotassium chloride 1% (wt/vol)/30 min+++++++++++Sodium citrate 1%(wt/vol)/30 min+++++++++++++++̶ ̶: no inhibition zone; + Inhibition zone<8mm; ++ have inhibition zone>10 mm.


### 3.2. Primary and Secondary Screening

Out of 118 actinomycete isolates subjected to primary screening, 36 (30.50%) isolates showed varying levels of antimicrobial activity against the six test organisms (Table1). During screening, the highest inhibition zones were recorded from three (RS48, AS89, and AS115) actinomycete strains (Figure 3).Figure 3
Primary screening ofStreptomyces isolates for antibacterial compound production against bacterial strains using the perpendicular streak plate method.The CFS of the eight potent isolates (RS18, RS25, RS37, RS48, RS72, AS89, AS97, and AS115) was subjected to secondary screening using the disc diffusion method (Table3 and Figure 4). The in vitro antibacterial activity ranges from 7 to 24 mm in diameter. During the secondary screening, as compared to the remaining isolates, the results of this study revealed that RS48, AS89, and AS115 showed the highest inhibition zones with broad-spectrum activity against all tested spoilage and foodborne bacterial pathogens.Figure 4
Secondary screening of someStreptomyces isolates and gentamicin (center) against spoilage and foodborne bacterial pathogens using the disc diffusion assay.


### 3.3. MIC and MBC of the Potent Isolates

The MIC and MBC of all broad-spectrum antibacterial compound-producingStreptomyces were done against six (3 Gram-positive and 3 Gram-negative) bacterial strains. The MIC value ranges from 0.10 to 0.25 μg/mL, while the MBC value ranges from 0.15 to 0.40 μg/mL. As compared to the other tested pathogens, L. monocytogenes, S. enterica, and P. aeruginosa required high μg/mL CFS to inhibit and kill them (Table 7).Table 7
The MIC and MBC of the isolates CFS inμg/mL against foodborne bacterial strains.
Isolates codeMICMBCSaBcEcLmSePaSaBcEcLmSePaRS180.150.100.200.250.250.250.300.200.300.400.400.40RS250.200.150.100.250.250.250.300.200.200.400.400.40RS370.100.150.200.250.250.250.200.300.300.400.400.40RS480.100.100.100.200.150.200.200.200.200.300.200.30RS720.200.100.200.250.250.250.300.200.300.400.400.40AS890.100.100.100.200.100.100.200.200.200.300.200.20AS970.200.100.200.250.200.250.300.300.300.300.400.40AS1150.100.100.100.200.100.200.200.200.200.300.200.30Keys: Sa =Staphylococcus aureus; Bc = Bacillus cereus; Ec = Escherichia coli; Lm = Listeria monocytogenes; Se = Salmonella enterica; Pa = Pseudomonas aeruginosa.


## 3.1. Isolation and Characterization of Potent Isolates

A total of 118 different actinomycete isolates were recovered from the rhizosphere 83 (70.30%) and agricultural 35 (29.70%) soil samples of PARC using starch casein agar growth medium. Of those, 36 (30.50%) isolates showed antibacterial activity against at least two of the test bacterial strains (Table1). Out of the 36 bioactive isolates, only eight strains (five from the rhizosphere and three from agricultural soil samples) demonstrated broad-spectrum antibacterial activity (Table 2 and Table 3). These potential isolates (RS18, RS25, RS37, RS48, RS72, AS89, AS97, and AS115) were characterized following morphological, physiological, biochemical, and molecular tests. The isolates showed red, gray, and white aerial mycelium, while the substrate mycelium showed yellow, gray, green, orange, and brown colors (Figure 1). All isolates showed positive Gram and catalase reactions, nitrate reduction, starch, casein, and urea hydrolysis. Negative indole and H2S production, motility, and lysine decarboxylase tests were observed (Table 2). According to molecular and a series of morphological, physiological, and biochemical characterizations, eight highly active and wide-spectrum actinomycete isolates were included under the genus Streptomyces (Table 4).Table 1
Primary screening of actinomycete isolates for antimicrobial compound production against spoilage and foodborne bacterial pathogens.
Gram-positive test bacterial strainsGram-negative test bacterial strainsIsolatesS. aureusB. cereusL. MonocytogenesE. coliS. EntericaP. aeruginosaRS1—++——+RS3+———+—RS7+———+—RS9—+++——+RS12+—+———RS14++—++—RS18++++++++++++RS19+++———RS21—+++———RS23——+—+—RS25++++++++++++RS26+——+——RS28—++——+RS29—++————RS30—++———RS31+——+——RS33++++———RS35+++——+—RS37++++++++++++RS40—++———RS42+——++—RS45+———+—RS46—+——+—RS48++++++++++++RS49+———+—RS72++++++++++++AS78+————+AS84+———+—AS89++++++++++++AS93+——++—AS97++++++++++++AS100+——+——AS104++————AS107—++—+——AS111—+++——AS115++++++++++++Keys: RS = rhizosphere soil; AS = agricultural soil. ̶ denotes no clear zone + inhibitionzone<8mm; ++ have inhibition zone>10mm.Table 2
The morphological, physiological, and biochemical characteristics of the isolates with broad-spectrum antagonistic activity.
CharacteristicsRS18RS25RS37RS48RS72AS89AS97AS115Aerial myceliumWhiteWhiteWhiteRedWhiteGrayWhiteWhiteSubstrate myceliumGrayBrownGrayOrangeGreenYellowYellowBrownConsistencyDryDryDryMoistDryMoistDryMoistGram reaction++++++++Starch hydrolysis++++++++Casein hydrolysis++++++++Nitrate reduction++++++++Gelatin hydrolysis+—+—++—+Urea hydrolysis++++++++Citrate utilization+—++—+—+Indole production————————H2S production————————Motility test————————Catalase reaction+++++—++Lysine decarboxylase test————————Presumptive identificationStreptomycesStreptomycesStreptomycesStreptomycesStreptomycesStreptomycesStreptomycesStreptomycesKey: + = positive for test; ̶ = test negative.Table 3
Secondary screening ofStreptomyces isolates in triplicate mean±SE of the clear zone in mm against spoilage and foodborne bacterial pathogens using the disc diffusion assay.
Test strainsStreptomyces isolates inhibition zone in millimeter (mm)S18±SERS25±SERS37±SERS48±SERS72±SEAS89±SEAS97±SEAS115±SECN±SEEASa14±0.7c16±0.1b15±0.2c23±1.0c14±0.4b22±0.6c15±0.3c24±1.4c19±1.4aNDBc13±0.4a11±0.8b10±0.1a20±1.4b12±0.6b22±0.5b9.5±1.4b21±0.5b24±0.5cNDEc12±0.6c7.0±0.5a11±1.4b20±0.8b10±0.5a18±1.4a7.6±0.6a19±1.4c23±1.4cNDLm12±0.3a8.5±0.5c10±0.4a21±0.5c14±1.4c22±0.3b7.8±0.4a21±0.3c23±0.8cNDSe11±0.7b12±0.6b13±0.8b23±0.3c10±0.5a22±1.4c12±0.7b21±0.7b22±1.4bNDPa10±0.5c7.5±0.5a9.0±0.3a17±0.6b11±0.8a18±0.8b12±0.8b19±0.1a21±0.0aNDKeys: Sa =Staphylococcus aureus; Bc = Bacillus cereus; Ec = Escherichia coli; Lm = Listeria monocytogenes; Se = Salmonella enterica; Pa = Pseudomonas aeruginosa; CN = gentamicin (10 μg/disc) as a positive control; EA = ethylene acetate (10 μl/disc) as a negative control; ND = not detected. Data are average of triplicates; numbers followed by the same letter superscripts in the same row do not vary significantly by Duncan’s multiple range test at P<0.05; SE: standard error.Figure 1
Colony characteristics of broad-spectrum bioactive compound producingStreptomyces isolates.Table 4
Identification ofStreptomyces strains through 16S rRNA gene analysis.
Isolates codeIdentifiedStreptomyces strainsPercent (%) of similarityRS18Streptomyces fasciculus85RS25Streptomyces roseochromogenes80RS37Streptomyces ruber80RS48Streptomyces glaucus98RS72Streptomyces griseus98AS89Streptomyces cellulosae98AS97Streptomyces griseoflavus85AS115Streptomyces xanthophaeus80The sequences of 16S rRNA gene products of the eightStreptomyces strains with antibacterial activities result were observed at 1500 bp of ladder sequence and compared with the GenBank databases using the NCBI BLAST and gave 98% similarity to Streptomyces glaucus, Streptomyces griseus, and Streptomyces cellulosae, 85% identical to Streptomyces fasciculus, and Streptomyces griseoflavus were observed, while 80% homology was observed for Streptomyces roseochromogenes, Streptomyces ruber, and Streptomyces xanthophaeus (Table 4 and Figure 2).Figure 2
PCR amplification of the 16S rRNA from the isolatedStreptomyces on a 0.8% agarose gel. M: marker. RS18, RS25, RS37, RS48, RS72, AS89, AS97, and AS115 refer to the isolates code denoted in the tables.The CFS was treated with different enzymes (Proteinase K, Papain,α-Amylase, and Lysozyme) and their antagonistic effects were examined after 24 h of incubation at 37 °C. The antibacterial effect of the CFS of strains was reduced after treatment with proteinase K and papain, while the lysozyme and α-amylase enzymes had no effect on the antagonistic activity of the CFS of the Streptomyces strains (Table 5).Table 5
Effects of different enzymes on the antibacterial activities of metabolites of the stains.
EnzymesRS18RS25RS37RS48RS72AS89AS97AS115Proteinase K++++++++Papain++++++++α-Amylase++++++++++++++++Lysozyme+++++++++++++++++ Inhibitionzone<8 mm; ++ have inhibition zone>10mm.Low and medium temperatures had no effect on the antibacterial activity of theStreptomyces CFS. However, high temperatures (100 and 121°C) had a high effect. pH of 4 and 6 is a preferable environment for the inhibitory effect of the CFS. Ethanol was a good solvent that enhanced the antibacterial effects of the CFS as compared to methanol. The eight Streptomyces strain CFS were treated with additives (sodium citrate and potassium chloride) to verify the effect of food additives and other chemicals on the antibacterial substances of Streptomyces. Sodium citrate was an effective additive with Streptomyces strain CFS as compared to potassium chloride (Table 6).Table 6
The antagonistic effects of temperature, pH, organic solvents, and additives on the CFS.
Concentrations used for treatmentRS18RS25RS37RS48RS72RS89RS97RS115Temperature/time45°C/30 min++++++++++++++++65°C/30 min++++++++++++++++85°C/30 min+++++++++++100°C/30 min̶̶—++—++̶++121°C/30 min̶̶—̶̶—̶̶pH/time2/30 min++++++++++++4/30 min++++++++++++++++6/30 min++++++++++++++++8/30 min+++++++++++10/30 min̶̶̶̶̶̶̶̶12/30 min̶̶—̶̶—̶̶Organic solvent/timeMethanol 10% (vol/vol)/30 min+++++++++++Ethanol 10% (vol/vol)/30 min++++++++++++++++Additive/timePotassium chloride 1% (wt/vol)/30 min+++++++++++Sodium citrate 1%(wt/vol)/30 min+++++++++++++++̶ ̶: no inhibition zone; + Inhibition zone<8mm; ++ have inhibition zone>10 mm.


## 3.2. Primary and Secondary Screening

Out of 118 actinomycete isolates subjected to primary screening, 36 (30.50%) isolates showed varying levels of antimicrobial activity against the six test organisms (Table1). During screening, the highest inhibition zones were recorded from three (RS48, AS89, and AS115) actinomycete strains (Figure 3).Figure 3
Primary screening ofStreptomyces isolates for antibacterial compound production against bacterial strains using the perpendicular streak plate method.The CFS of the eight potent isolates (RS18, RS25, RS37, RS48, RS72, AS89, AS97, and AS115) was subjected to secondary screening using the disc diffusion method (Table3 and Figure 4). The in vitro antibacterial activity ranges from 7 to 24 mm in diameter. During the secondary screening, as compared to the remaining isolates, the results of this study revealed that RS48, AS89, and AS115 showed the highest inhibition zones with broad-spectrum activity against all tested spoilage and foodborne bacterial pathogens.Figure 4
Secondary screening of someStreptomyces isolates and gentamicin (center) against spoilage and foodborne bacterial pathogens using the disc diffusion assay.


## 3.3. MIC and MBC of the Potent Isolates

The MIC and MBC of all broad-spectrum antibacterial compound-producingStreptomyces were done against six (3 Gram-positive and 3 Gram-negative) bacterial strains. The MIC value ranges from 0.10 to 0.25 μg/mL, while the MBC value ranges from 0.15 to 0.40 μg/mL. As compared to the other tested pathogens, L. monocytogenes, S. enterica, and P. aeruginosa required high μg/mL CFS to inhibit and kill them (Table 7).Table 7
The MIC and MBC of the isolates CFS inμg/mL against foodborne bacterial strains.
Isolates codeMICMBCSaBcEcLmSePaSaBcEcLmSePaRS180.150.100.200.250.250.250.300.200.300.400.400.40RS250.200.150.100.250.250.250.300.200.200.400.400.40RS370.100.150.200.250.250.250.200.300.300.400.400.40RS480.100.100.100.200.150.200.200.200.200.300.200.30RS720.200.100.200.250.250.250.300.200.300.400.400.40AS890.100.100.100.200.100.100.200.200.200.300.200.20AS970.200.100.200.250.200.250.300.300.300.300.400.40AS1150.100.100.100.200.100.200.200.200.200.300.200.30Keys: Sa =Staphylococcus aureus; Bc = Bacillus cereus; Ec = Escherichia coli; Lm = Listeria monocytogenes; Se = Salmonella enterica; Pa = Pseudomonas aeruginosa.


## 4. Discussion

Foodborne diseases have become more prevalent in recent years, posing a danger to the treatment of a growing number of pathogens. Antibiotics are the most important bioactive compounds for the treatment of infectious diseases. But now, because of the emergency of multidrug-resistant foodborne pathogens, there are basic challenges in the effective treatment of infectious diseases. Thus, due to the high frequency of multidrug-resistant pathogens around the world, there has been an increasing interest in searching soil actinomycetes for effective antibiotics from diversified ecological niches. Antimicrobial compounds from the soil environment, notably rhizosphereStreptomyces, have recently been discovered to be exploited in different food industries for biopreservation potential [5–7, 9, 20–22].A total of 118 actinomycete strains were recovered from the rhizosphere and agricultural soil samples. Of these, 83/118 (70.30%) of the actinomycetes were isolated from the rhizosphere of plants, whereas the remaining 35/118 (29.30%) of the isolates were recovered from farm soil. Geetanjali and Jain [22] also reported that greater percentages of actinomycetes are found in rhizosphere soils. This might be due to different plants producing different chemical metabolites that may be useful for the microbes around them or vice versa. In the present study, all (118) actinomycete isolates were primarily screened against test organisms using the perpendicular streak plate method. Of them, 36 (30.50%) showed inhibitory activity on at least two of the tested bacteria, and this was lower as compared to the previous two similar studies reported as 58.53% by Sapkota et al., [23] in Nepal and Rahman et al., [24] in Bangladesh (53.3%).During the secondary screening, the potent strains CFS showed broad-spectrum antagonistic effects against tested spoilage and foodborne bacterial strains. Similar findings were reported earlier by Schimel and Hattenschwiler [25] and Anupama et al. [26]. The antibacterial activity of the CFS inhibition zone varied widely across several studies. Singh et al. [27] have shown a maximum inhibition zone of 14 mm against tested bacteria. Gurung et al. [28] reported 0-8 mm inhibition zone against selected tested organisms. In the present study, 7-24 mm inhibition zone was observed from the CFS against test organisms, which is comparatively higher than the previous reports. This might be associated with the agrogeographical variation and the diverse antimicrobial metabolites produced by Streptomyces strains.In the present study, as compared to Gram-negatives, the Gram-positive bacteria were highly susceptible to the CFS of the isolates. This result was consistent with the report of Ilic et al. [29]. This might be due to the lipopolysaccharide outer membrane of Gram-negative pathogenic bacteria, which makes the cell wall impermeable to lipophilic extracts. Since, a Gram-positive bacteria lacks an outer membrane. In this study, there was also a marked difference betweenthe crude extracts and pure antibacterial drug (gentamicin). However, some of the potent crude extracts showed an equivalent zone of inhibition to the gentamicin antibiotic that is already in pharmaceutical use. This result was in agreement with the report of Rex et al. [30] as it showed that a significant difference was normally present in CFS compared with the pure drug that was already in clinical use. MIC and MBC value variations among CFS of Streptomyces were also observed against tested bacterial pathogens. This result was similar to the reports of Sibanda et al. [31]. This difference might be due to the concentration and type of inhibitory substances produced. Therefore, the CFS of soil Streptomyces could be a potent source for antibiotic production, which could lead to the development of novel drugs for the biopreservation of foods and the treatment of foodborne bacterial diseases.In this study, the potent CFS antimicrobial effect were partially inactivated after being treated byα-amylase and lysozyme enzymes, while the effect was slightly retained and inactivated in some strains after being treated with proteinase K and papain. Cocolin et al., [2], Ren et al., [32], and Girma and Aemiro [33] also demonstrated that the inhibitory effects of the bacteriocin isolated and produced by LAB from India, China, and Ethiopia were unstable after pepsin, trypsin, papain, and proteinase K enzymes were administered. These reports agreed with the results of this finding. This finding revealed that after acid and catalase were eliminated, the eight Streptomyces strains principal antimicrobial activity was also dependent on peptides. Moreover, this might be due to the inhibitory substances of some strains that may contain organic compounds like carbohydrates that promote inhibition to some extent.After treating the CFS at different temperatures (45, 65, 85, 100, and 121°C), high antagonistic effects were observed, and some strains like RS18, RS25, RS37, RS72, and AS97 CFS antibacterial activity were reduced when the temperature was increased. Girma and Aemiro [33] also demonstrated that the antibacterial activities of the CFS were resistant to diverse temperature intervals. This might be due to the low molecular weight and chemically diverse secondary structures of the crude extracts, which lead to the high-temperature resistance of most antimicrobial compounds. This implies that the antibacterial compounds produced by Streptomyces strains could be used as biological preservatives for high-temperature treatments of food.With regard to pH, the antibacterial activities of the CFS were active and stable in acidic (pH 2-6) environments. Also, someStreptomyces strains were active at pH 8, but the antibacterial effect was reduced when pH increased. This indicates the varying transport regulation and metabolism of Streptomyces isolates. The inhibitory effect of the CFS was completely lost at pH 10 and 12 (alkaline conditions), and this finding was inconsistent with Pinto et al. [20] and Ren et al. [32], who isolated LAB and lost its antibacterial activities at pH 12 and 14, respectively. While consistent results were reported by Girma and Aemiro [33], who isolated LAB from Ethiopian traditional fermented dairy products and lost its antibacterial activities at pH 10. This implies the main antibacterial effects of most strains, including Streptomyces, are dependent on acid. As a result, RS48, AS89, and AS115 strains of the CFS were the most potent and could be used as biopreservative agents for foods in acidic and alkaline environments.The majority ofStreptomyces CFS was soluble in organic solvents such as methyl and ethyl alcohols. Avaiyarasi et al. [3] and Girma and Aemiro [33] also demonstrated that the bacteriocin substances are soluble in organic solvents. As compared to ethanol, the antibacterial effect of methanol treatment on CFS was reduced. This was inconsistent with the findings of Avaiyarasi et al. [3]. This might be due to the chemical structures of the CFS secreted by Streptomyces strains being intolerable to methanol solvent. Streptomyces CFS showed a stable antagonistic effect against test organisms after being treated with additives (Na3C6H5O7 and KCl2). This is consistent with Ren et al. [32] and Girma and Aemiro [33] reports that suggested, the antibacterial activity of lactic acid bacteria isolated in fermented foods which were shown effective when potassium chloride and sodium citrate additives are added.


### 4.1. Limitations of the Study

Because of the COVID-19 pandemic, high-performance liquid chromatography (HPLC) analysis and purification of antagonistic metabolites were not performed. The effect of CFS on cell morphology and intracellular organization of the pathogens was also not performed due to the unavailability of a scanning electron microscope (SEM).


## 4.1. Limitations of the Study

Because of the COVID-19 pandemic, high-performance liquid chromatography (HPLC) analysis and purification of antagonistic metabolites were not performed. The effect of CFS on cell morphology and intracellular organization of the pathogens was also not performed due to the unavailability of a scanning electron microscope (SEM).


## 5. Conclusions

The present study revealed that eight (Streptomyces fasciculus, Streptomyces roseochromogenes, Streptomyces ruber, Streptomyces glaucus, Streptomyces griseus, Streptomyces cellulosae, Streptomyces griseoflavus, and Streptomyces xanthophaeus) potent Streptomyces strains possessed an in vitro broad-spectrum antibacterial activity against spoilage and foodborne bacterial pathogens with an inhibition zone ranging from 7 to 24 mm. The MIC and MBC values range from 0.10 to 0.25 μg/mL and 0.15 to 0.40 μg/mL, respectively. After the treatment of the Streptomyces CFS with proteinase K, papain, α-amylase, and lysozyme enzymes, their antagonistic effects against test organisms were also observed. Most Streptomyces CFS antibacterial activity was highly resistant to heat (45–100 °C), acidity (pH 2–8), organic solvents (methanol and ethanol), and additives (potassium chloride and sodium citrate). Thus, the results of this investigation revealed that soil actinomycetes could be a valuable source of novel antibacterial agents applicable for food biopreservation and the treatment of foodborne bacterial infections.



---

*Source: 5547406-2022-04-06.xml*