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Study of Soil Actynomycetes and Their Antagonastic Activity against Entric Fever Pathogens

Oliver Madhale*, Vinay Chougule, Ujwala Mane

Department of Microbiology, Rayat Institute of Research and Development, Satara, & Shivaji University, Kolhapur, Maharashtra, India

Abstract: Actinomycetes were isolated from agricultural and non-agricultural farm soil samples in the Sangli district of Maharashtra. 60 actinomycetes were isolated from 30 soil samples, with only three isolates from agricultural soil and three from non-agricultural soil displaying inhibitory activity. Soil samples from non-agriculture farms yielded a higher percentage of antagonistic actinomycetes (10%) than those from agriculture farms (2 %). Three non-agricultural isolates with high inhibitory activity were tested for their inhibitory profile against three human test pathogens, Salmonella typhi, S. paratyphi A, and paratyphi B. The actinomycete isolate INA - 53 exhibited a high degree of inhibitory activity against all the test pathogens both in terms of the number of test pathogens inhibited and the zone of inhibition as compared to the standard antibiotic used that is streptomycin. INA - 53 was identified to be belonging to the genus Streptomyces spp. The results suggest that non agriculture soil samples are rich in organic matter with a high C:N ratio are the potential source of antagonistic actinomycetes and a large number of antagonistic actinomycetes producing anti-microbial compounds can be isolated from non agriculture soil.

Keywords: Actinomycetes, antibacterial activity, antibiotics, human pathogens

Received January 20, 2022; Revised February 23, 2022; Accepted March 01, 2022

Citation: Oliver Madhale, Vinay Chougule, Ujwala Mane, Study of Soil Actynomycetes and Their Antagonastic Activity against Entric Fever Pathogens. Applied Ecology and Environmental Sciences, 10 (2). 60-64. January 2022. doi:10.12691/aees-10-2-5

1. Introduction

Actynomycetes are part of the normal soil microflora and are an important component of the soil microbial community because they produce secondary metabolites with antimicrobial properties. [1].

Soil microorganisms are a fantastic resource for isolating and identifying therapeutically important products. Actinomycetales are a significant group among them [2]

Actynomycetes have produced over 4,000 naturally occurring antibiotics, many of which are important in medicine [3].

One-third of the thousands of naturally occurring antibiotics are thought to have been produced by actinomycetes, and drug-resistant human pathogens pose a significant threat to disease treatment [4].

Because of their ability to produce antibiotics, actinomycetes have grown in popularity in recent years [5].

Actinomycetes are important bioactive compound producers. Gram-positive [6]. Actinomycetes, or filamentous bacteria, are common in soil and a good source of antibiotics. Actinomycetes are known to be the source of nearly 80% of the world's antibiotics [7].

Human infectious diseases are the leading cause of morbidity and mortality in developing countries such as India. Actinomycetes are important in the pharmaceutical and agricultural industries. In a previous study, actinomycetes isolated from Malaysian soil were found to have the ability to suppress the growth of many plant diseases [8].

Resistance to multiple drugs is a significant issue in the treatment of infectious diseases caused by pathogenic microorganisms. Actinomycetes are a potential source of many bioactive compounds) that have a wide range of clinical effects and important applications in medicine for treating a wide range of human diseases and disorders [9,10].

2. Materials and Method

Soil samples were taken and studied from various sections of a non-agricultural farm and an agricultural farm. Soil samples were taken by digging 3cm into the ground and then collecting them in a sterile container from various sections of the field. The samples were transferred to a lab for further examination.

For the growth of actinomycetes, procedures such as serial dilution, selective media, and the application of antifungal and antibiotic agents were used since soil includes a significant number of microflora. 1gm of soil taken from the sample location was added to 10ml of d/w using the serial dilution procedure. Using the spread plate method, 0.2ml of serially diluted sample was distributed on glycerol asparagines agar plate. The glycerol asparagines medium was employed as a selective media, and it was also mixed with antifungal and antibacterial agents to minimise the development of undesired microorganisms, and a correct temperature of 37°C was maintained for 14 days for actinomycetes to grow. A primary and secondary screening were performed on the isolated actinomycetes to determine their antagonistic activity.

2.1. Primary Screening of Actinomycetes

For primary screening, 60 actinomycete isolates were chosen. Using the cross streak technique, the isolates were first evaluated for antagonistic activity against enteric fever pathogens. isolates were streaked perpendicular to actinomycetes isolate on the nutrient agar plate. The plates were incubated for 24 to 48 hours at 37°C. For secondary screening, only the isolates that exhibited excellent sensitivity to the pathogens were chosen.

2.2. Secondary Screening of Actinomycetes

For secondary screening, six actinomycetes were chosen. The secondary isolates were grown for 14 days in glycerol asparagines broth at 37°C. For the secondary screening, the agar well diffusion technique was utilised to observe the zone of inhibition. The method was carried out on Nutrient agar plates, with each Enteric fever pathogen spread on separate agar plates, agar wells were made in the nutrient media plates containing enteric fever pathogen and 0.2ml of the selected actinomycetes isolates broth was added in the wells, the plates were kept in the refrigerator for 20 minutes and then incubated at 37°C for 24 hours.

2.3. Morphological and Physiological Characteristics of Potent Actinomycetes Strain

The Morphological and Physiological characteristics of the potent actinomycetes strain was carried out by studying its morphological characters, gram nature, motility various physiological characters like temperature, pH, and salt concentration.

2.4. Antagonestic Activity against Pathogens of Enteric Fever

The actinomycetes strain was used to treat enteric fever pathogens. The isolate was cultivated in glycerol asparagine broth at 37°C for 14 days, and antagonistic metabolites were extracted using ethanol in a 3:1 ratio. The extracted antagonistic metabolite was then tested against enteric fever bacteria, and the resulting zone of inhibition was compared to the conventional antibiotic streptomycin, which is used to treat enteric fever. With the use of a spreader, the pathogens Salmonella typhi, Salmonella parathyphi A, and Salmonella paratyphi B were spread on nutrient agar plates. The agar wells were drilled with a cork borer. In each inoculated pathogen plate, three wells were done. In the first well, 0.2ml of an antagonistic metabolite produced by actinomycetes was added, in the second well, 0.2ml streptomycin antibiotic was added and 0.2 ml of d/w was added as control. The plates were refrigerated for 20 minutes before being incubated at 37°C for 24 hours. For the most efficient antibiotic, the zone of inhibition was evaluated and compared.

3. Results

The soil samples were spread on glycerol asparagines media and incubated at 37°C for 14 days total of 60 actinomycetes were selected and were purified and isolated separately and then taken for the primary and secondary screening process.

3.1. Primary Screening of Actinomycetes

60 Actinomycetes were isolated and used for the following screening process by using cross streak method against the enteric fever pathogens.

Figure 1. Primary screening by cross streak method

As in Figure 1 Six isolates from non-agricultural farm soil, INA – 56, INA – 53, and INA – 59, as well as isolates IA – 16, IA – 24, and IA – 30 from agricultural farm soil, exhibited a substantial impact against the enteric fever pathogen.

The enteric fever pathogens Salmonella typhi, Salmonella Paratyhpi A, and Salmonella Paratyhpi B showed modest sensitivity or resistance to the other 54 actinomycetes isolates. Out of the six actinomycetes strains studied, the isolate INA – 53 demonstrated a strong inhibitory factor against enteric fever pathogenic strains.

3.2. Secondary Screening of Actinomycetes

Using the agar well diffusion technique, six actinomycetes from primary screening were exposed to secondary screening on nutrient agar plates. The test pathogens were Salmonella typhi, Salmonella paratyphi A, and Salmonella paratyphi B, incubation was at 37°C for 24 hours, the antagonistic activity of the chosen strains against the enteric fever pathogen were studied by analyzing the zone of inhibition, as shown in Figure 2 and Table 4.

As isolate INA 53 demonstrated considerable antagonistic potential against enteric fever pathogens, isolate INA-53 actinomycetes was chosen for further study. All other actinomycetes only inhibited one or two enteric fever pathogenic strains, and only in a small area. They were eliminated as a result of the secondary screening.

Figure 2. Secondary screening of Actinomycetes by Agar well diffusion method to observe zone of inhibition against s. typhi

The six actinomycetes isolates were investigated for their antagonistic activity against Salmonella typhi. The zone of inhibition was measured in order to choose the most potent actinomycetes strain. In comparison to the other five actinomycetes strains, Figure 2. Showed that INA – 53 exhibited the greatest zone of inhibition.

Figure 3. Secondary screening of Actinomycetes by Agar well diffusion method to observe zone of inhibition against s. paratyphi A

The antagonistic activity of the six actinomycetes isolates against Salmonella paratyphi A was studied. In order to choose the most potent actinomycetes strain, the zone of inhibition was assessed. Figure 3 shows that INA – 53 had the greatest zone of inhibition when compared to the other five actinomycetes strains.

The six actinomycetes isolates were investigated for their antagonistic activity against Salmonella paratyphi B. In order to find the most effective actinomycetes strain, the zone of inhibition was measured, and Figure 4. demonstrated that INA – 53 had the greatest zone of inhibition when compared to the other five actinomycetes strains.

Figure 4. Secondary screening of Actinomycetes by Agar well diffusion method to observe zone of inhibition against s. paratyphi B

The enteric fever pathogen was susceptible to the actinomycetes isolate INA – 53. As a consequence, it was determined that Actinomycetes Isolate INA 53 is a more potent actenomycetes isolate than the other isolates examined. As a result, isolate no. INA 53 was chosen to be investigated further.

Table 1. Zone of Inhibition against Pathogens of Enteric fever

As in Table 1 In comparison to the other isolates, isolate INA-53 had a greater zone of inhibition; the zones of inhibition for s.typhi, s.paratyphi A, and s.paratyphi B were 28mm, 19mm, and 14mm, respectively. This revealed that the isolate INA-53 was the most effective actinomycetes strain identified, with stronger antagonistic activity than the others.

3.3. Morphological and Physiological Characteristics of Potent Actinomycete Strain

The physiological and morphological properties of Actinomycetes strain INA – 53 were investigated. A variety of methods were used to observe and investigate the strain's properties.

Table 2. Colony Characters of INA – 53 isolate

The colony features of the potent actinomycetes isolate INA – 53 were visually detected on glycerol asparagines agar plate. The uniformity of the colony was assessed during its selection for gram staining and motility.

Table 3. Microscopic Characteristics of INA – 53 Isolate

The strain INA – 53 was identified as violet-colored rod-shaped bacteria that were gram positive in nature, the presence of spores, the presence of filaments, and the presence of motility was observed.

Physiological Characteristics of INA – 53 Isolate

The effect of temperature, pH, and salt concentration on the strain's ability to grow in these conditions was observed, and the strain's ability to develop in these conditions was investigated.

Table 4. Effect of Temperature on Growth of INA – 53 Isolate

Inoculating actinomycetes strain INA – 53 in glycerol asparagines broth tubes at 10°C, room temperature, 37°C, and 45°C for 7 days at each temperature was used to investigate the influence of temperature. According to the findings, growth occurred in the tubes at 30°C, 37°C, and 45°C, but not at 10°C. The turbidity of the broth was utilised to track the bacteria's growth.

Table 5. Effect of pH on Growth of INA – 53 Isolate

Inoculating the INA – 53 actinomycetes strain in glycerol asparagines broth with pH 7.4, pH 9, and pH 11 adjusted in each tube and incubating at 37°C for 7 days was used to examine the pH impact. The media turbidity was utilised to track growth at pH 7.4, pH 9, and pH 11, but at pH 5, there was no growth.

Table 6. Effect of Salt Concentration on Growth of INA – 53 Isolate

Inoculating the actinomycetes strain INA – 53 in glycerol asparagines broth with varied salt concentrations of 3% NaCl, 5 % NaCl, 8% NaCl, and 10% NaCl and incubating for 7 days at 37°C was used to investigate the impact of salt concentration. Growth was seen at 3% NaCl and 5% NaCl salt concentrations, but not at 8% NaCl or 10 % NaCl salt concentrations. Media turbidity was utilized to estimate growth.

3.4. Antagonistic Activity Against Pathogens of Enteric Fever

The produced substance was evaluated for antibacterial activity against enteric fever bacteria. The antibacterial substance was compared to the regular antibiotic streptomycin, as well as distilled water as a control. The strain was chosen for its ability to fight enteric fever pathogens in its intended use.

Each plate was divided into three wells, with D/w serving as the control, streptomycin serving as the standard antibiotic, and the metabolic product generated by isolate INA – 53 serving as the third well.

Figure 5. Antibacterial Activity of metabolite produced by INA-53 isolate compared with Streptomycin antibiotic tested against Pathogens of Enteric Fever by Well Diffusion Method

As in Figure 5. The antibacterial action was analyzed by looking at the inhibition zone on the plate. The zone of inhibition created by the examined metabolite was compared to the streptomycin zone of inhibition. Enteric fever pathogens were shown to be less sensitive to streptomycin but extremely sensitive to a metabolite generated by the actinomycetes strain INA – 53, the antibacterial metabolite compound produced by the INA – 53 strain was found to be more effective than the conventional antibiotic. The test isolate's zone of inhibition was 28mm, 20mm, 18mm against Salmonella typhi, Salmonella paratyphi A, and Salmonella paratyphi B pathogens, respectively, whereas the standard antibiotic zone of inhibition was 20mm, 17mm, 14mm against Salmonella typhi, Salmonella paratyphi A, and Salmonella paratyphi B pathogens, respectively.

4. Discussion

In comparison to study regions, the soil samples from the non-agriculture farm produced the greatest number of antagonistic soil actinomycetes; however, the INA 53 isolate exhibits a good zone of inhibition against. Enteric fever bacteria.

60 actinomycetes isolates were isolated from 30 soil samples, with only a few displaying the zone of inhibition, three from agricultural soil and three from non-agricultural soil. agricultural soil inhibited the enteric fever significantly less than compared with non-agricultural soil.

In agriculture soil isolates, the smallest zone of inhibition was observed for Salmonella paratyphi B was 9 mm and the largest zone of inhibition for Salmonella typhi was 18 mm. However, in the case of a non-agriculture soil sample the minimum zone of inhibition against Salmonella paratyphi B was 14 mm and the maximum zone of inhibition against Salmonella typhi was 28 mm. Only six actinomycete isolates from the two sampling areas inhibited the growth of Salmonella typhi, Salmonella parathypi A, and Salmonella parathypi B, as shown in Figure 2, Figure 3 & Figure 4.

The antagonistic activity of the six actinomycetes isolates against Enteric fever pathogen was investigated. In order to select the most potent actinomycetes strain, the zone of inhibition was measured. In comparison to the other five actinomycetes strains, INA – 53 exhibited the greatest zone of inhibition, as shown in Figure 2, Figure 3 & Figure 4.

The antibacterial activity of the isolated antagonistic metabolite of INA – 53 was tested using the agar well diffusion method. Enteric fever pathogens were used to test the strain's antibacterial activity. All three strains were found to be streptomycin sensitive. All of the strains tested for antibacterial activity were isolated on Nutrient agar plates. On comparison of streptomycin antibiotic and metabolite of INA 53 the isolated strain gave a larger zone of inhibition compared to the standard antibiotic used.

When compared with streptomycin antibiotic enteric fever pathogen were all more susceptible to the metabolite produced by actinomycetes isolate INA 53 having a larger zone of inhibition that is of 28mm, 20mm and 18mm compared to the zone of inhibition of streptomycin that is 20mm, 17mm, & 14mm by observing these readings metabolite of INA53 was discovered to be more potent against the pathogens of enteric fever.

5. Conclusion

As a result, soil samples are clearly the most abundant source of actinomycetes, as well as a complex source of antibacterial compounds. Because soil contains a high carbon: nitrogen ratio and contains a lot of organic matter, a lot of antagonistic actinomycetes can be isolated from the environment. This will result in the creation of antibacterial compounds that are effective against human pathogens.

The results of this study clearly show that actinomycetes have the ability to produce new antibiotics the INA 53 strain metabolite that shows antibacterial activity can be used as a new antibiotics against enteric fever. As soil acts as a rich source of actinomycetes more amount of research can be carried out to produce new antibiotics which is the need for today as the there is a increase in antibiotic resistance this need opens a door for newer and cheaper antibiotics in which actinomycetes spp. Can play a major role. samples and that actinomycetes are the most abundant source of antimicrobial compounds. Considering the findings, the active metabolites produced by the chosen strain could be considered as candidates for antibiotic discovery for enteric fever. The findings support the notion that the ability of an extracted metabolite of INA 53 to be used as an antibiotic source could be beneficial during the screening stage.

References

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