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Micro Environer 2021, 1(1): 54-62… https://doi.org/10.54458/mev.v1i01.6673

Analysis of the antimicrobial activity of Ashurak extracts prepared with different solvents on Klebsiella pneumoniae and Shigella dysentery isolated from poultry faeces.

1 Associate professor of clinical pathology, Department of Clinical Sciences, Faculty of Veterinary Medicine, University of Zabol, Iran

2 Agricultural Biotechnology Institute, University of Zabol, Zabol, Iran

*Corresponding Author: Mehdi Jahantigh ([email protected])

A B S T R A C T

Bacteria are more likely to cause disease than foodborne pathogens. Articles have shown that most plant extracts have antimicrobial properties. This article aimed to analyze the antimicrobial activity of Ashurak extract prepared with different solvents on K. pneumoniae and Sh. dysentery isolated from poultry faeces. In this article, antimicrobial effects of various extracts (ethyl acetate, aqueous, ethanolic, methanolic and hydroalcoholic) on K. pneumoniae bacteria Sh. dysentery isolated from poultry faeces was obtained by diffusion method in Müller Hinton agar medium using 6 mm paper discs according to the instructions. Mean comparison was performed using two-way analysis of variance and post hoc test with the least significant difference. The lowest inhibitory concentrations of aqueous, ethanolic, methanolic and hydroalcoholic extracts of Ashurak on K. pneumoniae were 12.5, 12.5, 12.5 and 3.1 ppm, respectively. The lowest inhibitory concentration of aqueous, ethanolic, methanolic and hydroalcoholic extracts of Ashurak on SchiegelDysentery was equal to 12.5, 12.5, 25 and 25 ppm. Among the mentioned extracts, the aqueous extract had the most significant effect on the inhibition of K. pneumoniae, but there was no difference between the extracts. According to the present study results and the increasing resistance to synthetic antibacterial substances, it seems that Ashurak can be considered an effective plant in cleansing some bacteria, including K. pneumoniae and Sh. dysentery. In addition, the results of this study showed that the Ashurak plant is more effective. In contrast, K. pneumoniae had dysentery compared to Shigella.

Article info

Received: 23 May 2021

Revised: 05 Sep 2021

Accepted: 19 Oct 2021

 

 

 

 

Keywords:

Shigellosis, Flavonoids, Ethyl acetate, Hydroalcohol, Methanol, Proanthocyanidin

  1. Introduction

One of the challenges of the food and drug industry is the development of microbial indices resistant to drugs and antibiotics. Today, due to the toxic and carcinogenic properties of chemical and synthetic compounds, the use of medicinal plants to treat chronic diseases has attracted the attention of many researchers. Therefore, the use of natural antimicrobial and antioxidant compounds such as organic acids, essential oils and plant extracts can be a suitable and safe alternative in food [1]. Also, in problems related to oral and arthritis that collagen is exposed to degradation, phenolic compounds and antioxidants of these plants can prevent it [2].

Flavonoids are phenolic compounds that directly inhibit the active molecules of superoxide, hydrogen peroxide, hydroxyl and peroxyl radicals[3].

Numerous studies have shown that plants containing flavonoid compounds exhibit high antioxidant activity [3]. This antioxidant activity is usually related to the ability of these compounds to give electrons or hydrogen atoms, and therefore. They are medically essential and significant [4]. It is noteworthy that some fruits’ seeds and skin parts have more antioxidant activity even than meat. For example, grape seeds and pomegranate peel have more antioxidant activity than meat and are rich in proanthocyanidins, potent inhibitors of reactive oxygen radicals [5].

Reactive oxygen radicals can attack the best cell compounds such as fatty acids, proteins, nucleic acids and pigments [6] to neutralize the toxic effect. Therefore, reactive oxygen radicals, antioxidant compounds are needed. Plant cells from two enzymatic antioxidant systems (superoxide dismutase, catalase, peroxidase, antioxidant metabolites phenyl, phenyl anhydride, carotene, phenyl, carthenol) [7, 8].

Since the second half of the last century, extensive research has been done on medicinal plants in most countries of the world, and after that, many herbal medicines have been prepared and marketed due to the rich flora of Iran, which is more than 7500 plant species, and ان‌هThey are medicinal [9, 10]. Therefore, the need to study the antibacterial properties of plants such as Ashurak on K. pneumoniae and Shigella has been significant.

Rhazya stricta is an important drug that is rich in alkaloids containing anti-cancer alkaloids. Herbal medicines are used locally to treat various diseases in South Asia (Pakistan, India and Afghanistan) and the Middle East (for example, Saudi Arabia, Qatar, the United Arab Emirates (UAE), Iran and Iraq). Some of its alkaloids also have anti-cancer properties has been reported. More than 100 alkaloids have been isolated from R. stricta leaves, but their therapeutic activities are known only for a few compounds. Very few ethnographic studies have been performed on R. stricta [11].

K. pneumoniae is an important opportunistic pathogen that frequently causes urinary tract infections and pneumonia in immunocompromised individuals. After Escherichia coli, the most common cause of sepsis is gram-negative bacteria and nosocomial infections. The global spread of multidrug-resistant strains of K. pneumoniae K is an important cause for concern. Antibiotic resistance is the ability of a microorganism to stop the effect of an antibiotic and is a significant cause of failure in the treatment of infectious diseases, which increases the incidence of disease, mortality and health care costs. High resistance of Klebsiella to antibiotics and their rapid spread in different parts of the hospital cause significant problems in treatment and cause septicemia and death of patients [12, 13].

Shigellosis is an intestinal infection whose symptoms range from watery diarrhoea to severe inflammation. This disease with pain severe abdominal pain, fever, bloody stools and mucus are known. This disease is usually self-limiting, except in cases where the patient has an immunodeficiency, or primary medical treatment is not available [14]. Sh. dysentery, buidi and Sonei are different species of this bacterium [15].

Shigella is highly contagious and is easily transmitted from person to person through faeces and mouth. Therefore, eating contaminated food and water and human faeces is the primary source of Shigella. Specific symptoms of dysentery include: anorexia, fever, intestinal inflammation, bloody stools, purulent, abdominal pain, And the feeling of incomplete emptying of the intestine is anal pain [16].

Antioxidants have many uses in addition to the treatment and prevention of cancer and atherosclerosis; for example, natural antioxidant compounds of the olive leaf to increase the storage of fats and oils, almond peel extract due to its antioxidant activity is used preserve chips and non-chips [17]. On the other hand, because different extracts have different abilities to extract active substances and, of course, have different properties on the lack of bacterial growth, so for this purpose, In the present study, we will try to extract the extract of the Ashurak plant with various solvents and then focus on the inactivity of K. pneumoniae and Shigella bacteria

  1. Materials and methods of work

2.1. Herbal materials

R.stricta was collected from Saravan (Coordinates: 27 ° 22′15 ″ N 62 ° 20′03 ″ E), and their species were identified in the botanical laboratory of University of Zabol (Fig. 1).

After collecting these samples in natural conditions and dry shade and then crushed, to prepare the extract, 40 g of dry plant powder was placed in half-litre Erlenmeyer flakes containing 200 ml of 96% ethanol, methanol, ethyl acetate, hydroalcoholic and water. The contents of the Erlenmeyer flask were mixed at room temperature for 24 hours with a shaker (at 130 rpm) and then filtered through Whatman 2 paper. The solvent was separated from the extract by a rotary apparatus using a vacuum pump (distillation in vacuum). The weighted extracts were then dissolved in DMSO solvent.

Figure 1. Appearance characteristics of leaves (A, B), Stem (B), and flowers (A, C) of R. stricta plant [18]

2.2 Culture and identification of K. pneumoniae and Sh. dysentery

To identify K. pneumoniae, the samples were cultured on agar medium by flame and incubated for 37-24 hours at 37 °C. After bacterial growth, the strains of K. pneumoniae were identified based on microbiological methods, including hot staining and microscopic observation of bacilli OF-TSI-SIM-MR-VP-citrate-urea-lysine decarboxylase and orrentine deoxyribose [19] tests. To identify Sh. dysentery from Salmonella Shigella agar specific culture media and selenite F is used [20].

Determining the level of inhibitory concentration and minimum lethal concentration of Ashurak extract prepared with different solvents: Sensitivity of bacterial isolates with multiple resistance to the extract was determined using the dilution method in the well. Seven wells were made in the solid culture medium, and 100 μl of Müller-Hinton nutrient medium (MHB) was added to each well. To the first well, 100 ml of diluted solution of essential oil or extract was added, and after mixing, 100 μl of the first well was removed and added to the second well. This was done until the last well was removed from the previous well of 100 μl of culture medium. 100 μl of microbial suspension containing 107 units/ml equivalent to 0.5 McFarland was added and incubated at 37 ° C. It was exposed to Celsius for 24 hours. The first tube to prevent the growth after incubation is considered, and to ensure clear wells, ten microliters are taken and transferred to the Müller Hinton agar medium. After 24 hours, the first dilution can be 99.9. The percentage of bacteria killed is shown as the minimum lethal concentration

  1. Results

This paper showed that the lowest inhibitory concentration of Ashurak ethyl acetate extract against K. pneumoniae was 25 ppm, in which two strains were inhibited. The lowest inhibitory concentrations of aqueous, ethanolic, methanolic and hydroalcoholic extracts were 12.5, 12.5, 12.5, 3.1 ppm, respectively, in which 2-3-1 and two strains were inhibited at these concentrations (Table 1).

The lowest inhibitory concentration of ethyl acetate against Sh. dysentery samples were 25 ppm. At the same time, the lowest inhibitory concentration of aqueous extract, ethanol, Methanolic and hydroalcoholic was equal to 12.5, 12.5, 25, 25 ppm. The highest inhibitory concentrations of ethyl acetate-aqueous-ethanol-methanolic and hydroalcoholic were 50, 25, 25, 50 and 50 ppm (Table 1).

The lowest lethal concentrations of ethyl acetate, aqueous, ethanolic, methanolic, hydroalcoholic extracts against K. pneumoniae were 50, 25, 25, 25 and 6.25 ppm, in which 2, 2, 3, 1 and 2 strains were inhibited at this concentration (Table 2).

Analysis of variance diameter of growth inhibition zone due to the effect of different solvents showed that among the mentioned extracts of the extractAqueous had the most significant impact on the inhibition of K. pneumoniae. Still, there was no difference between the extracts in the inhibition of Sh. dysentery (Fig. 2).

Table 1. Minimum bactericidal concentrations of Ashurak extract with different solvents on Klebsiella and Shigella (ppm)

Bacteria Strain Ethyl acetate Aqueous extract Ethanolic methanolic hydroalcoholic
K. neumoniae 1 50 25 12.5 12.5 12.5
K. pneumonia 2 50 25 12.5 50 25
K. neumoniae 3 50 25 25 50 12.5
K. neumoniae 4 50 12.5 25 50 25
K. neumoniae 5 50 25 25 50 3.1
K. neumoniae 6 50 25 25 50 3.1
K. neumoniae 7 25 25 12.5 25 25
K. neumoniae 8 25 12.5 25 25 50
Sh. Dysentery 1 25 25 12.5 25 25
Sh. Dysentery 2 25 25 25 25 25
Sh. Dysentery 3 50 12.5 25 50 50

Table 2. The minimum lethal concentration of Ashurak extract with different solvents on Klebsiella and Shigella (ppm)

Bacteria Strain Ethyl acetate Aqueous extract Ethanolic methanolic hydroalcoholic
K. neumoniae 1 100 50 25 25 25
K. neumoniae 2 100 50 25 100 50
K. neumoniae 3 100 50 50 100 25
K. neumoniae 4 100 25 50 100 50
K. neumoniae 5 100 50 50 100 6.25
K. neumoniae 6 100 50 50 100 6.25
K. neumoniae 7 50 50 25 50 50
K. neumoniae 8 50 25 50 50 100
Sh. Dysentery 1 50 50 25 50 50
Sh. Dysentery 2 50 50 50 50 50
Sh. Dysentery 3 100 25 50 100 100

 

 

Fig. 2. Inhibitory halo diameter of Ashurak extract on Klebsiella and Shigella bacteria (mm)

  1. Discussion

The antibacterial effects of whole fruit powder extract, pomegranate, and pumpkin juice (Bottle Gourd) on gram-positive and gram-negative bacteria such as Salmonella typhi, Escherichia coli, and K. pneumoniae, Bacillus cereus, Bacillus subtilis and Sh. dysentery have been investigated. It was concluded that pomegranate powder extract with 250 micrograms per disc could slightly inhibit the growth of the studied bacteria except for Bacillus subtilis. However, Pomegranate juice and pumpkin and pumpkin powder extract did not show antibacterial activity [21]. However, in the present study, it was found that the hydroalcoholic extract of Ashurak had the most significant effect on K. pneumoniae and Sh. dysentery, which showed that Ashurak was more effective than pomegranate peel.

The antimicrobial effect of aqueous and alcoholic extracts of chamomile, turmeric, peppermint and nettle on K. pneumoniae and comparing their impact with common antibiotics, and concluded that the alcoholic extract of chamomile with a maximum diameter of no growth zone of 14 mm against Klebsiella Has shown the best effect. The bacteria in question showed resistance to aqueous and alcoholic extracts of deciduous plants. An ethanolic extract of nettle and chamomile plants in 0.39 mg/ml had the best antibacterial effect. The minimum growth inhibitory concentration of blueberry extract against K. pneumoniae was 12.5 mg/ml and lower than other types of extracts [22]. In the present study, it was found that the most significant effect of the Ashurak plant was obtained from hydro-alkaline extract with a 10 mm diameter growth inhibition zone. And indicates greater power of growth inhibitionBacteria were caused by chamomile compared to Ashurak. Investigation of zinc oxide nanoparticles with antimicrobial and antibiotic activity against K. pneumoniae concluded that antibiofilm activity and biofilm removal by zinc oxide nanoparticles were obtained at 50 and 500 μg/ml, respectively [23]. The effect of iron oxide nanoparticles on the expression of biofilm production genes and antibiotic resistance in K. pneumoniae strains has been investigated, and it has been concluded that both types of iron oxide nanoparticles have an essential effect in inhibiting biofilm formation and resistance they had antibiotics for this bacterium [24]. In the present study, it was found that the minimum inhibitory concentration of hydrochloric acid extract of Ashurak against K. pneumoniae was equal to 3.1 ppm, which had a more significant effect than the mentioned plants.

The antiplasmid effects of extracts of safflower, safflower, licorice, eucalyptus on resistant strains of K. pneumoniae have been investigated, and it has been concluded that all extracts have antimicrobial properties and have been able to prevent the growth of resistant strains of K. pneumoniae. Eucalyptus leaf extract has the highest antimicrobial properties (average MIC 0.1 mg/ml) [25]. In the present study, it was found that the minimum inhibitory concentration of hydrochloric acid extract of Ashurak against K. pneumoniae was equal to 3.1 ppm, which was less effective than eucalyptus extract.

It has been reported that the ethanolic extract has a positive effect on Staphylococcus aureus [26, 27], but the affected oil has shown a more significant impact [27]. Also, the positive effect of other extracts of chloroform, ethyl acetate and methanol on the leaves and also the dependence of these extracts on the concentration they were more effective on Staphylococcus aureus [28] and more effective than methanolic extract than ethanolic extract of the leaves and branches of the plant on the bacteria Listeria monocytogenes, Sodomonas aeruginosa and Staphylococcus aureus[29]. In one study, it was found that although methanolic extract of the plant did not show a high effect on the diffusion activity of Escherichia coli, the minimum lethal concentration for Escherichia coli was reported above 40 mg/ml [30]. In other studies, no effect of the case extracts on Escherichia coli has been reported [27, 29]. In a study, Houshmand et al. Had the highest impact of the plant extract on Pseudomonas aeruginosa at a concentration of 2.5% with a growth inhibition zone diameter of about 16 mm and the lowest effectiveness of the plant extract at a concentration of 5% on lactobacilli with a growth inhibition zone diameter of about 6 mm reported and in general concluded that the extract with different concentrations has different effects on bacteria [31]. In general, it has been found that other extracts have different effects depending on the type of plant they inhibit bacteria [32, 33]. The present study found that the hydrocarbon solvent was the most effective solvent for extracting the active ingredients.

The study of Kouitcheu reports the in vitro and in vivo anti-shigellosis activity of the methanol extract of this plant on rats. The result shows that in vitro, the extract had an antimicrobial effect on 11 out of the 17 pathogenic strains tested. The values of CMI and CMB obtained against Shigella dysenteriae type I were 800 and 6400 μg/ml, respectively. In vivo, diarrhoea induction was effective, and we noticed an increase in faeces frequency and weight (p < 0.05), an increase in the percentage of diarrheic stool released as well as the mucus contained in the stool (p < 0.05), an increase in bacterial population in the stool (p < 0.05). Picralima nitida extract, like ciprofloxacin, markedly reduces the frequency of faeces released and sd1 density from 100% (diarrheic rats) to 47.22 and 61.69% (500 mg/kg), respectively. It also slowed down the movement of charcoal meal through the gastrointestinal tract, with the percentage of intestinal length covered at 60.54 (500 mg/kg)[34].

The study of René investigated the acute toxicity and effect of the aqueous ethanol extract of the plant on gastrointestinal propulsion, in vitro bacterial growth and in vivo bacillary dysentery. The aqueous ethanol extract of E. prostrata was not toxic. In vitro, the extract’s minimal inhibitory and minimal bactericidal concentrations were 3,500 and 12,000 µg/ml, respectively. In vivo, the diarrhoea went along with an increase in faeces frequency (P < 0.01 by the 3rd day), increase in the bacterial population to a maximum on the 2nd day after infection (P < 0.01). The death rate in the diarrheic control group was 100% by day 6. E. prostrata extracts (20 and 40 mg/kg), like norfloxacin, reduced the bacterial growth (P < 0.01) so that by the 6th day, Sd1 density was < 0.01) reduction in faeces frequencies. The extract exhibited notable (P < 0.01) inhibition of intestinal propulsion[35].

The study of Khosravi was to investigate the antibacterial effects of Tecomella undulata, and Momordica charantia extracts on Escherichia coliStaphylococcus aureusPseudomonas aeruginosa, and Shigella dysenteriae. The result shows that the minimum inhibitory concentrations of Tecomella undulata extract against the studied bacteria were 1.87 to 3.75 mg/ml. The highest lethal concentration of leaf extract was 15 mg/mL, and the lowest lethal concentration of Momordica charantiaextract was 1.87 to 3.75 mg/mL. The highest Momordica charantia extract concentration was 15 mg/mL, and Shigella dysenteriae were eliminated at this concentration[36].

The study of Saravani was to investigate the antimicrobial effects of Momordica charantia against pathogenic bacteria. This study showed that the lowest inhibitory concentration of extract against bacteria was 12.5 ppm (Vibrio cholera, Pseudomonas aeruginosa and E. coli). The results also showed that only one inhibited. However, the highest inhibitory concentration was estimated at 25 ppm against Shigella dysenteriae and Bacillus cereus and the highest bactericidal concentration was estimated at 25 and 50 ppm[37].

  1. Conclusion

Overall, the results of this study showed that Ashurak had a more significant effect against inhibition of K. pneumoniae than Sh. dysentery. Among the mentioned extracts, the hydroalcoholic extract had the most effect.

Considering that every research has its shortcomings and the present study is no exception, it is suggested that in the next stage, the active ingredients of Ashurak, which has played an effective role on antimicrobial properties, beBe evaluated directly to present the conclusion more accurately.

Abbreviation

Not applicable

Conflict of Interest

The authors declare there is no conflict of interest in this research study.

Consent for publications

All authors read and approved the final manuscript for publication.

Availability of data and material

All data generated during this study are included in this published article.

Ethics approval and consent to participate

No human or animals were used in the present research.

Ethics declarations.

The authors declare no conflict of interest in financial or any other sphere. All applicable international, national, and institutional guidelines for the care and use of animals were followed.

 Source of Funding

No funding was received against this research study.

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