INTRODUCTION

Rhodococcus equi is a Gram positive, pleomorphic, rod shaped bacteria. It is an important pathogen of young foals and commonly found in soil. R. equi infection can lead to chronic and severe pyogranulomatous pneumonia in young horses, subacute or chronic abscessating bronchopneumonia sometimes with ulcerative typhlocolitis and may include mesenteric lymphadenitis, osteomyelitis, purulent arthritis, reactive arthritis and ulcerative lymphangitis (Dedar et al., 2017). R. equi is a facultative intracellular pathogen susceptible to neutrophil mediated killing. It is able to surviving and replicating in macrophages, resists innate macrophage defenses and establishes residence within the intracellular environment of that phagocyte (Hondalus, 1997).

R. equi is an important cause of foal mortalities and about 17–20% foals are PCR positive on swab sampling from the upper respiratory tract in the studies carried out by Kishor Kumar and Irfan Ahmad Mir in Rajasthan and Jammu & Kashmir respectively (Kumar et al., 2014, Mir et al., 2015). At present time there are so many antibiotics are discovered. The most important advances of modern science considered the development of antibiotics (Marston et al., 2016). The combination of rifampin and erythromycin used to treat the disease (Sweeney et al., 1987, Hillidge, 1987). Recenly clarithromycin or azithromycin, newer generation macrolides replaces the erythromycin in combination with rifampin (Gigue`re et al., 2004).

Acquired resistance genes may enable a bacterium to produce enzymes that prevent the drug from reaching its intracellular target, to modify the drug’s target site, or to produce an alternative metabolic pathway that bypasses the action of the drug. Conjugation, transformation or transduction is method of acquisition of new genetic material by antimicrobial susceptible bacteria from resistant strains of bacteria (Tenover, 2006). The overuse & abuse of antibiotics, lack of information on pathogens and limited surveillance have contributed to the global epidemic of antibiotic resistance (Fletcher, 2015, Cisek et al., 2014).

It is stated that increased used of macrolides to control the disease have contributed to the emergence of resistance (Pauw and Eloff, 2014). Resistant strains to either of these drugs have also been encountered (McNeil and Brown, 1992, Fines et al., 2001, Kotze and Eloff, 2002, Asoh et al., 2003, Jacks, 2003, Gigue`re et al., 2010, Pauw and Eloff, 2014).  The lack of effective alternatives against R. equi makes it compulsiveto identify novel antimicrobial agents to control and treat R. equi infection in foals.

The discussion concludes that some of the common issues are often overlooked and whilst there are numerous opportunities for environmental factors to contribute to the growing burden of antimicrobial resistance, a renewed focus on innovative and traditional environmental approaches is needed to tackle the problem.

Herbal therapy (Rasayan Chikitsa) is an ancient process of traditional treatment (Basak et al., 2020). The variety of plant derived compounds provides very diverse chemical structures that may supply both the novel mechanisms of antimicrobial action. The rapid development of modern biotechnologies opens up the way for obtaining bioactive compounds in eco friendly and low toxic conditions (Gorlenko et al, 2020). Plants have many secondary metabolites like phenols, phenolic acids, quinones, flavones, flavonoids, flavonols, tannins, coumarins, glucosinolates, terpenoids, phenylpropanoids, alkaloids, camalexin, saponins, terpenes, glycosides, carbohydrates and steroids (Kliebenstein, 2004, Maneesha et al., 2021, Sharma et. al., 2021). The secondary metabolites are important source of antimicrobial substances and useful in the treatment of bacterial infections (Srivastava et al., 2014, Fernebro, 2011).

Bikaner has a large diversity of plant species. So we planned to screen the in vitro antibacterial activity of extracts of some plants in Bikaner region to identify in vitro antimicrobial activity against R. equi, which could be further exploited for isolation of phytochemicals for treatment of foals or disinfection of stables.

MATERIALS AND METHODS

2.1.  Initial screening

In the present study, the research work was carried out during June 2018 to March 2019 at ICAR-NRCE-EPC (Indian Council of Agricultural Research, National Research Centre on Equines,Equine Production Campus), Jorbeer, Bikaner (Rajasthan). In the initial screening, fresh leaves of Aegle marmelos (Bael), Morus alba (White mulberry / Sahtoot), Nerium indicum (Red kaner), Cascabela thevetia (L.) Lippold (Pilli kaner / yellow oleander / lucky nut / suicide tree), Plumeria alba L. (Nagchampa) and Azadirachta indica (Neem) were collected manually from campus of ICAR-NRCE, EPC, Jorbeer, Bikaner, dried in hot air oven at 50⁰C and grined in mixer grinder to powder formation. Prepared ethanolic extract by using 500 ml absolute ethanol (99.9%) in 50 gram of powder of plant leaves. Then it was incubated overnight at 37⁰C in shaker incubator, sonicated in sonicator and evaporated the filtrate of sonicated extract in the rotary evaporator machine. Weight of the ethanolic extract was measured against absolute ethanol in similar volume.

2.2.  Polarity based fractionation of the active compound

Further, polarity based fractionation was done to separate non-polar and polar compounds using chloroform and distill water sequential extraction using basic principles (Jeyaseelan et al., 2012).

2.2.1. Preparation of chloroformic extract for fractionation of non-polar compounds

500 ml chloroform (99.9% pure) was added in 50 g plant’s parts powder and incubated overnight at 37⁰C in shaker incubator. Then filtered and residual supernatant was washed with chloroform until clean chloroform was observed and evaporated the filtrate in the rotary evaporator machine. Weight of the chloroformic extract was measured against 99.9% pure chloroform in similar volume.

2.2.2.  Preparation of sequentially extracted water extract (SEWE) for fractionation of polar compounds

Chloroformic washed supernatant was spread on the blotting paper for complete drying. 500 ml distilled water was added in dried supernatant, incubated overnight at 37⁰C in shaker incubator, sonicated in sonicator and evaporated the filtrate of Sonicated extract in the rotary evaporator machine. Weight of the Sequentially Extracted Water Extract (SEWE) was measured against distilled water in same volume.

2.3.  Solubility based fractionations of polar compounds of SEWE

Further, solubility based fractionations of polar compounds of SEWE were done with sequentially in ethanol, methanol and distilled water and collected Ethanol Soluble Fraction (ESF), Methanol Soluble Fraction (MSF) and Water Soluble Fraction (WSF) respectively and tested for their in vitro antibacterial activity against R. equi.

2.4.  Evaluation of in vitro antibacterial activity

Disc diffusion method (Nostro et al., 2000, Salie et al., 1996)and agar well diffusion method (Irshad et al., 2012) were used to evaluate in vitro antibacterial activity of extracts of plant parts against Vap A and Vap C positive R. equi usingMuller Hinton Broth and Muller Hinton HiVeg Agar. Measured the Inhibition Zone (IZ) diameter to determine the degree of in vitro antibacterial activity of plant’s parts extract against R. equi were as followings:

Non Active- when IZ diameter is zero

Mild Active- when IZ is less than 10 mm diameter

Moderate Active- when IZ is greater than 10 mm and less than 15 mm diameter

Good Active- when IZ is greater than 15 mm diameter

2.5.  Control

Azythromicin and rifampicin 10 mg L^-1 were taken as control.

2.6.  Polymerase chain reaction (PCR) technique

Pure colony of R. equi was procured from NCVTC, Hisar and verified time to time for purity by using the PCR technique. We obtained the amplified 550 and 700 BP fragments of the R. equi pathogenic Vap Aand Vap C genes respectively.

RESULTS AND DISCUSSION

In present study, pure colony of R. equi was procured from National Center for Veterinary Type Cultures (NCVTC), National Research Centre on Equines (NRCE), Hisar and verified time to time for purity by using PCR based on pathogenic Vap A and Vap C genes. By the PCR technique, we obtained the amplification of 550 and 700 bp fragments of the R. equi pathogenic Vap Aand Vap C genes respectively. These pathogenic Vap A and Vap C genes indicated the colony of the R. equi was pure.

3.1.  Extract / Fraction of plant’s parts

In vitro antibacterial activity of ethanolic leaves extract of Aegle marmelos (Bael), Morus alba (White mulberry / Sahtoot), Plumeria alba L. (Nagchampa), Nerium indicum (Red kaner), Cascabela thevetia (L.) Lippold (Pilli kaner / yellow oleander / lucky nut / suicide tree) and Azadirachta indica (Neem) against R. equi and further polarity and solubility based fractionation are showing in table 1. In initial screening ethanolic leaves extract of all these plants except Azadirachta indica were non-active against R. equi (Figure 1). Chloroformic (Figure 2b) leaves extract of Azadirachta indica did not showed in vitro antibacterial activity against R. equi while Ethanolic (Figure 2a) and SEWE (Figure 2c) showed good in vitro antibacterial activity against R. equi. Further, solubility based fractionations, Ethanol Soluble Fraction (ESF), Methanol Soluble Fraction (MSF) and Water Soluble Fraction (WSF) of polar compounds of SEWE of Azadirachta indica leaves were did not showed in vitro antibacterial activity against R. equi (Figure 3).

Table 1: In vitro antibacterial activity of plant’s leaves extract / fraction against R. equi

Figure 1: In vitro antibacterial activity of Ethanolic Extract of leaves of (a) Aegle marmelos; (b) Morus alba; (c) Plumeria alba L.; (d) Nerium indicum and (e) Cascabela thevetia (L.) Lippold against R. equi

Figure 1: Continue...

Figure 2: In vitro antibacterial activity of leaves extract of Azadirachta indica against R. equi: (a) Ethanolic Extract; (b) Chloroformic Extract and (c) SEWE

Figure 3: In vitro antibacterial activity of solvent based fractionation of SEWE of Azadirachta indica leaves against R. equi: (a) ESF of SEWE; (b) MSF of SEWE and (c) WSF of SEWE

3.2.  Solvents

In the present study, the chemical solvents were used analytical grade. In disc diffusion method, discs were dip in solvents (ethyl alcohol and chloroform) and dry until the solvents were completely evaporate. So the concentration of these chemical solvents in the dry discs were zero. Ethanol is well known to dissolve both polar and non-polar compounds because of its polar nature due to its hydroxyl group (OH^-) and non-polar nature due to ethyl (C₂ H₅) group. Chloroform dissolves non-polar compounds and distilled water dissolves polar compounds.

3.3. Non-active plants

In initial screening the ethanolic leaves extract of Aegle marmelos (Bael), Morus alba (White mulberry / Sahtoot), Nerium indicum (Red kaner), Cascabela thevetia (L.) Lippold (Pilli kaner / yellow oleander / lucky nut / suicide tree) and Plumeria alba L. (Nagchampa) did not show in vitro antibacterial activity against R. equi (Figure 1). There are so many factors like environment, pH of the medium, temperature, water activity, oxygen availability, nutrient availability, choice of solvent, source of the organisms, biochemistry, physiology, metabolism, adaptation strategies of the microbes, plant species, age, parts, concentration of the plant extract and period of extraction, which affect the antimicrobial susceptibility pattern of plant extract (Izah, 2018).

3.4. Azadirachta indica (Neem)

In present study, ethanolic leaves extract of Azadirachta indica has shown good antibacterial activity against R. equi (Figure 2a). Antibacterial activity of alcoholic extract of A. indica have been reported against many bacteria and were found comparable to chlorhexidine (Mistry et al., 2014). In present study, on sequential extraction, chloroform extract was found ineffective (Figure 2b) while sequential extracted water extract (SEWE) found good effective against R. equi (Figure 2c). It shows that effective components of A. indica are polar in nature.

Ethanolic, methanolic, chloroformic and aqueous leaf extract of A. indica exhibited antimicrobial activity against different microorganisms (Reddy et al., 2013, Raut et al. 2014, Rajasekaran, 2008, Koona et al., 2011). Phytochemical analysis of Ethanolic, Methanolic, Acetonic and Aqueous leaves extract of A. indica gave positive results for lipid, steroids, triterpinoids, reducing sugars, alkaloids, phenolic compounds, flavonoids, tannins, proanthocyanidin, glycosides and coumarin (Susmitha et al., 2013, Vinoth et al., 2012). In the present study, aqueous leaves extract of A. indica has been reported for antimicrobial activity against pathogenic microorganism R. equi. Further, dilutions of SEWE concentrations up to 70 mg ml^-1 was found effective against R. equi.

3.5. Control: Azithromycin and Rifampicin

Azithromycin and Rifampicin were taken as control having concentration of 10 mg l^-1 and showed 25.0 mm (Figure 4) and 20.0 mm (Figure 5) diameter of inhibition zone respectively against R. equi using agar well diffusion method.

Figure 4: Control Azithromycin IZ- 25.0 mm conc.- 10.0 mg L-1 (Agar Well Diffusion Method)

Figure 5: Control Rifampicin IZ- 20.0 mm Conc.- 10.0 mg L-1 (Agar Well Diffusion Method)

3.6.  Comparison with antibiotics

The combination of Macrolides (erythromycin / azythromicin) and rifampicin is the most effective and prevalent treatment against R. equi in foals, but resistant strains of R. equi is also being observed (Cisek et al., 2014). In present experiment, commercially available azythromicin and rifamipicin was used @ 10 mg l^-1 and both the antibiotics have shown good zone of inhibition (Figure 4 and Figure 5). While most effective herbal fraction SEWE of A. indica leaves showed their minimum inhibitory concentration at 70 mg ml^-1. It shows that, quantitatively currently used antibiotics have more times antimicrobial efficacy than the fraction SEWE of A. indica leaves. It depicts that even if the extracts are considered nontoxic and not interfered by digestive and metabolic processes than there will be use as antimicrobial agent against R. equi in foals. So it suggests that there is need to find more purified compound of these extracts for to see the possibilities of in vivo use. However, there are possibilities of direct use of A. indica leaves and their water extract against R. equi as farm disinfectant.

CONCLUSION

On comparison with currently used antibiotics, required concentration of the most active SEWE fraction of A. indica leaves is too high for their possibilities for in vivo use. However, abundant availability of A. indica leaves and their activity against R. equi suggests their potential for use as disinfectant against R. equi.

ACKNOWLEDGEMENT

We would thankful and grateful to Prof. (Dr.) Rakesh Rao, Dean, College of Veterinary and Animal Science, RAJUVAS, Bikaner, Rajasthan, for rendering all the required facilities all times. It is a pleasant disposition to express gratitude to Dr. B. N. Tripathi, Director, ICAR-NRCE, Hisar, Haryana, for providing all the assistance for this project. We would also like to thank Dr. S. C. Mehta, Office In-charge, ICAR-NRCE, EPC, Bikaner, Rajasthan, for equipments and laboratory facilities. We would like to thank Dr. Sanjay Kumar, Principal Scientist, ICAR-NRCE, Hisar, Haryana. We also thank to Dr. Sanjay Barua and Dr. R. K. Vaid, Principal Scientist, NCVTC, Hisar, Haryana, for providing me pure colony of Rhodococcus equi for standardization of PCR.

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