In vitro antimicrobial activity of nine homoeopathic preparations in different volumes against Staphylococcus aureus and Escherichia coli

INTRODUCTION

The global incidence and mortality rate of anti-microbial resistance (AMR) in 2019 was 1.27 million and 5 million, respectively.^[1] Staphylococcus aureus and Escherichia coli are among the six pathogenic microbes that are responsible for AMR in India. The staphylococcus genus contains approximately 30 species of medical importance, mainly S. aureus which is responsible for hospital-acquired infections.^[2] It produces pyogenic infections in the skin and various other organs.^[3]

E. coli belongs to a group of diarrheagenic Enterobacteria.^[3] Enterobacteria contain five species of medical importance, mainly E. coli, which are normally present in the human intestinal flora. The virulent strain of E. coli produces urinary bladder infections, wound infections and diarrhoea, among other disorders. E. coli is most commonly isolated among the Enterobacteria and is also the main isolate from the urine samples.^[2]

Homoeopathy is one of the major alternative medicine systems used worldwide. Homoeopathic medicines are administered in ultra-diluted forms; they are effective in various disorders, which can be confirmed through clinical trials. However, there is a lack of in vitro and in vivo studies in homoeopathy.

Denny, in an in vitro study against various cell lines, observed that antiproliferative properties of polyketides can be isolated from Myristica sebifera leaves.^[4] A study by Sultana suggested that the methanolic extract of Atropa belladonna showed a significant degree of antimicrobial activity against medically important bacteria like S. aureus, E. coli and Klebsiella pneumonia.^[5] Munir et al., in another study, also observed that the ethanolic extract of A. belladonna showed more significant anti-microbial activity against S. aureus than E. coli.^[6] A study by Salari et al. suggests the antibacterial activity of Eucalyptus globulus leaf extract was determined for different isolates of S. aureus. The results suggest the possible therapeutic role of E. globulus leaf extract in the treatment of respiratory tract infection.^[7] A study was carried out at Maranhão State University and Paraíba Federal University to evaluate the antimicrobial activity of Ruta graveolens. Bacillus cereus and S. aureus were the two bacteria that were most responsive to the antibacterial activity of Ruta R. graveolens against Gram-positive and Gram-negative bacteria with inhibition zones.^[8]

Several homoeopathic remedies are indicated in cases of infections and inflammations. However, in homoeopathy, there is no specific remedy for specific infections. Therefore, homoeopathy is limited to symptomatology and Materia Medica descriptions. The selection of nine homoeopathic preparations is based on source books on homoeopathy and other traditional systems.

M. sebifera is indicated in inflammation of the skin, cellular tissue and periosteum and also in traumatic infections, parotitis and carbuncles. It has an almost specific action in panaritium.^[9] A. belladonna is indicated in erysipelatous inflammation with swelling, phlegmonous erysipelas, bright red and radiating. Vesicular erysipelas (when fever is violent). Intense erysipelatous fever, accompanied by inflamed swellings, passing even into gangrene.^[10] E. globulus is indicated for chronic desquamative nephritis and pyelonephritis.^[11] It is a powerful antiseptic and destructive to a low form of life. It is also useful in treating foul and indolent ulcers with glandular enlargement and nodular swellings.^[9] R. graveolens is a remedy for periostitis and erysipelas following bruises and mechanical injuries.^[12]

We aimed to evaluate the efficacy of antimicrobial activities of these nine homoeopathic preparations against S. aureus and E. coli through an in vitro study.

MATERIAL AND METHODS

Microorganisms

S. aureus (IDH-7473) and E. coli (IDH-13003) isolates were obtained from the National Institute of Cholera and Enteric Diseases, ICMR, Kolkata 700010. As shown in Figure 1, the cultural characteristics of E. coli include large, white, round and moist colonies.

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Figure 1:

Cultural characteristic of E. coli. Two nutrient agar plates for each microbe were prepared in a petri dish and media was incubated for 24 h at 37°C. The characteristics of E. coli shows large colonies (>2 mm), white, round and moist colonies. E. coli: Escherichia coli.

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Broth microdilution technique

The broth-microdilution assay was performed to identify the lowest volumes of homoeopathic preparation that could inhibit the microbes. The antimicrobial activities of nine homoeopathic preparations in different volumes were performed in a 96-well plate (Elisa plate flat bottom, HSN Code- 39233090, Maxome Labsciences Pvt Ltd.) having a total capacity of 400 μL volume with a flat bottom as described in CLSI with slight modifications (M07-A10).^[13-15] First, different volumes of homoeopathic preparations 5–30 μL (selection of volume is based on the pre-experiment assay) were used instead of serial dilution of antimicrobial agents. Second, gentamycin (Positive Control) and 91% ethyl alcohol (Negative Control) were used to identify the drug action. Total volume of 200 μL was used out of 400 μL. MH broth (185 μL) and bacterial suspensions (10 μL) were added to a 96-well microliter plate except for the 12^th column. The nine homoeopathic preparations (5 μL) were added up to the 9^th column. The gentamycin (5 μL) was added to the 10^th column as positive control and 91% of ethyl alcohol (5 μL) was added to the 11^th column as a negative control. The 12^th column contained 190 μL of MH broth and bacterial suspensions (10 μL) as control. All test tubes were incubated at 37°C for 24 h. The test was repeated with the same procedure for other five different volumes of homoeopathic preparations (10 μL, 15 μL, 20 μL, 25 μL and 30 μL) after withdrawing the same amounts of media from 96-well plates before adding bacterial suspensions (10 μL). All assays were performed in triplicate for accuracy and reproducibility. The 96 microplates were examined using a visible spectrophotometer (Automated ELISA Microstrip Analyser, Model: ELAN 30s) at an absorbance of 630 nm to find the optical density (OD)/ turbidity for identification of the inhibitory properties of homoeopathic preparations. The graphical abstract [Figure 2] depicts the key findings of the study.

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Figure 2:

Graphical abstract.

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RESULTS

The mean and standard deviation of control were observed as 1.334 ± 0.25 against S. aureus and 1.038 ± 0.22 against E. coli [Tables 1 and 2] respectively.

M. sebifera 12C in 10 μL, 15 μL and 30 μL has shown statistically significant results against S. aureus [Table 3] and in 15 μL, 20 μL against E. coli [Table 4]. M. sebifera 30C in 15 μL has shown statistically significant results against S. aureus [Table 3] and in 25 μL against E. coli [Table 4].

A. belladonna Q in 25 μL has shown statistically significant results against S. aureus [Table 3] and in 5 μL, 10 μL, 15 μL and 20 μL against E. coli [Table 4].

E. globulus 3C in 10 μL, 15 μL, 20 μL, 25 μL and 30 μL has shown statistically significant results against S. aureus [Table 3] and in 25 μL against E. coli [Table 4]. E. globulus 12C in 10 μL, 20 μL and 30 μL has shown statistically significant results against S. aureus [Table 3] and in 10 μL, 15 μL, 20 μL and 25 μL against E. coli [Table 4]. E. globulus 30C in 15 μL and 25 μL has shown statistically significant results against E. coli [Table 4].

R. graveolens 12C in 10 μL, 15 μL, 20 μL and 30 μL has shown statistically significant results against S. aureus [Table 3] and in 15 μL and 25 μL against E. coli [Table 4]. R. graveolens 30C in 10 μL, 20 μL and 30 μL has shown statistically significant results against S. aureus [Table 3] and in 15 μL, 20 μL and 25 μL against E. coli [Table 4]. R. graveolens 200C in 10 μL, 15 μL, 25 μL and 30 μL has shown statistically significant results against S. aureus [Table 3] and in 5 μL, 10 μL, 15 μL, 20 μL and 25 μL against E. coli [Table 4].

However, alcohol has shown more significant results in comparison to some homoeopathic medicines in certain volumes against both microbes [Tables 3 and 4].

DISCUSSION

The ultra-diluted nine homoeopathic preparations at different volumes, especially lower volumes, showed significant results against S. aureus (10 μL) and E. coli (15 μL) when compared to negative controls. This implies that homoeopathic medicines are useful for treating disorders caused by AMR bacteria such as S. aureus and E. coli at lower volumes.

Further, the macrodilution assay and disc diffusion assay will provide the minimum bactericidal concentration of homoeopathic medicines. Molecular docking is useful for the identification of compounds in homoeopathic medicines which bind to microbial structure for its inhibitory action. In vivo studies like acute toxicity studies in animal models and clinical trials may be carried out to confirm their effectiveness and mechanism of action of homoeopathic preparations.

CONCLUSION

The study suggests that ultra-diluted homoeopathic preparations at different volumes, particularly lower volumes, exhibited significant antimicrobial activity against S. aureus and E. coli compared to the negative controls. Therefore, it can be concluded that homoeopathic medicines may be useful in treating disorders caused by antimicrobial resistant bacteria such as S. aureus and E. coli at lower volumes.