INTRODUCTION:

Algae are very popular as a food product and embraced as food for centuries in south-eastern Asian countries¹. They are a rich source of proteins, carbohydrates, minerals, vitamins, and dietary fiber². It has been shown that algae and seaweeds are immune-booster and having anticancer and anti-diabetic properties³.Apart from source for food and feed supplements, algae find its use in biomass production, oils for fuels, and coproducts in biodiesel^{4, 5}. One of the effective antimicrobial activities is attributed to secondary metabolite chlorellin^6-9. Apart from Chlorella some of the plant with antimicrobial properties were also reported namely Morinda citrifolia¹⁰, Coriandrum sativum, Apium graveolens¹¹, Ficus racemose¹², Cyclamen hederifolium¹³, Givotia rottleriformis¹⁴.

However, a comprehensive study depicting the role of Chlorella against human opportunistic fungal pathogens is lacking. Opportunistic organisms include fungus, bacteria, or viruses which cause infection in immunosuppressed patients. This is due to the indiscriminate use of antibiotics, weakened immune systems due to cancer, diabetes, and organ transplantation^15,16. The opportunistic infection is a deadly disease owing to the lack of early detection/diagnosis system, the emergence of multidrug-resistant strains, lack of effective therapy especially in the case of human fungal infections¹⁷. The situation is alarming as most of the drugs used for treating fungal diseases are either outdated or expensive^{18, 19}. To explore the other possible solutions for the treatment, we have used the cell wall lysed Chlorella powder and studied its antibacterial and antifungal properties. Cell wall lysed Chlorella gave an added advantage as it can be directly consumed and easily digestible.

MATERIAL AND METHODS:

Media and Strain: All the media used in the study were procured from HiMedia or LobaChemie. The standard procedure of autoclaving and microbiological practice was followed. The strains were obtained from the MTCC culture collection center, enlisted in Table 1. The strains were revived in nutrient agar and yeast extract peptone dextrose media. Kirby–Bauer (disc-diffusion antibiotic susceptibility) assay was performed to test the efficacy of the crude extract ²⁰ using Muller Hinton and Sabouraud Dextrose media for bacteria and fungus respectively.

Table 1: List of the strains used in the present study

Strain	MTCC number*
Bacillus subtilis	MTCC121
Pseudomonas aeruginosa	MTCC2582
Enterococcus faecalis	MTCC2729
Klebsiella pneumonia	MTCC7028
Staphylococcus aureus	MTCC3160
Candida albicans	MTCC854
Candida parapsilosis	MTCC998
Candida tropicalis	MTCC184
Candida auris	PGI^†
Saccharomyces cerevisiae	MTCC172

*Microbial Type Culture Collection and Gene Bank, Chandigarh, India

† Post Graduate Institute of Medical Education and Research, Chandigarh, India

Extraction of active constituents: The extraction of the active constituents was performed by following the method described by Choudhury et al ^21.We have used water, ethyl acetate, methanol, and ethyl acetate solvents for the extraction. However, we found better results with methanol and ethyl acetate hence it was considered for further studies. It is also based on our previous experiences with the extraction procedure²². The extraction was performed using the Soxhlet method for 30 cycles, using 20 g of Chlorella powder and 200 mL of solvent at 65°C and 77°C for methanol and ethyl acetate respectively. The extract collected was evaporated in a rotary evaporator at 40 rpm at 45°C and further dried in hot air oven to obtain a dry powder. A stock solution of 10 mg/mL was prepared in dimethyl sulfoxide (DMSO) and used for disc diffusion assay.

Disc susceptibility assay: The assay was performed in Muller Hinton (MH) and Sabouraud Dextrose (SD) agar medium for bacteria and fungi respectively by following the method described²². An overnight grown culture equivalent to OD 600_nM 0.025 and 0.05 was spread plated on MH and SD agar plates for bacteria and fungi respectively. A saturated 6 mM sterile disc with crude lysate was placed over the plate. As a negative control DMSO and for positive control ofloxacin (5 μg/disc) and fluconazole (10 μg/disc) were used. The zone of inhibition was calculated after 24 h and 48 h incubation at 37°C and 28°C for bacterium and fungus respectively. The zone of inhibition is plotted against the concentration of the lysate (calculated based on stock solution and μL of crude lysate used).

RESULTS AND DISCUSSION:

Commensal organisms residing in the human body cause serious infection, contributing to a high mortality rate due to an immunocompromised state²³. In the United States every year more than 2 million bacterial infections occur and resulting in the death of more than 2300 patients per year²⁴. Antibiotic resistance in these organisms is due to the mutation in a genetic level that leads to the development of resistance. Apart from the United States, developing countries like India are also facing the emergence of higher multidrug drug-resistant strains²⁵. Most of the drugs used for controlling pathogenic microorganisms are not fruitful or having side effects²⁶. As an alternative strategy, edible Chlorella can be used as it is previously shown to be a health booster²⁷. Based on these principles we have selected five bacterial and five fungal strains that cause serious fatal diseases in humans.

Chlorella extracts antibacterial property: With an initial powder of 20 g subjected to the extraction process, we obtained a dried powder of ~100 mg. This is dissolved in DMSO at a concentration of 1 mg/mL and used for the disc diffusion assay. We have taken gram-negative Pseudomonas aeruginosa, Klebsiella pneumoniae, and gram-positive Enterococcus faecalis, Bacillus subtilis, Staphylococcus aureus strains, the list of the strains used is mentioned in Table 1. The representative zone of inhibition for two strains is shown in Figure 1 (A and B).

Figure 1: Antibacterial susceptibility assay of an extract of Chlorella. (A) E. faecalis (B) S. aureus. 1 to 4 represents methanolic and 5 to 8 represents ethyl acetate extract respectively. 4 and 8 is negative control without crude lysate. 1 to 3 and 5 to 7 represent 10, 20 and 30 µL (Stock solution of 4 mg/mL). (C) E. faecalis (D) S. aureus. 1 represents positive control ofloxacin 5 µg/disc, 2 and 3 corresponds to ~12 mg/mL methanolic extract and ethyl acetate respectively.

Based on the zone of inhibition and extract used the effective concentration was calculated and depicted in Figure 2 (C and D). The effective concentration was deduced to be 12 mg/mL. The results suggest that methanolic extract is more potent in inhibiting the growth of the bacteria. However, it is indifferent to P. aeruginosa, as we didn’t observe any inhibition. Based on the zone of the inhibition we find it is most effective for E. faecalis and S. aureus (Table 2). Most important it is equivalent to the ofloxacin a positive antibacterial used in the experiment.

Figure 2. MIC for bacteria. (A) Methanolic extract. (B) Ethanolic extract. The bar depicts the MIC based on the zone of the inhibition (X-axis) and concentration of extract (mg/mL), Pseudomonas aeruginosa (P.a), Klebsiella pneumoniae (K.p), Bacillus subtilis (B.s), Staphylococcus aureus (S. a), Enterococcus faecalis (E.f).

Table 2: Antibacterial property: Comparative zone of inhibition with the positive control.

Strains	Cell wall extract of Chlorella 12 mg/ml
	Methanol	Ethyl acetate	Ofloxacin (5µg/disc)
P. aeruginosa	NA*	NA	NA
K. pneumoniae	14mm	10mm	22mm
B. subtills	21mm	15mm	25mm
S. aureus	24mm	20mm	29mm
E. faecalis	25mm	16mm	28mm

* NA=Not applicable (as no zone of inhibition observed).

We observed more zone of inhibition with methanolic extract compared to ethanolic extract. It suggests that methanolic been a more polar solvent could able to successfully extract the active components more easily²⁸. Previously it has been suggested that methanolic extract could able to extract terpenoids, saponins, tannins, flavonoids, and polyphenolic compounds, similarly, ethyl acetate could able to extract flavonoids, alkaloids, and terpenoids from plants²⁹. Our results for P. aeruginosa are on similar lines as reported for other microalgae²⁹. Moreover, the antibacterial activity except for S. aureus, E. faecalis is similar to the earlier report³⁰.

Antifungal property: to study whether the extract is having any anti-fungal activity we have tested on Candida sps. Most of the Candida sps are commensals and reside in the gastrointestinal and mucus layer. However, under the invasive condition, it causes serious mortality up to 50 %³¹. In our study, we used S. cerevisiae as a positive control as we anticipated the extract will be effective against it. We have also used a multidrug resistance global pathogen Candida auri³². Other Candida species cause candidiasis and are showing resistance to the existing drugs^{33, 34, 35}. Our results suggest that both methanolic and ethanolic extracts are at par (Figure 3 A and B), the effective IC was 12 mg/mL and it is most applicable for S. cerevisiae (Figure 4). The most affected strain was C. tropicalis and C. albicans. It is indifferent towards MDR C. auris as anticipated. However, when compared with positive antifungal drug fluconazole the zone of inhibition is only 50 % as compared to anti-bacterial property (Table 3). We infer this is due fungi are more robust towards cell lytic or inhibitory active constituents compared to bacteria. In a similar study, the synergistic use of curcumin with other antifungals drugs were shown to be fruitful³¹.

Figure 3: Antifungal susceptibility assay of an extract of Chlorella. (A) C. albicans (B) C. tropicalis. 1 to 4 represents methanolic and 5 to 8 represents ethyl acetate extract respectively. 4 and 8 is negative control without crude lysate. 1 to 3 and 5 to 7 represent 10, 20 and 30 µL (Stock solution of 4 mg/mL). (C) C. albicans (D) C. tropicalis. 1 represents positive control fluconazole 10 µg/dis, 2 and 3 corresponds to ~120 mg/mL methanolic extract and ethyl acetate respectively.

Figure 4. MIC for fungi. (A) Methanolic extract. (B) Ethanolic extract. The bar depicts the MIC based on the zone of the inhibition (X-axis) and concentration of extract (mg/mL), Saccharomyces cerevisiae (S.c), Candida auris (C. a), Candida parapsilosis (C. p), Candida tropicalis (C. t), Candida albicans (C.al).

Table 3: Antifungal property: Comparative zone of inhibition with the positive control.

Strains	Cell wall extract of Chlorella 12 mg/ml
	Methanol	Ethyl acetate	Fluconazole (10 µg/disc)
S. cerevisiae	14 mm	12 mm	24 mm
C. auris	NA*	NA	25 mm
C. parapsilosis	10 mm	08 mm	22 mm
C. tropicalis	12 mm	10 mm	24 mm
C. albicans	12 mm	08 mm	24 mm

* NA=Not applicable (as no zone of inhibition observed).

Our study has taken three new fungal strains C. auris, C. parapsilosis, and C. tropicalis, not reported earlier^{29, 37, 38, 39}. Although the zone of inhibition was not equivalent to the positive antifungal agent, the study suggests that the alga has antifungal properties. Based on the solvents we used and the anticipated compounds which can be extracted we also performed the High-performance Liquid Chromatography (HP-TLC) using quercetin and gallic acid as control. However, it failed to report any active band except a band similar to quercetin (data not shown.). Similar to our study we find other plants and marine brown algae, Sargassum with antimicrobial properties⁴⁰. Hemalatha et al has reported the antimicrobial property of Calotropis gigantea root bark extracts with ethyl acetate and methanol solvents⁴¹. The aqueous leaves extract of Jatropha curcas indicates that it is have antimicrobial activity against E. coli, S. aureus, Bacillus. spp., P. aeruginosa⁴².

CONCLUSION:

Our study showed that cell wall lysed Chlorella powder is having anti-microbial properties. It is the most effective antibacterial as compared to antifungal. Four organisms E. faecalis, C. tropicalis, C. parapsilosis is not reported earlier and incorporated in our study suggest that it can be used as an antimicrobial agent against these organisms. It is most effective against E. faecalis and C. albicans and indifferent towards P. aeruginosa and C. auris. We surmise that apart from a good source of dietary fiber, vitamins, antioxidant properties Chlorella cell wall lysed powder can be used as food supplements. Further studies are required to delineate the essential and active bioactive compounds which can be used for therapeutics against the clinical isolates.

COMPETING INTEREST:

The authors have no competing interest to declare.

ACKNOWLEDGMENTS:

The lab funding from the Scientific and Engineering Research Board (SERB), India, file no. EMR/2017/002299 is duly acknowledged.

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Received on 27.05.2020 Modified on 15.07.2020

Research J. Pharm. and Tech. 2021; 14(7):3695-3699.

DOI: 10.52711/0974-360X.2021.00639