1. Introduction
Antimicrobial resistance amongst infective bacterial communities poses a serious threat related to increased morbidity and mortality [
1]. Resistance to popular antimicrobial therapies and the emergence of multidrug-resistant bacteria is growing at an unprecedented pace. There are challenges in combating bacterial infections and associated diseases due to acute shortage of effective drugs, lack of successful preventive measures, and presence of few new antibiotics in the clinical pipeline, which has inspired the development of novel treatment options and alternative antimicrobial therapies [
2]. According to WHO (World Health Organization), resistant microorganisms (such as bacteria, fungi, viruses, and parasites) are capable of resisting antimicrobial activity resulting in the existence and propagation of infections that cause numerous drug-resistant strains or organisms [
3]. Centers for Disease Control and Prevention (CDC) report that nosocomial infections contribute to at least 5% of the hospital patients clinical setting is reported to have had more than 2 million illnesses and 99,000 deaths per year [
4]. These morbid factors have cumulatively built the momentum towards the investigation of novel antimicrobial agents that are effective against pathogenic micro-organisms exhibiting resistant against conventional treatments [
5]. Nowadays, novel antimicrobial agents from natural sources such as microbes, fungi, and plants are flourishing, and plant-based innovative therapeutics are steadily increasing [
6].
Several natural resources have been explored for antimicrobial properties in the recent past, with mushrooms being one of the highly probed source [
5]. The intrinsic properties of some mushrooms have been noted to exhibit healthy, safe and effective medicinal effects in the treatment of human disease. Moderate to good antimicrobial activity against bacterial and fungal infections has been demonstrated by some mushrooms, such as
P. ostreatus,
P. sajor-caju,
P. eryngii,
P. florida and
Agaricus bisporus extract against several pathogenic bacteria [
7]. A large number of bioactive compounds have been isolated and identified in mushrooms that include terpenoids, flavonoids, tannins, alkaloids, and polysaccharides, etc. [
8]. To exploit the antimicrobial properties of mushroom, careful selection of solvents is critical, as they influence the type of bioactive components and the concentration that is extracted [
9]. These bioactive compounds are generally produced as secondary metabolites for the growth and survival of the mushrooms in a specific environment [
10]. Crude extracts usually contain more than one bioactive compound that tend to have synergistic effect thereby adding potential therapeutic value. Since mushrooms posess similar microbial antagonists to humans, bioprospecting mushrooms for antimoicrobial agents could provide some novel therapeutics. The antibacterial activities detected here warrant investigation for their potential to improve human health and application as dietary supplements and also as neutraceuticals [
11]. Different bioactives can be found during various stages of mushroom development. For instance, fruiting bodies and mycelium of mushrooms show distinct health-promoting values due to the nature and action of numerous bioactive compounds [
6].
Pleurotus species belong to
Phylum Basidiomycota that produce oyster shaped mushrooms (basidiocarps) and hence the name oyster mushrooms. They are are edible and among the most common mushrooms worldwide [
10]. The oyster mushroom,
Pleurotus spp., is commonly grown on a broad variety of substrates consisting of lignin and cellulose.
Pleurotus sp. is promising as a therapeutic mushroom with haematological, antiviral, antitumor, antifungal, antibacterial, hypocholesterolic, antioxidant and immunomodulation actions [
12,
13].
Pleurotus opuntiae is an essential mushroom of the xerophytic temperate regions of Mexico. Taxonomic relationships of
P. opuntiae show that
P. agaves is a synonym of
P. opuntiae and
P. levis are close species of
P. opuntiae. Traditional uses of
P. opuntiae as food and remedy for several health problems have been identified in literature [
14,
15]. The extensive cultivation of
Pleurotus species is carried out for its consumption as food and for medicinal properties. Literature data are mostly available for the most common sps. such as
P. ostreatus and
P. eryngii, etc., while very little is known about the potential therapeutic properties of taxa, specially
P. opuntiae. The medicinal value of
Pleurotus taxa is still under-investigated in most of the Mediterranean countries [
15]. In general, the bioprospecting studies concentrate almost exclusively on the screening of antibacterial properties of mushroom extracts without specifying the compounds responsible for their action. However, some of the compounds previously reported for antimicrobial effect are sesquiterpenes and other terpenes, hormones, benzoic acid derivatives, anthraquinones and quinolines, and peptides and proteins [
10]. The modes of action of organic compounds that may be associated with the antimicrobial function of mushroom extracts have been the subject of several studies [
9].
Thus, determining the compounds or secondary metabolites of antimicrobial action of extracts obtained from medicinally important mushroom will help to design alternate or combinational treatment strategies to treat infections caused by antibiotic resistant bacteria. In line with this, the current study will focus on the lesser studied Pleurotus opuntiae for evaluating their medicinal value (antimicrobial). The antibacterial action of the mushroom extracts with two different solvent system will be determined and the extracts will further by characterized based on the types of mycoconstituents present. Thus, by understanding the type of bioactives present, the components responsible for antimicrobial properties can be identified. In future this could lead towards new drug discovery in the field of antimicrobial chemotherapy by isolation and identification of these antimicrobial compounds.
4. Discussion
Mycoconstituents display a wide range of biological activities against pathogenic microorganisms [
26]. The findings obtained from present study affirm this further with a focus on antibacterial activity. Similar antimicrobial potentials have been observed in
P. ostreatus [
27] and many other species [
21] but have never been reported in
P. opuntiae despite having medicinal properties. Bacterial strains included in this study were chosen based on their significance and etiology in clinical microbiology and in different infectious processes [
28]. Eloff; 1998 [
29] studied the antimicrobial activity of different plant extracts on different test organisms by using colorimetric assays of various tetrazolium salts as a reagent. The result of his experiment indicated that the formation of formazan product was most stable, particularly in the case of INT formazan tetrazolium salt assay. Hence, in our study, we rationally used INT tetrazolium salt assay for the MIC determination of
P. opuntiae. Both extracts showed potential bactericidal activity against the tested pathogenic bacteria. Antimicrobial activity is usually regarded as bactericidal if the MBC/MIC ratio is ≤4 and bacteriostatic if MBC/MIC > 4 [
30]. The obtained results from both the extracts were bactericidal as their MBC/MIC value were ≤4.
Both extracts showed various degrees of antimicrobial activity against test pathogens. Variation in MIC of different extracts may arise from differences in their chemical constituents and volatile nature of their constituents [
22]. Furthermore, the type of extraction solvent, the origin of the mushrooms, their age, species, as well as the type of bacterial strain, can be attributed to the differences in the antimicrobial activity [
5]. The higher bioactive content in the mushroom extracts can also be one of the major factors correlating with different ranges of MIC values as some could exhibit synergistic effects as well. From the above result, it was concluded that ethanol extract gives the highest total antimicrobial activity with lower MIC and MBC values as compared to methanol extract of
P. opuntiae. During mycochemical screening, the ethanol extract of
P. opuntiae gave a large number of mycoconstituents compared to methanol extract. Such a difference between two extracts is generally correlated with the affinity of a particular bioactive compound towards a solvent which in turn depends on the polarity. By specific separation and purification strategies, the type of components specific to the solvents can be identified. The presence of a higher quantity of compounds in the ethanol extract of
P. opuntiae is responsible for the antimicrobial activity at lower MIC values against test microorganisms [
31,
32]. The preliminary screening tests can be helpful in the identification of bioactive compounds in the mushroom and can help in the discovery and production of drugs [
21]. Groups of mycochemical compounds are commonly associated with combating microbial resistance and having antimicrobial behavior in edible mushroom. Currently, many of the pathogenic bacteria exhibit multidrug-resistance to commonly used antibiotics causing different diseases [
33]. Due to these contributory factors, the search for a new drug is important in current times, and
P. opuntiae has been proven as a potential source of bioactive molecules. The isolation of these bioactive compounds needs different analytical methodologies which include thin layer chromatography as one of the procedures for preliminary screening [
33].
Since silica gel retains more polar compounds, the nonpolar compounds are eluted first and moved further up the TLC plate. Hence, the more polar the compound, shows lower Rf (Retention factor) value, and the less polar the compound, show larger the Rf value. Thus, it can be concluded that the mushroom extract of
P. opuntiae studied here has both polar and nonpolar compounds [
22]. This could also justify the difference in the extraction efficiency and antimicrobial activity between the two solvent extracts. Though methanol and ethanol exhibit closer polarity index, there is however a significant difference between these two solvents. As a result, bioactive compounds with a very specific affinity towards ethanol are extracted with ethanol, unlike methanol. According to Bubueanu (2017 [
34]) the
Pleurotus ostreatus extract was characterized by two major spots, one was ferulic acid (Rf ~0.9 and ~0.55) and other was coumarins (Rf~0.1, 0.6 and 0.8). The presence of ergosterol (T3) with four major spots (Rf~0.3–0.9) was also found in different solvent systems. Thus, the present study on
P. opuntiae elicits similar findings with major spots corresponding to the same Rf values as stated above. It was also found that in
P. opuntiae extract, the highest percentage area (76.75% and 49.37%) covered by the Rf value 0.3, matched to the Rf value of ergosterol as mentioned above. This can further be justified by the higher extraction efficiency of ethanol, as ergosterol shows relatively lower polarity and hence soluble to a greater extent in ethanol compared to methanol. So, these findings suggest strong evidence-based similarities for presence of ergosterol, ferulic acid and coumarins along with other compounds respectively. Further analysis by digital chromatographic techniques like HPTLC posted strong scientific evidence validating the TLC results. The differentiation in HPTLC profile is an important and powerful procedure that has often been used for detection of compounds [
35]. Huizhen Li [
36] in his HPTLC study of
Pleurotus ostreatus recorded Rf = 0.34 & Rf = 0.31 and found it to be corresponding with saponin which is also same with our HPTLC results as the maximum area at wavelength 366 nm is being Rf = 0.34, which is 49.37%, and the maximum area at wavelength 540 nm is being Rf = 0.34 which is 14.13%. This result is also being supported by the mycochemical screening test for the presence of saponins in the extract. As a result, the presence of saponin is confirmed in higher amounts in
P. opuntiae extract [
36].
As per the definition of antimicrobial coating from the literature, it should be some synthetic resin or epoxide or a nano-emulsion that may release certain chemicals to inhibit the growth of pathogens; hence, to prove this release of antimicrobial chemicals from the extract we used a TLC plate to show the separated antimicrobial compounds. We used mushroom extract for natural antimicrobial coating applications. The silica has been used as the antibacterial surface design with the incorporation or adhesion of mushroom extracts due to their properties of being non-toxic, eco-friendly, cheap, easily available and not containing heavy metals, presenting support to silica-based surfaces by natural adjuvants to promote its activity. Some papers had reported that the natural extracts need to be used in an optimized amount to have the best performance for the coating. The natural additives included biopolymers like chitosan, gluten, gelatin, etc. Mushroom extracts on the other hand presented itself as “self-sustained edible coatings”. Different biopolymers, such as proteins, lipids, polysaccharides or their combinations, have been used as carriers to produce edible coatings with antimicrobial properties. Since it is well reported that mushroom have large amount of polysaccharide (glucans, chitin, mannas, galactans, xylans, etc.) the purpose of edible coating is well fulfilled [
37].
The main coating techniques in food packaging are spraying, dipping or spreading, which have all been carried out by us at different stages of experimentation. In this context, we propose that
P. opuntiae, an edible mushroom, can act as an edible antimicrobial coating in future. Antimicrobial edible coatings are ready to provide an effective alternative in active packaging materials to improve the safety of processed food products for commercial purposes [
38,
39]. The mushroom antimicrobial coating can be used in two ways: one by immobilizing it on a packaging film or the other relatively easy method by spreading a layer of extract on the food material. The mushroom releases its active compounds by forming a matrix and releasing active mycochemicals presenting a layer or coat over the food. Our results have shown that mushroom extracts exhibit stronger antibacterial properties against several pathogens. They could be incorporated into an edible film and coating formulation as antimicrobial agents, which will lead to improved food safety [
40].
It is well known from the literature that basidiomycete comprises numerous bioactive compounds that facilitate a range of functional properties, acting individually and/or synergistically.
A. blazei includes several other compounds, such as ergosterol, other forms of fatty acids, polysaccharides and alkaline compounds, which can also play an important role in synergistic antimicrobial activity [
41]. Sudirman et al. [
42] purified the antimicrobial compound fraction of Rf value 0.73 from
Lentinus cladopus LC4, which is also similar with the results of our TLC but the structure of that compound is still unknown [
42]. The developed HPTLC method gave strong fingerprints for species authentication through direct band comparison and can confirm the existence of bioactive compounds. Thus, the present study provides sufficient information about the therapeutic efficacy of the medication and in the identification, standardization and quality assurance of the specific natural product. In conclusion, the findings of the qualitative assessment of the HPTLC fingerprint images should aid in the detection and quality assurance of the medication and ensure therapeutic efficacy [
43].
Our evidence-based study provides facts about the use of mushroom as a functional food. It contains different components (bioactives) which might be acting “synergistically” in an in-vivo physiological system to produce different medicinal attributes which may not be “individually” present in desired compounds if they are isolated and tested “in-vitro”. Since mushrooms, and specifically
Pleurotus opuntiae, are widely used as edible food supplement in the Indian sub-continent and the Asian diaspora worldwide, this study is a preliminary attempt to indicate the potential antimicrobial activity of the mushroom, identifying it as a super-food with tremendous medicinal properties [
3].
This research is one of the few studies identifying the antimicrobial action of the fungal extracts derived from their fruiting body. Our findings also support the possible application of fungal metabolites in pharmaceutical products and the pharmaceutical industry [
26]. With an increasing number of bacteria developing resistance to commercial antibiotics or MDR, extracts and derivatives from mushrooms hold a great promise for novel medicines in modern times [
2,
44,
45]. In the light of the current global pandemic, more multidisciplinary methods, such as phyto-micro/nanotechnology-enabled strategies, may become front-runners in the battle against infectious diseases and global initiative for the prevention and treatment of these infections [
3,
45]. Lastly, we need to scrutinize the recently evolving diverse cellular machinery of microbial flora [
46,
47,
48] and resistant viruses by extensive research into natural-based therapeutics to improve preventative foresight.