To detect the minimum inhibitory concentration and time-kill curve of shiitake mushroom on periodontal pathogens: An in vitro study.

INTRODUCTION: Shiitake (Lentinula edodes) is an Asian edible mushroom with the second-largest cultivation percentages among edible mushrooms in the world. Previous studies have shown its nutritional richness such as high quantities of proteins, minerals, and vitamins. Moreover, in vitro and animal studies have displayed the medical importance of shiitake extracts including antitumor, antiviral, antibiotic, and hypocholesterolemic actions and also shown to have antibacterial efficacy and has been used for the elimination of oral biofilms and as a substitute of current chemical-based treatments. This study aims to analyze the inhibitory and antibacterial efficacy of shiitake mushroom extracts on periodontal pathogens.
MATERIALS AND METHODS: Double extract, i.e., hot water and ethanol extract of shiitake were used for the assessments of minimum inhibitory concentrations on Fusobacterium nucleatum, Aggregatibacter actinomycetemcomitans and Porphyromonas gingivalis, and Prevotella intermedia using the tube-dilution method, and the time-kill curve was assessed for the above bacteria.
RESULTS: Shiitake has shown potent antibacterial effects at a concentration of 3.12 μg/ml and has shown no growth after 2 h of culture.
CONCLUSION: Shiitake mushroom extract has shown the potential antimicrobial effect on the certain periodontal pathogens.

INTRODUCTION

Adiverse species of microorganisms is found in human oral cavity both at supragingival and subgingival sites. These oral sites are basically the area where the bacteria accumulate as microbial communities called as dental plaque or dental biofilm.[1]

A disturbed host balance due to the altered composition of biofilm is a known cause of initiation of dental diseases, i.e., dental caries and periodontal diseases. Both dental caries and periodontal diseases have a different etiology which involves bacteria described as metabolic and geographic opposites. The most common site of dental caries is supragingivally whereas periodontal diseases are seen sub gingivally attacking the tooth supporting structures. Dynamics of microbiota causing these diseases may also affect the treatment of either.[2]

Dental caries caused by cariogenic plaque which comprises of mainly of low-pH streptococci, predominantly Streptococcus mutans, Streptococcus oralis, Streptococcus mitis, Rothia, actinomyces, Lactobacilli, and Bifidobacterium species whereas periodontal disease which is a cellular inflammatory response of gingiva, and surrounding connective tissue is caused due to the bacterial accumulations seen on the teeth. As it is known that the etiology of periodontitis is very complex (Ledder et al., 2007); however, researches have shown the involvement of several key species in the causation of periodontal diseases which include Porphyromonas gingivalis (Pg), Treponema denticola, Prevotella intermedia (Pi), Aggregatibacter actinomycetemcomitans (Aa), and Fusobacterium nucleatum (Fn).

Developing a better and effective diagnosis and a cost-effective means of curing periodontitis is required. These oral diseases can be prevented by the inhibition of bacterial plaque and biofilm on the tooth surfaces. This may include the mechanical debridement and the use of antimicrobial agents which are a quick and inexpensive means of augmenting mechanical periodontal debridement.[3]

Studies have shown the inhibitory effect of various antimicrobial agents such as antibiotics, fluorides, povidone-iodine, triclosan, and chlorhexidine on the bacteria grown on oral surfaces. However, these agents might have some side effects which include gastrointestinal irritation, tooth staining, and most importantly, risk of developing antimicrobial resistance; hence, a search of safe, novel and natural bioactive compounds that interfere with biofilm development is required.

Medicinal plants and edible food products having natural, antimicrobial properties, such as phenolic compounds and their subclasses, coumarins, flavonoids, and essential oils, have been studied in relation to pharmaceutical and therapeutical applications in different fields such as medicine and dentistry.[1]

Medicinal mushrooms, including Lentinula edodes or shiitake, have been used in Asia for centuries and is known to have numerous health benefits shiitake mushroom contain many chemical compounds that protect DNA from oxidative damage.[4]

They are unique in sense because they contain important essential amino acids and essential fatty acids, i.e., linolenic acid. This helps in various functions such as weight loss, boost immune system, destroy cancer cells, improve cardiovascular system health has antimicrobial properties, provide vitamin D, etc. However, still, shiitake has not as yet been assessed for its oral health benefits.

Hence, this study aims to analyze the inhibitory and antibacterial efficacy of shiitake mushroom extracts on periodontal pathogens (minimum inhibitory concentration [MIC]) and to determine the time-kill curve for the periodontal pathogens.

MATERIALS AND METHODS

Procurement of material

The dried shiitake mushroom weighing about 50 g was obtained from the food store [Figure 1]. The obtained dried mushroom was then crushed into a dried powder which was then used for the preparation of the double extract, i.e., alcohol and aqueous extract.

Figure 1

Shiitake mushroom (crude form)

Preparation of mushroom extract

The procedure for the preparation of mushroom extract was carried at a Pharmacy College.

Materials required

For the preparation of the double extract of mushroom following materials were used

Dried shiitake mushroom (50 g) (powdered form)

ethanol (100 ml) (99% pure)

Distilled water (100 ml)

Wide-mouth bottle

Filter stand

Filter paper

Electronic water bath (maintained at a temperature of 70°–80° centigrade).

A double extract of mushroom, i.e., aqueous and ethanol extract was prepared by a process called maceration. It involves dividing the dried mushroom powder into two equal halves. A 100 ml of 99% pure ethanol and 100 ml of distilled water were taken into two wide-mouth bottles separately. Then, equal divided halves of mushroom powder were added into the individual bottles and were kept for 24 h. Later on, this was filtered using the filter paper. The filtrate in electronic water bath at temperatures of 70-80 degree C, the filtrate dries off and the so obtained residue left behind is final mushroom extract [Figure 2].

Figure 2

Prepared shiitake mushroom extract

Determination of minimum inhibitory concentration and time-kill curve for the microorganisms

The obtained mushroom extract was then sent to the Department of Microbiology, for the microbiological procedures, i.e., determination of MIC and time-kill curve for the microorganisms included in the study.

Minimum inhibitory concentration procedural steps

Nine dilutions of the prepared extract were done with brain-heart infusion (BHI) for MIC

In the initial tube, 20 μl of drug was added into the 380 μl of BHI broth

For dilutions, 200 μl of BHI broth was added into the next nine tubes separately

Then, from the initial tube, 200 μl was transferred to the first tube containing 200 μl of BHI broth. This was considered as 10−1 dilution

From 10−1 diluted tube, 200 μl was transferred to the second tube to make 10−2 dilution

The serial dilution was repeated up to 10-9 dilution for each drug

From the maintained stock cultures of required organisms, 5 μl was taken and added into 2 ml of BHI broth

In each serially diluted tube, 200 μl of above culture suspension was added

The tubes were incubated for 24 h and observed for turbidity [Figure 3].

Figure 3

Minimum inhibitory concentration

RESULTS

The present study on the prepared extract of the shiitake mushroom has tested for the MIC for Pg, Pi, Fn, and Aa, wherein we found that MIC for Pg and Pi was 0.4 μl which was seen in the form of turbidity which concludes that Pg and Pi are highly sensitive for the prepared mushroom extract. At the same time, we found out that Aa showed sensitivity at 0.8 μl showing that it is less sensitive to shiitake mushroom in comparison to Pg and Pi whereas Fn showed the MIC values at 12.5 μl showing its sensitivity at this concentration [Table 1].

Table 1

Minimum inhibitory concentration of shiitake mushroom

Mushroom extract	100 µl	50	25	12.5	6.25	3.12	1.6	0.8	0.4	0.2
Pg	S	S	S	S	S	S	S	S	S	R
Pi	S	S	S	S	S	S	S	S	S	R
Fn	S	S	S	S	R	R	R	R	R	R
Aa	S	S	S	S	S	S	S	S	R	R

Pg – Porphyromonas gingivalis; Pi – Prevotella intermedia; Fn – Fusobacterium nucleatum; Aa – Aggregatibacter actinomycetemcomitans; S – Sensitive; R – Resistant; µl – Microliter

The time-kill curve for the following bacteria was then drawn at their MIC. We found out that Pg at 0.4 μl concentration showed no growth at all whereas Fn at MIC of 12.5 μl showed no growth which indicates Pg is more sensitive than Fn. Pi at its MIC of 0.4 μl showed a gradual decrease in its growth but failed to show no growth after 2-h duration. Aa at its MIC of 0.8 μl had shown a gradual decrease in its growth and after 2-h duration showed absolute no growth [Table 2 and Graph 1].

Table 2

Minimum time required to inhibit the growth of the periodontal pathogens in the study (time-kill curve)

Mushroom extract	0 min	5 min	10 min	30 min	2 h
Pg	NG	NG	NG	NG	NG
Pi	186	172	154	102	06
Fn	NG	NG	NG	NG	NG
Aa	138	124	106	56	NG

Pg – Porphyromonas gingivalis; Pi – Prevotella intermedia; Fn – Fusobacterium nucleatum; Aa – Aggregatibacter actinomycetemcomitans; NG – No growth; Min – Minutes; h – Hours

Graph 1

Time-kill curve: Plotted against X-axis and Y-axis with X-axis showing – number of microorganisms and Y-axis showing time (in min). Pg – Porphyromonas gingivalis; Pi – Prevotella intermedia; Fn – Fusobacterium nucleatum; Aa – Aggregatibacter actinomycetemcomitans; NG – No growth; Min – Minutes; Hrs – Hours

DISCUSSION

In the present study, the MIC and the time-kill curve of the prepared mushroom extract were studied on four periodontal pathogens, i.e., Pi, Pg, Fn, and Actinobacillus actinomycetemcomitans.

The MIC value in the present study ranged from 0.4 μl to 12.5 μl. Pg and Pi showed the least value for MIC, i.e., 0.4 μl whereas Fn and A. actinomycetemcomitans were found to be sensitive at 12.5 μl and 0.8 μl. The time-kill curve which demonstrates the bactericidal activity of shiitake mushroom showed absolute no growth of Pg and Fn whereas Pi and A. actinomycetemcomitans showed a gradual decrease in the growth.

In the study by Gulhas et al. concluded that shiitake essential oil is an effective antibacterial and antibiofilm agent on important oral pathogens. The study elaborated the importance of a major antimicrobial component, i.e., carvacrol an aromatic monoterpenes which showed to disintegrate the outer membrane of Gram-negative bacteria by releasing lipopolysaccharide from their cell wall and increasing membrane permeability.[1]

In one of the studies by Yano et al., showed that some components of mushrooms, including alpha-glucanases, might inhibit the sucrose-induced formation of oral biofilms.[5]

In another study by Hearst et al., it showed the potential antimicrobial effects of shiitake extracts.[6]

In an in vitro study by Ciric et al., which compared the effectiveness of shiitake mushroom extract to that of the active component in the leading gingivitis mouthwash, containing chlorhexidine, in an artificial mouth model (constant-depth film fermenter) found out that the shiitake mushroom extract lowered the numbers of some pathogenic taxa without affecting the taxa associated with health, unlike chlorhexidine which has a limited effect on all taxa associated with the health.[7]

CONCLUSION

Within the limits of the study, it can be concluded that shiitake mushroom extract has shown the potential antimicrobial effect on the certain periodontal pathogens. However, in the above-mentioned studies, it was found that some active compound of mushroom-like carvacrol has a significant role as an antimicrobial compound. Hence, further studies to isolate and to identify these active compounds require to be undertaken. Furthermore, the suitable pharmaceutical delivery system is needed to allow concentrated extract to be prepared and deliver optimally rather than the crude ingestion of the raw materials, which could promote further bacterial resistance.

Financial support and sponsorship

This study was self-funded.

Conflicts of interest

There are no conflicts of interest.