The anti-fungal and anti-bacterial effects of Psidium guajava on fungi Microsporum gypseum, Trichophyton mentagrophytes, bacteria Staphylococcus aureus, and Staphylococcus epidermidis



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The anti-fungal and anti-bacterial effects of Psidium guajava on fungi Microsporum gypseum, Trichophyton mentagrophytes, bacteria Staphylococcus aureus, and Staphylococcus epidermidis.

Joy Yamada

Advisor: Dr. Douglas Oba

December 13, 2002


ABSTRACT:

Psidium guajava is known to be a herbal remedy and is used in many countries to treat gastrointestinal and skin disorders. The anti-fungal and anti-bacterial properties of P. guajava were tested on two fungi and two bacteria. The bacteria were tested using a modified Kirby-Bauer method and the fungi were tested in broth cultures. Psidium guajava exhibited anti-fungal and anti-bacterial properties against fungi M. gypseum, T. mentagrophytes, & bacteria S. aureus, and S. epidermidis.
INTRODUCTION:

Psidium guajava commonly known, as Guava, is a member of the Myrtaceae family. It is a fruit found in tropical climates throughout the world. Although it is mainly used for food, the guava plant can be utilized medicinally. The leaves and bark of the guava plant have been used to treat diarrhea, other gastrointestinal disorders, toothaches, colds, and swelling, in areas such as Tonga (Weiner 1971), India (Dutta et al. 2000), Africa (Rabe & van Staden 1997, Tona et al. 1998,2000; Lin et al. 2002), Hawaii (Nagata 1971), Malaysia & Panama (Lutterodt 1989). In Tahiti (Weiner 1971), Guatemala (Caceres et al. 1990, 1993), the Philippines (Weiner 1971), Northeast India (Dutta et al. 2000), and West Bengal (Gupta & Banerjee 1972), guava is used for skin disorders such as an astringent for acne, rashes, and ringworm. The Southeast Nicaraguan indigenous communities of Cuna and Waunana, make a tea from the leaves and bark of the guava for treatment of diarrhea and dysentery, while the Sumu, Panamahka, Twahka, Ulwa, and Bawihka tribes use it for upset stomachs, vertigo, and to regulate menstrual cycles (Coe & Anderson 1999).

Many experiments have examined the antimicrobial properties of P. guajava. Lin et al. (2002) tested P. guajava extracts for anti-microbial activities against different species of diarrheagenic Escherichia coli, Salmonella, and Shigella. Psidium guajava showed inhibitory activities against two species of Salmonella, Shigella flexneri, Shigella virchow, and Shigella dysenteriae, and two varieties of enteropathgenic E. coli. Guava sprouts (young leaves of P. guajava) have been used in Brazilian medicine for gastrointestinal disorders, the microbicidal activity of P. guajava showed inhibition on E. coli and S. aureus, Salmonella typhi, Shigella flexneri and Shigella dysenteriae (Caceres et al. 1990, 1993).

Dutta et al. (2000) tested P. guajava against dermatophytes Trichophtyon tonsurans, Trichophyton rubum, and Microsporum fulvum. Almost all dermatophytes showed no growth with the exception of two; Trichosporon beigelli and Candida albicans. Extracts from both the bark and the leaves were used, although the extracts from the bark were more efficient in inhibiting the dermatophytes than the leaves. The leaves and bark also act as an antidiarrhoeic (Lutterodt 1989, 1992; Tona et al. 1998, 2000).

Jaiarj et al. (1999) tested the anticough and antimicrobial activities of P. guajava leaf extracts. The guava juice showed some positive effects on reducing coughs and the leaves demonstrated some antimicrobial activity on S. aureus and beta-streptococcus groups. Gnan and Demello (1999) also performed experiments on the inhibition of S. aureus by aqueous Gioba (P. guajava) extracts. The Gioba leaf and fruit extracts showed antimicrobial activity against the nine different strains of S. aureus.

Based on the experimental results reported in the literature, the P. guajava has demonstrated considerable anti-fungal and anti-bacterial effects. The purpose of this experiment was to determine the effects of P. guajava on organisms responsible for skin disorders, specifically the fungi Microsporum gypseum, Trichophyton mentagrophytes, & bacteria S. aureus, and Staphylococcus epidermidis.
MATERIALS AND METHODS:

Materials tested in this study were the leaves and bark of the P. guajava plant (Waimea Arboretum 74 E606). Aqueous solutions were obtained by grinding the leaves and the bark. After grinding, 100ml of ethyl alcohol was added. Two solutions were prepared using traditional methods. One solution was left out for two days at room temperature and then placed in the refrigerator. The other solution was immediately placed in the refrigerator. Both solutions were agitated twice daily for five minutes, after seven days both solutions were strained through sterile cheesecloth, and placed back inside of the refrigerator.

Mueller-Hinton agar was used to grow the bacteria S. aureus and S. epidermidis. Sabouraud Dextrose broth was used to grow the fungi Trichophyton mentagrophytes and Microsporum gypseum. A modified Kirby-Bauer method was performed to test the effectiveness of the guava extract on the bacteria S. aureus, and S. epidermis. Twenty-five micro-liters (µl) of each solution was added to sterile disks and placed on the Mueller-Hinton agar of each bacteria. A negative control blank disk, a positive Tetracycline control disk, and an ethanol control disk were also placed on the agar of each dish and were incubated for 24 hours. After incubation, the diameters of the inhibition zones were measured in millimeters. Each test was conducted with 15 replicates. An ANOVA Tukey’s test was performed.

Twenty- four tubes of Sabouraud Dextrose broth containing extract (1:400 v/v) were inoculated, twelve tubes of fungi M. gypseum and twelve tubes of T. mentagrophytes. As a negative control, a tube was inoculated with fungi only and as a positive control tube two drops of a Tolnaftate topical 1% anti-fungal solution was placed in the inoculated broth. The tubes were incubated in a shaking water bath for two days. After incubation, the tubes were observed for fungi growth, and a qualitative reading with the degree of growth represented from four plus (+) to one plus (+). Three replicates were performed for each test group.



RESULTS:

Staphylococcus aureus and S. epidermidis, and fungi M. gypseum and T. mentagrophyte were inhibited by P. guajava aqueous extracts. Staphylococcus aureus (Table 1) and S. epidermidis (Table 2) P. guajava solutions were effective in inhibiting bacterial growth. Neither extract was as effective as the positive control (Tetracycline), but both were more effective then the ethyl alcohol control and the negative control (blank disc). This confirms that the P. guajava extract was the active compound inhibiting the growth and not the solvent.

Table 1. Staphylococcus aureus growth inhibition diameter in millimeters (mm).




Average

St. Dev

B- Blank disc (negative control)

0

0

T- Tetracycline (positive control)

35.4

1.92

S1- Solution 1 (25l)

20.5

1.85

S2- Solution 2 (25l)

20.2

2.43

E- Ethyl alcohol control

4

3.61



Table 2. Staphylococcus epidermidis growth inhibition diameter in millimeters (mm).




Average

St. Dev

B- Blank disc (negative control)

0

0

T- Tetracycline (positive control)

38.4

2.50

S1- Solution 1 (25l)

24.9

3.07

S2- Solution 2 (25l)

24.6

2.56

E- Ethyl alcohol control

2.2

3.84

ANOVA Tukey’s test indicated a significant (p < 0.05) difference between the average of both controls and the two treatments for S. aureus and S. epidermidis (Figures 1 and 2).

There was no significant difference between the zones of inhibition for the two different preparations of P. guajava solutions, for both S. aureus and S. epidermidis.

Figure 1. The inhibition of Staphylococcus aureus.



Figure 2. The inhibition of Staphylococcus epidermidis.


Due to the rhizoid-type growth which remained clumped in broth culture, the growth of fungi M. gypseum (Table 4) and T. mentagrophyte (Table 5) could not be measured quantitatively, however, in the presence of the commercial anti-fungal solution and the two P. guajava solutions fungi growth was prevented.

Table 4. Microsporum gypseum growth results for each test group. Positive growth ranging from 4+ to 1+.

M.gypseum

M. gypseum w/ antifungal

M. gypseum w/ solution 1

M. gypseum w/ solution 2

Tube 1

++++

Tube 1

-

Tube 1

-

Tube 1

-

Tube 2

++

Tube 2

-

Tube 2

-

Tube 2

-

Tube 3

+++

Tube 3

-

Tube 3

-

Tube 3

-




Table 5. Trichophyton mentagrophytes growth results for each test group. Positive growth ranging from 4+ to 1+.

T. mentagrophyte

T. mentagrophyte w/ antifungal

T. mentagrophyte w/ solution 1

T. mentagrophyte w/ solution 2

Tube 1

++++

Tube 1

-

Tube 1

-

Tube 1

-

Tube 2

+++

Tube 2

-

Tube 2

-

Tube 2

-

Tube 3

++

Tube 3

-

Tube 3

-

Tube 3

-


DISCUSSION AND CONCLUSION:

In comparing the Tetracycline positive control to both solutions, Tetracycline had a significantly (p <0.05) stronger inhibition effect than both solutions. This could be due to the fact that the Tetracycline is a pure chemical while the P. guajava solutions were crude extracts.

Both P. guajava solutions were effective against inhibiting the growth of bacteria S. aureus and S. epidermidis, and fungi M. gypseum and T. mentagrophytes. This supports the reported use of P. guajava in many countries as a traditional herbal medicine.

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