Effect Of Topical Pomegranate Peel Extract on Methicillin Resistant Staphylococcus aureus Bacteria on Second Degree Burn Wound in Rat Strain Wistar

R. W. Prasanti.*, I.Dososaputro, L. Zarasade
Department of Plastic Reconstructive and Aesthetic Surgery
School of Medicine Airlangga University / Hospital Dr. Soetomo

Abstract

Introduction: Pomegranate (Punica granatum) has different chemical content associated with antibacterial properties, one of them is ellagic acid ellagitannin. The antibacterial properties of pomegranate have been studied in many bacteria, such asmethicillin resistant Staphylococcus aureus (MRSA). MRSA is a bacterial pathogen that plays an important role as a nosocomial pathogen and causes outbreaks of nosocomial infections which is become serious problems in the management of burns patients because many strains has changed become multiresistant to several classes of antibiotics. Treatment of MRSA bacterial infection in burns is still a problem because the cost are too high and there is an increasing trend of resistance, so we need to find alternatives to deal with this problem, one of them by using pomegranate peel extract topically.This study aimed to compare the topical administration of pomegranate peel extract with mupirocin and see the effects of combined administration of pomegranate peel extract with mupirocin against bacterial colonization of MRSA on the burn wound rat skin.

Materials and methods: Isolates of MRSA bacteria inoculated on burn wound in male rats strain Wistar. They were divided into three groups, each group was given treatment six hours after bacterial inoculation. The first group was given mupirocin in its burn wound, the second group is given pomegranate peel extract topically, and the third group received a combination mupirocin with pomegranate peel extract topically. Swab culture were taken on the third and fifth day post-treatment. The specimen was sent to the Microbiology Laboratory for microbiological examination to evaluate the efficacy of pomegranate peel extract in inhibiting bacterial colonization of MRSA burn wound.

Results:Topical pomegranate fruit peel extract is significantly inhibit MRSA bacterial colonization in rat burn wound{p sig.(2-tailed),0,045, P < 0,05). There is no significant result in bacterial count if topical pomegranate fruit peel extract administationis compared with topical mupirocin administration only or combination between topical pomegranate and mupirocin.

Discussion:Topical pomegranate fruit peel extract has antibacterial effect, but it is not more effective than mupirocin administration only or combination between pomegranate and mupirocin.

Keywords: pomegranate, antibacterial, methicillin resistant Staphylococcus aureus, mupirocin

INTRODUCTION

Thermal burns damages skin barrier which is normally prevents invasion by microorganisms.1 Burn patients vulnerable to infection due to loss of the protective barrier and decreased cellular and humoral immunity. It has been estimated that infections in burn patients are responsible for 70% and 30% mortality attributed to other causes(Naqvi, 2007)..

Methicillinresistant Staphylococcus aureus (MRSA) is an important pathogen among gram-positive coccus. The percentage of patients with MRSA infections has been shown increased dramatically in microbiology examination. Hospital’s burn unit become the main resevoir for MRSA which have unique characteristics that is rapidly spreading in the hospital environment. Therefore, it is considered asa pathogenic organism and causes outbreaks of nosocomial infections that causes serious problems in the patient burn management because many strains become multiresistant to several classes of antibiotics(Naqvi, 2007).

Indiscriminate use of antibiotics has led to an increase in antibiotic resistance to microorganisms, thus require the development of new antimicrobials. In recent years, there is increasing of many interest in exploring natural chemicals for antimicrobial materials. Pomegranate, which has antibacterial properties, as antibiotics are cheap, no side effects and has the advantage against antibiotic-resistant pathogens(Parseh H, 2012).In vitro studies demonstrates bactericidal activity against some highly pathogenic organisms and sometimes resistant to antibiotics. In vitro study showed that the effect of synergism between antibiotics tested with pomegranate extract against S. aureus. Based on initial research and the results of this study, ellagitanin punicalagin compound is thought to be responsible for the antibacterial effect(Jurenka J, 2008).

In vitro studies on this research will be a reference in conducting research to compare the pomegranate peel extract with mupirocin and evaluate the effects of administration of pomegranate peel extract combined with mupirocin against bacterial colonization of MRSA on burn wound rat skin.

MATERIALS AND METHODS

Method for making a topical extract

Materials used for this experimental study was pomegranate peel extract powder which was standarized to contains 40% ellagic acid. Pomegranate peel extract was produced by Xi’an Biof Bio-Technology Co., Ltd. (Room 1-1111, High-tech Venture Park, No. 69 Jinye Road, Gaoxin Distric of Xi’an, People Republic of China). Powder that has been separated from its can was transferred to round bottom flask and suspended with distilled water and a suspending agent sodium carboxy methyl cellulose (CMC) 0.5%. Suspension agent was made by boiling 0.5% sodium CMC to 100 ml of distilled water and stirred with magnetic stirrer until dissolved.

Animals and experimental protocol

This prospective experimental study was conducted on 27 male rats strain Wistar aged 3 months and performed at the Biochemistry Laboratory Airlangga University. Before it wasconducted, this research will be submitted to Ethical Committee for approval. Each rat was maintained in different cages in the same room, and was fed the same amount and same type food. Premedication (atrophine sulphat 0,04 mg/body weight intramuscular) was given, 10 minutes after that it was combined with ketamine 20 mg/body weight and xylazine 2% (10 mg/body weight) intramuscular. After the rats were anesthetized, each rat was shaved fur on the belly, creates one square shape design with length 2 cm and width 2 cm then second degree burn was made in the placing a nail that have been boiled in boiling water for 10 seconds. Each wound wasinoculated MRSA bacteria and covered with a transparent dressing to prevent contamination from surrounding area.

After 6 hours, the wound wasswab cultured with Levine technique as a control. The rats were randomized and divided into 3 groups. The first groupwas given mupirocin topically, the second group was given pomegranate peel extract topically, and the third group was given combination of pomegranate peel extract with mupirocin. On day 3 and day 5 post-treatment, swab cultures wastaken over. The specimens will be sent to Microbiology Laboratory Airlangga University and the results of each culture swab expressed in terms of the number of colonies of bacteria (Colony Forming Unit, CFU).

Data Processing and Analysis

The collected data were analyzed using the ANOVA statistical analysis method.

RESULT

MRSA colony count description in all group :

Group / Day 0 / Day 3 / Day 5
Mupirocin / 92,6 ± 116,6 / 6,3 ± 14,8 / 14,6 ± 26,9
Mupirocin + Pome peel * / 23,1 ± 43,8 / 88,2 ± 139,6 / 39,8 ± 81,7
Pome peel * / 45,2 ± 80,9 / 0 / 0

* : Pseudomonas aeruginosa contamination has been found

Clinical Appareance :

  1. Mupirocin administration group : In wound observation day 3 after mupirocin administration, the wound has been seen wet and slough became increasing in day 5.

Day 0 Day 3 Day 5

  1. Pomegranate peel extract administation group : In wound observation day 3, the wound has been seen wet. Eritrematous in wound edge and slough became increasing in day 5. Pseudomonas aeruginosa contamination has been found in microscopically examination.

Day 0 Day 3 Day 5

  1. Combination between pomegranate peel extract and mupirocin administration group : In wound observation day 3, the wound has been seen wet. Eritrematous in wound edge and slough became increasing in day 5. Pseudomonas aeruginosa contamination has been found in microscopically examination.

Day 0 Day 3 Day 5

Statistical Analysis :

  1. MRSA bacterial count changes in mupirocin group

There was decreasing bacterial colony count in day 3 if it was compared with day 0, but it was not statistically significant ( p > 0,05), also in day 5 if it was compared with day 3. Meanwhile bacterial colony count decreasing in day 5 was statistically significant (p < 0,05) if it was compared with day 0.

Observation day comparison / MRSA colony count difference / p value
Day 0 and 3 / -86,3 / 0,062
Day 0 and 5 / -78,1 / 0,048 *
Day 3 and 5 / 8,2 / 0,471

Note :

* : p < 0,05 means significant

  1. MRSA bacterial count changes in pomegranate peel extract

There was decreasing in bacterial colony count changesin day 3 if it was compared with day 0, and it was stabilize in day 5. This decreasing was statistically significant (p < 0,05).

Observation day comparison / MRSA colony count difference / p value
Day 0 and 3 / -45,2 / 0,008 *
Day 0 and 5 / -45,2 / 0,008 *
Day 3 and 5 / - / -

Note :

* : p < 0,05 means significant

  1. MRSA bacterial count changes in combination group

Bacterial colony count decreasing has been seen in day 5, but it was not statisticallt significant (p > 0,05).

Observation day comparison / MRSA colony count difference / p value
Day 0 and 3 / 65,1 / 0,250
Day 0 and 5 / 16,7 / 0,735
Day 3 and 5 / -48,4 / 0,109

Note :

* : p < 0,05 means significant

  1. MRSA bacterial count change between all group

In day 0, bacterial count change between all group were not statistically significant, also in day 3 and day 5 (p > 0,05).

Day observation / Mupirocin / Combination / Pome extract / p value
Day 0 / 92,6±116,6 / 23,1±43,8 / 45,2±80,9 / 0,233
Day 3 / 0 (0 - 44) / 0 (0 - 341 ) / 0 (0 - 0) / 0,162
Day 5 / 0 (0 - 80) / 0 (0 - 221 ) / 0 (0 - 0) / 0,221

REVIEW

In pomegranate peel extract administation group, MRSA bacterial count decrease (p = 0,008). It is support the evidence that pomegranate peel can inhibit MRSA bacterial growth (Jurenka, 2008). Pomegranate polyphenol (ellagic acid & punicalagin) is thought to be responsible for the antimicrobial effect (Jurenka, 2008). Antibacterial mechanism is probably due to effect on bacterial cell wall, enzim inhibition by oxydant agent, interaction with protein and disturbs microorganisme co-agregation (Abdollahzadeh, 2011)

In combination administration group, MRSA bacterial count doesn’t decrease significantly. Combination pomegranate peel extract with mupirocin administration has not been proven able to decrease MRSA bacterial count. Fluctuation pattern in bacterial colony count is probably due to antagonism between mupirocin and pomegranate peel extract, so that the administration did not reduce MRSA bacterial colonization.

There is no significant difference in MRSA bacterial count reduction between all group (p > 0,05). Pomegranate peel extract onlyadministration or combination with mupirocin has not been proven able to decrease MRSA bacterial count greater than mupirocin administration

In wound observation, slough & eritrema was found in all treatment groups and it increases over time, especially in group with pomegranate fruit peel extract administration. It may be due to Pseudomonas aeruginosa contamination. Pomegranate peel extract more sensitive to Gram-positive bacteria compared to gram-negative bacteria. Sensitivity is quite high in bacteria Staphylococcus aureus. Sensitivity to the bacteria Pseudomonas aeruginosa lacking when compared to other gram-negative bacteria. (Dahham, 2010)

CONCLUSION

  1. Pomegranate peel extract administration topically in rat burn woundcan inhibit bacterial colonization of MRSA
  2. Pomegranate peel extract topically in rat burn wound is not more effective than mupirocin in inhibiting MRSA bacterial colonization
  3. Combinationbetween pomegranate peel extract and mupirocin administration in rat burn wound is not more effective than mupirocin or pomegranate extract only administration in inhibiting MRSA bacterial colonization

SUGGESTIONS

  1. Further studies related to the treatment of pomegranate peel extract against various bacteria that cause burn infections in vivo
  2. Further studies related to pomegranate peel extract therapy effectiveness and safety, both in terms of dose, composition and form of packaging, as a topical cure for MRSA burn wound
  3. Further studies to research interaction between pomegranate peel extract and mupirocin

REFERENCES

  1. Abdollahzadeh, S, Mashouf, RY, Mortazavi, H, Moghaddam, MH, Roozbahani , N, Vahedi, M, 2011, ‘Antibacterial and Antifungal Activities of Punica Granatum Peel Extracts Against Oral Pathogens’, Journal of Dentistry, Vol. 8, No. 1. Pp 1-5
  2. Dahham, SS, et all, 2010, ‘Studies on Antibacterial and Antifungal Activity of Pemgranate (Punica granatum L.)’, Merican-Eurasian J Agriculture & Environment Science, Vol 9 (3), pp. 273-281
  3. Jurenka, J, 2008, ‘Therapeutic Applications Of Pomegranate (Punica Granatum L.): A Review’, Alternative Medicine Review, Volume 13, Number 2, Pp 128-141
  4. Naqvi, ZA; Hashmi K; Kharal, SA, 2007, ‘Methicillin Resistant Staphylococcus Aureus. (MRSA) In Burn Patients’, Pakistan Journal Of Pharmacology, Vol.24, No.2, July 2007, Pp.7-11.
  5. Parseh, H et al, 2012, Antimicrobial properties of Pomegranate (Punica granatum L.) as a Tannin rich Fruit: a review. The 1th International and The 4th National Congress on Recycling of Organic Waste in Agriculture. 26 – 27 April 2012 in Isfahan, Iran.