Antimicrobial Effects of Melittin on Bacterial Skin Infections in Dogs : 개 세균성 피부감염에 대한 멜리틴의 항균 효과

다국어 초록 (Multilingual Abstract)

Bacterial skin infections are major skin diseases caused by Staphylococcus pseudintermedius. The melittin peptide, the main active component of bee venom, has potent antibacterial activities. Therefore, this study was aimed to investigate whether melittin can have antimicrobial activities and treat bacterial skin infections caused by S. pseudintermedius as an alternative to antibiotics.
The agar dilution method was applied to determine the minimum inhibitory concentrations (MIC) of melittin, in accordance with the Clinical and Laboratory Standards Institute (CLSI) guidelines. Bacterial skin infections were induced via topical application of S. pseudintermedius (108 CFU/mL) with a dermaroller in dogs. Distilled water and mupirocin were used as negative and positive control, respectively. Evaluation was performed by gross lesion, cytological, and histopathological examinations.
As a result, The MIC of melittin towards six S. pseudintermedius strains ranged between 12.5 µg/mL and 50 µg/mL, suggesting that melittin has antimicrobial activities.
In the results of bacterial skin infections, gross lesions showed erythematous and noticeable pustule formations. Identified S. pseudintermedius from pustules of all dogs turned out to be the same as the inoculated bacteria. On the cytological examination, lots of inflammatory cells, a few keratinocytes and neutrophils with intracellular cocci were found. On the histopathological examination, marked pustule formations were demonstrated in S. pseudintermedius infected skin tissues with related inflammatory cell – mainly neutrophils, eosinophils and epitheloid macrophages infiltration, edematous changes – loosening and lysis of collagen fibers, and hyperplasia of epithelial cells. These are re-confirmed by histomorphometrical analysis; Significant (P < 0.01) increases of mean epithelial thicknesses, numbers of inflammatory cells infiltrated in the dermis, pustule numbers and diameters were observed in pyoderma skin tissues as compared to those of normal skin tissues with significantly (P < 0.01) decreases of collagen fiber occupied regions on the dermis, suggesting confirmed S. pseudintermedius-induced skin infection in dorsal back skin of beagle dogs.
In the results of melittin efficacy study, topical application of melittin at 100 µg/mL made all papules, pustules, crusts and epidermal collarettes resolve within 14 days after initiation of treatment. Lesions score in the melittin and mupirocin treated groups demonstrated significantly (P < 0.05) reduced compared to the negative control group. Histopathological changes were significantly (P < 0.01) inhibited by treatment of melittin as comparable to those of mupirocin ointment; the mean epithelial thicknesses, numbers of inflammatory cells, pustule numbers and diameters were significantly (P < 0.01) decreased in melittin and mupirocin ointment treated bacterial skin tissues, whereas mean collagen fiber occupied regions were significantly (P < 0.01) increased in melittin and mupirocin ointment treated bacterial skin tissues as compared to those control skin tissues, constantly as comparable to those of mupirocin ointment in melittin.
This study demonstrated that melittin might be used as an alternative to antibiotics for the treatment of bacterial skin infections, particularly by topical application.

국문 초록 (Abstract)

개에서 세균성 피부감염은 Staphylococcus pseudintermedius에 의해 다발하는 주요 피부 질환이다. Melittin peptide는 봉독의 주요 활성 성분으로서 강력한 항균 작용 및 항염 작용을 한다고 알려져 있다. 따라서 본 연구의 목적은 S. pseudintermedius에 대한 melittin의 항균 효과를 확인하고 세균성 피부감염에 대한 치료효과를 알아 보는 것이다.
한천 희석법을 사용하여 Clinical and Laboratory Standards Institute (CLSI) 지침에 따라 S. pseudintermedius에 대한 melittin의 최소 억제 농도 (MIC)를 측정하였다. 비글견의 등쪽 피부에 S. pseudintermedius (108 CFU/mL)를 도포한 후, 더마롤러를 적용하여 세균성 피부감염을 유발하였다. 증류수와 mupirocin을 음성 대조군과 양성 대조군으로 각각 사용하였으며, 육안 병변, 세포 및 조직 검사로 확인하였다.
S. pseudintermedius의 6 균주에 대한 melittin의 MIC는 12.5 ~ 50 μg/mL 범위였다. 세균성 피부감염을 유발한 결과, 육안적으로 발적 및 현저한 농포형성이 확인되었고 세포 검사에서 다수의 염증세포, 소수의 각화세포와 세균 탐식 호중구가 관찰되었다. 조직병리검사에서 호중구, 호산구 및 탐식세포의 침윤, 콜라겐 섬유의 융해와 상피 세포 증식과 같은 부종성 변화가 관찰되었다. 조직형태 분석 결과에서 정상 피부조직에 비해 S. pseudintermedius에 감염된 피부조직에서 진피 내 콜라겐 섬유가 차지하는 영역비율은 유의하게 (P < 0.01) 감소되었으나 평균 상피 두께, 침윤 염증세포 수, 형성 농포 수 및 직경은 유의하게 (P < 0.01) 증가되었다. 치료효과 실험에서 육안적 병변은 melittin치료에 의해 시간이 지남에 따라 농포가 사라지고 일시적으로 각질이 증가하였다. 또한 육안적 병변을 수치화한 결과 음성 대조군에 비해 유의하게 (P < 0.05) 감소되었다. 조직 병리학적 검사에서 세균성 피부감염이 유발된 피부 병변에 melittin적용은 mupirocin 연고와 유사한 평균 상피 두께, 침윤 염증세포 수, 형성 농포 수 및 직경의 유의한 (P < 0.01) 감소와 진피 내 콜라겐 섬유가 차지하는 영역비율의 유의한 (P < 0.01) 증가를 보였다.
따라서 melittin의 S. pseudintermedius에 대한 항균 효과를 확인하였고 개의 세균성 피부감염에 대한 국소 치료제로 적용할 수 있을 것으로 판단된다.

목차 (Table of Contents)

• INTRODUCTION 1
• MATERIALS AND METHODS 5
• Staphylococcus pseudintermedius strains and reagents 5
• Determination of minimum inhibitory concentrations 5
• Efficacy of melittin on bacterial skin infections 6
• Experimental animals 6
• Study design 6
• Staphylococcus pseudintermedius-induced skin infections 7
• Clinical evaluation of lesions 8
• Histopathological process 9
• Histomorphometrical analysis 10
• Statistical analysis 11
• RESULTS 13
• Minimum inhibitory concentrations 13
• Inducement of bacterial skin infections 13
• Gross findings 13
• Cytological findings 13
• Bacterial culture and species identification 14
• Histopathological findings 14
• Histomorphometrical findings 14
• Efficacy of melittin on bacterial skin infections 16
• Gross findings 16
• Clinical evaluation of lesions 16
• Histopathological findings 17
• Histomorphometrical findings 17
• DISCUSSION 44
• REFERENCES 51
• ABSTRACT 59
• ABSTRACT IN KOREAN 62