Exploring treatment strategies for methicillin-resistant Staphylococcus aureus (MRSA): A study on the synergistic effects of antibiotic and drug combination therapy = 메티실린 내성 황색포도상구균 (MRSA) 치료 전략 탐색: 항생제와 약물 병용 요법의 상승효과에 대한 연구

다국어 초록 (Multilingual Abstract)

Staphylococcus aureus is a major Gram-positive pathogen responsible for both community and hospital-acquired infections. It frequently infects human skin and soft tissue and can cause severe diseases such as pneumonia and bacteremia. Gram-positive bacteria are generally susceptible to β-lactam antibiotics because of their thick cell walls composed of peptidoglycan layers that are targeted by cell wall synthesis inhibitors. However, Methicillin-resistant Staphylococcus aureus (MRSA) exhibits resistance not only to methicillin and oxacillin but also to multiple other classes of antibiotics. This multidrug resistance poses a serious public health threat worldwide, complicating treatment strategies and emphasizing the urgent need for novel therapeutic approaches. To explore treatment strategies for MRSA, this study evaluated the synergistic effect of oxacillin, a β-lactam antibiotic, and 5-nitro-2-(3- phenylpropylamino) benzoic acid (NPPB), a chloride channel inhibitor. The synergistic effect of oxacillin and NPPB was confirmed in five MRSA strains (ATCC 33591, NCTC10442, N315, CNU-2601, and CNU-2617) using a checkerboard assay. Co-treatment with oxacillin and NPPB reduced the minimum inhibitory concentration (MIC) of oxacillin in five MRSA strains, demonstrating that NPPB enhances susceptibility of MRSA to oxacillin. In the reference strain MRSA ATCC 33591, the combination treatment was further validated by disc diffusion and time-kill assay. Real-time PCR analysis demonstrated that combined treatment significantly suppressed the expression of efflux pump genes (abcA and norA) compared to oxacillin and NPPB alone. In addition, NPPB markedly reduced oxacillin-induced biofilm formation in the ATCC 33591 strain. Field emission transmission electron microscopy showed severe damage to the bacterial cell wall and membrane in the combination group, ultimately leading to cell lysis. Toxicity evaluation in mice showed no adverse effects on hepatic and renal function after one week of combined administration of NPPB and oxacillin. Collectively, these findings suggest that NPPB enhances the antibacterial efficacy of oxacillin and represents a promising strategy to overcome MRSA resistance.

국문 초록 (Abstract)

황색포도상구균 (Staphylococcus aureus)은 지역사회 및 병원 감염을 유발하는 주요 그람 양성 병원균이다. 사람의 피부와 연조직을 자주 감염시키며 폐렴이나 균혈증과 같은 심각한 질병을 유발할 수 있다. 그람 양성균은 세포벽 합성 억제제의 표적이 되는 펩티도글리칸 층으로 구성된 두꺼운 세포벽을 가지고 있기 때문에 일반적으로 β-락탐 계열 항생제에 취약하다. 그러나 메티실린 내성 황색포도상구균 (Methicillin-resistant staphylococcus aureus)은 메티실린과 옥사실린뿐만 아니라 여러 계열의 항생제에도 내성을 나타낸다. 이러한 다제내성은 전 세계적으로 심각한 공중보건 위협을 초래하여 치료 전략을 복잡하게 만들고 새로운 치료법의 시급한 필요성을 강조한다. MRSA 치료 전략을 모색하기 위해 본 연구에서는 β-락탐 항생제인 옥사실린과 염화물 채널 억제제인 5-니트로-2-(3-페닐프로필아미노) 벤조산 (NPPB)의 상승 효과를 평가했다. 옥사실린과 NPPB의 상승 효과는 체커보드 분석을 통해 다섯 가지 MRSA 균주 (ATCC 33591, NCTC10442, N315, CNU-2601, CNU-2617)에서 확인했다. 옥사실린과 NPPB의 병용 투여는 다섯 가지 MRSA 균주에서 옥사실린의 최소 저해 농도 (Minimum inhibitory concentration)를 감소시켜 NPPB가 옥사실린에 대한 감수성을 증가시킨다는 것을 확인했다. 기준 균주인 MRSA ATCC 33591에서 병용 투여의 효과는 디스크 확산 분석과 시간-살상 분석을 통해 더욱 검증되었다. 실시간 PCR 분석 결과, 옥사실린과 NPPB 병용 투여는 유출 펌프 유전자 (abcA 및 norA)의 발현을 옥사실린과 NPPB 단독 투여에 비해 유의미하게 억제하는 것으로 나타났다. 또한, NPPB는 ATCC 33591 균주에서 옥사실린에 의해 유도된 바이오필름 형성을 현저히 감소시켰다. 전자현미경 (FE-TEM) 검사 결과, 병용 투여군에서 세균 세포벽과 세포막에 심각한 손상이 관찰되었으며, 궁극적으로 세포 용해가 발생했다. 마우스를 대상으로 한 독성 평가 결과, NPPB와 옥사실린 병용 투여 1주일 후 간 및 신장 기능에 대한 부작용은 나타나지 않았다. 이러한 결과는 NPPB가 옥사실린의 항균 효능을 향상시키고 MRSA 내성 극복을 위한 유망한 전략임을 시사한다.

목차 (Table of Contents)

• ABSTRACT 1
• INTRODUCTION 3
• 1. Resistance mechanisms and current therapeutic strategies of Methicillin-resistant
• Staphylococcus aureus (MRSA) 3
• 2. Strategies for repurposing existing drugs to overcome antibiotic resistance 11
• 3. Objective of this study 16
• MATERIALS AND METHODS 20
• 1. Chemicals and reagents 20
• 2. Bacteria strains and culture 20
• 3. Antibacterial susceptibility testing: Checkerboard assay and minimum inhibitory
• concentrations (MIC) 20
• 4. Minimum bactericidal concentration (MBC) assay 21
• 5. Time-kill curve assay 21
• 6. Kirby-Bauer disc diffusion assay 22
• 7. RNA extraction and quantitative real-time PCR (qPCR) 22
• 8. Crystal violet staining assay for biofilm quantification 23
• 9. Morphological characterization of MRSA by Field emission transmission electron
• microscope (FE-TEM) 24
• 10. Safety assessment of the combination treatment with oxacillin and NPPB 24
• 11. Statistical analysis 25
• RESULTS 27
• 1. Differential effects of NPPB and DIDS on methicillin susceptibility in MRSA 27
• 2. Vancomycin MIC is unchanged in the presence of NPPB or DIDS 30
• 3. Bactericidal activity of oxacillin and NPPB against MSSA and MRSA 33
• 4. NPPB enhances oxacillin susceptibility across MRSA strains 37
• 5. NPPB shows no modulatory effect on erythromycin, tetracycline, or kanamycin 41
• 6. Time-dependent antibacterial effect of oxacillin and NPPB combination 44
• 7. NPPB enhances oxacillin activity in disc diffusion assay 47
• 8. Downregulation of resistance genes by oxacillin and NPPB combination 50
• 9. Inhibition of oxacillin enhanced biofilm formation by NPPB 53
• 10. Morphological alterations induced by oxacillin and NPPB combination 56
• 11. Oxacillin and NPPB are non-cytotoxic to mouse splenocytes 59
• 12. Oxacillin and NPPB combination does not affect liver or kidney function 62
• DISCUSSION 65
• REFERENCES 72
• ABSTRACT (in Korean) 83
• ACKNOWLEDGEMENT 85