Mezenkimal kök hücre uygulamasının MRSA ile enfekte olmuş diyabetik ratlardaki antibakteriyel etkinliği: Deneysel çalışma

Öz

Amaç: Mezenkimal kök hücre (MSC) uygulamasının Metisiline dirençli Staphylococcus aureus (MRSA) kaynaklı yara enfeksiyonu üzerindeki antibakteriyel etkileri, diyabetik rat modelinde, vankomisin, tigesiklin ve daptomisin tedavisi ile karşılaştırıldı.

Yöntemler: 60 rat ile oluşturulan deney ve negatif-pozitif kontrol grupları oluşturuldu. MSC, vankomisin, tigesiklinin ve daptomisinin antibakteriyel etkinlikleri karşılaştırıldı. Ratların tümü streptozotosin ile diyabetik hale getirildi. Açlık kan glukozu takibiyle hiperglisemik oldukları gösterildi. Cerrahi olarak cilt altına keseler oluşturuldu. Grup-0 (negatif kontrol grubu) enfekte ve tedavi edilmedi. Diğer tüm gruplar MRSA ile enfekte edildi. Grup-1 (pozitif kontrol grubu) enfekte edildi fakat tedavi edilmedi. Diğer 4 grup tedavi grupları olarak belirlendi. Grup-MSC; MSC ile, Grup-Van; vankomisin ile, Grup-Tig; tigesiklin ile, Grup-Dap; daptomisin ile tedavi edildi. Bir haftalık tedavi sonrası ötenazi ile numuneler toplandı. Doku örnekleri histopatolojik olarak hematoksilen/eosin ile boyanarak değerlendirildi. Yara bölgesinde MSC varlığı immun floresan boyama ile kanıtlandı. Kantitatif olarak bakteri koloni sayıları tespit edildi. Alınan kan numunelerinden TNF-α, TGF-β, IL-1, PDGF, FGF, VEGF ve Kaspaz-3 seviyeleri ELISA yöntemiyle ölçüldü. 

Bulgular: Grup-0'da bakteri kolonizasyonu gözlenmez iken, Grup-1'de belirgin kolonizasyon saptandı. Grup-Tig ve Grup-Dap'ta tam eradikasyon sağlandı ve Grup-Van’da 1, Grup-MSC’de 4 ratta eradikasyon sağlanamadı. Kontamine edilmeyen negatif kontrol grubunda (Grup-0) minimal inflamasyon düzeyi izlenirken, en şiddetli inflamasyon enfekte edilmiş ve tedavi verilmemiş ratlarda (Grup-1) gözlendi (P<0,001). MSC, Vankomisin, Daptomisin ve Tigesiklin gruplarında orta düzeyde inflamasyon ve ödem gözlendi. MSC grubunda önemli vaskülarite artışı görüldü (P=0,001). Negatif kontrol grubunda ve MSC grubunda benzer oranda anlamlı olarak daha az adezyon artışı ve fibrozis görüldü (P<0,001, P<0,001).

Sonuç: Bu çalışma, diyabetik ratlarda MRSA kaynaklı yara enfeksiyonu tedavisinde MSC’nin antibakteriyel etkinliği sağlayabileceğini düşündürmektedir. Buna ilaveten MSC sayesinde yara bölgesinde sınırlandırılmış bir inflamasyon sağlanabileceği düşünülmüştür. Diyabetik yara enfeksiyonlarının tedavisinde antibiyoterapiyle birlikte MSC kullanımının sinerjistik etkisinin araştırılacağı klinik çalışmalara ihtiyaç vardır.

Anahtar Kelimeler

• Mezenkimal kök hücre,Metisiline dirençli Staphylococcus aureus,Diabetes Mellitus,Vankomisin,Tigesiklin,Daptomisin

Antibacterial efficacy of mesenchymal stem cell administration in diabetic rats infected with MRSA: An experimental study

Abstract

Aim: The antibacterial effects of mesenchymal stem cell (MSC) administration and vancomycin, tigecycline and daptomycin treatment were compared in a diabetic rat model with wound infection due to methicillin-resistant Staphylococcus aureus (MRSA).

Methods: Experiment, negative and positive control groups were created using 60 rats. The antibacterial efficacy of MSC, vancomycin, tigecycline and daptomycin were compared. All rats had diabetes induced with streptozotocin. They were shown to be hyperglycemic with fasting blood glucose monitoring. During surgery, subdermal pouches were created. Group 0 (negative control group) was not infected or treated. All other groups were infected with MRSA. Group 1 (positive control group) was infected but not treated. The other 4 groups were determined as treatment groups: Group MSC was treated with MSC, Group Van treated with vancomycin, Group Tig treated with tigecycline and Group Dap treated with daptomycin. After one week of treatment, samples were collected following euthanasia. Tissue samples were evaluated after histopathologic staining with hematoxylin/eosin. The presence of MSC in the wound region was shown by immunofluorescent staining. Bacterial colony counts were identified quantitatively. TNF-α, TGF-β, IL-1, PDGF, FGF, VEGF and Caspase-3 levels in blood samples were measured with the ELISA method.

Results: While bacterial colonization was not observed in Group 0, a clear colonization was identified in Group 1. Full eradication was achieved in Group Tig and Group Dap. Eradication could not be achieved for 1 rat in Group Van and 4 rats in Group MSC. The uncontaminated negative control group rats (Group 0) had minimal inflammation, while the most severe inflammation was observed in infected and untreated rats (Group 1) (P<0.001). Group-MSC, Group-Van, Group-Dap and Group-Tig had moderate levels of inflammation and edema. The MSC group showed significant increase in vascularity (P=0.001). Adhesion and fibrosis were observed significantly less in the negative control group and MSC groups, similarly (P<0.001, P<0.001).

Conclusion: MSC may exert antibacterial-like effects for MRSA-induced wound infection treatment in diabetic rats, and limit the inflammation in and around the wound. Further clinical studies researching the synergistic effects of MSC with antibiotherapy for treatment of diabetic wound infections are needed.

Keywords

• Mesenchymal stem cell,Vancomycin,Tigecycline,Daptomycin,Methicillin-resistant Staphylococcus aureus,Diabetes Mellitus

References

1. 1. Yang SY, Strong N, Gong X, Heggeness MH. Differentiation of nerve-derived adult pluripotent stem cells into osteoblastic and endothelial cells. Spine J. 2017;17:277-81.
2. 2. İnan S, Ozbilgin K. Kok Hucre Biyolojisi. Sağlıkta Birikim. 2009;1;5:11-23.
3. 3. Alcayaga-Miranda F, Cuenca J, Martin A, Contreras L, Figueroa FE, Khoury M. Combination therapy of menstrual derived mesenchymal stem cells and antibiotics ameliorates survival in sepsis. Stem Cell Res Ther. 2015;6:199.
4. 4. Sutton MT, Fletcher D, Ghosh SK, Weinberg A, Heeckeren R, Kaur S, et al. Antimicrobial properties of mesenchymal stem cells: therapeutic potential for cystic fibrosis infection and treatment. Stem Cells Int. 2016:12.
5. 5. Sayek İ, Ozmen MM, Temel Cerrahi El Kitabı, Guneş Tıp Kitabevleri, 2009.
6. 6. Brunicardi CF, Schwartz’s Principles Of Surgery, Mc Graw-Hill, 2005.
7. 7. Anderson DJ, Kaye KS, Chen LF, et al. Clinical and financial outcomes due to methicillin resistant Staphylococcus aureus surgical site infection: a multi-center matched outcomes study. PloS One. 2009;4:e8305.
8. 8. Akgün S, Sayiner HS. Using multiplex PCR as a diagnostic tool to detect methicillin resistant Staphylococcus aureus. J Surg Med. 2018;2(3):215-17.

9. 9. Gardete S, Tomasz A. Mechanisms of vancomycin resistance in Staphylococcus aureus. J Clin Invest 2014;124:2836.
10. 10. Yasim A, Gul M, Ciralik H, Ergun Y. Gelatin-sealed Dacron graft is not more susceptible to MRSA infection than PTFE graft. Eur J Vasc Endovasc Surg. 2006;32:425-30.
11. 11. Aybar Y, Ozaras R., Besirli K, Engin E, Karabulut E, Salihoglu T, Yilmaz MH. Efficacy of tigecycline and vancomycin in experimental catheter-related Staphylococcus epidermidis infection: microbiological and electron microscopic analysis of biofilm. Int J Antimicrob Agents. 2012;39:338-42.
12. 12. AL-Malki, Abdulrahman L, El Rabey, Haddad A. The antidiabetic effect of low doses of Moringa oleifera Lam. seeds on streptozotocin induced diabetes and diabetic nephropathy in male rats. BioMed Res Int. 2015;2015.
13. 13. Nagaya N, Kangawa K, Itoh T, Iwase T, Murakami S, Miyahara Y, Tokudome T. Transplantation of mesenchymal stem cells improves cardiac function in a rat model of dilated cardiomyopathy. Circulation 2005;112:1128-35.
14. 14. Lodise TP, Lomaestro B, Graves J, Drusano GL. Larger vancomycin doses (at least four grams per day) are associated with an increased incidence of nephrotoxicity. Antimicrobial agents and chemotherapy. 2008;52:1330-6.
15. 15. Tally, FP, DeBruin MF. Development of daptomycin for gram-positive infections. Journal of Antimicrobial Chemotherapy. 2000;46:523-6.
16. 16. Goessens WH, Mouton JW, Marian T, Sörgel F, Kinzig M, Bakker-Woudenberg IA. The therapeutic effect of tygecycline, unlike that of ceftazidime, is not influenced by whether the Klebsiella pneumoniae strain produces extended-spectrum β-lactamases in experimental pneumonia in rats. Antimicrob Agents Chemother. 2013;57:643-6.
17. 17. Hernandez-Richter T, Schardey HM, Wittmann F, Mayr S, Schmitt-Sody M, Blasenbreu S, Angele MK. Rifampin and Triclosan but not silver is effective in preventing bacterial infection of vascular dacron graft material. Eur J Vasc Endovasc Surg. 2003;26:550-7.
18. 18. Martin ET, Kaye KS, Knott C, Nguyen H, Santarossa M, Evans R, Jaber L. Diabetes and risk of surgical site infection: a systematic review and meta-analysis. infection control & hospital epidemiology. 2016;37:88-99.
19. 19. Meisel R, Brockers S, Heseler K, Degistirici O, Bülle H, Woite C, et al. Human but not murine multipotent mesenchymal stromal cells exhibit broad-spectrum antimicrobial effector function mediated by indoleamine 2, 3-dioxygenase. Leukemia. 2011;25:648-54.
20. 20. Guerra AD, Cantu DA, Vecchi JT, Rose WE, Hematti P, Kao WJ. Mesenchymal stromal/stem cell and minocycline-loaded hydrogels inhibit the growth of Staphylococcus aureus that evades immunomodulation of blood-derived leukocytes. AAPS J. 2015;17:620-30.
21. 21. Qian J, Hu Y Zhao L, Xia J, Li C, Shi L, Xu F. Protective Role of Adipose Derived Stem Cells in Staphylococcus aureus Induced Lung Injury is Mediated by RegIIIγ Secretion. Stem Cells. 2016;34:1947-56.
22. 22. Amy M. DiMarino, Arnold I. Caplan, and Tracey L. Bonfield. Mesenchymal Stem Cells in Tissue Repair. Front Immunol. 2013;4:201.
23. 23. Oh JY, Roddy GW, Choi H, Lee RH, Ylostalo JH, Rosa RH, Jr, et al. Anti-inflammatory protein TSG-6 reduces inflammatory damage to the cornea following chemical and mechanical injury. Proc Natl Acad Sci USA. 2010;107:16875–8010.
24. 24. Patel SA, Sherman L, Munoz J, Rameshwar P. Immunological properties of mesenchymal stem cells and clinical implications. Arch Immunol Ther Exp (Warsz) 2008;56:1–8.
25. 25. Matthay MA. Advances and challenges in translating stem cell therapies for clinical diseases. Transl Res. 2010;156:107–11.
26. 26. Kong D, Liu X, Li X, Hu J, Li X, Xiao J, et al. Mesenchymal stem cells significantly improved treatment effects of Linezolid on severe pneumonia in a rabbit model. Biosci Rep. 2019 Sep 4. pii: BSR20182455. doi: 10.1042/BSR20182455.
27. 27. Uccelli A, Rosbo NK. The immunomodulatory function of mesenchymal stem cells: mode of action and pathways. Ann NY Acad Sci. 2015;1351:114-26.
28. 28. Tian Y, Deng YB, Huang YJ, Wang Y. Bone marrow-derived mesenchymal stem cells decrease acute graft-versus-host disease after allogeneic hematopoietic stem cells transplantation. Immunol Invest. 2008;37:29–42.