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Yara Örneklerinden İzole Edilen Metisilin’e Dirençli Staphylococcus Aureus Suşlarında Virulans Genlerinin ve Klonal İlişkilerinin Araştırılması

Year 2020, Volume: 3 Issue: 3, 137 - 144, 01.11.2020

Abstract

Amaç: Staphylococcus aureus insanlarda deri ve yara enfeksiyonlarına neden olan en önemli patojen bakterilerdendir. Metisiline dirençli S. aureus (MRSA) suşlarının yaralarda varlığı hastaların tedavisini zorlaştırmaktadır. Çalışmamızda yara sürüntülerinden izole edilen ve MRSA olarak tanımlanan suşların antibiyotik duyarlılıkları, virulans gen içerikleri ve izolatlar arasındaki genetiksel ilişki araştırılmıştır.
Gereç ve Yöntem: Yara örneklerinden izole edilen suşlar biyokimyasal ve moleküler yöntemlerle tanımlanmıştır. Suşların 12 antibiyotiğe karşı duyarlılıkları agar disk difüzyon yöntemi uygulanarak, indüklenebilir klindamisin direnci D-testi ile ve virulans gen içerikleri PZR ile belirlenmiştir. Suşlar arasındaki genetiksel ilişkinin belirlenmesinde PFGE analizi uygulanmış, SmaI enzimi ile elde edilen band profillerinden dendrogram oluşturularak filogenetik analiz yapılmıştır.
Bulgular: Çalışmada 18 MRSA suşu tanımlanmış ve tümünün penisilin, tetrasiklin, rifampin ve gentamisin'e karşı dirençli oldukları bulunmuştur. Buna karşın tüm suşların linezolid, trimetoprim-sülfametoksazol, kloramfenikol ve quinupristin/dalfopristin'e karşı ise duyarlı oldukları tespit edilmiştir. Ayrıca 4 MRSA suşu indüklenebilir klindamisin direnci göstermiştir. PZR çalışmalarında tüm suşların ermA ve spc-ermA genlerini taşıdığı bulunmuştur. Diğer yandan tetK, tetL, ermB, dfrK, vgaC, ermT, msrA ve msrB genleri suşlarda tespit edilememiştir. ileS, mrm ve spc genleri suşların %94’ünde (17/18) ve ermC geni ise suşların %17’sinde (3/18) pozitif bulunmuştur. Sadece MRSA 50B suşu PVL geni taşıdığı tespit edilmiştir. PFGE analizinde izole edilen MRSA suşları arasında %100 ila %69.4 arasında genetiksel benzerlik olduğu filogenetik analiz ile ortaya konmuştur.
Sonuç: Yara sürüntülerinden izole edilen MRSA suşlarının farklı kökenlerden geldiği ve farklı antibiyotik direnci ve gen içeriklerine sahip olduğu tespit edilmiş ve yara enfeksiyonlarının tedavisinde suşların virulans özelliklerinin de değerlendirilmesinin son derece önemli olduğu sonucuna varılmıştır.

References

  • 1. Tong SYC, Davis JS, Eichenberger E, Holland TL, Fowler VG, Jr. Staphylococcus aureus infections: Epidemiology, pathophysiology, clinical manifestations, and management. Clinical Microbiology Reviews 2015;28(3):603-661.
  • 2. Kourtis AP ve ark. Vital Signs: Epidemiology and Recent Trends in Methicillin-Resistant and in Methicillin-Susceptible Staphylococcus aureus Bloodstream Infections-United States. Morbidity and Mortality Weekly Report 2019;68(9):214-219.
  • 3. Labandeira-Rey M, Couzon F, Boisset S,Brown EL, Bes M, Benito Y, Barbu EM, Vazquez V, Höök M, Etienne J, Vandenesch F, Bowden MG. Staphylococcus aureus Panton-Valentine leukocidin causes necrotizing pneumonia. Science 2007;315:1130-1133.
  • 4. Otter JA, French GL. Community-associated methicillin-resistant Staphylococcus aureus: The case for a genotypic definition. Journal of Hospital Infection 2012;81:143-148.
  • 5. Reducing Healthcare Associated Infection in Hospitals in England. 12 June 2009. National Audit Office. https://www.nao.org.uk/report/reducing-healthcare-associated-infections-in-hospitals-in-england/
  • 6. Palavecino EL. Clinical, Epidemiologic, and Laboratory Aspects of Methicillin-Resistant Staphylococcus aureus Infections. Yinduo Ji (ed.), Methicillin-Resistant Staphylococcus aureus (MRSA) Protocols, Methods in Molecular Biology 2014; 1085:1-24.
  • 7. Sudagidan M, Çavuşoğlu C, Bacakoğlu F. Investigation of the virulence genes in methicillin-resistant Staphylococcus aureus strains isolated from biomaterial surfaces. Mikrobiyoloji Bülteni 2008;42:29-39.
  • 8. Sudagidan M, Aydin A. Screening virulence properties of staphylococci isolated from meat and meat products. Veterinay Medicine Austria/Wiener Tierärztliche Monatsschrift 2009;96:128-134.
  • 9. Hookey JV, Richardson JF, Cookson BD. Molecular typing of Staphylococcus aureus based on PCR restriction fragment length polymorphism and DNA sequence analysis of the coagulase gene. Journal of Clinical Microbiology 1998;36:1083-1089.
  • 10. Aires-de-Sousa M, Boye K, de Lencastre H, Deplano A, Enright MC, Etienne J, Friedrich A, Harmsen D, Holmes A, Huijsdens XW, Kearns AM, Mellmann A, Meugnier H, Rasheed JK, Spalburg E, Strommenger B, Struelens MJ, Tenover FC, Thomas J, Vogel U, Westh H, Xu J, Witte W. High interlaboratory reproducibility of DNA sequence-based typing of bacteria in a multicenter study. Journal of Clinical Microbiology 2006;44:619-621.
  • 11. Clinical and Laboratory Standards Institute (CLSI), Performance Standards for Antimicrobial Susceptibility Testing 2020; 30th International Ed. document M100, Pennsylvania, USA.
  • 12. Levin TP, Suh B, Axelrod P, Truant AL, Fekete T. Potential clindamycin resistance in clindamycin-susceptible, erythromycin-resistant Staphylococcus aureus: Report of a clinical failure. Antimicrobial Agents and Chemotherapy 2005;49(3):1222-1224.
  • 13. Lem P, Spiegelman J, Toye B, Ramotar K. Direct detection of mecA, nuc and 16S rRNA genes in BacT/Alert blood culture bottles. Diagnostic Microbiology and Infectious Disease 2001;41(3):165-168.
  • 14. Lina G, Piemont Y, Godail-Gamot F, Bes M, Peter M-O, Gauduchon V, Vandenesch F, Etienne J. Involvement of Panton-Valentine leukocidin-producing Staphylococcus aureus in primary skin infections and pneumonia. Clinical Infectious Diseases 1999;29:1128-1132.
  • 15. Seah C, Alexander DC, Louie L, Simor A, Low DE, Longtin J, Melano RG. MupB, a New High-Level Mupirocin Resistance Mechanism in Staphylococcus aureus. Antimicrobial Agents and Chemotherapy 2012; 56(4): 1916-1920.
  • 16. Lina G, Quaglia A, Reverdy M-E, Leclercq R, Vandenesch F, Etienne J. Distribution of genes encoding resistance to macrolides, lincosamides and streptogramins among staphylococci. Antimicrobial Agents and Chemotherapy 1999; 43(5):1062-1066.
  • 17. Weigel LM, Donlan RM, Shin DH, Jensen B, Clark NC, McDougal LK, Zhu W, Musser KA, Thompson J, Kohlerschmidt D, Dumas N, Limberger RJ, Patel JB. High-Level Vancomycin-Resistant Staphylococcus aureus Isolates Associated with a Polymicrobial Biofilm. Antimicrobial Agents and Chemotherapy 2007;51(1):231-238.
  • 18. Feßler A, Scott C, Kadlec K, Ehricht R, Monecke S, Schwarz S. Characterization of methicillin-resistant Staphylococcus aureus ST398 from cases of bovine mastitis. Journal of Antimicrobial Chemotherapy 2010;65:619-625.
  • 19. Sudagidan M, Aydin A. Virulence properties of methicillin-susceptible S. aureus food isolates encoding Panton-Valentine Leukocidin gene. International Journal of Food Microbiology 2010;138:287-291.
  • 20. Durmaz R, Otlu B, Çalışkan A, Gürsoy N. Acinetobacter baumannii, Escherichia coli ve Klebsiella türlerinin moleküler tiplendirmesinde kullanılabilecek kısa süreli “pulsed-field gel” elektroforez (PFGE) protokolü. ANKEM Dergisi 2007;21:113-117.
  • 21. Pfalzgraff A, Brandenburg K, Weindl G. Antimicrobial peptides and their therapeutic potential for bacterial skin infections and wounds. Frontiers in Pharmacology 2018;9:281.
  • 22. Thangamani S, Nepal M, Chmielewski J, Seleem MN. Antibacterial activity and therapeutic efficacy of Fl-P(R)P(R)P(L)-5, a cationic amphiphilic polyproline helix, in a mouse model of staphylococcal skin infection. Drug Design, Development and Therapy 2015;9:5749-5754.
  • 23. Guillamet CV, Kollef MH. How to stratify patients at risk for resistant bugs in skin and soft tissue infections? Current Opinion in Infectious Diseases 2016;29:116-123.
  • 24. Russotto V, Cortegiani AC, Fasciana T, Iozzo P, Raineri SM, Gregoretti C, Giammanco A, Giarratano A. What healthcare workers should know about environmental bacterial contamination in the intensive care unit. Biomed Research International 2017;6905450.
  • 25. Long T ve ark. Methicillin-Resistant Staphylococcus aureus Skin Infections Among Tattoo Recipients-Ohio, Kentucky, and Vermont, 2004-2005. Morbidity and Mortality Weekly Report (MMWR) 2006;55(24):677-679.
  • 26. Udobi CE, Obajuluwa AF, Onaolapo JA. Prevalence and antibiotic resistance pattern of methicillin-resistant Staphylococcus aureus from an orthopaedic hospital in Nigeria. BioMed Research International 2013;860467.
  • 27. Lai PS, Bebell LM, Meney C, Valeri L, White MC. Epidemiology of antibiotic-resistant wound infections from six countries in Africa. BMJ Global Health 2018;2:e000475.
  • 28. Acquisto NM, Bodkin RP, Brown JE, Graman PS, Jones CMC, Li T, Hardy DJ, Ashley ED. MRSA nares swab is a more accurate predictor of MRSA wound infection compared with clinical risk factors in emergency department patients with skin and soft tissue infections. Emergency Medicine Journal 2018;35(6):357-360.
  • 29. Víquez-Molina G, Aragón-Sánchez J, Pérez-Corrales C, Murillo-Vargas C, López-Valverde ME, Lipsky BA. Virulence factor genes in Staphylococcus aureus isolated from diabetic foot soft tissue and bone infections. The International Journal of Lower Extremity Wounds 2018;17(1):36-41.
  • 30. Pardos de la Gandara M, Raygoza Garay JA, Mwangi M, Tobin JN, Tsang A, Khalida C, D’Orazio B, Kost RG, Leinberger-Jabari A, Coffran C, Evering TH, Coller BS, Balachandra S, Urban T, Parola C, Salvato S, Jenks N, Wu D, Burgess R, Chung M, de Lencastre H, Tomasz A. Molecular types of methicillin-resistant Staphylococcus aureus and methicillin-sensitive S. aureus strains causing skin and soft tissue infections and nasal colonization, identified in community health centers in New York City. Journal of Clinical Microbiology 2015;53:2648-2658.
  • 31. Yildirim F, Aydin A, Akyazi İ, Sudagidan M, Gurel A. Expression of IL-6, IL-8, IL-10 ve TNF-α in a pneumonia model induced by foodborne Staphylococcus aureus producing Panton-valentine leukocidin toxin. 9. Uluslararası Katılımlı Veteriner Patoloji Kongresi, 25-28 Ekim 2018, sayfa 29-30.

Determination Virulence Genes and Clonal Relationships of Methicillin Resistant Staphylococcus Aureus Strains Isolated from Wound Samples

Year 2020, Volume: 3 Issue: 3, 137 - 144, 01.11.2020

Abstract

Objective: Staphylococcus aureus is one of the most important pathogenic bacteria that cause skin and wound infections in humans. The presence of methicillin-resistant S. aureus (MRSA) strains in wounds makes the treatment of patients difficult. In this study, the antibiotic susceptibilities, virulence gene contents and genetic relationships among MRSA strains isolated from wounds were investigated.
Materials and Methods: The strains isolated from wound samples were identified by biochemical and molecular methods. The susceptibility of the strains against 12 antibiotics was determined using agar disk diffusion method, inducible clindamycin resistance by D-test and virulence gene contents by PCR. PFGE analysis was applied to determine the genetic relationships among the strains, phylogenetic analysis was performed by creating a dendrogram from the band patterns obtained with SmaI enzyme.
Results: The results showed that 18 MRSA strains were identified and all strains were found to be resistant to penicillin, tetracycline, rifampin and gentamicin. On the other hand, all strains were found to be susceptible to linezolid, trimethoprim-sulfamethoxazole, chloramphenicol and quinupristin/dalfopristin. In addition, 4 MRSA strains showed inducible clindamycin resistance. In PCR experiments, it was found that all strains carry ermA and spc-ermA genes. Whereas, tetK, tetL, ermB, dfrK, vgaC, ermT, msrA and msrB genes were not detected. ileS, mrm and spc genes were found positive in 94% (17/18) of the strains and the ermC gene in 17% (3/18) of the strains. Only MRSA 50B strain was found to carry the PVL gene. Phylogenetic analysis revealed that there was 100% to 69.4% genetic similarity between the MRSA strains in the PFGE analysis.
Conclusion: MRSA strains isolated from wound swabs came from different origins and had different antibiotic resistance and gene contents, and it was concluded that it is extremely important to evaluate the virulence properties of the strains in the treatment of wound infections.

References

  • 1. Tong SYC, Davis JS, Eichenberger E, Holland TL, Fowler VG, Jr. Staphylococcus aureus infections: Epidemiology, pathophysiology, clinical manifestations, and management. Clinical Microbiology Reviews 2015;28(3):603-661.
  • 2. Kourtis AP ve ark. Vital Signs: Epidemiology and Recent Trends in Methicillin-Resistant and in Methicillin-Susceptible Staphylococcus aureus Bloodstream Infections-United States. Morbidity and Mortality Weekly Report 2019;68(9):214-219.
  • 3. Labandeira-Rey M, Couzon F, Boisset S,Brown EL, Bes M, Benito Y, Barbu EM, Vazquez V, Höök M, Etienne J, Vandenesch F, Bowden MG. Staphylococcus aureus Panton-Valentine leukocidin causes necrotizing pneumonia. Science 2007;315:1130-1133.
  • 4. Otter JA, French GL. Community-associated methicillin-resistant Staphylococcus aureus: The case for a genotypic definition. Journal of Hospital Infection 2012;81:143-148.
  • 5. Reducing Healthcare Associated Infection in Hospitals in England. 12 June 2009. National Audit Office. https://www.nao.org.uk/report/reducing-healthcare-associated-infections-in-hospitals-in-england/
  • 6. Palavecino EL. Clinical, Epidemiologic, and Laboratory Aspects of Methicillin-Resistant Staphylococcus aureus Infections. Yinduo Ji (ed.), Methicillin-Resistant Staphylococcus aureus (MRSA) Protocols, Methods in Molecular Biology 2014; 1085:1-24.
  • 7. Sudagidan M, Çavuşoğlu C, Bacakoğlu F. Investigation of the virulence genes in methicillin-resistant Staphylococcus aureus strains isolated from biomaterial surfaces. Mikrobiyoloji Bülteni 2008;42:29-39.
  • 8. Sudagidan M, Aydin A. Screening virulence properties of staphylococci isolated from meat and meat products. Veterinay Medicine Austria/Wiener Tierärztliche Monatsschrift 2009;96:128-134.
  • 9. Hookey JV, Richardson JF, Cookson BD. Molecular typing of Staphylococcus aureus based on PCR restriction fragment length polymorphism and DNA sequence analysis of the coagulase gene. Journal of Clinical Microbiology 1998;36:1083-1089.
  • 10. Aires-de-Sousa M, Boye K, de Lencastre H, Deplano A, Enright MC, Etienne J, Friedrich A, Harmsen D, Holmes A, Huijsdens XW, Kearns AM, Mellmann A, Meugnier H, Rasheed JK, Spalburg E, Strommenger B, Struelens MJ, Tenover FC, Thomas J, Vogel U, Westh H, Xu J, Witte W. High interlaboratory reproducibility of DNA sequence-based typing of bacteria in a multicenter study. Journal of Clinical Microbiology 2006;44:619-621.
  • 11. Clinical and Laboratory Standards Institute (CLSI), Performance Standards for Antimicrobial Susceptibility Testing 2020; 30th International Ed. document M100, Pennsylvania, USA.
  • 12. Levin TP, Suh B, Axelrod P, Truant AL, Fekete T. Potential clindamycin resistance in clindamycin-susceptible, erythromycin-resistant Staphylococcus aureus: Report of a clinical failure. Antimicrobial Agents and Chemotherapy 2005;49(3):1222-1224.
  • 13. Lem P, Spiegelman J, Toye B, Ramotar K. Direct detection of mecA, nuc and 16S rRNA genes in BacT/Alert blood culture bottles. Diagnostic Microbiology and Infectious Disease 2001;41(3):165-168.
  • 14. Lina G, Piemont Y, Godail-Gamot F, Bes M, Peter M-O, Gauduchon V, Vandenesch F, Etienne J. Involvement of Panton-Valentine leukocidin-producing Staphylococcus aureus in primary skin infections and pneumonia. Clinical Infectious Diseases 1999;29:1128-1132.
  • 15. Seah C, Alexander DC, Louie L, Simor A, Low DE, Longtin J, Melano RG. MupB, a New High-Level Mupirocin Resistance Mechanism in Staphylococcus aureus. Antimicrobial Agents and Chemotherapy 2012; 56(4): 1916-1920.
  • 16. Lina G, Quaglia A, Reverdy M-E, Leclercq R, Vandenesch F, Etienne J. Distribution of genes encoding resistance to macrolides, lincosamides and streptogramins among staphylococci. Antimicrobial Agents and Chemotherapy 1999; 43(5):1062-1066.
  • 17. Weigel LM, Donlan RM, Shin DH, Jensen B, Clark NC, McDougal LK, Zhu W, Musser KA, Thompson J, Kohlerschmidt D, Dumas N, Limberger RJ, Patel JB. High-Level Vancomycin-Resistant Staphylococcus aureus Isolates Associated with a Polymicrobial Biofilm. Antimicrobial Agents and Chemotherapy 2007;51(1):231-238.
  • 18. Feßler A, Scott C, Kadlec K, Ehricht R, Monecke S, Schwarz S. Characterization of methicillin-resistant Staphylococcus aureus ST398 from cases of bovine mastitis. Journal of Antimicrobial Chemotherapy 2010;65:619-625.
  • 19. Sudagidan M, Aydin A. Virulence properties of methicillin-susceptible S. aureus food isolates encoding Panton-Valentine Leukocidin gene. International Journal of Food Microbiology 2010;138:287-291.
  • 20. Durmaz R, Otlu B, Çalışkan A, Gürsoy N. Acinetobacter baumannii, Escherichia coli ve Klebsiella türlerinin moleküler tiplendirmesinde kullanılabilecek kısa süreli “pulsed-field gel” elektroforez (PFGE) protokolü. ANKEM Dergisi 2007;21:113-117.
  • 21. Pfalzgraff A, Brandenburg K, Weindl G. Antimicrobial peptides and their therapeutic potential for bacterial skin infections and wounds. Frontiers in Pharmacology 2018;9:281.
  • 22. Thangamani S, Nepal M, Chmielewski J, Seleem MN. Antibacterial activity and therapeutic efficacy of Fl-P(R)P(R)P(L)-5, a cationic amphiphilic polyproline helix, in a mouse model of staphylococcal skin infection. Drug Design, Development and Therapy 2015;9:5749-5754.
  • 23. Guillamet CV, Kollef MH. How to stratify patients at risk for resistant bugs in skin and soft tissue infections? Current Opinion in Infectious Diseases 2016;29:116-123.
  • 24. Russotto V, Cortegiani AC, Fasciana T, Iozzo P, Raineri SM, Gregoretti C, Giammanco A, Giarratano A. What healthcare workers should know about environmental bacterial contamination in the intensive care unit. Biomed Research International 2017;6905450.
  • 25. Long T ve ark. Methicillin-Resistant Staphylococcus aureus Skin Infections Among Tattoo Recipients-Ohio, Kentucky, and Vermont, 2004-2005. Morbidity and Mortality Weekly Report (MMWR) 2006;55(24):677-679.
  • 26. Udobi CE, Obajuluwa AF, Onaolapo JA. Prevalence and antibiotic resistance pattern of methicillin-resistant Staphylococcus aureus from an orthopaedic hospital in Nigeria. BioMed Research International 2013;860467.
  • 27. Lai PS, Bebell LM, Meney C, Valeri L, White MC. Epidemiology of antibiotic-resistant wound infections from six countries in Africa. BMJ Global Health 2018;2:e000475.
  • 28. Acquisto NM, Bodkin RP, Brown JE, Graman PS, Jones CMC, Li T, Hardy DJ, Ashley ED. MRSA nares swab is a more accurate predictor of MRSA wound infection compared with clinical risk factors in emergency department patients with skin and soft tissue infections. Emergency Medicine Journal 2018;35(6):357-360.
  • 29. Víquez-Molina G, Aragón-Sánchez J, Pérez-Corrales C, Murillo-Vargas C, López-Valverde ME, Lipsky BA. Virulence factor genes in Staphylococcus aureus isolated from diabetic foot soft tissue and bone infections. The International Journal of Lower Extremity Wounds 2018;17(1):36-41.
  • 30. Pardos de la Gandara M, Raygoza Garay JA, Mwangi M, Tobin JN, Tsang A, Khalida C, D’Orazio B, Kost RG, Leinberger-Jabari A, Coffran C, Evering TH, Coller BS, Balachandra S, Urban T, Parola C, Salvato S, Jenks N, Wu D, Burgess R, Chung M, de Lencastre H, Tomasz A. Molecular types of methicillin-resistant Staphylococcus aureus and methicillin-sensitive S. aureus strains causing skin and soft tissue infections and nasal colonization, identified in community health centers in New York City. Journal of Clinical Microbiology 2015;53:2648-2658.
  • 31. Yildirim F, Aydin A, Akyazi İ, Sudagidan M, Gurel A. Expression of IL-6, IL-8, IL-10 ve TNF-α in a pneumonia model induced by foodborne Staphylococcus aureus producing Panton-valentine leukocidin toxin. 9. Uluslararası Katılımlı Veteriner Patoloji Kongresi, 25-28 Ekim 2018, sayfa 29-30.
There are 31 citations in total.

Details

Primary Language Turkish
Subjects Clinical Sciences
Journal Section Research article
Authors

Mert Sudağıdan 0000-0002-3980-8344

Samet Uçak 0000-0002-3461-2481

Orhan Yavuz 0000-0002-9263-657X

Mediha Nur Zafer Yurt This is me 0000-0002-3064-3811

Behiye Büşra Taşbaşı This is me 0000-0002-2076-3756

Elif Esma Acar This is me 0000-0002-6264-7550

Veli Cengiz Özalp This is me 0000-0002-7659-5990

Ali Aydın 0000-0002-4931-9843

Şöhret Aydemir This is me 0000-0001-8354-9100

Publication Date November 1, 2020
Acceptance Date January 19, 2021
Published in Issue Year 2020 Volume: 3 Issue: 3

Cite

APA Sudağıdan, M., Uçak, S., Yavuz, O., Yurt, M. N. Z., et al. (2020). Yara Örneklerinden İzole Edilen Metisilin’e Dirençli Staphylococcus Aureus Suşlarında Virulans Genlerinin ve Klonal İlişkilerinin Araştırılması. Tıp Fakültesi Klinikleri Dergisi, 3(3), 137-144.


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