Seminario biolo molecular


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Seminario biolo molecular

  1. 1. Methicillin-resistant Staphylococcus aureus ST30-SCCmec IVc clone as the major cause of community-acquired invasive infections in Argentina S. Fernandez a, L. de Vedia b, M.J. Lopez Furst c, N. Gardella a, S. Di Gregorio , M.C. Ganaha , S. Prieto f, E. Carbone g, N. Lista , F. Rotrying , M.E. Stryjewski , M. Mollerach . Aura María Vélez Granda Vanessa Morales Ospina III Semester
  2. 2. INTRODUCTION Generalities •Staphylé: “grape”, Coccus: “granule” • Gram positive bacteria. • Diameter of about 0.5-1 um • Includes at least 40 species, 24 subspecies. •Facultative anaerobes. Staphylococcus
  3. 3. INTRODUCTION Generalities • Grown in conditions with high concentrations of NaCl (10%) • Temperature 18-40 ° C. • Pathogens in humans. • The non-sporulating bacteria more resistant to heat, desiccation and salinity. • Has in its wall teichoic acid and is sensitive to the enzyme lysostaphin. Staphylococcus
  4. 4. INTRODUCTION Generalities • Facultative anaerobes bacteria. • Gram positive bacteria. • Production of coagulase, catalase, entero toxin, thermonuclease • Stationary and not sporulated. Staphylococcus aureus
  5. 5. INTRODUCTION • Scalded skin •syndrome • Food poisoning • Toxic Shock Syndrome • Skin infections • Bacteremia • Endocarditis • Pneumonia • Osteomyelitis Diseases caused by S. aureus
  6. 6. INTRODUCTION Community-acquired methicillin-resistant Staphylococcus aureus generally exhibit SCCmec IV or V commonly carry Panton- Valentine leukocidin (PVL) genes Associated with infections involve skin structures, abscess formation, necrotizing pneumonia, severe sepsis, osteomyelitis, meningitis and death shows greater virulence, spreading more rapidly and causing more severe disease
  7. 7. INTRODUCTION hospital associated methicillin-resistant Staphylococcus aureus generally exhibit SCCmec I, II, III Panton-Valentine leukocidin (PVL) genes, rarely identified Is much less sever than CA-MRSA infections that occur in hospitals and health centers for poor health care, washing techniques, contamination, immunosup pression. They may include surgical wound infections, urinary tract infections, bloodstream infections and pneumonia.
  8. 8. INTRODUCTION is a cytotoxin, a β-toxins pore formers. LPV presence is associated with increased virulence of certain strains of Staphylococcus aureus It is present in the majority of CA-MRSA
  9. 9. INTRODUCTION Staphylococcal cassette chromosome mec region of the chromosome of S. aureus, which is a series of genes encoding and regulate the resistance to methicillin
  10. 10. INTRODUCTION mecA gene Encoded protein PBP2a It has a low affinity for β-lactam Involved in resistance various types of SCCmec (I, II, III, IV, V, VI)
  11. 11. INTRODUCTION acts inhibiting the synthesis of the bacterial cell wall lactam antibiotic - penicillin group Prevents the formation of cross-links between the linear peptidoglycan polymer chains are a major component of the cell wall of Gram positive bacteria
  12. 12. INTRODUCTION Acts by binding and competitive inhibition of transpeptidase enzyme used by the bacteria to generate crosslinks (D-alanyl-alanine) used in peptidoglycan synthesis
  13. 13. INTRODUCTION Staphilococcus Staphilococcus aureus CA-MRSA HA-MRSA SCCmec Methicillin
  14. 14. INTRODUCTION Objetive Study and know the clinical characteristics and genotype of HA-MRSA and CA-MRSA and especially various infections, their prevalence in specific locations and what are the variants that may have some influence
  15. 15. MATERIALES Y METODOS ESTUDIO Multicentrico, prospectivo, observacional Evalúa características clínicas y moleculares de la invasión CA - MRSA Argentina De marzo de 2010 a diciembre de 2011
  16. 16. Pacientes inscritos de 11 hospitales de Argentina , un total de 55 pacientes Edad: ≥ 14 años Pacientes para incluirse en el estudio no debían presentar: .Dialisis .Cirugía . Presencia catéteres .Dispositivos médicos .Residido en un centro de cuidado por mucho tiempo. MATERIALES Y METODOS
  17. 17. Información demográfica y clínica por medio de formularios de in forme clínico RECOLECCIÓN Información socio económica a partir de previas revisiones, fuentes de aislamiento , pruebas de laboratorio MATERIALES Y METODOS
  18. 18. MATERIALES Y METODOS  PCR ( Reacción en cadena de la polimerasa) Es una técnica desarrollada por Karry Mullis en 1980 cuyo objetivo es la amplificación de genes o de un fragmento de DNA o indirectamente de uno de RNA. La amplificación permite que aumente el numero de copias de una secuencia particular de DNA
  19. 19. MATERIALES Y METODOS  PFGE ( Electroforesis en Gel de campo pulsado) La PFGE permite la separación de grandes fragmentos de DNA mediante la inducción de reorientación de una serie de cambios en el campo eléctrico, cuya duración permite saber el intervalo de tamaños que se pueden separar. Su utilidad es semejante a la electroforesis convencional, salvo que los fragmentos de DNA separados son de mayor tamaño.
  20. 20. MATERIALES Y METODOS  MLST (Tipificación multilocus de secuencias) es una técnica genética para la caracterización taxonómica de bacterias y microorganismos. Su técnica consiste en amplificación mediante PCR seguida de la secuenciación del ADN. Se pueden rastrear las diferencias en nucleótidos entre cepas en un número variable de genes en función del nivel de discriminación que se desee.
  21. 21. RESULTADOS
  22. 22. RESULTADOS
  23. 23. Los MRSA aislados fueron mas sensibles a la Vancomicina, además ninguno de los aislamientos fueron multiresistentes. RESULTADOS
  24. 24. RESULTADOS
  25. 25. Clon ST30 23 Aislamientos con subtipos C1 16 aislamientos C2 a C6, 7 aislamientos SCCmec Tipo IVa Spa T019 Agr IIIPFGE Type C RESULTADOS
  26. 26. Clon ST5 2 Aislamientos SCCmec Tipo IVa Spa t311 t2724 Agr IIPFGE Type A RESULTADOS
  27. 27. Clon ST72 2 Aislamientos SCCmec Tipo IV, IVvar Spa t1364 t148a Agr IPFGE Type F RESULTADOS
  28. 28. Clon STND 1 Aislamiento SCCmec Tipo IV Spa t002 Agr IIPFGE Type F RESULTADOS
  29. 29. RESULTADOS
  30. 30. DISCUSSION Investigator´s Principal Statement Agree or Disagree Ma et al, 2002 Antimicrobial resistance patterns have been used to distinguish between CA-MRSA and HA-MRSA strains Agree Gardellan et al., 2008 The molecular characteristics shared by the isolates this major clone largely that reported for a minor clone described in our previous study, Agree Gardella et al., 2008; Sola et al., 2008 One of the minor clones found in this study (n = 2), the CAA clone, (PFGE type A, ST5- SCCmec IV-spa t311), had been identified as prevalent in Argentina since 2004 Agree Deleteo et al. , 2010 In contrast to other clones which have been described to have a certain continent specificity, ST30 is distributed world wide Agree
  31. 31. CONCLUSIONS • Finally this study is very important for us because it allows us to know variation and the pathogen of these clones behavior, giving us excellent bases at the time of developing a gene therapy and have new therapeutic targets that avoids these develop their virulence. • Also the study allows also analyzing at the level of the community that things must take into account that levels of prevention should be developed within the communities to re- establish the infectious effects of strains.
  32. 32. CONCLUSIONS • The knowledge of this study brings us to the awareness and the recognition of the different infections caused by the different S. aureus clones and the consequences that has caused with its constant prevalence that is no longer only if no community hospital, by what has become a global concern and will continue to evolve • discover the importance of describing the clinical, molecular and epidemiological of current invasive infections caused by HA- MRSA and CA-MRSA