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PCR Heptaflex
- 1. María Salomé Hernández Ramírez Carlos Andrés Osorio Third Semester 2015
- 2. INTRODUCTION STAPHYLOCOCCUS • Gram-positive cocci • Spherical cells • Arranged in clusters like “cluster grapes” • They grow rapidly on many types of media and are metabolically active Ferment carbohydrates Produce pigments
- 3. • They are devoid of motility and not form spores. • Facultative anaerobes: They can grow in aerobic or anaerobic form. • Can be positive-coagulase ( Staphylococcus aureus) or negative- coagulase (Staphylococcus epidermidis). • Hydrogen peroxide H20 + O2 CATALASE STREPTOCOCCUS
- 4. • The genus Staphylococcus contains at least 35 species and 17 subspecies. Three of importance are Staphylococcus aureus , Staphylococcus epidermidis , Staphylococcus saprophyticus . • Cell wall: polysaccharides and antigenic proteins such as peptidoglycan , teichoic acids, protein A. • Can cause disease by their ability to multiply and spread extensively in the tissues and by the production of many extracellular substances (enzymes and toxins ) • Some are members of the normal flora of the skin and mucous membranes of humans; others cause suppuration , abscess formation , pyogenic infections or fatal septicemia.
- 5. GRAM STAINING • GRAM-positive bacteria • GRAM staining classified bacteria based on the structure of their cell walls. • Purple stain due to its thick wall peptidoglycan retaining the dye.
- 6. Staphylococcal lesions appear as grain , a hair follicle infection or abscess COMMON DISEASES Intense inflammatory reaction Drain pus Fibrin Cicatrization Originate a broad spectrum of systemic diseases that can endanger life , infections of the skin, soft tissues , bones and genitourinary system and opportunistic infections .
- 7. • Scalded skin syndrome • Food poisoning • Toxic shock • Impetigo • Folliculitis • Boils • Bacteremia and endocarditis • Pneumonia and empyema • Osteomyelitis • Wound, genitourinary , catheters and prostheses infections
- 8. Staphylococcus aureus Almost everyone suffers from an infection by this bacterium in his lifetime , ranging from food poisoning or minor skin infections to life- threatening severe skin infections.
- 9. • Gram-positive cocci , positive catalase. • Cause disease by producing toxin or through direct invasion and tissue destruction • Coagulase on the surface of the cell wall: binds to fibrinogen and bacterial aggregation occurs • Exposure to contaminated things. • Direct contact
- 10. Risk factors • Strange leather (prostheses , catheters ). • Previous surgery • Antibiotics which suppress the normal microbial flora • Immunosuppressed
- 11. Virulence factors Regulated by sensitive sistems to environmental cues Proteins Kinase Response regulator Accessory gene regulator (agr ) is essential for controlling gene expression regulatory functions in gene transcription
- 12. agr controlling expression of Surface adhesins Proteín A, coagulase, fibronectin binding protein Exoproteins production Toxins as TSST-1 depending on growth phase and bacterial density
- 13. Low bacterial density P2 promoter inactive Transcript of transmembrane proteins , AgrB , precursor peptide, AgrD, AgrC y AgrA. Increased bacterial densities AgrC + AgrA + P2 P3 Hemolisine δ and RNAIII transcription surface expression of adhesins exoproteins secretation
- 14. Bacterial resistance Quickly develop resistance to antibiotics after the introduction of penicillin 10% sensitive PENICILASE Β lactamase hydrolyzes the β lactam ring of penicillin The genetic information encoding the production of this enzyme is a transmissible plasmid It facilitates the rapid spread of resistance among Staphylococcus
- 15. Semisynthetic penicillins Resistant to hydrolysis by β-lactamases Methiciline Nafcillin Oxacillin Dicloxacillin Can also acquire resistance to these antibiotics 30- 50% Methicillin-resistant strains S. aureus are included under the acronym MRSA
- 16. Acquisition of resistance gene mecA Encoding PBP2 protein that binds to penicillin • Penicillins and other lactam antibiotics kill bacteria by their ability to bind proteins binding to penicillin , which are the enzymes responsible for building wall peptide glycan PBP2 protein does’nt bind to penicillin , but maintains its enzymatic activity Heterogeneous resistance not all bacteria from a resistant population expressing this protein binding to penicillin
- 17. The only antibiotic that had kept his uniform activity against staphylococcus was vancomycin Vancomycin resistance mechanisms Low level resistance Thickest and disorganized wall vancomycin molecules are trapped in the matrix of the cell wall and can’t reach the cytoplasmic membrane , in which alter the cell wall synthesis High-level resistance Encoded by the vanA gene operon These bacteria have a modified peptidoglycan layer that does’t fix the molecules of vancomycin
- 18. PCR • PCR-based assays are capable of identifying the genetic capacity for STAAR (staphylococcal toxigenicity and antibiotic-resistance) without the influence of variability in gene expression and are preferred for speed and sensitivity. Multiplex PCR assays capable of detection of multiple microbes or multiple genetic markers within the same PCR tube New heptaplex PCR assay Detected 7 DNA markers 16S rRNA spa tuf (genus Staphylococcus) cns (CoNS) pvl (PVL virulence factor)mecAvanA
- 19. In set Staphylococcus Staphylococcus aureus Gram-positive cocci Systemic diseases: skin infections, soft tissues , bones and genitourinary PCR Control of gene expression by accessory gene regulator (agr ) Bacterial resistence VRSA MRSA
- 20. General purpose Develop a new method of PCR assay to identify and differentiate objectively virulence caused by staphylococcus aureus or CoNS and mechanisms of antibiotic resistance.
- 21. Materiales y métodos • La aprobación de bioética y bioseguridad de este estudio fue dada por el Comité de investigación de la Universidad de Nottingham. • Se utilizaron aislados bacterianos y hemocultivos de pacientes no identificados para que no pudieran ser rastreados de nuevo. • Muchas de las cepas bacterianas utilizadas en este estudio se obtuvieron de la Red para la resistencia antibiótica de Staphylococcus aureus (NARSA ). Algunos eran multi-resistentes o altamente virulentos • Nivel 2 de bioseguridad: Para trabajo almacenamiento de agentes potencialmente peligrosos
- 22. Cepas n= 255 • Sub-cultivadas y almacenados (-80 ° C) en (BHI) y se sembraron en agar BHI. Cultivos de referencia n= 53 Para validar los 7 marcadores genéticos Cepas estafilocócicas locales n= 124 Cepas CoNS n= 31 S. auricularis (2) S capitis (2) S. caprae (2) S. chromogenes (2) S. cohnii (2) S. epidermidis (2) S. hemolyticus (1) S. hominis (2) S. hyicus (2) S. intermedius (2) S. lugdunensis (1) S. saprophyticus (3) S. sciuri (2) S. simulans (2) S. warneri (2) S. xylosus (2)
- 23. Otras bacterias no estafilococos n= 47 Escherichia coli, Pseudomonas, Klebsiella, Aeromonas, Salmonella, Citrobacteria, Proteus, estreptococos del grupo A (GAS).
- 24. NARSA (Network on Antimicrobial Resistance in Staphylococcus aureus) • Mantiene un depósito de cepas de Staphylococcus aureus y bacterias relacionadas, que son importantes para el estudio de la resistencia antibacteriana. TRITON X-100: Lisis celular Es un detergente no iónico usado para desnaturalizar membranas de células sin desnaturalizar la proteína.
- 25. Brain Heart Infusion (BHI) Agar PCR Amplificar un gen o un fragmento específico de DNA en varios millones de veces. • Es un medio de uso general adecuado para el cultivo de una amplia variedad de tipos de organismos (bacterias). • Obtiene los nutrientes de la infusión de cerebro y corazón, la peptona y la glucosa. Se utiliza fosfato disódico como tampón en el medio
- 26. Taq DNA polimerasa • Es la enzima usada en el procedimiento de la PCR • Producida por la bacteria termófila Thermus aquaticus. • Enzima termoestable que no pierde su actividad a temperaturas elevadas Termophol buffer • Proporciona condiciones de reacción superiores para otras polimerasas termófilas de ADN, incluyendo Taq ADN polimerasa, así como otros ADN y enzimas modificadoras de ARN. Electroforesis método de laboratorio en el que se utiliza una corriente eléctrica controlada, con la finalidad de separar biomoléculas según su tamaño y carga eléctrica a través de una matriz gelatinosa.
- 27. GenElute™ Kit que contiene todos los reactivos necesarios para aislar y purificar ADN genómico a partir de bacterias gram-negativas. En esta investigación se usó para purificar los productos de PCR. Prueba coagulasa tubo Para diferenciar S. aureus de CoNS
- 29. Results
- 30. Results
- 31. Discussion AUTHOR THEY SAID YES/ NO Guembe M To circumvent this, the new heptaplex PCR assay identifies all CoNS by detection of the cns marker. Similarly, the detection of the 16S rRNA in all (100 %) bacteria studied including staphylococcal and non- staphylococcal strains supports the use of this gene as a diagnostic marker for bacteria [43]. YES Pichon B The 0.5 mg/L oxacillin used in this study supports the recent report of low oxacillinresistant staphylococci (MIC = 0.5 mg/L) in the UK [45]. NO Geja DJ It has been suggested that mecA-positive staphylococci showing low-level oxacillin-resistance should be regarded as constitutively oxacillin-resistant [3]. YES Hill R The new assay completes cycling within an hour which compares favourably with a recent PVL/MRSA real-time PCR assay [45]. NO
- 32. Conclusions • The evolution of STAAR is such a relevant adverse aspect in Public Health, antibiotic threatments should take it as the main target.
- 33. Conclusions • Biomolecular studies most keep working together with health studies, as it focus the treatments to its more specific grade; representing less adverse effects for patients.
- 34. Conclusions • The most effective ways of controling the development of STAAR is making a razonable use of antibiotics.
- 35. Conclusions • Medical staff should try to make a good diferencial diagnostic before starting any antibiotic treatment, in order to dicrease bio resistance development. That is why the current assay used PCR heptaflex.
- 36. María Salomé Hernández R
- 37. Carlos Andrés Osorio G.
- 38. GRACIAS
- 39. BIBLIOGRAFÍA • Murray, P.R; Rosenthal K.S; Pfaller M.A. Staphylococcus y microorganismos relacionados. Microbiología médica. Edición 5. Madrid, España: Elsevier; 2007. p. 221- 236 • Brooks, G.F; Carroll, K.C; Butel, J.S; Morse, S.A. Estafilococos. Microbiología médica de Jawetz, Melnick y Adelberg. 19ª edición. México: Manual Moderno, S.A; 2008. p.235- 241
- 40. BIBLIOGRAFÍA • Clinical and Laboratory Standards Institute. Molecular Diagnostic Methods for Infectious Diseases. Approved Guideline. Second Editionth ed. Wayne, PA: Clinical and Laboratory Standards Institute; 2010. Document MM06-A2. • Panda S, Kar S, Choudhury R, Sharma S, Singh DV. Development and evaluation of hexaplex PCR for rapid detection of methicillin, cadmium/zinc and antiseptic resistant staphylococci, with simultaneous identification of PVL-positive and- negative Staphylococcus aureus and coagulase negative staphylococci. FEMS Microbiol Lett. 2014;352(1):114–22.