nosocomial infections with metallo-beta-lactamaseproducing pseudomonas aeruginosa: molecular...
TRANSCRIPT
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Nosocomial infections with metallo-beta-lactamase producing Pseudomonas aeruginosa: molecular epidemiology, risk factors, clinical features and
outcomes
A. Lucena, L.M. Dalla Costa, K.S. Nogueira, A.P. Matos, A.C. Gales, M.C. Paganini f, M.E.S. Castro f, S.M. Raboni.
Journal of Hospital Infection
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Laura Cristina CastrillónElizabeth Andrea García
Medicina, Tercer semestreDocente: Lina María Martínez
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INTRODUCTION
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Nosocomial infection
•A hospital-acquired infection (HAI) is an infection whose development is favored by a hospital environment, such as one acquired by a patient during a hospital visit or one developing among hospital staff
•According to WHO, including infections that would not have said or were in the incubation period, that is acquired during your stay and are not the cause of income
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Nosocomial infection
•Are commonly transmitted when hospital officials become complacent and personnel do not practice correct hygiene regularly
•The drug-resistant Gram-negative bacteria, for the most part, threaten only hospitalized patients whose immune systems are weak.
•They can survive for a long time on surfaces in the hospital and enter the body through wounds, catheters, and ventilators
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Nosocomial infection
Pseudomona aeruginosa
Staphylococcus aureus
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Nosocomial infection
Sterilization
Isolation
Handwashing
GlovesSurface sanitation
Antimicrobial surfaces
Aprons
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Pseudomona
• Some species synthesize exopolysaccharides capsule facilitating cell adhesion, biofilm formation, and protects against phagocytosis, antibody complement or increasing their pathogenicity
• 191 validly described species
• Rod-shaped• Gram-negative• One or more polar flagella,
providing motility• Aerobic• Non-spore forming• Positive catalase test• Positive oxidase test• Metabolic diversity• secretion of pyoverdine, a fluorescent
yellow-green sidephore
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Pseudomona
•Most Pseudomonas are naturally resistant to penicillin and the majority of related beta-lactam antibiotics
•This ability to thrive in harsh conditions is a result of their hardy cell wall that contains porins.
•Their resistance to most antibiotics is attributed to efflux pumps, which pump out some antibiotics before the antibiotics are able to act.
Scientific classificationDomain: BacteriaPhylum: Proteobacteria Class: GammaproteobacteriaOrder: PseudomonadalesFamily: PseudomonadaceaeGenus: Pseudomonas
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Pseudomona aeruginosa
Gram-negative, aerobic bacterium with unipolar motility. It is an opportunistic human pathogen and
plant
Secretes a variety of pigments such as pyocyanin (blue-green), fluorescein (fluorescent yellow-green)
and piorubina (brownish red).
Infects the pulmonary tract, urinary tract, tissues, wounds, and also causes other blood infections
Is naturally resistant to a large number of different families of antibiotics
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Metallo β lactamases
•Metallo B lactamases are enzymes that are capable of hydrolyzing a variety of B-lactam agents such as penicillins, cephalosporins and carbapenems.
• They are transferable, the majority are in cassettes genes located on integrons type 1 and causes in plasmids and transposons.
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Metallo β lactamases
•Usually are associated with other resistance genes located in the same gene cassettes allowing you to have multiple antibiotic resistance as: the B-lactam (oxyimino cephalosporins, cephamycins, carbapenems), aminoglycosides and quinolones, have varying sensitivity to aztreonam
•The acquired MBLs can be classified into 4 groups according to molecular structure are called IMP, VIM, GIM and SPM.
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Nosocomial infections
Immune vulnerability
Pseudomona aeruginosaResistant
Metallo β Lactamase
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Main objective
To assess the molecular epidemiology, risk factors and outcomes of nosocomial infections
caused by MBL-PA in a teaching hospital in Southern Brazil.
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MATERIALES Y MÉTODOS
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Datos generales
De enero de 2001 a diciembre de 2008 142 cepas de P. aeruginosa resistentes a carbapenems fueron aisladas de muestras clínicas distintas de los pacientes hospitalizados. Estos aislados fueron seleccionados para MBLs, y se sometieron a PCR, secuenciación y electroforesis en gel de campo pulsado (PFGE).
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Cepas
Tomar las cepas Prueba de difusión de disco
Resistencia completa o incompleta
Almacenamiento a -80º
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Prueba de difusión de disco
•Estudio de sensibilidad a los antimicrobianos
•No requiere un material especial
•Permite el estudio de una gran diversidad de antimicrobianos
•Rutinaria
•Alta utilidad
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Prueba de difusión de disco
1. Preparación del medio de cultivo 2. Preparación del inóculo3. Inoculación de las placas de agar4. Aplicación de los discos de antimicrobianos5. Incubación de las placas (16-18 horas)6. Lectura de las placas tras la incubación 7. Medida de los halos e interpretación de los resultados de
sensibilidad
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Pruebas de susceptibilidad
•Pruebas bioquímicas convencionales (no especificadas)
•Método de dilución en agar
ImipenemMeropenemPiperacilina/TazobactamCeftazidimaCiprofloxacinaGentamicinaAmikacinaAztreonamCefepimaB polimixina
Cepa de control: P. aeruginosa ATCC 27853
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Método de dilución en agar
•Considerado el método de referencia
•Placas con una determinada concentración de un antibiótico son inoculadas con el microorganismo en estudio y luego incubadas por 16 a 18 horas. Después de la incubación, se examina si el organismo crece o no en cada una de las placas
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Identificación de las MBLs
It is performed by agar diffusion using Mueller-Hinton plate inoculated with a bacterial suspension adjusted to the pattern of a 0.5 McFarland scale; placed thereon, standard load discs (30 mg) of cefotaxime, ceftazidime, cefuroxime and aztreonam arranged at a distance of 25-30 mm disc of amoxicillin / clavulanate (20/10 mg). Synergy was considered when an extension of the zone of inhibition for any of the cephalosporins or aztreonam is observed.
Prueba de sinergia de doble disco
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Identificación de las MBLs
Técnica de biología molecular, cuyo objetivo es obtener un gran número de copias de un fragmento de ADN particular. Se fundamenta en la propiedad natural de los ADN polimerasas para replicar hebras de ADN.
Reacción en Cadena de la Polimerasa (PCR)
Amplifica fragmento de
DNA
Identificación de virus y bacterias
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Identificación de las MBLs
•Clonación de ADN para la secuenciación
•Filogenia basada en ADN
•Análisis funcional de genes
•Diagnóstico de trastornos hereditarios
•Identificación de huellas genéticas
•Detección y diagnóstico de enfermedades infecciosas
Reacción en Cadena de la Polimerasa (PCR)
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Electroforesis en gel de campo pulsado
Separar moléculas grandes de DNA (hasta 10 Mb)
En este método, el DNA viaja a través de un gel de agarosa concentrado, bajo la influencia de dos campos eléctricos. Los dos ángulos de los campos eléctricos se encuentran cerca a la perpendicularidad, no son uniformes en la intensidad de campo y cambian de manera alterna (pulsos).
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Metallo β lactamases
Supone que en concentraciones altas de agarosa y con tensiones elevadas, las moléculas grandes de DNA deben ser elongadas a lo largo de la dirección del campo eléctrico con el fin de penetrar a través de los poros del gel. Cuanto más grande sea la molécula de DNA, mayor será el tiempo para encontrar la nueva orientación y la retención en el gel
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Estudio clínico
Estudio retrospectivo Factores de riesgo Estudio clínico
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Estudio clínico
ControlesPacientes infectados con no-
MBL-PA
CasosPacientes infectados con
MBL-PA
EdadSexo
Enfermedades de baseCondiciones comórbidas
Sitio de la infecciónProcedimiento quirúrgico anterior
Uso de dispositivos Ventilación mecánica
Inmunosupresión Uso previo de fármacos antimicrobianos
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RESULTADOS
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Análisis de resultados
Grupo de 99 cepas
87 SPM1
Tipo de MBL
Se clasificaron en 25 Subtipos
Patrones y dendrograma de Electroforesis en Gel de Campo Pulsado (PFGE) de 142 cepas de Pseudomona aeruginosa
resistentes a carbapenem aisladas de pacientes de un hospital de Brasil entre 2001- 2008
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Análisis de resultados
71
56
A
Patrones y dendrograma de Electroforesis en Gel de Campo Pulsado (PFGE)
Indicador Característica
Patrón de 8- Mayor %
Dos líneas constantes-Las mas notorias
Patrones diferentes de todo el subtipo
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Análisis de resultados
Indicador Característica
Nueva banda (vs subtipo A)
Desaparece (vs subtipo A
Diferente (A y B)
Patrón
Igual – anterior (constante A y B)
28
15
13
Patrones y dendrograma de Electroforesis en Gel de Campo Pulsado (PFGE)
B
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Análisis uni y multivariado de factores de riesgo por infección de MBL- PA
84 (59%)
58 (69%)MBL-PA
57->bla SPM-1
1->bla IMP-1626 (31%)Non MBL-PA
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Análisis de resultados
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Análisis de resultados
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Análisis de resultados
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Análisis de resultados
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Análisis de resultados
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DISCUSIÓN
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Discusión
Authors What did they say? Agree Desagree
10. Hirakata Y, Yamaguchi T, Nakano M, et al. 2003.12. Zavascki AP, Barth AL, Gaspareto PB, et al.2006.
Unlike other studies, antibiotic exposure was not a risk factor in this study.
31. Gales AC, Menezes LC, Silbert S, Sader HS. 2003.
The low sensitivity of MBL-PA to aztreonam can be explained by the presence of differentmechanisms of resistance in P. aeruginosa.
33. Cornaglia G, Giamarellou H, Rossolin GM. 2011.
Infection with MBL-producing bacteria often occurs in patients with multiple comorbidities, previous surgery and ICU admission.
12. Zavascki AP, Barth AL, Gaspareto PB, et al. J .2006.
Zavascki et al. found that MBL-PA infection resulted in a higher mortality ratethan non-MBL-PA infection (51.2% vs 32.1%, respectively).
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CONCLUSIONES
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Conclusiones
1. There are very specific bacterial strains associated to specific places, especially in hospitals, where in a minimum area, there are a lot immunosuppression people, this situation becomes this problem in an institutional responsibility and a public health issue. Investigate and search a possible solutions to avoid the complication of patients and their spread in the community are urgent issues.
2. For alternative measures of multiresistant infections spread be effective, it is necessary the participation of institutions, but is immediate have state laws for assure significant results.
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Conclusiones
3. This kind of investigations confirm that bacterians can be an invasive disease and alert about a problem, which should be manage by each health institution like a futurure problem wich could be prevent now.
4. Althoug bla SPM-1 gene was the most comun in patients, bla IMP-1 and bla IPM-16 were found in one strain, it isnt a significant outcome, but it is important for recognize that transmission is a topic intra and extra clinical.
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BIBLIOGRAFÍA
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Bilbiografía
1. Pérez I Alfonso, García C Patricia, Poggi M Helena, Braun J Stephanie, Castillo V Claudia, Román Juan Carlos et al . Presencia de metalo-ß-lactamasas en Pseudomonas aeruginosa resistente a imipenem. Rev. méd. Chile, 2008. 136( 4 ): 423-432. [on line] Consulta: Agosto 9. Disponible en: http://www.scielo.cl/scielo.php?script=sci_arttext&pid=S003498872008000400002&lng=es.
2. Soberón G. Pseudomonas aeruginosa. Instituto de Biotecnología, Universidad Nacional Autónoma de México. [online] Consulta: Agosto 9. Disponible en: http://www.biblioweb.tic.unam.mx/libros/microbios/Cap3/
3. Fresnadillo MJ, García MI, García E, García JE. Los carbapenems disponibles: Propiedades y diferencias. Rev. Enfermedades infecciosas microbiología clínica. Vol. 28. Núm. Septiembre 2010
4. https://www.seimc.org/contenidos/ccs/revisionestematicas/bacteriologia/kpbpea.pdf , ß-lactamasas plasmidicas de espectro ampliado. Consulta: Agosto 8
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Bibliografía
5. http://www.cdc.gov/ncidod/dbmd/gss/publications/documents/Argentina-LevelI/Manual_procedimientos.pdf Manual de procedimientos para la determinación de la sensibilidad a los antimicrobianos en bacterias aisladas de humanos. Consulta: Agosto 8.
6. Detección de metalobetalactamasas (MBLs) en Pseudomonas aeruginosa resistentes a los carbapenemas en un Hospital Nacional, en los meses de enero a octubre del año 2008. José Alberto Díaz Tello, Lima-Perú, 2008.
7. Pseudomonas aeruginosa: aportación al conocimiento de su estructura y al de los mecanismos que contribuyen a su resistencia a los antimicrobianos. Lídia Ruiz Martínez, Barcelona, Noviembre 2007.
8. http://coesant-seimc.org/documents/M%C3%A9todosB%C3%A1sicos_SensibilidadAntibi%C3%B3ticos.pdf Procedimientos en Microbiología Clínica. Consulta: Agosto 8
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MAPAS CONCEPTUALES
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