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Seminario biología molecular
Juan Pablo Ruiz
Dayana Quintero
2016
INTRODUCTION
Burn injury is a global public health problem with approximately 195,000 deaths annually . this injury remains the cause of morbidity and mortality in Iran with 100,000 burns . Multi drug resistance (MDR) bacterial infection in burn patients it`s the most common complication in treatment of burn patients and may lead to more mortality .Acinetobacter baumannii is one of the opportunistic Gram-negative bacteria considering the second cause of nosocomial infections in burn patients
OVERVIEWAcinetobacter: Gram-negative bacteria Scientific classification
Domain: BacteriaKingdom: EubacteriaPhylum: ProteobacteriaClass: GammaproteobacteriaOrder: PseudomonadalesFamily:MoraxellaceaeGenus: AcinetobacterSpecies: A. baumannii
Binomial nameAcinetobacter baumannii
INTRODUCTION
CARBAPENEMASEcarbapenemase–carbapenem inactivating enzymes-, is one of the most recent, but perhaps of the greatest concern because virtually inactivate the last therapeutic step against multidrug-resistant gram-negative organisms.
Function: enzymes capable of hydrolyzing carbapenems (imipenem, meropenem, ertapenem, doripenem)
INTRODUCTION
PULSED-FIELD GEL ELECTROPHORESIS
Pulsed field gel electrophoresis is a technique used for the separation of large deoxyribonucleic acid (DNA) molecules by applying to a gel matrix an electric field that periodically changes direction.
Applications: PFGE may be used for genotyping or genetic fingerprinting.Adventages: PFGE subtyping has been successfully applied to the subtyping of many pathogenic bacteria and has high concordance with epidemiological relatedness.-DNA restriction patterns generated by PFGE are stable and reproducible.
INTRODUCTION
VARIABLE NUMBER TANDEM REPEAT
loci are chromosomal regions in which a short DNA sequence is repeated a variable number of times end-to-end at a single location. These can be found on many chromosomes, and often show variations in length between individuals. Each variant acts as an inherited allele, allowing them to be used for personal or parental identification.
INTRODUCTION
Acinetobacter baumannii is a bacteria able to produce carbapenemaces, this enzymes provide the bacteria
resistance to medicaments. The most of these carbapenemases genes are located in mobile genetic element
and can transfer among bacteria. Multiple-locus variable-number tandem-repeat analysis (MLVA) is the PCR based method which is related to the population of VNTR and
different polymorphisms in bacterial genomecan be very useful for molecular epidemiology; On the other hand,
pulsed-field gel electrophoresis (PFGE) is the gold standard method for A. baumannii typing .
INTRODUCTION
General objective
• Determine the molecular epidemiology of carbapenemases producing A. baumannii isolates by MLVA and PFGE.
Aislamiento bacteriano
• Se recolectaron 50 A. baumannii resistentes a
carbapenem, de pacientes quemados
hospitalizados en el Hospital de Motahari en
Tehran.
• Identificación se hizo a través de test
bioquímicos
Control positivo
A. Baumannii ATCC 19606
MATERIALES Y MÉTODOS
Test de susceptibilidad Carbapenem• Los aislamientos con sensibilidad > 13 contra
imipenem, meropenem y ertapenem
Clasificación: Prueba de resistencia cruzada y un test de susceptibilidad antibiótica para:
Resistente carbapenem
Cefotaxima (30 µg) Ceftazidima (30 µg) Ticarcilina (75 µg)
MATERIALES Y MÉTODOS
Detección molecular de genes carbapenemases
• Extracción de DNA bacteriano a través de un plásmido.
• Detección molecular de genes carbapenemases:
• Amplificación de DNA a través de PCR.
bla VIM , bla IMP ,bla OXA-51 ,bla OXA-23 ,bla OXA-48 , bla NDM-1 ,bla SPM-1 y bla KPC
MATERIALES Y MÉTODOS
Tipificación molecular de cepas usando MLVA
VNTR ADN en:
Mediante amplificación por PCR, utilizando en cada caso diana:
A. baumannii cebadores
Oligonucleótidos adyacentes a los extremos 5’ y 3’.
A cada locus VNTR se le aplicó PCR:Desnaturalización inicialCiclos de desnaturalizaciónHibridación ElongaciónFinal elongación
• A. baumannii 2240• A. baumannii 3530• A. baumannii 3002• A. baumannii 3406
MATERIALES Y MÉTODOS
Tomado de CDC: Center for DiseasesControl and Prevention
MATERIALES Y MÉTODOS
La tipificación molecular de las cepas mediante PFGE
• Enzima de restricción ApaI.
• Segmentos de ADN se separaron en 2 bloques:
Bloque 1:13 h a 6 V / cm a 120 ° C con tiempos de pulso inicial 2s y
final 10s.
Bloque 2:6 h a 6 V / cm a 120 ° C con tiempos inicial de pulso 20s
y final 25s.
MATERIALES Y MÉTODOS
• Braenderup Salmonella: Marcador de tamaño de ADN.
• Patrones de banda fueron agrupados por UPGMA, utilizando software Gelcompare II versión 4.0 .
La tipificación molecular de las cepas mediante PFGE
MATERIALES Y MÉTODOS
Tomado de CDC: Center for DiseasesControl and Prevention
MATERIALES Y MÉTODOS
Resultados
Estos genes no se detectaron: bla IMP
bla OXA-48
bla NDM-1
bla SPM-1
5 (10%): bla KPC , bla OXA-23
2 (4%): bla VIM
5 (10%): bla VIM, bla OXA-23
34 (67%): bla OXA-23
Todos tenían bla OXA-51
Resultados
Resultados
Resultados
Resultados
DiscussionAuthor Opinion Agree or
disagree
Azimi. L. Et al. “Burn patients are in the high risk of
nosocomial infections considering the
loss of skin as a first protective barrier.
So, it can cause increasing rate of
morbidity and mortality among these
patients”
Yes.
Owlia. P. Et al. “Carbapenemase producing Gram-
negative bacteria including A.
baumannii, as a second cause of
nosocomial infection in burn patients
in Iran, can have important role in this
area”
Yes.
Author Opinion Agree or
disagree
Teo J. Et al. “In this regard molecular epidemiology may be very
important for source and molecular type
determination in carbapenemase producing A.
baumannii in burn care center. In addition, it can
determine the genetic relationship between specimens
that were isolated from different hospitalized patients
especially in different hospital wards. Saranathan et al.
in 2015 showed that eight clusters in their tested
carbapenem resistant A. baumannii by REP-PCR”
Yes
Rafei R. Et al. “The results of our study indicated more potency of
PFGE than MLVA for separating of molecular types in
our isolates. So, according to previous lectures PFGE
remains the gold standard for outbreak investigations
due to its higher discriminatory power”
Yes
Discussion
Conclusions
• PFGE continue being the gold standard method for A. baumannii typing, because it is more especific to determinate the infection.
• Nosocomial infection that are producing by A. baumannii in burn patients are increasing due multidrug resistance (MDR), because this bacteria has many carbapenemases. It means difficulty in thetreatment of bacterial infection and burn.
CONCLUSIONS
• PFGE it`s more effective in the separation of molecular types in their isolates(11 molecular clusters by PFGE ) compared with MLVA (5 molecular clusters by MLVA)
• Can be a lot of differences between the bacterias in latin america and Iran, and we need to study the pattern between their bacterias and ours.
MUCHAS GRACIAS.