Biología molecular

1. Seminario biología molecular
Juan Pablo Ruiz
Dayana Quintero
2016

3. 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

4. OVERVIEW
Acinetobacter: Gram-negative bacteriaScientific classification
Domain: Bacteria
Kingdom: Eubacteria
Phylum: Proteobacteria
Class: Gammaproteobacteria
Order: Pseudomonadales
Family:Moraxellaceae
Genus: Acinetobacter
Species: A. baumannii
Binomial name
Acinetobacter baumannii
INTRODUCTION

5. CARBAPENEMASE
carbapenemase–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

6. 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

7. 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

8. 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

9. General objective
• Determine the molecular epidemiology of
carbapenemases producing A. baumannii isolates by
MLVA and PFGE.

10. 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

11. 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

12. 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

13. 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 inicial
Ciclos de desnaturalización
Hibridación
Elongación
Final elongación
• A. baumannii 2240
• A. baumannii 3530
• A. baumannii 3002
• A. baumannii 3406
MATERIALES Y MÉTODOS

14. Tomado de CDC: Center for Diseases
Control and Prevention
MATERIALES Y
MÉTODOS

15. 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

16. • 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

17. Tomado de CDC: Center for Diseases
Control and Prevention
MATERIALES Y MÉTODOS

18. 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

19. Resultados

20. Resultados

21. Resultados

22. Resultados

23. Discussion
Author 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.

24. 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

25. 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 multi
drug resistance (MDR), because this bacteria has
many carbapenemases. It means difficulty in the
treatment of bacterial infection and burn.

26. 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.

29. MUCHAS GRACIAS.