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.