Transferable Vancomycin Resistancein a Community-Associated MRSA Lineage

Flávia Rossi, M.D., Ph.D., Lorena Diaz, Ph.D., Aye Wollam, B.Sc.,Diana Panesso, Ph.D., Yanjiao Zhou, Ph.D., Sandra Rincon, M.Sc.,

Apurva Narechania, M.A., Galen Xing, Thais S.R. Di Gioia, M.D., André Doi, M.D.,Truc T. Tran, Pharm.D., Jinnethe Reyes, M.Sc., Jose M. Munita, M.D.,

Lina P. Carvajal, B.Sc., Alejandra Hernandez-Roldan, M.Sc., Denise Brandão, M.D.,Inneke Marie van der Heijden, Ph.D., Barbara E. Murray, M.D.,

Paul J. Planet, M.D., Ph.D., George M. Weinstock, Ph.D.,and Cesar A. Arias, M.D., Ph.D.

By: Carolina Carvajal Miranda

Universidad Pontificia BolivarianaMedellín

INTRODUCTION

• Bacterial Antibiotic resistance: form of drug resistance whereby some populations of bacterial species are able to survive after exposure to one or more antibiotics.

INTRODUCTION

• Methicillin-resistant Staphylococcus aureus (MRSA): strain that has developed resistance to beta-lactam antibiotics, which include the penicillins (methicillin, dicloxacillin, nafcillin, oxacillin, etc.) and the cephalosporins

INTRODUCTION

(MRSA)

24-48

INTRODUCTION Methicillin:• Narrow-spectrum β-lactam antibiotic of the penicillin

class

• Inhibits the cross-linkage between the linear peptidoglycan polymer chains (wall).

• It binds and inhibits the transpeptidase enzymes. (PBPs).

INTRODUCTION

• PBPs crosslink glycopeptides (D-alanyl-alanine), forming the peptidoglycan cell wall.

• Meticillin is a structural analog of D-alanyl-alanine.

• Methicillin is a penicillinase-resistant B-lactam antibiotic.

• Methicillin is not bound and hydrolysed by penicillinase, meaning it can kill the bacteria, even if this enzyme is present.

INTRODUCTION

Resistance mecanism:• Resistance is mediated by the acquisition of

extrachromosomal genetic elements (resistance genes).

• Ability to thrive in the presence of penicillin-like antibiotics.

INTRODUCTION Resistance mecanism:

• mecA ----- resistance to methicillin/ encodes (PBP2a)

• Gene is integrated and localized in the S. aureus chromosome.

• PBP2a can catalyze the transpeptidation reaction required for peptidoglycan cross-linking

• Enableling cell wall synthesis

INTRODUCTION

• Enterococcus faecalis and E.faecium could be transferred by horizontal gene transfer to S. aureus

• A conjugative plasmid (pBRZ01) carrying the vanA

• MRSA + vanA = health concern.

GENERAL OBJETIVE

• Describe and characterize from a molecular point of view a case of patient with a bloodstream infection caused by a strain of methicillin-resistant Staphylococcus aureus (MRSA) that adquired vancomycin resistance and properly aislate the gene that is responsable for this resistance.

METODOS

Reporte de caso:

Paciente masculino de 35 años de edad, residente de Sao Paulo-Brazil, es inicialmente admitido al hospital psiquiátrico por depresión y tentativas de suicidio.

(AP)

P: Diabetes Mellitus, micosis fungoide, depresion.

H: Adicción a Cocaína

Evolucion: Retiro al tratamiento por estado terminal.

Reporte de caso:

METODOSIdentificación bacteriana, test de

susceptibilidad y escaneo molecular:

• PCR: S.aureus VREF (sangre)• Secuenciación 16S RNA ribosomal (recto)• Susceptibilidad: dilución agar• PFGE (BR-VSSA y BR-VRSA)• PCR (PVL y elemento móvil catabólico)

METODOS

• PCR (reacción en cadena de la polimerasa): Permite amplificar un fragmento de ADN,es decir, obtener un numero de copias de un fragmento de ADN en particular para identificar. (ADN polimerasas).

• PRGE (Electroforesis en gel de campo pulsado): Separación de grandes moléculas de DNA aplicando a un gel una carga eléctrica que periódicamente cambia de dirección.

METODOS

• Secuenciación: determinación del orden de los nucleótidos en un oligonucleótido de ADN.

METODOS

Plásmidos:• Transferabilidad: BR-VRSA y VREF (donantes)

BR-VSSA, S.aureus RN4220M S.aureus COL, y E. faecalis OG1RF (receptores)

• Digestión con nucleasa, PFGE e hibridación----vanA?

• PCR para plásmidos

RESULTADOS

• BR-VRSA (55kb) con vanA + aac(6) –aph(2)----enzimas resistencia bacteriana.

• Plásmido con vanA----S.aureus

• Secuencia del vanA VREF idéntica al BR-VRSA y las secuencias de E. faecalis

Origen enterococcico

DISCUSION

CONCLUTIONS

The use of tecnics such as PCR and PFGE have a mayor role on the identification an characterization in transferable antibiotic resistance genes.

A conjugative plasmid of 55,706bp carrying the canA cluster was identified and readily transferred to other staphylococci.

The presence of dissemination of MRSA containing the vanA cluster can became a mayor public health problem that may cause a serius resistant infection.