Transferable Vancomycin Resistance in a community-associated MRSA Lineage

1. Transferable Vancomycin Resistance
in 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 Bolivariana
Medellín

2. 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.

3. 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

4. INTRODUCTION
(MRSA)
24-48

5. 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).

6. 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.

7. 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.

8. 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

10. 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.

11. 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.

12. 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.

13. Reporte de caso:

14. METODOS
Identificación 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)

15. 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.

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

17. 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

18. 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

21. DISCUSION

22. 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.