The study characterized 270 MRSA isolates from Colombia to determine molecular epidemiology and virulence genes. PFGE analysis identified dominant clones related to the Chilean and USA300-0114 clones. MLST found sequence types ST1110 and ST1111 related to known clones. SCCmec typing showed types I, II, IVa-c. Virulence genes like enterotoxins, exfoliative toxins, and adhesins were present. Mortality was higher for HA-MRSA and infections like bacteremia. The presence of certain genes was associated with increased severity and pathology.

1. Molecular epidemiology and
characterization of virulence genes of
commnunity-acquired and hospital-acquired
methicillin-resistant Staphylococcus aureus
in Colombia
Bibiana Chavarro Portillo, Jaime Enrique Moreno, Nancy Yomayusa, Carlos
Arturo Álvarez, Betsy Esperanza Castro Cardozo, Javier Antonio Escobar
Pérez, Paula Lucía Díaz, Milciades Ibañez, Sebastián Mendez-Alvarez, Aura Lucía
Leal, Natasha Vanegas Gómez
Manuela Rendón Díez
Daniel Peña Tobón

2. *INTRODUCTION

3. Methicillin-resistant Staphylococcus aureus
Kind Virulence Factors
Staphylococcus
Strucutaral
components
Enzymes
Toxins
Genus Methicillin
Aureus Beta Lactam
antiobiotic
INTRODUCTION

4. INTRODUCTION
Staphylococcus From the Greck sthapylé meaning bunch of grapes
Gram Positive Bacteria
Facultative bacteria growth: growing in aerobic
and anaerobic enviroments
Microorganims in the skin and mucous of
humans and animals
Cell wall: pollysaccharide capsule to protect from
phagocytosis
Secreat liquid with monosaccharides, proteins and
peptides: to increase their bond sterenght

5. Aureus Facultative anaerobic bacteria
Gram Positive Bacteria
Produce diseases like
Main cause of nosocomial infections: passing
easily into the bloodstream
Pollysaccharide cell envelope: increase adhesion
capacity, prevents recognition, reinforces it´s
antiphagocytic effect
INTRODUCTION
Benign skin infections:
folliculitis
Benign mucosal
infections: conjunctivitis
Life threatening
diseases:
ostomyelitis, meningitis
, sepsis, pneumonia

6. INTRODUCTION
Methicillin
Beta-lactam
antiobiotic
Was used to
treat
infections
Caused by
gram positive
bacteria
That produce
beta-
lactamase

7. INTRODUCTION
Methicillin
MODE
OF
ACTION
Inhibiting the
synthesis of bacterial
cell walls
Inhibition
competitive of
transpeptidase
enzyme
Prevents
formation of
bonds between
the
peptidoglycan
chains

8. INTRODUCTION
Virulence factors
Structural components
Virulence factors Action
Capsule Inhibits chemotaxis and phagocytosis
Extracelular
pollysaccharides
Facilites adhesion
Peptidoglycan Stimulates cell lysis and production of endogenous pyrogens
Teichoic acid Staphylococcus adhesion to fibronectin
MSCRAMM Increase tissue adhesion
Protein A Inmune response decreases

9. INTRODUCTION
Virulence factors
Enzymes
Virulence factors Action
Coagulase Transformation of fibrinogen to fibrin (inmune response
decreases)
Hyaluronidase Destruction of hialuronic acid to the spread of s.aureus
Fibrinolysin Dissolves fibrin clots
Lipases Lipid hydrolisis: acumulation of s.aureus
Endonucleases/DNAsa DNA hydrolysis
Beta lactamases

10. INTRODUCTION
Virulence factors
Toxins
Virulence factors Action
Cytotoxins Destroys cells
Exfoliative toxins Cutaneous damages
Enterotoxins Gastrointestinal problems by openin channels
TSST-1 Toxic shock: lymphocyte activation for citokine production

11. Methicillin-resistant Staphylococcus aureus (MRSA)
Infection that doesnt improve with the first line of antibiotics
Acquired in the hospital or in the community
HA
Chilean clone
Brazilian clone
CA USA 300
Virulence factors: severity of infections
Proteolytic activity,
toxic and litic effects
Frequent in patients with weakened
inmune systems
INTRODUCTION

12. Methicillin-resistant Staphylococcus aureus (MRSA)
INTRODUCTION
Symptoms and sings
Skin: red,
swollen
and sore
Secretion
of pus
Wounds
that dont
heal
Chest
pain
Cough
Shortness
of breath
Fatigue

13. *OBJECTIVE

14. OBJECTIVE
To determine the molecular epidemilogy and presence of
virulence genes in community-acquired (CA) and hospital-
acquired (HA) methicillin-resistant Staphylococus aureus
(MRSA) insolates and their relationship to clinical outcomes.

15. *Materiales y métodos

16. MATERIALES Y MÉTODOS
Población de estudio
Junio 2006-Diciembre 2007
Mayores de 18 años
Hospitales de 3° nivel
Muestras clínicas
Enviadas a la laboratorio
de referencia
Datos clínicos
Infección
Resultado
Recaída
Mejora
Muerte
30 días
Sangre
Secreciones
Tejidos blandos
Heridas

17. Aislados
bacterianos
y cepas de
referencia
Mu50
TC146
USA 300-
0114
JCSC4744
JCSC2172
81/108
JCSC4469
NPS123
USA500
FRI1151
MATERIALES Y MÉTODOS

18. MATERIALES Y MÉTODOS
PRC: Reacción en cadena de polimerasa
*Kary Mullis --- > 80´s
*Amplificación directa de un gen o un
fragmento de DNA
*Incremento en el número de copias
de un DNA en corto tiempo

19. MATERIALES Y MÉTODOS
PRC
DESNATURALIACIÓN
HIBRIDACIÓN
REPLICACIÓN

20. MATERIALES Y MÉTODOS
PRC: ¿Qué necesito?
*DNA plantilla
*Primer
*DNA polimerasa
*Buffer
PRC: ¿Para qué?
*Clonación
*Establecer polimorfirmos
*Dx enfermedades genéticas
*Medicina Forense
*Detección de
microorganismos infecciosos

21. PRC y el staphylococcus aureus resistente a la meticilina???
MATERIALES Y MÉTODOS
Confirmar especie y resistencia a
meticilina.
*Enterotoxina
*Exfoliativa
*Hemolisina
*Shock Tóxico
*Protíenas adhesivas
Presencia de

22. PFGE: Electroforesis en gel de campo pulsátil
MATERIALES Y MÉTODOS
*Schwartz y Cantor: 1984

23. MATERIALES Y MÉTODOS
PFGE: Electroforesis en gel de campo pulsátil
ELECTROFORESIS
Utilización de corriente eléctrica
controlada
Separación de biomoléculas según:
Carga Tamaño

24. MATERIALES Y MÉTODOS
PFGE: Electroforesis en gel de campo pulsátil
*Reducción del movimiento de las
moléculas utilizando un medio que
oponga resistencia =GEL
Los fragmentos son sometidos a
cargas eléctricas en diferentes
direcciones.

25. MATERIALES Y MÉTODOS
PFGE: Electroforesis en gel de campo pulsátil
*Separación de grandes
fragmentos de DNA
Induce reordenamiento
por cambios eléctricos
periódicos constantes
Duración en el campo
eléctrico determina el tamaño
del fragmento.

26. MATERIALES Y MÉTODOS
Susceptibilidad antimicrobiana Métodos para determinar la
susceptibilidad de los organismos a
agentes antimicrobianos.
*Oxacilina
*Gentamicina
*Rifampicina
*Eritromicina
*Ciprofloxacina
*Vancomicina
*Linezoild
*Tetraciclina
*Clindamicina
*Trimetropim
*Sulfametoxazol

27. MATERIALES Y MÉTODOS
Susceptibilidad antimicrobiana
Recomendaciones para
establecer la terapia mas
adecuada para un paciente

28. MATERIALES Y MÉTODOS
Análisis estadístico
Basándose en:
Mejoría
Mortalidad

29. *RESULTADOS
RESULTADOS

30. Se obtuvieron diferentes patrones de los 270
aislados
• F, U, D, G, H, K,O y P
Por PFGE buscando relación
genética con
• Clon Chileno, Clon USA300-0114,
Clon pediátrico
RESULTADOS

31. • 162 (60 %)
• Clon Chileno -
relación genética
(>78,8%)
PATRÓN F
• 86(31,8%)
• USA300-0114 –
relación genética
(>79%)
PATRÓN U
• 4(1,5%)
• Clon pediátrico–
relación genética
(79%)
PATRÓN D
RESULTADOS

32. • ST1110 - 2 aislamientos
Clon Chileno – gen arcC
• ST1111 – 1 aislamiento
Clon Pediátrico – gen arcC
ANÁLISIS POR MLST
RESULTADOS

33. RESULTADOS
TIPIFICACION POR SCCmec
Patrón U
82 – Tipo IVc / 3 – Tipo IVa / 1 – Tipo
IVb
Patrones F y D
Tipo I y IV
Patrón G
9 – Tipo II
1- Tipo I

34. RESULTADOS
Factores de Virulencia
• Genes sem, sen y seo (184), seg (173), sei (154) ------- Grupo de genes de la
enterotoxina ( EGC )
• Toxina exfoliativa A (52 CA-MRSA y 171 HA-MSRA)
• Toxina exfoliativa B (4)
• Genes sej and seg (2 HA-MRSA)
• Genes sek, seq y lukF-PV/luckS-PS (CA –MRSA)
• Proteínas de unión a fibronectina A y B (CA-MRSA)

35. RESULTADOS

36. RESULTADOS

37. RESULTADOS
MORTALIDAD • Relación entre la mortalidad y la
infección por HA – MRSA en
comparación con CA-MRSA ( 21 vs
10,6 %)
• Aumento en mortalidad en pacientes
con bacteriemia (45,9%), neumonía (
43,8%), en comparación con infección
en el sitio quirúrgico (10,8%)
• Existen relación directos entre
factores de virulencia y patologías
desarrolladas

38. *DISCUSSION

39. DISCUSSION
INVESTIGATORS IDEA AGREE OR DISAGREE
Ferry T, Thomas D,
Genestier AL, Bes M, et al.
Described a greater
prevalence of the egc –
positive isolates recovered
from surfaces infections
compared to invasive
infections.
Ferry T, Thomas D,
Genestier AL, Bes M, et al
Observed a negative
relationship between the
egc cluster and clinical
severity

40. DISCUSSION
INVESTIGATORS IDEA AGREE OR DISAGREE
Van Belkun A, Melles DC,
Snijders SV et al
They did not find any
evidence of an association
of the egc cluster with
mortlity in patients with
MRSA- related bacteremia.
Bae IG, Tonthat GT,
Stryjewsky ME et al
They found a paradoxically
higther rate of clinical
healing through the
presence of some adhesive
genes

41. *CONCLUSIONS

42. CONCLUSIONS
*Staphylococcus aureus is a microorganism
that causes problems in hospitals, because
when people go to the hospital for many
surgeries can be infected with the organism
*The high quantity of isolates of MRSA are a major problem for a
treatment, this microorganism is difficult to eradicate because when we
want kill the pathogen agent, we found that each clones of MRSA has a
important number of isolates with genomic differences between its.

43. CONCLUSIONS
*The types of MRSA ( USA300-0114, pediatric y
Chilean ) has many genes that generate the
different between its, that to permite separate
the isolates in families. The genetic variability
is essential as a survival mechanism of the
organism.
*This article is very important because the investigators developed a
study for explain the molecular and genetic causes in Staphylococcus
aureus infection, based in identify the genetic variability, then when
they identified the genes that generate differences between clones, the
investigators found the relationship between genes and diseases

45. Manuela Rendón Díez