Presentación Tesis Doctoral 21-6-11

Epidemiologa molecular y bases genticas de la
resistencia y virulencia de Staphylococcus aureus

de diferente origen
Mara de los ngeles Argudn Regueiro

Tesis Doctoral
Universidad de Oviedo
Junio, 2011
Staphylococcus aureus
Objeto de estudio: Staphylococcus aureus
o Clasificacin taxonmica: Dominio Bacteria
Phylum XIII Firmicutes
Clase I Bacilli
Orden I Bacillales
Familia VIII Staphylococcaceae
Especie Staphylococcus aureus
o Caractersticas: Cocos (0,8-1 m ), Gram positivos, bajo contenido GC.

Agrupaciones en racimos irregulares, inmvil y sin esporas.
Anaerobio facultativo y mesofilo
Introduccin
Patologas:
o Infeccin directa (superficiales en piel y profundas)
o Infeccin invasiva (bacteriemias, sepsis e infeccin metastsica)
o Enfermedades mediadas por toxinas
Asociadas a hospital (HA)

Asociadas a comunidad (CA)
Estado de portador: Microbiota normal persistente o transente de la mucosa

nasofarngea (piel y otras reas corporales) Tasa de portadores 20-40%
S. aureus Virulencia
Amplia gama de determinantes de virulencia:
o Componentes de la pared celular:
Pptidoglicano
cidos Teicoicos
Protenas de superficie (protena A)
Cpsula externa
Introduccin
o Enzimas:
Catalasa
Coagulasas
Estafiloquinasas
Hialuronidasas
Lipasas
Proteasas
Termonucleasas
Protenas de superficie
Enzimas y toxinas
Coagulasa
Protena A
Protena de unin
a elastina
Protena fijadora
de colgeno
S. aureus
Protenas fijadora
de fibronectina
Factor de agregacin
Amplia gama de determinantes de virulencia:
o Toxinas:
Citotoxinas
Hemolisinas (genes hla, hlb, hld, hlg, hlg-v)
Leucotoxinas LukS-PV/LukF-PV (lukPV)
LukE/LukD (lukED)
LukM/LukF-PV (lukM)
Exfoliatinas Infecciones en piel y SSSS

ETA (eta)
ETB (etb)
ETD (etd)
Lesiones cutneas y neumona necrotizante

Antgeno
convencional
Superantgenos
Clula presentadora de
antgenos
Clase II MHC
Pptido de un
antgeno
convencional
Lisosoma
Superantgenos
Introduccin
TSST-1 (tst)
Enterotoxinas Intoxicacin alimentaria
SEs: SEA, SEB, SEC, SED, SEE, SEG, SEH, SEI, SER, SES, SET
SEls: SElJ, SElK, SElL, SElM, SElN, SElO, SElP, SElQ, SElU
Linfocito T
colaborador
Elementos genticos mviles (EGM):
o Islas genmicas: vSa (lukED egc)
vSa
II
Introduccin
III
Cluster sern-proteasas
2
2
sdS hsdM
h
1
1
G
A B C D F
sau
sau
spl spl spl spl spl
bsa
Cluster egc
E
luk
D
luk
2
2
dS
dM
1 h s a u 1 h s p lF p lC p lB p lA
u
a
s
s
s
s s s
S2
M2
h s d u 1 h s d lF lE
1
u
sa
sa
sp sp
o Islas de patogenicidad:
SaPI (etd)
SaPI1 (ear-tst-selk-selq)
SaPI2 (tst)
SaPI3 (ear-seb-selk-selq)
SaPI5 (ear-selk-selq)
SaPIn1/m1 (ear-tst-sec-sell)
SaPImw2 (ear-sec-sell)
SaPIbov1 (tst-sec-sell)
SaPI1
SaPI2
SaPI3
SaPI5
SaPIm1/n1
SaPImw2
SaPIbov1
n
seg sel
m o
sei sel sel tRNA
Cluster lantibiticos
G E F P
D C
bsa bsa bsa bsa bsa bsa
spl
attL
A2 A 1 E
D
bsa bsa luk luk
tRNA
n u
m o
seg sel sel sei sel sel tRNA
ear
ts t
te r
attL
tst
attL
e a r s e b te r
attL
ear
attL
sel
attL
l2 4
sel sec ear
attL
bsa
sel
ter
rep
sec
sec
ear
attR
q k
sel sel
int
q k
sel sel
rep
ts t
te r
te r
tst
ter
in t
int
rep
ter
l
q k
sel sel
rep
int
int
int
rep
attR
attR
rep
rep
attR
attR
attR
int
attR
Elementos genticos mviles (EGM):
o Profagos: Familia Siphoviridae, clasificacin:
Integrasa tipo Sa1: eta
Integrasa tipo Sa2: lukPV
Integrasa tipo Sa3: sea, sea-selk-selq, sep
Integrasa tipo Sa5: lukM
o Plsmidos:
pETB (etb)
pZA10 (seb)
pIB485-like (sed-selj-ser)
pF5-like (selj-ser-ses-set)
Introduccin
o Casetes cromosmicos estafiloccicos (SCC): MGEmw2/mssa476 (seh, seo)
MGEmssa476
MGEmw2
elo eh
s
elo eh
s
attL
attL
A1
ccr ccrB
attR
A1
ccr ccrB
2
I
127 e cR A
IS m mec
IS4
31
attR
Regulacin accessory gene regulation, agr
o Accin dual durante fase estacionaria:
Represin transcripcin genes que codifican protenas asociadas a la pared
Activacin transcripcin genes de exoprotenas (enzimas y toxinas)
B
agr
D
agr
C
agr
A
agr
P2 P3
Introduccin
o Diferencias definen 4 grupos: agrI, agrII, agrIII, agrIV (+ tipos mixtos y negativos)
o Enfermedades, resistencias y factores de virulencia determinado agr
Susceptibilidad reducida a vancomicina agrI y agrII
TSST-1 agrIII
LukPV + Neumona necrotizante agrIII
Exfoliatinas agrIV
S. aureus Resistencia
Tratamiento con agentes antimicrobianos:
Sntesis de cidos nucleicos

Quinolonas
Rifamicinas
Sntesis de la pared bacteriana

-lactmicos
Glucopptidos
ADN
THFA
ARNm
Membrana citoplasmtica
Lipopptidos
Introduccin
Rutas metablicas
Sulfonamidas
Diaminopirimidinas
DHFA
Ribosomas
PABA
50
30
50
30
50
30
Sntesis proteica
Aminoglicsidos
Fenicoles
Macrlidos
Lincosamidas
Estreptograminas
Mupirocina
Oxazolidinonas
Tetraciclinas
Glicilciclinas
Resistencia a antimicrobianos:
-lactamasa
1942: R-penicilina
EARSS MRSA 2008
S. aureus
60s: Penicilinas semisintticas

R- -lactamasas
(meticilina, oxacilina)
Penicillium
Introduccin
1928: Descubrimiento
penicilina
Otros
antimicrobianos
50
Era Pre-antibitica
Hospital HA-MRSA
(S. aureus R-meticilina) Comunidad CA-MRSA
60s: MRSA
R-tetraciclina
R-macrlidos, MLS
R-aminoglicsidos
Glucopptidos
R-quinolonas
R-rifampicina
1996: VISA
2002: VRSA
(I-vancomicina)(R-vancomicina)
Otros
antimicrobianos
60
70
80
Era Antibitica
90
00
?
oEnzimas inactivadoras y modificadoras
Resistencia a antimicrobianos:
blaZ-lactmicos
aphA-3 , aadD y aacA-aphDAminoglucsidos
catFenicoles
mphCMacrlidos
vatA y vatBEstreptograminas A
vgbA y vgbBEstreptograminas B
linA/linALincosamidas
Bomba
expulsin
Antibitico
oBombas de expulsin
Diana
alterada
fexAFenicoles
msrA y msrBMacrlidos
vgaA, vgaB y vgaCEstreptograminas A
tetK y tetLTetraciclinas
mepATigeciclina
norA, norB, norC, mepA y sdrMQuinolonas
Enzima
inactivadora
Antibitico
Enzima
modificadora
Antibitico
Introduccin
Antibitico
oAlteracin de la diana
mecA-lactmicos
vanAGlucopptidos
ermMacrlidos, lincosaminas y estreptograminas B
cfrFenicoles, estreptograminas A, lincosamidas,
oxazolidinonas
ileS y mupAMupirocina
tetM y tetOTetraciclinas
grlA, grlB, gyrA y gyrBQuinolonas
rpoBRifamicinas
dfrA (S1), dfrB (D), dfrK y dfrGDiaminopirimidinas
sulASulfonamidas
Resistencia a antimicrobianos: S. aureus resistente a meticilina (MRSA)-SCCmec
SCCmec I (34.3 kb)

clase B
J1
SCCmec II (53.0 kb)
J2
pUB110
J3
HA-MRSA
Tn554
HA-MRSA
Clase A
SCCmec III (66.9 kb)
Tn554
Operon mer
pT181
Tn554
Clase A
SCCHg
CA-MRSA
SCCmec IV (20.9-24.3 kb)

clase B
Complejo mec (mecA mecRI y mecI)

CA-MRSA
Complejo ccr con genes de recombinasas (ccrAB y/o ccrC)
SCCmec V (28 kb)

Clase C2
SCCmec VI (20.9 kb)
Junkyard or joining regions (J) determinan variantes
Introduccin
clase B
SCCmec VII (35.9 kb)

Clase C1
SCCmec VIII (32.2 kb)
Tn554
Clase A
S. aureus Linajes
Estructura poblacional altamente clonal: linajes o complejos clonales (CCs)
o MLST (multilocus sequence typing)
Perfil allico arcC, aroE, glpF, gmk, pta, tpi, yqiL: secuencia tipo (ST)
STs con 5-7 alelos en comn definen un CC
CC45
CC22
ST239
CC15
CC1
CC5
CC30
ST8
CC8
CC25
CC121
CC12
eBURST CC8
http://www.mlst.net/
Introduccin
o Importancia distincin CCs: Adquisicin SCCmec Clones HA-MRSA y CA-MRSA

o Subdivisin o diferenciacin intra-especie Tcnicas tipificacin
Tipificacin spa: secuenciacin del gen protena A
Macrorrestricin-electroforesis en campo pulsante (PFGE)
Objetivos
Diferentes poblaciones S. aureus:
oAislamientos clnicos de 2 hospitales:

81, periodo 2005-2006 HUCA
62, periodo 1997-2006 HMN
oAislamientos de portadores sanos (PS)
68, periodo 1997-2002
43, periodo 2004-2006
oAislamientos de alimentos/manipuladores (ALM)
64, periodo 1997-2008
oAislamientos de animales (AN) (periodo 2004-2008)

105, clon ST398
4, otros STs
Federal Institute for Risk Assessment

Berln
Total: 427 aislamientos
Objetivos
Objetivo 1. Identificar los linajes y clones de S. aureus de distinto origen

(hospitales, portadores sanos y relacionados con alimentos) que han
estado
circulando en el Principado de Asturias, y sub-tipificar el linaje
ST398 de origen
animal.
Objetivo 2. Identificar determinantes de virulencia en los linajes encontrados en el
Principado de Asturias y en el clon ST398, y establecer su relacin con
elementos genticos mviles.
Objetivo 3. Determinar los fenotipos y genotipos de resistencia (prestando
especial atencin al SCCmec) en los linajes encontrados en el Principado de
Asturias y en el clon ST398.
Objetivo 4. Caracterizar mediante tcnicas moleculares el plsmido hbrido de
virulencia-resistencia pUO-Sa-SED3.
Resultados y Discusin
Objetivo 1. Linajes
Linajes circulantes en el Principado de Asturias
o MLST 112 aislamientos (HUCA, HMN, PS, ALM)- 31 STs
o Secuenciacin spa 294 aislamientos (HUCA, HMN, PS, ALM)- 85 Tipos spa
CC
ST
Tipos spa
CC
ST
Tipos spa
CC1
ST1
ST3
t127, t3201
t177
CC25
ST25
ST26
t078, t081, t167, t1054

t7125
nd
t078, t081, t287, t6489
ST30
ST34
t012, t017, t018, t021, t122, t840

t136, t166, t6490
ST188 t189
CC5
ST5
ST6
t001, t002, t045, t179, t548, t2173, t6488

t701
ST125 t067, t3734
ST605 t275
ST146 t002, t1560
nd
t018, t012, t021, t253, t942, t1515, t6713, t7785
ST45
t015, t026, t040, t050, t065, t350, t1078, t1618
ST1619 t166
ST47
t383
nd
t001, t002, t067, t071, t242, t306, t1340
ST546 t604
ST8
t008, t024, t351
nd
ST228 t109
CC8
CC30
ST239 t037
ST247 t051
CC45
CC59
ST59
t015, t026, t050, t282, t798, t1618, t1494,

t1618
t216
nd
t3952
Objetivo 1. Linajes
Linajes circulantes en el Principado de Asturias:
CC1, CC5, CC8, CC9, CC12, CC15, CC22, CC25, CC30, CC45, CC59, CC72, CC88, CC97,
CC121
HUCA (N=81)
HMN (N=62)
PS (N=111)
ALM (N=64)
CC30
Objetivo 1. Linajes
Linajes circulantes en hospitales del Principado de Asturias:
CC1, CC5, CC8, CC12, CC15, CC25, CC30, CC45, CC59, CC72, CC88, CC121
HUCA (N=81)
HMN (N=62)
CC5
36%
CC5
57%
CC8
19%
2005-2006
1996-2006
100
CC121
CC97
CC88
CC72
CC59
CC45
CC30
CC25
CC22
CC15
CC12
CC9
CC8
CC5
90
80
70
60
50
40
30
20
10
0
1996
1997
1998
1999
2000
2001
2002
2003
2004
2005
2006
Objetivo 1. Linajes
Linajes circulantes en la comunidad del Principado de Asturias:
CC1, CC5, CC8, CC9, CC15, CC22, CC25, CC30, CC45, CC59, CC72, CC97, CC121
ALM (N=64)
PS (N=111)
CC45
16%
CC30
27%
CC5
19%
CC45
38%
CC5
20%
CC30
CC30
20%
kb
582.0
398.0
CC5
242.5
194.0
145.5
97.0
48.5
J
0.28
0.4
0.52
0.64
HUCA
0.76
0.88
CC5
CC45
CC8
CC72
CC1/9/97
J
0,28
0,4
0,52
0,64
0,76
PS
0,88
kb
145.5
97.0
48.5
PSS150
mecA
PSS149
PSS148
PSS147
194.0
PSS146
PSS145
582.0
398.0
242.5
CC45
HUCAS50
HUCAS49
HUCAS48
HUCAS47
HUCAS46
PS-MSSA
HUCAS44
HUCA-MRSA
HUCAS45
CC30
CC59 CC72
HUCA-S7
PS-S129
HUCA-S26
PS-S127
HUCA-S33
PS-S5
HUCA-S4
PS-S33
HUCA-S49
PS-S31
HUCA-S19
PS-S26
HUCA-S48
PS-S145
S0
HUCAS43
HUCAS42
HUCAS39
HUCAS40
HUCAS41
HUCAS37
HUCAS38
HUCAS36
HUCAS35
CC72
CC5
PSS144
PSS143
PSS142
PSS141
PSS140
PSS139
PSS138
PSS137
PSS136
PSS135
PSS134
PSS133
PSS132
PSS131
PSS130
CC25
S58
S57
S28
S143
S142
S136
S25
S13
S127 (3)
S5
S7
S11 (3)
S9
S4
S126 (2)
S3 (11)
S138
S75
S134
S27
S26
S146
S139
S62 (2)
S70 (6)
S73 (2)
S71 (2)
S65
S72
S148
S63
S23
S131 (2)
S56
S55 (4)
S149
S129
S128
S130
S52 (4)
S53
S51 (6)
S54
S42 (3)
S140
S132
S33 (2)
S32
S31 (11)
S18
S0
S144
S145
S141
S137
S74 (2)
S60
S133 (2)
S150
S147
S15
S135
HUCAS31
HUCAS32
HUCAS33
HUCAS34
HUCAS30
HUCAS29
HUCAS27
HUCAS28
HUCAS26
HUCAS22
HUCAS23
HUCAS24
HUCAS25
HUCAS20
HUCAS21
PSS129
HUCAS14
HUCAS15
HUCAS16
HUCAS17
HUCAS18
HUCAS19
CC15
PSS128
PSS33
CC12
PSS31
PS
PSS11
CC15
CC59
PSS127
CC45
48.5
CC30
CC30
PSS126
97.0
CC121
PSS70
CC5
PSS55
242.5
194.0
PSS52
HUCAS12
HUCAS13
CC30
582.0
388.0
145.5
S44 (1)
S43 (2)
S45 (1)
S40 (1)
S12 (1)
S31 (1)
S33 (1)
S37 (1)
S35 (1)
S42 (1)
S26 (6)
S5 (4)
S41 (2)
S19 (1)
S46 (1)
S15 (1)
S11 (1)
S14 (1)
S13 (1)
S10 (1)
S21 (1)
S49 (2)
S4 (1)
S0 (1)
S48 (1)
S32 (1)
S47 (2)
S3 (1)
S36 (1)
S17 (1)
S2 (1)
S25 (3)
S20 (1)
S30 (1)
S50 (1)
S28 (3)
S18 (3)
S23 (1)
S7 (3)
S39 (2)
S29 (1)
S27 (1)
S22 (1)
S6 (7)
S34 (3)
S16 (1)
S8 (2)
S38 (1)
S9 (1)
S24 (2)
S1 (1)
PSS51
kb
HUCAS11
HUCAS9
HUCAS10
HUCAS7
HUCAS8
CC45
HUCAS5
HUCAS6
HUCAS4
HUCAS3
HUCA
HUCAS1
HUCAS2
o Macrorrestriccin genmica SmaI y Electroforesis en Campo Pulsante (PFGE)

192 aislamientos (HUCA, PS)- 105 Pulsotipos
PSS3
Objetivo 1. Linajes
Objetivo 1. Linajes
o Test Restriccin-Modificacin (RM) 237 aislamientos (HMN, PS, ALM)- 7 tipos
RM test1
RMtest2
RMtest3
CC30/ST36
CC22
CC45
CC1
CC8/239
CC5
203
990
AF
sau1CC22hsdS1
AR22
AF 722
sau1CC45hsdS1
AR45
AF 1037
sau1CC1hsdS1
AR1
BF
sau1CC1hsdS2
BR8
BF
sau1CC8hsdS2
BR8
BF
sau1CC5hsdS2
BR5
680
680
1071
AF
sau1CC30hsdS1
AR30
Poblacin (%)
CCs
60-80%
sau1hsdS1
Identificacin correcta
No tipificacin
Falsos negativos
Asignaciones errneas
HMN (79%), PS (61,3%), ALM (79,7%)

HMN (21%), PS (20,7%), ALM (12,5%)
PS (5,4%)
HMN (3,2%), PS (12,6%), ALM (7,8%)
CC1, CC5, CC8, CC22, CC30, CC45

CC9, CC15, CC59, CC72, CC88, CC97, CC121
CC1, CC30, CC45
CC5, CC25
Objetivo 1. Linajes
Linajes en aislamientos de animales (AN)
o MLST 23 aislamientos- 4 STs

o Secuenciacin spa 109 aislamientos- 22 Tipos spa
CC
CC5
CC9
105 CC30
CC398
ST
Tipos spa
ST5
ST9
ST433
ST398
t002
t337, t1430
t318
t011, t034, t108, t571, t779, t1250, t1255, t1451, t1457, t1580, t1793, t1928, t1985, t2011, t2346, t2576, t2970, t5210
o Macrorrestriccin genmica SmaI-PFGE

105 aislamientos ST398
CC398
Objetivo 1. Linajes
Linajes en aislamientos de animales (AN)
o Macrorrestriccin genmica Cfr9I-PFGE
105 aislamientos CC398- 35 Pulsotipos
SCCmec V*
(n>1)
Perfil Cfr9I
SCCmec IVa
SCCmec V
SCCmec nt
(6) (3)
(3) (2)
(3)
N C9 C11 C13 C29 N C4 C15 C30 N C3 C17 C32 N
SCCmec V
(2)
N C23 C24 C25 C26 C27 N C28 C31 C33 C34 C35 N
ATCC
S3
S2
S4
NCTC
S1
C28
C22
C31
C19
C27
C35
C33
C34
C6 (16)
C21
C10 (2)
C12 (9)
C5 (8)
C25
C24 (2)
C23
C20 (2)
C18 (6)
C2 (2)
C8 (2)
C16 (4)
C14 (6)
C26
C1 (11)
C32
C15 (3)
C4
C7
C13
C29
C11 (3)
C9 (6)
C30 (2)
C17
C3 (3)
kb
kb
674
674
361
361
262
262
208
208
175
175
135
135
80
80
44
44
SCCmec V
SCCmec V
SCCmec V
(11) (2) (8) (16)

(2) (2) (9) (6) (4)
(6)
(2)
N C1 C2 C5 C6 C7 N C8 C10 C12 C14 C16 N C18 C19 C20 C21 C22 N
0,28
0,4
0,52
0,64
0,76
0,88
IVa
nt
V
V
V
V
V
V
V
V
V (16)
V
V (2)
V (9)
V (8)
V
V (2)
V
V (2)
V (6)
V (2)
V
V, nt (2), V (6
V
V (11)
IVa (2), nt
IVa
V
V*
V*
V* (3)
V* (6)
IVa (2)
nt
nt (3)
Objetivos

estado
ST398 de origen
animal.
Objetivos
Objetivo 2. Virulencia
359 aislamientos [HUCA, HMN, PS (2004-2006), ALM, AN]
Deteccin determinantes de virulencia
Citotoxinas
Hemolisinas (genes hla, hlb, hld, hlg, hlg-v)
Leucotoxinas (genes lukPV, lukED, lukM)
Exfoliatinas (eta, etb, etd)

Superantgenos
TSST-1 (tst)
Enterotoxinas SEs (sea, seb, sec, sed, see, seg, seh, sei, ser, ses, set)
SlEs (selj, selk, sell, selm, seln, selo, selp, selq, selu)
Marcadores vSa: splF, bsaB

Marcadores SaPIs: ear
Hemolisinas-ampliamente distribuidas
Hemolisinas-ampliamente distribuidas
vSa
I
II
III
spl
E
luk
spl
D
luk
n
seg sel
m o
sei sel sel
bsa
spl
n u
m o
seg sel sel sei sel sel
E
D
luk luk
vSa
I
II
III
spl
E
luk
spl
D
luk
n
seg sel
m o
sei sel sel
bsa
spl
n u
m o
E
D
luk luk
vSa
I
II
III
spl
E
luk
spl
D
luk
n
seg sel
m o
sei sel sel
bsa
spl
n u
m o
E
D
luk luk
vSa
I
II
III
spl
E
luk
spl
D
luk
n
seg sel
m o
sei sel sel
bsa
spl
n u
m o
E
D
luk luk
vSa
I
II
III
spl
E
luk
spl
D
luk
n
seg sel
m o
sei sel sel
bsa
spl
n u
m o
E
D
luk luk
vSa
I
II
III
spl
E
luk
spl
D
luk
n
seg sel
m o
sei sel sel
bsa
spl
n u
m o
E
D
luk luk
vSa
I
II
III
spl
E
luk
spl
D
luk
n
seg sel
m o
sei sel sel
b
spl
saB
n u
m o
CC5 y CC15
E
D
luk luk
CC30 y CC121
vSa
I
II
III
spl
E
luk
spl
D
luk
n
seg sel
m o
sei sel sel
b
spl
saB
n u
m o
CC5 y CC15
E
D
luk luk
CC30 y CC121
Otras combinaciones:
splF-bsaB-lukED-egc1
splF-bsaB-egc1 splF-bsaB lukED

splF-bsaB-egc2 splF-egc1 egc1
splF-lukED-egc2 splF-lukED egc2
bsaB-lukED-egc1 lukED-egc1
Exfoliatinas y Leucotoxinas
eta (Profago Sa1)
etb (Plsmido pETB)
eta (Profago Sa1)
etb (Plsmido pETB)
etd (SaPI)
eta (Profago Sa1)
lukM (Profago Sa5)
etb (Plsmido pETB)
etd (SaPI)
eta (Profago Sa1)
lukM (Profago Sa5)
etb (Plsmido pETB)
lukPV (Profago Sa2)
etd (SaPI)
Gen tst y genes de enterotoxinas en SaPIs
SaPI2 (tst)
CC30, CC59
CC1, CC5, CC8, CC9, CC12, CC15, CC25,
CC30, CC45, CC88, CC121
SaPI3 (ear-seb-selk-selq) CC5, CC8, CC59
CC5, CC8, CC59
CC25, CC45, CC59, CC72
SaPIn1/m1 (ear-tst-sec-sell) CC45, CC59
SaPI2 (tst)
CC30, CC59
CC1, CC5, CC8, CC9, CC12, CC15, CC25,
CC30, CC45, CC88, CC121
CC5, CC8, CC59
CC25, CC45, CC59, CC72

SaPI2 (tst)
CC30, CC59
CC1, CC5, CC8, CC9, CC12, CC15, CC25,
CC30, CC45, CC88, CC121
CC5, CC8, CC59
CC25, CC45, CC59, CC72
SaPI2 (tst)
CC30, CC59
CC1, CC5, CC8, CC9, CC12, CC15, CC25,
CC30, CC45, CC88, CC121
CC5, CC8, CC59
CC25, CC45, CC59, CC72
SaPI2 (tst)
CC30, CC59
CC1, CC5, CC8, CC9, CC12, CC15, CC25,
CC30, CC45, CC88, CC121
CC5, CC8, CC59
CC25, CC45, CC59, CC72
Genes de enterotoxinas en profagos Sa3
Genes de enterotoxinas en elementos SCC
Genes de enterotoxinas en plsmidos
pIB485 (sed-seljser) CC1, CC5, CC8, CC9, CC15, CC25, CC30, CC45, CC59, CC72, CC121
pF5 (selj-ser-ses-set) CC5
Distribucin agr
o 427 aislamientos (HUCA, HMN, PS, ALM, AN)
Distribucin agr
agrI
agrII
agrIII
agrIV
CC8, CC22, CC45, CC72, CC398

CC5, CC15
CC1, CC30, CC88
CC25, CC59, CC121
Distribucin agr
Excepciones:
agrI
agrII
agrIII
agrIV
agrI-agrIV
CC8, CC22, CC45, CC72, CC398

CC5, CC15
CC1, CC30, CC88
CC25, CC59, CC121
CC1, CC5, CC25, CC30, CC97

CC1, CC9, CC12, CC25, CC45, CC97, CC121
CC5, CC9, CC12, CC45, CC121
CC5, CC8, CC12, CC15, CC45
CC59, CC72
Objetivos

estado
ST398 de origen
animal.
Objetivo 3. Resistencia
Resistencia a meticilina
o 49 aislamientos MRSA (HUCA, HMN, ALM)
HUCA (81): 32.1%
HMN (62): 33.9%
PS (111): 0%
ALM (64): 3%
HUCA (81): 32.1%
HMN (62): 33.9%
PS (111): 0%
ALM (64): 3%
HUCA (81): 32.1%
HMN (62): 33.9%
PS (111): 0%
ALM (64): 3%
HUCA (81): 32.1%
HMN (62): 33.9%
PS (111): 0%
ALM (64): 3%
CC
Clon
ST
SCCmec
CC5
Nueva York/Japn (USA 100)

Peditrico (USA 800)
5
5
II
IVc
Peditrico (USA 800)
IVd
125
III
125
IVa
125
IVc
125
Alemania-Sur
228
Brasileo/Hngaro
239
III
CC8
HUCA (81): 32.1%
HMN (62): 33.9%
PS (111): 0%
ALM (64): 3%
CC
Clon
ST
SCCmec
CC5

Peditrico (USA 800)
5
5
II
IVc
Peditrico (USA 800)
IVd
125
III
125
IVa
125
IVc
125
Alemania-Sur
228
Brasileo/Hngaro
239
III
CC8
HUCA (81): 32.1%
HMN (62): 33.9%
PS (111): 0%
ALM (64): 3%
CC
Clon
ST
SCCmec
CC5

Peditrico (USA 800)
5
5
II
IVc
Peditrico (USA 800)
IVd
125
III
125
IVa
125
IVc
125
Alemania-Sur
228
Brasileo/Hngaro
239
III
CC8
HUCA (81): 32.1%
HMN (62): 33.9%
PS (111): 0%
ALM (64): 3%
CC
Clon
ST
SCCmec
CC5

Peditrico (USA 800)
5
5
II
IVc
Peditrico (USA 800)
IVd
125
III
125
IVa
125
IVc
125
Alemania-Sur
228
Brasileo/Hngaro
239
III
CC8
HUCA (81): 32.1%
HMN (62): 33.9%
PS (111): 0%
ALM (64): 3%
CC
Clon
ST
SCCmec
CC5

Peditrico (USA 800)
5
5
II
IVc
Peditrico (USA 800)
IVd
125
III
125
IVa
125
IVc
125
Alemania-Sur
228
Brasileo/Hngaro
239
III
CC8
HUCA (81): 32.1%
HMN (62): 33.9%
PS (111): 0%
ALM (64): 3%
CC
Clon
ST
SCCmec
CC5

Peditrico (USA 800)
5
5
II
IVc
Peditrico (USA 800)
IVd
125
III
125
IVa
125
IVc
125
Alemania-Sur
228
Brasileo/Hngaro
239
III
CC8
o 105 aislamientos MRSA (AN)
CC
ST
CC5
5
CC9
9
CC398 398
398
SCCmec
nd
IVa
IVa
V
398 V*
398 nd
o 105 aislamientos MRSA (AN)
CC
ST
CC5
5
CC9
9
CC398 398
398
SCCmec
nd
IVa
IVa
V
398 V*
398 nd
Resistencia a antimicrobianos
o No se encontraron resistencias frente a: Vancomicina, Linezolid, Tigeciclina
o No se encontraron resistencias frente a: Vancomicina, Linezolid, Tigeciclina

o Multirresitencia (a 4 o ms grupos): 44% aislamientos HUCA/HMN
2,5% aislamientos PS/ALM
68% aislamientos AN
o 284 aislamientos (PS, ALM, AN)
Resistencia
Gen(es)
Poblacin (%)
[PEN-AMP]
[AMK-GEN-KAN-TOB]
[AMK-KAN-TOB]
KAN
CHL
blaZ
aacA-aphD
aadD
aphA-3
catpC194
catpC221
PS (89,2), ALM (75), AN (93,6)

PS (5,4), ALM (1,6), AN (11,9)
PS (2,7), ALM (1,6), AN (35,8)
PS (24,3), ALM (3,1), AN (2,8)
PS (1,8)
PS (0,9)
fexA
AN (3,1)
cfr
AN (1,6)
ermA
ermB
AN (33,9)
PS (1,8), AN (30,3)
ermC
PS (7,2), AN (39,4)
[ERY-CLII]
ermA
ermC
ALM (3,1)
PS (10,8), ALM (12,5)
ERY
msrA
msrB
PS (4,5), ALM (3,1)

PS (20,7), ALM (4,7)
CLI
MUP
TET
linA/linA
mupA
tetK
tetL
PS (5,4), ALM (1,6), AN (3,7)

ALM (1,6)
PS (11,7), ALM (6,3), AN (69,7)
AN (38,5)
[ERY-CLIC]
Resistencia
Gen(es)
Poblacin (%)
[PEN-AMP]
[AMK-GEN-KAN-TOB]
[AMK-KAN-TOB]
KAN
CHL
blaZ
aacA-aphD
aadD
aphA-3
catpC194
catpC221
PS (89,2), ALM (75), AN (93,6)

PS (5,4), ALM (1,6), AN (11,9)
PS (2,7), ALM (1,6), AN (35,8)
PS (24,3), ALM (3,1), AN (2,8)
PS (1,8)
PS (0,9)
fexA
AN (3,1)
cfr
AN (1,6)
ermA
ermB
AN (33,9)
PS (1,8), AN (30,3)
ermC
PS (7,2), AN (39,4)
[ERY-CLII]
ermA
ermC
ALM (3,1)
PS (10,8), ALM (12,5)
ERY
msrA
msrB
PS (4,5), ALM (3,1)

PS (20,7), ALM (4,7)
CLI
MUP
TET
linA/linA
mupA
tetK
PS (5,4), ALM (1,6), AN (3,7)

ALM (1,6)
PS (11,7), ALM (6,3), AN (69,7)
[ERY-CLIC]
Resistencia
Gen(es)
Poblacin (%)
[PEN-AMP]
[AMK-GEN-KAN-TOB]
[AMK-KAN-TOB]
KAN
CHL
blaZ
aacA-aphD
aadD
aphA-3
catpC194
catpC221
PS (89,2), ALM (75), AN (93,6)

PS (5,4), ALM (1,6), AN (11,9)
PS (2,7), ALM (1,6), AN (35,8)
PS (24,3), ALM (3,1), AN (2,8)
PS (1,8)
PS (0,9)
fexA
AN (3,1)
cfr
AN (1,6)
ermA
ermB
AN (33,9)
PS (1,8), AN (30,3)
ermC
PS (7,2), AN (39,4)
[ERY-CLII]
ermA
ermC
ALM (3,1)
PS (10,8), ALM (12,5)
ERY
msrA
msrB
PS (4,5), ALM (3,1)

PS (20,7), ALM (4,7)
CLI
MUP
TET
linA/linA
mupA
tetK
PS (5,4), ALM (1,6), AN (3,7)

ALM (1,6)
PS (11,7), ALM (6,3), AN (69,7)
[ERY-CLIC]
Resistencia
Gen(es)
Poblacin (%)
[PEN-AMP]
[AMK-GEN-KAN-TOB]
[AMK-KAN-TOB]
KAN
CHL
blaZ
aacA-aphD
aadD
aphA-3
catpC194
catpC221
PS (89,2), ALM (75), AN (93,6)

PS (5,4), ALM (1,6), AN (11,9)
PS (2,7), ALM (1,6), AN (35,8)
PS (24,3), ALM (3,1), AN (2,8)
PS (1,8)
PS (0,9)
fexA
AN (3,1)
cfr
AN (1,6)
ermA
ermB
AN (33,9)
PS (1,8), AN (30,3)
ermC
PS (7,2), AN (39,4)
[ERY-CLII]
ermA
ermC
ALM (3,1)
PS (10,8), ALM (12,5)
ERY
msrA
msrB
PS (4,5), ALM (3,1)

PS (20,7), ALM (4,7)
CLI
MUP
TET
linA/linA
mupA
tetK
PS (5,4), ALM (1,6), AN (3,7)

ALM (1,6)
PS (11,7), ALM (6,3), AN (69,7)
[ERY-CLIC]
Resistencia
Gen(es)
Poblacin (%)
[PEN-AMP]
[AMK-GEN-KAN-TOB]
[AMK-KAN-TOB]
KAN
CHL
blaZ
aacA-aphD
aadD
aphA-3
catpC194
catpC221
PS (89,2), ALM (75), AN (93,6)

PS (5,4), ALM (1,6), AN (11,9)
PS (2,7), ALM (1,6), AN (35,8)
PS (24,3), ALM (3,1), AN (2,8)
PS (1,8)
PS (0,9)
fexA
AN (3,1)
cfr
AN (1,6)
ermA
ermB
AN (33,9)
PS (1,8), AN (30,3)
ermC
PS (7,2), AN (39,4)
[ERY-CLII]
ermA
ermC
ALM (3,1)
PS (10,8), ALM (12,5)
ERY
msrA
msrB
PS (4,5), ALM (3,1)

PS (20,7), ALM (4,7)
CLI
MUP
TET
linA/linA
mupA
tetK
PS (5,4), ALM (1,6), AN (3,7)

ALM (1,6)
PS (11,7), ALM (6,3), AN (69,7)
[ERY-CLIC]
Resistencia
Gen(es)
Poblacin (%)
[PEN-AMP]
[AMK-GEN-KAN-TOB]
[AMK-KAN-TOB]
KAN
CHL
blaZ
aacA-aphD
aadD
aphA-3
catpC194
catpC221
PS (89,2), ALM (75), AN (93,6)

PS (5,4), ALM (1,6), AN (11,9)
PS (2,7), ALM (1,6), AN (35,8)
PS (24,3), ALM (3,1), AN (2,8)
PS (1,8)
PS (0,9)
fexA
AN (3,1)
cfr
AN (1,6)
ermA
ermB
AN (33,9)
PS (1,8), AN (30,3)
ermC
PS (7,2), AN (39,4)
[ERY-CLII]
ermA
ermC
ALM (3,1)
PS (10,8), ALM (12,5)
ERY
msrA
msrB
PS (4,5), ALM (3,1)

PS (20,7), ALM (4,7)
CLI
MUP
TET
linA/linA
mupA
tetK
PS (5,4), ALM (1,6), AN (3,7)

ALM (1,6)
PS (11,7), ALM (6,3), AN (69,7)
[ERY-CLIC]
Resistencia
Gen(es)
Poblacin (%)
[PEN-AMP]
[AMK-GEN-KAN-TOB]
[AMK-KAN-TOB]
KAN
CHL
blaZ
aacA-aphD
aadD
aphA-3
catpC194
catpC221
PS (89,2), ALM (75), AN (93,6)

PS (5,4), ALM (1,6), AN (11,9)
PS (2,7), ALM (1,6), AN (35,8)
PS (24,3), ALM (3,1), AN (2,8)
PS (1,8)
PS (0,9)
fexA
AN (3,1)
cfr
AN (1,6)
ermA
ermB
AN (33,9)
PS (1,8), AN (30,3)
ermC
PS (7,2), AN (39,4)
[ERY-CLII]
ermA
ermC
ALM (3,1)
PS (10,8), ALM (12,5)
ERY
msrA
msrB
PS (4,5), ALM (3,1)

PS (20,7), ALM (4,7)
CLI
MUP
TET
linA/linA
mupA
tetK
PS (5,4), ALM (1,6), AN (3,7)

ALM (1,6)
PS (11,7), ALM (6,3), AN (69,7)
[ERY-CLIC]
Resistencia
Gen(es)
Poblacin (%)
[PEN-AMP]
[AMK-GEN-KAN-TOB]
[AMK-KAN-TOB]
KAN
CHL
blaZ
aacA-aphD
aadD
aphA-3
catpC194
catpC221
PS (89,2), ALM (75), AN (93,6)

PS (5,4), ALM (1,6), AN (11,9)
PS (2,7), ALM (1,6), AN (35,8)
PS (24,3), ALM (3,1), AN (2,8)
PS (1,8)
PS (0,9)
fexA
AN (3,1)
cfr
AN (1,6)
ermA
ermB
AN (33,9)
PS (1,8), AN (30,3)
ermC
PS (7,2), AN (39,4)
[ERY-CLII]
ermA
ermC
ALM (3,1)
PS (10,8), ALM (12,5)
ERY
msrA
msrB
PS (4,5), ALM (3,1)

PS (20,7), ALM (4,7)
CLI
MUP
TET
linA/linA
mupA
tetK
PS (5,4), ALM (1,6), AN (3,7)

ALM (1,6)
PS (11,7), ALM (6,3), AN (69,7)
[ERY-CLIC]
Resistencia
Gen(es)
Poblacin (%)
[PEN-AMP]
[AMK-GEN-KAN-TOB]
[AMK-KAN-TOB]
KAN
CHL
blaZ
aacA-aphD
aadD
aphA-3
catpC194
catpC221
PS (89,2), ALM (75), AN (93,6)

PS (5,4), ALM (1,6), AN (11,9)
PS (2,7), ALM (1,6), AN (35,8)
PS (24,3), ALM (3,1), AN (2,8)
PS (1,8)
PS (0,9)
fexA
AN (3,1)
cfr
AN (1,6)
ermA
ermB
AN (33,9)
PS (1,8), AN (30,3)
ermC
PS (7,2), AN (39,4)
[ERY-CLII]
ermA
ermC
ALM (3,1)
PS (10,8), ALM (12,5)
ERY
msrA
msrB
PS (4,5), ALM (3,1)

PS (20,7), ALM (4,7)
CLI
MUP
TET
linA/linA
mupA
tetK
PS (5,4), ALM (1,6), AN (3,7)

ALM (1,6)
PS (11,7), ALM (6,3), AN (69,7)
[ERY-CLIC]
Resistencia
Gen(es)
Poblacin (%)
[PEN-AMP]
[AMK-GEN-KAN-TOB]
[AMK-KAN-TOB]
KAN
CHL
blaZ
aacA-aphD
aadD
aphA-3
catpC194
catpC221
PS (89,2), ALM (75), AN (93,6)

PS (5,4), ALM (1,6), AN (11,9)
PS (2,7), ALM (1,6), AN (35,8)
PS (24,3), ALM (3,1), AN (2,8)
PS (1,8)
PS (0,9)
fexA
AN (3,1)
cfr
AN (1,6)
ermA
ermB
AN (33,9)
PS (1,8), AN (30,3)
ermC
PS (7,2), AN (39,4)
[ERY-CLII]
ermA
ermC
ALM (3,1)
PS (10,8), ALM (12,5)
ERY
msrA
msrB
PS (4,5), ALM (3,1)

PS (20,7), ALM (4,7)
CLI
MUP
TET
linA/linA
mupA
tetK
PS (5,4), ALM (1,6), AN (3,7)

ALM (1,6)
PS (11,7), ALM (6,3), AN (69,7)
[ERY-CLIC]
Resistencia
Gen(es)
Poblacin (%)
[PEN-AMP]
[AMK-GEN-KAN-TOB]
[AMK-KAN-TOB]
KAN
CHL
blaZ
aacA-aphD
aadD
aphA-3
catpC194
catpC221
PS (89,2), ALM (75), AN (93,6)

PS (5,4), ALM (1,6), AN (11,9)
PS (2,7), ALM (1,6), AN (35,8)
PS (24,3), ALM (3,1), AN (2,8)
PS (1,8)
PS (0,9)
fexA
AN (3,1)
cfr
AN (1,6)
ermA
ermB
AN (33,9)
PS (1,8), AN (30,3)
ermC
PS (7,2), AN (39,4)
[ERY-CLII]
ermA
ermC
ALM (3,1)
PS (10,8), ALM (12,5)
ERY
msrA
msrB
PS (4,5), ALM (3,1)

PS (20,7), ALM (4,7)
CLI
MUP
TET
linA/linA
mupA
tetK
PS (5,4), ALM (1,6), AN (3,7)

ALM (1,6)
PS (11,7), ALM (6,3), AN (69,7)
[ERY-CLIC]
Resistencia
Gen(es)
Poblacin (%)
[PEN-AMP]
[AMK-GEN-KAN-TOB]
[AMK-KAN-TOB]
KAN
CHL
blaZ
aacA-aphD
aadD
aphA-3
catpC194
catpC221
PS (89,2), ALM (75), AN (93,6)

PS (5,4), ALM (1,6), AN (11,9)
PS (2,7), ALM (1,6), AN (35,8)
PS (24,3), ALM (3,1), AN (2,8)
PS (1,8)
PS (0,9)
fexA
AN (3,1)
cfr
AN (1,6)
ermA
ermB
AN (33,9)
PS (1,8), AN (30,3)
ermC
PS (7,2), AN (39,4)
[ERY-CLII]
ermA
ermC
ALM (3,1)
PS (10,8), ALM (12,5)
ERY
msrA
msrB
PS (4,5), ALM (3,1)

PS (20,7), ALM (4,7)
CLI
MUP
TET
linA/linA
mupA
tetK
PS (5,4), ALM (1,6), AN (3,7)

ALM (1,6)
PS (11,7), ALM (6,3), AN (69,7)
[ERY-CLIC]
Resistencia
Gen(es)
Poblacin (%)
[PEN-AMP]
[AMK-GEN-KAN-TOB]
[AMK-KAN-TOB]
KAN
CHL
blaZ
aacA-aphD
aadD
aphA-3
catpC194
catpC221
PS (89,2), ALM (75), AN (93,6)

PS (5,4), ALM (1,6), AN (11,9)
PS (2,7), ALM (1,6), AN (35,8)
PS (24,3), ALM (3,1), AN (2,8)
PS (1,8)
PS (0,9)
fexA
AN (3,1)
cfr
AN (1,6)
ermA
ermB
AN (33,9)
PS (1,8), AN (30,3)
ermC
PS (7,2), AN (39,4)
[ERY-CLII]
ermA
ermC
ALM (3,1)
PS (10,8), ALM (12,5)
ERY
msrA
msrB
PS (4,5), ALM (3,1)

PS (20,7), ALM (4,7)
CLI
MUP
TET
linA/linA
mupA
tetK
PS (5,4), ALM (1,6), AN (3,7)

ALM (1,6)
PS (11,7), ALM (6,3), AN (69,7)
[ERY-CLIC]
Resistencia
Gen(es)
Poblacin (%)
[PEN-AMP]
[AMK-GEN-KAN-TOB]
[AMK-KAN-TOB]
KAN
CHL
blaZ
aacA-aphD
aadD
aphA-3
catpC194
catpC221
PS (89,2), ALM (75), AN (93,6)

PS (5,4), ALM (1,6), AN (11,9)
PS (2,7), ALM (1,6), AN (35,8)
PS (24,3), ALM (3,1), AN (2,8)
PS (1,8)
PS (0,9)
fexA
AN (3,1)
cfr
AN (1,6)
ermA
ermB
AN (33,9)
PS (1,8), AN (30,3)
ermC
PS (7,2), AN (39,4)
[ERY-CLII]
ermA
ermC
ALM (3,1)
PS (10,8), ALM (12,5)
ERY
msrA
msrB
PS (4,5), ALM (3,1)

PS (20,7), ALM (4,7)
CLI
MUP
TET
linA/linA
mupA
tetK
PS (5,4), ALM (1,6), AN (3,7)

ALM (1,6)
PS (11,7), ALM (6,3), AN (69,7)
[ERY-CLIC]
Resistencia
Gen(es)
Poblacin (%)
[PEN-AMP]
[AMK-GEN-KAN-TOB]
[AMK-KAN-TOB]
KAN
CHL
blaZ
aacA-aphD
aadD
aphA-3
catpC194
catpC221
PS (89,2), ALM (75), AN (93,6)

PS (5,4), ALM (1,6), AN (11,9)
PS (2,7), ALM (1,6), AN (35,8)
PS (24,3), ALM (3,1), AN (2,8)
PS (1,8)
PS (0,9)
fexA
AN (3,1)
cfr
AN (1,6)
ermA
ermB
AN (33,9)
PS (1,8), AN (30,3)
ermC
PS (7,2), AN (39,4)
[ERY-CLI ]
ermA
ermC
ALM (3,1)
PS (10,8), ALM (12,5)
ERY
msrA
msrB
PS (4,5), ALM (3,1)

PS (20,7), ALM (4,7)
CLI
MUP
TET
linA/linA
mupA
tetK
PS (5,4), ALM (1,6), AN (3,7)

ALM (1,6)
PS (11,7), ALM (6,3), AN (69,7)
[ERY-CLIC]
Tipo Resistencia (CMI)
Bajo nivel (8-64 g/ml)

Nivel intermedio
Alto nivel ( 512 g/ml)
65,2
8,3
25
Poblacin
HUCA, HMN, PS, ALM
HMN
HUCA, ALM
111 aislamientos (PS) Plsmidos de resistencia
Perfil pR
kb
9 10 11 12 13 14 15 16 17 18 19 20 21
14.211.5z
t
5.14.5-
z
t
z
t
2.82.52.11.7-
z
t
z
t
z
t
22
t
z
tz
z
t
z
t
z
b
tz
bc c
tz
zt
bc bc ctz bctz
z
1.1-
Perfil pR
kb
24 25 26 27 28 29 30
31 32 33 34 35 36 37 38 39 40
5.14.52.82.52.11.71.1-
z z
hpt
t
t
pz
tz
btz tz
ab
t
t
k
ctz
ktz
k
k hkp k
bc
pz
z
t
z
pR CC
1
2
3
4
5
6
7
8
9
10
11
12
13
14
28
29
30
31
32
33
34
35
36
37
38
39
40
CC5, CC30
CC5
CC25, CC45, CC97
CC15, CC30, CC45
CC15, CC25
CC5
CC59
CC30
CC15
CC5, CC25, CC30
CC5
CC5, CC59
CC5
CC59
z
14.211.5-
pR CC
23
b
1
ktz
t
k
bc k
1
c
15 CC1
t, p480 Tn552; z, blaZ
d, aadD; h, accA+aphD; p, aphA
a; msrA; b, msrB; c, ermC
1, cat::pC194; 2, cat::pC221
k, tetK
16 CC1, CC45, CC72

17 CC15
18 CC25
19 CC1
20 CC5
21 CC5
CC15
CC97
CC30
CC30, CC45
CC8
CC30
CC1
CC25
CC45
CC15
CC97
CC9
CC25
Objetivos

estado
ST398 de origen
animal.
Objetivo 4. pUO-Sa-SED3
Plsmidos portadores de genes de enterotoxinas
pIB485 (sed-seljser)
repA
cad
HUCA (23.5%), HMN (16%),

PS (25.6%), ALM (10.9%)
HMN (1,6%)
pF5 (selj-ser-set-ses)
ftsK
Perfil pUO-SA-SED
kb
14.211.5-
pIB485-like
abiK
blaZ
sed
r
d jl rd jl r
d jl
r
d jl
jl r
5.1d jl r d jl r
selj
ser
AD
MarR sin
bin
blaR1
blaI
2.8-
jl r
2.52.11.7-
repA
ses
set
pF5-like
selj
ser
AD
MarR
sin
cad
bin blaI
blaR1 blaZ
opUO-Sa-SED1 sed-selj-ser
opUO-Sa-SED3 sed-selj
opUO-Sa-SED4 selj-ser-ses-
Plsmidos sed/selj/ser/ses/set
pIB485-like
repAFw3
pUO-Sa-SED1
pUO-Sa-SED2
pUO-Sa-SED3
pUO-Sa-SED4
2156 pb
1219 pb
1458 pb
blaZblaRI-Rv2 AD-Fw2
ser-Rv2
966 pb
ftsK-Rv Fw2
bin-Fw
sin-Rv
868 pb
1016 pb
1560 pb abiK-Rv2
blaRI-Fw
ser-Fw
selj-Rv
ftsK-Fw
blaI-Rv
1876 pb
1115 pb
MarR-Rv
abiK-Fw
blaZ- sin-Fw
1411 pb
Rv2 935 pb
blaI-Fw
bin-Rv selj-Fw2
sed-Rv2
908 pb
MarR-Fw
AD-Rv
cad-Rv2
2485 pb
cad-Fw2
+
+
+
+
+
+
+
+
pF5
pUO-Sa-SED1
pUO-Sa-SED2
pUO-Sa-SED3
pUO-Sa-SED4
+
+
+
+
+
+
+
+
+
bact-Rv2
1387 pb
bact-Fw2
ABCtranp-Rv2
+
+
pUO-Sa-SED1
pUO-Sa-SED2
pUO-Sa-SED3
pUO-Sa-SED4
repA-Fw4
+
+
-
2271 pb
ses-Fw2
pF5-like
+
+
-
4175 pb
TnaseIS431-Rv
1135 pb
cad-Fw2
TnaseIS431-Rv
1180 pb
TnaseIS431-Fw
blaZ1-Rv2
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
-
pIB485-like:pUO-Sa-SED1
pUO-Sa-SED2
pF5-like:pUO-Sa-SED4
Nuevo pIB485-like:pUO-Sa-SED3
1379pb
selj-Fw
set-Rv
1429 pb
set-Fw
ses-Rv
3800 pb
ses-Fw2
+ +
+
ABCtransp-Rv2
pUO-Sa-SED3 (53,5 kb)
repA
cad
ftsK
repA
pIB485-like
abiK
cad
blaZ
sed
selj
ser
blaR1
blaI
ADMarRsinbin
ftsK
pIB485-like
Regin27,4
mer kb
pIB485-like
sed
ORF-1 ORF-2 ORF-3

IS431
merR
abiK
pIB485-like
blaZ
ORF-4
selj
merT merA
merB IS431
blaR1
msrA
AD
MarR
sin
pIB485-like
ORF-6 ORF-7
traG
AD
16,3 kb
bin
Elemento traG-ftsK
ftsK
sin
blaI
ser
ORF-5
mphC
ORF-8
IS30B/C/D
ser
selj
sed
T7SS/ESAT-6 Mycobacterium Sistema Esx S. aureus Newman-Patogenicidad

Elemento traG/ftsK S. aureus D30-Persitencia estado portador
10,4 kb
Molecular epidemiology and genetic basis of

resistance and virulence of Staphylococcus aureus
from different origin

Doctoral Thesis
University of Oviedo
June, 2011
Objectives
Different S. aureus populations:
University of Oviedo
oNosocomial isolates from 2 hospitals :

81, 2005-2006 period HUCA
62,1997-2006 period HMN
oHealthy carrier isolates (HC)
68, 1997-2002 period
43, 2004-2006 period
oFood-borne/Food handlers isolates (FBH)
64, 1997-2008 period
oAnimal isolates (AN) (2004-2008 period)

105, clone ST398
4, other STs
Federal Institute for Risk Assessment

Berlin
Total: 427 isolates
Objectives
Objective 1. Identification of S. aureus lineages and clones from different origins

(hospitals, healthy carriers, and food-borne) which are circulating in the Principality of
Asturias, and sub-typing the animal clone ST398.
Objective 2. Identification of virulence determinants in lineages from the
Principality of Asturias and the ST398 clone, and establishment of their relationships
with mobile genetic elements.
Objective 3. Phenotypes and genetic basis of resistance in lineages from the
Principality of Asturias and the ST398 clone.
Objective 4. Characterization of the virulence-resistance hybrid plasmid pUO-SaSED3 using molecular techniques.
Results and Discussion
Objective 1. Lineages
CCs circulating among the Principality of Asturias
CC1, CC5, CC8, CC9, CC12, CC15, CC22, CC25, CC30, CC45, CC59, CC72, CC88, CC97, CC121
o MLST 112 isolates (HUCA, HMN, HC, FBH)- 31 STs
o spa typing 294 isolates (HUCA, HMN, HC, FBH)- 85 spa types
o Restriction-Modification (RM) test 237 isolates (HUCA, HC, FBH)- 7 types
HUCA (N=81)
CC5
57%
HMN (N=62)
CC5
36%
CC8
19.4%
100
90
80
70
60
50
40
30
20
10
0
HC (N=111)
CC45
16%
CC30
27%
CC5
19%
FBH (N=64)
CC45
38%
CC5
20%
CC30
CC3
0
20%
CC121
CC97
CC88
CC72
CC59
CC45
CC30
CC25
CC22
CC15
CC12
CC9
CC8
CC5
CCs circulating among the Principality of Asturias
kb
kb
582.0
398.0
582.0
388.0
242.5
242.5
194.0
194.0
145.5
145.5
97.0
97.0
48.5
S44 (1)
S43 (2)
S45 (1)
S40 (1)
S12 (1)
S31 (1)
S33 (1)
S37 (1)
S35 (1)
S42 (1)
S26 (6)
S5 (4)
S41 (2)
S19 (1)
S46 (1)
S15 (1)
S11 (1)
S14 (1)
S13 (1)
S10 (1)
S21 (1)
S49 (2)
S4 (1)
S0 (1)
S48 (1)
S32 (1)
S47 (2)
S3 (1)
S36 (1)
S17 (1)
S2 (1)
S25 (3)
S20 (1)
S30 (1)
S50 (1)
S28 (3)
S18 (3)
S23 (1)
S7 (3)
S39 (2)
S29 (1)
S27 (1)
S22 (1)
S6 (7)
S34 (3)
S16 (1)
S8 (2)
S38 (1)
S9 (1)
S24 (2)
S1 (1)
CC45
CC30
CC5
CC45
CC15
CC12
CC5
J
0.28
0.4
0.52
0.64
HUCA
0.76
0.88
CC121
CC30
CC30
CC59
CC15
CC25
CC5
CC45
CC8
CC72
CC1/9/97
J
0,28
0,4
0,52
0,64
HC
0,76
0,88
S58
S57
S28
S143
S142
S136
S25
S13
S127 (3)
S5
S7
S11 (3)
S9
S4
S126 (2)
S3 (11)
S138
S75
S134
S27
S26
S146
S139
S62 (2)
S70 (6)
S73 (2)
S71 (2)
S65
S72
S148
S63
S23
S131 (2)
S56
S55 (4)
S149
S129
S128
S130
S52 (4)
S53
S51 (6)
S54
S42 (3)
S140
S132
S33 (2)
S32
S31 (11)
S18
S0
S144
S145
S141
S137
S74 (2)
S60
S133 (2)
S150
S147
S15
S135
1
HUCA
HC
MRSA MSSA
CC5
CC30
kb
582.0
398.0
242.5
194.0
145.5
97.0
48.5
CC45
CC59 CC72
HUCA-S7
HC-S129
HUCA-S26
HC-S127
HUCA-S33
HC-S5
HUCA-S4
HC-S33
HUCA-S49
HC-S31
HUCA-S19
HC-S26
HUCA-S48
HC-S145
S0
48.5
mecA
HCS150
HCS149
HCS148
HCS147
HCS146
HCS145
HCS144
HCS143
HCS142
HCS141
HCS140
HCS139
HCS138
HCS137
HCS136
HCS135
HCS134
HCS133
HCS132
HCS131
HCS130
HCS129
HCS128
HCS127
HCS126
HCS70
HCS55
HCS52
HCS51
HCS33
HCS31
HCS11
HCS3
HUCAS50
HUCAS49
HUCAS48
HUCAS47
HUCAS46
HUCAS45
HUCAS44
HUCAS43
HUCAS42
HUCAS41
HUCAS40
HUCAS39
HUCAS38
HUCAS37
HUCAS36
HUCAS35
HUCAS34
HUCAS33
HUCAS32
HUCAS31
HUCAS30
HUCAS29
HUCAS28
HUCAS27
HUCAS26
HUCAS25
HUCAS24
HUCAS23
HUCAS22
HUCAS21
HUCAS20
HUCAS19
HUCAS18
HUCAS17
HUCAS16
HUCAS15
HUCAS14
HUCAS13
HUCAS12
HUCAS11
HUCAS10
HUCAS9
HUCAS8
HUCAS7
HUCAS6
HUCAS5
HUCAS4
HUCAS3
HUCAS2
HUCAS1
o SmaI Genomic macrorestriction and pulsed-field gel electrophoresis (PFGE)

192 isolates (HUCA, HC)- 105 types
CCs among animal isolates (AN)

o MLST 23 isolates- 4 STs
o spa typing 109 isolates- 22 spa types
o SmaI-PFGE 109 isolates 5 types
CC5, CC9 y CC30
CC398
kb
582.0
388.0
242.5
194.0
145.5
97.0
48.5
o 105 ST398 isolates Cfr9I Genomic macrorestriction and PFGE- 35 types

SCCmec V*
(n>1)
Cfr9I profile
kb
SCCmec V
SCCmec IVa
SCCmec nt
(6 (3)
(3) (2)
) C11 C13 C29 N C4 C15 C30
C
9
(3)
N C C17 C32
3
ATCC
S3
S2
S4
NCTC
S1
C28
C22
C31
C19
C27
C35
C33
C34
C6 (16)
C21
C10 (2)
C12 (9)
C5 (8)
C25
C24 (2)
C23
C20 (2)
C18 (6)
C2 (2)
C8 (2)
C16 (4)
C14 (6)
C26
C1 (11)
C32
C15 (3)
C4
C7
C13
C29
C11 (3)
C9 (6)
C30 (2)
C17
C3 (3)
SCCmec V
(2)
N C23 C24 C25 C26 C27 N C28 C31 C33 C34 C35 N
674
361
262
208
175
135
80
44
9
SCCmec V
SCCmec V
SCCmec V
(11) (2 (8) (16)

(2) (2) (9) (6) (4)
(6)
(2)
N C1 )C C C6 C7 N C8 C10 C12 C14 C16 N C18 C19 C20 C21 C22 N
2 5
J
0,28
0,4
0,52
0,64
0,76
0,88
IVa
nt
V
V
V
V
V
V
V
V
V (16)
V
V (2)
V (9)
V (8)
V
V (2)
V
V (2)
V (6)
V (2)
V
V, nt (2), V (6
V
V (11)
IVa (2), nt
IVa
V
V*
V*
V* (3)
V* (6)
IVa (2)
nt
nt (3)
Objectives

Objective 2. Virulence
359 isolates [HUCA, HMN, HC (2004-2006), FBH, AN]
oHaemolysins (hla, hlb, hld, hlg, hlg-v)-widely spread
HUCA
HC
HMN
FBH
AN
HUCA
HC
HMN
FBH
oExfoliatins (eta, etb, etd)-more frequent in hospital isolates

eta (Prophage Sa1)
etb (Plasmid pETB)
etd (SaPI)
AN
HUCA
HC
HMN
FBH
oExfoliatins (eta, etb, etd)-more frequent in hospital isolates

eta (Prophage Sa1)
etb (Plasmid pETB)
etd (SaPI)
AN
ovSa (lukED, egc 1 or egc2, splF, bsaB)
I
II
III
spl
E
luk
spl
D
luk
n
seg sel
m o
sei sel sel
bsa
spl
n u
m o
E
D
luk luk
HUCA
HC
HMN
FBH
AN
I
II
III
spl
E
luk
spl
D
luk
n
seg sel
m o
sei sel sel
bsa
spl
n u
m o
E
D
luk luk
HUCA
HC
HMN
FBH
AN
I
II
III
spl
E
luk
spl
D
luk
n
seg sel
m o
sei sel sel
bsa
spl
n u
m o
E
D
luk luk
HUCA
HC
HMN
FBH
AN
I
II
III
spl
E
luk
spl
D
luk
n
seg sel
m o
sei sel sel
bsa
spl
n u
m o
E
D
luk luk
HUCA
HC
HMN
FBH
AN
HUCA
HC
HMN
FBH

I
II
III
spl
E
luk
D
luk
n
seg sel
m o
sei sel sel
B
bsa
F
spl
spl
n u
m o
CC5 y CC15
E
D
luk luk
CC30 y CC121
AN
HUCA
HC
HMN
FBH

I
II
III
spl
E
luk
spl
D
luk
n
seg sel
m o
sei sel sel
bsa
spl
E
D
luk luk
n u
m o
Other arrangements:
splF-bsaB-egc1 splF-bsaB lukED

splF-bsaB-egc2 splF-egc1 egc1
splF-lukED-egc2 splF-lukED egc2
bsaB-lukED-egc1 lukED-egc1
AN
oLeukotoxins
lukM (Prophage Sa5)
lukPV (Prophage Sa2)
HUCA
HC
HMN
FBH
AN
oLeukotoxins
lukM (Prophage Sa5)
HUCA
HC
HMN
FBH
AN
oLeukotoxins
lukM (Prophage Sa5)
otst gene and enterotoxins genes among SaPIs

SaPI2 (tst)
CC30, CC59
CC1, CC5, CC8, CC9, CC12, CC15, CC25,
CC30, CC45, CC88, CC121
CC5, CC8, CC59
CC25, CC45, CC59, CC72
HUCA
HC
HMN
FBH
AN
oLeukotoxins
lukM (Prophage Sa5)

SaPI2 (tst)
CC30, CC59
CC1, CC5, CC8, CC9, CC12, CC15, CC25,
CC30, CC45, CC88, CC121
CC5, CC8, CC59
CC25, CC45, CC59, CC72
HUCA
HC
HMN
FBH
AN
oLeukotoxins
lukM (Prophage Sa5)

SaPI2 (tst)
CC30, CC59
CC1, CC5, CC8, CC9, CC12, CC15, CC25,
CC30, CC45, CC88, CC121
CC5, CC8, CC59
CC25, CC45, CC59, CC72
HUCA
HC
HMN
FBH
AN
oLeukotoxins
lukM (Prophage Sa5)

SaPI2 (tst)
CC30, CC59
CC1, CC5, CC8, CC9, CC12, CC15, CC25,
CC30, CC45, CC88, CC121
CC5, CC8, CC59
CC25, CC45, CC59, CC72
HUCA
HC
HMN
FBH
AN
oLeukotoxins
lukM (Prophage Sa5)

SaPI2 (tst)
CC30, CC59
CC1, CC5, CC8, CC9, CC12, CC15, CC25,
CC30, CC45, CC88, CC121
CC5, CC8, CC59
CC25, CC45, CC59, CC72
HUCA
HC
HMN
FBH
AN
oEnterotoxins genes in prophages
HUCA
HC
HMN
FBH
AN
HUCA
HC
HMN
FBH
AN
HUCA
HC
HMN
FBH
AN
oEnterotoxins genes in SCC elements
HUCA
HC
HMN
FBH
AN
HUCA
HC
HMN
FBH
oEnterotoxins genes in SCC elements
oEnterotoxins genes in plasmids
pIB485 (sed-seljser) CC1, CC5, CC8, CC9, CC15, CC25, CC30, CC45, CC59, CC72, CC121
pF5 (selj-ser-ses-set) CC5
AN
Virulence regulatory system agr
o 427 isolates (HUCA, HMN, HC, FBH, AN)
agrI
agrII
agrIII
agrIV
CC8, CC22, CC45, CC72, CC398

CC5, CC15
CC1, CC30, CC88
CC25, CC59, CC121
Virulence regulatory system agr
Exceptions:
agrI
agrII
agrIII
agrIV
agrI-agrIV
CC8, CC22, CC45, CC72, CC398

CC5, CC15
CC1, CC30, CC88
CC25, CC59, CC121
CC1, CC5, CC25, CC30, CC97

CC1, CC9, CC12, CC25, CC45, CC97, CC121
CC5, CC9, CC12, CC45, CC121
CC5, CC8, CC12, CC15, CC45
CC59, CC72
Objectives

Objective 3. Resistance
Methicillin resistance
o 154 isolates (HUCA, HMN, FBH, AN)
HUCA
HC
HMN
FBH
CC
Clone
ST
SCCmec
CC5
NewYork/Japan (USA 100)

Pediatric (USA 800)
5
5
II
IVc
Pediatric (USA 800)
IVd
125
III
125
IVa
125
IVc
125
Southern Germany
228
CC8
CC
Brasilian/Hungarian
Iberian
239
ST
247
III
SCCmec
I
CC15
CC5
CC45
CC9
CC398
Berlin (USA 600)

-
14
545
945
398
398
47
V
nd
III
IVa
IVc
IVa
VIVc
USA700
398
72
V*
IVc
CC72
AN
HUCA
HC
HMN
FBH
CC
Clone
ST
SCCmec
CC5

Pediatric (USA 800)
5
5
II
IVc
Pediatric (USA 800)
IVd
125
III
125
IVa
125
IVc
125
Southern Germany
228
CC8
CC
Brasilian/Hungarian
Iberian
239
ST
247
III
SCCmec
I
CC15
CC5
CC45
CC9
CC398
Berlin (USA 600)

-
14
545
945
398
398
47
V
nd
III
IVa
IVc
IVa
VIVc
USA700
398
72
V*
IVc
CC72
AN
HUCA
HC
HMN
FBH
CC
Clone
ST
SCCmec
CC5

Pediatric (USA 800)
5
5
II
IVc
Pediatric (USA 800)
IVd
125
III
125
IVa
125
IVc
125
Southern Germany
228
CC8
CC
Brasilian/Hungarian
Iberian
239
ST
247
III
SCCmec
I
CC15
CC5
CC45
CC9
CC398
Berlin (USA 600)

-
14
545
945
398
398
47
V
nd
III
IVa
IVc
IVa
VIVc
USA700
398
72
V*
IVc
CC72
AN
HUCA
HC
HMN
FBH
CC
Clone
ST
SCCmec
CC5

Pediatric (USA 800)
5
5
II
IVc
Pediatric (USA 800)
IVd
125
III
125
IVa
125
IVc
125
Southern Germany
228
CC8
CC
Brasilian/Hungarian
Iberian
239
ST
247
III
SCCmec
I
CC15
CC5
CC45
CC9
CC398
Berlin (USA 600)

-
14
545
945
398
398
47
V
nd
III
IVa
IVc
IVa
VIVc
USA700
398
72
V*
IVc
CC72
AN
HUCA
HC
HMN
FBH
CC
Clone
ST
SCCmec
CC5

Pediatric (USA 800)
5
5
II
IVc
Pediatric (USA 800)
IVd
125
III
125
IVa
125
IVc
125
Southern Germany
228
CC8
CC
Brasilian/Hungarian
Iberian
239
ST
247
III
SCCmec
I
CC15
CC5
CC45
CC9
CC398
Berlin (USA 600)

-
14
545
945
398
398
47
V
nd
III
IVa
IVc
IVa
VIVc
USA700
398
72
V*
IVc
CC72
AN
HUCA
HC
HMN
FBH
CC
Clone
ST
SCCmec
CC5

Pediatric (USA 800)
5
5
II
IVc
Pediatric (USA 800)
IVd
125
III
125
IVa
125
IVc
125
Southern Germany
228
CC8
CC
Brasilian/Hungarian
Iberian
239
ST
247
III
SCCmec
I
CC15
CC5
CC45
CC9
CC398
Berlin (USA 600)

-
14
545
945
398
398
47
V
nd
III
IVa
IVc
IVa
VIVc
USA700
398
72
V*
IVc
CC72
AN
HUCA
HC
HMN
FBH
CC
Clone
ST
SCCmec
CC5

Pediatric (USA 800)
5
5
II
IVc
Pediatric (USA 800)
IVd
125
III
125
IVa
125
IVc
125
Southern Germany
228
CC8
CC
Brasilian/Hungarian
Iberian
239
ST
247
III
SCCmec
I
CC15
CC5
CC45
CC9
CC398
Berlin (USA 600)

-
14
545
945
398
398
47
V
nd
III
IVa
IVc
IVa
VIVc
USA700
398
72
V*
IVc
CC72
AN
Resistance to antimicrobial agents
HUCA
HC
HMN
FBH
AN
HUCA
HC
HMN
FBH
AN
HUCA
HC
HMN
FBH
AN
HUCA
HC
HMN
FBH
AN
HUCA
HC
HMN
FBH
AN
HUCA
HC
HMN
FBH
AN
o No resistance against: Vancomycin, Linezolid, Tigecycline

o Multidrug Resistance (to 4 or more groups): 44% isolates HUCA/HMN
2.5% isolates HC/FBH
68% isolates AN
HUCA
HC
HMN
FBH
AN
o 284 isolates (HC, FBH, AN)
Resistance
Gene(s)
Population (%)
[PEN-AMP]
[AMK-GEN-KAN-TOB]
[AMK-KAN-TOB]
KAN
CHL
blaZ
aacA-aphD
aadD
aphA-3
catpC194
catpC221
HC (89.2), FBH (75), AN (93.6)

HC (5.4), FBH (1.6), AN (11.9)
HC (2.7), FBH (1.6), AN (35.8)
HC (24.3), FBH (3.1), AN (2.8)
HC (1.8)
HC (0.9)
fexA
AN (3.1)
cfr
AN (1.6)
ermA
ermB
AN (33.9)
HC (1.8), AN (30.3)
ermC
HC (7.2), AN (39.4)
[ERY-CLII]
ermA
ermC
FBH (3.1)
HC (10.8), FBH (12.5)
ERY
msrA
msrB
HC (4.5), FBH (3.1)

HC (20.7), FBH (4.7)
CLI
MUP
TET
linA/linA
mupA
tetK
HC (5.4), FBH (1.6), AN (3.7)

FBH (1.6)
HC (11.7), FBH (6.3), AN (69.7)
[ERY-CLIC]
HUCA
HC
HMN
FBH
AN
Resistance
Gene(s)
Population (%)
[PEN-AMP]
[AMK-GEN-KAN-TOB]
[AMK-KAN-TOB]
KAN
CHL
blaZ
aacA-aphD
aadD
aphA-3
catpC194
catpC221
HC (89.2), FBH (75), AN (93.6)

HC (5.4), FBH (1.6), AN (11.9)
HC (2.7), FBH (1.6), AN (35.8)
HC (24.3), FBH (3.1), AN (2.8)
HC (1.8)
HC (0.9)
fexA
AN (3.1)
cfr
AN (1.6)
ermA
ermB
AN (33.9)
HC (1.8), AN (30.3)
ermC
HC (7.2), AN (39.4)
[ERY-CLII]
ermA
ermC
FBH (3.1)
HC (10.8), FBH (12.5)
ERY
msrA
msrB
HC (4.5), FBH (3.1)

HC (20.7), FBH (4.7)
CLI
MUP
TET
linA/linA
mupA
tetK
HC (5.4), FBH (1.6), AN (3.7)

FBH (1.6)
HC (11.7), FBH (6.3), AN (69.7)
[ERY-CLIC]
HUCA
HC
HMN
FBH
AN
Resistance
Gene(s)
Population (%)
[PEN-AMP]
[AMK-GEN-KAN-TOB]
[AMK-KAN-TOB]
KAN
CHL
blaZ
aacA-aphD
aadD
aphA-3
catpC194
catpC221
HC (89.2), FBH (75), AN (93.6)

HC (5.4), FBH (1.6), AN (11.9)
HC (2.7), FBH (1.6), AN (35.8)
HC (24.3), FBH (3.1), AN (2.8)
HC (1.8)
HC (0.9)
fexA
AN (3.1)
cfr
AN (1.6)
ermA
ermB
AN (33.9)
HC (1.8), AN (30.3)
ermC
HC (7.2), AN (39.4)
[ERY-CLII]
ermA
ermC
FBH (3.1)
HC (10.8), FBH (12.5)
ERY
msrA
msrB
HC (4.5), FBH (3.1)

HC (20.7), FBH (4.7)
CLI
MUP
TET
linA/linA
mupA
tetK
HC (5.4), FBH (1.6), AN (3.7)

FBH (1.6)
HC (11.7), FBH (6.3), AN (69.7)
[ERY-CLIC]
HUCA
HC
HMN
FBH
AN
Resistance
Gene(s)
Population (%)
[PEN-AMP]
[AMK-GEN-KAN-TOB]
[AMK-KAN-TOB]
KAN
CHL
blaZ
aacA-aphD
aadD
aphA-3
catpC194
catpC221
HC (89.2), FBH (75), AN (93.6)

HC (5.4), FBH (1.6), AN (11.9)
HC (2.7), FBH (1.6), AN (35.8)
HC (24.3), FBH (3.1), AN (2.8)
HC (1.8)
HC (0.9)
fexA
AN (3.1)
cfr
AN (1.6)
ermA
ermB
AN (33.9)
HC (1.8), AN (30.3)
ermC
HC (7.2), AN (39.4)
[ERY-CLII]
ermA
ermC
FBH (3.1)
HC (10.8), FBH (12.5)
ERY
msrA
msrB
HC (4.5), FBH (3.1)

HC (20.7), FBH (4.7)
CLI
MUP
TET
linA/linA
mupA
tetK
HC (5.4), FBH (1.6), AN (3.7)

FBH (1.6)
HC (11.7), FBH (6.3), AN (69.7)
[ERY-CLIC]
HUCA
HC
HMN
FBH
AN
Resistance
Gene(s)
Population (%)
[PEN-AMP]
[AMK-GEN-KAN-TOB]
[AMK-KAN-TOB]
KAN
CHL
blaZ
aacA-aphD
aadD
aphA-3
catpC194
catpC221
HC (89.2), FBH (75), AN (93.6)

HC (5.4), FBH (1.6), AN (11.9)
HC (2.7), FBH (1.6), AN (35.8)
HC (24.3), FBH (3.1), AN (2.8)
HC (1.8)
HC (0.9)
fexA
AN (3.1)
cfr
AN (1.6)
ermA
ermB
AN (33.9)
HC (1.8), AN (30.3)
ermC
HC (7.2), AN (39.4)
[ERY-CLII]
ermA
ermC
FBH (3.1)
HC (10.8), FBH (12.5)
ERY
msrA
msrB
HC (4.5), FBH (3.1)

HC (20.7), FBH (4.7)
CLI
MUP
TET
linA/linA
mupA
tetK
HC (5.4), FBH (1.6), AN (3.7)

FBH (1.6)
HC (11.7), FBH (6.3), AN (69.7)
[ERY-CLIC]
HUCA
HC
HMN
FBH
AN
Resistance
Gene(s)
Population (%)
[PEN-AMP]
[AMK-GEN-KAN-TOB]
[AMK-KAN-TOB]
KAN
CHL
blaZ
aacA-aphD
aadD
aphA-3
catpC194
catpC221
HC (89.2), FBH (75), AN (93.6)

HC (5.4), FBH (1.6), AN (11.9)
HC (2.7), FBH (1.6), AN (35.8)
HC (24.3), FBH (3.1), AN (2.8)
HC (1.8)
HC (0.9)
fexA
AN (3.1)
cfr
AN (1.6)
ermA
ermB
AN (33.9)
HC (1.8), AN (30.3)
ermC
HC (7.2), AN (39.4)
[ERY-CLII]
ermA
ermC
FBH (3.1)
HC (10.8), FBH (12.5)
ERY
msrA
msrB
HC (4.5), FBH (3.1)

HC (20.7), FBH (4.7)
CLI
MUP
TET
linA/linA
mupA
tetK
HC (5.4), FBH (1.6), AN (3.7)

FBH (1.6)
HC (11.7), FBH (6.3), AN (69.7)
[ERY-CLIC]
Mupirocin resitance (CMI) %

Low level (8-64 g/ml)
Intermediate level
High level ( 512 g/ml)
HUCA
HC
HMN
FBH
Population
65.2 HUCA, HMN, HC, FBH

8.3 HMN
25 HUCA, FBH
AN
Resistance plasmids 111 isolates HC
pR profile
kb
9 10 11 12 13 14 15 16 17 18 19 20 21
14.211.5z
t
5.14.5-
z
t
z
t
2.82.52.11.7-
z
t
z
t
z
t
22
t
z
tz
z
t
z
t
z
b
tz
bc c
tz
zt
bc bc ctz bctz
z
1.1-
pR profile
kb
24 25 26 27 28 29 30
31 32 33 34 35 36 37 38 39 40
5.14.52.82.52.11.71.1-
z z
hpt
t
t
pz
tz
btz tz
ab
t
t
k
ctz
ktz
k
k hkp k
bc
pz
z
t
z
pR CC
1
2
3
4
5
6
7
8
9
10
11
12
13
14
28
29
30
31
32
33
34
35
36
37
38
39
40
CC5, CC30
CC5
CC25, CC45, CC97
CC15, CC30, CC45
CC15, CC25
CC5
CC59
CC30
CC15
CC5, CC25, CC30
CC5
CC5, CC59
CC5
CC59
z
14.211.5-
pR CC
23
b
1
ktz
t
k
bc k
1
c
15 CC1
t, p480 Tn552; z, blaZ
d, aadD; h, accA+aphD; p, aphA
a; msrA; b, msrB; c, ermC
1, cat::pC194; 2, cat::pC221
k, tetK
16 CC1, CC45, CC72

17 CC15
18 CC25
19 CC1
20 CC5
21 CC5
CC15
CC97
CC30
CC30, CC45
CC8
CC30
CC1
CC25
CC45
CC15
CC97
CC9
CC25
Objectives

Objective 4. pUO-Sa-SED3
Plasmids with enterotoxins
pIB485-like
repA
repAFw3
cadpb
2156
1219 pb
1458 pb
blaZcad-Rv2
blaRI-Rv2 AD-Fw2
ser-Rv2
2485 pb
966 pb
ftsK-Rv Fw2
repA
bin-Fw
sin-Rv
ftsK
868 pb
1016 pb
1560ses
pb abiK-Rv2
blaRI-Fw
ser-Fw
selj-Rv
ftsK-Fw
blaI-Rv
set
1876 pb
1115 pb
MarR-Rv
abiK-Fw
blaZ- sin-Fw
selj
pF5-like
1411 pb
abiK
Rv2 935 pb
blaI-Fw
bin-Rv selj-Fw2
sed-Rv2
ser
908 pb
MarR-Fw
AD-Rv
cad-Fw2
pIB485-like
sed
pUO-Sa-SED1
pUO-Sa-SED2
selj
pUO-Sa-SED3 ser
pUO-Sa-SED4
pF5
+
+ blaR1
+ blaI
bin
+
AD
MarR sin
blaZ
+
+
+
+
+
+
-
AD
+
+
+
+
+
+
sin
- blaI bin
- blaR1+blaZ +
MarR
+
+
+
+
+
+
+
cad
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
pF5-like
repAFw4
pUO-Sa-SED1
pUO-Sa-SED2
pUO-Sa-SED3
pUO-Sa-SED4
4175 pb
d jl r d jl r r
d jl
r
d jl
jl r
5.1d jl r d jl r
2.82.52.1-
jl r
1.7-
opUO-Sa-SED3 sed-selj
opUO-Sa-SED4 selj-ser-ses-set
TnaseIS431-Rv
1135 pb
cad-Fw2
TnaseIS431-Rv
1180 pb
TnaseIS431-Fw
blaZ1-Rv2
+
+
+
+
+
+
-
pIB485 (sed-seljser) HUCA (23.5%), HMN (16%),

2271 pb
PSbact-Rv2
(25.6%), FBH (10.9%)
ses-Fw2
1387 pb
pF5 (selj-ser-set-ses)
HMN (1,6%)
bact-Fw2
ABCtranp-Rv2
pUO-Sa-SED1
pUO-Sa-SED2
pUO-Sa-SED3
pUO-Sa-SED4
pUO-SA-SED
kb
14.211.5-
1379pb
selj-Fw
set-Rv
1429 pb
set-Fw
ses-Rv
3800 pb
ses-Fw2
+ +
+
ABCtransp-Rv2
Objective 4. pUO-Sa-SED3
pUO-Sa-SED3 (53,5 kb)
repA
cad
ftsK
repA
pIB485-like
abiK
cad
blaZ
sed
selj
ser
blaR1
blaI
ADMarRsinbin
ftsK
pIB485-like
mer region
(27.4 kb)
pIB485-like
sed
ORF-1 ORF-2 ORF-3

IS431
merR
abiK
pIB485-like
blaZ
ORF-4
selj
merT merA
merB IS431
blaR1
msrA
AD
MarR
sin
pIB485-like
ORF-6 ORF-7
traG
AD
16,3 kb
bin
traG-ftsK element
ftsK
sin
blaI
ser
ORF-5
mphC
ORF-8
IS30B/C/D
ser
selj
sed
T7SS/ESAT-6 Mycobacterium Esx system S. aureus Newman-Patogenicity

traG/ftsK element S. aureus D30-Carrier state
10,4 kb

de diferente origen

Tesis Doctoral
Junio, 2011

de diferente origen

Tesis Doctoral
Junio, 2011
Introduccin
S. aureus-Virulencia
Regulacin Gen regulador accesorio (accessory gene regulation, agr)
AIP
Pptido seal
autoinducible
AgrA
AgrC
AgrB
Pi
Pi
AgrA
AgrD
B
agr
agr
RNAII
C
agr
A
agr
P2 P3
RNAIII
Regulacin
expresin gnica
Exoprotenas
ej. Hemolisinas, TSST-1
Protenas asociadas a la pared
ej. Protena A
Conclusiones
1.
La aplicacin de distintas tcnicas de tipificacin molecular: macrorrestriccin genmica con SmaI

seguida de electroforesis en campo pulsante (PFGE), secuenciacin del gen de la protena A (spa),
secuenciacin de multiples loci (MLST) y amplificacin por PCR de los genes sau1hsdS1 y sau1hsdS2
del sistema de restriccin-modificacin Sau1, permiti establecer la estructura poblacional de los
aislamientos de Staphylococcus aureus que han estado circulando en el Principado de Asturias (PA),
tanto en hospitales como en la comunidad.
2.
Se detectaron quince linajes o complejos clonales (CCs): CC1, CC5, CC8, CC9, CC12, CC15, CC22,
CC25, CC30, CC45, CC59, CC72, CC88, CC97, CC121, la mayora presentes, aunque en proporciones
variables, en todas las poblaciones analizadas. Estas incluyeron aislamientos de dos hospitales:
Hospital Universitario Central de Asturias y Hospital Monte Naranco; de portadores sanos, de alimentos
y manipuladores de alimentos. En todos los casos, CC5, CC30 y CC45 fueron linajes ms frecuentes,
poniendo de manifiesto su xito en diferentes ambientes.
3.
La tipificacin molecular demostr el intercambio de aislamientos entre el medio hospitalario y la

comunidad. Esto se puso de manifiesto, por ejemplo, mediante la tcnica SmaI-PFGE, que fue
altamente discriminativa y mostr una elevada correlacin con las tipificaciones spa y MLST.
4.
En las poblaciones analizadas la mayor parte de los linajes se asoci con un tipo concreto del sistema
agr regulador de virulencia. Sin embargo, se encontraron excepciones que apoyan la existencia de
intercambio gentico entre linajes, lo que podra introducir variaciones en la expresin de las funciones
de virulencia.
5.
Todas las poblaciones del PA estudiadas presentaron altos porcentajes de genes de hemolisinas,
lukED, genes sea, sec y de los clsters egc1 y egc2 de enterotoxinas. Los genes de exfoliatinas y de la
leucotoxina Panto-Valentine fueron ms frecuentes en aislamientos clnicos que en aislamientos
comunitarios.
Conclusiones
6.
La combinacin de genes de virulencia y marcadores de distintos elementos genticos mviles (EGMs)

revel la presencia de distintos tipos de isla genmica Sa, de islas de patogenicidad (SaPIs), de
profagos y de plsmidos en los aislamientos del PA, y apoy la existencia de nuevas islas o variantes
que pudieron surgir por procesos de delecin y/o recombinacin.
7.
Algunos genes y/o EGMs fueron ms frecuentes en determinados linajes, destacando las asociaciones
etd-CC25, sep-CC5, tst-CC30, SaPImw2-CC45, SaPI3-CC59, Sa I-CC5 y CC15, Sa III-CC30 y
CC121. Sin embargo, la amplia dispersin de determinantes de virulencia entre los aislamientos
analizados indica la existencia de fenmenos de transferencia horizontal, no slo dentro de un mismo
linaje sino tambin entre linajes.
8.
Todas las poblaciones del PA presentaron altos porcentajes de resistencia a ampicilina-penicilina,

kanamicina, eritromicina y clindamicina, pero la multirresistencia fue ms frecuente en los aislamientos
nosocomiales que en los comunitarios (44 y 2,5%, respectivamente). Adems, los aislamientos CC5 y
CC8 de procedencia hospitalaria mostraron elevados porcentajes de resistencia a gentamicina y
ciprofloxacina.
9.
La frecuencia total de aislamientos de S. aureus resistentes a meticilina (MRSA) fue del 15,4%, siendo
la mayora de origen hospitalario (95,9%). Los restantes se hallaron en muestras de manipuladores de
alimentos (4,1%).
10. En las poblaciones del PA se identificaron 15 clones MRSA, siendo tres ellos: ST14-SCCmec V, ST125SCCmec III y ST125-SCCmec V, de nueva descripcin. Los clones MRSA ms frecuentes fueron el
Peditrico (ST5, SCCmec IV) y el ST125-SCCmec IV, coincidiendo con lo descrito en Espaa durante el
periodo de estudio.
Conclusiones
11. En S. aureus de origen comunitario destaca el elevado porcentaje del gen aphA (24,3%), que confiere
resistencia a kanamicina, en aislamientos de portadores sanos y la redundancia de genes de
resistencia a antimicrobianos del grupo MLS en poblaciones procedentes de portadores sanos,
alimentos y manipuladores de alimentos.
12. En todas las poblaciones se encontraron aislamientos resistentes a mupirocina, el antibitico de
eleccin para la erradicacin del estado de portador. La resistencia a mupirocina fue de nivel bajo
(65,2%), intermedio (8,3%) o alto (25%).
13. El 64,9% de los aislamientos presentaron plsmidos de resistencia, muchos de ellos compartidos por
distintos linajes. Mediante experimentos de hibridacin se comprob la localizacin plasmdica de la
mayora de los determinantes de resistencia encontrados en S. aureus de portadores sanos.
14. Los plsmidos hbridos de virulencia-resistencia pUO-Sa-SED constituyen un ejemplo de evolucin
bacteriana y plasmdica, destacando pUO-Sa-SED3 que contiene, no slo genes de virulencia y
resistencia, sino tambin genes implicados en colonizacin y procesos de conjugacin. Estos ltimos
facilitaran la persistencia de la bacteria en el hospedador y la dispersin del plsmido entre bacterias.
La co-seleccin de las distintas funciones codificadas por el mismo EGM constituye un importante
problema de salud pblica.
15. La sustitucin del enzima de restriccin SmaI por el isosquizmero Cfr9I permiti la tipificacin del clon
ST398 (CC398) mediante PFGE. A pesar de su reciente emergencia, la tipificacin Cfr9I-PFGE puso de
manifiesto la existencia de variabilidad intra-clon, asociada al tipo de SCCmec. Una C5-citosinametiltransferasa es responsable de la resistencia del genoma de ST398 a la digestin por SmaI.
Conclusiones
16. La mayora de los aislamientos MRSA del CC398 contienen SCCmec V y ms de la mitad fueron
multiresistentes. Destaca la redundancia de genes de resistencia a antibiticos del grupo MLS y
tetraciclina y la presencia de genes de resistencia a aminoglicsidos en cepas susceptibles.
17. Por el momento, el potencial toxignico del CC398 es muy limitado. De 30 genes de virulencia
analizados slo se detect el gen de la enterotoxina SEB en dos aislamientos.

Presentación Tesis Doctoral 21-6-11

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Epidemiologa molecular y bases genticas de la

resistencia y virulencia de Staphylococcus aureus

Mara de los ngeles Argudn Regueiro

o Caractersticas: Cocos (0,8-1 m ), Gram positivos, bajo contenido GC.

Asociadas a hospital (HA)

Estado de portador: Microbiota normal persistente o transente de la mucosa

Exfoliatinas Infecciones en piel y SSSS

Lesiones cutneas y neumona necrotizante

o Casetes cromosmicos estafiloccicos (SCC): MGEmw2/mssa476 (seh, seo)

Sntesis de cidos nucleicos

Sntesis de la pared bacteriana

EARSS MRSA 2008

60s: Penicilinas semisintticas

SCCmec I (34.3 kb)

SCCmec II (53.0 kb)

SCCmec III (66.9 kb)

SCCmec IV (20.9-24.3 kb)

Complejo mec (mecA mecRI y mecI)

SCCmec V (28 kb)

SCCmec VI (20.9 kb)

Junkyard or joining regions (J) determinan variantes

SCCmec VII (35.9 kb)

SCCmec VIII (32.2 kb)

o Importancia distincin CCs: Adquisicin SCCmec Clones HA-MRSA y CA-MRSA

Diferentes poblaciones S. aureus:

oAislamientos clnicos de 2 hospitales:

oAislamientos de animales (AN) (periodo 2004-2008)

Federal Institute for Risk Assessment

Total: 427 aislamientos

Objetivo 1. Identificar los linajes y clones de S. aureus de distinto origen

t078, t081, t167, t1054

t078, t081, t287, t6489

t012, t017, t018, t021, t122, t840

t001, t002, t045, t179, t548, t2173, t6488

ST125 t067, t3734

ST146 t002, t1560

t018, t012, t021, t253, t942, t1515, t6713, t7785

t015, t026, t040, t050, t065, t350, t1078, t1618

t001, t002, t067, t071, t242, t306, t1340

t008, t024, t351

t015, t026, t050, t282, t798, t1618, t1494,

Linajes circulantes en el Principado de Asturias:

o Macrorrestriccin genmica SmaI y Electroforesis en Campo Pulsante (PFGE)

HMN (79%), PS (61,3%), ALM (79,7%)

CC1, CC5, CC8, CC22, CC30, CC45

o MLST 23 aislamientos- 4 STs

o Macrorrestriccin genmica SmaI-PFGE

(11) (2) (8) (16)

Objetivo 1. Identificar los linajes y clones de S. aureus de distinto origen

Exfoliatinas (eta, etb, etd)

Marcadores vSa: splF, bsaB

splF-bsaB-egc1 splF-bsaB lukED

etb (Plsmido pETB)

etb (Plsmido pETB)

lukM (Profago Sa5)

etb (Plsmido pETB)

lukM (Profago Sa5)

etb (Plsmido pETB)

lukPV (Profago Sa2)

Gen tst y genes de enterotoxinas en SaPIs

Genes de enterotoxinas en elementos SCC

CC8, CC22, CC45, CC72, CC398