Doctoral Thesis Speach 28-6-12 CODA

Molecular epidemiology and genetic basis of
resistance and virulence of Staphylococcus

aureus from different origin
M. ngeles Argudn Regueiro
Doctoral Thesis-University of Oviedo

(June, 2011)
28/06/2012
Staphylococcus aureus
Object of study: Staphylococcus aureus
o Taxonomic Classification: Dominio Bacteria
Phylum XIII Firmicutes

Clase I Bacilli
Orden I Bacillales
Familia VIII Staphylococcaceae
Especie Staphylococcus aureus
o Characteristics: Gram positive coccal bacterium (0,8-1m), low GC content

www.coda-cerva.be
Grape-like clusters, non-motile, non spore former

Facultative anaerobe and mesofile
Several pathologies:
o
o
o
Hospital associated-HA
Community associated-CA
Direct infection (superficial in skin and deep)

Invasive infection (bacteremia, sepsis and metastatic infection)
Toxin-mediated diseases
Carrier state: Normal and transient microbiota of nasopharyngeal mucosa

(skin and other corporal areas) General carrier rate 20-40%
9/5/16
S. aureus Virulence
Diversity of virulence factors:
o Cell wall components:
Peptidoglycan
Teichoic acids
Surface proteins (protein A)
External capsule
Surface proteins
Coagulase
Protein A
Elastin-binding
protein
www.coda-cerva.be
Collagenbinding
protein
o Enzymes:
Catalase
Coagulases
Staphylokinases
Hyaluronidases
Lipases
Proteases
Thermonucleases
9/5/16
Enzymes and Toxins
S. aureus
Fibronectinbinding
protein
Clumping factor
S. aureus Virulence
Diversity of virulence factors:
o Toxins:
Cytotoxins
Haemolysins (hla, hlb, hld, hlg, hlg-v genes)
Leukotoxins
Skin lesions and necrotizing

pneumonia
LukS-PV/LukF-PV (lukPV)
www.coda-cerva.be
LukE/LukD (lukED)
LukM/LukF-PV (lukM)
9/5/16
Superantigens
Exfoliatins Skin infections and SSSS

ETA (eta)
ETB (etb)
ETD (etd)
Conventional
antigen
Surface class
II MHC
Antigen presenting
cell
Conventional
antigen peptide
Superantigen
TSST-1 (tst)
Enterotoxins Food poisoning
T cell
Lysosome
S. aureus Virulence
Mobile genetic elements (MGEs):
o Genomic islands: vSa (lukED egc)
vSa
I
S2 M2 Serine-proteases cluster
hsd u1hsd lA lB lC lD lF
1
G
u
sa sa
sp sp sp sp sp
bsa
II
2
2
sdS 1hsdM
h
1
F C B A
sau sau spl spl spl spl
2
2
sdS1hsdM
h
1
F
E
sau sau spl spl
www.coda-cerva.be
III
SaPI1
SaPI2
SaPI3
SaPI5
SaPIm1/n1
SaPImw2
o Pathogenicity islands:
9/5/16 SaPI (etd)
egc cluster
n
m o
g
e
s sel
sei sel sel tRNA
E
D
luk luk
SaPIbov1
Lantibiotics cluster
B
E
G a aF aP aD aC
bsa
bsa bs bs bs bs bs
C
spl
attL
tRNA
n u
m o
seg sel sel sei sel sel tRNA
ear
tst ter
int
rep
attL
ear seb ter
rep
attL
ear
rep
attL
l c3 r
sel se ea
l2 4
sel sec ear
attL sell sec
q k
sel sel
rep
tst ter
attL
attL
A2 A1 E D
bsa bsa luk luk
ter
int
attR
q k t
sel sel in
q k t
sel sel in
tst ter
ter
int
rep
tst ter
rep
attR
attR
int
rep
attR
attR
attR
int attR
S. aureus Virulence
Mobile genetic elements (MGEs):
www.coda-cerva.be
o Prophages: Siphoviridae family, classification:

Integrase type Sa1: eta
Integrase type Sa2: lukPV
Integrase type Sa3: sea, sea-selk-selq, sep
Integrase type Sa5: lukM
o Plasmids:
pETB (etb)
pZA10 (seb)
pIB485-like (sed-selj-ser)
pF5-like (selj-ser-ses-set)
lo
A B
e
att
ccr ccr
s seh
o Staphylococcal
chromosomal
cassette
(SCC): att
MGEmw2/mssa476
MGEmssa476
MGEmw2
9/5/16
e lo
s seh
attL
A1 B
ccr ccr
seo)
2 (seh,
I
127 ecR cA
31
S
I
attR
m me
IS4
S. aureus Virulence
Regulation accessory gene regulation, agr
o Dual action in stationary phase:
Transcription repression of wall associated protein coding genes
Transcription activation of exoprotein genes (enzymes and toxins)
www.coda-cerva.be
B
agr
agr
C
agr
A
agr
P2 P3
o Differences define 4 groups: agrI, agrII, agrIII, agrIV

(+ mixed types and negatives)
o Diseases, resistances and virulence factors certain agr
Vancomycin reduced susceptibility agrI y agrII
TSST-1 agrIII
LukPV + Necrotizing pneumonia agrIII
Exfoliatins agrIV
9/5/16
S. aureus Resistance
Treatment with antimicrobial agents:
Nucleic acid synthesis

Quinolones
Rifamicins
Inhibition of Cell wall synthesis

-lactams
Glycopeptides
ADN
THFA
ARNm
www.coda-cerva.be
Cytoplasmatic membrane
Lipopeptides
Metabolic routes
Sulfonamides
Diaminopyrimidines
9/5/16
DHFA
Ribosomes
PABA
50
30
50
30
50
30
Protein synthesis
Aminoglycosides
Fenicols
Macrolides
Lincosamides
Streptogramins
Mupirocin
Oxazolidinones
Tetracyclines
Glicilciclines
Antimicrobial resistance:
-lactamase
1942: R-penicillin
EARSS MRSA 2008
www.coda-cerva.be
S. aureus
60s: Semisynthetic penicillin

R- -lactamases
(methicilin, oxacilin)
Penicillium
1928: Penicillin discover

Other antimicrobial
agents
Pre-antibiotic Era
9/5/16
50
60s: MRSA
HospitalHA-MRSA
(S. aureus R-methicilin) CommunityCA-MRSA
R-tetracyclin
R-macrolides, MLS
R-aminoglycosides
Glycopeptides
R-quinolones
R-rifampicin
60
1996: VISA
2002: VRSA
(I-vancomycine)(R-vancomycine)
Other antimicrobial
agents
70
80
Antibiotic Era
90
00
9
Antimicrobial resistance:
o Inactivating and modifying enzymes

blaZ-lactams
aphA-3 , aadD y aacA-aphDAminoglycosides
catPhenicols
mphCMacrolides
vatA y vatBStreptogramins A
vgbA y vgbBStreptogramins B
linA/linALincosamides
Efflux
pump
Antibiotic
Altered
target
Inactivating
enzyme
www.coda-cerva.be
Antibiotic
Antibiotic
9/5/16
Modifying
enzyme
Antibiotic
o Efflux pumps
fexAPhenicols
msrA y msrBMacrolides
vgaA, vgaB y vgaCStreptogramins A
tetK y tetLTetracyclines
mepATigecyclines
norA, norB, norC, mepA y sdrMQuinolones
o Altered target
mecA-lactams
vanAGlycopeptides
ermMacrlidos, lincosamides y streptogramins B
cfrPhenicols, streptogramins A, lincosamides,
oxazolidinones
ileS y mupAMupirocin
tetM y tetOTetracyclines
grlA, grlB, gyrA y gyrBQuinolones
rpoBRifamycins
dfrA(S1), dfrB(D), dfrK y dfrGDiaminopyrimidines
sulASulfonamides
10
Antimicrobial resistance: Methicillin resistant S. aureus (MRSA)-SCCmec
SCCmec I (34.3 kb)
Class B
J1
SCCmec II (53.0 kb)
J2
J3
HA-MRSA
Tn554
pUB110
HA-MRSA
Class A
SCCmec III (66.9 kb)
mer
operon
Tn554
Tn554
pT181
Class A
SCCHg
CA-MRSA
www.coda-cerva.be
SCCmec IV (20.9-24.3 kb)

Class B
SCCmec V (28 kb)
CA-MRSA
Class
C2
SCCmec VI (20.9 kb)

Class B
SCCmec VII (35.9 kb)
mec complex (mecA mecRI and mecI)

ccr complex with recombinase genes (ccrAB and/or ccrC
Junkyard or joining regions (J) determine variants
Class
C1
SCCmec VIII (32.2 kb)
Tn554
Class A
9/5/16
11
S. aureus Lineages
Clonal population structure: lineages or clonal complexes (CCs)
o MLST (multilocus sequence typing)
Allelic profile arcC, aroE, glpF, gmk, pta, tpi, yqiL: sequence type (ST)
STs with 5-7 common alleles define a CC
CC45
CC22
ST239
CC15
CC30
CC1
CC5
ST8
www.coda-cerva.be
CC8
CC12
CC25
CC121
eBURST CC8
http://www.mlst.net/
9/5/16
o
Importance CCs distinction: SCCmec acquisition HA-MRSA12y CA-MRSA clones
Objectives
Different S. aureus populations:
www.coda-cerva.be
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

9/5/16
13
Objectives
www.coda-cerva.be
Objective 1. Identification of S. aureus lineages and clones

from different origins (hospitals, healthy carriers, and foodborne) 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-Sa-SED3 using molecular techniques.
9/5/16
14
Lineages among Asturias

Lineages circulating among the Principality of Asturias
o
o
o
MLST 112 isolates (HUCA, HMN, HC, FBH)- 31 STs

spa typing 294 isolates (HUCA, HMN, HC, FBH)- 85 spa types
Restriction-Modification test (RM) 237 isolates (HMN, HC, FBH)- 7 CCs identified
CC
CC1
ST
ST1
ST3
spa types
t127, t3201
t177
CC
CC25
ST188 t189
www.coda-cerva.be
CC5
ST5
ST6
t001, t002, t045, t179, t548, t2173, t6488

t701
nd
t078, t081, t287, t6489
ST30
ST34
t012, t017, t018, t021, t122, t840

t136, t166, t6490
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
ST239 t037
CC45
CC59
ST247 t051
9/5/16
spa types
t078, t081, t167, t1054
t7125
ST125 t067, t3734
ST228 t109
CC8
CC30
ST
ST25
ST26
nd
t008, t051
CC72
ST59
t015, t026, t050, t282, t798, t1618, t1494,

t1618
t216
nd
t3952
ST72
t148
15

Lineages circulating among the Principality of Asturias
www.coda-cerva.be
CC1, CC5, CC8, CC9, CC12, CC15, CC22, CC25, CC30, CC45, CC59, CC72, CC88, CC97, CC121
HUCA (N=81)
HMN (N=62)
HC (N=111)
FBH (N=64)
CC30
9/5/16
16

Lineages circulating among hospitals in the Principality of Asturias
CC1, CC5, CC8, CC12, CC15, CC25, CC30, CC45, CC59, CC72, CC88, CC121
HUCA (N=81)
HMN (N=62)
CC5
36%
CC5
57%
www.coda-cerva.be
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
9/5/16
0
1996
1997
1998
1999
2000
2001
2002
2003
2004
2005
2006
17

Lineages circulating among the community in the Principality of Asturias
CC1, CC5, CC8, CC9, CC15, CC22, CC25, CC30, CC45, CC59, CC72, CC97, CC121
FBH (N=64)
HC (N=111)
CC45
16%
www.coda-cerva.be
CC30
27%
9/5/16
CC5
19%
CC45
38%
CC5
20%
CC30
CC30
20%
18

Lineages circulating in the Principality of Asturias
kb
582.0
398.0
242.5
CC5
194.0
145.5
97.0
48.5
J
0.28
9/5/16
0.52
0.4
0.64
HUCA
0.76
0.88
CC5
CC45
CC8
CC72
CC1/9/97
J
0,28
0,4
0,52
0,64
HC
0,76
0,88
CC30
CC45
kb
145.5
97.0
48.5
19
HCS150
mecA
HCS149
HCS148
HCS147
194.0
HCS146
582.0
398.0
242.5
HUCAS50
HUCAS49
HUCAS48
HUCAS47
HUCAS45
HC-MSSA
HUCAS46
HUCAS43
HUCAS44
HUCA-MRSA
HUCAS42
CC72
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
HCS145
HUCAS36
HUCAS35
HUCAS39
HUCAS40
HUCAS41
CC5
HCS144
HCS143
HCS141
HCS140
HCS139
HCS138
HCS137
HCS136
HCS135
HCS134
HCS133
HCS132
HCS131
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
HUCAS20
HUCAS21
HUCAS15
HUCAS16
HUCAS17
HUCAS18
HUCAS19
HUCAS14
HUCAS22
HUCAS23
HUCAS24
HUCAS25
HCS130
CC12
HCS129
CC15
HCS128
HCS3
HCS33
CC15
HC
HCS31
CC45
HCS11
www.coda-cerva.be
48.5
HCS127
97.0
CC59
HCS126
CC5
145.5
CC121
CC30
CC30
HCS70
242.5
194.0
HCS55
CC30
HCS52
HUCAS12
HUCAS13
kb
582.0
388.0
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)
HCS51
CC45
HUCAS11
HUCAS9
HUCAS10
HUCAS7
HUCAS8
HUCAS5
HUCAS6
HUCAS4
HUCAS3
HUCA
HUCAS1
HUCAS2
192 isolates (HUCA, HC)- 105 PFGE types
HUCAS37
HUCAS38
SmaI Macrorestriction + pulsed-field gel electrophoresis (PFGE)
HCS142
Lineages among animal isolates

Lineages in animal isolates (AN)
o
o
MLST 23 isolates-4 STs

spa typing 109 isolates-22 spa types
CC
spa types
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
www.coda-cerva.be
CC5
CC9
105 CC30
CC398
ST
CC5, CC9 y CC30
CC398
kb
582.0
388.0
242.5
o
9/5/16
SmaI Macrorestriction-PFGE
109 isolates- 5 types
(105 ST398-Nontypeable type)
194.0
145.5
97.0
48.5
20
Lineages among animal isolates

Lineages in animal isolates (AN)
o
Cfr9I Macrorestriction-PFGE
105 ST398 isolates- 35 types
SCCmec V*
(n>1)
Profile 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
www.coda-cerva.be
M.SauST398
(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
C5-citosine
methyltransferase
SCCmec V
SCCmec V
SCCmec V
0,28
9/5/16
0,4
0,52
0,64
0,76
21
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
www.coda-cerva.be

elements.
9/5/16
22
Virulence
359 isolates [HUCA, HMN, HC (2004-2006), FBH, AN]
Virulence factors detection
Cytotoxins
Haemolysins (hla, hlb, hld, hlg, hlg-v)
www.coda-cerva.be
Leukotoxins (lukPV, lukED, lukM)
Exfoliatins (eta, etb, etd)
Superantigens
TSST-1 (tst)
Enterotoxins SEs (sea, seb, sec, sed, see, seg, seh, sei, ser, ses, set)
SlEs (selj, selk, sell, selm, seln, selo, selp, selq, selu)
9/5/16
vSa markers: splF, bsaB

23
Virulence
HUCA
HC
HMN
FBH
oHaemolysins (hla, hlb, hld, hlg, hlg-v)-widely spread
hla
hlb
www.coda-cerva.be
hlg
eta (Prophage Sa1)
hld
hlg-v
etb (Plasmid pETB)
etd (SaPI)
oExfoliatins (eta, etb, etd)-more frequent in hospital isolates

9/5/16
24
AN
Virulence
ovSa (lukED, egc 1 or egc2, splF, bsaB)
I
F
spl
n
seg sel
F
spl
III
HMN
FBH
E D
luk luk
n u
m o
seg sel sel sei sel sel
CC30 y CC121
splF
egc1
bsaB
Other arrangements:
splF-bsaB-lukED-egc1 splF-bsaB-egc1
splF-bsaB-lukED-egc2 splF-bsaB-egc2
splF-lukED-egc2
9/5/16
AN
CC5 y CC15
lukED
egc2
HC
m o
sei sel sel
B
b sa
F
spl
II
www.coda-cerva.be
E
D
luk luk
HUCA
25
splF-bsaB
splF-egc1
splF-lukED
lukED
egc1
egc2
Virulence
oLeukotoxins
lukM (Prophage Sa5)
www.coda-cerva.be
SaPI1 (ear-tst-selk-selq)
HUCA
HC
HMN
FBH
lukPV (Prophage Sa2)
CC30, CC59
ear
SaPI2 (tst)
CC1, CC5, CC8, CC9, CC12, CC15, CC25,

CC30, CC45, CC88, CC121
otst gene and enterotoxins
genes among SaPIs
SaPI3 (ear-seb-selk-selq)
CC5, CC8, CC59
SaPI5 (ear-selk-selq)
CC5, CC8, CC59
SaPImw2 (ear-sec-sell)
CC25, CC45, CC59, CC72
SaPIn1/m1 (ear-tst-sec-sell) CC45, CC59
tst
seb
9/5/16
sec
selk
sell
selq
26
AN
Virulence
oEnterotoxins genes in prophages
sea
HUCA
HC
HMN
FBH
see
AN
selp
www.coda-cerva.be
seh
oEnterotoxins genes in SCC elements
pIB485 (sed-seljser)
pF5 (selj-ser-ses-set)
sed
CC1, CC5, CC8, CC9, CC15, CC25,

CC30, CC45, CC59, CC72, CC121
CC5
selj
ser
oEnterotoxins genes in plasmids

9/5/16
27
Virulence
Virulence regulatory system agr
www.coda-cerva.be
427 isolates (HUCA, HMN, HC, FBH, AN)
Exceptions:
agrI
agrII
agrIII
agrIV
CC8, CC22, CC45, CC72, CC398 CC1, CC5, CC25, CC30, CC97
CC5, CC15
CC1, CC9, CC12, CC25, CC45, CC97, CC121
CC1, CC30, CC88
CC25, CC59, CC121
CC5, CC9, CC12, CC45, CC121
CC5, CC8, CC12, CC15, CC45
agrI-agrIV
9/5/16
CC59, CC72
28
Objectives
www.coda-cerva.be

elements.
9/5/16
29
Resistance
Methicillin resistance
o
154 isolates (HUCA, HMN, FBH, AN)

CC
CC5
www.coda-cerva.be
MET-OXA
HMN
FBH
ST
5
5
SCCmec
II
IVc
Pediatric (USA 800)
IVd
125
III
125
IVa
125
IVc
125
Southern Germany
228
CC8
Brasilian/Hungarian
Iberian
239
247
III
I
CC15
CC
CC45
CC5
Berlin (USA 600)

-
14
ST
545
945
398
47
398
V
SCCmec
III
nd
IVc
IVa
IVa
VIVc
USA700
72
398
IVc
V*
CC9
CC398
CC72
9/5/16
HC
Clone
NewYork/Japan (USA 100)
Pediatric (USA 800)
MET-OXA
HUCA (81): 32.1%

HMN (62): 33.9%
PS (111): 0%
ALM (64): 3%
HUCA
398 nd
30
AN
Resistance
Resistance to antimicrobial agents
o
HUCA
HC
HMN
FBH
www.coda-cerva.be
AMPICILLIN-PENICILLIN
9/5/16
KANAMYCIN
ERYTHROMYCIN
CLINDAMYCIN
CIPROFLOXACIN
TETRACYCLINE
31
AN
Resistance
o
HUCA
HC
HMN
FBH
GENTAMICIN
TRIMETHOPRIMSULFAMETHOXAZOLE
RIFAMPICIN
MUPIROCIN
www.coda-cerva.be
TRIMETHOPRIM
CHLORAMPHENICOL
9/5/16
No resistance against: Vancomycin, Linezolid, Tigecycline

32
AN
Resistance
o
HC
HMN
FBH
284 isolates (HC, FBH, AN)

Resistance
[PEN-AMP]
[AMK-GEN-KAN-TOB]
[AMK-KAN-TOB]
KAN
CHL
[ERY-CLIC]
www.coda-cerva.be
HUCA
[ERY-CLII]
ERY
CLI
MUP
TET
9/5/16
Gene(s)
blaZ
aacA-aphD
aadD
aphA-3
catpC194
catpC221
Population (%)
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)
ermA
ermC
FBH (3.1)
HC (10.8), FBH (12.5)
msrA
msrB
HC (4.5), FBH (3.1)

HC (20.7), FBH (4.7)
linA/linA
mupA
tetK
tetL
HC (5.4), FBH (1.6), AN (3.7)

FBH (1.6)
HC (11.7), FBH (6.3), AN (69.7)
AN (38.5)
tetM
AN (98.2)
AMP-PEN
blaZ
TRIMETHOPRIM
AN
AMINOGLYCOSIDES
MUPIROCIN
MLS GROUP
Mupirocin resitance
(CMI)
Population
Low level TETRACYCLINE

(8-64 g/ml)
65.2 HUCA, HMN, HC, FBH
Intermediate level
8.3 HMN
High level ( 512 g/ml) 25 HUCA, FBH
33
Resistance
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
www.coda-cerva.be
1.1-
pR profile
kb
24 25 26 27 28 29 30
31 32 33 34 35 36 37 38 39 40
5.14.52.82.52.1-
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
1.7-
18 CC25
1.1-
19 CC1
9/5/16
20 CC5
34
CC15
CC97
CC30
CC30, CC45
CC8
CC30
CC1
CC25
CC45
CC15
CC97
CC9
CC25
Objectives
www.coda-cerva.be

elements.
9/5/16
35
pUO-Sa-SED3
Plasmids with enterotoxins
pIB485 (sed-seljser)
repA
cad
pF5 (selj-ser-set-ses)
ftsK
14.211.5-
pIB485-like
abiK
selj
ser
r
d jl rd jl r
d jl
r
d jl
jl r
5.1-
blaZ
sed
www.coda-cerva.be
Perfil pUO-SA-SED
kb
HUCA (23.5%), HMN (16%),

PS (25.6%), ALM (10.9%)
HMN (1,6%)
d jl r d jl r
blaR1
blaI
ADMarRsinbin
2.8-
jl r
2.52.11.7-
repA
ses
set
selj
pF5-like
ser
9/5/16
AD
MarR
sin
binblaI
blaR1blaZ
cad
o
o
o
o
pUO-Sa-SED1
pUO-Sa-SED2
pUO-Sa-SED3
pUO-Sa-SED4
sed-selj-ser
sed-selj-ser
sed-selj
selj-ser-ses-set
36
pUO-Sa-SED3
Plasmids with enterotoxins
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
+
+
+
+
+
+
+
+
www.coda-cerva.be
pF5
pUO-Sa-SED1
pUO-Sa-SED2
pUO-Sa-SED3
pUO-Sa-SED4
9/5/16
+
+
+
+
+
+
+
+
+
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
New pIB485-like:pUO-Sa-SED3
1379pb
selj-Fw
set-Rv
1429 pb
set-Fw
ses-Rv
3800 pb
ses-Fw2
+ +
+
ABCtransp-Rv2
37
pUO-Sa-SED3
pUO-Sa-SED3 (53,5 kb)
repA
cad
ftsK
repA
cad
pIB485-like
abiK
blaZ
sed
selj
ser
blaR1
blaI
ADMarRsinbin
pIB485-like
(27.4 kb)
mer region
pIB485-like
www.coda-cerva.be
ftsK
sed
ORF-1 ORF-2 ORF-3

IS431
merR
abiK
pIB485-like
blaZ
ORF-4
selj
merT merA
blaR1
merB IS431
msrA
ser
AD
MarR
sin
ftsK
sin
pIB485-like
ORF-6 ORF-7
traG
ORF-8
IS30B/C/D
ser
selj
sed
10,4 kb
T7SS/ESAT-6 Mycobacterium Esx system S. aureus Newman-Patogenicity

traG/ftsK element S. aureus D30-Carrier state
9/5/16
AD
16,3 kb
bin
traG-ftsK element
ORF-5
mphC
blaI
38
Conclusions
www.coda-cerva.be
Originally 17 conclusions
1.
The application of different molecular typing techniques established the population structure
of Staphylococcus aureus isolates that have been circulating in the Principality of Asturias
(PA). Fifteen lineages have been detected, being the most frequent: CC5, CC30 and CC45. It
has been demonstrated the strains exchange between hospital and community settings.
2.
We identified a C5-cytosine-methyltransferase responsible for the SmaI genome digestion

resistance of CC398 isolates, and we developed a PFGE alternative typing method using the
SmaI isoesquizomer Cfr9I.
3.
There was a wide dispersion of virulence genes, mobile genetic elements and agr types in S.
aureus from different lineages and origin, but some elements were clearly more frequent in
certain lineages or populations.
4.
Fifteen methicillin resistant S. aureus clones were identified in the PA, being three of new
description (ST14-SCCmec V, ST125-SCCmec III and ST125-SCCmec V), and the Pediatric
(ST5, SCCmec IV) and ST125-SCCmec IV clones the most frequent ones.
5.
All S. aureus populations showed high rates of antimicrobial resistance being multidrug
resistance more common in hospitals than in community populations. Some resistance genes
have detected in the community being some of them associated to plasmids.
9/5/16
39
Acknowledgments
Prof. M. Rosario Rodicio

Prof. M. Carmen Mendoza
Dr. M. Cruz Martn
Lab M1 team
Dr. Beatriz Guerra

Salm team
Staph team

(June, 2011)
Molecular epidemiology and genetic basis of

resistance and virulence of Staphylococcus
aureus from different origin
M. ngeles Argudn Regueiro

(June, 2011)
28/06/2012

Doctoral Thesis Speach 28-6-12 CODA

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Doctoral Thesis Speach 28-6-12 CODA

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Copyright:

Verfügbare Formate

Molecular epidemiology and genetic basis of

resistance and virulence of Staphylococcus

M. ngeles Argudn Regueiro

Doctoral Thesis-University of Oviedo

Phylum XIII Firmicutes

o Characteristics: Gram positive coccal bacterium (0,8-1m), low GC content

Grape-like clusters, non-motile, non spore former

Direct infection (superficial in skin and deep)

Carrier state: Normal and transient microbiota of nasopharyngeal mucosa

Enzymes and Toxins

Skin lesions and necrotizing

Exfoliatins Skin infections and SSSS

9/5/16 SaPI (etd)

ear seb ter

attL sell sec

o Prophages: Siphoviridae family, classification:

o Differences define 4 groups: agrI, agrII, agrIII, agrIV

Nucleic acid synthesis

Inhibition of Cell wall synthesis

EARSS MRSA 2008

60s: Semisynthetic penicillin

1928: Penicillin discover

o Inactivating and modifying enzymes

SCCmec II (53.0 kb)

SCCmec III (66.9 kb)

SCCmec IV (20.9-24.3 kb)

SCCmec V (28 kb)

SCCmec VI (20.9 kb)

SCCmec VII (35.9 kb)

mec complex (mecA mecRI and mecI)

SCCmec VIII (32.2 kb)

Importance CCs distinction: SCCmec acquisition HA-MRSA12y CA-MRSA clones

oNosocomial isolates from 2 hospitals :

oAnimal isolates (AN) (2004-2008 period)

Total: 427 isolates

Objective 1. Identification of S. aureus lineages and clones

Lineages among Asturias

MLST 112 isolates (HUCA, HMN, HC, FBH)- 31 STs

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

t078, t081, t287, t6489

t012, t017, t018, t021, t122, t840

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

ST125 t067, t3734

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

Lineages among Asturias

Lineages among Asturias

Lineages among Asturias

Lineages among Asturias

192 isolates (HUCA, HC)- 105 PFGE types

SmaI Macrorestriction + pulsed-field gel electrophoresis (PFGE)

Lineages among animal isolates

MLST 23 isolates-4 STs

CC5, CC9 y CC30

Lineages among animal isolates

Objective 1. Identification of S. aureus lineages and clones

Leukotoxins (lukPV, lukED, lukM)

Exfoliatins (eta, etb, etd)

vSa markers: splF, bsaB

oHaemolysins (hla, hlb, hld, hlg, hlg-v)-widely spread

eta (Prophage Sa1)