View
4
Download
0
Category
Preview:
Citation preview
PantonPanton––Valentine leukocidin (PVL) Role in the Induction, Valentine leukocidin (PVL) Role in the Induction, Maintenance and Local Extension of Maintenance and Local Extension of
CommunityCommunity--Associated MethicillinAssociated Methicillin--Resistant Resistant Staphylococcus aureusStaphylococcus aureus (CA(CA--MRSA) Rabbit OsteomyelitisMRSA) Rabbit Osteomyelitis
UNIVERSITE DE VERSAILLES SAINT QUENTIN-EN-YVELINES
A. SALEHA. SALEH--MGHIRMGHIR11, O. DIMITRESCU, O. DIMITRESCU22, G. LINA, G. LINA22, C. VALLEE, C. VALLEE11, , F. VANDENESHF. VANDENESH22, J.F. COTE, J.F. COTE11, J. ETIENNE, J. ETIENNE22, A.C. CREMIEUX*, A.C. CREMIEUX*11
1 1 --
EA 3647 UniversitEA 3647 Universitéé
Versailles StVersailles St--Quentin, Quentin, Hôpital Raymond PoincarHôpital Raymond Poincaréé, France, France
2 2 --
INSERM U851, Centre National de RINSERM U851, Centre National de Rééfféérence des Staphylocoques rence des Staphylocoques UniversitUniversitéé
Lyon 1, FacultLyon 1, Facultéé
Laennec, France.Laennec, France.
BackgroundBackground
The role of PVL in CAThe role of PVL in CA--MRSA pathogenicity remains MRSA pathogenicity remains controversial and might depend on the infection controversial and might depend on the infection site and/or experimental modelsite and/or experimental model
Osteomyelitis has long been recognized as a major Osteomyelitis has long been recognized as a major clinical syndrome of invasive clinical syndrome of invasive S. aureusS. aureus disease.disease.
The impact of PVL on the course of acute The impact of PVL on the course of acute osteomyelitis in children and young adultsosteomyelitis in children and young adults
was already suspected during the prewas already suspected during the pre--antibiotic eraantibiotic era(Specifi(Specificc immunity in acute staphylococcal osteomyelitis. immunity in acute staphylococcal osteomyelitis. Valentine and Butler Lancet 1939;1: 973Valentine and Butler Lancet 1939;1: 973--8)8)
was recently readdressed in the era of CAwas recently readdressed in the era of CA--MRSAMRSA(CE Bocchini et al. Pediatrics 2006; 117:433(CE Bocchini et al. Pediatrics 2006; 117:433--4040Dohin et al. Pediatr Infect Dis J. 2007; 26:1042Dohin et al. Pediatr Infect Dis J. 2007; 26:1042--8)8)
Study ObjectiveStudy Objective
Assess the specific role of PVL in a in a CA-MRSA rabbit osteomyelitis model
By comparing the outcomes of infections caused by
PVL-positive MRSA USA 300: LACPVL-negative isogenic strain: LACΔ pvl(kindly provided by Frank R. DeLeo)
Rabbit osteomyelitis modelRabbit osteomyelitis model (Norden’s model J Infect Dis 1970)
New Zealand white rabbits
Intramedullary injection of a sclerosing agent (0.1 mL of 3% sodium tetradecyl sulfate) into the tibia, followed by 0.2 mL of inoculum
Inocula: 8×105 (low) or 4×108 (high) CA-MRSA CFU (cultured in CCY and diluted in PBS)
On D7 and D28, rabbits were sacrificedmacroscopic findings: noted and photographed, infected tibias were removed and bacteria in crushed bones were counted.Serum samples : Anti PVLantibody and CRP titers
Imaging and histopathological examinations
Low inoculum: Serial MRI (Philips INTERA 1.5T) performed on 6 rabbits (D7, D14, D21)
High inoculum: Plain film +MRI + histopathological examination of 6 rabbits at the time of sacrifice (D7 or D28)
MRI (C. Vallee)
MuscleBone marrow(JF Cotte)
Joint space
Results Low Inoculum (8 x 105
CA-MRSA CFU)
0
10
20
30
40
50
60
70
80
90
100
D7 + D28
% in
fect
ed a
nim
als
LAC LAC D pvl
n = 19 n = 19
p = 0.04 vs LAC D pvl*
* Fisher’s Exact test
ResultsResults Low Inoculum (Low Inoculum (88××10105 5 CACA--MRSA CFU)MRSA CFU)
0
1
2
3
4
5
6
7
8
D7 D28
Log
10 C
FU/g
of b
one
LAC LAC D pv l
n = 10 n = 9n = 9n = 10
p = 0.03 vs D28*
* Mann-Whitney non-parametric U-test
p = 0.07 vs D28*
MRI PVLMRI PVL++
D7 D14
PVL+
D21
4.6 log10 CFU/g
Control D28
MRI PVLMRI PVL--
6.0 log10 CFU/g
D21D14D7
Control D28
ResultsResults High Inoculum (High Inoculum (4x108
CACA--MRSA CFU)MRSA CFU)
% infected animals
100
8690
100
0
20
40
60
80
100
120
D7 D28
LAC LAC D pvl
n = 11
n = 12
n = 10
n = 7
ResultsResults High Inoculum (High Inoculum (4 x 108
CACA--MRSA CFU)MRSA CFU)
0
1
2
3
4
5
6
7
8
D7 D28
Log
10 C
FU/g
of b
one
LAC
LAC D pvl
n=12n=11n=10
n=7
P = 0.004 vs D28*
* Mann-Whitney non-parametric U-test
P = 0.26 vs D28* NS
ResultsResults High Inoculum (High Inoculum (4 x108
CACA--MRSA CFU)MRSA CFU)
Macroscopic findingsMacroscopic findings
***
% in
fect
ed a
nim
als
100% 100% 100% 100%
67%
0%
60% 57%60%
7%
57%
9%
LAC LACΔpvl
Day 7 Day 28LAC LACΔpvl
12 12 15 12 12 15 10 10 14 7 7 11n =
P=0.001vs LAC
P=0.03vs LACP=0.005
vs LAC
bone marrowinvolvement
abnormalbone architecture
muscle or jointinvolvement% of rabbits with
High Inoculum (High Inoculum (4x108
CACA--MRSA)MRSA) Radiographic and histological findings in a PVLRadiographic and histological findings in a PVL++--infected rabbit on D28infected rabbit on D28
Bone
Abscess
Sequestrum
Deformation and widening of the entire diaphysis
Control PVL+
High Inoculum High Inoculum MRI and histological findings in a PVLMRI and histological findings in a PVL++
--infected rabbit on D28infected rabbit on D28
Soft tissue Abscess 3x2x1(cm)
x10
x1
High Inoculum High Inoculum MRI and histological findings in a PVLMRI and histological findings in a PVL++
--
infected rabbit that died on D8infected rabbit that died on D8
Abscess in the joint cavityPyomyositis
Distribution of C-reactive protein (High inoculum)
Distribution of anti-PVL antibody
levels in sera
ConclusionConclusion
Our results showed that PVL could contribute to the pathogenesisof early and late phase of CA-MRSA rabbit osteomyelitis by enabling :
Better bacterial persistence (low inoculum)Local extension during the infection’s early phase (highinoculum)
Concordant with clinical studies
Differences with previous experimental explorations of the role of PVL could be explained by
Location of infectionThe experimental model used: similar to the human situation; allows evaluation at different times post-infectionSelection of clinically relevant experimental readouts
This model may be suitable to select optimal therapies for PVL+CA-MRSA osteomyelitis
Recommended