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