Staphylococcus and biofilms (and foreign body infections) Johan Van - - PowerPoint PPT Presentation

Staphylococcus and biofilms (and foreign body infections)

Johan Van Eldere K.U.Leuven

Staphylococci and staphylococcal biofilms in foreign body infections

• CoNS are the leading cause of nosocomial

bloodstream infections – 20-32% of all bloodstream isolates

• CoNS most frequent isolates in implanted devices

associated infections

• High level of antibiotic resistance in FBI

associated CoNS

Staphylococci and staphylococcal biofilms in foreign body infections

• Biofilms represent a

protected growth mode

– Against changing environmental conditions (pH, hypoxia, …), against disinfectants and disinfecting techniques, against host defence mechanisms, against antibiotics

» Costerton, Science, 99, 284, 1318; De Beer, AEM, 94, 60, 4339; Cochrance, J Med Microbiol., 92, 36, 255; Khoury, Am Soc Artif Int Organs J, 92, 38, 174; Chen, Environ Sci Technol, 96, 30, 2078; Bolister, JAC, 89, 24, 619; Dunne, AAC, 93, 37, 2522; Anderl, AAC, 00, 44, 1818

Molecular events in staphylococcal biofilm formation

• Three stages:

– Attachment:

• Uncoated, coated material

– Biofilm formation

• Intercellular adhesion and accumulation of multi-

layered cell clusters

• Generation of slime glycocalix

– Persistence and detachment (phase variation)

Molecular events in staphylococcal biofilm formation

Vuong, Micr Infect, 02, 4, 481

Molecular events in staphylococcal biofilm formation

• Initial attachment to uncoated plastic material
• primary adhesion: within seconds after exposure, aspecific, dependent
• n physicochemical interactions and surface properties of foreign

body surface

• Main bacterial parameter: hydrophobicity of bacterial surface

– Role of AtlE (autolysin) in surface hydrophobicity » Direct binding, loss of surface proteins, binding to host-matrix proteins – Role of lipoteichoic-like acids? » Altered surface charge, decreased binding of surface proteins – Role of fimbria-like polymers » SSP1 and SSP2

• Significance for biofilm formation considering rapid (seconds)

coating of foreign body?

» Ferreiros, FEMS Microbiol Lett, 89, 51, 89; Vacheethasanee, J Biomed Mat, 98, 42, 425; Vacheethasanee, J

Biomed Mat, 00, 50, 302; Heilmann, Infect Immun, 96, 46, 277; Gross, Infect Immun, 01, 69, 3423; Lambert, FEMS Immun Med Micro, 00, 29, 195

Molecular events in staphylococcal biofilm formation

• (Secondary) attachment to material coated with host-derived

proteins

• Promoted by surface irregularities, host-derived substances

(fibronectin, collagen, laminin, vitronectin, fibrinogen, fibrin thrombi, activated platelets)

• MSCRAMM’s:

– fibrinogen-binding protein (Fbe) – Few peptidoglycan-bound surface proteins – Role of non-covalently linked surface proteins (AtlE)

• Polysaccharide Intercellular Adhesin (PIA) or PNAG (S.

aureus)

– Encoded by icaABCD

» Gross, Infect Immun, 01, 69, 3423; Franson, JCM, 84, 20, 500;; Heilman, Mol Microbiol, 97, 24, 1013; Timmerman, Infect Immun, 91, 59, 4187; Tojo, JID, 88, 157, 713; Mc Kenney, Infect Immun, 98, 66, 4711; Dunne, CMR, 02, 15, 155; Nilsson, Infect Immun, 98, 66, 2666

Molecular events in staphylococcal biofilm formation

• Intercellular adhesion and accumulation

– Within 40 to 60 min after adhesion – Formation of multi-layered clusters of interconnected cells – Several polymeric carbohydrates and proteins involved

• Accumulation Associated Protein (AAP)
• Polysaccharide Intercellular Adhesin (PIA) or

PNAG (S. aureus)

– encoded by icaABCD

» Mack, J Bac, 96, 178, 175; Heilmann, Mol Microbiol, 97, 24, 1013; Husain, Infect Immun, 97, 65, 519; Stewart, Lancet, 01, 358, 135; Zimmerli, JID, 82, 146, 487

Molecular events in staphylococcal biofilm formation

20% residues deacetylated PIA: linear homoglycan of β-1,6-linked N-acetylglucosamine

Molecular events in staphylococcal biofilm formation

• Generation of extra cellular slime

– Composition extra cellular slime

• Teichoic acid
• Bacterial proteins
• Host proteins
• Polysaccharide Intercellular Adhesin (PIA)

Molecular events in staphylococcal biofilm formation

Model for PIA – PNAG biosynthesis

Gotz, Mol Micro, 02, 43, 1367; McKenney, Infect Immun, 98, 66, 4711

PS/A

Regulation of ica operon expression

• Anaerobic growth conditions
• Low concentrations of antibiotics
• Osmotic stress
• Environmental regulation via icaR
• TcaR in S. aureus

Molecular events in staphylococcal biofilm formation

• Persistence:

– Deficiencies in local host immune response

• Due to bacterial products
• Due to the foreign body

» Chuard, JID, 91, 163, 1369; Chuard, AAC, 93, 37, 625; BaddourJID, 88, 157, 757; Zimmerli, JID, 82, 146, 487; Francois, 9, 17, 514;

– Intrinsic resistance to antimicrobial compounds

Molecular events in staphylococcal biofilm formation

Development of an in vivo rat model of FBI

– First generation offspring from ex-germfree inbred Fisher rats – Subcutaneous implantation of catheter fragments inoculated with defined number of S. epidermidis CFU – Explantation of catheters after varying time intervals and study of adherent staphylococci – Use of low (physiological) inocula of S. epidermidis and pre-implantation inoculation – Development of FBI with S. epidermidis in 90-100% if no prophylaxis given

» Van Wijngaerden, JAC, 99, 44, 669; Van Eldere, Micro Ther, 99, 28, 307

Molecular events in staphylococcal biofilm formation

persistence of CoNS biofilms:

• resistance against host immune defences

blood foreign body Numbers log10 CFU 6.3 / 1.7 6.2 / 2.1 Chemotactic index 2.4 / 0.2 2.0 / 0.5 phagocytosis 410 / 74 60 / 8.9 respiratory burst activity 12.2 / 0.5 4.4 / 2.8 expression of ICAM-1 3.7 / 2.4 43 / 9.8

Van Eldere, Micro Ther, 99, 28, 307

Molecular events in staphylococcal biofilm formation

• Quantification of gene expression in FBI-associated
• S. epidermidis
• rapid disruption and isolation nucleic acids (< 3 min)
• ⇒ use of instant mechanical disruption (FastPrepTM)

in presence of lytic solution combined with Qiagen RNeasy kit + RNase treatment to reduce gDNA contamination

• Cheung, Anal.Biochem,1994,222,511-514; Vandecasteele, J Bac, 01, 183, 7094
• use of gDNA as a measure of the initial amount of

bacteria

• Vandecasteele,BBRS 02, 291, 528
• .

Molecular events in staphylococcal biofilm formation

30 60 90 120 150 180

• 1.0
• 0.5

0.0 0.5 1.0 1.5

time (min) log (16S expression)

Red: sessile Blue: planktonic 16S expression in vitro

Molecular events in staphylococcal biofilm formation

2880 5760 8640 11520 14400 17280 20160

time (in minutes)

• 1,0
• 0,5

0,0 0,5 1,0 1,5

l

• g

( 1 6 S e x p r e s s i

• n

)

] ] ] ] ] ] ] ] ] ] ]

240 480 720 960 1200 1440

time (in minutes)

• 1,0
• 0,5

0,0 0,5 1,0 1,5

] ] ] ] ] ] ] ]

16S expression in vivo

Molecular events in staphylococcal biofilm formation

5000 10000 15000 20000

time (min)

• 3,00
• 2,50
• 2,00
• 1,50
• 1,00
• 0,50

l

• g

1 ( i c a C e x p r e s s i

• n

)

] ] ] ]] ] ] ] ] ] ] Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω

icaC expression in vivo

In vitro expression of biofilm-associated genes

In vitro expression of biofilm-associated genes

In vivo expression of biofilm-associated genes

Contents

• Foreign body infections and role of

staphylococci

• Molecular events in staphylococcal

biofilm formation

– Different stages in biofilm formation – Cellular control of biofilm formation

Molecular events in staphylococcal biofilm formation

• Cellular control of biofilm formation

– Role of agr quorum-sensing system:

• Stimulates expression virulence factors
• Down regulates expression of surface –proteins

(including AtlE)

– Role of additional regulatory loci:

• sar: SarA co-stimulates with AgrA~P transcription

RNAIII

• SigB: stimulates PIA and biofilm production

» Otto, FEBS Lett, 98, 424, 89; Rachid, AAC, 00, 44, 3357; Fluckiger, Infect Immun, 98, 66, 2871; Vuong, Infect Immun, 00, 68, 1048; Otto, Pept, 01, 22, 1603

Bronner

Bronner

Molecular events in staphylococcal biofilm formation

/ +

?

Global regulation of protein expression in S. epidermidis

Vuong, Micr Infect, 02, 4, 481

Knobloch

In vitro expression of S. epidermidis genes in sessile versus planktonic bacteria

• 3,20
• 2,40
• 1,60
• 0,80

0,00 0,80 60 120 180 time(min)

log10 agrA

• 3,20
• 2,40
• 1,60
• 0,80

0,00 0,80 60 120 180 time(min)

log10 agrA

• 3,20
• 2,40
• 1,60
• 0,80

0,00 0,80 60 120 180 time(min)

log10 RNAIII

• 3,20
• 2,40
• 1,60
• 0,80

0,00 0,80 60 120 180 time(min)

log10 RNAIII

BHI NaCl

In vitro expression of S. epidermidis genes in sessile versus planktonic bacteria

• 3,20
• 2,40
• 1,60
• 0,80

0,00 0,80 60 120 180 time(min)

log10 sarA

• 3,20
• 2,40
• 1,60
• 0,80

0,00 0,80 60 120 180 t ime(min)

log10 sarA

• 3,20
• 2,40
• 1,60
• 0,80

0,00 0,80 60 120 180 t ime(min)

log10 sigB

• 3,20
• 2,40
• 1,60
• 0,80

0,00 0,80 60 120 180 time (min)

log10 sigB

BHI NaCl

In vitro expression of S. epidermidis genes in sessile versus planktonic bacteria

• 3,20
• 2,40
• 1,60
• 0,80

0,00 0,80 60 120 180 t ime(min)

log10 rsbU

• 3,20
• 2,40
• 1,60
• 0,80

0,00 0,80 60 120 180 t ime(min)

log10 rsbU

• 3,20
• 2,40
• 1,60
• 0,80

0,00 0,80 60 120 180 t ime(min)

log10 rsbV

• 3,20
• 2,40
• 1,60
• 0,80

0,00 0,80 60 120 180 t ime(min)

log10 rsbV

In vivo expression of S. epidermidis genes in sessile bacteria

• 3 ,0
• 2 ,0
• 1 ,0

6 0 120 18 0 2 40 3 00 3 60 time(ho urs)

• 3,0
• 2,0
• 1,0

2880 5760 8640 11520 14400 17280 2 time(hours)

• 2,0
• 1,0

0,0 1,0 2,0 60 120 180 240 300 360 time(hours)

• 2,0
• 1,0

0,0 1,0 2,0 2880 5760 8640 11520 14400 17280 2 time(hours)

In vivo expression of S. epidermidis genes in sessile bacteria

• 3,5
• 2,5
• 1,5
• 0,5

0,5 60 120 180 240 300 360 time(hours)

• 3,5
• 2,5
• 1,5
• 0,5

0,5 2880 5760 8640 11520 14400 17280 20160 time(hours)

• 3 ,0
• 2 ,0
• 1 ,0

0 ,0 6 0 1 2 0 1 8 0 2 4 0 3 0 0 3 6 0 time(ho urs )

• 3,0
• 2,0
• 1,0

0,0 2880 5760 8640 11520 14400 17280 201 time(hours)

In vivo expression of S. epidermidis genes in sessile bacteria

• 3,0
• 2,0
• 1,0

60 120 180 240 300 360 time(hours)

• 3,0
• 2,0
• 1,0

2880 5760 8640 11520 14400 17280 20160 time(hours)

• 3 ,0
• 2 ,0
• 1 ,0

6 0 1 2 0 1 8 0 2 4 0 3 0 0 3 6 0 time(ho urs)

• 3,0
• 2,0
• 1,0

2880 5760 8640 11520 14400 17280 20160 time(hours)

• E. Van Wijngaerden
• J. Vandersmissen
• S. Vandecasteele
• Valerie Pintens
• Caroline Massonet
• R. Merckx