Individuel eller i flok

Os og bakterierne

Forskellen

Bakterier er flok dyr

I og på vores krop

Det humane hud mikrobiom

Hår follikle

Akutte infektioner Necroticerende fasciitis af S. pyogenes gruppe A

Antibiotika

MBEC and MBIC MBIC MBEC Minimal Biofilm Inhibitory Concentration Minimal Biofilm Eradication Concentration Mus behandlet intraperitonalt med 1x colistin (16 mg per kg) eller imipenem (64 mg per kg). Bjarnsholt et al Nature Review Drug Discovery. 2013 Oct 1;12(10):791-808

Aggregering Voksende celler Stationære celler (tolerante) Immunforsvaret og antibiotika virkningsløse mod Immunforsvaret og antibiotika virker (for det meste)

Individuel eller i flok Studie fra 1956. 7.500.000 Staphylococcus aureus bakterier sprøjtes ind under huden på frivillige = kun 50% inficeres, alle kommer sig < 100 bakterier på stof under huden = 100% inficeres, INGEN kommer sig Ann N Y Acad Sci. 1956 Aug 31;65(3):85-90. Experimental staphylococcal infections in the skin of man. ELEK SD.

Det humane hud mikrobiom Courtesy of Lise Christensen Bjarnsholt et al. 2008

nyt 4 dage efter steroid Courtesy of Lise Christensen

site Type of PAAG Time since inj Initial treatment Time with AE Cheek Aquamid 2 years Steroid inj Later AB inj Lip Aquamid ½ year Steroid inj Later ABs Lip Aquamid 1 month Steroid + ABs Later ABs Breast Amazing gel 2 years ABs liposuction Tear-trough Aquamid 2 years Steroid inj Later ABs Naso-labial fold Aquamid 14 days Steroid ABs+surgery Lip Interfall gel ½ year Steroid ABs+surgery Penis Aquamid 2 years Steroid AB inj+surgery 7 months ½ year 2 years 5 months ½ year 1½ year 2½ years 1½ years 4 days after steroid Courtesy of Lise Christensen

Bjarnsholt et al; Dermatol.surg. 2009 (Aug). Followup: Clin Infect Dis. 2013 and animal experiments Pathog Dis. 2014

Biofilm i kroniske sår Bjarnsholt et al; Wound Repair and Regeneration, 2008 Jan-Feb;16(1):2-10.

Cystisk fibrose det klassiske eksempel CF mand, 28 års kronisk bakterieinfektion 4 x 2 ugers/år IV antibiotika behandling 20 års daglig inhalering af antibiotika 1 kg tobramycin, 10 kg beta-lactam antibiotika og 1 kg inhaleret colistin Bjarnsholt et al; Pseudomonas aeruginosa biofilms in the Respiratory Tract of Cystic Fibrosis Patients; Pediatr Pulmonol. 2009 Jun;44(6):547-58

Kronisk mellemørebetændelse

Biofilm infektioner Kronisk langstrakt infektion Ofte virker antibiotika ikke Modstår immunforsvaret Behandlingssvigt eneste mulighed er ofte at fjerne implantatet Svært at diagnosticere

Biofilm klinisk Hvad? Hvor? Hvordan skal der tages prøver? Hvordan skal der diagnosticeres? Hvad er sværest ved at diagnosticere en biofilm infektion?

Hvor? McConoughey et al. 2014

Fordeling af specier S. aureus P. aeruginosa

Forskellige områder

Heterogen fordeling af bakterier i kroniske sår qpcr Pseudomonas aeruginosa 9 12 Positio n Wound 1 Wound 2 C C 510±18% 920±9% 6 3 3 No sample 300±13% 6 760±7% 8200±8% 9 47±9% 800±10% 12 280±3% 15±5% Thomsen TR, Aasholm MS, Bjarnsholt T, Givskov M, Kirketerp-Møller K, and Nielsen PH. 2010. The bacteriology of chronic venous leg ulcer examined by culture-independent molecular methods. Wound Repair and Regeneration: Jan-Feb;18(1):38-49 Picture from homepage of Montana State University

Hvordan genkender man en biofilm? A. Bakterier på en overflade B. Store biofilm C. Bakterier og inflammation

in vivo Biofilmen Sår mushrooms Små microkolonier værtsmateriale Inflammation Heterogen fordeling Uafhængig af overflade CF lunge Implantat muse model Bjarnsholt et al Trends in Microbiology Trends Microbiol. 2013 Sep;21(9):466-74

Biofilm demonstrated in Visualization method Approximately diameter size lung infections Light microscopy ~4-8 µm Light microscopy ~5-100 µm FISH ~5-100 µm FISH ~5-50 µm Chronic wounds FISH ~5-200 µm Light and electron microscopy ~35-55 µm Reference Høiby 1977 5, Baltimore 1989 72 Kirketerp-Møller 73 Lefmann 74 Bjarnsholt 2008 64 James 2008 75 Soft tissue fillers FISH ~5-25 µm Bjarnsholt 65 Otitis media FISH FISH FISH ~15-25 µm ~10-80 µm ~4-40 µm Hall-Stoodley 2006 76 Nistico 2011 77 Homoe 67 Implant etc associated Electron microscopy FISH Electron microscopy FISH ~500 µm ~50 µm ~5-15 µm ~5-30 µm Marrie1982 78, Waar 2005 79 Costerton 2010 80 Veeh 2003 81 Catheter and shunt Electron microscopy ~5-1000 µm Marrie 1983 82 associated Electron microscopy ~20-500 µm Marrie 1984 83 Fluorescence microscopy ~20-1200 µm Stoodley 2010 84 FISH and electron microscopy >1000 µm Parsa 2010 85 Chronic osteomylistis Electron microscopy ~25 µm Gristina 1985 86, Electron microscopy ~25 µm Marrie 1985 87 Light and electron microscopy ~5-50 µm Sedghizadeh 2009 88 Chronic Rhinosinusitis Electron microscopy ~5-30 µm Cryer 2004 89 Fluorescence microscopy ~5-20 µm Li 2011 90 Contact linses Electron microscopy ~50-100 µm Stapleton 1995 91

Surface or not to surface that is not the question Alhede M et al.; PLoS One. 2011;6(11):e27943

Diagnosis Method Advantages Pitfalls and difficulties Culturing Bacterial presence is confirmed Antibiotic susceptibility Direct quantification Heterogeneous distribution Finding the focus Pathogens vs. contamination Biofilms or planktonic samples can be culture-negative PCR etc Microscopy Fast results even when culture is negative Low cut-off (used to be 10 5-6 ) Biofilms are confirmed Interactions with tissues Inflammatory cells Results even when culture-negative Heterogeneous distribution Finding the focus Pathogens vs. contamination Biofilm or planktonic Heterogeneous distribution Finding the focus

MEN Hvordan opstår kroniske infektioner?

Det humane hud mikrobiom

bakterier?

Eller manglende diversitet? Normal skin flora er ændret hos patienter med Hidradenitis Suppurativa

Aggregater har en fordel! 1 A Single Cells B Aggregate 2 C D 3 Kragh et al MBio. 2016 Mar 22;7(2):e00237

Modstand mod immunforsvaret

Planktoniske bakterier Af Maria Alhede

PMNer mod biofilm bakterier Flow celle Biofilm

Hvad hvis man ikke kan finde noget? Medical history of biofilm predisposing condition (implanted medical device, CF, IE, chronic OM)(See Table 1) Recurrence of the infection (particularly if evidence is provided that the same organism is responsible at multiple time points) Documented evidence/history of antibiotic failure or persistent infection despite adequate choice of antibiotic agent Evidence of local or systemic signs and symptoms of infection that resolve with antibiotic therapy, only to recur after therapy has ceased such as: ** Hall-Stoodley L, Stoodley P, Kathju S, Høiby N, Moser C, William Costerton J, Moter A, Bjarnsholt T.; Towards Diagnostic Guidelines for Biofilm-Associated Infections.; FEMS Immunol Med Microbiol. 2012 Apr

Forebyggelse? Stratum corneum Epidermis Bacteria primarily found in the stratum corneum (1 exsample of bacteria in hair follicle) Litterature: 85 % in stratum corneum 25 % in hair follicle Lange-Asschenfeldt et al. 2011

Udfordringen Biofilm INGEN diagnostik INGEN behandling MEN VI ARBEJDER PÅ DET

Acknowledgments University of Copenhagen: Maria Alhede Kasper N Kragh Steffen R Eickhardt-Sørensen Anne K Nielsen Stephanie G Crone Majken Sønderholm Blaine Fritz Camilla Stavnsbjerg Marie Thaysen Lasse Kvich Lene Bay Anne Hesselvig -------------- Michael Givskov, Klaus Qvortrup Oana Ciofu, Søren Sørensen Michael Kühl, Mette Burmølle Hans Petter Hougen, Henrik Elvang Jensen Louise Kruse Johansen Rigshospitalet: Niels Høiby Peter Østrup Jensen Claus Moser Kim Thomsen Lars Christophersen Michael Tvede Claus B Andersen Preben Homøe Others: Klaus Kirketerp-Møller Lise H Christensen Trine Rolighed Thomsen Claus Sternberg Christine R Hansen Tanja Pressler Mark Shirtliff Claus Manniche Benny Dahl Anders Odgaard Søren Orth-Nissen

Funding Sources -Human Frontier Science project -Capital Region Research Foundation for Health Research -Novo Nordisk A/S -AdvanDx Inc. -Contura

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