Ifit’sonthepitch,isitinterferingwithplay?

CraigWilliams

Therearelotsofbugsinulcers!

•  Labreport

Ifit’sonthepitch,isitinterferingwithplay?

Thedifferencebetweenmicrobiologyandcardiology

Doubtisnotapleasantcondition,butcertaintyisabsurd

Voltaire

DiabeticfootulcersareaproblemMorethan60,000peoplewithdiabetesinEnglandarethoughttohavefootulcersatanygiventime.In2014-15theannualcostofdiabeticfootdiseasetotheNHSinEnglandwasestimatedat£1billion,inadditiontothepersonal/socialcostsofreducedmobilityandsicknessabsence.Onlyaroundhalfofpeoplewithdiabeteswhohavehadadiabeticfootulcersurvivefor5years.Treatmentfordiabeticfootdiseasemayinvolveamputation.Around7,000peopleeachyear. NationalDiabetesFootCareAudit-

2014-2016

Table 8: Factors associated with being alive and ulcer-free at 12 and 24 weeks, England and Wales, 2014-2016

Factors associated with healing

Outcome at ___ weeks

12 24

Associated with better healing

Patient is female p p

Patient is from a less deprived area of the country tu p

Patient has Black or Asian ethnicity p tu

Patient has Type 1 diabetes p tu

Patient has had diabetes for less than 5 years p p

Patient has had diabetes at least 5 and less than 10 years p tu

Patient self-referred to the specialist foot care service p tu

Associated with worse healing

Patient has mixed or ‘other’ ethnicity tu q

Patient currently smokes q tu

Patient presented with Charcot foot disease q q

…with Site/Ischaemia/Neuropathy/Area/Depth q q

…with Bacterial infection q tu

Patient waited more than 2 months for expert assessment q q

Patient has not had all 8 NICE recommended processes q q

Key: Strength of models (c-statistic) = poor. See Glossary (Statistical terms) in the main report for explanation of terms.

▲ = Associated with better healing; ▼ = associated with worse healing;◄► = no association found. Tested at the 0.05 level.

Table 10: Ulcer factors associated with ulcer healing at 12 and 24 weeks, England and Wales, 2014-2016 12 weeks 24 weeks Strength of model (c-statistic)1 Poor 0.697 Poor 0.672

Ulcer characteristicOdds ratios1

12 weeks 24 weeks SINBAD element: Site (on hindfoot) q 0.810 q 0.702 SINBAD element: Ischaemia q 0.494 q 0.482 SINBAD element: Neuropathy q 0.637 q 0.756 SINBAD element: Bacterial infection q 0.792 tu SINBAD element: Area (≥1cm2) q 0.504 q 0.594 SINBAD element: Depth (to tendon or bone) q 0.670 q 0.684 Charcot foot disease = present2 q 0.722 q 0.620 Charcot foot disease = possible2 p 1.600 tu Charcot foot disease = unknown2 p 1.248 tu Time to expert assessment = self-referred3 p 1.191 tu Time to expert assessment = >2 months3 q 0.557 q 0.595

Notes: 1. See Glossary (Statistical terms) in the main report for explanation of terms. 2. Vs. Charcot foot disease = not present 3. Vs. Time to expert assessment = ≤2 days.

Key: Strength of models (c-statistic) = poor. See Glossary (Statistical terms) in the main report for explanation of terms. ▲ = Associated with better healing; ▼ = associated with worse healing;◄► = no association found. Tested at the 0.05 level.

Rememberthechangebetween12and24weeks

There are lots of bugs everywhere!

Especiallyontheskin•  The skin, our largest organ is completely covered with microbes,

with an estimate of about 1 billion microbial cells per cm2 of skin covering its surface and extending down into the appendages and glands

•  Variability between individuals and variability between different body sites is common

•  the dominant genera of skin bacteria are relatively stable and include Staphylococcus, Propionibacterium, and Corynebacterium,

•  Streptococcus and Pseudomonas are isolated less frequently but account for most variability

KongJinvestDermatol2012Mar;132(3Pt2):933-9

Arediabeticsdifferent?

Thediabeticmicrobiota

phylumActinobacteria,(Corynebacterium),ismoreprevalentindiabeticfootskinNofollowupsonoextrapolationtopossibleDFUprognosticmicrobialsignatureswasattainablewiththisstudy.

RedelJInfDis2013Apr;207(7):1105-14

Isthereabadmicrobiome

Smith K. BMC Microbiol 2016 Mar 22;16:54

Eachulcerisanexperimentinbacteriology

Don’tforgetthefungi

KalanMbio2016

Sofar

•  Complexmixtureofbacteriaandfungi•  Nosmokinggunsofar

HowdoorganismsgrowinulcersBiofilms are present in most, if not all, chronic non-healing wounds 78% of chronic wounds contain a biofilm. Micro-organisms in biofilms are not only located at the wound surface but may also be present in deeper tissues

Malone J Wound Care 2017;26:20-5 Schaber Infect Immun2007;75:3715-21

Howisthebiofilmgrowing?

EurJOralSci.2007,115:459–467.JClinMicro2008,8:2717-22

Streptococci Actinomycesnaeslundii

FunctionallyEquivalentPathogroup:consortiaofgenotypicallydistinctbacteriathatsymbioticallyproduceapathogeniccommunity.

Whatdoescompetitionmean?

•  Mixturesinbiofilmsbehavedifferentlytoplanktonicmixtures

•  StaphaureusandPseudomonas

•  Biofilmsoveralldeveloptoreduceimmunogenicity

•  Complexinter-kingdomandinter-speciesinteractionsoccurinbiofilm

Bacterial interaction: Staph and Pseudomonas

Armbruster et al. mBio 2016; doi:10.1128/mBio.00538-16

ProteinAfromSaureusprotectsPsaeruginosafromneutrophilphagocytosis

Bacterial interaction: Staph and Pseudomonas

Limoli et al. mBio 2017; doi:10.1128/mBio.00186-17

Ps aeruginosa isolates from coinfected CF patients are less competitive with S. aureus.

Inter-kingdominteractions:CandidaandStaph

Kean.FrontMicrobiol.2017Feb23;8:258

Inter-kingdominteractions:CandidaandStaph

Kean.FrontMicrobiol.2017Feb23;8:258

Bacterialinteractionsinbiofilms:Mousemodel

P.aeruginosa

S.aureus E.faecalis,F.magna,andhostcellDNA

DaltonPLoSOne2011;6(11):

Mixedinfectionsdelayhealing

Doesthe“immunogenicity”ofthemixedbiofilmchangeovertimeThemiddleperiod(12weeks)seemstobeimportant

DaltonPLoSOne2011;6(11):

Rememberthechangebetween12and24weeks

Sofar

•  Thebugsgrowasbiofilms,probablyindiscreteclumpsof“competing”bacteriain“unhappy”biofilms

•  Bacterialspeciescan•  interacttomodifyhostresponse•  Interactwitheachothertoallowotherspeciestothrive

•  Interactwithfungitoincreasepathogenicity

Thisisallverycomplicated

The hydrogel-cellulose matrix (3D) model

C the cellulose matrix containing the microorganisms B 50% serum gel Allows the form of a 3D biofilm

structure.

A B

2Dv3Dmodel

C a S a P a10 0

10 1

10 2

10 3

10 4

10 5

10 6

10 7

10 8

O rg an ism

CFU

/mL

U n trea ted

P V P - I

C HX

# # # #

****

***

***

C a S a P a10 0

10 1

10 2

10 3

10 4

10 5

10 6

10 7

10 8

10 9

10 10

O rg an ism

CFU

/mL

U n trea ted

P V P - I

C HX

#* #

***

******

***

IndividualorganismsgrownonThermanoxcoverslips(2D)

IndividualorganismsgrowninCellulosematrix(3D)

CHXPVICONT

TownsendBiofouling2016Nov;32(10):1259-1270

2Dv3Dmodelwithantiseptics

TownsendBiofouling2016Nov;32(10):1259-1270

Viabilityin2Dand3DsystemsU n tre a te d

T o ta l L ive0

2 0

4 0

6 0

8 0

1 0 0

C a

S a

P a

Pe

rce

nta

ge

(%

)

1 .2 3 x 1 0 7 1 .5 3 x 1 0 6

P V P -I

T o ta l L ive0

2 0

4 0

6 0

8 0

1 0 0

C a

S a

P a

Pe

rce

nta

ge

(%

)

9 .2 1 x 1 0 5 3 .4 5 x 1 0 4

C H X

T o ta l L ive0

2 0

4 0

6 0

8 0

1 0 0

C a

S a

P a

Pe

rce

nta

ge

(%

)

3 .0 2 x 1 0 7 1 .2 5 x 1 0 6

UNTREATED

UNTREATED

PVI

PVI CHX

CHX

3Dmodelwithantimicrobials

TownsendAntimicrobAgentsChemother.2017Aug24;61(9).

Flucloxacillin

Ciprofloxacin

Fluconazole

TownsendAntimicrobAgentsChemother.2017Aug24;61(9).

https://woundsinternational.wordpress.com/tag/biofilm/

Muchofthefocusonpathogenesishasbeenonalteredneutrophilfunctionsuggestingthatinefficientchemotaxis,pooractivation,orfailuretoresolveinflammationpreventanappropriateresponsetomicrofloraandpromotetissuedestruction

MuchofthefocusonpathogenesisofLAPhasbeenonalteredneutrophilfunctionsuggestingthatinefficientchemotaxis,pooractivation,orfailuretoresolveinflammationpreventanappropriateresponsetomicrofloraandpromotetissuedestruction

Whataboutthehost

Biofilmstatenotbiofilmsource

•  Biofilmsource(i.e.derivedfromahealthyordiseasedsite)doesnotinfluencepatternofimmuneresponse,

•  Biofilmstate(i.e.homeostaticvs.disequilibrium)doesinfluencethepatternofimmuneresponse

VelskoOralMicrobiol2017Jun12;9(1):

Samebiofilmdifferentresponse

Intactbiofilmsarelessrecognizableandmoredifficulttophagocytosebymacrophagesduetotheextracellularmatrixthatcoatsand‘hides’thebacteriainthebiofilm.Differencesintheresponsestointactversusdispersedbiofilmsinboththemagnitudeandthepatternofthecytokineresponse,MacrophagesareskewedtoanM2phenotypewhenexposedtoS.aureusinabiofilm,renderingthemlessinflammatoryandlesscapableofclearingthebiofilmthanM1-typemacrophages

ThurlowJImmunol2011,186;11:6585-96.

Doesthehostselectthebacteria

E.coli(×),S.aureus(◆)(◊)A.baumannii(─),P.aeruginosa(●),(ο),(▪)

GonzalezmSphere2016Apr27;1(2).

Conclusion•  Dynamicsnotconstituentsofbiofilmmaydetermineoutcome

•  Windowofopportunityforbacteriatohaveanyinfluenceonulcerswhichisoverby24weeks.

•  Blackboxbacteriologyvstampcollecting

•  Antibioticstreatinfectionbutwilnothealwoundsandmayhaveunexpectedeffectsonbiofilm.

SoshouldwetreatthePseudomonas?

•  Labreport

Thanks

GordonRamageRanjithRanjendranLeighannSherry

KarenSmithEleanorTownsendRyanKean

AndyCollier