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Staphylococcal bacteraemia: finding the path of least persistence Nisha Ranganathan, Rebecca Johnson, Andrew M. Edwards Background • Despite the falling incidence of MRSA bacteraemia, the rate of MSSA bacteraemia is rising 1 • In a significant propor@on of cases, bacteraemia persists despite appropriate therapy 2 • Challenges to treatment arise as under the twin threats of an@bio@cs and the immune system, bacteria become dormant, metabolically inac@ve and are thus an@bio@c tolerant 3 • The regulator of the stress response in S. aureus , SigB, regulates entry into dormancy 4 The host environment modulates an7bio7c suscep7bility A B C Figure 2. (A) Titra7ng concentra7on of AHT in the complemented sigB mutant resulted in increasing sigB expression, as evidenced by increase in pigment produc7on and (B)fibronec7n binding (both sigB dependent phenotypes). (C)Increasing survival of S. aureus in blood on increasing induc7on of sigB expression with AHT in complemented mutant but not in empty plasmid vector. Figure 1. Blood modulates an7bio7c suscep7bility of S. aureus on exposure to Cloxacillin (A) Gentamicin (B), Vancomycin (C), Ciprofloxacin (D) Clindamycin (E). With the excep7on of (B) the addi7on of an7bio7c failed to significantly enhance killing compared to blood alone, however an7bio7cs func7oned in serum. A SigB modulates an7bio7c suscep7bility in the host context Figure 4. (A) sigB effectors SA2440 and SA2452 promote survival in blood infec7on model. Increasing expression of SA2452 but not SA2440 increases survival in blood alone (A) and in blood plus cloxacillin (B) in a dose dependent manner. Increasing expression of both genes increases survival on exposure to ciprofloxacin (C). SA2440 but not SA2452 promotes survival on exposure to gentamicin (D). Conclusions • Blood appears to have an inhibitory effect on the ac@vity of certain an@bio@cs. • SigB promotes survival in both blood alone plus in combina@on with cloxacillin, ciprofloxacin and gentamicin. • Two SigB regulated genes have been iden@fied that promote survival in an an@bio@c specific manner. • Rethinking therapy in the context of the host-pathogen-drug interac@ons may lead to innova@ve solu@ons to enhance exis@ng treatment strategies. References 1. Health Protec@on Agency. Voluntary Repor2ng of Staphylococcus aureus Bacteraemia in England, Wales and Northern Ireland, 2010. London: Health Protec@on Agency, 2011 2. Kha@b R, et al. 2006. Persistence in Staphylococcus aureus bacteremia: incidence, characteris@cs of pa@ents and outcome. Scand. J. Infect. Dis. 38: 7–14 3. K Lewis Mul@drug tolerance of biofilms and persister cells Curr Top Microbiol Immunol, 322 (2008), pp. 107–131 Bischoff M, Dunman P, Kormanec J, Macapagal D, Murphy E, Mounts W, Berger- Bachi B, Projan S. 2004. Microarray-based analysis of the Staphylococcus aureus σB regulon. J Bacteriol 186:4085–4099. Acknowledgements Members of the fi‘h floor of the Centre for Molecular Bacteriology and Infec@on at Imperial College London, and the Edwards group in par@cular, are acknlowedged for their invaluable support. This work is funded by the MRC as part of a clinical research training fellowship SigB promotes survival in blood 0 10 100 0 10 100 AHT WT ΔsigB ΔsigBpEmpty ΔsigBpEmpty ΔsigBpEmpty ΔsigBpsigB ΔsigBpsigB ΔsigBpsigB 0 10 20 30 40 50 % Survival 0 10 100 0 10 100 AHT WT ΔsigB ΔsigBpEmpty ΔsigBpEmpty ΔsigBpEmpty ΔsigBpsigB ΔsigBpsigB ΔsigBpsigB 0.0 0.2 0.4 0.6 0.8 gentamicin % Survival WT ΔsigB ΔsigBpEmpty ΔsigBpEmpty ΔsigBpEmpty ΔsigBpsigB ΔsigBpsigB ΔsigBpsigB 0 2 4 6 8 cloxacillin % Survival WT ΔsigB ΔsigBpEmpty ΔsigBpEmpty ΔsigBpEmpty ΔsigBpsigB ΔsigBpsigB ΔsigBpsigB 0 2 4 6 8 10 ciprofloxacin % Survival WT ΔsigB ΔsigBpEmpty ΔsigBpEmpty ΔsigBpEmpty ΔsigBpsigB ΔsigBpsigB ΔsigBpsigB 0 100 200 300 clindamycin % Survival Blood Serum WT ΔsigB ΔsigBpEmpty ΔsigBpEmpty ΔsigBpEmpty ΔsigBpsigB ΔsigBpsigB ΔsigBpsigB 0 50 100 150 vancomycin % Survival SigB regulated genes modulate survival in blood +/- an7bio7cs D WT ΔsigB p2440 p2440 p2440 2440pEmpty 2440pEmpty 2440pEmpty p2452 p2452 p2452 2452pEmpty 2452pEmpty 2452pEmpty 0 5 10 15 Blood Alone % Survival WT ΔsigB p2440 p2440 p2440 2440pEmpty 2440pEmpty 2440pEmpty p2452 p2452 p2452 2452pEmpty 2452pEmpty 2452pEmpty 0 5 10 15 20 25 % Survival Blood + Cloxacillin WT ΔsigB p2440 p2440 p2440 2440pEmpty 2440pEmpty 2440pEmpty p2452 p2452 p2452 2452pEmpty 2452pEmpty 2452pEmpty 0 2 4 6 % Survival Blood + Ciprofloxacin WT ΔsigB p2440 p2440 p2440 2440pEmpty 2440pEmpty 2440pEmpty p2452 p2452 p2452 2452pEmpty 2452pEmpty 2452pEmpty 0.00 0.05 0.10 0.15 0.20 % Survival Blood + Gentamicin B D C A E c B 0 2 4 6 10 100 1000 Cloxacillin Time (h) % Survival 0 2 4 6 1 10 100 1000 Cloxacillin Time (h) % Survival 0 2 4 6 1 10 100 1000 Cloxacillin Time (h) % Survival 0 2 4 6 1 10 100 1000 Cloxacillin Time (h) % Survival 0 2 4 6 0.001 0.01 0.1 1 10 100 1000 Gentamicin Time (h) % Survival 0 2 4 6 1 10 100 1000 Vancomycin Time (h) 0 2 4 6 0.001 0.01 0.1 1 10 100 1000 Ciprofloxacin Time (h) % Survival 0 2 4 6 10 100 1000 Clindamycin Time (h) % Survival B Figure 3. Increasing sigB expression increases survival of S. aureus in blood plus cloxacillin ciprofloxacin and gentamicin (A, B, C) but not on exposure to blood plus vancomycin or clindamycin (D,E) . A • Aim: To characterise the role of SigB and its effector molecules in the survival of S. aureus in a clinical bacteraemia model Lewis et al 3 C D E

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Page 1: Nisha Ranganathan, Rebecca Johnson, Andrew M. Edwardsevent.federationinfectionsocieties.com/wp-content/... · 2017. 12. 18. · Nisha Ranganathan, Rebecca Johnson, Andrew M. Edwards

Staphylococcal bacteraemia: finding the path of least persistence NishaRanganathan,RebeccaJohnson,AndrewM.Edwards

Background•  DespitethefallingincidenceofMRSAbacteraemia,therateofMSSAbacteraemiaisrising1

•  Inasignificantpropor@onofcases,bacteraemiapersistsdespiteappropriatetherapy2

•  Challengestotreatmentariseasunderthetwinthreatsofan@bio@csandtheimmune

system,bacteriabecomedormant,metabolicallyinac@veandarethusan@bio@ctolerant3

•  TheregulatorofthestressresponseinS.aureus,SigB,regulatesentryintodormancy4

Thehostenvironmentmodulatesan7bio7c

suscep7bility

A B

C

Figure2.(A)Titra7ngconcentra7onofAHTinthecomplementedsigBmutantresultedinincreasingsigB expression, as evidencedby increase inpigmentproduc7onand (B)fibronec7nbinding (bothsigBdependentphenotypes). (C)IncreasingsurvivalofS.aureusinbloodonincreasinginduc7onofsigBexpressionwithAHTincomplementedmutantbutnotinemptyplasmidvector.

Figure1.Bloodmodulatesan7bio7csuscep7bilityofS.aureusonexposuretoCloxacillin(A)Gentamicin(B),Vancomycin(C),Ciprofloxacin(D)Clindamycin(E).Withtheexcep7onof(B)theaddi7onofan7bio7cfailedtosignificantlyenhancekillingcomparedtobloodalone,howeveran7bio7csfunc7onedinserum.

A

SigBmodulatesan7bio7csuscep7bilityinthehostcontext

Figure 4. (A) sigB effectors SA2440 and SA2452 promote survival in blood infec7onmodel.IncreasingexpressionofSA2452butnotSA2440increasessurvival inbloodalone(A)andinbloodplus cloxacillin (B) inadosedependentmanner. Increasingexpressionofbothgenesincreasessurvivalonexposuretociprofloxacin(C).SA2440butnotSA2452promotessurvivalonexposuretogentamicin(D).

Conclusions•  Bloodappearstohaveaninhibitoryeffectontheac@vityofcertainan@bio@cs.

•  SigBpromotessurvivalinbothbloodaloneplusincombina@onwithcloxacillin,ciprofloxacinandgentamicin.

•  TwoSigBregulatedgeneshavebeeniden@fiedthatpromotesurvivalinanan@bio@cspecificmanner.

•  Rethinkingtherapyinthecontextofthehost-pathogen-druginterac@onsmayleadtoinnova@vesolu@onstoenhanceexis@ngtreatmentstrategies.

References1.  HealthProtec@onAgency.VoluntaryRepor2ngofStaphylococcusaureusBacteraemiainEngland,WalesandNorthernIreland,2010.London:HealthProtec@onAgency,20112.  Kha@bR,etal.2006.PersistenceinStaphylococcusaureusbacteremia:incidence,characteris@[email protected]:7–143.  KLewisMul@drugtoleranceofbiofilmsandpersistercellsCurrTopMicrobiolImmunol,322(2008),pp.107–131BischoffM,DunmanP,KormanecJ,MacapagalD,MurphyE,MountsW,Berger-BachiB,ProjanS.2004.Microarray-basedanalysisoftheStaphylococcusaureusσBregulon.JBacteriol186:4085–4099.

AcknowledgementsMembersofthefi`hflooroftheCentreforMolecularBacteriologyandInfec@onatImperialCollegeLondon,andtheEdwardsgroupinpar@cular,areacknlowedgedfortheir invaluable support. This work is funded by the MRC as part of a clinicalresearchtrainingfellowship

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Figure3.IncreasingsigBexpressionincreasessurvivalofS.aureusinbloodpluscloxacillinciprofloxacinandgentamicin(A,B,C)butnotonexposuretobloodplusvancomycinorclindamycin(D,E).

A

•  Aim:TocharacterisetheroleofSigBanditseffectormoleculesinthesurvivalofS.aureusinaclinicalbacteraemiamodelLewisetal3

C D E

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