antimicrobial salvage therapy for persistent...

Clinical Therapeutics/Volume ], Number ], 2014

Antimicrobial Salvage Therapy for Persistent StaphylococcalBacteremia Using Daptomycin Plus Ceftaroline

George Sakoulas, MD1; Pamela A. Moise, PharmD2; Anthony M. Casapao, PharmD3;Poochit Nonejuie, PhD1; Joshua Olson, BS1; Cheryl Y.M. Okumura, PhD1;Michael J. Rybak, PharmD3; Ravina Kullar, PharmD4,*; Abhay Dhand, MD5;Warren E. Rose, PharmD6; Debra A. Goff, PharmD7; Adam M. Bressler, MD8;Yuman Lee, PharmD9; Joseph Pogliano, PharmD1; Scott Johns, PharmD10;Glenn W. Kaatz, MD11; John R. Ebright, MD11; and Victor Nizet, MD1

1University of California San Diego School of Medicine, La Jolla, California; 2Cubist Pharmaceuticals,Lexington, Massachusetts; 3Eugene Applebaum College of Pharmacy and Health Sciences, School ofMedicine, Wayne State University, Detroit, Michigan; 4Oregon State University/Oregon Health & ScienceUniversity, Portland, Oregon; 5New York Medical College, Valhalla, New York; 6University of WisconsinMadison School of Pharmacy, Madison, Wisconsin; 7The Ohio State University Wexner Medical Center,Columbus, Ohio; 8Dekalb Medical Center, Decatur, Georgia; 9Maimonides Medical Center, Brooklyn,New York; 10VA San Diego Healthcare System, San Diego, California; and 11John D. Dingell VA MedicalCenter, Wayne State University School of Medicine, Detroit, Michigan

Accepted for publication May 23, 2014.

ABSTRACT

Purpose: Guidelines recommend daptomycin com-bination therapy as an option for methicillin-resistantStaphylococcus aureus (MRSA) bacteremia after van-comycin failure. Recent data suggest that combiningdaptomycin with a β-lactam may have unique benefits;however, there are very limited clinical data regardingthe use of ceftaroline with daptomycin.

Methods: All 26 cases from the 10 medical centersin which ceftaroline plus daptomycin was used fortreatment of documented refractory staphylococcalbacteremia from March 2011 to November 2012were included. In vitro (synergy studies, bindingassays, cathelicidin LL-37 killing assays), and in vivo(virulence assays using a murine subcutaneous infec-tion model) studies examining the effects of ceftarolinewith daptomycin were also performed.

Findings: Daptomycin plus ceftaroline was used in26 cases of staphylococcal bacteremia (20 MRSA, 2vancomycin-intermediate S aureus, 2 methicillin-susceptible S aureus [MSSA], 2 methicillin-resistantS epidermidis). Bacteremia persisted for a median of10 days (range, 3–23 days) on previous antimicrobial

*Current affiliation: Cubist Pharmaceuticals, Lexington, Mas-sachusetts.

] 2014

therapy. After daptomycin plus ceftaroline was started,the median time to bacteremia clearance was 2 days(range, 1–6 days). In vitro studies showed ceftarolinesynergy against MRSA and enhanced MRSA killing bycathelicidin LL-37 and neutrophils. Ceftaroline alsoinduced daptomycin binding in MSSA and MRSA to acomparable degree as nafcillin. MRSA grown in sub-inhibitory concentrations of ceftaroline showed attenu-ated virulence in a murine subcutaneous infection model.

Implications: Ceftaroline plus daptomycin may bean option to hasten clearance of refractory staph-ylococcal bacteremia. Ceftaroline offers dual benefitvia synergy with both daptomycin and sensitization toinnate host defense peptide cathelicidin LL37, whichcould attenuate virulence of the pathogen. (Clin Ther.2014;]:]]]–]]]) & 2014 Elsevier HS Journals, Inc. Allrights reserved.

Key Words: ceftaroline, daptomycin, MRSA,MRSE bacteremia, MSSA, VISA.

http://dx.doi.org/10.1016/j.clinthera.2014.05.0610149-2918/$ - see front matter

& 2014 Elsevier HS Journals, Inc. All rights reserved.

1

dx.doi.org/10.1016/j.clinthera.2014.05.061



Clinical Therapeutics

INTRODUCTIONBacteremia due to methicillin-resistant Staphylococcusaureus (MRSA) poses significant surgical and medicalchallenges to clinicians and the health care system.1

The most difficult cases are those that persist despiteappropriate antimicrobial therapy and without aneasily identified and removable focus, or cases inwhich an infected biomedical device is identified butcannot be removed without extreme risks to thepatient.2 We have previously shown S aureus cross-resistance between cationic antimicrobial host defensepeptides (HDPs) of the human innate immune systemand vancomycin and daptomycin,3,4 the only anti-biotics approved by the US Food and Drug Admin-istration for the treatment of MRSA bacteremia.These data portend a worrisome scenario in clinicalcases in which the pathogen resists eradication andresistance to these agents could develop simultane-ously under continuous selective pressures, not just byadministered antibiotics but also by HDPs. Thus, thesense of clinical urgency in eradicating these infectionsis just becoming realized.

We have previously described very successful outcomesin patients with refractory MRSA bacteremia usingcombination therapy with daptomycin and an antistaphy-lococcal β-lactam.5 In addition, development of dapto-mycin resistance by MRSA in vitro was suppressed in thepresence of antistaphylococcal β-lactams.6

Ceftaroline was approved by the US Food andDrug Administration in 2010, and it became the firstβ-lactam available in the United States in 2011 within vitro and in vivo MRSA activity for the treatmentof bacterial skin and skin structure infections.7 Weanticipated that the combination of daptomycin plusceftaroline therapy might exhibit superior activityagainst MRSA given the following: (1) the demon-strated synergy between daptomycin and β-lactams5;(2) the intrinsic activity of ceftaroline against MRSA7;(3) the observed decrease in S aureus ceftaroline MICin S aureus upon loss of daptomycin susceptibility8;and (4) a recent case in which daptomycin plusceftaroline was used successfully in salvage therapywith supporting in vitro data.9

In the present article, we report the use of dapto-mycin and ceftaroline as a salvage antimicrobialregimen in the treatment of refractory staphylococcalbacteremia at 10 US medical centers. In vitro synergyof ceftaroline plus daptomycin against MRSA isdemonstrated and correlated to enhanced daptomycin

2

binding induced by ceftaroline. Finally, we show thatantistaphylococcal activity of human cathelicidinHDPs and neutrophils of the innate immune systemare significantly increased by ceftaroline.

MATERIALS AND METHODSClinical Cases

All 26 cases from the 10 medical centers in whichceftaroline plus daptomycin was used for the treatmentof documented refractory staphylococcal bacteremiafrom March 2011 to November 2012 were included(Figures 1 and 2). Additional case details are outlined inTable I. Participating institutions included thefollowing: Sharp Memorial Hospital, San Diego,California; Detroit Medical Center, Detroit, Michigan;Oregon Health & Science University Hospital,Portland, Oregon; Westchester Medical Center,Valhalla, New York; University of WisconsinHospital, Madison, Wisconsin; The Ohio StateUniversity Wexner Medical Center, Columbus, Ohio;Dekalb Medical Center, Decatur, Georgia; MaimonidesMedical Center, Brooklyn, New York; VA San DiegoHealthcare System, San Diego, California; and John D.Dingell VA Medical Center, Detroit, Michigan.Approval or waiver from each center’s institutionalreview board was obtained where appropriate based onthe number of cases per site.

Bacterial IsolatesOne MRSA isolate (SA1, case 23, isolate 2, dapto-

mycin MIC 1.0–2.0 mg/L; ceftaroline MIC 1 mg/L;nafcillin MIC 8 mg/L) and 1 methicillin-susceptibleS aureus (MSSA) isolate (LUC77, case 22, daptomycinMIC 1.0 mg/L; ceftaroline MIC 0.25 mg/L; nafcillinMIC 0.5 mg/L) available from the case series werechosen for further in vitro analyses. SA1 was deter-mined to have a daptomycin MIC of 1 mg/L by usingbroth microdilution testing but an MIC of 2 mg/L byEpsilometer test (Etest, BioMerieux, Durham, NC). Invivo mouse studies described in the following textwere performed on previously published strain MRSASanger 252 in which this infection model was wellestablished in our laboratory.10

In Vitro AssaysKill curves were performed in Mueller-Hinton

broth supplemented with 50 mg/L Ca2þ by using astarting inoculum of 107 CFU/mL. Samples wereobtained at 0, 4, 24, and (in some cases) 48 hours,

Volume ] Number ]

Case#1: L-sided IE

Case#2: L-sided IE withverterbral osteomyelitis

Case#3: L-sided IE withseptic lung emboli

Case#4: IE with osteomyelitisand discitis

Case#5: L-sided IEMRSAbacteremia

withendocarditis

MRSAbacteremia

withoutendocarditis

Case#6: L-sided IE and ICD

Case#7: L-sided IE withsplenic emboli

Case#7: L-sided IE, withendopthalmitis

Case#10:L-sided IE and ICD

Case#11:Epidural abcess

Case#12:Sacroiliac bone/joint

Case#13:Epidural abcess andseptic brain emboli

Case#14:Unknown source

Case#15:Verterbral osteomyelitis

Case#16:AV graft and SepticThrombophlebitis

Case#17:Vertebral osteomyelitis

Case#18:osteomyelitis and chronic

foot wounds

Case#19:Tunneled venous catheter with multiple soft tissue

infection foci

Case#20:Prepatellar bursitis VAN failure

VAN failure

VAN failure

VAN failure

VAN failure

VAN failure

VAN failure

VAN failure

VAN failure

VAN + LINfailure

VAN failure

VAN failure

VAN failure

VAN failure

VAN failure

VAN + CLINfailure

VAN + CLINfailure

VAN + CLIN + RIFfailure

VAN + GENfailure

DAP 6failure

DAP 6failure

DAP 6failure

DAP 4failure

DAP 10failure

DAP 10failure

DAP 8 + LINfailure

DAP 10failure

DAP 10failure

DAP 10failure

DAP 10failure

DAP8 + CLINfailure

DAP 10failure

DAP 10 + GENfailure

DAP 9failure

DAP 6failure

DAP 6failure

DAP 10 + CPT 600 q12Clear 5d






DAP6 + CPT 600 q12Clear 6d











DAP9 + CPT 600 q12 + RIFClear 1d



Case#9: L-sided IE, withhematogenous

osteomyelitis/sternoclavicularseptic arthritis

Figure 1. Summary of cases in which daptomycin (DAP) þ ceftaroline (CPT) was used to clear persistentmethicillin-resistant Staphylococcus aureus (MRSA) bacteremia, stratified according to the presence (topstrata) or the absence (bottom strata) of endocarditis. Additional details can be found in the Table 1.L ¼ left; IE ¼ infective endocarditis; VAN ¼ vancomycin; LIN¼ linezolid; ICD ¼ infected cardiac device;RIF ¼ rifampin; CLIN ¼ clindamycin; GEN ¼ gentamicin; AV ¼ arteriovenous.

G. Sakoulas et al.

] 2014 3

Case #21:LVAD-

associatedR-sided IE

VAN + PIP/TAZfailure

AMP/SULfailure

DAP 8 +AMP/SUL

failure

DAP 8 + NAFfailure

DAP 8 + CPT 600 q12clear 2d

DAP 8 + CPT 600 q12clear 1d

NAF + GENfailure

VANfailure

VANfailure

VANfailure

VAN + GENfailure

VAN + GEN + RIFfailure

DAP 6 + CPT 400 Q12clear 4d

DAP 10 + CPT 200 q12cleared (further

supression with PO DOX)

DAP 10 + CPT 200 q12clear (no qd cultures to

document date)

DAP 10 + CPT 400 q12clear* (no qd cultures to documented date)

further supression with PO DOX(relapsed after DOX discontinued)

LINthrombocytopenia

LIN + GENfailure

VAN + CTX +TMP/SMX +RIF failure

Case #22:Complex

bacteremiawith bone &joint, softtissue &

meningitis

Case #23:R-sided IE;

not a surgicalcandidate

Case #24:Epidural abcess

withosteomyelitis

Case #25:R-sided IE

prosthetic valvenot a surgical

candidate

Case #26:R-sided IE

prosthetic valve

MSSAbacteremia

withendicarditis

MSSAbacteremia

withoutendicarditis

VISA(and MRSA)bacteremia

withendicarditis

VISAbacteremia

withoutendocarditis

MRSEbacteremia

withendocarditis

Figure 2. Summary of cases in which daptomycin (DAP) þ ceftaroline (CPT) was used to clear either (A)methicillin-susceptible Staphylococcus aureus (MSSA), (B) vancomycin-intermediate-susceptible S aureus(VISA), or (C) methicillin-resistant S epidermidis (MRSE). Additional details can be found in the Table 1.Case 23: VISA infective endocarditis (IE) case relapsed with bacteremia and endophthalmitis afterdoxycycline (DOX) was discontinued; relapse was re-treated with DAP þ CPT. LVAD ¼ left ventricularassist device; L ¼ left; VAN ¼ vancomycin; PIP/TAZ ¼ piperacillin/tazobactam; AMP/SUL ¼ ampicillin/sulbactam; CTX ¼ ceftriaxone; TMP/SMX ¼ trimethoprim/sulfamethoxazole; RIF ¼ rifampin; NAF ¼nafcillin; GEN ¼ gentamicin; R ¼ right; PO ¼ oral; LIN ¼ linezolid.


serially diluted 1:10 to 1:107, and 10 mL plated induplicate on Todd Hewitt agar (THA) plates. Assayswere performed in duplicate in each experiment, andexperiments were performed twice on separate days.Colonies were enumerated after 24 hours and log10CFU/mL calculated for graphical presentation. One

4

representative experiment is shown. Limit of detectionwas 1000 CFU/mL (log10 ¼ 3).

Susceptibility testing to daptomycin in varying con-centrations of ceftaroline or nafcillin was performed byusing broth microdilution methods established by theClinical and Laboratory Standards Institute.11

Volume ] Number ]

Table I. Clinical details of cases included in this study.

PatientAge/Sex Pathogen

(MICs) Comorbidities Diagnostic Findings Site(s) of Infection

Antimicrobial Therapies

Comments1st Line 2nd Line 3rd Line 4th Line

MRSA cases1 44 M

MRSA(VAN 2, DAP 1,CPT 0.5)

HIV/AIDS (CD4count 0

Viral load41 million), Afib

Mitral valve IE L-sided IE VANd 1-3

DAP 10d 4–9

DAP 10 þ CPT600 Q12d 10–43Clear 5 d

None

2 75 MMRSA(VAN 2, DAPo0.5, CPT 2)

ESRD on HD, HTN, DM AV 3 � 5 mmvegetation

IE (side unspecified)with discitis,osteomyelitis

VANd 1

DAP 8 þCPT 600 q24

d 2–3Clear 2 d

DAP 10 þCPT 600 q24

d 4

DAP 10 þ LINd 5-9

Changed to DAP monotherapy for 6weeks once stabilized

3 67 MMRSA(VAN 1, DAP0.25)

Asthma MRSA bacteremia; EBV pro-liferative disorder; pulmon-ary septic emboli; TTE AVvegetation

Splenic infarcts; sepsis

Aortic valve IE Pulmon-ary septic emboli

VANd 1–5

VAN þ LINd 6–12

DAP 8 þ LINd 13–18

DAP 8 þ CPT600 q8

d 19-35Clear 5 day

Changed to DAP 8 þ VAN þ MERO þCIPRO, and then DAP 8 þ CPT 600q12þ MOXI

DAP stopped d 43 due to suspectedpneumonitis (CT chest [groundglass infiltrates]) þ peripheraleosinophilia

Discharged on VAN þ MOXI4 69 M

MRSA(VAN 2, DAP0.25)

HTN, CKD, Obesity, DM,anemia of CKD, HTN,stroke, T6-L4 spinal fu-sion 1 month previous

Complicated by early MRSAsurgical site infection withosteomyelitis/ hardware withMRSA bacteremia

Osteomyelitis anddiscitisIE

VANd 1–2

DAP 6d 3–6

DAP 10d 7–9

DAP 10 þ CPT400 q8

d 10-17Clear 2 day

Patient provided comfort care anddied

5 51 MMRSA

CHF, DM, HTN, CKD, Afib Echodensity along RV wire Left-sided IE VANd 1–3

DAP 10d 3–10

DAP 10 þCPT 600 q12d 10–52Clear 1 d

None Pacer removed d 4; sent home on d 18of DAP þ CPT

6 73 MMRSA

Prostate cancer, HTN, DM,Afib, CHF

2.4-cm mass RV andRA leads

L-sided IE and ICD(pacemaker)

DAP 10d 1–7


DAP 10d 29–51

None Pacemaker removed d 13

7 55 MMRSA(VAN 1.5,DAP 3)

Hepatitis C, IVDU, COPD,stroke

MV 0.7 X 1.3 cm vegetation L-sided IE (MV) withsplenic emboli

VANd 1–7

DAP 10d 8–11

DAP 10 þCPT 600 q8d 12–72Clear 4 day

None

Patient had MV replacement withprosthetic valve on d 29. Tissuevalve culture was negative

8 43 FMRSA

ESRD on HDDM

Endophthalmitis PresumedL-sided IE

VANd 1–3

DAP 6 q48,CPT 200 q12d 4–14Clear 2 d

DAP 6 q48d 15–28

None Suspected initial source: HD catheter

9 47 MMRSA

DM, HTN,ETOH abuse

Complicated soft tissueinfection

Sternoclavicular septicarthritis

L-sided IE (MV) withhematogenousbone/joint

VAN þ CLINd 1–3

VAN þ CLINþ RIF d 4–10

DAP 6 þCPT 600 q12

d 10–37Clear 6 d

None

10 86 MMRSA(VAN 2, DAP1, CPT 1)

Afib, Dementia,HTN, GERD, venous stasis,glaucoma, blind

TEE negative, culture of pace-maker wires (þ) MRSA VANMIC 2

PresumedL-sided IE; infectedpacemaker

VANd 1–3

DAP 9d 4–19

DAP 9þ CPT 600q12þ RIF

d 20–41Clear 1 day

LINd 41-48DOXd 48-57

Pacemaker removed d 13; clearedbacteremia d 20 (7 d after pace-maker removal and 1 d after DAPþ CPT); discharged on DOX PO

(continued)

G.Sak

oulas

etal.

]2014

5

Table I. (continued).





11 55 MMRSA(VAN 1, DAPo0.5)

DM, HTN MRI epidural abscess50 cm � 20 cm � 3 cm

Epidural abscess VAN þ CLINd 1–3

DAP 8 þ CLINd 4–8

DAP 10 þ CPT600 q8

d 9–12Clear 1 d

DAP 10 þTMP-SMXd 13-50

Abscess drainage d 2

12 27 MMRSA(VAN 2, DAP1, CPT 1)

HIV with disseminatedMAC, asthma

MRI of sacroiliac joint abscess2.4 � 2 cm periarticularfluid; TTE negative

Septic sacroiliac jointwith adjacentabscess

VANd 1–9


None None

13 29 FMRSA

IVDU; anemia Spinal MRI: T12-L3 epiduraland paraspinal abscesses;Septic emboli to brain withresulting cranial nerve III, IV,and VI palsies; left pleuralempyema

Bilateral psoas abscessand epidural abscesswith septic brainemboli

VANd 1

DAP 6d 2–3


VANd 20-62

VAN � 6 wk was started at discharge

14 76 MMRSA(VAN 2,DAP r0.5)

CAD, DM, HTN,ESRD on HD

COPD, morbid obesity

TEE negativeMRI/CT negative

Unknown source VANd 1

DAP 10d 2–7


DAP 10d 18-56

15 63 MMRSA(VAN 2, DAP 2)

DM, morbid obesityVentral hernia with colonnecrosis/ perforation; pre-vious PICC-associatedMRSA bacteremia

CT: Psoas and iliopsoasabscesses, vertebralosteomyelitis

TEE negative

Vertebral osteomyelitis DAP 4d 1–2

DAP 4þCPT 600 q12d 3–5Clear 2 d

LINd 6–10

CPT 600 q12d 11-42

DAP 4 based on actual body weight,DAP 6 based on ideal body weight

16 66 FMRSA(VAN 1, DAPr0.5, CPT 0.5)

HIV, DM, HTN, ESRD onHD, CHF, asthma, HCV

TEE negative, US of LUE:heterogeneous echogenicfoci noted

IV catheter; AV graftand septicthrombophlebitis

VANd 1–2

DAP 10d 3–5

DAP 10 þ GEN1 mg/kg

d 6–7

DAP 10 þ CPT400 q24

d 8-22Clear 6 d

Source of bacteremia unknown for 10days; Removal of AV graft d 11;cleared 72 h after graft removal.De-escalated to DAP 10 as out-patient treatment for 6 wk with HD

17 60 MMRSA(VAN 2, DAP 1)

IVDU admitted forNSTEMI, AKI, andseptic shock, HD

TEE negative; CT scan smallthigh abscess; MRI of spinelumbar osteomyelitis withoutepidural abscess

Small abscesses in theright thigh; Osteo-myelitis of C2–C6

VANd 1–7

DAP 10 þCPT 200 q12

d 8–14Clear 1 d

DAP 10d 15–53

None Patient had AKI and septic shock;started HD on day 8 of admission;De-escalated to DAP 10

18 63 MMRSA

ESRD on HD,DM

Chronic wounds on feet withosteomyelitis

HD catheter VAN þ GEN� 5 d 1–5

DAP 6 � 5 dd 6–10

DAP 6 þ CPT400 q 12

d 11–26Clear 3 d

None Recent 6 wk previous course of VAN

19 63 FMRSA

T-cell lymphoma Multiple skin infection foci Venous access tunneledvenous catheter

DAP 6d 1–5


VANd 16–22

LINd 23-28

20 49 MMRSA

DM, HTN, BPH No other foci Prepatellar bursitis VANd 1–8

DAP 6d 9–13


CPT 600 q12d 22–42

(continued)

Clin

icalTherap

eutics

6Volume]Number

]

Table I. (continued).





MSSA cases21 50 M

MSSANonischemic

cardiomyopathyLVAD 3 y previously

TEE negativeCT abdomen: No abscess

LVAD infectionWound culture:MSSA/Escherichia coli

Blood culture:MSSA only

VAN þ PIP/TAZd 1–3

AMP/SULd 3–5

DAP 8 þ AMP/SULd 5–6

DAP 8þ CPT600 q12d 6-12Clear 2 day

Patient signed out AMA on d 12 oftherapy; given a prescription for POcephalexin þ RIF;

Returned with E coli bacteremia twice(1 mo and 3 mo later);died 4 mo later(MSSA bacteremia had neverreturned)

22 54 MMSSA

DM, hepatitis C, ETOHabuse

TEE-negativeCT/MRI: RetroperitonealPhlegmon, L3–L5 Discitis/osteomyelitis/epidural ab-scess. Psoas pyomyositis

CSF: 2186/mm3 WBC (89%PMN), Glucose 31 mg/dL,protein 240 mg/dL

Retroperitonealinfection

VAN/CTX/TMP-SMX/RIFd 1-3

NAF þ GENd 3–6

DAP 8 þ NAFd 6–10

DAP 8þ CPT600 q12d 10-17Clear 1 d

Treated purely with medical therapyDAP 8 þ NAF d 17–24NAF d 24–56Complete cure 6-mo follow-up

VISA cases23 60 F

VISA/MRSA(isolate 1:VAN 4, DAP 3,CPT 0.75)(isolate 2:VAN 2, DAP 1-2[see text],CPT 0.75)

DM, HTN, s/pCABGPrior MRSAsternal woundInfection � 2

TEE: atrial appendage 33-mm thrombus

NM scan: no increasedfocal uptake

CT/MRI spine chest/abdo-men/pelvis: no osteomyeli-tis, no abscess

RA appendageseptic thrombus

Poor surgicalcandidate

VANd 1–30

DAP 10 þCPT 400 q1242 days

PO DOX(noncompliant)

Retreatment forrelapse

Discharge on suppression on DOXre-admitted with bacteremia and en-

dophthalmitis after stoppedtaking DOX

retreated with DAP þ CPT anddischarged on DOX

24 71 FVISA(VAN 3-4; DAP 2,CPT 0.38)

ESRD on HDs/p laminectomy

TEE negativeUS AV graft: no fluidcollection

CT spine: lumbar osteomye-litis, Discitis

Epidural abscess

Epidural abscessOsteomyelitis

VAN LIN þ GEN DAP 10 þCPT 200 q1242 days

DOX90 days

DOX � 3 months; no sign ofrecurrence

LIN caused thrombocytopeniaRIF resistance emerged

MRSE cases25 83 F

MRSE(VAN 4, DAP 0.5,CPT 0.25)

ESRD on HD,CABG, AVR

CirrhosisPrior MRSE bacteremia3 times in past

TEE negative, mild MVthickening

US AV graft: no fluidcollection

NM scan: no increasedfocal uptake

CT/MRI spine Chest/Abdo-men/Pelvis: no osteomyeli-tis, no abscess

Probable IE

VAN þ GENd 1–9

LINd 10–16

DAP 10þCPT 200 q12d 17–42 d

DOX Suppressiontherapy

LIN caused thrombocytopeniaRIF resistantTMP-SMX severe allergy2 prior breakthrough bacteremia onDOX and on wk 6 DAP

Poor surgical candidate

(continued)

G.Sak

oulas

etal.

]2014

7

Tab

leI.

(continued).

Patient

Age/Sex

Pathog

en(M

ICs)

Com

orbidities

Diagn

ostic

Find

ings

Site(s)of

Infection

Antim

icrobial

Therap

ies

Com

ments

1stLine

2ndLine

3rdLine

4thLine

2660

MMRSE

ESRD

onHD

DM

AV,

MVbiop

rosthetic

valve

endo

carditis

Left-sided

IE,prosthetic

valve

VAN

d1–3

VAN

þGEN

þRIF

d4–14

DAP6þ

CPT

400q1

2d15

–46

Clear

4d

Non

eSu

spectedsource:HD

catheter

MRSA

¼methicillin-resistan

tStaphylococcus

aureus;M

¼male;

VAN

¼vancom

ycin;DAP¼

daptom

ycin;CPT

¼ceftaroline;

IE¼

infective

endocarditis;Afib¼

atrial

fibrillation;

ESRD¼

end-stagerena

ldisease;H

D¼

hemod

ialysis;HTN

¼hypertension

;DM

¼diab

etes

mellitus;A

V¼

arteriovenou

s;LIN¼

linezolid;E

BV¼

Epstein-

Barrvirus;

TEE¼

tran

sesop

hageal

echo

cardiograp

hy;MER

O¼

merop

enem

;CIPRO

¼ciprofloxacin

;MOXI

¼moxifloxacin;CT¼

compu

tedtomog

raph

y;CKD

¼chronickidn

eydisease;

CHF¼

cong

estiveheartfailu

re;RV¼

righ

tventricular;

RA

¼righ

tatrial;ICD

¼infected

cardiacdevice;IVDU

¼intravenou

sdrug

use;

COPD

¼chronicob

structivepu

lmon

arydisease;MV¼

mitral

valve;F¼

female;ET

OH

¼alcoho

l;RIF

¼rifampin;

GER

D¼

gastroesop

hageal

reflux

disease;DOX¼

doxycycline;MRI¼

magnetic

resona

nceim

aging;

CLIN

¼clinda

mycin;TM

P-SM

X¼

trim

etho

prim

-sulfametho

xazole;CAD

¼corona

ryartery

disease;

PICC

¼periph

erallyinserted

centralcatheter;HCV¼

hepa

titisCvirus;US¼

ultrasou

nd;GEN

¼gentam

icin;NST

EMI¼

non–ST

-segmentelevationmyocardialinfarctio

n;AKI¼

acutekidn

eyinjury;MSSA¼

methicillin-susceptib

leSaureus;LVAD

¼left

ventricularassist

device;AMP/SU

L¼

ampicillin/sulbactam

;PIP/TA

Z¼

piperacillin/

tazoba

ctam

;AMA¼

againstmedical

advice;C

SF¼

cerebrospina

lfluid;

WBC¼

white

bloo

dcellcoun

t;PM

N¼

polymorph

onuclear

cell;

CTX

¼ceftriaxon

e;VISA

¼vancom

ycin-in

term

ediate

Saureus;CABG

¼corona

ryartery

bypa

ssgraft;

NM

¼nu

clearmedicine;

MRSE

¼methicillin-resistan

tSepidermidis;

AVR

¼aorticvalve

replacem

ent.


8

Human cathelicidin LL-37 susceptibility testingand killing assays were performed in RPMI mediasupplemented with 5% Luria broth (LB) as previouslydescribed.5,12

Daptomycin binding assays were performed, aspreviously described, after bacteria were grown toOD600nm of 0.6, exposed for 1 hour with ceftaroline 1mg/L or nafcillin 10 mg/L, and then labeled with 4 mg/Lbodipy–daptomycin (Cubist Pharmaceuticals, Lexing-ton, Massachusetts) for 15 minutes.5,12 The antimicro-bial activity of bodipy–daptomycin is slightly reducedsuch that the MIC is 1 dilution higher than unlabeleddaptomycin. Cytochrome c binding was performed aspreviously described.13 Ceftaroline was provided byForest Pharmaceuticals (New York, New York).

Neutrophil Killing AssaysNeutrophils were freshly isolated from the blood of

healthy donors by using the PolyMorphPrep kit (Frese-nius Kabi, Homburg, Germany), and erythrocytes werelysed with sterile H2O as previously described.14 MRSASanger 252 was grown to log phase in ceftaroline 0.1μg/mL or media alone (untreated), washed, andresuspended in phosphate-buffered saline (PBS). At thisconcentration of ceftaroline, there was no appreciableeffect on growth rate. Bacteria were inoculated at amultiplicity of infection ¼ 1 with 5 � 105 polymor-phonuclear cells in RPMI þ 2% 701C heat-inactivatedfetal bovine serum in suspension culture plates.15 After90 minutes of incubation at 371C/5% CO2, cells werelysed with 0.025% Triton X-100, and the total numberof remaining bacteria were enumerated on THA plates.Survival was calculated as the percentage of the initialinoculum. Experiments were performed by using bloodfrom at least 3 healthy donors. Use and procedureswere approved by the University of California SanDiego Human Research Protections Program.

Animal Model of MRSA Cutaneous InfectionMRSA Sanger 252 has been used in our laboratory

to study the effect of β-lactam antibiotics on MRSAvirulence. The strain was grown overnight to thestationary phase in 40 mL of antibiotic-free LB orLB containing ceftaroline 0.1 mg/L. Bacteria werewashed in PBS 40 mL, and resuspended in 2 mL ofPBS and 2 mL of Cytodex beads (Sigma-Aldrich, St.Louis, MO) (1 mg/L), yielding 1010 CFU/mL. Next,0.1 mL of the bacterial suspension was injectedsubcutaneously into flanks of 25-g female CD1 mice.

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G. Sakoulas et al.

To control for animal-to-animal variability, pairedlesions were created, with ceftaroline-treated MRSAon one side and antibiotic-free grown MRSA on thecontralateral side using a total of 12 mice. Right andleft sides were alternated for antibiotic-free grown andceftaroline-grown MRSA.

Lesion sizes were measured at 2 days and comparedby using a paired-sample Wilcoxon signed-rank test.A P value of o0.05 was considered significant. Allanimal studies were performed under protocols thatwere reviewed and approved by the University ofCalifornia San Diego Institutional Animal Use andCare Committee. All animal research in this inves-tigation was performed in accordance with nationaland local guidelines that are in place to maximizehumane animal treatment.

RESULTSCase SeriesCharacteristics of the Clinical Cases

Daptomycin plus ceftaroline was used in 26 casesof staphylococcal bacteremia from 10 geographicallyand demographically distinct US hospitals. Clinicaldetails are provided in Table I, and a summary ofrelevant clinical characteristics are provided in Table II.The pathogens consisted of MRSA in 20 cases(Figure 1), MSSA in 2 cases, vancomycin-intermediateS aureus (VISA) in 2 cases, and methicillin-resistantS epidermidis in 2 cases (Figure 2). As expected,endocarditis (confirmed echocardiographically) washighly represented (54% [14 of 26]), and included 12patients with left-sided, 1 side unspecified, and 1 right-sided endocarditis. The mean patient age was 60 years,and 73% were males. Nine (35%) of the patients hadend-stage renal disease and were on hemodialysis, and15 (58%) patients had diabetes mellitus.

Daptomycin Plus Ceftaroline SalvageThe daptomycin plus ceftaroline combination was

used most often as third-line therapy in 13 cases(50%), followed by second-line therapy in 8 cases(31%), and fourth-line therapy in 5 cases (19%). Themedian duration of daptomycin plus ceftaroline com-bination therapy was 16 days. Once deemed stable,some patients received additional de-escalated, step-down antibiotics replacing daptomycin plus ceftaro-line (eg, daptomycin monotherapy, oral doxycycline),to complete a median total duration of 42 days oftherapy for staphylococcal bacteremia (Tables I and

] 2014

II). Twenty-five (96%) of the patients survived com-pletion of therapy; 1 patient died when medical carewas withdrawn due to multiple medical problemsupon which the MRSA bacteremia was superimposed.

The use of daptomycin plus ceftaroline combina-tion therapy was mostly for documented failure ofbacteremia clearance. For 23 of the 26 cases for whichdetailed microbiologic information was available viaserial daily blood culture results, bacteremia persistedfor a median of 10 days (range, 3–23 days) onprevious antimicrobial therapy, and the bacteremiacleared in a median of 2 days (range, 1–6 days) afterdaptomycin plus ceftaroline was started. It is criticalto note that in all but 1 case (case 21; Table I), thebacteremia clearance on daptomycin plus ceftarolinetherapy was not temporally related to a surgicalprocedure such as device removal.

Endocarditis CasesAs anticipated from a case series examining salvage

therapy for staphylococcal bacteremia, a large per-centage of patients (14 of 26 [54%]) had bacterialendocarditis. These cases warrant closer scrutinybecause bacteremia persistence is a salient feature oftheir clinical and microbiologic course. These 14patients comprised 10 with MRSA, 1 with MSSA, 1with VISA, and 2 with methicillin-resistant S epider-midis endocarditis. Daptomycin plus ceftaroline wasused as second-line therapy for 4 patients, third-linetherapy for 7, and fourth-line therapy for 2. Amongthe 10 patients with endocarditis who had serial bloodcultures collected, bacteremia cleared a median of 2days (range, 1–6 days) after daptomycin plus ceftaro-line therapy was started.

Nonsusceptible OrganismsOther salient features in these cases are the involve-

ment of nonsusceptible organisms to daptomycin in 4cases and ceftaroline in 1 case. The ceftaroline-resistant isolate was successfully cleared after 2 dayswith the combination therapy using daptomycin 8 mg/kg per day plus ceftaroline 600 mg every 24 hours.The cases with daptomycin nonsusceptibility involved:(1) 2 cases with VISA (cases 23 and 24); (2) one casewith 7 days of vancomycin therapy followed bydaptomycin 10 mg/kg (case 7); and (3) 1 case treatedwith daptomycin 6 mg/kg based on ideal body weightthat was 4 mg/kg actual body weight (case 15).

9

Table II. Summary of patient and infectioncharacteristics. Values are given asmedian (range) or number (%).

CharacteristicValue

(n ¼ 26)

Age, y 60 (27–86)Male 19 (73)PathogenMRSA 20 (77)MSSA 2 (8)VISA 2 (8)MRSE 2 (8)

Common comorbiditiesDiabetes 15 (58)Hemodialysis 9 (35)HIV/AIDS 3 (12)Liver disease 5 (19)Malignancy 2 (8)

Sites of infection*Endocarditis 14 (54)Left-sided IE 12 (46)Right-sided IE 1 (4)Side not specified 1 (4)Left ventricular assist device 1 (4)Pacemaker/defibrillator 2 (8)Osteoarticular 11 (42)Discitis/vertebral osteomyelitis/epidural abscess 8 (31)Sternoclavicular septic arthritis 1 (4)Sacroiliac joint 1 (4)Osteomyelitis and chronic foot wounds 1 (4)Other deep tissue 3 (12)Tunneled venous catheter with soft tissueinfection foci

1 (4)

AV graft with septic thrombophlebitis 1 (4)Prepatellar bursitis 1 (4)Septic brain emboli/meningitis 2 (8)Unknown bacteremia source 1 (4)

Bacteremia duration before DAP þ CPT, d 10 (3–23)Bacteremia duration after DAP þ CPT, d 2 (1–6)DAP þ CPT salvageSecond-line 8 (31)Third-line 13 (50)Fourth-line 5 (19)

Daptomycin dosing, mg/kg4 1 (4)6 7 (27)48 18 (69)

CPT dosingq8 h 5 (19)q12 h 19 (73)q24 h 2 (8)

Duration of DAP þ CPT, d 16 (3–61)47 23 (88)428 7 (28)

(continued)

Table II. (continued).

CharacteristicValue

(n ¼ 26)

Duration of DAP þ CPT plus follow-upantibiotics, d

42 (8–132)

414 25 (96)442 14 (54)

Antimicrobial resistanceDaptomycin nonsusceptible 4 (15)

MRSA ¼ methicillin-resistant Staphylococcus aureus; MSSA¼ methicillin-susceptible S aureus; VISA ¼ vancomycin-intermediate S aureus; MRSE ¼ methicillin-resistant Sepidermidis; IE ¼ infective endocarditis; AV ¼ arteriovenous;DAP ¼ daptomycin; CPT ¼ ceftaroline.*May have 41 site, so total 4 100%.


10

Refractory MSSA BacteremiaOf note, 2 of the 5 cases in which the combination

therapy was used as fourth-line therapy involvedpersistent bacteremia due to MSSA that failed toclear promptly with daptomycin in combination withnafcillin or ampicillin/sulbactam. One case (case 22;Table I) involved a massive retroperitoneal phlegmonlocalized between the vertebral spine posteriorly andthe descending aorta anteriorly. The patient’s bac-teremia resolved, and he made a complete clinical,microbiologic, and radiographic recovery without anysurgical intervention. The MSSA from this patient(LUC77) was subjected to in vitro study (describedlater). The second of the MSSA bacteremia caseswarrants mention because the patient had an infectednonremovable left ventricular assist device, withsuperficial cultures growing MSSA and Escherichiacoli and persistent blood culture specimens growingMSSA (case 21). The patient ultimately cleared theMSSA bacteremia after 8 days, including 48 hours onceftaroline plus daptomycin. However, the patient hada history of medical noncompliance and abusivebehavior toward medical staff, eliminating hischances for device exchange, and he signed himselfout of the hospital against medical advice 4 days afterbacteremia clearance (12 days of total parenteralantimicrobial therapy). He was prescribed cephalexinplus rifampin orally with unknown compliance. Re-markably, while the patient was readmitted 2 sub-sequent times with E coli bacteremia and concomitant

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Table III. Daptomycin (DAP) susceptibilitiesof methicillin-resistant Staphylococcusaureus (MRSA) SA1 and methicillin-susceptible S aureus (MSSA) LUC77in broth media containing varyingconcentrations of ceftaroline ornafcillin.

Antibiotic in BrothMedia

LUC 77(MSSA) SA1 (MRSA)DAP MIC(mg/L)

DAP MIC(mg/L)

Ceftaroline, mg/L0 1 20.125 0.25 10.25 0.50.50 0.1251.0

Nafcillin, mg/L0 1 20.125 0.5 20.25 0.25 20.5 21.0 22.0 24.0 18.0

G. Sakoulas et al.

left ventricular assist device driveline infection, theMSSA bacteremia never recurred for 4 months untilthe patient died.

Antibiotic-related Adverse EffectsCharacteristic adverse effects related to specific

antimicrobial agents requiring alternative therapywere observed among patients in this case series.These adverse effects included hepatotoxicity andinterstitial nephritis from rifampin, thrombocytopeniafrom linezolid, eosinophilic pneumonitis from dapto-mycin, and hypersensitivity reactions from trimetho-prim/sulfamethoxazole.

In Vitro Synergy TestingTo determine in vitro synergy between daptomycin

and ceftaroline, checkerboard (Table III) and time-killstudies were performed against MSSA LUC77 fromcase 22 (Figure 3A) and MRSA SA1 from case 23(Figure 3B). Against MSSA LUC77, nafcillin 20 mg/Land ceftaroline 5 mg/L alone achieved comparablebactericidal killing at 24 hours. These concentrationswere chosen because they approximate the free Cmax

achieved in vivo with standard dosing regimens.7,16,17

When co-incubated with a subinhibitory daptomycinconcentration of 0.5 mg/L, the ceftaroline combinationproduced further killing than ceftaroline alone, but thenafcillin combination allowed significant regrowth.Against MRSA, the combination of daptomycin 1mg/L plus ceftaroline 0.1 mg/L demonstrated consid-erable and obvious synergy over each drug alone.

Daptomycin BindingOur previous studies have demonstrated β-lactam–

induced binding of daptomycin to MRSA5 andvancomycin-resistant Enterococcus faecium.12 Figure 4microscopically and quantitatively demonstrates thatceftaroline also induces daptomycin binding in MSSAand MRSA comparably to nafcillin. Under these con-ditions of ceftaroline exposure, there was no significantdifference in cytochrome c binding for either MRSA SA1or MSSA LUC77, suggesting that the mechanism ofenhanced daptomycin binding was not mediated byreduction in net surface charge (data not shown).

Ceftaroline Effects on Innate StaphylocidalImmunity

We have demonstrated that antistaphylococcalβ-lactams and ampicillin enhance killing of MRSA5

] 2014

and vancomycin-resistant E faecium,12 respectively, bycationic antimicrobial peptides. MRSA SA1 and MSSALUC77 demonstrated MIC to LL-37 of 16 mM. In thepresence of 0.1 mg/L of ceftaroline or nafcillin, the LL-37 MIC was reduced to 8 mM for both isolates.Figure 5A shows that against MRSA SA1, growth insubinhibitory concentrations of ceftaroline results in aconcentration-dependent hypersensitization to killingby the human cathelicidin LL-37 (16 mM). We thenexamined the effects of ceftaroline on killing of MRSASanger 252 by using freshly isolated human neutro-phils, which produce LL-37 abundantly. These assaysdemonstrated a significant enhancement of neutrophilkilling of MRSA after the growth in ceftaroline 0.1 mg/L compared with the same strain grown in antibiotic-free LB broth (Figure 5B).

MRSA Sanger 252 was cultured under the sameconditions and subsequently injected subcutaneouslyinto CD1 mice. The exact inoculum was 2.95 � 109

11

10ControlNAF 20CPT 5DAP 0.5DAP 0.5 + NAF 20

MSSA

MRSA

DAP 0.5 + CPT 5

ControlNAF 0.1CPT 0.1DAP 2DAP 2 + NAF 0.1DAP 2 + CPT 0.1

8

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L

6

4

10

8

Log 1

0 C

FU/m

L

6

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0 10 20Time (h)

30

0 10 20Time (h)

30

Figure 3. Kill curves demonstrating the effect ofdaptomycin (DAP), nafcillin (NAF),and ceftaroline (CPT) alone or incombination at the specified concen-trations (milligrams per liter) against(A) methicillin-susceptible Staphylococ-cus aureus (MSSA) LUC77 and (B)methicillin-resistant S aureus (MRSA)SA1.


CFU for the control antibiotic-free grown MRSA and2.90 � 109 CFU for the ceftaroline-grown MRSA in0.1 mL, confirming minimal growth differences ofMRSA Sanger 252 under these conditions. If theenhanced LL-37 and white blood cell activity renderedby exposure to ceftaroline was functionally significantin vivo, then attenuation in lesion sizes would beexpected. Indeed, this outcome was observed asshown in Figure 5C, with MRSA Sanger 252 grownin ceftaroline 0.1 mg/L before mouse challengeproducing significantly smaller lesion sizes comparedwith the same strain cultured in antibiotic-free mediaand injected on the contralateral side.

DISCUSSIONRefractory MRSA bacteremia is a very challengingclinical situation, especially when source control isnot possible by virtue of an elusive or irremovable

12

focus. Although such refractory infections have beenappreciated to pose increased risks of adverse individ-ual patient outcomes such as mortality and increasedhealth care resource utilization, recent emerging datasuggest more global implications of such infections.1,2

It has long been appreciated that selection pressure onbacteria in a persistent high-inoculum focus of infec-tion (particularly by suboptimally dosed antibiotics)can drive antibiotic resistance and therefore highlightsthe importance of surgical source control of suchinfections.18,19 However, it is becoming increasinglyapparent that antimicrobial resistance in staphylococcidoes not emerge in a closed system driven only by theantimicrobial agents themselves. We have shown thatimportant cationic HDPs of the human innate immunesystem can select for resistance to vancomycin3 anddaptomycin4 that clinicians subsequently administerto patients for therapy. The longer these infectionspersist, HDPs plus administered antibiotics may co-select drug-resistant bacteria if the bacterial inoculumremains high. A more recent study has even shown theemergence of daptomycin resistance in vivo underHDP selection pressure without the additional selec-tive pressure of antibiotic therapy.20 Although clear-ing these infections in the fastest possible mannerreduces the risk of antibiotic resistance, the data arelimited identifying the best possible approaches toachieve this goal.

Based on previous data,21–25 we have examined andshown that the addition of antistaphylococcal β-lactamsto daptomycin may prove to be helpful in bacteremiaclearance.5 In addition, β-lactams seem to reduce thedevelopment of daptomycin (and therefore possiblyHDP) resistance in S aureus.6 Through research collabo-rative discussions of this phenomenon, we identifiedcenters where the novel cephalosporin ceftaroline wasused in combination with daptomycin. These centerscontributed all their cases in which the daptomycin plusceftaroline combination was used as treatment ofpersistent staphylococcal bacteremia to this series.

As in the previous case series, the combination ofdaptomycin plus ceftaroline was highly successful, clear-ing the bacteremia in a median of 2 days after persistingfor a median of 10 days. Although there is no way ofknowing how this time to bacteremia clearance wouldcompare with either agent alone (particularly withceftaroline monotherapy), 2 very recent studies suggestthat the combination therapy may clear bacteremia morerapidly than monotherapy. One study showed a mean

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1.0

Control+ CPT 1

+ NAF 10

Control

+ CPT 1

+ NAF 10

0.8

0.6

Rel

ativ

e Fr

eque

ncy

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ativ

e Fr

eque

ncy

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Control NAF 10CPT 1

0

00.0

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0.4

0.6

0.8

1.0

40 80 120 160 200+

200 400 600Bodipy-Daptomycin Intensity

Bodipy-Daptomycin Intensity

800 1000+

Figure 4. Effect of bodipy-labeled daptomycin (4 mg/mL) binding to methicillin-resistant Staphylococcus aureusisolate SA1 (top panel) or methicillin-susceptible S aureus isolate LUC77 (bottom panel) afterexposure for 1 hour to either ceftaroline (CPT) 1 mg/L or nafcillin (NAF) 10 mg/L compared withno antibiotic. The accompanying histograms on the right quantitatively demonstrate that CPT andNAF result in higher intensity binding.

60 150 15

10

5

0

100

50

00 0.1

P < 0.02P < 0.05 P = 0.0340

% S

urvi

val (

2 h)

% S

urvi

val

Lesi

on s

ize

(mm

3 )

20

00 0.06

CPT (�g/mL) CPT (�g/mL)Untreated CPT0.25 0.50

Figure 5. (A) Methicillin-resistant Staphylococcus aureus (MRSA) SA1 grown in antibiotic-free Luria broth (LB)or LB containing 0.06, 0.25, and 0.5 mg/L of ceftaroline (CPT), washed in phosphate-bufferedsaline, and subjected to LL37 16 mM. Percent survival at 2 hours in shown (Kruskal-Wallis ANOVA,P ¼ 0.02). (B) CPT-sensitized MRSA Sanger 252 is more susceptible to killing by neutrophils. MRSASanger 252 grown in 0.1 mg/L of CPT is more susceptible to polymorphonuclear cell killing thanbacteria grown in media containing no antibiotic (Mann-Whitney U test, P o 0.05). (C) CPT-sensitized MRSA Sanger 252 produces smaller lesions when injected subcutaneously into CD1 micecompared with the same strain grown in antibiotic-free media (paired-sample Wilcoxon signed-ranktest, P ¼ 0.03).

G. Sakoulas et al.

] 2014 13


bacteremia duration of 5 days when salvage ceftarolinemonotherapy was used in staphylococcal bacteremiaafter glycopeptide failure.26 In another recent studythat examined outcomes of 31 patients with persistentMRSA bacteremia treated with ceftaroline, 0% failure (0of 10) was reported among the 10 patients who receivedceftaroline combination therapy (5 with daptomycin),compared with 38% failure (8 of 21) among patientswho received ceftaroline monotherapy (Fisher exact text,P ¼ 0.0317).27

Against a MRSA from 1 case in our study, in vitrosynergy was clearly demonstrated between daptomy-cin and ceftaroline. When evaluating MSSA fromanother case, synergy was found between daptomycinand ceftaroline; however, the addition of subinhibi-tory concentrations of daptomycin to nafcillin provedless effective in killing than nafcillin alone. Thisfinding may potentially explain why nafcillin plusdaptomycin failed to clear the bacteremia as a third-line regimen in MSSA bacteremia, yet the combinationof daptomycin and ceftaroline was subsequently suc-cessful as a fourth-line regimen. It also highlights thecomplex pharmacodynamic interactions of antibioticsin vivo, with potential negative effects of combinationantibiotic therapy against susceptible organisms.Although daptomycin plus nafcillin has shown greatpromise in the treatment of MRSA bacteremia, thiscombination needs to be examined further againstMSSA. Based on previous work, we hypothesized thatthe differences between nafcillin and ceftaroline withrespect to synergy with daptomycin rests on relativebinding to penicillin-binding protein 1 (PBP1).28

Ceftaroline seems to bind PBP1, as well as all otherPBPs of S aureus with the exception of PBP4.29

However, there is considerable heterogeneity in the50% inhibitory concentrations on PBP1 and otherPBPs between different S aureus strains, and theserelative differences need to be explored further, likelythrough sequencing on PBP genes of different strains.

Treatment of the strains with ceftaroline 1 mg/Lresulted in an enhancement of daptomycin binding toMRSA SA1 and MSSA LUC-77 comparable to naf-cillin 10 mg/L (Figure 4). Interestingly, we did not seethis effect when we reduced ceftaroline to 0.1 mg/Land grew the bacteria overnight. Thus, we “pulsed”the bacteria with ceftaroline 1 mg/L or nafcillin 10mg/L for 1 hour and searched for changes indaptomycin binding, with no appreciable effect onbacterial viability. This finding suggests that binding

14

to PBPs by ceftaroline must be 40.1 mg/L to producethe necessary physiologic changes that will result indaptomycin synergy, consistent with previous data onbinding affinity of ceftaroline for S aureus PBPs.29

With respect to the enhancement of innate immune-mediated staphylocidal activity, however, growingbacteria overnight in ceftaroline 0.1 mg/L was sufficientto show enhancement of cathelicidin LL-37 killing,enhancement of neutrophil killing, and reduced viru-lence in skin lesion generation when injected into mice.We suspect that the reduced lesion sizes were due to theincreased effectiveness of the host cathelicidin andneutrophil defenses in bacteria exposed to ceftaroline.

This study provides encouraging data to clinicianstreating serious infections due to staphylococci, par-ticularly cases of MRSA bacteremia refractory tostandard forms of therapy due to unidentifiable orunremovable foci of infection. In addition, althoughthe combination of daptomycin plus antistaphylococ-cal β-lactams has shown successful outcomes in a fewcases of refractory MRSA bacteremia and substanti-ated by recent additional in vitro studies, one clinicalcase in this series of MSSA bacteremia and subsequentin vitro studies in this report suggest that thiscombination may be less potent than the antistaphy-lococcal agent alone for some MSSA strains, as seenfor strain LUC77 (case 22; Table I). However,ceftaroline plus daptomycin exhibited excellentactivity against this particular organism in vitro, andthis combination was ultimately successful in clearingthe bacteremia. The patient ultimately was de-escalated to daptomycin plus nafcillin and then tocefazolin monotherapy to complete a prolonged pa-renteral course of therapy, with an excellent clinicaloutcome. Thus, it seems that once an infection isadequately controlled, and there are no outstandingsurgical management issues, it may not be necessary tocomplete an entire parenteral course of antimicrobialtherapy with daptomycin plus the β-lactam or ceftaro-line, as combination therapy can be very cumbersomeand expensive outside an acute care hospital setting.However, we caution against early de-escalation inpatients with left-sided endocarditis for which acardiac surgical indication remains despite bacteremiaclearance, and we suggest continuation of combina-tion therapy until the patient can be bridged tosurgery (G.S., unpublished data).

The present study is clearly limited in its retrospec-tive, noncomparative design. Furthermore, although

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G. Sakoulas et al.

bacteremia cleared a median of 2 days after daptomycinplus ceftaroline therapy, it is impossible to know howlong the bacteremia would have taken to clear had theprevious therapies been continued without reliance ondaptomycin plus ceftaroline salvage. This questioncould only have been answered in a prospective com-parative study. In addition, follow-up of 43 monthswas available for only 2 of the 25 surviving patients,and the durability of this therapy is unknown.

Nevertheless, there are promising data to suggest aclinical utility of daptomycin plus ceftaroline in the acuteperiod to quench refractory bacteremia and potentiallywiden the time window to obtain prompt source controland reduce HDP-driven resistance to daptomycin andvancomycin. The medical centers that contributed casesto this series included all cases in which daptomycin plusceftaroline was used, not just cases with successfuloutcome. This understanding, along with our compan-ion in vitro studies, suggests that the combination ofdaptomycin and ceftaroline warrants further investiga-tion. In more global terms, this study, as well asnumerous previously in vitro,21–24,28 animal models,25

and human data,5,26,27 suggest that daptomycin plus β-lactam therapy may be an appropriate candidate forformal large-scale clinical trials. Although direct com-parisons of the current MRSA bacteremia treatmentstandards daptomycin and vancomycin monotherapyversus these agents with nafcillin added are probably themost anticipated among infectious disease clinicians,exploration of ceftaroline in combination with theseagents is also warranted, probably as a follow-up study.We believe that combination therapy using β-lactams ispoised to represent the new treatment paradigm forMRSA bacteremia in the future.

CONCLUSIONSCeftaroline plus daptomycin may be an option tohasten clearance of refractory staphylococcal bacter-emia. Ceftaroline offers a dual benefit via synergy withboth daptomycin and sensitization to innate hostdefense peptide cathelicidin LL37, which could attenu-ate virulence of the pathogen.

ACKNOWLEDGMENTSThis work was supported by a grant from the Na-tional Institute of Health, Division of National In-stitute of Child Health and Human Development(grant U54 HD071600-01 [September 26, 2011–June 30, 2016] Developmental and Translational

] 2014

Pharmacology of Pediatric Antimicrobial Therapy(Drs. Sakoulas and Nizet)]. Additional funding wasprovided by Forest Pharmaceuticals (New York, NY).

G. Sakoulas and P.A. Moise was responsible insearching of the literature. G. Sakoulas, P.A. Moiseand V. Nizet was responsible in figure creation. G.Sakoulas and V. Nizet was responsible in designingthe study. G. Sakoulas, A.M. Casapao, P. Nonejuie, J.Olson, C.Y.M. Okumura, M.J. Rybak, R. Kullar, A.Dhand, W.E. Rose, D.A. Goff, A.M. Bressler, Y. Lee,J. Pogliano, S. Johns, G.W. Kaatz and J.R. Ebrightwas responsible in data collection. G. Sakoulas, P.A.Moise and V. Nizet was responsible in data interpre-tation. And all the authors were responsible forwriting of this paper.

CONFLICTS OF INTERESTDr. Sakoulas has received research grant support fromCubist Pharmaceuticals and speaking honoraria fromCubist, Pfizer, Forest, Novartis, and Astellas Pharma-ceuticals. Dr. Moise is an employee and shareholder ofCubist. Dr. Casapao has received grant support fromCubist, Forest, and the Michigan Department ofCommunity Health. Dr. Rybak has received grantsupport from, consulted for, or provided lectures forCubist, Forest, Durata, Cepheid, and Novartis. Dr.Kullar is an employee and shareholder of Cubist; thisresearch was conducted during her prior employmentat Oregon State University. At that time, Dr. Kullarreceived speaking honoraria from Cubist and Forest,and served on the advisory board of Optimer. Dr.Dhand has received speaker’s honoraria from Cubistand Pfizer. Dr. Rose has received grant support andspeaking honoraria from Cubist and is a consultantfor The Medicines Company and Visante, Inc. Dr.Goff has received grant support from Merck andserves on the advisory board of Optimer, Cubist,Merck, Rempex, and Forest. Dr. Pogliano has re-ceived research grants and consulting fees from Cub-ist. Dr. Nizet was formerly on the advisory board ofTrius Therapeutics (acquired by Cubist). The authorshave indicated that they have no other conflicts ofinterest regarding the content of this article.

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] 2014

Address correspondence to: George Sakoulas, MD, University of Cal-ifornia San Diego School of Medicine, Center for Immunity, Infection &Inflammation, Biomedical Research Facility II, Room 4114, 9500 GilmanDrive, Mail Code 0760, La Jolla, CA 92093-0760. E-mail: [email protected]

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