Impact of cefazolin co-administration with vancomycin to reduce development of vancomycin-intermediate Staphylococcus aureus

Accepted Manuscript Impact of Cefazolin Co-administration with Vancomycin to Reduce Development of Vancomycin Intermediate Staphylococcus aureus

Nivedita B. Singh, Juwon Yim, Seyedehameneh Jahanbakhsh, George Sakoulas, Michael J. Rybak PII: DOI: Reference:

S0732-8893(18)30113-5 doi:10.1016/j.diagmicrobio.2018.03.020 DMB 14572

To appear in: Received date: Revised date: Accepted date:

2 October 2017 16 March 2018 29 March 2018

Please cite this article as: Nivedita B. Singh, Juwon Yim, Seyedehameneh Jahanbakhsh, George Sakoulas, Michael J. Rybak , Impact of Cefazolin Co-administration with Vancomycin to Reduce Development of Vancomycin Intermediate Staphylococcus aureus. The address for the corresponding author was captured as affiliation for all authors. Please check if appropriate. Dmb(2018), doi:10.1016/j.diagmicrobio.2018.03.020

This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT Impact of Cefazolin Co-administration with Vancomycin to Reduce Development of Vancomycin Intermediate Staphylococcus aureus

T

Nivedita B. Singh1, Juwon Yim1, Seyedehameneh Jahanbakhsh1, George

Anti-Infective Research Laboratory, Department of Pharmacy Practice, Eugene

CR

1

IP

Sakoulas4, Michael J. Rybak1,2,3

Applebaum College of Pharmacy and Health Sciences, Wayne State University,

US

Detroit, MI, USA; 2 Department of Medicine, Division of Infectious Diseases,

AN

School of Medicine, Wayne State University, Detroit, MI, USA; 3 Department of Pharmacy Services, Detroit Medical Center, Detroit, MI, USA; 4 Division of Host-

M

Microbe Systems and Therapeutics Center for Immunity, Infection and

ED

Inflammation, University of California San Diego School of Medicine, La Jolla,

PT

California, USA

CE

Key words: MSSA, MRSA; VISA; Cefazolin; Vancomycin; Resistance

AC

Running title: Cefazolin Vancomycin combination prevent VISA Corresponding author: Michael J. Rybak, Pharm.D., M.P.H., Ph.D., AntiInfective Research Laboratory, Department of Pharmacy Practice, Eugene Applebaum College of Pharmacy and Health Sciences, Wayne State University, 259 Mack Ave, Detroit, MI 48201, [email protected], (313) 577-4376.

ACCEPTED MANUSCRIPT ABSTRACT Objective: Development of antimicrobial resistance during monotherapy of complicated methicillin-resistant Staphylococcus aureus bacteremia is problematic due to cross-resistance between vancomycin (VAN) and

T

daptomycin, the only approved agents for this condition. Our objective was to

IP

demonstrate that development of resistance under conditions of sub-optimal VAN

CR

(200 mg q 12h) exposure in S. aureus can be attenuated by addition of cefazolin (CFZ).

US

Methods: Two strains of S. aureus, one MSSA (RN9120) and one MRSA

AN

(JH1) were evaluated. The organisms were exposed to sub-therapeutic VAN concentrations in a 1-compartment pharmacokinetic/pharmacodynamic (PK/PD)

M

model combined with recycling in the presence and absence of CFZ. Changes in

ED

MIC to glyco/lipopeptides and β-lactams along with susceptibility to human cathelicidin LL-37 killing were studied. Population analysis profiles (PAP) were

PT

performed to detect changes in VAN heteroresistance.

CE

Results: VAN MIC of both organisms increased from 1 to 4 mg/L within 144h under sub-therapeutic VAN exposure. Increase in VAN MIC was associated

AC

with increased glyco/lipopeptides MICs. Additionally, increased survival in LL-37 killing assays from 40% to >90% accompanied the increase in VAN MIC. Addition of CFZ prevented the emergence of VISA. PAP demonstrated an attenuation of VAN area under the curve (AUC) shift (reduced organism selection with higher MICs values) when suboptimal VAN exposure was accompanied with CFZ compared to VAN alone (MSSA 17.81 vs 36.027, MRSA -0.35 vs 17.92,

ACCEPTED MANUSCRIPT respectively). Given the emerging data on the clinical benefits of -lactam adjunctive therapy in refractory MRSA bacteremia, additional studies on a larger collection of clinical isolates is needed to establish the utility of VAN plus CFZ for

AC

CE

PT

ED

M

AN

US

CR

IP

T

treatment of MRSA bacteremia.

ACCEPTED MANUSCRIPT INTRODUCTION

Staphylococcus aureus is a major cause of both hospital and community acquired infections, ranging from minor skin infections to fatal infective endocarditis and septicemia.(Stapleton and Taylor, 2002) A significant challenge

T

in treating S. aureus bloodstream infections is the pathogen’s ability to

IP

simultaneously co-evolve cross-resistance to both administered antibiotics and

CR

innate immune defense mechanisms.(Schito, 2006, Segal, 2005) For methicillin resistant Staphylococcus aureus infection (MRSA), or for

US

patients with methicillin-susceptible S. aureus infections (MSSA) having an

AN

anaphylactic type penicillin allergy, vancomycin (VAN) is considered the treatment of choice. Unfortunately, low-level VAN resistance is an emerging

M

problem in hospital settings. These include VAN-Intermediate Staphylococcus

ED

aureus (VISA, VAN MIC 4-8 mg/L) and heterogeneous vancomycin-intermediate Staphylococcus aureus (hVISA). Strains of hVISA, which have become more

PT

prevalent in recent years have VAN MIC values in the susceptible range ≤2 mg/L

CE

but harbor VISA sub-populations (Hiramatsu et al., 1997, Zhang et al., 2015). These strains are more commonly found in high-inoculum infections, such as

AC

complicated bloodstream infections including infective endocarditis.(Casapao et al., 2014, Fridkin, 2001) Due to its large structure and relative poor tissue penetration, administration of VAN may result in sub-therapeutic concentrations at the site of infection. For example, only about 41% of the serum concentrations are achieved in epithelial lining fluid of lungs.(Rybak M. J., 2006) Subtherapeutic vancomycin concentrations have been observed in a variety of patient populations including

ACCEPTED MANUSCRIPT critically ill patients and those that demonstrate augmented renal clearance and in patients receiving hemodialysis.(Bakke et al., 2017, Baptista et al., 2012, Hirai et al., 2016, Wilson and Berns, 2012) It has been shown that sub-therapeutic vancomycin exposure at the site of infection can lead to development of

T

resistance and treatment failure. (Deresinski, 2007, Estes and Derendorf, 2010,

IP

Lamer et al., 1993, Rybak Michael J., 2006)

CR

Daptomycin (DAP) is often used as second line therapy in MRSA bacteremia after VAN has failed. (Larry M. Baddour, 2015) Many studies have

US

demonstrated the co-emergence of low-level VAN resistance and the

AN

development of DAP non-susceptible strains leading to secondary DAP treatment failure.(Barber K. E. et al., 2014, Cui et al., 2006, Keel et al., 2010,

M

Patel et al., 2006, Sakoulas et al., 2006) Even more concerning is that VAN-DAP

ED

co-resistance also frequently accompanied by increased resistance to cationic antimicrobial peptides (CAMPs) produced by the host innate immune system,

PT

such as platelet microbicidal proteins (PMPs), cathelicidins (LL-37), and

CE

defensins (alpha and beta defensin) due to persistent bacteremia.(Fowler et al., 2004, Mishra et al., 2011, Sakoulas G. et al., 2005) Therefore, alternative

AC

treatment regimens are needed to prevent this triad of VAN-DAP-CAMP resistance from emerging in MRSA during failed monotherapy treatment.(Kullar et al., 2014) Recent studies have focused on use of combination antimicrobial agents for the treatment of VAN and DAP non-susceptible strains in complicated infections (Claeys et al., 2015, Dhand et al., 2011, Dhand and Sakoulas, 2014).

ACCEPTED MANUSCRIPT β-lactam and VAN combination has been a topic of interest in many in vitro and animal studies demonstrating synergistic activity against most S. aureus strains. (Barr et al., 1990, Climo et al., 1999, Fox et al., 2006, Hagihara et al., 2012, Leonard, 2012, Rochon-Edouard et al., 2000) Furthermore the combination of β-

T

lactam with VAN combination has been shown to shorten the duration of MRSA

IP

bacteremia.(Davis et al., 2016) Cefazolin (CFZ) has demonstrated in vitro

CR

synergy with VAN and is associated with less side effects compared to other βlactams antibiotics with VAN.(Rochon-Edouard et al., 2000, Sy Cheng Len et al.,

US

2016)

AN

The primary aim of this study was to establish whether CFZ could prevent the development of i) VISA, ii) the loss of DAP susceptibility, and iii) resistance to

ED

M

killing by human cathelicidin LL-37 secondary to suboptimal VAN exposure.

MATERIALS AND METHODS:

PT

Bacterial Strains: S. aureus strains selected were RN9120 a MSSA agr group II-null strain and JH1, a well described clinical MRSA strain. (Sakoulas et al.,

CE

2003, Sakoulas G. et al., 2002) Both of these strains have been extensively

AC

studied and have a proclivity to gain resistance to VAN and become VISA upon VAN exposure.(McAleese Fionnuala et al., 2006, Sakoulas George et al., 2005, Sakoulas George et al., 2002, Tsuji et al., 2007, Vidaillac Celine et al., 2013).

Antimicrobials: All antimicrobials were purchased commercially. VAN was purchased from Pfizer, New York City, NY; CFZ from Sandoz, Princeton, NJ; oxacillin(OXA) from Sigma Aldrich, St Louis, MO. Teicoplanin(TEI) was

ACCEPTED MANUSCRIPT purchased from Sanofi Aventis, Bridgewater, NJ and DAP from Merck Pharmaceuticals, Lexington, MA.

Susceptibility testing. Antimicrobial minimum inhibitory concentration (MIC)

T

values were determined in duplicate by broth microdilution as per CLSI

IP

guidelines.(2014) VAN MICs were also determined in the presence of CFZ either

CR

at half MIC or peak unbound serum concentration whichever was lesser, to

US

determine the ability of the β-lactams to reduce the VAN MIC.

A one-compartment in vitro

AN

In vitro one-compartment PK/PD Models:

pharmacokinetic/pharmacodyamic (PK/PD) model of 250-ml capacity previously

M

described was used. (Smith et al., 2015) The elimination rate for the combination

ED

models was set for the antibiotic with the shorter half-life, and the antibiotic with the longer half-life was supplemented into the second chamber. (Blaser, 1985)

PT

Procedures for evaluating the impact of CFZ on the prevention of VISA

CE

were 1) establish a suboptimal VAN dose and exposure that would reproducibly produce VISA in a shortest period of time, 2) determine the CFZ dose that

AC

prevents the emergence of VISA and 3) use of population analysis to further establish the impact of CFZ on the prevention of VISA. Additionally, the impact of CFZ on the survival of the Cathelicidin LL-37 was also examined. Regimens simulating bacterial exposure of VAN ranging from 62.5 to 500mg q 12h (fAUC/MIC of 118.8-16.8) that have been previously demonstrated to select for VISA were evaluated in the present study.(Mishra et al., 2012, Tsuji

ACCEPTED MANUSCRIPT et al., 2007) While these exposures are not the clinically administered doses of VAN, they were utilized in order to create conditions whereby VISA would be selected promptly, and to determine if this selection process could be reduced or eliminated with CFZ. A modified model procedure that included the recycling of

T

organisms after each model 72h run was incorporated to increase the rate and

IP

reproducibility of VISA production to test our primary hypothesis. Recycling

CR

entailed selecting organisms from the resistance screening plates, at the end of each model run, and then reinserting these colonies as the starting organisms for

US

the next model run.(Berti et al., 2012, Friedman et al., 2006, Mishra et al., 2012)

AN

This procedure was repeated until we recovered an organism with a VAN MIC of 4 mg/L or greater from VAN monotherapy of 200 mg q 12 h. This specific VAN

M

regimen was selected on the ability to consistently produce VISA in a 72-144h

ED

time frame. All PK/PD models were run in duplicate to ensure reproducibility. Samples from each time point 0-240h were plated on Tryptic Soy Agar (TSA) for

PT

colony enumeration and on Brain Heart Infusion Agar (BHIA) plates containing 3x

CE

VAN MIC for resistance screening. In a similar fashion, various dosage regimens of CFZ were tested in combination with VAN to determine what dosage/exposure

AC

of CFZ provided therapeutic enhancement but still allowed enough colonies to survive for subsequent model recycling experiments. For combination models, the dose of cefazolin was selected depending on the organism under question. The dose selected should allow some organisms to survive for characterization. For MSSA RN9120, continuous infusion of CFZ at ½ MIC the concentration of 0.125 mg/l was used. Since JH1 was a MRSA isolate and therefore resistant to

ACCEPTED MANUSCRIPT CFZ (MIC=16mg/l), a therapeutic dose of CFZ i.e. 1g q 8h, was initially administered with VAN regimen of 200mg q 12h. (Data not shown). This PK/PD model run resulted in organism colony counts below detection limits within 8 hours and no surviving organism at the end of first run of 72h. Therefore, the

T

CFZ concentration was decreased and the dosing interval was increased until

IP

organisms survived at end of first model run to be recycled. The final dosage

CR

administration for JH1 selected for the model was VAN 200 mg q 12h + CFZ 125mg q 24h. In addition, a PK/PD model with VAN 200 mg q 12h+ CFZ 125mg

US

q 24h for JH1 was studied for an extended period of 21 days to evaluate the

AN

impact of long term exposure of combination therapy on emergence of

M

resistance.

ED

Pharmacodynamic analysis. Samples from each model were collected at each cycle of 72h at 0, 4, 8, 24, 32, 48, 72h in duplicate. Colony counts were

PT

determined by spiral plating appropriate dilutions using an automatic spiral plater

CE

(Whitely Automatic Spiral Plater; DW Scientific, West Yorkshire, England). Post incubation, the colonies were quantified using a laser colony counter (ProtoCOL;

AC

Synoptics Limited, Frederick, MD). Bacteria were plated on Tryptic soy agar (TSA, Difco, Detroit, MI). These methods have a low limit of reliable detection of 2 log10 CFU/ml. The total reduction in log10 CFU/ml over 72h was determined by plotting model time-kill curves based on the number of remaining organisms over the time period.

ACCEPTED MANUSCRIPT Selection of Antimicrobial Resistance: The emergence of VAN resistance was evaluated by plating 120-μl samples from the model, at each sampling point, on BHIA plates supplemented with VAN at 3 x the MIC. The plates were examined for growth after 48h of incubation at 35°C. Resistant colonies grown on screening

T

plates were evaluated for VAN MIC susceptibility changes by broth microdilution

IP

method. If resistance was detected, the organism at the end of the model was

CR

also characterized for susceptibility changes to other antibiotics.

US

Pharmacokinetic analysis: VAN and CFZ pharmacokinetics were determine

AN

from model samples (1 ml) drawn from each model over 0, 1, 2, 4, 6, 8 and 24h. Both VAN and CFZ concentrations were determined by bioassay using Kocuria

M

rhizophila ATCC 9341 and Bacillus subtilis ATCC 6633 as test organisms on

ED

antibiotic medium 11. (Fields et al., 1993)The samples were incubated for 24h before measuring the zone of inhibition for assay standards and model samples.

PT

The half-life and peak concentrations were determined using the PK Analyst

CE

software (version 1.10; MicroMath Scientific Software, Salt Lake City, UT).

AC

Cathelicidin LL-37 microbicidal assay. The RN9120 and the JH1 parent strain were grown on TSA while the mutant obtained from the models i.e. RN9120-2 and JH1-2 were grown on BHIA supplemented with 1mg/l VAN for 16 to 20h, and exposed at an inoculum of 105 CFU/ml to 16 µM, LL-37, in Roswell Park Memorial Institute medium (RPMI) +5% Lysogeny broth (LB). The percentage of surviving bacteria (%standard deviation [SD]) after 2 h of incubation at 35°C was calculated by plating on TSA plates, as previously described. (Sakoulas G. et al.,

ACCEPTED MANUSCRIPT

2014, Smith et al., 2015)

Population Analysis Profile.

T

The VAN PAP AUC was measured for isolates obtained at the end of

IP

216h or at the end of last run where any organism survived, along with the parent

CR

strain. A 2 McFarland suspension for the organism being tested containing ~10 9 CFU/ml was prepared. Serial dilutions of the test organism were plated using the

AN

decreasing concentrations of VAN BHI.

US

automatic spiral plater (WASP, DW Scientific, West Yorkshire, England) onto

The VAN agar concentrations in the range 0 to 16mg/l was used. Colony

M

growth at 48h was measured using a laser colony counter (ProtoCOL, Synoptics

concentration.

ED

limited, Frederick, MD) and graphed as log10 CFU/ml versus the VAN The parent strain was included in the run for comparison

PT

purposes. The area under the curve was calculated and compared for the parent

AC

RESULTS:

CE

and mutant derived organisms.

Selection of VAN heteroresistance in the presence or absence of CFZ. Baseline MIC values for all organisms against all antimicrobials are listed in Table 1. The VAN MIC in the presence of ½ MIC CFZ decreased >4 fold for MSSA RN9120 and 3-fold for MRSA JH1. The PK/PD recycle model simulating VAN 200mg q 12h produced VISA in both RN9120 and JH1 within 120-128h of

ACCEPTED MANUSCRIPT suboptimal vancomycin exposure. The plots for models are shown in Figure 1 for RN9120 and JH1. The changes in VAN MIC versus time are shown in Table 2. Table 1 Parent MSSA (RN9120) and MRSA (JH1)susceptibility to VAN, DAP,TEI, OX and CFZ prior and post exposure of VAN 200 mg q 12 h

DAP MIC mg/l

TEI MIC mg/l

OXA MIC mg/l

CFZ MIC mg/l

RN9120

1

0.125

0.125

0.25

0.25

JH1

1

0.0625

0.25

>32

16

RN9120-2

4

1

4

0.25

0.25

JH1-2

4

0.5

8

2

1

US

CR

IP

T

VAN MIC mg/l

Treatment

AN

Table 2 Sample Time points depicting MIC change for MSSA RN9120 and JH1 in PK/PD model

Change in MIC

RN9120-1

Parent VAN MIC 1mg/l

0h

JH1-2

Mutant 1

Mutant 2

Parent

Mutant 1

Mutant 2

VAN MIC 2mg/l

VAN MIC 4mg/l

VAN MIC 1mg/l

VAN MIC 2mg/l

VAN MIC 4mg/l

48-56 h

*NC = no change in MIC

AC

JH1-1

ED

VAN + CFZ

JH1

PT

0h

CE

VAN monotherapy

RN9120-2

M

RN9120

72 h

120-128 h

0h

48-56 h

120-128 h

>216h-NC*

0h

>216h-NC*

>216h-NC*

ACCEPTED MANUSCRIPT

AC

CE

PT

ED

M

AN

US

CR

IP

T

Figure 1 Representative activity of Vancomycin, Cefazolin and Vancomycin in combination with Cefazolin at simulated regimen against RN9120 and JH1. Error bars indicate standard deviations

ACCEPTED MANUSCRIPT The VAN MIC for RN9120 and JH1 increased from 1 to 2 mg/l within the first 72h cycle of the PK/PD model simulating VAN 200 mg q 12 h, generating mutants RN9120-1 and JH1-1. When the strains were recycled for the next 72h model run, the VAN MIC to both organisms increased to 4mg/l, yielding VISA

T

(RN9120-2, JH1-2) within 144h of suboptimal VAN exposure. Upon further

IP

exposure up to 216h, the mutants RN9120-2 and JH1-2 remained stable for 3

CR

passages onto antibiotic free media, maintaining their VAN MIC of 4 mg/L. These models were repeated three more times resulting in the VISA phenotype

US

for both strains within 144h of VAN 200 mg q 12h exposure reproducibly.

AC

CE

PT

ED

M

AN

Figure 2 Growth Curve of Parent (initial strain) and Mutant organism (VISA obtained from models).

In order to determine if the VISA isolated obtained by the recycling method were still as biologically fit as those in its parent population growth curve characteristics of parent vs mutant strains was evaluated. No appreciable difference of viability was seen between the parent and the mutant as seen in

ACCEPTED MANUSCRIPT figure 2. Thus confirming that there was no significant difference in the biological fitness of the parent and mutant population. The mean observed fCmax and fAUC for VAN were 3.715± 0.092 mg/l (target 3.7mg/l), and 31.14± 1.15 mg.h/l, (target 41.1 mg.h/l) respectively. The

T

average T1/2h obtained was 5.81± 0.071 h (target, 6h). The mean observed

IP

fCmax for CFZ in 0.125mg/l infusion used in RN9120 was 0.15± 0.029 mg/l

CR

(target 0.15mg/l). While in CFZ 125mg q 24h model used against JH1 the

obtained was 1.66± 0.071 h (target, 1.8h).

US

observed fCmax was 3.03 ± 0.042 mg/l, (target 3 mg.h/l) and the average T1/2h

AN

The PK/PD model for VAN regimen of 200mg q 12h with CFZ continuous infusion of CFZ at half (½ MIC) at 0.125mg/l against RN9120 is shown in Figure

M

1. For MRSA JH1, some organisms survived at 72h exposure to VAN 200mg q

ED

12h + CFZ 125mg q 24h. However, no growth was observed on VAN resistance plates indicating no development of resistance. The colonies obtained at 72h

PT

were then recycled through the model for additional runs up to 168h to determine

CE

whether the addition of CFZ could prevent the emergence of VISA. For both RN9120 and JH1 strains, the addition of CFZ to the VAN 200mg

AC

q 12h regimen prevented the emergence of VISA. These results are shown in Table 2.

Of note, the VAN MIC RN9120 increased from 1-2 mg/L in the

presence of CFZ, but no further MIC changes were noted running the models of VAN + CFZ for up to 9 days, supporting our hypothesis. The extended model with VAN 200 mg q 12h+ CFZ 125mg q 24h for JH1 ran over a period of 21 days demonstrated no emergence of resistance and

ACCEPTED MANUSCRIPT CFU/ml counts for the model remained at detection limits for the duration of the study. The results are shown in Figure 3. No growth was observed on VAN resistance plates over the duration of the study.

PT

ED

M

AN

US

CR

IP

T

Figure 3- JH1 in presence of VAN 200 mg q 12 h in presence of CFZ in a 21 day model. The open circle represents exposure of JH1 to VAN 200mg q 12h in presence of CFZ 125mg q 24h

Population Analysis Profile. The population analysis profiles demonstrated

CE

increased VAN hetero-resistance with VAN monotherapy exposure. The addition

AC

of CFZ to VAN reduced VAN hetero-resistance. The population analysis profiles for RN9120 and JH1 are depicted in Figure 4. For RN9120, the VAN population analysis AUC increased from 38.53 log10CFU.g/ml2 to 74.56 log10CFU.g/ml2 in presence of VAN alone, whereas the addition of CFZ

attenuated the AUC

increase to only 56.34 log10CFU.g/ml2 limiting the shift of the organism to a more vancomycin resistant population. Similarly, for JH1, the AUC of the parent increased from 38.43 log10CFU.g/ml2 to 56.36 log10CFU.g/ml2 in presence of

ACCEPTED MANUSCRIPT VAN alone, but the addition of CFZ resulted in AUC to 38.09 log10CFU.g/ml2 thus demonstrating a lack of shift in the organisms’ population by the addition of CFZ.

M

AN

US

CR

IP

T

Figure 4 Population analysis profile for RN9120 and JH1 along with their respective counterparts obtained from models exposing them to sub-therapeutic vancomycin and sub-therapeutic vancomycin in presence of cefazolin.

ED

Change in MIC to other antimicrobials and LL-37. The MIC’s of VAN, DAP,

PT

teicoplanin, CFZ and oxacillin were compared between the parent strains (VAN MIC 1 mg/l) and those recovered from the VAN mono PK/PD model at 144h

CE

(VAN MIC 4 mg/l) to check for the potential for cross-resistance (Table 1). As the

AC

vancomycin MIC increased the corresponding increase in MIC for DAP and TEI was observed in both organisms. A decrease in MIC to beta-lactams was observed for JH1 with increase in VAN MIC but the RN9120 MIC remained unchanged to beta-lactams as VAN MIC increased. In addition, the LL-37 MIC for the mutants RN9120-2 and JH1-2 was > 32 mg/L, notably increased from MIC 16 mg/L of RN9120 and JH1 parent strains.

ACCEPTED MANUSCRIPT Cathelicidin LL-37 microbicidal assay. The LL-37 killing at 2h reduced upon emergence of VAN hetero-resistance, as shown by higher survival of mutants obtained following exposure to VAN monotherapy as compared to the parent baseline isolates. The survival of RN9120 increased from 42.53% to 95.96%

T

while for JH1 it increased from 39.62% to 100%(Figure 5)

PT

ED

M

AN

US

CR

IP

Figure 5 Percent survival of S.aureus MSSA parent RN9120, MRSA parent JH1, MSSA RN9120 exposed to VAN 200mg q 12h (RN9120-2) and MRSA JH1 exposed to VAN 200 mg q 12h (JH1-2) against 16mcgM LL-37. Error bars indicate standard deviation.

CE

Note A > 100 % survival is possible when the survival with LL-37 exceeds the survival of the growth control

AC

DISCUSSION:

In this study, we confirmed that suboptimal VAN dosing in S. aureus leads to VISA for both MSSA (RN9120) and MRSA (JH1). The organism JH1 in this experiment has been previously documented to develop the VISA phenotype while on VAN therapy in a patient with infective endocarditis (McAleese F. et al., 2006, Mwangi et al., 2007, Sieradzki et al., 2003) We have previously demonstrated in a simulated endocardial vegetation PK/PD model that over a

ACCEPTED MANUSCRIPT period of 30 and 60 days of VAN dosing at 500 mg q 12h or 1g q 12h, respectively, the VSSA MRSA strain JH1 develops a VISA phenotype, characterized by cell wall thickening and genotypically by various mutations associated with the cell wall envelope.(Vidaillac Celine et al., 2013, Vidaillac C,

T

2016)

IP

Reduced VAN susceptibility with suboptimal VAN monotherapy exposure

CR

has been reported for MSSA in clinical strains as well.(Hu et al., 2013, Rybak et al., 2005) MSSA with dysfunctional AGR group have a high proclivity towards

US

developing resistance under these conditions.(Tsuji et al., 2007) Using our

AN

method of recycling at suboptimal VAN concentrations, we were able to generate VISA within 144h, more rapidly and reproducibly as compared to other reported

M

methods for both MSSA (RN9120) and MRSA (JH1).(Tsuji et al., 2007, Vidaillac

ED

C. et al., 2013)

Clinical investigations have demonstrated that suboptimal VAN exposure

PT

can lead to VISA and treatment failure.(Rybak et al., 2009) The acquisition of

CE

VISA is important since these strains are often cross resistant to DAP which is one of the primary alternatives to VAN therapy, especially after VAN failure.

AC

(Chen et al., 2015) We found that an increase in VAN resistance resulted in an increase in MIC not only to other glycopeptide and lipopeptides such as teicoplanin and DAP, but also to the endogenous CAMP human cathelicidin LL37. Resistance to human cathelicidin LL-37 and increased DAP MIC may be associated with increased mortality in patients with complicated MRSA bloodstream infections (Sakoulas George et al., 2014). This finding is of interest

ACCEPTED MANUSCRIPT since we observed an increase in the survival of the test S. aureus strains to LL37 as the organism became more resistant to VAN (Figure 5), as had been previously shown for the platelet derived CAMP, platelet microbicial protein (PMP) and vancomycin exposure (Sakoulas G. et al., 2005). The cross-

T

resistance triad between VAN, DAP and CAMP also raises concern that in vivo

IP

exposure to CAMP as occurs in an uncontrolled MRSA infection may select for

CR

DAP or VAN heteroresistance even in the absence of these administered antibiotics, as has been shown in a rabbit MRSA osteomyelitis model (Mishra et

US

al., 2013). Taken together, these results validate the concern for the

AN

development of cross-resistance by prior VAN exposure not only to other glycopeptides and lipopeptides, but to CAMPS of the human innate immune

M

system.

ED

Previous studies have evaluated combination of VAN and a variety of beta-lactams for synergy. VAN and nafcillin have been shown to be synergistic

PT

in more than 90% strains by time kill studies with an overall increase in the

CE

magnitude of activity observed in PK/PD models. (Leonard, 2012)Similarly, synergy of VAN with beta-lactams such as cefepime, cefpirome, carbapenems,

AC

cefotaxime and many others has been documented. (Climo et al., 1999, Deresinski, 2009, Sy C. L. et al., 2016) CFZ in combination to VAN has been evaluated for synergy against S. epidermidis and had been found effective in about 40% of the cases.(Siebert et al., 1979) However, there is very little information about the combination of VAN and CFZ against S. aureus. CFZ is reported to be one of the most consistently synergistic beta-lactam in

ACCEPTED MANUSCRIPT combination with VAN. (Claeys et al., 2015, Dhand and Sakoulas, 2014, RochonEdouard et al., 2000)

In accordance, we found VAN and CFZ combination

caused reduction in MIC for VAN for all organisms tested as compared to either drug alone. Additionally CFZ had fewer side effects as compared to drugs like

T

nafcillin.(Lee et al., 2011) These advantages of CFZ paved the way for the use of

IP

CFZ in our study.

CR

We employed experimental conditions of VAN exposure to rapidly select for VISA in order to determine if CFZ could attenuate this selective process.

US

These experimental conditions were repeated a minimum of two times in

AN

duplicate in order to verify the reproducibility. Under these conditions, CFZ coadministered with VAN prevented an increase in VAN MIC for MRSA JH1, even

M

in the event of long term exposure. A one dilution increase in VAN MIC was

ED

detected in MSSA RN9120, likely because high-susceptibility to CFZ of RN9120 did not allow for such high concentrations of CFZ to be utilized as in the MRSA

PT

model. CFZ when added to VAN regimen was able to reduce or eliminate the

CE

shift of population to higher MIC values and reduce the area under curve for both the organisms. The mechanism by which CFZ prevents the emergence of VISA

AC

is not known. However, a potential explanation may be associated with the so called “Seesaw Effect” whereby S. aureus exposure to vancomycin has been shown to improve beta-lactam susceptibility potentially by the alteration of penicillin binding proteins. (Barber Katie E. et al., 2014, Jousselin et al., 2015) We point out to clinically-minded readers that the doses simulated by the low VAN exposures employed here are never intentionally administered to

ACCEPTED MANUSCRIPT patients in practice, and were chosen to rigorously challenge CFZ in preventing the emergence of VISA. Nevertheless, it is conceivable that VAN exposures as low as the ones employed may have clinical relevance in compartments of poor VAN penetration or augmented VAN clearance in vivo (eg. upper airway mucosa,

T

respiratory tract, biofilm associated medical device infections, critically ill patients

IP

etc.), where S. aureus may colonize or infect.

CR

Thus, data would suggest that application of a beta-lactams like CFZ and VAN as initial therapy may prevent the emergence of VISA on VAN therapy.

US

This has potential important clinical implications since the VAN failure often leads

AN

to subsequent failure with alternative (salvage) antibiotics such as DAP. The prevention of resistance by using a combination of VAN and CFZ up front could

M

curtail the need to escalate therapy.

ED

Limitations of this study include the small number of evaluated strains, the short duration of antibiotic exposure experiments and non-standard dosing. In

PT

clinical settings, a patient may receive these antibiotics for longer durations of

CE

time. While only one beta-lactam (CFZ) was explored in our study, parallel studies should be considered for other clinically available beta-lactams such as

AC

nafcillin, ceftaroline, and cefepime should be evaluated in combination studies with VAN to evaluate their potential in the prevention of resistance. In summation, the addition of CFZ to the regimen prevented of emergence of VISA for both MSSA and MRSA, with the collateral benefit of preventing resistance to killing by cathelicidin LL-37. Additional studies on a wider range of

ACCEPTED MANUSCRIPT isolates, more antibiotic combinations along with experiments on animal models will further validate the utility of this antibiotic combination for clinical use. Acknowledgements: None

IP

T

Funding:

CR

This project received no external funding support.

US

Disclosures:

MJR received research support, consulted or is part of a speaker bureau for

AN

Allergan, Bayer, Cempra, Merck, The Medicine Company, Sunovian and

M

Theravance Biopharmaceutics and is support in part by NIAID R01 AI121400

ED

and R21 AI109266.

GS has received speaking honoraria from Allergan, The Medicines Company,

PT

Sunovion and consulting fees from Allergan.

AC

CE

NS has nothing to disclose.

ACCEPTED MANUSCRIPT

AC

CE

PT

ED

M

AN

US

CR

IP

T

REFERENCES: CLSI. Performance Standards for Antimicrobial Susceptibility Testing; Twenty-Fourth Informational Supplement. CLSI document M100-S24. Wayne, PA: Clinical and Laboratory Standards Institute. 2014. Bakke V, Sporsem H, Von der Lippe E, Nordoy I, Lao Y, Nyrerod HC, et al. Vancomycin levels are frequently subtherapeutic in critically ill patients: a prospective observational study. Acta Anaesthesiol Scand 2017;61(6):627-35. Baptista JP, Sousa E, Martins PJ, Pimentel JM. Augmented renal clearance in septic patients and implications for vancomycin optimisation. Int J Antimicrob Agents 2012;39(5):420-3. Barber KE, Ireland CE, Bukavyn N, Rybak MJ. Observation of "seesaw effect" with vancomycin, teicoplanin, daptomycin and ceftaroline in 150 unique MRSA strains. Infect Dis Ther 2014;3(1):35-43. Barber KE, Ireland CE, Bukavyn N, Rybak MJ. Observation of “Seesaw Effect” with Vancomycin, Teicoplanin, Daptomycin and Ceftaroline in 150 Unique MRSA Strains. Infectious Diseases and Therapy 2014;3(1):35-43. Barr JG, Smyth ETM, Hogg GM. In vitro antimicrobial activity of imipenem in combination with vancomycin or teicoplanin againstStaphylococcus aureus andStaphylococcus epidermidis. European Journal of Clinical Microbiology and Infectious Diseases 1990;9(11):804-9. Berti AD, Wergin JE, Girdaukas GG, Hetzel SJ, Sakoulas G, Rose WE. Altering the proclivity towards daptomycin resistance in methicillin-resistant Staphylococcus aureus using combinations with other antibiotics. Antimicrob Agents Chemother 2012;56(10):5046-53. Blaser J. In-vitro model for simultaneous simulation of the serum kinetics of two drugs with different half-lives. Journal of Antimicrobial Chemotherapy 1985;15(suppl A):125-30. Casapao AM, Davis SL, McRoberts JP, Lagnf AM, Patel S, Kullar R, et al. Evaluation of Vancomycin Population Susceptibility Analysis Profile as a Predictor of Outcomes for Patients with Infective Endocarditis Due to Methicillin-Resistant Staphylococcus aureus. Antimicrobial Agents and Chemotherapy 2014;58(8):4636-41. Chen CJ, Huang YC, Chiu CH. Multiple pathways of cross-resistance to glycopeptides and daptomycin in persistent MRSA bacteraemia. J Antimicrob Chemother 2015;70(11):296572. Claeys KC, Smith JR, Casapao AM, Mynatt RP, Avery L, Shroff A, et al. Impact of the combination of daptomycin and trimethoprim-sulfamethoxazole on clinical outcomes in methicillin-resistant Staphylococcus aureus infections. Antimicrob Agents Chemother 2015;59(4):1969-76. Climo MW, Patron RL, Archer GL. Combinations of Vancomycin and β-Lactams Are Synergistic against Staphylococci with Reduced Susceptibilities to Vancomycin. Antimicrobial Agents and Chemotherapy 1999;43(7):1747-53. Cui L, Tominaga E, Neoh H-m, Hiramatsu K. Correlation between Reduced Daptomycin Susceptibility and Vancomycin Resistance in Vancomycin-Intermediate Staphylococcus aureus. Antimicrobial Agents and Chemotherapy 2006;50(3):1079-82. Davis JS, Sud A, O'Sullivan MVN, Robinson JO, Ferguson PE, Foo H, et al. Combination of Vancomycin and β-Lactam Therapy for Methicillin-Resistant Staphylococcus aureus Bacteremia: A Pilot Multicenter Randomized Controlled Trial. Clinical Infectious Diseases 2016;62(2):173-80. Deresinski S. Counterpoint: Vancomycin and Staphylococcus aureus—An Antibiotic Enters Obsolescence. Clinical Infectious Diseases 2007;44(12):1543-8. Deresinski S. Vancomycin in Combination with Other Antibiotics for the Treatment of Serious Methicillin-Resistant Staphylococcus aureus Infections. Clinical Infectious Diseases 2009;49(7):1072-9.

ACCEPTED MANUSCRIPT

AC

CE

PT

ED

M

AN

US

CR

IP

T

Dhand A, Bayer AS, Pogliano J, Yang SJ, Bolaris M, Nizet V, et al. Use of antistaphylococcal beta-lactams to increase daptomycin activity in eradicating persistent bacteremia due to methicillin-resistant Staphylococcus aureus: role of enhanced daptomycin binding. Clin Infect Dis 2011;53(2):158-63. Dhand A, Sakoulas G. Daptomycin in combination with other antibiotics for the treatment of complicated methicillin-resistant Staphylococcus aureus bacteremia. Clin Ther 2014;36(10):1303-16. Estes KS, Derendorf H. Comparison of the pharmacokinetic properties of vancomycin, linezolid, tigecyclin, and daptomycin. European Journal of Medical Research 2010;15(12):533-43. Fields MT, Herndon BL, Bamberger DM. beta-Lactamase-mediated inactivation and efficacy of cefazolin and cefmetazole in Staphylococcus aureus abscesses. Antimicrob Agents Chemother 1993;37(2):203-6. Fowler JVG, Sakoulas G, McIntyre LM, Meka VG, Arbeit RD, Cabell CH, et al. Persistent Bacteremia Due to Methicillin-Resistant Staphylococcus aureus Infection Is Associated with agr Dysfunction and Low-Level In Vitro Resistance to Thrombin-Induced Platelet Microbicidal Protein. The Journal of Infectious Diseases 2004;190(6):1140-9. Fox PM, Lampen RJ, Stumpf KS, Archer GL, Climo MW. Successful Therapy of Experimental Endocarditis Caused by Vancomycin-Resistant Staphylococcus aureus with a Combination of Vancomycin and β-Lactam Antibiotics. Antimicrobial Agents and Chemotherapy 2006;50(9):2951-6. Fridkin SK. Vancomycin-intermediate and -resistant Staphylococcus aureus: what the infectious disease specialist needs to know. Clin Infect Dis 2001;32(1):108-15. Friedman L, Alder JD, Silverman JA. Genetic changes that correlate with reduced susceptibility to daptomycin in Staphylococcus aureus. Antimicrob Agents Chemother 2006;50(6):2137-45. Hagihara M, Wiskirchen DE, Kuti JL, Nicolau DP. In Vitro Pharmacodynamics of Vancomycin and Cefazolin Alone and in Combination against Methicillin-Resistant Staphylococcus aureus. Antimicrobial Agents and Chemotherapy 2012;56(1):202-7. Hirai K, Ishii H, Shimoshikiryo T, Shimomura T, Tsuji D, Inoue K, et al. Augmented Renal Clearance in Patients With Febrile Neutropenia is Associated With Increased Risk for Subtherapeutic Concentrations of Vancomycin. Ther Drug Monit 2016;38(6):706-10. Hiramatsu K, Aritaka N, Hanaki H, Kawasaki S, Hosoda Y, Hori S, et al. Dissemination in Japanese hospitals of strains of Staphylococcus aureus heterogeneously resistant to vancomycin. Lancet 1997;350(9092):1670-3. Hu J, Ma XX, Tian Y, Pang L, Cui LZ, Shang H. Reduced Vancomycin Susceptibility Found in Methicillin-Resistant and Methicillin-Sensitive Staphylococcus aureus Clinical Isolates in Northeast China. PLoS ONE 2013;8(9):e73300. Jousselin A, Manzano C, Biette A, Reed P, Pinho MG, Rosato AE, et al. The Staphylococcus aureus Chaperone PrsA Is a New Auxiliary Factor of Oxacillin Resistance Affecting Penicillin-Binding Protein 2A. Antimicrob Agents Chemother 2015;60(3):1656-66. Keel RA, Sutherland CA, Aslanzadeh J, Nicolau DP, Kuti JL. Correlation between vancomycin and daptomycin MIC values for methicillin-susceptible and methicillin-resistant Staphylococcus aureus by 3 testing methodologies. Diagn Microbiol Infect Dis 2010;68(3):326-9. Kullar R, McKinnell JA, Sakoulas G. Avoiding the perfect storm: the biologic and clinical case for reevaluating the 7-day expectation for methicillin-resistant Staphylococcus aureus bacteremia before switching therapy. Clin Infect Dis 2014;59(10):1455-61.

ACCEPTED MANUSCRIPT

AC

CE

PT

ED

M

AN

US

CR

IP

T

Lamer C, de Beco V, Soler P, Calvat S, Fagon JY, Dombret MC, et al. Analysis of vancomycin entry into pulmonary lining fluid by bronchoalveolar lavage in critically ill patients. Antimicrobial Agents and Chemotherapy 1993;37(2):281-6. Larry M. Baddour M, FAHA, Chair; Walter R. Wilson, MD; Arnold S. Bayer, MD; Vance G. Fowler, Jr, MD, MHS; Imad M. Tleyjeh, MD, MSc; Michael J. Rybak, PharmD, MPH; Bruno Barsic, MD, PhD; Peter B. Lockhart, DDS; Michael H. Gewitz, MD, FAHA; Matthew E. Levison, MD; Ann F. Bolger, MD, FAHA; James M. Steckelberg, MD; Robert S. Baltimore, MD; Anne M. Fink, PhD, RN; Patrick O’Gara, MD, FAHA; Kathryn A. Taubert, PhD, FAHA. Infective Endocarditis in Adults: Diagnosis, Antimicrobial Therapy, and Management of Complications. American Heart Association, Inc 2015:16-20. Lee S, Choe PG, Song K-H, Park S-W, Kim HB, Kim NJ, et al. Is Cefazolin Inferior to Nafcillin for Treatment of Methicillin-Susceptible Staphylococcus aureus Bacteremia? Antimicrobial Agents and Chemotherapy 2011;55(11):5122-6. Leonard SN. Synergy between Vancomycin and Nafcillin against Staphylococcus aureus in an In Vitro Pharmacokinetic/Pharmacodynamic Model. PLoS ONE 2012;7(7):e42103. McAleese F, Wu SW, Sieradzki K, Dunman P, Murphy E, Projan S, et al. Overexpression of genes of the cell wall stimulon in clinical isolates of Staphylococcus aureus exhibiting vancomycin-intermediate- S. aureus-type resistance to vancomycin. J Bacteriol 2006;188(3):1120-33. McAleese F, Wu SW, Sieradzki K, Dunman P, Murphy E, Projan S, et al. Overexpression of Genes of the Cell Wall Stimulon in Clinical Isolates of Staphylococcus aureus Exhibiting Vancomycin-Intermediate- S. aureus-Type Resistance to Vancomycin. Journal of Bacteriology 2006;188(3):1120-33. Mishra NN, McKinnell J, Yeaman MR, Rubio A, Nast CC, Chen L, et al. In Vitro CrossResistance to Daptomycin and Host Defense Cationic Antimicrobial Peptides in Clinical Methicillin-Resistant Staphylococcus aureus Isolates. Antimicrobial Agents and Chemotherapy 2011;55(9):4012-8. Mishra NN, Rubio A, Nast CC, Bayer AS. Differential Adaptations of Methicillin-Resistant Staphylococcus aureus to Serial In Vitro Passage in Daptomycin: Evolution of Daptomycin Resistance and Role of Membrane Carotenoid Content and Fluidity. Int J Microbiol 2012;2012:683450. Mishra NN, Yang S-J, Chen L, Muller C, Saleh-Mghir A, Kuhn S, et al. Emergence of Daptomycin Resistance in Daptomycin-Naïve Rabbits with Methicillin-Resistant Staphylococcus aureus Prosthetic Joint Infection Is Associated with Resistance to Host Defense Cationic Peptides and mprF Polymorphisms. PLoS ONE 2013;8(8):e71151. Mwangi MM, Wu SW, Zhou Y, Sieradzki K, de Lencastre H, Richardson P, et al. Tracking the in vivo evolution of multidrug resistance in Staphylococcus aureus by whole-genome sequencing. Proc Natl Acad Sci U S A 2007;104(22):9451-6. Patel JB, Jevitt LA, Hageman J, McDonald LC, Tenover FC. An Association between Reduced Susceptibility to Daptomycin and Reduced Susceptibility to Vancomycin in Staphylococcus aureus. Clinical Infectious Diseases 2006;42(11):1652-3. Rochon-Edouard S, Pestel-Caron M, Lemeland J-F, Caron F. In Vitro Synergistic Effects of Double and Triple Combinations of β-Lactams, Vancomycin, and Netilmicin against Methicillin-Resistant Staphylococcus aureus Strains. Antimicrobial Agents and Chemotherapy 2000;44(11):3055-60. Rybak M, Lomaestro B, Rotschafer JC, Moellering R, Jr., Craig W, Billeter M, et al. Therapeutic monitoring of vancomycin in adult patients: a consensus review of the American Society of Health-System Pharmacists, the Infectious Diseases Society of America, and the Society of Infectious Diseases Pharmacists. Am J Health Syst Pharm 2009;66(1):82-98.

ACCEPTED MANUSCRIPT

AC

CE

PT

ED

M

AN

US

CR

IP

T

Rybak MJ. The pharmacokinetic and pharmacodynamic properties of vancomycin. Clin Infect Dis 2006;42 Suppl 1:S35-9. Rybak MJ. The Pharmacokinetic and Pharmacodynamic Properties of Vancomycin. Clinical Infectious Diseases 2006;42(Supplement_1):S35-S9. Rybak MJ, Cha R, Cheung CM, Meka VG, Kaatz GW. Clinical isolates of Staphylococcus aureus from 1987 and 1989 demonstrating heterogeneous resistance to vancomycin and teicoplanin. Diagn Microbiol Infect Dis 2005;51(2):119-25. Sakoulas G, Alder J, Thauvin-Eliopoulos C, Moellering RC, Eliopoulos GM. Induction of Daptomycin Heterogeneous Susceptibility in Staphylococcus aureus by Exposure to Vancomycin. Antimicrobial Agents and Chemotherapy 2006;50(4):1581-5. Sakoulas G, Eliopoulos GM, Fowler VG, Jr., Moellering RC, Jr., Novick RP, Lucindo N, et al. Reduced susceptibility of Staphylococcus aureus to vancomycin and platelet microbicidal protein correlates with defective autolysis and loss of accessory gene regulator (agr) function. Antimicrob Agents Chemother 2005;49(7):2687-92. Sakoulas G, Eliopoulos GM, Fowler VG, Moellering RC, Novick RP, Lucindo N, et al. Reduced Susceptibility of Staphylococcus aureus to Vancomycin and Platelet Microbicidal Protein Correlates with Defective Autolysis and Loss of Accessory Gene Regulator (agr) Function. Antimicrobial Agents and Chemotherapy 2005;49(7):2687-92. Sakoulas G, Eliopoulos GM, Moellering RC, Jr., Novick RP, Venkataraman L, Wennersten C, et al. Staphylococcus aureus accessory gene regulator (agr) group II: is there a relationship to the development of intermediate-level glycopeptide resistance? J Infect Dis 2003;187(6):929-38. Sakoulas G, Eliopoulos GM, Moellering RC, Jr., Wennersten C, Venkataraman L, Novick RP, et al. Accessory gene regulator (agr) locus in geographically diverse Staphylococcus aureus isolates with reduced susceptibility to vancomycin. Antimicrob Agents Chemother 2002;46(5):1492-502. Sakoulas G, Eliopoulos GM, Moellering RC, Wennersten C, Venkataraman L, Novick RP, et al. Accessory Gene Regulator (agr) Locus in Geographically Diverse Staphylococcus aureus Isolates with Reduced Susceptibility to Vancomycin. Antimicrobial Agents and Chemotherapy 2002;46(5):1492-502. Sakoulas G, Guram K, Reyes K, Nizet V, Zervos M. Human cathelicidin LL-37 resistance and increased daptomycin MIC in methicillin-resistant Staphylococcus aureus strain USA600 (ST45) are associated with increased mortality in a hospital setting. J Clin Microbiol 2014;52(6):2172-4. Sakoulas G, Guram K, Reyes K, Nizet V, Zervos M. Human Cathelicidin LL-37 Resistance and Increased Daptomycin MIC in Methicillin-Resistant Staphylococcus aureus Strain USA600 (ST45) Are Associated with Increased Mortality in a Hospital Setting. Journal of Clinical Microbiology 2014;52(6):2172-4. Schito GC. The importance of the development of antibiotic resistance in Staphylococcus aureus. Clinical Microbiology and Infection 2006;12, Supplement 1:3-8. Segal AW. How Neutrophils Kill Microbes. Annual review of immunology 2005;23:197-223. Siebert WT, Moreland N, Williams TW. Synergy of Vancomycin plus Cefazolin or Cephalothin against Methicillin-Resistant Staphylococcus epidermidis. Journal of Infectious Diseases 1979;139(4):452-7. Sieradzki K, Leski T, Dick J, Borio L, Tomasz A. Evolution of a vancomycin-intermediate Staphylococcus aureus strain in vivo: multiple changes in the antibiotic resistance phenotypes of a single lineage of methicillin-resistant S. aureus under the impact of antibiotics administered for chemotherapy. J Clin Microbiol 2003;41(4):1687-93. Smith JR, Barber KE, Raut A, Rybak MJ. beta-Lactams enhance daptomycin activity against vancomycin-resistant Enterococcus faecalis and Enterococcus faecium in in vitro

ACCEPTED MANUSCRIPT

AC

CE

PT

ED

M

AN

US

CR

IP

T

pharmacokinetic/pharmacodynamic models. Antimicrob Agents Chemother 2015;59(5):2842-8. Stapleton PD, Taylor PW. Methicillin resistance in Staphylococcus aureus: mechanisms and modulation. Science progress 2002;85(Pt 1):57-72. Sy CL, Huang T-S, Chen CS, Chen Y-S, Tsai H-C, Wann S-R, et al. Synergy of β-Lactams with Vancomycin against Methicillin-Resistant Staphylococcus aureus: Correlation of Disk Diffusion and Checkerboard Methods. Journal of Clinical Microbiology 2016;54(3):565-8. Sy CL, Huang TS, Chen CS, Chen YS, Tsai HC, Wann SR, et al. Synergy of beta-Lactams with Vancomycin against Methicillin-Resistant Staphylococcus aureus: Correlation of Disk Diffusion and Checkerboard Methods. J Clin Microbiol 2016;54(3):565-8. Tsuji BT, Rybak MJ, Lau KL, Sakoulas G. Evaluation of accessory gene regulator (agr) group and function in the proclivity towards vancomycin intermediate resistance in Staphylococcus aureus. Antimicrobial agents and chemotherapy 2007;51(3):1089-91. Vidaillac C, Gardete S, Tewhey R, Sakoulas G, Kaatz GW, Rose WE, et al. Alternative mutational pathways to intermediate resistance to vancomycin in methicillin-resistant Staphylococcus aureus. J Infect Dis 2013;208(1):67-74. Vidaillac C, Gardete S, Tewhey R, Sakoulas G, Kaatz GW, Rose WE, et al. Alternative Mutational Pathways to Intermediate Resistance to Vancomycin in Methicillin-Resistant Staphylococcus aureus. The Journal of Infectious Diseases 2013;208(1):67-74. Vidaillac C SG, Tewey, R, Karoma, T, Bernardin, F, Muller, A., Kaatz, GW, Rose, WE, Constanceias, F, Vallar L, Rybak MJ. Additional insights towards a better understanding of low-level of vancomycin resistance in methicillin resistant Staphylococcus aureus. ASM Microbe 2016, Boston, MA June 18 Abstract #416. Boston, MA 2016. Wilson FP, Berns JS. Vancomycin levels are frequently subtherapeutic during continuous venovenous hemodialysis (CVVHD). Clin Nephrol 2012;77(4):329-31. Zhang S, Sun X, Chang W, Dai Y, Ma X. Systematic Review and Meta-Analysis of the Epidemiology of Vancomycin-Intermediate and Heterogeneous Vancomycin-Intermediate Staphylococcus aureus Isolates. PLoS One 2015;10(8):e0136082.