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1

Sequential Evolution of Vancomycin-Intermediate Resistance Alters Virulence in 2

Staphylococcus aureus: PK/PD Targets for Vancomycin Exposure 3

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Authors: Justin R. Lenhard1,2, Tanya Brown1,2, Michael J. Rybak3, Calvin J. Meaney1,2, 5

Nichols B. Norgard1,2,, Zackery P. Bulman1,2, Daniel Brazeau1,2, Steven R. Gill4, Brian T. 6

Tsuji1,2* 7

8

Affiliations: Laboratory for Antimicrobial Pharmacodynamics, School of Pharmacy and 9

Pharmaceutical Sciences1 and the New York State Center of Excellence in Life 10

Sciences and Bioinformatics, Buffalo, NY2, Anti-Infective Research Laboratory, Eugene 11

Applebaum College of Pharmacy and Health Sciences, Detroit, MI, USA3, Department 12

of Microbiology, University of Rochester Medical Center, Rochester, NY, 142264 13

14

*Corresponding Author: Brian T. Tsuji, Pharm.D., University at Buffalo, School of 15

Pharmacy and Pharmaceutical Sciences. Phone: (716) 881-7543 Fax: (716) 849-6890 16

Email: btsuji@buffalo.edu 17

18

Running Title: Evolution of Vancomycin Resistance and Virulence 19

Keywords: Evolutionary Dynamics, Staphyolococcus aureus, vancomycin resistance, 20

pharmacodynamics, G. mellonella, accessory gene regulator 21

22

Abstract word count: 243 23

Manuscript word count: 3,345 24

AAC Accepted Manuscript Posted Online 28 December 2015Antimicrob. Agents Chemother. doi:10.1128/AAC.02657-15Copyright © 2015, American Society for Microbiology. All Rights Reserved.

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Abstract: 25

Background: Staphylococcus aureus possesses exceptional virulence and a 26

remarkable ability to adapt in the face of antibiotic therapy. We examined the in vitro 27

evolution of S. aureus in response to escalating vancomycin exposure by evaluating 28

bacterial killing and the progression of resistance. 29

Methods: A hollow fiber infection model was utilized to simulate human doses of 30

vancomycin increasing from 0.5g to 4g q12h vs. a high inoculum (108 CFU/ml) of MRSA 31

USA300 and USA400. Host pathogen interactions using Galleria mellonella and 32

accessory gene regulator (agr) expression were studied in serially obtained isolates. 33

Results: In both USA300 and USA400 MRSA isolates, vancomycin exposure up to 2g 34

q12h resulted in persistence and regrowth, whereas 4g q12h achieved sustained killing 35

against both strains. As vancomycin exposure increased from 0.5g to 2g q12h, the 36

bacterial population shifted toward vancomycin-intermediate resistance, and collateral 37

increases in the MICS of daptomycin and televancin were observed over 10 days. 38

Guideline recommended exposure of an fAUC/MIC of 200 displayed a 0.344 log 39

bacterial reduction in area, whereas fAUC/MICs of 371 and 554 were needed to achieve 40

a 1.00 and 2.00 log reduction in area, respectively. The step wise increase in 41

resistance paralleled a decrease in G. mellonella mortality (p=0.021) and a gradual 42

decline of RNAIII expression over 10 days. 43

Conclusions: Currently recommended doses of vancomycin resulted in amplification 44

of resistance and collateral damage to other antibiotics. Decreases in agr expression 45

and virulence during therapy may be an adaptive mechanism of S. aureus persistence. 46

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Introduction 47

Staphylococcus aureus is a primary human pathogen capable of exceptional virulence 48

and an array of life threatening infections ranging from necrotizing pneumonia to 49

endocarditis(1). S. aureus also has a remarkable ability to adapt in the face of 50

antimicrobial therapy via a plethora of resistance mechanisms(2-6). Although there is a 51

large body of information on the mechanisms of antibiotic resistance in S. aureus, the 52

interplay between virulence and antibiotic resistance is not completely understood. The 53

results of genome wide sequencing analyses have been confounded by the discovery 54

that multiple genetic pathways may lead to similar levels of antibiotic resistance(7-9) 55

Also, while the sequential development of resistance mutations during the course of an 56

infection has been analyzed, the temporal link between resistance and virulence is 57

poorly defined(10). 58

59

Despite the successful use of vancomycin to combat community-associated (CA) MRSA 60

for several decades, the spread of vancomycin intermediate-resistance (VISA) has 61

brought the utility of vancomycin into question(4, 11). The genetic basis for VISA is 62

unknown, although reduced vancomycin susceptibility has been associated with 63

dysfunction of the accessory gene regulator (agr), the master regulator of pathogenicity 64

in S. aureus(12-14). Isolates of S. aureus that are defective in agr have significantly 65

reduced virulence profiles and low level vancomycin resistance conferred through a 66

number of mechanisms including biofilm formation, stationary phase growth, and 67

alterations in autolysis(15-17). At present, it is not known how modulating vancomycin 68

dosing regimens will alter the timescale of agr expression or S. aureus virulence. Here, 69

we attempted to resolve the link between vancomycin resistance and virulence by 70

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simulating human dosing in an in vitro hollow fiber infection model (HFIM) to study the 71

step wise evolution of vancomycin resistance, and also determine the impact that these 72

mutations have on virulence. 73

74

MATERIALS AND METHODS 75

Bacterial isolates 76

To represent the predominant clones of MRSA in the United States, Europe, and 77

Canada, we selected the two pulsed field gel electrophoresis–type community acquired 78

strains USA300 (FPR 3757) and USA400 (MW2), of which the complete genome 79

sequences have been established(18-20). Both isolates were obtained from the 80

network of antimicrobial resistance in Staphylococcus (NARSA). 81

82

Antibiotics and medium 83

Vancomycin, ciprofloxacin, levofloxacin, gentamicin, nafcillin, and rifampicin analytical 84

grade powders were commercially purchased (Sigma Chemical Company, St. Louis, 85

Missouri). Daptomycin was obtained from Cubist (Lexington, MA), telavancin was 86

obtained commercially from the University at Buffalo Pharmacy, and linezolid was 87

obtained from Pfizer (Groton, CT). Brain Heart Infusion (BHI) broth and BHI agar 88

(Difco Laboratories, Detroit, Michigan) were used for all in vitro hollow experiments. 89

90

Determining alterations in MIC 91

Antimicrobial agents commonly used to treat MRSA were tested with CLSI broth 92

microdilution methods to evaluate the potential collateral damage of vancomycin on 93

other antibiotics. Isolates were initially evaluated for MICs at baseline prior to 94

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vancomycin exposure. Selective post-exposure mutants obtained from the HFIM were 95

also evaluated for MICs. Mueller–Hinton broth adjusted to contain physiological levels 96

of calcium (50 mg/L) was used as test medium. CLSI interpretive criteria were used to 97

categorize the isolates as susceptible, intermediate, or resistant. S. aureus ATCC 98

29213 was utilized as quality control (QC) organisms. All QC results were within 99

published limits. 100

101

Hollow Fiber Infection Model. As previously described, a HFIM was used to evaluate 102

how clinically relevant vancomycin regimens alter the bacterial burden of MRSA and 103

influence the amplification of antibiotic resistance over 240h(21). The HFIM employed a 104

cellulosic cartridge C3008 (FiberCell Systems, Frederick, MD, USA). Bacteria were 105

trapped in the extracapillary space of the cartridge and the large surface area from the 106

hollow fibers was used to facilitate nutrient exchange and antibiotic exposure. Each 107

vancomycin dosing regimen was administered in a manner to achieve the same AUC 108

over 10 days as would be expected in humans. The clinical scenario of a high bacterial 109

burden infection such as a bi-lobar pneumonia or endocarditis was simulated using a 110

108CFU/ml inoculum achieved from overnight MRSA cultures. Samples were taken from 111

the 10 day HFIM experiment at 0, 24, 48, 72, 96, 120, 144, 168, 192, 216, and 240h 112

and subsequently incubated and quantified to assess bacterial growth. 113

114

To simulate human antibiotic dosing, four different vancomycin regimens were 115

administered in the HFIM assuming a free (ƒ) fraction of 50% and a 6 hour half-life. The 116

following dosing schemes were chosen to target the pharmacokinetic parameters of 117

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maximal concentration (Cmax), minimum concentration (Cmin), and an area under the 118

curve to MIC ratio (AUC:MIC) expected in human plasma. 119

500mg q12h (ƒCmax of 10 mg/L, ƒCmin of 2.5mg/L, and ƒAUC:MIC of 112.5) 120

1000mg q12h (ƒCmax of 20 mg/L, ƒCmin of 5mg/L, and ƒAUC:MIC of 225) 121

2000mg q12h (ƒCmax of 40 mg/L, ƒCmin of 10mg/L, and ƒAUC:MIC of 450) 122

4000mg q12h (ƒCmax of 80 mg/L, ƒCmin of 20mg/L, and ƒAUC:MIC of 900) 123

124

Samples from the HFIM were also obtained serially for confirmation of vancomycin 125

pharmacokinetics using a standard agar diffusion bioassay procedure. MHA with 126

Micrococcus luteus ATCC 9341 as an indicator organism was utilized to validate 127

vancomycin concentrations as previously described. All observed pharmacokinetic 128

parameters were within 12% of targeted values. 129

130

Population Analysis Profiles 131

Quantitative cultures from the Hollow Fiber Infection Model were determined ‘real time’ 132

in mini-population analysis profiles (PAPs) that utilized BHI agar containing 0, 2, 4, and 133

6 mg/L of vancomycin, which enabled the daily detection of the total bacterial population 134

as well as vancomycin resistant subpopulations in all 4 dosing regimens. Full PAPs 135

were also completed using 0, 0.5, 1, 2, 3, 4, 6, 8, and 16 mg/L of vancomycin to quantify 136

daily evolution of the resistant subpopulations in the 2g q12h regimen, as well as the 137

240h time point of all 4 dosing regimens. PAPs cultures were incubated for 48h, and 138

the colonies were then counted and plotted against either vancomycin concentration or 139

time. 140

141

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RNA Extraction and Quantitative Real Time PCR 142

Cells were collected at 240h from the hollow fiber infection model for MRSA USA300 143

before and after exposure to the four vancomycin regimens. RNAIII expression was 144

then assessed with real-time PCR as described previously.(22) Briefly, the collected 145

samples were centrifuged at 14,000rpm for 5 minutes at room temperature. The 146

supernatant was aspirated and the pellet was immediately frozen at -80ºC until RNA 147

isolation. Total RNA was isolated from the pellet (SV Total RNA Isolation System, 148

Promega, Madison WI) following the manufacturer’s protocol for gram positive bacteria. 149

From the total RNA pool, messenger RNA (mRNA) was purified (MICROBExpress, 150

Ambion, Austin TX). Reverse transcription of the mRNA (315ng) was carried out using 151

random hexamer primers and the AccuScript High Fidelity RT-PCR System (Stratagene, 152

La Jolla CA). 153

154

G. mellonella Virulence assays 155

G. mellonella was utilized to investigate the pathogenicity of resistant mutants which 156

evolved in the HFIM as detailed previously(23, 24). In the 10 day HFIM utilizing a 157

vancomycin dose of 2g q12h, USA300 mutants were collected daily and stored at -80°C 158

prior to the virulence assessment. Twenty randomly chosen caterpillars 200–300 mg in 159

weight in the final instar larval stage (Vanderhorst, Inc., St. Mary’s, OH) were used in 160

each group. A 10-µL Hamilton syringe was used to inject 10-µL aliquots of the inoculum 161

into the hemocoel of each caterpillar via the last left proleg. Bacterial colony counts 162

were used to confirm all inocula, and appropriate control arms with caterpillars receiving 163

no injection or an injection of phosphate buffered saline were included. 164

165

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RESULTS 166

MRSA USA 300 and USA 400 HFIMs 167

To study the temporal profile of MRSA USA 300’s response to increasing antibiotic 168

exposure, a HFIM was utilized to simulate human vancomcyin dosing over a 10 day 169

period (Figure 1). Despite high doses of up to 2g q12h, mimicking exposure profiles in 170

administered humans, USA300 demonstrated persistence and tolerance, maintaining 171

bacterial counts >1010 CFU/ml with growth similar to control by the 240h endpoint. The 172

lower dose vancomycin regimen of 500mg q12h was nearly identical to control 173

throughout the 240h. The 1g q12h scheme, which is the regimen administered to the 174

majority of patients with S. aureus bloodstream infections, demonstrated initial stasis at 175

~5x107 CFU/ml for approximately 24h, followed by gradual regrowth that plateaued at 176

~3.5x1010 CFU/ml by 120h. Similarly, the 2g q12h regimen demonstrated step wise 177

increases in bacterial counts beginning with an initial stasis phase from 0 to 48h at ~108 178

CFU/ml, a regrowth phase from 48h to 96h at ~109 CFU/ml, followed by a final regrowth 179

phase at 144h to ~1010 CFU/ml which continued to 240h. Unlike the lower dose 180

regimens, the 4g q12h scheme achieved a >3 log reduction in bacterial counts by 72h, 181

and after 240h of continuous killing, resulted in a final population of ~3x102 CFU/ml. 182

183

An additional HFIM analysis was conducted on USA 400 to confirm the activity of 184

vancomycin is comparable amongst common CA-MRSA strains (Figure 2). Similar to 185

USA 300, vancomycin doses up to 2g q12h were unable to prevent USA 400 from 186

regrowing by 48h. While the 500mg and 1g q12h regimens mirrored the growth control 187

by 96h, the 2g q12h scheme maintained counts below 1010 CFU/ml for the duration of 188

the experiment. The 4g q12h regimen was once again the only simulated vancomycin 189

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therapy capable of achieving bactericidal activity, with a >3 log reduction conferred by 190

120h and a final population count of ~104 CFU/ml at 240h. Co-modelling the reduction 191

in the Log Ratio Area of both USA 300 and USA 400 as a function of vancomycin 192

exposure confirmed that vancomycin’s performance was similar between the two strains 193

(Figure 3). In both USA 300 and USA 400, fAUC24/MICs of approximately 200, 371, 554, 194

and 757 achieved a reduction in the total population’s Log Ratio Area of 0.344, 1.00, 195

2.00, and 3.00 respectively (R2 = 0.990), as described by a Hill-type function utilized 196

previously (25). 197

198

Sequential Emergence of Antibiotic Resistance 199

Throughout the 10 day HFIM experiments, subpopulations capable of growing on 2, 4, 200

and 6mg/L of vancomycin were tracked for both USA 300 and USA 400 (Figure 1 and 201

Figure 2). In both investigational strains, lower vancomycin doses of 500mg and 1g 202

q12h did not substantially amplify vancomycin resistance, with < 1% of the population 203

ever growing on 2 mg/L of vancomycin. However, when the dose of vancomycin was 204

increased to 2g q12h, ~10% of USA 300’s population and ~50% of USA 400’s total 205

population were capable of growing on 2 mg/L of vancomycin by 240h. Increasing the 206

vancomycin dose even further to 4g q12h resulted in drastic killing of the total bacterial 207

populations, with resistant subpopulations growing on 2 mg/L of vancomycin comprising 208

< 1% of the population for the duration of the experiments. 209

210

A more comprehensive PAP scheme utilizing vancomycin concentrations up to 16 mg/L 211

was also performed for the entire 2g q12h regimen and the 240h terminal time points of 212

all the USA 300 HFIMs (Figure 4). After several days of vancomycin exposure, 213

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subpopulations of USA 300 began to grow on 6 mg/L of vancomycin beginning at 120h. 214

Resistance to vancomycin intensified as the 2g q12h regimen continued, eventually 215

resulting in isolates capable of growing on 8 mg/L of vancomycin by 240h. Comparing 216

the dose response among different regimens, doses of 500mg up to 2g q12h produced 217

similar resistance profiles by 240h, characterized by bacterial growth on 8 mg/L of 218

vancomcyin. In contrast, the 4g q12h regimen prevented the growth of any colonies in 219

the presence of ≥ 4mg/L of vancomycin at 240h. 220

221

In order to track the emergence of resistance to other agents during vancomycin 222

therapy, the MICs of common antibiotics were determined for USA 300 isolates 223

collected every 24h during the 2g q12h HFIM experiment and are presented in Table 1. 224

Secondary to vancomycin exposure, significant alterations in the susceptibility to the 225

lipopeptide daptomycin and lipoglycopeptide telavancin were observed. For daptomycin, 226

sequential increases were noted from 0.125 to 0.25 on Day 4, to 0.5 on day 7, and 227

1.0mg/L on day 10. For telavancin, sequential increases from 1.0 to 2.0 on day 2, and 228

to 4.0mg/L on day 5 occurred. There was also a consistent trend of decreases in 229

rifampicin susceptibility from 0.015 to 0.03mg/L, albeit only a one fold difference 230

231

G. Mellonella Survival 232

To determine what impact vancomycin resistance has on S. aureus virulence, G. 233

mellonella were inoculated with USA 300 isolates collected daily throughout the 2g q12h 234

regimen (Figure 5 Panel A), and also isolates obtained at the 240h terminal time point of 235

each USA 300 HFIM experiment (Figure 5 Panel B). In the 2g q12h regimen, isolates 236

collected after ≥ 96h of vancomycin exposure displayed attenuated virulence that 237

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paralleled the agr dysfunctional control, with significantly higher G. mellonella survival 238

rates after 6 days compared to the baseline isolate (G. mellonella survival ≥ 80% vs. 239

45% on day 6, p=0.021, Log-Rank Test). Conversely, isolates obtained prior to 96h of 240

vancomycin exposure demonstrated killing that mirrored the profile of the baseline 241

isolate (G. mellonella survival ≤ 60% on day 6). When all 4 vancomycin regimens were 242

compared to one another, doses of 500mg to 2g q12h resulted in significantly better G. 243

mellonella survival by day 6 relative to the baseline isolate (G. mellonella survival ≥ 80% 244

vs. 45% on day 6, p=0.029), whereas the 4g q12h regimen resulted in an isolate with 245

comparable virulence to the baseline isolate (p=0.231). 246

247

RNA III Expression and Co-modelling with Total Counts, Virulence, and 248

Resistance Plots 249

Real time PCR of RNA III was performed on all USA 300 isolates recovered from 0 to 250

240h during the 2g q12h regimen to delineate the temporal pattern of agr expression 251

during antibiotic therapy and its relationship to resistance and virulence in S. aureus 252

(Figure 6, Panel A). The quantity of RNA III initially surged to >10x baseline between 0 253

and 24h, then following a steep decline to almost undetectable amounts of RNAIII at 254

96h, after which agr expression hovered around baseline until 240h. When agr 255

expression was co-modelled with bacterial counts and G. mellonella mortality, a trend 256

was observed in which the initial surge in agr expression coincided with higher levels of 257

G. mellonella mortality (~50%), whereas the low levels of agr expression beginning at 258

96h corresponded to attenuated mortality rates of ≤ 20% (Figure 6, Panel B). The 259

relationship between bacterial counts and agr expression was not as clearly defined in 260

the 2g of vancomycin q12h regimen. However, when agr expression and bacterial 261

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counts were plotted as a function of vancomycin exposure, an inverse relationship was 262

observed in which a fAUC24/MIC of 900 mg*h/L resulted in a bacterial count of ~3x102 263

CFU/ml and agr expression >10x baseline at 240h (Figure 6, Panel C). 264

265

DISCUSSION 266

Although vancomycin was the drug of choice for MRSA infections for several decades, 267

increased treatment failure rates coinciding with the spread of VISA have casted doubt 268

on the reliable use of vancomycin(26-30). As S. aureus MICs continue to creep for 269

vancomycin, understanding how drug exposure influences both resistance and 270

virulence is critical to the judicious use of anti-MRSA agents. Here, we investigated 271

clinically relevant vancomycin regimens in a 10 day HFIM to translate how vancomycin 272

dosing alters MRSA population dynamics and glycopeptide resistance. In both of the 273

investigational CA-MRSA strains, administration of 500mg or 1g of vancomycin q12h 274

was unable to achieve bacteriostatic activity. When the dose of vancomycin was 275

increased to 2g q12h, a regimen yielding twice the drug exposure of the most common 276

vancomycin scheme, bacterial counts still rose steadily in the face of intensified 277

antibiotic dosing. Only the 4g q12h regimen achieved bactericidal activity, with 278

sustained killing over 10 days for both investigational strains. 279

280

Unlike the AUC/MIC target of 400 (fAUC/MIC = 200) advocated by current vancomycin 281

guidelines, higher vanomycin exposures were needed to kill the CA-MRSA strains 282

investigated in the present study.(31) As current guidelines recommend vancomycin 283

use for MRSA infections in which the causative organism’s vancomycin MIC is ≤ 1mg/L, 284

the isolates investigated in the current study are an accurate representation of MRSA 285

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strains commonly treated with vancomycin (MIC = 1mg/L for USA 300 and USA 400). 286

However, the 2g q12h vancomycin regimen produced a fAUC/MIC > 2x the suggested 287

AUC/MIC of 400 (fAUC/MIC of 200) and negligible killing was achieved in both 288

investigational strains. An alarmingly high fAUC/MIC of 554 was necessary to achieve a 289

2 log bacterial reduction in area. Vancomycin regimens commonly used in the clinic 290

may therefore be unable to fully overcome S. aureus resistance mechanisms at high 291

inocula, and alternative regimens may need to be considered even when an organism’s 292

vancomycin MIC is deceptively below 2mg/L. 293

294

Not only were the 500mg to 2g of vancomycin q12h regimens incapable of killing S. 295

aureus, but the suboptimal vancomycin exposure also amplified antibiotic resistance. 296

Similar to other investigations concerning antibiotic resistance, an inverted “U” 297

phenomenon was observed in which rising vancomycin concentrations resulted in 298

higher levels of antibiotic resistance until extreme concentrations were capable of killing 299

the entire population.(32) Vancomycin exposure was also found to augment resistance 300

to other antimicrobials, including daptomycin, telavancin, and rifampin. Taken together, 301

these results emphasize the importance of utilizing optimal vancomycin dosing practices 302

against MRSA that is capable of being killed by vancomycin. If a high enough 303

vancomycin exposure cannot be achieved to confer bactericidal activity, resistance to 304

glycopeptides and other antibiotic classes will increase in a manner that is proportionate 305

to the amount of vancomycin exposure. 306

307

While the amplification of antibiotic resistance in concerning, agr expression and G. 308

mellonella mortality demonstrated that the ability of S. aureus to resist glycopeptide 309

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treatment appears to come at a cost to its virulence. Lower vancomycin dosing 310

schemes of 500mg to 2g q12h resulted in reduced agr activity and a higher survival rate 311

of the G. mellonella, whereas the bactericidal 4g q12h regimen suppressed vancomycin 312

resistance and correspondingly did not significantly alter the survival rate of G. 313

mellonella. Although the emergence of vancomycin resistance appears to be a 314

heterogenous process, two observations commonly made upon rising vancomycin MICs 315

are thicker cell walls and dysfunctional agr profiles.(4, 16, 33, 34) It has been proposed 316

that alterations in the cell wall of S. aureus may interfere with the cell’s ability to bind the 317

auto-inducing peptide of the agr system used for quorum sensing and activation of S. 318

aureus’ toxins and other virulence factors.(35) It is likely that suboptimal vancomycin 319

dosing in the clinic will drive MRSA from a more antibiotic susceptible and virulent state 320

towards a more resistant but less virulent population. 321

322

Another consequence of vancomycin exposure is the potential conversion of S. aureus 323

from a virulent phenotype into a persistent state better adapted to survive antimicrobial 324

therapy and the host immune response. In the present study, the survival of G. 325

mellonella improved as the expression of agr decreased, suggesting that the reduction 326

in virulence factor release reduced the pathogenicity of S. aureus. A prior investigation 327

that compared S. aureus isolates collected from a patient before and after 6 weeks of 328

vancomycin treatment found that vancomycin exposure resulted in downregulated agr, 329

slower autolysis, increased cell wall thickness, and a reduction in the secretion of α-330

toxin and phenol soluble modulins(36). It has been established that α-toxin release 331

activates the NLRP3-inflammasome and induces interleukin-1 and interleukin-6 332

secretion, while the VISA phenotype has been shown to provoke less tumor necrosis 333

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factor-α and interleukin release than its vancomycin-susceptible counterparts(9, 37, 38). 334

An investigation of the selective pressure of vancomycin on MRSA also found that 335

vancomycin exposure amplifies the relative abundance of the small colony variant 336

phenotype, which is a slow growing phenotype implicated in chronic and recurrent S. 337

aureus infections as well as intracellular persistence(25, 39). The consequence of 338

suboptimal vancomycin exposure is therefore not restricted to proliferating antibiotic 339

resistance, but also shifting the population dynamics of S. aureus toward a persistent 340

state that is capable of enduring host countermeasures and exogenous antimicrobials. 341

342

The current study has several meaningful limitations to consider before the results can 343

be fully translated into the clinical setting. Similar to other in vitro investigations, not only 344

does the HFIM fail to account for host defenses against S. aureus, but the use of 345

Mueller-Hinton broth provides a nutrient-rich environment that may improve bacterial 346

survival relative to in vivo experiments. Most importantly, the poor performance of 347

standard vancomycin regimens is likely ascribed to the high inoculum investigated in the 348

present study. A previous in vitro investigation not only demonstrated a large inoculum 349

effect for vancomycin against heteroresistant-VISA, but also found that simulated doses 350

of vancomycin up to 5g q12h were unable to achieve sustained killing(40). A murine 351

thigh infection model was later used to characterize the magnitude of inocula effects for 352

MRSA and heteroresistant-VISA exposed to several antimicrobials(41). The authors 353

concluded that the inoculum effect was much more severe for vancomycin in 354

comparison to other anti-staphylococcal agents such as daptomycin and linezolid. It is 355

therefore prudent to view the results of the current investigation as representative of a 356

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worst-case clinical scenario, as vancomycin may achieve much better activity in more 357

favorable conditions. 358

359

In closing, clinically relevant vancomycin regimens simulated in a HFIM were largely 360

ineffective against two common CA-MRSA strains, with increasing vancomycin 361

exposure conferring more substantial antibiotic resistance and severe losses in 362

virulence. The only investigational regimen capable of killing USA 300 and USA 400 363

was the 4g q12h regimen, which is a dosing scheme neglected clinically due to 364

vancomycin’s propensity for dose related nephrotoxicity.(42) In the model in vitro 365

system, simply obtaining the suggested AUC/MIC of 400 was not enough to overcome 366

the resistance mechanims of S. aureus. Owing to vancomycin’s inability to achieve 367

bactericidal activity at clinical concentrations, clinicians are cautioned about selecting 368

vancomycin for MRSA infections containing S. aureus strains with questionable 369

vancomycin susceptibility. Aggressive stewardship and the use of alternative 370

antimicrobials may be necessary to stem S. aureus’ progressive MIC creep to 371

vancomycin, as seemingly optimal vancomycin regimens may actually exacerbate 372

vancomycin resistance. It will likely take many years to fully define where the niche of 373

vancomycin now lies in the context of new anti-MRSA agents that offer clinicians a more 374

diverse armamentarium to select alternative agents from. 375

376

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Acknowledgements 377

We thank Dr. Alan Forrest for insight into PK/PD and statistical analyses. B.T.s 378

participation in this study was supported by National Institute of Allergy and Infectious 379

Diseases of the National Institutes award number R01AI111990. The content is solely 380

the responsibility of the authors and does not necessarily represent the official views of 381

the National Institutes of Health. 382

383

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Table 1. Bacterial Isolates of MRSA USA300 collected during the 10d HFIM 384

investigating a 2g q12h vancomycin regimen. Minimum inhibitory concentrations (mg/L) 385

to vancomycin and other antibiotic of interest are listed for each isolate. 386

Time of sample secondary

Strain Vanco 2g q12h exposure VAN LZD DAP RIF LEV CIP GEN NAF

LAD201

Baseline 0h 1 2 0.5 0.015 8 32 1 32

LAD202 24h 1 2 0.5 0.015 8 32 2 32

LAD203 48h 1 2 0.5 0.015 8 32 2 32

LAD204 72h 2 2 0.5 0.015 16 32 2 32

LAD205 96h 2 2 0.25 0.015 8 32 2 32

LAD206 120h 4 2 0.5 0.03 8 32 2 32

LAD207 144h 4 2 1 0.03 16 32 0.5 32

LAD208 168h 4 2 1 0.03 16 16 2 32

LAD209 192h 4 2 2 0.03 8 16 2 16

LAD210 216h 4 2 2 0.03 8 16 1 32

LAD211 240h 4 2 2 0.03 8 16 2 32

387 NOTE: VAN, vancomycin; LZD, linezolid; DAP, daptomycin; RIF, rifampicin, LEV, 388

levofloxacin; CIP, ciprofloxacin; GEN, gentamicin; NAF, nafcillin 389

390

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Figure 1. Humanized dosing regimens of vancomycin against MRSA USA300 391

quantifying the total population (Blue), and the sequential emergence of resistance 392

secondary to drug exposure (Red, Gray, and Pink) over a 10 day period. 393

0 24 48 72 96 120 144 168 192 216 2400

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ControlTotal Population2 x MIC Mutants4 x MIC Mutants6 x MIC Mutants

A. Vanco 500mg every 12h B. Vanco 1000mg every 12h

C. Vanco 2000mg every 12h D. Vanco 4000mg every 12h

Time (Hour)

Log 1

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Figure 2. Humanized dosing regimens of vancomycin against MRSA USA400 396

quantifying the total population (Blue), and the sequential emergence of resistance 397

secondary to drug exposure (Red, Gray and Pink) over a 10 day period. 398

Time (Hour)

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A. Vanco 500mg every 12h B. Vanco 1000mg every 12h

C. Vanco 2000mg every 12h D. Vanco 4000mg every 12h

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Figure 3. The bacterial reductions (represented as Log Ratio Areas) of MRSA USA300 408

and USA400 are plotted as a function of vancomycin exposure. The fAUC/MIC needed 409

to confer a reduction in the Log Ratio Area of 0.344, 1, 2, and 3 (black, blue, green, and 410

purple, respectively) are listed above the corresponding vancomycin exposures. The 411

data were described by a Hill-type function as described previously.(25) 412

0 100 200 300 400 500 600 700 800 900 1000-5

-4

-3

-2

-1

0

R2=0.990

fAUC/MIC

log reduction

200 371 554 757

-0.344 -1.00 -2.00 -3.00

Vancomycin Exposure (fAUC/MIC)

Bacterial Strain:● MRSA USA300○ MRSA USA400

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Figure 4. A.) Step wise evolution of resistance as shown in population analysis profiles 422

of vancomycin quantified every 24h over a 10 day period in response to simulated 423

human exposure of vancomycin 2g q12h. B) Comparative population analysis profiles 424

among different vancomycin dosing regimens of 500mg, 1000mg, 2000mg and 4000mg 425

q12h at the 240h study endpoint. 426

427

Vancomycin Concentration (mg/L)

0 2 4 6 8 10 12 14 16

Log 10

CFU

/ml

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10 LAD188 (240h 500mg every 12h)LAD200 (240h 1000mg every 12h)LAD211 (240h 2000mg every 12h)LAD349 (240h 4000mg every 12h)

Vancomycin Concentration (mg/L)

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10LAD201 (0h)LAD202 (24h)LAD203 (48h)LAD204 (72h)LAD205 (96h)LAD206 (120h)LAD207 (144h)LAD208 (168h)LAD209 (192h)LAD210 (216h)LAD211 (240h)

A. Evolution of Resistance B. PAPs and Drug Exposure

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Figure 5. A.) G. mellonella virulence assay in response to simulated human 428

vancomycin exposure. Worms were inoculated with MRSA USA 300 isolates collected 429

throughout the 2g q12h vanomycin regimen in the HFIM, and the subsequent survival of 430

the worms was recorded. G. mellonella inoculated with isolates collected after ≥ 96h of 431

vancomycin treatment displayed significantly better survival in comparison to worms 432

inoculated with the baseline isolate (p=0.021, Log-Rank Test). B.) The assay was 433

repeated for the terminal MRSA USA 300 isolates collected at the end of the 10d HFIM 434

experiments for each vancomycin regimen. Vancomycin doses of ≤ 2g q12h resulted in 435

significantly better survival of the G. mellonella relative to the baseline isolate (p=0.029), 436

whereas the isolate collected after 240h exposure to the 4g q12h regimen was 437

comparably virulent to the baseline isolate (p=0.231). 438

439

LAD200 (240h of vanco 1g q12h)

LAD201 (Baseline) LAD202 (24h of vanco 2g q12h)LAD203 ((48h of vanco 2g q12h)LAD204 (72h of vanco 2g q12g)LAD205 (96h of vanco 2g q12h)LAD 206 (120h of vanco 2g 12h)LAD 207 (144h of vanco 2g q12h)LAD208 (168h of vanco 2g q12h)LAD209 (192h of vanco 2g q12h)LAD210 (216h of vanco 2g q12h)LAD211 (240h of vanco 2g q12h)RN6607 (agr+ S. aureus control) RN9120 (agr- S. aureus control)

Time (Day)

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Legend:LAD205 (96h of vanco 2g q12h)LAD206 (120h of vanco 2g 12h)LAD207 (144h of vanco 2g q12h)LAD208 (168h of vanco 2g q12h)LAD209 (192h of vanco 2g q12h)LAD210 (216h of vanco 2g q12h)LAD211 (240h of vanco 2g q12h)

RN9120 (agr- S. aureus control)

Time (Day)

0 1 2 3 4 5 6

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ival

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LAD201 (Baseline)

LAD188 (240h of vanco 0.5 q12h)LAD211 (240h of vanco 2g q12g)

LAD349 (240h of vanco 4g q12h)

A. B.

RN6607 (agr+ S. aureus control)

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Figure 6. A.) RNAIII profiling to measure activity of agr, the primary quorum sensing 440

response regulator of virulence in S. aureus, quantified every 24h in response to USA 441

MRSA 300 exposed to vancomycin 2g q12h for 10 days. B.) Co-modelling of the agr 442

expression in panel A (pink) with MRSA USA 300’s total population (black), 443

vancomycin-resistant subpopulations growing on 4.0 mg/L of vancomycin (red), and G. 444

mellonella mortality (blue). C.) The same analysis is repeated using the 240h terminal 445

time point for vancomycin doses of 500 mg to 4g q12h. 446

Vancomycin Exposure (fAUC24/MIC)

0 100 200 300 400 500 600 700 800 900 1000Bact

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Total PopulationResistant Sub-populationRelative agr ExpressionG. mellonella Mortality

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