1

Rapid detection and simultaneous antibiotic susceptibility analysis of Yersinia 1

pestis directly from clinical specimens using reporter phage 2

3

Vandamm, J. P1., Rajanna, C2., Sharp, N. J1., Molineux, I. J3., and Schofield, D. A1# 4

1Guild BioSciences, Charleston, South Carolina, 29407 5

2Department of Molecular Genetics and Microbiology, University of Florida, Gainesville 6

32610 7

3Molecular Biosciences, Institute for Cellular and Molecular Biology, University of Texas 8

at Austin, Texas, 78712 9

#Corresponding author. Mailing address: Guild BioSciences, 1313B Ashley River Road, 10

Charleston, SC29407. Phone: (843) 573 0095. Fax: (843) 573 0707. 11

E-mail: dschofield@guildbiosciences.com 12

13

Running title: Phage-mediated detection of Y. pestis 14

Keywords: Plague, diagnosis, reporter phage, bioluminescence, septicemia, blood 15

culture 16

17

JCM Accepts, published online ahead of print on 11 June 2014J. Clin. Microbiol. doi:10.1128/JCM.00316-14Copyright © 2014, American Society for Microbiology. All Rights Reserved.

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Yersinia pestis is a Tier 1 agent due to its contagious pneumopathogenicity, extremely 18

rapid progression and high mortality rate. As the disease is usually fatal without 19

appropriate therapy, rapid detection from clinical matrices is critical to patient outcomes. 20

We previously engineered the diagnostic phage ΦA1122 with luxAB to create a ‘light-21

tagged’ reporter phage. ΦA1122::luxAB rapidly detects Y. pestis in pure culture and 22

human serum by transducing a bioluminescent signal response. In this report, we 23

assessed the analytical specificity of the reporter phage and investigate diagnostic utility 24

(detection and antibiotic susceptibility analysis) directly from spiked whole blood. The 25

bioreporter displayed 100% (n=59) inclusivity for Y. pestis and consistent intra-specific 26

signal transduction levels. False-positives were not obtained from species typically 27

associated with bacteremia or those relevant to plague diagnosis. However, some non-28

pestis Yersinia strains and Enterobacteriaceae did elicit signals, albeit at highly 29

attenuated transduction levels. Diagnostic performance was assayed in simple broth-30

enriched blood samples and standard aerobic culture bottles. In blood, <102 CFU/mL 31

was detected within 5 h. In addition, Y. pestis was identified directly from positive blood 32

cultures within 20-45 min without further processing. Importantly, co-incubation of blood 33

samples with antibiotics facilitated simultaneous antimicrobial susceptibility profiling. 34

Consequently, the reporter phage demonstrated rapid detection and antibiotic 35

susceptibility profiling directly from clinical samples, features that may improve patient 36

prognosis during plague outbreaks. 37

38

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INTRODUCTION 39

Yersinia pestis, the causative agent of the plague, is a Tier 1 agent carrying great 40

risk for deliberate misuse and a significant socio-economic, public health threat. Plague 41

has the ability to spread person-to-person and is typically fatal in the absence of 42

expedient antibiotic therapy. Over the last 20 years, there has been a global rise in 43

plague incidence and strains have been isolated from patients with bubonic plague that 44

are streptomycin- and multidrug-resistant (MDR) (1-3). However, Y. pestis is typically 45

not drug resistant, and recent data indicates pneumonic plague may not be as 46

transmissible as previously believed (4). A zoonotic pathogen, Y. pestis circulates 47

among rodents and lagomorph populations, with endemic foci found in Asia, Africa, the 48

Americas, and the former Soviet Union. Most commonly spread to humans by fleas, 49

plague typically begins by bacterial multiplication at the site of dermal inoculation, 50

followed by spread to the draining lymph node via lymphatic vessels, and the formation 51

of characteristic buboes. Bubonic plague can rapidly progress to secondary septicemia 52

and in some cases, the bacteria can spread systemically and infiltrate alveoli, resulting 53

in the highly lethal pneumonic form. Because of the potential for pneumonia ensuing 54

from bubonic or septicemic plague, the CDC recommends quarantine of all plague 55

patients (5). Of greatest concern in bioterrorism is primary pneumonic plague contracted 56

via inhalation of aerosolized bacilli, which frequently leads to septicemia. Pneumonic 57

plague can be spread person-to-person and, like septicemic plague, is nearly always 58

fatal without appropriate antibiotic therapy and aggressive supportive care within 18 to 59

24 h of symptom onset (6). 60

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Plague is definitively diagnosed by the isolation and identification of the organism 61

from clinical specimens or by demonstrating a 4-fold or greater change in antibody titre 62

against the F1 antigen in paired serum specimens. Because Y. pestis grows slowly 63

(doubling time of 1.25 h at 28˚C), culturing from clinical specimens can involve 64

incubation for 24 to 72 h. Antibiotic susceptibility analysis from colony isolates requires 65

an additional 24 to 48 h (7, 8) and thus can extend the total diagnostic timeline to 66

between 2 and 5 days. However, because of the fulminant nature of plague infection, 67

rapid appropriate antibiotic therapy is paramount to patient prognosis. Therefore, faster 68

methods of detection and antimicrobial susceptibility profiling of Y. pestis directly from 69

clinical matrices may be of valuable. 70

Classical phage lysis assays using the plague diagnostic phage ΦA1122 are utilized 71

by the WHO, CDC and public health laboratories for Y. pestis identification. Thought to 72

target receptors likely essential for growth and virulence (9-11), ΦA1122 infects and 73

lyses all but two out of thousands of Y. pestis strains (12). In order to increase the time 74

to detection and bypass the necessity for pure cultures, ΦA1122 was previously 75

genetically engineered with the genes encoding bacterial luciferase (luxA and luxB) to 76

create a reporter phage capable of transducing bioluminescence to infected cells (13). 77

The reporter phage ΦA1122::luxAB was able to rapidly transduce a bioluminescent 78

phenotype (≤20 min) and sensitively detect Y. pestis in pure culture and in spiked 79

human serum, thereby displaying promise as a diagnostic tool. In addition, as phage 80

infection and gene expression is proportional to cell fitness, the reporter phage can 81

rapidly generate antimicrobial susceptibility data from pure cultures, which correlate 82

closely to results obtained using standard methods (14). In this report, we examine 83

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diagnostic performance of the Y. pestis reporter phage directly from blood and from 84

commercial blood culture bottles, and evaluate critical diagnostic parameters. 85

86

MATERIALS AND METHODS 87

Bacterial strains. Bacillus anthracis (attenuated strain) was kindly provided by the 88

CDC, Atlanta (Dr. Eilke Saile). Escherichia coli (5 various O antigenic and 89

uropathogenic strains), Enterococcus faecalis (5 strains), Enterococcus faecium (2 90

strains), Klebsiella pneumoniae (2 strains), Francisella tularensis (2 attenuated strains), 91

Klebsiella oxytoca (1 strain), Klebsiella spp. (2 unspeciated strains), Listeria 92

monocytogenes (5 strains), Salmonella enterica (10 strains), Shigella flexneri (3 93

strains), Shigella boydii (1 strain), Shigella sonnei (3 strains), Staphylococcus aureus (4 94

strains), Staphylococcus epidermidis (2 strains), Streptococcus pneumoniae (3 strains), 95

Yersinia enterocolitica (10 strains), Yersinia pseudotuberculosis (10 strains) and the 96

attenuated Y. pestis strain A1122 were obtained from the Biodefense and Emerging 97

Infections Research Resources Repository (BEI resources) or the American Type 98

Culture Collection (ATCC). Experiments involving virulent Y. pestis strains were 99

performed at the University of Florida under BSL3 conditions. A collection of 58 strains 100

including the classical strains KIM and CO92, as well as strains isolated from diverse 101

sources (human, rodents and fleas) and different geographical locations (e.g. U.S., 102

former Soviet Union, Brazil, Zimbabwe, India and Nepal) were analyzed. A list of strains 103

is provided in Supplementary Tables S1-S4. 104

Phage. The Y. pestis diagnostic phage ΦA1122::luxAB was described previously 105

(14). Reporter phage stocks were prepared from Y. pestis A1122 using agar overlays. 106

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Phage were eluted from the top agar in SM buffer, clarified by centrifugation (4,000 x g 107

for 10 min) and vacuum-filter sterilized (0.22 µM) twice before treating with 1M NaCl and 108

precipitating with 8% polyethylene glycol (BioUltra 8000, Sigma-Aldrich, St. Louis, MO). 109

The phage precipitate was resuspended in ~1/10th original volume SM buffer and 110

adjusted to a concentration of 1 x 1010 to 1011 plaque forming units (PFU)/mL. Filter 111

sterilization was repeated for concentrated preparations, and confirmed by negative 112

culture on Luria-Bertani (LB) agar. Preparations were stored at 4°C. 113

Bacterial growth and manipulation. Frozen stocks (in 25% glycerol at -70°C) of Y. 114

pestis A1122 and the 58 virulent strains were individually streaked onto Brain Heart 115

Infusion (BHI) agar with 6% sheep blood agar (SBA) and LB agar, respectively, and 116

incubated for 48 h at 28°C. Isolates were subcultured in LB broth at 28°C with shaking 117

(250 rpm). Assays were performed using early exponential phase cells (A600 of ~0.2). B. 118

anthracis, E. coli, L. monocytogenes, Y. enterocolitica, Y. pseudotuberculosis and 119

Enterococcus and Staphylococcus spp. were cultured using BHI media at 37˚C. 120

Klebsiella, Salmonella and Shigella spp. were cultured in Tryptic Soy media, and F. 121

tularensis strains on BHI or Cystine Heart Agar (CHA) supplemented with 2% 122

hemoglobin. Cultures at an A600 of 0.2 ± 0.03 were used for experiments. All 123

manipulations were performed within a Class II biosafety cabinet. 124

Bioluminescence assays directly from blood and blood culture bottles. Fresh 125

(≤1 week old) whole human blood collected with sodium citrate (Research Blood 126

Components, LLC, Boston, MA) was spiked with Y. pestis A1122 at the indicated 127

concentrations (see Fig. 2 legend). Spiked blood was diluted 1:20 with LB (50 µL into 128

950 µL) in 14 mL culture tubes and incubated at 28°C with shaking (250 rpm). After a 4 129

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h incubation, blood cells were collected by centrifugation (250 x g for 1 to 2 min) and the 130

resulting supernatants extracted and re-centrifuged at 10,000 x g for 4 min. Bacterial 131

cell pellets were resuspended in 225 µL of remaining supernatant and mixed with the 132

ΦA1122::luxAB reporter phage (1.1 x 109 PFU/assay). 133

Spike blood samples were also syringe-transferred (5 mL) into blood culture bottles 134

(BacT/Alert® Standard Aerobic, Biomérieux, Inc., Durham, NC or Bactec™ Plus 135

Aerobic/F Culture Vials, BD and Co., Sparks, MD) (final blood dilution factor; 1:9 in 136

BacT/Alert®, 1:7 in Bactec™ bottles). Spiked samples were incubated at 35 to 37°C 137

within BacT/Alert® and Bactec™ Blood Culture Systems. Aliquots from seeded (~101 138

CFU/mL) culture system-positive Bactec™ bottles were also assayed without the need 139

for the aforementioned processing and concentration steps. Phage-infected samples 140

were incubated with shaking (250 rpm) for 20 to 45 min. Aliquots (195 µL) were added 141

to 96-well microtiter plates and measured for bioluminescence (Glomax 96 Microplate 142

Luminometer 9101-001, Promega Corp., Madison, WI) for 10 s following injection of 2% 143

n-decanal. 144

Antibiotic resistance/susceptibility profiling from blood. To assess the ability of 145

the reporter phage to detect and simultaneously determine antibiotic susceptibility of Y. 146

pestis in blood, spiked blood samples were mixed with LB containing chloramphenicol, 147

streptomycin or tetracycline at increasing concentrations throughout known activity 148

ranges and minimum inhibitory concentrations (MIC). Samples were treated as 149

described above for bioluminescence assays directly from blood. Antibiotic efficacy was 150

confirmed against the quality control S. aureus strain ATCC29213 according to the 151

Clinical and Laboratory Standards Institute (CLSI) broth microdilution method (7, 8). 152

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Specificity and selectivity assays. Selectivity of the recombinant ΦA1122::luxAB 153

reporter was assayed for bioluminescence against 59 strains of Y. pestis. Recombinant 154

phage specificity was also determined against 47 strains of closely related genera (i.e. 155

Enterobacteriaceae) and 26 strains of non-related but clinically relevant bacterial 156

pathogens through bioluminescence assays. Briefly, log-phase cultures (1 mL) were 157

placed into 14 mL snap cap tubes (n = 3), infected with ΦA1122::luxAB and assayed for 158

bioluminescence over time. Relative bioluminescence (RB) was calculated by dividing 159

the average relative light units (RLU) of Y. pestis A1122 by the average RLU of the test 160

strain. Efficiency of plating (EOP), as defined by the phage titer on a test strain/species 161

compared to the maximum titer, was also analyzed where appropriate using Y. pestis 162

A1122 as the standard (12). Specific strain data for EOP and relative bioluminescence 163

is provided in Supplementary Tables S2 and S3. 164

Statistics. Reported as CFU/mL or PFU/mL, all bacterial cell or phage titer 165

concentrations were quantified as the average of duplicate colony or plaque plate 166

counts, respectively. Bioluminescence, presented as relative light units (RLU), are the 167

averages of three infections (n=3) ± standard deviations (SD). All experiments were 168

performed in duplicate. Statistical significance was determined using unpaired Student’s 169

t-tests. Tukey’s-adjusted ANOVAs were performed for comparisons between multiple 170

data sets, and Pearson’s correlation coefficient (r) was used to evaluate dependencies 171

between data sets. Significance was assigned at p ≤ 0.05. Statistical analyses were 172

performed using Microsoft excel 2010, and GraphPad Prism 5.0 on Windows 7. 173

174

RESULTS 175

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Selectivity and specificity. We previously analyzed the ability of ΦA1122::luxAB to 176

detect Y. pestis A1122, an attenuated exempt select agent strain. However, functionality 177

of the reporter phage against a range of virulent strains had not been assessed. 178

Therefore, performance was tested against a diverse library of Y. pestis strains (n=59). 179

In addition, non-Y. pestis Yersinia spp., other Enterobacteriaceae, and distantly-related 180

but clinically relevant bacterial pathogens were also analyzed. Of the 59 Y. pestis 181

strains, all (100%) elicited rapid bioluminescence of similar signal intensities and 182

kinetics (Fig. 1 and data not shown). For example, after 20 min of ΦA1122::luxAB 183

infection Y. pestis CO92 elicited a 100-fold increase in signal compared to controls, with 184

peak light signal manifesting at 40 to 60 min post infection (Fig. 1). Dose response 185

analysis of 10-fold serially diluted cells indicated that a bioluminescent signal (RLU) was 186

detectable from 2400, 240, and 24 CFU/mL after 20, 40, and 60 min of infection, 187

respectively (Fig. 1, p<0.05). Similar data was obtained with 4 other Y. pestis strains 188

(A1122, 2095G, NR-20, and NR-641). The data also indicates that: (i) ΦA1122::luxAB 189

exhibits the same broad intra-species as its parent ΦA1122, and (ii) results obtained 190

using the attenuated Y. pestis A1122 strain are translatable to virulent strains. 191

The reporter phage was challenged against 73 non-Y. pestis strains for phage-192

mediated bioluminescence, and in some cases, for phage amplification (EOP assays). 193

These included closely related Yersiniae, members of the Enterobacteriaceae, 194

enterococci and staphylococci and B. anthracis and F. tularensis. Three of 10 Y. 195

enterocolitica strains and 7 of 10 Y. pseudotuberculosis strains elicited phage-mediated 196

bioluminescence (Table 1). Of these, only one Y. pseudotuberculosis strain expressed a 197

signal as high as one log below that of Y. pestis, while the remainder were generally 104 198

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to 105-fold lower. In general, there was a correlation between phage-mediated 199

bioluminescence and EOP; strains that elicited low bioluminescence yielded low EOP. 200

Due to temperature-dependent differences in LPS structure, ΦA1122 does not form 201

plaques on Y. pseudotuberculosis below 28°C (9, 12). As expected, phage-mediated 202

bioluminescence assays at 20˚C revealed an attenuated (102 to 104-fold lower) signal in 203

5 Y. pseudotuberculosis strains compared to assays performed at 37˚C. Unexpectedly, 204

one strain elicited a 103-fold increase at the reduced temperature (data not shown). 205

Among other species, none of the E. coli, Klebsiella, S. boydii or S. flexneri strains 206

elicited phage-mediated bioluminescence (Table 1). However, bioluminescence was 207

detected from four S. enterica and two S. sonnei strains, although the signal response 208

was extremely low. As may be expected, none of the phylogenetically distant but 209

clinically relevant strains elicited reporter phage-mediated bioluminescence. 210

Rapid detection of Y. pestis from blood and blood culture bottles. Because 211

bacteremia is common in bubonic, pneumonic and septicemic plague, blood is an 212

important matrix for laboratory diagnosis. Blood culture systems can be used to screen 213

for bacteremia. Therefore, the efficacy of reporter phage-mediated detection of Y. pestis 214

A1122 was assessed directly from blood and blood culture bottles. Y. pestis was 215

detected in diluted blood within 5 h, without the need for culture isolation (Fig. 2A, 216

p<0.05). The signal response exhibited dose-dependent characteristics with a sensitivity 217

limit of detection (LoD) in the 102 CFU/mL range, peaking 45 min after phage infection. 218

Similar results were obtained from BacT/Alert® and BactecTM culture bottles, indicating 219

a functional compatibility of this detection platform with established clinical procedures 220

for processing blood samples. Although ‘light’ signal measurements were somewhat 221

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attenuated in blood, signal production was extremely rapid. Seeded Bactec™ bottles 222

(n=6) registered positive for bacterial growth after 24 to 30 h (blood alone controls did 223

not register positive as expected), and aliquots from these bottles were diagnosed by 224

the reporter phage 20 to 45 min after infection without sample processing or 225

concentration (Fig. 2B & C, p<0.05). 226

Rapid antibiotic susceptibility profiling from blood. Antimicrobial susceptibility 227

profiling using the reporter phage has been previously demonstrated using pure cultures 228

(14). To assess the ability of ΦA1122::luxAB to profile antibiotic susceptibility of Y. 229

pestis without the need for culture isolation, blood samples were spiked with ca. 102 230

CFU/mL Y. pestis and diluted in LB containing increasing concentrations of 231

chloramphenicol, streptomycin, or tetracycline along the known MIC ranges (Fig. 3A-C). 232

These antibiotics were selected because they are the standard therapeutics and 233

prophylactics recommended for plague in humans (6). Samples were incubated for 4 h, 234

infected with ΦA1122::luxAB, and then assayed for bioluminescence after 45 min. For 235

each antibiotic, bioluminescence was inversely proportional to antibiotic concentration. 236

High antibiotic concentrations completely nulified signal production whereas low 237

antibiotic concentrations resulted in strong signals. Thus, rapid detection and 238

simultaneous antibiotic susceptibility profiling of low levels of Y. pestis is achieved 239

through use of the reporter phage detection platform. 240

241

DISCUSSION 242

Plague is definitively diagnosed by the isolation and identification of the organism 243

from clinical specimens or by demonstrating a 4-fold or greater change in antibody titre 244

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against the F1 antigen in paired serum specimens. As phage A1122 can be used to 245

identify Y. pestis using classical lysis assays, ΦA1122::luxAB has the potential to 246

expedite this process. Data obtained using strain A1122 indicated detection was 247

achieved with blood spiked with 100 CFU/mL within 5 h, with similar results from blood 248

culture. Of note, samples from culture-positive blood bottles were ‘diagnosed’ by the 249

reporter with no preparation, processing, or concentration steps, within 20 to 45 min. 250

Because massive plague bacteremia generally foreshadows a fatal prognosis, and 251

bacteremic concentrations of ≥100 CFU/mL are significantly correlated with mortality, 252

detection of plague bacilli below this level is crucial for a clinically actionable plague 253

diagnostic (6, 15, 16). 254

ΦA1122::luxAB efficiently detected all Y. pestis strains tested, and likely possesses 255

the same specificity of its parent phage, which infects all but two of thousands of 256

isolates. However, the broad intraspecific host range is counterbalanced by suboptimal 257

cross-reactivity, as the reporter transduced bioluminescence to 16 of 47 strains of 258

phylogenetically related species (i.e. Enterobacteriaceae). In cross-reactive strains, 259

amplitude of transduced light was 10 to 106-fold lower (overall median RB = 1.13 x 10-5) 260

RLU/assay and time to peak signal generally longer than for Y. pestis. In receptive Y. 261

pseudotuberculosis strains, assays at sub-diagnostic temperature (20°C) revealed 262

signal attenuation by up to 104-fold in most, but not all strains evaluated. Therefore, the 263

reporter phage may require complementary diagnostic testing to reduce the likelihood of 264

false positives, and particularly to discriminate between Y. pestis and Y. 265

pseudotuberculosis. Importantly, none of the assayed pathogens integral in the 266

differential diagnosis for plague (i.e. B. anthracis, F. tularensis, and Klebsiella 267

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pneumoniae) or those most frequently associated with bacteremia (i.e. Enterococcus 268

spp., E. coli, coagulase-negative Staphylococcus spp., and Streptococcus spp.) (17-19) 269

were positive for bioluminescence. However, as the reporter phage diagnostic requires 270

laboratory equipment and up to 5 h to elicit a response from clinical specimens, it is not 271

usable in its current form as a point-of-care test. In contrast, the plague dipstick tests 272

which targets the F1 antigen are specific (no other Yersiniae are detected), sensitive, 273

and are particularly useful for field applications as they are rapid (less than 15 min) and 274

do not require equipment (20, 21). Therefore, dipstick tests have many advantages over 275

the reporter phage technology. However, as F1-negative isolates can occur albeit rarely 276

(22), the reporter phage may be a useful alternative to tests that rely on the F1 antigen. 277

Appropriate antibiotic therapy within 18 to 24 h of symptom onset is essential for a 278

positive prognosis in plague patients (23, 24). Chloramphenicol, streptomycin, and 279

tetracycline are the recommended primary level treatments (1, 6). In addition to the 280

potential for engineered resistance in bioweapons, naturally occurring antibiotic-281

resistant strains of Y. pestis, although extremely rare, have been isolated (2, 3, 25). 282

Thus the ability to rapidly generate an antibiotic susceptibility profile is of value. 283

Immunoassays and most PCR-based methods are unable to distinguish viable from 284

dead cells and are therefore of limited use in determining antibiotic susceptibility. A 285

method employing flow cytometry to determine antibiotic susceptibility following the 286

separation of plague bacilli from spiked blood cultures has been described, although the 287

need for pre-enrichment extends the assay time to ca. 39 h. The reporter phage 288

diagnostic described here has the ability to detect Y. pestis and to simultaneously 289

determine its susceptibility to antibiotics directly from blood within 5 h. This diagnostic 290

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therefore has the potential to provide critical information, timely enough to augment 291

treatment and improve patient outcomes in both bioterrorism and naturally acquired 292

cases of plague. 293

294

ACKNOWLEDGEMENTS 295

This research was supported by award numbers R43AI082698, R44AI082698, and 296

R01AI111535 from the National Institute of Allergy and Infectious Diseases (awarded to 297

D.A.S. of Guild BioSciences). We thank Dr. Sulakvelidze (University of Florida) and Dr. 298

Westwater (Medical University of South Carolina) for their advice and support. 299

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301

Table 1: Specificity of ΦA1122::luxAB among non-pestis Yersiniae, closely-related

species and non-related but clinically relevant bacterial pathogens.

Positive strains/total

tested

Relative

bioluminescenc

e (range)a

Efficiency of

platingb

Yersiniae

Y. enterocolitica 3 / 10 10-6 to 10-4 ≤ 3.6 x 10-6

Y. pseudotuberculosis 7 / 10 10-6 to 10-1 < 1 x 10-6

Enterobacteriaceae

E. colic 0 / 5 background nd

Klebsiella spp.d 0 / 5 background nd

S. entericae 4 / 10 10-6 to 10-3 < 1 x 10-6

S. boydii 0 / 1 background nd

S. flexneri 0 / 3 background nd

S. sonnei 2 / 3 10-6 to 10-5 ≤1.2x10-5

Non-

Enterobacteriaceae

B. anthracis 0 / 1 background nd

E. faecalis 0 / 5 background nd

E. faecium 0 / 2 background nd

F. tularensis 0 / 2 background nd

L. monocytogenes 0 / 5 background nd

Staphylococcus spp.f 0 / 5 background nd

S. pneumoniae 0 / 3 background nd

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302

aRelative to Y. pestis A1122; relative bioluminescence (RB) = RLU of non-Y. pestis 303

strain / RLU Y. pestis A1122 at 28˚C. 304

bEfficiency of plating (EOP), relative to Y. pestis A1122 at 28˚C. 305

cIncludes O91:H21, O157:H7, O145:nm, and a uropathogenic strain (serotype 306

unknown). 307

dIncludes K. oxytoca and K. pneumoniae. 308

eIncludes both Typhi and non-Typhi S enterica subspecies enterica strains. 309

fIncludes S. aureus and S. epidermidis. 310

nd, not determined 311

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FIG. 1. Bioluminescent response kinetics for the detection of Y. pestis CO92. Actively 313

growing (A600 of 0.2) cells were serially diluted, infected with 1.4 x 108 PFU/mL 314

ΦA1122::luxAB and assayed for bioluminescence (RLU) following the addition of n-315

decanal substrate. (*) denotes a significant increase (p < 0.05) compared to phage-316

negative (cells only) controls at the designated times. Values are means (n=3) ± SD. 317

Similar results were obtained with 58 other Y. pestis strains. 318

319

FIG. 2. Bioluminescent detection of Y. pestis from whole human blood. 320

A. 1 mL blood aliquots harboring Y. pestis A1122 (5.5 x 106 to 5.5 x 102 CFU/mL), were 321

diluted 1:20 in LB, enriched for 4 h, and analyzed 45 min post phage-infection for a 322

bioluminescent signal response. 323

B. Blood (5 mL) was spiked with 12 CFU/mL Y. pestis A1122 and transferred to 324

Standard Aerobic BacT/Altert® culture bottles. Seeded bottles were machine 325

(BacT/Alert 3D 60) incubated until a colorimetric positive reading (24 to 27 h). Neat or 326

diluted aliquots were analyzed 20 and 45 min post phage-infection for a bioluminescent 327

signal response. 328

C. Blood (5 mL) was spiked with 4.1 CFU/mL Y. pestis A1122 and transferred to Plus 329

Aerobic/F BactecTM culture bottles. Bottles were machine-incubated until a colorimetric 330

positive reading (24 to 30 h). Neat or diluted aliquots were analyzed 20 to 45 min post 331

phage-infection for a bioluminescent signal response. 332

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Values are means ± SD from 3 independent spiked blood samples. Graphs presented 333

are representative of 2 independent experiments. Asterisks (*) denote a significant 334

increase (p < 0.05) compared to phage negative controls (cells alone in blood). 335

336

FIG. 3. Phage-mediated antibiotic susceptibility profiles of Y. pestis A1122 directly from 337

blood incubated in the presence of chloramphenicol (A), streptomycin (B), or 338

tetracycline (C). Antibiotics were prepared using the CLSI microdilution method. Whole 339

blood was spiked with 75, 44, and 105 CFU/mL for A, B, and C, respectively, diluted 340

1:20 and incubated at 35°C for 4 h before being infected with the reporter phage. 341

Values are means ± SD from 3 independent infections of spiked blood. Graph 342

presented are representative of 2 independent experiments. Asterisks (*) denote a 343

significant difference increase (p < 0.05) between the assigned value and the next 344

highest antibiotic concentration. 345

346

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347

REFERENCES 348

1. Galimand M, Carniel E, Courvalin P. 2006. Resistance of Yersinia pestis to 349

antimicrobial agents. Antimicrob. Agents Chemother. 50:3233-3236. 350

2. Galimand M, Guiyoule A, Gerbaud G, Rasoamanana B, Chanteau S, Carniel 351

E, Courvalin P. 1997. Multidrug resistance in Yersinia pestis mediated by a 352

transferable plasmid. New Engl. J. Med. 337:677-680. 353

3. Guiyoule A, Gerbaud G, Buchrieser C, Galimand M, Rahalison L, Chanteau 354

S, Courvalin P, Carniel E. 2001. Transferable plasmid-mediated resistance to 355

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