Bacillus anthracis diagnostic detection and rapid ...

Journal of Microbiological Methods 95 (2013) 156–161

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Bacillus anthracis diagnostic detection and rapid antibiotic susceptibility determination using ‘bioluminescent’ reporter phage David A. Schofield a,⁎, Natasha J. Sharp a, Joshua Vandamm a, Ian J. Molineux b, Krista A. Spreng c, Chythanya Rajanna d, Caroline Westwater e, George C. Stewart c a

Guild Associates, Inc., Charleston, SC 29407, United States Molecular Genetics and Microbiology, Institute for Cellular and Molecular Biology, University of Texas at Austin, TX 78712, United States Department of Veterinary Pathobiology, University of Missouri, Columbia, MO 65211, United States d Department of Molecular Genetics and Microbiology, University of Florida, Gainesville 32610, United States e Department of Craniofacial Biology, Medical University of South Carolina, Charleston, SC 29425, United States b c

a r t i c l e

i n f o

Article history: Received 9 May 2013 Received in revised form 1 August 2013 Accepted 8 August 2013 Available online 28 August 2013 Keywords: Bacillus anthracis Diagnosis Anthrax Reporter phage Detection Bioluminescence

a b s t r a c t Genetically modified phages have the potential to detect pathogenic bacteria from clinical, environmental, or food-related sources. Herein we assess an engineered ‘bioluminescent’ reporter phage (Wß::luxAB) as a clinical diagnostic tool for Bacillus anthracis, the etiological agent of anthrax. Wß::luxAB is able to rapidly (within minutes) detect a panel of B. anthracis strains by transducing a bioluminescent phenotype. The reporter phage displays species specificity by its inability, or significantly reduced ability, to detect members of the closely related Bacillus cereus group and other common bacterial pathogens. Using spiked clinical specimens, Wß::luxAB detects B. anthracis within 5 h at clinically relevant concentrations, and provides antibiotic susceptibility information that mirrors the CLSI method, except that data are obtained at least 5-fold faster. Although anthrax is a treatable disease, a positive patient prognosis is dependent on timely diagnosis and appropriate therapy. Wß::luxAB rapidly detects B. anthracis and determines antibiotic efficacy, properties that will help patient outcome. © 2013 Elsevier B.V. All rights reserved.

1. Introduction Anthrax is caused by inhalation, and cutaneous, or gastrointestinal (GI) exposure to Bacillus anthracis (Beatty et al., 2003; Inglesby et al., 2002). Naturally-acquired anthrax occurs infrequently in the US with approximately 20 cases of inhalation anthrax reported in the last 100 years, mostly from goat hair or wool mill employees, or by accidental contamination of laboratory researchers (Inglesby et al., 2002). However, B. anthracis is a Tier 1 pathogen and a potential bioterrorist weapon (Greenfield and Bronze, 2003). Tier 1 pathogens are classified as agents that can be easily disseminated or transmitted from person to person, result in high mortality rates, may cause public panic and social disruption, and require special actions for public health preparedness. The deliberate release of B. anthracis spores, either via aerosols or by deliberate contamination of the food or water supply (Khan et al., 2001; Sobel et al., 2002), could lead to a massive outbreak of anthrax. Early diagnosis is problematic since both GI and inhalational anthrax present symptoms that are difficult to distinguish from other less serious ailments (Inglesby et al., 2002; Shafazand et al., 1999; Woods, 2005). Disease progression is fairly rapid and usually ⁎ Corresponding author at: Guild Associates Inc., 1313B Ashley River Road, Charleston, SC 29407, United States. Tel.: +1 843 573 0095; fax: +1 843 573 0707. E-mail address: dschofi[email protected] (D.A. Schofield). 0167-7012/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.mimet.2013.08.013

fatal if not treated within the first 24 h following symptom onset (Woods, 2005). Rapid detection, diagnosis and the administration of appropriate antimicrobial therapy are therefore essential for a positive prognosis. B. anthracis isolates can be identified by microbiological and morphological methods (Edwards et al., 2006; Klee et al., 2006). B. anthracis is a large Gram-positive rod (1–1.5 × 3–10 μm) that is non-motile (an unusual feature of Bacillus species), sensitive to penicillin, selectively grows on polymyxin B/lysozyme/EDTA/thallium acetate (PLET) agar, and is not ß-hemolytic on sheep- or horse-blood agar plates. Capsule formation, which occurs during infection and is mediated by the virulence plasmid pXO2, is verified by staining clinical specimens (e.g. blood smears, cerebrospinal fluid [CSF]) with India ink and visualization by light microscopy). The M'Fadyean stain and the direct fluorescence assay (DFA) for capsular antigen may also be used for the detection of encapsulated bacilli. Molecular assays such as multiplex PCR and real-time PCR targeting chromosomal markers (e.g. Ba813) or plasmid markers (pXO1 or pXO2) have been developed to sensitively and rapidly detect B. anthracis (Bell et al., 2002; Edwards et al., 2006). As such, the PCR based JBAIDS anthrax detection kit is FDA-cleared for the presumptive identification of B. anthracis and it exhibits a limit of detection of 1000 CFU/mL from blood (FDA, 2005; Lingenfelter et al., 2006). Due to the potential release of deliberately engineered antibiotic resistant strains, molecular assays

D.A. Schofield et al. / Journal of Microbiological Methods 95 (2013) 156–161

are also being developed to rapidly provide an antibiotic susceptibility profile (Loveless et al., 2010; Weigel et al., 2010). The conventional Clinical Laboratory Standards Institute (CLSI) microdilution method requires 16–20 h (Clinical, 2010), which is somewhat at odds with fulminant disease progression. The use of qPCR to identify the fluorescence threshold cycle of amplified DNA from cells incubated with each drug concentration has enabled antibiotic susceptibility tests to be performed within 6 h (Weigel et al., 2010). Bacteriophages (phages) may also provide a means to rapidly diagnose and provide antibiotic susceptibility information. The γ phage assay is FDA-approved (US Army, 2005) and used by the CDC and Laboratory Response Network (LRN) as a standard for confirmatory identification of B. anthracis (Abshire et al., 2005; Inglesby et al., 2002). This assay takes advantage of a naturally occurring phage, which is specific and lytic for B. anthracis. After overnight growth on laboratory media, the presence of plaques provides a positive identification of B. anthracis. In order to improve the time to detection and simplify the assay, we developed a genetically engineered B. anthracis reporter phage (Schofield and Westwater, 2009). The reporter phage was generated by integrating genes encoding bacterial luciferase into the genome of the temperate Wß phage, the parent phage of γ (Brown and Cherry, 1955). Using the attenuated B. anthracis Sterne strain, the reporter phage Wß::luxAB was able to transduce a bioluminescent phenotype to recipient cells. In this report, we demonstrate the ability of the reporter phage to function as a clinical tool for the diagnostic detection of B. anthracis, and also as a means of rapidly determining antibiotic susceptibility. Using a panel of wildtype B. anthracis strains, and non-anthracis Bacillus species, the analytical specificity of the reporter was evaluated, as well as the diagnostic detection performance of B. anthracis from mock-infected blood specimens. We demonstrate that the reporter phage can be used to detect B. anthracis at clinically relevant concentrations. In addition, the phage-mediated bioluminescent signal response, which is correlated to the fitness of the cell, may be used to determine antibiotic sensitivity in a significantly faster timeframe compared to the standard CLSI methodology. 2. Materials and methods 2.1. Bacterial strains and cultivation Bacterial strains were obtained from the American Type Culture Collection (ATCC), the Bacillus Group Stock Center (BGSC), the Biodefense and Emerging Infections Research Resources Repository (BEI resources) and the Centers for Disease Control and Prevention (kindly provided by Dr. Elke Saile at CDC Atlanta). Bacillus species (B. anthracis [38 strains], Bacillus cereus [62 strains], Bacillus thuringiensis [45 strains], Bacillus mycoides [9 strains], Bacillus subtilis [3 strains], Bacillus megaterium [4 strains] and Bacillus weihenstephanensis [3 strains]), Listeria monocytogenes (10 strains) and Yersinia enterocolitica were grown in Brain Heart Infusion (BHI) media at 37 °C, unless otherwise stated. Salmonella enterica (10 strains), and Shigella species (Shigella flexneri [3 strains], Shigella dysenteriae [1 strain], Shigella sonnei [3 strains], and Shigella boydii [1 strain]) were grown in Luria Bertani (LB) media at 37 °C. Clostridium spp. (Clostridium innocuum [1 strain], Clostridium orbiscindens [1 strain], Clostridium clostridioforme [1 strain], Clostridium aldenense [1 strain], Clostridium difficile [2 strains], Clostridium bolteae [1 strain], Clostridium perfringens [1 strain], Clostridium citroniae [1 strain]) were grown on tryptic soy agar (TSA) supplemented with 5% sheep's blood at 37 °C under anaerobic conditions (atmosphere of b1% O2, N 13% CO2). Experiments with B. anthracis strains, with the exception of B. anthracis Sterne (exempt select agent, attenuated strain) were performed in Biosafety Level (BSL) 3 facilities at the University of Missouri or the University of Florida. Encapsulated vegetative cells of B. anthracis Ames were prepared by growing cells with BHI containing

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0.8% sodium bicarbonate at 37 °C, and with 10% CO2 (Scorpio et al., 2007; Wilson et al., 2008). Capsule formation was assessed by staining with India ink and by the observation of the characteristic mucoid growth. 2.2. B. anthracis reporter phage The Wß::luxAB reporter phage (Schofield and Westwater, 2009) was prepared from the B. anthracis Sterne lysogen. The reporter phage was previously generated by replacing the non-essential Wß wp40/41 genes with a luxAB expression cassette by homologous recombination. luxAB harbored a ribosome binding site to initiate translation and an upstream heterologous promoter containing a consensus −35 region and a 1 bp mismatch −10 region to drive transcriptional expression (Schofield and Westwater, 2009). Consequently, luxAB expression was expected to be independent of phage gene expression. Culture supernatants harboring the reporter phage were clarified by centrifugation (4000 ×g, 15 min) and passed through a 0.2 μm sterile nylon filtration system. Phage lysates were concentrated by adding 0.75 M NaCl at 4 °C for 60 min, followed by PEG8000 to a final concentration of 8% for 3 h. Precipitated phages were collected by centrifugation (11,000 ×g, 30 min). Phages were resuspended with SM buffer (50 mM Tris–HCl [pH 7.5], 0.1 M NaCl, 8 mM MgSO4∙7H2O, 0.01% gelatin) and stored at 4 °C. Reporter phage lysates typically harbored 108 plaque forming units (PFU)/mL. Unless otherwise stated, actively growing cultures (A600 of 0.2) were mixed with the reporter phage (final concentration of ~5 × 106 PFU/mL) and assayed for bioluminescence at the times indicated. 2.3. Detection of B. anthracis from blood Pooled whole human blood in sodium citrate (BioChemed) was mixed with an actively growing culture of B. anthracis Sterne (990 μL blood plus 10 μL bacteria). Blood-only samples served as negative controls. Blood samples (50 μL) were diluted 1:20 in BHI media and incubated at 37 °C for 4 h. Samples were centrifuged (250 ×g, 1 min) to collect red blood cells, the supernatant was transferred to a fresh tube and recentrifuged at 10,000 ×g for 4 min. Dilution of the blood, and centrifugation were necessary in order to minimize the inhibitory effects of blood on phage infection and of hemoglobinmediated quenching of the bioluminescent signal. The pellet was resuspended in 250 μL of supernatant, and reporter phage was added (final concentration of 2.5 × 107 PFU/mL). Bioluminescence was measured following a 60 min incubation at 37 °C. 2.4. Antimicrobial susceptibility assays The broth microdilution method was used to determine antimicrobial susceptibility of B. anthracis Sterne by the standard growth method (Clinical, 2009), and by the reporter phage. Antibiotics (penicillin G [potassium salt], doxycycline, tetracycline, ciprofloxacin, erythromycin) were purchased from Sigma-Aldrich and prepared according to the CLSI recommendations (Clinical, 2009). B. anthracis Sterne and the quality control (QC) strain Staphylococcus aureus ATCC 29213 were grown on Mueller Hinton agar (MHA) at 35 °C. Cells from freshly grown plates were prepared in cation-adjusted Mueller Hinton broth (CAMHB) to a final concentration of 2 to 4 × 105 CFU/mL, and used for each experiment. To determine antibiotic susceptibility profiles according to the standard CLSI method, cells were incubated with a range of antibiotic concentrations in 96 well round bottom microtiter plates and incubated at 35 °C. Growth was assessed at A625 after 16–20 h at 35 °C. To determine phage susceptibility, reporter phages were added (final concentration of 5 × 106 PFU/mL) immediately (tetracycline, doxycycline, erythromycin), 30 min (penicillin) or 80 min (ciprofloxacin) after the addition of the antibiotics. Bioluminescence was measured following a further 80 min incubation at 35 °C.

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2.5. Bioluminescence assays ‘Flash’ bioluminescence was measured using a Biotek Synergy II multiplate detection reader. Cultures (195 μL) were mixed with ndecanal (67 μL of a 2% solution) in 96 well white microtiter plates and read for 10 s. Controls included cells alone and phage alone. Bioluminescence is depicted as relative light units (RLU) and results are presented as the average of three experiments ± SD. Statistical significance was determined using Student's t-test (p b 0.05).

3. Results 3.1. B. anthracis reporter phage specificity Wild-type Wß phage infects and lyses a wide range of B. anthracis strains. For example, in two separate studies, Wß was shown to grow on all 171 (McCloy, 1951) and all 41 (Brown and Cherry, 1955) B. anthracis strains tested. We demonstrated previously the ability of the Wß::luxAB reporter phage to detect B. anthracis Sterne (Schofield and Westwater, 2009), an attenuated Biosafety Level 2 strain which lacks the pXO2 virulence plasmid, but its functionality with fully virulent strains was not previously tested. Therefore, the ability of the Wß::luxAB reporter phage to confer a bioluminescent signal response to 38 B. anthracis strains was assessed. All strains (e.g. Ames, Vollum, 46-PY-5, WNA, Kruger B, Graves, CNEVA 9066) were detected (Table 1). The signal response was rapid. A 104-fold increase in signal over background controls was detectable within 10 min after infection, and the signal was constant for 150 min (Fig. 1A). The signal response displayed dose-dependent characteristics as expected; high CFU/mL concentrations produced the strongest signals. As few as 3000 CFU/mL was detectable within 20 min (Fig. 1B). The parental Wß phage displays species specificity to B. anthracis since the phage does not lyse 242 out of the 244 strains (99% specificity) analyzed from 17 different non-anthracis Bacillus species (McCloy, 1951). However, ‘unusual’ B. cereus strains that manifest phenotypes of both B. anthracis and B. cereus have been identified that are susceptible to phage infection (Schuch and Fischetti, 2006). The ability of Wß::luxAB to confer a bioluminescent signal to B. cereus, B. thuringiensis, B. weihenstephanensis and B. mycoides was assessed. These species were chosen because they are part of the closely related B. cereus group. Of the 119 strains analyzed, 6 non-anthracis Bacillus strains displayed a signal above background controls (95% specificity, Table 1). The six false positives consisted of 1 B. cereus, 2 B. thuringiensis, and 3 B. mycoides strains. Of these, 5 of the 6 strains produced a signal

Table 1 Analytical specificity of the reporter phage. Species

Number of strains detected/number of strains tested

Comments

B. anthracis

38/38

Bioluminescent signal within ~20 min. 1 strain elicited a signal that was 100-fold lower than B. anthracis. 2 strains elicited signals that were 10 to 100-fold lower than B. anthracis. 1 of the 3 positive strains elicited a signal that was 100-fold lower than B. anthracis.

B. cereus

1/62

B. thuringiensis

2/45

B. mycoides

3/9

B. weihenstephanensis B. megaterium B. subtilis Non-Bacillus spp.

0/3 0/4 0/3 0/47

Listeria monocytogenes, Salmonella enterica, Shigella species, Clostridium species, and Yersinia enterocolitica were tested.

Fig. 1. Reporter phage detection of B. anthracis. A. Signal response time. B. anthracis Ames was grown in BHI with agitation (225 rpm) at 37 °C until an A600 of ~ 0.2 (4.7 × 106 CFU/mL). Reporter phages were added (5 × 106 PFU/mL, final concentration), and bioluminescence (relative light units) was monitored over time. Numbers are the mean (n = 3) ± SD. B. Sensitivity levels of detection. B. anthracis Ames was grown in BHI at 37 °C until an A600 of ~ 0.2. The culture was serially diluted in fresh BHI, reporter phage was added (5 × 106 PFU/mL, final concentration), and bioluminescence was monitored over time. Bacterial cell numbers depicted of 3.1 × 10 2 to 3.1 × 106 CFU/mL are the mean (n = 3) ± SD. *p b 0.05 compared to controls.

that was 10 to 100-fold lower than for B. anthracis. B. subtilis (3 strains), B. megaterium (4 strains), and the non-Bacillus spp. (15 spp. comprising 47 strains) were also unable to elicit a bioluminescent signal with the reporter phage. 3.2. Detection of ‘mock’-infected clinical specimens One of the main differences between Wß and γ is Wß's inability to infect encapsulated forms of B. anthracis (Brown and Cherry, 1955; McCloy, 1951). Although capsule formation occurs exclusively during infection, encapsulation can be induced in vitro by growing the cells under conditions that mimic the host; for example, by the addition of serum or sodium bicarbonate, and growing the cells at 37 °C with CO2 (Fouet and Mesnage, 2002). The ability of Wß::luxAB to transduce a bioluminescent phenotype to encapsulated bacilli when grown under nonencapsulation conditions was assessed. Encapsulated B. anthracis Ames cells were generated by growth on BHI agar containing 0.8% sodium bicarbonate at 37 °C with 10% CO2 (Scorpio et al., 2007; Wilson et al., 2008); the characteristic mucoid growth was observed. B. anthracis Ames was then grown under encapsulation conditions in liquid media, cells were harvested, and then incubated under non-encapsulating conditions (BHI, atmospheric CO2) in the presence of reporter phage.


Encapsulated cells were initially resistant to phage (Fig. 2A); however, within 20 min, a bioluminescent signal response was evident. After a further 60 min, comparable signal strengths were obtained from both encapsulated and non-encapsulated cultures. These results suggest that encapsulated bacilli rapidly become phage-sensitive when grown under non-encapsulating conditions. It also suggests that encapsulated cells, as would be found in clinical samples, do not preclude Wß::luxAB reporter phage mediated detection of B. anthracis. Blood cultures are the standard clinical specimen for cutaneous, gastrointestinal and inhalational anthrax (Beatty et al., 2003; Jernigan et al., 2001; Shafazand et al., 1999). The ability of the reporter phage to detect B. anthracis from spiked human blood was therefore assessed (Fig. 2B). As the CFU/mL increased from 102 to 105, the signal increased correspondingly, indicating a dose-dependent response. 710 CFU/mL could be detected from spiked blood with a total time to detection of 5 h (4 h outgrowth and 1 h detection). 3.3. Rapid determination of B. anthracis antibiotic susceptibility using reporter phage

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Wß::luxAB could be used to generate an antibiotic susceptibility profile, assays were performed in parallel to the standard CLSI microdilution method. Freshly prepared B. anthracis Sterne cells were incubated in the presence of tetracycline, doxycycline, erythromycin, penicillin, and ciprofloxacin; these antimicrobial agents are recommended for susceptibility testing (Clinical, 2010), as they are used for the treatment of anthrax, and prophylaxis management following exposure (Bartlett et al., 2002; Inglesby et al., 2002). S. aureus strain ATCC 29213 was used as a control, producing MICs that were within the reported ranges (data not shown) (Clinical, 2010). Bioluminescent signal response profiles mirrored inhibition of growth by the antibiotics (Fig. 3). At antibiotic concentrations that had minimal effect on growth, the bioluminescent signal response from the reporter phage remained maximal. Conversely, at antibiotic concentrations that were at the MIC or higher, signal responses were significantly reduced. These data show that phage-induced bioluminescence assays yield comparable antibiotic susceptibility profiles to the standard growth method. However, the phage assay is 5 to 10-fold faster. 4. Discussion

Reporter phage may facilitate antibiotic susceptibility testing since the signal response is dependent on host cell fitness. Thus, in the presence of an antibiotic, resistant cells are expected to elicit strong bioluminescent signal responses. In contrast, antibiotic-susceptible cells are expected to elicit attenuated responses. To determine if

Fig. 2. Detection of ‘clinical’-like samples. A. Reporter phage detection of encapsulated cells. B. anthracis Ames was grown in BHI under atmospheric conditions, or BHI supplemented with 0.8% sodium bicarbonate in the presence of 10% CO2 (encapsulated cells) at 37 °C. Encapsulated and non-capsulated cells were harvested, incubated with reporter phage and incubated in BHI under atmospheric conditions. Bioluminescence was assessed over time following the addition of n-decanal. Numbers are the mean (n = 3) ± SD. *p b 0.05 compared to controls. B. Detection of B. anthracis from ‘spiked’ blood. Human blood (in sodium citrate) was spiked with B. anthracis Sterne, diluted with BHI, and allowed to grow for 4 h at 37 °C. Following centrifugation and resuspension, reporter phages were added and bioluminescence was measured following a 60 min incubation at 37 °C. Cell numbers shown are 7.1 × 102 to 7.1 × 105 CFU/mL from the original blood samples. Phage only controls are blood samples without B. anthracis. Numbers are the mean (n = 3) ± SD. *p b 0.05 compared to controls.

In a previous study, Wß was shown to exhibit a broad strain range by its ability to grow on all 171 B. anthracis strains (McCloy, 1951). The reporter phage Wß::luxAB displays a similar trend by its ability to rapidly confer a strong bioluminescent signal response to all 38 B. anthracis strains tested. Wild-type Wß also displays species specificity for B. anthracis, with the exception of some ‘atypical’ Bacillus strains (McCloy, 1951; Schuch and Fischetti, 2006). However, the mechanisms for determining wild-type phage specificity and reporter phage specificity are not analogous, as wild-type specificity is determined by the ability of the phage to infect, replicate in, and lyse cells. In contrast, reporter phage host-susceptibility is measured by a bioluminescent signal that requires fewer steps, i.e. infection and luxAB expression. Nevertheless, the reporter phage maintains genus specificity; no non-Bacillus species yielded a signal response. Six of 126 (95% specificity) non-anthracis Bacillus strains did generate a signal but five of these generated 10 to 100-fold lower signals compared to B. anthracis. B. cereus can cause bacteremia and gastrointestinal infection (Bottone, 2010), and rare B. thuringiensis infections have been associated with burn wounds and eye infections (Damgaard et al., 1997; Peker et al., 2010). Aside from a temperate versus lytic lifestyle, one of the main differences between Wß and γ is the inability of Wß to infect encapsulated forms of B. anthracis (Brown and Cherry, 1955; McCloy, 1951). Capsule formation occurs only during vegetative growth in vivo or under specific environmental conditions that mimic the mammalian host (Brown and Cherry, 1955; Fouet and Mesnage, 2002; Koehler, 2002; Meynell and Meynell, 1964). The capsule is a principal virulence factor during infection because it inhibits phagocytosis (Makino et al., 1989). One potential disadvantage of using Wß is its inability to infect encapsulated cells. However, encapsulated cells rapidly become phage-susceptible when grown under non-encapsulating conditions. Given that the standard method for determining bacterial sepsis is microbiological culturing for up to 5 days, a 20 min period of growth before encapsulated cells become susceptible to the reporter phage is not significant. Using human blood spiked with B. anthracis, b800 CFU/mL is detectable with the reporter phage within 5 h. As B. anthracis has a rapid mean generation time (De Siano et al., 2006), the sensitivity limits of detection could be improved further by a longer outgrowth period. Nevertheless, the level of sensitivity and time to detection are comparable to the FDA-cleared PCR diagnostic test for B. anthracis (1000 CFU/mL and 3 h, respectively). The reporter phage, however, also has the ability to rapidly determine antibiotic susceptibility. It should be noted that levels of 10–104 CFU/mL B. anthracis in bacteremic rhesus monkeys are common within 13–72 h after aerosol challenge; these numbers can rise to as high as 109 CFU/mL

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Fig. 3. Antibiotic susceptibility profiles using the standard microdilution method or the reporter phage. Antibiotic susceptibility profiles of B. anthracis Sterne were generated with tetracycline (A), doxycycline (B), erythromycin (C), penicillin G (D), or ciprofloxacin (E). Inocula (final concentrations of 2.7 (A), 2.5 (B), 2.8 (C), 3.7 (D), and 2.1 (E) × 105 CFU/mL were prepared directly with colonies from a freshly grown plate) and antibiotics were prepared according to the CLSI microdilution method. Cells were incubated at 35 °C and assessed for growth (A625) after 16–20 h (right axis). Reporter phages were mixed (final concentration of 5 × 106 PFU/mL) with cells at time 0 (A, B, C), 30 min (D) or 90 min (E) after antibiotic addition. Bioluminescence was measured 80 min following the addition of n-decanal. Numbers are the mean (n = 3) ± SD.

in moribund animals (Friedlander et al., 1993; Lincoln et al., 1965). Moreover, blood cultures from all patients of the 2001 anthrax letter attacks who had not received prior antibiotic therapy were positive for B. anthracis even in the initial phase of the illness (Jernigan et al., 2001). Therefore, the sensitivity and rapid detection of B. anthracis from blood specimens by the reporter phage may accelerate diagnosis during early stages of infection. This is critical as once symptoms appear, there is a narrow window of opportunity during which patients need to be diagnosed and treated. Traditional microbiological culturing and identification can take days to complete and are inadequate to deal with the rapid course of disease. One prerequisite of phage-mediated detection is that target cells are viable. In contrast, most in vitro tests do not distinguish between live and dead cells or even between cells and free DNA unless the assay is modified to include a growth step or a specific viability dye such as propidium iodide (Smartt and Ripp, 2011). The need for metabolically active cells may be exploited in determining resistance of an isolate to a particular drug. This approach has also been utilized with other phage-based diagnostics for Mycobacterium tuberculosis and S. aureus (Banaiee et al., 2001; Piuri et al., 2009; Schofield et al., 2012). Although B. anthracis isolates are not typically drug resistant, it is conceivable that

strains may be deliberately engineered to be multi-drug resistant and subsequently released for malicious purposes. Because it is well known which antibiotics will be prescribed, and because inhalational anthrax is usually fatal if not treated within 24 h after symptom onset (Woods, 2005), the consequences of prescribing ineffective drugs could be severe. As the standard CLSI methodology for determining sensitivity requires 16–20 h using isolated cultures, methods that yield a more rapid determination of an isolate's antibiotic susceptibility are invaluable. The Wß::luxAB reporter phage provides an antibiotic susceptibility profile that mirrors the CLSI growth profile, except that bioluminescence data are acquired at least 5-fold faster. Consequently, diagnostic tools such as the reporter phage that can both diagnose and determine antibiotic efficacy, should help improve patient prognosis following natural or deliberate exposure to B. anthracis. Acknowledgments This research was supported in part by the USDA National Institute of Food and Agriculture (NIFA, 2009-33610-20028) awarded to D.A.S. of Guild Associates, Inc. We thank Drs. Michael G. Schmidt, Alvin Fox and Alexander Sulakvelidze for their advice and support.


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