Comparison of false-negative rates and limits of detection following macrofoam-swab sampling of Bacillus anthracis surrogates via Rapid Viability PCR and plate culture.

AIMS: We evaluated the effects of Bacillus anthracis surrogates, low surface concentrations, surface materials and assay methods on false-negative rate (FNR) and limit of detection (LOD95 ) for recovering Bacillus spores using a macrofoam-swab sampling procedure. METHODS AND RESULTS: Bacillus anthracis Sterne or Bacillus atrophaeus Nakamura spores were deposited over a range of low target concentrations (2-500 per coupon) onto glass, stainless steel, vinyl tile and plastic. Samples were assayed using a modified Rapid Viability-PCR (mRV-PCR) method and the traditional plate culture method to obtain FNR and LOD95 results. CONCLUSIONS: Mean FNRs tended to be lower for mRV-PCR compared to culturing, and increased as spore concentration decreased for all surface materials. Surface material, but not B. anthracis surrogate, influenced FNRs with the mRV-PCR method. The mRV-PCR LOD95 was lowest for glass and highest for vinyl tile. LOD95 values overall were lower for mRV-PCR than for the culture method. SIGNIFICANCE AND IMPACT OF STUDY: This study adds to the limited data on FNR and LOD95 for mRV-PCR and culturing methods with low concentrations of B. anthracis sampled from various surface materials by the CDC macrofoam-swab method. These are key inputs for planning characterization and clearance studies for low contamination levels of B. anthracis.

Proteomic signatures differentiating Bacillus anthracis Sterne sporulation on soil relative to laboratory media.

The process of sporulation is vital for the stability and infectious cycle of Bacillus anthracis. The spore is the infectious form of the organism and therefore relevant to biodefense. While the morphological and molecular events occurring during sporulation have been well studied, the influence of growth medium and temperature on the proteins expressed in sporulated cultures is not well understood. Understanding the features of B. anthracis sporulation specific to natural vs. laboratory production will address an important question in microbial forensics. In an effort to bridge this knowledge gap, a system for sporulation on two types of agar-immobilized soils was used for comparison to cultures sporulated on two common types of solid laboratory media, and one liquid sporulation medium. The total number of proteins identified as well as their identity differed between samples generated in each medium and growth temperature, demonstrating that sporulation environment significantly impacts the protein content of the spore. In addition, a subset of proteins common in all of the soil-cultivated samples was distinct from the expression profiles in laboratory medium (and vice versa). These differences included proteins involved in thiamine and phosphate metabolism in the sporulated cultures produced on soils with a notable increase in expression of ATP binding cassette (ABC) transporters annotated to be for phosphate and antimicrobial peptides. A distinct set of ABC transporters for amino acids, sugars and oligopeptides were found in cultures produced on laboratory media as well as increases in carbon and amino acid metabolism-related proteins. These protein expression changes indicate that the sporulation environment impacts the protein profiles in specific ways that are reflected in the metabolic and membrane transporter proteins present in sporulated cultures.

False-negative rate, limit of detection and recovery efficiency performance of a validated macrofoam-swab sampling method for low surface concentrations of Bacillus anthracis Sterne and Bacillus atrophaeus spores.

AIMS: We sought to evaluate the effects of Bacillus species, low surface concentrations, and surface material on recovery efficiency (RE), false-negative rate (FNR) and limit of detection for recovering Bacillus spores using a validated macrofoam-swab sampling procedure. METHODS AND RESULTS: The performance of a macrofoam-swab sampling method was evaluated using Bacillus anthracis Sterne (BAS) and Bacillus atrophaeus Nakamura (BG) spores applied at nine low target surface concentrations (2 to 500 CFU per plate or coupon) to positive-control plates and test coupons (25·8064 cm(2) ) of four surface materials (glass, stainless steel, vinyl tile and plastic). The Bacillus species and surface material had statistically significant effects on RE, but surface concentration did not. Mean REs were the lowest for vinyl tile (50·8% with BAS and 40·2% with BG) and the highest for glass (92·8% with BAS and 71·4% with BG). FNR values (which ranged from 0 to 0·833 for BAS and from 0 to 0·806 for BG) increased as surface concentration decreased in the range tested. Surface material also had a statistically significant effect on FNR, with FNR the lowest for glass and highest for vinyl tile. Finally, FNR tended to be higher for BG than for BAS at lower surface concentrations, especially for glass. CONCLUSIONS: Concentration and surface material had significant effects on FNR, with Bacillus species having a small effect. Species and surface material had significant effects on RE, with surface concentration having a nonsignificant effect. SIGNIFICANCE AND IMPACT OF THE STUDY: The results provide valuable information on the performance of the macrofoam-swab method for low surface concentrations of Bacillus spores, which can be adapted to assess the likelihood that there is no contamination when all macrofoam-swab samples fail to detect B. anthracis.

Bacillus anthracis spores germinate extracellularly at air-liquid interface in an in vitro lung model under serum-free conditions.

AIMS: To better understand the parameters that govern spore dissemination after lung exposure using in vitro cell systems. METHODS AND RESULTS: We evaluated the kinetics of uptake, germination and proliferation of Bacillus anthracis Sterne spores in association with human primary lung epithelial cells, Calu-3 and A549 cell lines. We also analysed the influence of various cell culture medium formulations related to spore germination. CONCLUSIONS: We found negligible spore uptake by epithelial cells, but germination and proliferation of spores in the serum-free extracellular environment was evident. Spore germination was appreciably higher in immortalized cell cultures than in primary epithelial cells. Additionally, spores still germinated apically at a mucus-secreting air-liquid interface lung barrier that was devoid of cell culture medium much earlier than medium-only controls. SIGNIFICANCE AND IMPACT OF THE STUDY: The role of lung epithelial cells in B. anthracis spore dissemination after inhalation remains poorly defined and rather controversial. These results are novel as they show spore germination is appreciably enhanced in the presence of lung cells in vitro, however, the cell line and cell state (air-liquid interface vs submerged in medium) dictates the extent of germination and in some cases proliferation.

Defining cell culture conditions to improve human norovirus infectivity assays.

Significant difficulties remain for determining whether human noroviruses (hNoV) recovered from water, food, and environmental samples are infectious. Three-dimensional (3-D) tissue culture of human intestinal cells has shown promise in developing an infectivity assay, but reproducibility, even within a single laboratory, remains problematic. From the literature and our observations, we hypothesized that the common factors that lead to more reproducible hNoV infectivity in vitro requires that the cell line be (1) of human gastrointestinal origin, (2) expresses apical microvilli, and (3) be a positive secretor cell line. The C2BBe1 cell line, which is a brush-border producing clone of Caco-2, meets these three criteria. When challenged with Genogroup II viruses, we observed a 2 Log(10) increase in viral RNA titer. A passage experiment with GII viruses showed evidence of the ability to propagate hNoV by both quantitative reverse transcription polymerase chain reaction (qRT-PCR) and microscopy. In our hands, using 3-D C2BBe1 cells improves reproducibility of the infectivity assay for hNoV, but the assay can still be variable. Two sources of variability include the cells themselves (mixed phenotypes of small and large intestine) and initial titer measurements using qRT-PCR that measures all RNA vs. plaque assays that measure infectious virus.

Evaluation of the FilmArray® system for detection of Bacillus anthracis, Francisella tularensis and Yersinia pestis.

AIMS: To evaluate the sensitivity and specificity of the BioFire Diagnostics FilmArray(®) system in combination with their Biothreat Panel for the detection of Bacillus anthracis (Ba), Francisella tularensis (Ft) and Yersinia pestis (Yp) DNA, and demonstrate the detection of Ba spores. METHODS AND RESULTS: DNA samples from Ba, Ft and Yp strains and near-neighbours, and live Ba spores were analysed using the FilmArray(®) Biothreat Panel, a multiplexed PCR-based assay for 17 pathogens and toxins. Sensitivity studies with DNA indicate that the limit of detection is 250 genome equivalents (GEs) per sample or lower. Furthermore, the identification of Ft, Yp or Bacillus species was made in 63 of 72 samples tested at 25 GE or less. With samples containing 25 CFU of Ba Sterne spores, at least one of the two possible Ba markers was identified in all samples tested. We observed no cross-reactivity with near-neighbour DNAs. CONCLUSIONS: Our results indicate that the FilmArray(®) Biothreat Panel is a sensitive and selective assay for detecting the genetic signatures of Ba, Ft and Yp. SIGNIFICANCE AND IMPACT OF THE STUDY: The FilmArray(®) platform is a complete sample-to-answer system, combining sample preparation, PCR and data analysis. This system is particularly suited for biothreat testing where samples need to be analysed for multiple biothreats by operators with limited training.