Timothy W. Janzen

Immunomagnetic capture of Bacillus anthracis spores from food.

Food is a vulnerable target for potential bioterrorist attacks; therefore, a critical mitigation strategy is needed for the rapid concentration and detection of biothreat agents from food matrices. Magnetic beads offer a unique advantage in that they have a large surface area for efficient capture of bacteria. We have demonstrated the efficient capture and concentration of Bacillus anthracis (Sterne) spores using immunomagnetic beads for a potential food application. Magnetic beads from three different sources, with varying sizes and surface chemistries, were functionalized with monoclonal antibodies and polyclonal antibodies from commercial sources and used to capture and concentrate anthrax spores from spiked food matrices, including milk, apple juice, bagged salad, processed meat, and bottled water. The results indicated that the Pathatrix beads were more effective in the binding and capture of anthrax spores than the other two bead types investigated. Furthermore, it was observed that the use of polyclonal antibodies resulted in a more efficient recovery of anthrax spores than the use of monoclonal antibodies. Three different magnetic capture methods, inversion, the Pathatrix Auto system, and the new i CropTheBug system, were investigated. The i CropTheBug system yielded a much higher recovery of spores than the Pathatrix Auto system. Spore recoveries ranged from 80 to 100% for the i CropTheBug system when using pure spore preparations, whereas the Pathatrix Auto system had recoveries from 20 to 30%. Spore capture from food samples inoculated at a level of 1 CFU/ml resulted in 80 to 100% capture for milk, bottled water, and juice samples and 60 to 80% for processed meat and bagged salad when using the i CropTheBug system. This efficient capture of anthrax spores at very low concentrations without enrichment has the potential to enhance the sensitivity of downstream detection technologies and will be a useful method in a foodborne bioterrorism response.

Evaluation of DNA extraction methods for Bacillus anthracis spores isolated from spiked food samples

Nine commercial DNA extraction kits were evaluated for the isolation of DNA from 10‐fold serial dilutions of Bacillus anthracis spores using quantitative real‐time PCR (qPCR). The three kits determined by qPCR to yield the most sensitive and consistent detection (Epicenter MasterPure Gram Positive; MoBio PowerFood; ABI PrepSeq) were subsequently tested for their ability to isolate DNA from trace amounts of B. anthracis spores (approx. 6·5 × 101 and 1·3 × 102 CFU in 25 ml or 50 g of food sample) spiked into complex food samples including apple juice, ham, whole milk and bagged salad and recovered with immunomagnetic separation (IMS).

Rapid detection and identification of Bacillus anthracis in food using pyrosequencing technology.

The development of advanced methodologies for the detection of Bacillus anthracis has been evolving rapidly since the release of the anthrax spores in the mail in 2001. Recent advances in detection and identification techniques could prove to be an essential component in the defense against biological attacks. Sequence based such as pyrosequencing, which has the capability to determine short DNA stretches in real-time using biotinylated PCR amplicons, has potential biodefense applications. Using markers from the virulence plasmids (pXO1 and pXO2) and chromosomal regions, we have demonstrated the power of this technology in the rapid, specific and sensitive detection of B. anthracis spores in food matrices including milk, juice, bottled water, and processed meat. The combined use of immunomagnetic separation and pyrosequencing showed positive detection when liquid foods (bottled water, milk, juice), and processed meat were experimentally inoculated with 6CFU/mL and 6CFU/g, respectively, without an enrichment step. Pyrosequencing is completed in about 60min (following PCR amplification) and yields accurate and reliable results with an added layer of confidence. The entire assay (from sample preparation to sequencing information) can be completed in about 7.5h. A typical run on food samples yielded 67-80bp reads with 94-100% identity to the expected sequence. This sequence based approach is a novel application for the detection of anthrax spores in food with potential application in foodborne bioterrorism response and biodefense involving the use of anthrax spores.

Rapid detection and antimicrobial resistance gene profiling of Yersinia pestis using pyrosequencing technology.

When a bioterrorism attack is attempted or perpetrated there is considerable risk for public health and large scale socioeconomic consequences. It is imperative that we possess established assays for the rapid identification of biothreat agents with high sensitivity and specificity to ensure emergency response measures can be deployed appropriately. Highly trustworthy information within a relevant timeframe is required to make a rapid and informed decision. Obtaining DNA sequence data from a suspected agent provides an added layer of confidence compared to a presumptive positive PCR amplicon. Sequencing based technologies, such as pyrosequencing, have sufficient discrimination potential to be used for microbial identification and can also be used to identify antimicrobial resistance (AMR) genes. We have shown in this study the power of pyrosequencing in the unambiguous detection and identification of nine Yersinia pestis strains based on virulence genes. Furthermore, we developed assays to characterize their AMR gene profiles. Sequence results ranging from 40 to 84bp were generated in about 60 min following initial PCR amplification and provide a rapid method for determining the AMR profile as compared to the conventional plate method which takes several days. The high sequence identities (95-100%) and specificity observed indicate the high level of accuracy of pyrosequencing technology. In addition, the read lengths of up to 84 bp observed in this study are unprecedented for pyrosequencing using the Pyromark Q24. We propose this method as a novel, rapid, sequence based detection and identification tool for Y. pestis with a potential application in biodefence.

Rapid Detection and Identification of Yersinia pestis from Food Using Immunomagnetic Separation and Pyrosequencing

Interest has recently been renewed in the possible use of Y. pestis, the causative agent of plague, as a biological weapon by terrorists. The vulnerability of food to intentional contamination coupled with reports of humans having acquired plague through eating infected animals that were not adequately cooked or handling of meat from infected animals makes the possible use of Y. pestis in a foodborne bioterrorism attack a reality. Rapid, efficient food sample preparation and detection systems that will help overcome the problem associated with the complexity of the different matrices and also remove any ambiguity in results will enable rapid informed decisions to be made regarding contamination of food with biothreat agents. We have developed a rapid detection assay that combines the use of immunomagnetic separation and pyrosequencing in generating results for the unambiguous identification of Y. pestis from milk (0.9 CFU/mL), bagged salad (1.6 CFU/g), and processed meat (10 CFU/g). The low detection limits demonstrated in this assay provide a novel tool for the rapid detection and confirmation of Y. pestis in food without the need for enrichment. The combined use of the iCropTheBug system and pyrosequencing for efficient capture and detection of Y. pestis is novel and has potential applications in food biodefence.

Rapid detection method for Bacillus anthracis using a combination of multiplexed real-time PCR and pyrosequencing and its application for food biodefense.

Bacillus anthracis, the causative agent of anthrax, has the capacity to form highly resilient spores as part of its life cycle. The potential for the dissemination of these spores using food as a vehicle is a huge public health concern and, hence, requires the development of a foodborne bioterrorism response approach. In this work, we address a critical gap in food biodefense by presenting a novel, combined, sequential method involving the use of real-time PCR and pyrosequencing for the rapid, specific detection of B. anthracis spores in three food matrices: milk, apple juice, and bottled water. The food samples were experimentally inoculated with 40 CFU ml(-1), and DNA was extracted from the spores and analyzed after immunomagnetic separation. Applying the combination of multiplex real-time PCR and pyrosequencing, we successfully detected the presence of targets on both of the virulence plasmids and the chromosome. The results showed that DNA amplicons generated from a five-target multiplexed real-time PCR detection using biotin-labeled primers can be used for single-plex pyrosequencing detection. The combined use of multiplexed real-time PCR and pyrosequencing is a novel, rapid detection method for B. anthracis from food and provides a tool for accurate, quantitative identification with potential biodefense applications.

Single nucleotide repeat analysis of B. anthracis isolates in Canada through comparison of pyrosequencing and Sanger sequencing.

Several technology platforms have been developed to resolve the phylogenetic placement of B. anthracis. However, these methods lack the resolution to identify differences between closely related strains within an outbreak due to the highly clonal nature of B. anthracis. Single Nucleotide Repeats (SNRs) are a type of rapidly evolving genetic marker that can be used to track epidemiological distribution in the event of an outbreak. Four SNR targets were used to detect and type 35 B. anthracis isolates in our collection; 18 from across Canada obtained between 1972 and 2005 and 17 from the 2006 Anthrax outbreak in north eastern Saskatchewan. A control sequence was developed for pyrosequencing which yielded consistent and accurate reads of SNRs. However, when DNA from the isolates was tested using pyrosequencing the results were inconsistent and did not reflect the number of SNRs obtained by Sanger sequencing. The SNR numbers derived from the Sanger sequencing show two of the four SNR loci could provide information on subtype, whereas the other two were not discriminatory. There is variation in SNRs between strains isolated from different outbreaks, the subset of 2006 outbreak strains showed very little difference in SNR number, and thus suggests low diversity among the strains sampled from the same outbreak.

Rapid SNP Detection and Genotyping of Bacterial Pathogens by Pyrosequencing

Bacterial identification and typing are fixtures of microbiology laboratories and are vital aspects of our response mechanisms in the event of foodborne outbreaks and bioterrorist events. Whole genome sequencing (WGS) is leading the way in terms of expanding our ability to identify and characterize bacteria through the identification of subtle differences between genomes (e.g. single nucleotide polymorphisms (SNPs) and insertions/deletions). Modern high-throughput technologies such as pyrosequencing can facilitate the typing of bacteria by generating short-read sequence data of informative regions identified by WGS analyses, at a fraction of the cost of WGS. Thus, pyrosequencing systems remain a valuable asset in the laboratory today. Presented in this chapter are two methods developed in the Amoako laboratory that detail the identification and genotyping of bacterial pathogens. The first targets canonical single nucleotide polymorphisms (canSNPs) of evolutionary importance in Bacillus anthracis, the causative agent of Anthrax. The second assay detects Shiga-toxin (stx) genes, which are associated with virulence in Escherichia coli and Shigella spp., and differentiates the subtypes of stx-1 and stx-2 based on SNP loci. These rapid methods provide end users with important information regarding virulence traits as well as the evolutionary and biogeographic origin of isolates.

Rapid detection and serovar identification of common Salmonella enterica serovars in Canada using a new pyrosequencing assay

Serotyping of Salmonella enterica subsp. enterica is a critical step for foodborne salmonellosis investigation. To identify Salmonella enterica subsp. enterica serovars, we have developed a new assay based on a triplex polymerase chain reaction (PCR) with pyrosequencing for amplicon confirmation and phylogenetic discrimination of strains. The top 54 most prevalent serovars of S. enterica in Canada were examined with a total of 23 single-nucleotide polymorphisms (SNPs) and (or) single-nucleotide variations (SNVs) located on 3 genes (fliD, sopE2, and spaO). Seven of the most common serovars, Newport, Typhi, Javiana, Infantis, Thompson, Heidelberg, and Enteritidis, were successfully distinguished from the other serovars based on their unique SNP-SNV combinations. The remaining serovars, including Typhimurium, ssp I:4,[5],12:i:-, and Saintpaul, were further divided into 47 subgroups that demonstrate the relatedness to phylogenetic classifications of each serovar. This pyrosequencing assay is not only cost-effective, rapid, and user-friendly, but also provides phylogenetic information by analyzing 23 selected SNPs. With the added layer of confidence in the PCR results and the accuracy and speed of pyrosequencing, this novel method would benefit the food industry and provides a tool for rapid outbreak investigation through quick detection and identification of common S. enterica serovars in Canada.

Development of a novel multiplexed qPCR and Pyrosequencing method for the detection of human pathogenic yersiniae

The purpose of this study was to develop a novel and robust molecular assay for the detection of human pathogenic yersiniae (i.e. Yersinia enterocolitica, Y. pseudotuberculosis and Y. pestis) in complex food samples. The assay combines multiplexed real-time PCR (qPCR) and Pyrosequencing for detecting and differentiating human pathogenic yersiniae with high confidence through sequence based confirmation. The assay demonstrated 100% specificity and inclusivity when tested against a panel of 14 Y. enterocolitica, 22 Y. pestis, 24 Y. pseudotuberculosis and a diverse selection of 17 other non-Yersinia bacteria. Pyrosequencing reads ranged from 28 to 40bp in length and had 94-100% sequence identity to the correct species in the GenBank nr database. Microbial enrichments of 48 ready-to-eat foods collected in the Greater Toronto Area from March 2014 to May 2014, including 46 fresh sprout and 2 salad products, were then tested using the assay. All samples were negative for Y. pestis and Y. pseudotuberculosis. Both salads (n=2) and 35% of sprout products (n=46) including 7.1% of alfalfa sprouts (n=14), 81% of bean sprouts (n=16), 12% of mixed sprouts (n=8) tested positive for Y. enterocolitica which was not detected in broccoli sprouts (n=5), onion sprouts (n=1), and pea sprouts (n=2). Cycle thresholds (Ct) of positive samples for Y. enterocolitica were between 23.0 and 37.9 suggesting post enrichment concentrations of approximately 1×102 to 1×106Y. enterocolitica per 1mL of enriched broth. An internal amplification control which was coamplified with targets revealed PCR inhibition in five samples which was resolved following a one in ten dilution. Pyrosequencing of qPCR amplicons suggests monoclonality and revealed a single nucleotide polymorphism that is present in Y. enterocolitica biotype 1A suggesting low pathogenicity of the detected strains. This study is the first to combine Pyrosequencing and qPCR for the detection of human pathogenic yersiniae and is applicable to a broad range of complex samples including ready-to-eat food samples.