A comparison of fourteen fully characterized mammalian-associated Campylobacter fetus isolates suggests that loss of defense mechanisms contribute to high genomic plasticity and subspecies evolution.

Campylobacter fetus is currently classified into three main subspecies, but only two of these, C. fetus subspecies fetus and C. fetus subsp. venerealis originate principally from ruminants where they inhabit different niches and cause distinct pathogenicity. Their importance as pathogens in international trade and reporting is also different yet the criteria defining these properties have never been fully substantiated nor understood. The situation is further compromised because the ability to differentiate between these two closely related C. fetus subspecies has traditionally been performed by phenotypic characterisation of isolates, methods which are limited in scope, time-consuming, tedious, and often yield inconsistent results, thereby leading to isolate misidentification. The development of robust genetic markers that could enable rapid discrimination between C. fetus subsp. fetus and subsp. venerealis has also been challenging due to limited differences in the gene complement of their genomes, high levels of sequence repetition, the small number of closed genome sequences available and the lack of standardisation of the discriminatory biochemical tests employed for comparative purposes. To yield a better understanding of the genomic differences that define these C. fetus strains, seven isolates were exhaustively characterised phenotypically and genetically and compared with seven previously well characterised isolates. Analysis of these 14 C. fetus samples clearly illustrated that adaption by C. fetus subsp. venerealis to the bovine reproductive tract correlated with increasing genome length and plasticity due to the acquisition and propagation of several mobile elements including prophages, transposons and plasmids harbouring virulence factors. Significant differences in the repertoire of the CRISPR (clustered regularly interspersed short palindromic repeats)-cas system of all C. fetus strains was also found. We therefore suggest that a deficiency in this adaptive immune system may have permitted the emergence of extensive genome plasticity and led to changes in host tropism through gene disruption and/or changes in gene expression. Notable differences in the sub-species complement of DNA adenine methylase genes may also have an impact. These data will facilitate future studies to better understand the precise genetic differences that underlie the phenotypic and virulence differences between these animal pathogens and may identify additional markers useful for diagnosis and sub-typing.

Decontamination of Bacillus anthracis Spores at Subzero Temperatures by Complete Submersion.

Introduction: Bacillus anthracis, the etiological agent of anthrax, produces long-lived spores, which are resistant to heat, cold, pH, desiccation, and chemical agents. The spores maintain their ability to produce viable bacteria even after decades, and when inhaled can cause fatal disease in over half of the clinical cases. Owing to these characteristics, anthrax has been repeatedly selected for both bioweapon and bioterrorism use. In the event of a bioterrorism attack, surfaces in the vicinity of the attack will be contaminated, and recovering from such an event requires rapid and effective decontamination. Previous decontamination method development has focused mainly on temperatures >0°C, and have shown poor efficacy at subzero temperatures. Methods: In this study, we demonstrate the use of calcium chloride (CaCl2) as a freezing point depression agent for pH-adjusted sodium hypochlorite (NaOCl) for the effective and rapid decontamination of B. anthracis Sterne strain spores at subzero temperatures. Results: We show the complete decontamination of 106 B. anthracis Sterne strain spores at temperatures as low as -20°C within 2.5 min by submersion in solution containing 25% (w/v) CaCl2, 0.50% NaOCl, and 0.40% (v/v) acetic acid. We also demonstrate significant reduction in number of spores at -28°C. Conclusions: The results show promise for rapidly decontaminating equipment and materials used in the response to bioterrorism events using readily available consumer chemicals. Future study should examine the efficacy of these results on complex surfaces.

Author Correction: Surveillance of Enterococcus spp. reveals distinct species and antimicrobial resistance diversity across a One-Health continuum.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Whole Genome Sequencing Differentiates Presumptive Extended Spectrum Beta-Lactamase Producing Escherichia coli along Segments of the One Health Continuum.

Antimicrobial resistance (AMR) has important implications for the continued use of antibiotics to control infectious diseases in both beef cattle and humans. AMR along the One Health continuum of the beef production system is largely unknown. Here, whole genomes of presumptive extended-spectrum ß-lactamase E. coli (ESBL-EC) from cattle feces (n = 40), feedlot catch basins (n = 42), surrounding streams (n = 21), a beef processing plant (n = 4), municipal sewage (n = 30), and clinical patients (n = 25) are described. ESBL-EC were isolated from ceftriaxone selective plates and subcultured on ampicillin selective plates. Agreement of genotype-phenotype prediction of AMR ranged from 93.2% for ampicillin to 100% for neomycin, trimethoprim/sulfamethoxazole, and enrofloxacin resistance. Overall, ß-lactam (100%; blaEC, blaTEM-1, blaSHV, blaOXA, blaCTX-M-), tetracycline (90.1%; tet(A), tet(B)) and folate synthesis (sul2) antimicrobial resistance genes (ARGs) were most prevalent. The ARGs tet(C), tet(M), tet(32), blaCTX-M-1, blaCTX-M-14, blaOXA-1, dfrA18, dfrA19, catB3, and catB4 were exclusive to human sources, while blaTEM-150, blaSHV-11-12, dfrA12, cmlA1, and cmlA5 were exclusive to beef cattle sources. Frequently encountered virulence factors across all sources included adhesion and type II and III secretion systems, while IncFIB(AP001918) and IncFII plasmids were also common. Specificity and prevalence of ARGs between cattle-sourced and human-sourced presumptive ESBL-EC likely reflect differences in antimicrobial use in cattle and humans. Comparative genomics revealed phylogenetically distinct clusters for isolates from human vs. cattle sources, implying that human infections caused by ESBL-EC in this region might not originate from beef production sources.

Surveillance of Enterococcus spp. reveals distinct species and antimicrobial resistance diversity across a One-Health continuum.

For a One-Health investigation of antimicrobial resistance (AMR) in Enterococcus spp., isolates from humans and beef cattle along with abattoirs, manured fields, natural streams, and wastewater from both urban and cattle feedlot sources were collected over two years. Species identification of Enterococcus revealed distinct associations across the continuum. Of the 8430 isolates collected, Enterococcus faecium and Enterococcus faecalis were the main species in urban wastewater (90%) and clinical human isolates (99%); Enterococcus hirae predominated in cattle (92%) and feedlot catch-basins (60%), whereas natural streams harbored environmental Enterococcus spp. Whole-genome sequencing of E. faecalis (n = 366 isolates) and E. faecium (n = 342 isolates), revealed source clustering of isolates, indicative of distinct adaptation to their respective environments. Phenotypic resistance to tetracyclines and macrolides encoded by tet(M) and erm(B) respectively, was prevalent among Enterococcus spp. regardless of source. For E. faecium from cattle, resistance to ß-lactams and quinolones was observed among 3% and 8% of isolates respectively, compared to 76% and 70% of human clinical isolates. Clinical vancomycin-resistant E. faecium exhibited high rates of multi-drug resistance, with resistance to all ß-lactam, macrolides, and quinolones tested. Differences in the AMR profiles among isolates reflected antimicrobial use practices in each sector of the One-Health continuum.

Comparative genomics of multidrug-resistant Enterococcus spp. isolated from wastewater treatment plants.

BACKGROUND: Wastewater treatment plants (WWTPs) are considered hotspots for the environmental dissemination of antimicrobial resistance (AMR) determinants. Vancomycin-Resistant Enterococcus (VRE) are candidates for gauging the degree of AMR bacteria in wastewater. Enterococcus faecalis and Enterococcus faecium are recognized indicators of fecal contamination in water. Comparative genomics of enterococci isolated from conventional activated sludge (CAS) and biological aerated filter (BAF) WWTPs was conducted. RESULTS: VRE isolates, including E. faecalis (n = 24), E. faecium (n = 11), E. casseliflavus (n = 2) and E. gallinarum (n = 2) were selected for sequencing based on WWTP source, species and AMR phenotype. The pangenomes of E. faecium and E. faecalis were both open. The genomic fraction related to the mobilome was positively correlated with genome size in E. faecium (p < 0.001) and E. faecalis (p < 0.001) and with the number of AMR genes in E. faecium (p = 0.005). Genes conferring vancomycin resistance, including vanA and vanM (E. faecium), vanG (E. faecalis), and vanC (E. casseliflavus/E. gallinarum), were detected in 20 genomes. The most prominent functional AMR genes were efflux pumps and transporters. A minimum of 16, 6, 5 and 3 virulence genes were detected in E. faecium, E. faecalis, E. casseliflavus and E. gallinarum, respectively. Virulence genes were more common in E. faecalis and E. faecium, than E. casseliflavus and E. gallinarum. A number of mobile genetic elements were shared among species. Functional CRISPR/Cas arrays were detected in 13 E. faecalis genomes, with all but one also containing a prophage. The lack of a functional CRISPR/Cas arrays was associated with multi-drug resistance in E. faecium. Phylogenetic analysis demonstrated differential clustering of isolates based on original source but not WWTP. Genes related to phage and CRISPR/Cas arrays could potentially serve as environmental biomarkers. CONCLUSIONS: There was no discernible difference between enterococcal genomes from the CAS and BAF WWTPs. E. faecalis and E. faecium have smaller genomes and harbor more virulence, AMR, and mobile genetic elements than other Enterococcus spp.

Clonal expansion of environmentally-adapted Escherichia coli contributes to propagation of antibiotic resistance genes in beef cattle feedlots.

Livestock wastewater lagoons represent important environmental reservoirs of antibiotic resistance genes (ARGs), although factors contributing to their proliferation within these reservoirs remain poorly understood. Here, we characterized Escherichia coli from feedlot cattle feces and associated wastewater lagoons using CRISPR1 subtyping, and demonstrated that while generic E. coli were genetically diverse, populations were dominated by several 'feedlot-adapted' CRISPR types (CTs) that were widely distributed throughout the feedlot. Moreover, E. coli bearing beta-lactamase genes, which confer reduced susceptibility to third-generation cephalosporin's, predominantly belonged to these feedlot-adapted CTs. Remarkably, the genomic region containing the CRISPR1 allele was more frequently subject to genetic exchange among wastewater isolates compared to fecal isolates, implicating this region in environmental adaptation. This allele is proximal to the mutS-rpoS-nlpD region, which is involved in regulating recombination barriers and adaptive stress responses. There were no loss-of-function mutS or rpoS mutations or beneficial accessory genes present within the mutS-rpoS-nlpD region that would account for increased environmental fitness among feedlot-adapted isolates. However, comparative sequence analysis revealed that protein sequences within this region were conserved among most feedlot-adapted CTs, but not transient fecal CTs, and did not reflect phylogenetic relatedness, implying that adaptation to wastewater environments may be associated with genetic variation related to stress resistance. Collectively, our findings suggest adaptation of E. coli to feedlot environments may contribute to propagation of ARGs in wastewater lagoons.

First Complete Genome Sequence of Yersinia massiliensis.

Using a combination of Illumina paired-end sequencing, Pacific Biosciences RS II sequencing, and OpGen Argus whole-genome optical mapping, we report here the first complete genome sequence of Yersinia massiliensis The completed genome consists of a 4.99-Mb chromosome, a 121-kb megaplasmid, and a 57-kb plasmid.

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.

Comparative genomics of Enterococcus spp. isolated from bovine feces.

BACKGROUND: Enterococcus is ubiquitous in nature and is a commensal of both the bovine and human gastrointestinal (GI) tract. It is also associated with clinical infections in humans. Subtherapeutic administration of antibiotics to cattle selects for antibiotic resistant enterococci in the bovine GI tract. Antibiotic resistance genes (ARGs) may be present in enterococci following antibiotic use in cattle. If located on mobile genetic elements (MGEs) their dissemination between Enterococcus species and to pathogenic bacteria may be promoted, reducing the efficacy of antibiotics. RESULTS: We present a comparative genomic analysis of twenty-one Enterococcus spp. isolated from bovine feces including Enterococcus hirae (n = 10), Enterococcus faecium (n = 3), Enterococcus villorum (n = 2), Enterococcus casseliflavus (n = 2), Enterococcus faecalis (n = 1), Enterococcus durans (n = 1), Enterococcus gallinarum (n = 1) and Enterococcus thailandicus (n = 1). The analysis revealed E. faecium and E. faecalis from bovine feces share features with human clinical isolates, including virulence factors. The Tn917 transposon conferring macrolide-lincosamide-streptogramin B resistance was identified in both E. faecium and E. hirae, suggesting dissemination of ARGs on MGEs may occur in the bovine GI tract. An E. faecium isolate was also identified with two integrative conjugative elements (ICEs) belonging to the Tn916 family of ICE, Tn916 and Tn5801, both conferring tetracycline resistance. CONCLUSIONS: This study confirms the presence of enterococci in the bovine GI tract possessing ARGs on MGEs, but the predominant species in cattle, E. hirae is not commonly associated with infections in humans. Analysis using additional complete genomes of E. faecium from the NCBI database demonstrated differential clustering of commensal and clinical isolates, suggesting that these strains may be specifically adapted to their respective environments.

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.

Subtyping Escherichia coli Virulence Genes Isolated from Feces of Beef Cattle and Clinical Cases in Alberta.

Clinical outcomes of Shiga toxin (stx)-producing Escherichia coli infection are largely determined by virulence gene subtypes. This study used a polymerase chain reaction (PCR)-pyrosequencing assay to analyze single-nucleotide polymorphisms for subtyping three major virulence genes (stx1, stx2, eae) of pathogenic E. coli (O157, O26, O111, and O103) isolated from cattle over a 2-year interval (n = 465) and human clinical cases (n = 42) in western Canada. Most bovine isolates were PCR positive for at least one target virulence gene (367/465), whereas 100% of human isolates harbored eae in combination with at least one stx gene. Four Shiga toxin (1a, 2a, 2c, and 2e) and four eae (λ/γ1-eae, ɛ-eae, θ/γ2-eae, and ß-eae) subtypes were identified in over 25 distinct virulence genotypes. Among cattle isolates, every serogroup, but O103, presented a dominant genotype (O157: stx1a+stx2a+λ/γ1-eae, O26: ß-eae alone, and O111: stx1a+θ/γ2-eae). Similar patterns were found in human isolates, although it was not possible to establish a clear genotypic association between the two sources. Many O157 and non-O157 cattle isolates lacked stx genes; the absence was greater in non-O157 (75/258) and O157:non-H7 (19/40) than in O157:H7 strains (1/164). In addition, there was a greater diversity of virulence genotypes of E. coli isolated from cattle than those of human diseases, which could be due to sample characteristics (e.g., source and clinical condition). However, the majority of cattle strains had virulence profiles identical to those of clinical cases. Consequently, determining the presence of certain stx (stx1a and stx2a) and eae (λ/γ1-eae) subtypes known to cause human disease would be a valuable tool for risk assessment and prediction of disease outcome along the farm-to-fork continuum.

Draft Genome Sequence of an Enterococcus thailandicus Strain Isolated from Bovine Feces.

Here, we report the first draft genome sequence of Enterococcus thailandicus isolated from the feces of feedlot cattle in Southern Alberta.

Comparative Evaluation of Genomic and Laboratory Approaches for Determination of Shiga Toxin Subtypes in Escherichia coli.

The determination of Shiga toxin (ST) subtypes can be an important element in the risk characterization of foodborne ST-producing Escherichia coli (STEC) isolates for making risk management decisions. ST subtyping methods include PCR techniques based on electrophoretic or pyrosequencing analysis of amplicons and in silico techniques based on whole genome sequence analysis using algorithms that can be readily incorporated into bioinformatics analysis pipelines for characterization of isolates by their genetic composition. The choice of technique will depend on the performance characteristics of the method and an individual laboratory's access to specialized equipment or personnel. We developed two whole genome sequence-based ST subtyping tools: (i) an in silico PCR algorithm requiring genome assembly to replicate a reference PCR-based method developed by the Statens Serum Institut (SSI) and (ii) an assembly-independent routine in which raw sequencing results are mapped to a database of known ST subtype sequence variants (V-Typer). These tools were evaluated alongside the SSI reference PCR method and a recently described PCR-based pyrosequencing technique. The V-Typer method results corresponded closely with the reference method in the analysis of 67 STEC cultures obtained from a World Health Organization National Reference Laboratory. In contrast, the in silico PCR method failed to detect ST subtypes in several cases, a result which we attribute to assembly-induced errors typically encountered with repetitive gene sequences. The V-Typer can be readily integrated into bioinformatics protocols used in the identification and characterization of foodborne STEC isolates.

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.

A new pyrosequencing assay for rapid detection and genotyping of Shiga toxin, intimin and O157-specific rfbE genes of Escherichia coli.

Shiga toxin (stx)-producing Escherichia coli (STEC) contamination in food and water is one of the most recognized concerns and a major financial burden in human hygiene control worldwide. Rapid and highly reliable methods of detecting and identifying STEC causing gastroenteric illnesses are crucial to prevent foodborne outbreaks. A number of tests have been developed and commercialized to detect STEC using molecular microbiology techniques. Most of these are designed to identify virulence factors such as Shiga toxin and intimin as well as E. coli O and H antigen serotype specific genes. In order to screen pathogenic STEC without relying on O:H serotyping, we developed a rapid detection and genotyping assay for STEC virulence genes using a PCR-pyrosequencing application. We adapted the PyroMark Q24 Pyrosequencing platform for subtyping 4 major virulence genes, Shiga toxin 1 and 2 (stx1 and stx2), intimin (eae) and O157-antigen gene cluster target rfbE, using Single Nucleotide Polymorphism (SNP) analysis. A total of 224 E. coli strains including isolates from Canadian environment, food and clinical cases were examined. Based on the multiple alignment analysis of 30-80 base nucleotide pyrogram reads, three alleles of the Shiga toxin 1a gene (stx1a) (stx1a-I, stx1a-II, stx1a-III) were identified. Results of the stx1, stx2, eae and rfbE genotyping revealed that each group of O:H serotype shares distinctive characteristics that could be associated with the virulence of each genotype. O157:H7/NM carries stx1a-II (94%), stx2a (82%), λ/γ1-eae (100%) and rfbE type-H7/NM (100%). Whereas isolates of the "Top-6" serotypes (O26, O45, O103, O111, O121, O145) had a high incidence of stx1a-I (90%) and stx2a (100%). stx1a-III (60%) was only observed in non Top-7 (Top-6 plus O157) STEC and Shigella spp. The entire assay, from extracting DNA from colonies on a plate to the generation of sequence information, can be completed in 5h. The method of profiling these 4 STEC pathogenic genotypes as demonstrated in this paper is rapid, easily performed, informative and cost-effective, and thus has a potential to be deployed in the food industry for the routine screening of potentially pathogenic STEC isolates.

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 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.

A New Generation Microarray for the Simultaneous Detection and Identification of Yersinia pestis and Bacillus anthracis in Food.

The use of microarrays as a multiple analytic system has generated increased interest and provided a powerful analytical tool for the simultaneous detection of pathogens in a single experiment. A wide array of applications for this technology has been reported. A low density oligonucleotide microarray was generated from the genetic sequences of Y. pestis and B. anthracis and used to fabricate a microarray chip. The new generation chip, consisting of 2,240 spots in 4 quadrants with the capability of stripping/rehybridization, was designated as "Y-PESTIS/B-ANTHRACIS 4x2K Array." The chip was tested for specificity using DNA from a panel of bacteria that may be potentially present in food. In all, 37 unique Y. pestis-specific and 83 B. anthracis-specific probes were identified. The microarray assay distinguished Y. pestis and B. anthracis from the other bacterial species tested and correctly identified the Y. pestis-specific oligonucleotide probes using DNA extracted from experimentally inoculated milk samples. Using a whole genome amplification method, the assay was able to detect as low as 1 ng genomic DNA as the start sample. The results suggest that oligonucleotide microarray can specifically detect and identify Y. pestis and B. anthracis and may be a potentially useful diagnostic tool for detecting and confirming the organisms in food during a bioterrorism event.

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.