Mark R. Wilson

Bacillus anthracis comparative genome analysis in support of the Amerithrax investigation

Before the anthrax letter attacks of 2001, the developing field of microbial forensics relied on microbial genotyping schemes based on a small portion of a genome sequence. Amerithrax, the investigation into the anthrax letter attacks, applied high-resolution whole-genome sequencing and comparative genomics to identify key genetic features of the letters’ Bacillus anthracis Ames strain. During systematic microbiological analysis of the spore material from the letters, we identified a number of morphological variants based on phenotypic characteristics and the ability to sporulate. The genomes of these morphological variants were sequenced and compared with that of the B. anthracis Ames ancestor, the progenitor of all B. anthracis Ames strains. Through comparative genomics, we identified four distinct loci with verifiable genetic mutations. Three of the four mutations could be directly linked to sporulation pathways in B. anthracis and more specifically to the regulation of the phosphorylation state of Spo0F, a key regulatory protein in the initiation of the sporulation cascade, thus linking phenotype to genotype. None of these variant genotypes were identified in single-colony environmental B. anthracis Ames isolates associated with the investigation. These genotypes were identified only in B. anthracis morphotypes isolated from the letters, indicating that the variants were not prevalent in the environment, not even the environments associated with the investigation. This study demonstrates the forensic value of systematic microbiological analysis combined with whole-genome sequencing and comparative genomics.

On the Evolutionary History, Population Genetics and Diversity among Isolates of Salmonella Enteritidis PFGE Pattern JEGX01.0004

Facile laboratory tools are needed to augment identification in contamination events to trace the contamination back to the source (traceback) of Salmonella enterica subsp. enterica serovar Enteritidis (S. Enteritidis). Understanding the evolution and diversity within and among outbreak strains is the first step towards this goal. To this end, we collected 106 new S. Enteriditis isolates within S. Enteriditis Pulsed-Field Gel Electrophoresis (PFGE) pattern JEGX01.0004 and close relatives, and determined their genome sequences. Sources for these isolates spanned food, clinical and environmental farm sources collected during the 2010 S. Enteritidis shell egg outbreak in the United States along with closely related serovars, S. Dublin, S. Gallinarum biovar Pullorum and S. Gallinarum. Despite the highly homogeneous structure of this population, S. Enteritidis isolates examined in this study revealed thousands of SNP differences and numerous variable genes (nu200a=u200a366). Twenty-one of these genes from the lineages leading to outbreak-associated samples had nonsynonymous (causing amino acid changes) changes and five genes are putatively involved in known Salmonella virulence pathways. While chromosome synteny and genome organization appeared to be stable among these isolates, genome size differences were observed due to variation in the presence or absence of several phages and plasmids, including phage RE-2010, phage P125109, plasmid pSEEE3072_19 (similar to pSENV), plasmid pOU1114 and two newly observed mobile plasmid elements pSEEE1729_15 and pSEEE0956_35. These differences produced modifications to the assembled bases for these draft genomes in the size range of approximately 4.6 to 4.8 mbp, with S. Dublin being larger (∼4.9 mbp) and S. Gallinarum smaller (4.55 mbp) when compared to S. Enteritidis. Finally, we identified variable S. Enteritidis genes associated with virulence pathways that may be useful markers for the development of rapid surveillance and typing methods, potentially aiding in traceback efforts during future outbreaks involving S. Enteritidis PFGE pattern JEGX01.0004.

The Complete Genome Sequence of Bacillus anthracis Ames “Ancestor”

The pathogenic bacterium Bacillus anthracis has become the subject of intense study as a result of its use in a bioterrorism attack in the United States in September and October 2001. Previous studies suggested that B. anthracis Ames Ancestor, the original Ames fully virulent plasmid-containing isolate, was the ideal reference. This study describes the complete genome sequence of that original isolate, derived from a sample kept in cold storage since 1981.

Draft Genome Sequences of 21 Salmonella enterica Serovar Enteritidis Strains

Salmonella enterica subsp. enterica serovar Enteritidis is a common food-borne pathogen, often associated with shell eggs and poultry. Here, we report draft genomes of 21 S. Enteritidis strains associated with or related to the U.S.-wide 2010 shell egg recall. Eleven of these genomes were from environmental isolates associated with the egg outbreak, and 10 were reference isolates from previous years, unrelated to the outbreak. The whole-genome sequence data for these 21 human pathogen strains are being released in conjunction with the newly formed 100K Genome Project.

The forensic analysis of foodborne bacterial pathogens in the age of whole-genome sequencing

The forensic evaluation of bacterial pathogens presents new challenges to the forensic science community. This review examines bacterial pathogens as objects of forensic comparison, focusing on their nucleic acid sequences as an important aspect of the comparison process. Because of the clonal propagation of most bacterial pathogens, a phylogenetic approach to understanding the diversity and using this understanding to address common forensic questions is explored. As a general phylogenetic framework is now employed in human mitochondrial DNA analysis, we will use the relevant concepts and approaches common in this area to develop this approach further. We also address the impact of the current ease and prevalence of whole‐genome DNA sequence analysis in the forensic comparison process.

Aedes pertinax, a Newly Recognized Mosquito Species in the United States

Abstract Specimens of a mosquito new to the continental USA, Aedes pertinax, were retrospectively identified from 2 collections made in 2011 in Indian River County, FL. Routine mosquito surveillance in subsequent years yielded more than 700 specimens appearing in 100 collections. The distribution of this mosquito in Florida and the United States is currently unknown, and recognition of the adult female is likely hampered by morphological similarities to Ae. atlanticus and Ae. tormentor.

Legal, Technical, and Interpretational Considerations in the Forensic Analysis of Viruses

The forensic evaluation of viruses presents new challenges to the forensic science community. Although many criminal cases have been adjudicated involving the deliberate transmission of viruses, especially HIV, this review provides a general approach to viral forensics, especially in light of significant biodefense challenges. Newly emerging techniques of nucleic acid sequencing are discussed in a forensic context. Human mitochondrial DNA analysis, wherein mixed profiles are routinely assessed in a forensic context, provides the groundwork for an interpretational approach to the issue of mixed DNA sequences. The importance of phylogenetic classification is discussed as both providing an integrated graphical depiction of the structure of viral nucleic acid variation as well as offering a tool that can be used to assess the relatedness of complex populations of nucleic acids.

Microbial Forensic Investigations in the Context of Bacterial Population Genetics

Publisher Summary This chapter focuses on microbial forensic investigations. Genetic analysis created a revolution in the field of forensics, and its application to microbial forensics is a major part of many investigations involving a biothreat agent. The utility and importance of genetic analysis are not surprising given that genomes contain extensive and varied information content. It is exploited to precisely characterize and identify biological evidentiary material and support other investigative efforts. In human forensic DNA analysis, molecular biology tools have become incredibly powerful due to a great understanding of human biology, the human genome, and human population-level genetics. One of the early scientific legal challenges to DNA fingerprinting was the lack of high-quality human population genetic data on the forensically relevant genetic markers. Over the past two decades these data have been generated and represent an invaluable resource to forensic analyses, as they are a point of reference against which forensic DNA profiles are considered for weighing the significance of an observation. Genetic and genomic analyses of forensic evidence are interpreted properly in the context of a specific pathogen’s population genetic structure, diversity, and reproductive mechanisms. Genetic and genomic analyses should lead to quantitative similarity data where evidentiary materials may match, nearly match, or exclude, which represents just three points along a nondiscrete continuum of possibilities.

Differentiation of Aedes atlanticus and Aedes tormentor by restriction fragment length polymorphisms of the second internal transcribed spacer.

Abstract Using novel DNA sequence data, we designed a restriction enzyme assay that distinguishes Aedes atlanticus and Ae. tormentor, based on size polymorphisms. The restriction endonuclease Hpy188I digests polymerase chain reaction–amplified 2nd internal transcribed spacer products once for Ae. atlanticus and twice for Ae. tormentor, thus providing a useful method for identifying adult female collections that are generally considered morphologically indistinguishable.

Successful nuclear DNA profiling of rootless hair shafts: a novel approach

Historically, rootless hair shaft samples submitted to a forensic laboratory for DNA analysis are reserved for mitochondrial DNA (mtDNA) analysis due to the presence of highly degraded as well as insufficient amounts of nuclear DNA. Although mtDNA has been very successful in obtaining results from rootless hair, this system has its limitations, namely, it is a lineage marker that cannot differentiate between maternally related genotypes. Given the high incidence of hairs as forensic evidence, there is a need for the use of a nuclear DNA test system capable of producing reliable results for hair shaft forensic evidence. This study reports the utilization of an enhanced DNA extraction methodology for hairs, in combination with a recently developed novel, nuclear DNA typing assay, InnoTyper® 21, to improve the success rate for obtaining informative results from highly compromised, degraded, and trace forensic samples such as rootless hair shafts. The InnoTyper 21 kit is a small amplicon retrotransposon marker typing system compatible with currently used capillary electrophoresis platforms. This system contains 20 Alu element markers, ranging in size from 60 to 125xa0bp, making the assay highly sensitive for extremely degraded forensic samples and thus enabling recovery of nuclear DNA profiles from samples that would otherwise require mtDNA sequencing. A subset of samples was also tested with the GlobalFiler kit with less success due to the larger amplicon sizes in comparison with InnoTyper 21. Results were variable but very promising, with approximately 40% of the total number of hairs tested producing interpretable nuclear DNA profiles with InnoTyper 21. These results demonstrate the ability of the utilized methodologies to produce nuclear DNA results with high statistical power from rootless hair shafts.