A likelihood ratio-based approach for improved source attribution in microbiological forensic investigations

A common objective in microbial forensic investigations is to identify the origin of a recovered pathogenic bacterium by DNA sequencing. However, there is currently no consensus about how degrees of belief in such origin hypotheses should be quantified, interpreted, and communicated to wider audiences. To fill this gap, we have developed a concept based on calculating probabilistic evidential values for microbial forensic hypotheses. The likelihood-ratio method underpinning this concept is widely used in other forensic fields, such as human DNA matching, where results are readily interpretable and have been successfully communicated in juridical hearings. The concept was applied to two case scenarios of interest in microbial forensics: (1) identifying source cultures among series of very similar cultures generated by parallel serial passage of the Tier 1 pathogen Francisella tularensis, and (2) finding the production facilities of strains isolated in a real disease outbreak caused by the human pathogen Listeria monocytogenes. Evidence values for the studied hypotheses were computed based on signatures derived from whole genome sequencing data, including deep-sequenced low-frequency variants and structural variants such as duplications and deletions acquired during serial passages. In the F. tularensis case study, we were able to correctly assign fictive evidence samples to the correct culture batches of origin on the basis of structural variant data. By setting up relevant hypotheses and using data on cultivated batch sources to define the reference populations under each hypothesis, evidential values could be calculated. The results show that extremely similar strains can be separated on the basis of amplified mutational patterns identified by high-throughput sequencing. In the L. monocytogenes scenario, analyses of whole genome sequence data conclusively assigned the clinical samples to specific sources of origin, and conclusions were formulated to facilitate communication of the findings. Taken together, these findings demonstrate the potential of using bacterial whole genome sequencing data, including data on both low frequency SNP signatures and structural variants, to calculate evidence values that facilitate interpretation and communication of the results. The concept could be applied in diverse scenarios, including both epidemiological and forensic source tracking of bacterial infectious disease outbreaks.

Introduction and persistence of tularemia in Bulgaria

Introduction: Outbreaks of the zoonotic disease tularemia occurred in north-east Bulgaria in the 1960s. Then came 30 years of epidemiological silence until new outbreaks occurred in west Bulgaria in the 1990s. To investigate how bacterial strains of Francisella tularensis causing tularemia in wildlife and humans in the 1960s and the 1990s were related, we explored their genetic diversity. Material and methods: TenF. tularensis genomes from the 1960s (n=3) and the 1990s (n=7) were sequenced, assigned to canonical single-nucleotide polymorphism (canSNP) clades, and compared to reference genomes. We developed four new canSNP polymerase chain reaction (PCR) assays based on the genome sequence information. Results and discussion: The genetic analysis showed that the outbreaks in the 1960s as well as in the 1990s involved multiple clones and new genetic diversity. The smallest genetic difference found between any of the Bulgarian strains was five SNPs between the strains L2 and 81 isolated 43 years apart, indicating that F. tularensis may persist locally over long time periods without causing outbreaks. The existence of genetically highly similar strain-pairs isolated the same year in the same area from different hosts supports a hypothesis of local expansion of clones during outbreaks. Close relationship (two SNPs) was found between one strain isolated 1961 in northeast Bulgaria and one strain isolated 5 years before in USSR. Historical data coinciding with the actual time point describe the introduction of water rats from USSR into the Bulgarian outbreak area, which may explain the close genetic relationship and the origin of the outbreak. Conclusion: Genome analysis of strains from two outbreaks in the 1960s and the 1990s provided valuable information on the genetic diversity and persistence of F. tularensis in Bulgaria

Authors’ reply

Original article in Infection Ecology &amp; Epidemiology, Volume 6, 2016 - Issue 1, DOI: 10.3402/iee.v6.32838 Reply in Infection Ecology &amp; Epidemiology, Volume 7, 2017 - Issue 1. DOI: 10.1080/20008686.2017.1323532</p

Hare-to-Human Transmission of Francisella tularensis subsp. holarctica, Germany

In November 2012, a group of 7 persons who participated in a hare hunt in North Rhine-Westphalia, Germany, acquired tularemia. Two F. tularensis subsp. holarctica isolates were cultivated from human and hare biopsy material. Both isolates belonged to the FTN002–00 genetic subclade (derived for single nucleotide polymorphisms B.10 and B.18), thus indicating likely hare-to-human transmission

Complete Genome Sequence of Francisella guangzhouensis Strain 08HL01032(T), Isolated from Air-Conditioning Systems in China

We present the complete genome sequence of Francisella guangzhouensis strain 08HL01032(T), which consists of one chromosome (1,658,482 bp) and one plasmid ( 3,045 bp) with G+C contents of 32.0% and 28.7%, respectively

Genomic characterization of Francisella tularensis and other diverse Francisella species from complex samples

Francisella tularensis, the bacterium that causes the zoonosis tularemia, and its genetic near neighbor species, can be difficult or impossible to cultivate from complex samples. Thus, there is a lack of genomic information for these species that has, among other things, limited the development of robust detection assays for F. tularensis that are both specific and sensitive. The objective of this study was to develop and validate approaches to capture, enrich, sequence, and analyze Francisella DNA present in DNA extracts generated from complex samples. RNA capture probes were designed based upon the known pan genome of F. tularensis and other diverse species in the family Francisellaceae. Probes that targeted genomic regions also present in non-Francisellaceae species were excluded, and probes specific to particular Francisella species or phylogenetic clades were identified. The capture-enrichment system was then applied to diverse, complex DNA extracts containing low-level Francisella DNA, including human clinical tularemia samples, environmental samples (i.e., animal tissue and air filters), and whole ticks/tick cell lines, which was followed by sequencing of the enriched samples. Analysis of the resulting data facilitated rigorous and unambiguous confirmation of the detection of F. tularensis or other Francisella species in complex samples, identification of mixtures of different Francisella species in the same sample, analysis of gene content (e.g., known virulence and antimicrobial resistance loci), and high-resolution whole genome-based genotyping. The benefits of this capture-enrichment system include: even very low target DNA can be amplified; it is culture-independent, reducing exposure for research and/or clinical personnel and allowing genomic information to be obtained from samples that do not yield isolates; and the resulting comprehensive data not only provide robust means to confirm the presence of a target species in a sample, but also can provide data useful for source attribution, which is important from a genomic epidemiology perspective

Eight New Genomes and Synthetic Controls Increase the Accessibility of Rapid Melt-MAMA SNP Typing of <i>Coxiella burnetii</i>

<div><p>The case rate of Q fever in Europe has increased dramatically in recent years, mainly because of an epidemic in the Netherlands in 2009. Consequently, there is a need for more extensive genetic characterization of the disease agent <i>Coxiella burnetii</i> in order to better understand the epidemiology and spread of this disease. Genome reference data are essential for this purpose, but only thirteen genome sequences are currently available. Current methods for typing <i>C. burnetii</i> are criticized for having problems in comparing results across laboratories, require the use of genomic control DNA, and/or rely on markers in highly variable regions. We developed in this work a method for single nucleotide polymorphism (SNP) typing of <i>C. burnetii</i> isolates and tissue samples based on new assays targeting ten phylogenetically stable synonymous canonical SNPs (canSNPs). These canSNPs represent previously known phylogenetic branches and were here identified from sequence comparisons of twenty-one <i>C. burnetii</i> genomes, eight of which were sequenced in this work. Importantly, synthetic control templates were developed, to make the method useful to laboratories lacking genomic control DNA. An analysis of twenty-one <i>C. burnetii</i> genomes confirmed that the species exhibits high sequence identity. Most of its SNPs (7,493/7,559 shared by >1 genome) follow a clonal inheritance pattern and are therefore stable phylogenetic typing markers. The assays were validated using twenty-six genetically diverse <i>C. burnetii</i> isolates and three tissue samples from small ruminants infected during the epidemic in the Netherlands. Each sample was assigned to a clade. Synthetic controls (vector and PCR amplified) gave identical results compared to the corresponding genomic controls and are viable alternatives to genomic DNA. The results from the described method indicate that it could be useful for cheap and rapid disease source tracking at non-specialized laboratories, which requires accurate genotyping, assay accessibility and inter-laboratory comparisons.</p></div

Phylogenetic tree.

<p>Branch lengths are proportional to the total number of SNPs, which are indicated above or below the branches. Genomes sequenced in this study are in bold face. The genomic groups (I–VII) and plasmid types are denoted as described <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0085417#pone.0085417-Beare1" target="_blank">[21]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0085417#pone.0085417-Glazunova1" target="_blank">[26]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0085417#pone.0085417-Hornstra1" target="_blank">[28]</a>. CanSNP markers are depicted in red (C.1–C.15) and the corresponding MST markers are depicted in grey. The genomes Cb109, Cb175 and Z3055 became available after the time of assay design resulting in four new branches (C.12 to C.15). Although not included in this work, it demonstrates that the systematics of canSNPs easily can be extended when new genomes become available. The ancestral and derived state for C.1 and C.2 could not be determined due to the lack of sequence from a near neighbor (a root). Therefore, we could only design one assay C.1 that could be on either the C.1 or C.2 branches.</p