Strategy for Sensitive and Specific Detection of <i>Yersinia pestis</i> in Skeletons of the Black Death Pandemic

<div><p><i>Yersinia pestis</i> has been identified as the causative agent of the Black Death pandemic in the 14^th century. However, retrospective diagnostics in human skeletons after more than 600 years are critical. We describe a strategy following a modern diagnostic algorithm and working under strict ancient DNA regime for the identification of medieval human plague victims. An initial screening and DNA quantification assay detected the <i>Y. pestis</i> specific <i>pla</i> gene of the high copy number plasmid pPCP1. Results were confirmed by conventional PCR and sequence analysis targeting both <i>Y. pestis</i> specific virulence plasmids pPCP1 and pMT1. All assays were meticulously validated according to human clinical diagnostics requirements (ISO 15189) regarding efficiency, sensitivity, specificity, and limit of detection (LOD). Assay specificity was 100% tested on 41 clinically relevant bacteria and 29 <i>Y. pseudotuberculosis</i> strains as well as for DNA of 22 <i>Y. pestis</i> strains and 30 previously confirmed clinical human plague samples. The optimized LOD was down to 4 gene copies. 29 individuals from three different multiple inhumations were initially assessed as possible victims of the Black Death pandemic. 7 samples (24%) were positive in the pPCP1 specific screening assay. Confirmation through second target pMT1 specific PCR was successful for 4 of the positive individuals (14%). A maximum of 700 and 560 copies per µl aDNA were quantified in two of the samples. Those were positive in all assays including all repetitions, and are candidates for future continuative investigations such as whole genome sequencing. We discuss that all precautions taken here for the work with aDNA are sufficient to prevent external sample contamination and fulfill the criteria of authenticity. With regard to retrospective diagnostics of a human pathogen and the uniqueness of ancient material we strongly recommend using a careful strategy and validated assays as presented in our study.</p> </div

qPCR targeting <i>Y. pestis pla</i> gene screening ancient samples.

<p>qPCR targeting <i>Y. pestis pla</i> gene screening ancient samples.</p

Schematic workflow for the proceeding through highly optimized and validated protocols for the detection of <i>Y. pestis</i> from ancient samples.

<p>Schematic workflow for the proceeding through highly optimized and validated protocols for the detection of <i>Y. pestis</i> from ancient samples.</p

Amino acid profile of specimen MP 59-I, channel 1 (570 nm) and 2 (440 nm).

<p>The profile represents a typical collagenous pattern with a high amino acid yield. However, aspartic and glutamic acid concentrations are significantly reduced. At 72 min an unidentified peak was detected that did not show up in the other samples. </p

CRISPR genotypes.

<p>CRISPR genotypes.</p

MLVA clustering and SNP branch assignment of 66 previously published <i>Y. pestis microtus</i> and <i>pestis</i> 0, 1 and 3 branches.

<p><i>Microtus</i> and strains from the 0 and 1 branches so far investigated by MLVA25 and by SNP analysis are shown <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030624#pone.0030624-Li1" target="_blank">[5]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030624#pone.0030624-Morelli1" target="_blank">[6]</a>. Three Ulegeica, two Hissarica and nine Altaica strains not investigated by SNP analysis are also included. For completion, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030624#pone-0030624-t001" target="_blank">Table 1</a> gives further information about assignment of biovar, genotype, and origin. Colors reflect MLVA clustering as suggested by Li et al. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030624#pone.0030624-Li1" target="_blank">[5]</a>. The SNP branch assignment of each strain as defined by Morelli et al. is indicated (column <i>Morelli2010</i>) together with the strain ID and biovar designation <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030624#pone.0030624-Morelli1" target="_blank">[6]</a>. The results of CRISPR analysis according to Cui et al. are shown in column <i>group </i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030624#pone.0030624-Cui1" target="_blank">[18]</a>. Bootstrap support values are indicated. The figure shows the satisfying terminal branches clustering achieved by MLVA but the sometimes incorrect and usually low bootstrap values of deep branching nodes illustrating the complementarity of the two methods.</p

MLVA25 assignment of four clusters of the investigated Mongolian <i>Y. pestis</i> strains.

<p>MLVA25 tree of 16 investigated Mongolian <i>Y. pestis</i> strains (marked with color and boxes) representing four of the 6 clusters, and various <i>Y. pestis</i> strains originating from <i>microtus</i> and <i>pestis</i> biovars. For each strain, the tentative SNP branch or node according to Morelli et al. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030624#pone.0030624-Morelli1" target="_blank">[6]</a> as deduced by the presence of a linking strain in the same MLVA cluster is indicated by a question mark. Strain name, CRISPR profile as investigated in this study, and the sampling site (Focus) are listed.</p

MLVA clustering and SNP branch assignment of 68 previously published <i>Y. pestis pestis</i> branches 1 and 2.

<p>Sixty-eight strains from the 1 and 2 branches previously investigated by both MLVA25 and SNP analysis are displayed <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030624#pone.0030624-Li1" target="_blank">[5]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030624#pone.0030624-Morelli1" target="_blank">[6]</a>. For completion, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030624#pone-0030624-t001" target="_blank">Table 1</a> gives further information about assignment of biovar, genotype, and origin. Colors reflect MLVA clustering as suggested by Li et al. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030624#pone.0030624-Li1" target="_blank">[5]</a>. The SNP branch assignment of each strain as defined by Morelli et al. is indicated (column <i>Morelli2010</i>) together with the strain ID and biovar designation <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030624#pone.0030624-Morelli1" target="_blank">[6]</a>. Bootstrap support values are indicated for each node. The results of CRISPR analysis according to Cui et al. are given in column <i>group </i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030624#pone.0030624-Cui1" target="_blank">[18]</a>. * This strain shows a Medievalis phenotype due to a different mutation in the napA gene compared to the mutation causing the Medievalis phenotype in the Medievalis biovar, as demonstrated by Pourcel et al. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030624#pone.0030624-Pourcel1" target="_blank">[13]</a>.</p

Protospacers for newly identified spacers a6′, a85–88, and b48–49.

<p>Protospacers for newly identified spacers a6′, a85–88, and b48–49.</p

Overview of <i>Y. pestis</i> subspecies, biovar, genotype, and natural foci as suggested by different authors [4], [5], [6], [7], [18], and as deduced in this study.

<p>*abbreviations as defined by Achtman et al. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030624#pone.0030624-Achtman1" target="_blank">[7]</a> and Morelli et al. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030624#pone.0030624-Morelli1" target="_blank">[6]</a>: PE – pestoides (<i>microtus</i>), ANT – Antiqua, IN – Intermedium, ORI – Orientalis, and MED – Medievalis; Intermedium in Morelli et al. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030624#pone.0030624-Morelli1" target="_blank">[6]</a> has not the same meaning as intermedium defined by Li et al. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030624#pone.0030624-Li1" target="_blank">[5]</a> which refers to Rhamnose positive <i>Y. pestis pestis</i> isolates.</p><p># prefix refers to foci as described by Anisimov et al. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030624#pone.0030624-Anisimov1" target="_blank">[4]</a>. Numbers without # refer to Mongolian foci as shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030624#pone-0030624-g004" target="_blank">Figure 4</a>.</p