Evolution of a pathogen: a comparative genomics analysis identifies a genetic pathway to pathogenesis in acinetobacter.

Acinetobacter baumannii is an emergent and global nosocomial pathogen. In addition to A. baumannii, other Acinetobacter species, especially those in the Acinetobacter calcoaceticus-baumannii (Acb) complex, have also been associated with serious human infection. Although mechanisms of attachment, persistence on abiotic surfaces, and pathogenesis in A. baumannii have been identified, the genetic mechanisms that explain the emergence of A. baumannii as the most widespread and virulent Acinetobacter species are not fully understood. Recent whole genome sequencing has provided insight into the phylogenetic structure of the genus Acinetobacter. However, a global comparison of genomic features between Acinetobacter spp. has not been described in the literature. In this study, 136 Acinetobacter genomes, including 67 sequenced in this study, were compared to identify the acquisition and loss of genes in the expansion of the Acinetobacter genus. A whole genome phylogeny confirmed that A. baumannii is a monophyletic clade and that the larger Acb complex is also a well-supported monophyletic group. The whole genome phylogeny provided the framework for a global genomic comparison based on a blast score ratio (BSR) analysis. The BSR analysis demonstrated that specific genes have been both lost and acquired in the evolution of A. baumannii. In addition, several genes associated with A. baumannii pathogenesis were found to be more conserved in the Acb complex, and especially in A. baumannii, than in other Acinetobacter genomes; until recently, a global analysis of the distribution and conservation of virulence factors across the genus was not possible. The results demonstrate that the acquisition of specific virulence factors has likely contributed to the widespread persistence and virulence of A. baumannii. The identification of novel features associated with transcriptional regulation and acquired by clades in the Acb complex presents targets for better understanding the evolution of pathogenesis and virulence in the expansion of the genus

Using Whole Genome Analysis to Examine Recombination across Diverse Sequence Types of Staphylococcus aureus

Staphylococcus aureus is an important clinical pathogen worldwide and understanding this organism\u27s phylogeny and, in particular, the role of recombination, is important both to understand the overall spread of virulent lineages and to characterize outbreaks. To further elucidate the phylogeny of S. aureus, 35 diverse strains were sequenced using whole genome sequencing. In addition, 29 publicly available whole genome sequences were included to create a single nucleotide polymorphism (SNP)-based phylogenetic tree encompassing 11 distinct lineages. All strains of a particular sequence type fell into the same clade with clear groupings of the major clonal complexes of CC8, CC5, CC30, CC45 and CC1. Using a novel analysis method, we plotted the homoplasy density and SNP density across the whole genome and found evidence of recombination throughout the entire chromosome, but when we examined individual clonal lineages we found very little recombination. However, when we analyzed three branches of multiple lineages, we saw intermediate and differing levels of recombination between them. These data demonstrate that in S. aureus, recombination occurs across major lineages that subsequently expand in a clonal manner. Estimated mutation rates for the CC8 and CC5 lineages were different from each other. While the CC8 lineage rate was similar to previous studies, the CC5 lineage was 100-fold greater. Fifty known virulence genes were screened in all genomes in silico to determine their distribution across major clades. Thirty-three genes were present variably across clades, most of which were not constrained by ancestry, indicating horizontal gene transfer or gene loss

A whole genome phylogeny of 136 sequenced genomes in the genus <i>Acinetobacter</i>.

<p>The phylogeny was inferred with FastTree2 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054287#pone.0054287-Price1" target="_blank">[52]</a> on a single nucleotide polymorphism (SNP) matrix alignment calculated with kSNP <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054287#pone.0054287-Gardner1" target="_blank">[50]</a> and filtered with noisy <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054287#pone.0054287-Dress1" target="_blank">[51]</a>. The phylogeny was rooted with <i>A. radioresistens</i>. Genomes sequenced in the current study are shown in red. Genomes in the <i>Acinetobacter calcoaceticus-baumannii</i> (<i>Acb</i>) complex are colored by clade.</p

A heatmap of blast score ratio (BSR) [<b>57</b>] values for efflux pump and beta-lactamase genes identified in <i>Acinetobacter</i>.

<p>BSR values were visualized with the multi-experiment viewer <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054287#pone.0054287-Saeed1" target="_blank">[58]</a>. Accession details for each gene are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054287#pone.0054287.s005" target="_blank">Table S3</a>, with raw data shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054287#pone.0054287.s007" target="_blank">Table S5</a>.</p

Annotation details of lost and acquired genes in the evolution of <i>A. baumannii</i>.

*<p><i>Acb</i> = <i>Acinetobacter calcoaceticus-baumannii</i>, b = <i>baumannii</i>, n = <i>nosocomialis</i>.</p

A heatmap of blast score ratio (BSR) [<b>57</b>] values for branch specific regions in the <i>Acb</i> complex.

<p>BSR values were visualized with the multi-experiment viewer <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054287#pone.0054287-Saeed1" target="_blank">[58]</a>. Samples were clustered using an average linkage clustering algorithm. Numbers for each feature correlate with features described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054287#pone-0054287-t001" target="_blank">Table 1</a>. Raw data values are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054287#pone.0054287.s007" target="_blank">Table S5</a>.</p

Best Practices for Evaluating Single Nucleotide Variant Calling Methods for Microbial Genomics

Innovations in sequencing technologies have allowed biologists to make incredible advances in understanding biological systems. As experience grows, researchers increasingly recognize that analyzing the wealth of data provided by these new sequencing platforms requires careful attention to detail for robust results. Thus far, much of the scientific communities’ focus for use in bacterial genomics has been on evaluating genome assembly algorithms and rigorously validating assembly program performance. Missing, however, is a focus on critical evaluation of variant callers for these genomes. Variant calling is essential for comparative genomics as it yields insights into nucleotide-level organismal differences. Variant calling is a multistep process with a host of potential error sources that may lead to incorrect variant calls. Identifying and resolving these incorrect calls is critical for bacterial genomics to advance. The goal of this review is to provide guidance on validating algorithms and pipelines used in variant calling for bacterial genomics. First, we will provide an overview of the variant calling procedures and the potential sources of error associated with the methods. We will then identify appropriate datasets for use in evaluating algorithms and describe statistical methods for evaluating algorithm performance. As variant calling moves from basic research to the applied setting, standardized methods for performance evaluation and reporting are required; it is our hope that this review provides the groundwork for the development of these standards

SupportinglnformationS2

S2. Maximum parsimony tree using seven parsimony informative (synapomorphic) SNPs with loci shared with closest outgroup species. Only one SNP locus (out of 11,386 total SNPs among C. burnetii genomes) was present in all genomes. The remaining six loci were found by relaxing the requirement that all loci are shared among all C. burnetii genomes as they were not present in the Q177 genome. All seven SNP loci were present in Pseudomonas syringae and Legionella pneumophila. Four of the seven loci were present in Ricketsiella grylli. Consistency index = 1.0. Numbers on branches indicate bootstrap support percentages from 1000 bootstrap replicates

Data from: When outgroups fail; phylogenomics of rooting the emerging pathogen, Coxiella burnetii

Rooting phylogenies is critical for understanding evolution, yet the importance, intricacies and difficulties of rooting are often overlooked. For rooting, polymorphic characters among the group of interest (ingroup) must be compared to those of a relative (outgroup) that diverged before the last common ancestor (LCA) of the ingroup. Problems arise if an outgroup does not exist, is unknown, or is so distant that few characters are shared, in which case duplicated genes originating before the LCA can be used as proxy outgroups to root diverse phylogenies. Here, we describe a genome-wide expansion of this technique that can be used to solve problems at the other end of the evolutionary scale: where ingroup individuals are all very closely related to each other, but the next closest relative is very distant. We used shared orthologous single nucleotide polymorphisms (SNPs) from 10 whole genome sequences of Coxiella burnetii, the causative agent of Q fever in humans, to create a robust, but unrooted phylogeny. To maximize the number of characters informative about the rooting, we searched entire genomes for polymorphic duplicated regions where orthologs of each paralog could be identified so that the paralogs could be used to root the tree. Recent radiations, such as those of emerging pathogens, often pose rooting challenges due to a lack of ingroup variation and large genomic differences with known outgroups. Using a phylogenomic approach, we created a robust, rooted phylogeny for C. burnetii