Whole-genome analysis of mycobacteria from birds at the San Diego Zoo

<div><p>Methods</p><p>Mycobacteria isolated from more than 100 birds diagnosed with avian mycobacteriosis at the San Diego Zoo and its Safari Park were cultured postmortem and had their whole genomes sequenced. Computational workflows were developed and applied to identify the mycobacterial species in each DNA sample, to find single-nucleotide polymorphisms (SNPs) between samples of the same species, to further differentiate SNPs between as many as three different genotypes within a single sample, and to identify which samples are closely clustered genomically.</p><p>Results</p><p>Nine species of mycobacteria were found in 123 samples from 105 birds. The most common species were <i>Mycobacterium avium</i> and <i>Mycobacterium genavense</i>, which were in 49 and 48 birds, respectively. Most birds contained only a single mycobacterial species, but two birds contained a mixture of two species. The <i>M</i>. <i>avium</i> samples represent diverse strains of <i>M</i>. <i>avium avium</i> and <i>M</i>. <i>avium hominissuis</i>, with many pairs of samples differing by hundreds or thousands of SNPs across their common genome. By contrast, the <i>M</i>. <i>genavense</i> samples are much closer genomically; samples from 46 of 48 birds differ from each other by less than 110 SNPs. Some birds contained two, three, or even four genotypes of the same bacterial species. Such infections were found in 4 of 49 birds (8%) with <i>M</i>. <i>avium</i> and in 11 of 48 birds (23%) with <i>M</i>. <i>genavense</i>. Most were mixed infections, in which the bird was infected by multiple mycobacterial strains, but three infections with two genotypes differing by ≤ 10 SNPs were likely the result of within-host evolution. The samples from 31 birds with <i>M</i>. <i>avium</i> can be grouped into nine clusters within which any sample is ≤ 12 SNPs from at least one other sample in the cluster. Similarly, the samples from 40 birds with <i>M</i>. <i>genavense</i> can be grouped into ten such clusters. Information about these genomic clusters is being used in an ongoing, companion study of mycobacterial transmission to help inform management of bird collections.</p></div

Mycobacterial species found by WGS in 123 samples from 105 birds.

<p>Mycobacterial species found by WGS in 123 samples from 105 birds.</p

Tree for 46 <i>M</i>. <i>genavense</i> samples.

<p>Each sample or subsample in one of the colored clusters is ≤ 12 SNPs from at least one other sample in the cluster. Although not shown on the tree, the bootstrap support values are between 97 and 100% for most but not all of the nodes outside of the colored clusters. Thus the overall tree topology is not quite as robust as that for the <i>M</i>. <i>avium</i> trees, presumably because the relevant branch lengths measured in SNPs are much shorter in the <i>M</i>. <i>genavense</i> tree.</p

Number of genotypes per bird showing the presence of mixed infections and within-host evolution.

<p>Number of genotypes per bird showing the presence of mixed infections and within-host evolution.</p

Concordance in species identification between WGS and conventional tests for 132 samples.

<p>Concordance in species identification between WGS and conventional tests for 132 samples.</p

Tree for 53 <i>M</i>. <i>avium</i> samples.

<p>Each sample or subsample in one of the colored clusters is ≤ 12 SNPs from at least one other sample in the cluster. The two clades above and below the arbitrary root have very different shapes. The upper clade is spread out with some samples separated from each other by nearly 10,000 SNPs. By contrast, the separation between the samples in the lower clade is much less and is better resolved in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0173464#pone.0173464.g002" target="_blank">Fig 2</a>. Based on matches to the NCBI database, the 39 samples in the lower clade are all <i>M</i>. <i>avium avium</i>, while the 14 samples in the upper clade all seem to be <i>M</i>. <i>avium hominissuis</i>.</p

Tree for 39 <i>M</i>. <i>avium avium</i> samples.

<p>Each sample or subsample in one of the colored clusters is ≤ 12 SNPs from at least one other sample in the cluster. Although not shown on the trees, the bootstrap support values are 100% at all of the nodes outside of the colored clusters. Thus the overall tree topology is very robust.</p