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WGS of Klebsiella Pathogen IDs Species Evolution, Antimicrobial Resistance and Virulence Genes

NEW YORK (GenomeWeb) – Researchers from the University of Melbourne and the Wellcome Trust Sanger Institute have sequenced the whole genomes of 300 isolates of the pathogen Klebsiella pneumoniae in order to gain a population-wide understanding of the pathogen and how it is evolving and developing antimicrobial resistance.

The sequencing and analysis was published today in the Proceedings of the National Academy of the Sciences. In the study, the researchers found that K. pneumoniae has split into three distinct species and that the species most commonly associated with human infection has developed multiple hypervirulent or multi-drug resistant clones.

Multi-drug resistant pathogens have become an increasing problem particularly in hospital settings. According to a 2013 report by the US Centers for Disease Control and Prevention, an estimated 7,900 individuals were infected with drug-resistant Klebsiella in healthcare settings in the US, leading to 520 deaths. The CDC now classifies drug-resistant Klebsiella as an "urgent threat."

In order to better understand how the pathogen has evolved and its population structure, the researchers sequenced 300 human and animal isolates collected from four different continents. They also conducted a pangenome-wide association study to look for links between the gene repertoire and disease potential and/or outcome and also to identify genes associated with virulence.

Their sequencing data supports the theory that Klebsiella has split into three different species — K. pneumoniae (KpI), K. quasipneumoniae (KpII), and K. variicola (KpIII). In addition, for KpI, the species most associated with human infection, the team identified more than 150 lineages, including numerous multidrug resistant and hypervirulent clones. They also identified nearly 30,000 genes, including genes specific to hospital-acquired infections and isolates carried by humans, as well as different genes that were associated with invasive disease.

The team sequenced 288 isolates and compared them to an additional 40 publicly available whole-genome sequences. They identified 1,743 core genes that were conserved in all genomes with over 175,000 SNPs. A network analysis of the SNPs identified four phylogroups — KpI, KpII-A, KpII-B, and KpIII.

The team identified a pangenome of nearly 30,000 genes, including 1,888 genes that were shared in more than 95 percent of the genomes from each phylogroup. Each individual isolate also carried a median of 3,817 accessory genes for a total median of 5,705 genes per genome.

Looking at the relationship between the phylogroups, the team found that nucleotide divergence between the phylogroups was 3 percent to 4 percent, while divergence within the phylogroups was just .5 percent. In addition, there was little evidence of homologous recombination between the phylogroups, providing "whole-genome support for the proposal that KpI, KpII, and KpIII are distinct species … that are evolving independently," the authors wrote.

Next, the team looked more closely at the KpI isolates, which are most often associated with human infection. Genetic loci have previously been identified as virulent, including gene clusters associated with siderophore systems; the rmpA and rmpA2 genes, which upregulate capsule production; an allantoinase gene cluster; the operon kfuABC, which is associated with iron transportation; and the regulator kvgAS. All but the kvgAS gene were detected solely in the KpI isolates.

The PGWAS found that the rmpA/2 and siderophore genes, and five additional genes predicted to be involved in iron metabolism were the most closely associated with human infection. These genes were present in 75 percent of isolates from community-acquired infections, but rare among other isolates like ones from hospital-acquired infections, present in less than 10 percent of those isolates, suggesting that iron access may be required for the bacteria to cause infection in humans that are not immunocompromised. The team then identified several KpI clones that were significantly enriched for siderophores and/or rmpA genes, including a clone called ST23 that is known to be able to cause especially severe disease in otherwise healthy individuals.

The team also focused in on antimicrobial-resistant genes, identifying 84 genes that conferred antimicrobial resistance. A total of 150 out of 288 isolates carried at least one resistance gene. The distribution of the genes varied, with many associated with geographic location, likely due to the specific drugs used in various locations.

Both isolates from hospital-acquired infections as well as those that were simply colonizers without causing infection contained antimicrobial-resistant genes.

Given the wide range of antimicrobial-resistant genes and the pool of clones that are virulent, there is the "potential for the emergence of an extremely drug-resistant (XDR), hypervirulent K. pneumoniae clone capable of causing severe, untreatable, infections in healthy individuals," the authors wrote. Indeed, there is already evidence of such clones emerging, including hypervirulent clones that have developed antimicrobial resistant genes in China, Madagascar, and Brazil.

The authors deposited the genomic data into the BIGSdb database to facilitate further study. And while the study is a first step, it will "provide a critical foundation and practical support for future studies investigating ecological niche adaptation, pathogenicity, and lineage diversification in K. pneumoniae and will facilitate more deeply informed genomic tracking and surveillance for the emergence and convergence of virulence and AMR in this increasingly important pathogen," the authors wrote.