Methylation analysis of Klebsiella pneumoniae from Portuguese hospitals

Anton Spadar,James Charleston,Susana Campino,Rita Elias,Martin L Hibberd,Paola Florez De Sessions,João Perdigão,Aida Duarte,Jody Phelan,Ana Modesto,Taane G Clark

doi:10.1038/s41598-021-85724-2

Abstract

Klebsiella pneumoniae is an important nosocomial infectious agent with a high antimicrobial resistance (AMR) burden. The application of long read sequencing technologies is providing insights into bacterial chromosomal and putative extra-chromosomal genetic elements (PEGEs) associated with AMR, but also epigenetic DNA methylation, which is thought to play a role in cleavage of foreign DNA and expression regulation. Here, we apply the PacBio sequencing platform to eight Portuguese hospital isolates, including one carbapenemase producing isolate, to identify methylation motifs. The resulting assembled chromosomes were between 5.2 and 5.5Mbp in length, and twenty-six PEGEs were found. Four of our eight samples carry blaCTX-M-15, a dominant Extended Spectrum Beta Lactamase in Europe. We identified methylation motifs that control Restriction–Modification systems, including GATC of the DNA adenine methylase (Dam), which methylates N6-methyladenine (m6A) across all our K. pneumoniae assemblies. There was a consistent lack of methylation by Dam of the GATC motif downstream of two genes: fosA, a locus associated with low level fosfomycin resistance, and tnpB transposase on IncFIB(K) plasmids. Overall, we have constructed eight high quality reference genomes of K. pneumoniae, with insights into horizontal gene transfer and methylation m6A motifs.

Highlights

Klebsiella pneumoniae is an important nosocomial infectious agent with a high antimicrobial resistance (AMR) burden
To put our samples in a broader context we constructed a maximum likelihood phylogenetic tree based on the alignment of seven multi-locus sequence types (MLSTs) informative and nineteen core genome loci, which contained 83 representative Klebsiella pneumoniae (Kp) MLST groups sourced from the NCBI database (Fig. 1)[21]
Across seven Kp isolates with high quality PacBio data, we identified the common DNA adenine methylase (Dam) based methylation mechanism involving GATC motifs, as well as several type I R–M systems

Summary

Introduction

Klebsiella pneumoniae is an important nosocomial infectious agent with a high antimicrobial resistance (AMR) burden. The application of long read sequencing technologies is providing insights into bacterial chromosomal and putative extra-chromosomal genetic elements (PEGEs) associated with AMR, and epigenetic DNA methylation, which is thought to play a role in cleavage of foreign DNA and expression regulation. We identified methylation motifs that control Restriction–Modification systems, including GATC of the DNA adenine methylase (Dam), which methylates N6-methyladenine (m6A) across all our K. pneumoniae assemblies. Klebsiella pneumoniae (Kp) are Gram-negative bacteria that are found in the normal flora of the mouth, intestines, skin and faeces, but in other parts of the body, such as the lungs, can cause severe morbidity with a diverse disease spectrum that can culminate in complicated invasive infections This pathogen is increasingly recognized as an important etiological agent of healthcare associated infections. While not part of complete type II R–M, Dam is the most common MTase in Gamma-proteobacteria and it recognises palindromic 5′-GATC-3′ motifs[4,8]

Methods

Results

Conclusion