Limited Genetic Diversity of blaCMY-2-Containing IncI1-pST12 Plasmids from Enterobacteriaceae of Human and Broiler Chicken Origin in The Netherlands.

Evert Drijver,Marjolein Bergh,Francisca Velkers,Joep Stohr,Arjan Stegeman,Jaco Verweij,Carlo Verhulst,Jan Kluytmans,I---Health Group

doi:10.3390/microorganisms8111755

Abstract

Distinguishing epidemiologically related and unrelated plasmids is essential to confirm plasmid transmission. We compared IncI1–pST12 plasmids from both human and livestock origin and explored the degree of sequence similarity between plasmids from Enterobacteriaceae with different epidemiological links. Short-read sequence data of Enterobacteriaceae cultured from humans and broilers were screened for the presence of both a blaCMY-2 gene and an IncI1–pST12 replicon. Isolates were long-read sequenced on a MinION sequencer (OxfordNanopore Technologies). After plasmid reconstruction using hybrid assembly, pairwise single nucleotide polymorphisms (SNPs) were determined. The plasmids were annotated, and a pan-genome was constructed to compare genes variably present between the different plasmids. Nine Escherichia coli sequences of broiler origin, four Escherichia coli sequences, and one Salmonella enterica sequence of human origin were selected for the current analysis. A circular contig with the IncI1–pST12 replicon and blaCMY-2 gene was extracted from the assembly graph of all fourteen isolates. Analysis of the IncI1–pST12 plasmids revealed a low number of SNP differences (range of 0–9 SNPs). The range of SNP differences overlapped in isolates with different epidemiological links. One-hundred and twelve from a total of 113 genes of the pan-genome were present in all plasmid constructs. Next generation sequencing analysis of blaCMY-2-containing IncI1–pST12 plasmids isolated from Enterobacteriaceae with different epidemiological links show a high degree of sequence similarity in terms of SNP differences and the number of shared genes. Therefore, statements on the horizontal transfer of these plasmids based on genetic identity should be made with caution.

Highlights

Antimicrobial resistance in Gram-negative bacteria is a worldwide growing public health problem [1,2]
The objective of the current study is to determine the relatedness between Incompatibility group I1 (IncI1)–pST12 plasmids of epidemiologically related and unrelated Enterobacteriaceae isolates from humans and livestock, and we explore the possibility of accurately distinguishing related from unrelated samples based on plasmid sequencing data alone
A small single nucleotide polymorphisms (SNPs) difference was seen between epidemiologically related strains with a maximum difference of two SNPs

Summary

Introduction

Antimicrobial resistance in Gram-negative bacteria is a worldwide growing public health problem [1,2]. Often shared between plasmid and chromosomal DNA, hinder the assembly of the bacterial genome from short-read data, often resulting in contigs of which the origin, either plasmid or chromosomal, cannot be resolved [17] This limits the interpretation of plasmid transmission by not providing accurate prediction of the total plasmid sequence. Everything considered, the amount of studies using combined short- and long-read sequencing data of IncI1-pST12 plasmids from human and livestock origin is still limited. We hypothesize that a combination of short- and long-read sequence data of blaCMY-2 containing IncI1–pST12 plasmids reveal highly conserved plasmid sequencing, which complicates distinguishing plasmid transmission between epidemiologically related and unrelated isolates. The objective of the current study is to determine the relatedness between IncI1–pST12 plasmids of epidemiologically related and unrelated Enterobacteriaceae isolates from humans and livestock, and we explore the possibility of accurately distinguishing related from unrelated samples based on plasmid sequencing data alone

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Conclusion