Outcome of Different Sequencing and Assembly Approaches on the Detection of Plasmids and Localization of Antimicrobial Resistance Genes in Commensal Escherichia coli

Abstract

Antimicrobial resistance (AMR) is a major threat to public health worldwide. Currently, AMR typing changes from phenotypic testing to whole-genome sequence (WGS)-based detection of resistance determinants for a better understanding of the isolate diversity and elements involved in gene transmission (e.g., plasmids, bacteriophages, transposons). However, the use of WGS data in monitoring purposes requires suitable techniques, standardized parameters and approved guidelines for reliable AMR gene detection and prediction of their association with mobile genetic elements (plasmids). In this study, different sequencing and assembly strategies were tested for their suitability in AMR monitoring in Escherichia coli in the routines of the German National Reference Laboratory for Antimicrobial Resistances. To assess the outcomes of the different approaches, results from in silico predictions were compared with conventional phenotypic- and genotypic-typing data. With the focus on (fluoro)quinolone-resistant E.coli, five qnrS-positive isolates with multiple extrachromosomal elements were subjected to WGS with NextSeq (Illumina), PacBio (Pacific BioSciences) and ONT (Oxford Nanopore) for in depth characterization of the qnrS1-carrying plasmids. Raw reads from short- and long-read sequencing were assembled individually by Unicycler or Flye or a combination of both (hybrid assembly). The generated contigs were subjected to bioinformatics analysis. Based on the generated data, assembly of long-read sequences are error prone and can yield in a loss of small plasmid genomes. In contrast, short-read sequencing was shown to be insufficient for the prediction of a linkage of AMR genes (e.g., qnrS1) to specific plasmid sequences. Furthermore, short-read sequencing failed to detect certain duplications and was unsuitable for genome finishing. Overall, the hybrid assembly led to the most comprehensive typing results, especially in predicting associations of AMR genes and mobile genetic elements. Thus, the use of different sequencing technologies and hybrid assemblies currently represents the best approach for reliable AMR typing and risk assessment.

Complete genome for Actinobacillus pleuropneumoniae serovar 8 reference strain 405: comparative analysis with draft genomes for different laboratory stock cultures indicates little genetic variation

“…Our comparison of the Illumina-and PacBio-generated genomes for the original stock 405D culture revealed no SNPs between these sequences, indicating comparable accuracy with both platforms. It has also been previously reported that small plasmid sequences may not be identified using the PacBio platform [52,53], and this was seen in our results, with sequences for two small plasmids (pARD3079 and p405tetH) found in all of the draft genomes, but not the PacBio sequence. It should be noted that the presence of the two plasmids will not affect the use of the strains as serovar controls, but has the potential to introduce variation into other types of studies, e.g.…”

Phenotypic and Genotypic Properties of Fluoroquinolone-Resistant, qnr-Carrying Escherichia coli Isolated from the German Food Chain in 2017

“…Detection of qnr gene-carrying fragments was performed by Southern blotting and DNA-DNA hybridisation of S1-PFGE agarose gels. Hybridisation was conducted using digoxigenin-labelled (Roche Diagnostics, Mannheim-Penzberg, Germany) PCR probes of qnr genes and were prepared as previously described [ 23 ] ( Table S1 ).…”

Antibiotic-resistant bacteria, antibiotic resistance genes, and antibiotic residues in wastewater from a poultry slaughterhouse after conventional and advanced treatments

“…Clinically associated fluoroquinolone resistance is mainly caused by chromosomal mutations in the quinolone resistance-determining regions (QRDRs) of gyrA and parC 22 , 23 . Furthermore, the cooccurrence of plasmid-mediated quinolone resistance (PMQR) genes and QRDR mutations can lead to high resistance (fluoro)quinolone phenotypes 24 . Recently, high percentages of ciprofloxacin resistance in ESBL-producing E. coli (50.5%) and K. pneumoniae (67.9%) from process and wastewater of poultry slaughterhouses have been reported 17 .…”