Plasmidome-Analysis of ESBL-Producing Escherichia coli Using Conventional Typing and High-Throughput Sequencing

Abstract

Infections caused by Extended spectrum β-lactamase (ESBL)-producing E. coli are an emerging global problem, threatening the effectiveness of the extensively used β-lactam antibiotics. ESBL dissemination is facilitated by plasmids, transposons, and other mobile elements. We have characterized the plasmid content of ESBL-producing E. coli from human urinary tract infections. Ten diverse isolates were selected; they had unrelated pulsed-field gel electrophoresis (PFGE) types (<90% similarity), were from geographically dispersed locations and had diverging antibiotic resistance profiles. Three isolates belonged to the globally disseminated sequence type ST131. ESBL-genes of the CTX-M-1 and CTX-M-9 phylogroups were identified in all ten isolates. The plasmid content (plasmidome) of each strain was analyzed using a combination of molecular methods and high-throughput sequencing. Hidden Markov Model-based analysis of unassembled sequencing reads was used to analyze the genetic diversity of the plasmid samples and to detect resistance genes. Each isolate contained between two and eight distinct plasmids, and at least 22 large plasmids were identified overall. The plasmids were variants of pUTI89, pKF3-70, pEK499, pKF3-140, pKF3-70, p1ESCUM, pEK204, pHK17a, p083CORR, R64, pLF82, pSFO157, and R721. In addition, small cryptic high copy-number plasmids were frequent, containing one to seven open reading frames per plasmid. Three clustered groups of such small cryptic plasmids could be distinguished based on sequence similarity. Extrachromosomal prophages were found in three isolates. Two of them resembled the E. coli P1 phage and one was previously unknown. The present study confirms plasmid multiplicity in multi-resistant E. coli. We conclude that high-throughput sequencing successfully provides information on the extrachromosomal gene content and can be used to generate a genetic fingerprint of possible use in epidemiology. This could be a valuable tool for tracing plasmids in outbreaks.

Characterization of ESBL disseminating plasmids

“…Second, when several related but compatible plasmids are present in the same cell it is not always possible to discriminate which plasmid a shared repeat region belongs to using only sequencing data. Combination of S1/PFgE of plasmids to calculate the plasmid number and sizes and thereafter Southern blotting or PCr to sort out which plasmid is of what replicon type and perhaps which of the plasmids that encode a particular gene of interest can then be used [59]. To cope with repetitive regions sequencing techniques that generate long sequence reads are preferred over high coverage.…”

Whole-Genome Sequencing Identifies In Vivo Acquisition of a bla CTX-M-27 -Carrying IncFII Transmissible Plasmid as the Cause of Ceftriaxone Treatment Failure for an Invasive Salmonella enterica Serovar Typhimurium Infection

“…The ST882S/ST931R virulence plasmid is 100% identical to eight contigs (99.8% coverage) for CVM N42450, covering all of the above SNPs and the deletion; unfor-tunately, coverage for the repeat sequence is lacking. The smaller plasmid was identical to pEC147-3 (JX238454), a cryptic plasmid discovered in a plasmidome analysis of a set of ESBL-producing Escherichia coli isolates from urinary tract infections (52).…”

Direct identification of antibiotic resistance genes on single plasmid molecules using CRISPR/Cas9 in combination with optical DNA mapping

“…Plasmid pEC019 has previously been characterized by traditional methods such as S1/PFGE and whole genome sequencing20. By S1/PFGE the isolate was shown to contain one plasmid with an estimated size of 150 kbp (+/−5 kbp) and the technique required four full working days for completion.…”