The presence of multiple variants of IncF plasmid alleles in a single genome sequence can hinder accurate replicon sequence typing using in silico pMLST tools.

IncF plasmids are mobile genetic elements found in bacteria from the Enterobacteriaceae family and often carry critical antibiotic and virulence gene cargo. The classification of IncF plasmids using the plasmid Multi-Locus Sequence Typing (pMLST) tool from the Center for Genomic Epidemiology (CGE; https://www.genomicepidemiology.org/) compares the sequences of IncF alleles against a database to create a plasmid sequence type (ST). Accurate identification of plasmid STs is useful as it enables an assessment of IncF plasmid lineages associated with pandemic enterobacterial STs. Our initial observations showed discrepancies in IncF allele variants reported by pMLST in a collection of 898 Escherichia coli ST131 genomes. To evaluate the limitations of the pMLST tool, we interrogated an in-house and public repository of 70,324 E. coli genomes of various STs and other Enterobacteriaceae genomes (n = 1247). All short-read assemblies and representatives selected for long-read sequencing were used to assess pMLST allele variants and to compare the output of pMLST tool versions. When multiple allele variants occurred in a single bacterial genome, the Python and web versions of the tool randomly selected one allele to report, leading to limited and inaccurate ST identification. Discrepancies were detected in 5,804 of 72,469 genomes (8.01%). Long-read sequencing of 27 genomes confirmed multiple IncF allele variants on one plasmid or two separate IncF plasmids in a single bacterial cell. The pMLST tool was unable to accurately distinguish allele variants and their location on replicons using short-read genome assemblies, or long-read genome assemblies if the same allele variant was present more than once.

Importance: Plasmid sequence type is crucial for describing IncF plasmids due to their capacity to carry important antibiotic and virulence gene cargo and consequently due to their association with disease-causing enterobacterial lineages exhibiting resistance to clinically relevant antibiotics in humans and food-producing animals. As a result, precise reporting of IncF allele variants in IncF plasmids is necessary. Comparison of the FAB formulae generated by the pMLST tool with annotated long-read genome assemblies identified inconsistencies, including examples where multiple IncF allele variants were present on the same plasmid but missing in the FAB formula, or in cases where two IncF plasmids were detected in one bacterial cell, and the pMLST output provided information only about one plasmid. Such inconsistencies may cloud interpretation of IncF plasmid replicon type in specific bacterial lineages or inaccurate assumptions of host strain clonality.