Multiple variants of IncF plasmid alleles discovered within single bacterial cells challenge previous assumptions

IncF plasmids are diverse 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 compares the sequences of IncF alleles against a database to create a plasmid sequence type (ST). Accurate identification of plasmid STs is epidemiologically useful because it enables an assessment of the crucial IncF plasmid lineages associated with pandemic and emerging enterobacterial sequence types, inferring important information about specific bacterial lineages. 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 publicly available repository of 70324 E. coli genomes of various STs and other Enterobacterales genomes (n=1247). All short-read genomes and representatives selected for long-read sequencing were used to assess allele variants and to compare the output with the real biological situation. 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 5804 of 72469 genomes (8.01%). Long read sequencing of 27 carefully selected genomes confirmed multiple IncF allele variants present on one plasmid, or two separate IncF plasmids present in a single bacterial cell. The pMLST tool was unable to accurately distinguish allele variants and their location on replicons using short-read nor long-read genome sequences.