Empirical genomic methods for tracking plasmid spread among healthcare-associated bacteria

Abstract

Background: Healthcare-associated bacterial pathogens frequently carry plasmids that contribute to antibiotic resistance and virulence. The horizontal transfer of plasmids in healthcare settings has been previously documented, but genomic and epidemiologic methods to study this phenomenon remain underdeveloped. The objectives of this study were to develop a method to systematically resolve and track plasmids circulating in a single hospital, and to identify epidemiologic links that indicated likely horizontal plasmid transfer. Methods: We derived empirical thresholds of plasmid sequence similarity from comparisons of plasmids carried by bacterial isolates infecting individual patients over time, or involved in hospital outbreaks. We then applied those metrics to perform a systematic screen of 3,074 genomes of nosocomial bacterial isolates from a single hospital for the presence of 89 plasmids. We also collected and reviewed data from electronic health records for evidence of geotemporal associations between patients infected with bacteria encoding plasmids of interest. Findings: Our analyses determined that 95% of analyzed genomes maintained roughly 95% of their plasmid genetic content at a nucleotide identity at least 99.985%. Applying these similarity thresholds to identify horizontal plasmid transfer identified 45 plasmids circulating among clinical isolates. Ten plasmids met criteria for geotemporal links associated with horizontal transfer. Several plasmids with shared backbones also encoded different additional mobile genetic element content, and these elements were variably present among the sampled clinical isolate genomes. Interpretation: The horizontal transfer of plasmids among nosocomial bacterial pathogens is frequent within hospitals and can be monitored with whole genome sequencing and comparative genomics approaches. These approaches should incorporate both nucleotide identity and reference sequence coverage to study the dynamics of plasmid transfer in the hospital. Funding: This research was supported by the US National Institute of Allergy and Infectious Disease (NIAID) and the University of Pittsburgh School of Medicine.

Intensive care unit sinks are persistently colonized with multidrug resistant bacteria and mobilizable, resistance-conferring plasmids

“…To identify potential HGT events, we developed a computational pipeline for identifying horizontally transferred sequences in our entire data set using a BLASTn-based clustering method to identify families of common plasmidic sequences ( 25 , 35 ). To reduce the likelihood of hits resulting from nearly identical, vertically acquired sequences, we first selected contigs originating from plasmids using Platon on all genomes ( 34 ).…”

“…5B ) of highly similar plasmid families. To identify plasmid sharing within each family, we employed an SNV-based analysis used for tracking plasmid spread within healthcare-associated bacteria ( 35 ). Within each family, a reference sequence was chosen based on the first appearance of that plasmid, and Illumina reads from each sample within that family were aligned to that reference to quantify SNVs.…”

“…Within each family, a reference sequence was chosen based on the first appearance of that plasmid, and Illumina reads from each sample within that family were aligned to that reference to quantify SNVs. A threshold of 99.985% (<15 SNVs per 100 kbp) was chosen to determine if the shared sequences were likely due to HGT ( 35 ).…”

Genomic Epidemiologic Investigation of a Multispecies Hospital Outbreak of NDM-5-Producing Enterobacterales Infections

“…The outbreak plasmid was defined as an IncX3 family conjugative plasmid harboring blaNDM-5 that had sequence coverage values (i.e., the proportion of each plasmid's sequence that was found in the Illumina genome) of at least 95•0%, and sequence identity of <15 SNPs per 100kb of plasmid sequence compared with the fully resolved, circular plasmid sequence recovered from an early outbreak isolate. 22 An intrapatient horizontal plasmid transfer event was defined as a patient from whom two different bacterial species containing the outbreak plasmid were isolated, either on the same or different days. An interpatient horizontal plasmid transfer event was defined as two patients from whom different bacterial species, or same species but different genetic lineages, carrying the outbreak plasmid were isolated, supported by epidemiologic evidence of transfer.…”