Lab Matters Fall 2020 - Page 47

APHL 2020 POSTER ABSTRACTS elements like plasmids and bacteriophages . The Virginia Division of Consolidated Laboratory Services ( DCLS ) performs susceptibility testing and targeted AMR gene profiling of carbapenem-resistant organisms ( CRO ) isolated from hospitalized patients in Virginia . A genomics approach to AMR genotyping provides additional insights into genes associated with resistance , prediction of drug resistance , and mobile genetic elements conferring AMR genes . To develop high-confidence predictive bioinformatic workflows for identifying resistance markers , 20 CRO isolates were sequenced at DCLS using the Illumina MiSeq and characterized for AMR determinants using traditional wet lab methods . The laboratory analyzed sequence reads for the presence of AMR determinants using Shovill v1.0.4 for genome assembly and StarAMR v0.5.1 to screen assemblies for plasmid-borne AMR determinants .
Resistance genes identified in the dataset included KPC ( Klebsiella pneumoniae carbanepenemase ), NDM ( New Delhi metallo-betalactamase ), OXA-48 ( oxacillinase ), and IMP ( Imipenemase Metallobeta-lactamase ). Plasmids were predicted in 15 of the 20 strains tested . These results suggest that plasmid-borne AMR genes are highly mobile and are associated with drug-resistant infections in Virginia . Genes that confer antibiotic resistance were called at drug class level .
While these predictions are dependable , they utilize short-read assemblies that may miss entire genes if the locus is in a region with highly repetitive nucleotide sequences , or if the gene is split across contiguous reads . To determine the complete genome , identify additional AMR genes , and elucidate plasmids with AMR genes , Nanopore MinION long read data will be combined with the short read data produced by the Illumina MiSeq using the bioinformatic tool Unicycler . Complete genomes for strains predicted to have plasmids will be annotated to identify other potential AMR genes . Accurate plasmid prediction and mapping of AMR genes to these mobile elements is important in studying healthcare facility infectious disease outbreaks , as plasmids conferring AMR genes may be transferred among bacteria .
Presenter : Rachael St . Jacques , Virginia Division of Consolidated Laboratory Services , Richmond , VA , Rachael . stjacques @ dgs . virginia . edu
Identifying Drivers of KPC Transmission in CRE Using Long Read Sequence Data
A . Shockey 1 , K . Florek 1 , A . Bateman 1 , M . Lasure 2 ; 1 Wisconsin State Laboratory of Hygiene , Madison , WI , 2 Wisconsin Department of Health Services , Madison , WI
Background : Carbapenem-resistant Enterobacteriaceae ( CRE ) are an urgent threat to global public health . CRE infections are associated with high mortality rates and difficult to treat . In the United States there were an estimated 13,100 infections and 1,100 deaths due to CRE in 2017 . CRE are susceptible to few , if any , antibiotics , and approximately 30 % of CRE carry a carbapenemase that can spread between species and across genera via mobile elements . The most common carbapenemase in the United States is the Klebsiella pneumoniae carbapenemase ( KPC ). Understanding the spread of KPC among CRE is critical to decreasing the number of infections and deaths due to CRE . Here , we use whole genome sequencing to develop a retrospective picture of KPC diversity and transmission dynamics in Wisconsin .
Methods : In partnership with the Wisconsin Department of Public Health , we selected 147 CRE isolates collected from 2011 – 2018 in Wisconsin for sequencing . In order to accurately determine if carbapenem resistance was encoded on the chromosome and / or a mobile element , we used a hybrid approach and sequenced each isolate on the Pacific Biosciences ( PacBio ) and Oxford Nanopore Technologies ( ONT ) MinION sequencing platforms . We used ONT long reads for genome assembly and highly accurate PacBio circular consensus sequences to polish the assemblies . We identified putative plasmids using Mash with PLSDB , a database of > 15,000 complete bacterial plasmids . Additionally , we used Abricate to screen for plasmid replicons and KPC genes using the PlasmidFinder database and the Comprehensive Antibiotic Resistance Database , respectively .
Results : We were able circularize 99 % of the chromosomal sequences in our sample of CRE . Mash matched 98 % of the remaining circular sequences with at least one plasmid in PLSDB . Abricate identified plasmid replicons in 91 % of these putative plasmids . These results indicate our sample of CRE has 422 putative plasmids from 12 different plasmid incompatibility groups . We found several variants of KPC in our sample , including KPC 1-5 . KPC-3 was most prevalent . The majority of KPC genes present in our sample were plasmid encoded ( 90 %). However , we also identified a transposable element ( Tn4401 ) containing a KPC gene in 25 % of the putative plasmids . Additionally , we found Tn4401 containing a KPC gene in the chromosome of six of our CRE isolates , two of which also had Tn4401 encoding a KPC gene integrated into a plasmid .
Conclusions : Our results suggest the plasmid diversity of CRE in Wisconsin is extensive and mobile elements play a major role in the spread of KPC among these isolates . While additional work is needed to elucidate the transmission method of KPC in CRE , this work provides new insights into the drivers of KPC spread in Wisconsin .
Presenter : Abigail Shockey , Wisconsin State Laboratory of Hygiene , Madison , WI , abigail . shockey @ slh . wisc . edu
Informatics Curriculum Framework R . Shepherd , Association of Public Health Laboratories , Silver Spring , MD
Over the past several years , APHL , in cooperation with CDC , has developed materials in support of a public health informatics curriculum , as well as a series of web-based introductory courses to the topic of laboratory informatics . Since February of 2019 , approximately 20 subject matter experts from the public health community , CDC , and APHL have been working across workgroups to develop a robust curriculum framework , currently in the final stages of development .
This poster will serve as an opportunity to share the completed framework , which is intended to serve as a visual map , cataloging the necessary skills and competencies required to advance in the field of laboratory informatics by defining objectives and goals in the following categories ranging from beginner to expert levels :
• Data Concepts
• Data Systems
• Interoperability
• IT Infrastructure
• Laboratory Activities
• Standards and Regulations
Infectious Disease / Informatics
Fall 2020 LAB MATTERS 45