Lab Matters Fall 2024 | Page 105

APHL 2024 POSTER ABSTRACTS that 30x coverage was sufficient for performing AMR profiling and Salmonella serotyping with 100 % accuracy . Additionally , because DNA methylation has been implicated in virulence in a variety of bacterial pathogens , we used MicrobeMod to identify a total of 75 motifs methylated with 6-methyladenine , 5-methylcytosine , or 4-methylcytosine across all samples .

We also investigated the performance of Nanopore sequencing for metagenomic assembly and composition profiling using the ZymoBIOMICS Microbial Community Standard ( MCS ) and the ZymoBIOMICS Fecal Reference ( FR ) samples . We demonstrate complete and accurate reconstruction of all eight bacteria in the MCS sample . We also generated over 200 high-quality meta-genome assembled genomes ( MAGs ) from the FR sample using Nanopore Duplex sequencing , including 97 recovered as single-contigs . We compared bioinformatics pipelines for taxonomic profiling , finding that tools varied widely in both computational performance and accuracy , with some level of tradeoff between these two metrics .

Together , our findings demonstrate that Nanopore sequencing can enable routine complete and accurate genome assembly for microbial isolates and metagenomic samples , as well as accurate profiling of microbial communities , all on a single platform .

Presenter : Priyesh Rughani , priyesh . rughani @ nanoporetech . com

Implementation of Nextstrain and MicrobeTrace Pipeline for Mpox Genomic Surveillance and Rapid Public Health Action

R . Cintron 1 , C . Gigante 1 , Y . Li 1 , R . Kelly 2 , J . Caravas 1 , W . Switzer 1 , Centers for Disease Control and Prevention 1 , General Dynamics Information Technology 2

The 2022-2023 mpox outbreak in the United States included 40 % of persons with HIV coinfections and required novel bioinformatic tools to inform mpox and HIV transmission . Due to the sensitive nature of HIV-mpox data , we implemented a local Nextstrain build to integrate mpox and HIV lab data in MicrobeTrace for cluster visualization and analysis . MicrobeTrace , a secure and free outbreak detection and characterization tool , is compatible with cloud platforms that use Nextstrain to visualize genomic and spatiotemporal data . The mpox virus ( mpxv ) Nextstrain build uses the lineage A Rivers , Nigeria sequence as reference for the alignment , mutation calling analysis and to root the phylogenies . We modified the mpxv build to root the tree on lineage A . 1.1 , include only US mpxv sequences and export the pairwise genetic distances for analysis in MicrobeTrace . The local Nextstrain build was tested with curated mpxv sequences ( n = 5,873 ). Sequence subsampling was done based on virus lineage , sampling date and US state-level location . We used the Auspice server to visualize the Nextstrain results and the mpxv pairwise genetic distances were securely integrated with HIV infection status for clustering analyses of HIV-mpox coinfections . We analyzed in MicrobeTrace 251 mpox samples that were tested for HIV and the genetic network was overlayed on the US map to show the mpxv lineage distribution . From the 251 mpox samples , about 47 % were HIV- positive and 19 % had detectable HIV viral load . However , about 80 % of the mpox samples tested for HIV had no mpox sequence data available when the analyses were done in August 2023 . HIV sequences were not available to infer clustering by HIV genotype . During the mpox outbreak , we leveraged the use of freely accessible bioinformatics tools , Nextstrain and MicrobeTrace , for secure genomic surveillance . Sequence subsampling facilitates selection of representative viruses and the addition of new sequences will help identify emerging variants or hotspots for early outbreak detection and response .

Presenter : Roxana Cintron , vrw7 @ cdc . gov

Longitudinal Analysis of ESBL ESKAPEE Isolates using Whole Genome Sequencing

J . Monk 1 , P . Sankey 2 , C . Wong 2 , A . Galicia 3 , M . Crumpler 3 , M . Zahn 4 , Palmona Pathogenomics 1 , Avellino Lab 2 , Orange County Public Health Lab 3 , Orange County Health Care Agency 4

Overview : A set of 381 ESKAPEE isolates possessing extended spectrum beta lactamase ( ESBL ) genes were collected from the Orange County Public Health Laboratory from 2017-2023 . The strains whole-genome sequenced ( WGS ) using Illumina sequencing technologies , assembled and annotated for presence of virulence factors and antibiotic resistance ( AR ) genes . The resulting data showed a varied distribution of genes leading to ESBL activity .

Results : Across the 381 isolates a total of 107 unique AR genes and 216 virulence factor genes were detected . A total of 19 different gene types with ESBL activity were identified . The most common ESBL conferring gene was blaCTX-M-15 ( 188 strains ) followed by blaOXA-1 ( 103 strains ) and blaCTX-M-27 ( 101 strains ). More rare genes were also found including blaCMY-4 ( 1 strain ), blaCMY-42 ( 1 strain ) and blaCTX-M-1 ( 1 strain ). The strains spanned phylogroups with the most coming from E . coli Clermont type B2 of these most were of serotype H4 : O25 . This presentation will examine the clinical and epidemiological features associated with ESBL carrying strains and will explore changes over time in presence and type of ESBL genes .

Methods : Single colonies of the clinical ESKAPE pathogens were isolated on blood agar plates and grown in Mueller Hinton broth ( MHB ) overnight at 37 ° C shaking . Cells in the overnight culture were pelleted prior to lysis . Total DNA was extracted from 1 mL of each overnight culture using the PureLink Pro 96 well Genomic DNA Purification Kit . RNase solution was added prior to a protein precipitation solution . GS libraries were prepared using the seqWill plexWell 96 kit protocol . Samples were normalized to 12.5 ng input using the Tecan Freedom Evo prior to sample barcoding , plate barcoding , library amplification and purification steps . Pooled libraries were diluted to a final concentration of 2nM and loaded onto an Illumina NextSeq1000 using a NextSeq 1000 / 2000 P2 reagent kit ( 300 Cycles ). DNA sample and library QC was done using the Varioskan LUX and Agilent Tapestation 4200 System . Read QC / QA was conducted with FastQC v0.11.9 for both platforms . Illumina genomes were assembled using Unicycler v0.4.8 with Spades v3.13 . Genome assemblies were evaluated using Quast v5.2.0 . All genomes were annotated with Prokka v 1.14.6 . AR genes were called using AMRfinder v 1.0.13 and virulence factors were identified using Abritamr v1.0.1 .

Presenter : Jonathan Monk , jonathan . monk @ palmona . com