Lab Matters Summer 2019 - Page 67

APHL 2019 POSTER ABSTRACTS these investigations provided a unique real-case scenario in which to compare the capabilities of traditional culture methods to the IDEXX LegiolertTM test kit for Legionella pneumophila (Lp) detection and isolation. and the WC AR bioinformatics pipeline that identifies over 6,000 AR gene variants. Both methods employed quality control (QC) steps including coverage cutoffs and removal of low-quality sequences; all 24 isolates passed QC. To maximize the likelihood that Lp1 could be isolated from a given water sample, we employed both traditional culture methods and Legiolert during both investigations. For traditional culture, 0.1 ml of samples was plated directly or heat-treated (50oC for 30 min) before plating on buffered charcoal yeast extract (BCYE) agar or BCYE with antibiotic supplement agar followed by incubation at 36oC for up to 10 days. In contrast, Legiolert involved a sample pretreatment step prior to combining 1.0 ml of sample with the Legiolert culture reagent and subsequent incubation of sealed trays at 37oC for 7 days. After 7 days, positive Legiolert tray wells were loop-transferred to BCYE agar for colony isolation and further characterization. Using a combination of culture and Legiolert methods, Lp1 was isolated from 45% (18/40) of the samples. A total of 10 samples were positive by both methods and Lp1 was isolated exclusively using either Legiolert or traditional culture in a total of 8 samples; 4 each using Legiolert or traditional culture. Isolation of Lp1 exclusively with one method or the other was not solely due to being at the detection limits for the comparative methods. Subsequent characterization of cultured isolates that involved identification of serogroup, pulsed-field gel electrophoresis pattern, and whole genome sequence single nucleotide polymorphism analysis, revealed that both Legiolert and traditional culture yielded the same Lp1 strains from a given sample. Results from this study support the combined use of both methods for the isolation of Lp from cooling tower water samples to increase the overall sensitivity of culture for Lp1 from cooling tower water samples. Results: WC WGS analysis identified over 100 different AR genes belonging to numerous antibiotic classes across all 24 isolates, with up to 24 genes found in a single isolate. In comparison, AmpliSeq™ AMR identified about 75 different AR genes, with up to 22 different AR genes detected in a single isolate. Both methods were able to identify beta-lactamase gene families with 100% concordance in all isolates. However, the WC WGS results had higher resolution for variant level gene identification, possibly due to the larger AR gene database used by the WC AR pipeline. Variant level inconsistencies most commonly arose with genes associated with beta-lactam, quinolone and aminoglycoside resistance. Naming convention differences between databases also contributed to discrepancies observed. Presenter: Scott Hughes, New York City Public Health Laboratory, New York, NY, hughes@health.nyc.gov Whole-Genome Sequencing and Characterization of a Cluster of Carbapenemase-Producing Pseudomonas aeruginosa Isolates Identified in New York State Is the Full Picture Necessary? Targeted Sequencing vs. Whole Genome Sequencing E. Snavely, E. Nazarian, W. Haas, S. Kogut, J. Greenko, R. Giardina, E. Adams, N. Singh, C. Wagner, J. Bodnar, K. Cummings, S. Morris, K. Mitchell, E. Lutterloh and K. Musser, New York State Department of Health-Wadsworth Center N. Peinovich 1 , E. Snavely 1 , Y. Wang 2 , A. Shah 2 , K. Mitchell 1 , K. Musser 1 ; 1 New York State Department of Health-Wadsworth Center, 2 Thermo Fisher Scientific Background: Whole genome sequencing (WGS) is currently being validated at the Wadsworth Center (WC) for clinical isolate testing to comprehensively identify acquired antimicrobial resistance (AR) genes. WC’s WGS approach utilizes Illumina sequencing and an in-house developed bioinformatic pipeline. ThermoFisher’s next generation sequencing (NGS) panel, AmpliSeq™ Antimicrobial Resistance (AMR), uses targeted sequencing of specific regions of the genome, simultaneously sequencing hundreds of genes rather than the whole genome. These analyses utilize different approaches to provide AR gene profiles. We chose to compare these methods on the same set of clinical isolates to evaluate AR prediction. Methods: Twenty-four clinical isolates that were previously characterized at WC were selected for targeted sequencing with Ampliseq™ AMR assay. Isolate characterization included bacterial identification, detection of carbapenemase-encoding gene(s) using real-time PCR, carbapenemase production and WGS. Library preparation, templating, and sequencing followed manufacturer protocols. The Ampliseq™ AMR assay can identify up to 478 AR genes related to 28 different antibiotic classes using 2 pools of 815 total amplicons for the library preparation. WC WGS utilized Nextera XT library prep, Miseq 2 x 250 bp read length sequencing PublicHealthLabs @APHL APHL.org Conclusion: The Ampliseq™ AMR assay is easy to use and has a fast turnaround time (TAT) of 3 days from library prep to sequencing. Additionally, sequencing from both isolates and primary samples is possible. Conversely, the WC WGS has a longer TAT (~4 days), however, WGS data can be used to identify AR genes and determine isolate relatedness. Both sequencing methodologies provide valuable information but have strengths and weaknesses depending on their intended use. Presenter: Nadine Peinovich, AR Fellow, New York State Department of Health-Wadsworth Center, Albany, NY, nadine.peinovich@health.ny.gov Background: The New York State Department of Health (NYSDOH) Wadsworth Center (WC) investigates and responds to infectious disease threats in the Northeast region as part of the Antibiotic Resistance Laboratory Network (AR Lab Network). The emergence of carbapenemase-producing, carbapenem-resistant Pseudomonas aeruginosa (CP-CRPA) is of particular concern given the limited therapeutic options and easy transfer of resistance mechanisms between bacterial species. In this study we identified and characterized an unrecognized regional cluster of CP-CRPA in New York State, resulting in an epidemiologic investigation. Methods: CRPA testing at the WC included antimicrobial susceptibility testing, detection of carbapenemase production, and multiplexed real-time PCR assays to identify carbapenemase- encoding genes. Whole-genome sequencing (WGS) was performed with Illumina MiSeq and Oxford Nanopore MinION. WC-developed bioinformatic pipelines were used to identify AR genes, screen for mobile genetic elements, and determine isolate relatedness through single nucleotide polymorphism (SNP) analysis. Results: As part of the AR Lab Network, the WC tested over 1,400 P. aeruginosa isolates in 2018. Of the 56 CP-CRPA isolates identified, 32 contained a VIM-family carbapenemase. Nearly all (97%) VIM-positive isolates were multi-drug resistant and harbored Summer 2019 LAB MATTERS 65