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, [email protected] 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
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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,
[email protected]
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
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