Lab Matters Summer 2021 | Page 64

APHL 2021 Poster Abstracts
Infectious Disease all K . pneumoniae isolates were multi-locus sequence type 147 and harbored the blaNDM-1 gene . Additional genes conferring resistance to β-lactams , aminoglycosides , fluoroquinolones , phenicol , sulphonamide , and trimethoprim were identified in all nine isolates . Antimicrobial susceptibility testing by broth microdilution confirmed resistance to β-lactams , fluoroquinolones , and aminoglycosides per CLSI guidelines ; and found MICs ≤0.25 μg / mL for colistin , polymyxin B , and tigecycline ( no available CLSI breakpoints ). Bioinformatics analysis identified two IncF family plasmids ( IncFIB ( pKPHS1 ), IncFIB ( pQil )) and an IncR plasmid in all the isolates . IncF family plasmids have been associated with extended-spectrum β-lactamases and genes encoding resistance to quinolones and aminoglycoside . Detailed epidemiological and WGS data point to clonal spread of NDM-producing K . pneumoniae in this outbreak . However , since NDM is typically found on a plasmid , there is potential for horizontal gene transfer of antibiotic resistance between different bacterial genera . We did find two different genera of NDM-1-producing organisms ( K . pneumoniae and E . coli ) with a potential common plasmid . Additional analysis including long-read sequencing is required to demonstrate the presence of plasmids and potential relatedness . Genomic characterization of outbreak isolates is useful in understanding the mechanisms of transmission and the scope of spread . This can inform outbreak response and assessment of infection control interventions .
Presenter : Sarah Namugenyi , Minnesota Public Health Laboratory , sarah . namugenyi @ state . mn . us
Detection of Urethritis-associated Neisseria meningitidis ST11 in Washington , DC
S . Nguyen , D . Edwards , E . Zelaya , J . Doss , M . McCarroll , J . Kiehlbauch , A . Tran and J . Hauser , DC Department of Forensic Sciences , Washington , DC
Neisseria gonorrhoeae , which colonizes both the male and female urogenital tract mucosa , is the second most common sexuallytransmitted bacterial infection in the US . In men , gonococcal infections usually present as urethritis demonstrated by dysuria and a purulent urethral discharge . Although atypical , cases of urethritis associated with Neisseria meningitidis , which typically colonizes the nasopharyngeal mucosa and causes meningitis , have been described . An expansion of a non-groupable urethritis-associated Neisseria meningitidis clade ( NmNg , ST11 ), which had acquired genes found in Neisseria gonorrhoeae , was first reported in Columbus , Ohio . As part of the District of Columbia ( DC ) Gonococcal Isolate Surveillance Program ( GISP ), 360 urethral swabs collected from symptomatic males between March 2019 and December 2020 were cultured for N . gonorrhoeae at the DC Public Health Laboratory . N . gonorrhoeae was isolated from 43 % ( 154 / 360 ) of cultures . Among these , eleven isolates of urethritis-associated N . meningitidis were identified . Whole genome sequencing of these isolates confirmed the N . meningitidis isolates were closely related to the Ohio NmNg clade . Core genome analysis revealed that all NmNg isolates had acquired insertion sequences that have disrupted the capsule ( cps ) loci , which encodes the genes necessary for capsule production , as well as gonococcal denitrification alleles necessary for nitrite dependent anaerobic growth in a urethral niche . Phylogenomic analyses of the DC NmNg show the isolates were highly related to each other . Additionally , pan-genomic analysis shows that the DC NmNg clade had acquired additional gonococcal genes which indicate a recent genetic recombination event . Notable alleles identified include a ferrochelatase ( hemH ) and tonB receptor involved in iron acquisition and DNA repair genes such as alkA and tag . While it is unknown if these gonococcal alleles contribute to fitness in urethritis for N . meningitidis , whole genome sequencing demonstrates the need to monitor novel urethral isolates to detect potential emerging pathogens .
Presenter : Scott Nguyen , Washington , DC Department of Forensic Sciences , scott . nguyen @ dc . gov
Evaluation of a Blood Filtration Device for Use in Molecular Detection Applications
K . Parker 1 , B . Knight 1 , B . Peterson 1 , K . Yeh 1 , P . Luk 2 , M . Zubaidi 2 , M . McNeely 2 ; 1 MRIGlobal , Kansas City , MO , 2 Gatta Co ., Murrieta , CA
The optimization of sample preparation methods for next-generation sequencing is important when working with challenging samples or samples with a high host background . The abundance of human nucleic acids in clinically relevant samples often prevents the detection of low-level pathogens . There are various methods available for sample dehosting , including differential lysis and enzymatic digestion . One novel concept involves removal of red and white blood cells from whole blood by filtration to provide some degree of dehosting . The GattaCo Sipon-60 device is capable of removing red blood cells and white blood cells from a 250 µ L whole blood sample with a 60 µ L output of plasma . We evaluated this device alongside two extraction methods to generate proof of concept data . The data presented here demonstrates the feasibility of this device for downstream applications such as qPCR and nextgeneration sequencing detection of blood-borne pathogens .
Presenter : Kyle Parker , MRIGlobal , kparker @ mriglobal . org
Getting to the Core : Single-cell Dynamics and Subcellular Trafficking of Hepatitis B Virus Core Protein
S . Romero , N . Unchwaniwala , D . Loeb and N . Sherer , University of Wisconsin-Madison , Madison , WI
Hepatitis B virus ( HBV ) is a reverse-transcribing DNA virus that infects the liver and is a leading cause of hepatocellular carcinoma . Although a vaccine offers protection against infection , it is not therapeutic and the incidence of HBV infections in the US has increased over the last decade . Thus , determining novel HBV-host interactions that support infection is needed to develop curative anti-HBV strategies . During replication , HBV pregenomic ( pg ) RNA is packaged by Core protein ( Cp ) into nucleocapsids where pgRNA is reversetranscribed to generate relaxed-circular ( rc ) DNA . Here , we performed a comprehensive analysis of Cp subcellular trafficking over time in Huh7 hepatocellular carcinoma cells using immunofluorescence , live-cell imaging , and biochemical assays for three replication conditions : ( I ) WT HBV genomes , ( II ) genomes encoding an assembly incompetent Cp ( Y132A ), and ( III ) genomes encoding functional Cp but lack the pgRNA packaging signal ( Eps- ). Although HBV cores are thought to form in the cytoplasm , we unexpectedly observed high levels of high-order Cp assemblages accumulating preferentially in the nucleus at early time points ( 24h ) followed by a marked shift to the cytoplasm at 48h . This transition was not observed for either Cp ( Y132A ) or WT Cp encoded by Eps- pgRNA . Live cell imaging revealed Cp nucleus-to-cytoplasm re-localization occurs through two mechanisms ; ( I ) predominantly by nuclear escape during mitosis followed by cytoplasmic retention or ( II ) punctuated release of nuclear
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LAB MATTERS Summer 2021