APHL 2021 Poster Abstracts blood cultures flagged positive for bacterial growth are subcultured for enrichment and isolation , and isolates are identified by matrix-assisted laser desorption ionization-time of flight mass spectroscopy . Metagenomics offers the potential to characterize pathogens directly from positive blood culture and eliminate biases inherent to sub-culturing to laboratory growth media for enrichment and isolation . Bloodstream infections are usually a monoculture dominated by a single pathogen but metagenomics allows for characterization of subpopulations and antimicrobial resistance ( AR ) genes in one assay . Residual material from 12 positive cadaveric blood culture bottles was selected to evaluate a metagenomics method for pathogen identification . DNA was extracted using the Qiagen BiOstic Bacteremia kit , which yielded DNA of sufficient quality for library preparation using Illumina DNA Flex kit and MiSeq . Each library averaged 950,000 bacterial reads , with > 90 % of total reads mapped to bacteria and < 10 % to host cells on average . Sequence analysis results were compared between a PHL-developed pipeline built from open-source programs and the commercial One Codex platform . Both analysis methods resulted in good concordance with the 20 species identified by traditional culture . The PHL pipeline using Kraken mapped reads to all 20 species and One Codex matched 19 of 20 . The highest proportion of mapped reads were concordant with the traditional culture-based identifications for 75 % of the samples . Additionally , metagenomics analysis identified bacteria not recovered through sub-culture and therefore provided a more comprehensive representation of the microbial population present in the sample . The preliminary data from this pilot study highlights the potential for metagenomics as a tool for characterization of pathogens direct from positive blood culture bottles and the extraction method was effective for removal of host cells , but further study is required to establish data analysis threshold criteria for reporting species identifications and AR genes . Overall metagenomics results showed good concordance with traditional culture methods and identification of more than one pathogen is not uncommon for cadaveric specimens . Turnaround time to actionable results and cost per sample are limiting factors for implementation of metagenomics for routine use in a clinical setting , but alternative sequencing platforms with shorter run times and lower cost per sample could be evaluated in future studies .
Presenter : Stephen LaVoie , New York City Public Health Laboratory , slavoie5 @ gmail . com
A Whole Genome Sequencing Approach to Antimicrobial Susceptibility Testing in Mycobacterium tuberculosis Complex
A . Lopez 1 , P . Valois 1 , B . Jones 1 , S . Schmedes 1 , P . Lapierre 2 , K . Musser 2 , M . Soehnlen 3 , J . Blanton 1 , M-C . Rowlinson 1 ; 1 Florida Bureau of Public Health Laboratories , Jacksonville , FL , 2 New York Department of Health / Wadsworth Center , Albany , NY , 3 Michigan Bureau of Laboratories , Lansing , MI
Antimicrobial susceptibility testing ( AST ) of the Mycobacterium tuberculosis complex ( MTBC ) is a key component for the appropriate treatment of TB . Characterization of the organism ’ s susceptibility to antimicrobial drugs informs clinicians of which treatment regimen will likely prove most effective and identifies cases of multi-drug resistant tuberculosis ( MDR-TB ) and extensively drug-resistant tuberculosis ( XDR-TB ), both of which cause significant morbidity and mortality . The Florida Department of Health , Bureau of Public Health Laboratories ( BPHL ) performs AST on clinical isolates of MTBC , utilizing a culture-based , minimum inhibitory concentration ( MIC ) method . MTBC is a slow growing organism and MIC uses cultures grown on solid media for inoculation which reduces contamination but means a longer turnaround time compared to liquid culture methods . Therefore , molecular methods play an essential role in providing information in a timelier manner . Rapid molecular methods may also be complemented by WGS which provides additional information to the clinician and for surveillance purposes before MIC results may be available . WGS can be performed on specimens grown in liquid culture and provides comprehensive genotypic information . The New York State Department of Health Wadsworth Center ( WC ) has developed a WGS protocol and bioinformatics pipeline that analyzes sequencing output and detects mutations associated with antimicrobial resistance to nine drugs used to treat TB , as well as provides species identification and genotype . This ongoing study aims to evaluate the WC protocol and implement it in the Florida BPHL as a diagnostic assay for the molecular detection of mutations associated with resistance . A retrospective study was conducted on 445 Florida TB patients in which WGS data generated by the Michigan Department of Health and Human Services were analyzed by the WC pipeline and compared to Florida MIC data . This study found 99 % concordance between the WGS and MIC data and demonstrated that WGS is an accurate method for detection of mutations associated with resistance . Following the retrospective study , 57 Florida MTBC isolates were sequenced and analyzed using the WC bioinformatics pipeline , along with output from 100 previously sequenced specimens . 85.7 % of drug susceptibility results derived from WGS were concordant with results from other AST methods . Contamination or coinfection with other mycobacteria was found to be a factor in discrepant results . Overall , WGS is a promising tool for antimicrobial susceptibility testing , potentially improving patient outcomes , reducing laboratory workload , and benefiting public health .
Presenter : Andrea Lopez , Florida Bureau of Laboratories , andrea . lopez @ flhealth . gov
Molecular Epidemiology of the First NDM-1-producing Klebsiella pneumoniae Outbreak in Minnesota
S . Namugenyi 1 , 3 , S . O ’ Malley 2 , B . VonBank 2 , R . Lynfield 2 , P . Snippes Vagnone 3 ; 1 Association of Public Health Laboratories , Silver Spring , MD , 2 Minnesota Department of Health , St . Paul , MN , 3 Minnesota Public Health Laboratory , St . Paul , MN
New Delhi-metallo-β-lactamase ( NDM ) is an enzyme , found in gram negative bacteria , capable of hydrolyzing penicillin , cephalosporin , monobactam , and carbapenem classes of antibiotics ; which contributes to bacterial resistance to these antimicrobials . Though uncommon in the United States , NDMproducing Enterobacteriaceae are more common in healthcare settings in other countries and are an emerging public health threat . In Minnesota , fewer than 10 cases were reported annually from 2012-2018 most of whom received recent healthcare abroad . Between December 2018 and May 2019 , the first outbreak of NDM-producing Klebsiella pneumoniae occurred in Minnesota among eleven cases with an epidemiologic link to one long-term care facility , and without patient history of international travel . One case also harbored an NDM-producing Escherichia coli . Isolates were obtained from 9 / 11 cases . WGS , using the Illumina platform and single nucleotide polymorphism ( SNP ) analysis , demonstrated relatedness between all nine K . pneumoniae isolates with SNP differences ranging from 1-18 . WGS analysis also determined that
Infectious Disease
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Summer 2021 LAB MATTERS 61 |