Lab Matters Fall 2024 | Page 96

APHL 2024 POSTER ABSTRACTS

( MycoSNP ) that identifies closely related strains by tabulating differences in Single Nucleotide Polymorphisms ( SNPs ). Clusters of isolates that differed by 12 or fewer SNPs were further examined for epidemiological links .

Results : Our findings revealed a predominance of Clade 1 and Clade 3 C . auris strains within the healthcare network . MycoSNP identified several independent clusters of C . auris that differed by five or fewer SNPs , many of which had epidemiologic links suggestive of nosocomial transmission . There was a high degree of genetic diversity observed within patients longitudinally colonized with C . auris ( including a maximum difference of 15 SNPs ) and , in some cases , sequencing of multiple isolates was key to establishing a microbiologic link between two patients .

Conclusions : The MycoSNP pipeline identified several independent C . auris clusters , consistent with the rise in C . auris cases observed within the hospital system . Application of existing thresholds for C . auris outbreak detection (< 12 SNPs ) are complicated by the high degree of diversity observed in some colonized patients , highlighting a potential need to sequence multiple isolates from the same patient during an investigation . Overall , these findings emphasize the critical role of WGS in identifying nosocomial spread of emerging fungal pathogens .

Presenter : Shannon Murphy , shannon . murphy . microbe @ gmail . com

Detection of Antimicrobial Resistance in Carbapenemresistant Gram-negative Bacteria : Development of a Sequencing Workflow in the State of Illinois

D . Hendricks , I . Heimler , V . Dhiman , Illinois Department of Public Health ( IDPH )

Carbapenems are an important class of antibiotic drugs often used for the treatment of severe bacterial infections , especially those that are multidrug-resistant . However , recent years have seen the rise of carbapenem-resistant Gram-negative bacteria ( CR-GNB ), which often render these vital antibiotics ineffective for the treatment of serious infections . The resulting increase in global morbidity and mortality from infections with CR-GNB is a current major area of public health concern , as is the lack of sequencing-based surveillance in many state and national public health laboratories for carbapenemase genes . As a result , there is a significant need for public health laboratories to evolve from standard PCR-based detection and develop a more comprehensive sequencing-based workflow for identifying carbapenemase genes for state- and nationwide surveillance . For this reason , the CDC Antibiotic Resistance Laboratory Network ( AR Lab Network ) provides recommendations on whole genome sequencing of antimicrobialresistant ( AMR ) pathogens . The purpose of this effort was to develop a workflow for carbapenemase gene identification at the Illinois Dept of Public Health ( IDPH ) laboratory , beginning at DNA extraction and ending with accurate sequencing and identification of carbapenemase genes of concern ( based on AR Lab Network guidance ). The workflow explores the use of either the ClearDX platform , which is fully automated from DNA extraction to FASTQ generation , or manual DNA extraction and library preparation followed by sequencing on the Illumina NextSeq 2000 system . Quality control ( QC ) metrics were assessed for the output reads by running them through the FastQC tool . Next , all reads were run through two publicly available bioinformatics pipelines that were used for the purpose of identifying AMR genes — including carbapenemase genes — post-sequencing . The programs utilized were Argonne National Laboratory ’ s Bacterial and Viral Bioinformatics Resource Center ( BV-BRC ) and the Short Read Sequence Typing ( SRST2 ) tool available on Illumina ’ s BaseSpace platform . To compare the range and accuracy of carbapenemase genes identified by these bioinformatics programs , we used A ) the CarbaNP panel of CR-GNB from the CDC and FDA ’ s Antibiotic Resistance Isolate Bank ( ARIsolateBank ) as positive controls for the presence of carbapenemase genes and B ) CR-GNBs with PCRconfirmed carbapenemase genes that were submitted to IDPH . Each organism on this panel possesses some level of carbapenem resistance and at least one identified carbapenemase gene . Results suggest that both manual and automated approaches may be an effective workflow for accurate identification of carbapenemase genes in CR-GNB , though with slight differences resulting from the choice of bioinformatics program used . Future directions should include efforts to optimize this workflow in terms of efficiency and further explore alternative bioinformatic options , including PHoeNIx . Reducing sequencing turn-around time will aid in timely identification and acquisition of data on CR-GNB that will aid actionable public health decisions .

Presenter : Dylan Hendricks , dylanlhendricksxc @ gmail . com

Detection of Mobilized Colistin Resistance mcr-9.2 gene and Associated IncHI2 / IncHI2A Plasmids in Carbapenemresistant Enterobacter hormaechei using Whole Genome Sequencing

S . Nallani 1 , S . Ceesay 1 , A . Iyo 1 , C . Hoover 1 , L . Nichols 1 , D . Ware 1 , B . Fowler 2 , J . Narcy 2 , C . Allard 2 , B . Karolewicz 1 , Mississippi Public Health Laboratory 1 , Mississippi Department of Health 2

Carbapenems are a class of very effective antibiotic agents . However , with the widespread use of antibiotics , carbapenemresistant Enterobacteriaceae have become common and pose a great threat to public health . New Delhi metallo-β-lactamase ( NDM ) is a bacterial enzyme that renders all current β-lactam antibiotics inactive . The NDM-encoding genes , blaNDM-1 to blaNDM-24 , have been reported in conjugative plasmids belonging to several incompatibility ( Inc ) groups . Dissemination of blaNDM carried on the incompatibility group plasmids , has been widely reported .

Colistin ( polymyxin E ) is an important antibiotic agent and is often the last line of defense in the treatment of carbapenemresistant Enterobacteriaceae such as Escherichia , Klebsiella and Enterobacter . Because of its importance as a drug of last resort , global communities have been carefully monitoring the emergence and spread of mobilized colistin resistance ( mcr ) genes in carbapenem-resistant bacteria . To date , ten mcr variants have been described and reported in conjugative plasmids belonging to several incompatibility groups . The IncHI2 / 2A and other incompatibility group plasmids such as IncN or IncX have been shown to carry the mcr genes .

In this study , bacterial DNA was extracted using the Qiagen DNeasy Blood and Tissue kit and subjected to WGS workflow . DNA libraries were prepared using an Illumina DNA Prep library preparation kit and sequenced on a MiSeq sequencing platform . Bioinformatic analysis was performed using HiPerGator . Resistance genes were identified using AMRFinderPlus and reconfirmed using CDC ’ s PHoeNIx tool . Plasmids were detected with the MobileElementFinder ( MGE ) tool from the Center for Genomic Epidemiology and