INDUSTRY MATTERS
Emerging Infectious Diseases and Antimicrobial Resistance Genes in Wastewater
By Kayley Janssen , Wisconsin State Laboratory of Hygiene ; Morgan Roos , Molecular Quantification Core , Colorado State University ; Jim Huang , Molecular Quantification Core , Colorado State University ; Jacob Bierstedt , Illumina , Inc .; Irina Khrebtukova , Illumina , Inc .; Evelyn Doolittle , Wisconsin State Laboratory of Hygiene ; Griffin Knuth , Wisconsin State Laboratory of Hygiene ; Dash Cooper , Wisconsin State Laboratory of Hygiene ; Ellie Olsen , Wisconsin State Laboratory of Hygiene ; Adelaide Roguet , Wisconsin State Laboratory of Hygiene ; Jeffrey Koble , Illumina , Inc .; Ying Lin , Illumina , Inc .; Kate Broadbent , Illumina , Inc .; Courtney Gonzalez , Illumina , Inc .; Robert Schlaberg , Illumina , Inc .; Gary Schroth , Illumina , Inc .; Carol Wilusz , Molecular Quantification Core , Colorado State University ; and Scott Kuersten , Illumina , Inc .
The SARS-CoV-2 pandemic accelerated the integration of genomics into pathogen surveillance . It demonstrated the value of monitoring wastewater for infectious diseases and antimicrobial resistance ( AR ) as an important means to aid public health professionals in predicting upcoming trends with potentially large impacts on communities . Increased SARS-CoV-2 nucleic acid detection in wastewater by molecular methods precedes increases in COVID-19 hospital cases by days to weeks , providing an opportunity for non-invasive monitoring of community-level prevalence trends . 1 , 2 , 3 , 4 As hospitalization rates have decreased and at-home testing has replaced pointof-care or centralized laboratory testing , wastewater data may be a more reliable indicator of SARS-CoV-2 circulation in the community . 6
Method
Digital droplet polymerase chain reaction ( ddPCR ) methods to detect and quantify pathogens in wastewater have become routine in wastewater surveillance and provide a sensitive and targeted means to track increases in SARS-CoV-2 and other infections . 5 This approach has distinct advantages for both sensitivity and throughput . However , it is limited to a single ( or , at best , a small number of ) pathogens for any assay . 7 Next-generation sequencing ( NGS ) offers a potent alternative to monitor large numbers of distinct pathogens simultaneously . It has the added benefit of providing strain level and potentially full genome coverage of the pathogens . Importantly , there are a myriad of methods for sample collection , concentrating the microbes and extracting the nucleic acids from wastewater samples . However , additional studies to identify how pre-analytical factors impact the number and types of microorganisms detected in such samples are an important prerequisite to routine implementation . Through the end of 2022 and the beginning of 2023 , Illumina collaborated with researchers at the Wisconsin State Laboratory of Hygiene and Colorado State University to evaluate sample collection approaches and , more importantly , sample concentration and extraction methods for wastewater-based surveillance by NGS .
Testing
Utilizing influent , the effects of sample storage , concentration technique and extraction method on pathogen detection were compared . Sample concentration methods evaluated in these studies including the Concentrating Pipette from InnovaPrep and Nanotrap ® Microbiome Particles from Ceres Nanosciences . Furthermore , four different extraction techniques were performed on samples from different wastewater sources , including college dorms and water treatment plants in Colorado and Wisconsin . Following concentration and isolation , the nucleic acids were sequenced with various NGS protocols to evaluate their ability to detect and characterize a broad range of pathogens and AR genes . These included shotgun metagenomic approaches and shotgun metatranscriptomics with ribosomal RNA ( rRNA ) depletion using Illumina ’ s RiboZero™ Plus Microbiome . In addition , two precision targeted NGS assays , the Urinary Pathogen ID / AR Panel ( UPIP ) and the Viral Surveillance Panel ( VSP )— both products available through Illumina — were utilized to enrich and profile a broad spectrum of microbial sequences of interest . Both bait-capture panels utilize biotinylated probes that hybridize to the genomic regions of interest for specific targets . UPIP targets 174 genitourinary pathogens and > 3700 AR markers , while VSP targets 66 DNA and RNA viruses .
Samples were sequenced very deeply ( 100 million reads per sample ) and then downsampled to 8-20M reads for most of the analysis to compare the performance of enrichment vs . deep shotgun sequencing .
Results
The samples prepped using InnovaPrep and Ceres Nanosciences products showed negligible differences in viral pathogen detection yield by ddPCR and by NGS methods as well as genome coverage by NGS . However , the microbial content of concentrated wastewater samples changed over time and with the number of individuals contributing to the wastewater system . Additionally , the specific NGS method used for detection affected the type of pathogens detected and the genomic coverage of those pathogens . In general , shotgun DNA and RNA approaches demonstrated high detection yield for viruses that are abundant in wastewater , such as hCoV-OC43 and human polyomaviruses . Compared to traditional shotgun methods , VSP enrichment enhanced the number of viruses observed ( mainly enteric , oncolytic or respiratory in nature ), allowed for more granular strain identification , and increased the callable positions and coverage of viruses ( e . g ., BK polyomavirus ( DNA ), aichivirus A ( RNA ), astrovirus ( RNA ) and hCoV-OC43 ( RNA )) in wastewater samples . Inclusion of a bait-capture step with UPIP prior to sequencing improved AR marker detection , coverage and depth ; enriched samples show a > 2X increase in average detection and a 409X increase in median depth at 5X fewer reads than shotgun methods ( Figure 1 ).
Overall , bait-capture enrichment provides an expansive and bioinformatically economical NGS approach for surveillance of viral and bacterial pathogens in wastewater , a complex
12 LAB MATTERS Winter 2023
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