SARS-CoV-2 wastewater variant surveillance: pandemic response leveraging FDA's GenomeTrakr network.

Wastewater surveillance has emerged as a crucial public health tool for population-level pathogen surveillance. Supported by funding from the American Rescue Plan Act of 2021, the FDA's genomic epidemiology program, GenomeTrakr, was leveraged to sequence SARS-CoV-2 from wastewater sites across the United States. This initiative required the evaluation, optimization, development, and publication of new methods and analytical tools spanning sample collection through variant analyses. Version-controlled protocols for each step of the process were developed and published on protocols.io. A custom data analysis tool and a publicly accessible dashboard were built to facilitate real-time visualization of the collected data, focusing on the relative abundance of SARS-CoV-2 variants and sub-lineages across different samples and sites throughout the project. From September 2021 through June 2023, a total of 3,389 wastewater samples were collected, with 2,517 undergoing sequencing and submission to NCBI under the umbrella BioProject, PRJNA757291. Sequence data were released with explicit quality control (QC) tags on all sequence records, communicating our confidence in the quality of data. Variant analysis revealed wide circulation of Delta in the fall of 2021 and captured the sweep of Omicron and subsequent diversification of this lineage through the end of the sampling period. This project successfully achieved two important goals for the FDA's GenomeTrakr program: first, contributing timely genomic data for the SARS-CoV-2 pandemic response, and second, establishing both capacity and best practices for culture-independent, population-level environmental surveillance for other pathogens of interest to the FDA. IMPORTANCE: This paper serves two primary objectives. First, it summarizes the genomic and contextual data collected during a Covid-19 pandemic response project, which utilized the FDA's laboratory network, traditionally employed for sequencing foodborne pathogens, for sequencing SARS-CoV-2 from wastewater samples. Second, it outlines best practices for gathering and organizing population-level next generation sequencing (NGS) data collected for culture-free, surveillance of pathogens sourced from environmental samples.

Genomic analysis of a cAmpC (CMY-41)-producing Citrobacter freundii ST64 isolated from patient.

Antibiotic resistance in Citrobacter freundii is a public health concern. This study evaluated the closed genome of a C. freundii isolated from the stool of a hospitalized patient initially related to a Salmonella outbreak. Confirmation of the isolate was determined by whole-genome sequencing. Nanopore sequencing was performed using a MinION with a Flongle flow cell. Assembly using SPAdes and Unicycler yielded a closed genome annotated by National Center for Biotechnology Information Prokaryotic Genome Annotation Pipeline. Genomic analyses employed MLST 2.0, ResFinder4.1, PlasmidFinder2.1, and VFanalyzer. Phylogenetic comparison utilized the Center for Food Safety and Applied Nutrition (CFSAN)-single nucleotide polymorphism pipeline and Genetic Algorithm for Rapid Likelihood Inference. Antimicrobial susceptibility was tested by broth microdilution following Clinical and Laboratory Standards Institute criteria. Multi-locus sequence type in silico analysis assigned the C. freundii as sequence type 64 and the blaCMY-41 gene was detected in resistome investigation. The susceptibility to antibiotics, determined using Sensititre® plates, revealed resistance to aztreonam, colistin, cefoxitin, amoxicillin/clavulanic acid, sulfisoxazole, ampicillin, and streptomycin. The genetic relatedness of the C. freundii CFSAN077772 with publicly available C. freundii genomes revealed a close relationship to a C. freundii SRR1186659, isolated in 2009 from human stool in Tanzania. In addition, C. freundii CFSAN077772 is nested in the same cluster with C. freundii clinical strains isolated in Denmark, Mexico, Myanmar, and Canada, suggesting a successful intercontinental spread.

Draft Genome Sequences of 17 Campylobacter coli Strains Isolated from Animal and Food Sources in Brazil.

Campylobacter spp. have been reported as one of the most frequent causes of acute gastroenteritis in humans worldwide. Here, we report 17 draft genome sequences of C. coli strains isolated from animal and food sources in Brazil. These data will improve our understanding of this species in Brazil.

Genomic and phylogenetic analysis of Salmonella enterica serovar Enteritidis strains linked to multiple outbreaks in Brazil.

Salmonella enterica subsp. enterica serovar Enteritidis (SE) has become the prevalent serovar isolated from gastroenteritis cases in Brazil since the 1990s. To better understand the genomic diversity and phylogenetic relationship amongst SE epidemic isolates from Brazil, 30 SE isolates from a variety of implicated foods and case patients of outbreaks between 1999 and 2006 were selected for genome comparison analyses. SE genomes were also compared against publicly available Brazilian SE isolates from pre- and postepidemic period. MLST analysis revealed that all isolates belong to sequence type (ST) 11. A total of seven Salmonella pathogenicity islands (SPIs) (SPI-1, SPI-3-5, SPI-13, SPI14, and C63PI) were identified in the evaluated genomes and all studied SE genomes carried similar prophage profiling. Resistome analysis revealed the presence of resistance genes to aminoglycosides [aac(6')laa, aph(3")-lb, aph(6)-ld], as well as point mutations in gyrA. Phylogenetic analysis demonstrated that certain isolates have circulated in Brazil for years and been involved in distinct outbreaks.

Interpretative Labor and the Bane of Nonstandardized Metadata in Public Health Surveillance and Food Safety.

Open-source DNA sequence databases have long been touted as beneficial to public health, including the facilitation of earlier detection and response to infectious disease outbreaks. Of critical importance to harnessing these benefits is the metadata that describe general and other domain-specific attributes (eg, collection location, isolate type) of a sample. Unlike the sequence data, metadata are often incomplete and lack adherence to an international standard. Here, we describe the problem posed by such variable and incomplete metadata in terms of interpretative labor costs (the time and energy necessary to make sense of the signal in the genetic data) and the impact such metadata have on foodborne outbreak detection and response. Improving the quality of sequence-associated metadata would allow for earlier detection of emerging food safety hazards and allow faster response to foodborne outbreaks.

Draft Genome Sequences of 80 Salmonella enterica Serovar Infantis Strains Isolated from Food, Environmental, Human, and Veterinary Sources in Brazil.

Salmonella enterica serovar Infantis is a broadly distributed serovar infecting humans and animal reservoirs globally. Here, we report 80 draft genome sequences of S. Infantis strains isolated from diverse sources in Brazil. These data will improve our understanding of the specific traits of S. Infantis isolated in this country.

Draft Genome Sequences of 62 Staphylococcus aureus Isolates Associated with Four Foodborne Outbreaks in the United States.

Staphylococcus aureus bacteria are ranked among the top five foodborne pathogens in the United States. Here, we report the draft genome sequences of 62 S. aureus isolates that originated from the manufacturing environment of an Illinois bakery and were associated with outbreaks between 2010 and 2011 in the United States.

Genomic investigation of antimicrobial resistance determinants and virulence factors in Salmonella enterica serovars isolated from contaminated food and human stool samples in Brazil.

This study investigated the antimicrobial resistance determinants, virulence factors and identified serovars in 37 Salmonella enterica strains isolated from human stool and contaminated foods linked to outbreaks that occurred in Brazil over 7 years using whole genome sequencing (WGS). Phylogenetic analysis of selected serovars (S. Typhimurium, S. Infantis, S. London, and S. Johannesburg) was performed. Ten distinct serovars were identified and, 51% of the tested strains (n = 19) showed disagreement with the previous conventional serotyping. The antimicrobial resistance (AMR) determinants or plasmids varied among the strains. Resistome analysis revealed the presence of resistance genes to aminoglycosides [aac (6')-laa, aph (3″)-lb, aph (6)-ld, aadA1 and aadA2], sulfonamides (sul1), trimethoprin (dfrA8), fosfomycin (fosA7) and tetracyclines (tetA, tetB, tetC), as well as point mutations in parC (T57S) and gyrA (S83F). Plasmidome showed the presence of IncHI2, IncHI2A, IncFIB (S), IncFII (S), IncI1 and p0111 plasmids. Eight Salmonella pathogenicity islands and up to 102 stress and/or virulence genes were identified in the evaluated genomes. Virulence genes of K88 fimbrial adhesin were first reported in S. enterica (S. Pomona, S. Bredeney and S. Mbandaka strains). pilW gene was first identified in S. Pomona. Phylogenetic analysis showed that some serovars circulated in Brazil for decades, primarily within the poultry production chain. Findings highlighted the virulence and AMR determinants in strains that may lead to recurring food outbreaks.

Gen-FS coordinated proficiency test data for genomic foodborne pathogen surveillance, 2017 and 2018 exercises.

The US PulseNet and GenomeTrakr laboratory networks work together within the Genomics for Food Safety (Gen-FS) consortium to collect and analyze genomic data for foodborne pathogen surveillance (species include Salmonella enterica, Listeria monocytogenes, Escherichia coli (STECs), and Campylobactor). In 2017 these two laboratory networks started harmonizing their respective proficiency test exercises, agreeing on distributing a single strain-set and following the same standard operating procedure (SOP) for genomic data collection, running a jointly coordinated annual proficiency test exercise. In this data release we are publishing the reference genomes and raw data submissions for the 2017 and 2018 proficiency test exercises.

Nipah virus dynamics in bats and implications for spillover to humans.

Nipah virus (NiV) is an emerging bat-borne zoonotic virus that causes near-annual outbreaks of fatal encephalitis in South Asia-one of the most populous regions on Earth. In Bangladesh, infection occurs when people drink date-palm sap contaminated with bat excreta. Outbreaks are sporadic, and the influence of viral dynamics in bats on their temporal and spatial distribution is poorly understood. We analyzed data on host ecology, molecular epidemiology, serological dynamics, and viral genetics to characterize spatiotemporal patterns of NiV dynamics in its wildlife reservoir, Pteropus medius bats, in Bangladesh. We found that NiV transmission occurred throughout the country and throughout the year. Model results indicated that local transmission dynamics were modulated by density-dependent transmission, acquired immunity that is lost over time, and recrudescence. Increased transmission followed multiyear periods of declining seroprevalence due to bat-population turnover and individual loss of humoral immunity. Individual bats had smaller host ranges than other Pteropus species (spp.), although movement data and the discovery of a Malaysia-clade NiV strain in eastern Bangladesh suggest connectivity with bats east of Bangladesh. These data suggest that discrete multiannual local epizootics in bat populations contribute to the sporadic nature of NiV outbreaks in South Asia. At the same time, the broad spatial and temporal extent of NiV transmission, including the recent outbreak in Kerala, India, highlights the continued risk of spillover to humans wherever they may interact with pteropid bats and the importance of limiting opportunities for spillover throughout Pteropus's range.

Optimizing open data to support one health: best practices to ensure interoperability of genomic data from bacterial pathogens.

The holistic approach of One Health, which sees human, animal, plant, and environmental health as a unit, rather than discrete parts, requires not only interdisciplinary cooperation, but standardized methods for communicating and archiving data, enabling participants to easily share what they have learned and allow others to build upon their findings. Ongoing work by NCBI and the GenomeTrakr project illustrates how open data platforms can help meet the needs of federal and state regulators, public health laboratories, departments of agriculture, and universities. Here we describe how microbial pathogen surveillance can be transformed by having an open access database along with Best Practices for contributors to follow. First, we describe the open pathogen surveillance framework, hosted on the NCBI platform. We cover the current community standards for WGS quality, provide an SOP for assessing your own sequence quality and recommend QC thresholds for all submitters to follow. We then provide an overview of NCBI data submission along with step by step details. And finally, we provide curation guidance and an SOP for keeping your public data current within the database. These Best Practices can be models for other open data projects, thereby advancing the One Health goals of Findable, Accessible, Interoperable and Re-usable (FAIR) data.

Draft Genome Sequences of 81 Salmonella enterica Strains from Informal Markets in Cambodia.

Salmonella enterica is an important global pathogen due to its contribution to human morbidity and death. The presence of S. enterica in Southeast Asian informal markets is amplified by cross-contamination between market surfaces and food products. Here, we describe the draft genome sequences of 81 Salmonella enterica isolates from informal markets in Cambodia.

Draft Whole-Genome Sequences of 51 Campylobacter jejuni and 12 Campylobacter coli Clinical Isolates from Chile.

Campylobacter species are the leading cause of gastroenteritis worldwide and an emerging threat in developing countries. Here, we report the draft whole-genome sequences of 51 Campylobacter jejuni and 12 Campylobacter coli strains isolated from patients with gastroenteritis in Santiago, Chile.

Closed Genome Sequences of 28 Foodborne Pathogens from the CFSAN Verification Set, Determined by a Combination of Long and Short Reads.

Foodborne pathogens have been implicated in illnesses worldwide. Here, we report the complete closed genome sequences of 28 bacterial strains belonging to 18 different species. These genomes belong to known foodborne pathogens. The genomes were closed by a combination of long-read and short-read sequencing.

Draft Genome Sequences of 26 Salmonella Isolates Associated with Multiple Outbreaks in Brazil.

Salmonella enterica subsp. enterica isolates are the leading cause of foodborne illness worldwide. Here, we report the draft genomes of 26 Salmonella isolates of food and clinical origin, belonging to four serovars, associated with outbreaks from 1999 to 2006 in the south of Brazil.

Draft Genome Sequences of 30 Salmonella enterica Serovar Enteritidis Isolates Associated with Multiple Outbreaks in Brazil.

Salmonella enterica subsp. enterica serovar Enteritidis has been the prevalent serovar isolated from gastroenteritis cases in Brazil since the 1990s. Here, we report the draft genomes of 30 S Enteritidis isolates originating from a variety of patients and implicated foods during outbreaks between 1999 and 2006 in Brazil.

Five Closed Salmonella enterica Genome Sequences from a 2017-2018 Multistrain, Multistate Kratom Outbreak.

We report here the closed genomes of Salmonella enterica strains from the 2017-2018 multistrain, multistate kratom outbreak using single-molecule real-time DNA sequencing. Four of the genomes consist of one circular chromosome, and the fifth has a circular chromosome and a single plasmid.