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.

Persistence comparison of two Shiga-toxin producing Escherichia coli (STEC) serovars during long-term storage and thermal inactivation in various wheat flours.

Foodborne outbreaks associated with Shiga toxin-producing Escherichia coli (STEC) contaminated wheat flour have been an increasing food safety concern in recent decades. However, there is little literature aimed at investigating the impact of different flour types on the persistence of STEC during storage and thermal inactivation. Therefore, two serovars of STEC, O121 and O157, were selected to inoculate each of five different types of common wheat flours: whole wheat, bleached, unbleached, bread, and self-rising. Inoculated flours were examined for the stability of STEC during storage for up to 42 days at room temperature (RT) and aw ~0.56. Additionally, the thermal resistance of O121 and O157 under isothermal conditions at 60, 70, 80, and 90°C was analyzed for the inoculated flours. STEC storage persistence at RT was generally not affected by flour type, however, decreases of 1.2 and 2.4 log CFU/day within whole wheat flour for O121 and O157, respectively, were significantly lower than other flours. Though few differences were identified in relation to flour type, O121 exhibited significantly better survival rates than O157 during both equilibrium and storage periods. Compared to an approximate 6 log reduction in the population of O157, O121 population levels were reduced by a significantly lower amount (~3 log) during the entire storage period at RT. At each isothermal temperature, the impact of flour type on the thermal resistance capabilities of O121 or O157 was not a significant factor and resulted in similar survival curves regardless of serovar. Instead of exhibiting linear survival curves, both O121 and O157 displayed nonlinear curves with some shoulder/tail effect. Similar for both O121 and O157, the predicted decimal reduction time (D-value) decreased from approximately 25 min to around 8 min as the isothermal temperature increased from 60°C to 90°C. Results reported here can contribute to risk assessment models concerning contamination of STEC in wheat flour and add to our understanding of the impacts of flour type and STEC serovar on desiccation stability during storage and isothermal inactivation during thermal treatment.

Infection biology of Salmonella enterica.

Salmonella enterica is the leading cause of bacterial foodborne illness in the USA, with an estimated 95% of salmonellosis cases due to the consumption of contaminated food products. Salmonella can cause several different disease syndromes, with the most common being gastroenteritis, followed by bacteremia and typhoid fever. Among the over 2,600 currently identified serotypes/serovars, some are mostly host-restricted and host-adapted, while the majority of serotypes can infect a broader range of host species and are associated with causing both livestock and human disease. Salmonella serotypes and strains within serovars can vary considerably in the severity of disease that may result from infection, with some serovars that are more highly associated with invasive disease in humans, while others predominantly cause mild gastroenteritis. These observed clinical differences may be caused by the genetic make-up and diversity of the serovars. Salmonella virulence systems are very complex containing several virulence-associated genes with different functions that contribute to its pathogenicity. The different clinical syndromes are associated with unique groups of virulence genes, and strains often differ in the array of virulence traits they display. On the chromosome, virulence genes are often clustered in regions known as Salmonella pathogenicity islands (SPIs), which are scattered throughout different Salmonella genomes and encode factors essential for adhesion, invasion, survival, and replication within the host. Plasmids can also carry various genes that contribute to Salmonella pathogenicity. For example, strains from several serovars associated with significant human disease, including Choleraesuis, Dublin, Enteritidis, Newport, and Typhimurium, can carry virulence plasmids with genes contributing to attachment, immune system evasion, and other roles. The goal of this comprehensive review is to provide key information on the Salmonella virulence, including the contributions of genes encoded in SPIs and plasmids during Salmonella pathogenesis.

Efficacy of iSupport-Brasil for unpaid caregivers of people living with dementia: protocol for a randomized and controlled clinical trial.

Unpaid caregivers of people living with dementia tend to suffer mental health problems as a result of the negative effects associated with the care tasks. Thus, psychosocial interventions for this population group are necessary. iSupport is an online support program for caregivers that was created by the World Health Organization. Objective: To describe the design of a randomized clinical trial to measure the efficacy of the iSupport-Brasil version on caregivers' mental health and well-being. Methods: The participants will be randomized into Intervention Group (IG) (n=195) and Control Group (CG) (n=195). For three months, the IG will access the iSupport-Brasil platform, the CG will enter the electronic page of the Brazilian Alzheimer's Association, and both groups will be emailed the preliminary version of the "Guia de cuidados para a pessoa idosa" e-book (a guide to providing care to the elderly) from the Ministry of Health. The data will be collected at three moments: baseline, and three and six months after the beginning of the intervention. Results: It is expected that it will be possible to provide diverse validity evidence about iSupport-Brasil as an online and free intervention alternative, as a preventive means and as a way to promote mental health among caregivers of people living with dementia. Conclusion: Through the evaluation protocol of this randomized clinical trial on the effects of the iSupport-Brasil program, it may become a reference for countries that plan to adapt and improve the iSupport program using digital health solutions.

Cuidadores informais de pessoas que vivem com demência tendem a sofrer problemas de saúde mental como resultado dos efeitos negativos associados às tarefas de cuidado. Assim, intervenções psicossociais para essa população são necessárias. O iSupport é um programa de apoio online para cuidadores criado pela Organização Mundial da Saúde. Objetivo: Descrever o desenho de um ensaio clínico randomizado para medir a eficácia da versão iSupport-Brasil na saúde mental e no bem-estar de cuidadores. Métodos: Os participantes serão randomizados em Grupo Intervenção (GI) (n=195) e Grupo Controle (GC) (n=195). Durante três meses, o GI acessará a plataforma iSupport-Brasil, o CG entrará na página eletrônica da Associação Brasileira de Alzheimer e ambos os grupos receberão por e-mail a versão preliminar do e-book Guia de cuidados para a pessoa idosa, do Ministério da Saúde. Os dados serão coletados em três momentos: linha de base, três e seis meses após o início da intervenção. Resultados: Espera-se que seja possível fornecer diversas evidências sobre os efeitos do iSupport-Brasil como alternativa de intervenção online e gratuita, de forma a promover a saúde mental entre os cuidadores de pessoas que vivem com demência. Conclusão: O protocolo de avaliação deste ensaio clínico randomizado sobre os efeitos do programa iSupport-Brasil pode se tornar uma referência para os países que planejam adaptar e melhorar esta intervenção, usando soluções digitais de saúde.

Results of the 2020 Genomic Proficiency Test for the network of European Union Reference Laboratory for Antimicrobial Resistance assessing whole-genome-sequencing capacities.

The global surveillance and outbreak investigation of antimicrobial resistance (AMR) is amidst a paradigm shift from traditional biology to bioinformatics. This is due to developments in whole-genome-sequencing (WGS) technologies, bioinformatics tools, and reduced costs. The increased use of WGS is accompanied by challenges such as standardization, quality control (QC), and data sharing. Thus, there is global need for inter-laboratory WGS proficiency test (PT) schemes to evaluate laboratories' capacity to produce reliable genomic data. Here, we present the results of the first iteration of the Genomic PT (GPT) organized by the Global Capacity Building Group at the Technical University of Denmark in 2020. Participating laboratories sequenced two isolates and corresponding DNA of Salmonella enterica, Escherichia coli and Campylobacter coli, using WGS methodologies routinely employed at their laboratories. The participants' ability to obtain consistently good-quality WGS data was assessed based on several QC WGS metrics. A total of 21 laboratories from 21 European countries submitted WGS and meta-data. Most delivered high-quality sequence data with only two laboratories identified as overall underperforming. The QC metrics, N50 and number of contigs, were identified as good indicators for high-sequencing quality. We propose QC thresholds for N50 greater than 20 000 and 25 000 for Campylobacter coli and Escherichia coli, respectively, and number of contigs >200 bp greater than 225, 265 and 100 for Salmonella enterica, Escherichia coli and Campylobacter coli, respectively. The GPT2020 results confirm the importance of systematic QC procedures, ensuring the submission of reliable WGS data for surveillance and outbreak investigation to meet the requirements of the paradigm shift in methodology.

The Interaction of Borrelia burgdorferi with Human Dendritic Cells: Functional Implications.

Dendritic cells bridge the innate and adaptive immune responses by serving as sensors of infection and as the primary APCs responsible for the initiation of the T cell response against invading pathogens. The naive T cell activation requires the following three key signals to be delivered from dendritic cells: engagement of the TCR by peptide Ags bound to MHC molecules (signal 1), engagement of costimulatory molecules on both cell types (signal 2), and expression of polarizing cytokines (signal 3). Initial interactions between Borrelia burgdorferi, the causative agent of Lyme disease, and dendritic cells remain largely unexplored. To address this gap in knowledge, we cultured live B. burgdorferi with monocyte-derived dendritic cells (mo-DCs) from healthy donors to examine the bacterial immunopeptidome associated with HLA-DR. In parallel, we examined changes in the expression of key costimulatory and regulatory molecules as well as profiled the cytokines released by dendritic cells when exposed to live spirochetes. RNA-sequencing studies on B. burgdorferi-pulsed dendritic cells show a unique gene expression signature associated with B. burgdorferi stimulation that differs from stimulation with lipoteichoic acid, a TLR2 agonist. These studies revealed that exposure of mo-DCs to live B. burgdorferi drives the expression of both pro- and anti-inflammatory cytokines as well as immunoregulatory molecules (e.g., PD-L1, IDO1, Tim3). Collectively, these studies indicate that the interaction of live B. burgdorferi with mo-DCs promotes a unique mature DC phenotype that likely impacts the nature of the adaptive T cell response generated in human Lyme disease.

Assessment of plasmids for relating the 2020 Salmonella enterica serovar Newport onion outbreak to farms implicated by the outbreak investigation.

BACKGROUND: The Salmonella enterica serovar Newport red onion outbreak of 2020 was the largest foodborne outbreak of Salmonella in over a decade. The epidemiological investigation suggested two farms as the likely source of contamination. However, single nucleotide polymorphism (SNP) analysis of the whole genome sequencing data showed that none of the Salmonella isolates collected from the farm regions were linked to the clinical isolates-preventing the use of phylogenetics in source identification. Here, we explored an alternative method for analyzing the whole genome sequencing data driven by the hypothesis that if the outbreak strain had come from the farm regions, then the clinical isolates would disproportionately contain plasmids found in isolates from the farm regions due to horizontal transfer. RESULTS: SNP analysis confirmed that the clinical isolates formed a single, nearly-clonal clade with evidence for ancestry in California going back a decade. The clinical clade had a large core genome (4,399 genes) and a large and sparsely distributed accessory genome (2,577 genes, at least 64% on plasmids). At least 20 plasmid types occurred in the clinical clade, more than were found in the literature for Salmonella Newport. A small number of plasmids, 14 from 13 clinical isolates and 17 from 8 farm isolates, were found to be highly similar (> 95% identical)-indicating they might be related by horizontal transfer. Phylogenetic analysis was unable to determine the geographic origin, isolation source, or time of transfer of the plasmids, likely due to their promiscuous and transient nature. However, our resampling analysis suggested that observing a similar number and combination of highly similar plasmids in random samples of environmental Salmonella enterica within the NCBI Pathogen Detection database was unlikely, supporting a connection between the outbreak strain and the farms implicated by the epidemiological investigation. CONCLUSION: Horizontally transferred plasmids provided evidence for a connection between clinical isolates and the farms implicated as the source of the outbreak. Our case study suggests that such analyses might add a new dimension to source tracking investigations, but highlights the need for detailed and accurate metadata, more extensive environmental sampling, and a better understanding of plasmid molecular evolution.

Prospective Genomic Surveillance Reveals Cryptic MRSA Outbreaks with Local to International Origins among NICU Patients.

Methicillin-resistant Staphylococcus aureus (MRSA) infections cause substantive morbidity and mortality in neonates. Using publicly available resources from the National Center of Biotechnology Information (NCBI) and Food and Drug Administration's (FDA) GalaxyTrakr pipeline, we illustrate the dynamics of MRSA colonization and infection in neonates. Over 217 days of prospective surveillance, analyses revealed concurrent MRSA transmission chains affecting 11 of 17 MRSA-colonized patients (65%), with two clusters that demonstrated intervals of more than a month among the appearance of isolates. All MRSA infected neonates (n = 3) showed previous colonization with the infecting strain. GalaxyTrakr clustering of the NICU strains, in the context of 21,521 international isolates deposited in NCBI's Pathogen Detection Resource, revealed NICU isolates to be distinct from adult MRSA strains seen locally and internationally. Clustering of the NICU strains within an international context enhanced the resolution of strain clusters and supported the rule-out of suspected, local transmission events within the NICU. Analyses also identified sequence type 1535 isolates, emergent in the Middle East, carrying a unique SCCmec with fusC and aac(6')-Ie/aph(2'')-1a that provided a multidrug-resistant phenotype. NICU genomic pathogen surveillance, leveraging public repositories and outbreak detection tools, supports rapid identification of cryptic MRSA clusters, and can inform infection prevention interventions for this vulnerable patient population. Results demonstrate that sporadic infections in the NICU may be indicative of hidden chains of asymptomatic transmission best identified with sequenced-based approaches.

Screening of new adsorbents to remove algal organic matter from aqueous solutions: kinetic analyses and reduction of disinfection by-products formation.

The algal organic matter (AOM) is a problem in water treatment. Although the adsorption process is extensively applied to drinking water treatment, little information is known about the potential of new adsorbents to remove AOM. Herein, this work evaluated the removal of AOM and its main compounds (dissolved organic carbon (DOC), carbohydrate, and protein) by new adsorbents-mesoporous silica (SBA-16), graphene oxide material from citric acid (CA), and sugar (SU), and a composite of CA immobilized on sand (GSC). In general, the removal efficiencies followed the order of SBA-16 > CA > SU or GSC for DOC, carbohydrate, and protein. At environmental condition (5 mg DOC·L-1 and pH 8), high removals were reported for SBA-16 (88.8% DOC, 80.0% carbohydrate, and 99.6% protein) and CA (70.0% DOC, 66.7% carbohydrate, and 89.7% protein), while moderate removals were found for SU (60.5% DOC, 47.9% carbohydrate, and 66.5% protein) and GSC (67.4% DOC, 60.8% carbohydrate, and 57.4% protein). Based on these results, further analyses were done with SBA-16 and CA. Both adsorbents' efficiencies decayed with the pH increment of the test water. Disinfection by-products reductions found using SBA-16 - trihalomethanes (58.2 to 94.7%) and chloral hydrate (48.7 to 78.8%) - were higher than the ones using CA-trihalomethanes (45.2 to 82.4%) and chloral hydrate (40.1 to 70.8%). This study showed the potential of applying these adsorbents for AOM removal, and further investigations are suggested to increase the adsorption capacity of these adsorbents.

Efficacy of iSupport-Brasil for unpaid caregivers of people living with dementia: protocol for a randomized and controlled clinical trial / Eficácia do iSupport-Brasil para cuidadores informais de pessoas que vivem com demência: protocolo de um ensaio clínico randomizado e controlado

ABSTRACT. Unpaid caregivers of people living with dementia tend to suffer mental health problems as a result of the negative effects associated with the care tasks. Thus, psychosocial interventions for this population group are necessary. iSupport is an online support program for caregivers that was created by the World Health Organization. Objective: To describe the design of a randomized clinical trial to measure the efficacy of the iSupport-Brasil version on caregivers' mental health and well-being. Methods: The participants will be randomized into Intervention Group (IG) (n=195) and Control Group (CG) (n=195). For three months, the IG will access the iSupport-Brasil platform, the CG will enter the electronic page of the Brazilian Alzheimer's Association, and both groups will be emailed the preliminary version of the "Guia de cuidados para a pessoa idosa" e-book (a guide to providing care to the elderly) from the Ministry of Health. The data will be collected at three moments: baseline, and three and six months after the beginning of the intervention. Results: It is expected that it will be possible to provide diverse validity evidence about iSupport-Brasil as an online and free intervention alternative, as a preventive means and as a way to promote mental health among caregivers of people living with dementia. Conclusion: Through the evaluation protocol of this randomized clinical trial on the effects of the iSupport-Brasil program, it may become a reference for countries that plan to adapt and improve the iSupport program using digital health solutions.

RESUMO. Cuidadores informais de pessoas que vivem com demência tendem a sofrer problemas de saúde mental como resultado dos efeitos negativos associados às tarefas de cuidado. Assim, intervenções psicossociais para essa população são necessárias. O iSupport é um programa de apoio online para cuidadores criado pela Organização Mundial da Saúde. Objetivo: Descrever o desenho de um ensaio clínico randomizado para medir a eficácia da versão iSupport-Brasil na saúde mental e no bem-estar de cuidadores. Métodos: Os participantes serão randomizados em Grupo Intervenção (GI) (n=195) e Grupo Controle (GC) (n=195). Durante três meses, o GI acessará a plataforma iSupport-Brasil, o CG entrará na página eletrônica da Associação Brasileira de Alzheimer e ambos os grupos receberão por e-mail a versão preliminar do e-book Guia de cuidados para a pessoa idosa, do Ministério da Saúde. Os dados serão coletados em três momentos: linha de base, três e seis meses após o início da intervenção. Resultados: Espera-se que seja possível fornecer diversas evidências sobre os efeitos do iSupport-Brasil como alternativa de intervenção online e gratuita, de forma a promover a saúde mental entre os cuidadores de pessoas que vivem com demência. Conclusão: O protocolo de avaliação deste ensaio clínico randomizado sobre os efeitos do programa iSupport-Brasil pode se tornar uma referência para os países que planejam adaptar e melhorar esta intervenção, usando soluções digitais de saúde.

Trabajar en salud durante la pandemia: Experiencias de vinculación con la comunidad relativas a la producción social del cuidado en Mar del Plata, Argentina / Work in health during the pandemic: Experiences of linking with the community related to the social production of care in Mar del Plata, Argentina

Resumen En este artículo proponemos visibilizar las estrategias de vinculación comunitaria de trabajadores/as de salud pública relativas a la producción del cuidado en el contexto crítico de la pandemia de la covid-19, signado por la profundización de la pobreza, la restricción de los servicios de salud y las tensiones del sistema sanitario en Argentina. Con un diseño metodológico cualitativo y enfoque etnográfico, entrevistamos a trabajadoras/es de distintas disciplinas, ocupaciones y géneros que se desempeñan en centros de salud y hospitales públicos en Mar del Plata y zonas rurales circundantes, entre los meses de marzo y diciembre de 2021. A partir de sus narrativas, analizamos las dimensiones de acceso a la alimentación, medicación y terapias holísticas, las dificultades encontradas y su contribución a la producción social del cuidado en salud. Concluimos que las estrategias generadas conllevan el potencial instituyente para la reorientación de los servicios públicos de salud de acuerdo con los desafíos del derecho a la salud desde la perspectiva de género y derechos en el tránsito a la postpandemia.

Abstract In this article we propose to make visible the strategies of community engagement of public health workers related to the production of care in the critical context of the covid-19 pandemic, marked by the deepening of poverty, the restriction of health services and the tensions of the health system in Argentina. With a qualitative methodological design and ethnographic approach, we interviewed workers from different disciplines, occupations and genders, who work in health centers and public hospitals in Mar del Plata and surrounding rural areas, between the months of March and December 2021. Based on their narratives, we analyze the dimensions of access to food, medication and holistic therapies, the difficulties encountered and their contribution to the social production of health care. We conclude that the strategies generated carry the instituting potential for the reorientation of public health services in accordance with the challenges of the right to health from the perspective of gender and rights in the transition to the post-pandemic.

Pathogenome comparison and global phylogeny of Escherichia coli ST1485 strains.

Escherichia coli ST1485 strains belong to the clinically important phylogroup F and have disseminated worldwide in humans, animals, and the environment. Here, we elucidated the pathogenome of a global collection of E. coli ST1485 isolates from diverse sources retrieved from public databases and a high-quality sequenced complete genome of colistin-resistant E. coli strain CFSAN061771 isolated from raw milk cheese which designated as a reference strain. CFSAN061771 belongs to O83:H42-ST1485 pathotype and carries a conjugative ColV plasmid, pCFSAN061771_01, combining extraintestinal virulence genes (ompt, sitA, iroN, etsC, traT, cvaC, hylF, iss, tsh, mchf, iucC, iutA) with a multidrug resistance island (blaTEM-1, aph(6)-Id, aph(3″)-Ib, sul2, dfrA14). Comparative genomic analysis revealed a high frequency of pCFSAN061771_01-like plasmids in E. coli ST1485. A notable evolutionary genetic event in E. coli ST1485 strains is the acquisition of a pCFSAN061771_02-like plasmid, which confers resistance to several antimicrobials, tellurium, and quaternary ammonium compounds. The identical virulence and antibiotic resistance profiles identified in some human and animal strains are worrisome. This is the first study to emphasize the significance of E. coli ST1485 as a global high-risk virulent and multidrug-resistant clone with zoonotic potential.

Changes in the genomes and methylomes of three Salmonella enterica serovars after long-term storage in ground black pepper.

Low moisture foods (LMFs) have traditionally been recognized as safe for consumption, as most bacteria require higher water content to grow. However, outbreaks due to LMF foods are increasing, and the microbial pathogen Salmonella enterica is frequently implicated. S. enterica can survive in LMFs for years, but few serovars have been studied, and the mechanisms which underlie this longevity are not well understood. Here, we determine that S. enterica serovars S. Tennessee, S. Anatum, and S. Reading but not S. Oranienburg can survive in the ground black pepper for 6 years. S. Reading was not previously associated with any LMF. Using both Illumina and Pacific Biosciences sequencing technologies, we also document changes in the genomes and methylomes of the surviving serovars over this 6-year period. The three serovars acquired a small number of single nucleotide polymorphisms (SNPs) including seven substitutions (four synonymous, two non-synonymous, and one substitution in a non-coding region), and two insertion-deletions. Nine distinct N6-methyladenine (m6A) methylated motifs across the three serovars were identified including five which were previously known, G m6ATC, CAG m6AG, BATGC m6AT, CRT m6AYN6CTC, and CC m6AN7TGAG, and four novel serovar-specific motifs, GRT m6AN8TTYG, GA m6ACN7GTA, GAA m 6A CY, and CAA m6ANCC. Interestingly, the BATGCAT motif was incompletely methylated (35-64% sites across the genome methylated), suggesting a possible role in gene regulation. Furthermore, the number of methylated BATGC m6AT motifs increased after storage in ground black pepper for 6 years from 475 to 657 (S. Tennessee), 366 to 608 (S. Anatum), and 525 to 570 (S. Reading), thus warranting further study as an adaptive mechanism. This is the first long-term assessment of genomic changes in S. enterica in a low moisture environment, and the first study to examine the methylome of any bacteria over a period of years, to our knowledge. These data contribute to our understanding of S. enterica survival in LMFs, and coupled with further studies, will provide the information necessary to design effective interventions which reduce S. enterica in LMFs and maintain a healthy, safe food supply.

Complete genome sequences and genomic characterization of five plasmids harbored by environmentally persistent Cronobacter sakazakii strains ST83 H322 and ST64 GK1025B obtained from powdered infant formula manufacturing facilities.

BACKGROUND: Cronobacter sakazakii is a foodborne pathogen that causes septicemia, meningitis, and necrotizing enterocolitis in neonates and infants. The current research details the full genome sequences of two extremely persistent C. sakazakii strains (H322 and GK1025B) isolated from powdered infant formula (PIF) manufacturing settings. In addition, the genetic attributes associated with five plasmids, pH322_1, pH322_2, pGK1025B_1, pGK1025B_2, and pGK1025B_3 are described. MATERIALS AND METHODS: Using PacBio single-molecule real-time (SMRT®) sequencing technology, whole genome sequence (WGS) assemblies of C. sakazakii H322 [Sequence type (ST)83, clonal complex [CC] 83) and GK1025B (ST64, CC64) were generated. Plasmids, also sequenced, were aligned with phylogenetically related episomes to determine, and identify conserved and missing genomic regions. RESULTS: A truncated ~ 13 Kbp type 6 secretion system (T6SS) gene cluster harbored on virulence plasmids pH322_2 and pGK1025B_2, and a second large deletion (~ 6 Kbp) on pH322_2, which included genes for a tyrosine-type recombinase/integrase, a hypothetical protein, and a phospholipase D was identified. Within the T6SS of pH322_2 and pGK1025B_2, an arsenic resistance operon was identified which is in common with that of plasmids pSP291_1 and pESA3. In addition, PHASTER analysis identified an intact 96.9 Kbp Salmonella SSU5 prophage gene cluster in pH322_1 and pGK1025B_1 and showed that these two plasmids were phylogenetically related to C. sakazakii plasmids: pCS1, pCsa767a, pCsaC757b, pCsaC105731a. Plasmid pGK1025B_3 was identified as a novel conjugative Cronobacter plasmid. Furthermore, WGS analysis identified a ~ 16.4 Kbp type 4 secretion system gene cluster harbored on pGK1025B_3, which contained a phospholipase D gene, a key virulence factor in several host-pathogen diseases. CONCLUSION: These data provide high resolution information on C. sakazakii genomes and emphasizes the need for furthering surveillance studies to link genotype to phenotype of strains from previous investigations. These results provide baseline data necessary for future in-depth investigations of C. sakazakii that colonize PIF manufacturing facility settings and genomic analyses of these two C. sakazakii strains and five associated plasmids will contribute to a better understanding of this pathogen's survival and persistence within various "built environments" like PIF manufacturing facilities.

Closed Genome Sequences of Salmonella enterica Serovar Javiana Isolates from Various Sources.

Salmonella enterica serovar Javiana is a major Salmonella serovar that causes human Salmonella infection in the United States. The complete genomic sequences of 9 S. Javiana isolates collected from food, environmental, and kratom sources in the United States were determined by hybrid assembly using Nanopore long-read sequencing and MiSeq short-read sequencing.

Whole-Genome Sequences of Two Listeria monocytogenes Biofilm Formers.

Listeria monocytogenes strains from different lineages show different biofilm-forming abilities. In this study, two strains of L. monocytogenes were whole genome sequenced using single-molecule real-time (SMRT) technology and characterized.

Use of Whole Genome Sequencing by the Federal Interagency Collaboration for Genomics for Food and Feed Safety in the United States.

ABSTRACT: This multiagency report developed by the Interagency Collaboration for Genomics for Food and Feed Safety provides an overview of the use of and transition to whole genome sequencing (WGS) technology for detection and characterization of pathogens transmitted commonly by food and for identification of their sources. We describe foodborne pathogen analysis, investigation, and harmonization efforts among the following federal agencies: National Institutes of Health; Department of Health and Human Services, Centers for Disease Control and Prevention (CDC) and U.S. Food and Drug Administration (FDA); and the U.S. Department of Agriculture, Food Safety and Inspection Service, Agricultural Research Service, and Animal and Plant Health Inspection Service. We describe single nucleotide polymorphism, core-genome, and whole genome multilocus sequence typing data analysis methods as used in the PulseNet (CDC) and GenomeTrakr (FDA) networks, underscoring the complementary nature of the results for linking genetically related foodborne pathogens during outbreak investigations while allowing flexibility to meet the specific needs of Interagency Collaboration partners. We highlight how we apply WGS to pathogen characterization (virulence and antimicrobial resistance profiles) and source attribution efforts and increase transparency by making the sequences and other data publicly available through the National Center for Biotechnology Information. We also highlight the impact of current trends in the use of culture-independent diagnostic tests for human diagnostic testing on analytical approaches related to food safety and what is next for the use of WGS in the area of food safety.

Genomic evidence of environmental and resident Salmonella Senftenberg and Montevideo contamination in the pistachio supply-chain.

Pistachios have been implicated in two salmonellosis outbreaks and multiple recalls in the U.S. This study performed an in-depth retrospective data analysis of Salmonella associated with pistachios as well as a storage study to evaluate the survivability of Salmonella on inoculated inshell pistachios to further understand the genetics and microbiological dynamics of this commodity-pathogen pair. The retrospective data analysis on isolates associated with pistachios was performed utilizing short-read and long-read sequencing technologies. The sequence data were analyzed using two methods: the FDA's Center for Food Safety and Applied Nutrition Single Nucleotide Polymorphism (SNP) analysis and Whole Genome Multilocus Sequence Typing (wgMLST). The year-long storage study evaluated the survival of five strains of Salmonella on pistachios stored at 25 °C at 35% and 54% relative humidity (RH). Our results demonstrate: i) evidence of persistent Salmonella Senftenberg and Salmonella Montevideo strains in pistachio environments, some of which may be due to clonal resident strains and some of which may be due to preharvest contamination; ii) presence of the Copper Homeostasis and Silver Resistance Island (CHASRI) in Salmonella Senftenberg and Montevideo strains in the pistachio supply chain; and iii) the use of metagenomic analysis is a novel tool for determining the composition of serovar survival in a cocktail inoculated storage study.

Whole-Genome Assessment of Clinical Acinetobacter baumannii Isolates Uncovers Potentially Novel Factors Influencing Carbapenem Resistance.

Carbapenems-one of the important last-line antibiotics for the treatment of gram-negative infections-are becoming ineffective for treating Acinetobacter baumannii infections. Studies have identified multiple genes (and mechanisms) responsible for carbapenem resistance. In some A. baumannii strains, the presence/absence of putative resistance genes is not consistent with their resistance phenotype-indicating the genomic factors underlying carbapenem resistance in A. baumannii are not fully understood. Here, we describe a large-scale whole-genome genotype-phenotype association study with 349 A. baumannii isolates that extends beyond the presence/absence of individual antimicrobial resistance genes and includes the genomic positions and pairwise interactions of genes. Ten known resistance genes exhibited statistically significant associations with resistance to imipenem, a type of carbapenem: blaOXA-23, qacEdelta1, sul1, mphE, msrE, ant(3")-II, aacC1, yafP, aphA6, and xerD. A review of the strains without any of these 10 genes uncovered a clade of isolates with diverse imipenem resistance phenotypes. Finer resolution evaluation of this clade revealed the presence of a 38.6 kbp conserved chromosomal region found exclusively in imipenem-susceptible isolates. This region appears to host several HTH-type DNA binding transcriptional regulators and transporter genes. Imipenem-susceptible isolates from this clade also carried two mutually exclusive plasmids that contain genes previously known to be specific to imipenem-susceptible isolates. Our analysis demonstrates the utility of using whole genomes for genotype-phenotype correlations in the context of antibiotic resistance and provides several new hypotheses for future research.