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.

Unveiling the genomic landscape of Salmonella enterica serotypes Typhimurium, Newport, and Infantis in Latin American surface waters: a comparative analysis.

Surface waters are considered ecological habitats where Salmonella enterica can persist and disseminate to fresh produce production systems. This study aimed to explore the genomic profiles of S. enterica serotypes Typhimurium, Newport, and Infantis from surface waters in Chile, Mexico, and Brazil collected between 2019 and 2022. We analyzed the whole genomes of 106 S. Typhimurium, 161 S. Newport, and 113 S. Infantis isolates. Our phylogenetic analysis exhibited distinct groupings of isolates by their respective countries except for a notable case involving a Chilean S. Newport isolate closely related to two Mexican isolates, showing 4 and 13 single nucleotide polymorphisms of difference, respectively. The patterns of the most frequently detected antimicrobial resistance genes varied across countries and serotypes. A strong correlation existed between integron carriage and genotypic multidrug resistance (MDR) across serotypes in Chile and Mexico (R > 0.90, P < 0.01), while integron(s) were not detected in any of the Brazilian isolates. By contrast, we did not identify any strong correlation between plasmid carriage and genotypic MDR across diverse countries and serotypes.IMPORTANCEUnveiling the genomic landscape of S. enterica in Latin American surface waters is pivotal for ensuring public health. This investigation sheds light on the intricate genomic diversity of S. enterica in surface waters across Chile, Mexico, and Brazil. Our research also addresses critical knowledge gaps, pioneering a comprehensive understanding of surface waters as a reservoir for multidrug-resistant S. enterica. By integrating our understanding of integron carriage as biomarkers into broader MDR control strategies, we can also work toward targeted interventions that mitigate the emergence and dissemination of MDR in S. enterica in surface waters. Given its potential implications for food safety, this study emphasizes the critical need for informed policies and collaborative initiatives to address the risks associated with S. enterica in surface waters.

Evaluation of Virulence Determinants Using Whole-Genome Sequencing and Phenotypic Biofilm Analysis of Outbreak-Linked Staphylococcus aureus Isolates.

Biofilms are a frequent cause of food contamination of potentially pathogenic bacteria, such as Staphylococcus aureus. Given its vast role in human disease, the possible impact of biofilm-producing S. aureus isolates in a food processing environment is evident. Sixty-nine S. aureus isolates collected from one firm following multiple staphylococcal food poisoning outbreak investigations were utilized for this analysis. Strain evaluations were performed to establish virulence determinants and the evolutionary relationships using data generated by shotgun whole-genome sequencing (WGS), along with end point polymerase chain reaction (PCR) and in vitro phenotypic assessments. S. aureus isolates were grouped into six well-supported clades in the phylogenetic tree, with the relationships within the clades indicating a strong degree of clonal structure. Our analysis identified four major sequence types 47.8% ST1, 31.9% ST45, 7.2% ST5, and 7.2% ST30 and two major spa types 47.8% t127 and 29.0% t3783. Extrapolated staphylococcal enterotoxin (SE) analysis found that all isolates were positive for at least 1 of the 23 SEs and/or SE-like toxin genes. Enterotoxigenic assessments found that 93% of the isolates expressed a classical SE(A-E). SE gene concurrence was observed at 96.2%, based on PCR and WGS results. In total, 46 gene targets were distinguished. This included genes that encode for adhesion and biofilm synthesis such as clfA, clfB, bbp, ebpS, ica, bap and agr. Our evaluation found agr group III to be the most prevalent at 55%, followed by 35% for agr group I. All isolates harbored the complete intercellular adhesion operon that is recognized to contain genes responsible for the adhesion step of biofilm formation by encoding proteins involved in the syntheses of the biofilm matrix. Phenotypic characterization of biofilm formation was evaluated three times, with each test completed in triplicate and accomplished utilizing the microtiter plate method and Congo red agar (CRA). The microtiter plate results indicated moderate to high biofilm formation for 96% of the isolates, with 4% exhibiting weak to no biofilm development. CRA results yielded all positive to intermediate results. The potential to inadvertently transfer pathogenic bacteria from the environment into food products creates challenges to any firm and may result in adulterated food.

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.

Characterization of Salmonella enterica Isolates from Selected U.S. Swine Feed Mills by Whole-Genome Sequencing.

Every year salmonellosis is responsible for $2.3 billion in costs to the U.S. food industry, with nearly 6% of the reported cases associated with pork and/or pork products. Several studies have demonstrated the role of pigs as Salmonella reservoirs. Furthermore, this pathogen has been identified as a potential biological hazard in many livestock feeds. The overall objective of this research was to characterize Salmonella enterica isolates in selected U.S. swine feed mills by whole-genome sequencing (WGS) and evaluate isolates in association with the season and feed production stages. Salmonella isolates were collected from 11 facilities during a previous study. Samples were analyzed for Salmonella prevalence following the U.S. Department of Agriculture guidelines and confirmed by PCR. WGS was carried out on either the MiSeq or NextSeq sequencer. De novo genome assemblies were obtained with the Shovill pipeline, version 0.9. ResFinder and SPIFinder were used to identify antibiotic resistance genes and pathogenicity islands. Finally, their phylogenetic relationship and diversity were determined by core genome multilocus sequence typing. Overall, our analysis showed the presence of S. enterica in the feed mill environment. Isolates belonged to 16 different serotypes. Salmonella Agona, Salmonella Mbandaka, Salmonella Senfenberg, and Salmonella Scharzengrund were the most frequently found, and 18 single-nucleotide polymorphism clusters were identified. In silico analysis showed that 40% of the strains carried at least one antimicrobial resistance gene. All isolates in this study could be considered of public health concern and pathogenic potential. Our findings underscore the potential role of the feed mill environment as the pathogen entry route into the human food value chain.

Draft Genome Sequences of 57 Salmonella enterica Strains from Selected U.S. Swine Feed Mills.

The number of Salmonella infection cases linked to pork products has increased. Pathogen presence in the feed mill environment is one of the many potential transmission routes into the food production chain. Here, we describe the draft genome sequences of 57 Salmonella enterica isolates from selected U.S. swine feed mills.

Analysis of the VIDAS® Staph Enterotoxin III (SET3) for Detection of Staphylococcal Enterotoxins G, H, and I in Foods.

BACKGROUND: Staphylococcal food poisoning (SFP) frequently causes illnesses worldwide. SFP occurs from the ingestion of staphylococcal enterotoxins (SEs) preformed in foods by enterotoxigenic strains of Staphylococcus species, primarily S. aureus. SEG, SEH, and SEI induce emesis and have been implicated in outbreaks. Immunological-based methods are deemed the most practical methods for the routine analysis of SEs in foods given their ease of use, sensitivity, specificity, and commercial availability. These kits are routinely used to test for SEA-SEE. However, only recently has a kit been developed to detect SEG, SEH, and SEI. OBJECTIVE: Our research examined the performance of the novel VIDAS® Staph Enterotoxin III (SET3) for the detection of staphylococcal enterotoxins SEG, SEH, and SEI in foods. METHODS: Here we assess the sensitivity and specificity of SET3 using duplicate test portions of six foods at varying concentrations of inclusivity and exclusivity inocula: pure SEG, SEH, SEI, S. aureus strain extracts positive for seg, seh, and sei, as well as SEA, SEB, SEC, SED, and SEE. RESULTS: The overall detection limit was less than 2.09 ng/mL for foods inoculated with SEG, SEH, and SEI, with no cross reactivity observed. HIGHLIGHTS: Integrating concurrent testing to detect the presence of SEA-SEE and SEG-SEI utilizing the SET3 along with the VIDAS SET2, Ridascreen® SET total, or other comparable kits will be instrumental for the future food assessments in our laboratory and may become the new standard for SE analysis of foods.

From Commensal to Consumer: Staphylococcus aureus Toxins, Diseases, and Detection Methods.

Staphylococcus aureus is a Gram-positive bacterium capable of causing a wide array of infections. Generally a commensal organism, S. aureus encodes several virulence mechanisms that contribute to disease progression. This review highlights toxins as a secreted virulence factor by S. aureus, the diseases that manifest as a result, and the methods used to detect them. In particular, the advantages and limitations of current toxin detection methods are discussed.

Screening, detection, and serotyping methods for toxin genes and enterotoxins in Staphylococcus strains.

Staphylococcus aureus continues to play a significant role in foodborne outbreak investigations, with numerous individuals sickened each year after ingesting assorted foods contaminated with staphylococcal enterotoxins. The purpose of this study was to evaluate the use of several methods for the screening, detection, and enterotoxin serotyping of staphylococcal bacterial strains for classical staphylococcal enterotoxins (SEs; SEA, SEB, SEC, SED, and SEE) and the newly described SE and SE-like enterotoxin genes (seg, seh, sei, sej, sek, sel, sem, sen, seo, sep, seq, ser, ses, set, and seu). Inclusivity and exclusivity panels of staphylococcal strains were tested using a multiplex PCR method in addition to three polyvalent commercially prepared ELISA systems for the detection of SEA-SEE and one monovalent assay for the identification of classical SE serotypes. The results indicate an overall agreement between serological detection methods with a few exceptions, and molecular characterization identified an abundance of SE and SE-like enterotoxin genes including several potentially enterotoxigenic isolates that would have otherwise been missed by ELISA-based methods. These findings demonstrate the significance of PCR for future screening purposes and the use of ELISA systems for the detection and enterotoxin serotyping of staphylococcal bacterial strains.

Staphylococcal enterotoxin B-specific electrochemiluminescence and lateral flow device assays cross-react with staphylococcal enterotoxin D.

Guam school children and faculty members experienced symptoms of vomiting, nausea, abdominal cramps, and diarrhea shortly after eating breakfast prepared by contracted caterers. The first illness was reported within an hour after breakfast, affecting 295 students and two faculty members. Local hospitals treated 130 people, and 61 were admitted for further treatment. Reported symptoms were consistent with staphylococcal food poisoning. Initial food testing using a lateral flow device and electrochemiluminescence method incorrectly implicated staphylococcal enterotoxin B as the causative agent, prompting partial activation of Guam's Emergency Response Center. Traditional ELISAs proved that the food poisoning agent was staphylococcal enterotoxin D. More specific and sensitive assays would have alleviated the issues and confusion that surrounded the reporting and investigation of this outbreak.

Novel platform for the detection of Staphylococcus aureus enterotoxin B in foods.

Staphylococcal contamination of food products and staphylococcal food-borne illnesses continue to be a problem worldwide. Screening of food for the presence of Staphylococcus aureus and/or enterotoxins using traditional methods is laborious. Reliable and rapid multiplex detection methods from a single food extract or culture supernatant would simplify testing. A fluorescence-based cytometric bead array was developed for the detection of staphylococcal enterotoxin B (SEB), using magnetic microspheres coupled with either an engineered, enterotoxin-specific Vß domain of the T-cell receptor (Vß-TCR) or polyclonal antibodies. The binding affinity of the Vß-TCR for SEB has been shown to be in the picomolar range, comparable to the best monoclonal antibodies. The coupled beads were validated with purified enterotoxins and tested in a variety of food matrices spiked with enterotoxins. The Vß-TCR or antibody was shown to specifically bind SEB in four different food matrices, including milk, mashed potatoes, vanilla pudding, and cooked chicken. The use of traditional polyclonal antibodies and Vß-TCR provides a redundant system that ensures accurate identification of the enterotoxin, and the use of labeled microspheres permits simultaneous testing of multiple enterotoxins from a single sample.

Efficient isolation and identification of Bacillus cereus group.

Bacillus cereus is a group of ubiquitous facultative anaerobic sporeforming Gram-positive rods commonly found in soil. The spores frequently contaminate a variety of foods, including produce, meat, eggs, and dairy products. Foodborne illnesses associated with toxins produced by B. cereus can result in self-limiting diarrhea or vomiting. Plate enumeration methods recommended by recognized food authorities to detect the presence of B. cereus in potentially contaminated food products do not inhibit other Gram-positive competitive bacteria. This study evaluated the use of Bacara, a new chromogenic agar, as an efficient method to identify and enumerate B. cereus group from food matrixes, even in the presence of background flora. Inclusivity and exclusivity testing was performed using four different selective and differential media for B. cereus, including Mannitol Egg Yolk Polymyxin (MYP), Polymyxin Pyruvate Egg-Yolk Mannitol Bromothymol Blue Agar, Bacillus Chromogenic Media, Brilliance, and Bacara. MYP and Bacara were also used in plate enumeration studies to isolate B. cereus from artificially contaminated foods.

Moonlighting bacteriophage proteins derepress staphylococcal pathogenicity islands.

Staphylococcal superantigen-carrying pathogenicity islands (SaPIs) are discrete, chromosomally integrated units of approximately 15 kilobases that are induced by helper phages to excise and replicate. SaPI DNA is then efficiently encapsidated in phage-like infectious particles, leading to extremely high frequencies of intra- as well as intergeneric transfer. In the absence of helper phage lytic growth, the island is maintained in a quiescent prophage-like state by a global repressor, Stl, which controls expression of most of the SaPI genes. Here we show that SaPI derepression is effected by a specific, non-essential phage protein that binds to Stl, disrupting the Stl-DNA complex and thereby initiating the excision-replication-packaging cycle of the island. Because SaPIs require phage proteins to be packaged, this strategy assures that SaPIs will be transferred once induced. Several different SaPIs are induced by helper phage 80alpha and, in each case, the SaPI commandeers a different non-essential phage protein for its derepression. The highly specific interactions between different SaPI repressors and helper-phage-encoded antirepressors represent a remarkable evolutionary adaptation involved in pathogenicity island mobilization.

Specificity of staphylococcal phage and SaPI DNA packaging as revealed by integrase and terminase mutations.

SaPI1 and SaPIbov1 are chromosomal pathogenicity islands in Staphylococcus aureus that carry tst and other superantigen genes. They are induced to excise and replicate by certain phages, are efficiently encapsidated in SaPI-specific small particles composed of phage virion proteins and are transferred at very high frequencies. In this study, we have analysed three SaPI genes that are important for the phage-SaPI interaction, int (integrase) terS (phage terminase small subunit homologue) and pif (phage interference function). SaPI1 int is required for SaPI excision, replication and packaging in a donor strain, and is required for integration in a recipient. A SaPI1 int mutant, following phage induction, produces small SaPI-specific capsids which are filled with partial phage genomes. SaPIbov1 DNA is efficiently packaged into full-sized phage heads as well as into SaPI-specific small ones, whereas SaPI1 DNA is found almost exclusively in the small capsids. TerS, however, determines DNA packaging specificity but not the choice of large versus small capsids. This choice is influenced by SaPIbov1 gene 12, which prevents phage DNA packaging into small capsids, and which is also primarily responsible for interference by SaPIbov1 with phage reproduction.

Transducing particles of Staphylococcus aureus pathogenicity island SaPI1 are comprised of helper phage-encoded proteins.

The relationship between the composition of SaPI1 transducing particles and those of helper phage 80alpha was investigated by direct comparison of virion proteins. Twelve virion proteins were identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and mass spectrometry; all were present in both 80alpha and SaPI1 virions, and all were encoded by 80alpha. No SaPI1-encoded proteins were detected. This confirms the prediction that SaPI1 is encapsidated in a virion assembled from helper phage-encoded proteins.

Nosocomial bloodstream infections in US hospitals: analysis of 24,179 cases from a prospective nationwide surveillance study.

BACKGROUND: Nosocomial bloodstream infections (BSIs) are important causes of morbidity and mortality in the United States. METHODS: Data from a nationwide, concurrent surveillance study (Surveillance and Control of Pathogens of Epidemiological Importance [SCOPE]) were used to examine the secular trends in the epidemiology and microbiology of nosocomial BSIs. RESULTS: Our study detected 24,179 cases of nosocomial BSI in 49 US hospitals over a 7-year period from March 1995 through September 2002 (60 cases per 10,000 hospital admissions). Eighty-seven percent of BSIs were monomicrobial. Gram-positive organisms caused 65% of these BSIs, gram-negative organisms caused 25%, and fungi caused 9.5%. The crude mortality rate was 27%. The most-common organisms causing BSIs were coagulase-negative staphylococci (CoNS) (31% of isolates), Staphylococcus aureus (20%), enterococci (9%), and Candida species (9%). The mean interval between admission and infection was 13 days for infection with Escherichia coli, 16 days for S. aureus, 22 days for Candida species and Klebsiella species, 23 days for enterococci, and 26 days for Acinetobacter species. CoNS, Pseudomonas species, Enterobacter species, Serratia species, and Acinetobacter species were more likely to cause infections in patients in intensive care units (P<.001). In neutropenic patients, infections with Candida species, enterococci, and viridans group streptococci were significantly more common. The proportion of S. aureus isolates with methicillin resistance increased from 22% in 1995 to 57% in 2001 (P<.001, trend analysis). Vancomycin resistance was seen in 2% of Enterococcus faecalis isolates and in 60% of Enterococcus faecium isolates. CONCLUSION: In this study, one of the largest multicenter studies performed to date, we found that the proportion of nosocomial BSIs due to antibiotic-resistant organisms is increasing in US hospitals.

Nosocomial bloodstream infections in pediatric patients in United States hospitals: epidemiology, clinical features and susceptibilities.

BACKGROUND: We identified the predominant pathogens and antimicrobial susceptibilities of nosocomial bloodstream isolates in pediatric patients in the US Prospective surveillance for nosocomial bloodstream infections at 49 hospitals during a 6-year period [Surveillance and Control of Pathogens of Epidemiologic Importance (SCOPE)] detected 22 609 bloodstream infections, of which 3432 occurred in patients < or =16 years of age. RESULTS: Gram-positive organisms accounted for 65% of cases, Gram-negative organisms accounted for 24% of cases and 11% were caused by fungi. The overall crude mortality was 14% (475 of 3432) but notably higher for infections caused by Candida spp. and Pseudomonas aeruginosa, 20 and 29%, respectively. The most common organisms were coagulase-negative staphylococci (43%), enterococci, Staphylococcus aureus and Candida spp. (each, 9%). The mean interval between admission and infection averaged 21 days for coagulase-negative staphylococci, 25 days for S. aureus and Candida spp., 32 days for Klebsiella spp. and 34 days for Enterococcus spp. The proportion of methicillin-resistant S. aureus increased from 10% in 1995 to 29% in 2001. Vancomycin-resistance was seen in 1% of Enterococcus faecalis and in 11% of Enterococcus faecium isolates. CONCLUSION: Nosocomial BSI occurred predominantly in very young and/or critically ill children. Gram-positive pathogens predominated across all ages, and increasing antimicrobial resistance was observed in pediatric patients.