Global transcriptomic analyses of Salmonella enterica in Iron-depleted and Iron-rich growth conditions.

BACKGROUND: Salmonella enterica possess several iron acquisition systems, encoded on the chromosome and plasmids. Recently, we demonstrated that incompatibility group (Inc) FIB plasmid-encoded iron acquisition systems (Sit and aerobactin) likely play an important role in persistence of Salmonella in human intestinal epithelial cells (Caco-2). In this study, we sought to determine global transcriptome analyses of S. enterica in iron-rich (IR) and iron-depleted (ID) growth conditions. RESULTS: The number of differentially-expressed genes were substantially higher for recipient (SE819) (n = 966) and transconjugant (TC) (n = 945) compared to the wild type (WT) (SE163A) (n = 110) strain in ID as compared to IR growth conditions. Several virulence-associated factors including T3SS, flagellin, cold-shock protein (cspE), and regulatory genes were upregulated in TC in ID compared to IR conditions. Whereas, IS1 and acrR/tetR transposases located on the IncFIB plasmid, ferritin and several regulatory genes were downregulated in TC in ID conditions. Enterobactin transporter (entS), iron ABC transporter (fepCD), colicin transporter, IncFIB-encoded enolase, cyclic di-GMP regulator (cdgR) and other regulatory genes of the WT strain were upregulated in ID compared to IR conditions. Conversely, ferritin, ferrous iron transport protein A (feoA), IncFIB-encoded IS1 and acrR/tetR transposases and ArtA toxin of WT were downregulated in ID conditions. SDS-PAGE coupled with LC-MS/MS analyses revealed that siderophore receptor proteins such as chromosomally-encoded IroN and, IncFIB-encoded IutA were upregulated in WT and TC in ID growth conditions. Both chromosome and IncFIB plasmid-encoded SitA was overexpressed in WT, but not in TC or recipient in ID conditions. Increased expression of flagellin was detected in recipient and TC, but not in WT in ID conditions. CONCLUSION: Iron concentrations in growth media influenced differential gene expressions both at transcriptional and translational levels, including genes encoded on the IncFIB plasmid. Limited iron availability within the host may promote pathogenic Salmonella to differentially express subsets of genes encoded by chromosome and/or plasmids, facilitating establishment of successful infection.

Cross-talk among flesh-eating Aeromonas hydrophila strains in mixed infection leading to necrotizing fasciitis.

Necrotizing fasciitis (NF) caused by flesh-eating bacteria is associated with high case fatality. In an earlier study, we reported infection of an immunocompetent individual with multiple strains of Aeromonas hydrophila (NF1-NF4), the latter three constituted a clonal group whereas NF1 was phylogenetically distinct. To understand the complex interactions of these strains in NF pathophysiology, a mouse model was used, whereby either single or mixed A. hydrophila strains were injected intramuscularly. NF2, which harbors exotoxin A (exoA) gene, was highly virulent when injected alone, but its virulence was attenuated in the presence of NF1 (exoA-minus). NF1 alone, although not lethal to animals, became highly virulent when combined with NF2, its virulence augmented by cis-exoA expression when injected alone in mice. Based on metagenomics and microbiological analyses, it was found that, in mixed infection, NF1 selectively disseminated to mouse peripheral organs, whereas the other strains (NF2, NF3, and NF4) were confined to the injection site and eventually cleared. In vitro studies showed NF2 to be more effectively phagocytized and killed by macrophages than NF1. NF1 inhibited growth of NF2 on solid media, but ExoA of NF2 augmented virulence of NF1 and the presence of NF1 facilitated clearance of NF2 from animals either by enhanced priming of host immune system or direct killing via a contact-dependent mechanism.

Deep-sea hydrothermal vent bacteria related to human pathogenic Vibrio species.

Vibrio species are both ubiquitous and abundant in marine coastal waters, estuaries, ocean sediment, and aquaculture settings worldwide. We report here the isolation, characterization, and genome sequence of a novel Vibrio species, Vibrio antiquarius, isolated from a mesophilic bacterial community associated with hydrothermal vents located along the East Pacific Rise, near the southwest coast of Mexico. Genomic and phenotypic analysis revealed V. antiquarius is closely related to pathogenic Vibrio species, namely Vibrio alginolyticus, Vibrio parahaemolyticus, Vibrio harveyi, and Vibrio vulnificus, but sufficiently divergent to warrant a separate species status. The V. antiquarius genome encodes genes and operons with ecological functions relevant to the environment conditions of the deep sea and also harbors factors known to be involved in human disease caused by freshwater, coastal, and brackish water vibrios. The presence of virulence factors in this deep-sea Vibrio species suggests a far more fundamental role of these factors for their bacterial host. Comparative genomics revealed a variety of genomic events that may have provided an important driving force in V. antiquarius evolution, facilitating response to environmental conditions of the deep sea.

Nested Russian Doll-Like Genetic Mobility Drives Rapid Dissemination of the Carbapenem Resistance Gene blaKPC.

The recent widespread emergence of carbapenem resistance in Enterobacteriaceae is a major public health concern, as carbapenems are a therapy of last resort against this family of common bacterial pathogens. Resistance genes can mobilize via various mechanisms, including conjugation and transposition; however, the importance of this mobility in short-term evolution, such as within nosocomial outbreaks, is unknown. Using a combination of short- and long-read whole-genome sequencing of 281 blaKPC-positive Enterobacteriaceae isolates from a single hospital over 5 years, we demonstrate rapid dissemination of this carbapenem resistance gene to multiple species, strains, and plasmids. Mobility of blaKPC occurs at multiple nested genetic levels, with transmission of blaKPC strains between individuals, frequent transfer of blaKPC plasmids between strains/species, and frequent transposition of blaKPC transposon Tn4401 between plasmids. We also identify a common insertion site for Tn4401 within various Tn2-like elements, suggesting that homologous recombination between Tn2-like elements has enhanced the spread of Tn4401 between different plasmid vectors. Furthermore, while short-read sequencing has known limitations for plasmid assembly, various studies have attempted to overcome this by the use of reference-based methods. We also demonstrate that, as a consequence of the genetic mobility observed in this study, plasmid structures can be extremely dynamic, and therefore these reference-based methods, as well as traditional partial typing methods, can produce very misleading conclusions. Overall, our findings demonstrate that nonclonal resistance gene dissemination can be extremely rapid, presenting significant challenges for public health surveillance and achieving effective control of antibiotic resistance.

Enrichment dynamics of Listeria monocytogenes and the associated microbiome from naturally contaminated ice cream linked to a listeriosis outbreak.

BACKGROUND: Microbiota that co-enrich during efforts to recover pathogens from foodborne outbreaks interfere with efficient detection and recovery. Here, dynamics of co-enriching microbiota during recovery of Listeria monocytogenes from naturally contaminated ice cream samples linked to an outbreak are described for three different initial enrichment formulations used by the Food and Drug Administration (FDA), the International Organization of Standardization (ISO), and the United States Department of Agriculture (USDA). Enrichment cultures were analyzed using DNA extraction and sequencing from samples taken every 4 h throughout 48 h of enrichment. Resphera Insight and CosmosID analysis tools were employed for high-resolution profiling of 16S rRNA amplicons and whole genome shotgun data, respectively. RESULTS: During enrichment, other bacterial taxa were identified, including Anoxybacillus, Geobacillus, Serratia, Pseudomonas, Erwinia, and Streptococcus spp. Surprisingly, incidence of L. monocytogenes was proportionally greater at hour 0 than when tested 4, 8, and 12 h later with all three enrichment schemes. The corresponding increase in Anoxybacillus and Geobacillus spp.indicated these taxa co-enriched in competition with L. monocytogenes during early enrichment hours. L. monocytogenes became dominant after 24 h in all three enrichments. DNA sequences obtained from shotgun metagenomic data of Listeria monocytogenes at 48 h were assembled to produce a consensus draft genome which appeared to have a similar tracking utility to pure culture isolates of L. monocytogenes. CONCLUSIONS: All three methods performed equally well for enrichment of Listeria monocytogenes. The observation of potential competitive exclusion of L. mono by Anoxybacillus and Geobacillus in early enrichment hours provided novel information that may be used to further optimize enrichment formulations. Application of Resphera Insight for high-resolution analysis of 16S amplicon sequences accurately identified L. monocytogenes. Both shotgun and 16S rRNA data supported the presence of three slightly variable genomes of L. monocytogenes. Moreover, the draft assembly of a consensus genome of L. monocytogenes from shotgun metagenomic data demonstrated the potential utility of this approach to expedite trace-back of outbreak-associated strains, although further validation will be needed to confirm this utility.

Cilantro microbiome before and after nonselective pre-enrichment for Salmonella using 16S rRNA and metagenomic sequencing.

BACKGROUND: Salmonella enterica is a common cause of foodborne gastroenteritis in the United States and is associated with outbreaks in fresh produce such as cilantro. Salmonella culture-based detection methods are complex and time consuming, and improvments to increase detection sensitivity will benefit consumers. In this study, we used 16S rRNA sequencing to determine the microbiome of cilantro. We also investigated changes to the microbial community prior to and after a 24-hour nonselective pre-enrichment culture step commonly used by laboratory analysts to resuscitate microorganisms in foods suspected of contamination with pathogens. Cilantro samples were processed for Salmonella detection according to the method in the United States Food and Drug Administration Bacteriological Analytical Manual. Genomic DNA was extracted from culture supernatants prior to and after a 24-hour nonselective pre-enrichment step and 454 pyrosequencing was performed on 16S rRNA amplicon libraries. A database of Enterobacteriaceae 16S rRNA sequences was created, and used to screen the libraries for Salmonella, as some samples were known to be culture positive. Additionally, culture positive cilantro samples were examined for the presence of Salmonella using shotgun metagenomics on the Illumina MiSeq. RESULTS: Time zero uncultured samples had an abundance of Proteobacteria while the 24-hour enriched samples were composed mostly of Gram-positive Firmicutes. Shotgun metagenomic sequencing of Salmonella culture positive cilantro samples revealed variable degrees of Salmonella contamination among the sequenced samples. CONCLUSIONS: Our cilantro study demonstrates the use of high-throughput sequencing to reveal the microbiome of cilantro, and how the microbiome changes during the culture-based protocols employed by food safety laboratories to detect foodborne pathogens. Finding that culturing the cilantro shifts the microbiome to a predominance of Firmicutes suggests that changing our culture-based methods will improve detection sensitivity for foodborne enteric pathogens.

Analysis of the cellulose synthase operon genes, bcsA, bcsB, and bcsC in Cronobacter species: Prevalence among species and their roles in biofilm formation and cell-cell aggregation.

Cronobacter species are emerging food-borne pathogens that cause severe sepsis, meningitis, and necrotizing entercolitis in neonates and infants. Bacterial pathogens such as Escherichia coli and Salmonella species produce extracellular cellulose which has been shown to be involved in rugosity, biofilm formation, and host colonization. In this study the distribution and prevalence of cellulose synthase operon genes (bcsABZC) were determined by polymerase chain reaction (PCR) analysis in 231 Cronobacter strains isolated from clinical, food, environmental, and unknown sources. Furthermore, bcsA and bcsB isogenic mutants were constructed in Cronobacter sakazakii BAA894 to determine their roles. In calcofluor binding assays bcsA and bcsB mutants did not produce cellulose, and their colonial morphotypes were different to that of the parent strain. Biofilm formation and bacterial cell-cell aggregation were significantly reduced in bcsA and bcsB mutants compared to the parental strain. bcsA or bcsAB PCR-negative strains of C. sakazakii did not bind calcofluor, and produced less biofilm and cell-cell aggregation compared to strains possessing bcsAB genes. These data indicated that Cronobacter bcsABZC were present in all clinical isolates and most of food and environmental isolates. bcsA and bcsB genes of Cronobacter were necessary to produce cellulose, and were involved in biofilm formation and cell-cell aggregation.

Functional genomic characterization of virulence factors from necrotizing fasciitis-causing strains of Aeromonas hydrophila.

The genomes of 10 Aeromonas isolates identified and designated Aeromonas hydrophila WI, Riv3, and NF1 to NF4; A. dhakensis SSU; A. jandaei Riv2; and A. caviae NM22 and NM33 were sequenced and annotated. Isolates NF1 to NF4 were from a patient with necrotizing fasciitis (NF). Two environmental isolates (Riv2 and -3) were from the river water from which the NF patient acquired the infection. While isolates NF2 to NF4 were clonal, NF1 was genetically distinct. Outside the conserved core genomes of these 10 isolates, several unique genomic features were identified. The most virulent strains possessed one of the following four virulence factors or a combination of them: cytotoxic enterotoxin, exotoxin A, and type 3 and 6 secretion system effectors AexU and Hcp. In a septicemic-mouse model, SSU, NF1, and Riv2 were the most virulent, while NF2 was moderately virulent. These data correlated with high motility and biofilm formation by the former three isolates. Conversely, in a mouse model of intramuscular infection, NF2 was much more virulent than NF1. Isolates NF2, SSU, and Riv2 disseminated in high numbers from the muscular tissue to the visceral organs of mice, while NF1 reached the liver and spleen in relatively lower numbers on the basis of colony counting and tracking of bioluminescent strains in real time by in vivo imaging. Histopathologically, degeneration of myofibers with significant infiltration of polymorphonuclear cells due to the highly virulent strains was noted. Functional genomic analysis provided data that allowed us to correlate the highly infectious nature of Aeromonas pathotypes belonging to several different species with virulence signatures and their potential ability to cause NF.

Re-examination of the taxonomic status of Enterobacter helveticus, Enterobacter pulveris and Enterobacter turicensis as members of the genus Cronobacter and their reclassification in the genera Franconibacter gen. nov. and Siccibacter gen. nov. as Franconibacter helveticus comb. nov., Franconibacter pulveris comb. nov. and Siccibacter turicensis comb. nov., respectively.

Recently, a taxonomical re-evaluation of the genus Enterobacter, based on multi-locus sequence typing (MLST) analysis, has led to the proposal that the species Enterobacter pulveris, Enterobacter helveticus and Enterobacter turicensis should be reclassified as novel species of the genus Cronobacter. In the present work, new genome-scale analyses, including average nucleotide identity, genome-scale phylogeny and k-mer analysis, coupled with previously reported DNA-DNA hybridization values and biochemical characterization strongly indicate that these three species of the genus Enterobacter are not members of the genus Cronobacter, nor do they belong to the re-evaluated genus Enterobacter. Furthermore, data from this polyphasic study indicated that all three species constitute two new genera. We propose reclassifying Enterobacter pulveris and Enterobacter helveticus in the genus Franconibacter gen. nov. as Franconibacter pulveris comb. nov. (type strain 601/05(T) = LMG 24057(T) = DSM 19144(T)) and Franconibacter helveticus comb. nov. (type strain 513/05(T) = LMG 23732(T) = DSM 18396(T)), respectively, and Enterobacter turicensis in the genus Siccibacter gen. nov. as Siccibacter turicensis comb. nov. (type strain 508/05(T) = LMG 23730(T) = DSM 18397(T)).

Cronobacter: an emergent pathogen causing meningitis to neonates through their feeds.

The recognition of Cronobacter as a public health concern was raised when powdered infant formula (PIF) was linked to several neonatal meningitis outbreaks. It is an opportunistic pathogen that causes necrotising enterocolitis, infantile septicaemia, and meningitis which carries a high mortality rate among neonates. It has been also linked with cases of infection in adults and elderly. Over the past decade, much focus has been made on developing sensitive and specific characterisation, detection, and isolation methods to ascertain the quality of foods, notably contamination of PIF with Cronobacter and to understand its ability to cause disease. Whole genome sequencing has unveiled several putative virulence factors, yet the full capacity of the pathogenesis of Cronobacter has not yet been elucidated.

Advancing metagenome-assembled genome-based pathogen identification: unraveling the power of long-read assembly algorithms in Oxford Nanopore sequencing.

Oxford Nanopore sequencing is one of the high-throughput sequencing technologies that facilitates the reconstruction of metagenome-assembled genomes (MAGs). This study aimed to assess the potential of long-read assembly algorithms in Oxford Nanopore sequencing to enhance the MAG-based identification of bacterial pathogens using both simulated and mock communities. Simulated communities were generated to mimic those on fresh spinach and in surface water. Long reads were produced using R9.4.1+SQK-LSK109 and R10.4 + SQK-LSK112, with 0.5, 1, and 2 million reads. The simulated bacterial communities included multidrug-resistant Salmonella enterica serotypes Heidelberg, Montevideo, and Typhimurium in the fresh spinach community individually or in combination, as well as multidrug-resistant Pseudomonas aeruginosa in the surface water community. Real data sets of the ZymoBIOMICS HMW DNA Standard were also studied. A bioinformatic pipeline (MAGenie, freely available at https://github.com/jackchen129/MAGenie) that combines metagenome assembly, taxonomic classification, and sequence extraction was developed to reconstruct draft MAGs from metagenome assemblies. Five assemblers were evaluated based on a series of genomic analyses. Overall, Flye outperformed the other assemblers, followed by Shasta, Raven, and Unicycler, while Canu performed least effectively. In some instances, the extracted sequences resulted in draft MAGs and provided the locations and structures of antimicrobial resistance genes and mobile genetic elements. Our study showcases the viability of utilizing the extracted sequences for precise phylogenetic inference, as demonstrated by the consistent alignment of phylogenetic topology between the reference genome and the extracted sequences. R9.4.1+SQK-LSK109 was more effective in most cases than R10.4+SQK-LSK112, and greater sequencing depths generally led to more accurate results.IMPORTANCEBy examining diverse bacterial communities, particularly those housing multiple Salmonella enterica serotypes, this study holds significance in uncovering the potential of long-read assembly algorithms to improve metagenome-assembled genome (MAG)-based pathogen identification through Oxford Nanopore sequencing. Our research demonstrates that long-read assembly stands out as a promising avenue for boosting precision in MAG-based pathogen identification, thus advancing the development of more robust surveillance measures. The findings also support ongoing endeavors to fine-tune a bioinformatic pipeline for accurate pathogen identification within complex metagenomic samples.

Draft genome sequence of multidrug-resistant Vibrio navarrensis strain DA9 isolated from a coastal canal in the Florida Keys (USA).

A presumptive Vibrio isolate with a multidrug resistance profile was isolated from surface seawater collected from a coastal canal in 2014 and identified as Vibrio navarrensis, designated as strain DA9. Here, we report a 5.1-Mb draft genome sequence of strain DA9 with a G + C content of 47.5%.

Complete genome sequences of 17 Salmonella enterica serovar Schwarzengrund isolates carrying an IncFIB-IncFIC (FII) fusion plasmid.

Seventeen Salmonella enterica serovar Schwarzengrund isolates from chicken (n = 9) and clinical samples including stool (n = 6), urine (n = 1), and gallbladder (n = 1) were sequenced and found to carry an IncFIB-IncFIC (FII) fusion plasmid of approximately 145 Kb. This information provides reference genomic data for comparative studies of S. Schwarzengrund pathogenicity and plasmid genetics.

Metagenomic survey of antimicrobial resistance (AMR) in Maryland surface waters differentiated by high and low human impact.

Here, we examine surface waters as a modality to better understand baseline antimicrobial resistance (AMR) across the environment to supplement existing AMR monitoring in pathogens associated with humans, foods, and animals. Data from metagenomic and quasimetagenomic (shotgun sequenced enrichments) are used to describe AMR in Maryland surface waters from high and low human impact classifications.

Genetic relatedness and virulence potential of Salmonella Schwarzengrund strains with or without an IncFIB-IncFIC(FII) fusion plasmid isolated from food and clinical sources.

A total of 55 food and clinical S. Schwarzengrund isolates were assayed for plasmid content, among which an IncFIB-IncFIC(FII) fusion plasmid, conferring streptomycin resistance, was detected in 17 isolates. Among the 17 isolates, 9 were food isolates primarily collected from poultry meat, and 8 clinical isolates collected from stool, urine, and gallbladder. SNP-based phylogenetic analyses showed that the isolates carrying the fusion plasmid formed a subclade indicating the plasmid was acquired and is now maintained by the lineage. Phylogenetic analysis of the plasmid suggested it is derived from avian pathogenic plasmids and might confer an adaptive advantage to the S. Schwarzengrund isolates within birds. IncFIB-IncFIC(FII) fusion plasmids from all food and three clinical isolates were self-conjugative and successfully transferred into E. coli J53 by conjugation. Food and clinical isolates had similar virulome profiles and were able to invade human Caco-2 cells. However, the IncFIB-IncFIC(FII) plasmid did not significantly add to their invasion and persistence potential in human Caco-2 cells.

A multicenter genomic epidemiological investigation in Brazil, Chile, and Mexico reveals the diversity and persistence of Salmonella populations in surface waters.

This study examined the diversity and persistence of Salmonella in the surface waters of agricultural regions of Brazil, Chile, and Mexico. Research groups (three in 2019-2020 and five in 2021-2022) conducted a long-term survey of surface water across 5-8 months annually (n = 30 monthly). On-site, each team filtered 10-L water samples with modified Moore Swabs to capture Salmonella, which were then isolated and identified using conventional microbiological techniques. Salmonella isolates were sequenced on Illumina platforms. Salmonella was present in 1,493/3,291 water samples (45.8%), with varying isolation rates across countries and years. Newport, Infantis, and Typhimurium were the most frequent among the 128 different serovars. Notably, 22 serovars were found in all three countries, representing almost half of the 1,911 different isolates collected. The resistome comprised 72 antimicrobial resistance (AMR) genes and six point mutations in three genes. At least one AMR determinant was observed in 33.8% (646/1,911) of the isolates, of which 47.4% (306/646) were potentially multidrug resistant. Phylogeny based on core genome multilocus sequence typing (cgMLST) showed that most isolates clustered according to sequence type and country of origin. Only 14 cgMLST multi-country clusters were detected among the 275 clusters. However, further analysis confirmed that close genetic relatedness occurred mostly among isolates from the same country, with three exceptions. Interestingly, isolates closely related phylogenetically were recovered over multiple years within the same country, indicating the persistence of certain Salmonella in those areas. In conclusion, surface waters in these regions are consistently contaminated with diverse Salmonella, including strains that persist over time.IMPORTANCESalmonella is a leading foodborne pathogen responsible for millions of illnesses, hospitalizations, and deaths annually. Although Salmonella-contaminated water has now been recognized as an important contamination source in the agrifood chain, there is a lack of knowledge on the global occurrence and diversity of Salmonella in surface water. Moreover, there has been insufficient research on Salmonella in surface waters from Latin American countries that are major producers and exporters of agricultural products. Incorporating genetic profiling of Salmonella isolates from underrepresented regions, such as Latin America, enhances our understanding of the pathogen's ecology, evolution, antimicrobial resistance, and pathogenicity. Moreover, leveraging genomic data derived from pathogens isolated from diverse geographical areas is critical for assessing the potential public health risk posed by the pathogen and expediting investigations of foodborne outbreaks. Ultimately, global efforts contribute significantly to reducing the incidence of foodborne infections.

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.

A one health approach for monitoring antimicrobial resistance: developing a national freshwater pilot effort.

Antimicrobial resistance (AMR) is a world-wide public health threat that is projected to lead to 10 million annual deaths globally by 2050. The AMR public health issue has led to the development of action plans to combat AMR, including improved antimicrobial stewardship, development of new antimicrobials, and advanced monitoring. The National Antimicrobial Resistance Monitoring System (NARMS) led by the United States (U.S) Food and Drug Administration along with the U.S. Centers for Disease Control and U.S. Department of Agriculture has monitored antimicrobial resistant bacteria in retail meats, humans, and food animals since the mid 1990's. NARMS is currently exploring an integrated One Health monitoring model recognizing that human, animal, plant, and environmental systems are linked to public health. Since 2020, the U.S. Environmental Protection Agency has led an interagency NARMS environmental working group (EWG) to implement a surface water AMR monitoring program (SWAM) at watershed and national scales. The NARMS EWG divided the development of the environmental monitoring effort into five areas: (i) defining objectives and questions, (ii) designing study/sampling design, (iii) selecting AMR indicators, (iv) establishing analytical methods, and (v) developing data management/analytics/metadata plans. For each of these areas, the consensus among the scientific community and literature was reviewed and carefully considered prior to the development of this environmental monitoring program. The data produced from the SWAM effort will help develop robust surface water monitoring programs with the goal of assessing public health risks associated with AMR pathogens in surface water (e.g., recreational water exposures), provide a comprehensive picture of how resistant strains are related spatially and temporally within a watershed, and help assess how anthropogenic drivers and intervention strategies impact the transmission of AMR within human, animal, and environmental systems.

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.

Pan-genome analysis of the emerging foodborne pathogen Cronobacter spp. suggests a species-level bidirectional divergence driven by niche adaptation.

BACKGROUND: Members of the genus Cronobacter are causes of rare but severe illness in neonates and preterm infants following the ingestion of contaminated infant formula. Seven species have been described and two of the species genomes were subsequently published. In this study, we performed comparative genomics on eight strains of Cronobacter, including six that we sequenced (representing six of the seven species) and two previously published, closed genomes. RESULTS: We identified and characterized the features associated with the core and pan genome of the genus Cronobacter in an attempt to understand the evolution of these bacteria and the genetic content of each species. We identified 84 genomic regions that are present in two or more Cronobacter genomes, along with 45 unique genomic regions. Many potentially horizontally transferred genes, such as lysogenic prophages, were also identified. Most notable among these were several type six secretion system gene clusters, transposons that carried tellurium, copper and/or silver resistance genes, and a novel integrative conjugative element. CONCLUSIONS: Cronobacter have diverged into two clusters, one consisting of C. dublinensis and C. muytjensii (Cdub-Cmuy) and the other comprised of C. sakazakii, C. malonaticus, C. universalis, and C. turicensis, (Csak-Cmal-Cuni-Ctur) from the most recent common ancestral species. While several genetic determinants for plant-association and human virulence could be found in the core genome of Cronobacter, the four Cdub-Cmuy clade genomes contained several accessory genomic regions important for survival in a plant-associated environmental niche, while the Csak-Cmal-Cuni-Ctur clade genomes harbored numerous virulence-related genetic traits.