Weather factors, soil microbiome, and bacteria-fungi interactions as drivers of the epiphytic phyllosphere communities of romaine lettuce.

Lettuce is associated with seasonal outbreaks of Shiga toxin-producing Escherichia coli (STEC) infections. Little is known about how various biotic and abiotic factors affect the lettuce microbiome, which in turn impacts STEC colonization. We characterized the lettuce phyllosphere and surface soil bacterial, fungal, and oomycete communities at harvest in late-spring and -fall in California using metagenomics. Harvest season and field type, but not cultivar, significantly influenced the microbiome composition of leaves and surface soil near plants. Phyllosphere and soil microbiome compositions were correlated with specific weather factors. The relative abundance of Enterobacteriaceae, but not E. coli, was enriched on leaves (5.2%) compared to soil (0.4%) and correlated positively with minimum air temperature and wind speed. Co-occurrence networks revealed seasonal trends in fungi-bacteria interactions on leaves. These associations represented 39%-44% of the correlations between species. All significant E. coli co-occurrences with fungi were positive, while all negative associations were with bacteria. A large proportion of the leaf bacterial species was shared with those in soil, indicating microbiome transmission from the soil surface to the canopy. Our findings provide new insight into factors that shape lettuce microbial communities and the microbial context of foodborne pathogen immigration events in the lettuce phyllosphere.

Development and validation of an improved method for the detection of Salmonella in cinnamon bark and oregano leaves using the adsorbent beta zeolite in the pre-enrichment media.

The detection of Salmonella in spices is challenging due to the presence of antibacterial components. In this study, we evaluated the use of an adsorbent beta zeolite in pre-enrichment media to improve the recovery of Salmonella from cinnamon bark and oregano leaves. Samples (25 g) were spiked with varying levels of S. Montevideo or S. Senftenberg. After 2 weeks of stabilization at RT, betazeolite was added to cinnamon and oregano samples prior to the addition of 225 mL or 475 mL of pre-enrichment media, respectively. Detection sensitivity and rate of the test method were compared to the FDA Bacteriological Analytical Manual (BAM) method which requires the use of 2.5 L pre-enrichment broth. While Salmonella could not be detected in the test method using the reduced volume of pre-enrichment media alone, the addition of beta zeolite resulted in a positivity rate of 62% and 72.6% for cinnamon bark and oregano leaves respectively (all spike levels and both serovars combined). Furthermore, while there were differences in the LOD50 compared to the BAM method, there was no significant difference in the minimum level of detection between the betazeolite and the BAM methods. Our results demonstrate that the use of betazeolite in the pre-enrichment media offers a method with reduced media volumes without compromising on the sensitivity or efficiency of Salmonella detection in cinnamon bark and oregano leaves.

Insular Microbiogeography: Three Pathogens as Exemplars.

Traditional taxonomy in biology assumes that life is organized in a simple tree. Attempts to classify microorganisms in this way in the genomics era led microbiologists to look for finite sets of 'core' genes that uniquely group taxa as clades in the tree. However, the diversity revealed by large-scale whole genome sequencing is calling into question the long-held model of a hierarchical tree of life, which leads to questioning of the definition of a species. Large-scale studies of microbial genome diversity reveal that the cumulative number of new genes discovered increases with the number of genomes studied as a power law and subsequently leads to the lack of evidence for a unique core genome within closely related organisms. Sampling 'enough' new genomes leads to the discovery of a replacement or alternative to any gene. This power law behaviour points to an underlying self-organizing critical process that may be guided by mutation and niche selection. Microbes in any particular niche exist within a local web of organism interdependence known as the microbiome. The same mechanism that underpins the macro-ecological scaling first observed by MacArthur and Wilson also applies to microbial communities. Recent metagenomic studies of a food microbiome demonstrate the diverse distribution of community members, but also genotypes for a single species within a more complex community. Collectively, these results suggest that traditional taxonomic classification of bacteria could be replaced with a quasispecies model. This model is commonly accepted in virology and better describes the diversity and dynamic exchange of genes that also hold true for bacteria. This model will enable microbiologists to conduct population-scale studies to describe microbial behaviour, as opposed to a single isolate as a representative.

Global transcriptomic response of Listeria monocytogenes during growth on cantaloupe slices.

Understanding a pathogen's response to food environments is imperative to develop effective control strategies as well as to elucidate the impact of foods on virulence potential. The purpose of this study was to assess transcriptional response of Listeria monocytogenes after growth in cantaloupe, as well as its impact on survival in synthetic gastric fluid (SGF). The transcriptional profiles of L. monocytogenes grown in cantaloupe or Brain Heart Infusion (BHI) under refrigeration were compared by a custom-designed microarray. A total of 286 and 175 genes were significantly up- and down-regulated, respectively, in L. monocytogenes grown in cantaloupe as compared to BHI (fold change ≥ 2.5 and adj. P < 0.05). The majority of upregulated genes belonged to functions related to amino acid and nucleotide metabolism, flagellar biosynthesis, and iron acquisition, while most downregulated genes belonged to carbohydrate metabolism. Notably, the branched chain amino acid (BCAA: leucine, isoleucine, valine) biosynthesis operon was shown to be highly upregulated as well as the purine and pyrimidine biosynthesis pathways. Transcript levels of several stress- and virulence-related genes were significantly altered, implying an impact of growth in cantaloupe on the virulence potential of L. monocytogenes. Enhanced survival of L. monocytogenes in SGF following growth in cantaloupe further demonstrated the impact of cantaloupe-associated growth on the pathogen's subsequent response to a host relevant stress.

A Vegetable Fermentation Facility Hosts Distinct Microbiomes Reflecting the Production Environment.

Fermented vegetables are highly popular internationally in part due to their enhanced nutritional properties, cultural history, and desirable sensorial properties. In some instances, fermented foods provide a rich source of the beneficial microbial communities that could promote gastrointestinal health. The indigenous microbiota that colonize fermentation facilities may impact food quality, food safety, and spoilage risks and maintain the nutritive value of the product. Here, microbiomes within sauerkraut production facilities were profiled to characterize variance across surfaces and to determine the sources of these bacteria. Accordingly, we used high-throughput sequencing of the 16S rRNA gene in combination with whole-genome shotgun analyses to explore biogeographical patterns of microbial diversity and assembly within the production facility. Our results indicate that raw cabbage and vegetable handling surfaces exhibit more similar microbiomes relative to the fermentation room, processing area, and dry storage surfaces. We identified biomarker bacterial phyla and families that are likely to originate from the raw cabbage and vegetable handling surfaces. Raw cabbage was identified as the main source of bacteria to seed the facility, with human handling contributing a minor source of inoculation. Leuconostoc and Lactobacillaceae dominated all surfaces where spontaneous fermentation occurs, as these taxa are associated with the process. Wall, floor, ceiling, and barrel surfaces host unique microbial signatures. This study demonstrates that diverse bacterial communities are widely distributed within the production facility and that these communities assemble nonrandomly, depending on the surface type.IMPORTANCE Fermented vegetables play a major role in global food systems and are widely consumed by various global cultures. In this study, we investigated an industrial facility that produces spontaneous fermented sauerkraut without the aid of starter cultures. This provides a unique system to explore and track the origins of an "in-house" microbiome in an industrial environment. Raw vegetables and the surfaces on which they are handled were identified as the likely source of bacterial communities rather than human contamination. As fermented vegetables increase in popularity on a global scale, understanding their production environment may help maintain quality and safety goals.

Laryngeal Mask Airway for Surfactant Administration in Neonates: A Randomized, Controlled Trial.

OBJECTIVE: To determine if preterm infants with moderate respiratory distress syndrome on continuous positive airway pressure (CPAP) who received surfactant via a laryngeal mask airway (LMA) would have a decreased rate of intubation and mechanical ventilation compared with those on CPAP who did not receive surfactant. STUDY DESIGN: In this prospective, multicenter, randomized controlled trial, 103 premature infants 280/7-356/7 weeks gestation, ≥1250 g and ≤36 hours old on CPAP requiring fraction of inspired oxygen 0.30-0.40 were assigned to receive surfactant administered through an LMA then placed back on CPAP (LMA group) or maintained on CPAP with no surfactant administered (control group). The primary outcome was treatment failure necessitating intubation and mechanical ventilation in the first 7 days of life. RESULTS: Surfactant administration through an LMA (n = 50) significantly decreased the rate of intubation and mechanical ventilation compared with controls (n = 53): 38% vs 64%, respectively, OR 0.30 (95% CI 0.13, 0.70), P = .006, number needed to treat: 4). There were no serious adverse events associated with placement of the LMA or surfactant administration. CONCLUSIONS: In premature neonates with moderate respiratory distress syndrome, surfactant administered through an LMA decreased the rate of intubation and mechanical ventilation. This intervention may have significant impact on clinical care in both high and low resource settings. TRIAL REGISTRATION: ClinicalTrials.gov: NCT01116921.

The Randomized, Controlled Trial of Late Surfactant: Effects on Respiratory Outcomes at 1-Year Corrected Age.

OBJECTIVE: To determine the effects of late surfactant on respiratory outcomes determined at 1-year corrected age in the Trial of Late Surfactant (TOLSURF), which randomized newborns of extremely low gestational age (≤28 weeks' gestational age) ventilated at 7-14 days to late surfactant and inhaled nitric oxide vs inhaled nitric oxide-alone (control). STUDY DESIGN: Caregivers were surveyed in a double-blinded manner at 3, 6, 9, and 12 months' corrected age to collect information on respiratory resource use (infant medication use, home support, and hospitalization). Infants were classified for composite outcomes of pulmonary morbidity (no PM, determined in infants with no reported respiratory resource use) and persistent PM (determined in infants with any resource use in ≥3 surveys). RESULTS: Infants (n = 450, late surfactant n = 217, control n = 233) were 25.3 ± 1.2 weeks' gestation and 713 ± 164 g at birth. In the late surfactant group, fewer infants received home respiratory support than in the control group (35.8% vs 52.9%, relative benefit [RB] 1.28 [95% CI 1.07-1.55]). There was no benefit of late surfactant for No PM vs PM (RB 1.27; 95% CI 0.89-1.81) or no persistent PM vs persistent PM (RB 1.01; 95% CI 0.87-1.17). After adjustment for imbalances in baseline characteristics, relative benefit of late surfactant treatment increased: RB 1.40 (95% CI 0.89-1.80) for no PM and RB 1.24 (95% CI 1.08-1.42) for no persistent PM. CONCLUSION: Treatment of newborns of extremely low gestational age with late surfactant in combination with inhaled nitric oxide decreased use of home respiratory support and may decrease persistent pulmonary morbidity. TRIAL REGISTRATION: ClinicalTrials.gov: NCT01022580.

Hybrid Shiga Toxin-Producing and Enterotoxigenic Escherichia sp. Cryptic Lineage 1 Strain 7v Harbors a Hybrid Plasmid.

UNLABELLED: Hybrid isolates of Shiga toxin-producing Escherichia coli (STEC) and enterotoxigenic E. coli (ETEC) encoding heat-stable enterotoxin (ST) are being reported with increasing frequency from a variety of sources. However, information regarding the plasmids that these strains harbor is scarce. In this study, we sequence and characterize a plasmid, p7v, from the STEC/ETEC hybrid strain 7v. Whole-genome phylogenetic analyses of STEC/ETEC hybrid strains and prototype E. coli isolates of other pathotypes placed 7v in the Escherichia sp. cryptic lineage 1 (CL1) clade. The complete plasmid, p7v, was determined to be 229,275 bp and encodes putative virulence factors that are typically carried on STEC plasmids as well as those often carried on ETEC plasmids, indicating that the hybrid nature of the strain extends beyond merely encoding the two toxins. Plasmid p7v carries two copies of sta with identical sequences, which were discovered to be divergent from the sta sequences found in the prototype human ETEC strains. Using a nomenclature scheme based on a phylogeny constructed from sta and stb sequences, the sta encoded on p7v is designated STa4. In silico analysis determined that p7v also encodes the K88 fimbria, a colonization factor usually associated with porcine ETEC plasmids. The p7v sequence and the presence of plasmid-encoded virulence factors are compared to those of other STEC/ETEC CL1 hybrid genomes and reveal gene acquisition/loss at the strain level. In addition, the interrogation of 24 STEC/ETEC hybrid genomes for identification of plasmid replicons, colonization factors, Stx and ST subtypes, and other plasmid-encoded virulence genes highlights the diversity of these hybrid strains. IMPORTANCE: Hybrid Shiga toxin-producing Escherichia coli/enterotoxigenic Escherichia coli (STEC/ETEC) strains, which have been isolated from environmental, animal, and human clinical samples, may represent an emerging threat as food-borne pathogens. Characterization of these strains is important for assessing virulence potential, aiding in the development of pathogen detection methods, and understanding how the hybrid strains evolve to potentially have a greater impact on public health. This study represents, to our knowledge, both the first characterization of a closed plasmid sequence from a STEC/ETEC hybrid strain and the most comprehensive phylogenetic analysis of available STEC/ETEC hybrid genomes to date. The results demonstrate how the mobility of plasmid-associated virulence genes has resulted in the creation of a diverse plasmid repertoire within the STEC/ETEC hybrid strains.

FDA Escherichia coli Identification (FDA-ECID) Microarray: a Pangenome Molecular Toolbox for Serotyping, Virulence Profiling, Molecular Epidemiology, and Phylogeny.

UNLABELLED: Most Escherichia coli strains are nonpathogenic. However, for clinical diagnosis and food safety analysis, current identification methods for pathogenic E. coli either are time-consuming and/or provide limited information. Here, we utilized a custom DNA microarray with informative genetic features extracted from 368 sequence sets for rapid and high-throughput pathogen identification. The FDA Escherichia coli Identification (FDA-ECID) platform contains three sets of molecularly informative features that together stratify strain identification and relatedness. First, 53 known flagellin alleles, 103 alleles of wzx and wzy, and 5 alleles of wzm provide molecular serotyping utility. Second, 41,932 probe sets representing the pan-genome of E. coli provide strain-level gene content information. Third, approximately 125,000 single nucleotide polymorphisms (SNPs) of available whole-genome sequences (WGS) were distilled to 9,984 SNPs capable of recapitulating the E. coli phylogeny. We analyzed 103 diverse E. coli strains with available WGS data, including those associated with past foodborne illnesses, to determine robustness and accuracy. The array was able to accurately identify the molecular O and H serotypes, potentially correcting serological failures and providing better resolution for H-nontypeable/nonmotile phenotypes. In addition, molecular risk assessment was possible with key virulence marker identifications. Epidemiologically, each strain had a unique comparative genomic fingerprint that was extended to an additional 507 food and clinical isolates. Finally, a 99.7% phylogenetic concordance was established between microarray analysis and WGS using SNP-level data for advanced genome typing. Our study demonstrates FDA-ECID as a powerful tool for epidemiology and molecular risk assessment with the capacity to profile the global landscape and diversity of E. coli IMPORTANCE: This study describes a robust, state-of-the-art platform developed from available whole-genome sequences of E. coli and Shigella spp. by distilling useful signatures for epidemiology and molecular risk assessment into one assay. The FDA-ECID microarray contains features that enable comprehensive molecular serotyping and virulence profiling along with genome-scale genotyping and SNP analysis. Hence, it is a molecular toolbox that stratifies strain identification and pathogenic potential in the contexts of epidemiology and phylogeny. We applied this tool to strains from food, environmental, and clinical sources, resulting in significantly greater phylogenetic and strain-specific resolution than previously reported for available typing methods.

Randomized Trial of Late Surfactant Treatment in Ventilated Preterm Infants Receiving Inhaled Nitric Oxide.

OBJECTIVE: To assess whether late surfactant treatment in extremely low gestational age (GA) newborn infants requiring ventilation at 7-14 days, who often have surfactant deficiency and dysfunction, safely improves survival without bronchopulmonary dysplasia (BPD). STUDY DESIGN: Extremely low GA newborn infants (GA ≤28 0/7 weeks) who required mechanical ventilation at 7-14 days were enrolled in a randomized, masked controlled trial at 25 US centers. All infants received inhaled nitric oxide and either surfactant (calfactant/Infasurf) or sham instillation every 1-3 days to a maximum of 5 doses while intubated. The primary outcome was survival at 36 weeks postmenstrual age (PMA) without BPD, as evaluated by physiological oxygen/flow reduction. RESULTS: A total of 511 infants were enrolled between January 2010 and September 2013. There were no differences between the treated and control groups in mean birth weight (701 ± 164 g), GA (25.2 ± 1.2 weeks), percentage born at GA <26 weeks (70.6%), race, sex, severity of lung disease at enrollment, or comorbidities of prematurity. Survival without BPD did not differ between the treated and control groups at 36 weeks PMA (31.3% vs 31.7%; relative benefit, 0.98; 95% CI, 0.75-1.28; P = .89) or 40 weeks PMA (58.7% vs 54.1%; relative benefit, 1.08; 95% CI, 0.92-1.27; P = .33). There were no between-group differences in serious adverse events, comorbidities of prematurity, or severity of lung disease to 36 weeks. CONCLUSION: Late treatment with up to 5 doses of surfactant in ventilated premature infants receiving inhaled nitric oxide was well tolerated, but did not improve survival without BPD at 36 or 40 weeks. Pulmonary and neurodevelopmental assessments are ongoing. TRIAL REGISTRATION: ClinicalTrials.gov: NCT01022580.

Application of metagenomic sequencing to food safety: detection of Shiga Toxin-producing Escherichia coli on fresh bagged spinach.

Culture-independent diagnostics reduce the reliance on traditional (and slower) culture-based methodologies. Here we capitalize on advances in next-generation sequencing (NGS) to apply this approach to food pathogen detection utilizing NGS as an analytical tool. In this study, spiking spinach with Shiga toxin-producing Escherichia coli (STEC) following an established FDA culture-based protocol was used in conjunction with shotgun metagenomic sequencing to determine the limits of detection, sensitivity, and specificity levels and to obtain information on the microbiology of the protocol. We show that an expected level of contamination (∼10 CFU/100 g) could be adequately detected (including key virulence determinants and strain-level specificity) within 8 h of enrichment at a sequencing depth of 10,000,000 reads. We also rationalize the relative benefit of static versus shaking culture conditions and the addition of selected antimicrobial agents, thereby validating the long-standing culture-based parameters behind such protocols. Moreover, the shotgun metagenomic approach was informative regarding the dynamics of microbial communities during the enrichment process, including initial surveys of the microbial loads associated with bagged spinach; the microbes found included key genera such as Pseudomonas, Pantoea, and Exiguobacterium. Collectively, our metagenomic study highlights and considers various parameters required for transitioning to such sequencing-based diagnostics for food safety and the potential to develop better enrichment processes in a high-throughput manner not previously possible. Future studies will investigate new species-specific DNA signature target regimens, rational design of medium components in concert with judicious use of additives, such as antibiotics, and alterations in the sample processing protocol to enhance detection.

Inhaled nitric oxide increases urinary nitric oxide metabolites and cyclic guanosine monophosphate in premature infants: relationship to pulmonary outcome.

OBJECTIVE: Inhaled nitric oxide (iNO) has been tested to prevent bronchopulmonary dysplasia (BPD) in premature infants, however, the role of cyclic guanosine monophosphate (cGMP) is not known. We hypothesized that levels of NO metabolites (NOx) and cGMP in urine, as a noninvasive source for biospecimen collection, would reflect the dose of iNO and relate to pulmonary outcome. STUDY DESIGN: Studies were performed on 125 infants who required mechanical ventilation at 7 to 14 days and received 24 days of iNO at 20-2 ppm. A control group of 19 infants did not receive iNO. RESULTS: In NO-treated infants there was a dose-dependent increase of both NOx and cGMP per creatinine (maximal 3.1- and 2-fold, respectively, at 10-20 ppm iNO) compared with off iNO. NOx and cGMP concentrations at both 2 ppm and off iNO were inversely related to severity of lung disease during the 1st month, and the NOx levels were lower in infants who died or developed BPD at term. NOx was higher in Caucasian compared with other infants at all iNO doses. CONCLUSION: Urinary NOx and cGMP are biomarkers of endogenous NO production and lung uptake of iNO, and some levels reflect the severity of lung disease. These results support a role of the NO-cGMP pathway in lung development.

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)).

Genomic anatomy of Escherichia coli O157:H7 outbreaks.

The rapid emergence of Escherichia coli O157:H7 from an unknown strain in 1982 to the dominant hemorrhagic E. coli serotype in the United States and the cause of widespread outbreaks of human food-borne illness highlights a need to evaluate critically the extent to which genomic plasticity of this important enteric pathogen contributes to its pathogenic potential and its evolution as well as its adaptation in different ecological niches. Aimed at a better understanding of the evolution of the E. coli O157:H7 pathogenome, the present study presents the high-quality sequencing and comparative phylogenomic analysis of a comprehensive panel of 25 E. coli O157:H7 strains associated with three nearly simultaneous food-borne outbreaks of human disease in the United States. Here we present a population genetic analysis of more than 200 related strains recovered from patients, contaminated produce, and zoonotic sources. High-resolution phylogenomic approaches allow the dynamics of pathogenome evolution to be followed at a high level of phylogenetic accuracy and resolution. SNP discovery and study of genome architecture and prophage content identified numerous biomarkers to assess the extent of genetic diversity within a set of clinical and environmental strains. A total of 1,225 SNPs were identified in the present study and are now available for typing of the E. coli O157:H7 lineage. These data should prove useful for the development of a refined phylogenomic framework for forensic, diagnostic, and epidemiological studies to define better risk in response to novel and emerging E. coli O157:H7 resistance and virulence phenotypes.

Evolution of mechanical ventilation of the newborn infant.

Artificial ventilation of the newborn infant is the foundation of neonatology. Early practitioners included pediatricians, anesthesiologists, cardiologists, respiratory therapists, and engineers. The discovery of surfactant, followed by the death of Patrick Kennedy, jump-started the new area, with investment and research rapidly expanding. The ever more complex design of mechanical ventilators necessitated a more thorough understanding of newborn pulmonary physiology in order to provide support with minimal associated injury. This piece briefly reviews and highlights this history.

Detection of Cyclospora cayetanensis in Food and Water Samples: Optimized Protocols for Specific and Sensitive Molecular Methods from a Regulatory Agency Perspective.

Cyclospora cayetanensis is a coccidian parasite of the phylum Apicomplexa that causes cyclosporiasis, a human-specific gastrointestinal disease. Unlike most enteric pathogens, C. cayetanensis does not infect via direct fecal-oral transmission between humans because shed oocysts must be exposed to environmental triggers prior to becoming infectious. The development of specific and sensitive detection methods for C. cayetanensis is crucial to effectively address data gaps and provide regulatory support during outbreak investigations. In this study, new more specific molecular markers for the detection of C. cayetanensis were developed based on updated genomic databases of Apicomplexa mitochondrial sequences. Novel alternative reagents and supplies, as well as optimization protocols, were tested in spiked produce and agricultural water samples. The selected Mit1C primers and probe combined showed at least 13 mismatches to other related species. The new optimized qualitative real-time PCR assay with modifications to sample processing and replacement of discontinued items produced results comparable to the previously validated methods. In conclusion, the new optimized qualitative Mit1C real-time PCR assay demonstrated an increase in its specificity in comparison to other detection methods previously published, while it showed to be robust and as sensitive as the previously validated method at the FDA. This study has also expanded the array of PCR reagents that can be used to detect C. cayetanensis in produce and agricultural water samples and provided several improvements to the method for detection in agricultural water including replacements for discontinued items and a new dialysis filter for water filtration.

Fecal microbiomes of laboratory beagles receiving antiparasitic formulations in an experimental setting.

Here, we describe the fecal microbiome of laboratory beagles in a non-invasive experiment designed to contrast in vivo versus in vitro bioequivalence in response to antiparasitic drug administration. The experiment provided a unique opportunity to evaluate metagenomic profiles of canine feces before and after anti-parasitic drug exposure.

Paired metagenomic and chemical evaluation of aflatoxin-contaminated dog kibble.

Introduction: Identification of chemical toxins from complex or highly processed foods can present 'needle in the haystack' challenges for chemists. Metagenomic data can be used to guide chemical toxicity evaluations by providing DNA-based description of the wholistic composition (eukaryotic, bacterial, protozoal, viral, and antimicrobial resistance) of foods suspected to harbor toxins, allergens, or pathogens. This type of information can focus chemistry-based diagnostics, improve hazard characterization and risk assessment, and address data gaps. Additionally, there is increasing recognition that simultaneously co-occurring mycotoxins, either from single or multiple species, can impact dietary toxicity exposure. Metagenomic data provides a way to address data gaps related to co-occurrence of multiple fungal species. Methods: Paired metagenomic and chemical data were used to evaluate aflatoxin-contaminated kibble with known levels of specific mycotoxins. Kibble was ground to a fine powder for both chemical and molecular analyses. Chemical analyses were performed with Liquid Chromatography Mass Spectrometry (LCMS) and according to the AOAC Official method 2005.08: Aflatoxins in Corn, Raw Peanuts, and Peanut Butter using Liquid Chromatography with Post-Column Photochemical Derivatization. Metagenomes were created from DNA extracted from ground kibble and sequenced on an Illumina NextSeq 2000 with an average sequence depth of 180 million reads per replicate. Results and discussion: Metagenomic data demonstrated that the abundance of DNA from putative aflatoxigenic Aspergillus spp. correlated with the levels of aflatoxin quantified by LCMS. Metagenomic data also identified an expansive range of co-occurring fungal taxa which may produce additional mycotoxins. DNA data paired with chemical data provides a novel modality to address current data gaps surrounding dietary mycotoxin exposure, toxigenic fungal taxonomy, and mycotoxins of emerging concern.

Evaluation of the Increased Genetic Resolution and Utility for Source Tracking of a Recently Developed Method for Genotyping Cyclospora cayetanensis.

Cyclospora cayetanensis is a foodborne parasite that causes cyclosporiasis, an enteric illness in humans. Genotyping methods are used to genetically discriminate between specimens from cyclosporiasis cases and can complement source attribution investigations if the method is sufficiently sensitive for application to food items. A very sensitive targeted amplicon sequencing (TAS) assay for genotyping C. cayetanensis encompassing 52 loci was recently designed. In this study, we analyzed 66 genetically diverse clinical specimens to assess the change in phylogenetic resolution between the TAS assay and a currently employed eight-marker scheme. Of the 52 markers, ≥50 were successfully haplotyped for all specimens, and these results were used to generate a hierarchical cluster dendrogram. Using a previously described statistical approach to dissect hierarchical trees, the 66 specimens resolved into 24 and 27 distinct genetic clusters for the TAS and an 8-loci scheme, respectively. Although the specimen composition of 15 clusters was identical, there were substantial differences between the two dendrograms, highlighting the importance of both inclusion of additional genome coverage and choice of loci to target for genotyping. To evaluate the ability to genetically link contaminated food samples with clinical specimens, C. cayetanensis was genotyped from DNA extracted from raspberries inoculated with fecal specimens. The contaminated raspberry samples were assigned to clusters with the corresponding clinical specimen, demonstrating the utility of the TAS assay for traceback efforts.