Application of quasimetagenomics methods to define microbial diversity and subtype Listeria monocytogenes in dairy and seafood production facilities.

IMPORTANCE: In developed countries, the human diet is predominated by food commodities, which have been manufactured, processed, and stored in a food production facility. Little is known about the application of metagenomic sequencing approaches for detecting foodborne pathogens, such as L. monocytogenes, and characterizing microbial diversity in food production ecosystems. In this work, we investigated the utility of 16S rRNA amplicon and quasimetagenomic sequencing for the taxonomic and phylogenetic classification of Listeria culture enrichments of environmental swabs collected from dairy and seafood production facilities. We demonstrated that single-nucleotide polymorphism (SNP) analyses of L. monocytogenes metagenome-assembled genomes (MAGs) from quasimetagenomic data sets can achieve similar resolution as culture isolate whole-genome sequencing. To further understand the impact of genome coverage on MAG SNP cluster resolution, an in silico downsampling approach was employed to reduce the percentage of target pathogen sequence reads, providing an initial estimate of required MAG coverage for subtyping resolution of L. monocytogenes.

Draft Genome Sequences of 18 Streptococcus Strains Isolated from Live Dietary Supplements and Cultured Food Products.

We present the genome sequences of 18 Streptococcus isolates from 8 different dietary supplements and 9 cultured food products. Strains from this species naturally colonize the human mouth and upper respiratory tract. Studies have shown that S. thermophilus and S. salivarius strains confer oral health benefits to their host with little to no risk of pathogenic infection.

Draft Genome Sequences of Nine Bacillus Strains and Two Weizmannia Strains Isolated from Live Dietary Supplements and a Cultured Food Product.

We present the genome sequences of nine Bacillus isolates and two Weizmannia isolates from 10 different dietary supplements and one cultured food product. Strains of these species have been associated with health benefits when ingested by humans, due to their ability to survive the stomach's acidic environment and colonize the intestinal tract.

Variability in the Occupancy of Escherichia coli O157 Integration Sites by Shiga Toxin-Encoding Prophages.

Escherichia coli O157:H7 strains often produce Shiga toxins encoded by genes on lambdoid bacteriophages that insert into multiple loci as prophages. O157 strains were classified into distinct clades that vary in virulence. Herein, we used PCR assays to examine Shiga toxin (Stx) prophage occupancy in yehV, argW, wrbA, and sbcB among 346 O157 strains representing nine clades. Overall, yehV was occupied in most strains (n = 334, 96.5%), followed by wrbA (n = 213, 61.6%), argW (n = 103, 29.8%), and sbcB (n = 93, 26.9%). Twelve occupancy profiles were identified that varied in frequency and differed across clades. Strains belonging to clade 8 were more likely to have occupied sbcB and argW sites compared to other clades (p < 0.0001), while clade 2 strains were more likely to have occupied wrbA sites (p < 0.0001). Clade 8 strains also had more than the expected number of occupied sites based on the presence of stx variants (p < 0.0001). Deletion of a 20 kb non-Stx prophage occupying yehV in a clade 8 strain resulted in an ~18-fold decrease in stx2 expression. These data highlight the complexity of Stx prophage integration and demonstrate that clade 8 strains, which were previously linked to hemolytic uremic syndrome, have unique Stx prophage occupancy profiles that can impact stx2 expression.

Development and Validation of a Quantitative PCR Method for Species Verification and Serogroup Determination of Listeria monocytogenes Isolates.

ABSTRACT: Listeria monocytogenes (Lm) is one of the leading causes of death because of foodborne illness, affecting the elderly, pregnant women, neonates, and people who are immunocompromised. Serologically, Lm can be classified into 13 serotypes, although only 4 are typically linked with food contamination and illness. Since 2000, a shift in serotypes involved in listeriosis outbreaks has been observed, suggesting that tracking of serotypes could help identify emerging trends. A PCR method developed in 2004 allowed detection of the four major serotypes as molecular serogroups, corresponding to broad phylogenetic groups. In this study, a novel quantitative PCR (qPCR) method was developed that uses two multiplex qPCRs, one to confirm the Listeria genus and Lm species and the second for Lm molecular serogrouping. This method was compared with the U.S. Food and Drug Administration Bacteriological Analytical Manual (BAM) method for Lm and the seroagglutination method, using a 208-strain panel. Comparison of the genus and species qPCR assay with the BAM methods found an equal or slightly higher accuracy for the qPCR method (>98%), compared with the BAM protocol (>96%), when evaluated against independent characterization data. Molecular serogrouping using the qPCR method (96.6%) was more accurate than the seroagglutination assay (75.6%). The qPCR method identified Lm 4bV strains, which could not be resolved using seroagglutination. The qPCR could not identify lineage III and IV serotype 4b strains but did correctly identify 16 of 18 lineage III and IV strains. The qPCR method performed genus identification for the Listeria species Lm, L. innocua, L. welshimeri, L. ivanovii, and L. seeligeri. In addition, the method performed species identification for Lm and classified Lm into six molecular serogroups: 2A, 2B, 2C, 4B, NT, and 4bV. This method provided a rapid and accurate confirmation of Lm and serogroup determinations; furthermore, it could help identify otherwise unlinked strains by enabling whole genome sequencing analysis based on broad phylogeny, independent of other information.

Comparative Transcriptomics of Shiga Toxin-Producing and Commensal Escherichia coli and Cytokine Responses in Colonic Epithelial Cell Culture Infections.

Ingestion of Shiga toxin-producing Escherichia coli (STEC) can result in a range of illness severity from asymptomatic to hemorrhagic colitis and death; thus risk assessment of STEC strains for human pathogenicity is important in the area of food safety. Illness severity depends in part on the combination of virulence genes carried in the genome, which can vary between strains even of identical serotype. To better understand how core genes are regulated differently among strains and to identify possible novel STEC virulence gene candidates that could be added to the risk assessment repertoire, we used comparative transcriptomics to investigate global gene expression differences between two STEC strains associated with severe illness and a commensal E. coli strain during in vitro intestinal epithelial cell (IEC) infections. Additionally, we compared a wide array of concomitant cytokine levels produced by the IECs. The cytokine expression levels were examined for a pattern representing STEC pathogenicity; however, while one STEC strain appeared to elicit a proinflammatory response, infection by the other strain produced a pattern comparable to the commensal E. coli. This result may be explained by the significant differences in gene content and expression observed between the STEC strains. RNA-Seq analysis revealed considerable disparity in expression of genes in the arginine and tryptophan biosynthesis/import pathways between the STEC strains and the commensal E. coli strain, highlighting the important role some amino acids play in STEC colonization and survival. Contrasting differential expression patterns were observed for genes involved in respiration among the three strains suggesting that metabolic diversity is a strategy utilized to compete with resident microflora for successful colonization. Similar temporal expression results for known and putative virulence genes were observed in the STEC strains, revealing strategies used for survival prior to and after initial adherence to IECs. Additionally, three genes encoding hypothetical proteins located in mobile genetic elements were, after interrogation of a large set of E. coli genomes, determined to likely represent novel STEC virulence factors.

Draft Genome Sequences of Isolates of Diverse Host Origin from the E. coli Reference Center at Penn State University.

Escherichia coli strains present a vast genomic diversity. We report the draft genome sequences of 1,000 isolates from the E. coli Reference Center at Penn State University. These strains were originally isolated from multiple animal and environmental sources over the past 50 years.

gndDb, a Database of Partial gnd Sequences To Assist with Analysis of Escherichia coli Communities Using High-Throughput Sequencing.

The use of culture methods to detect Escherichia coli diversity does not provide sufficient resolution to identify strains present at low levels. Here, we target the hypervariable gnd gene and describe a database containing 534 distinct partial gnd sequences and associated O groups for use with culture-independent E. coli community analysis.

Microbiota of the Hickey Run Tributary of the Anacostia River.

Water from the Hickey Run Tributary of the Anacostia River is being collected quarterly (beginning August 2018) and analyzed to create high-resolution baseline taxonomic profiles of microbiota associated with this important aquatic ecosystem, which has a long history of exposure to residential and commercial effluents from Washington, DC. These United States National Arboretum Microbial Observatory data are available under NCBI BioProject number PRJNA498951.

Phenotypic and Genotypic Characterization of Escherichia coli and Salmonella enterica from Dairy Cattle Farms in the Wakiso District, Uganda: A Cross-Sectional Study.

Enterobacteriaceae producing ß-lactamases have spread rapidly worldwide and pose a serious threat to human-animal-environment interface. In this study, we present the presence of Salmonella enterica (1.3%) and commensal Escherichia coli (96.3%) isolated from 400 environmental fecal dairy cattle samples over 20 farms in Uganda. Among E. coli isolates, 21% were resistant to at least one antimicrobial tested and 7% exhibited multidrug resistance. Four E. coli isolates displayed extended-spectrum beta-lactamase (ESBL)-producing genes, including blaCTX-M-15 (n = 2/4), blaCTX-M-27 (n = 1/4), blaSHV-12 (n = 1/4), and blaTEM-1B (n = 2/4). Whole genome sequencing confirmed the presence of the plasmid-mediated quinolone resistance qnrS1 gene among three ESBL isolates. No statistically significant differences in seasonal prevalence for E. coli and S. enterica among dairy cattle sampling periods were observed. Furthermore, to our knowledge, this is the first report of E. coli carrying blaCTX-M-15, blaCTX-M-27, blaSHV-12, or qnrS1 isolated from dairy cattle in Uganda. We conclude that the presence of globally disseminated blaCTX-M-15 and blaCTX-M-27 warrants further study to prevent further spread. In addition, the presence of fluoroquinolone resistant ESBL-producing E. coli on dairy farms highlights the potential risk among the human-livestock-environment interaction. This study can be used as a baseline for implementation of a more robust national integrated surveillance system throughout Uganda.

Draft Genome Sequences of the Escherichia coli Reference (ECOR) Collection.

Here, we report the genomes of all 72 isolates belonging to the Escherichia coli reference (ECOR) collection. Strains in this collection were isolated from diverse hosts and geographic locations and have been used for more than 30 years to represent the phylogenetic diversity of E. coli.

Draft Genome Sequences of 72 Isolates from All Four Species of Shigella.

Shigella is a genus of Gram-negative enteric pathogenic bacteria which has four species, Shigella dysenteriae, S. flexneri, S. boydii, and S. sonnei. Shigella species are clinically important bacteria because they cause shigellosis or dysentery. Here we report the genome sequences of 72 Shigella isolates from these four species.

Interlaboratory Evaluation of the U.S. Food and Drug Administration Escherichia coli Identification Microarray for Profiling Shiga Toxin-Producing Escherichia coli.

The U.S. Food and Drug Administration Escherichia coli Identification (FDA-ECID) microarray provides rapid molecular characterization of E. coli. The effectiveness of the FDA-ECID for characterizing Shiga toxin-producing E. coli (STEC) was evaluated by three federal laboratories and one reference laboratory with a panel of 54 reference E. coli strains from the External Quality Assurance program. Strains were tested by FDA-ECID for molecular serotyping (O and H antigens), Shiga toxin subtyping, and the presence of the ehxA and eae genes for enterohemolysin and intimin, respectively. The FDA-ECID O typing was 96% reproducible among the four laboratories and 94% accurate compared with the reference External Quality Assurance data. Discrepancies were due to the absence of O41 target loci on the array and to two pairs of O types with identical target sequences. H typing was 96% reproducible and 100% accurate, with discrepancies due to two strains from one laboratory that were identified as mixed by FDA-ECID. Shiga toxin (Stx) type 1 subtyping was 100% reproducible and accurate, and Stx2 subtyping was 100% reproducible but only 64% accurate. FDA-ECID identified most Stx2 subtypes but had difficulty distinguishing among stx2a, stx2c, and stx2d genes because of close similarities of these sequences. FDA-ECID was 100% effective for detecting ehxA and eae and accurately subtyped the eae alleles. This interlaboratory study revealed that FDA-ECID for STEC characterization was highly reproducible for molecular serotyping, stx and eae subtyping, and ehxA detection. However, the array was less useful for distinguishing among the highly homologous O antigen genes and the stx2a, stx2c, and stx2d subtypes.

Draft Genome Sequences of Bifidobacterium Strains Isolated from Dietary Supplements and Cultured Food Products.

Here, we present the genome sequences of 23 Bifidobacterium isolates from several commercially available dietary supplements and cultured food products. Strains of this genus are natural inhabitants of the mammalian mouth, gastrointestinal tract, and vagina. Some species are considered beneficial to human health.

Fifty-Six Draft Genome Sequences of 10 Lactobacillus Species from 22 Commercial Dietary Supplements.

Here, we present the genome sequences of 56 isolates of 10 species of the genus Lactobacillus that are considered beneficial components of the gut microbiota. The isolates examined were found in commercially available dietary supplements in the U.S. market.

Draft Genome Sequences of Escherichia albertii, Escherichia fergusonii, and Strains Belonging to Six Cryptic Lineages of Escherichia spp.

We report here the genome sequences of 55 strains belonging to the genus Escherichia from multiple animal and environmental sources. These strains include representatives of Escherichia albertii, Escherichia fergusonii, and six additional genetically distinct lineages of Escherichia spp., one of which is newly discovered and is being reported for the first time here.

Species-Wide Collection of Escherichia coli Isolates for Examination of Genomic Diversity.

Pathogenic and nonpathogenic Escherichia coli strains present a vast genomic diversity. We report the genome sequences of 2,244 E. coli isolates from multiple animal and environmental sources. Their phylogenetic relationships and potential risk to human health were examined.

Shiga Toxin-Producing Serogroup O91 Escherichia coli Strains Isolated from Food and Environmental Samples.

Shiga toxin-producing Escherichia coli (STEC) strains of the O91:H21 serotype have caused severe infections, including hemolytic-uremic syndrome. Strains of the O91 serogroup have been isolated from food, animals, and the environment worldwide but are not well characterized. We used a microarray and other molecular assays to examine 49 serogroup O91 strains (environmental, food, and clinical strains) for their virulence potential and phylogenetic relationships. Most of the isolates were identified to be strains of the O91:H21 and O91:H14 serotypes, with a few O91:H10 strains and one O91:H9 strain being identified. None of the strains had the eae gene, which codes for the intimin adherence protein, and many did not have some of the genetic markers that are common in other STEC strains. The genetic profiles of the strains within each serotype were similar but differed greatly between strains of different serotypes. The genetic profiles of the O91:H21 strains that we tested were identical or nearly identical to those of the clinical O91:H21 strains that have caused severe diseases. Multilocus sequence typing and clustered regularly interspaced short palindromic repeat analyses showed that the O91:H21 strains clustered within the STEC 1 clonal group but the other O91 serotype strains were phylogenetically diverse.IMPORTANCE This study showed that food and environmental O91:H21 strains have similar genotypic profiles and Shiga toxin subtypes and are phylogenetically related to the O91:H21 strains that have caused hemolytic-uremic syndrome, suggesting that these strains may also have the potential to cause severe illness.

Characterization and Virulence Potential of Serogroup O113 Shiga Toxin-Producing Escherichia coli Strains Isolated from Beef and Cattle in the United States.

Shiga toxin-producing Escherichia coli (STEC) of serotype O113:H21 have caused severe diseases but are unusual in that they do not produce the intimin protein required for adherence to intestinal epithelial cells. Strains of serogroup O113 are one of the most common STEC found in ground beef and beef products in the United States, but their virulence potential is unknown. We used a microarray to characterize 65 O113 strains isolated in the United States from ground beef, beef trim, cattle feces, and fresh spinach. Most were O113:H21 strains, but there were also nine strains of O113:H4 serotype. Although strains within the same serotype had similar profiles for the genes that were tested on the array, the profiles were distinct between the two serotypes, and the strains belonged to different clonal groups. Analysis by clustered regularly interspaced short palindromic repeat analysis showed that O113:H4 strains are conserved genetically, but the O113:H21 strains showed considerable polymorphism and genetic diversity. In comparison to the O113:H21 strains from Australia that were implicated in severe disease, the U.S. isolates showed similar genetic profiles to the known pathogens from Australia, suggesting that these may also have the potential to cause infections.

Strain-Level Discrimination of Shiga Toxin-Producing Escherichia coli in Spinach Using Metagenomic Sequencing.

Consumption of fresh bagged spinach contaminated with Shiga toxin-producing Escherichia coli (STEC) has led to severe illness and death; however current culture-based methods to detect foodborne STEC are time consuming. Since not all STEC strains are considered pathogenic to humans, it is crucial to incorporate virulence characterization of STEC in the detection method. In this study, we assess the comprehensiveness of utilizing a shotgun metagenomics approach for detection and strain-level identification by spiking spinach with a variety of genomically disparate STEC strains at a low contamination level of 0.1 CFU/g. Molecular serotyping, virulence gene characterization, microbial community analysis, and E. coli core gene single nucleotide polymorphism (SNP) analysis were performed on metagenomic sequence data from enriched samples. It was determined from bacterial community analysis that E. coli, which was classified at the phylogroup level, was a major component of the population in most samples. However, in over half the samples, molecular serotyping revealed the presence of indigenous E. coli which also contributed to the percent abundance of E. coli. Despite the presence of additional E. coli strains, the serotype and virulence genes of the spiked STEC, including correct Shiga toxin subtype, were detected in 94% of the samples with a total number of reads per sample averaging 2.4 million. Variation in STEC abundance and/or detection was observed in replicate spiked samples, indicating an effect from the indigenous microbiota during enrichment. SNP analysis of the metagenomic data correctly placed the spiked STEC in a phylogeny of related strains in cases where the indigenous E. coli did not predominate in the enriched sample. Also, for these samples, our analysis demonstrates that strain-level phylogenetic resolution is possible using shotgun metagenomic data for determining the genomic relatedness of a contaminating STEC strain to other closely related E. coli.