Whole genome sequencing analyses revealed that Salmonella enterica serovar Dublin strains from Brazil belonged to two predominant clades.

Salmonella Dublin is a cattle-associated serovar sporadically causing disease in humans. S. Dublin strains isolated in Brazil and in other countries were analyzed to determine their phylogenetic relationships, the presence of genes, plasmids, genomic regions related to virulence and antimicrobial resistance genes repertoire, using WGS analyses. Illumina was used to sequence the genome of 112 S. Dublin strains isolated in Brazil from humans (n = 82) and animals (n = 30) between 1983 and 2016. Furthermore, 87 strains from other countries were analyzed. WGSNP analysis revealed three different clades, in which the strains from Brazil belonged to two clades, A and C. Most of the genes and genomic regions searched varied among the strains studied. The siderophore genes iroB and iroC were exclusively found in strains from Brazil and pegD gene, related to fimbrial adherence determinants, were positive in 124 strains from clades A and B but absent in all the strains from clade C (n = 71). Eleven plasmid replicons were found in the strains from Brazil, and nine were exclusively found in strains from other countries. The antimicrobial resistance genes mdsA and mdsB, that encode an efflux pump, were found in all the strains studied. The strains from Brazil carried other resistance genes, such as tet(A) (n = 11), tet(B) (n = 4) and tet(C) (n = 4), blaTEM-1 (n = 4), catA1 (n = 1), aadA1 (n = 1), and sul1 (n = 1). In conclusion, S. Dublin strains isolated in Brazil presented some few unique genes not found in strains from other countries and were allocated into two distinct clades with strains of human and animal origin epidemiologically related. This fact stresses the zoonotic potential of S. Dublin circulating in Brazil for more than 30 years.

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.

Draft Genome Sequences of 62 Staphylococcus aureus Isolates Associated with Four Foodborne Outbreaks in the United States.

Staphylococcus aureus bacteria are ranked among the top five foodborne pathogens in the United States. Here, we report the draft genome sequences of 62 S. aureus isolates that originated from the manufacturing environment of an Illinois bakery and were associated with outbreaks between 2010 and 2011 in the United States.

Draft Whole-Genome Sequences of 51 Campylobacter jejuni and 12 Campylobacter coli Clinical Isolates from Chile.

Campylobacter species are the leading cause of gastroenteritis worldwide and an emerging threat in developing countries. Here, we report the draft whole-genome sequences of 51 Campylobacter jejuni and 12 Campylobacter coli strains isolated from patients with gastroenteritis in Santiago, Chile.

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.

In-depth comparative analysis of Illumina® MiSeq run metrics: Development of a wet-lab quality assessment tool.

Whole genome sequencing of bacterial isolates has become a daily task in many laboratories, generating incredible amounts of data. However, data acquisition is not an end in itself; the goal is to acquire high-quality data useful for understanding genetic relationships. Having a method that could rapidly determine which of the many available run metrics are the most important indicators of overall run quality and having a way to monitor these during a given sequencing run would be extremely helpful to this effect. Therefore, we compared various run metrics across 486 MiSeq runs, from five different machines. By performing a statistical analysis using principal components analysis and a K-means clustering algorithm of the metrics, we were able to validate metric comparisons among instruments, allowing for the development of a predictive algorithm, which permits one to observe whether a given MiSeq run has performed adequately. This algorithm is available in an Excel spreadsheet: that is, MiSeq Instrument & Run (In-Run) Forecast. Our tool can help verify that the quantity/quality of the generated sequencing data consistently meets or exceeds recommended manufacturer expectations. Patterns of deviation from those expectations can be used to assess potential run problems and plan preventative maintenance, which can save valuable time and funding resources.

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.

Changing of the Genomic Pattern of Salmonella Enteritidis Strains Isolated in Brazil Over a 48 year-period revealed by Whole Genome SNP Analyses.

Salmonella Enteritidis became the main serovar isolated from gastroenteritis cases in Brazil after the 90's. In this study we used whole genome sequence analysis to determine the phylogenetic relationships among a collection of strains isolated in Brazil to identify possible genomic differences between the strains isolated in the pre and post-epidemic period. Also, we compared our data from strains isolated in Brazil to strains available in the public domain from other South American countries. Illumina technology was used to sequence the genome of 256 Salmonella Enteritidis strains isolated over a 48 year-period in Brazil, comprising the pre- and post-epidemic period. Phylogenetic analyses revealed distinct lineages for strains isolated before and after 1994. Moreover, the phage region SE20 that may be related to the emergence of Salmonella Enteritidis worldwide was present only in strains of the post-epidemic cluster. In conclusion, our results showed that the genomic profile of Salmonella Enteritidis strains isolated in Brazil shifted after 1994, replaced by a global epidemic group of strains. It may be hypothesized that the presence of the prophage SE20 might have conferred to these strains a better ability to colonize chicken and consequently to infect and cause disease in humans, which might better explain the increase in the number of S. Enteritidis cases in Brazil and other South American countries. However, to verify this hypothesis further studies are needed.

Draft Genome Sequences of 112 Salmonella enterica Serovar Dublin Strains Isolated from Humans and Animals in Brazil.

Salmonella enterica serovar Dublin is a strongly adapted serovar that causes enteritis and/or systemic disease in cattle and results in high rates of mortality. Here, we report the draft genome sequences of 112 S. Dublin strains isolated from humans and animals in Brazil. These draft genome sequences will help enhance our understanding of this serovar in Brazil.

Draft Genome Sequences of 510 Listeria monocytogenes Strains from Food Isolates and Human Listeriosis Cases from Northern Italy.

Listeriosis outbreaks are frequently multistate/multicountry outbreaks, underlining the importance of molecular typing data for several diverse and well-characterized isolates. Large-scale whole-genome sequencing studies on Listeria monocytogenes isolates from non-U.S. locations have been limited. Herein, we describe the draft genome sequences of 510 L. monocytogenes isolates from northern Italy from different sources.

Draft Genome Sequences of 23 Salmonella enterica Strains Isolated from Cattle in Ibadan, Nigeria, Representing 21 Salmonella Serovars.

To provide a better understanding of the diversity of Salmonella enterica, we report the assembled genome sequences of 23 Salmonella enterica strains isolated from fecal samples of cattle in Nigeria comprising 21 different Salmonella serovars.

Draft Genome Sequences of 64 Salmonella enterica Serotype Enteritidis Isolates Obtained from Wild Mice.

Salmonella enterica serotype Enteritidis is a foodborne pathogen of global concern, because it is frequently isolated from foods and patients. Draft genome sequences are reported here for 64 S Enteritidis strains isolated from the intestines and spleens of mice caught live on chicken farms in the U.S. Northeast. The availability of these genomes provides baseline information on the genomic diversity of S Enteritidis during the 1990s, when foodborne outbreaks traced to internal contamination of eggs were prevalent.

Draft Genome Sequences of Yersinia frederiksenii, Yersinia intermedia, and Yersinia kristensenii Strains from Brazil.

Yersinia enterocolitica-like strains are usually understudied. In this work, we reported the draft genome sequences of two Yersinia frederiksenii, two Yersinia intermedia, and two Yersinia kristensenii strains isolated from humans, animals, food, and the environment in Brazil. These draft genomes will provide better molecular characterizations of these species.

Draft Genome Sequences of 256 Salmonella enterica subsp. enterica Serovar Enteritidis Strains Isolated from Humans, Food, Chickens, and Farm Environments in Brazil.

Salmonella enterica subsp. enterica serovar Enteritidis emerged in the late 1980s as the most isolated Salmonella serovar worldwide. Here, we report the draft genomes of 256 S Enteritidis strains isolated from humans, food, chickens, and farm environments in Brazil. These draft genomes will help enhance our understanding of this serovar in Brazil.

Whole Genome and Core Genome Multilocus Sequence Typing and Single Nucleotide Polymorphism Analyses of Listeria monocytogenes Isolates Associated with an Outbreak Linked to Cheese, United States, 2013.

Epidemiological findings of a listeriosis outbreak in 2013 implicated Hispanic-style cheese produced by company A, and pulsed-field gel electrophoresis (PFGE) and whole genome sequencing (WGS) were performed on clinical isolates and representative isolates collected from company A cheese and environmental samples during the investigation. The results strengthened the evidence for cheese as the vehicle. Surveillance sampling and WGS 3 months later revealed that the equipment purchased by company B from company A yielded an environmental isolate highly similar to all outbreak isolates. The whole genome and core genome multilocus sequence typing and single nucleotide polymorphism (SNP) analyses results were compared to demonstrate the maximum discriminatory power obtained by using multiple analyses, which were needed to differentiate outbreak-associated isolates from a PFGE-indistinguishable isolate collected in a nonimplicated food source in 2012. This unrelated isolate differed from the outbreak isolates by only 7 to 14 SNPs, and as a result, the minimum spanning tree from the whole genome analyses and certain variant calling approach and phylogenetic algorithm for core genome-based analyses could not provide differentiation between unrelated isolates. Our data also suggest that SNP/allele counts should always be combined with WGS clustering analysis generated by phylogenetically meaningful algorithms on a sufficient number of isolates, and the SNP/allele threshold alone does not provide sufficient evidence to delineate an outbreak. The putative prophages were conserved across all the outbreak isolates. All outbreak isolates belonged to clonal complex 5 and serotype 1/2b and had an identical inlA sequence which did not have premature stop codons.IMPORTANCE In this outbreak, multiple analytical approaches were used for maximum discriminatory power. A PFGE-matched, epidemiologically unrelated isolate had high genetic similarity to the outbreak-associated isolates, with as few as 7 SNP differences. Therefore, the SNP/allele threshold should not be used as the only evidence to define the scope of an outbreak. It is critical that the SNP/allele counts be complemented by WGS clustering analysis generated by phylogenetically meaningful algorithms to distinguish outbreak-associated isolates from epidemiologically unrelated isolates. Careful selection of a variant calling approach and phylogenetic algorithm is critical for core-genome-based analyses. The whole-genome-based analyses were able to construct the highly resolved phylogeny needed to support the findings of the outbreak investigation. Ultimately, epidemiologic evidence and multiple WGS analyses should be combined to increase confidence levels during outbreak investigations.

Listeria monocytogenes in Stone Fruits Linked to a Multistate Outbreak: Enumeration of Cells and Whole-Genome Sequencing.

In 2014, the identification of stone fruits contaminated with Listeria monocytogenes led to the subsequent identification of a multistate outbreak. Simultaneous detection and enumeration of L. monocytogenes were performed on 105 fruits, each weighing 127 to 145 g, collected from 7 contaminated lots. The results showed that 53.3% of the fruits yielded L. monocytogenes (lower limit of detection, 5 CFU/fruit), and the levels ranged from 5 to 2,850 CFU/fruit, with a geometric mean of 11.3 CFU/fruit (0.1 CFU/g of fruit). Two serotypes, IVb-v1 and 1/2b, were identified by a combination of PCR- and antiserum-based serotyping among isolates from fruits and their packing environment; certain fruits contained a mixture of both serotypes. Single nucleotide polymorphism (SNP)-based whole-genome sequencing (WGS) analysis clustered isolates from two case-patients with the serotype IVb-v1 isolates and distinguished outbreak-associated isolates from pulsed-field gel electrophoresis (PFGE)-matched, but epidemiologically unrelated, clinical isolates. The outbreak-associated isolates differed by up to 42 SNPs. All but one serotype 1/2b isolate formed another WGS cluster and differed by up to 17 SNPs. Fully closed genomes of isolates from the stone fruits were used as references to maximize the resolution and to increase our confidence in prophage analysis. Putative prophages were conserved among isolates of each WGS cluster. All serotype IVb-v1 isolates belonged to singleton sequence type 382 (ST382); all but one serotype 1/2b isolate belonged to clonal complex 5. IMPORTANCE: WGS proved to be an excellent tool to assist in the epidemiologic investigation of listeriosis outbreaks. The comparison at the genome level contributed to our understanding of the genetic diversity and variations among isolates involved in an outbreak or isolates associated with food and environmental samples from one facility. Fully closed genomes increased our confidence in the identification and comparison of accessory genomes. The diversity among the outbreak-associated isolates and the inclusion of PFGE-matched, but epidemiologically unrelated, isolates demonstrate the high resolution of WGS. The prevalence and enumeration data could contribute to our further understanding of the risk associated with Listeria monocytogenes contamination, especially among high-risk populations.

Tracing Origins of the Salmonella Bareilly Strain Causing a Food-borne Outbreak in the United States.

BACKGROUND: Using a novel combination of whole-genome sequencing (WGS) analysis and geographic metadata, we traced the origins of Salmonella Bareilly isolates collected in 2012 during a widespread food-borne outbreak in the United States associated with scraped tuna imported from India. METHODS: Using next-generation sequencing, we sequenced the complete genome of 100 Salmonella Bareilly isolates obtained from patients who consumed contaminated product, from natural sources, and from unrelated historically and geographically disparate foods. Pathogen genomes were linked to geography by projecting the phylogeny on a virtual globe and produced a transmission network. RESULTS: Phylogenetic analysis of WGS data revealed a common origin for outbreak strains, indicating that patients in Maryland and New York were infected from sources originating at a facility in India. CONCLUSIONS: These data represent the first report fully integrating WGS analysis with geographic mapping and a novel use of transmission networks. Results showed that WGS vastly improves our ability to delimit the scope and source of bacterial food-borne contamination events. Furthermore, these findings reinforce the extraordinary utility that WGS brings to global outbreak investigation as a greatly enhanced approach to protecting the human food supply chain as well as public health in general.

Draft Genome Sequence of Salmonella enterica subsp. enterica Serovar Give, Isolated from an Imported Chili Powder Product.

We report the genome sequence of Salmonella enterica subsp. enterica serovar Give (CFSAN012622), isolated from imported chili powder in 2014. This genome contains genes previously reported to be specific only to S. enterica serovar Enteritidis. This strain shows a unique pulsed-field gel electrophoresis (PFGE) pattern clustering with serovar Enteritidis (JEG X01.0005).

The role of dystroglycan in PDGF-BB-dependent migration of activated hepatic stellate cells/myofibroblasts.

Hepatic stellate cells are embedded in the loose connective tissue matrix within the space of Disse. This extracellular matrix contains several basement membrane components including laminin, but its composition changes during liver injury because of the production of extracellular matrix components found in scar tissue. These changes in extracellular matrix composition and in cell-extracellular matrix interactions may play a key role in hepatic stellate cell transdifferentiation. In this communication we used early passages of mouse hepatic stellate cells (activated HSC/myofibroblasts) to study the platelet-derived growth factor BB (PDGF-BB)-dependent expression and regulation of ß-dystroglycan and its role in activated HSC/myofibroblast migration. We used Northern and Western analysis to study dystroglycan expression and confocal microscopy to investigate changes in subcellular distribution of the protein. Activated HSC migration was investigated using an in vitro wound-healing assay. PDGF-BB induced significant changes in dystroglycan regulation and subcellular distribution of the protein. Whereas steady-state levels of dystroglycan mRNA remained constant, PDGF-BB increased dystroglycan transcription but shortened the t(1/2) by 50%. Moreover, PDGF-BB changed dystroglycan and α5-integrin cellular distribution. Cell migration experiments revealed that PDGF-BB-dependent migration of activated HSC/myofibroblasts was completely blocked by neutralizing antibodies to fibronectin, α5-integrin, laminin, and ß-dystroglycan. Overall, these findings suggest that both laminin and fibronectin and their receptors play a key role in PDGF-BB-induced activated HSC migration.