Developing isotopic proxies to reconstruct the metabolic rates and thermal histories of octopus.

Understanding an animal's metabolic rate and thermal history is pivotal for ecological research. Recent studies have proposed the use of stable carbon and oxygen isotopes (δ13C and δ18O) in biogenic carbonates as proxies of metabolic rate and experienced temperature, respectively, to overcome the challenges of directly measuring these data in the field. Our study represents the first experimental investigation to develop δ13C and δ18O proxies in octopus. Octopus berrima hatchlings were raised in captivity, at varying water temperatures, for up to 110 days. O. berrima statoliths were then subsequently analysed for δ13C and δ18O values. The proportion of metabolically derived carbon, or respired carbon (Cresp), increased as the octopus grew (slope = 0.076, R2 = 0.72), suggesting an influence of somatic growth rate and body mass on δ13C values. Additionally, we identified an inverse correlation between δ18O values and environmental temperature (slope = -0.163, R2 = 0.91), which was subsequently used to develop a thermal reconstruction model. Our experiment aids in interpreting stable isotopic values in statoliths and their application as temperature and metabolic proxies in wild-caught octopus. Such proxies will increase our monitoring capabilities of these ecologically and commercially significant cephalopods and contribute to their conservation and effective management.

Mapping Phosphorus Availability in Soil at a Large Scale and High Resolution Using Novel Diffusive Gradients in Thin Films Designed for X-ray Fluorescence Microscopy.

A novel binding layer (BL) as part of the diffusive gradients in thin films (DGT) technique was developed for the two-dimensional visualization and quantification of labile phosphorus (P) in soils. This BL was designed for P detection by synchrotron-based X-ray fluorescence microscopy (XFM). It differs from the conventional DGT BL as the hydrogel is eliminated to overcome the issue that the fluorescent X-rays of P are detected mainly from shallow sample depths. Instead, the novel design is based on a polyimide film (Kapton) onto which finely powdered titanium dioxide-based P binding agent (Metsorb) was applied, resulting in superficial P binding only. The BL was successfully used for quantitative visualization of P diffusion from three conventional P fertilizers applied to two soils. On a selection of samples, XFM analysis was confirmed by quantitative laser-ablation inductively coupled plasma mass spectrometry (LA-ICP-MS). The XFM method detected significant differences in labile P concentrations and P diffusion zone radii with the P fertilizer incubation, which were explained by soil and fertilizer properties. This development paves the way for fast XFM analysis of P on large DGT BLs to investigate in situ diffusion of labile P from fertilizers and to visualize large-scale P cycling processes at high spatial resolution.

GalaxyTrakr: a distributed analysis tool for public health whole genome sequence data accessible to non-bioinformaticians.

BACKGROUND: Processing and analyzing whole genome sequencing (WGS) is computationally intense: a single Illumina MiSeq WGS run produces ~ 1 million 250-base-pair reads for each of 24 samples. This poses significant obstacles for smaller laboratories, or laboratories not affiliated with larger projects, which may not have dedicated bioinformatics staff or computing power to effectively use genomic data to protect public health. Building on the success of the cloud-based Galaxy bioinformatics platform ( http://galaxyproject.org ), already known for its user-friendliness and powerful WGS analytical tools, the Center for Food Safety and Applied Nutrition (CFSAN) at the U.S. Food and Drug Administration (FDA) created a customized 'instance' of the Galaxy environment, called GalaxyTrakr ( https://www.galaxytrakr.org ), for use by laboratory scientists performing food-safety regulatory research. The goal was to enable laboratories outside of the FDA internal network to (1) perform quality assessments of sequence data, (2) identify links between clinical isolates and positive food/environmental samples, including those at the National Center for Biotechnology Information sequence read archive ( https://www.ncbi.nlm.nih.gov/sra/ ), and (3) explore new methodologies such as metagenomics. GalaxyTrakr hosts a variety of free and adaptable tools and provides the data storage and computing power to run the tools. These tools support coordinated analytic methods and consistent interpretation of results across laboratories. Users can create and share tools for their specific needs and use sequence data generated locally and elsewhere. RESULTS: In its first full year (2018), GalaxyTrakr processed over 85,000 jobs and went from 25 to 250 users, representing 53 different public and state health laboratories, academic institutions, international health laboratories, and federal organizations. By mid-2020, it has grown to 600 registered users and processed over 450,000 analytical jobs. To illustrate how laboratories are making use of this resource, we describe how six institutions use GalaxyTrakr to quickly analyze and review their data. Instructions for participating in GalaxyTrakr are provided. CONCLUSIONS: GalaxyTrakr advances food safety by providing reliable and harmonized WGS analyses for public health laboratories and promoting collaboration across laboratories with differing resources. Anticipated enhancements to this resource will include workflows for additional foodborne pathogens, viruses, and parasites, as well as new tools and services.

Phylogenetic and Biogeographic Patterns of Vibrio parahaemolyticus Strains from North America Inferred from Whole-Genome Sequence Data.

Vibrio parahaemolyticus is the most common cause of seafood-borne illness reported in the United States. The draft genomes of 132 North American clinical and oyster V. parahaemolyticus isolates were sequenced to investigate their phylogenetic and biogeographic relationships. The majority of oyster isolate sequence types (STs) were from a single harvest location; however, four were identified from multiple locations. There was population structure along the Gulf and Atlantic Coasts of North America, with what seemed to be a hub of genetic variability along the Gulf Coast, with some of the same STs occurring along the Atlantic Coast and one shared between the coastal waters of the Gulf and those of Washington State. Phylogenetic analyses found nine well-supported clades. Two clades were composed of isolates from both clinical and oyster sources. Four were composed of isolates entirely from clinical sources, and three were entirely from oyster sources. Each single-source clade consisted of one ST. Some human isolates lack tdh, trh, and some type III secretion system (T3SS) genes, which are established virulence genes of V. parahaemolyticus Thus, these genes are not essential for pathogenicity. However, isolates in the monophyletic groups from clinical sources were enriched in several categories of genes compared to those from monophyletic groups of oyster isolates. These functional categories include cell signaling, transport, and metabolism. The identification of genes in these functional categories provides a basis for future in-depth pathogenicity investigations of V. parahaemolyticusIMPORTANCEVibrio parahaemolyticus is the most common cause of seafood-borne illness reported in the United States and is frequently associated with shellfish consumption. This study contributes to our knowledge of the biogeography and functional genomics of this species around North America. STs shared between the Gulf Coast and the Atlantic seaboard as well as Pacific waters suggest possible transport via oceanic currents or large shipping vessels. STs frequently isolated from humans but rarely, if ever, isolated from the environment are likely more competitive in the human gut than other STs. This could be due to additional functional capabilities in areas such as cell signaling, transport, and metabolism, which may give these isolates an advantage in novel nutrient-replete environments such as the human gut.

Patellofemoral Pain Syndrome and Pain Severity Is Associated With Asymmetry of Gluteus Medius Muscle Activation Measured Via Ultrasound.

OBJECTIVE: The aim of the study was to determine whether gluteus medius muscle thickness or activation differed between left and right sides and was associated with patellofemoral pain presence or severity. DESIGN: Males and females were recruited and screened by a physiotherapist for inclusion in the control or patellofemoral pain syndrome group. Bilateral measures were obtained for Q angle and gluteus medius muscle thickness at rest and on contraction via standing hip external rotation, using ultrasound. Muscle activation was calculated as the percentage change in muscle thickness on contraction relative to at rest. Patellofemoral pain syndrome participants completed the anterior knee pain scale and a visual analog pain scale. RESULTS: Gluteus medius muscle thickness at rest and on contraction, muscle activation, and Q angle were not different between control (n = 27, 63% female) and patellofemoral pain syndrome (n = 27, 59% female) groups. However, patellofemoral pain syndrome participants had a significantly larger left-right side imbalance in gluteus medius muscle activation than controls (15.9 ± 19.3% vs. 4.4 ± 21.9%, P < 0.05). Among patellofemoral pain syndrome participants, the magnitude of asymmetry of gluteus medius muscle activation was correlated with knee pain score (r = 0.425, P = 0.027). CONCLUSIONS: Asymmetry of gluteus medius muscle activation was associated with patellofemoral pain syndrome and pain severity. This is clinically relevant for patellofemoral pain syndrome prevention and treatment, particularly because this was quantifiable using ultrasound.

In Silico Serotyping Based on Whole-Genome Sequencing Improves the Accuracy of Shigella Identification.

Bacteria of the genus Shigella, consisting of 4 species and >50 serotypes, cause shigellosis, a foodborne disease of significant morbidity, mortality, and economic loss worldwide. Classical Shigella identification based on selective media and serology is tedious, time-consuming, expensive, and not always accurate. A molecular diagnostic assay does not distinguish Shigella at the species level or from enteroinvasive Escherichia coli (EIEC). We inspected genomic sequences from 221 Shigella isolates and observed low concordance rates between conventional designation and molecular serotyping: 86.4% and 80.5% at the species and serotype levels, respectively. Serotype determinants for 6 additional serotypes were identified. Examination of differentiation gene markers commonly perceived as characteristic hallmarks in Shigella showed high variability among different serotypes. Using this information, we developed ShigaTyper, an automated workflow that utilizes limited computational resources to accurately and rapidly determine 59 Shigella serotypes using Illumina paired-end whole-genome sequencing (WGS) reads. Shigella serotype determinants and species-specific diagnostic markers were first identified through read alignment to an in-house curated reference sequence database. Relying on sequence hits that passed a threshold level of coverage and accuracy, serotype could be unambiguously predicted within 1 min for an average-size WGS sample of ∼500 MB. Validation with WGS data from 380 isolates showed an accuracy rate of 98.2%. This pipeline is the first step toward building a comprehensive WGS-based analysis pipeline of Shigella spp. in a field laboratory setting, where speed is essential and resources need to be more cost-effectively dedicated.IMPORTANCEShigella causes diarrheal disease with serious public health implications. However, conventional Shigella identification methods are laborious and time-consuming and can be erroneous due to the high similarity between Shigella and enteroinvasive Escherichia coli (EIEC) and cross-reactivity between serotyping antisera. Further, serotype interpretation is complicated for inexperienced users. To develop an easier method with higher accuracy based on whole-genome sequencing (WGS) for Shigella serotyping, we systematically examined genomic information of Shigella isolates from 53 serotypes to define rules for differentiation and serotyping. We created ShigaTyper, an automated pipeline that accurately and rapidly excludes non-Shigella isolates and identifies 59 Shigella serotypes using Illumina paired-end WGS reads. A serotype can be unambiguously predicted at a data processing speed of 538 MB/min with 98.2% accuracy from a regular laptop. Once it is installed, training in bioinformatics analysis and Shigella genetics is not required. This pipeline is particularly useful to general microbiologists in field laboratories.

Sequence Analysis of IncA/C and IncI1 Plasmids Isolated from Multidrug-Resistant Salmonella Newport Using Single-Molecule Real-Time Sequencing.

Multidrug-resistant (MDR) plasmids play an important role in disseminating antimicrobial resistance genes. To elucidate the antimicrobial resistance gene compositions in A/C incompatibility complex (IncA/C) plasmids carried by animal-derived MDR Salmonella Newport, and to investigate the spread mechanism of IncA/C plasmids, this study characterizes the complete nucleotide sequences of IncA/C plasmids by comparative analysis. Complete nucleotide sequencing of plasmids and chromosomes of six MDR Salmonella Newport strains was performed using PacBio RSII. Open reading frames were assigned using prokaryotic genome annotation pipeline (PGAP). To understand genomic diversity and evolutionary relationships among Salmonella Newport IncA/C plasmids, we included three complete IncA/C plasmid sequences with similar backbones from Salmonella Newport and Escherichia coli: pSN254, pAM04528, and peH4H, and additional 200 draft chromosomes. With the exception of canine isolate CVM22462, which contained an additional IncI1 plasmid, each of the six MDR Salmonella Newport strains contained only the IncA/C plasmid. These IncA/C plasmids (including references) ranged in size from 80.1 (pCVM21538) to 176.5 kb (pSN254) and carried various resistance genes. Resistance genes floR, tetA, tetR, strA, strB, sul, and mer were identified in all IncA/C plasmids. Additionally, blaCMY-2 and sugE were present in all IncA/C plasmids, excepting pCVM21538. Plasmid pCVM22462 was capable of being transferred by conjugation. The IncI1 plasmid pCVM22462b in CVM22462 carried blaCMY-2 and sugE. Our data showed that MDR Salmonella Newport strains carrying similar IncA/C plasmids clustered together in the phylogenetic tree using chromosome sequences and the IncA/C plasmids from animal-derived Salmonella Newport contained diverse resistance genes. In the current study, we analyzed genomic diversities and phylogenetic relationships among MDR Salmonella Newport using complete plasmids and chromosome sequences and provided possible spread mechanism of IncA/C plasmids in Salmonella Newport Lineage II.

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.

Draft Genome Sequences of 40 Salmonella enterica Serovar Typhimurium Strains Isolated from Humans and Food in Brazil.

Salmonellosis is an important health problem worldwide and Salmonella enterica serovar Typhimurium is one of the most common isolated serovars. Here, we reported the draft genomes of 40 S Typhimurium strains isolated from humans and food in Brazil. These draft genomes will improve phylogenetic analysis and will help enhance our understanding of strains of this serovar isolated in Brazil.

Quantifying the Sensitivity of Soil Microbial Communities to Silver Sulfide Nanoparticles Using Metagenome Sequencing.

Soils are a sink for sulfidised-silver nanoparticles (Ag2S-NPs), yet there are limited ecotoxicity data for their effects on microbial communities. Conventional toxicity tests typically target a single test species or function, which does not reflect the broader community response. Using a combination of quantitative PCR, 16S rRNA amplicon sequencing and species sensitivity distribution (SSD) methods, we have developed a new approach to calculate silver-based NP toxicity thresholds (HCx, hazardous concentrations) that are protective of specific members (operational taxonomic units, OTUs) of the soil microbial community. At the HC20 (80% of species protected), soil OTUs were significantly less sensitive to Ag2S-NPs compared to AgNPs and Ag+ (5.9, 1.4 and 1.4 mg Ag kg-1, respectively). However at more conservative HC values, there were no significant differences. These trends in OTU responses matched with those seen in a specific microbial function (rate of nitrification) and amoA-bacteria gene abundance. This study provides a novel molecular-based framework for quantifying the effect of a toxicant on whole soil microbial communities while still determining sensitive genera/species. Methods and results described here provide a benchmark for microbial community ecotoxicological studies and we recommend that future revisions of Soil Quality Guidelines for AgNPs and other such toxicants consider this approach.

Draft Genome Sequences of Salmonella enterica subsp. enterica Serovars Typhimurium and Nottingham Isolated from Food Products.

A quantitative real-time PCR (qPCR) designed to detect Salmonella enterica subsp. enterica serovar Enteritidis, targeting the sdf gene, generated positive results for S. enterica subsp. enterica serovar Typhimurium (CFSAN033950) and S. enterica subsp. enterica serovar Nottingham (CFSAN006803) isolated from food samples. Both strains show pulsed-field gel electrophoresis (PFGE) patterns distinct from those of S Enteritidis. Here, we report the genome sequences of these two strains.

Complete Genome and Methylome Sequences of Salmonella enterica subsp. enterica Serovar Panama (ATCC 7378) and Salmonella enterica subsp. enterica Serovar Sloterdijk (ATCC 15791).

Salmonella enterica spp. are pathogenic bacteria commonly associated with food-borne outbreaks in human and animals. Salmonella enterica spp. are characterized into more than 2,500 different serotypes, which makes epidemiological surveillance and outbreak control more difficult. In this report, we announce the first complete genome and methylome sequences from two Salmonella type strains associated with food-borne outbreaks, Salmonella enterica subsp. enterica serovar Panama (ATCC 7378) and Salmonella enterica subsp. enterica serovar Sloterdijk (ATCC 15791).

Complete Genome and Methylome Sequences of Two Salmonella enterica spp.

Salmonella enterica is responsible for major foodborne outbreaks worldwide. It can cause gastroenteritis characterized by diarrhea, vomiting, and fever. Salmonella infections raise public health concerns along with consequential economic impacts. In this report, we announce the first complete genome sequences of Salmonella enterica subsp. enterica serovar Choleraeuis (S. Choleraeuis) ATCC 10708 and Salmonella enterica subsp. enterica serovar Pullorum (S. Pullorum) ATCC 9120, isolated from patients with diarrhea.

Complete Genome Sequence of Enteroinvasive Escherichia coli O96:H19 Associated with a Severe Foodborne Outbreak.

We present here the complete genome sequence of a strain of enteroinvasive Escherichia coli O96:H19 from a severe foodborne outbreak in a canteen in Italy in 2014. The complete genome may provide important information about the acquired pathogenicity of this strain and the transition between commensal and pathogenic E. coli.

Complete Genome Sequence of Salmonella enterica subsp. enterica Serovar Agona 460004 2-1, Associated with a Multistate Outbreak in the United States.

Within the last several years, Salmonella enterica subsp. enterica serovar Agona has been among the 20 most frequently isolated serovars in clinical cases of salmonellosis. In this report, the complete genome sequence of S. Agona strain 460004 2-1 isolated from unsweetened puffed-rice cereal during a multistate outbreak in 2008 was sequenced using single-molecule real-time DNA sequencing.

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).

Genome-wide methylation patterns in Salmonella enterica Subsp. enterica Serovars.

The methylation of DNA bases plays an important role in numerous biological processes including development, gene expression, and DNA replication. Salmonella is an important foodborne pathogen, and methylation in Salmonella is implicated in virulence. Using single molecule real-time (SMRT) DNA-sequencing, we sequenced and assembled the complete genomes of eleven Salmonella enterica isolates from nine different serovars, and analysed the whole-genome methylation patterns of each genome. We describe 16 distinct N6-methyladenine (m6A) methylated motifs, one N4-methylcytosine (m4C) motif, and one combined m6A-m4C motif. Eight of these motifs are novel, i.e., they have not been previously described. We also identified the methyltransferases (MTases) associated with 13 of the motifs. Some motifs are conserved across all Salmonella serovars tested, while others were found only in a subset of serovars. Eight of the nine serovars contained a unique methylated motif that was not found in any other serovar (most of these motifs were part of Type I restriction modification systems), indicating the high diversity of methylation patterns present in Salmonella.

Complete Sequences of Six IncA/C Plasmids of Multidrug-Resistant Salmonella enterica subsp. enterica Serotype Newport.

Multidrug-resistant (MDR) Salmonella enterica subsp. enterica serotype Newport has been a long-standing public health concern in the United States. We present the complete sequences of six IncA/C plasmids from animal-derived MDR S. Newport ranging from 80.1 to 158.5 kb. They shared a genetic backbone with S. Newport IncA/C plasmids pSN254 and pAM04528.

First Fully Closed Genome Sequence of Salmonella enterica subsp. enterica Serovar Cubana Associated with a Food-Borne Outbreak.

Salmonella enterica subsp. enterica serovar Cubana (Salmonella serovar Cubana) is associated with human and animal disease. Here, we used third-generation, single-molecule, real-time DNA sequencing to determine the first complete genome sequence of Salmonella serovar Cubana CFSAN002050, which was isolated from fresh alfalfa sprouts during a multistate outbreak in 2012.