High-Resolution Identification of Multiple Salmonella Serovars in a Single Sample by Using CRISPR-SeroSeq

Salmonella enterica is represented by \u3e2,600 serovars that can differ in routes of transmission, host colonization, and in resistance to antimicrobials. S. enterica is the leading bacterial cause of foodborne illness in the United States, with well-established detection methodology. Current surveillance protocols rely on the characterization of a few colonies to represent an entire sample; thus, minority serovars remain undetected. Salmonella contains two CRISPR loci, CRISPR1 and CRISPR2, and the spacer contents of these can be considered serovar specific. We exploited this property to develop an amplicon-based and multiplexed sequencing approach, CRISPR-SeroSeq (serotyping by sequencing of the CRISPR loci), to identify multiple serovars present in a single sample. Using mixed genomic DNA from two Salmonella serovars, we were able to confidently detect a serovar that constituted 0.01% of the sample. Poultry is a major reservoir of Salmonella spp., including serovars that are frequently associated with human illness, as well as those that are not. Numerous studies have examined the prevalence and diversity of Salmonella spp. in poultry, though these studies were limited to culture-based approaches and therefore only identified abundant serovars. CRISPR-SeroSeq was used to investigate samples from broiler houses and a processing facility. Ninety-one percent of samples harbored multiple serovars, and there was one sample in which four different serovars were detected. In another sample, reads for the minority serovar comprised 0.003% of the total number of Salmonella spacer reads. The most abundant serovars identified were Salmonella enterica serovars Montevideo, Kentucky, Enteritidis, and Typhimurium. CRISPR-SeroSeq also differentiated between multiple strains of some serovars. This high resolution of serovar populations has the potential to be utilized as a powerful tool in the surveillance of Salmonella species

Molecular Diversity of Bacteroidales in Fecal and Environmental Samples and Swine-Associated Subpopulations

Several swine-specific microbial source tracking methods are based on PCR assays targeting Bacteroidales 16S rRNA gene sequences. The limited application of these assays can be explained by the poor understanding of their molecular diversity in fecal sources and environmental waters. In order to address this, we studied the diversity of 9,340 partial (\u3e600bp in length) Bacteroidales 16S rRNA gene sequences from 13 fecal sources and nine feces-contaminated watersheds. The compositions of major Bacteroidales populations were analyzed to determine which host and environmental sequences were contributing to each group. This information allowed us to identify populations which were both exclusive to swine fecal sources and detected in swine-contaminated waters. Phylogenetic and diversity analyses revealed that some markers previously believed to be highly specific to swine populations are shared by multiple hosts, potentially explaining the cross-amplification signals obtained with nontargeted hosts. These data suggest that while many Bacteroidales populations are cosmopolitan, others exhibit a preferential host distribution and may be able to survive different environmental conditions. This study further demonstrates the importance of elucidating the diversity patterns of targeted bacterial groups to develop more inclusive fecal source tracking applications

Using Phosphorus Recovery from Wastewater as a Context for Teaching Sustainable Development with USEPA P3 Support

Phosphorus is an essential element required for agriculture. Current practices include one-way use of phosphorus: that is mining; production of fertilizer; land application; and ultimately loss to aquatic sediments. Once in the aquatic environment, phosphorus stimulates eutrophication resulting in the death of water bodies. With financial support from a People, Prosperity, and Planet program grant of the U.S. Environmental Protection Agency, an interdisciplinary team of faculty at the University of Cincinnati used phosphorus recovery from sewage and re-utilization as a struvite fertilizer as the context for a year-long course of study integrating graduate and undergraduate students in Environmental Engineering and Science as well as Environmental Studies. This presentation will highlight the original course format, results of student assessment from the 2004-2005 academic year, modifications incorporated for the 2005-2006 academic year, and the subsequent findings of student perceptions and learning. The challenge of integrating across two colleges within a comprehensive university system will be discussed, and our approaches for meeting the learning needs and course expectations of a diverse student population will be included

Comparative fecal metagenomics unveils unique functional capacity of the swine gut

<p>Abstract</p> <p>Background</p> <p>Uncovering the taxonomic composition and functional capacity within the swine gut microbial consortia is of great importance to animal physiology and health as well as to food and water safety due to the presence of human pathogens in pig feces. Nonetheless, limited information on the functional diversity of the swine gut microbiome is available.</p> <p>Results</p> <p>Analysis of 637, 722 pyrosequencing reads (130 megabases) generated from Yorkshire pig fecal DNA extracts was performed to help better understand the microbial diversity and largely unknown functional capacity of the swine gut microbiome. Swine fecal metagenomic sequences were annotated using both MG-RAST and JGI IMG/M-ER pipelines. Taxonomic analysis of metagenomic reads indicated that swine fecal microbiomes were dominated by Firmicutes and Bacteroidetes phyla. At a finer phylogenetic resolution, <it>Prevotella </it>spp. dominated the swine fecal metagenome, while some genes associated with <it>Treponema </it>and <it>Anareovibrio </it>species were found to be exclusively within the pig fecal metagenomic sequences analyzed. Functional analysis revealed that carbohydrate metabolism was the most abundant SEED subsystem, representing 13% of the swine metagenome. Genes associated with stress, virulence, cell wall and cell capsule were also abundant. Virulence factors associated with antibiotic resistance genes with highest sequence homology to genes in Bacteroidetes, Clostridia, and <it>Methanosarcina </it>were numerous within the gene families unique to the swine fecal metagenomes. Other abundant proteins unique to the distal swine gut shared high sequence homology to putative carbohydrate membrane transporters.</p> <p>Conclusions</p> <p>The results from this metagenomic survey demonstrated the presence of genes associated with resistance to antibiotics and carbohydrate metabolism suggesting that the swine gut microbiome may be shaped by husbandry practices.</p

Engineering Education Collaboration: Innovative Pedagogical Methods for High School and University Environmentalists

This paper presents an innovative teaching approach, how it is implemented, student response results of the implementation, and the assessment of impact on student learning. The findings are based on surveys given to the students after each lab lesson taught in partnership with university (Project STEP) and community members. The purpose of this paper is to showcase authentic molecular technology research methods that have been incorporated into a high school level water quality study in cooperation with a watershed restoration program. Typically, water quality studies focus on chemical analysis such as pH, dissolved oxygen, biochemical oxygen demand, orthophosphates, nitrates, temperature, turbidity, macro-invertebrate survey and fecal coliform cultures. This paper shows that by using molecular technology, the source of pollution in the watershed can be determined. Students in these high school science classes are engaged in authentic experiences to identify and analyze human impact on the environment and local ecosystems. Students also are able to collect and analyze data using computer and molecular technology. With help from the local watershed managers, the AP high school students filter bacteria, isolate their DNA, use the polymerase chain reaction (PCR) to amplify the DNA, and finally use gel electrophoresis to trace the DNA to its source (human, cow or intestinal bacteria). In this way, both AP and Physical Science students can extend the water quality study to trace the pollution to a point source. This is a unique approach to high school science laboratory activities. All watershed data is collected and organized using Microsoft Excel spreadsheets and graphing software. Students are able to form conclusions using technology that is used in today\u27s workplace. Initial findings regarding student response to this innovative teaching approach indicate that the actual application of molecular technology methods, employed to solve a problem with an unknown conclusion, is very meaningful to students. Unlike other traditional classroom labs, neither the teacher nor the students know what the results of the watershed tests are before-hand. This type of innovative teaching approach, supported by research on inquiry lessons, provides a more memorable experience for the students - actually performing technology that they would otherwise only read about in textbooks and articles. This paper will provide other instructors with a kind of roadmap, but one where there are experiences of many partners and students that highlight both successes and challenges

Bacterial Bio-indicators of Marcellus Shale Activities in Pennsylvania: A Molecular Ecology Survey

The practice of hydraulic fracking has increased over the years especially in Pennsylvania where most of the subterraneous gas-rich Marcellus Shale formations are located. Our previous work showed that headwater streams in proximity to hydraulic fracking operations have significantly different bacterial assemblages as compared to un-impacted streams in central PA. Aquatic bacterial communities are of great importance because they are often the ‘first-responders’ to environmental perturbations. We are interested in which bacteria become enriched, as this might serve as robust biomarkers of fracking, and can potentially biodegrade constituents of fracking fluids. In this study, we plan to expand upon our previous work to identify additional sentinel bacterial taxa in other areas in PA (Northeast and Southwest) heavily impacted by fracking. Water and sediment samples have been collected from Northern Pennsylvania (n=31) and Southwestern (n=11) regions upstream and downstream of fracking activities. Bacterial community profiles of these samples were generated via high-throughput sequencing of the 16S rRNA, a robust phylogenetic marker for bacterial identification. The data generated provide a snapshot of all bacteria taxa present and their relative abundance. Thus, differences in bacterial community structure between impacted and un-impacted environments can help glean which bacterial taxa are responding to environmental perturbations associated with fracking. This research can help us generate a list of potential bioindicators of nascent fracking activities and can be used to help track impacts and bioremediation potential within environmental scenarios

Evaluation of Swine-Specific PCR Assays Used for Fecal Source Tracking and Analysis of Molecular Diversity of Swine-Specific bacteroidales Populations

In this study, we evaluated the specificity, distribution, and sensitivity of Prevotella strain-based (PF163 and PigBac1) and methanogen-based (P23-2) PCR assays proposed to detect swine fecal pollution in environmental waters. The assays were tested against 222 fecal DNA extracts derived from target and nontarget animal hosts and against 34 groundwater and 15 surface water samples from five different sites. We also investigated the phylogenetic diversity of 1,340 Bacteroidales 16S rRNA gene sequences derived from swine feces, swine waste lagoons, swine manure pits, and waters adjacent to swine operations. Most swine fecal samples were positive for the host-specific Prevotella-based PCR assays (80 to 87%), while fewer were positive with the methanogen-targeted PCR assay (53%). Similarly, the Prevotella markers were detected more frequently than the methanogen-targeted assay markers in waters historically impacted with swine fecal contamination. However, the PF163 PCR assay cross-reacted with 23% of nontarget fecal DNA extracts, although Bayesian statistics suggested that it yielded the highest probability of detecting pig fecal contamination in a given water sample. Phylogenetic analyses revealed previously unknown swine-associated clades comprised of clones from geographically diverse swine sources and from water samples adjacent to swine operations that are not targeted by the Prevotella assays. While deeper sequencing coverage might be necessary to better understand the molecular diversity of fecal Bacteroidales species, results of sequence analyses supported the presence of swine fecal pollution in the studied watersheds. Overall, due to nontarget cross amplification and poor geographic stability of currently available host-specific PCR assays, development of additional assays is necessary to accurately detect sources of swine fecal pollution

Early-life formula feeding is associated with infant gut microbiota alterations and an increased antibiotic resistance load

Background Infants are at a high risk of acquiring fatal infections, and their treatment relies on functioning antibiotics. Antibiotic resistance genes (ARGs) are present in high numbers in antibiotic-naive infants' gut microbiomes, and infant mortality caused by resistant infections is high. The role of antibiotics in shaping the infant resistome has been studied, but there is limited knowledge on other factors that affect the antibiotic resistance burden of the infant gut. Objectives Our objectives were to determine the impact of early exposure to formula on the ARG load in neonates and infants born either preterm or full term. Our hypotheses were that diet causes a selective pressure that influences the microbial community of the infant gut, and formula exposure would increase the abundance of taxa that carry ARGs. Methods Cross-sectionally sampled gut metagenomes of 46 neonates were used to build a generalized linear model to determine the impact of diet on ARG loads in neonates. The model was cross-validated using neonate metagenomes gathered from public databases using our custom statistical pipeline for cross-validation. Results Formula-fed neonates had higher relative abundances of opportunistic pathogens such as Staphylococcus aureus, Staphylococcus epidermidis, Klebsiella pneumoniae, Klebsiella oxytoca, and Clostridioides difficile. The relative abundance of ARGs carried by gut bacteria was 69% higher in the formula-receiving group (fold change, 1.69; 95% CI: 1.12-2.55; P = 0.013; n = 180) compared to exclusively human milk-fed infants. The formula-fed infants also had significantly less typical infant bacteria, such as Bifidobacteria, that have potential health benefits. Conclusions The novel finding that formula exposure is correlated with a higher neonatal ARG burden lays the foundation that clinicians should consider feeding mode in addition to antibiotic use during the first months of life to minimize the proliferation of antibiotic-resistant gut bacteria in infants.Peer reviewe

FOAM (functional ontology assignments for metagenomes):a hidden markov model (HMM) database with environmental focus

A new functional gene database, FOAM (Functional Ontology Assignments for Metagenomes), was developed to screen environmental metagenomic sequence datasets. FOAM provides a new functional ontology dedicated to classify gene functions relevant to environmental microorganisms based on Hidden Markov Models (HMMs). Sets of aligned protein sequences (i.e. ‘profiles’) were tailored to a large group of target KEGG Orthologs (KOs) from which HMMs were trained. The alignments were checked and curated to make them specific to the targeted KO. Within this process, sequence profiles were enriched with the most abundant sequences available to maximize the yield of accurate classifier models. An associated functional ontology was built to describe the functional groups and hierarchy. FOAM allows the user to select the target search space before HMM-based comparison steps and to easily organize the results into different functional categories and subcategories. FOAM is publicly available at http://portal.nersc.gov/project/m1317/FOAM/

Evaluation of Swine-Specific PCR Assays Used for Fecal Source Tracking and Analysis of Molecular Diversity of Swine-Specific “Bacteroidales” Populations

In this study, we evaluated the specificity, distribution, and sensitivity of Prevotella strain-based (PF163 and PigBac1) and methanogen-based (P23-2) PCR assays proposed to detect swine fecal pollution in environmental waters. The assays were tested against 222 fecal DNA extracts derived from target and nontarget animal hosts and against 34 groundwater and 15 surface water samples from five different sites. We also investigated the phylogenetic diversity of 1,340 “Bacteroidales” 16S rRNA gene sequences derived from swine feces, swine waste lagoons, swine manure pits, and waters adjacent to swine operations. Most swine fecal samples were positive for the host-specific Prevotella-based PCR assays (80 to 87%), while fewer were positive with the methanogen-targeted PCR assay (53%). Similarly, the Prevotella markers were detected more frequently than the methanogen-targeted assay markers in waters historically impacted with swine fecal contamination. However, the PF163 PCR assay cross-reacted with 23% of nontarget fecal DNA extracts, although Bayesian statistics suggested that it yielded the highest probability of detecting pig fecal contamination in a given water sample. Phylogenetic analyses revealed previously unknown swine-associated clades comprised of clones from geographically diverse swine sources and from water samples adjacent to swine operations that are not targeted by the Prevotella assays. While deeper sequencing coverage might be necessary to better understand the molecular diversity of fecal Bacteroidales species, results of sequence analyses supported the presence of swine fecal pollution in the studied watersheds. Overall, due to nontarget cross amplification and poor geographic stability of currently available host-specific PCR assays, development of additional assays is necessary to accurately detect sources of swine fecal pollution