Regina Lamendella

Comparative fecal metagenomics unveils unique functional capacity of the swine gut

BackgroundUncovering 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.ResultsAnalysis 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, Prevotella spp. dominated the swine fecal metagenome, while some genes associated with Treponema and Anareovibrio 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 Methanosarcina 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.ConclusionsThe 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.

Identification of bacterial populations in drinking water using 16S rRNA-based sequence analyses

Intracellular RNA is rapidly degraded in stressed cells and is more unstable outside of the cell than DNA. As a result, RNA-based methods have been suggested to study the active microbial fraction in environmental matrices. The aim of this study was to identify bacterial populations in drinking water by analyzing 16S rRNA-based clone libraries. Hollow-fiber ultrafiltration was used to concentrate bacterial communities from 40l of tap water collected at 12 different times during three different summer months from a single point-of-use. Total RNA was extracted from the microbial concentrates and used to develop 16S rRNA-based clone libraries. Phylogenetic analyses of 1231 partial 16S rRNA gene sequences showed that difficult-to-classify bacterial sequences were the most predominant clones, representing 57.6% of the sequences analyzed. Within these unclassified clades, most sequences were closely related to sequences retrieved from previous DNA- and RNA-based drinking water studies. Other bacterial groups represented in this study included Proteobacteria, cyanobacteria, Actinobacteria, Bacteroidetes, and Planctomycetes. Overall, the results suggest that these bacterial groups are amongst potentially active bacteria in drinking water. Diversity analyses of clones generated show that while overall diversity is similar amongst the different months, membership changes with respect to time. The results from this study further improve our understanding of the molecular diversity and bacterial population dynamics of drinking water microbial communities. Moreover, these results provide the sequence foundation for the development of molecular assays that target active drinking water bacteria.

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

ABSTRACT 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.

Identification of Naegleria fowleri in warm ground water aquifers.

The free-living amoeba Naegleria fowleri was identified as the etiological agent of primary amoebic meningoencephalitis that caused the deaths of two children in Peoria, Arizona, in autumn of 2002. It was suspected that the source of N. fowleri was the domestic water supply, which originates from ground water sources. In this study, ground water from the greater Phoenix Metropolitan area was tested for the presence of N. fowleri using a nested polymerase chain reaction approach. Phylogenetic analyses of 16S rRNA sequences of bacterial populations in the ground water were performed to examine the potential link between the presence of N. fowleri and bacterial groups inhabiting water wells. The results showed the presence of N. fowleri in five out of six wells sampled and in 26.6% of all ground water samples tested. Phylogenetic analyses showed that beta- and gamma-proteobacteria were the dominant bacterial populations present in the ground water. Bacterial community analyses revealed a very diverse community structure in ground water samples testing positive for N. fowleri.

Microbial Characterization of Drinking Water Systems Receiving Groundwater and Surface Water as the Primary Sources of Water

Earlier descriptions of water distribution systems (WDS) microbial communities have relied on culturing techniques. These techniques are known to be highly selective in nature, but more importantly, they tend to grossly underestimate the microbial diversity of most environments. The goal of this ongoing study is to compare the microbial composition of a WDS receiving different sources of water. To circumvent some of the problems associated with culture-based techniques, sequence analysis of total community 16S rDNA clone libraries was performed. A total of 260 16S rDNA clones were analyzed in this study. Sequence comparisons with existing databases revealed that α-Proteobacteria and Mycobacterium sp. represented nearly 43% and 48% of the total clones examined, respectively. Other bacterial groups identified included members of the genera Legionella, Pseudomonas, and Agrobacterium. Seventy percent of the clones analyzed in this study showed at least 97% sequence identity with sequences available in current databases. While α-Proteobacteria is a numerically dominant group in chlorinated drinking water systems, the abundance of mycobacterial sequences in this study indicates that there are significant differences in microbial community structure between the WDS analyzed. These differences could be attributed to differences in water treatment or receiving water sources between the distribution systems. While some of the genera identified in this study have been associated with some public health risks, it should be noted that analysis of 16S rDNA clones does not confirm the presence of pathogenic strains of any organisms identified in this study, as current methods for detection and identification require several steps including selective enrichment, isolation, and final confirmation via in vitro studies. Future studies will focus on expanding sequencing databases to more accurately characterize these potential differences. This paper was presented at the 8th Annual Water Distribution Systems Analysis Symposium which was held with the generous support of Awwa Research Foundation (AwwaRF).

STEPing to sustainability in a graduate K-12 partnership

The Science and Technology Enhancement Program (STEP) at the University of Cincinnati (UC) links university faculty, graduate students, secondary teachers, and secondary students in an effort to enhance student competence and promote awareness in STEM disciplines. Graduate-student Fellows gain valuable experience assisting and teaching in urban high schools. This NSF-funded project has generated hundreds of STEM lessons suitable for use by secondary school teachers. The sustainability of the program is the focus of this paper as viewed through the lessons, students, teachers, website, and the STEP Fellows. Over the past eight years, Project STEP has interacted with all of these groups to create and implement a successful community partnership. Conclusions are presented and concerns addressed. Project STEP students, teachers, and Fellows demonstrate increased awareness of the requirements and challenges of effective STEM education.