QPCR analysis of functional genes in iron-rich microbial mats at an active hydrothermal vent system Loihi Seamount, Hawaii

The recently discovered Zetaproteobacteria represent a novel class of Proteobacteria which oxidize Fe(II) to Fe(III), driving CO2 fixation at hydrothermal vents. These chemolithoautotrophs are the dominant bacterial population in iron-rich microbial mats, and represent a unique opportunity to investigate the connection between deep-sea geochemical processes and the dark microbial world. Zetaproteobacteria were first discovered at Loihi Seamount, located 35 km southeast off the big island of Hawaii and characterized by low-temperature diffuse hydrothermal venting. These vents are surrounded by luxuriant, iron-rich microbial mats that are colonized and often dominated by Zetaproteobacteria. Five novel non-degenerate QPCR assays were designed using sequence data derived from microbial iron-mat samples collected at Loihi in March 2013. Genes of interest were nifH, nirK, and arsC, associated with microbial nitrogen fixation, denitrification and arsenic detoxification, respectively. We also examined carbon fixation genes cbbM and aclB, which are indicators for the Calvin Benson Bassham (CBB) and reductive tricarboxylic acid (rTCA) cycles, respectively. All functional genes were found to be present at Loihi Seamount with the exception of nifH, which was undetectable with our method. Functional genes arsC and nirK were detected in all samples assayed, indicating that both arsenic detoxification and denitrification processes are likely occurring across all hydrothermal mat habitats. cbbM and aclB were also detectable in all samples assayed, indicating the co-occurence of these two modes of carbon fixation. T-RFLP analysis indicates that the communities in iron-rch mat samples collected in 2013 are very similar to one another. T-RFLP Group 1 had high Zetaproteobacteria abundance and low aclB relative to cbbM, indicating that the CBB cycle is the major mode of carbon fixation in Zetaproteobacteria-rich mat communities. T-RFLP Group 2 had low Zetaproteobacteria abundance and high aclB gene copy numbers, suggesting that the rTCA cycle is operating in non-Zetaproteobacteria taxa and plays an important role in carbon fixation in these communities. Based on these results, we conclude that aclB may be an important functional gene indicator of community composition. QPCR variance was explained by mat morphology but not temperature or sample site. Gene aclB was significantly associated with mat morphology, and may contribute to the significant relationship between the QPCR data and mat type. Fe(II) was significant with mat morphology. Geochemistry data was significantly associated with sample site and mat morphology, indicating that there is a range of chemistries in which these iron-rich microbial communities can thrive, and/or that the abundance of functional genes in these mat communities changes gradually in response to more dynamic chemical variation over time. Together, these QPCR assays constitute a ‘functional gene signature’ for iron mat samples across a broad array of temperatures, mat types, chemistries, and sampling sites in and around Pele’s Pit at Loihi Seamount

Clustering of Vibrio parahaemolyticus Isolates Using MLST and Whole-Genome Phylogenetics and Protein Motif Fingerprinting

Vibrio parahaemolyticus is a ubiquitous and abundant member of native microbial assemblages in coastal waters and shellfish. Though V. parahaemolyticus is predominantly environmental, some strains have infected human hosts and caused outbreaks of seafood-related gastroenteritis. In order to understand differences among clinical and environmental V. parahaemolyticus strains, we used high quality DNA sequencing data to compare the genomes of V. parahaemolyticus isolates (n = 43) from a variety of geographic locations and clinical and environmental sample matrices. We used phylogenetic trees inferred from multilocus sequence typing (MLST) and whole-genome (WG) alignments, as well as a novel classification and genome clustering approach that relies on protein motif fingerprints (MFs), to assess relationships between V. parahaemolyticus strains and identify novel molecular targets associated with virulence. Differences in strain clustering at more than one position were observed between the MLST and WG phylogenetic trees. The WG phylogeny had higher support values and strain resolution since isolates of the same sequence type could be differentiated. The MF analysis revealed groups of protein motifs that were associated with the pathogenic MLST type ST36 and a large group of clinical strains isolated from human stool. A subset of the stool and ST36-associated protein motifs were selected for further analysis and the motif sequences were found in genes with a variety of functions, including transposases, secretion system components and effectors, and hypothetical proteins. DNA sequences associated with these protein motifs are candidate targets for future molecular assays in order to improve surveys of pathogenic V. parahaemolyticus in the environment and seafood

Why are so many enteric pathogen infections asymptomatic? Pathogen and gut microbiome characteristics associated with diarrhea symptoms and carriage of diarrheagenic E. coli in northern Ecuador

ABSTRACTA high proportion of enteric infections, including those caused by diarrheagenic Escherichia coli (DEC), are asymptomatic for diarrhea. The factors responsible for the development of diarrhea symptoms, or lack thereof, remain unclear. Here, we used DEC isolate genome and whole stool microbiome data from a case–control study of diarrhea in Ecuador to examine factors associated with diarrhea symptoms accompanying DEC carriage. We investigated i) pathogen abundance, ii) gut microbiome characteristics, and iii) strain-level pathogen characteristics from DEC infections with diarrhea symptoms (symptomatic infections) and without diarrhea symptoms (asymptomatic infections). We also included data from individuals with and without diarrhea who were not infected with DEC (uninfected cases and controls). i) E. coli relative abundance in the gut microbiome was highly variable, but higher on-average in individuals with symptomatic compared to asymptomatic DEC infections. Similarly, the number and relative abundances of virulence genes in the gut were higher in symptomatic than asymptomatic DEC infections. ii) Measures of microbiome diversity were similar regardless of diarrhea symptoms or DEC carriage. Proteobacterial families that have been described as pathobionts were enriched in symptomatic infections and uninfected cases, whereas potentially beneficial taxa, including the Bacteroidaceae and Bifidobacteriaceae, were more abundant in individuals without diarrhea. An analysis of high-level gene functions recovered in metagenomes revealed that genes that were differentially abundant by diarrhea and DEC infection status were more abundant in symptomatic than asymptomatic DEC infections. iii) DEC isolates from symptomatic versus asymptomatic individuals showed no significant differences in virulence or accessory gene content, and there was no phylogenetic signal associated with diarrhea symptoms. Together, these data suggest signals that distinguish symptomatic from asymptomatic DEC infections. In particular, the abundance of E. coli, the virulence gene content of the gut microbiome, and the taxa present in the gut microbiome have an apparent role

Quantifying Enteropathogen Contamination along Chicken Value Chains in Maputo, Mozambique: A Multidisciplinary and Mixed-Methods Approach to Identifying High Exposure Settings

Background: Small-scale poultry production is widespread and increasing in low- and middle-income countries (LMICs). Exposure to enteropathogens in poultry feces increases the hazard of human infection and related sequela, and the burden of disease due to enteric infection in children < 5 y in particular is substantial. Yet, the containment and management of poultry-associated fecal waste in informal settings in LMICs is largely unregulated. Objectives: To improve the understanding of potential exposures to enteropathogens carried by chickens, we used mixed methods to map and quantify microbial hazards along production value chains among broiler, layer, and indigenous chickens in Maputo, Mozambique. Methods: To map and describe the value chains, we conducted 77 interviews with key informants working in locations where chickens and related products are sold, raised, and butchered. To quantify microbial hazards, we collected chicken carcasses (n = 75) and fecal samples (n = 136) from chickens along the value chain and assayed them by qPCR for the chicken-associated bacterial enteropathogens C. jejuni/coli and Salmonella spp. Results: We identified critical hazard points along the chicken value chains and identified management and food hygiene practices that contribute to potential exposures to chicken-sourced enteropathogens. We detected C. jejuni/coli in 84 (76%) of fecal samples and 52 (84%) of carcass rinses and Salmonella spp. in 13 (11%) of fecal samples and 16 (21%) of carcass rinses. Prevalence and level of contamination increased as chickens progressed along the value chain, from no contamination of broiler chicken feces at the start of the value chain to 100% contamination of carcasses with C. jejuni/coli at informal markets. Few hazard mitigation strategies were found in the informal sector. Discussion: High prevalence and concentration of C. jejuni/coli and Salmonella spp. contamination along chicken value chains suggests a high potential for exposure to these enteropathogens associated with chicken production and marketing processes in the informal sector in our study setting. We identified critical control points, such as the carcass rinse step and storage of raw chicken meat, that could be intervened in to mitigate risk, but regulation and enforcement pose challenges. This mixed-methods approach can also provide a model to understand animal value chains, sanitary risks, and associated exposures in other settings