Standard Protocols for the 3 MST Markers Evaluated

This protocol outlines the procedure developed for the EPA Gulf of Mexico grant to Harwood, Lepo and Wang to process environmental water samples via DNA extraction from membrane filters. DNA is then used in PCR-based microbial source tracking assays for human-associated Bacteroides (HBac), M. smithii (Msmithii) and human polyomavirus BK and JC (HPyVs) targets

Year 1 Report

This report covers the project from its inception (08/03/07) through May 2008

Year 2 Report

This report covers the project from the end of the preliminary year 1 report, May 31, 2008 through May 31, 2009

Project Overview

The overarching goal of this study is to identify useful method(s) for detecting human sewage pollution in Gulf of Mexico waters and to produce protocols that are readily transferable across laboratories (standard operating procedures), such that these microbial source tracking methods can be used by laboratories across the Gulf of Mexico states. This project is a collaborative effort among several universities, including the University of South Florida, University of West Florida and University of Southern Mississippi (co‐principal investigators) as well as Texas A&M University at Corpus Christi, Texas A&M University at El Paso and Nicholls State University (collaborators)

Year 3 Final Report

This report covers the project from the end of the preliminary year 2 report, June 1, 2009 through August 31, 2010. It also provides a synopsis of the accomplishments for the duration of the grant (3 years)

Multiple approaches to microbial source tracking in tropical northern Australia

© The Author(s), 2014. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in MicrobiologyOpen 3 (2014): 860–874, doi:10.1002/mbo3.209.Microbial source tracking is an area of research in which multiple approaches are used to identify the sources of elevated bacterial concentrations in recreational lakes and beaches. At our study location in Darwin, northern Australia, water quality in the harbor is generally good, however dry-season beach closures due to elevated Escherichia coli and enterococci counts are a cause for concern. The sources of these high bacteria counts are currently unknown. To address this, we sampled sewage outfalls, other potential inputs, such as urban rivers and drains, and surrounding beaches, and used genetic fingerprints from E. coli and enterococci communities, fecal markers and 454 pyrosequencing to track contamination sources. A sewage effluent outfall (Larrakeyah discharge) was a source of bacteria, including fecal bacteria that impacted nearby beaches. Two other treated effluent discharges did not appear to influence sites other than those directly adjacent. Several beaches contained fecal indicator bacteria that likely originated from urban rivers and creeks within the catchment. Generally, connectivity between the sites was observed within distinct geographical locations and it appeared that most of the bacterial contamination on Darwin beaches was confined to local sources

Identification of Host-Specific Bacteroidales 16S rDNA Sequences from Human Sewage and Ruminant Feces

The need to identify the source of fecal contamination of water has led to the development of various fecal source identification methods, a field known as microbial source tracking (MST). One promising method of MST focuses on fecal members of the order Bacteroidales, some of which exhibit a high degree of host-specificity. In order to identify host-specific Bacteroidales genetic markers, a ∼1060 bp section of Bacteroidales 16S rDNA was amplified from human sewage (n = 6), and bovine (n = 6) and ovine fecal (n = 5) samples and used for the generation of three clone libraries. Phylogenetic analysis of sequences from the three clone libraries revealed that the Bacteroidales species found in both human sewage and bovine and ovine feces were a highly diverse group of organisms, many of which were not represented by previously characterised 16S rDNA. Ovine and bovine feces appear to host similar populations of Bacteroidales species and these species were more diverse and less closely related to cultivated species than the Bacteroidales population found in human sewage. Species of Bacteroidales from the ruminant and human feces formed isolated clusters containing putatively host-specific sequences. These sequences were subsequently exploited for the design of host-specific primers which were used in MST studies

Geography and Location Are the Primary Drivers of Office Microbiome Composition.

In the United States, humans spend the majority of their time indoors, where they are exposed to the microbiome of the built environment (BE) they inhabit. Despite the ubiquity of microbes in BEs and their potential impacts on health and building materials, basic questions about the microbiology of these environments remain unanswered. We present a study on the impacts of geography, material type, human interaction, location in a room, seasonal variation, and indoor and microenvironmental parameters on bacterial communities in offices. Our data elucidate several important features of microbial communities in BEs. First, under normal office environmental conditions, bacterial communities do not differ on the basis of surface material (e.g., ceiling tile or carpet) but do differ on the basis of the location in a room (e.g., ceiling or floor), two features that are often conflated but that we are able to separate here. We suspect that previous work showing differences in bacterial composition with surface material was likely detecting differences based on different usage patterns. Next, we find that offices have city-specific bacterial communities, such that we can accurately predict which city an office microbiome sample is derived from, but office-specific bacterial communities are less apparent. This differs from previous work, which has suggested office-specific compositions of bacterial communities. We again suspect that the difference from prior work arises from different usage patterns. As has been previously shown, we observe that human skin contributes heavily to the composition of BE surfaces. IMPORTANCE Our study highlights several points that should impact the design of future studies of the microbiology of BEs. First, projects tracking changes in BE bacterial communities should focus sampling efforts on surveying different locations in offices and in different cities but not necessarily different materials or different offices in the same city. Next, disturbance due to repeated sampling, though detectable, is small compared to that due to other variables, opening up a range of longitudinal study designs in the BE. Next, studies requiring more samples than can be sequenced on a single sequencing run (which is increasingly common) must control for run effects by including some of the same samples in all of the sequencing runs as technical replicates. Finally, detailed tracking of indoor and material environment covariates is likely not essential for BE microbiome studies, as the normal range of indoor environmental conditions is likely not large enough to impact bacterial communities