Biomedical subjects

Jorge W Santo Domingo

Publications and source records attributed to Jorge W Santo Domingo.

10 recordsLinked to original sources

PubMed2007

Identification of bacterial DNA markers for the detection of human fecal pollution in water.

We used genome fragment enrichment and bioinformatics to identify several microbial DNA sequences with high potential for use as markers in PCR assays for detection of human fecal contamination in water. Following competitive solution-phase hybridization of total DNA from human and pig fecal samples, 351 plasmid clones were sequenced and were determined to define 289 different genomic DNA regions. These putative human-specific fecal bacterial DNA sequences were then analyzed by dot blot hybridization, which confirmed that 98% were present in the source human fecal microbial community and absent from the original pig fecal DNA extract. Comparative sequence analyses of these sequences suggested that a large number (43.5%) were predicted to encode bacterial secreted or surface-associated proteins. Deoxyoligonucleotide primers capable of annealing to a subset of 26 of the candidate sequences predicted to encode factors involved in interactions with host cells were then used in the PCR and did not amplify markers in DNA from any additional pig fecal specimens. These 26 PCR assays exhibited a range of specificity in tests with 11 other animal sources, with more than half amplifying markers only in specimens from dogs or cats. Four assays were more specific, detecting markers only in specimens from humans, including those from 18 different human populations examined. We then demonstrated the potential utility of these assays by using them to detect human fecal contamination in several impacted watersheds.

Animals↗

PubMed2006

Assessment of fecal pollution sources in a small northern-plains watershed using PCR and phylogenetic analyses of Bacteroidetes 16S rRNA gene.

We evaluated the efficacy, sensitivity, host-specificity, and spatial/temporal dynamics of human- and ruminant-specific 16S rRNA gene Bacteroidetes markers used to assess the sources of fecal pollution in a fecally impacted watershed. Phylogenetic analyses of 1271 fecal and environmental 16S rRNA gene clones were also performed to study the diversity of Bacteroidetes in this watershed. The host-specific assays indicated that ruminant feces were present in 28-54% of the water samples and in all sampling seasons, with increasing frequency in downstream sites. The human-targeted assays indicated that only 3-5% of the water samples were positive for human fecal signals, although a higher percentage of human-associated signals (19-24%) were detected in sediment samples. Phylogenetic analysis indicated that 57% of all water clones clustered with yet-to-be-cultured Bacteroidetes species associated with sequences obtained from ruminant feces, further supporting the prevalence of ruminant contamination in this watershed. However, since several clusters contained sequences from multiple sources, future studies need to consider the potential cosmopolitan nature of these bacterial populations when assessing fecal pollution sources using Bacteroidetes markers. Moreover, additional data is needed in order to understand the distribution of Bacteroidetes host-specific markers and their relationship to water quality regulatory standards.

Animals↗

PubMed2006

Use of competitive DNA hybridization to identify differences in the genomes of bacteria.

Although recent technological advances in DNA sequencing and computational biology now allow scientists to compare entire microbial genomes, comparisons of closely related bacterial species and individual isolates by whole-genome sequencing approaches remains prohibitively expensive for most laboratories. Here we report the development and testing of a biochemical approach for targeted sequencing of only those chromosomal regions that differ between two DNA preparations. The method, designated GFE (genome fragment enrichment) uses competitive solution hybridization and positive selection to obtain genomic DNA fragments that are present in one pool of fragments but not another. Repeated comparisons of the genomes of Enterococcus faecalis and E. faecium led to the identification of 225 putative genome-specific DNA fragments. Species and strain variations within these fragments were confirmed by both experimental and bioinformatic analyses. The E. faecalis genome-specific sequences identified included both a preponderance of those predicted to encode surface-exposed proteins, as well as several previously described unique marker regions embedded within highly conserved rrn operons. The GFE strategy we describe efficiently identified genomic differences between two enterococcal genomes, and will be widely applicable for studying genetic variation among closely related bacterial species.

DNA, Bacterial↗

PubMed2006

Development and applications of microbial ecogenomic indicators for monitoring water quality: report of a workshop assessing the state of the science, research needs and future directions.

This article brings forth recommendations from a workshop sponsored by the U.S. Environmental Protection Agency's Science to Achieve Results (STAR) and Environmental Monitoring and Assessment (EMAP) Programs and by the Council of State Governments, held during May 2002 in Kansas City, Kansas. The workshop assembled microbial ecologists and environmental scientists to determine what research and science is needed to bring existing molecular biological approaches and newer technologies arising from microbial genomic research into environmental monitoring and water quality assessments. Development of genomics and proteomics technologies for environmental science is a very new area having potential to improve environmental water quality assessments. The workshop participants noted that microbial ecologists are already using molecular biological methods well suited for monitoring and water quality assessments and anticipate that genomics-enabled technologies could be made available for monitoring within a decade. Recommendations arising from the workshop include needs for (i) identification of informative microbial gene sequences, (ii) improved understandings of linkages between indicator taxa, gene expression and environmental condition, (iii) technological advancements towards field application, and (iv) development of the appropriate databases.

Animals↗

PubMed2006

Identification of active bacterial communities in a model drinking water biofilm system using 16S rRNA-based clone libraries.

Recent phylogenetic studies have used DNA as the target molecule for the development of environmental 16S rRNA gene clone libraries. As DNA may persist in the environment, DNA-based libraries cannot be used to identify metabolically active bacteria in water systems. In this study, an annular reactor was used to generate model drinking water biofilms grown on polycarbonate slides. High-quality RNA was extracted from 2-month-old biofilms and used to generate 16S rRNA-based clones. Sequencing analyses of 16S rRNA-based clones suggested that the active bacterial fraction consisted of a few dominant bacterial groups related to Nevskia ramosa and to uncultured bacteria. Several of these bacterial groups were closely related to clones characterized in a DNA-based clone library also generated in this study. Altogether, these results suggest that some of the predominant drinking water bacteria identified using DNA-based techniques are indeed active.

Bacteria↗

PubMed2006

Competitive metagenomic DNA hybridization identifies host-specific microbial genetic markers in cow fecal samples.

Several PCR methods have recently been developed to identify fecal contamination in surface waters. In all cases, researchers have relied on one gene or one microorganism for selection of host-specific markers. Here we describe the application of a genome fragment enrichment (GFE) method to identify host-specific genetic markers from fecal microbial community DNA. As a proof of concept, bovine fecal DNA was challenged against a porcine fecal DNA background to select for bovine-specific DNA sequences. Bioinformatic analyses of 380 bovine enriched metagenomic sequences indicated a preponderance of Bacteroidales-like regions predicted to encode membrane-associated and secreted proteins. Oligonucleotide primers capable of annealing to select Bacteroidales-like bovine GFE sequences exhibited extremely high specificity (>99%) in PCR assays with total fecal DNAs from 279 different animal sources. These primers also demonstrated a broad distribution of corresponding genetic markers (81% positive) among 148 different bovine sources. These data demonstrate that direct metagenomic DNA analysis by the competitive solution hybridization approach described is an efficient method for identifying potentially useful fecal genetic markers and for characterizing differences between environmental microbial communities.

Animals↗

PubMed2005

Population diversity in model potable water biofilms receiving chlorine or chloramine residual.

Most water utilities use chlorine or chloramine to produce potable water. These disinfecting agents react with water to produce residual oxidants within a water distribution system (WDS) to control bacterial growth. While monochloramine is considered more stable than chlorine, little is known about the effect it has on WDS biofilms. Community structure of 10-week old WDS biofilms exposed to disinfectants was assessed after developing model biofilms from unamended distribution water. Four biofilm types were developed on polycarbonate slides within annular reactors while receiving chlorine, chloramine, or inactivated disinfectant residual. Eubacteria were identified through 16S rDNA sequence analysis. The model WDS biofilm exposed to chloramine mainly contained Mycobacterium and Dechloromonas sequences, while a variety of alpha- and additional beta-proteobacteria dominated the 16S rDNA clone libraries in the other three biofilms. Additionally, bacterial clones distantly related to Legionella were found in one of the biofilms receiving water with inactivated chlorine residual. The biofilm reactor receiving chloraminated water required increasing amounts of disinfectant after 2 weeks to maintain chlorine residual. In contrast, free chlorine residual remained steady in the reactor that received chlorinated water. The differences in bacterial populations of potable water biofilms suggest that disinfecting agents can influence biofilm development. These results also suggest that biofilm communities in distribution systems are capable of changing in response to disinfection practices.

Bacteria↗

PubMed2005

Host distributions of uncultivated fecal Bacteroidales bacteria reveal genetic markers for fecal source identification.

The purpose of this study was to examine host distribution patterns among fecal bacteria in the order Bacteroidales, with the goal of using endemic sequences as markers for fecal source identification in aquatic environments. We analyzed Bacteroidales 16S rRNA gene sequences from the feces of eight hosts: human, bovine, pig, horse, dog, cat, gull, and elk. Recovered sequences did not match database sequences, indicating high levels of uncultivated diversity. The analysis revealed both endemic and cosmopolitan distributions among the eight hosts. Ruminant, pig, and horse sequences tended to form host- or host group-specific clusters in a phylogenetic tree, while human, dog, cat, and gull sequences clustered together almost exclusively. Many of the human, dog, cat, and gull sequences fell within a large branch containing cultivated species from the genus Bacteroides. Most of the cultivated Bacteroides species had very close matches with multiple hosts and thus may not be useful targets for fecal source identification. A large branch containing cultivated members of the genus Prevotella included cloned sequences that were not closely related to cultivated Prevotella species. Most ruminant sequences formed clusters separate from the branches containing Bacteroides and Prevotella species. Host-specific sequences were identified for pigs and horses and were used to design PCR primers to identify pig and horse sources of fecal pollution in water. The primers successfully amplified fecal DNAs from their target hosts and did not amplify fecal DNAs from other species. Fecal bacteria endemic to the host species may result from evolution in different types of digestive systems.

Animals↗

PubMed2003

Real-time PCR method to detect Enterococcus faecalis in water.

A 16S rDNA real-time PCR method was developed to detect Enterococcus faecalis in water samples. The dynamic range for cell detection spanned five logs and the detection limit was determined to be 6 cfu/reaction. The assay was capable of detecting E. faecalis cells added to biofilms from a simulator of a water distribution system and in freshwater samples. Nucleic acid extraction was not required, permitting the detection of E. faecalis cells in less than 3 h.

DNA, Bacterial↗

PubMed2002

Microbial source tracking: state of the science.

Although water quality of the Nation's lakes, rivers and streams has been monitored for many decades and especially since the passage of the Clean Water Act in 1972, many still do not meet the Act's goal of "fishable and swimmable". While waterways can be impaired in numerous ways, the protection from pathogenic microbe contamination is most important for waters used for human recreation, drinking water and aquaculture. Typically, monitoring methods used for detecting potential pathogenic microorganisms in environmental waters are based upon cultivation and enumeration of fecal indicator bacteria (i.e. fecal coliforms, E. coli, and fecal enterococci). Currently, there is increasing interest in the potential for molecular fingerprinting methods to be used not only for detection but also for identification of fecal contamination sources. Molecular methods have been applied to study the microbial ecology of environmental systems for years and are now being applied to help improve our waters by identifying problem sources and determining the effect of implemented remedial solutions. Management and remediation of water pollution would be more cost-effective if the correct sources could be identified. This review provides an outline of the main methods that either have been used or have been suggested for use in microbial source tracking and some of the limitations associated with those methods.

Electrophoresis, Gel, Pulsed-Field↗