Bina Nayak

The importance of sediment and submerged aquatic vegetation as potential habitats for persistent strains of enterococci in a subtropical watershed

Recent evidence of extended survival of fecal indicator bacteria in sediments and submerged aquatic vegetation (SAV) has raised concerns about using indicator bacteria to reliably detect fecal contamination. We monitored enterococci densities and population structure in water, sediment and SAV simultaneously at sites across a subtropical watershed (Tampa Bay, FL, USA) over one year to determine the extent to which each matrix serves as a potential reservoir of enterococci. SAV harbored significantly higher mean densities of enterococci than sediments, which harbored higher densities than water. Mean enterococci densities were also greater at sites located further upstream in the watershed. The population structure assessed by BOX-PCR genotyping was relatively dissimilar in each sample, although some similarities among samples suggested grouping by location. Strain diversity ranged from very high to negligible, with lowest overall diversity in lake samples taken during the summer. Several strains were highly abundant and cosmopolitan (found across sites, seasons, and matrices) and were identified by 16S rRNA gene sequencing as the Enterococcus species casseliflavus, faecalis, faecium, hirae, and mundtii. The proportional dominance of certain strains suggests the existence of persistent and possibly naturalized indicator bacteria populations that are not directly related to pollution events.

Microbial population dynamics in laboratory-scale solid waste bioreactors in the presence or absence of biosolids.

Aims:  Decomposition of solid waste is microbially mediated, yet little is known about the associated structure and temporal changes in prokaryotic communities. Bioreactors were used to simulate landfill conditions and archaeal and bacterial community development in leachate was examined over 8 months.

A Novel Microbial Source Tracking Microarray for Pathogen Detection and Fecal Source Identification in Environmental Systems

Pathogen detection and the identification of fecal contamination sources are challenging in environmental waters. Factors including pathogen diversity and ubiquity of fecal indicator bacteria hamper risk assessment and remediation of contamination sources. A custom microarray targeting pathogens (viruses, bacteria, protozoa), microbial source tracking (MST) markers, and antibiotic resistance genes was tested against DNA obtained from whole genome amplification (WGA) of RNA and DNA from sewage and animal (avian, cattle, poultry, and swine) feces. Perfect and mismatch probes established the specificity of the microarray in sewage, and fluorescence decrease of positive probes over a 1:10 dilution series demonstrated semiquantitative measurement. Pathogens, including norovirus, Campylobacter fetus, Helicobacter pylori, Salmonella enterica, and Giardia lamblia were detected in sewage, as well as MST markers and resistance genes to aminoglycosides, beta-lactams, and tetracycline. Sensitivity (percentage true positives) of MST results in sewage and animal waste samples (21-33%) was lower than specificity (83-90%, percentage of true negatives). Next generation DNA sequencing revealed two dominant bacterial families that were common to all sample types: Ruminococcaceae and Lachnospiraceae. Five dominant phyla and 15 dominant families comprised 97% and 74%, respectively, of sequences from all fecal sources. Phyla and families not represented on the microarray are possible candidates for inclusion in subsequent array designs.

Comparison of Genotypic and Phylogenetic Relationships of Environmental Enterococcus Isolates by BOX-PCR Typing and 16S rRNA Gene Sequencing

ABSTRACT Environmental Enterococcus spp. were compared by BOX-PCR genotyping and 16S rRNA gene sequencing to clarify the predictive relationship of BOX-PCR fingerprints to species designation. BOX-PCR and 16S rRNA gene relationships agreed for 77% of strains. BOX-PCR provided superior intraspecies discrimination but incorrectly identified some strains to the species level and divided some species into multiple groups.

Lysimeter comparison of the role of waste characteristics in the formation of mineral deposits in leachate drainage systems

A common operational problem in leachate collection systems is clogging due to the formation of deposits within pore spaces and collection pipes. The role of co-disposal of municipal solid waste (MSW) and combustion residues from waste-to-energy (WTE) facilities in clogging is evaluated in this paper. Five parallel lysimeters were operated in monofill or co-disposal mode using MSW, WTE combustion residues, and water/wastewater treatment byproducts. Leachate was applied to each lysimeter to simulate sequential flooding and draining and leachates were characterized over a 7-month period. Waste composition and the presence/absence of biological activity influenced the redox potential, pH, and alkalinity, which impacted the rate and extent of biological degradation and chemical solubility. Calcium carbonate was identified as the most abundant chemical precipitate. Leachates from ash mono-fills were highly alkaline (pH > 11) and had higher ionic strength due to relatively higher levels of calcium and other minerals, while carbonate levels were limited due to the lack of biological activity. The MSW monofill generated leachates with high levels of biological activity, lower concentrations of calcium, and a rich carbonate system. Co-disposal of MSW, combustion and treatment process residues generated leachates that were not limited in either calcium or carbonate, creating ideal conditions for formation of precipitates.

Ultrafiltration and Microarray for Detection of Microbial Source Tracking Marker and Pathogen Genes in Riverine and Marine Systems

ABSTRACT Pathogen identification and microbial source tracking (MST) to identify sources of fecal pollution improve evaluation of water quality. They contribute to improved assessment of human health risks and remediation of pollution sources. An MST microarray was used to simultaneously detect genes for multiple pathogens and indicators of fecal pollution in freshwater, marine water, sewage-contaminated freshwater and marine water, and treated wastewater. Dead-end ultrafiltration (DEUF) was used to concentrate organisms from water samples, yielding a recovery efficiency of >95% for Escherichia coli and human polyomavirus. Whole-genome amplification (WGA) increased gene copies from ultrafiltered samples and increased the sensitivity of the microarray. Viruses (adenovirus, bocavirus, hepatitis A virus, and human polyomaviruses) were detected in sewage-contaminated samples. Pathogens such as Legionella pneumophila, Shigella flexneri, and Campylobacter fetus were detected along with genes conferring resistance to aminoglycosides, beta-lactams, and tetracycline. Nonmetric dimensional analysis of MST marker genes grouped sewage-spiked freshwater and marine samples with sewage and apart from other fecal sources. The sensitivity (percent true positives) of the microarray probes for gene targets anticipated in sewage was 51 to 57% and was lower than the specificity (percent true negatives; 79 to 81%). A linear relationship between gene copies determined by quantitative PCR and microarray fluorescence was found, indicating the semiquantitative nature of the MST microarray. These results indicate that ultrafiltration coupled with WGA provides sufficient nucleic acids for detection of viruses, bacteria, protozoa, and antibiotic resistance genes by the microarray in applications ranging from beach monitoring to risk assessment.

LA35 Poultry Fecal Marker Persistence Is Correlated with That of Indicators and Pathogens in Environmental Waters.

ABSTRACT Disposal of fecally contaminated poultry litter by land application can deliver pathogens and fecal indicator bacteria (FIB) into receiving waters via runoff. While water quality is regulated by FIB enumeration, FIB testing provides inadequate information about contamination source and health risk. This microbial source tracking (MST) study compared the persistence of the Brevibacterium sp. strain LA35 16S rRNA gene (marker) for poultry litter with that of pathogens and FIB under outdoor, environmentally relevant conditions in freshwater, marine water, and sediments over 7 days. Salmonella enterica, Campylobacter jejuni, Campylobacter coli, Bacteroidales, and LA35 were enumerated by quantitative PCR (qPCR), and Enterococcus spp. and E. coli were quantified by culture and qPCR. Unlike the other bacteria, C. jejuni was not detectable after 48 h. Bacterial levels in the water column consistently declined over time and were highly correlated among species. Survival in sediments ranged from a slow decrease over time to growth, particularly in marine microcosms and for Bacteroidales. S. enterica also grew in marine sediments. Linear decay rates in water (k) ranged from −0.17 day−1 for LA35 to −3.12 day−1 for C. coli. LA35 levels correlated well with those of other bacteria in the water column but not in sediments. These observations suggest that, particularly in the water column, the fate of LA35 in aquatic environments is similar to that of FIB, C. coli, and Salmonella, supporting the hypothesis that the LA35 marker gene can be a useful tool for evaluating the impact of poultry litter on water quality and human health risk.

Vancomycin resistant enterococci and bacterial community structure following a sewage spill into an aquatic environment

ABSTRACT Sewage spills can release antibiotic-resistant bacteria into surface waters, contributing to environmental reservoirs and potentially impacting human health. Vancomycin-resistant enterococci (VRE) are nosocomial pathogens that have been detected in environmental habitats, including soil, water, and beach sands, as well as wildlife feces. However, VRE harboring vanA genes that confer high-level resistance have infrequently been found outside clinical settings in the United States. This study found culturable Enterococcus faecium harboring the vanA gene in water and sediment for up to 3 days after a sewage spill, and the quantitative PCR (qPCR) signal for vanA persisted for an additional week. Culturable levels of enterococci in water exceeded recreational water guidelines for 2 weeks following the spill, declining about five orders of magnitude in sediments and two orders of magnitude in the water column over 6 weeks. Analysis of bacterial taxa via 16S rRNA gene sequencing showed changes in community structure through time following the sewage spill in sediment and water. The spread of opportunistic pathogens harboring high-level vancomycin resistance genes beyond hospitals and into the broader community and associated habitats is a potential threat to public health, requiring further studies that examine the persistence, occurrence, and survival of VRE in different environmental matrices. IMPORTANCE Vancomycin-resistant enterococci (VRE) are harmful bacteria that are resistant to the powerful antibiotic vancomycin, which is used as a last resort against many infections. This study followed the release of VRE in a major sewage spill and their persistence over time. Such events can act as a means of spreading vancomycin-resistant bacteria in the environment, which can eventually impact human health.

Evidence for Extraintestinal Growth of Bacteroidales Originating from Poultry Litter

ABSTRACT Water quality monitoring techniques that target microorganisms in the order Bacteroidales are potential alternatives to conventional methods for detection of fecal indicator bacteria. Bacteroidales and members of the genus Bacteroides have been the focus of microbial source tracking (MST) investigations for discriminating sources of fecal pollution (e.g., human or cattle feces) in environmental waters. For accurate source apportionment to occur, one needs to understand both the abundance of Bacteroides in host feces and the survival of these host-associated microbial markers after deposition in the environment. Studies were undertaken to evaluate the abundance, persistence, and potential for growth of Bacteroidales originating from poultry litter under oxic and anoxic environmental conditions. Bacteroidales abundance, as determined by quantitative PCR (qPCR) with GenBac primers and probe, increased 2 to 5 log gene copies ml−1 and 2 log gene copies g litter−1 under most conditions during incubation of poultry litter in a variety of laboratory microcosm and field mesocosm studies. DNA sequencing of the Bacteroidales organisms in the litter identified taxa with sequences corresponding exactly to the GenBac primer and probe sequences and that were closely related to Bacteroides uniformis, B. ovatus, and B. vulgatus. These results suggest that MST studies using qPCR methods targeting Bacteroidales in watersheds that are affected by poultry litter should be interpreted cautiously. Growth of Bacteroidales originating from poultry litter in environmental waters may occur while Bacteroidales growth from other fecal sources declines, thus confounding the interpretation of MST results.

Determination of wild animal sources of fecal indicator bacteria by microbial source tracking (MST) influences regulatory decisions

Fecal indicator bacteria (FIB) are used to assess fecal pollution levels in surface water and are among the criteria used by regulatory agencies to determine water body impairment status. While FIB provide no information about pollution source, microbial source tracking (MST) does, which contributes to more direct and cost effective remediation efforts. We studied a watershed in Florida managed for wildlife conservation that historically exceeded the state regulatory guideline for fecal coliforms. We measured fecal coliforms, enterococci, a marker gene for avian feces (GFD), and a marker gene for human-associated Bacteroides (HF183) in sediment, vegetation, and water samples collected monthly from six sites over two years to: 1) assess the influence of site, temporal factors, and habitat (sediment, vegetation, and water) on FIB and MST marker concentrations, 2) test for correlations among FIB and MST markers, and 3) determine if avian feces and/or human sewage contributed to FIB levels. Sediment and vegetation had significantly higher concentrations of FIB and GFD compared to water and thus may serve as microbial reservoirs, providing unreliable indications of recent contamination. HF183 concentrations were greatest in water samples but were generally near the assay limit of detection. HF183-positive results were attributed to white-tailed deer (Odocoileus virginianus) feces, which provided a false indication of human sewage in this water body. FIB and GFD were positively correlated while FIB and HF183 were negatively correlated. We demonstrated that birds, not sewage, were the main source of FIB, thus avoiding implementation of a total maximum daily load program (TMDL). Our results demonstrate that the concomitant use of FIB and MST can improve decision-making and provide direction when water bodies are impaired, and provides a strategy for natural source exclusion in water bodies impacted by wild animal feces.