Jennifer Weidhaas

Identification of a Brevibacterium marker gene specific to poultry litter and development of a quantitative PCR assay.

Aim:  To identify a DNA sequence specific to a bacterium found in poultry litter that was indicative of faecal contamination by poultry sources.

Biodegradation of IMX-101 explosive formulation constituents: 2,4-dinitroanisole (DNAN), 3-nitro-1,2,4-triazol-5-one (NTO), and nitroguanidine.

Defense agencies are increasingly using insensitive munitions (IM) in place of explosives such as 2,4,6-trinitrotoluene. In this study simultaneous aerobic degradation of the IMX-101 formulation constituents 2,4-dinitroanisole (DNAN), 3-nitro-1,2,4-triazol-5-one (NTO), and nitroguanidine (NQ) was observed and degradation products were examined. Degradation products over four days of incubation included: nitrourea, 1,2-dihydro-3H-1,2,4-triazol-3-one, and 2,4-dinitrophenol. The enrichment culture maximum specific growth rate of 0.12h(-1) and half saturation constant of 288 mg L(-1) during degradation of IMX-101 as a sole nitrogen source suggest that enrichment culture growth kinetics may closely relate to those of other explosive and nitroaromatic compounds.

Correlation of Quantitative PCR for a Poultry-Specific Brevibacterium Marker Gene with Bacterial and Chemical Indicators of Water Pollution in a Watershed Impacted by Land Application of Poultry Litter

ABSTRACT The impact of fecal contamination from human and agricultural animal waste on water quality is a major public health concern. Identification of the dominant source(s) of fecal pollution in a watershed is necessary for assessing the safety of recreational water and protecting water resources. A field study was conducted using quantitative PCR (qPCR) for the 16S rRNA gene of Brevibacterium sp. LA35 to track feces-contaminated poultry litter in environmental samples. Based on sensitivity and specificity characteristics of the qPCR method, the Bayesian conditional probability that detection of the LA35 marker gene in a water sample represented a true-positive result was 93%. The markers covariance with fecal indicator bacteria (FIB) and metals associated with poultry litter was also assessed in litter, runoff, surface water, and groundwater samples. LA35 was detected in water and soil samples collected throughout the watershed, and its concentration covaried with concentrations of Escherichia coli, enterococci, As, Cu, P, and Zn. Significantly greater concentrations of FIB, As, Cu, P, and Zn were observed in edge-of-field runoff samples in which LA35 was detected, compared to samples in which it was not detected. Furthermore, As, Cu, P, and Zn concentrations covaried in environmental samples in which LA35 was detected and typically did not in samples in which the marker gene was not detected. The covariance of the poultry-specific LA35 marker gene with these known contaminants from poultry feces provides further evidence that it is a useful tool for assessing the impact of poultry-derived fecal pollution in environmental waters.

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.

Dissolution, sorption, and phytoremediation of IMX-101 explosive formulation constituents: 2,4-dinitroanisole (DNAN), 3-nitro-1,2,4-triazol-5-one (NTO), and nitroguanidine.

The insensitive munition, IMX-101 approved for use in the USA, contains 2,4-dinitroanisole (DNAN), 3-nitro-1,2,4-triazol-5-one (NTO), and nitroguanidine (NQ) and is designed to be less sensitive to shock and sympathetic detonation. Given the estimated future use of IMX-101, an understanding of IMX-101 constituent attenuation mechanisms on testing and training ranges is needed. Studies were conducted to determine (1) the rates of IMX-101 fragment dissolution during simulated rainfall, (2) DNAN and NTO soil sorption coefficients, (3) ability of grasses to germinate in and phytoremediate IMX-101 contaminated soil, and (4) effect of the addition of IMX-101 degrading enrichment cultures on phytoremediation. The IMX-101 particles were found to dissolve slowly under simulated rainfall conditions with NQ and NTO dissolving first, leaving DNAN crystals. DNAN and NTO sorption to soils fit Freundlich isotherms and limited desorption was observed. DNAN and NQ were shown to be taken up into the roots and shoots of a mixture of big bluestem grass (Andropogon gerardii), Nash Indiangrass (Sorghastrum nutans), and switchgrass (Panicum virgatum) during phytoremediation of soils contaminated with up to 50 mg kg(-1) IMX-101. Complete degradation of IMX-101 to below detection limits occurred over 225 days. The addition of an IMX-101 degrading enrichment culture to the treatments significantly increased the root and shoot mass.

Kinetics and microbial ecology of batch sulfidogenic bioreactors for co-treatment of municipal wastewater and acid mine drainage

The kinetics and microbial ecology in sulfidogenic bioreactors used in a novel two-stage process for co-treatment of acid mine drainage (AMD) and municipal wastewater (MWW) were investigated. Michaelis-Menten modeling of COD oxidation by sulfate reducing bacteria (SRB) (Vmax=0.33mgL(-1)min(-1), Km=4.3mgL(-1)) suggested that the Vmax can be reasonably achieved given the typical COD values in MWW and anticipated mixing with AMD. Non-competitive inhibition modeling (Ki=6.55mgL(-1)) indicated that excessive iron level should be avoided to limit its effects on SRB. The COD oxidation rate was positively correlated to COD/sulfate ratio and SRB population, as evidenced by dsrA gene copies. Phylogenetic analysis revealed diverse microbial communities dominated by sulfate reducing delta-proteobacteria. Microbial community and relative quantities of SRB showed significant differences under different COD/sulfate ratios (0.2, 1 and 2), and the highest dsrA gene concentration and most complex microbial diversity were observed under COD/sulfate ratio 2. Major species were associated with Desulfovirga, Desulfobulbus, Desulfovibrio, and Syntrophus sp. The reported COD kinetics, SRB abundances and the phylogenetic profile provide insights into the co-treatment process and help identify the parameters of concerns for such technology development.

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.

A new method for tracking poultry litter in the Potomac Basin headwaters of West Virginia

To validate the distribution of a poultry litter‐specific marker gene in faecally contaminated environmental waters of an intensive poultry litter rearing region.

Run-off studies demonstrate parallel transport behaviour for a marker of poultry fecal contamination and Staphylococcus aureus

To determine whether poultry litter marker gene LA35 is correlated with pathogens and fecal indicator bacteria (FIB) in run‐off from poultry litter‐amended plots.

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.