Emily Viau

Pyrosequencing of the 16S rRNA gene to reveal bacterial pathogen diversity in biosolids

Given the potential for a variety of bacterial pathogens to occur in variably stabilized sewage sludge (biosolids), an understanding of pathogen diversity and abundance is necessary for accurate assessment of infective risk when these products are land applied. 16S rDNA was PCR amplified from genomic DNA extracted from municipal wastewater residuals (mesophilic- and thermophilic-phased anaerobic digestion (MAD and TPAD), composting (COM)), and agricultural soil (SOIL), and these amplicons were sequenced using massively parallel pyrosequencing technology. Resulting libraries contained an average of 30,893 16S rDNA sequences per sample with an average length of 392 bases. FASTUNIFRAC-based comparisons of population phylogenetic distance demonstrated similarities between the populations of different treatment plants performing the same stabilization method (e.g. different MAD samples), and population differences among samples from different biosolids stabilization methods (COM, MAD, and TPAD). Based on a 0.03 Jukes-Cantor distance to 80 potential bacterial pathogens, all samples contained pathogens and enrichment ranged from 0.02% to 0.1% of sequences. Most (61%) species identified were opportunistic pathogens of the genera Clostridium and Mycobacterium. As risk sciences continue to evolve to address scenarios that include multiple pathogen exposure, the analysis described here can be used to determine the diversity of pathogens in an environmental sample. This work provides guidance for prioritizing subsequent culturable and quantitative analysis, and for the first time, ensuring that potentially significant pathogens are not left out of risk estimations.

Survey of Wastewater Indicators and Human Pathogen Genomes in Biosolids Produced by Class A and Class B Stabilization Treatments

ABSTRACT Accurate modeling of the infectious aerosol risk associated with the land application of biosolids requires an in-depth knowledge of the magnitudes and changes in pathogen concentrations for a variety of class A and class B stabilization methods. The following survey used quantitative PCR (qPCR) and culture assays to detect environmentally resistant bacterial and viral pathogens and biosolid indicator organisms for 36 biosolid grab samples. Biosolids were collected from 14 U.S. states and included 16 class B mesophilic anaerobic digestion (MAD) samples and 20 class A biosolid samples from temperature-phased anaerobic digestion (TPAD), MAD plus composting (COM), and MAD plus heat pelletization processes. The indicator concentrations of fecal coliforms and male-specific coliphages as well as pathogen genome concentrations for human adenovirus species, Legionella pneumophila, Staphylococcus aureus, and Clostridium difficile were significantly lower in the class A samples, and a multivariate analysis of variance ranked the stabilization processes from the lowest pathogen/indicator load to the highest as (i) class A COM, (ii) class A TPAD, and (iii) class B MAD. Human adenovirus genomes were found in 88% of the class B samples and 70 to 100% of the class A samples. L. pneumophila, S. aureus, and C. difficile genomes were detected at the qPCR assay detection limits in 19 to 50% of the class B and class A anaerobic digestion samples, while L. pneumophila was detected in 50% of the class A compost samples. When considering all the stabilization methods, both the fecal coliform and the male-specific coliphage concentrations show a significant linear correlation with the pathogen genome concentrations. This survey provides the necessary pathogen concentrations to add to biosolid aerosol risk and pathogen exposure analyses and clarifies the effectiveness of class A stabilization methods with the pathogen and indicator loads in biosolids.

Source Tracking Aerosols Released from Land-Applied Class B Biosolids during High-Wind Events

ABSTRACT DNA-based microbial source tracking (MST) methods were developed and used to specifically and sensitively track the unintended aerosolization of land-applied, anaerobically digested sewage sludge (biosolids) during high-wind events. Culture and phylogenetic analyses of bulk biosolids provided a basis for the development of three different MST methods. They included (i) culture- and 16S rRNA gene-based identification of Clostridium bifermentans, (ii) direct PCR amplification and sequencing of the 16S rRNA gene for an uncultured bacterium of the class Chloroflexi that is commonly present in anaerobically digested biosolids, and (iii) direct PCR amplification of a 16S rRNA gene of the phylum Euryarchaeota coupled with terminal restriction fragment length polymorphism to distinguish terminal fragments that are unique to biosolid-specific microorganisms. Each method was first validated with a broad group of bulk biosolids and soil samples to confirm the targets exclusive presence in biosolids and absence in soils. Positive responses were observed in 100% of bulk biosolid samples and in less than 11% of the bulk soils tested. Next, a sampling campaign was conducted in which all three methods were applied to aerosol samples taken upwind and downwind of fields that had recently been land applied with biosolids. When average wind speeds were greater than 5 m/s, source tracking results confirmed the presence of biosolids in 56% of the downwind samples versus 3% of the upwind samples. During these high-wind events, the biosolid concentration in downwind aerosols was between 0.1 and 2 μg/m3. The application of DNA-based source tracking to aerosol samples has confirmed that wind is a possible mechanism for the aerosolization and off-site transport of land-applied biosolids.

Viral metagenome analysis to guide human pathogen monitoring in environmental samples.

Aims:  The aim of this study was to develop and demonstrate an approach for describing the diversity of human pathogenic viruses in an environmentally isolated viral metagenome.

Source Bioaerosol Concentration and rRNA Gene-Based Identification of Microorganisms Aerosolized at a Flood Irrigation Wastewater Reuse Site

ABSTRACT Reuse of partially treated domestic wastewater for agricultural irrigation is a growing practice in arid regions throughout the world. A field sampling campaign to determine bioaerosol concentration, culturability, and identity at various wind speeds was conducted at a flooded wastewater irrigation site in Mexicali, Baja California, Mexico. Direct fluorescent microscopy measurements for total microorganisms, culture-based assays for heterotrophs and gram-negative enteric bacteria, and small-subunit rRNA gene-based cloning were used for microbial characterizations of aerosols and effluent wastewater samples. Bioaerosol results were divided into two wind speed regimens: (i) below 1.9 m/s, average speed 0.5 m/s, and (ii) above 1.9 m/s, average speed 4.5 m/s. Average air-borne concentration of total microorganisms, culturable heterotrophs, and gram-negative enteric bacteria were, respectively, 1.1, 4.2, and 6.2 orders of magnitude greater during the high-wind-speed regimen. Small-subunit rRNA gene clone libraries processed from samples from air and the irrigation effluent wastewater during a high-wind sampling event indicate that the majority of air clone sequences were more than 98% similar to clone sequences retrieved from the effluent wastewater sample. Overall results indicate that wind is a potential aerosolization mechanism of viable wastewater microorganisms at flood irrigation sites.

Evaluation of the enterococci indicator in biosolids using culture-based and quantitative PCR assays

The utility of the enterococci indicator for measuring biosolids quality was evaluated in biosolids from 22 U.S. wastewater treatment facilities. Enterococci were enumerated using 23S rRNA quantitative PCR (qPCR) and membrane filtration with mEI-agar culture analyses in biosolids collected after mesophilic anaerobic digestion (MAD, class B, 13 treatment plants), composting (class A, 10 treatment plants), and temperature-phased anaerobic digestion (TPAD, class A, six treatment plants). Enterococci qPCR and culture values were not significantly different for a given treatment (P>0.05, paired t-test) and both assays showed differences in biosolid treatment effectiveness-anaerobic digestion treatments averaged 5-5.5log genomic units (GU) and colony forming units (CFU)/dry g while composting decreased enterococci on average to 3.7logGU and 3.8logCFU/dry g. Only in class A TPAD biosolids dewatered with a belt-filter press were culture values significantly lower than qPCR values (1.7logCFU/dryg vs. 5GU/dryg). Further investigation of compost inactivation was compared for enterococci and other fecal indicators (n=5 treatment plants)-the enterococci indicator was more resistant to compost treatment than fecal coliforms, with reductions averaging only 1-2.5 logs for enterococci, male-specific coliphages, and sulfite-reducing Clostridia while 5-log reductions were observed for fecal coliforms. Lastly, biosolid isolates from culture-based methods were identified using DNA sequencing-these results revealed that non-enterococci, including Bacillus spp. and Vagococcus spp., were commonly isolated from compost and TPAD biosolids using mEI agar. Given the equivalency of culture- and qPCR-based enterococci concentrations in biosolids and the more conservative inactivation noted for both assays during class A composting, the use of enterococci qPCR monitoring could bypass non-specificity issues with culture-based methods while providing an improved description of pathogen fate in biosolids.

Respiratory Toxicity and Inflammatory Response in Human Bronchial Epithelial Cells Exposed to Biosolids, Animal Manure, and Agricultural Soil Particulate Matter

This study investigated cytotoxicity and inflammation caused by human bronchial epithelial cells exposed to respirable aerosols produced during the land application of stabilized sewage sludges (biosolids). BEAS-2B cells were exposed to respirable aerosols (PM(10)) derived from soils, biosolids stabilized by mesophilic anaerobic digestion (MAD), temperature-phased anaerobic digestion (TPAD), and composting (COM) as well as animal manures stabilized by mesophilic anaerobic digestion (AMAD) and composting (ACOM). Anaerobically digested particles (MAD, TPAD, AMAD) induced the highest cytotoxicity with LD(50) levels of 70 microg/cm(2), 310 microg/cm(2) for, and 375 microg/cm(2) for MAD, AMAD, and TPAD, respectively. Conversely, there was no observed cytotoxicity for soils, composted biosolids, or composted manures at the in vitro doses tested. Inflammatory responses, measured by interleukin (IL)-6 and IL-8 release, were 2- to 15-fold greater in biosolids and manures than for equivalent doses in soils. Biosolids treatment rankings for human bronchial epithelial cell toxicity and inflammation were similar to the rankings found in recent biosolids pathogen content studies-from lowest pathogen content or toxicity to highest, rankings were as follows: COM < TPAD < MAD. Coupling in vitro responses with modeled tracheobronchial lung surface doses that may occur during a biosolids land application event suggests that an inflammatory aerosol exposure in the TB region could only occur under worst case scenarios (exercising human with reduced lung capacity at <65 m set backs), but examination of lower in vitro doses as well as consideration of the head and lower lung respiratory tract regions are needed to more definitively describe the links between biosolids aerosols and the potential for respiratory inflammation.