Elsevier

Water Research

Volume 44, Issue 14, July 2010, Pages 4252-4260
Water Research

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

https://doi.org/10.1016/j.watres.2010.05.039Get rights and content

Abstract

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.

Introduction

More than 7 million dry tons of sewage sludge are produced annually in the U.S., Bastian (1997). Globally, this waste stream continues to increase as urban populations increase, municipal wastewater treatment facilities move toward biological nutrient removal, and urban areas of developing nations build sewer systems and centralized treatment works. In the U.S., sewage sludges that have been stabilized by digestion or composting are termed “biosolids” if there is a resulting beneficial use. Greater than 60% of stabilized sewage sludges are reused through application to agricultural land, Bastian, 1997, Spicer, 2002. While the agricultural benefits of land applying biosolids are well documented, Tenenbaum (1997), the potential pathogen content of biosolids and associated health complaints from residents living near biosolids land application sites have resulted in widespread public health concerns and community opposition to this practice, NRC (2002).

The pathogen content of biosolids is likely diverse. In large municipalities, centralized wastewater treatment facilities commonly serve over 1 million residents. Although enteric pathogens are the traditional focus of biosolids management practice, all viral and bacterial pathogens can be excreted in urine and feces, Sinclair et al. (2008). Potential exposure routes to these pathogens during land application include ingestion, inhalation, and dermal contact, NRC (2002). Surveys of pathogens in biosolids have thus far only included a limited suite of known infectious agents and indicators, Rusin et al., 2003, Viau and Peccia, 2009b. Given the potential for such diverse pathogen content, past approaches that have targeted only a limited number of organisms may underestimate the pathogen diversity and total pathogen content, thus limiting the ability to fully understand potential infectious risk from human exposure during land application, Brooks et al., 2005, Eisenberg et al., 2008, Low et al., 2007.

To properly define the risks posed by biosolids land application, a more complete understanding of pathogen abundance and diversity is required. Previous analyses of the dominant biosolids aerosol exposure pathway have estimated a maximum inhalation dose of respirable biosolids material to be 1.4 μg at 500 m, Low et al. (2007). At a bulk concentration of 5 × 1010 total cells per dry gram of biosolids, Paez-Rubio et al. (2007), this dose corresponds to inhalation of 7 × 104 biosolids-derived cells respectively during a land application event. Methods to examine microbial community diversity in such an environmental sample—primarily construction of phylogenetic clone libraries—are typically limited to less than 103 sequence identifications per sample. This does not reach the sampling depth required to identify pathogens, which are less common and typically account for far less than 1% of an environmental microbial population. Recently developed massively parallel sequencing technologies can provide a large number of short read sequences, Margulies et al. (2005), and significantly improve researcher’s ability to investigate community members that are not highly enriched, Sogin et al. (2006). These studies have been performed with sequence reads of 250 bp and less, McKenna et al., 2008, Sanapareddy et al., 2009. However, technology for improved read lengths that average up to 400 bp is now available, allowing for a more definitive phylogenetic-based classification of individual reads.

We hypothesize that massively parallel sequencing technology will allow for sequencing deeply enough into a biosolids populations such that pathogen diversity can be explored. To test this hypothesis, 454 FLX Titanium series technology was used to produce large 16S rRNA encoding gene libraries (average 30,893 sequences per sample with average 390 base read length). Samples included mesophilic anaerobically digested municipal wastewater sludge (MAD), thermophilic temperature-phased anaerobically digested municipal sludges (TPAD), and composted municipal sludges (COM), as well as an unamended agricultural soil for comparison. The bacterial pathogen content and diversity of each sample were determined, and microbial population structures from different treatments were compared.

Section snippets

Sample collection

Biosolids were collected from three anonymous U.S. wastewater treatment facilities. All treatments process influent wastes were from municipal wastewater treatment facilities and all samples obtained from processes were final products. Sampling occurred in November 2008. At two separate facilities (Texas and California, USA), biosolids representing a Class B (Class B biosolids are expected to contain pathogens, USEPA (1999)) quality product were sampled after mesophilic anaerobic digestion

Sequencing results

A total of 238,718 raw sequences were generated. After trimming, sorting, and quality control, 185,358 or 78% of the sequences were used in our analysis. A summary of trimmed sequence information is included in Table 3 and a characteristic histogram of trimmed read lengths is shown in Figure 1. Based on previous studies that indicate short reads can be used to adequately classify microbial communities, Liu et al. (2007), a minimum read length of 50 bases was set to include all potentially

Conclusions

Efforts to determine pathogen concentration and exposure in environmental air, water, and wastewater samples are biased by the requirement to select a single or limited group of potential pathogens for analysis. Massively parallel sequencing technology coupled with continually increasing read lengths and sequence quantities can potentially remove these biases by sequencing deeply enough into populations to describe the true diversity of pathogens and include both established and emerging

Acknowledgements

This work was supported by the National Science Foundation grant BES0348455. KJB is partially supported by a fellowship from the Environmental Research and Education Foundation.

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