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| Content Provider | Springer Nature Link |
|---|---|
| Author | Frankenhuyzen, Jessica K. Trevors, Jack T. Flemming, Cecily A. Lee, Hung Habash, Marc B. |
| Copyright Year | 2013 |
| Abstract | Biosolids result from treatment of sewage sludge to meet jurisdictional standards, including pathogen reduction. Once government regulations are met, materials can be applied to agricultural lands. Culture-based methods are used to enumerate pathogen indicator microorganisms but may underestimate cell densities, which is partly due to bacteria existing in a viable but non-culturable physiological state. Viable indicators can also be quantified by real-time polymerase chain reaction (qPCR) used with propidium monoazide (PMA), a dye that inhibits amplification of DNA found extracellularly or in dead cells. The objectives of this study were to test an optimized PMA-qPCR method for viable pathogen detection in wastewater solids and to validate it by comparing results to data obtained by conventional plating. Reporter genes from genetically marked Pseudomonas sp. UG14Lr and Agrobacterium tumefaciens 542 cells were spiked into samples of primary sludge, and anaerobically digested and Lystek-treated biosolids as cell-free DNA, dead cells, viable cells, and mixtures of live and dead cells, followed by DNA extraction with and without PMA, and qPCR. The protocol was then used for Escherichia coli quantification in the three matrices, and results compared to plate counts. PMA-qPCR selectively detected viable cells, while inhibiting signals from cell-free DNA and DNA found in membrane-compromised cells. PMA-qPCR detected 0.5–1 log unit more viable E. coli cells in both primary solids and dewatered biosolids than plate counts. No viable E. coli was found in Lystek-treated biosolids. These data suggest PMA-qPCR may more accurately estimate pathogen cell numbers than traditional culture methods. |
| Starting Page | 1251 |
| Ending Page | 1261 |
| Page Count | 11 |
| File Format | |
| ISSN | 13675435 |
| Journal | Journal of Industrial Microbiology and Biotechnology |
| Volume Number | 40 |
| Issue Number | 11 |
| e-ISSN | 14765535 |
| Language | English |
| Publisher | Springer Berlin Heidelberg |
| Publisher Date | 2013-08-20 |
| Publisher Place | Berlin, Heidelberg |
| Access Restriction | Subscribed |
| Subject Keyword | Biosolids Escherichia coli Propidium monoazide Real-time PCR Reporter gene Viable pathogen Microbiology Biochemistry Inorganic Chemistry Genetic Engineering Biotechnology Bioinformatics |
| Content Type | Text |
| Resource Type | Article |
| Subject | Medicine Bioengineering Applied Microbiology and Biotechnology Biotechnology |
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