Evaluation of the Use of PCR and Reverse Transcriptase PCR for Detection of Pathogenic Bacteria in Biosolids from Anaerobic Digestors and Aerobic Composters

Abstract

ABSTRACT A PCR-based method and a reverse transcriptase PCR (RT-PCR)-based method were developed for the detection of pathogenic bacteria in organic waste, using Salmonella spp., Listeria monocytogenes , Yersinia enterocolitica , and Staphylococcus aureus as model organisms. In seeded organic waste samples, detection limits of less than 10 cells per g of organic waste were achieved after one-step enrichment of bacteria, isolation, and purification of DNA or RNA before PCR or RT-PCR amplification. To test the reproducibility and reliability of the newly developed methods, 46 unseeded samples were collected from diverse aerobic (composting) facilities and anaerobic digestors and analyzed by both culture-based classical and newly developed PCR-based procedures. No false-positive but some false-negative results were generated by the PCR- or RT-PCR-based methods after one-step enrichment when compared to the classical detection methods. The results indicated that the level of activity of the tested bacteria in unseeded samples was very low compared to that of freshly inoculated cells, preventing samples from reaching the cell density required for PCR-based detection after one-step enrichment. However, for Salmonella spp., a distinct PCR product could be obtained for all 22 nonamended samples that tested positive for Salmonella spp. by the classical detection procedure when a selective two-step enrichment (20 h in peptone water at 37°C and 24 h in Rappaport Vassiliadis medium at 43°C) was performed prior to nucleic acid extraction and PCR. Hence, the classical procedure was shortened, since cell plating and further differentiation of isolated colonies can be omitted, substituted for by highly sensitive and reliable detection based on nucleic acid extraction and PCR. Similarly, 2 of the 22 samples in which Salmonella spp. were detected also tested positive for Listeria monocytogenes according to a two-step enrichment procedure followed by PCR, compared to 3 samples that tested positive when classical isolation procedures were followed. The study shows that selective two-step enrichment is useful when very low numbers of bacterial pathogens must be detected in organic waste materials, such as biosolids. There were no false-positive results derived from DNA of dead cells in the waste sample, suggesting that it is not necessary to perform RT-PCR analyses when PCR is combined with selective enrichment. Large numbers of added nontarget bacteria did not affect detection of Salmonella spp., L. monocytogenes , and Y. enterocolitica but increased the detection limit of Staphylococcus aureus from <10 to 10 4 CFU/g of organic waste. Overall, the detection methods developed using seeded organic waste samples from one waste treatment facility (WTF) needed to be modified for satisfactory detection of pathogens in samples from other WTFs, emphasizing the need for extensive field testing of laboratory-derived PCR protocols. A survey of 13 WTFs in Germany revealed that all facilities complied with the German Biowaste Ordinance , which mandates that the end product after anaerobic digestion or aerobic composting be free of Salmonella . In addition, all biosolids were free of L. monocytogenes , Staphylococcus aureus , and Y. enterocolitica , as evidenced by both classical and PCR-based detection methods.