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Rapid MPN-Qpcr Screening for Pathogens in Air, Soil, Water, and Agricultural Produce

Abstract

A sensitive, high-throughput, and cost-effective method for screening bacterial pathogens in the environment was developed. A variety of environmental samples, including aerosols, soil of various types (sand, sand/clay mix, and clay), wastewater, and vegetable surface (modeled by tomato), were concomitantly spiked with Salmonella enterica and/or Pseudomonas aeruginosa to determine recovery rates and limits of detection. The various matrices were first enriched with a general pre-enrichment broth in a dilution series and then enumerated by most probable number (MPN) estimation using quantitative PCR for rapid screening of amplicon presence. Soil and aerosols were then tested in non-spiked environmental samples, as these matrices are prone to large experimental variation. Limit of detection in the various soil types was 1–3 colony-forming units (CFU) g−1; on vegetable surface, 5 CFU per tomato; in treated wastewater, 5 CFU L−1; and in aerosols, >300 CFU mL−1. Our method accurately identified S. enterica in non-spiked environmental soil samples within a day, while traditional methods took 4 to 5 days and required sorting through biochemically and morphologically similar species. Likewise, our method successfully identified P. aeruginosa in non-spiked aerosols generated by a domestic wastewater treatment system. The obtained results suggest that the developed method presents a broad approach for the rapid, efficient, and reliable detection of relatively low densities of pathogenic organisms in challenging environmental samples.

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Acknowledgments

This research was supported by Research Grant No. CP-9033-09 from The US–Israel Binational Agricultural Research and Development Fund. EO and MB were supported by the Israeli Water Authority graduate fellowships (87227611 and 874130, respectively) and the Kreitman School for Graduate Studies. MB was also supported by the Zuck Maccabi Fund. MD was supported by a fellowship from Kraft food group and the MIT International Science and Technology Initiatives (MIT-MISTI). We acknowledge Ahuva Vonshak, Omar Bawab, and Wa’d Odeh for invaluable assistance.

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Correspondence to Ezra Orlofsky or Osnat Gillor.

Electronic Supplementary Materials

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ESM 1

Table S1. MPN index of soil samples spiked with Salmonella enterica and Pseudomonas aeruginosa at concentrations covering three orders of magnitude and detected by qPCR preceded by pre-enrichment. Table S2. Average threshold cycle (Ct) and ending relative fluoresence units (RFU) obtained from qPCR assays of three soil types spiked with Salmonella enterica at concentrations covering three orders of magnitude and pre-enriched in a dilution series amenable to MPN quantification. Table S3. Average threshold cycle (Ct) and ending relative fluoresence units (RFU) obtained from qPCR assays of three soil types spiked with Pseudomonas aeruginosa at concentrations covering three orders of magnitude and pre-enriched in a dilution series amenable to MPN quantification. Table S4. MPN index of liquid impinger collected air samples after spiked Pseudomonas aeruginosa into nebulizer and allowed to aerosolize the bacteria after 1 h. Concentrations covering three orders of magnitude and detected by qPCR preceded by pre-enrichment. Table S5. Results from MPN-qPCR targeting Salmonella enterica in environmental soil samples amended with composted poultry manure. (PDF 140 kb)

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Orlofsky, E., Benami, M., Gross, A. et al. Rapid MPN-Qpcr Screening for Pathogens in Air, Soil, Water, and Agricultural Produce. Water Air Soil Pollut 226, 303 (2015). https://doi.org/10.1007/s11270-015-2560-x

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Keywords

  • MPN-qPCR
  • Nonspecific enrichment
  • Pathogen
  • Detection
  • Vegetable
  • Aerosol
  • Soil
  • Wastewater