Detection and enumeration of Pseudomonas aeruginosa in soil and manure assessed by an ecfX qPCR assay

Abstract

To develop a qPCR approach for the detection of Pseudomonas aeruginosa in soil and manure and explore its efficacy and limitations compared with that of a classical culture-dependent approach. A Ps.aeruginosa ecfX qPCR assay was developed. This assay was optimized for soils of contrasting physico-chemical properties and evidenced a three-log dynamic range of detection [5×10(4) -5× 10(6) cells(gdrywtsoil)(-1) ] in inoculated microcosms. Sensitivity was determined to be around 5×10(4) cells(gdrywtsoil)(-1) . In parallel, the minimum detection limit was estimated in the range of 10-100CFU(gdrywtsoil)(-1) using a culture-dependent approach based on the use of a selective medium (cetrimide agar base medium supplemented with nalidixic acid), coupled to ecfX gene amplification to confirm isolate identity. These soil samples led to the growth of abundant non-Ps.aeruginosa colonies mainly belonging to other Pseudomonas species but also some beta-Proteobacteria. These bacteria strongly impacted the detection threshold of this approach. Efficacy of these approaches was compared for Ps.aeruginosa enumeration among manure and agricultural soil samples from various sites in France, Tunisia and Burkina Faso. The developed qPCR assay enabled a specific detection of Ps.aeruginosa in soil and manure samples. The culture-based approach was usually found more sensitive than the qPCR assay. However, abundance of non-Ps.aeruginosa species among the indigenous communities able to grow on the selective medium affected the sensitivity of this latter approach. This study describes the first specific and sensitive qPCR assay for the detection and enumeration of Ps.aeruginosa in soil and manure and shows its complementarity with a culture-based approach.