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

AIMS: 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. METHODS AND RESULTS: 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 (g drywt soil)(-1) ] in inoculated microcosms. Sensitivity was determined to be around 5 × 10(4)  cells (g drywt soil)(-1) . In parallel, the minimum detection limit was estimated in the range of 10-100 CFU (g drywt soil)(-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. CONCLUSIONS: 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. SIGNIFICANCE AND IMPACT OF THE STUDY: 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.

Identification of Stenotrophomonas maltophilia strains isolated from environmental and clinical samples: a rapid and efficient procedure.

AIMS: Aim of the study is to identify accurately Stenotrophomonas maltophilia isolates recovered from environmental and clinical samples. METHODS AND RESULTS: Recovery of Sten. maltophilia-like isolates from soil samples using the vancomycin, imipenem, amphotericin B (VIA) selective agar medium enabled distinction of various morphotype colonies. A set of soil and clinical isolates was tested for species identification using different methods. 16S rDNA analyses showed the dark green with a blue halo morphotype to be typical Sten. maltophilia strains. The API-20NE, Vitek-2 and Biolog phenotypic analyses typically used for the identification of clinical isolates did not perform well on these soil isolates. The species-specific PCR screening targeting Sten. maltophilia 23S rDNA and the multiplex smeD/ggpS PCR, differentiating Sten. maltophilia from Stenotrophomonas rhizophila, were tested for improvement of these identification schemes. The latter multiplex PCR identified all isolates tested in this study, whatever be their origin. CONCLUSIONS: Isolation on VIA medium and confirmation of Sten. maltophilia species membership by smeD PCR is proposed to identify environmental and clinical isolates of Sten. maltophilia. SIGNIFICANCE AND IMPACT OF THE STUDY: The proposed approach enables isolation and identification of Sten. maltophilia from different environments in an easy and rapid way. This approach will be useful to accurately manage studies on the abundance and distribution of Sten. maltophilia in hospital and nonhospital environments.