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.

Freshwater selenium-methylating bacterial thiopurine methyltransferases: diversity and molecular phylogeny.

The diversity of bacterial thiopurine methyltransferases (bTPMT) among five natural Se-methylating freshwaters was investigated by polymerase chain reaction (PCR) screenings and sequencings. DNA sequence analyses confirmed the cloned products' identity and revealed a broad diversity of freshwater TPMTs. Neighbour-joining (NJ) phylogenetic analyses combining these sequences, all GenBank entries closely related to these sequences and deduced TPMTs obtained in this work from selected gamma-proteobacteria showed TPMTs to form a distinct radiation, closely related to UbiG methyltransferases. Inside the TPMT phylogenetic cluster, eukaryote sequences diverged early from the bacterial ones, and all the bacterial database entries belonged to a subgroup of gamma-proteobacteria, with an apparent lateral transfer of a particular allele to beta-proteobacteria of Bordetella. The NJ phylogenetic tree revealed 22 bTPMT lineages, 10 of which harboured freshwater sequences. All lineages showed deep and long branches indicative of major genetic drifts outside regions encoding highly conserved domains. Selected residues among these highly variable domains could reflect adaptations for particular ecological niches. PCR lineage-specific primers differentiated Se-methylating freshwaters according to their 'tpm lineage' signatures. Most freshwater tpm alleles were found to be distinct from those available in the databases, but a group of tpm was found encoding TPMTs identical to an Aeromonas veronii TPMT characterized in this work.