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Detection and enumeration of P seudomonas aeruginosa in soil and manure assessed by an ecfX q PCR assay
Author(s) -
Coli C.,
Deredjian A.,
Hien E.,
Brothier E.,
Bouziri L.,
Cournoyer B.,
Hartman A.,
Henry S.,
Jolivet C.,
Ranjard L.,
Nazaret S.
Publication year - 2013
Publication title -
journal of applied microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.889
H-Index - 156
eISSN - 1365-2672
pISSN - 1364-5072
DOI - 10.1111/jam.12189
Subject(s) - pseudomonas aeruginosa , manure , enumeration , agar , biology , detection limit , microbiology and biotechnology , bacteria , soil microbiology , pseudomonas , veterinary medicine , nalidixic acid , enrichment culture , food science , chemistry , chromatography , antibiotics , ciprofloxacin , agronomy , mathematics , medicine , genetics , combinatorics
Aims To develop a q PCR approach for the detection of P seudomonas aeruginosa in soil and manure and explore its efficacy and limitations compared with that of a classical culture‐dependent approach. Methods and Results A P s. aeruginosa ecfX q PCR 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‐ P s. aeruginosa colonies mainly belonging to other P seudomonas species but also some beta‐ P roteobacteria. These bacteria strongly impacted the detection threshold of this approach. Efficacy of these approaches was compared for P s. aeruginosa enumeration among manure and agricultural soil samples from various sites in F rance, T unisia and B urkina F aso. Conclusions The developed q PCR assay enabled a specific detection of P s. aeruginosa in soil and manure samples. The culture‐based approach was usually found more sensitive than the q PCR assay. However, abundance of non‐ P s. 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 q PCR assay for the detection and enumeration of P s. aeruginosa in soil and manure and shows its complementarity with a culture‐based approach.