Improved reliability of Pseudomonas aeruginosa PCR detection by the use of the species-specific ecfX gene target

Abstract

Reliability of the most widely used PCR screenings for the human opportunistic pathogen Pseudomonas aeruginosa was evaluated. Specificity analyses showed the gyrB, toxA, and 16S-23S rDNA internal transcribed spacer (ITS) but not the 16S rDNA, oprI, oprL, and fliC PCR screenings to discriminate P. aeruginosa cells from a collection of fifteen Pseudomonas species. Sensitivity analyses showed all these PCR except the toxA one to be reliable for 100% of the P. aeruginosa strains tested in this study. Specificity of the ITS and gyrB PCR screenings were further investigated on 9 soils and 29 freshwater DNA extracts of different origins, and on DNA extracted from 3 horse manures. The ITS PCR showed the highest efficacy on water and soil DNA extracts but only the gyrB one detected P. aeruginosa DNA in horse manure. DNA sequence analyses of ITS and gyrB PCR products revealed uncertainties and false positive results in these P. aeruginosa identification schemes. A novel PCR screening, targeting the ecfX gene, was thus developed. ecfX encodes an ECF (extracytoplasmic function) sigma factor which is restricted to P. aeruginosa, and might play a role in haem-uptake and virulence. Specificity and sensitivity analyses showed the ecfX PCR screening to be highly reliable, giving PCR products of the expected size for all P. aeruginosa strains tested and not amplifying DNA from any of the other Pseudomonas species tested. The ecfX PCR screening was validated on environmental DNA extracts. DNA sequence analyses of the ecfX PCR products confirmed their identity and allocation to P. aeruginosa. These investigations suggest a preferential colonization of water rather than soil environments by P. aeruginosa. Detection limits of P. aeruginosa in environmental samples were improved by the ecfX PCR screening.