Resumo

Pseudomonas aeruginosa is a metabolically versatile bacterium that can adapt to a wide range of habitats. This adaptability accounts for their constant presence in the environment. It is an opportunistic pathogen, particularly in patients who are immunocompromised and outbreaks of P. aeruginosa has been associated with contamination of recreational waters and tap water. According to national legislation (Uruguay) potable water must not contain this bacterium. In this work we used the method of SMEWW (9213F) to detect P. aeruginosa in potable water. The confirmation in this method relies on the particularity that this bacterium can grow in a medium that contains ammonia and acetate as nitrogen and carbon sources (acetamide) after preliminary enrichment in asparagine broth. However a positive result due to a color change of the phenol red indicator to alkaline pH is not always clear and doubtful readings are frequent. The aim of this work was to assess the confirmation of P. aeruginosa by a molecular method. For this purpose we amplified the gyrB gene by PCR from presumptive positive asparagine broths and from positive acetamide broths. Amplification was done directly from the centrifuged biomass and from DNA extracted from the biomass with a kit (Wizard® Genomic DNA Purification kit). DNA extraction from asparagine broth was difficult and only one sample gave a visible band by gel electrophoresis, however amplifiable DNA was obtained for all other samples. PCR detection from biomass or DNA gave similar results indicating that direct amplification from biomass is a valid alternative to DNA extraction. 10 out of 21 samples gave a negative result by PCR from acetamide and asparagine broths, revealing a strong discordance between methods. Asparagine broths of negative PCR samples were used for the purpose of isolating P. aeruginosa in milk agar with cetrimide. It was isolated only in one sample out of ten; moreover all other samples revealed the presence of other fluorescent non P. aeruginosa, suggesting some false positive results by the culture method. We also tested 3 samples that were presumptive positive for asparagine but negative for acetamide by the culture method: one sample was positive by PCR and confirmed by sequencing. The data obtained indicated that a revision of the acetamide confirmation method should be performed and compared to other culture and molecular methods.