Identification of Pseudomonas aeruginosa in water-bottling plants on the basis of procedures included in ISO 16266:2006

Abstract

ISO 16266:2006 provides a standardized procedure for the isolation and identification of Pseudomonas aeruginosa in waters. In some cases the method described in this ISO is not conclusive enough to confirm or discard the presence of this opportunistic human pathogen. In this study the capacity of the procedure described in ISO 16266:2006 to identify presumptive P. aeruginosa isolates was evaluated. Forty-one presumptive P. aeruginosa strains, previously isolated from water-bottling plants following ISO 16266:2006, were submitted to all the tests recommended by ISO 16266:2006 (Cetrimide agar with nalidixic acid, King B agar, Acetamide broth and Oxidase test). Additional tests that have been widely used for the identification of P. aeruginosa were also performed (Asparagine broth and King A agar). Furthermore, we also conducted the non-compulsory ISO 16266:2006 assay to study the capacity of the strains to grow at 4 degrees C and 42 degrees C. Finally, all the strains were biochemically phenotyped with PhP-48 plates (Bactus AB, Sweden) and API 20NE galleries (Biomérieux, France), and their 16 rRNA gene was sequenced. ISO 16266:2006 correctly identified 27 out of 29 genotypically confirmed P. aeruginosa isolates, although two false negative identifications were obtained. Growth in Asparagine broth should be discarded as a confirmative test as it showed false negatives and false positives. In contrast, API 20NE galleries correctly identified all the confirmed isolates. King A medium and growth tests at 4 degrees C and 42 degrees C correctly discriminated all the studied strains, even the two that were not identified with the basic ISO 16266:2006 tests. Given that King A medium and growth tests at 4 degrees C and 42 degrees C are straightforward, rapid, and inexpensive, it is strongly recommended that they be used for routine confirmation of P. aeruginosa when applying ISO 16266:2006.