Genomic typing of Pseudomonas aeruginosa isolates by comparison of Riboprinting and PFGE: correlation of experimental results with those predicted from the complete genome sequence of isolate PAO1 Jeannie Botes a , G. Williamson a , V. Sinickas a , V. Gu ¨rtler b, * a Department of Microbiology, Melbourne Health Shared Pathology Services, C/O Post Office, The Royal Melbourne Hospital, Parkville 3050, Australia b Department of Microbiology, Austin and Repatriation Medical Centre, P.O. Box 5555, Studley Road Heidelberg, Victoria 3084, Australia Received 6 January 2003; received in revised form 3 April 2003; accepted 18 April 2003 Abstract The whole genomic typing of 21 isolates of Pseudomonas aeruginosa from 15 intensive care unit (ICU) patients was performed by pulsed-field gel electrophoresis (PFGE using SpeI) and Riboprinting (using EcoRI and PvuII), and then the results were compared with predictions made from the whole genome sequence of P. aeruginosa PAO1. The analysis of electronic images from PFGE and Riboprinting by GelComparII demonstrated similar discrimination between PFGE and Riboprinting with PvuII enzyme; however, Riboprinting by EcoRI had reduced banding patterns and was shown to be of lower discrimination than PvuII. When analyzing isolates from patients, both PFGE and Riboprinting using PvuII enzyme gave equivalent results, with the exception of two isolates that were closely related by PvuII Riboprinting and unrelated by PFGE. These discrepancies in typing results can be explained and adjusted for by comparisons with the rrn properties and the SpeI restriction fragments predicted from the whole genome of P. aeruginosa PAO1. Properties of the rrn operon that need to be taken into account include: (i) restriction enzyme sites that produce one or two fragments for each rrn operon; (ii) genomic variability in ISR sequence length; (iii) different enzymes need to be used to determine differences in rrn operon copy number from Riboprints; and (iv) choice of a restriction enzyme that produces riboprinter bands derived from rrn operon regions that are highly variable within the genome and between isolates. This knowledge has ramifications for PFGE and Riboprinter design and analysis so that for each new species to be typed comparisons can be made using the whole genome sequence. Crown Copyright D 2003 Published by Elsevier Science B.V. All rights reserved. Keywords: Pseudomonas aeruginosa; Whole genome band pattern analysis; PFGE; Ribotyping; Intergenic 16S – 23S rDNA spacer region 1. Introduction Pseudomonas aeruginosa is one of the most com- mon nosocomial pathogens in humans and is often a major problem in intensive care units (ICU) (Bodey et al., 1983). Many of the epidemiological studies to date 0167-7012/03/$ - see front matter. Crown Copyright D 2003 Published by Elsevier Science B.V. All rights reserved. doi:10.1016/S0167-7012(03)00156-8 Abbreviations: RFLP, restriction fragment length polymor- phism; ISR, intergenic 16S – 23S rDNA spacer region; RAPD, randomly amplified polymorphic DNA; ICU, intensive care unit. * Corresponding author. Tel.: +61-3-9496-3136; fax: +61-3- 9457-2590. E-mail address: volker@austin.unimelb.edu.au (V. Gu ¨rtler). www.elsevier.com/locate/jmicmeth MIMET-01854 Journal of Microbiological Methods 55 (2003) 231 – 240