Can laboratory reference strains mirror ‘real-world’ pathogenesis? C.A. Fux 1 , M. Shirtliff 2 , P. Stoodley 1,3 and J.W. Costerton 1 1 Center for Biofilm Engineering, Montana State University, 366 EPS Building - P.O. Box 173980, Bozeman, MT 59717, USA 2 Department of Biomedical Sciences, Dental School, University of Maryland, 666 W. Baltimore Street, Baltimore, MA 21201, USA 3 Center for Genomic Sciences, Allegheny-Singer Research Institute, 11th Floor South Tower, 320 East North Avenue, Pittsburgh, PA 15212-4772, USA The extraordinary plasticity of bacterial genomes raises concerns about the adequacy of laboratory-adapted reference strains for the study of ‘real world’ patho- genesis. Some laboratory strains have been sub- cultured for decades since their first isolation and might have lost important pathophysiological charac- teristics. Evidence is presented that bacteria rapidly adapt to in vitro conditions. Genomic differences between laboratory reference strains and corresponding low-passage clinical isolates are reviewed. It appears that no bacterial strain can truly represent its species. For DNA microarray and proteomic studies, this limi- tation might be overcome by the summation of individual genomes to produce a species-specific virtual supragenome. Introduction Bacterial growth conditions in vitro are strikingly differ- ent from in vivo ecosystems, including bacterial infections. Growth within multispecies communities in complex and changing environments of the ‘real world’ contrasts with the standardized and idealized conditions in laboratory monocultures (Figure 1). Despite these differences, microbial research is largely based on laboratory-adapted reference strains that are essentially non-pathogenic [1–3]. As outlined in Box 1, laboratory strains, such as K12 of Escherichia coli, PAO1 of Pseudomonas aeruginosa and COL of Staphylococcus aureus, have been sub-cultured for decades since their first isolation. A variety of genotypes have consequently evolved over time [4]. As a result, current PAO1 strains from various laboratories express notably different biofilm phenotypes under ostensibly identical growth conditions (P. Stoodley, unpublished). The extraordinary plasticity of bacterial genomes, which might be best illustrated by the diversity in genome sizes within one species [5,6], is only now being fully appreciated. This high degree of genetic flexibility, however, raises concerns about the adequacy of labora- tory-adapted reference strains for the study of ‘real-world’ pathogenesis. In the course of sequential in vitro passage, laboratory reference strains might have significantly differentiated from non-passaged clinical samples. There- fore, any research conducted on the basis of current laboratory strains and their genome sequence could miss important pathophysiological mechanisms that only are present in clinical strains. This limitation obviously affects DNA microarray studies, but it also influences proteomic studies that virtually reconstruct proteins on the basis of the sequenced genome of a laboratory Figure 1. Conventional sampling and passage techniques enrich for planktonic populations that have adapted to idealized in vitro growth conditions. (a) In vivo, bacteria predominantly grow as biofilms – complex multispecies communities in a continually changing milieu. (b) The first selection for planktonic populations occurs during sampling, in which predominantly detached biofilm and planktonically grown cells are collected. (c) The sequential passage of broth cultures selects for planktonic cells, whereas surface-adherent biofilm phenotypes are left behind. (d) By the time bacteria are maintained in a surface-adherent manner on agar media, many of the biofilm phenotypes that enable survival in the ‘real world’ – and their specific genetic information - might have disappeared. Corresponding author: Fux, C.A. (christophf@erc.montana.edu). Available online 2 December 2004 Opinion TRENDS in Microbiology Vol.13 No.2 February 2005 www.sciencedirect.com 0966-842X/$ - see front matter Q 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.tim.2004.11.001