J Antimicrob Chemother 1999; 43: 164- 165 Sir, Recently, there has been much interest in an aspect of the stationary phase of non-sporulating environmental bacteria termed the general stress response (GSR), which results in cells resistant to numerous physical and chemical stresses. 1 The final, non-replicating stages have been variously described as quiescent, resting, or dormant. We wished to test the hypothesis that this response to stress could be a critical event in chronic infection, resulting in at least a subpopulation of cells contributing to the characteristic antibiotic resistance of chronic infections. Using RT-PCR we have found strong mRNA expression of the master regulator of GSR in sputum from cystic fibrosis (CF) patients with chronic Pseudomonas aeruginosa lung infection. A general tendency for nutrient depletion and slow or zero growth to be associated with antibiotic resistance 2 has long been established. In retrospect, it seems probable that, in addition to the particular consequences of adaptation to specific nutrient depletion or starvation and of reduced growth rate, the GSR played a significant role in resistance. There is also evidence that nutrient depletion has major effects on the susceptibility of P. aeruginosa3 and of Burkholderia cepacia4 to phagocytosis (and cationic proteins) and to serum killing. Nevertheless, there are as yet few papers on the effects of the general stress response on the susceptibility of microbes to host defences and antibiotics. The rpoS-encoded sigma factor σs is a master regulator in a complex network of stationary-phase-responsive genes in Escherichia coli, 1 whereas in P. aeruginosa it seems likely that at least two sigma factors, RpoS and AlgU, and density-dependent N-acyl homoserine lactone (AHL) quorum sensing molecules act in concert to orchestrate the organism's remarkable persistence in the CF lung. There is a hierarchical link between AHLs and RpoS expression. 5 Not only do AHLs influence virulence factor production by P. aeruginosa, they also have immunomodulatory activity. 6 Furthermore, AHLs influence biofilm development. 7 Thus, the density-dependent regulation of virulence determinants (and secondary metabolites) in P. aeruginosa is integrated with biofilm formation and adaptation and survival in the stationary phase. In the CF lung, colonization by P. aeruginosa, expressing an iron-restricted phenotype, 8 is due to cells present as biomasses or adherent biofilms 9 which are slow growing or non-growing, from which cells may break away to cause acute exacerbations of infection. For prescribing purposes it is these latter, growing cells that are typically characterized in vitro in terms of antibiotic susceptibility. Cell density will be relatively high in the biomass or adherent biofilm fraction of the population and such populations would be expected to demonstrate density-related effects at a stage when their free-growing planktonic counterparts of equivalent population numbers would not. Sputum samples from CF patients undergoing routine physiotherapy were collected and immediately resuspended in a 0.05% solution of sodium azide and stored in dry ice before transport and long-term storage at −20°C. This azide step was included to disrupt the production of ATP and prevent the synthesis of mRNA resulting from any general stress response associated with sample preparation and processing. A multiplex PCR (M-PCR) test based on the simultaneous amplification of two P. aeruginosa outer membrane lipoproteins was performed on these samples. M-PCR detected five positives out of 19 sputum samples. RNA extracted from these samples using an RNeasy Extraction Kit (Qiagen, Crawley, UK) was used as the template for RT-PCR. P. aeruginosa specific primers designed to amplify a 528 bp rpoS product were used in conjunction with Titan One-Tube RT-PCR kits (Boehringer, Mannheim, Germany) to measure gene expression. In all cases rpoS was found to be strongly expressed in vivo by the clinical isolates. Its identity was confirmed by sequence analysis. Biofilm cultures of bacteria, including P. aeruginosa, are much more resistant than equivalent cells grown in suspension. The reasons for the extreme resistance of biofilms are not clear. We propose that biofilm growth leads to an early accumulation of density-dependent signals and to an early general stress response and possibly a more complete expression of the response relative to that in conventional planktonic culture. This may partially explain both the exceptional antibiotic resistance of P. aeruginosa in CF lung infection in particular and of biofilms generally.
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Foley et al. (1999) studied this question.