With a carrier rate of 1 in 25 and an incidence of 1 in 2,500 live births, cystic fibrosis (CF) is the most common life-threatening, recessively inherited disease amongst Caucasians. During chronic pulmonary infection, CF patients suffer decline in lung function associated with periodic exacerbations triggered by bacterial products.

The most widespread CF pathogen is Pseudomonas aeruginosa, which causes infections that, once established, are impossible to eradicate.

The blue, redox-active phenazine pyocyanin is secreted by P. aeruginosa under the control of its cell-density-dependent quorum sensing (QS) system 1. Pyocyanin and its precursor phenazine-1-carboxylic acid (PCA) have a number of toxic effects relevant to CF 23. Pyocyanin and PCA inhibit the beating of human respiratory cilia in vitro 45, and alter expression of immunomodulatory proteins by human airway epithelial cells 6. For example, increases in IL-8 and intercellular adhesion molecule-1 (ICAM-1) expression by oxidant-dependent mechanisms have been demonstrated in vitro and in vivo 67. It has also been shown that pyocyanin production by P. aeruginosa induces neutrophil apoptosis and impairs neutrophil-mediated host defenses in vitro 8 and in vivo 9. Furthermore, Kong et al. 10 have demonstrated that physiologically relevant concentrations of pyocyanin inactivate the airway vacuolar ATPase, resulting in reduced expression of CF transmembrane conductance regulator (CFTR) in cultured lung and nasal epithelial cells.

In 1996 Cheng et al. 11 used molecular typing to demonstrate the spread of a drug-resistant strain of P. aeruginosa [named the Liverpool Epidemic Strain (LES)] amongst patients in a children's CF centre in Liverpool, UK. Analysis of post-2000 patient samples identified the LES in 79% of 80 P. aeruginosa-colonised CF patients in the Liverpool adult CF centre, confirming the spread and longevity of LES infections 12. Furthermore, a survey of 31 CF centres in England and Wales, involving the analysis of more than 1200 isolates of P. aeruginosa, identified the LES as the most common clone, being present in 48% of CF centres and accounting for 11% of the isolates 13.

In addition to its transmissibility, the LES appears to be more aggressive than other P. aeruginosa strains. It has been shown to replace previously established strains of P. aeruginosa (superinfection) 14, and it has infected both non-CF parents of a CF patient, causing significant morbidity 15. In addition, there is evidence for greater morbidity amongst CF patients colonised with the LES compared to those carrying non-epidemic strains of P. aeruginosa 16.

Recently, we demonstrated that some LES isolates express an unusual phenotype characterized by the early and over-expression of QS-regulated virulence genes, including those encoding the secretion of pyocyanin, LasA and elastase 17. The unusual phenotype was called hypervirulence because of increased virulence in a Drosophila infection model 17. Here we refer to it as the over-production (of pyocyanin; OP) phenotype. We reported previously that this phenotype was detected in isolates from both non-CF parents and the CF patient of the unusual infection episode 15 but was lacking from an isolate (LES400) associated with a chronic infection 17.

In this paper, we determine the prevalence of LES isolates exhibiting the OP phenotype amongst adult CF patients in Liverpool, and report a wider analysis of LES isolates with respect to phenotypic and genotypic variations.

We have demonstrated that an unusual phenotype, characterised by early- and over-production of QS-regulated virulence factors such as the exoproducts pyocyanin and LasA protease, is widespread amongst CF isolates of the LES. In a simple broth test for pyocyanin production, whereby cultures were incubated overnight in 5 ml cultures, the levels of pyocyanin were lower than those measured during exponential growth in 50 ml cultures. It is likely that this was either due to some breakdown of pyocyanin or because of greater aeration during growth of the 50 ml cultures in 250 ml flasks. Hence, although the simple broth test is a useful screening tool, the full assay gives a better indication of actual levels of pyocyanin production.

A recent study sought to assess the genetic adaptation of P. aeruginosa strains during CF infections by whole genome analysis of sequential isolates 18. The authors concluded that virulence factors required for initiation of acute infections are often selected against during chronic infection. Amongst those mutations leading to the loss of virulence-associated phenotypes, lasR mutations were the most common 18. Indeed, the observation that lasR mutants are common amongst clinical and environmental isolates of P. aeruginosa has led to the suggestion that the QS system may have a negative impact on the organism's fitness 19. If this is the case, then the unusual OP phenotype of the LES might be expected to amplify any negative impact. However, we found evidence that the OP phenotype can persist amongst isolates within a patient for periods in excess of five years (patients A, B and C) and up to seven years (patients B and C). To our knowledge there are no other reported instances of natural isolates of P. aeruginosa that exhibit the OP phenotype. Given that the OP phenotype places a considerable metabolic burden on the bacteria, the retention of such a high-maintenance phenotype within LES populations suggests that there may be some selective advantage. The fact that the OP phenotype is retained increases the possibility that it may play a role in the transmissibility of the strain. Interestingly, we also found that the same lasR mutant can persist within a patient for up to six years (patient A). We did not find complete correlation between pyocyanin over-production and high levels of LasA activity. This suggests that secondary mutations not affecting the whole QS regulon also lead to diverse phenotypes in LES populations.

CF isolates are often non-motile or auxotrophic. In one study, 33% of CF patients had a mean percentage auxotroph count of more than 50% total cfu/ml 20. Oliver et al. 21 reported an overall prevalence for the hypermutable phenotype amongst CF isolates of P. aeruginosa as 20%. In our study, we found that LES isolates exhibiting the hypermutable phenotype comprised 18% of the isolates from the six CF patients. It has been suggested that the hypermutator phenotype plays an important role in the development of multiple-antimicrobial resistance in P. aeruginosa strains causing chronic lung infections 22. However, we found little evidence that hypermutable isolates were less susceptible to antimicrobials than other non-hypermutable isolates from the same patient (Table 1).

Variations in colony morphology are also commonplace. Normal laboratory practice when dealing with sputum samples is to select a limited number of the most numerous colony morphotypes for storage, antimicrobial susceptibity profiling and occasionally strain typing. Morphology has been linked to increased virulence in the case of small colony variants (SCV) 23. However, it has also been demonstrated that identical colony morphotypes sharing the same genotype can vary in activities such as antimicrobial susceptibility 24. In our study, isolates obtained from an individual patient at the same sampling time differed in morphology, but there are a number of examples with sequential isolates where the same colony morphotype and genotype exhibited variations in antimicrobial susceptibity and/or QS-regulated activities. If pulsotype variation is discounted there are further examples suggesting that colony morphotype is an imperfect basis for the selection of isolate variants (Table 1). It has already been reported that the antibiotic susceptibilities of LES isolates vary widely 1217.

The LES was first identified in a CF unit where ceftazidime monotherapy was being used as standard antipseudomonal treatment for respiratory exacerbations 11. The authors suggested that the strategy of monotherapy had contributed to the success of the ceftazidime-resistant LES. Blázquez and colleagues 25 have not only suggested that ceftazidime concentrations thought previously to be lethal are not, but that ceftazidime treatment can increase the frequency at which P. aeruginosa mutates. Thus, it is entirely plausible that the strategy of ceftazidime therapy may have contributed to the emergence of the OP phenotype exhibited by the LES.

Although first identified because of the spread of a multiresistant clone in a CF unit 11, it is clear that isolates of the LES vary considerably in their antimicrobial susceptibility profiles. We found an interesting correlation between the OP phenotype and resistance to the β-lactams ceftazidime, aztreonam and meropenem. It has been demonstrated recently that pyocyanin can act as a signalling molecule in P. aeruginosa strains PA01 and PA14, upregulating in late exponential or stationary phase, a limited number of genes from the QS network, including the mexGHI-opmD genes involved in efflux, redox processes and iron acquisition 26. We have reported previously that these genes are up-regulated in late exponential phase in the OP-positive isolate LES431 when compared to either an OP-negative LES isolate or strain PA01 17. Not only does this highlight another possible consequence of the early and over-production of pyocyanin in LES populations, but it suggests that pyocyanin itself may play a direct role in the observed link between antimicrobial susceptibilities and the OP phenotype.

Our data clearly support the notion that bacterial populations in the sputum samples of CF patients are mixed with respect to both genotype and phenotype, even when isolates represent the same clone. We saw many minor variations in PFGE banding patterns between isolates of the LES, even between isolates taken from the same sputum sample. The LES seems to have an unstable genotype 1327, suggesting that individuals within LES populations undergo deletions, insertions and rearrangements. As well as these quite large genetic events, there are often point mutations leading to significant phenotypic changes. A study analysing greater numbers of isolates from each sputum sample may give a far better insight into the diversity within populations of P. aeruginosa in CF than the snapshot obtained when analysing relatively small numbers.

Isolates of a CF epidemic strain of P. aeruginosa exhibit considerable phenotypic and genotypic diversity. The OP phenotype, characterized by de-regulation of the QS system leading to increased production of exoproducts including pyocyanin, is common amongst LES isolates and can persist within CF patients for several years. The presence and widespread nature of such an unusual phenotype amongst populations of a highly successful CF epidemic strain, suggests that the OP phenotype may play an important role in the success of the LES.

JLF and SP carried out most of the laboratory work leading to data acquisition. CAH, MJW and TLP participated in the design, supervision and coordination of the study, and in revision of the manuscript. CW participated in the design, supervision and coordination of the study, and drafted the manuscript. All authors read and approved the final manuscript.