Characterisation of persister cells in biofilms and planktonic bacterial populations

Persister cells are described as a small phenotypically different subpopulation of specialized survivor cells found within biofilms and planktonic bacterial populations. They have been observed within a wide variety of pathogenic species and have been shown to be highly tolerant to antimicrobials. Persister cells found within biofilms have been reported to be the cause of persistent and difficult to treat infections. This study was aimed at understanding the tolerance of persister cells to antimicrobials. Repeated challenges of Escherichia coli K12 with antibiotics for 3 h followed by regrowth in fresh medium revealed that a similar number of persister cells were obtained from cultures treated with ampicillin (0.001%) and cefotaxime (0.01%) after each treatment indicating a phenotypic rather than a genotypic tolerance. However survival increased after each challenge with repeated treatment with gentamicin. Surviving cell numbers increased from 0.0001% following the first gentamicin treatment to 1% after the third phase of treatment. In contrast to previous studies, when persister cells collected after an initial antibiotic treatment were immediately re-treated with the same concentration of all three antibiotics, no surviving cells could be detected. Further, when persister cells were treated continuously with these antibiotics over several days, no survivors were found after the first 48 h. Continuous culture was used to examine the relationship between specific growth rate and the formation of persister cells. Irrespective of the antibiotic used, the specific growth rate of the cells was shown to determine the number of persister cells formed within a culture. Biofilms were observed to produce persister cells at a similar percentage of the population to that of the slowest specific growth rate tested. Treatment with cefotaxime (32 μg/ml for 24 h) resulted in 12.7% persister cells in biofilms and 11.8% persisters at the slowest specific growth rate of 0.09 Lh-1. The results of these experiments suggested that slow growing cells in continuous culture may be used as persister cells for experimentation. To investigate the effect of microbial metabolites in spent culture fluid on the formation of persister cells, cells were grown in a growth medium containing spent culture fluid (conditioned medium). When E. coli was grown in its own conditioned medium, or that of other E. coli strains, there was a 2-3 log10 increase in the number of persister cells. However, in the conditioned medium of Pseudomonas aeruginosa or Klebsiella pneumoniae no survivors were found. Microarray studies were carried out to compare the expression of genes in cells grown in these conditioned media. Gene expression profiles of E. coli cells growing in the P. aeruginosa conditioned medium showed a much greater change in expression than E. coli grown in its own spent medium. Of the genes differentially expressed (by 2-fold or more), in cells growing in their own conditioned medium 62 were down regulated and 16 were up regulated. Several stress response genes were observed to be differentially expressed in both conditioned media. The cspA cold shock gene was up regulated by 2.0 fold. This gene has been identified as a possible persister gene, and is reported to be induced by indole. When grown in the P. aeruginosa conditioned medium, 155 genes were down regulated and 164 were up regulated. Differential gene expression indicated the possible presence of hydroxyindole in the conditioned medium. Hydroxyindole can be produced as a result of the alteration of indole produced by the E. coli cells, by monooxygenases and dioxygenases reported to be produced by P. aeruginosa. The presence of hydroxyindole in this conditioned medium is hypothesised to have prevented the survival or development of the persister phenotype. The gene expression profiles also indicated that AI-2, a known signaling molecule in E. coli, was not the factor causing the increase of persister cells in the E. coli conditioned medium. The connection of microbial metabolites or signalling molecules with the formation of persister cells has not previously been reported. These results suggest that indole may explain both the increase and decrease in the number of persister cells in E. coli in the conditioned media and this finding may indicate possible drug targets (as discussed in Conclusions) for the treatment of persistent infections