The majority of cystic fibrosis (CF) patients succumb to a chronic infection of the airway with Pseudomonas aeruginosa. Paradoxically, pathogenic strains are often susceptible to antibiotics, but the infection cannot be eradicated with antimicrobial therapy. We find that in a majority of patients with airway infections, late isolates of P. aeruginosa produce increased levels of drug-tolerant persister cells. The genomes of a clonal pair of early/late isolates from a single patient have been previously sequenced, and the late isolate (obtained at age 96 months) showed a 100-fold increase in persister levels. The 96-month isolate carries a large number of mutations, including a mutation in mutS that confers a hypermutator phenotype. There is also a mutation in the mexZ repressor controlling the expression of the MexXY-OprM multidrug pump, which results in a moderate increase in the ofloxacin, carbenicillin, and tobramycin MICs. Knocking out the mexXY locus restored the resistance to that of the parent strain but did not affect the high levels of persisters formed by the 96-month isolate. This suggests that the late isolate is a high-persister (hip) mutant. Increased persister formation was observed in exponential phase, stationary phase, and biofilm populations of the 96-month isolate. Analysis of late isolates from 14 additional patients indicated that 10 of them are hip mutants. Most of these hip mutants did not have higher drug resistance. Increased persister formation appears to be their sole mechanism for surviving chemotherapy. Taken together, these findings suggest a link between persisters and recalcitrance of CF infection and identify an overlooked culprit-high-persister mutants producing elevated levels of drugtolerant cells. Persisters may play a similarly critical role in the recalcitrance of other chronic infections.The majority of cystic fibrosis (CF) patients succumb to a chronic untreatable Pseudomonas aeruginosa infection of the airways (18). The strains responsible for these infections are largely clonal and show evidence of positive selection for mutations leading to adaptation to the airway environment (39). Mutants of P. aeruginosa resistant to antibiotics are selected in CF patients, which can contribute to therapy failure. These include overexpression of multidrug resistance pumps, target modification, and plasmid-mediated resistance (18). It is important to note, however, that isolates of P. aeruginosa from CF patients do not necessarily develop high-level antibiotic resistance (4,19,21). This creates a paradox: antibiotics that are effective in inhibiting the growth of the pathogen in vitro are unable to cure the infection. We suggested that persister cells are responsible for maintaining untreatable infections (36). Persisters are dormant phenotypic variants rather than mutants and have been produced by all microbial pathogens studied to date (28,34,44,48). Persisters make up a small fraction of the population but are the only cells to survive treatment with high doses of bactericidal ant...

Bactericidal antibiotics kill by modulating their respective targets. This traditional view has been challenged by studies that propose an alternative, unified mechanism of killing, whereby toxic reactive oxygen species (ROS) are produced in the presence of antibiotics. We found no correlation between an individual cell's probability of survival in the presence of antibiotic and its level of ROS. An ROS quencher, thiourea, protected cells from antibiotics present at low concentrations, but the effect was observed under anaerobic conditions as well. There was essentially no difference in survival of bacteria treated with various antibiotics under aerobic or anaerobic conditions. This suggests that ROS do not play a role in killing of bacterial pathogens by antibiotics.

Pseudomonas aeruginosa is a ubiquitous organism that is the focus of intense research because of its prominent role in disease. Due to its relatively large genome and flexible metabolic capabilities, this organism exploits numerous environmental niches. It is an opportunistic pathogen that sets upon the human host when the normal immune defenses are disabled. It’s deadliness is most apparent in cystic fibrosis patients, but it also is a major problem in burn wounds, chronic wounds, chronic obstructive pulmonary disorder (COPD), surface growth on implanted biomaterials, and within hospital surface and water supplies where it poses a host of threats to vulnerable patients [1,2]. Once established in the patient, P. aeruginosa can be especially difficult to treat. The genome encodes a host of resistance genes, including multidrug efflux pumps [3]and enzymes conferring resistance to beta-lactam and aminoglycoside antibotics [4], making therapy against this gram-negative pathogen particularly challenging due to the lack of novel antimicrobial therapeutics [5]. This challenge is compounded by the ability of P. aerugionsa to grow in a biofilm, which may enhance its ability to cause infections by protecting bacteria from host defenses and chemotherapy. Here we review recent studies of P. aeruginosa biofilms with a focus on how this unique mode of growth contributes to its ability to cause recalcitrant infections.