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Microbes are subjected to selective pressures during chronic infections of host tissues. Pseudomonas aeruginosa isolates with inactivating mutations in the transcriptional regulator LasR are frequently selected within the airways of people with cystic fibrosis (CF), and infection with these isolates has been associated with poorer lung function outcomes. The mechanisms underlying selection for lasR mutation are unknown but have been postulated to involve the abundance of specific nutrients within CF airway secretions. We characterized lasR mutant P. aeruginosa strains and isolates to identify conditions found in CF airways that select for growth of lasR mutants. Relative to wild-type P. aeruginosa, lasR mutants exhibited a dramatic metabolic shift, including decreased oxygen consumption and increased nitrate utilization, that is predicted to confer increased fitness within the nutrient conditions known to occur in CF airways. This metabolic shift exhibited by lasR mutants conferred resistance to two antibiotics used frequently in CF care, tobramycin and ciprofloxacin, even under oxygen-dependent growth conditions, yet selection for these mutants in vitro did not require preceding antibiotic exposure. The selection for loss of LasR function in vivo, and the associated adverse clinical impact, could be due to increased bacterial growth in the oxygen-poor and nitrate-rich CF airway, and from the resulting resistance to therapeutic antibiotics. The metabolic similarities among diverse chronic infection-adapted bacteria suggest a common mode of adaptation and antibiotic resistance during chronic infection that is primarily driven by bacterial metabolic shifts in response to nutrient availability within host tissues., Author Summary Chronic infections are distinguished from many other infections in that they are difficult to eradicate with antibiotics. Thus, the microbes that cause chronic infections persist within host tissues for long periods despite our best treatment efforts. During the course of these chronic infections, the causative microbes often change genetically. For example, a bacterium that commonly infects the lungs of people with the genetic disease cystic fibrosis (CF) undergoes several known changes that affect the growth of this pathogen. However, the causes and clinical impact of the changes undergone by this and other chronically infecting microbes are unclear. We show that a common, early mutation found in bacteria isolated from chronically infected CF airways renders these bacteria better able to grow in the nutrients found in CF lung secretions. Interestingly, these same changes also confer resistance to several antibiotics used commonly to treat CF patients. Many of the characteristics conferred by this mutation are exhibited by other microbes found in chronic infections, suggesting that adaptation of these microbes to host tissue nutrient environments may be a common mechanism of antibiotic resistance in chronic infections.

Intracellular pathogens must withstand nitric oxide (NO·) generated by host phagocytes. Salmonella enterica serovar Typhimurium interferes with intracellular trafficking of inducible nitric oxide synthase (iNOS) and possesses multiple systems to detoxify NO·. Consequently, the level of NO· stress encountered by S. Typhimurium during infection in vivo has been unknown. The Base Excision Repair (BER) system recognizes and repairs damaged DNA bases including cytosine and guanine residues modified by reactive nitrogen species. Apurinic/apyrimidinic (AP) sites generated by BER glycosylases require subsequent processing by AP endonucleases. S. Typhimurium xth nfo mutants lacking AP endonuclease activity exhibit increased NO· sensitivity resulting from chromosomal fragmentation at unprocessed AP sites. BER mutant strains were thus used to probe the nature and extent of nitrosative damage sustained by intracellular bacteria during infection. Here we show that an xth nfo S. Typhimurium mutant is attenuated for virulence in C3H/HeN mice, and virulence can be completely restored by the iNOS inhibitor L-NIL. Inactivation of the ung or fpg glycosylase genes partially restores virulence to xth nfo mutant S. Typhimurium, demonstrating that NO· fluxes in vivo are sufficient to modify cytosine and guanine bases, respectively. Mutants lacking ung or fpg exhibit NO·–dependent hypermutability during infection, underscoring the importance of BER in protecting Salmonella from the genotoxic effects of host NO·. These observations demonstrate that host-derived NO· damages Salmonella DNA in vivo, and the BER system is required to maintain bacterial genomic integrity., Author Summary The host innate immune system represents the first line of defense against invading microorganisms. An integral part of this response involves the production of nitric oxide (NO·), a potent antimicrobial effector. Bacterial pathogens have evolved a variety of systems to counteract the inhibitory effects of host NO·. Here we describe the role of a complex DNA repair pathway in the bacterium Salmonella Typhimurium that minimizes the mutagenic nature of host NO·. We have determined the mutation rate of bacteria during a model infection. Our results reveal that the Salmonella Base Excision Repair system (BER), comprised of DNA glycosylases and AP endonucleases, is able to eliminate excess mutations that accumulate in NO·–exposed cells during their interaction with the host. Thus, during Salmonella infections, the BER system protects the bacterium against potentially detrimental DNA damage arising from NO·–exposure. This provides genomic stability to a pathogenic microorganism that has evolved to survive within the genotoxic intracellular environment of host phagocytes.