In the mid-1800's, Louis Pasteur advised microbiologists to think of the human body as a “culture vessel” for microbes, in the context of understanding immunity 
. Pasteur's approach has been revised and updated several times 
, with a recent review encouraging researchers to be attentive to the effects of different in vivo
carbon sources on bacterial metabolism and physiology 
. Pasteur's advice is particularly relevant for an understanding of the evolution of disease-causing microbes. Natural selection may be imposed by the particular nutritional and metabolic resources available in a given tissue, the innate and adaptive immune systems, and, in the past 80 or so years, by antibiotics or anti-virals. Many pathogens – particularly opportunistic pathogens, emerging pathogens, and microbes causing chronic disease – are faced with a novel and hostile growth environment to which they must adapt or face extinction. Colonization and establishment of an infection in a new host or host species can thus be interpreted as a specific instance of a more general process of adaptation to a novel environment.
Understanding adaptive processes in pathogen populations, and in particular characterizing the variety of genetic routes to adaptation, is important for developing effective treatment strategies. Take as an example the management of antibiotic resistance. Resistance is often thought to be costly, in the sense that resistant strains should be less fit than susceptible strains in the absence of antibiotic. If so, then attempts to reduce the frequency of resistance in patient populations by stopping the use of an antibiotic should afford sensitive strains an advantage, and so prolong the utility of an antibiotic for treatment. Antibiotic cessation has met with mixed success (e.g., 
), however, either because some resistance mutations actually pay little or no cost, or because second site mutations that restore fitness without compromising resistance are common. The management of antibiotic resistance in patient populations depends crucially on which of these two mechanisms is more often responsible for the persistence of resistance.
The last 15 years have seen a number of studies of in vivo
genome evolution in select pathogens, primarily viruses (e.g., 
) and bacteria (e.g., 
), that shed vital insight onto the genetic changes that occur during epidemics or chronic infections. The importance of these changes for pathogen fitness in a host can be difficult to ascertain, however, because it is rarely possible to establish with certainty that the observed mutations are adaptive, since some neutral or deleterious mutations may accumulate through drift or by hitchhiking with adaptive mutations. Moreover, it can be difficult to obtain sufficient in vivo
samples to ask questions about the repeatability of in vivo
evolution – that is, how often pathogens take the same adaptive routes in independent patients or populations.
For these reasons we have turned to a complementary approach, laboratory selection experiments, to provide an understanding of the broad patterns and principles of pathogen evolution. In a typical microbial experimental evolution protocol, many populations are founded from a single genotype, and are propagated serially or in a chemostat for tens, hundreds, or thousands of generations (reviewed in 
). By maintaining multiple replicate populations in each of two or more environments (e.g., antibiotic treated vs. not antibiotic treated), the effects of a treatment can be systematically investigated in a manner that is often inaccessible with in vivo
samples. Experimental evolution has by now a rich history in studying basic evolutionary processes (e.g., 
for reviews), as well as more applied topics such as the evolution of antibiotic resistance 
and of virulence 
. In addition, experimental evolution has significant potential as an investigative tool for elucidating basic biological processes 
. With the development of technologies that allow the rapid and affordable sequencing of entire bacterial genomes, an increasing number of studies have sought to describe the genomic basis of laboratory adaptation (reviewed in 
Here we use a combination of experimental evolution and whole-genome sequencing (WGS) to investigate the initial stages of pathogen adaptation using the bacterium Pseudomonas aeruginosa
. This gram-negative bacterium is widely distributed in nature 
, and is an important opportunistic pathogen. P. aeruginosa
can cause acute infections of wounds, burns and of lungs, and is frequently implicated in nosocomial infections. Moreover, P. aeruginosa
is an important pathogen of individuals with cystic fibrosis (CF), with approximately 60–70% of Canadian adults with CF harbouring this bacterium 
. P. aeruginosa
chronically infects the CF lung, and once the infection is established, it is virtually impossible to eradicate: Intensive antibiotic regimens are effective at reducing symptoms, but almost never succeed in clearing the infection entirely.
populations that have persisted for long periods of time in the lungs of individuals with CF show characteristic signatures of adaptation to this novel culture environment. Recent studies have documented patterns of parallel evolution at the level of phenotype, gene expression, and genotype 
, indicating repeatable patterns of long-term adaptation to the CF lung. For example, CF lung sputum is highly viscous, and P. aeruginosa
typically grows as an unattached biofilm, or microcolony, in this environment 
. While environmental isolates of P. aeruginosa
are motile, long-term CF colonists show evidence of adaptation to the sessile lifestyle of the microcolony, including reduced motility, and a morphological shift to small colony variants (SCVs) on agar plates 
. Increased intracellular levels of cyclic di-GMP are thought to be important for this adaptive shift 
, but the causative mutations have yet to be fully elucidated. Other characteristic changes include mutations associated with reduced virulence, presumably to avoid detection by the host immune system, and increased small molecule efflux that can afford resistance to antibiotics commonly used with CF patients 
Given evidence of long-term adaptation during chronic infection in P. aeruginosa, we have examined the genomic basis of adaptation to CF-like culture conditions and to fluoroquinolone antibiotics through WGS of experimentally evolved P. aeruginosa isolates. Our primary aim is to describe the genetic changes underlying adaptation to this novel environment, and to ask how repeatable these changes are. In addition, we also investigate the genetic architecture of the costs of resistance: When antibiotic resistance evolves, how often is it costly, and what mutations underlie those costs? Our data allow us to quantify the nature and extent of parallel genomic evolution and, in so doing, provide a unique view of the variety of genetic routes taken during adaptation to a medically relevant novel environment.