Hello. My name is Lars Jelsbak and I am Associate professor at the Technical University in Denmark. In this module, called "Evolution in Chronic Infections", we will discuss how bacterial pathogen adapt to the human host environment during long-term, chronic infections. More specifically, we will discuss how the opportunistic pathogen, Pseudomonas aeruginosa – evolves during adaptation to the airways of cystic fibrosis patients. You have already encountered both the pathogen and the disease in previous modules, so I will just briefly summarize what you already know: Cystic fibrosis is a genetically inherited disease for which we have no cure. The genetic disorder makes cystic fibrosis patients unable to clear bacteria from their lungs in an effective manner. This means that they are prone to obtain chronic lung infections from a range of pathogens including P. aeruginosa. Most CF patients will become chronically infected with P. aeruginosa despite aggressive antibiotic treatments, and this organism is associated with most of the morbidity and mortality for the patients. Clone typing of P. aeruginosa isolates sampled from cystic fibrosis patients have shown that individual patients can be infected with the same clone type over several years. And also, it has been shown that some clone types are able to become transmitted between patients. This is illustrated on this slide where we can follow the infection history of the red DK1 clone type and the blue DK2 clone type as they disseminate within a patient population and cause chronic infections in individual patients for an extended time period. The two clone types on this illustration – DK1 and DK2 – do exist in real life. They have transmitted among Danish CF patients since the early seventies and are still found in certain patients today. I will focus on the DK2 clone type, and use this as an example of how to study bacterial adaptation to the human host environment in relation to chronic infections. At this point, it is important to emphasize that the CF airways is a highly complex environment and that the infecting P. aeruginosa bacteria are faced with a range of challenging conditions – conditions that they must adapt to in order to persist in this environment. Some of challenging conditions are listed on this illustration and include the antibiotics administered by the clinicians during the course of the infection, the cells and effector molecules of host immune response mounted in response to infection, and the presence other microorganisms (such as other bacteria, fungi and bacteriophages) that may co-infect the CF airways. A key question is how P. aeruginosa genetically adapt to these selective forces in the CF hosts. The answer to this question is of course important from a clinical perspective as it could potentially aid the development of new treatment strategies. The answers are also of more general relevance as they may enhance our understanding of how organisms genetically adapt to new environments. We can begin to adress this question by investigating the DK2 clone type. As I have mentioned previously, the DK2 clone type has been isolated from multiple CF patients in Denmark. The earliest sampled clone dates back to the early seventies and since then it has evolved into a successful colonizer of CF airways that – despite aggressive antibiotic therapy and in the presence of these other challenging conditions - has spread among patients, and is still found in CF patients today. So to specifically answer the question we need to analyze the genomes of longitudinal collected DK2 clones, and then follow the genomic evolution of this human pathogen as it disseminates through the cohorte of patients over more than 40 years. As you can guess, availability of culture collections of P. aeruginosa strains sampled from infected CF patients over time is absolutely essential if we are to understand how these bacteria adapt to the host environment. Fortunately, the clinical microbiologists in Copenhagen have sampled and stored infecting strains over many years – a small portion of the culture collection is shown here as tubes with bacteria frozen at -80C. And it is now possible to revive these frozen DK2 isolates that was sampled from CF patients decades ago, and compare them with present day isolates. In a sense, because of the systematic sampling, it is now possible to rewind the tape of evolution and inspect how ancestral isolates change though time. On this slide you see a top view microscope image of two DK2 colonies grown on agar plates culture. The two particular DK2 isolates where sampled 35 years apart. Although you might notice small differences in the morphology of the bacterial colonies, this type of visual inspection of morphology is not detailed enough – we need to look at the genomes of each of the isolates to identify the molecular details of the evolutionary events. In the next couple of slides, I will discuss how to use genomic information to understand how these bacteria has genetically adapted. In this example, we have sequenced 4 P. aeruginosa strains sampled from a CF patient. Based on the genome sequences we can then find the differences between the 4 genomes. Here, isolate 1 has a particular sequence in the 8 loci on the genome labeled A though H. On the other hand, isolate 2 has 5 has a different sequence – a mutation – in 5 of the 8 loci (here indicated by red color), isolate 3 has 4 mutations – 3 of theme are identical to the ones observed in isolate 2, and 1 different. Finally, isolate 4 has 2 mutations not observed in any of the other isolates. Based on this information, we can build a simple model – a phylogenetic tree – that ilustrates the genetic relationship between the isolates: Isolate 2 and 3 share a common branch in the tree composed of 3 mutations before they branch of in separate branches. Isolate 4 – on the other hand – form its own branch and share little evolutionary history with isolates 2 and 3. We have already discussed that the P. aeruginosa clone type DK2 has spread among different CF patients for a time period of more than 40 years. This is illustrated with 3 patients in green, blue and red. Each of these patients have DK2 infections (DK2 is illustrated with circles). By sequencing and analysing the genomes of these stored DK2 isolates – as I just described in the previous slide – we can outline the evolutionary history of the DK2 clone type. We can use this phylogenetic tree to identify genes that undergo adaptive evolution – that is genes that are mutated to provide a fitness advantage in relation to host adaptation. We can identify such genes by inspecting the phylogenetic tree for recurrent patterns of mutations. In the example on the slide, we observe that gene “x” has been mutated in parallel in all three DK2 sub-lineages in the three CF patients. That this gene is mutated in three DK2 sub-lineages that evolve independently in separate patients strongly suggest that mutations in this gene confer beneficial changes to DK2 We can extent this analysis to include other additional clone types – illustrated here by the gray X clone type and the pink Y clone type. Again, we look for recurrent patterns of mutations in the same bacterial genes in individual patients and clone types. I will not shown the entire list of genes targeted by pathoadaptive mutations, but we can group the majority of the genes in either of the following categories: Antibiotic resistance, Cell membrane and cell surface, and regulation. The fitness advantage of the first category is self-explanatory and the genes are for example the gyrase genes conferring resistance towards fluoroquinolones. The second category are genes with functions related to the makeup of the cell surface, and possible these mutations have selected to evade the host immune response and may also be involved in antibiotic resistance. The third category includes regulatory genes, and the reason that these genes are targeted is that their mutations facilitate adaptation by a remodeling of the regulatory networks. In some cases the literature can be used to predict the selective advantage of the identified mutations. Nonetheless, we can also pick out genes ourselves to investigate how their mutation confer some kind of selective advantage. One example case is the observation of multiple independent SNP mutations in the 23s rRNA. These particular mutations have been found in other bacteria to confer resistance to macrolide antibiotic such as Azithromycin. Systematic long-term, low-dose azithromycin treatment has been used for cystic fibrosis (CF) patients chronically infected with Pseudomonas aeruginosa in the Copenhagen CF center in Denmark since 2001. Even though the use of azithromycin on CF lung infections has been found to have beneficial clinical effects, it is unclear how azithromycin works on P. aeruginosa and if macrolide resistance can emerge. And resistance mutations in 23S rRNA have not previously been found. To investigate these mutations further in relation to macrolide resistance, we first showed that clinical DK2 isolates with the mutations were more resistant than isolates without the mutations. We confirmed this finding by introduction of the specific mutations into a laboratory wild-type strain of Pseudomonas aeruginosa Culture collections of microbial species have been assembled by microbiologists and clinical laboratories since the use of solid media for single colony isolation was invented and such microbial strain collections have been instrumental for our understanding of the epidemiology and evolution of bacterial infectious diseases. Because we are now able to sequence the genome of these bacterial pathogen at a reasonable price and speed we can now analyze these strain collections, with unprecedented resolution to determine their relatedness among the bacterial isolates, to determine their mutations, that have occured in their genome. I've tried to explain how this work schedule has helped us understand how a specific pathogen evolves during CF infections and how this has helped us pinpoint key mutations, that have made this pathogen so succesful as it is. This information will help us develop better diagnostic tool, and to better tailor the treatment of the patients It is important to emphasize, that the strategy I have talked about here in relation to pseudomonas aeruginosa infections in CF patients, can be generalized to other bacterial pathogen. In fact, strategies outlined here, has significantly enhanced our understandig of populations structures of bacterial pathogens and their global dissemination as well as their evolution.
