From asymptomatic colonization to acute infection: comparative genomics and proteomics approaches in the study of Pseudomonas aeruginosa pathogenicity

Pseudomonas aeruginosa is an opportunistic pathogen that can cause chronic and acute infections in immunocompromised individuals and in cystic fibrosis patients. It represents a nosocomial concern in that is frequently involved in hospital infections, severe infections and often shows resistance to conventional antimicrobial treatment. P. aeruginosa typically infects the pulmonary tract, urinary tract, burns, wounds, causes blood infections and frequently colonizes medical devices. P. aeruginosa possesses an arsenal of virulence factors and displays a remarkable range of virulence, from non-virulent to highly virulent broad spectrum strains. Most of the studies regarding the pathogenic potential of Pseudomonas aeruginosa have been conducted in clinical strains isolated in cases of acute or chronic infections and specially in strains from cystic fibrosis patients. A number of virulence factors and regulators involved in the development of an acute or a chronic infectious disease have been also identified and deeply analyzed in clinical bacterial strain collections. However, few is known about the factors addressing the final fate, as a colonizer or an agent of infectious disease, of Pseudomonas strains entering in contact with a susceptible host. To study Pseudomonas pathogenicity potential resulting in colonization or acute infection, a collection of Pseudomonas clinical strains deriving both from acute infections and from suspected body colonization, was analyzed. The final goal was to individuate potential differences in virulence factors, in particular those associated to secretion systems, between these two clinical Pseudomonas roles in infected patients. The analysis of a series of characteristics, such as the health state and clinical symptoms of the patient, the clinical sample examined, the results from microbiological tests, allowed to associate to each Pseudomonas strain a possible role as colonizer or cause of acute infection. The antibiotic susceptibility tests applied to all the strains lead to the identification in the strain collection of other two categories, multi-resistant and susceptible clinical isolates. On the basis of the analysis conducted from this point of view, a significant prevalence of antibiotic multiresistance strains in colonized subjects in comparison to infected patients has been found. This finding supports the hypothesis that the persistence of a bacterial strain in the colonization state arises the importance of bacterial factors conferring defense against the environment and the host immune system. Bacterial molecular typing has been used as tool for a rapid and cheap characterization of strains of clinical interest. In this study, and extensive and accurate molecular typing enabled to characterize and compare the strains originating from different patients and hospital units and to define genetic clones including closely related isolates. The association between genetic clones and clinical aspects indicated that ERIC-PCR led to more reliable results with respect to the other two methods although it has to be noted that PFGE was only used as a reference method to confirm or complement data obtained by rep-PCR. Secretion systems are mechanisms that Gram negative bacteria evolved for secretion of many factors as those involved directly in host colonization and invasion. I have reported in this thesis the distribution of selected genes coding for both structural and secreted proteins of type I to VI secretion systems in the clinical strain collection. It has been reported in literature that the prevalence of one or another secretion system is significantly associated to a given type of infection and that the lack of expression of particular genes belonging to secretion systems is associated to changes in virulence potential, formation of biofilm, and the capability of causing acute or chronic infections. As an example, the type III secretion phenotype is significantly more represented in acutely infected patients rather than in chronically infected CF patients. This secretion system is regulated in an opposite way with respect to T6 secretion system which has been instead proposed as an indicator of chronic infections. In this study, no significant correlation between any secretion system, their specific genes or SS gene profiles and acute infection or colonization cases have been established. However, prevalence of certain SS genes and/or SS gene profiles in acute infection or colonization cases have been observed. Some of the data obtained are similar to those reported in literature. For instance, the incidence of the genes belonging to T3SS found in the Verona bacterial strains collection are comparable to those previously found on the distribution of exoS and exoU gene in other clinical strain collections. Moreover, these two genes resulted to be present with a significant higher frequency in antibiotic multiresistant strains, in particular those showing ciprofloxacin resistance. This indication has been previously reported for strains with T3SS effectors and quinolones-resistance and showing high virulence potential. The proteomic approach has demonstrated an useful tool to detect differences among the secretome of two clonal strains differing in their pathogenic potential. It has been possible to compare the expression profiles and to relate the differences in the level of expression of some proteins with the specific infectious role of each one of the two Pseudomonas clinical strains belonging to the same clone. These data are preliminary because corresponding only to two clinical strains, but the application of this approach to all the strains of the collection would contribute to better understand which genetic mechanisms are associated to the colonization and acute infection states induced by Pseudomonas. Similarly, data obtained and analyzed during my stage in Germany confirm, in its application to Pseudomonas pathogenicity, that pool sequencing is a rapid and useful approach which could globally describe the distribution of more or less conserved genomic traits at a wide genome or single gene level in order to identify region or genes that could be associated with phenotypic traits.