Stenotrophomonas maltophilia isolated from patients affected by cystic fibrosis : genotyping analysis and molecular characterisation of virulence determinants

Abstract

Stenotrophomonas maltophilia is a an environmental organism, that in recent years has been isolated with increasing frequency as an opportunistic pathogen in nosocomial infections as well as from the airways of cystic fibrosis (CF) patients. Despite its growing clinical importance, the mechanisms adopted by this organism to establish persistent infections have not been clarified yet. The availability of information on genome sequence of two S. maltophilia strains (K279a and R551-3), allow development of new post-genomic approaches direct to understanding of key factors involved in the colonization of respiratory tract of CF patients and of cellular and molecular mechanisms of its pathogenicity. Identification of S. maltophilia genes expressed inside host, is essential for development of new antimicrobial compounds. Moreover, molecular and genetic characterization of S. maltophilia clinical strains is essential to understand pathogenicity mechanism of these organisms and to build detailed epidemiological maps to draw their spread in CF patients. The present study is aimed at determining the genomic and epidemiological relatedness of 52 S. maltophilia strains. Strains included 43 clinical collected, between January 2003 and December 2005, from 43 independent patients (41 were from CF patients, and two from blood cultures of two non-CF patients) attending the Paediatric Hospital “Bambino Gesù” of Rome. Other strains were: K279a and LMG958, two S. maltophilia reference clinical strains, and seven strains of environmental origin (two isolated within the Hospital “Bambino Gesù”). In these strains the presence and expression of the virulence-associated genes were evaluated and their contribution to virulence was assessed using larvae of the wax moth Galleria mellonella as an infection model. To confirm a role of the putative virulence factors identified we constructed K279a (the clinical reference strain whose genome sequence is available) mutants and performed complementation assay with the wild type functional gene. Moreover, to study genomic evolution of S. maltophilia toward a pathogenic lifestyle, we compare the genome of an environmental strain with that of the clinical strain K279a. Genotyping analysis. Data that we obtained indicates PFGE fingerprinting as the most discriminatory technique to characterize genomospecies present in S. maltophilia. All strains produced well-defined PFGE profiles: forty-seven different PFGE pulsotypes were identified, indicating a remarkable genomic diversity among the strains analysed. Three clusters (pulsotype 1, 17 and 33) showed a banding pattern identical to at least 1 other isolate likely indicating either cross-transmission among patients or infection from a common source. However, the results obtained with the RFLP of gyrB gene and with the variable regions of the 16S rRNA gene clearly indicated that certain phylogenetic groups are likely better able to cause infection than others. Identification and characterization of virulence genes involved in S. maltophilia pathogenicity. All 52 S. maltophilia strains were characterized for the presence and expression of type-1 fimbria, esterase, proteases (StmPr1 and StmPr2). Regarding the type-1 fimbria, only the clinical strains showed the presence of the gene coding for this surface structure, indicating that it might be an important virulence factor of this opportunistic pathogen. The expression of esterase activity by most CF-derived S. maltophilia strains, reinforces the hypothesis that esterase, such as fimbria, may play some role in the virulence. In order to verify the role of these potential virulence factors in S. maltophilia pathogenesis, representative S. maltophilia strains unable to express proteases or esterase were assayed in G. mellonella infection. The results indicate that proteases may be relevant virulence factors of S. maltophilia. Moreover, the type-1 fimbria could play an important role since the environmental strains, lacking of smf-1, show the higher lethal dose (LD50). To analyze the relative role of the two proteases (StmPr1 and StmPr2), the OBGTC23 strain, naturally impaired in protease expression, was complemented with the StmPr1 or StmPr2 functional genes and tested in G. mellonella assay. Data obtained showed that StmPr1 protease play a major role in S. maltophilia pathogenesis. Analysis of the role of S. maltophilia virulence factors by mutants construction. In the complex, the results that we obtained using different clinical OBGTC strains, impaired in the expression of one or more hypothetical virulence factors, indicates proteases as important virulence determinants for S. maltophilia. Nevertheless, these natural mutant strains could lack of many others virulence determinants that may be involved in pathogenesis process. Then, to study the effect of the single factors identified and to confirm their role, deletions encompassing different putative virulence genes are introduced by allelic exchange in reference strain K279a, whose genome sequence is available. The proteases mutants showed a strong reduction of the protease activity and were less virulent, compared to wild-type, in G. mellonella larvae, confirming that proteases, particularly StmPr1, are important virulence factors for S. maltophilia. The proteases expression is controlled by the diffusible signal factor (DSF) a quorum sensing signal molecule that controls the expression of several virulence factors. Moreover, the DSF mutant reveals a greater reduction of virulence in G. mellonella infection model, compared to that of proteases mutants, suggesting a DSF involvement in the control of proteases but also of other virulence factors. Finally, despite fimbriae are important to establish chronic infection in airways CF patients, this surface structures may be not important in G. mellonella infection model. Genomic evolution of S. maltophilia toward a pathogenic lifestyle. To identify the specific genetic determinants acquired from S. maltophilia during its evolution towards a more pathogenic lifestyle, we performed a Suppression Subtractive Hybridization: this technique allowed us to identify the genetic sequences present in the clinical strain K279a and absent in the environmental strain LMG959. The majority of the subtracted sequence may have been acquired from other organisms by horizontal gene transfers, since their different G+C content and since the presence of several IS elements. Among the sequence, particularly interesting are genes coding for haemagglutinin, Clp protease, TonB dependent receptor protein, and a putative ankyrin repeat-containing protein, which are known to be important virulence factors in many gram-negative bacteria. Moreover, many of the subtracted sequence represent genes involved in metabolism, DNA restriction/modification system, transmembrane proteins, hypothetical proteins and proteins with unknown function. These differences between S. maltophilia K279a and S. maltophilia LMG959 could be related to niche adaptation or host preference and this accessory genome may represents an advantage for pathogen evolution driven by the need for continuous adaptation to the host in order to evade or suppress coevolving host defense mechanisms, making it an emergent opportunistic pathogen in nosocomial infections.