Valentina Crocetta

Phenotypic and genotypic characterization of Stenotrophomonas maltophilia isolates from patients with cystic fibrosis: Genome diversity, biofilm formation, and virulence

BackgroundStenotrophomonas maltophilia is emerging as one of the most frequently found bacteria in cystic fibrosis (CF) patients. In the present study, phenotypic and genotypic traits of a set of 98 isolates of S. maltophilia obtained from clinical (CF and non-CF patients) and environmental sources were comparatively evaluated.ResultsS. maltophilia exhibited a high level of genomic diversity in both CF and non-CF group, thus possibly allowing this bacterium to expand its pathogenic potentials. Strains sharing the same pulsotype infected different patients, thus likely indicating the occurrence of clonal spread or acquisition by a common source. CF isolates differed greatly in some phenotypic traits among each other and also when compared with non-CF isolates, demonstrating increased mean generation time and susceptibility to oxidative stress, but reduced ability in forming biofilm. Furthermore, in CF isolates flagella- and type IV pili-based motilities were critical for biofilm development, although not required for its initiation. Sequential isogenic strains isolated from the same CF patient displayed heterogeneity in biofilm and other phenotypic traits during the course of chronic infection. CF and non-CF isolates showed comparable virulence in a mouse model of lung infection.ConclusionsOverall, the phenotypic differences observed between CF and non-CF isolates may imply different selective conditions and persistence (adaptation) mechanisms in a hostile and heterogeneous environment such as CF lung. Molecular elucidation of these mechanisms will be essential to better understand the selective adaptation in CF airways in order to design improved strategies useful to counteract and eradicate S. maltophilia infection.

Adhesion to and biofilm formation on IB3-1 bronchial cells by Stenotrophomonas maltophilia isolates from cystic fibrosis patients.

BackgroundStenotrophomonas maltophilia has recently gained considerable attention as an important emerging pathogen in cystic fibrosis (CF) patients. However, the role of this microorganism in the pathophysiology of CF lung disease remains largely unexplored. In the present study for the first time we assessed the ability of S. maltophilia CF isolates to adhere to and form biofilm in experimental infection experiments using the CF-derived bronchial epithelial IB3-1cell line. The role of flagella on the adhesiveness of S. maltophilia to IB3-1 cell monolayers was also assessed by using fliI mutant derivative strains.ResultsAll S. maltophilia CF isolates tested in the present study were able, although at different levels, to adhere to and form biofilm on IB3-1 cell monolayers. Scanning electron and confocal microscopy revealed S. maltophilia structures typical of biofilm formation on bronchial IB3-1 cells. The loss of flagella significantly (P < 0.001) decreased bacterial adhesiveness, if compared to that of their parental flagellated strains. S. maltophilia CF isolates were also able to invade IB3-1 cells, albeit at a very low level (internalization rate ranged from 0.01 to 4.94%). Pre-exposure of IB3-1 cells to P. aeruginosa PAO1 significantly increased S. maltophilia adhesiveness. Further, the presence of S. maltophilia negatively influenced P. aeruginosa PAO1 adhesiveness.ConclusionsThe main contribution of the present study is the finding that S. maltophilia is able to form biofilm on and invade CF-derived IB3-1 bronchial epithelial cells, thus posing a rationale for the persistence and the systemic spread of this opportunistic pathogen in CF patients. Experiments using in vivo models which more closely mimic CF pulmonary tissues will certainly be needed to validate the relevance of our results.

Potential novel therapeutic strategies in cystic fibrosis: antimicrobial and anti-biofilm activity of natural and designed α-helical peptides against Staphylococcus aureus, Pseudomonas aeruginosa, and Stenotrophomonas maltophilia.

BackgroundTreatment of cystic fibrosis-associated lung infections is hampered by the presence of multi-drug resistant pathogens, many of which are also strong biofilm producers. Antimicrobial peptides, essential components of innate immunity in humans and animals, exhibit relevant in vitro antimicrobial activity although they tend not to select for resistant strains.ResultsThree α-helical antimicrobial peptides, BMAP-27 and BMAP-28 of bovine origin, and the artificial P19(9/B) peptide were tested, comparatively to Tobramycin, for their in vitro antibacterial and anti-biofilm activity against 15 Staphylococcus aureus, 25 Pseudomonas aeruginosa, and 27 Stenotrophomonas maltophilia strains from cystic fibrosis patients. All assays were carried out in physical-chemical experimental conditions simulating a cystic fibrosis lung. All peptides showed a potent and rapid bactericidal activity against most P. aeruginosa, S. maltophilia and S. aureus strains tested, at levels generally higher than those exhibited by Tobramycin and significantly reduced biofilm formation of all the bacterial species tested, although less effectively than Tobramycin did. On the contrary, the viability-reducing activity of antimicrobial peptides against preformed P. aeruginosa biofilms was comparable to and, in some cases, higher than that showed by Tobramycin.ConclusionsThe activity shown by α-helical peptides against planktonic and biofilm cells makes them promising “lead compounds” for future development of novel drugs for therapeutic treatment of cystic fibrosis lung disease.

Cooperative pathogenicity in cystic fibrosis: Stenotrophomonas maltophilia modulates Pseudomonas aeruginosa virulence in mixed biofilm.

The present study was undertaken in order to understand more about the interaction occurring between S. maltophilia and P. aeruginosa, which are frequently co-isolated from CF airways. For this purpose, S. maltophilia RR7 and P. aeruginosa RR8 strains, co-isolated from the lung of a chronically infected CF patient during a pulmonary exacerbation episode, were evaluated for reciprocal effect during planktonic growth, adhesion and biofilm formation onto both polystyrene and CF bronchial cell monolayer, motility, as well as for gene expression in mixed biofilms. P. aeruginosa significantly affected S. maltophilia growth in both planktonic and biofilm cultures, due to an inhibitory activity probably requiring direct contact. Conversely, no effect was observed on P. aeruginosa by S. maltophilia. Compared with monocultures, the adhesiveness of P. aeruginosa on CFBE41o- cells was significantly reduced by S. maltophilia, which probably acts by reducing P. aeruginosas swimming motility. An opposite trend was observed for biofilm formation, confirming the findings obtained using polystyrene. When grown in mixed biofilm with S. maltophilia, P. aeruginosa significantly over-expressed aprA, and algD—codifying for protease and alginate, respectively—while the quorum sensing related rhlR and lasI genes were down-regulated. The induced alginate expression by P. aeruginosa might be responsible for the protection of S. maltophilia against tobramycin activity we observed in mixed biofilms. Taken together, our results suggest that the existence of reciprocal interference of S. maltophilia and P. aeruginosa in CF lung is plausible. In particular, S. maltophilia might confer some selective “fitness advantage” to P. aeruginosa under the specific conditions of chronic infection or, alternatively, increase the virulence of P. aeruginosa thus leading to pulmonary exacerbation.

Evolution of Stenotrophomonas maltophilia in Cystic Fibrosis Lung over Chronic Infection: A Genomic and Phenotypic Population Study

Stenotrophomonas maltophilia has been recognized as an emerging multi-drug resistant opportunistic pathogen in cystic fibrosis (CF) patients. We report a comparative genomic and phenotypic analysis of 91 S. maltophilia strains from 10 CF patients over a 12-year period. Draft genome analyses included in silico Multi-Locus Sequence Typing (MLST), Single-Nucleotide Polymorphisms (SNPs), and pangenome characterization. Growth rate, biofilm formation, motility, mutation frequency, in vivo virulence, and in vitro antibiotic susceptibility were determined and compared with population structure over time. The population consisted of 20 different sequence types (STs), 11 of which are new ones. Pangenome and SNPs data showed that this population is composed of three major phylogenetic lineages. All patients were colonized by multiple STs, although most of them were found in a single patient and showed persistence over years. Only few phenotypes showed some correlation with population phylogenetic structure. Our results show that S. maltophilia adaptation to CF lung is associated with consistent genotypic and phenotypic heterogeneity. Stenotrophomonas maltophilia infecting multiple hosts likely experiences different selection pressures depending on the host environment. The poor genotype-phenotype correlation suggests the existence of complex regulatory mechanisms that need to be explored in order to better design therapeutic strategies.

Stenotrophomonas maltophilia Phenotypic and Genotypic Diversity during a 10-year Colonization in the Lungs of a Cystic Fibrosis Patient

The present study was carried out to understand the adaptive strategies developed by Stenotrophomonas maltophilia for chronic colonization of the cystic fibrosis (CF) lung. For this purpose, 13 temporally isolated strains from a single CF patient chronically infected over a 10-year period were systematically characterized for growth rate, biofilm formation, motility, mutation frequencies, antibiotic resistance, and pathogenicity. Pulsed-field gel electrophoresis (PFGE) showed over time the presence of two distinct groups, each consisting of two different pulsotypes. The pattern of evolution followed by S. maltophilia was dependent on pulsotype considered, with strains belonging to pulsotype 1.1 resulting to be the most adapted, being significantly changed in all traits considered. Generally, S. maltophilia adaptation to CF lung leads to increased growth rate and antibiotic resistance, whereas both in vivo and in vitro pathogenicity as well as biofilm formation were decreased. Overall, our results show for the first time that S. maltophilia can successfully adapt to a highly stressful environment such as CF lung by paying a “biological cost,” as suggested by the presence of relevant genotypic and phenotypic heterogeneity within bacterial population. S. maltophilia populations are, therefore, significantly complex and dynamic being able to fluctuate rapidly under changing selective pressures.

Stenotrophomonas maltophilia Virulence and Specific Variations in Trace Elements during Acute Lung Infection: Implications in Cystic Fibrosis

Metal ions are necessary for the proper functioning of the immune system, and, therefore, they might have a significant influence on the interaction between bacteria and host. Ionic dyshomeostasis has been recently observed also in cystic fibrosis (CF) patients, whose respiratory tract is frequently colonized by Stenotrophomonas maltophilia. For the first time, here we used an inductively mass spectrometry method to perform a spatial and temporal analysis of the pattern of changes in a broad range of major trace elements in response to pulmonary infection by S. maltophilia. To this, DBA/2 mouse lungs were comparatively infected by a CF strain and by an environmental one. Our results showed that pulmonary ionomic profile was significantly affected during infection. Infected mice showed increased lung levels of Mg, P, S, K, Zn, Se, and Rb. To the contrary, Mn, Fe, Co, and Cu levels resulted significantly decreased. Changes of element concentrations were correlated with pulmonary bacterial load and markers of inflammation, and occurred mostly on day 3 post-exposure, when severity of infection culminated. Interestingly, CF strain – significantly more virulent than the environmental one in our murine model - provoked a more significant impact in perturbing pulmonary metal homeostasis. Particularly, exposure to CF strain exclusively increased P and K levels, while decreased Fe and Mn ones. Overall, our data clearly indicate that S. maltophilia modulates pulmonary metal balance in a concerted and virulence-dependent manner highlighting the potential role of the element dyshomeostasis during the progression of S. maltophilia infection, probably exacerbating the harmful effects of the loss of CF transmembrane conductance regulator function. Further investigations are required to understand the biological significance of these alterations and to confirm they are specifically caused by S. maltophilia.

Identification, antimicrobial resistance and molecular characterization of the human emerging pathogen Streptococcus gallolyticus subsp. pasteurianus.

This study aimed to retrospectively identify 22Streptococcus bovis clinical strains based on the new taxonomy, as well as to investigate their antibiotic-resistance and clonality. Strains were identified by Phoenix100 system, 16S rRNA sequencing, and two MALDI-TOF MS platforms (Bruker Biotyper, Vitek MS). Antibiotic resistance was determined both phenotypically and genotypically, and clonality was assessed by PFGE. Most of strains (63.6%) were isolated from urine, and diabetes was the most common underlying disease (31.8%). Phoenix100 system revealed all strains belonged to biotype II, and 16S rRNA sequencing identified all strains as S. gallolyticus subsp pasteurianus (SGSP). Although both MALDI-TOF MS systems correctly identified isolates to the species level, only Bruker Biotyper accurately identified to the subspecies level. Erythromycin-resistant strains (31.8%) were also clindamycin-resistant and positive for erm(B). Strains resistant to tetracycline (68.2%) were also resistant to erythromycin. PFGE showed high genetic variability identifying 17 different pulsotypes, most of which single.

Exposure to extremely low-frequency magnetic field affects biofilm formation by cystic fibrosis pathogens

AIMS To evaluate the in vitro effects of extremely low-frequency magnetic field (ELF-MF) on growth and biofilm formation by Staphylococcus aureus, Pseudomonas aeruginosa, Burkholderia cepacia and Stenotrophomonas maltophilia strains from cystic fibrosis patients. MATERIALS & METHODS The motion of selected ions (Fe, Ca, Cu, Zn, Mg, K, Na) was stimulated by the ion resonance effect, then influence on growth and biofilm formation/viability was assessed by spectrophotometry or viability count. RESULTS Generally, exposure to ELF-MF significantly increased bacterial growth and affected both biofilm formation and viability, although with differences with regard to ions and species considered. CONCLUSION Exposure to ELF-MF represents a possible new approach for treatment of biofilm-associated cystic fibrosis lung infections.

Phylogenetic relationships, biofilm formation, motility, antibiotic resistance and extended virulence genotypes among Escherichia coli strains from women with community-onset primitive acute pyelonephritis

The present work set out to search for a virulence repertoire distinctive for Escherichia coli causing primitive acute pyelonephritis (APN). To this end, the virulence potential of 18 E. coli APN strains was genotypically and phenotypically assessed, comparatively with 19 strains causing recurrent cystitis (RC), and 16 clinically not significant (control, CO) strains. Most of the strains belong to phylogenetic group B1 (69.8%; p<0.01), and APN strains showed unique features, which are the presence of phylogroup A, and the absence of phylogroup B2 and non-typeable strains. Overall, the most dominant virulence factor genes (VFGs) were ecpA and fyuA (92.4 and 86.7%, respectively; p<0.05), and the mean number of VFGs was significantly higher in uropathogenic strains. Particularly, papAH and malX were exclusive for uropathogenic strains. APN and RC strains showed a significantly higher prevalence of fyuA, usp, and malX than of CO strains. Compared to RC strains, APN ones showed a higher prevalence of iha, but a lower prevalence of iroN, cnf1, and kpsMT-II. Hierarchical cluster analysis showed a higher proportion of two gene clusters (malX and usp, and fyuA and ecpA) were detected in the APN and RC groups than in CO, whereas iutA and iha clusters were detected more frequently in APN strains. The motility level did not differ among the study-groups and phylogroups considered, although a higher proportion of swarming strains was observed in APN strains. Antibiotic-resistance rates were generally low except for ampicillin (37.7%), and were not associated with specific study- or phylogenetic groups. APN and RC strains produced more biofilm than CO strains. In APN strains, iha was associated with higher biofilm biomass formation, whereas iroN and KpSMT-K1 were associated with a lower amount of biofilm biomass. Further work is needed to grasp the virulence and fitness mechanisms adopted by E. coli causing APN, and hence develop new therapeutic and prophylactic approaches.