Achromobacter spp. adaptation in cystic fibrosis infection and candidate biomarkers of antimicrobial resistance

Achromobacter spp. can establish occasional or chronic lung infections in patients with cystic fibrosis (CF). Chronic colonization has been associated with worse prognosis highlighting the need to identify markers of bacterial persistence. To this purpose, we analyzed phenotypic features of 95 Achromobacter spp. isolates from 38 patients presenting chronic or occasional infection. Virulence was tested in Galleria mellonella larvae, cytotoxicity was tested in human bronchial epithelial cells, biofilm production in static conditions was measured by crystal violet staining and susceptibility to selected antibiotics was tested by the disk diffusion method. The presence of genetic loci associated to the analyzed phenotypic features was evaluated by a genome-wide association study. Isolates from occasional infection induced significantly higher mortality of G. mellonella larvae and showed a trend for lower cytotoxicity than chronic infection isolates. No significant difference was observed in biofilm production among the two groups. Additionally, antibiotic susceptibility testing showed that isolates from chronically-infected patients were significantly more resistant to sulfonamides and meropenem than occasional isolates. Candidate genetic biomarkers associated with antibiotic resistance or sensitivity were identified. Achromobacter spp. strains isolated from people with chronic and occasional lung infection exhibit different virulence and antibiotic susceptibility features, which could be linked to persistence in CF lungs. This underlines the possibility of identifying predictive biomarkers of persistence that could be useful for clinical purposes

In silico analysis and theratyping of an ultra-rare CFTR genotype (W57G/A234D) in primary human rectal and nasal epithelial cells

Mutation targeted therapy in cystic fibrosis (CF) is still not eligible for all CF subjects, especially for cases carrying rare variants such as the CFTR genotype W57G/A234D (c.169T>G/c.701C>A). We performed in silico analysis of the effects of these variants on protein stability, which we functionally characterized using colonoids and reprogrammed nasal epithelial cells. The effect of mutations on cystic fibrosis transmembrane conductance regulator (CFTR) protein was analyzed by western blotting, forskolin-induced swelling (FIS), and Ussing chamber analysis. We detected a residual CFTR function that increases following treatment with the CFTR modulators VX661±VX445±VX770, correlates among models, and is associated with increased CFTR protein levels following treatment with CFTR correctors. In vivo treatment with VX770 reduced sweat chloride concentration to non-CF levels, increased the number of CFTR-dependent sweat droplets, and induced a 6% absolute increase in predicted FEV1% after 27 weeks of treatment indicating the relevance of theratyping with patient-derived cells in CF

Computing organoids’ volume in medical images: the case study of cystic fibrosis

We present CORVO (Computing Organoids VOlume in medical images), a tool for calculating the volume of complex time-changing 3D structures from medical videos. In order to identify anisotropies in volume variation over time, CORVO is equipped with a module implementing an advanced regression-based statistical analysis. We tested CORVO for the analysis of the variation of rectal organoids volume, whose anisotropic or missing expansion indicates a pathological state and a non-response to a pharmacological treatment, respectively

Virulence and antibiotic resistance of Achromobacter spp. isolates from chronic and occasional lung infection in cystic fibrosis patients

Background: Achromobacter spp. are opportunistic pathogens that can establish both chronic or occasional infections in the lungs of cystic fibrosis (CF) patients. Chronic infections caused by these bacterial species have been associated with decline in respiratory function and lung inflammation, highlighting the need to identify markers of persistence. To this purpose, in this study virulence and antibiotic resistance of isolates from CF chronic and occasional infection were analyzed. Methods: Ninety-five Achromobacter spp. clinical isolates were collected from 38 patients followed at the CF centers of Verona and Rome (Italy): 24 patients presented a chronic colonization and 14 an occasional infection. A number of features were evaluated in vitro: virulence potential through inoculation of bacteria in Galleria mellonella larvae; susceptibility to selected antibiotics by Kirby-Bauer disk diffusion test; cytotoxicity by quantitative measurement of lactate dehydrogenase; biofilm formation by crystal violet staining of surface‐attached bacteria cultured in static conditions. Statistical analysis was performed to compare chronic and occasional isolates and ascertain the significance of results. Results: Virulence testing showed that isolates from occasionally infected patients induced significantly higher mortality of G. mellonella larvae than chronic infection isolates (Kaplan-Meier survival estimate p-value=0.02; Cox hazard ratio=1.32; 95% confidence interval (CI)=1.04-1.66). As concerns antibiotic susceptibility, isolates from chronically infected patients were significantly more resistant to sulfonamide and meropenem (respectively: Fisher’s exact test p-value=0.04 and 0.01 after 10000 permutations; CI=0.04-0.62 and 0-0.34; odds ratio=0.17 and 0) than occasional isolates. Cytotoxicity was tested in human bronchial epithelial cells: although no statistically significant difference was found, we observed that chronic infection isolates induced greater cytotoxicity than occasional isolates (Wilcoxon Mann Whitney p-value=0.05). Finally, no significant difference was observed in biofilm production among the two groups. Conclusion: Our results show that Achromobacter spp. isolates from chronic and occasional lung infection exhibit different virulence and antibiotic resistance characteristics, some of which might be linked to persistence in CF lungs. This highlights the potential to identify predictive markers of persistence that could be translated into the clinical setting. Acknowledgements: This study was supported by the Italian Cystic Fibrosis Research Foundation (project FFC#18/2019)