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).
Virulence and antibiotic resistance of Achromobacter spp. isolates from chronic and occasional lung infection in cystic fibrosis patients
Laura Veschetti;Angela Sandri;Giulia Maria Saitta;Rebeca Passarelli Mantovani;Gloria Burlacchini;Sara Preato;Claudio Sorio;Paola Melotti;Caterina Signoretto;Marzia Boaretti;Giovanni Malerba;Maria M. Lleò
2021-01-01
Abstract
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).
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