Pseudomonas Aeruginosa Colonising Patients with Cystic Fibrosis: Antibiotic Resistance and Growth Conditions Determined by in vivo and in vitro Gene Expression
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Abstract
Background: Pseudomonas aeruginosa is the main pathogen responsible for lung destruction in cystic fibrosis, becoming difficult to eradicate in chronic infection.
Aims: To describe antibiotic resistance among strains of P.aeruginosa isolated from sputa of patients with cystic fibrosis. To investigate in vivo and in vitro expression of genes related to antibiotic resistance and anaerobic growth.
Methods: Sputa (in vivo) and strains (in vitro) from 26 patients were obtained during 17 months. Genotypes were compared by random polymorphic DNA amplification. Expression of nirS (anaerobic respiration) and mexY (MexXY efflux pump) were measured by quantitative real time polymerase chain reaction. Expression levels of nirS in aerobiosis and anaerobiosis were compared to estimate oxygenation status within lungs. Mutations in the regulator gene mexZ were investigated in sputa expressing mexY and were correlated with strains’ antibiotic resistance.
Results: Nine patients and 56 sputa were finally analysed. Seven patients carried a single genotype. Gene mexY was detected in all the sputa; expression levels were higher in sputa with mexZ mutations. Multi-resistance was frequent. Resistance profiles not always correlated with mexY expression levels or mexZ mutations. Comparison of in vivo and in vitro nirS expression indicated mainly aerobic and microaerophilic environments within sputa.
Discussion: Mutations in e mexZ are frequent in strains of P.aeruginosa colonising patients with cystic fibrosis. Presence of these mutations correlates with increased expression of mexY in vivo and in vitro, but no with in vivo antibiotic resistance. Results of nirS expression suggest that the lungs represent heterogeneous environments regarding oxygenation status. This complexity explains that mechanisms of growth and antibiotic resistance within the lungs of these patients are still largely unknown.
Conclusions: After many years of research few studies, including the present, revealed different aspects of in vivo growth of P. aeruginosa. We determined a cut-off to discriminate between sputa containing mexZ wild type and mutated alleles and showed that comparison of in vivo and in vitro nirS expression allows to predict oxygenation status. So far, none of the studies can explain all the factors influencing the behaviour of P.aeruginosa colonising cystic fibrosis patients making it difficult to design new therapeutic strategies.
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