Objectives: Recent studies have shown that a complex microbiota can be detected in the lungs of cystic fibrosis (CF) patients, raising the possibility that pathogenic processes in CF airways represent polymicrobial activities. P. aeruginosa, the main responsible for CF chronic lung infections, is frequently co-isolated with S. maltophilia, whose pathogenic role in CF has not yet been clarified. The main aim of this study was to evaluate the effect of exposure to S. maltophilia on P. aeruginosa virulence - namely biofilm formation, susceptibility to oxidative stress, motility, and antibiotic-resistance - as assessed by phenotypic and genotypic assays. Methods: Two strains of P. aeruginosa (RR8, RR11) and one of S. maltophilia (RR7), co-isolated in lung of a chronically colonized CF patient, were tested. Kinetic of planktonic growth and biofilm formation by P. aeruginosa, tested both as alone or co-cultured with S. maltophilia, was evaluated by viable cell counts. The activity of tobramycin against P. aeruginosa biofilms, formed alone and in combination, was similarly assayed. The influence of S. maltophilia supernatants against P. aeruginosa motility (swimming, twitching, swarming) and resistance to oxidative stress was also evaluated. The transcriptional profile of selected P. aeruginosa virulence-related genes (rhlR, lasI, aprA, toxA, algD, exoS, mexC, mexE) was also assayed by RT-PCR in the presence of S. maltophilia. Finally, the ultrastructure of biofilms, grown in mono- and co-culture, was evaluated by multiphoton confocal microscopy. Results: The planktonic growth rate of both species was significantly reduced when co-cultured, compared to mono-culture. Both P. aeruginosa strains significantly reduced S. maltophilia viability during biofilm formation, as confirmed by microscopic analysis. Susceptibility of biofilms formed by P. aeruginosa RR8 and RR11 to tobramycin was not affected by the presence of S. maltophilia. Exposure to S. maltophilia reduced P. aeruginosa RR8 swimming (17.0±1.2 vs 20.4±2.1mm; exposed vs unexposed, respectively; p<0.0001), while increased P. aeruginosa RR11 swimming and swarming (swimming: 16.4±1.5 vs 11.7±1.2mm, p<0.01; swarming: 20.4±2.0 vs 17.0±1.2mm, p<0.05; exposed vs ctrl, respectively). S. maltophilia increased the sensitivity to oxidative stress of both P. aeruginosa strains (RR8: 18.8±0.8 vs 17.4±0.5mm; RR11: 18.0±1.0 vs 16.2±0.8mm; exposed vs ctrl, respectively, p<0.05). Analysis of the transcriptional profile of P. aeruginosa biofilm formed in co-culture with S. maltophilia showed a significant over-expression of aprA, algD, toxA mexC (p<0.05), but a down-expression of rhlR and lasI (p<0.001). Conclusions: Our results clearly show that S. maltophilia is able to significantly modulate P. aeruginosa virulence, thus suggesting that studying bacterial interactions may be more useful to evaluate CF disease progression than the classic approach based on “stand-alone” opportunistic pathogens. Further in vitro and in vivo studies are warranted to define the mechanisms underlying these interactions and their impact both on status and management of CF patients.

Microbial cooperation in cystic fibrosis: Stenotrophomonas maltophilia significantly modulates Pseudomonas aeruginosa virulence.

Valentina Crocetta;Arianna Pompilio;Serena De Nicola;Fabio Verginelli;Simone Guarnieri;Giovanni Di Bonaventura
2014

Abstract

Objectives: Recent studies have shown that a complex microbiota can be detected in the lungs of cystic fibrosis (CF) patients, raising the possibility that pathogenic processes in CF airways represent polymicrobial activities. P. aeruginosa, the main responsible for CF chronic lung infections, is frequently co-isolated with S. maltophilia, whose pathogenic role in CF has not yet been clarified. The main aim of this study was to evaluate the effect of exposure to S. maltophilia on P. aeruginosa virulence - namely biofilm formation, susceptibility to oxidative stress, motility, and antibiotic-resistance - as assessed by phenotypic and genotypic assays. Methods: Two strains of P. aeruginosa (RR8, RR11) and one of S. maltophilia (RR7), co-isolated in lung of a chronically colonized CF patient, were tested. Kinetic of planktonic growth and biofilm formation by P. aeruginosa, tested both as alone or co-cultured with S. maltophilia, was evaluated by viable cell counts. The activity of tobramycin against P. aeruginosa biofilms, formed alone and in combination, was similarly assayed. The influence of S. maltophilia supernatants against P. aeruginosa motility (swimming, twitching, swarming) and resistance to oxidative stress was also evaluated. The transcriptional profile of selected P. aeruginosa virulence-related genes (rhlR, lasI, aprA, toxA, algD, exoS, mexC, mexE) was also assayed by RT-PCR in the presence of S. maltophilia. Finally, the ultrastructure of biofilms, grown in mono- and co-culture, was evaluated by multiphoton confocal microscopy. Results: The planktonic growth rate of both species was significantly reduced when co-cultured, compared to mono-culture. Both P. aeruginosa strains significantly reduced S. maltophilia viability during biofilm formation, as confirmed by microscopic analysis. Susceptibility of biofilms formed by P. aeruginosa RR8 and RR11 to tobramycin was not affected by the presence of S. maltophilia. Exposure to S. maltophilia reduced P. aeruginosa RR8 swimming (17.0±1.2 vs 20.4±2.1mm; exposed vs unexposed, respectively; p<0.0001), while increased P. aeruginosa RR11 swimming and swarming (swimming: 16.4±1.5 vs 11.7±1.2mm, p<0.01; swarming: 20.4±2.0 vs 17.0±1.2mm, p<0.05; exposed vs ctrl, respectively). S. maltophilia increased the sensitivity to oxidative stress of both P. aeruginosa strains (RR8: 18.8±0.8 vs 17.4±0.5mm; RR11: 18.0±1.0 vs 16.2±0.8mm; exposed vs ctrl, respectively, p<0.05). Analysis of the transcriptional profile of P. aeruginosa biofilm formed in co-culture with S. maltophilia showed a significant over-expression of aprA, algD, toxA mexC (p<0.05), but a down-expression of rhlR and lasI (p<0.001). Conclusions: Our results clearly show that S. maltophilia is able to significantly modulate P. aeruginosa virulence, thus suggesting that studying bacterial interactions may be more useful to evaluate CF disease progression than the classic approach based on “stand-alone” opportunistic pathogens. Further in vitro and in vivo studies are warranted to define the mechanisms underlying these interactions and their impact both on status and management of CF patients.
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Utilizza questo identificativo per citare o creare un link a questo documento: http://hdl.handle.net/11564/720760
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