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Response to Gaseous NO2 Air Pollutant of P. fluorescens Airborne Strain MFAF76a and Clinical Strain MFN1032.

Kondakova T, Catovic C, Barreau M, Nusser M, Brenner-Weiss G, Chevalier S, Dionnet F, Orange N, Poc CD - Front Microbiol (2016)

Bottom Line: Human exposure to nitrogen dioxide (NO2), an air pollutant of increasing interest in biology, results in several toxic effects to human health and also to the air microbiota.Finally, NO2-exposed P. fluorescens strains were found to be more resistant to ciprofloxacin and chloramphenicol.Taken together, our study provides evidences for the bacterial response to NO2 toxicity.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Microbiology Signals and Microenvironment EA 4312, Normandy University, University of Rouen, SéSa, IRIBEvreux, France; Aerothermic and Internal Combustion Engine Technological Research CentreSaint Etienne du Rouvray, France.

ABSTRACT
Human exposure to nitrogen dioxide (NO2), an air pollutant of increasing interest in biology, results in several toxic effects to human health and also to the air microbiota. The aim of this study was to investigate the bacterial response to gaseous NO2. Two Pseudomonas fluorescens strains, namely the airborne strain MFAF76a and the clinical strain MFN1032 were exposed to 0.1, 5, or 45 ppm concentrations of NO2, and their effects on bacteria were evaluated in terms of motility, biofilm formation, antibiotic resistance, as well as expression of several chosen target genes. While 0.1 and 5 ppm of NO2did not lead to any detectable modification in the studied phenotypes of the two bacteria, several alterations were observed when the bacteria were exposed to 45 ppm of gaseous NO2. We thus chose to focus on this high concentration. NO2-exposed P. fluorescens strains showed reduced swimming motility, and decreased swarming in case of the strain MFN1032. Biofilm formed by NO2-treated airborne strain MFAF76a showed increased maximum thickness compared to non-treated cells, while NO2 had no apparent effect on the clinical MFN1032 biofilm structure. It is well known that biofilm and motility are inversely regulated by intracellular c-di-GMP level. The c-di-GMP level was however not affected in response to NO2 treatment. Finally, NO2-exposed P. fluorescens strains were found to be more resistant to ciprofloxacin and chloramphenicol. Accordingly, the resistance nodulation cell division (RND) MexEF-OprN efflux pump encoding genes were highly upregulated in the two P. fluorescens strains. Noticeably, similar phenotypes had been previously observed following a NO treatment. Interestingly, an hmp-homolog gene in P. fluorescens strains MFAF76a and MFN1032 encodes a NO dioxygenase that is involved in NO detoxification into nitrites. Its expression was upregulated in response to NO2, suggesting a possible common pathway between NO and NO2 detoxification. Taken together, our study provides evidences for the bacterial response to NO2 toxicity.

No MeSH data available.


Related in: MedlinePlus

NO2 effect on P. fluorescens biofilm and intracellular c-di-GMP level. (A) Airborne MFAF76a and (B) clinical MFN1032 P. fluorescens strains were exposed in triplicate to 45 ppm of NO2. Biofilm formation was analyzed in static conditions after 24 h development using confocal laser scanning microscope. The biofilm biomass and the maximum thickness were estimated from 6 fields on 3 independent experiments using COMSTAT software. Intracellular c-di-GMP concentrations (C) were measured in triplicate by LC-MS/MS for control () and 45 ppm of NO2 treated () MFAF76a and MFN1032. Obtained results are presented as average values ± SEM. Statistical significance was calculated by the non-parametric Mann-Whitney-Test. n.s., non-significant.
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Figure 2: NO2 effect on P. fluorescens biofilm and intracellular c-di-GMP level. (A) Airborne MFAF76a and (B) clinical MFN1032 P. fluorescens strains were exposed in triplicate to 45 ppm of NO2. Biofilm formation was analyzed in static conditions after 24 h development using confocal laser scanning microscope. The biofilm biomass and the maximum thickness were estimated from 6 fields on 3 independent experiments using COMSTAT software. Intracellular c-di-GMP concentrations (C) were measured in triplicate by LC-MS/MS for control () and 45 ppm of NO2 treated () MFAF76a and MFN1032. Obtained results are presented as average values ± SEM. Statistical significance was calculated by the non-parametric Mann-Whitney-Test. n.s., non-significant.

Mentions: In order to test the NO2 effect on P. fluorescens biofilm, both airborne MFAF76a and clinical MFN1032 were exposed to gaseous NO2 and synthetic air and grown for 4 h in static conditions. In the control condition, the airborne strain MFAF76a produced only a poorly structured biofilm with low biomass and thickness (Figure 2A). To the best of the authors' knowledge this is the first time that the biofilm of airborne P. fluorescens strain was investigated. On the opposite, the clinical strain MFN1032 was able to form a structured mushroom-like biofilm, with about 2 and 3 fold more biomass and thickness than the airborne strain MFAF76a, respectively (Figure 2B, control). These data are consistent with previous studies showing that clinical strains can strongly adhere and form structured biofilms (Rossignol et al., 2008; Ma et al., 2009). After NO2 exposition,the airborne strain MFAF76a produced biofilms with about 3 fold increase of the maximal thickness, while the biomass was similar (Figure 2A), when compared to synthetic air treatment. These data suggest that NO2 led to induce biofilm formation in this strain. Accordingly, similar NO concentrations were previously found to promote an increase of biofilm formation in P. aeruginosa (Barraud et al., 2006), suggesting a common effect between NO and NO2 treatment. On the other hand, NO2 exposure of the clinical strain MFN1032 led to a 1.7 fold increase in biofilm production in terms of biomass, while the maximal thickness was unchanged (Figure 2B). Taken together these data suggest that the NO2-mediated biofilm modifications are strain-dependent. Since we have shown previously that airborne MFAF76a expresses a virulence activity toward A549 epithelial pulmonary cells (Duclairoir Poc et al., 2014), these data suggest that elevated concentrations of NO2 increases biofilm formation in potentially virulent airborne strain and may represent a sanitary risk.


Response to Gaseous NO2 Air Pollutant of P. fluorescens Airborne Strain MFAF76a and Clinical Strain MFN1032.

Kondakova T, Catovic C, Barreau M, Nusser M, Brenner-Weiss G, Chevalier S, Dionnet F, Orange N, Poc CD - Front Microbiol (2016)

NO2 effect on P. fluorescens biofilm and intracellular c-di-GMP level. (A) Airborne MFAF76a and (B) clinical MFN1032 P. fluorescens strains were exposed in triplicate to 45 ppm of NO2. Biofilm formation was analyzed in static conditions after 24 h development using confocal laser scanning microscope. The biofilm biomass and the maximum thickness were estimated from 6 fields on 3 independent experiments using COMSTAT software. Intracellular c-di-GMP concentrations (C) were measured in triplicate by LC-MS/MS for control () and 45 ppm of NO2 treated () MFAF76a and MFN1032. Obtained results are presented as average values ± SEM. Statistical significance was calculated by the non-parametric Mann-Whitney-Test. n.s., non-significant.
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Related In: Results  -  Collection

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Figure 2: NO2 effect on P. fluorescens biofilm and intracellular c-di-GMP level. (A) Airborne MFAF76a and (B) clinical MFN1032 P. fluorescens strains were exposed in triplicate to 45 ppm of NO2. Biofilm formation was analyzed in static conditions after 24 h development using confocal laser scanning microscope. The biofilm biomass and the maximum thickness were estimated from 6 fields on 3 independent experiments using COMSTAT software. Intracellular c-di-GMP concentrations (C) were measured in triplicate by LC-MS/MS for control () and 45 ppm of NO2 treated () MFAF76a and MFN1032. Obtained results are presented as average values ± SEM. Statistical significance was calculated by the non-parametric Mann-Whitney-Test. n.s., non-significant.
Mentions: In order to test the NO2 effect on P. fluorescens biofilm, both airborne MFAF76a and clinical MFN1032 were exposed to gaseous NO2 and synthetic air and grown for 4 h in static conditions. In the control condition, the airborne strain MFAF76a produced only a poorly structured biofilm with low biomass and thickness (Figure 2A). To the best of the authors' knowledge this is the first time that the biofilm of airborne P. fluorescens strain was investigated. On the opposite, the clinical strain MFN1032 was able to form a structured mushroom-like biofilm, with about 2 and 3 fold more biomass and thickness than the airborne strain MFAF76a, respectively (Figure 2B, control). These data are consistent with previous studies showing that clinical strains can strongly adhere and form structured biofilms (Rossignol et al., 2008; Ma et al., 2009). After NO2 exposition,the airborne strain MFAF76a produced biofilms with about 3 fold increase of the maximal thickness, while the biomass was similar (Figure 2A), when compared to synthetic air treatment. These data suggest that NO2 led to induce biofilm formation in this strain. Accordingly, similar NO concentrations were previously found to promote an increase of biofilm formation in P. aeruginosa (Barraud et al., 2006), suggesting a common effect between NO and NO2 treatment. On the other hand, NO2 exposure of the clinical strain MFN1032 led to a 1.7 fold increase in biofilm production in terms of biomass, while the maximal thickness was unchanged (Figure 2B). Taken together these data suggest that the NO2-mediated biofilm modifications are strain-dependent. Since we have shown previously that airborne MFAF76a expresses a virulence activity toward A549 epithelial pulmonary cells (Duclairoir Poc et al., 2014), these data suggest that elevated concentrations of NO2 increases biofilm formation in potentially virulent airborne strain and may represent a sanitary risk.

Bottom Line: Human exposure to nitrogen dioxide (NO2), an air pollutant of increasing interest in biology, results in several toxic effects to human health and also to the air microbiota.Finally, NO2-exposed P. fluorescens strains were found to be more resistant to ciprofloxacin and chloramphenicol.Taken together, our study provides evidences for the bacterial response to NO2 toxicity.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Microbiology Signals and Microenvironment EA 4312, Normandy University, University of Rouen, SéSa, IRIBEvreux, France; Aerothermic and Internal Combustion Engine Technological Research CentreSaint Etienne du Rouvray, France.

ABSTRACT
Human exposure to nitrogen dioxide (NO2), an air pollutant of increasing interest in biology, results in several toxic effects to human health and also to the air microbiota. The aim of this study was to investigate the bacterial response to gaseous NO2. Two Pseudomonas fluorescens strains, namely the airborne strain MFAF76a and the clinical strain MFN1032 were exposed to 0.1, 5, or 45 ppm concentrations of NO2, and their effects on bacteria were evaluated in terms of motility, biofilm formation, antibiotic resistance, as well as expression of several chosen target genes. While 0.1 and 5 ppm of NO2did not lead to any detectable modification in the studied phenotypes of the two bacteria, several alterations were observed when the bacteria were exposed to 45 ppm of gaseous NO2. We thus chose to focus on this high concentration. NO2-exposed P. fluorescens strains showed reduced swimming motility, and decreased swarming in case of the strain MFN1032. Biofilm formed by NO2-treated airborne strain MFAF76a showed increased maximum thickness compared to non-treated cells, while NO2 had no apparent effect on the clinical MFN1032 biofilm structure. It is well known that biofilm and motility are inversely regulated by intracellular c-di-GMP level. The c-di-GMP level was however not affected in response to NO2 treatment. Finally, NO2-exposed P. fluorescens strains were found to be more resistant to ciprofloxacin and chloramphenicol. Accordingly, the resistance nodulation cell division (RND) MexEF-OprN efflux pump encoding genes were highly upregulated in the two P. fluorescens strains. Noticeably, similar phenotypes had been previously observed following a NO treatment. Interestingly, an hmp-homolog gene in P. fluorescens strains MFAF76a and MFN1032 encodes a NO dioxygenase that is involved in NO detoxification into nitrites. Its expression was upregulated in response to NO2, suggesting a possible common pathway between NO and NO2 detoxification. Taken together, our study provides evidences for the bacterial response to NO2 toxicity.

No MeSH data available.


Related in: MedlinePlus