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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 exposure affects Pseudomonas fluorescens growth with aminoglycosides. After 2 h exposure to 45 ppm of NO2, growth of airborne MFAF76a (A) and clinical MFN1032 (B) in presence of tobramycin (1.55 μg/mL; ) and kanamycin (3.1 μg/mL; ) was tested. A580 was recorded at indicated time points. The control sample was bacteria exposed to synthetic air, and grown in presence of antibiotics in indicated concentrations. The data are presented as percentages of growth relative to air-exposed control. Pooled data from three independent experiments in duplicate ± SEM are reported. Statistical significance was calculated by the non-parametric Mann-Whitney-Test p < 0.05 (*), < 0.01 (**); n.s. non-significant. Dotted line shows the control (100%).
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Figure 6: NO2 exposure affects Pseudomonas fluorescens growth with aminoglycosides. After 2 h exposure to 45 ppm of NO2, growth of airborne MFAF76a (A) and clinical MFN1032 (B) in presence of tobramycin (1.55 μg/mL; ) and kanamycin (3.1 μg/mL; ) was tested. A580 was recorded at indicated time points. The control sample was bacteria exposed to synthetic air, and grown in presence of antibiotics in indicated concentrations. The data are presented as percentages of growth relative to air-exposed control. Pooled data from three independent experiments in duplicate ± SEM are reported. Statistical significance was calculated by the non-parametric Mann-Whitney-Test p < 0.05 (*), < 0.01 (**); n.s. non-significant. Dotted line shows the control (100%).

Mentions: MexEF-OprN-overproducing mutants with enhanced fluoroquinolone resistance often increase bacterial susceptibility to aminoglycosides apparently owing to impairment of the MexXY system (Sobel et al., 2005; Morita et al., 2015). The effect of NO2 on tobramycin and kanamycin sensitivity was then assayed by performing MICs. As shown in Table 2, NO2 treatment led to reduce the MICs of the two tested antibiotics, suggesting that NO2 increases P. fluorescens sensitivity to aminoglycosides. Tobramycin and kanamycin, at subinhibitory concentration of 1.55 and 3.1 μg/mL respectively, were found to decrease the growth of NO2-exposed bacteria (Figure 6). This effect was observed only from 6 to 10 h of growth for MFN1032 and from 6 to 18 h of growth for MFAF76a, highlighting the time-limited NO2 effect on bacterial antibiotic sensitivity. Altogether, our data show that NO2 increases P. fluorescens sensitivity to tobramycin and kanamycin, accordingly its homolog NO is also found to increase P. aeruginosa sensitivity to tobramycin (Barraud et al., 2006). Noticeably, this phenotype is consistent with previously published data supporting decreasing resistance to aminoglycosides of MexEF-OprN-overproducing mutant (Sobel et al., 2005; Morita et al., 2015). Since this phenotype is often associated with the impairment of the MexXY-OprM efflux pump, we next assayed the effect of NO2 on the expression of the mexXY genes. As shown in Figure 4, NO2 treatment had an opposite effect on mexXY gene expression. While NO2 increased the expression of mexXY in the airborne strain MFAF76a, it drastically reduced production of mexXY mRNA in the clinical strain MFN1032. While this latter phenotype is often described in the literature (Sobel et al., 2003) as leading to increased aminoglycoside susceptibility, the overproduction of the two RND efflux pumps MexEF-OprN and MexXY-OprM is remarkable and found in very few strains, among which the multiresistant strain PA7 (Morita et al., 2015). Nevertheless, the increased expression of mexXY in the airborne strain cannot be related to the increased susceptibility to aminoglycosides, which we observed. Taken together, our data indicate that the NO2 effect on bacterial aminoglycoside resistance is complex and strain-dependent, and the up- or down- production of mexXY cannot account solely to explain the increased susceptibility to aminoglycosides of the two studied strains. Another hypothesis may arise related to the effects of NO2 on membrane properties. Indeed, the NO2 effect on P. fluorescens membrane was recently investigated, demonstrating the NO2-mediated modifications in both the membrane glycerophospholipids composition (i.e., ratio zwitterionic/anionic glycerophospholipids) and in the membrane electron-accepting properties (Kondakova, personal communication). It is thus conceivable that these membrane modifications would alter bacterial membrane permeability, facilitating the aminoglycoside entry into the bacterial cell.


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 exposure affects Pseudomonas fluorescens growth with aminoglycosides. After 2 h exposure to 45 ppm of NO2, growth of airborne MFAF76a (A) and clinical MFN1032 (B) in presence of tobramycin (1.55 μg/mL; ) and kanamycin (3.1 μg/mL; ) was tested. A580 was recorded at indicated time points. The control sample was bacteria exposed to synthetic air, and grown in presence of antibiotics in indicated concentrations. The data are presented as percentages of growth relative to air-exposed control. Pooled data from three independent experiments in duplicate ± SEM are reported. Statistical significance was calculated by the non-parametric Mann-Whitney-Test p < 0.05 (*), < 0.01 (**); n.s. non-significant. Dotted line shows the control (100%).
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Related In: Results  -  Collection

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Figure 6: NO2 exposure affects Pseudomonas fluorescens growth with aminoglycosides. After 2 h exposure to 45 ppm of NO2, growth of airborne MFAF76a (A) and clinical MFN1032 (B) in presence of tobramycin (1.55 μg/mL; ) and kanamycin (3.1 μg/mL; ) was tested. A580 was recorded at indicated time points. The control sample was bacteria exposed to synthetic air, and grown in presence of antibiotics in indicated concentrations. The data are presented as percentages of growth relative to air-exposed control. Pooled data from three independent experiments in duplicate ± SEM are reported. Statistical significance was calculated by the non-parametric Mann-Whitney-Test p < 0.05 (*), < 0.01 (**); n.s. non-significant. Dotted line shows the control (100%).
Mentions: MexEF-OprN-overproducing mutants with enhanced fluoroquinolone resistance often increase bacterial susceptibility to aminoglycosides apparently owing to impairment of the MexXY system (Sobel et al., 2005; Morita et al., 2015). The effect of NO2 on tobramycin and kanamycin sensitivity was then assayed by performing MICs. As shown in Table 2, NO2 treatment led to reduce the MICs of the two tested antibiotics, suggesting that NO2 increases P. fluorescens sensitivity to aminoglycosides. Tobramycin and kanamycin, at subinhibitory concentration of 1.55 and 3.1 μg/mL respectively, were found to decrease the growth of NO2-exposed bacteria (Figure 6). This effect was observed only from 6 to 10 h of growth for MFN1032 and from 6 to 18 h of growth for MFAF76a, highlighting the time-limited NO2 effect on bacterial antibiotic sensitivity. Altogether, our data show that NO2 increases P. fluorescens sensitivity to tobramycin and kanamycin, accordingly its homolog NO is also found to increase P. aeruginosa sensitivity to tobramycin (Barraud et al., 2006). Noticeably, this phenotype is consistent with previously published data supporting decreasing resistance to aminoglycosides of MexEF-OprN-overproducing mutant (Sobel et al., 2005; Morita et al., 2015). Since this phenotype is often associated with the impairment of the MexXY-OprM efflux pump, we next assayed the effect of NO2 on the expression of the mexXY genes. As shown in Figure 4, NO2 treatment had an opposite effect on mexXY gene expression. While NO2 increased the expression of mexXY in the airborne strain MFAF76a, it drastically reduced production of mexXY mRNA in the clinical strain MFN1032. While this latter phenotype is often described in the literature (Sobel et al., 2003) as leading to increased aminoglycoside susceptibility, the overproduction of the two RND efflux pumps MexEF-OprN and MexXY-OprM is remarkable and found in very few strains, among which the multiresistant strain PA7 (Morita et al., 2015). Nevertheless, the increased expression of mexXY in the airborne strain cannot be related to the increased susceptibility to aminoglycosides, which we observed. Taken together, our data indicate that the NO2 effect on bacterial aminoglycoside resistance is complex and strain-dependent, and the up- or down- production of mexXY cannot account solely to explain the increased susceptibility to aminoglycosides of the two studied strains. Another hypothesis may arise related to the effects of NO2 on membrane properties. Indeed, the NO2 effect on P. fluorescens membrane was recently investigated, demonstrating the NO2-mediated modifications in both the membrane glycerophospholipids composition (i.e., ratio zwitterionic/anionic glycerophospholipids) and in the membrane electron-accepting properties (Kondakova, personal communication). It is thus conceivable that these membrane modifications would alter bacterial membrane permeability, facilitating the aminoglycoside entry into the bacterial cell.

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