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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 protects Pseudomonas fluorescens from chloramphenicol toxicity. After 2 h exposure to 45 ppm of NO2, growth of airborne MFAF76a (A) and clinical MFN1032 (B)P. fluorescens with ciprofloxacin () and chloramphenicol () was assayed. Growth curves were performed with ciprofloxacin (3.125 μg/mL for MFAF76a and 1.156 μg/mL for MFN1032) and chloramphenicol (25 and 100 μg/mL respectively), and A580 was recorded at the indicated time points. The control sample was bacteria exposed to synthetic air, and grown in presence of antibiotics in indicated concentrations. The data are shown as percentages of growth relative to synthetic 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 (*); n.s., non-significant. Dotted line shows the control (100%).
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Figure 5: NO2 protects Pseudomonas fluorescens from chloramphenicol toxicity. After 2 h exposure to 45 ppm of NO2, growth of airborne MFAF76a (A) and clinical MFN1032 (B)P. fluorescens with ciprofloxacin () and chloramphenicol () was assayed. Growth curves were performed with ciprofloxacin (3.125 μg/mL for MFAF76a and 1.156 μg/mL for MFN1032) and chloramphenicol (25 and 100 μg/mL respectively), and A580 was recorded at the indicated time points. The control sample was bacteria exposed to synthetic air, and grown in presence of antibiotics in indicated concentrations. The data are shown as percentages of growth relative to synthetic 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 (*); n.s., non-significant. Dotted line shows the control (100%).

Mentions: In order to study the effect of NO2 on MexEF-OprN efflux pump, the transcription levels of mexE, mexF and oprN genes (KT070324, KT070321 and KT070325 for MFAF76a; KT070323, KT070322 and KT186432 for MFN1032, respectively) were compared using qRT-PCR in two P. fluorescens strains exposed or not to 45 ppm of NO2. In airborne and clinical strains, the mexE mRNA level was increased by almost 14- and 100-fold respectively; that of mexF almost 3.5- and 47-fold respectively and that of oprN almost 4.6- and 73-fold respectively (Figure 4). These data show that NO2 promoted mexEF-oprN expression, potentially causing modifications in P. fluorescens antibiotic resistance. We next tested the functionality of this pump. Since the MexEF-OprN RND efflux pump is involved in fluoroquinolone resistance, we next assayed bacterial sensitivity against ciprofloxacin by evaluating their MICs. As shown in Table 1, both the P. fluorescens strains were more resistant to this antibiotic after exposure to NO2 than to synthetic air. Chloramphenicol is a nitroaromatic antimicrobial that is a substrate for MexEF-OprN (Köhler et al., 1997; Sobel et al., 2005). Accordingly, NO2-exposed P. fluorescens strains MFAF76a and MFN1032 were about 2 fold more resistant to this antibiotic than synthetic air-treated bacteria (Table 1). Taken together, these data suggest a possible higher activity of this efflux pump in response to NO2 exposure. We next followed the growth of the NO2-exposed P. fluorescens strains in DMB medium containing ciprofloxacin or chloramphenicol at the higher antibiotic concentration leading to bacterial growth (Figure 5). Data were standardized with the control, the synthetic air treated cells growth. While ciprofloxacin had no effect on NO2-exposed bacteria, chloramphenicol at a concentration of 25 and 100 μg/mL for strain MFAF76a and MFN1032, respectively, led to an increase in growth for the two NO2-exposed P. fluorescens strains (Figure 5). Remarkably, the statistically significant increase of bacterial growth was maintained from 2 to 10 h, suggesting a possible NO2 protective effect that would be conserved for 8 h after exposure. Taken together, our data show that NO2 induced mexEF-oprN gene expression, and consequently increased the resistance to ciprofloxacin and chloramphenicol.


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 protects Pseudomonas fluorescens from chloramphenicol toxicity. After 2 h exposure to 45 ppm of NO2, growth of airborne MFAF76a (A) and clinical MFN1032 (B)P. fluorescens with ciprofloxacin () and chloramphenicol () was assayed. Growth curves were performed with ciprofloxacin (3.125 μg/mL for MFAF76a and 1.156 μg/mL for MFN1032) and chloramphenicol (25 and 100 μg/mL respectively), and A580 was recorded at the indicated time points. The control sample was bacteria exposed to synthetic air, and grown in presence of antibiotics in indicated concentrations. The data are shown as percentages of growth relative to synthetic 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 (*); n.s., non-significant. Dotted line shows the control (100%).
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Related In: Results  -  Collection

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Figure 5: NO2 protects Pseudomonas fluorescens from chloramphenicol toxicity. After 2 h exposure to 45 ppm of NO2, growth of airborne MFAF76a (A) and clinical MFN1032 (B)P. fluorescens with ciprofloxacin () and chloramphenicol () was assayed. Growth curves were performed with ciprofloxacin (3.125 μg/mL for MFAF76a and 1.156 μg/mL for MFN1032) and chloramphenicol (25 and 100 μg/mL respectively), and A580 was recorded at the indicated time points. The control sample was bacteria exposed to synthetic air, and grown in presence of antibiotics in indicated concentrations. The data are shown as percentages of growth relative to synthetic 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 (*); n.s., non-significant. Dotted line shows the control (100%).
Mentions: In order to study the effect of NO2 on MexEF-OprN efflux pump, the transcription levels of mexE, mexF and oprN genes (KT070324, KT070321 and KT070325 for MFAF76a; KT070323, KT070322 and KT186432 for MFN1032, respectively) were compared using qRT-PCR in two P. fluorescens strains exposed or not to 45 ppm of NO2. In airborne and clinical strains, the mexE mRNA level was increased by almost 14- and 100-fold respectively; that of mexF almost 3.5- and 47-fold respectively and that of oprN almost 4.6- and 73-fold respectively (Figure 4). These data show that NO2 promoted mexEF-oprN expression, potentially causing modifications in P. fluorescens antibiotic resistance. We next tested the functionality of this pump. Since the MexEF-OprN RND efflux pump is involved in fluoroquinolone resistance, we next assayed bacterial sensitivity against ciprofloxacin by evaluating their MICs. As shown in Table 1, both the P. fluorescens strains were more resistant to this antibiotic after exposure to NO2 than to synthetic air. Chloramphenicol is a nitroaromatic antimicrobial that is a substrate for MexEF-OprN (Köhler et al., 1997; Sobel et al., 2005). Accordingly, NO2-exposed P. fluorescens strains MFAF76a and MFN1032 were about 2 fold more resistant to this antibiotic than synthetic air-treated bacteria (Table 1). Taken together, these data suggest a possible higher activity of this efflux pump in response to NO2 exposure. We next followed the growth of the NO2-exposed P. fluorescens strains in DMB medium containing ciprofloxacin or chloramphenicol at the higher antibiotic concentration leading to bacterial growth (Figure 5). Data were standardized with the control, the synthetic air treated cells growth. While ciprofloxacin had no effect on NO2-exposed bacteria, chloramphenicol at a concentration of 25 and 100 μg/mL for strain MFAF76a and MFN1032, respectively, led to an increase in growth for the two NO2-exposed P. fluorescens strains (Figure 5). Remarkably, the statistically significant increase of bacterial growth was maintained from 2 to 10 h, suggesting a possible NO2 protective effect that would be conserved for 8 h after exposure. Taken together, our data show that NO2 induced mexEF-oprN gene expression, and consequently increased the resistance to ciprofloxacin and chloramphenicol.

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