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Lung Microbiota Changes Associated with Chronic Pseudomonas aeruginosa Lung Infection and the Impact of Intravenous Colistimethate Sodium.

Collie D, Glendinning L, Govan J, Wright S, Thornton E, Tennant P, Doherty C, McLachlan G - PLoS ONE (2015)

Bottom Line: Such exacerbations are treated with antibiotics, which generally lead to an improvement in lung function and reduced sputum P. aeruginosa density.With only one exception the reduction was seen in both direct and remote lung segments.This reduction, coupled with generally increasing or stable levels of Pseudomonadales, meant that the proportion of the latter relative to total Gram negative bacteria increased in all bar one direct and one remote lung segment.

View Article: PubMed Central - PubMed

Affiliation: The Roslin Institute and Royal (Dick) School of Veterinary Studies, University of Edinburgh, Edinburgh, United Kingdom.

ABSTRACT

Background: Exacerbations associated with chronic lung infection with Pseudomonas aeruginosa are a major contributor to morbidity, mortality and premature death in cystic fibrosis. Such exacerbations are treated with antibiotics, which generally lead to an improvement in lung function and reduced sputum P. aeruginosa density. This potentially suggests a role for the latter in the pathogenesis of exacerbations. However, other data suggesting that changes in P. aeruginosa sputum culture status may not reliably predict an improvement in clinical status, and data indicating no significant changes in either total bacterial counts or in P. aeruginosa numbers in sputum samples collected prior to pulmonary exacerbation sheds doubt on this assumption. We used our recently developed lung segmental model of chronic Pseudomonas infection in sheep to investigate the lung microbiota changes associated with chronic P. aeruginosa lung infection and the impact of systemic therapy with colistimethate sodium (CMS).

Methodology/principal findings: We collected protected specimen brush (PSB) samples from sheep (n = 8) both prior to and 14 days after establishment of chronic local lung infection with P aeruginosa. Samples were taken from both directly infected lung segments (direct) and segments spatially remote to such sites (remote). Four sheep were treated with daily intravenous injections of CMS between days 7 and 14, and four were treated with a placebo. Necropsy examination at d14 confirmed the presence of chronic local lung infection and lung pathology in every direct lung segment. The predominant orders in lung microbiota communities before infection were Bacillales, Actinomycetales and Clostridiales. While lung microbiota samples were more likely to share similarities with other samples derived from the same lung, considerable within- and between-animal heterogeneity could be appreciated. Pseudomonadales joined the aforementioned list of predominant orders in lung microbiota communities after infection. Whilst treatment with CMS appeared to have little impact on microbial community composition after infection, or the change undergone by communities in reaching that state, when Gram negative organisms (excluding Pseudomonadales) were considered together as a group there was a significant decrease in their relative proportion that was only observed in the sheep treated with CMS. With only one exception the reduction was seen in both direct and remote lung segments. This reduction, coupled with generally increasing or stable levels of Pseudomonadales, meant that the proportion of the latter relative to total Gram negative bacteria increased in all bar one direct and one remote lung segment.

Conclusions/significance: The proportional increase in Pseudomonadales relative to other Gram negative bacteria in the lungs of sheep treated with systemic CMS highlights the potential for such therapies to inadvertently select or create a niche for bacteria seeding from a persistent source of chronic infection.

No MeSH data available.


Related in: MedlinePlus

Microbiota sampling protocol.During a baseline examination lung health was confirmed in the form of direct visualisation of the airway tree and later cytological analysis of bronchoalveolar lavage fluid (BALF). Protected specimen brush (PSB) samples were collected from the segmental bronchus serving the right apical (RA) lobe, the first ventral diaphragmatic (RVD1) segment of the right caudal diaphragmatic lobe, and the left cardiac (LC) segment of the apicocardiac lobe (Pre-samples). BALF was subsequently collected from RA. At least two weeks later P. aeruginosa agar beads were instilled into the right cardiac (RC) lobe, the second ventral diaphragmatic (RVD2) segment of the right caudal diaphragmatic lobe, the left cardiac (LC) segment of the apicocardiac lobe, and the second ventral diaphragmatic (LVD2) segment of the left caudal diaphragmatic lobe. Three days later these instillations were repeated. Four days after the second instillation sheep were randomly assigned to daily intravenous injections of either CMS or placebo. Eleven days after the second instillation–after one week of daily injections—PSB specimens were obtained from the previously sampled lobes (RA, RVD1 and LC) (Post-samples). After the sheep was killed and the lungs removed for further analysis, BALF was derived from each segment under study (RA, RC, RVD1, RVD2, LC and LVD2) prior to further dissection, sampling and recording.
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pone.0142097.g001: Microbiota sampling protocol.During a baseline examination lung health was confirmed in the form of direct visualisation of the airway tree and later cytological analysis of bronchoalveolar lavage fluid (BALF). Protected specimen brush (PSB) samples were collected from the segmental bronchus serving the right apical (RA) lobe, the first ventral diaphragmatic (RVD1) segment of the right caudal diaphragmatic lobe, and the left cardiac (LC) segment of the apicocardiac lobe (Pre-samples). BALF was subsequently collected from RA. At least two weeks later P. aeruginosa agar beads were instilled into the right cardiac (RC) lobe, the second ventral diaphragmatic (RVD2) segment of the right caudal diaphragmatic lobe, the left cardiac (LC) segment of the apicocardiac lobe, and the second ventral diaphragmatic (LVD2) segment of the left caudal diaphragmatic lobe. Three days later these instillations were repeated. Four days after the second instillation sheep were randomly assigned to daily intravenous injections of either CMS or placebo. Eleven days after the second instillation–after one week of daily injections—PSB specimens were obtained from the previously sampled lobes (RA, RVD1 and LC) (Post-samples). After the sheep was killed and the lungs removed for further analysis, BALF was derived from each segment under study (RA, RC, RVD1, RVD2, LC and LVD2) prior to further dissection, sampling and recording.

Mentions: A baseline examination was conducted in which each sheep was subject to clinical examination and a blood sample taken from the jugular vein for routine haematological analysis. Thereafter each sheep was anaesthetised to facilitate bronchoscopic examination and sample collection according to standard protocol [16]. During this examination lung health was confirmed in the form of direct visualisation of the airway tree and later cytological analysis of bronchoalveolar lavage fluid (BALF). Protected specimen brush (PSB) samples were collected from the segmental bronchus serving the right apical (RA) lobe, the first ventral diaphragmatic (RVD1) segment of the right caudal diaphragmatic lobe, and the left cardiac (LC) segment of the apicocardiac lobe (Fig 1). BALF was subsequently collected from RA. After a recovery period of not less than two weeks (23 [15–34] days), the sheep were re-anaesthetised and Pseudomonas in agar beads (2.5x109 cfu in 2.5ml) instilled into the right cardiac (RC) lobe, the second ventral diaphragmatic (RVD2) segment of the right caudal diaphragmatic lobe, the left cardiac (LC) segment of the apicocardiac lobe, and the second ventral diaphragmatic (LVD2) segment of the left caudal diaphragmatic lobe. The method of instillation followed that previously described [16]. Three days later the sheep were blood sampled, anaesthetised again, and these instillations repeated. Four days after the second instillation the sheep assigned to the CMS group commenced daily treatment with intravenously administered systemic antibiotic (an intravenous dose of 50,000 international units (IU) kg-1 of colistimethate sodium (Colomycin®, Forest Laboratories UK Ltd, Dartford, Kent) every 24 h), with the placebo group commencing daily injections of saline. Eleven days after the second instillation–after one week of daily injections—blood samples were acquired before the sheep were anaesthetised and PSB specimens obtained from the previously sampled lobes (RA, RVD1 and LC). The sheep were then euthanized by intravenous injection of baribiturate, and the heart and lungs carefully removed from the carcase following standard necropsy protocols before the heart was dissected away and the lungs presented for further sampling and analysis. BALF was derived from each segment under study (RA, RC, RVD1, RVD2, LC and LVD2) prior to further dissection, sampling and recording using previously described methodology [16]. This experimental protocol and sampling scheme therefore allowed us to evaluate the effects of local lung infection with P aer., both within the direct segments (RC, LC, RVD2 and LVD2), within non-infected segments remote to the sites of direct infection (RA and RVD1), and systemically.


Lung Microbiota Changes Associated with Chronic Pseudomonas aeruginosa Lung Infection and the Impact of Intravenous Colistimethate Sodium.

Collie D, Glendinning L, Govan J, Wright S, Thornton E, Tennant P, Doherty C, McLachlan G - PLoS ONE (2015)

Microbiota sampling protocol.During a baseline examination lung health was confirmed in the form of direct visualisation of the airway tree and later cytological analysis of bronchoalveolar lavage fluid (BALF). Protected specimen brush (PSB) samples were collected from the segmental bronchus serving the right apical (RA) lobe, the first ventral diaphragmatic (RVD1) segment of the right caudal diaphragmatic lobe, and the left cardiac (LC) segment of the apicocardiac lobe (Pre-samples). BALF was subsequently collected from RA. At least two weeks later P. aeruginosa agar beads were instilled into the right cardiac (RC) lobe, the second ventral diaphragmatic (RVD2) segment of the right caudal diaphragmatic lobe, the left cardiac (LC) segment of the apicocardiac lobe, and the second ventral diaphragmatic (LVD2) segment of the left caudal diaphragmatic lobe. Three days later these instillations were repeated. Four days after the second instillation sheep were randomly assigned to daily intravenous injections of either CMS or placebo. Eleven days after the second instillation–after one week of daily injections—PSB specimens were obtained from the previously sampled lobes (RA, RVD1 and LC) (Post-samples). After the sheep was killed and the lungs removed for further analysis, BALF was derived from each segment under study (RA, RC, RVD1, RVD2, LC and LVD2) prior to further dissection, sampling and recording.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4636361&req=5

pone.0142097.g001: Microbiota sampling protocol.During a baseline examination lung health was confirmed in the form of direct visualisation of the airway tree and later cytological analysis of bronchoalveolar lavage fluid (BALF). Protected specimen brush (PSB) samples were collected from the segmental bronchus serving the right apical (RA) lobe, the first ventral diaphragmatic (RVD1) segment of the right caudal diaphragmatic lobe, and the left cardiac (LC) segment of the apicocardiac lobe (Pre-samples). BALF was subsequently collected from RA. At least two weeks later P. aeruginosa agar beads were instilled into the right cardiac (RC) lobe, the second ventral diaphragmatic (RVD2) segment of the right caudal diaphragmatic lobe, the left cardiac (LC) segment of the apicocardiac lobe, and the second ventral diaphragmatic (LVD2) segment of the left caudal diaphragmatic lobe. Three days later these instillations were repeated. Four days after the second instillation sheep were randomly assigned to daily intravenous injections of either CMS or placebo. Eleven days after the second instillation–after one week of daily injections—PSB specimens were obtained from the previously sampled lobes (RA, RVD1 and LC) (Post-samples). After the sheep was killed and the lungs removed for further analysis, BALF was derived from each segment under study (RA, RC, RVD1, RVD2, LC and LVD2) prior to further dissection, sampling and recording.
Mentions: A baseline examination was conducted in which each sheep was subject to clinical examination and a blood sample taken from the jugular vein for routine haematological analysis. Thereafter each sheep was anaesthetised to facilitate bronchoscopic examination and sample collection according to standard protocol [16]. During this examination lung health was confirmed in the form of direct visualisation of the airway tree and later cytological analysis of bronchoalveolar lavage fluid (BALF). Protected specimen brush (PSB) samples were collected from the segmental bronchus serving the right apical (RA) lobe, the first ventral diaphragmatic (RVD1) segment of the right caudal diaphragmatic lobe, and the left cardiac (LC) segment of the apicocardiac lobe (Fig 1). BALF was subsequently collected from RA. After a recovery period of not less than two weeks (23 [15–34] days), the sheep were re-anaesthetised and Pseudomonas in agar beads (2.5x109 cfu in 2.5ml) instilled into the right cardiac (RC) lobe, the second ventral diaphragmatic (RVD2) segment of the right caudal diaphragmatic lobe, the left cardiac (LC) segment of the apicocardiac lobe, and the second ventral diaphragmatic (LVD2) segment of the left caudal diaphragmatic lobe. The method of instillation followed that previously described [16]. Three days later the sheep were blood sampled, anaesthetised again, and these instillations repeated. Four days after the second instillation the sheep assigned to the CMS group commenced daily treatment with intravenously administered systemic antibiotic (an intravenous dose of 50,000 international units (IU) kg-1 of colistimethate sodium (Colomycin®, Forest Laboratories UK Ltd, Dartford, Kent) every 24 h), with the placebo group commencing daily injections of saline. Eleven days after the second instillation–after one week of daily injections—blood samples were acquired before the sheep were anaesthetised and PSB specimens obtained from the previously sampled lobes (RA, RVD1 and LC). The sheep were then euthanized by intravenous injection of baribiturate, and the heart and lungs carefully removed from the carcase following standard necropsy protocols before the heart was dissected away and the lungs presented for further sampling and analysis. BALF was derived from each segment under study (RA, RC, RVD1, RVD2, LC and LVD2) prior to further dissection, sampling and recording using previously described methodology [16]. This experimental protocol and sampling scheme therefore allowed us to evaluate the effects of local lung infection with P aer., both within the direct segments (RC, LC, RVD2 and LVD2), within non-infected segments remote to the sites of direct infection (RA and RVD1), and systemically.

Bottom Line: Such exacerbations are treated with antibiotics, which generally lead to an improvement in lung function and reduced sputum P. aeruginosa density.With only one exception the reduction was seen in both direct and remote lung segments.This reduction, coupled with generally increasing or stable levels of Pseudomonadales, meant that the proportion of the latter relative to total Gram negative bacteria increased in all bar one direct and one remote lung segment.

View Article: PubMed Central - PubMed

Affiliation: The Roslin Institute and Royal (Dick) School of Veterinary Studies, University of Edinburgh, Edinburgh, United Kingdom.

ABSTRACT

Background: Exacerbations associated with chronic lung infection with Pseudomonas aeruginosa are a major contributor to morbidity, mortality and premature death in cystic fibrosis. Such exacerbations are treated with antibiotics, which generally lead to an improvement in lung function and reduced sputum P. aeruginosa density. This potentially suggests a role for the latter in the pathogenesis of exacerbations. However, other data suggesting that changes in P. aeruginosa sputum culture status may not reliably predict an improvement in clinical status, and data indicating no significant changes in either total bacterial counts or in P. aeruginosa numbers in sputum samples collected prior to pulmonary exacerbation sheds doubt on this assumption. We used our recently developed lung segmental model of chronic Pseudomonas infection in sheep to investigate the lung microbiota changes associated with chronic P. aeruginosa lung infection and the impact of systemic therapy with colistimethate sodium (CMS).

Methodology/principal findings: We collected protected specimen brush (PSB) samples from sheep (n = 8) both prior to and 14 days after establishment of chronic local lung infection with P aeruginosa. Samples were taken from both directly infected lung segments (direct) and segments spatially remote to such sites (remote). Four sheep were treated with daily intravenous injections of CMS between days 7 and 14, and four were treated with a placebo. Necropsy examination at d14 confirmed the presence of chronic local lung infection and lung pathology in every direct lung segment. The predominant orders in lung microbiota communities before infection were Bacillales, Actinomycetales and Clostridiales. While lung microbiota samples were more likely to share similarities with other samples derived from the same lung, considerable within- and between-animal heterogeneity could be appreciated. Pseudomonadales joined the aforementioned list of predominant orders in lung microbiota communities after infection. Whilst treatment with CMS appeared to have little impact on microbial community composition after infection, or the change undergone by communities in reaching that state, when Gram negative organisms (excluding Pseudomonadales) were considered together as a group there was a significant decrease in their relative proportion that was only observed in the sheep treated with CMS. With only one exception the reduction was seen in both direct and remote lung segments. This reduction, coupled with generally increasing or stable levels of Pseudomonadales, meant that the proportion of the latter relative to total Gram negative bacteria increased in all bar one direct and one remote lung segment.

Conclusions/significance: The proportional increase in Pseudomonadales relative to other Gram negative bacteria in the lungs of sheep treated with systemic CMS highlights the potential for such therapies to inadvertently select or create a niche for bacteria seeding from a persistent source of chronic infection.

No MeSH data available.


Related in: MedlinePlus