lptG contributes to changes in membrane permeability and the emergence of multidrug hypersusceptibility in a cystic fibrosis isolate of Pseudomonas aeruginosa
Abstract Purpose In the lungs of cystic fibrosis patients, Pseudomonas aeruginosa is exposed to a myriad of antibiotics leading to alterations in antibiotic susceptibility. This study identifies mutations resulting in hypersusceptibility in isogenic mutants of a P. aeruginosa clinical isolate, PA34....
| Main Authors: | , , , , |
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| Format: | Article |
| Language: | English |
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Wiley
2019-11-01
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| Series: | MicrobiologyOpen |
| Subjects: | |
| Online Access: | https://doi.org/10.1002/mbo3.844 |
| _version_ | 1819111417274433536 |
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| author | Lucas B. Harrison Randal C. Fowler Baha Abdalhamid Anna Selmecki Nancy D. Hanson |
| author_facet | Lucas B. Harrison Randal C. Fowler Baha Abdalhamid Anna Selmecki Nancy D. Hanson |
| author_sort | Lucas B. Harrison |
| collection | DOAJ |
| description | Abstract Purpose In the lungs of cystic fibrosis patients, Pseudomonas aeruginosa is exposed to a myriad of antibiotics leading to alterations in antibiotic susceptibility. This study identifies mutations resulting in hypersusceptibility in isogenic mutants of a P. aeruginosa clinical isolate, PA34. Methods PA34 was exposed to subinhibitory concentrations of doripenem or meropenem during growth to mid‐log phase. Antibiotic susceptibility of surviving colonies was determined by agar dilution. Two carbapenem‐resistant colonies hypersusceptible to non‐carbapenem antibiotics were selected for further analysis. Antibiotic resistance gene expression was evaluated by RT‐rtPCR and OprD production by SDS‐PAGE. PA34 and isogenic mutants were evaluated with whole genome sequencing. Sequence variants were confirmed by Sanger sequencing, and cognate genes in eight carbapenem‐resistant clinical isolates hypersusceptible to non‐carbapenem antibiotics were sequenced. Lipopolysaccharide preparations of PA34 and hypersusceptible mutants were evaluated with ProQ‐Emerald stain. Results Isogenic mutants showed 4‐ to 8‐fold MIC increase for imipenem, meropenem, and doripenem. However, they were hypersusceptible (≥4‐fold MIC decrease) to aminoglycosides, fluoroquinolones, and non‐carbapenem β‐lactams. Expression of ampC or mex‐opr efflux pumps was unchanged, but OprD production was decreased. Mutations causing Q86H AlgU and G77C LptG amino acid substitutions and nonsense mutations within OprD were observed in both mutants. Lipopolysaccharide modifications were observed between isogenic mutants and PA34. Non‐synonymous mutations in LptF or LptG were observed in 6/8 hypersusceptible clinical isolates resistant to carbapenem antibiotics. Conclusion Evaluation of hypersusceptible mutants identified the association between lptG and a hypersusceptible phenotype. Modifications in lipopolysaccharide profiles suggests LptG modification interferes with lipopolysaccharide transport and contributes to hypersusceptibility. |
| first_indexed | 2024-12-22T03:57:17Z |
| format | Article |
| id | doaj.art-1165e968cbc74244b6e154498d4ed19c |
| institution | Directory Open Access Journal |
| issn | 2045-8827 |
| language | English |
| last_indexed | 2024-12-22T03:57:17Z |
| publishDate | 2019-11-01 |
| publisher | Wiley |
| record_format | Article |
| series | MicrobiologyOpen |
| spelling | doaj.art-1165e968cbc74244b6e154498d4ed19c2022-12-21T18:39:50ZengWileyMicrobiologyOpen2045-88272019-11-01811n/an/a10.1002/mbo3.844lptG contributes to changes in membrane permeability and the emergence of multidrug hypersusceptibility in a cystic fibrosis isolate of Pseudomonas aeruginosaLucas B. Harrison0Randal C. Fowler1Baha Abdalhamid2Anna Selmecki3Nancy D. Hanson4Department of Medical Microbiology and Immunology Creighton University Omaha NebraskaDepartment of Medical Microbiology and Immunology Creighton University Omaha NebraskaDepartment of Pathology and Microbiology University of Nebraska Medical Center OmahaDepartment of Medical Microbiology and Immunology Creighton University Omaha NebraskaDepartment of Medical Microbiology and Immunology Creighton University Omaha NebraskaAbstract Purpose In the lungs of cystic fibrosis patients, Pseudomonas aeruginosa is exposed to a myriad of antibiotics leading to alterations in antibiotic susceptibility. This study identifies mutations resulting in hypersusceptibility in isogenic mutants of a P. aeruginosa clinical isolate, PA34. Methods PA34 was exposed to subinhibitory concentrations of doripenem or meropenem during growth to mid‐log phase. Antibiotic susceptibility of surviving colonies was determined by agar dilution. Two carbapenem‐resistant colonies hypersusceptible to non‐carbapenem antibiotics were selected for further analysis. Antibiotic resistance gene expression was evaluated by RT‐rtPCR and OprD production by SDS‐PAGE. PA34 and isogenic mutants were evaluated with whole genome sequencing. Sequence variants were confirmed by Sanger sequencing, and cognate genes in eight carbapenem‐resistant clinical isolates hypersusceptible to non‐carbapenem antibiotics were sequenced. Lipopolysaccharide preparations of PA34 and hypersusceptible mutants were evaluated with ProQ‐Emerald stain. Results Isogenic mutants showed 4‐ to 8‐fold MIC increase for imipenem, meropenem, and doripenem. However, they were hypersusceptible (≥4‐fold MIC decrease) to aminoglycosides, fluoroquinolones, and non‐carbapenem β‐lactams. Expression of ampC or mex‐opr efflux pumps was unchanged, but OprD production was decreased. Mutations causing Q86H AlgU and G77C LptG amino acid substitutions and nonsense mutations within OprD were observed in both mutants. Lipopolysaccharide modifications were observed between isogenic mutants and PA34. Non‐synonymous mutations in LptF or LptG were observed in 6/8 hypersusceptible clinical isolates resistant to carbapenem antibiotics. Conclusion Evaluation of hypersusceptible mutants identified the association between lptG and a hypersusceptible phenotype. Modifications in lipopolysaccharide profiles suggests LptG modification interferes with lipopolysaccharide transport and contributes to hypersusceptibility.https://doi.org/10.1002/mbo3.844carbapenem resistancecystic fibrosishypersusceptibilitylipopolysaccharideslptGsubinhibitory |
| spellingShingle | Lucas B. Harrison Randal C. Fowler Baha Abdalhamid Anna Selmecki Nancy D. Hanson lptG contributes to changes in membrane permeability and the emergence of multidrug hypersusceptibility in a cystic fibrosis isolate of Pseudomonas aeruginosa MicrobiologyOpen carbapenem resistance cystic fibrosis hypersusceptibility lipopolysaccharides lptG subinhibitory |
| title | lptG contributes to changes in membrane permeability and the emergence of multidrug hypersusceptibility in a cystic fibrosis isolate of Pseudomonas aeruginosa |
| title_full | lptG contributes to changes in membrane permeability and the emergence of multidrug hypersusceptibility in a cystic fibrosis isolate of Pseudomonas aeruginosa |
| title_fullStr | lptG contributes to changes in membrane permeability and the emergence of multidrug hypersusceptibility in a cystic fibrosis isolate of Pseudomonas aeruginosa |
| title_full_unstemmed | lptG contributes to changes in membrane permeability and the emergence of multidrug hypersusceptibility in a cystic fibrosis isolate of Pseudomonas aeruginosa |
| title_short | lptG contributes to changes in membrane permeability and the emergence of multidrug hypersusceptibility in a cystic fibrosis isolate of Pseudomonas aeruginosa |
| title_sort | lptg contributes to changes in membrane permeability and the emergence of multidrug hypersusceptibility in a cystic fibrosis isolate of pseudomonas aeruginosa |
| topic | carbapenem resistance cystic fibrosis hypersusceptibility lipopolysaccharides lptG subinhibitory |
| url | https://doi.org/10.1002/mbo3.844 |
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