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Proc Natl Acad Sci USA. Enhanced in vivo fitness of carbapenem-resistant oprD mutants of Pseudomonas aeruginosa revealed through high-throughput sequencing
[Source: Proceedings of the National Academy of Sciences of the United States of America, full page: (LINK). Abstract, edited.]
Enhanced in vivo fitness of carbapenem-resistant oprD mutants of Pseudomonas aeruginosa revealed through high-throughput sequencing
David Skurnik<SUP>a</SUP>,<SUP>1</SUP>,<SUP>2</SUP>, Damien Roux<SUP>a</SUP>,<SUP>1</SUP>, Vincent Cattoir<SUP>b</SUP>,<SUP>c</SUP>,<SUP>3</SUP>, Olga Danilchanka<SUP>b</SUP>,<SUP>3</SUP>, Xi Lu<SUP>a</SUP>, Deborah R. Yoder-Himes<SUP>d</SUP>, Kook Han<SUP>b</SUP>, Thomas Guillard<SUP>a</SUP>, Deming Jiang<SUP>a</SUP>, Charlotte Gaultier<SUP>a</SUP>, Fran?ois Guerin<SUP>c</SUP>, Hugues Aschard<SUP>e</SUP>, Roland Leclercq<SUP>c</SUP>, John J. Mekalanos<SUP>b</SUP>, Stephen Lory<SUP>b</SUP>, and Gerald B. Pier<SUP>a</SUP>,<SUP>2</SUP>
<SUP></SUP>
Author Affiliations: <SUP>a</SUP>Division of Infectious Diseases, Department of Medicine, Brigham and Women's Hospital, Harvard Medical School, Boston, MA 02115; <SUP>b</SUP>Department of Microbiology and Immunobiology, Harvard Medical School, Boston, MA 02115; <SUP>c</SUP>?quipe Antibior?sistance 4655, Facult? de M?decine, Universit? de Caen Basse-Normandie, F-14032 Caen, France; <SUP>d</SUP>Department of Biology, University of Louisville, Louisville, KY 40202; and <SUP>e</SUP>Department of Epidemiology, Harvard School of Public Health, Boston, MA 02115
Edited* by Frederick M. Ausubel, Harvard Medical School and Massachusetts General Hospital, Boston, MA, and approved October 11, 2013 (received for review December 11, 2012)
Significance
It is thought antibiotic resistance carries a fitness cost and reduces microbial virulence. Using high-throughput sequencing analysis of a transposon insertion bank in Pseudomonas aeruginosa, we found enhanced fitness for in vivo mucosal colonization and systemic spread of strains with transposon insertions in the oprD gene. This conferred resistance to carbapenem antibiotics as well as enhanced resistance to killing at acidic pH and by normal human serum along with increased cytotoxicity against murine macrophages. RNA-sequencing analysis revealed that oprD deficiency led to transcriptional changes in numerous genes that may contribute to the enhanced in vivo fitness observed. Thus, if carbapenem resistance develops during antibiotic therapy of P. aeruginosa infections, it may lead to enhanced fitness and virulence in infected hosts.
Abstract
An important question regarding the biologic implications of antibiotic-resistant microbes is how resistance impacts the organism?s overall fitness and virulence. Currently it is generally thought that antibiotic resistance carries a fitness cost and reduces virulence. For the human pathogen Pseudomonas aeruginosa, treatment with carbapenem antibiotics is a mainstay of therapy that can lead to the emergence of resistance, often through the loss of the carbapenem entry channel OprD. Transposon insertion-site sequencing was used to analyze the fitness of 300,000 mutants of P. aeruginosa strain PA14 in a mouse model for gut colonization and systemic dissemination after induction of neutropenia. Transposon insertions in the oprD gene led not only to carbapenem resistance but also to a dramatic increase in mucosal colonization and dissemination to the spleen. These findings were confirmed in vivo with different oprD mutants of PA14 as well as with related pairs of carbapenem-susceptible and -resistant clinical isolates. Compared with OprD<SUP>+</SUP> strains, those lacking OprD were more resistant to killing by acidic pH or normal human serum and had increased cytotoxicity against murine macrophages. RNA-sequencing analysis revealed that an oprD mutant showed dramatic changes in the transcription of genes that may contribute to the various phenotypic changes observed. The association between carbapenem resistance and enhanced survival of P. aeruginosa in infected murine hosts suggests that either drug resistance or host colonization can cause the emergence of more pathogenic, drug-resistant P. aeruginosa clones in a single genetic event.
Footnotes
<SUP>1</SUP>D.S. and D.R. contributed equally to this work.
<SUP>2</SUP>To whom correspondence may be addressed. E-mail: dskurnik@rics.bwh.harvard.edu or gpier@rics.bwh.harvard.edu.
<SUP>3</SUP>V.C. and O.D. contributed equally to this work.
Author contributions: D.S., D.R., V.C., O.D., and G.B.P. designed research; D.S., D.R., V.C., O.D., X.L., D.R.Y.-H., K.H., T.G., D.J., C.G., and F.G. performed research; D.S., D.R., O.D., H.A., R.L., J.J.M., S.L., and G.B.P. analyzed data; and D.S. and G.B.P. wrote the paper.
The authors declare no conflict of interest.
*This Direct Submission article had a prearranged editor.
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1221552110/-/DCSupplemental.
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Enhanced in vivo fitness of carbapenem-resistant oprD mutants of Pseudomonas aeruginosa revealed through high-throughput sequencing
David Skurnik<SUP>a</SUP>,<SUP>1</SUP>,<SUP>2</SUP>, Damien Roux<SUP>a</SUP>,<SUP>1</SUP>, Vincent Cattoir<SUP>b</SUP>,<SUP>c</SUP>,<SUP>3</SUP>, Olga Danilchanka<SUP>b</SUP>,<SUP>3</SUP>, Xi Lu<SUP>a</SUP>, Deborah R. Yoder-Himes<SUP>d</SUP>, Kook Han<SUP>b</SUP>, Thomas Guillard<SUP>a</SUP>, Deming Jiang<SUP>a</SUP>, Charlotte Gaultier<SUP>a</SUP>, Fran?ois Guerin<SUP>c</SUP>, Hugues Aschard<SUP>e</SUP>, Roland Leclercq<SUP>c</SUP>, John J. Mekalanos<SUP>b</SUP>, Stephen Lory<SUP>b</SUP>, and Gerald B. Pier<SUP>a</SUP>,<SUP>2</SUP>
<SUP></SUP>
Author Affiliations: <SUP>a</SUP>Division of Infectious Diseases, Department of Medicine, Brigham and Women's Hospital, Harvard Medical School, Boston, MA 02115; <SUP>b</SUP>Department of Microbiology and Immunobiology, Harvard Medical School, Boston, MA 02115; <SUP>c</SUP>?quipe Antibior?sistance 4655, Facult? de M?decine, Universit? de Caen Basse-Normandie, F-14032 Caen, France; <SUP>d</SUP>Department of Biology, University of Louisville, Louisville, KY 40202; and <SUP>e</SUP>Department of Epidemiology, Harvard School of Public Health, Boston, MA 02115
Edited* by Frederick M. Ausubel, Harvard Medical School and Massachusetts General Hospital, Boston, MA, and approved October 11, 2013 (received for review December 11, 2012)
Significance
It is thought antibiotic resistance carries a fitness cost and reduces microbial virulence. Using high-throughput sequencing analysis of a transposon insertion bank in Pseudomonas aeruginosa, we found enhanced fitness for in vivo mucosal colonization and systemic spread of strains with transposon insertions in the oprD gene. This conferred resistance to carbapenem antibiotics as well as enhanced resistance to killing at acidic pH and by normal human serum along with increased cytotoxicity against murine macrophages. RNA-sequencing analysis revealed that oprD deficiency led to transcriptional changes in numerous genes that may contribute to the enhanced in vivo fitness observed. Thus, if carbapenem resistance develops during antibiotic therapy of P. aeruginosa infections, it may lead to enhanced fitness and virulence in infected hosts.
Abstract
An important question regarding the biologic implications of antibiotic-resistant microbes is how resistance impacts the organism?s overall fitness and virulence. Currently it is generally thought that antibiotic resistance carries a fitness cost and reduces virulence. For the human pathogen Pseudomonas aeruginosa, treatment with carbapenem antibiotics is a mainstay of therapy that can lead to the emergence of resistance, often through the loss of the carbapenem entry channel OprD. Transposon insertion-site sequencing was used to analyze the fitness of 300,000 mutants of P. aeruginosa strain PA14 in a mouse model for gut colonization and systemic dissemination after induction of neutropenia. Transposon insertions in the oprD gene led not only to carbapenem resistance but also to a dramatic increase in mucosal colonization and dissemination to the spleen. These findings were confirmed in vivo with different oprD mutants of PA14 as well as with related pairs of carbapenem-susceptible and -resistant clinical isolates. Compared with OprD<SUP>+</SUP> strains, those lacking OprD were more resistant to killing by acidic pH or normal human serum and had increased cytotoxicity against murine macrophages. RNA-sequencing analysis revealed that an oprD mutant showed dramatic changes in the transcription of genes that may contribute to the various phenotypic changes observed. The association between carbapenem resistance and enhanced survival of P. aeruginosa in infected murine hosts suggests that either drug resistance or host colonization can cause the emergence of more pathogenic, drug-resistant P. aeruginosa clones in a single genetic event.
Footnotes
<SUP>1</SUP>D.S. and D.R. contributed equally to this work.
<SUP>2</SUP>To whom correspondence may be addressed. E-mail: dskurnik@rics.bwh.harvard.edu or gpier@rics.bwh.harvard.edu.
<SUP>3</SUP>V.C. and O.D. contributed equally to this work.
Author contributions: D.S., D.R., V.C., O.D., and G.B.P. designed research; D.S., D.R., V.C., O.D., X.L., D.R.Y.-H., K.H., T.G., D.J., C.G., and F.G. performed research; D.S., D.R., O.D., H.A., R.L., J.J.M., S.L., and G.B.P. analyzed data; and D.S. and G.B.P. wrote the paper.
The authors declare no conflict of interest.
*This Direct Submission article had a prearranged editor.
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1221552110/-/DCSupplemental.
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