Diversity of regulatory systems and their contribution to colistin and tigecycline resistance in Acinetobacter baumannii
Diversity of regulatory systems and their contribution to colistin and tigecycline resistance in Acinetobacter baumannii
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DissertationDate of Exam
14.10.2021Date of Publication
06.12.2021Advisor
Seifert, HaraldCo-Referee
Deppenmeier, UweMetadata
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Abstract
Acinetobacter baumannii is a nosocomial pathogen which causes a wide range of serious infections in critically ill patients in the intensive care unit. Besides its high desiccation tolerance, the ability to form biofilms, and multiple other virulence factors, A. baumannii can acquire and upregulate antimicrobial resistance determinants to overcome all known antimicrobial drugs, so that multidrug- and even pandrug-resistant isolates have emerged.
This thesis focuses on the investigation of tigecycline and colistin resistance mechanisms in clinical multidrug-resistant A. baumannii isolates as part of the MagicBullet clinical trial. An algorithm to identify genetic differences associated with antimicrobial resistance based on whole-genome sequences was created and resulted in the identification of several novel mutations in tigecycline and colistin resistance determinants, as well as a more in-depth analysis of the bacterial resistome and the prevalence of mutations and/or resistance mechanisms. Further analysis of these mutations and resistance mechanisms was conducted by measuring gene expression, creating gene knockouts, using an in vivo infection model, and mass spectrometry.
In the first part of this thesis, tigecycline resistance mechanisms were investigated with a focus on the resistance-nodulation-cell division (RND) efflux pumps and with an emphasis on their regulators adeRS, adeN and adeL. The frequent disruption of the regulators adeS and adeN by insertion sequences (e.g. ISAba1) was revealed and was often associated with an increase in efflux pump expression, efflux activity, and virulence in the Galleria mellonella infection model as well as reduced susceptibility to several antimicrobial classes. Furthermore, three different nucleotide deletions, a nucleotide insertion, and a premature stop codon were identified in adeN, while several point mutations leading to amino acid substitutions in adeRS, adeN and adeL were also found to be associated with reduced tigecycline susceptibility in this thesis.
In the second part of this thesis, colistin resistance mechanisms were analysed in A. baumannii, focusing on the pmrCAB operon and the pmrC-homologue eptA, which are known to be involved in the modification of lipid A in the bacterial outer membrane. Novel mutations in pmrCAB were detected and were associated with elevated pmrC expression, unchanged fitness, and increased virulence in G. mellonella, but not necessarily with lipid A modification and a decrease in colistin susceptibility. This suggests the involvement of other yet unknown factors in colistin resistance. Moreover, first indications were found in this study that A. baumannii acquired pmrCAB mutations by homologous recombination across different clonal lineages. The presence of eptA was lineage-specific and often associated with insertion sequences in the proximity of the gene, however no correlation with eptA overexpression and lipid A modification or reduced colistin susceptibility was observed.
Combining the results of both parts of this thesis, several findings were made in both tigecycline and colistin resistance mechanisms in A. baumannii. First, both the regulatory genes adeR and pmrB harbour mutational hotspots, which might serve as genetic marker for the prediction of bacterial antimicrobial resistance and the virulence phenotype. Second, an additive effect of mutations is suggested, as a combination of different mutations in adeRS or pmrAB often resulted in reduced tigecycline or colistin susceptibility. Finally, mutations in adeRS or pmrAB can increase bacterial virulence and pathogenicity in G. mellonella.
In conclusion, tigecycline and colistin resistance mechanisms are diverse and multifactorial, with several factors often working in tandem to enable resistance against antimicrobial agents. Both two-component systems adeRS and pmrAB are among the most frequently mutated genes, indicating their importance in the development of antimicrobial resistance in A. baumannii. The use of a genomic approach highlights the importance and potential of whole-genome sequencing as a tool in the study of antimicrobial resistance in clinically relevant pathogens, but also revealed that a genotype does not always correlate with the predicted phenotype. Additional factors (e.g. environmental factors) and clonal lineage specific polymorphisms need to be considered when investigating novel mutations and resistance mechanisms, respectively. Overall, this thesis opens a window into the complexity of antimicrobial resistance mechanisms in A. baumannii, but future work is needed to understand them more comprehensively.
This thesis focuses on the investigation of tigecycline and colistin resistance mechanisms in clinical multidrug-resistant A. baumannii isolates as part of the MagicBullet clinical trial. An algorithm to identify genetic differences associated with antimicrobial resistance based on whole-genome sequences was created and resulted in the identification of several novel mutations in tigecycline and colistin resistance determinants, as well as a more in-depth analysis of the bacterial resistome and the prevalence of mutations and/or resistance mechanisms. Further analysis of these mutations and resistance mechanisms was conducted by measuring gene expression, creating gene knockouts, using an in vivo infection model, and mass spectrometry.
In the first part of this thesis, tigecycline resistance mechanisms were investigated with a focus on the resistance-nodulation-cell division (RND) efflux pumps and with an emphasis on their regulators adeRS, adeN and adeL. The frequent disruption of the regulators adeS and adeN by insertion sequences (e.g. ISAba1) was revealed and was often associated with an increase in efflux pump expression, efflux activity, and virulence in the Galleria mellonella infection model as well as reduced susceptibility to several antimicrobial classes. Furthermore, three different nucleotide deletions, a nucleotide insertion, and a premature stop codon were identified in adeN, while several point mutations leading to amino acid substitutions in adeRS, adeN and adeL were also found to be associated with reduced tigecycline susceptibility in this thesis.
In the second part of this thesis, colistin resistance mechanisms were analysed in A. baumannii, focusing on the pmrCAB operon and the pmrC-homologue eptA, which are known to be involved in the modification of lipid A in the bacterial outer membrane. Novel mutations in pmrCAB were detected and were associated with elevated pmrC expression, unchanged fitness, and increased virulence in G. mellonella, but not necessarily with lipid A modification and a decrease in colistin susceptibility. This suggests the involvement of other yet unknown factors in colistin resistance. Moreover, first indications were found in this study that A. baumannii acquired pmrCAB mutations by homologous recombination across different clonal lineages. The presence of eptA was lineage-specific and often associated with insertion sequences in the proximity of the gene, however no correlation with eptA overexpression and lipid A modification or reduced colistin susceptibility was observed.
Combining the results of both parts of this thesis, several findings were made in both tigecycline and colistin resistance mechanisms in A. baumannii. First, both the regulatory genes adeR and pmrB harbour mutational hotspots, which might serve as genetic marker for the prediction of bacterial antimicrobial resistance and the virulence phenotype. Second, an additive effect of mutations is suggested, as a combination of different mutations in adeRS or pmrAB often resulted in reduced tigecycline or colistin susceptibility. Finally, mutations in adeRS or pmrAB can increase bacterial virulence and pathogenicity in G. mellonella.
In conclusion, tigecycline and colistin resistance mechanisms are diverse and multifactorial, with several factors often working in tandem to enable resistance against antimicrobial agents. Both two-component systems adeRS and pmrAB are among the most frequently mutated genes, indicating their importance in the development of antimicrobial resistance in A. baumannii. The use of a genomic approach highlights the importance and potential of whole-genome sequencing as a tool in the study of antimicrobial resistance in clinically relevant pathogens, but also revealed that a genotype does not always correlate with the predicted phenotype. Additional factors (e.g. environmental factors) and clonal lineage specific polymorphisms need to be considered when investigating novel mutations and resistance mechanisms, respectively. Overall, this thesis opens a window into the complexity of antimicrobial resistance mechanisms in A. baumannii, but future work is needed to understand them more comprehensively.
Subjects
Acinetobacter baumannii, Tigecyclin, Colistin, Genomanalyse, Virulenz, Effluxpumpen, Antibiotikaresistenz, Colistin, Tigecycline, Acinetobacter baumannii, antimicrobial resistance, genome analysis, virulence, efflux pumps
Classification (DDC)
570 Biowissenschaften, BiologieGerson, Stefanie: Diversity of regulatory systems and their contribution to colistin and tigecycline resistance in Acinetobacter baumannii. - Bonn, 2021. - Dissertation, Rheinische Friedrich-Wilhelms-Universität Bonn.
Online-Ausgabe in bonndoc: https://nbn-resolving.org/urn:nbn:de:hbz:5-64741
Online-Ausgabe in bonndoc: https://nbn-resolving.org/urn:nbn:de:hbz:5-64741
@phdthesis{handle:20.500.11811/9448,
urn: https://nbn-resolving.org/urn:nbn:de:hbz:5-64741,
author = {{Stefanie Gerson}},
title = {Diversity of regulatory systems and their contribution to colistin and tigecycline resistance in Acinetobacter baumannii},
school = {Rheinische Friedrich-Wilhelms-Universität Bonn},
year = 2021,
month = dec,
note = {Acinetobacter baumannii is a nosocomial pathogen which causes a wide range of serious infections in critically ill patients in the intensive care unit. Besides its high desiccation tolerance, the ability to form biofilms, and multiple other virulence factors, A. baumannii can acquire and upregulate antimicrobial resistance determinants to overcome all known antimicrobial drugs, so that multidrug- and even pandrug-resistant isolates have emerged.
This thesis focuses on the investigation of tigecycline and colistin resistance mechanisms in clinical multidrug-resistant A. baumannii isolates as part of the MagicBullet clinical trial. An algorithm to identify genetic differences associated with antimicrobial resistance based on whole-genome sequences was created and resulted in the identification of several novel mutations in tigecycline and colistin resistance determinants, as well as a more in-depth analysis of the bacterial resistome and the prevalence of mutations and/or resistance mechanisms. Further analysis of these mutations and resistance mechanisms was conducted by measuring gene expression, creating gene knockouts, using an in vivo infection model, and mass spectrometry.
In the first part of this thesis, tigecycline resistance mechanisms were investigated with a focus on the resistance-nodulation-cell division (RND) efflux pumps and with an emphasis on their regulators adeRS, adeN and adeL. The frequent disruption of the regulators adeS and adeN by insertion sequences (e.g. ISAba1) was revealed and was often associated with an increase in efflux pump expression, efflux activity, and virulence in the Galleria mellonella infection model as well as reduced susceptibility to several antimicrobial classes. Furthermore, three different nucleotide deletions, a nucleotide insertion, and a premature stop codon were identified in adeN, while several point mutations leading to amino acid substitutions in adeRS, adeN and adeL were also found to be associated with reduced tigecycline susceptibility in this thesis.
In the second part of this thesis, colistin resistance mechanisms were analysed in A. baumannii, focusing on the pmrCAB operon and the pmrC-homologue eptA, which are known to be involved in the modification of lipid A in the bacterial outer membrane. Novel mutations in pmrCAB were detected and were associated with elevated pmrC expression, unchanged fitness, and increased virulence in G. mellonella, but not necessarily with lipid A modification and a decrease in colistin susceptibility. This suggests the involvement of other yet unknown factors in colistin resistance. Moreover, first indications were found in this study that A. baumannii acquired pmrCAB mutations by homologous recombination across different clonal lineages. The presence of eptA was lineage-specific and often associated with insertion sequences in the proximity of the gene, however no correlation with eptA overexpression and lipid A modification or reduced colistin susceptibility was observed.
Combining the results of both parts of this thesis, several findings were made in both tigecycline and colistin resistance mechanisms in A. baumannii. First, both the regulatory genes adeR and pmrB harbour mutational hotspots, which might serve as genetic marker for the prediction of bacterial antimicrobial resistance and the virulence phenotype. Second, an additive effect of mutations is suggested, as a combination of different mutations in adeRS or pmrAB often resulted in reduced tigecycline or colistin susceptibility. Finally, mutations in adeRS or pmrAB can increase bacterial virulence and pathogenicity in G. mellonella.
In conclusion, tigecycline and colistin resistance mechanisms are diverse and multifactorial, with several factors often working in tandem to enable resistance against antimicrobial agents. Both two-component systems adeRS and pmrAB are among the most frequently mutated genes, indicating their importance in the development of antimicrobial resistance in A. baumannii. The use of a genomic approach highlights the importance and potential of whole-genome sequencing as a tool in the study of antimicrobial resistance in clinically relevant pathogens, but also revealed that a genotype does not always correlate with the predicted phenotype. Additional factors (e.g. environmental factors) and clonal lineage specific polymorphisms need to be considered when investigating novel mutations and resistance mechanisms, respectively. Overall, this thesis opens a window into the complexity of antimicrobial resistance mechanisms in A. baumannii, but future work is needed to understand them more comprehensively.},
url = {https://hdl.handle.net/20.500.11811/9448}
}
urn: https://nbn-resolving.org/urn:nbn:de:hbz:5-64741,
author = {{Stefanie Gerson}},
title = {Diversity of regulatory systems and their contribution to colistin and tigecycline resistance in Acinetobacter baumannii},
school = {Rheinische Friedrich-Wilhelms-Universität Bonn},
year = 2021,
month = dec,
note = {Acinetobacter baumannii is a nosocomial pathogen which causes a wide range of serious infections in critically ill patients in the intensive care unit. Besides its high desiccation tolerance, the ability to form biofilms, and multiple other virulence factors, A. baumannii can acquire and upregulate antimicrobial resistance determinants to overcome all known antimicrobial drugs, so that multidrug- and even pandrug-resistant isolates have emerged.
This thesis focuses on the investigation of tigecycline and colistin resistance mechanisms in clinical multidrug-resistant A. baumannii isolates as part of the MagicBullet clinical trial. An algorithm to identify genetic differences associated with antimicrobial resistance based on whole-genome sequences was created and resulted in the identification of several novel mutations in tigecycline and colistin resistance determinants, as well as a more in-depth analysis of the bacterial resistome and the prevalence of mutations and/or resistance mechanisms. Further analysis of these mutations and resistance mechanisms was conducted by measuring gene expression, creating gene knockouts, using an in vivo infection model, and mass spectrometry.
In the first part of this thesis, tigecycline resistance mechanisms were investigated with a focus on the resistance-nodulation-cell division (RND) efflux pumps and with an emphasis on their regulators adeRS, adeN and adeL. The frequent disruption of the regulators adeS and adeN by insertion sequences (e.g. ISAba1) was revealed and was often associated with an increase in efflux pump expression, efflux activity, and virulence in the Galleria mellonella infection model as well as reduced susceptibility to several antimicrobial classes. Furthermore, three different nucleotide deletions, a nucleotide insertion, and a premature stop codon were identified in adeN, while several point mutations leading to amino acid substitutions in adeRS, adeN and adeL were also found to be associated with reduced tigecycline susceptibility in this thesis.
In the second part of this thesis, colistin resistance mechanisms were analysed in A. baumannii, focusing on the pmrCAB operon and the pmrC-homologue eptA, which are known to be involved in the modification of lipid A in the bacterial outer membrane. Novel mutations in pmrCAB were detected and were associated with elevated pmrC expression, unchanged fitness, and increased virulence in G. mellonella, but not necessarily with lipid A modification and a decrease in colistin susceptibility. This suggests the involvement of other yet unknown factors in colistin resistance. Moreover, first indications were found in this study that A. baumannii acquired pmrCAB mutations by homologous recombination across different clonal lineages. The presence of eptA was lineage-specific and often associated with insertion sequences in the proximity of the gene, however no correlation with eptA overexpression and lipid A modification or reduced colistin susceptibility was observed.
Combining the results of both parts of this thesis, several findings were made in both tigecycline and colistin resistance mechanisms in A. baumannii. First, both the regulatory genes adeR and pmrB harbour mutational hotspots, which might serve as genetic marker for the prediction of bacterial antimicrobial resistance and the virulence phenotype. Second, an additive effect of mutations is suggested, as a combination of different mutations in adeRS or pmrAB often resulted in reduced tigecycline or colistin susceptibility. Finally, mutations in adeRS or pmrAB can increase bacterial virulence and pathogenicity in G. mellonella.
In conclusion, tigecycline and colistin resistance mechanisms are diverse and multifactorial, with several factors often working in tandem to enable resistance against antimicrobial agents. Both two-component systems adeRS and pmrAB are among the most frequently mutated genes, indicating their importance in the development of antimicrobial resistance in A. baumannii. The use of a genomic approach highlights the importance and potential of whole-genome sequencing as a tool in the study of antimicrobial resistance in clinically relevant pathogens, but also revealed that a genotype does not always correlate with the predicted phenotype. Additional factors (e.g. environmental factors) and clonal lineage specific polymorphisms need to be considered when investigating novel mutations and resistance mechanisms, respectively. Overall, this thesis opens a window into the complexity of antimicrobial resistance mechanisms in A. baumannii, but future work is needed to understand them more comprehensively.},
url = {https://hdl.handle.net/20.500.11811/9448}
}
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