Induced Heteroresistance in Carbapenem-Resistant <i>Acinetobacter baumannii</i> (CRAB) via Exposure to Human Pleural Fluid (HPF) and Its Impact on Cefiderocol Susceptibility
Infections caused by Carbapenem-resistant <i>Acinetobacter baumannii</i> (CRAB) isolates, such as hospital-acquired pneumonia (HAP), bacteremia, and skin and soft tissue infections, among others, are particularly challenging to treat. Cefiderocol, a chlorocatechol-substituted siderophore...
| Main Authors: | , , , , , , , , , , , , , , |
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| Format: | Article |
| Language: | English |
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MDPI AG
2023-07-01
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| Series: | International Journal of Molecular Sciences |
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| Online Access: | https://www.mdpi.com/1422-0067/24/14/11752 |
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| author | Vyanka Mezcord Jenny Escalante Brent Nishimura German M. Traglia Rajnikant Sharma Quentin Vallé Marisel R. Tuttobene Tomás Subils Ingrid Marin Fernando Pasteran Luis A. Actis Marcelo E. Tolmasky Robert A. Bonomo Gauri Rao María S. Ramirez |
| author_facet | Vyanka Mezcord Jenny Escalante Brent Nishimura German M. Traglia Rajnikant Sharma Quentin Vallé Marisel R. Tuttobene Tomás Subils Ingrid Marin Fernando Pasteran Luis A. Actis Marcelo E. Tolmasky Robert A. Bonomo Gauri Rao María S. Ramirez |
| author_sort | Vyanka Mezcord |
| collection | DOAJ |
| description | Infections caused by Carbapenem-resistant <i>Acinetobacter baumannii</i> (CRAB) isolates, such as hospital-acquired pneumonia (HAP), bacteremia, and skin and soft tissue infections, among others, are particularly challenging to treat. Cefiderocol, a chlorocatechol-substituted siderophore antibiotic, was approved by the U.S. Food and Drug Administration (FDA) in 2019 and prescribed for the treatment of CRAB infections. Despite the initial positive treatment outcomes with this antimicrobial, recent studies reported a higher-than-average all-cause mortality rate in patients treated with cefiderocol compared to the best available therapy. The cause(s) behind these outcomes remains unconfirmed. A plausible hypothesis is heteroresistance, a phenotype characterized by the survival of a small proportion of cells in a population that is seemingly isogenic. Recent results have demonstrated that the addition of human fluids to CRAB cultures leads to cefiderocol heteroresistance. Here, we describe the molecular and phenotypic analyses of CRAB heteroresistant bacterial subpopulations to better understand the nature of the less-than-expected successful outcomes after cefiderocol treatment. Isolation of heteroresistant variants of the CRAB strain AMA40 was carried out in cultures supplemented with cefiderocol and human pleural fluid (HPF). Two AMA40 variants, AMA40 IHC1 and IHC2, were resistant to cefiderocol. To identify mutations and gene expression changes associated with cefiderocol heteroresistance, we subjected these variants to whole genome sequencing and global transcriptional analysis. We then assessed the impact of these mutations on the pharmacodynamic activity of cefiderocol via susceptibility testing, EDTA and boronic acid inhibition analysis, biofilm formation, and static time-kill assays. Heteroresistant variants AMA40 IHC1 and AMA40 IHC2 have 53 chromosomal mutations, of which 40 are common to both strains. None of the mutations occurred in genes associated with high affinity iron-uptake systems or β-lactam resistance. However, transcriptional analyses demonstrated significant modifications in levels of expression of genes associated with iron-uptake systems or β-lactam resistance. The <i>bla</i><sub>NDM-1</sub> and <i>bla</i><sub>ADC-2,</sub> as well as various iron-uptake system genes, were expressed at higher levels than the parental strain. On the other hand, the <i>carO</i> and <i>ompA</i> genes’ expression was reduced. One of the mutations common to both heteroresistant strains was mapped within <i>ppiA</i>, a gene associated with iron homeostasis in other species. Static time-kill assays demonstrated that supplementing cation-adjusted Mueller–Hinton broth with human serum albumin (HAS), the main protein component of HPF, considerably reduced cefiderocol killing activity for all three strains tested. Notably, collateral resistance to amikacin was observed in both variants. We conclude that exposing CRAB to fluids with high HSA concentrations facilitates the rise of heteroresistance associated with point mutations and transcriptional upregulation of genes coding for β-lactamases and biofilm formation. The findings from this study hold significant implications for understanding the emergence of CRAB resistance mechanisms against cefiderocol treatment. This understanding is vital for the development of treatment guidelines that can effectively address the challenges posed by CRAB infections. |
| first_indexed | 2024-03-11T00:59:22Z |
| format | Article |
| id | doaj.art-173835b1945e48288db2fe12d4b1c45e |
| institution | Directory Open Access Journal |
| issn | 1661-6596 1422-0067 |
| language | English |
| last_indexed | 2024-03-11T00:59:22Z |
| publishDate | 2023-07-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | International Journal of Molecular Sciences |
| spelling | doaj.art-173835b1945e48288db2fe12d4b1c45e2023-11-18T19:44:27ZengMDPI AGInternational Journal of Molecular Sciences1661-65961422-00672023-07-0124141175210.3390/ijms241411752Induced Heteroresistance in Carbapenem-Resistant <i>Acinetobacter baumannii</i> (CRAB) via Exposure to Human Pleural Fluid (HPF) and Its Impact on Cefiderocol SusceptibilityVyanka Mezcord0Jenny Escalante1Brent Nishimura2German M. Traglia3Rajnikant Sharma4Quentin Vallé5Marisel R. Tuttobene6Tomás Subils7Ingrid Marin8Fernando Pasteran9Luis A. Actis10Marcelo E. Tolmasky11Robert A. Bonomo12Gauri Rao13María S. Ramirez14Center for Applied Biotechnology Studies, Department of Biological Science, College of Natural Sciences and Mathematics, California State University Fullerton, Fullerton, CA 92831, USACenter for Applied Biotechnology Studies, Department of Biological Science, College of Natural Sciences and Mathematics, California State University Fullerton, Fullerton, CA 92831, USACenter for Applied Biotechnology Studies, Department of Biological Science, College of Natural Sciences and Mathematics, California State University Fullerton, Fullerton, CA 92831, USAUnidad de Genómica y Bioinformática, Departamento de Ciencias Biológicas, CENUR Litoral Norte, Universidad de la República, Salto 50000, UruguayUNC Eshelman School of Pharmacy, University of North Carolina, Chapel Hill, NC 27599, USAUNC Eshelman School of Pharmacy, University of North Carolina, Chapel Hill, NC 27599, USAÁrea Biología Molecular, Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario, Rosario 2000, ArgentinaInstituto de Procesos Biotecnológicos y Químicos de Rosario (IPROBYQ, CONICET-UNR), Rosario 2000, ArgentinaCenter for Applied Biotechnology Studies, Department of Biological Science, College of Natural Sciences and Mathematics, California State University Fullerton, Fullerton, CA 92831, USANational Regional Reference Laboratory for Antimicrobial Resistance (NRL), Servicio Antimicrobianos, Instituto Nacional de Enfermedades Infecciosas, ANLIS Dr. Carlos G. Malbrán, Buenos Aires 1282, ArgentinaDepartment of Microbiology, Miami University, Oxford, OH 45056, USACenter for Applied Biotechnology Studies, Department of Biological Science, College of Natural Sciences and Mathematics, California State University Fullerton, Fullerton, CA 92831, USAResearch Service and GRECC, Louis Stokes Cleveland Department of Veterans Affairs Medical Center, Cleveland, OH 44106, USAUNC Eshelman School of Pharmacy, University of North Carolina, Chapel Hill, NC 27599, USACenter for Applied Biotechnology Studies, Department of Biological Science, College of Natural Sciences and Mathematics, California State University Fullerton, Fullerton, CA 92831, USAInfections caused by Carbapenem-resistant <i>Acinetobacter baumannii</i> (CRAB) isolates, such as hospital-acquired pneumonia (HAP), bacteremia, and skin and soft tissue infections, among others, are particularly challenging to treat. Cefiderocol, a chlorocatechol-substituted siderophore antibiotic, was approved by the U.S. Food and Drug Administration (FDA) in 2019 and prescribed for the treatment of CRAB infections. Despite the initial positive treatment outcomes with this antimicrobial, recent studies reported a higher-than-average all-cause mortality rate in patients treated with cefiderocol compared to the best available therapy. The cause(s) behind these outcomes remains unconfirmed. A plausible hypothesis is heteroresistance, a phenotype characterized by the survival of a small proportion of cells in a population that is seemingly isogenic. Recent results have demonstrated that the addition of human fluids to CRAB cultures leads to cefiderocol heteroresistance. Here, we describe the molecular and phenotypic analyses of CRAB heteroresistant bacterial subpopulations to better understand the nature of the less-than-expected successful outcomes after cefiderocol treatment. Isolation of heteroresistant variants of the CRAB strain AMA40 was carried out in cultures supplemented with cefiderocol and human pleural fluid (HPF). Two AMA40 variants, AMA40 IHC1 and IHC2, were resistant to cefiderocol. To identify mutations and gene expression changes associated with cefiderocol heteroresistance, we subjected these variants to whole genome sequencing and global transcriptional analysis. We then assessed the impact of these mutations on the pharmacodynamic activity of cefiderocol via susceptibility testing, EDTA and boronic acid inhibition analysis, biofilm formation, and static time-kill assays. Heteroresistant variants AMA40 IHC1 and AMA40 IHC2 have 53 chromosomal mutations, of which 40 are common to both strains. None of the mutations occurred in genes associated with high affinity iron-uptake systems or β-lactam resistance. However, transcriptional analyses demonstrated significant modifications in levels of expression of genes associated with iron-uptake systems or β-lactam resistance. The <i>bla</i><sub>NDM-1</sub> and <i>bla</i><sub>ADC-2,</sub> as well as various iron-uptake system genes, were expressed at higher levels than the parental strain. On the other hand, the <i>carO</i> and <i>ompA</i> genes’ expression was reduced. One of the mutations common to both heteroresistant strains was mapped within <i>ppiA</i>, a gene associated with iron homeostasis in other species. Static time-kill assays demonstrated that supplementing cation-adjusted Mueller–Hinton broth with human serum albumin (HAS), the main protein component of HPF, considerably reduced cefiderocol killing activity for all three strains tested. Notably, collateral resistance to amikacin was observed in both variants. We conclude that exposing CRAB to fluids with high HSA concentrations facilitates the rise of heteroresistance associated with point mutations and transcriptional upregulation of genes coding for β-lactamases and biofilm formation. The findings from this study hold significant implications for understanding the emergence of CRAB resistance mechanisms against cefiderocol treatment. This understanding is vital for the development of treatment guidelines that can effectively address the challenges posed by CRAB infections.https://www.mdpi.com/1422-0067/24/14/11752<i>Acinetobacter baumannii</i>human pleural fluidcefiderocolNDM-1carbapenem-resistancewhole-genome sequencing |
| spellingShingle | Vyanka Mezcord Jenny Escalante Brent Nishimura German M. Traglia Rajnikant Sharma Quentin Vallé Marisel R. Tuttobene Tomás Subils Ingrid Marin Fernando Pasteran Luis A. Actis Marcelo E. Tolmasky Robert A. Bonomo Gauri Rao María S. Ramirez Induced Heteroresistance in Carbapenem-Resistant <i>Acinetobacter baumannii</i> (CRAB) via Exposure to Human Pleural Fluid (HPF) and Its Impact on Cefiderocol Susceptibility International Journal of Molecular Sciences <i>Acinetobacter baumannii</i> human pleural fluid cefiderocol NDM-1 carbapenem-resistance whole-genome sequencing |
| title | Induced Heteroresistance in Carbapenem-Resistant <i>Acinetobacter baumannii</i> (CRAB) via Exposure to Human Pleural Fluid (HPF) and Its Impact on Cefiderocol Susceptibility |
| title_full | Induced Heteroresistance in Carbapenem-Resistant <i>Acinetobacter baumannii</i> (CRAB) via Exposure to Human Pleural Fluid (HPF) and Its Impact on Cefiderocol Susceptibility |
| title_fullStr | Induced Heteroresistance in Carbapenem-Resistant <i>Acinetobacter baumannii</i> (CRAB) via Exposure to Human Pleural Fluid (HPF) and Its Impact on Cefiderocol Susceptibility |
| title_full_unstemmed | Induced Heteroresistance in Carbapenem-Resistant <i>Acinetobacter baumannii</i> (CRAB) via Exposure to Human Pleural Fluid (HPF) and Its Impact on Cefiderocol Susceptibility |
| title_short | Induced Heteroresistance in Carbapenem-Resistant <i>Acinetobacter baumannii</i> (CRAB) via Exposure to Human Pleural Fluid (HPF) and Its Impact on Cefiderocol Susceptibility |
| title_sort | induced heteroresistance in carbapenem resistant i acinetobacter baumannii i crab via exposure to human pleural fluid hpf and its impact on cefiderocol susceptibility |
| topic | <i>Acinetobacter baumannii</i> human pleural fluid cefiderocol NDM-1 carbapenem-resistance whole-genome sequencing |
| url | https://www.mdpi.com/1422-0067/24/14/11752 |
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