scholarly journals Phosphoethanolamine Modification of Lipid A in Colistin-Resistant Variants of Acinetobacter baumannii Mediated by the pmrAB Two-Component Regulatory System

2011 ◽  
Vol 55 (7) ◽  
pp. 3370-3379 ◽  
Author(s):  
Alejandro Beceiro ◽  
Enrique Llobet ◽  
Jesús Aranda ◽  
José Antonio Bengoechea ◽  
Michel Doumith ◽  
...  
Keyword(s):  
Acinetobacter Baumannii ◽  
Clinical Isolate ◽  
Lipid A ◽  
Gene Knockout ◽  
Regulatory System ◽  
Clinical Isolates ◽  
Strain Atcc ◽  
Colistin Resistance ◽  
Content Type ◽  
Knockout Mutants

ABSTRACTColistin resistance is rare inAcinetobacter baumannii, and little is known about its mechanism. We investigated the role of PmrCAB in this trait, using (i) resistant and susceptible clinical strains, (ii) laboratory-selected mutants of the type strain ATCC 19606 and of the clinical isolate ABRIM, and (iii) a susceptible/resistant pair of isogenic clinical isolates, Ab15/133 and Ab15/132, isolated from the same patient.pmrABsequences in all the colistin-susceptible isolates were identical to reference sequences, whereas resistant clinical isolates harbored one or two amino acid replacements variously located in PmrB. Single substitutions in PmrB were also found in resistant mutants of strains ATCC 19606 and ABRIM and in the resistant clinical isolate Ab15/132. No mutations in PmrA or PmrC were found. Reverse transcriptase (RT)-PCR identified increased expression ofpmrA(4- to 13-fold),pmrB(2- to 7-fold), andpmrC(1- to 3-fold) in resistant versus susceptible organisms. Matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry showed the addition of phosphoethanolamine to the hepta-acylated form of lipid A in the resistant variants and in strain ATCC 19606 grown under low-Mg2+induction conditions.pmrBgene knockout mutants of the colistin-resistant ATCC 19606 derivative showed >100-fold increased susceptibility to colistin and 5-fold decreased expression ofpmrC; they also lacked the addition of phosphoethanolamine to lipid A. We conclude that the development of a moderate level of colistin resistance inA. baumanniirequires distinct genetic events, including (i) at least one point mutation inpmrB, (ii) upregulation ofpmrAB, and (iii) expression ofpmrC, which lead to addition of phosphoethanolamine to lipid A.

scholarly journals A Prospective Study of Acinetobacter baumannii Complex Isolates and Colistin Susceptibility Monitoring by Mass Spectrometry of Microbial Membrane Glycolipids

2018 ◽  
Vol 57 (3) ◽  
Author(s):  
Lisa M. Leung ◽  
Christi L. McElheny ◽  
Francesca M. Gardner ◽  
Courtney E. Chandler ◽  
Sarah L. Bowler ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Acinetobacter Baumannii ◽  
Lipid A ◽  
Membrane Lipids ◽  
Clinical Isolates ◽  
Colistin Resistance ◽  
Hospital System ◽  
Content Type ◽  
Acinetobacter Baumannii Complex ◽  
Gram Negative Organisms

ABSTRACT Acinetobacter baumannii is a prevalent nosocomial pathogen with a high incidence of multidrug resistance. Treatment of infections due to this organism with colistin, a last-resort antibiotic of the polymyxin class, can result in the emergence of colistin-resistant strains. Colistin resistance primarily occurs via modifications of the terminal phosphate moieties of lipopolysaccharide-derived lipid A, which reduces overall membrane electronegativity. These modifications are readily identified by mass spectrometry (MS). In this study, we prospectively collected Acinetobacter baumannii complex clinical isolates from a hospital system in Pennsylvania over a 3-year period. All isolates were evaluated for colistin resistance using standard MIC testing by both agar dilution and broth microdilution, as well as genospecies identification and lipid A profiling using MS analyses. Overall, an excellent correlation between colistin susceptibility and resistance, determined by MIC testing, and the presence of a lipid A modification, determined by MS, was observed with a sensitivity of 92.9% and a specificity of 94.0%. Additionally, glycolipid profiling was able to differentiate A. baumannii complex organisms based on their membrane lipids. With the growth of MS use in clinical laboratories, a reliable MS-based glycolipid phenotyping method that identifies colistin resistance in A. baumannii complex clinical isolates, as well as other Gram-negative organisms, represents an alternative or complementary approach to existing diagnostics.

scholarly journals Investigation of Novel pmrB and eptA Mutations in Isogenic Acinetobacter baumannii Isolates Associated with Colistin Resistance and Increased Virulence In Vivo

2019 ◽  
Vol 63 (3) ◽  
Author(s):  
Stefanie Gerson ◽  
Jonathan W. Betts ◽  
Kai Lucaßen ◽  
Carolina Silva Nodari ◽  
Julia Wille ◽  
...  
Keyword(s):  
Acinetobacter Baumannii ◽  
Lipid A ◽  
Galleria Mellonella ◽  
Resistance Mechanism ◽  
Resistant Isolate ◽  
Infection Model ◽  
Colistin Resistance ◽  
Strain Specificity ◽  
Content Type

ABSTRACT Colistin resistance in Acinetobacter baumannii is of great concern and is a threat to human health. In this study, we investigate the mechanisms of colistin resistance in four isogenic pairs of A. baumannii isolates displaying an increase in colistin MICs. A mutation in pmrB was detected in each colistin-resistant isolate, three of which were novel (A28V, I232T, and ΔL9-G12). Increased expression of pmrC was shown by semi-quantitative reverse transcription-PCR (qRT-PCR) for three colistin-resistant isolates, and the addition of phosphoethanolamine (PEtN) to lipid A by PmrC was revealed by mass spectrometry. Interestingly, PEtN addition was also observed in some colistin-susceptible isolates, indicating that this resistance mechanism might be strain specific and that other factors could contribute to colistin resistance. Furthermore, the introduction of pmrAB carrying the short amino acid deletion ΔL9-G12 into a pmrAB knockout strain resulted in increased pmrC expression and lipid A modification, but colistin MICs remained unchanged, further supporting the strain specificity of this colistin resistance mechanism. Of note, a mutation in the pmrC homologue eptA and a point mutation in ISAba1 upstream of eptA were associated with colistin resistance and increased eptA expression, which is a hitherto undescribed resistance mechanism. Moreover, no cost of fitness was observed for colistin-resistant isolates, while the virulence of these isolates was increased in a Galleria mellonella infection model. Although the mutations in pmrB were associated with colistin resistance, PEtN addition appears not to be the sole factor leading to colistin resistance, indicating that the mechanism of colistin resistance is far more complex than previously suspected and is potentially strain specific.

Molecular characterization of lpxACD and pmrA/B two-component regulatory system in the colistin resistance Acinetobacter baumannii clinical isolates

Gene Reports ◽  
2020 ◽  
Vol 21 ◽  
pp. 100952
Author(s):  
Mohammad Reza Kandehkar Ghahraman ◽  
Hossein Hosseini-Nave ◽  
Omid Azizi ◽  
Mohammad Reza Shakibaie ◽  
Hamid Reza Mollaie ◽  
...  
Keyword(s):  
Acinetobacter Baumannii ◽  
Molecular Characterization ◽  
Regulatory System ◽  
Clinical Isolates ◽  
Colistin Resistance ◽  
Two Component

scholarly journals Colistin resistance in Acinetobacter baumannii is driven by multiple genomic traits: Evaluating the role of ISAba1-driven eptA overexpression among Indian isolates

2021 ◽  
Author(s):  
Saranya Vijayakumar ◽  
Jobin John Jacob ◽  
Karthick Vasudevan ◽  
Baby Abirami Shankar ◽  
Maria Lincy Francis ◽  
...  
Keyword(s):  
Acinetobacter Baumannii ◽  
Lipid A ◽  
Resistance Mechanisms ◽  
Clinical Isolates ◽  
Colistin Resistance ◽  
Two Component System ◽  
Synonymous Mutations ◽  
Insertional Inactivation ◽  
Indian Isolates

Colistin resistance in Acinetobacter baumannii is mediated by multiple mechanisms. Recently, mutations within pmrAB two component system and overexpression of eptA due to upstream insertion of ISAba1 play a major role. To characterize colistin resistance mechanisms among the clinical isolates of A. baumannii in India. A total of 224 clinical isolates of A. baumannii collected from 2016 to 2019 were included in this study. Mutations within lipid A biosynthesis and pmrAB genes were characterized by Whole Genome Shotgun sequencing. Twenty eight complete genomes were further characterized for insertional inactivation of lpx genes and the association of ISAba1-eptA using hybrid assembly approach. Non-synonymous mutations like M12I in pmrA, A138T and A444V in pmrB and E117K in lpxD were identified. Four of the five colistin resistant A. baumannii isolates had insertion of ISAba1 upstream eptA. No mcr genes were identified. Overall, the present study highlights the diversity of colistin resistance mechanisms in A. baumannii. ISAba1-driven eptA overexpression could be responsible for colistin resistance among Indian isolates of colistin resistant A. baumannii.

scholarly journals Genomic and Transcriptomic Analyses of Colistin-Resistant Clinical Isolates of Klebsiella pneumoniae Reveal Multiple Pathways of Resistance

2014 ◽  
Vol 59 (1) ◽  
pp. 536-543 ◽  
Author(s):  
Meredith S. Wright ◽  
Yo Suzuki ◽  
Marcus B. Jones ◽  
Steven H. Marshall ◽  
Susan D. Rudin ◽  
...  
Keyword(s):  
Klebsiella Pneumoniae ◽  
Lipid A ◽  
Transcriptional Profiling ◽  
Membrane Integrity ◽  
Regulatory System ◽  
Multidrug Resistant ◽  
Clinical Isolates ◽  
Kinase Gene ◽  
Content Type ◽  
Genetic Mechanisms

ABSTRACTThe emergence of multidrug-resistant (MDR)Klebsiella pneumoniaehas resulted in a more frequent reliance on treatment using colistin. However, resistance to colistin (Colr) is increasingly reported from clinical settings. The genetic mechanisms that lead to ColrinK. pneumoniaeare not fully characterized. Using a combination of genome sequencing and transcriptional profiling by RNA sequencing (RNA-Seq) analysis, distinct genetic mechanisms were found among nine Colrclinical isolates. Colrwas related to mutations in three different genes inK. pneumoniaestrains, with distinct impacts on gene expression. Upregulation of thepmrHoperon encoding 4-amino-4-deoxy-l-arabinose (Ara4N) modification of lipid A was found in all Colrstrains. Alteration of themgrBgene was observed in six strains. One strain had a mutation inphoQ. Common among these seven strains was elevated expression ofphoPQand unaltered expression ofpmrCAB, which is involved in phosphoethanolamine addition to lipopolysaccharide (LPS). In two strains, separate mutations were found in a previously uncharacterized histidine kinase gene that is part of a two-component regulatory system (TCRS) now designatedcrrAB. In these strains, expression ofpmrCAB,crrAB, and an adjacent glycosyltransferase gene, but not that ofphoPQ, was elevated. Complementation with the wild-type allele restored colistin susceptibility in both strains. ThecrrABgenes are present in mostK. pneumoniaegenomes, but not inEscherichia coli. Additional upregulated genes in all strains include those involved in cation transport and maintenance of membrane integrity. Because thecrrABgenes are present in only some strains, Colrmechanisms may be dependent on the genetic background.

scholarly journals RND-Type Efflux Pumps in Multidrug-Resistant Clinical Isolates of Acinetobacter baumannii: Major Role for AdeABC Overexpression and AdeRS Mutations

2013 ◽  
Vol 57 (7) ◽  
pp. 2989-2995 ◽  
Author(s):  
Eun-Jeong Yoon ◽  
Patrice Courvalin ◽  
Catherine Grillot-Courvalin
Keyword(s):  
Acinetobacter Baumannii ◽  
Efflux Pump ◽  
Regulatory System ◽  
Multidrug Resistant ◽  
Clinical Isolates ◽  
Clinical Settings ◽  
Two Component System ◽  
Content Type ◽  
High Incidence ◽  
Two Component

ABSTRACTIncreased expression of chromosomal genes for resistance-nodulation-cell division (RND)-type efflux systems plays a major role in the multidrug resistance (MDR) ofAcinetobacter baumannii. However, the relative contributions of the three most prevalent pumps, AdeABC, AdeFGH, and AdeIJK, have not been evaluated in clinical settings. We have screened 14 MDR clinical isolates shown to be distinct on the basis of multilocus sequence typing (MLST) and pulsed-field gel electrophoresis (PFGE) for the presence and overexpression of the three Ade efflux systems and analyzed the sequences of the regulators AdeRS, a two-component system, for AdeABC and AdeL, a LysR-type regulator, for AdeFGH. GeneadeBwas detected in 13 of 14 isolates, andadeGand the intrinsicadeJgene were detected in all strains. Significant overexpression ofadeBwas observed in 10 strains, whereas only 7 had moderately increased levels of expression of AdeFGH, and none overexpressed AdeIJK. Thirteen strains had reduced susceptibility to tigecycline, but there was no correlation between tigecycline MICs and the levels of AdeABC expression, suggesting the presence of other mechanisms for tigecycline resistance. No mutations were found in the highly conserved LysR regulator of the nine strains expressing AdeFGH. In contrast, functional mutations were found in conserved domains of AdeRS in all the strains that overexpressed AdeABC with two mutational hot spots, one in AdeS near histidine 149 suggesting convergent evolution and the other in the DNA binding domain of AdeR compatible with horizontal gene transfer. This report outlines the high incidence of AdeABC efflux pump overexpression in MDRA. baumanniias a result of a variety of single mutations in the corresponding two-component regulatory system.

scholarly journals Synergy between Colistin and the Signal Peptidase Inhibitor MD3 Is Dependent on the Mechanism of Colistin Resistance in Acinetobacter baumannii

2016 ◽  
Vol 60 (7) ◽  
pp. 4375-4379 ◽  
Author(s):  
Marta Martínez-Guitián ◽  
Juan C. Vázquez-Ucha ◽  
Joshua Odingo ◽  
Tanya Parish ◽  
Margarita Poza ◽  
...  
Keyword(s):  
Acinetobacter Baumannii ◽  
Lipid A ◽  
Growth Curves ◽  
Resistance Mechanisms ◽  
Signal Peptidase ◽  
Colistin Resistance ◽  
Content Type ◽  
Peptidase Inhibitor ◽  
Resistant Strains ◽  
Isogenic Mutants

ABSTRACTSynergy between colistin and the signal peptidase inhibitor MD3 was tested against isogenic mutants and clinical pairs ofAcinetobacter baumanniiisolates. Checkerboard assays and growth curves showed synergy against both colistin-susceptible strains (fractional inhibitory concentration index [FICindex] = 0.13 to 0.24) and colistin-resistant strains with mutations inpmrBand phosphoethanolamine modification of lipid A (FICindex= 0.14 to 0.25) but not against colistin-resistant Δlpxstrains with loss of lipopolysaccharide (FICindex= 0.75 to 1). A colistin/MD3 combination would need to be targeted to strains with specific colistin resistance mechanisms.

scholarly journals pmrCAB Recombination Events among Colistin-Susceptible and -Resistant Acinetobacter baumannii Clinical Isolates Belonging to International Clone 7

mSphere ◽  
2021 ◽  
Author(s):  
Carolina Silva Nodari ◽  
Sebastian Alexander Fuchs ◽  
Kyriaki Xanthopoulou ◽  
Rodrigo Cayô ◽  
Harald Seifert ◽  
...  
Keyword(s):  
Acinetobacter Baumannii ◽  
Clinical Isolates ◽  
Colistin Resistance ◽  
Content Type ◽  
Resistance Rates

Colistin resistance rates among Acinetobacter baumannii clinical isolates have increased over the last 20 years. Despite reports of the spread of plasmid-mediated colistin resistance among Enterobacterales , the presence of mcr -type genes in Acinetobacter spp. remains rare, and reduced colistin susceptibility is mainly associated with the acquisition of nonsynonymous mutations in pmrCAB .

scholarly journals Insertion Sequence ISAba11Is Involved in Colistin Resistance and Loss of Lipopolysaccharide in Acinetobacter baumannii

2011 ◽  
Vol 55 (6) ◽  
pp. 3022-3024 ◽  
Author(s):  
Jennifer H. Moffatt ◽  
Marina Harper ◽  
Ben Adler ◽  
Roger L. Nation ◽  
Jian Li ◽  
...  
Keyword(s):  
Multidrug Resistance ◽  
Acinetobacter Baumannii ◽  
Insertion Sequence ◽  
Lipid A ◽  
Complete Loss ◽  
Colistin Resistance ◽  
Content Type ◽  
Lipid A Biosynthesis ◽  
High Level

ABSTRACTInfections caused byAcinetobacter baumanniiare of increasing concern, largely due to the multidrug resistance of many strains. Here we show that insertion sequence ISAba11movement can result in inactivation of theA. baumanniilipid A biosynthesis geneslpxAandlpxC, resulting in the complete loss of lipopolysaccharide production and high-level colistin resistance.

scholarly journals Emergence of High-Level Colistin Resistance in an Acinetobacter baumannii Clinical Isolate Mediated by Inactivation of the Global Regulator H-NS

2018 ◽  
Vol 62 (7) ◽  
Author(s):  
Deanna Deveson Lucas ◽  
Bethany Crane ◽  
Amy Wright ◽  
Mei-Ling Han ◽  
Jennifer Moffatt ◽  
...  
Keyword(s):  
Acinetobacter Baumannii ◽  
Lipid A ◽  
Transcriptional Regulator ◽  
Therapy Resistance ◽  
Multidrug Resistant ◽  
Missense Mutations ◽  
Colistin Resistance ◽  
Extra Copy ◽  
Sequence Element ◽  
Content Type

ABSTRACT Colistin is a crucial last-line drug used for the treatment of life-threatening infections caused by multidrug-resistant strains of the Gram-negative bacterium Acinetobacter baumannii . However, colistin-resistant A. baumannii isolates can still be isolated following failed colistin therapy. Resistance is most often mediated by the addition of phosphoethanolamine (pEtN) to lipid A by PmrC, following missense mutations in the pmrCAB operon encoding PmrC and the two-component signal transduction system PmrA/PmrB. We recovered a pair of A. baumannii isolates from a single patient before (6009-1) and after (6009-2) failed colistin treatment. These strains displayed low and very high levels of colistin resistance (MICs, 8 to 16 μg/ml and 128 μg/ml), respectively. To understand how increased colistin resistance arose, we sequenced the genome of each isolate, which revealed that 6009-2 had an extra copy of the insertion sequence element IS Aba125 within a gene encoding an H-NS family transcriptional regulator. To confirm the role of H-NS in colistin resistance, we generated an hns deletion mutant in 6009-1 and showed that colistin resistance increased upon the deletion of hns . We also provided 6009-2 with an intact copy of hns and showed that the strain was no longer resistant to high concentrations of colistin. Transcriptomic analysis of the clinical isolates identified more than 150 genes as being differentially expressed in the colistin-resistant hns mutant 6009-2. Importantly, the expression of eptA , encoding a second lipid A-specific pEtN transferase but not pmrC , was increased in the hns mutant. This is the first time an H-NS family transcriptional regulator has been associated with a pEtN transferase and colistin resistance.

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