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 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.

scholarly journals Nonclonal Emergence of Colistin Resistance Associated with Mutations in the BasRS Two-Component System in Escherichia coli Bloodstream Isolates

mSphere ◽  
2020 ◽  
Vol 5 (2) ◽  
Author(s):  
Axel B. Janssen ◽  
Toby L. Bartholomew ◽  
Natalia P. Marciszewska ◽  
Marc J. M. Bonten ◽  
Rob J. L. Willems ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Lipid A ◽  
Gram Negative Bacteria ◽  
Colistin Resistance ◽  
Anionic Lipid ◽  
Gram Negative ◽  
Content Type ◽  
E Coli ◽  
Two Component ◽  
Resistant Strains

ABSTRACT Infections by multidrug-resistant Gram-negative bacteria are increasingly common, prompting the renewed interest in the use of colistin. Colistin specifically targets Gram-negative bacteria by interacting with the anionic lipid A moieties of lipopolysaccharides, leading to membrane destabilization and cell death. Here, we aimed to uncover the mechanisms of colistin resistance in nine colistin-resistant Escherichia coli strains and one Escherichia albertii strain. These were the only colistin-resistant strains of 1,140 bloodstream Escherichia isolates collected in a tertiary hospital over a 10-year period (2006 to 2015). Core-genome phylogenetic analysis showed that each patient was colonized by a unique strain, suggesting that colistin resistance was acquired independently in each strain. All colistin-resistant strains had lipid A that was modified with phosphoethanolamine. In addition, two E. coli strains had hepta-acylated lipid A species, containing an additional palmitate compared to the canonical hexa-acylated E. coli lipid A. One E. coli strain carried the mobile colistin resistance (mcr) gene mcr-1.1 on an IncX4-type plasmid. Through construction of chromosomal transgene integration mutants, we experimentally determined that mutations in basRS, encoding a two-component signal transduction system, contributed to colistin resistance in four strains. We confirmed these observations by reversing the mutations in basRS to the sequences found in reference strains, resulting in loss of colistin resistance. While the mcr genes have become a widely studied mechanism of colistin resistance in E. coli, sequence variation in basRS is another, potentially more prevalent but relatively underexplored, cause of colistin resistance in this important nosocomial pathogen. IMPORTANCE Multidrug resistance among Gram-negative bacteria has led to the use of colistin as a last-resort drug. The cationic colistin kills Gram-negative bacteria through electrostatic interaction with the anionic lipid A moiety of lipopolysaccharides. Due to increased use in clinical and agricultural settings, colistin resistance has recently started to emerge. In this study, we used a combination of whole-genome sequence analysis and experimental validation to characterize the mechanisms through which Escherichia coli strains from bloodstream infections can develop colistin resistance. We found no evidence of direct transfer of colistin-resistant isolates between patients. The lipid A of all isolates was modified by the addition of phosphoethanolamine. In four isolates, colistin resistance was experimentally verified to be caused by mutations in the basRS genes, encoding a two-component regulatory system. Our data show that chromosomal mutations are an important cause of colistin resistance among clinical E. coli isolates.

scholarly journals Unique Structural Modifications Are Present in the Lipopolysaccharide from Colistin-Resistant Strains of Acinetobacter baumannii

2013 ◽  
Vol 57 (10) ◽  
pp. 4831-4840 ◽  
Author(s):  
Mark R. Pelletier ◽  
Leila G. Casella ◽  
Jace W. Jones ◽  
Mark D. Adams ◽  
Daniel V. Zurawski ◽  
...  
Keyword(s):  
Acinetobacter Baumannii ◽  
Lipid A ◽  
Opportunistic Pathogen ◽  
Structure Characterization ◽  
Resistant Isolate ◽  
Patient Treatment ◽  
Cationic Antimicrobial Peptide ◽  
Content Type ◽  
Lipid A Structure ◽  
Resistant Strains

ABSTRACTAcinetobacter baumanniiis a nosocomial opportunistic pathogen that can cause severe infections, including hospital-acquired pneumonia, wound infections, and sepsis. Multidrug-resistant (MDR) strains are prevalent, further complicating patient treatment. Due to the increase in MDR strains, the cationic antimicrobial peptide colistin has been used to treatA. baumanniiinfections. Colistin-resistant strains ofA. baumanniiwith alterations to the lipid A component of lipopolysaccharide (LPS) have been reported; specifically, the lipid A structure was shown to be hepta-acylated with a phosphoethanolamine (pEtN) modification present on one of the terminal phosphate residues. Using a tandem mass spectrometry platform, we provide definitive evidence that the lipid A isolated from colistin-resistantA. baumanniiMAC204 LPS contains a novel structure corresponding to a diphosphoryl hepta-acylated lipid A structure with both pEtN and galactosamine (GalN) modifications. To correlate our structural studies with clinically relevant samples, we characterized colistin-susceptible and -resistant isolates obtained from patients. These results demonstrated that the clinical colistin-resistant isolate had the same pEtN and GalN modifications as those seen in the laboratory-adaptedA. baumanniistrain MAC204. In summary, this work has shown complete structure characterization including the accurate assignment of acylation, phosphorylation, and glycosylation of lipid A fromA. baumannii, which are important for resistance to colistin.

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 Lysin LysMK34 of Acinetobacter baumannii Bacteriophage PMK34 Has a Turgor Pressure-Dependent Intrinsic Antibacterial Activity and Reverts Colistin Resistance

2020 ◽  
Vol 86 (19) ◽  
Author(s):  
Karim Abdelkader ◽  
Diana Gutiérrez ◽  
Dennis Grimon ◽  
Patricia Ruas-Madiedo ◽  
Cédric Lood ◽  
...  
Keyword(s):  
Antibacterial Activity ◽  
Acinetobacter Baumannii ◽  
Osmotic Shock ◽  
Turgor Pressure ◽  
Bacterial Pathogen ◽  
Intrinsic Activity ◽  
Attractive Alternative ◽  
Colistin Resistance ◽  
Content Type ◽  
Resistant Strains

ABSTRACT The prevalence of extensively and pandrug-resistant strains of Acinetobacter baumannii leaves little or no therapeutic options for treatment for this bacterial pathogen. Bacteriophages and their lysins represent attractive alternative antibacterial strategies in this regard. We used the extensively drug-resistant A. baumannii strain MK34 to isolate the bacteriophage PMK34 (vB_AbaP_PMK34). This phage shows fast adsorption and lacks virulence genes; nonetheless, its narrow host spectrum based on capsule recognition limits broad application. PMK34 is a Fri1virus member of the Autographiviridae and has a 41.8-kb genome (50 open reading frames), encoding an endolysin (LysMK34) with potent muralytic activity (1,499.9 ± 131 U/μM), a typical mesophilic thermal stability up to 55°C, and a broad pH activity range (4 to 10). LysMK34 has an intrinsic antibacterial activity up to 4.8 and 2.4 log units for A. baumannii and Pseudomonas aeruginosa strains, respectively, but only when a high turgor pressure is present. The addition of 0.5 mM EDTA or application of an osmotic shock after treatment can compensate for the lack of a high turgor pressure. The combination of LysMK34 and colistin results in up to 32-fold reduction of the MIC of colistin, and colistin-resistant strains are resensitized in both Mueller-Hinton broth and 50% human serum. As such, LysMK34 may be used to safeguard the applicability of colistin as a last-resort antibiotic. IMPORTANCE A. baumannii is one of the most challenging pathogens for which development of new and effective antimicrobials is urgently needed. Colistin is a last-resort antibiotic, and even colistin-resistant A. baumannii strains exist. Here, we present a lysin that sensitizes A. baumannii for colistin and can revert colistin resistance to colistin susceptibility. The lysin also shows a strong, turgor pressure-dependent intrinsic antibacterial activity, providing new insights in the mode of action of lysins with intrinsic activity against Gram-negative bacteria.

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 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 Aminoarabinosylation of Lipid A Is Critical for the Development of Colistin Resistance in Pseudomonas aeruginosa

2017 ◽  
Vol 62 (3) ◽  
Author(s):  
Alessandra Lo Sciuto ◽  
Francesco Imperi
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Experimental Evolution ◽  
Reverse Genetics ◽  
Lipid A ◽  
Resistance Mechanisms ◽  
Colistin Resistance ◽  
Content Type ◽  
Promising Target

ABSTRACT Lipid A aminoarabinosylation is invariably associated with colistin resistance in Pseudomonas aeruginosa ; however, the existence of alternative aminoarabinosylation-independent colistin resistance mechanisms in this bacterium has remained elusive. By combining reverse genetics with experimental evolution assays, we demonstrate that a functional lipid A aminoarabinosylation pathway is critical for the acquisition of colistin resistance in reference and clinical P. aeruginosa isolates. This highlights lipid A aminoarabinosylation as a promising target for the design of colistin adjuvants against P. aeruginosa .

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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