scholarly journals Detection of Colistin Resistance in Pseudomonas aeruginosa Using the MALDIxin Test on the Routine MALDI Biotyper Sirius Mass Spectrometer

2021 ◽  
Vol 12 ◽  
Author(s):  
Katy Jeannot ◽  
Katheryn Hagart ◽  
Laurent Dortet ◽  
Markus Kostrzewa ◽  
Alain Filloux ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Lipid A ◽  
Response Regulator ◽  
Multidrug Resistant ◽  
Positive Control ◽  
Colistin Resistance ◽  
Clinical Strains ◽  
Efficient Detection ◽  
Maldi Biotyper ◽  
Resistant Strains

Colistin is frequently a last resort treatment for Pseudomonas aeruginosa infections caused by multidrug-resistant (MDR) and extensively drug resistant (XDR) strains, and detection of colistin resistance is essential for the management of infected patients. Therefore, we evaluated the recently developed MALDIxin test for the detection of colistin resistance in P. aeruginosa clinical strains using the routine matrix-assisted laser desorption ionization (MALDI) Biotyper Sirius system. The test is based on the detection by mass spectrometry of modified lipid A by the addition of 4-amino-l-arabinose (l-ara4N) molecules on one or two phosphate groups, in strains resistant to colistin. Overproduction of l-Ara4N molecules is mainly due to the constitutive activation of the histidine kinase (PmrB) or the response regulator (PmrA) following an amino-acid substitution in clinical strains. The performance of the test was determined on a panel of 14 colistin-susceptible and 14 colistin-resistant P. aeruginosa clinical strains, the reference strain PAO1 and positive control mutants PmrB (V28G), PmrB (D172), PhoQ (D240–247), and ParR (M59I). In comparison with the broth microdilution (BMD) method, all the susceptible strains (n=14) and 8/14 colistin-resistant strains were detected in less than 1h, directly on whole bacteria. The remaining resistant strains (n=6) were all detected after a short pre-exposure (4h) to colistin before sample preparation. Validation of the method on a larger panel of strains will be the next step before its use in diagnostics laboratories. Our data showed that the MALDIxin test offers rapid and efficient detection of colistin resistant P. aeruginosa and is thus a valuable diagnostics tool to control the spread of these emerging resistant strains.

scholarly journals Microevolution of acquired colistin resistance in Enterobacteriaceae from ICU patients receiving selective decontamination of the digestive tract

2020 ◽  
Author(s):  
Axel B. Janssen ◽  
Denise van Hout ◽  
Marc J.M. Bonten ◽  
Rob J.L. Willems ◽  
Willem van Schaik
Keyword(s):  
Digestive Tract ◽  
Response Regulator ◽  
Multidrug Resistant ◽  
Selective Decontamination ◽  
Opportunistic Pathogens ◽  
Colistin Resistance ◽  
Gram Negative ◽  
Routine Surveillance ◽  
Resistant Strains ◽  
Prophylactic Use

AbstractColistin is an antibiotic that targets the lipopolysaccharides present in the membranes of Gram-negative bacteria. It is used as last-resort drug to treat infections with multidrug-resistant strains. Colistin is also used in selective decontamination of the digestive tract (SDD), a prophylactic therapy used in patients hospitalised in intensive care units (ICUs) to selectively eradicate opportunistic pathogens in the oropharyngeal and gut microbiota. In this study, we aimed to unravel the mechanisms of acquired colistin resistance in Gram-negative opportunistic pathogens obtained from SDD-treated patients.Routine surveillance of 428 SDD-treated patients resulted in thirteen strains with acquired colistin resistance (Escherichia coli n=9; Klebsiella aerogenes, n=3; Enterobacter asburiae, n=1) from five patients. Genome sequence analysis showed that these isolates represented multiple distinct colistin-resistant clones, but that within the same patients, colistin-resistant strains were clonally related. We identified previously described mechanisms that lead to colistin resistance, i.e. a G53 substitution in the response regulator PmrA/BasR, and the acquisition of the mobile colistin resistance gene mcr-1.1, but we also observed novel variants of basR with an 18-bp deletion, and a G19E substitution in the sensor histidine kinase BasS. We experimentally confirmed these variants to contribute to reduced colistin susceptibility. In a single patient, we observed that colistin resistance in a single E. coli clone evolved through two unique variants in basRS.We show that prophylactic use of colistin during SDD can select for colistin resistance in species that are not intrinsically colistin-resistant. This highlights the importance of continued surveillance for the emergence of colistin resistance in patients treated with SDD.

scholarly journals Exogenous and Endogenous Phosphoethanolamine Transferases Differently Affect Colistin Resistance and Fitness in Pseudomonas aeruginosa

2021 ◽  
Vol 12 ◽  
Author(s):  
Matteo Cervoni ◽  
Alessandra Lo Sciuto ◽  
Chiara Bianchini ◽  
Carmine Mancone ◽  
Francesco Imperi
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Lipid A ◽  
Cell Envelope ◽  
Resistance Mechanism ◽  
Multidrug Resistant ◽  
Transferase Activity ◽  
Colistin Resistance ◽  
Phosphoethanolamine Transferases ◽  
Positively Charged ◽  
Inducible Gene

Colistin represents a last-line treatment option for infections caused by multidrug resistant Gram-negative pathogens, including Pseudomonas aeruginosa. Colistin resistance generally involves the modification of the lipid A moiety of lipopolysaccharide (LPS) with positively charged molecules, namely phosphoethanolamine (PEtN) or 4-amino-4-deoxy-L-arabinose (Ara4N), that reduce colistin affinity for its target. Several lines of evidence highlighted lipid A aminoarabinosylation as the primary colistin resistance mechanism in P. aeruginosa, while the contribution of phosphoethanolamination remains elusive. PEtN modification can be due to either endogenous (chromosomally encoded) PEtN transferase(s) (e.g., EptA in P. aeruginosa) or plasmid borne MCR enzymes, commonly found in enterobacteria. By individually cloning eptA and mcr-1 into a plasmid for inducible gene expression, we demonstrated that MCR-1 and EptA have comparable PEtN transferase activity in P. aeruginosa and confer colistin resistance levels similar to those provided by lipid A aminoarabinosylation. Notably, EptA, but not MCR-1, negatively affects P. aeruginosa growth and, to a lesser extent, cell envelope integrity when expressed at high levels. Mutagenesis experiments revealed that PEtN transferase activity does not account for the noxious effects of EptA overexpression, that instead requires a C-terminal tail unique to P. aeruginosa EptA, whose function remains unknown. Overall, this study shows that both endogenous and exogenous PEtN transferases can promote colistin resistance in P. aeruginosa, and that PEtN and MCR-1 mediated resistance has no impact on growth and cell envelope homeostasis, suggesting that there may be no fitness barriers to the spread of mcr-1 in P. aeruginosa.

scholarly journals Evolved resistance to colistin and its loss due to genetic reversion in Pseudomonas aeruginosa

2016 ◽  
Vol 6 (1) ◽  
Author(s):  
Ji-Young Lee ◽  
Young Kyoung Park ◽  
Eun Seon Chung ◽  
In Young Na ◽  
Kwan Soo Ko
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Bacterial Infections ◽  
Lipid A ◽  
Treatment Options ◽  
Genomic Analysis ◽  
Multidrug Resistant ◽  
Genetic Alterations ◽  
Comparative Genomic ◽  
Colistin Resistance

Abstract The increased reliance on colistin for treating multidrug-resistant Gram-negative bacterial infections has resulted in the emergence of colistin-resistant Pseudomonas aeruginosa. We attempted to identify genetic contributors to colistin resistance in vitro evolved isogenic colistin-resistant and -susceptible strains of two P. aeruginosa lineages (P5 and P155). Their evolutionary paths to acquisition and loss of colistin resistance were also tracked. Comparative genomic analysis revealed 13 and five colistin resistance determinants in the P5 and P155 lineages, respectively. Lipid A in colistin-resistant mutants was modified through the addition of 4-amino-L-arabinose; this modification was absent in colistin-susceptible revertant strains. Many amino acid substitutions that emerged during the acquisition of colistin resistance were reversed in colistin-susceptible revertants. We demonstrated that evolved colistin resistance in P. aeruginosa was mediated by a complicated regulatory network that likely emerges through diverse genetic alterations. Colistin-resistant P. aeruginosa became susceptible to the colistin upon its withdrawal because of genetic reversion. The mechanisms through which P. aeruginosa acquires and loses colistin resistance have implications on the treatment options that can be applied against P. aeruginosa infections, with respect to improving bactericidal efficacy and preventing further resistance to antibiotics.

scholarly journals Rapid and consistent evolution of colistin resistance in Pseudomonas aeruginosa during morbidostat culture

2016 ◽  
Author(s):  
Bianca Regenbogen ◽  
Matthias Willmann ◽  
Matthias Steglich ◽  
Boyke Bunk ◽  
Ulrich Nübel ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Molecular Targets ◽  
Multidrug Resistant ◽  
Signaling System ◽  
Colistin Resistance ◽  
Resistance Evolution ◽  
Two Component ◽  
Evolution Of Resistance ◽  
Resistant Strains

AbstractColistin is a last resort antibiotic commonly used against multidrug-resistant strains of Pseudomonas aeruginosa. To investigate the potential for in-situ evolution of resistance against colistin and map the molecular targets of colistin resistance, we exposed two P. aeruginosa isolates to colistin using a continuous culture device known as morbidostat. Colistin resistance emerged within two weeks along with highly stereotypic yet strain specific mutation patterns. The majority of mutations hit the prmAB two component signaling system and genes involved in lipopolysaccharide synthesis, including lpxC, pmrE, and migA. In seven out of 18 cultures, we observed mutations in mutS along with a mutator phenotype that seemed to facilitate resistance evolution.

scholarly journals Rapid and Consistent Evolution of Colistin Resistance in Extensively Drug-Resistant Pseudomonas aeruginosa during Morbidostat Culture

2017 ◽  
Vol 61 (9) ◽  
Author(s):  
Bianca Dößelmann ◽  
Matthias Willmann ◽  
Matthias Steglich ◽  
Boyke Bunk ◽  
Ulrich Nübel ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Multidrug Resistant ◽  
Colistin Resistance ◽  
Content Type ◽  
Resistance Evolution ◽  
Extensively Drug Resistant ◽  
Evolution Of Resistance ◽  
Resistant Strains ◽  
Detailed Picture

ABSTRACT Colistin is a last-resort antibiotic commonly used against multidrug-resistant strains of Pseudomonas aeruginosa. To investigate the potential for in situ evolution of resistance against colistin and to map the molecular targets of colistin resistance, we exposed two P. aeruginosa isolates to colistin using a continuous-culture device known as a morbidostat. As a result, colistin resistance reproducibly increased 10-fold within 10 days and 100-fold within 20 days, along with highly stereotypic yet strain-specific mutation patterns. The majority of mutations hit the pmrAB two-component signaling system and genes involved in lipopolysaccharide (LPS) synthesis, including lpxC, pmrE, and migA. We tracked the frequencies of all arising mutations by whole-genome deep sequencing every 3 to 4 days to obtain a detailed picture of the dynamics of resistance evolution, including competition and displacement among multiple resistant subpopulations. In 7 out of 18 cultures, we observed mutations in mutS along with a mutator phenotype that seemed to facilitate resistance evolution.

scholarly journals Evolution of Colistin Resistance in the Klebsiella pneumoniae Complex Follows Multiple Evolutionary Trajectories with Variable Effects on Fitness and Virulence Characteristics

2020 ◽  
Vol 65 (1) ◽  
pp. e01958-20
Author(s):  
Axel B. Janssen ◽  
Dennis J. Doorduijn ◽  
Grant Mills ◽  
Malbert R. C. Rogers ◽  
Marc J. M. Bonten ◽  
...  
Keyword(s):  
Klebsiella Pneumoniae ◽  
Lipid A ◽  
Multidrug Resistant ◽  
Sensu Stricto ◽  
Colistin Resistance ◽  
Content Type ◽  
Evolutionary Trajectories ◽  
Resistant Strains ◽  
Phosphate Groups

ABSTRACTThe increasing prevalence of multidrug-resistant Klebsiella pneumoniae has led to a resurgence in the use of colistin as a last-resort drug. Colistin is a cationic antibiotic that selectively acts on Gram-negative bacteria through electrostatic interactions with anionic phosphate groups of the lipid A moiety of lipopolysaccharides (LPSs). Colistin resistance in K. pneumoniae is mediated through loss of these phosphate groups, their modification by cationic groups, and by the hydroxylation of acyl groups of lipid A. Here, we study the in vitro evolutionary trajectories toward colistin resistance in four clinical K. pneumoniae complex strains and their impact on fitness and virulence characteristics. Through population sequencing during in vitro evolution, we found that colistin resistance develops through a combination of single nucleotide polymorphisms, insertions and deletions, and the integration of insertion sequence elements, affecting genes associated with LPS biosynthesis and modification and capsule structures. Colistin resistance decreased the maximum growth rate of one K. pneumoniaesensu stricto strain, but not those of the other three K. pneumoniae complex strains. Colistin-resistant strains had lipid A modified through hydroxylation, palmitoylation, and l-Ara4N addition. K. pneumoniaesensu stricto strains exhibited cross-resistance to LL-37, in contrast to the Klebsiella variicola subsp. variicola strain. Virulence, as determined in a Caenorhabditis elegans survival assay, was increased in two colistin-resistant strains. Our study suggests that nosocomial K. pneumoniae complex strains can rapidly develop colistin resistance through diverse evolutionary trajectories upon exposure to colistin. This effectively shortens the life span of this last-resort antibiotic for the treatment of infections with multidrug-resistant Klebsiella.

scholarly journals Resistance and Heteroresistance to Colistin in Pseudomonas aeruginosa Isolates from Wenzhou, China

2019 ◽  
Vol 63 (10) ◽  
Author(s):  
Jie Lin ◽  
Chunquan Xu ◽  
Renchi Fang ◽  
Jianming Cao ◽  
Xiucai Zhang ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Lipid A ◽  
Population Analysis ◽  
Colistin Resistance ◽  
Content Type ◽  
Maldi Tof Ms ◽  
Expression Levels ◽  
Maldi Tof ◽  
Broth Microdilution Method ◽  
Tof Ms

ABSTRACT The goal was to investigate the mechanisms of colistin resistance and heteroresistance in Pseudomonas aeruginosa clinical isolates. Colistin resistance was determined by the broth microdilution method. Colistin heteroresistance was evaluated by population analysis profiling. Time-kill assays were also conducted. PCR sequencing was performed to detect the resistance genes among (hetero)resistant isolates, and quantitative real-time PCR assays were performed to determine their expression levels. Pulsed-field gel electrophoresis and multilocus sequence typing were performed. Lipid A characteristics were determined via matrix-assisted laser desorption–ionization time of flight mass spectrometry (MALDI-TOF MS). Two resistant isolates and 9 heteroresistant isolates were selected in this study. Substitutions in PmrB were detected in 2 resistant isolates. Among heteroresistant isolates, 8 of 9 heteroresistant isolates had nonsynonymous PmrB substitutions, and 2 isolates, including 1 with a PmrB substitution, had PhoQ alterations. Correspondingly, the expression levels of pmrA or phoP were upregulated in PmrB- or PhoQ-substituted isolates. One isolate also found alterations in ParRS and CprRS. The transcript levels of the pmrH gene were observed to increase across all investigated isolates. MALDI-TOF MS showed additional 4-amino-4-deoxy-l-arabinose (l-Ara4N) moieties in lipid A profiles in (hetero)resistant isolates. In conclusion, both colistin resistance and heteroresistance in P. aeruginosa in this study mainly involved alterations of the PmrAB regulatory system. There were strong associations between mutations in specific genetic loci for lipid A synthesis and regulation of modifications to lipid A. The transition of colistin heteroresistance to resistance should be addressed in future clinical surveillance.

scholarly journals A Novel Cecropin D-Derived Short Cationic Antimicrobial Peptide Exhibits Antibacterial Activity Against Wild-Type and Multidrug-Resistant Strains of Klebsiella pneumoniae and Pseudomonas aeruginosa

2020 ◽  
Vol 16 ◽  
pp. 117693432093626
Author(s):  
Iván Darío Ocampo-Ibáñez ◽  
Yamil Liscano ◽  
Sandra Patricia Rivera-Sánchez ◽  
José Oñate-Garzón ◽  
Ashley Dayan Lugo-Guevara ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Antibacterial Activity ◽  
Klebsiella Pneumoniae ◽  
Multidrug Resistant ◽  
Clinical Isolates ◽  
Health Concern ◽  
Wild Type ◽  
Cationic Antimicrobial Peptide ◽  
Promising Alternative ◽  
Resistant Strains

Infections caused by multidrug-resistant (MDR) Pseudomonas aeruginosa and Klebsiella pneumoniae are a serious worldwide public health concern due to the ineffectiveness of empirical antibiotic therapy. Therefore, research and the development of new antibiotic alternatives are urgently needed to control these bacteria. The use of cationic antimicrobial peptides (CAMPs) is a promising candidate alternative therapeutic strategy to antibiotics because they exhibit antibacterial activity against both antibiotic susceptible and MDR strains. In this study, we aimed to investigate the in vitro antibacterial effect of a short synthetic CAMP derived from the ΔM2 analog of Cec D-like (CAMP-CecD) against clinical isolates of K pneumoniae (n = 30) and P aeruginosa (n = 30), as well as its hemolytic activity. Minimal inhibitory concentrations (MICs) and minimal bactericidal concentrations (MBCs) of CAMP-CecD against wild-type and MDR strains were determined by the broth microdilution test. In addition, an in silico molecular dynamic simulation was performed to predict the interaction between CAMP-CecD and membrane models of K pneumoniae and P aeruginosa. The results revealed a bactericidal effect of CAMP-CecD against both wild-type and resistant strains, but MDR P aeruginosa showed higher susceptibility to this peptide with MIC values between 32 and >256 μg/mL. CAMP-CecD showed higher stability in the P aeruginosa membrane model compared with the K pneumoniae model due to the greater number of noncovalent interactions with phospholipid 1-Palmitoyl-2-oleyl-sn-glycero-3-(phospho-rac-(1-glycerol)) (POPG). This may be related to the boosted effectiveness of the peptide against P aeruginosa clinical isolates. Given the antibacterial activity of CAMP-CecD against wild-type and MDR clinical isolates of P aeruginosa and K pneumoniae and its nonhemolytic effects on human erythrocytes, CAMP-CecD may be a promising alternative to conventional antibiotics.

scholarly journals Draft Genome Sequence of Pseudomonas aeruginosa Strain BWH047, a Sequence Type 235 Multidrug-Resistant Clinical Isolate Expressing High Levels of Colistin Resistance

2019 ◽  
Vol 8 (29) ◽  
Author(s):  
Kelly E. R. Bachta ◽  
Egon A. Ozer ◽  
Alisha Pandit ◽  
Francisco M. Marty ◽  
John J. Mekalanos ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Clinical Isolate ◽  
Genome Sequence ◽  
Draft Genome ◽  
Multidrug Resistant ◽  
Sequence Type ◽  
Draft Genome Sequence ◽  
Colistin Resistance ◽  
Content Type ◽  
Global Epidemic

The Gram-negative bacterium Pseudomonas aeruginosa is often multidrug resistant, associated with global epidemic outbreaks, and responsible for significant morbidity and mortality in hospitalized patients. Here, we present the draft genome sequence of BWH047, a multidrug-resistant P. aeruginosa clinical isolate belonging to the epidemic sequence type 235 and demonstrating high levels of colistin resistance.

scholarly journals In Vitro and In Vivo Anti-Salmonella Evaluation of Pectin Extracts and Hydrolysates from “Cas Mango” (Spondias dulcis)

2019 ◽  
Vol 2019 ◽  
pp. 1-10 ◽  
Author(s):  
Denis Zofou ◽  
Golda Lum Shu ◽  
Josepha Foba-Tendo ◽  
Merveille Octavie Tabouguia ◽  
Jules-Clement N. Assob
Keyword(s):  
Multidrug Resistant ◽  
Positive Control ◽  
Resistant Bacteria ◽  
Therapeutic Effects ◽  
Diffusion Technique ◽  
Resistant Strains ◽  
Almost All ◽  
Broth Dilution

Background. The threat to human health posed by multidrug-resistant strains of Salmonella typhi (S. typhi) and Salmonella paratyphi (S. paratyphi) is of growing concern. Generally, there has been increasing resistance and even multidrug resistance to almost all classes of antibiotics. This has rendered treatment with antibiotics difficult and costly. The present study investigated the bioactivity of pectin and pectin hydrolysates derived from a local fruit, Spondias dulcis, against four strains of Salmonellae. Methods. Pectin was extracted from alcohol extractives-free peel by acidic hydrolysis at a temperature of 80°C for one hour at pH 2 and 4. The pectin was precipitated with 95% alcohol at an extract to alcohol ratio of 1:10 v/v. Antimicrobial activity was determined using agar well diffusion technique. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) values were determined using the broth dilution technique. An in vivo study was then carried out with the bioactive extracts against the most resistant bacteria strain, to fully establish the therapeutic effect of these extracts. Balb/C mice were used, and ciprofloxacin was the positive control antibiotic. The extracts were administered to mice at two doses, 5mg/Kg and 10mg/Kg. The efficacy of extracts in the treatment of typhoid was evaluated based on survival rate, change in body weight, and change in bacteria load. Results. Only one of the extracts (crude pectin pH 2.5) was active against all the Salmonellae by well diffusion, and the growth inhibition varied from 12mm to 15mm at100 μg/ml. Three of the extracts (crude pectin pH 2.5, pH 4, 12h hydrolysate, and pH 4, 1h hydrolysate) had MIC and MBC against all four Salmonellae strains with MIC ranging from 5.68 to 44.45 μg/ml and MBC from 11.36 to 44.45 μg/mL. Three treatments, namely, the pH4-12 hr, hydrolysate at 10mg/Kg and 5mg/Kg, and the pH4-1hr, hydrolysate at 10mg/Kg, had therapeutic effects against Salmonella infection in mice. Conclusion. The present study highlights the potential of pectin oligosaccharides as new source of anti-Salmonella drugs. Further investigations including exploration of mechanism of action of the most active pectin extracts/hydrolysates are envisaged.

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