A whole cell-based Matrix-assisted laser desorption/ionization mass spectrometry lipidomic assay for the discovery of compounds that target lipid a modifications

Introduction: Matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) is a powerful analytical technique that has been applied to a wide variety of applications ranging from proteomics to clinical diagnostics. One such application is its use as a tool for discovery assays, such as monitoring the inhibition of purified proteins. With the global threat from antimicrobial-resistant (AMR) bacteria, new and innovative solutions are required to identify new molecules that could revert bacterial resistance and/or target virulence factors. Here, we used a whole cell-based MALDI-TOF lipidomic assay using a routine MALDI Biotyper Sirius system operating in linear negative ion mode combined with the MBT Lipid Xtract kit to discover molecules targeting bacteria that are resistant to polymyxins, which are considered last-resort antibiotics. Methods: A library of 1200 natural compounds was tested against an E. coli strain expressing mcr-1, which is known to modify lipid A by adding phosphoethanolamine (pETN), making the strain resistant to colistin. Results and Discussion: Using this approach, we identified 8 compounds that led to a decrease in this lipid A modification by MCR-1 and could potentially be employed to revert resistance. Taken together, as-proof-of-principle, the data we report here represent a new workflow based on the analysis of bacterial lipid A by routine MALDI-TOF for the discovery of inhibitors that could target bacterial viability and/or virulence

Discrimination of bovine milk from non-dairy milk by lipids fingerprinting using routine matrix-assisted laser desorption ionization mass spectrometry

An important sustainable development goal for any country is to ensure food security by producing a sufficient and safe food supply. This is the case for bovine milk where addition of non-dairy milks such as vegetables (e.g., soya or coconut) has become a common source of adulteration and fraud. Conventionally, gas chromatography techniques are used to detect key lipids (e.g., triacylglycerols) has an effective read-out of assessing milks origins and to detect foreign milks in bovine milks. However, such approach requires several sample preparation steps and a dedicated laboratory environment, precluding a high throughput process. To cope with this need, here, we aimed to develop a novel and simple method without organic solvent extractions for the detection of bovine and non-dairy milks based on lipids fingerprint by routine MALDI-TOF mass spectrometry (MS). The optimized method relies on the simple dilution of milks in water followed by MALDI-TOF MS analyses in the positive linear ion mode and using a matrix consisting of a 9:1 mixture of 2,5-dihydroxybenzoic acid and 2-hydroxy-5-methoxybenzoic acid (super-DHB) solubilized at 10 mg/mL in 70% ethanol. This sensitive, inexpensive, and rapid method has potential for use in food authenticity applications

Detection of colistin resistance in Salmonella enterica using MALDIxin test on the routine MALDI Biotyper Sirius mass spectrometer

Resistance to polymyxins in most Gram-negative bacteria arises from chemical modifications to the lipid A portion of their lipopolysaccharide (LPS) mediated by chromosomally-encoded mutations or the recently discovered plasmid-encoded mcr genes that have further complicated the landscape of colistin resistance. Currently, minimal inhibitory concentration (MIC) determination by broth microdilution, the gold standard for the detection of polymyxin resistance, is time consuming (24 hours) and challenging to perform in clinical and veterinatryveterinary laboratories. Here we present the use of the MALDIxin to detect colistin resistant Salmonella enterica using the MALDxin test on the routine matrix-assisted laser desorption ionization (MALDI) Biotyper Sirius system

Emergent expression of fitness-conferring genes by phenotypic selection

Genotypic and phenotypic adaptation is the consequence of ongoing natural selection in populations and is key to predicting and preventing drug resistance. Whereas classic antibiotic persistence is all-or-nothing, here we demonstrate that an antibiotic resistance gene displays linear dose-responsive selection for increased expression in proportion to rising antibiotic concentration in growing E. coli populations. Furthermore, we report the potentially wide-spread nature of this form of emergent gene expression by instantaneous phenotypic selection process under bactericidal and bacteriostatic antxibiotic treatment, as well as an amino acid synthesis pathway enzyme under a range of auxotrophic conditions. We propose an analogy to Ohm’s law in electricity (V=IR) where selection pressure acts similarly to voltage (V), gene expression to current (I), and resistance (R) to cellular machinery constraints and costs. Lastly, mathematical modelling using agent-based models of stochastic gene expression in growing populations and Bayesian model selection reveal that the emergent gene expression mechanism requires variability in gene expression within an isogenic population, and a cellular ‘memory’ from positive feedbacks between growth and expression of any fitness-conferring gene. Finally, we discuss the connection of the observed phenomenon to a previously described general fluctuation-response relationship in biology

Two-site study on performances of a commercially available MALDI-TOF MS-based assay for the detection of colistin resistance in Escherichia coli

Colistin is a last resort drug for the treatment of multiple drug-resistant (MDR) Gram-negative bacterial infections. Rapid methods to detect resistance are highly desirable. Here, we evaluated the performance of a commercially available MALDI-TOF MS-based assay for colistin resistance testing in Escherichia coli at two different sites. Ninety clinical E. coli isolates were provided by France and tested in Germany and UK using a MALDI-TOF MS-based colistin resistance assay. Lipid A molecules of the bacterial cell membrane were extracted using the MBT Lipid Xtract Kit™ (RUO; Bruker Daltonics, Germany). Spectra acquisition and evaluation were performed by the MBT HT LipidART Module of MBT Compass HT (RUO; Bruker Daltonics) on a MALDI Biotyper® sirius system (Bruker Daltonics) in negative ion mode. Phenotypic colistin resistance was determined by broth microdilution (MICRONAUT MIC-Strip Colistin, Bruker Daltonics) and used as a reference. Comparing the results of the MALDI-TOF MS-based colistin resistance assay with the data of the phenotypic reference method for the UK, sensitivity and specificity for the detection of colistin resistance were 97.1% (33/34) and 96.4% (53/55), respectively. Germany showed 97.1% (33/34) sensitivity and 100% (55/55) specificity for the detection of colistin resistance by MALDI-TOF MS. Applying the MBT Lipid Xtract™ Kit in combination with MALDI-TOF MS and dedicated software showed excellent performances for E. coli. Analytical and clinical validation studies must be performed to demonstrate the performance of the method as a diagnostic tool

Detection of colistin resistance in Pseudomonas aeruginosa using the MALDIxin test on the routine MALDI Biotyper Sirius mass spectrometer

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 Pseudomonas 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 Pseudomonas 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 1 hour, 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 Pseudomonas aeruginosa and is thus a valuable diagnostics tool to control the spread of these emerging resistant strains

Optimization of the MALDIxin test for the rapid identification of colistin resistance in Klebsiella pneumoniae using MALDI-TOF-MS

Background. With the dissemination of carbapenemase producers, a revival of colistin was observed for the treatment of infections caused by multidrug-resistant Gram-negatives. Unfortunately, the increasing usage of colistin led to the emergence of resistance. In Klebsiella pneumoniae, colistin resistance arises through addition of L-arabinose-4N (L-Ara4N) or phosphoethanolamine (pEtN) on the native lipid A. The underlying mechanisms involve numerous chromosome-encoded genes or the plasmid-encoded phosphoethanolamine transferase MCR. Currently, detection of colistin resistance is time consuming since it still relies on MIC determination by broth microdilution. Recently, a rapid diagnostic test based on MALDI-TOF detection of modified lipid A was developed (the MALDIxin test) and tested on Escherichia coli and Acinetobacter baumannii. Objectives. Optimize the MALDIxin test for the rapid detection of colistin resistance in Klebsiella pneumoniae. Methods. This optimization consists on an additional mild-acid hydrolysis of 15 min in 1% acetic acid. The optimized method was tested on a collection of 81 clinical K. pneumoniae isolates including 49 colistin resistant strains among which 45 correspond to chromosome-encoded resistance, 3 MCR-related resistance and one isolate harbouring both mechanisms. Results. The optimized method allowed the rapid (< 30 min) identification of L-Ara4N and pEtN modified lipid A of K. pneumoniae which are known to be the real triggers of polymyxin resistance. In the same time, it discriminates between chromosome-encoded and MCR-related polymyxin resistance. Conclusions. The MALDIxin test has the potential to become an accurate tool for the rapid diagnostic of colistin resistance in clinically-relevant Gram negative bacteria

Inhibition of the Niemann-Pick C1 protein is a conserved feature of multiple strains of pathogenic mycobacteria

Mycobacterium tuberculosis (Mtb) survives and replicates within host macrophages (MΦ) and subverts multiple antimicrobial defense mechanisms. Previously, we reported that lipids shed by pathogenic mycobacteria inhibit NPC1, the lysosomal membrane protein deficient in the lysosomal storage disorder Niemann-Pick disease type C (NPC). Inhibition of NPC1 leads to a drop in lysosomal calcium levels, blocking phagosome-lysosome fusion leading to mycobacterial survival. We speculated that the production of specific cell wall lipid(s) that inhibit NPC1 could have been a critical step in the evolution of pathogenicity. We therefore investigated whether lipid extracts from clinical Mtb strains from multiple Mtb lineages, Mtb complex (MTBC) members and non-tubercular mycobacteria (NTM) inhibit the NPC pathway. We report that inhibition of the NPC pathway was present in all clinical isolates from Mtb lineages 1, 2, 3 and 4, Mycobacterium bovis and the NTM, Mycobacterium abscessus and Mycobacterium avium. However, lipid extract from Mycobacterium canettii, which is considered to resemble the common ancestor of the MTBC did not inhibit the NPC1 pathway. We conclude that the evolution of NPC1 inhibitory mycobacterial cell wall lipids evolved early and post divergence from Mycobacterium canettii-related mycobacteria and that this activity contributes significantly to the promotion of disease