From Our Blog

Pataigin Director Matthew Sorensen Presents Key Technology at ASMS

Pataigin Director of Development Matthew Sorensen made an oral presentation at the 2020 American Society for Mass Spectrometry conference. The subject of Sorensen’s talk was Rigel, a technology developed at Pataigin under Sorensen’s leadership for mass spectrometry-based antimicrobial susceptibility testing. … Continue readingPataigin Director Matthew Sorensen Presents Key Technology at ASMS

Entrepreneur of The Year: Pataigin Founder Prof. Robert Ernst

Professor Robert Ernst, a founder of Pataigin, has been named the 2019 University of Maryland, Baltimore Entrepreneur of the Year. Prof. Ernst is Professor and Vice Chair, Department of Microbial Pathogenesis, School of Dentistry, University of Maryland, Baltimore (UMB). This prestigious award was made in part for Ernst’s work in translating his research into Pataigin.

In 2017, Ernst was honored as UMB’s Researcher of the Year. Thus over the course of three years, Ernst has received important University awards for both basic research and transnational science. … Continue readingEntrepreneur of The Year: Pataigin Founder Prof. Robert Ernst

Bacterial medium-chain 3-hydroxy fatty acid metabolites trigger immunity in Arabidopsis plants

Kutschera A, Dawid C, Gisch N, Schmid C, et al. Science 2019 Apr 12

In plants, cell-surface immune receptors sense molecular non–self-signatures. Lipid A of Gram-negative bacterial lipopolysaccharide is considered such a non–self-signature. The receptor kinase LIPOOLIGOSACCHARIDE-SPECIFIC REDUCED ELICITATION (LORE) mediates plant immune responses to Pseudomonas and Xanthomonas but not enterobacterial lipid A or lipopolysaccharide preparations. Here, we demonstrate that synthetic and bacterial lipopolysaccharide-copurified medium-chain 3-hydroxy fatty acid (mc-3-OH-FA) metabolites elicit LORE-dependent immunity. The mc-3-OH-FAs are sensed in a chain length– and hydroxylation-specific manner, with free (R)-3-hydroxydecanoic acid [(R)-3-OH-C10:0] representing the strongest immune elicitor. By contrast, bacterial compounds comprising mc-3-OH-acyl building blocks but devoid of free mc-3-OH-FAs—including lipid A or lipopolysaccharide, rhamnolipids, lipopeptides, and acyl-homoserine-lactones—do not trigger LORE-dependent responses. Hence, plants sense low-complexity bacterial metabolites to trigger immune responses. … Continue readingBacterial medium-chain 3-hydroxy fatty acid metabolites trigger immunity in Arabidopsis plants

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

Leung LM, McElheny CL, Gardner FM, et al. J Clin Microbiol. 2019 Feb.

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. … Continue readingA Prospective Study of Acinetobacter baumannii Complex Isolates and Colistin Susceptibility Monitoring by Mass Spectrometry of Microbial Membrane Glycolipids

Rapid Microbial Identification and Antibiotic Resistance Detection by Mass Spectrometric Analysis of Membrane Lipids

Liang T, Leung LM, Opene B, et al. Analytical Chemistry 2019 Jan.

Infectious diseases have a substantial global health impact. Clinicians need rapid and accurate diagnoses of infections to direct patient treatment and improve antibiotic stewardship. Current technologies employed in routine diagnostics are based on bacterial culture followed by morphological trait differentiation and biochemical testing, which can be time-consuming and labor-intensive. With advances in mass spectrometry (MS) for clinical diagnostics, the U.S. Food and Drug Administration has approved two microbial identification platforms based on matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) MS analysis of microbial proteins. We recently reported a novel and complementary approach by comparing MALDI-TOF mass spectra of microbial membrane lipid fingerprints to identify ESKAPE pathogens. However, this lipid-based approach used a sample preparation method that required more than a working day from sample collection to identification. Here, we report a new method that extracts lipids efficiently and rapidly from microbial membranes using an aqueous sodium acetate (SA) buffer that can be used to identify clinically relevant Gram-positive and -negative pathogens and fungal species in less than an hour. The SA method also has the ability to differentiate antibiotic-susceptible and antibiotic-resistant strains, directly identify microbes from biological specimens, and detect multiple pathogens in a mixed sample. These results should have positive implications for the manner in which bacteria and fungi are identified in general hospital settings and intensive care units.
Continue readingRapid Microbial Identification and Antibiotic Resistance Detection by Mass Spectrometric Analysis of Membrane Lipids

Pathogen Identification Direct From Polymicrobial Specimens Using Membrane Glycolipids

Fondrie WE, Liang T, Oyler BL, et al. Scientific Reports 2018 Oct.

With the increased prevalence of multidrug-resistant Gram-negative bacteria, the use of colistin and other last-line antimicrobials is being revisited clinically. As a result, there has been an emergence of colistin-resistant bacterial species, including Acinetobacter baumannii and Klebsiella pneumoniae. The rapid identification of such pathogens is vitally important for the effective treatment of patients. We previously demonstrated that mass spectrometry of bacterial glycolipids has the capacity to identify and detect colistin resistance in a variety of bacterial species. In this study, we present a machine learning paradigm that is capable of identifying A. baumannii, K. pneumoniae and their colistin-resistant forms using a manually curated dataset of lipid mass spectra from 48 additional Gram-positive and -negative organisms. We demonstrate that these classifiers detect A. baumannii and K. pneumoniae in isolate and polymicrobial specimens, establishing a framework to translate glycolipid mass spectra into pathogen identifications. … Continue readingPathogen Identification Direct From Polymicrobial Specimens Using Membrane Glycolipids

Optimized surface acoustic wave nebulization facilitates bacterial phenotyping

Liang T, Schneider T, Yoon SH, et al. Int J Mass Spectrom. 2018 Apr.

We report on the characterization of three different Surface Acoustic Wave Nebulization (SAWN) chip designs for use in mass spectrometric (MS) analysis of the bacterial glycolipid known as lipid A. We used three different statistical methods to objectively calculate MS noise level and, subsequently, signal-to-noise ratio for the purpose of choosing the optimum SAWN chip between three different designs. The best performing standing wave SAWN chip enabled MS detection of 125 fmol of the commercial standard monophosphoryl lipid A. All three chips allowed detection of lipid A extracted from 9 × 104 CFU of Francisella novicida. Finally, we show that SAWN-MS could be used to distinguish between different Gram-negative bacterial species based on their lipid A MS profiles, which has implications in the field of bacterial phenotyping. … Continue readingOptimized surface acoustic wave nebulization facilitates bacterial phenotyping

Lipid A structural modifications in extreme conditions and identification of unique modifying enzymes to define the Toll-like receptor 4 structure-activity relationship

Scott AJ, Oyler BL, Goodlett DR, Ernst RK. Biochim Biophys Acta Mol Cell Biol Lipids. 2017 Nov

Strategies utilizing Toll-like receptor 4 (TLR4) agonists for treatment of cancer, infectious diseases, and other targets report promising results. Potent TLR4 antagonists are also gaining attention as therapeutic leads. Though some principles for TLR4 modulation by lipid A have been described, a thorough understanding of the structure-activity relationship (SAR) is lacking. Only through a complete definition of lipid A-TLR4 SAR is it possible to predict TLR4 signaling effects of discrete lipid A structures, rendering them more pharmacologically relevant. A limited ‘toolbox’ of lipid A-modifying enzymes has been defined and is largely composed of enzymes from mesophile human and zoonotic pathogens. Expansion of this ‘toolbox’ will result from extending the search into lipid A biosynthesis and modification by bacteria living at the extremes. Here, we review the fundamentals of lipid A structure, advances in lipid A uses in TLR4 modulation, and the search for novel lipid A-modifying systems in extremophile bacteria. This article is part of a Special Issue entitled: Bacterial Lipids edited by Russell E. Bishop. … Continue readingLipid A structural modifications in extreme conditions and identification of unique modifying enzymes to define the Toll-like receptor 4 structure-activity relationship

Structural modification of LPS in colistin-resistant, KPC-producing Klebsiella pnemoniae

Leung LM, Cooper VS, Rasko DA, et al. J Antimicrob Chemother. 2017 Nov.

Colistin resistance in Klebsiella pneumoniae typically involves inactivation or mutations of chromosomal genes mgrB, pmrAB or phoPQ, but data regarding consequent modifications of LPS are limited.

Objectives
To examine the sequences of chromosomal loci implicated in colistin resistance and the respective LPS-derived lipid A profiles using 11 pairs of colistin-susceptible and -resistant KPC-producing K. pneumoniae clinical strains.

Methods
The strains were subjected to high-throughput sequencing with Illumina HiSeq. The mgrB gene was amplified by PCR and sequenced. Lipid profiles were determined using MALDI-TOF MS.

Results
All patients were treated with colistimethate prior to the isolation of colistin-resistant strains (MIC >2 mg/L). Seven of 11 colistin-resistant strains had deletion or insertional inactivation of mgrB. Three strains, including one with an mgrB deletion, had non-synonymous pmrB mutations associated with colistin resistance. When analysed by MALDI-TOF MS, all colistin-resistant strains generated mass spectra containing ions at m/z 1955 and 1971, consistent with addition of 4-amino-4-deoxy-l-arabinose (Ara4N) to lipid A, whereas only one of the susceptible strains displayed this lipid A phenotype.

Conclusions
The pathway to colistin resistance in K. pneumoniae primarily involves lipid A modification with Ara4N in clinical settings. … Continue readingStructural modification of LPS in colistin-resistant, KPC-producing Klebsiella pnemoniae