Deep-sea microbes as tools to refine the rules of innate immune pattern recognition

Gauthier AE, Chandler CE, Poli V, Gardner FW, et al. Sci Immunol. 2021 Mar 12

The assumption of near-universal bacterial detection by pattern recognition receptors is a foundation of immunology. The limits of this pattern recognition concept, however, remain undefined. As a test of this hypothesis, we determined whether mammalian cells can recognize bacteria that they have never had the natural opportunity to encounter. These bacteria were cultivated from the deep Pacific Ocean, where the genus Moritella was identified as a common constituent of the culturable microbiota. Most deep-sea bacteria contained cell wall lipopolysaccharide (LPS) structures that were expected to be immunostimulatory, and some deep-sea bacteria activated inflammatory responses from mammalian LPS receptors. However, LPS receptors were unable to detect 80% of deep-sea bacteria examined, with LPS acyl chain length being identified as a potential determinant of immunosilence. The inability of immune receptors to detect most bacteria from a different ecosystem suggests that pattern recognition strategies may be defined locally, not globally. … Continue readingDeep-sea microbes as tools to refine the rules of innate immune pattern recognition

Colistin heteroresistance is largely undetected among carbapenem-resistant Enterobacterales in the United States

Band V, Satola S, Smith R, et al. mBio 2021 Jan 26

Heteroresistance is a form of antibiotic resistance where a bacterial strain is comprised of a minor resistant subpopulation and a majority susceptible subpopulation. We showed previously that colistin heteroresistance can mediate the failure of colistin therapy in an in vivo infection model, even for isolates designated susceptible by clinical diagnostics. We sought to characterize the extent of colistin heteroresistance among the highly drug-resistant carbapenem-resistant Enterobacterales (CRE). We screened 408 isolates for colistin heteroresistance. These isolates were collected between 2012 and 2015 in eight U.S. states as part of active surveillance for CRE. Colistin heteroresistance was detected in 10.1% (41/408) of isolates, and it was more common than conventional homogenous resistance (7.1%, 29/408). Most (93.2%, 38/41) of these heteroresistant isolates were classified as colistin susceptible by standard clinical diagnostic testing. The frequency of colistin heteroresistance was greatest in 2015, the last year of the study. This was especially true among Enterobacter isolates, of which specific species had the highest rates of heteroresistance. Among Klebsiella pneumoniae isolates, which were the majority of isolates tested, there was a closely related cluster of colistin-heteroresistant ST-258 isolates found mostly in Georgia. However, cladistic analysis revealed that, overall, there was significant diversity in the genetic backgrounds of heteroresistant K. pneumoniae isolates. These findings suggest that due to being largely undetected in the clinic, colistin heteroresistance among CRE is underappreciated in the United States. … Continue readingColistin heteroresistance is largely undetected among carbapenem-resistant Enterobacterales in the United States

Rapid microbial identification and colistin resistance detection via MALDI-TOF MS using a novel on-target extraction of membrane lipids

Sorensen M, Chandler CE, Gardner F, et al. Scientific Reports 2020 Dec.

Rapid infection diagnosis is critical to improving patient treatment and outcome. Recent studies have shown microbial lipids to be sensitive and selective biomarkers for identifying bacterial and fungal species and antimicrobial resistance. Practical procedures for microbial lipid biomarker analysis will therefore improve patient outcomes and antimicrobial stewardship. However, current lipid extraction methods require significant hands-on time and are thus not suited for direct adoption as a clinical assay for microbial identification. Here, we have developed a method for lipid extraction directly on the surface of stainless-steel matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) plates, termed fast lipid analysis technique or FLAT, which facilitates the identification of bacterial and fungal species using a sub-60-minute workflow. Additionally, our method detects lipid A modifications in Gram-negative bacteria that are associated with antimicrobial resistance, including to colistin.
Continue readingRapid microbial identification and colistin resistance detection via MALDI-TOF MS using a novel on-target extraction of membrane lipids

Early evolutionary loss of the lipid A modifying enzyme PagP resulting in innate immune evasion in Yersinia pestis

Chandler CE, Harberts EM, Pelletier MR, et al. Proc Natl Acad Sci U S A 2020 Sep 15.

Immune evasion through membrane remodeling is a hallmark of Yersinia pestis pathogenesis. Yersinia remodels its membrane during its life cycle as it alternates between mammalian hosts (37 °C) and ambient (21 °C to 26 °C) temperatures of the arthropod transmission vector or external environment. This shift in growth temperature induces changes in number and length of acyl groups on the lipid A portion of lipopolysaccharide (LPS) for the enteric pathogens Yersinia pseudotuberculosis (Ypt) and Yersinia enterocolitica (Ye), as well as the causative agent of plague, Yersinia pestis (Yp). Addition of a C16 fatty acid (palmitate) to lipid A by the outer membrane acyltransferase enzyme PagP occurs in immunostimulatory Ypt and Ye strains, but not in immune-evasive Yp Analysis of Yp pagP gene sequences identified a single-nucleotide polymorphism that results in a premature stop in translation, yielding a truncated, nonfunctional enzyme. Upon repair of this polymorphism to the sequence present in Ypt and Ye, lipid A isolated from a Yp pagP+ strain synthesized two structures with the C16 fatty acids located in acyloxyacyl linkage at the 2′ and 3′ positions of the diglucosamine backbone. Structural modifications were confirmed by mass spectrometry and gas chromatography. With the genotypic restoration of PagP enzymatic activity in Yp, a significant increase in lipid A endotoxicity mediated through the MyD88 and TRIF/TRAM arms of the TLR4-signaling pathway was observed. Discovery and repair of an evolutionarily lost lipid A modifying enzyme provides evidence of lipid A as a crucial determinant in Yp infectivity, pathogenesis, and host innate immune evasion. … Continue readingEarly evolutionary loss of the lipid A modifying enzyme PagP resulting in innate immune evasion in Yersinia pestis

Streamlined Analysis of Cardiolipins in Prokaryotic and Eukaryotic Samples Using a Norharmane Matrix by MALDI-MSI

Yang H, Jackson SN, Woods AS, et al. J Am Soc Mass Spectrom. 2020 Oct 5.

Cardiolipins (CLs) are an important, regulated lipid class both in prokaryotic and eukaryotic cells, yet they remain largely unexplored by matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) in tissues. To date, no in-depth optimization studies of label-free visualization of CLs in complex biological samples have been reported. Here we report a streamlined modification to our previously reported MALDI-MSI method for detection of endogenous CLs in prokaryotic and eukaryotic cells based on preparation with norharmane (NRM) matrix. Notably, the use of NRM matrix permitted sensitive detection (4.7 pg/mm2) of spotted CL synthetic standards. By contrast, four other MALDI matrices commonly used for lipid analysis failed to generate CL ions. Using this NRM-based method, endogenous CLs were detected from two types of complex biological samples: dried bacterial arrays and mouse tissue sections. In both cases, using NRM resulted in a better signal/noise for CL ions than the other matrices. Furthermore, inclusion of a washing step improved CL detection from tissue and this combined tissue preparation method (washing and NRM matrix) was used to profile normal mouse lung. Mouse lung yielded 26 unique CLs that were mapped and identified. Consistent with previous findings, CLs containing polyunsaturated fatty acids (PUFAs) were found in abundance in the airway and vascular features of the lung. This work represents a comprehensive investigation of detection conditions for CL using MALDI-MSI in complex biological samples that resulted in a streamlined method that enables future studies of the biological role(s) of CL in tissue. … Continue readingStreamlined Analysis of Cardiolipins in Prokaryotic and Eukaryotic Samples Using a Norharmane Matrix by MALDI-MSI

On-tissue derivatization of lipopolysacharide for detection of lipid A using MALDI-MSI

Yang H, Chandler CE, Jackson SN, et al. Analytical Chemistry 2020 Sep.

We developed a method to directly detect and map the Gram-negative bacterial virulence factor lipid A derived from lipopolysaccharide (LPS) by coupling acid hydrolysis with matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI). As the structure of lipid A (endotoxin) determines the innate immune outcome during infection, the ability to map its location within an infected organ or animal is needed to understand localized inflammatory responses that results during host–pathogen interactions. We previously demonstrated detection of free lipid A from infected tissue; however detection of lipid A derived from intact (smooth) LPS from host–pathogen MSI studies, proved elusive. Here, we detected LPS-derived lipid A from the Gram-negative pathogens, Escherichia coli (Ec, m/z 1797) and Pseudomonas aeruginosa (Pa, m/z 1446) using on-tissue acid hydrolysis to cleave the glycosidic linkage between the polysaccharide (core and O-antigen) and lipid A moieties of LPS. Using accurate mass methods, the ion corresponding to the major Ec and lipid A species (m/z 1797 and 1446, respectively) were unambiguously discriminated from complex tissue substrates. Further, we evaluated potential delocalization and signal loss of other tissue lipids and found no evidence for either, making this LPS-to-Lipid A-MSI (LLA-MSI) method, compatible with simultaneous host–pathogen lipid imaging following acid hydrolysis. This spatially sensitive technique is the first step in mapping host-influenced de novo lipid A modifications, such as those associated with antimicrobial resistance phenotypes, during Gram-negative bacterial infection and will advance our understanding of the host–pathogen interface. … Continue readingOn-tissue derivatization of lipopolysacharide for detection of lipid A using MALDI-MSI

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