Publications related to Pataigin and BACLIB

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.

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.

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.

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.

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

Background
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.

Leung LM, Fondrie WE, Doi Y, et al. Sci Rep. 2017 Jul

Rapid diagnostics that enable identification of infectious agents improve patient outcomes, antimicrobial stewardship, and length of hospital stay. Current methods for pathogen detection in the clinical laboratory include biological culture, nucleic acid amplification, ribosomal protein characterization, and genome sequencing. Pathogen identification from single colonies by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) analysis of high abundance proteins is gaining popularity in clinical laboratories. Here, we present a novel and complementary approach that utilizes essential microbial glycolipids as chemical fingerprints for identification of individual bacterial species. Gram-positive and negative bacterial glycolipids were extracted using a single optimized protocol. Extracts of the clinically significant ESKAPE pathogens: Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter spp. were analyzed by MALDI-TOF-MS in negative ion mode to obtain glycolipid mass spectra. A library of glycolipid mass spectra from 50 microbial entries was developed that allowed bacterial speciation of the ESKAPE pathogens, as well as identification of pathogens directly from blood bottles without culture on solid medium and determination of antimicrobial peptide resistance. These results demonstrate that bacterial glycolipid mass spectra represent chemical barcodes that identify pathogens, potentially providing a useful alternative to existing diagnostics.

Liu YY, Chandler CE, Leung LM, et al. Antimicrob Agents Chemother. 2017 May 24

mcr-1 was initially reported as the first plasmid-mediated colistin resistance gene in clinical isolates of Escherichia coli and Klebsiella pneumoniae in China and has subsequently been identified worldwide in various species of the family Enterobacteriaceae. mcr-1 encodes a phosphoethanolamine transferase, and its expression has been shown to generate phosphoethanolamine-modified bis-phosphorylated hexa-acylated lipid A in E. coli. Here, we investigated the effects of mcr-1 on colistin susceptibility and on lipopolysaccharide structures in laboratory and clinical strains of the Gram-negative ESKAPE (Enterococcus faecium, Staphylococcus aureus, K. pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species) pathogens, which are often treated clinically by colistin. The effects of mcr-1 on colistin resistance were determined using MIC assays of laboratory and clinical strains of E. coli, K. pneumoniae, A. baumannii, and P. aeruginosa Lipid A structural changes resulting from MCR-1 were analyzed by mass spectrometry. The introduction of mcr-1 led to colistin resistance in E. coli, K. pneumoniae, and A. baumannii but only moderately reduced susceptibility in P. aeruginosa. Phosphoethanolamine modification of lipid A was observed consistently for all four species. These findings highlight the risk of colistin resistance as a consequence of mcr-1 expression among ESKAPE pathogens, especially in K. pneumoniae and A. baumannii. Furthermore, the observation that lipid A structures were modified despite only modest increases in colistin MICs in some instances suggests more sophisticated surveillance methods may need to be developed to track the dissemination of mcr-1 or plasmid-mediated phosphoethanolamine transferases in general.

Scott AJ, Flinders B, Cappell J, Liang T, Pelc RS, et al. Pathog Dis. 2016 Nov

The discovery of novel pathogenic mechanisms engaged during bacterial infections requires the evolution of advanced techniques. Here, we evaluate the dual polarity matrix norharmane (NRM) to improve detection of bacterial lipid A (endotoxin), from host and vector tissues infected with Francisella novicida(Fn). We evaluated NRM for improved detection and characterization of a wide range of lipids in both positive and negative polarities, including lipid A and phospholipids across a range of matrix-assisted laser desorption-ionization-coupled applications. NRM matrix improved the limit of detection (LOD) for monophosphoryl lipid A (MPLA) down to picogram level representing a 10-fold improvement of LOD versus 2,5-dihydroxybenzoic acid and 100-fold improvement of LOD versus 9-aminoacridine (9-AA). Improved LOD for lipid A subsequently facilitated detection of the Fn lipid A major ion (m/z 1665) from extracts of infected mouse spleen and the temperature-modified Fn lipid A at m/z 1637 from infected Dermacentor variabilis ticks. Finally, we simultaneously mapped bacterial phospholipid signatures within anFn-infected spleen along with an exclusively host-derived inositol-based phospholipid (m/z 933) demonstrating coprofiling of the host-pathogen interaction. Expanded use of NRM matrix in other infection models and endotoxin-targeting imaging experiments will improve our understanding of the lipid interactions at the host-pathogen interface.

Hittle LE, Powell DA, Jones JW, Tofigh M, Goodlett DR, Moskowitz SM, Ernst RK. Pathog Dis. 2015 Nov

Pseudomonas aeruginosa (PA) is an opportunistic Gram-negative pathogen associated with nosocomial infections, acute infections and chronic lung infections in patients with cystic fibrosis. The ability of PA to cause infection can be attributed to its ability to adapt to a multitude of environments. Modification of the lipid A portion of lipopolysaccharide (LPS) is a vital mechanism Gram-negative pathogens use to remodel the outer membrane in response to environmental stimuli. Lipid A, the endotoxic moiety of LPS, is the major component of the outer leaflet of the outer membrane of Gram-negative bacteria making it a critical factor for bacterial adaptation. One way PA modifies its lipid A is through the addition of laurate and 2-hydroxylaurate. This secondary or late acylation is carried out by the acyltransferase, HtrB (LpxL). Analysis of the PA genome revealed the presence of two htrB homologs, PA0011 (htrB1) and PA3242 (htrB2). In this study, we were able to show that each gene identified is responsible for site-specific modification of lipid A. Additionally, deletions of either gene altered resistance to specific classes of antibiotics, cationic antimicrobial peptides and increased membrane permeability suggesting a role for these enzymes in maintaining optimal membrane organization and integrity.

Pelletier MR, Casella LG, Jones JW, et al. Antimicrob Agents Chemother. 2013 Oct

Acinetobacter baumannii is 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 treat A. baumannii infections. Colistin-resistant strains of A. baumannii with 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-resistant A. baumannii MAC204 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-adapted A. baumannii strain MAC204. In summary, this work has shown complete structure characterization including the accurate assignment of acylation, phosphorylation, and glycosylation of lipid A from A. baumannii, which are important for resistance to colistin.