From Our Blog

Pataigin Introduces First Multiplex Lipid Microbiology Kit

VIENNA, Austria, BALTIMORE Maryland, and Seattle, Washington – 13 July 2021: At the 31st European Congress of Clinical Microbiology & Infectious Diseases (ECCMID, www.eccmid.org), Pataigin introduced the BACLIB Rapid Lipid Extraction Kit for microbial identification and colistin resistance detection.

The patented BACLIB Kit is unique in determining bacterial species ID and colistin resistance with a single assay. Superior to competing lipid microbiology kits, BACLIB requires less than a minute of hands-on time per sample, with no centrifugation or complex handling steps.

In contrast to competing kits that only identify a single species, the multiplex BACLIB Kit identifies and determines colistin resistance for all the ESKAPE pathogens. The BACLIB Kit includes access to custom software for ID/resistance determination. Time-to-result is only 30 minutes.

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Pataigin Awarded NIH Grant to Improve Antimicrobial Stewardship

Today, the US NIH awarded Pataigin a research grant to further development of Pataigin’s Rigel antimicrobial susceptibility test.

“We thank the NIH for supporting this research,” said Pataigin CEO Erik Nilsson. “Rigel promises to reduce medical costs while saving lives now lost to infectious disease.”

The grant, entitled Isotopic Labeling Rapid Antimicrobial Susceptibility Testing, furthers development of a rapid and sensitive Rigel AST test using lipid microbiology technology. Rigel can determine appropriate antibiotic therapy in six hours, compared to days for conventional tests.

“Rigel is based on my lab’s longstanding research into lipid microbiology,” said Prof. Robert Ernst, the Dr. Paul and Mrs. Jean Corcoran Professor and Chair, Department of Microbial Pathogenesis, School of Dentistry, University of Maryland, Baltimore. “In this research, we extend lipid microbiology beyond microbial ID to antimicrobial susceptibility testing.” Prof. Ernst is a collaborator on the grant.

Bacteremia and sepsis remain major health problems with high mortality. Progression is often rapid, so that a single hour saved in diagnosis time can significantly reduce septicemia death rates. Faster tests are therefore needed to save lives. By significantly accelerating test results, Rigel assay offers the potential of improved clinical outcomes at lower cost to hospitals.

Continue readingPataigin Awarded NIH Grant to Improve Antimicrobial Stewardship

Pataigin Founder Ernst Presents Rapid Colistin Assay at World Microbe Forum

Today, Pataigin founder Prof. Robert Ernst presented results of a new assay that determines microbial ID and colistin resistance in one hour, direct from clinical specimens. The assay uses lipid microbiology with the FLAT extraction technique to allow colistin resistance to be rapidly determined without culture.

Polymyxins – polymyxin B and polymyxin E (colistin) are increasingly used worldwide to treat patients with multidrug-resistant (MDR) Gram-negative bacterial infections. However, there currently is no reliable test for colistin resistance, complicating colistin treatment and increasing risks to patients. Prof. Ernst demonstrated that Pataigin’s FLAT technique can be reliably used to identify colistin resistance. The rapid and accurate test presented by Prof. Ernst promises to improve the safety and efficacy of colistin therapy.

The talk was entitled “Rapid Lipid-Based MALDI-TOF Assay for the Rapid Detection of Colistin Resistance,” and was presented in World Microbe Forum session AAR104. Prof. Ernst is the Dr. Paul and Mrs. Jean Corcoran Professor and Chair, Department of Microbial Pathogenesis, School of Dentistry, University of Maryland, Baltimore.

Pataigin is developing lipid microbiology tests for bacterial ID, antimicrobial resistance, and antimicrobial susceptibility.

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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

Published results: BACLIB Method IDs Pathogens and Colistin Resistance in One Hour

Scientific Reports today published a paper describing the FLAT rapid extraction method that allows identification of bacteria and fungi in one hour. Determination of colistin resistance in Gram-negative bacteria is also demonstrated. The paper was authored by Pataigin personnel in collaboration with the labs of BACLIB inventors Prof. Robert Ernst and Prof. David Goodlett. … Continue readingPublished results: BACLIB Method IDs Pathogens and Colistin Resistance in One Hour

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