Next Generation Chemiluminescent Probes for Antimalarial Drug Discovery.

Malaria is caused by parasites of the Plasmodium genus and remains one of the most pressing human health problems. The spread of parasites resistant to or partially resistant to single or multiple drugs, including frontline antimalarial artemisinin and its derivatives, poses a serious threat to current and future malaria control efforts. In vitro drug assays are important for identifying new antimalarial compounds and monitoring drug resistance. Due to its robustness and ease of use, the [3H]-hypoxanthine incorporation assay is still considered a gold standard and is widely applied, despite limited sensitivity and the dependence on radioactive material. Here, we present a first-of-its-kind chemiluminescence-based antimalarial drug screening assay. The effect of compounds on P. falciparum is monitored by using a dioxetane-based substrate (AquaSpark ß-D-galactoside) that emits high-intensity luminescence upon removal of a protective group (ß-D-galactoside) by a transgenic ß-galactosidase reporter enzyme. This biosensor enables highly sensitive, robust, and cost-effective detection of asexual, intraerythrocytic P. falciparum parasites without the need for parasite enrichment, washing, or purification steps. We are convinced that the ultralow detection limit of less than 100 parasites of the presented biosensor system will become instrumental in malaria research, including but not limited to drug screening.

Validation of N-Light™ Salmonella Risk Test Kit for Detection of Salmonella spp. on Environmental Surfaces: AOAC Performance Tested MethodSM 072204.

BACKGROUND: The NEMIS N-Light™ Salmonella Risk method uses chemiluminescence designed for the qualitative detection of Salmonella spp. from environmental surface samples. OBJECTIVE: To validate the N-Light Salmonella Risk assay using independent and method developer validation studies according to the AOAC Performance Tested MethodsSM (PTM) program for the detection of Salmonella spp. on stainless-steel, polystyrene, and ceramic environmental surfaces. METHOD: The N-Light Salmonella Risk assay was evaluated in a matrix study in comparison to the ISO 6579-1:2017 method ("Microbiology of the Food Chain-Horizontal Method for the Detection, Enumeration, and Serotyping of Salmonella-Part 1: Detection of Salmonella spp.") using an unpaired study design. Additional PTM studies performed were inclusivity/exclusivity, robustness, product consistency, and stability. RESULTS: The N-Light Salmonella Risk assay demonstrated a specific detection of all Salmonella strains tested. In the matrix study, the N-Light Salmonella Risk assay showed no significant differences between presumptive and confirmed results or between candidate and reference method results on the three surfaces evaluated. Data for additional PTM studies met acceptance criteria requirements. CONCLUSIONS: The NEMIS Technologies N-Light Salmonella Risk assay is an effective method for the qualitative detection of Salmonella on stainless-steel, polystyrene, and ceramic environmental surfaces. HIGHLIGHTS: The NEMIS Technologies N-Light Salmonella Risk assay, which is the first chemiluminescence-based detection system that uses a novel, patented dioxetane compound, allowing for easy and rapid detection of Salmonella.

Validation of N-LightTM L. monocytogenes for Detection of Listeria monocytogenes on Environmental Surfaces: AOAC Performance Tested MethodSM 122002.

BACKGROUND: The NEMIS Technologies N-LightTM L. monocytogenes assay uses chemiluminescence designed for the qualitative detection of Listeria monocytogenes from environmental surface samples. OBJECTIVE: To validate the NEMIS Technologies N-Light L. monocytogenes assay as part of the AOAC Performance Tested MethodSM program for the detection of L. monocytogenes on stainless steel, plastic (polystyrene), and ceramic environmental surfaces. METHOD: Using the Vitl Life Science Solutions Lu-mini luminometer, the NEMIS Technologies N-Light L. monocytogenes assay was compared to the ISO 11290-1:2017: Microbiology of the Food Chain-Horizontal method for the detection and enumeration of Listeria monocytogenes and of Listeria spp. - Part 1 using a 1" × 1" stainless steel test area in an unpaired study design. RESULTS: The NEMIS Technologies N-Light L. monocytogenes assay using the Vitl Life Science Solutions Lu-mini luminometer demonstrated no statistically significant differences between presumptive and confirmed results or between candidate and reference method results. Data for additional Performance Tested MethodSM studies met acceptance criteria requirements. CONCLUSIONS: The NEMIS Technologies N-Light L. monocytogenes assay is an effective method for the qualitative detection of L. monocytogenes from stainless steel, plastic (polystyrene), and ceramic environmental surface samples. HIGHLIGHTS: The NEMIS method is the first chemiluminescence detection system based on a novel, patented, dioxetane compound.

AquaSparkTM - Rapid Environmental Monitoring of Listeria monocytogenes.

In order to prevent microbial contamination of food, monitoring of the production environment, together with the rapid detection of foodborne pathogens have proven to be of utmost importance for Food Safety. Environmental monitoring should detect harmful pathogens at the earliest point in time in order for the necessary interventions to be taken. However, current detection methods fall short with regards to speed, ease of use, and cost. This article aims to present the idea behind NEMIS Technologies, a startup company making use of the novel AquaSparkTM technology for the development of a new generation of bacterial detection methods. These methods utilize chemiluminescence in order to detect live target bacteria in a short period of time compared to that of conventional methods. We show that dry-stressed Listeria monocytogenes can be detected within 24 hours, using small-molecule chemiluminescent probes, together with a bacteria-specific proprietary enrichment broth containing a cocktail of bacteriophages, which enhance the specificity and sensitivity. This novel platform technology has the potential to extend beyond environmental monitoring towards food analyses as well as veterinary and human health.

Glycotyping and Specific Separation of Listeria monocytogenes with a Novel Bacteriophage Protein Tool Kit.

The Gram-positive pathogen Listeria monocytogenes can be subdivided into at least 12 different serovars, based on the differential expression of a set of somatic and flagellar antigens. Of note, strains belonging to serovars 1/2a, 1/2b, and 4b cause the vast majority of foodborne listeriosis cases and outbreaks. The standard protocol for serovar determination involves an agglutination method using a set of sera containing cell surface-recognizing antibodies. However, this procedure is imperfect in both precision and practicality, due to discrepancies resulting from subjective interpretation. Furthermore, the exact antigenic epitopes remain unclear, due to the preparation of the absorbed sera and the complex nature of polyvalent antibody binding. Here, we present a novel method for quantitative somatic antigen differentiation using a set of recombinant affinity proteins (cell wall-binding domains and receptor-binding proteins) derived from a collection of Listeria bacteriophages. These proteins enable rapid, objective, and precise identification of the different teichoic acid glycopolymer structures, which represent the O-antigens, and allow a near-complete differentiation. This glycotyping approach confirmed serovar designations of over 60 previously characterized Listeria strains. Using select phage receptor-binding proteins coupled to paramagnetic beads, we also demonstrate the ability to specifically isolate serovar 1/2 or 4b cells from a mixed culture. In addition, glycotyping led to the discovery that strains designated serovar 4e actually possess an intermediate 4b-4d teichoic acid glycosylation pattern, underpinning the high discerning power and precision of this novel technique.IMPORTANCEListeria monocytogenes is a ubiquitous opportunistic pathogen that presents a major concern to the food industry due to its propensity to cause foodborne illness. The Listeria genus contains 15 different serovars, with most of the variance depending on the wall-associated teichoic acid glycopolymers, which confer somatic antigenicity. Strains belonging to serovars 1/2 and 4b cause the vast majority of listeriosis cases and outbreaks, meaning that regulators, as well as the food industry itself, have an interest in rapidly identifying isolates of these particular serovars in food processing environments. Current methods for phenotypic serovar differentiation are slow and lack accuracy, and the food industry could benefit from new technologies allowing serovar-specific isolation. Therefore, the novel method described here for rapid glycotype determination could present a valuable asset to detect and control this bacterium.

Ultrasensitive Detection of Salmonella and Listeria monocytogenes by Small-Molecule Chemiluminescence Probes.

Detection of Salmonella and L. monocytogenes in food samples by current diagnostic methods requires relatively long time to results (2-6 days). Furthermore, the ability to perform environmental monitoring at the factory site for these pathogens is limited due to the need for laboratory facilities. Herein, we report new chemiluminescence probes for the ultrasensitive direct detection of viable pathogenic bacteria. The probes are composed of a bright phenoxy-dioxetane luminophore masked by triggering group, which is activated by a specific bacterial enzyme, and could detect their corresponding bacteria with an LOD value of about 600-fold lower than that of fluorescent probes. Moreover, we were able to detect a minimum of 10 Salmonella cells within 6 h incubation. The assay allows for bacterial enrichment and detection in one test tube without further sample preparation. We anticipate that this design strategy will be used to prepare analogous chemiluminescence probes for other enzymes relevant to specific bacteria detection and point-of-care diagnostics.

Structure and transformation of bacteriophage A511 baseplate and tail upon infection of Listeria cells.

Contractile injection systems (bacteriophage tails, type VI secretions system, R-type pyocins, etc.) utilize a rigid tube/contractile sheath assembly for breaching the envelope of bacterial and eukaryotic cells. Among contractile injection systems, bacteriophages that infect Gram-positive bacteria represent the least understood members. Here, we describe the structure of Listeria bacteriophage A511 tail in its pre- and post-host attachment states (extended and contracted, respectively) using cryo-electron microscopy, cryo-electron tomography, and X-ray crystallography. We show that the structure of the tube-baseplate complex of A511 is similar to that of phage T4, but the A511 baseplate is decorated with different receptor-binding proteins, which undergo a large structural transformation upon host attachment and switch the symmetry of the baseplate-tail fiber assembly from threefold to sixfold. For the first time under native conditions, we show that contraction of the phage tail sheath assembly starts at the baseplate and propagates through the sheath in a domino-like motion.

Molecular Basis of Bacterial Host Interactions by Gram-Positive Targeting Bacteriophages.

The inherent ability of bacteriophages (phages) to infect specific bacterial hosts makes them ideal candidates to develop into antimicrobial agents for pathogen-specific remediation in food processing, biotechnology, and medicine (e.g., phage therapy). Conversely, phage contaminations of fermentation processes are a major concern to dairy and bioprocessing industries. The first stage of any successful phage infection is adsorption to a bacterial host cell, mediated by receptor-binding proteins (RBPs). As the first point of contact, the binding specificity of phage RBPs is the primary determinant of bacterial host range, and thus defines the remediative potential of a phage for a given bacterium. Co-evolution of RBPs and their bacterial receptors has forced endless adaptation cycles of phage-host interactions, which in turn has created a diverse array of phage adsorption mechanisms utilizing an assortment of RBPs. Over the last decade, these intricate mechanisms have been studied intensely using electron microscopy and X-ray crystallography, providing atomic-level details of this fundamental stage in the phage infection cycle. This review summarizes current knowledge surrounding the molecular basis of host interaction for various socioeconomically important Gram-positive targeting phage RBPs to their protein- and saccharide-based receptors. Special attention is paid to the abundant and best-characterized Siphoviridae family of tailed phages. Unravelling these complex phage-host dynamics is essential to harness the full potential of phage-based technologies, or for generating novel strategies to combat industrial phage contaminations.

A functional type II-A CRISPR-Cas system from Listeria enables efficient genome editing of large non-integrating bacteriophage.

CRISPR-Cas systems provide bacteria with adaptive immunity against invading DNA elements including bacteriophages and plasmids. While CRISPR technology has revolutionized eukaryotic genome engineering, its application to prokaryotes and their viruses remains less well established. Here we report the first functional CRISPR-Cas system from the genus Listeria and demonstrate its native role in phage defense. LivCRISPR-1 is a type II-A system from the genome of L. ivanovii subspecies londoniensis that uses a small, 1078 amino acid Cas9 variant and a unique NNACAC protospacer adjacent motif. We transferred LivCRISPR-1 cas9 and trans-activating crRNA into Listeria monocytogenes. Along with crRNA encoding plasmids, this programmable interference system enables efficient cleavage of bacterial DNA and incoming phage genomes. We used LivCRISPR-1 to develop an effective engineering platform for large, non-integrating Listeria phages based on allelic replacement and CRISPR-Cas-mediated counterselection. The broad host-range Listeria phage A511 was engineered to encode and express lysostaphin, a cell wall hydrolase that specifically targets Staphylococcus peptidoglycan. In bacterial co-culture, the armed phages not only killed Listeria hosts but also lysed Staphylococcus cells by enzymatic collateral damage. Simultaneous killing of unrelated bacteria by a single phage demonstrates the potential of CRISPR-Cas-assisted phage engineering, beyond single pathogen control.

Genome Sequences of the Listeria ivanovii subsp. ivanovii Type Strain and Two Listeria ivanovii subsp. londoniensis Strains.

We present the complete genomes of Listeria ivanovii subsp. ivanovii WSLC 3010 (ATCC 19119(T)), Listeria ivanovii subsp. londoniensis WSLC 30151 (SLCC 8854), and Listeria ivanovii subsp. londoniensis WSLC 30167 (SLCC 6032), representing the type strain of the species and two strains of the same serovar but different properties, respectively.

Genome Sequences of Three Frequently Used Listeria monocytogenes and Listeria ivanovii Strains.

We present the complete de novo assembled genome sequences of Listeria monocytogenes strains WSLC 1001 (ATCC 19112) and WSLC 1042 (ATCC 23074) and Listeria ivanovii WSLC 3009, three strains frequently used for the propagation and study of bacteriophages because they are presumed to be free of inducible prophages.