Identification by High-Throughput Real-Time PCR of 30 Major Circulating Listeria monocytogenes Clonal Complexes in Europe.

Listeria monocytogenes is a ubiquitous bacterium that causes a foodborne illness, listeriosis. Most strains can be classified into major clonal complexes (CCs) that account for the majority of outbreaks and sporadic cases in Europe. In addition to the 20 CCs known to account for the majority of human and animal clinical cases, 10 CCs are frequently reported in food production, thereby posing a serious challenge for the agrifood industry. Therefore, there is a need for a rapid and reliable method to identify these 30 major CCs. The high-throughput real-time PCR assay presented here provides accurate identification of these 30 CCs and eight genetic subdivisions within four CCs, splitting each CC into two distinct subpopulations, along with the molecular serogroup of a strain. Based on the BioMark high-throughput real-time PCR system, our assay analyzes 46 strains against 40 real-time PCR arrays in a single experiment. This European study (i) designed the assay from a broad panel of 3,342 L. monocytogenes genomes, (ii) tested its sensitivity and specificity on 597 sequenced strains collected from 24 European countries, and (iii) evaluated its performance in the typing of 526 strains collected during surveillance activities. The assay was then optimized for conventional multiplex real-time PCR for easy implementation in food laboratories. It has already been used for outbreak investigations. It represents a key tool for assisting food laboratories to establish strain relatedness with human clinical strains during outbreak investigations and for helping food business operators by improving their microbiological management plans. IMPORTANCE Multilocus sequence typing (MLST) is the reference method for Listeria monocytogenes typing but is expensive and takes time to perform, from 3 to 5 days for laboratories that outsource sequencing. Thirty major MLST clonal complexes (CCs) are circulating in the food chain and are currently identifiable only by sequencing. Therefore, there is a need for a rapid and reliable method to identify these CCs. The method presented here enables the rapid identification, by real-time PCR, of 30 CCs and eight genetic subdivisions within four CCs, splitting each CC into two distinct subpopulations. The assay was then optimized on different conventional multiplex real-time PCR systems for easy implementation in food laboratories. The two assays will be used for frontline identification of L. monocytogenes isolates prior to whole-genome sequencing. Such assays are of great interest for all food industry stakeholders and public agencies for tracking L. monocytogenes food contamination.

New insights into the epidemiology of Listeria monocytogenes - A cross-sectoral retrospective genomic analysis in the Netherlands (2010-2020).

Introduction: Listeriosis, caused by infection with Listeria monocytogenes (Lm), is a relatively rare but severe disease with one of the highest mortality rates among bacterial foodborne illnesses. A better understanding on the degree of Lm clustering, the temporal distribution of the clusters, and their association with the various food sources is expected to lead to improved source tracing and risk-based sampling. Methods: We investigated the genomic epidemiology of Lm in the Netherlands between 2010 and 2020 by analyzing whole-genome-sequencing (WGS) data of isolates from listerioss patients and food sources from nationwide integrated surveillance and monitoring. WGS data of 756 patient and 770 food/environmental isolates was assessed using core-genome multi-locus sequence typing (cgMLST) with Hamming distance as measure for pairwise distances. Associations of genotype with the epidemiological variables such as patient's age and gender, and systematic use of specific drugs were tested by multinomial logistic regressions. Genetic differentiation of the Lm within and between food categories was calculated based on allele frequencies at the 1701 cgMLST loci in each food category. Results: We confirmed previous results that some clonal complexes (CCs) are overrepresented among clinical isolates but could not identify any epidemiological risk factors. The main findings of this study include the observation of a very weak attribution of Lm types to food categories and a much better attribution to the producer level. In addition, we identified a high degree of temporal persistence of food, patient and mixed clusters, with more than half of the clusters spanning over more than 1 year and up to 10 years. Discussion: Taken together this would indicate that identifying persistent contamination in food production settings, and producers that process a wide variety of raw food produce, could significantly contribute to lowering the Lm disease burden.

The Microbial Diversity of Cabbage Pest Delia radicum Across Multiple Life Stages.

The cabbage root fly Delia radicum is a worldwide pest that causes yield losses of many common cabbage crops. The bacteria associated with D. radicum are suggested to influence the pest status of their host. In this study, we characterized insect-associated bacteria of D. radicum across multiple life stages and of their diet plant (turnip, Brassica rapa subsp. rapa) by sequencing the V3-V4 region of 16S rRNA genes using the Illumina MiSeq platform. In total, over 1.2M paired-end reads were obtained, identifying 1006 bacterial amplicon sequence variants (ASVs) in samples obtained from the eggs, larvae, pupae and adults of D. radicum, as well as turnips that were either fresh or infested with D. radicum larvae. The microbial community in D. radicum was dominated by Wolbachia, a common endosymbiont of arthropods which we found in all of the investigated insect samples, with the pupal stage having the highest relative abundance. Moderate amounts of Firmicutes were found only in adult D. radicum flies, but not in previous life stages. Actinobacteria were mostly found on the eggs and on the skin of fresh plants on which the eggs were deposited. These plants also harbored a large amount of Pseudomonas. The bacterial diversity of the healthy turnip was low, whereas the microbial community of decaying turnips that were heavily infested by D. radicum larvae and showing symptoms of advanced soft rot was characterized by a high bacterial diversity. Taken together, this work provides insights into the bacterial communities associated with the cabbage pest D. radicum and its associated disease symptoms.

Single gene enables plant pathogenic Pectobacterium to overcome host-specific chemical defence.

Plants of the Brassicales order, including Arabidopsis and many common vegetables, produce toxic isothiocyanates to defend themselves against pathogens. Despite this defence, plant pathogenic microorganisms like Pectobacterium cause large yield losses in fields and during storage of crops. The bacterial gene saxA was previously found to encode isothiocyanate hydrolase that degrades isothiocyanates in vitro. Here we demonstrate in planta that saxA is a virulence factor that can overcome the chemical defence system of Brassicales plants. Analysis of the distribution of saxA genes in Pectobacterium suggests that saxA from three different phylogenetic origins are present within this genus. Deletion of saxA genes representing two of the most common classes from P. odoriferum and P. versatile resulted in significantly reduced virulence on Arabidopsis thaliana and Brassica oleracea. Furthermore, expressing saxA from a plasmid in a potato-specific P. parmentieri strain that does not naturally harbour this gene significantly increased the ability of the strain to macerate Arabidopsis. These findings suggest that a single gene may have a significant role in defining the host range of a plant pathogen.

Co-cultivation of the strictly anaerobic methanogen Methanosarcina barkeri with aerobic methanotrophs in an oxygen-limited membrane bioreactor.

Wetlands contribute to 30% of global methane emissions due to an imbalance between microbial methane production and consumption. Methanogenesis and methanotrophy have mainly been studied separately, and little is known about their potential interactions in aquatic environments. To mimic the interaction between methane producers and oxidizers in the environment, we co-cultivated the methanogenic archaeon Methanosarcina barkeri with aerobic Methylocystaceae methanotrophs in an oxygen-limited bioreactor using acetate as methanogenic substrate. Methane, acetate, dissolved oxygen, available nitrogen, pH, temperature, and cell density were monitored to follow system stability and activity. Stable reactor operation was achieved for two consecutive periods of 2 months. Fluorescence in situ hybridization micrographs indicated close association between both groups of microorganisms. This association suggests that the methanotrophs profit from direct access to the methane that is produced from acetate, while methanogens are protected by the concomitant oxygen consumption of the methanotrophs. This proof of principle study can be used to set up systems to study their responses to environmental changes.

Functional Profiling and Crystal Structures of Isothiocyanate Hydrolases Found in Gut-Associated and Plant-Pathogenic Bacteria.

Isothiocyanates (ITCs) are produced by cruciferous plants to protect them against herbivores and infection by microbes. These compounds are of particular interest due to their antimicrobial and anticarcinogenic properties. The breakdown of ITCs in nature is catalyzed by isothiocyanate hydrolases (ITCases), a novel family within the metallo-ß-lactamase (MBL)-fold superfamily of proteins. saxA genes that code for ITCases are particularly widespread in insect- and plant-associated bacteria. Enzymatic characterization of seven phylogenetically related but distinct ITCases revealed similar activities on six selected ITCs, suggesting that phylogenetic diversity does not determine the substrate specificity of ITCases. X-ray crystallography studies of two ITCases sharing 42% amino acid sequence identity revealed a highly conserved tertiary structure. Notable features of ITCases include a hydrophobic active site with two Zn2+ ions coordinating water/hydroxide and a flexible cap that is implicated in substrate recognition and covers the active site. This report reveals the function and structure of the previously uncharacterized family of isothiocyanate hydrolases within the otherwise relatively well-studied superfamily of metallo-ß-lactamases.IMPORTANCE This study explores a newly discovered protein in the ß-lactamase superfamily, namely, SaxA, or isothiocyanate hydrolase. Isothiocyanates are defensive compounds found in many cabbage-related crop plants and are currently being investigated for their antimicrobial and anticarcinogenic properties. We show that isothiocyanate hydrolases are responsible for the breakdown of several of these plant defensive chemicals in vitro and suggest their potential for mitigating the beneficial effects of isothiocyanates in crop protection and cancer prevention.

Detoxifying symbionts in agriculturally important pest insects.

Pest insects lead to excessive agricultural and therefore economical losses on crops worldwide. These insects have to withstand toxic molecules that are inherent to plant defences, as well as those that are produced and introduced by humans in the form of insecticides. In recent years, research on insect-microbe symbioses has recognized that microbial symbionts may play a role protecting against these toxins, leading to a form of defensive symbiosis between the pest insect and different types of microorganisms that we term detoxifying symbioses. In this minireview, we will highlight well-characterized and emerging insect model systems of detoxifying symbioses and assess how the microorganisms influence the host's success.

Plasmids from the gut microbiome of cabbage root fly larvae encode SaxA that catalyses the conversion of the plant toxin 2-phenylethyl isothiocyanate.

Cabbage root fly larvae (Delia radicum) cause severe crop losses (≥ 50%) of rapeseed/ canola and cabbages used in the food and biofuel industries. These losses occur despite the fact that cabbages produce insecticidal toxins such as isothiocyanates. Here we describe the cabbage root fly larval gut microbiome as a source of isothiocyanate degrading enzymes. We sequenced the microbial gut community of the larvae and analysed phylogenetic markers and functional genes. We combined this with the isolation of several microbial strains representing the phylogenetic distribution of the metagenome. Eleven of those isolates were highly resistant towards 2-phenylethyl isothiocyanate, a subset also metabolized 2-phenylethyl isothiocyanate. Several plasmids appeared to be shared between those isolates that metabolized the toxin. One of the plasmids harboured a saxA gene that upon transformation gave resistance and enabled the degradation of 2-phenylethyl isothiocyanate in Escherichia coli. Taken together, the results showed that the cabbage root fly larval gut microbiome is capable of isothiocyanate degradation, a characteristic that has not been observed before, and may help us understand and design new pest control strategies.