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Chemische gewasbeschermingsmiddelen (GWBs) worden nauwgezet gevolgd door het publiek, ngo’s en autoriteiten (b.v. neonicotinoïden, fipronil). De verwachting is dat de toepassing van chemische GWBs de komende jaren verder beperkt zal worden. Ter bestrijding van plagen en ziektes zullen de chemische middelen in toenemende mate vervangen worden door “groene” producten, op basis van b.v. bacteriën, schimmels of virussen. Uit een eerdere survey (uitgevoerd 17-7-2019) naar groene GWBs is gebleken dat er 51 van dit soort middelen als GWB in Nederland geregistreerd staan op basis van 11 bacteriële, 14 schimmel- en drie virale biologische agentia. WFSR heeft tot dusver geen methoden operationeel voor monitoring en controle van deze GWBs, noch voor identificatie noch voor kwantificering van de biologische agentia in deze GWBs. Ook zijn er geen methoden voor het aantonen van de werkzame stoffen van deze middelen in voeding en diervoeder beschikbaar. Binnen dit project is een verkennende literatuurstudie uitgevoerd naar bestaande identificatie methoden voor het testen van groene GWBs en is hun praktische haalbaarheid beoordeeld. De EFSA-assessments welke zijn opgesteld bij de aanvraag voor toelating van de producten op de markt zijn hierbij als basis gebruikt. Het resultaat laat een grote diversiteit zien in de kwaliteit en effectiviteit van de analysemethoden. Slechts voor een beperkt aantal van de door EFSA geëvalueerde agentia blijken geschikte identificatie methoden beschikbaar. Dit betreft methoden gebaseerd op DNA sequentie informatie. Beschreven methoden gebaseerd op DNA- en eiwit “fingerprinting” technieken worden ongeschikt geacht omdat het onderscheidend vermogen van deze methoden te gering is voor onweerlegbare identificatie. Voor een aantal producten wordt in de EFSA assessments door de producenten geen duidelijke identificatiemethode aangedragen. Dit is opvallend omdat competente autoriteiten dit vereisen bij de registratie van een GWB. Als proof of principle zijn

AIMS: The aim of our study was to investigate the virulence and resistance of STEC from small ruminants farms in The Netherlands. Moreover, the potential transmission of STEC between animals and humans on farms was evaluated. METHODS AND RESULTS: From 182 farms, in total, 287 unique STEC isolates were successfully recovered from animal samples. In addition, STEC was isolated from eight out of 144 human samples. The most detected serotype was O146:H21; however, among other serotypes also O26:H11, O157:H7, and O182:H25 isolates were present. Whole genome sequencing covering all human isolates and 50 of the animal isolates revealed a diversity of stx1, stx2, and eae sub-types and an additional 57 virulence factors. The assessed antimicrobial resistance phenotype, as determined by microdilution, was concordant with the genetic profiles identified by WGS. WGS also showed that three of the human isolates could be linked to an animal isolate from the same farm. CONCLUSIONS: The obtained STEC isolates showed great diversity in serotype, virulence, and resistance factors. Further analysis by WGS allowed for an in-depth assessment of the virulence and resistance factors present and to determine the relatedness of human and animal isolates.

Listeria monocytogenes (Lm) is ubiquitous in nature and known for its ability to contaminate foods during production processes. Near real-time monitoring of whole genome sequences from food and human isolates, complemented with epidemiological data, has been used in the Netherlands since 2019 to increase the speed and success rate of source finding in the case of (active) clusters. Nine clusters with 4 to 19 human cases investigated between January 2019 and May 2023 are described. Fish production sites were most often linked to outbreaks of listeriosis (six clusters), though other types of food businesses can face similar Lm problems, as the production processes and procedures determine risk. The results showed that low levels of Lm in food samples can still be linked to disease. Therefore, the investigation of a cluster of cases and deployment of the precautionary principle helps to focus on safe food and to prevent further cases. Good practice of environmental monitoring within a food business allows early detection of potential issues with food safety and helps food businesses to take appropriate measures such as cleaning to prevent regrowth of Lm and thus future outbreaks.

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.

Genome mining of the bacterial strains Pseudomonas sp. SH-C52 and Pseudomonas fluorescens DSM 11579 showed that both strains contained a highly similar gene cluster encoding an octamodular nonribosomal peptide synthetase (NRPS) system which was not associated with a known secondary metabolite. Insertional mutagenesis of an NRPS component followed by comparative profiling led to the discovery of the corresponding novel linear octalipopeptide thanafactin A, which was subsequently isolated and its structure determined by two-dimensional NMR and further spectroscopic and chromatographic methods. In bioassays, thanafactin A exhibited weak protease inhibitory activity and was found to modulate swarming motility in a strain-specific manner.

The study of the virulence and anti-microbial resistance of Shiga toxin-producing Escherichia coli from goat and sheep dairy farms in the Netherlands with whole genome sequencing analysis, The study of the virulence and anti-microbial resistance of Shiga toxin-producing Escherichia coli from goat and sheep dairy farms in the Netherlands with whole genome sequencing analysis

Pseudomonas fluorescens DSM 11579 is known to be a producer of the lipopeptides brabantamide and thanamycin. Its draft genome gives insight into the complete secondary metabolite production capacity of the strain and builds the basis for a comparative study with Pseudomonas sp. strain SH-C52, a lipopeptide-producing strain involved in natural disease-suppressive soils.

A large European multi-country Salmonella enterica serovar Enteritidis outbreak associated with Polish eggs was character-ized by whole-genome sequencing (WGS)-based analysis, with various European institutes using different analysis workflows to identify isolates potentially related to the outbreak. The objective of our study was to compare the output of six of these different typing workflows (distance matrices of either SNP-based or allele-based workflows) in terms of cluster detection and concordance. To this end, we analysed a set of 180 isolates coming from confirmed and probable outbreak cases, which were representative of the genetic variation within the outbreak, supplemented with 22 unrelated contemporaneous S. enterica serovar Enteritidis isolates. Since the definition of a cluster cut-off based on genetic distance requires prior knowledge on the evolutionary processes that govern the bacterial populations in question, we used a variety of hierarchical clustering methods (single, average and complete) and selected the optimal number of clusters based on the consensus of the silhouette, Dunn2, and McClain–Rao internal validation indices. External validation was done by calculating the concordance with the WGS-based case definition (SNP-address) for this outbreak using the Fowlkes–Mallows index. Our analysis indicates that with complete-linkage hierarchical clustering combined with the optimal number of clusters, as defined by three internal validity indices, the six different allele-and SNP-based typing workflows generate clusters with similar compositions. Furthermore, we show that even in the absence of coordinated typing procedures, but by using an unsupervised machine learning methodology for cluster delineation, the various workflows that are currently in use by six European public-health authorities can identify concordant clusters of genetically related S. enterica serovar Enteritidis isolates; thus, providing public-health researchers with compara

In plant-associated Pseudomonas species, the production of several secondary metabolites and exoenzymes is regulated by the GacS/GacA two-component regulatory system (the Gac-system). Here, we investigated if a mutation in the GacS sensor kinase affects the production of volatile organic compounds (VOCs) in P. fluorescens SBW25 (Pf.SBW25) and how this impacts on VOCs-mediated growth promotion and induced systemic resistance of Arabidopsis and tobacco. A total of 205 VOCs were detected by Gas Chromatography Mass Spectrometry (GC-MS) for Pf. SBW25 and the gacS-mutant grown on 2 different media for 3 and 6 days. Discriminant function analysis followed by hierarchical clustering revealed 24 VOCs that were significantly different in their abundance between Pf.SBW25 and the gacS-mutant, which included three acyclic alkenes (3-nonene, 4-undecyne, 1-undecene). These alkenes were significantly reduced by the gacS mutation independently of the growth media and of the incubation time. For Arabidopsis, both Pf.SBW25 and the gacS-mutant enhanced, via VOCs, root and shoot biomass, induced systemic resistance against leaf infections by P. syringae and rhizosphere acidification to the same extent. For tobacco, however, VOCs-mediated effects on shoot and root growth were significantly different between Pf.SBW25 and the gacS-mutant. While Pf.SBW25 inhibited tobacco root growth, the gacS-mutant enhanced root biomass and lateral root formation relative to the non-treated control plants. Collectively these results indicate that the sensor kinase GacS is involved in the regulation of VOCs production in Pf.SBW25, affecting plant growth in a plant species-dependent manner.

Emerging fungal and oomycete pathogens are increasingly threatening animals and plants globally. Amongst oomycetes, Saprolegnia species adversely affect wild and cultivated populations of amphibians and fish, leading to substantial reductions in biodiversity and food productivity. With the ban of several chemical control measures, new sustainable methods are needed to mitigate Saprolegnia infections in aquaculture. Here, PhyloChip-based community analyses showed that the Pseudomonadales, particularly Pseudomonas species, represent one of the largest bacterial orders associated with salmon eggs from a commercial hatchery. Among the Pseudomonas species isolated from salmon eggs, significantly more biosurfactant producers were retrieved from healthy salmon eggs than from Saprolegnia-infected eggs. Subsequent in vivo activity bioassays showed that Pseudomonas isolate H6 significantly reduced salmon egg mortality caused by Saprolegnia diclina. Live colony mass spectrometry showed that strain H6 produces a viscosin-like lipopeptide surfactant. This biosurfactant inhibited growth of Saprolegnia in vitro, but no significant protection of salmon eggs against Saprolegniosis was observed. These results indicate that live inocula of aquatic Pseudomonas strains, instead of their bioactive compound, can provide new (micro) biological and sustainable means to mitigate oomycete diseases in aquaculture., QC 20151006

Protozoan predation of bacteria can significantly affect soil microbial community composition and ecosystem functioning. Bacteria possess diverse defense strategies to resist or evade protozoan predation. For soil-dwelling Pseudomonas species, several secondary metabolites were proposed to provide protection against different protozoan genera. By combining whole-genome transcriptome analyses with (live) imaging mass spectrometry (IMS), we observed multiple changes in the molecular and chemical dialogues between Pseudomonas fluorescens and the protist Naegleria americana. Lipopeptide (LP) biosynthesis was induced in Pseudomonas upon protozoan grazing and LP accumulation transitioned from homogeneous distributions across bacterial colonies to site-specific accumulation at the bacteria-protist interface. Also putrescine biosynthesis was upregulated in P. fluorescens upon predation. We demonstrated that putrescine induces protozoan trophozoite encystment and adversely affects cyst viability. This multifaceted study provides new insights in common and strain-specific responses in bacteria-protozoa interactions, including responses that contribute to bacterial survival in highly competitive soil and rhizosphere environments.

BACKGROUND: The plant microbiome represents an enormous untapped resource for discovering novel genes and bioactive compounds. Previously, we isolated Pseudomonas sp. SH-C52 from the rhizosphere of sugar beet plants grown in a soil suppressive to the fungal pathogen Rhizoctonia solani and showed that its antifungal activity is, in part, attributed to the production of the chlorinated 9-amino-acid lipopeptide thanamycin (Mendes et al. 2011. Science). To get more insight into its biosynthetic repertoire, the genome of Pseudomonas sp. SH-C52 was sequenced and subjected to in silico, mutational and functional analyses. The sequencing revealed a genome size of 6.3 Mb and 5,579 predicted ORFs. Phylogenetic analysis placed strain SH-C52 within the Pseudomonas corrugata clade. In silico analysis for secondary metabolites revealed a total of six nonribosomal peptide synthetase (NRPS) gene clusters, including the two previously described NRPS clusters for thanamycin and the 2-amino acid antibacterial lipopeptide brabantamide. Here we show that thanamycin also has activity against an array of other fungi and that brabantamide A exhibits anti-oomycete activity and affects phospholipases of the late blight pathogen Phytophthora infestans. Most notably, mass spectrometry led to the discovery of a third LP, designated thanapeptin, with a 22-amino-acid peptide moiety. Seven structural variants of thanapeptin were found with varying degrees of activity against P. infestans. Of the remaining four NRPS clusters, one was predicted to encode for yet another and unknown lipopeptide with a predicted peptide moiety of 8-amino acids. Collectively, these results show an enormous metabolic potential for Pseudomonas sp. SH-C52, with at least three structurally diverse lipopeptides, each with a different antimicrobial activity spectrum.

In Pseudomonas species, production of secondary metabolites and exoenzymes is regulated by the GacS/GacA two-component regulatory system. In P. fluorescens SBW25, mutations in the Gac-system cause major transcriptional changes and abolished production of the lipopeptide viscosin and of an exoprotease. In contrast to many other Pseudomonas species and strains, inactivation of the Gac-system in strain SBW25 significantly enhanced its antimicrobial activities against oomycete, fungal and bacterial pathogens. Here, random plasposon mutagenesis of the gacS mutant led to the identification of seven mutants with reduced or loss of antimicrobial activity. In four mutants, the plasposon insertion was located in genes of the pyrroloquinoline quinone (PQQ) biosynthesis pathway. Genetic complementation, ectopic expression, activity bioassays and RP-HPLC analyses revealed that a gacS mutation in SBW25 leads to enhanced expression of pqq genes, resulting in an increase in gluconic and 2-ketogluconic acid production, which in turn acidified the extracellular medium to levels that inhibit growth of other microorganisms. We also showed that PQQ-mediated acidification comes with a growth penalty for the gacS mutant in the stationary phase. In conclusion, PQQ-mediated acidification compensates for the loss of several antimicrobial traits in P. fluorescens SBW25 and may help gac mutants to withstand competitors.

Background The antimetabolite mangotoxin is a key factor in virulence of Pseudomonas syringae pv. syringae strains which cause apical necrosis of mango trees. Previous studies showed that mangotoxin biosynthesis is governed by the mbo operon. Random mutagenesis led to the identification of two other gene clusters that affect mangotoxin biosynthesis. These are the gacS/gacA genes and mgo operon which harbors the four genes mgoBCAD. Results The current study shows that disruption of the nonribosomal peptide synthetase (NRPS) gene mgoA resulted in loss of mangotoxin production and reduced virulence on tomato leaves. Transcriptional analyses by qPCR and promoter reporter fusions revealed that mbo expression is regulated by both gacS/gacA and mgo genes. Also, expression of the mgo operon was shown to be regulated by gacS/gacA. Heterologous expression under the native promoter of the mbo operon resulted in mangotoxin production in non-producing P. syringae strains, but not in other Pseudomonas species. Also introduction of the mbo and mgo operons in nonproducing P. protegens Pf-5 did not confer mangotoxin production but did enhance transcription of the mbo promoter. Conclusions From the data obtained in this study, we conclude that both mbo and mgo operons are under the control of the gacS/gacA two-component system and that the MgoA product acts as a positive regulator of mangotoxin biosynthesis.