Whole genome sequence analysis of the 2018 Persian onager isolate suggests sublineages within the Taylorella asinigenitalis species.

In 2018, a T. asinigenitalis strain (MCE663) was isolated in a Persian onager tested for contagious equine metritis (CEM) in a United Kingdom (UK) zoo. This bacterium had never been reported in the UK and Multilocus Sequence Typing described a new atypically divergent ST (ST60). Although the causative agent of CEM is the bacterium Taylorella equigenitalis, a first natural outbreak of endometritis caused by T. asinigenitalis ST70 was reported in 2019, putting its pathogenic potential into question. In this context, we aimed to further sequence the T. asinigenitalis MCE663 genome and characterize the strain using phenotypical and genetic approaches. Results showed that it gathered all identification characteristics of T. asinigenitalis with smaller colonies and it was susceptible to all tested antibiotics. Genome-level phylogeny showed that the genome MCE663 formed a distinct phylogroup, and only shared ≈ 96.1% of average nucleotide identity (ANI) with the three published T. asinigenitalis genomes, which together shared ≈ 98.3% ANI. According to current cut-offs consensus for species and subspecies delineation (95% and 98%, respectively), our results support the first insights of a sublineage delineation within the T. asinigenitalis species.

First report of enterotoxigenic Staphylococcus argenteus as a foodborne pathogen.

Staphylococcal enterotoxins preformed in food are the causative agents of staphylococcal food poisoning outbreaks (SFPO). In this study we characterised in depth two coagulase-positive non-pigmented staphylococci involved in two independent outbreaks that occurred in France. While indistinguishable from Staphylococcus aureus using PCR methods and growth phenotype comparisons, both isolates were identified as Staphylococcus argenteus by whole genome sequencing. The genomes were analysed for the presence of enterotoxin genes, whose expression was determined in laboratory medium and, for the first time, in artificially-contaminated milk samples by using liquid chromatography-mass spectrometry and ELISA methods. The concentration measured for the SEB toxin in milk (0.67 ng/ml) was comparable to concentrations reported for other types of enterotoxins behind SFPO. From a collection of publicly available genomes, we performed an unprecedented systematic investigation of the enterotoxin gene set of S. argenteus, including variants and pseudogenes. The most prevalent genes were sex, followed by sel26, sel27 and sey. The egc cluster was less frequent and most of the time carried a dysfunctional seg gene. Our results shed light on the enterotoxigenic properties of S. argenteus, and emphasize the importance in monitoring of S. argenteus as an emerging foodborne pathogen.

Top-Down Mass Spectrometry for Trace Level Quantification of Staphylococcal Enterotoxin A Variants.

We addressed here the need for improved sensitivity of top-down mass spectrometry for identification, differentiation, and absolute quantification of sequence variants of SEA, a bacterial toxin produced by Staphylococcus aureus and regularly involved in food poisoning outbreaks (FPO). We combined immunoaffinity enrichment, a protein internal standard, and optimized acquisition conditions, either by full-scan high-resolution mass spectrometry (HRMS) or multiplex parallel reaction monitoring (PRM) mode. Deconvolution of full-scan HRMS signal and PRM detection of variant-specific fragment ions allowed confident identification of each SEA variant. Summing the PRM signal of variant-common fragment ions was most efficient for absolute quantification, illustrated by a sensitivity down to 2.5 ng/mL and an assay variability below 15%. Additionally, we showed that relative PRM fragment ion abundances constituted a supplementary specificity criterion in top-down quantification. The top-down method was successfully evaluated on a panel of enterotoxin-producing strains isolated during FPO, in parallel to the conventional whole genome sequencing, ELISA, and bottom-up mass spectrometry methods. Top-down provided at the same time correct identification of the SEA variants produced and precise determination of the toxin level. The raw files generated in this study can be found on PASSEL (Peptide Atlas) under data set identifier PASS01710.

Quantitative Determination of Staphylococcus aureus Enterotoxins Types A to I and Variants in Dairy Food Products by Multiplex Immuno-LC-MS/MS.

Staphylococcal enterotoxins (SEs) are responsible for frequent food poisoning outbreaks worldwide. Specific identification of SEs is crucial for confirmation of food poisoning, tracking of the incriminated foods or food ingredients, and removal from the food chain. Here, we report on a new food testing protocol addressing the challenge of low abundance of SEs in contaminated food and high sequence heterogeneity. Multiplex ability of targeted high-resolution mass spectrometry was succesfully applied to the simultaneous and quantitative determination of the eight most frequent SEs including sequence variants. In this aim, between three and eight proteotypic peptides of each SE were selected by carefully considering amino acid variations within each type, and sequence homology between types. Quantification of trace levels of SEs directly in food samples was reached by immunoaffinity enrichment and optimized analytical conditions. The assay was validated in dairy food products with a lower limit of quantification down to 0.1 ng/g (in milk), and quantification of SEs was successfully demonstrated in real-life samples collected during staphylococcal food poisoning outbreaks. Importantly, the ability of the method to detect diverse sequence variants was also illustrated. By enabling for the first time the simultaneous quantification of the eight most frequent SEs, the new mass spectrometry-based assay would facilitate the laboratory confirmation of positive samples in situation of food poisoning outbreaks.

NAuRA: Genomic Tool to Identify Staphylococcal Enterotoxins in Staphylococcus aureus Strains Responsible for FoodBorne Outbreaks.

Food contamination by staphylococcal enterotoxins (SEs) is responsible for many food poisoning outbreaks (FPOs) each year, and they represent the third leading cause of FPOs in Europe. SEs constitute a protein family with 27 proteins. However, enzyme immunoassays can only detect directly in food the five classical SEs (SEA-SEE). Thus, molecular characterization methods of strains found in food are now used for FPO investigations. Here, we describe the development and implementation of a genomic analysis tool called NAuRA (Nice automatic Research of alleles) that can detect the presence of 27 SEs genes in just one analysis- and create a database of allelic data and protein variants for harmonizing analyses. This tool uses genome assembly data and the 27 protein sequences of SEs. To include the different divergence levels between SE-coding genes, parameters of coverage and identity were generated from 10,000 simulations and a dataset of 244 assembled genomes from strains responsible for outbreaks in Europe as well as the RefSeq reference database. Based on phylogenetic inference performed using maximum-likelihood on the core genomes of the strains in this collection, we demonstrated that strains responsible for FPOs are distributed throughout the phylogenetic tree. Moreover, 71 toxin profiles were obtained using the NAuRA pipeline and these profiles do not follow the evolutionary history of strains. This study presents a pioneering method to investigate strains isolated from food at the genomic level and to analyze the diversity of all 27 SE-coding genes together.

Genome-Wide Profiling of Enterotoxigenic Staphylococcus aureus Strains Used for the Production of Naturally Contaminated Cheeses.

Staphylococcus aureus is a major human pathogen and an important cause of livestock infections. More than 20 staphylococcal enterotoxins with emetic activity can be produced by specific strains responsible for staphylococcal food poisoning, one of the most common food-borne diseases. Whole genome sequencing provides a comprehensive view of the genome structure and gene content that have largely been applied in outbreak investigations and genomic comparisons. In this study, six enterotoxigenic S. aureus strains were characterised using a combination of molecular, phenotypical and computational methods. The genomes were analysed for the presence of virulence factors (VFs), where we identified 110 genes and classified them into five categories: adherence (n = 31), exoenzymes (n = 28), genes involved in host immune system evasion (n = 7); iron uptake regulatory system (n = 8); secretion machinery factors and toxins' genes (n = 36), and 39 genes coding for transcriptional regulators related to staphylococcal VFs. Each group of VFs revealed correlations among the six enterotoxigenic strains, and further analysis revealed their accessory genomic content, including mobile genetic elements. The plasmids pLUH02 and pSK67 were detected in the strain ProNaCC1 and ProNaCC7, respectively, carrying out the genes sed, ser, and selj. The genes carried out by prophages were detected in the strain ProNaCC2 (see), ProNaCC4, and ProNaCC7 (both positive for sea). The strain ProNaCC5 resulted positive for the genes seg, sei, sem, sen, seo grouped in an exotoxin gene cluster, and the strain ProNaCC6 resulted positive for seh, a transposon-associated gene. The six strains were used for the production of naturally contaminated cheeses which were tested with the European Screening Method for staphylococcal enterotoxins. The results obtained from the analysis of toxins produced in cheese, combined with the genomic features represent a portrait of the strains that can be used for the production of staphylococcal enterotoxin-positive cheese as reference material.

Ancestral acquisitions, gene flow and multiple evolutionary trajectories of the type three secretion system and effectors in Xanthomonas plant pathogens.

Deciphering the evolutionary history and transmission patterns of virulence determinants is necessary to understand the emergence of novel pathogens. The main virulence determinant of most pathogenic proteobacteria is the type three secretion system (T3SS). The Xanthomonas genus includes bacteria responsible for numerous epidemics in agroecosystems worldwide and represents a major threat to plant health. The main virulence factor of Xanthomonas is the Hrp2 family T3SS; however, this system is not conserved in all strains and it has not been previously determined whether the distribution of T3SS in this bacterial genus has resulted from losses or independent acquisitions. Based on comparative genomics of 82 genome sequences representing the diversity of the genus, we have inferred three ancestral acquisitions of the Hrp2 cluster during Xanthomonas evolution followed by subsequent losses in some commensal strains and re-acquisition in some species. While mutation was the main force driving polymorphism at the gene level, interspecies homologous recombination of large fragments expanding through several genes shaped Hrp2 cluster polymorphism. Horizontal gene transfer of the entire Hrp2 cluster also occurred. A reduced core effectome composed of xopF1, xopM, avrBs2 and xopR was identified that may allow commensal strains overcoming plant basal immunity. In contrast, stepwise accumulation of numerous type 3 effector genes was shown in successful pathogens responsible for epidemics. Our data suggest that capacity to intimately interact with plants through T3SS would be an ancestral trait of xanthomonads. Since its acquisition, T3SS has experienced a highly dynamic evolutionary history characterized by intense gene flux between species that may reflect its role in host adaptation.

Recombination-prone bacterial strains form a reservoir from which epidemic clones emerge in agroecosystems.

The acquisition of virulence-related genes through horizontal gene transfer can modify the pathogenic profiles of strains and lead to the emergence of new diseases. Xanthomonas arboricola is a bacterial species largely known for the damage it causes to stone and nut fruit trees worldwide. In addition to these host-specific populations called pathovars, many nonpathogenic strains have been identified in this species. Their evolutionary significance in the context of pathogen emergence is unknown. We looked at seven housekeeping genes amplified from 187 pathogenic and nonpathogenic strains isolated from various plants worldwide to analyze population genetics and recombination dynamics. We also examined the dynamics of the gains and losses of genes associated with life history traits (LHTs) during X. arboricola evolution. We discovered that X. arboricola presents an epidemic population structure. Successful pathovars of trees (i.e. pruni, corylina and juglandis) are epidemic clones whose emergence appears to be linked to the acquisition of eight genes coding for Type III effectors. The other strains of this species are part of a recombinant network, within which LHT-associated genes might have been lost. We suggest that nonpathogenic strains, because of their high genetic diversity and propensity for recombination, may promote the emergence of pathogenic strains.