Placental cartography of NADPH oxidase (NOX) family proteins: Involvement in the pathophysiology of preeclampsia.

The placenta is an essential organ for fetal development. During the first trimester, it undergoes dramatic changes as it develops in an environment poor in oxygen (around 2-3%). From about 10 gestational weeks, oxygen levels increase to 8% in the intervillous chamber. These changes are accompanied by modulation of the activity of NADPH oxidase, a major source of production of reactive oxygen species in the first trimester of pregnancy. The NOX complex is composed of seven different proteins (NOX1-5 and DUOX1-2) whose placental involvements during physiological and pathological pregnancies are largely unknown. The aim of the study was to produce a cartography of NOX family proteins, in terms of RNA, protein expression, and localization during physiological pregnancy and in the case of preeclampsia (PE), in a cohort of early-onset PE (n = 11) and late-onset PE (n = 7) cases. NOX family proteins were mainly expressed in trophoblastic cells (NOX4-5, DUOX1) and modulated during physiological pregnancy. NOX4 underwent an unexpected and hitherto unreported nuclear translocation at term. In the case of PE, two groups stood out: NOX1-3, superoxide producers, were down-regulated (p < 0.05) while NOX4-DUOX1, hydrogen peroxide producers, were up-regulated (p < 0.05), compared to the control group. Mapping of placental NOX will constitute a reference and guide for future investigations concerning its involvement in the pathophysiology of PE.

Benzo(a)pyrene and Cerium Dioxide Nanoparticles in Co-Exposure Impair Human Trophoblast Cell Stress Signaling.

Human placenta is a multifunctional interface between maternal and fetal blood. Studying the impact of pollutants on this organ is crucial because many xenobiotics in maternal blood can accumulate in placental cells or pass into the fetal circulation. Benzo(a)pyrene (BaP) and cerium dioxide nanoparticles (CeO2 NP), which share the same emission sources, are found in ambient air pollution and also in maternal blood. The aim of the study was to depict the main signaling pathways modulated after exposure to BaP or CeO2 NP vs. co-exposure on both chorionic villi explants and villous cytotrophoblasts isolated from human term placenta. At nontoxic doses of pollutants, BaP is bioactivated by AhR xenobiotic metabolizing enzymes, leading to DNA damage with an increase in γ-H2AX, the stabilization of stress transcription factor p53, and the induction of its target p21. These effects are reproduced in co-exposure with CeO2 NP, except for the increase in γ-H2AX, which suggests a modulation of the genotoxic effect of BaP by CeO2 NP. Moreover, CeO2 NP in individual and co-exposure lead to a decrease in Prx-SO3, suggesting an antioxidant effect. This study is the first to identify the signaling pathways modulated after co-exposure to these two pollutants, which are common in the environment.

Salmonella enterica subsp. enterica Welikade: guideline for phylogenetic analysis of serovars rarely involved in foodborne outbreaks.

BACKGROUND: Salmonella spp. is a major foodborne pathogen with a wide variety of serovars associated with human cases and food sources. Nevertheless, in Europe a panel of ten serovars is responsible for up to 80% of confirmed human cases. Clustering studies by single nucleotide polymorphism (SNP) core-genome phylogenetic analysis of outbreaks due to these major serovars are simplified by the availability of many complete genomes in the free access databases. This is not the case for outbreaks due to less common serovars, such as Welikade, for which no reference genomes are available. In this study, we propose a method to solve this problem. We propose to perform a core genome MLST (cgMLST) analysis based on hierarchical clustering using the free-access EnteroBase to select the most suitable genome to use as a reference for SNP phylogenetic analysis. In this study, we applied this protocol to a retrospective analysis of a Salmonella enterica serovar Welikade (S. Welikade) foodborne outbreak that occurred in France in 2016. Finally, we compared the cgMLST and SNP analyses. SNP phylogenetic reconstruction was carried out considering the effect of recombination events identified by the ClonalFrameML tool. The accessory genome was also explored by phage content and virulome analyses. RESULTS: Our findings revealed high clustering concordance using cgMLST and SNP analyses. Nevertheless, SNP analysis allowed for better assessment of the genetic distance among strains. The results revealed epidemic clones of S. Welikade circulating within the poultry and dairy sectors in France, responsible for sporadic and non-sporadic human cases between 2012 and 2019. CONCLUSIONS: This study increases knowledge on this poorly described serovar and enriches public genome databases with 42 genomes from human and non-human S. Welikade strains, including the isolate collected in 1956 in Sri Lanka, which gave the name to this serovar. This is the first genomic analysis of an outbreak due to S. Welikade described to date.

Disentangling a complex nationwide Salmonella Dublin outbreak associated with raw-milk cheese consumption, France, 2015 to 2016.

On 18 January 2016, the French National Reference Centre for Salmonella reported to Santé publique France an excess of Salmonella enterica serotype Dublin (S. Dublin) infections. We investigated to identify the source of infection and implement control measures. Whole genome sequencing (WGS) and multilocus variable-number tandem repeat analysis (MLVA) were performed to identify microbiological clusters and links among cases, animal and food sources. Clusters were defined as isolates with less than 15 single nucleotide polymorphisms determined by WGS and/or with identical MLVA pattern. We compared different clusters of cases with other cases (case-case study) and controls recruited from a web-based cohort (case-control study) in terms of food consumption. We interviewed 63/83 (76%) cases; 2,914 controls completed a questionnaire. Both studies' findings indicated that successive S. Dublin outbreaks from different sources had occurred between November 2015 and March 2016. In the case-control study, cases of distinct WGS clusters were more likely to have consumed Morbier (adjusted odds ratio (aOR): 14; 95% confidence interval (CI): 4.8-42) or Vacherin Mont d'Or (aOR: 27; 95% CI: 6.8-105), two bovine raw-milk cheeses. Based on these results, the Ministry of Agriculture launched a reinforced control plan for processing plants of raw-milk cheeses in the production region, to prevent future outbreaks.

Bacillus cereus-induced food-borne outbreaks in France, 2007 to 2014: epidemiology and genetic characterisation.

The aim of this study was to identify and characterise Bacillus cereus from a unique national collection of 564 strains associated with 140 strong-evidence food-borne outbreaks (FBOs) occurring in France during 2007 to 2014. Starchy food and vegetables were the most frequent food vehicles identified; 747 of 911 human cases occurred in institutional catering contexts. Incubation period was significantly shorter for emetic strains compared with diarrhoeal strains A sub-panel of 149 strains strictly associated to 74 FBOs and selected on Coliphage M13-PCR pattern, was studied for detection of the genes encoding cereulide, diarrhoeic toxins (Nhe, Hbl, CytK1 and CytK2) and haemolysin (HlyII), as well as panC phylogenetic classification. This clustered the strains into 12 genetic signatures (GSs) highlighting the virulence potential of each strain. GS1 (nhe genes only) and GS2 (nhe, hbl and cytK2), were the most prevalent GS and may have a large impact on human health as they were present in 28% and 31% of FBOs, respectively. Our study provides a convenient molecular scheme for characterisation of B. cereus strains responsible for FBOs in order to improve the monitoring and investigation of B. cereus-induced FBOs, assess emerging clusters and diversity of strains.

Perinatal, metabolic, and reproductive features in PPARG-related lipodystrophy.

OBJECTIVE: The adipogenic PPARG-encoded PPARγ nuclear receptor also displays essential placental functions. We evaluated the metabolic, reproductive, and perinatal features of patients with PPARG-related lipodystrophy. METHODS: Current and retrospective data were collected in patients referred to a National Rare Diseases Reference Centre. RESULTS: 26 patients from 15 unrelated families were studied (18 women, median age 43 years). They carried monoallelic PPARG variants except a homozygous patient with congenital generalized lipodystrophy. Among heterozygous patients aged 16 or more (n = 24), 92% had diabetes, 96% partial lipodystrophy (median age at diagnosis 24 and 37 years), 78% hypertriglyceridaemia, 71% liver steatosis, and 58% hypertension. The mean BMI was 26 ± 5.0 kg/m2. Women (n = 16) were frequently affected by acute pancreatitis (n = 6) and/or polycystic ovary syndrome (n = 12). Eleven women obtained one or several pregnancies, all complicated by diabetes (n = 8), hypertension (n = 4), and/or hypertriglyceridaemia (n = 10). We analysed perinatal data of patients according to the presence (n = 8) or absence (n = 9) of a maternal dysmetabolic environment. The median gestational age at birth was low in both groups (37 and 36 weeks of amenorrhea, respectively). As expected, the birth weight was higher in patients exposed to a foetal dysmetabolic environment of maternal origin. In contrast, 85.7% of non-exposed patients, in whom the variant is, or is very likely to be, paternally-inherited, were small for gestational age. CONCLUSIONS: Lipodystrophy-related PPARG variants induce early metabolic complications. Our results suggest that placental expression of PPARG pathogenic variants carried by affected foetuses could impair prenatal growth and parturition. This justifies careful pregnancy monitoring in affected families.

InhA1, NprA, and HlyII as candidates for markers to differentiate pathogenic from nonpathogenic Bacillus cereus strains.

Bacillus cereus is found in food, soil, and plants, and the ability to cause food-borne diseases and opportunistic infection presumably varies among strains. Therefore, measuring harmful toxin production, in addition to the detection of the bacterium itself, may be key for food and hospital safety purposes. All previous studies have focused on the main known virulence factors, cereulide, Hbl, Nhe, and CytK. We examined whether other virulence factors may be specific to pathogenic strains. InhA1, NprA, and HlyII have been described as possibly contributing to B. cereus pathogenicity. We report the prevalence and expression profiles of these three new virulence factor genes among 57 B. cereus strains isolated from various sources, including isolates associated with gastrointestinal and nongastrointestinal diseases. Using PCR, quantitative reverse transcriptase PCR, and virulence in vivo assays, we unraveled these factors as potential markers to differentiate pathogenic from nonpathogenic strains. We show that the hlyII gene is carried only by strains with a pathogenic potential and that the expression levels of inhA1 and nprA are higher in the pathogenic than in the nonpathogenic group of strains studied. These data deliver useful information about the pathogenicity of various B. cereus strains.

Semi-automated repetitive-sequence-based polymerase chain reaction compared to pulsed-field gel electrophoresis for Listeria monocytogenes subtyping.

Listeriosis is a severe infection that mainly affects pregnant women, neonates, and immuno-compromised adults. The commercially available semi-automated repetitive-sequence-based polymerase chain reaction assay system, DiversiLab, has been successfully used for subtyping several species of bacteria. In this article we compare the DiversiLab System with macrorestriction analysis by pulsed-field gel electrophoresis (PFGE), which is currently the gold standard for molecular subtyping of Listeria monocytogenes. We used a panel of 116 human and food L. monocytogenes isolates for the comparative evaluation. Among these isolates, there were 4 pairs of duplicates, 13 strains were epidemiologically related, and the remaining food isolates were epidemiologically unrelated. The isolates of different serotypes represented distinct DiversiLab types (DTs) and ApaI/AscI-PFGE types except for one DT-containing isolates of two serotypes, 4b and 1/2b. The four duplicates displayed the same DT and ApaI/AscI PFGE type demonstrating the good reproducibility of the two methods. The epidemiologically related strains were clustered in the same DT and PFGE type. The Simpson's index of diversity was 0.954; 0.988; 0.994; and 0.998 for DiversiLab, AscI-PFGE, ApaI-PFGE, and AscI/ApaI-PFGE, respectively. Thus, PFGE was more discriminating than DiversiLab. However, for 1/2a serotype strains, six AscI-PFGE, three ApaI-PFGE, and one ApaI/AscI PFGE type were divided into different DTs. DiversiLab enabled a good discrimination between serotype 1/2a strains. DiversiLab is less labor intensive than PFGE and provides results in <24 hours compared with 30 hours to 3 days for PFGE from the time a pure culture of the bacteria has been obtained. On the basis of these results, DiversiLab may be useful for tracking the source of contamination in food-processing facilities and their environments. Also, DiversiLab may be more appropriate for long-term epidemiological studies where less discrimination is needed.

AB_SA: Accessory genes-Based Source Attribution - tracing the source of Salmonella enterica Typhimurium environmental strains.

The partitioning of pathogenic strains isolated in environmental or human cases to their sources is challenging. The pathogens usually colonize multiple animal hosts, including livestock, which contaminate the food-production chain and the environment (e.g. soil and water), posing an additional public-health burden and major challenges in the identification of the source. Genomic data opens up new opportunities for the development of statistical models aiming to indicate the likely source of pathogen contamination. Here, we propose a computationally fast and efficient multinomial logistic regression source-attribution classifier to predict the animal source of bacterial isolates based on 'source-enriched' loci extracted from the accessory-genome profiles of a pangenomic dataset. Depending on the accuracy of the model's self-attribution step, the modeller selects the number of candidate accessory genes that best fit the model for calculating the likelihood of (source) category membership. The Accessory genes-Based Source Attribution (AB_SA) method was applied to a dataset of strains of Salmonella enterica Typhimurium and its monophasic variant (S. enterica 1,4,[5],12:i:-). The model was trained on 69 strains with known animal-source categories (i.e. poultry, ruminant and pig). The AB_SA method helped to identify 8 genes as predictors among the 2802 accessory genes. The self-attribution accuracy was 80 %. The AB_SA model was then able to classify 25 of the 29 S. enterica Typhimurium and S. enterica 1,4,[5],12:i:- isolates collected from the environment (considered to be of unknown source) into a specific category (i.e. animal source), with more than 85 % of probability. The AB_SA method herein described provides a user-friendly and valuable tool for performing source-attribution studies in only a few steps. AB_SA is written in R and freely available at https://github.com/lguillier/AB_SA.

Draft Genome Sequences of Salmonella enterica subsp. enterica Serovar Dublin Strains from St. Nectaire and Morbier Cheeses Characterized by Multilocus Variable-Number Tandem-Repeat Analysis Profiles Associated with Two Fatal Outbreaks in France.

We report here the draft genome sequences of 2 Salmonella enterica subsp. enterica serovar Dublin strains from St. Nectaire and Morbier cheeses having multilocus variable-number tandem-repeat analysis (MLVA) profiles identified during the fatal outbreaks that occurred in France in 2012 and 2015 to 2016, respectively. These draft genome sequences will help uncover the virulence determinants in invasive S. Dublin strains.

Whole-Genome Sequences of Two Salmonella enterica Serovar Dublin Strains That Harbor the viaA, viaB, and ompB Loci of the Vi Antigen.

Here, we report the genome sequences of two Salmonella enterica serovar Dublin strains, 03EB8736SAL and 03EB8994SAL, isolated from raw-milk cheese and milk filtrate, respectively. Analysis of the draft genomes of the two isolates reveals the presence of the viaA, viaB, and ompB loci of the Vi capsular polysaccharide antigen (Vi antigen).

Polyphyletic Nature of Salmonella enterica Serotype Derby and Lineage-Specific Host-Association Revealed by Genome-Wide Analysis.

In France, Salmonella Derby is one of the most prevalent serotypes in pork and poultry meat. Since 2006, it has ranked among the 10 most frequent Salmonella serotypes isolated in humans. In previous publications, Salmonella Derby isolates have been characterized by pulsed field gel electrophoresis (PFGE) and antimicrobial resistance (AMR) profiles revealing the existence of different pulsotypes and AMR phenotypic groups. However, these results suffer from the low discriminatory power of these typing methods. In the present study, we built a collection of 140 strains of S. Derby collected in France from 2014 to 2015 representative of the pork and poultry food sectors. The whole collection was characterized using whole genome sequencing (WGS), providing a significant contribution to the knowledge of this underrepresented serotype, with few genomes available in public databases. The genetic diversity of the S. Derby strains was analyzed by single-nucleotide polymorphism (SNP). We also investigated AMR by both genome and phenotype, the main Salmonella pathogenicity island (SPI) and the fimH gene sequences. Our results show that this S. Derby collection is spread across four different lineages genetically distant by an average of 15k SNPs. These lineages correspond to four multilocus sequence typing (MLST) types (ST39, ST40, ST71, and ST682), which were found to be associated with specific animal hosts: pork and poultry. While the ST71 and ST682 strains are pansusceptible, ST40 isolates are characterized by the multidrug resistant profile STR-SSS-TET. Considering virulence determinants, only ST39 and ST40 present the SPI-23, which has previously been associated with pork enterocyte invasion. Furthermore, the pork ST682 isolates were found to carry mutations in the fimH sequence that could participate in the host tropism of this group. Our phylogenetic analysis demonstrates the polyphyletic nature of the Salmonella serotype Derby and provides an opportunity to identify genetic factors associated with host adaptation and markers for the monitoring of these different lineages within the corresponding animal sectors. The recognition of these four lineages is of primary importance for epidemiological surveillance throughout the food production chains and constitutes the first step toward refining monitoring and preventing dispersal of this pathogen.

MLVA for Salmonella enterica subsp. enterica Serovar Dublin: Development of a Method Suitable for Inter-Laboratory Surveillance and Application in the Context of a Raw Milk Cheese Outbreak in France in 2012.

Salmonella enterica subspecies enterica serovar Dublin (S. Dublin) figures among the most frequently isolated Salmonella strains in humans in France. This serovar may affect production and animal health mainly in cattle herds with corresponding high economic losses. Given that the current gold standard method, pulsed-field gel electrophoresis (PFGE), provides insufficient discrimination for epidemiological investigations, we propose a standard operating procedure in this study for multiple-locus variable number tandem repeat analysis (MLVA) of S. Dublin, suitable for inter-laboratory surveillance. An in silico analysis on the genome of S. Dublin strains CT_02021853 was performed to identify appropriate microsatellite regions. Of 21 VNTR loci screened, six were selected and 401 epidemiologically unrelated and related strains, isolated from humans, food and animals were analyzed to assess performance criteria such as typeability, discriminatory power and epidemiological concordance. The MLVA scheme developed was applied to an outbreak involving Saint-Nectaire cheese for which investigations were conducted in France in 2012, making it possible to discriminate between epidemiologically related strains and sporadic case strains, while PFGE assigned only a single profile. The six loci selected were sequenced on a large set of strains to determine the sequence of the repeated units and flanking regions, and their stability was evaluated in vivo through the analysis of the strains investigated from humans, food and the farm environment during the outbreak. The six VNTR selected were found to be stable and the discriminatory power of the MLVA method developed was calculated to be 0.954 compared with that for PFGE, which was only 0.625. Twenty-four reference strains were selected from the 401 examined strains in order to represent most of the allele diversity observed for each locus. This reference set can be used to harmonize MLVA results and allow data exchange between laboratories. This original MLVA protocol could be used easily and routinely for monitoring of serovar Dublin isolates and for conducting outbreak investigations.

The overgrowth of Listeria monocytogenes by other Listeria spp. in food samples undergoing enrichment cultivation has a nutritional basis.

The isolation of Listeria monocytogenes from food is carried out using a double enrichment. In cases where multiple Listeria species are present within the original sample, L. monocytogenes can be overgrown during enrichment by other species of listeria present in the original sample. From a practical perspective, this can result in a false negative or complicate the ability of public health investigators to match food and clinical isolates. We have further investigated this phenomenon by analysing the growth kinetics of single species and pairs of different species over the ISO 11290-1 enrichment process. The overgrowth of a strain of L. monocytogenes by a strain of Listeria innocua resulted primarily from interactions which occurred in late exponential phase, where it was observed that growth of both strains stopped when the dominant strain reached stationary phase. In a second mixed culture, the dominant L. monocytogenes strain suppressed the exponential growth rate of the second Listeria welshimeri strain. Both findings suggest that the overgrowth could partially be explained in terms of a nutritional competition. Multi-factor analysis of Fraser broth constituents and growth temperatures using both stressed and non-stressed inoculants failed to identify any single factor in the ISO 11290-1 methodology which would contribute to the overgrowth phenomenon in our model system. Furthermore, species was not a significant factor in observed differences in growth parameters among a wider array of strains which had been stressed or not stressed prior to grown in Fraser broths, even though some strains had significantly faster growth rates than others. Limiting diffusion in Fraser broth through the addition of agar significantly reduced the extent of the overgrowth in experiments using mixtures of strains originally isolated from foods where overgrowth had been previously observed. Taken together, these findings support that the overgrowth phenomenon in most instances has a nutritional basis.