F74 plasmids are major vectors of virulence genes in bovine NTEC2.

Necrotoxigenic Escherichia coli 2 (NTEC2) are defined as E. coli producing the toxin known as cytotoxic necrotizing factor 2 (CNF2), a potent toxin primarily found in bovine but also in humans. NTEC2 are mostly associated with bovine, and cnf2 is known to be carried by pVir-like plasmids. In this study, we looked for NTEC2 in a collection of E. coli collected between 2011 and 2018 in French bovine. Thirty-two isolates, collected from both sick (n = 19) and healthy (n = 13) animals, were identified and characterized using whole-genome sequencing. One F74 plasmid of this bacterial collection was long-read sequenced: its size was 138 121 bp and it carried the cnf2, F17cA-eG, cdtB, iutA, iucC and ompP virulence factors (VFs), but no resistance gene. A large variety of genetic backgrounds was observed, but all cnf2-carrying plasmids belonged to the IncF family, and most of them (78·1%) were of the F74 group. Similar F74 plasmids were also reported from bovine in the United Kingdom and the United States, as identified in the publically available databases. Consequently, these F74 plasmids, which are widely disseminated among E. coli from cattle in the French territory, are vectors of virulence determinants that largely went unnoticed until now.

Molecular Profiling of Shiga Toxin-Producing Escherichia coli and Enteropathogenic E. coli Strains Isolated from French Coastal Environments.

UNLABELLED: Shiga toxin-producing Escherichia coli (STEC) and enteropathogenic E. coli (EPEC) strains may be responsible for food-borne infections in humans. Twenty-eight STEC and 75 EPEC strains previously isolated from French shellfish-harvesting areas and their watersheds and belonging to 68 distinguishable serotypes were characterized in this study. High-throughput real-time PCR was used to search for the presence of 75 E. coli virulence-associated gene targets, and genes encoding Shiga toxin (stx) and intimin (eae) were subtyped using PCR tests and DNA sequencing, respectively. The results showed a high level of diversity between strains, with 17 unique virulence gene profiles for STEC and 56 for EPEC. Seven STEC and 15 EPEC strains were found to display a large number or a particular combination of genetic markers of virulence and the presence of stx and/or eae variants, suggesting their potential pathogenicity for humans. Among these, an O26:H11 stx1a eae-ß1 strain was associated with a large number of virulence-associated genes (n = 47), including genes carried on the locus of enterocyte effacement (LEE) or other pathogenicity islands, such as OI-122, OI-71, OI-43/48, OI-50, OI-57, and the high-pathogenicity island (HPI). One O91:H21 STEC strain containing 4 stx variants (stx1a, stx2a, stx2c, and stx2d) was found to possess genes associated with pathogenicity islands OI-122, OI-43/48, and OI-15. Among EPEC strains harboring a large number of virulence genes (n, 34 to 50), eight belonged to serotype O26:H11, O103:H2, O103:H25, O145:H28, O157:H7, or O153:H2. IMPORTANCE: The species E. coli includes a wide variety of strains, some of which may be responsible for severe infections. This study, a molecular risk assessment study of E. coli strains isolated from the coastal environment, was conducted to evaluate the potential risk for shellfish consumers. This report describes the characterization of virulence gene profiles and stx/eae polymorphisms of E. coli isolates and clearly highlights the finding that the majority of strains isolated from coastal environment are potentially weakly pathogenic, while some are likely to be more pathogenic.

Fast detection of both O157 and non-O157 shiga-toxin producing Escherichia coli by real-time optical immunoassay.

UNLABELLED: Among bacterial pathogens involved in food-illnesses, seven serogroups (O26, O45, O103, O111, O121, O145 and O157) of Shiga-toxin producing Escherichia coli (STEC), are frequently identified. During such outbreak, and due to the perishable property of most foodstuff, the time laps for the identification of contaminated products and pathogens is thus critical to better circumvent their spread. Traditional detection methods using PCR or culture plating are time consuming and may present some limitations. In this study, we present a multiplexed immunoassay for the optical detection of most commonly enterohemorrhagic E. coli serogroups: O26, O45, O103, O111, O121, O145 and O157:H7 in a single device. The use of Surface Plasmon Resonance imaging not only enabled the label-free analysis of the samples but gave results in a real-time manner. A dedicated protocol was set up for the detection of both low contaminating bacterial concentrations of food samples (5 CFU per 25 g) and postenrichment aliquots. By combining one single device for the detection of O157 and non-O157 STEC in a label-free manner, this rapid approach may have an important economic and societal impact. SIGNIFICANCE AND IMPACT OF THE STUDY: This article presents a simple-to-operate immunoassay for the specific detection of Shiga-toxin producing Escherichia coli (STEC). This approach consists in the on-chip assay detection of viable cells on a specifically designed antibody microarray. By skipping any enrichment step and avoiding the use of labelling agent, this approach based on the Surface Plasmon Resonance imaging of the microarrays turns out to be much faster and more cost effective by comparison with standardized methods.

Virulence Genes in Expanded-Spectrum-Cephalosporin-Resistant and -Susceptible Escherichia coli Isolates from Treated and Untreated Chickens.

This study investigated antimicrobial resistance, screened for the presence of virulence genes involved in intestinal infections, and determined phylogenetic groups of Escherichia coli isolates from untreated poultry and poultry treated with ceftiofur, an expanded-spectrum cephalosporin. Results show that none of the 76 isolates appeared to be Shiga toxin-producing E. coli or enteropathogenic E. coli. All isolates were negative for the major virulence factors/toxins tested (ehxA, cdt, heat-stable enterotoxin [ST], and heat-labile enterotoxin [LT]). The few virulence genes harbored in isolates generally did not correlate with isolate antimicrobial resistance or treatment status. However, some of the virulence genes were significantly associated with certain phylogenetic groups.

Simultaneous enrichment and optical detection of low levels of stressed Escherichia coli O157:H7 in food matrices.

AIMS: Rapid detection of enterohaemorrhagic E. coli O157:H7 in large range of stress conditions occurring in food processing. METHODS AND RESULTS: Detection of E. coli O157:H7 in various food processing stress conditions using surface plasmon resonance imaging (SPRi) technique on an antibody microarray was evaluated. The direct detection method based on the culture/capture/measure (CCM) process consists of detecting bacteria during an enrichment step, which significantly decreases the overall assay duration. In optimized culture conditions, this method allows the specific detection of low CFU ml(-1) in <7 h. Detection of bacteria directly in contaminated food samples was also conducted. CONCLUSIONS: The CCM technique using an antibody microarray is a label-free immunoassay that allows rapid detection of E. coli O157:H7 in both food processing stress conditions and complex food matrices. SIGNIFICANCE AND IMPACT OF THE STUDY: The assay is promising for detecting E. coli O157:H7 at different steps of food and drink processing and during storage. SPRi appears to be a suitable and powerful detection method for routine quality controls in food industry with important economic and societal impact.

Evidence for the existence of a new genus Chlamydiifrater gen. nov. inside the family Chlamydiaceae with two new species isolated from flamingo (Phoenicopterus roseus): Chlamydiifrater phoenicopteri sp. nov. and Chlamydiifrater volucris sp. nov.

The family Chlamydiaceae currently comprises a single genus Chlamydia, with 11 validly published species and seven more taxa. It includes the human pathogens Chlamydia (C.) trachomatis, C. pneumoniae and C. psittaci, a zoonotic agent causing avian chlamydiosis and human psittacosis, as well as other proven or potential pathogens in ruminants, birds, snakes, reptiles and turtles. During routine testing of 15 apparently healthy captive flamingos in a zoo in 2011, an atypical strain of Chlamydiaceae was detected by real-time PCR of cloacal swab samples. Sequence analysis of the 16S rRNA gene revealed high similarity to the uncultured Chlamydiales bacterium clone 122, which previously had been found in gulls. As more samples were collected during annual campaigns of the flamingo ringing program in southern France from 2012 to 2015, Chlamydiaceae-specific DNA was detected by PCR in 30.9% of wild birds. From these samples, three strains were successfully grown in cell culture. Ultrastructural analysis, comparison of 16S and 23S rRNA gene sequences, whole-genome analysis based on de novo hybrid-assembled sequences of the new strains as well as subsequent calculation of taxonomic parameters revealed that the relatedness of the flamingo isolates to established members of the family Chlamydiaceae was sufficiently distant to indicate that the three strains belong to two distinct species within a new genus. Based on these data, we propose the introduction of Chlamydiifrater gen. nov., as a new genus, and Chlamydiifrater phoenicopteri sp. nov. and Chlamydiifrater volucris sp. nov., as two new species of the genus.

Molecular characterization of O157:H7, O26:H11 and O103:H2 Shiga toxin-producing Escherichia coli isolated from dairy products.

Pathogenic Shiga toxin-producing E. coli (STEC) are recognized worldwide as environment and foodborne pathogens which can be transmitted by ingestion of ready-to-eat food such as raw milk-derived products. STEC show a prevalence rate in dairy products of 0.9%, yet comparably few outbreaks have been related to dairy products consumption. In this study, we used rt-qPCR to identify the virulence potential of O157, O26 and O103 STEC strains isolated from raw-milk dairy products by analyzing virulence-related gene frequencies and associations with O-island (OI) 44, OI-48, OI-50, OI-57, OI-71 and OI-122. Results showed that 100% of STEC strains investigated harbored genes associated with EHEC-related virulence profile patterns (eae and stx, with either espK, espV, ureD and/or Z2098). We also found similarities in virulence-related gene content between O157:H7 and O103:H2 dairy and non-dairy STEC strains, especially isolates from human cases. The O26:H11-serotype STEC strains investigated harbor the arcA-allele 2 gene associated with specific genetic markers. These profiles are associated with high-virulence seropathotype-A STEC. However, the low frequency of stx2 gene associated with absence of other virulence genes in dairy isolates of O26:H11 remains a promising avenue of investigation to estimate their real pathogenicity. All O26:H11 attaching-effacing E. coli (AEEC) strains carried CRISPRO26:H11SP_O26_E but not genetic markers espK, espV, ureD and/or Z2098 associated with the emerging potentially high-virulence "new French clone". These strains are potentially as "EHEC-like" strains because they may acquire (or have lost) stx gene. In this study, O157:H7, O103:H2 and O26:H11 STEC strains isolated from dairy products were assigned as potential pathogens. However, research now needs to investigate the impact of dairy product environment and dairy processing on the expression of their pathogenicity.

Shiga toxin-producing Escherichia coli strains isolated from dairy products - Genetic diversity and virulence gene profiles.

Shiga toxin-producing Escherichia coli (STEC) are widely recognized as pathogens causing food borne disease. Here we evaluate the genetic diversity of 197 strains, mainly STEC, from serotypes O157:H7, O26:H11, O103:H2, O111:H8 and O145:28 and compared strains recovered in dairy products against strains from human, meat and environment cases. For this purpose, we characterized a set of reference-collection STEC isolates from dairy products by PFGE DNA fingerprinting and a subset of these by virulence-gene profiling. PFGE profiles of restricted STEC total DNA showed high genomic variability (0.9976 on Simpson's discriminatory index), enabling all dairy isolates to be differentiated. High-throughput real-time PCR screening of STEC virulence genes were applied on the O157:H7 and O26:H11 STEC isolates from dairy products and human cases. The virulence gene profiles of dairy and human STEC strains were similar. Nevertheless, frequency-wise, stx1 was more prevalent among dairy O26:H11 isolates than in human cases ones (87% vs. 44%) while stx2 was more prevalent among O26:H11 human isolates (23% vs. 81%). For O157:H7 isolates, stx1 (0% vs. 39%), nleF (40% vs 94%) and Z6065 (40% vs 100%) were more prevalent among human than dairy strains. Our data point to differences between human and dairy strains but these differences were not sufficient to associate PFGE and virulence gene profiles to a putative lower pathogenicity of dairy strains based on their lower incidence in disease. Further comparison of whole-genome expression and virulence gene profiles should be investigated in cheese and intestinal tract samples.

Transcriptional activities of the orphan nuclear receptor ERR alpha (estrogen receptor-related receptor-alpha).

Estrogen receptor-related receptor alpha (ERR alpha) is an orphan nuclear receptor closely related to the estrogen receptor (ER), whose expression covers various stages of embryonic development and persists in certain adult tissues. We show that ERR alpha binds as a homodimer on a specific target sequence, the SFRE (SF-1 response element), already known to respond to the orphan nuclear receptor SF-1. Target sequences that are related to the SFRE and that discriminate between ERR alpha and SF-1 were identified. We have also analyzed the transcriptional properties of the ERR alpha originating from various species. All ERR alpha orthologs act as potent transactivators through the consensus SFRE. ERR alpha activity depends on the putative AF2AD domain, as well as on a serum compound that is withdrawn by charcoal treatment, suggesting the existence of a critical regulating factor brought by serum.

Fibrates increase human REV-ERBalpha expression in liver via a novel peroxisome proliferator-activated receptor response element.

Fibrates are widely used hypolipidemic drugs that act by modulating the expression of genes involved in lipid and lipoprotein metabolism. Whereas the activation of gene transcription by fibrates occurs via the nuclear receptor peroxisome proliferator-activated receptor-alpha (PPARalpha) interacting with response elements consisting of a direct repeat of the AGGTCA motif spaced by one nucleotide (DR1), the mechanisms of negative gene regulation by fibrates and PPARalpha are largely unknown. In the present study, we demonstrate that fibrates induce the expression of the nuclear receptor Rev-erbalpha, a negative regulator of gene transcription. Fibrates increase Rev-erbalpha mRNA levels both in primary human hepatocytes and in HepG2 hepatoblastoma cells. In HepG2 cells, fibrates furthermore induce Rev-erbalpha protein synthesis rates. Transfection studies with reporter constructs driven by the human Rev-erbalpha promoter revealed that fibrates induce Rev-erbalpha expression at the transcriptional level via PPARalpha. Site-directed mutagenesis experiments identified a PPAR response element that coincides with the previously identified Rev-erbalpha negative autoregulatory Rev-DR2 element. Electromobility shift assay experiments indicated that PPARalpha binds as heterodimer with 9-cis-retinoic acid receptor to a subset of DR2 elements 5' flanked by an A/T-rich sequence such as in the Rev-DR2. PPARalpha and Rev-erbalpha bind with similar affinities to the Rev-DR2 site. In conclusion, these data demonstrate human Rev-erbalpha as a PPARalpha target gene and identify a subset of DR2 sites as novel PPARalpha response elements. Finally, the PPARalpha and Rev-erbalpha signaling pathways cross-talk through competition for binding to those response elements.

A specific and unusual nuclear localization signal in the DNA binding domain of the Rev-erb orphan receptors.

The orphan receptors Rev-erbalpha and Rev-erbbeta are members of the nuclear receptors superfamily and act as transcriptional repressors. Rev-erbalpha is expressed with a robust circadian rhythm and is involved in liver metabolism through repression of the ApoA1 gene, but no role has been yet defined for Rev-erbbeta. To gain better understanding of their function and mode of action, we characterized the proteins encoded by these two genes. Both Rev-erbalpha and Rev-erbbeta proteins were nuclear when transiently transfected in COS-1 cells. The major nuclear location signal (NLS) of Rev-erbalpha is in the amino-terminal region of the protein. Fusion of green fluorescent protein (GFP) to the amino terminus of Rev-erbalpha deletion mutants showed that the NLS is located within a 53 amino acid segment of the DNA binding domain (DBD). The homologous region of Rev-erbbeta fused to GFP also targeted the fusion protein to the nucleus, suggesting that the location of this NLS is conserved among all the Rev-erb group members. Interestingly, members of the phylogenetically closest nuclear orphan receptor group (ROR), which exhibit 58% amino acid identity with Rev-erb in the DBD, do not have their NLS located within the DBD. GFP/DBD. RORalpha or GFP/DBD.RORbeta remained cytoplasmic, in contrast to GFP/DBD. Rev-erb fusion proteins. Alignment of human Rev-erb and ROR DBD amino acid sequences predicted that the two basic residues, K167 and R168, located just upstream from the second zinc finger, could play a critical part in the nuclear localization of Rev-erb proteins. Substitution of these two residues with those found in ROR, in the GFP/DBD. Rev-erb context, resulted in cytoplasmic proteins. In contrast, the reverse mutation of the GFP/DBD. RORalpha towards the Rev-erbalpha residues targeted the fusion protein to the nucleus. Our data demonstrate that Rev-erb proteins contain a functional NLS in the DBD. Its location is unusual within the nuclear receptor superfamily and suggests that Rev-erb orphan receptors control their intracellular localization via a mechanism different from that of other nuclear receptors.

Prevalence of Clostridium botulinum and thermophilic heat-resistant spores in raw carrots and green beans used in French canning industry.

Two categories of vegetables (carrots and green beans) that are widely used in the manufacture of canned food were surveyed for their spore contamination. Samples were recovered from 10 manufactures spread over all producing areas in France. Two samples over 316 raw vegetables collected were found positive for botulinum neurotoxin producing Clostridia spores as tested by PCR-based GeneDisc assay. Both positive samplestested positive for the type B neurotoxin gene (bont/B). In parallel, heat-resistant spores of thermophilic bacteria that are likely to be associated with canned food spoilage after prolonged incubation at 55 °C were surveyed after specific enrichment. Prevalence varied between 1.6% for Moorella thermoacetica/thermoautotrophica in green bean samples and 8.6% for either Geobacillus stearothermophilus or Thermoanaerobacterium spp. in carrot samples. Vegetable preparation, e.g. washing and edge cutting, considerably reduced spore contamination levels. These data constitute the first wide examination of vegetables specifically cultivated for industrialpurposes for their contamination by spores of thermophilic bacterial species.

The nuclear receptors peroxisome proliferator-activated receptor alpha and Rev-erbalpha mediate the species-specific regulation of apolipoprotein A-I expression by fibrates.

Fibrates are widely used hypolipidemic drugs which activate the nuclear peroxisome proliferator-activated receptor (PPAR) alpha and thereby alter the transcription of genes controlling lipoprotein metabolism. Fibrates influence plasma high density lipoprotein and its major protein, apolipoprotein (apo) A-I, in an opposite manner in man (increase) versus rodents (decrease). In the present study we studied the molecular mechanisms of this species-specific regulation of apoA-I expression by fibrates. In primary rat and human hepatocytes fenofibric acid, respectively, decreased and increased apoA-I mRNA levels. The absence of induction of rat apoA-I gene expression by fibrates is due to 3 nucleotide differences between the rat and the human apoA-I promoter A site, rendering a positive PPAR-response element in the human apoA-I promoter nonfunctional in rats. In contrast, rat, but not human, apoA-I transcription is repressed by the nuclear receptor Rev-erbalpha, which binds to a negative response element adjacent to the TATA box of the rat apoA-I promoter. In rats fibrates increase liver Rev-erbalpha mRNA levels >10-fold. In conclusion, the opposite regulation of rat and human apoA-I gene expression by fibrates is linked to differences in cis-elements in their respective promoters leading to repression by Rev-erbalpha of rat apoA-I and activation by PPARalpha of human apoA-I. Finally, Rev-erbalpha is identified as a novel fibrate target gene, suggesting a role for this nuclear receptor in lipid and lipoprotein metabolism.