Virulence gene profiling of enterohemorrhagic (EHEC) and enteropathogenic (EPEC) Escherichia coli strains: a basis for molecular risk assessment of typical and atypical EPEC strains

<p>Abstract</p> <p>Background</p> <p>Enterohaemorrhagic <it>E. coli </it>(EHEC) can cause severe disease such as bloody diarrhoea and haemolytic uraemic syndrome in humans. Besides production of Shiga toxins, the presence of LEE (<it>eae</it>-gene) and non-LEE (<it>nle</it>) encoded effector genes harboured on O-islands OI-122, OI-71 and OI-57 is associated with EHEC virulence and their frequency in outbreaks. Genes encoded by the EHEC-plasmid are putative virulence markers of EHEC. EHEC-plasmids, LEE and non-LEE effector genes have also been detected in some strains of enteropathogenic <it>E. coli </it>(EPEC). The objective of this study was to analyze the relationship between EHEC and EPEC for virulence genes encoded by genomic O-islands and by the EHEC-plasmids.</p> <p>Results</p> <p>Nle genes <it>ent/espL2</it>, <it>nleB </it>and <it>nleE </it>(OI-122), <it>nleA</it>, <it>nleF </it>and <it>nleH1-2 </it>(OI-71), <it>nleG5-2 </it>and <it>nleG6-2 </it>(OI-57), <it>espK </it>(CP-933N) and the EHEC-plasmid encoded genes <it>ehxA</it>, <it>espP</it>, <it>etpD </it>and <it>katP </it>were searched in 73 typical and in 235 atypical enteropathogenic <it>E. coli </it>(EPEC) strains. Typical and atypical EPEC each fall into two clusters. Cluster 1 typical (n = 46) and atypical (n = 129) EPEC strains were characterized by the presence of OI-122 encoded genes and grouped together with 64 investigated EHEC strains. Cluster 2 typical (n = 27) and atypical (n = 106) strains grouped together with 52 LEE-negative, Shiga toxin-producing <it>E. coli </it>(STEC) and with 21 apathogenic <it>E. coli </it>strains. Typical EPEC Cluster 1 strains belonged to serotypes frequently involved in severe illness and outbreaks in children (O111:H2, O114:H2, O55:H6, O127:H6 and O142:H6). Atypical EPEC Cluster 1 strains were characterized by serotypes related to EHEC (O26:H11, O55:H7, O145:H28, O103:H2 and O103:H25).</p> <p>Conclusion</p> <p>The OI-122 encoded <it>nleB </it>gene was found to be most closely associated with Cluster 1 strains and may serve as a diagnostic tool for the identification of virulent EHEC and EPEC seropathotypes. OI-71 encoded genes <it>nleA</it>, <it>nleF </it>and <it>nleH1-2 </it>are less associated with Cluster 1 strains. EHEC-plasmid, OI-57 and CP-933 associated genes showed only weak similarities with virulent Cluster 1 EHEC and EPEC strains.</p

Inventory and First Assessment of Oil and Gas Wells Conversion for Geothermal Heat Recovery in France

International audienceThe repurposing of oil and gas wells for geothermal energy production and resource assessment can provide sustainable solutions to meet the objectives of renewable energy balance targeted within 2030 by the French Parliament in the "energy transition law for a green growth" promulgated in August 2015. Approximately 12 500 wells have been drilled in France since the 19th century for hydrocarbon reservoir exploration and exploitation. Most of them are closed and abandoned or nearing the end of production due to the planned end of exploitation of hydrocarbons in France by 2040. Several sustainable cases of conversion for geothermal energy production have been reported in France and abroad, demonstrating the possibility of using former wells for heat extraction from aquifers or coaxial heat exchangers. This paper presents an overview of the wells drilled in France and the methodology proposed to identify and rank them according to the a priori feasibility of open and closed loop conversion. To this purpose, wells data, geological and hydrothermal information acquired by the BRGM (geometry and dynamic aquifer properties from models) and land occupation have been cross-referenced. The quantitative overview should be followed by a detailed analysis of selected wells to assess their conversion potential for geothermal energy production (possible use at surface, well drilling and abandonment reports, hydrodynamic properties of the reservoir, technology to be implemented, etc.)

A multiplex real-time PCR assay targeting virulence and resistance genes in Salmonella enterica serotype Typhimurium

<p>Abstract</p> <p>Background</p> <p>Typhimurium is the main serotype of <it>Salmonella enterica </it>subsp. <it>enterica </it>implicated in food-borne diseases worldwide. This study aimed to detect the prevalence of ten markers combined in a macro-array based on multiplex real-time PCR. We targeted characteristic determinants located on pathogenicity islands (SPI-2 to -5, virulence plasmid <it>pSLT </it>and <it>Salmonella </it>genomic island 1 (SGI1)) as well as a specific 16S-23S rRNA intergenic spacer sequence of definitive type 104 (DT104). To investigate antimicrobial resistance, the study also targeted the presence of genes involved in sulfonamide (<it>sul1</it>) and beta-lactam (<it>bla</it>_TEM) resistance. Finally, the <it>intI1 </it>determinant encoding integrase from class 1 integron was also investigated.</p> <p>Results</p> <p>A total of 538 unrelated <it>S</it>. Typhimurium strains isolated between 1999 and 2009 from various sources, including food animals, food products, human and environmental samples were studied. Based on the combined presence or absence of these markers, we distinguished 34 different genotypes, including three major genotypes encountered in 75% of the studied strains, Although SPI determinants were almost always detected, SGI1, <it>intI1</it>, <it>sul1 </it>and <it>bla</it>_TEMdeterminants were found 47%, 52%, 54% and 12% of the time respectively, varying according to isolation source. Low-marker patterns were most often detected in poultry sources whereas full-marker patterns were observed in pig, cattle and human sources.</p> <p>Conclusion</p> <p>The GeneDisc^®assay developed in this study madeit easier to explore variability within serotype Typhimurium by analyzing ten relevant gene determinants in a large collection of strains. This real-time multiplex method constitutes a valuable tool for strains characterization on epidemiological purposes.</p

CO2-Dissolved : A Novel Approach to Combining CCS and Geothermal Heat Recovery

International audienceThis paper presents the outline of the CO2-DISSOLVED project whose objective is to assess the technical-economic feasibility of a novel CCS concept integrating geothermal energy recovery, aqueous dissolution of CO2 and injection via a doublet system, and an innovative post-combustion CO2 capture technology. Compared to the use of a supercritical phase, this approach offers substantial benefits in terms of storage safety, due to lower brine displacement risks, lower CO2 escape risks, and the potential for more rapid mineralization. However, the solubility of CO2 in brine will be a limiting factor to the amount of CO2 that can be injected. Consequently, and as another contributing novel factor, this proposal targets low to medium range CO2-emitters (ca. 10-100 kt/yr), that could be compatible with a single doublet installation. Since it is intended to be a local solution, the costs related to CO2 transport would then be dramatically reduced, provided that the local underground geology is favorable. Finally, this project adds the potential for energy and/or revenue generation through geothermal heat recovery. This constitutes an interesting way of valorization of the injection operations, demonstrating that an actual synergy between CO2 storage and geothermal activities may exist

Genetic Diversity and Pathogenic Potential of Attaching and Effacing Escherichia coli O26:H11 Strains Recovered from Bovine Feces in the United States

Escherichia coli O26 has been identified as the most common non-O157 Shiga toxin-producing E. coli (STEC) serogroup to cause human illnesses in the United States and has been implicated in outbreaks around the world. E. coli has high genomic plasticity, which facilitates the loss or acquisition of virulence genes. Attaching and effacing E. coli (AEEC) O26 strains have frequently been isolated from bovine feces, and there is a need to better characterize the relatedness of these strains to defined molecular pathotypes and to describe the extent of their genetic diversity. High-throughput real-time PCR was used to screen 178 E. coli O26 isolates from a single U.S. cattle feedlot, collected from May to July 2011, for the presence or absence of 25 O26 serogroup-specific and virulence-associated markers. The selected markers were capable of distinguishing these strains into molecularly defined groups (yielding 18 unique marker combinations). Analysis of the clustered regularly interspaced short palindromic repeat 1 (CRISPR1) and CRISPR2a loci further discriminated isolates into 24 CRISPR types. The combination of molecular markers and CRISPR typing provided 20.8% diversity. The recent CRISPR PCR target SP_O26-E, which was previously identified only in stx 2-positive O26:H11 human clinical strains, was identified in 96.4% (161/167 [95% confidence interval, 99.2 to 93.6%]) of the stx-negative AEEC O26:H11 bovine fecal strains. This supports that these stx-negative strains may have previously contained a prophage carrying stx or could acquire this prophage, thus possibly giving them the potential to become pathogenic to humans. These results show that investigation of specific genetic markers may further elucidate our understanding of the genetic diversity of AEEC O26 strains in bovine feces

Carbapenem-Resistant Bacteria Recovered from Faeces of Dairy Cattle in the High Plains Region of the USA

OBJECTIVE:A study was conducted to recover carbapenem-resistant bacteria from the faeces of dairy cattle and identify the underlying genetic mechanisms associated with reduced phenotypic susceptibility to carbapenems. METHODS:One hundred and fifty-nine faecal samples from dairy cattle were screened for carbapenem-resistant bacteria. Phenotypic screening was conducted on two media containing ertapenem. The isolates from the screening step were characterised via disk diffusion, Modified Hodge, and Carba NP assays. Carbapenem-resistant bacteria and carbapenemase-producing isolates were subjected to Gram staining and biochemical testing to include Gram-negative bacilli. Whole genome sequencing was performed on bacteria that exhibited either a carbapenemase-producing phenotype or were not susceptible to ertapenem and were presumptively Enterobacteriaceae. RESULTS:Of 323 isolates collected from the screening media, 28 were selected for WGS; 21 of which were based on a carbapenemase-producing phenotype and 7 were presumptively Enterobacteriaceae and not susceptible to ertapenem. Based on analysis of WGS data, isolates included: 3 Escherichia coli harbouring blaCMY-2 and truncated ompF genes; 8 Aeromonas harbouring blacphA-like genes; 1 Acinetobacter baumannii harbouring a novel blaOXA gene (blaOXA-497); and 6 Pseudomonas with conserved domains of various carbapenemase-producing genes. CONCLUSIONS:Carbapenem resistant bacteria appear to be rare in cattle. Nonetheless, carbapenem-resistant bacteria were detected across various genera and were found to harbour a variety of mechanisms conferring reduced susceptibility. The development and dissemination of carbapenem-resistant bacteria in livestock would have grave implications for therapeutic treatment options in human medicine; thus, continued monitoring of carbapenem susceptibility among enteric bacteria of livestock is warranted

The Unconventional Xer Recombination Machinery of Streptococci/Lactococci

Homologous recombination between circular sister chromosomes during DNA replication in bacteria can generate chromosome dimers that must be resolved into monomers prior to cell division. In Escherichia coli, dimer resolution is achieved by site-specific recombination, Xer recombination, involving two paralogous tyrosine recombinases, XerC and XerD, and a 28-bp recombination site (dif) located at the junction of the two replication arms. Xer recombination is tightly controlled by the septal protein FtsK. XerCD recombinases and FtsK are found on most sequenced eubacterial genomes, suggesting that the Xer recombination system as described in E. coli is highly conserved among prokaryotes. We show here that Streptococci and Lactococci carry an alternative Xer recombination machinery, organized in a single recombination module. This corresponds to an atypical 31-bp recombination site (difSL) associated with a dedicated tyrosine recombinase (XerS). In contrast to the E. coli Xer system, only a single recombinase is required to recombine difSL, suggesting a different mechanism in the recombination process. Despite this important difference, XerS can only perform efficient recombination when difSL sites are located on chromosome dimers. Moreover, the XerS/difSL recombination requires the streptococcal protein FtsKSL, probably without the need for direct protein-protein interaction, which we demonstrated to be located at the division septum of Lactococcus lactis. Acquisition of the XerS recombination module can be considered as a landmark of the separation of Streptococci/Lactococci from other firmicutes and support the view that Xer recombination is a conserved cellular function in bacteria, but that can be achieved by functional analogs

A Systematic Approach to Identify and Characterize the Effectiveness and Safety of Novel Probiotic Strains to Control Foodborne Pathogens

A total of 44 lactic acid bacteria (LAB) strains originally isolated from cattle feces and different food sources were screened for their potential probiotic features. The antimicrobial activity of all isolates was tested by well-diffusion assay and competitive exclusion on broth against Salmonella Montevideo, Escherichia coli O157:H7 and Listeria monocytogenes strain N1-002. Thirty-eight LAB strains showed antagonistic effect against at least one of the pathogens tested in this study. Improved inhibitory effect was observed against L. monocytogenes with zones of inhibition up to 24 mm when LAB overnight cultures were used, and up to 21 mm when cell-free filtrates were used. For E. coli O157:H7 and Salmonella maximum inhibitions of 12 and 11.5 mm were observed, respectively. On broth, 43 strains reduced L. monocytogenes up to 9.06 log10 CFU/ml, 41 reduced E. coli O157:H7 up to 0.84 log10 CFU/ml, and 32 reduced Salmonella up to 0.94 log10 CFU/ml 24 h after co-inoculation. Twenty-eight LAB isolates that exhibited the highest inhibitory effect among pathogens were further analyzed to determine their antimicrobial resistance profile, adhesion potential, and cytotoxicity to Caco-2 cells. All LAB strains tested were susceptible to ampicillin, linezolid, and penicillin. Twenty-six were able to adhere to Caco-2 cells, five were classified as highly adhesive with &gt; 40 bacterial cells/Caco-2 cells. Low cytotoxicity percentages were observed for the candidate LAB strains with values ranging from -5 to 8%. Genotypic identification by whole genome sequencing confirmed all as members of the LAB group; Enterococcus was the genus most frequently isolated with 21 isolates, followed by Pediococcus with 4, and Lactobacillus with 3. In this study, a systematic approach was used for the improved identification of novel LAB strains able to exert antagonistic effect against important foodborne pathogens. Our findings suggest that the selected panel of LAB probiotic strains can be used as biocontrol cultures to inhibit and/or reduce the growth of L. monocytogenes, Salmonella, and E. coli O157:H7 in different matrices, and environments