Development of Clostridium difficile R20291ΔPaLoc model strains and in vitro methodologies reveals CdtR is required for the production of CDT to cytotoxic levels

Assessing the regulation of Clostridium difficile transferase (CDT), is complicated by the presence of a Pathogenicity locus (PaLoc) which encodes Toxins A and B. Here we developed R20291ΔPaLoc model strains and cell-based assays to quantify CDT-mediated virulence. Their application demonstrated that the transcriptional regulator, CdtR, was required for CDT-mediated cytotoxicity

Functional significance of active site residues in the enzymatic component of the Clostridium difficile binary toxin

© 2016 Clostridium difficile binary toxin (CDT) is an ADP-ribosyltransferase which is linked to enhanced pathogenesis of C. difficile strains. CDT has dual function: domain a (CDTa) catalyses the ADP-ribosylation of actin (enzymatic component), whereas domain b (CDTb) transports CDTa into the cytosol (transport component). Understanding the molecular mechanism of CDT is necessary to assess its role in C. difficile infection. Identifying amino acids that are essential to CDTa function may aid drug inhibitor design to control the severity of C. difficile infections. Here we report mutations of key catalytic residues within CDTa and their effect on CDT cytotoxicity. Rather than an all-or-nothing response, activity of CDTa mutants vary with the type of amino acid substitution; S345A retains cytotoxicity whereas S345Y was sufficient to render CDT non-cytotoxic. Thus CDTa cytotoxicity levels are directly linked to ADP-ribosyltransferase activity

Characteristics of Lipo-Oligosaccharide Loci of Campylobacter jejuni Isolates Associated with Guillain-Barré Syndrome from Hebei, China

Ganglioside mimicry by C.jejuni lipo-oligosaccharides (LOS) could induce the production of autoantibodies against gangliosides and the development of Guillain-Barré syndrome (GBS). The LOS biosynthesis region exhibits significant variation with different strains. Using PCR amplifications of genes from published LOS loci and sequencing the LOS biosynthesis loci, the eight GBS-associated C. jejuni strains from HeBei could be classified into four classes. The expression of sialylated LOS structures (class A) or non-sialylated LOS structures(class F, H and P) in the C. jejuni LOS is considered to be two different factors for the induction of GBS

Propidium Monoazide Integrated with qPCR Enables the Detection and Enumeration of Infectious Enteric RNA and DNA Viruses in Clam and Fermented Sausages

The increase of foodborne viral outbreaks highlights the need for a rapid and sensitive method for the prediction of viral infectivity in food samples. This study assesses the use of propidium monoazide (PMA) coupled with real-time PCR methods (RT-qPCR or qPCR for RNA or DNA viruses, respectively) in the determination of viral infectivity in complex animal-related food matrices. Clam and Spanish fermented sausage (“chorizo”) samples were spiked with infectious and heat-inactivated human adenovirus-2 (HAdV-2) and mengovirus (vMC0). PMA-qPCR/RT-qPCR discriminated infective virus particles, with significant reductions (>2.7 log10 or 99.7%). Additionally, infectious HAdV-2 and vMC0 were quantified by plaque assay (in plaque forming units, PFU), and compared with those in virus genomes copies (GCs) quantified by PMA-qPCR/RT-qPCR. A consistent correlation (R2 > 0.92) was showed between PFU and GCs along serial 10-fold dilutions in both DNA and RNA virus and in both food matrices. This study shows the use of PMA coupled to qPCR/RT-qPCR as a promising alternative for prediction of viral infectivity in food samples in comparison to more expensive and time-consuming methods and for those viruses that are not able to grow under available cell culture techniques.ThisstudywasfinanciallysupportedbytheRTA2014-00024- C04-01 fromtheSpanishMinistryofEconomyandInnovation and theBrazilianCNPqProjectnumber472804/2013-8,andby CAPES/PNPD andCAPES/PDSE

Towards a Pathogenic Escherichia coli Detection Platform Using Multiplex SYBR®Green Real-Time PCR Methods and High Resolution Melting Analysis

Escherichia coli is a group of bacteria which has raised a lot of safety concerns in recent years. Five major intestinal pathogenic groups have been recognized amongst which the verocytotoxin or shiga-toxin (stx1 and/or stx2) producing E. coli (VTEC or STEC respectively) have received a lot of attention recently. Indeed, due to the high number of outbreaks related to VTEC strains, the European Food Safety Authority (EFSA) has requested the monitoring of the “top-five” serogroups (O26, O103, O111, O145 and O157) most often encountered in food borne diseases and addressed the need for validated VTEC detection methods. Here we report the development of a set of intercalating dye Real-time PCR methods capable of rapidly detecting the presence of the toxin genes together with intimin (eae) in the case of VTEC, or aggregative protein (aggR), in the case of the O104:H4 strain responsible for the outbreak in Germany in 2011. All reactions were optimized to perform at the same annealing temperature permitting the multiplex application in order to minimize the need of material and to allow for high-throughput analysis. In addition, High Resolution Melting (HRM) analysis allowing the discrimination among strains possessing similar virulence traits was established. The development, application to food samples and the flexibility in use of the methods are thoroughly discussed. Together, these Real-time PCR methods facilitate the detection of VTEC in a new highly efficient way and could represent the basis for developing a simple pathogenic E. coli platform

Sequence-based typing of genetic targets encoded outside of the O-antigen gene cluster is indicative of Shiga toxin-producing Escherichia coli serogroup lineages

Serogroup classifications based upon the O-somatic antigen of Shiga toxin-producing Escherichia coli (STEC) provide significant epidemiological information on clinical isolates. Each O-antigen determinant is encoded by a unique cluster of genes present between the gnd and galF chromosomal genes. Alternatively, serogroup-specific polymorphisms might be encoded in loci that are encoded outside of the O-antigen gene cluster. Segments of the core bacterial loci mdh, gnd, gcl, ppk, metA, ftsZ, relA and metG for 30 O26 STEC strains have previously been sequenced, and comparative analyses to O157 distinguished these two serogroups. To screen these loci for serogroup-specific traits within a broader range of clinically significant serogroups, DNA sequences were obtained for 19 strains of 10 additional STEC serogroups. Unique alleles were observed at the gnd locus for each examined STEC serogroup, and this correlation persisted when comparative analyses were extended to 144 gnd sequences from 26 O-serogroups (comprising 42 O : H-serotypes). These included O157, O121, O103, O26, O5 : non-motile (NM), O145 : NM, O113 : H21, O111 : NM and O117 : H7 STEC; and furthermore, non-toxin encoding O157, O26, O55, O6 and O117 strains encoded distinct gnd alleles compared to STEC strains of the same serogroup. DNA sequencing of a 643 bp region of gnd was, therefore, sufficient to minimally determine the O-antigen of STEC through molecular means, and the location of gnd next to the O-antigen gene cluster offered additional support for the co-inheritance of these determinants. The gnd DNA sequence-based serogrouping method could improve the typing capabilities for STEC in clinical laboratories, and was used successfully to characterize O121 : H19, O26 : H11 and O177 : NM clinical isolates prior to serological confirmation during outbreak investigations

Nanolitre real-time PCR detection of bacterial, parasitic, and viral agents from patients with diarrhoea in Nunavut, Canada

Background. Little is known about the microbiology of diarrhoeal disease in Canada's Arctic regions. There are a number of limitations of conventional microbiology testing techniques for diarrhoeal pathogens, and these may be further compromised in the Arctic, given the often long distances for specimen transport. Objective. To develop a novel multiple-target nanolitre real-time reverse transcriptase (RT)-PCR platform to simultaneously test diarrhoeal specimens collected from residents of the Qikiqtani (Baffin Island) Region of Nunavut, Canada, for a wide range of bacterial, parasitic and viral agents. Study design/methods. Diarrhoeal stool samples submitted for bacterial culture to Qikiqtani General Hospital in Nunavut over an 18-month period were tested with a multiple-target nanolitre real-time PCR panel for major diarrhoeal pathogens including 8 bacterial, 6 viral and 2 parasitic targets. Results. Among 86 stool specimens tested by PCR, a total of 50 pathogens were detected with 1 or more pathogens found in 40 (46.5%) stool specimens. The organisms detected comprised 17 Cryptosporidium spp., 5 Clostridium difficile with toxin B, 6 Campylobacter spp., 6 Salmonella spp., 4 astroviruses, 3 noroviruses, 1 rotavirus, 1 Shigella spp. and 1 Giardia spp. The frequency of detection by PCR and bacterial culture was similar for Salmonella spp., but discrepant for Campylobacter spp., as Campylobacter was detected by culture from only 1/86 specimens. Similarly, Cryptosporidium spp. was detected in multiple samples by PCR but was not detected by microscopy or enzyme immunoassay. Conclusions. Cryptosporidium spp., Campylobacter spp. and Clostridium difficile may be relatively common but possibly under-recognised pathogens in this region. Further study is needed to determine the regional epidemiology and clinical significance of these organisms. This method appears to be a useful tool for gastrointestinal pathogen research and may also be helpful for clinical diagnostics and outbreak investigation in remote regions where the yield of routine testing may be compromised

Tailored ß-Cyclodextrin Blocks the Translocation Pores of Binary Exotoxins from C. Botulinum and C. Perfringens and Protects Cells from Intoxication

International audienceBackgroundClostridium botulinum C2 toxin and Clostridium perfringens iota toxin are binary exotoxins, which ADP-ribosylate actin in the cytosol of mammalian cells and thereby destroy the cytoskeleton. C2 and iota toxin consists of two individual proteins, an enzymatic active (A-) component and a separate receptor binding and translocation (B-) component. The latter forms a complex with the A-component on the surface of target cells and after receptor-mediated endocytosis, it mediates the translocation of the A-component from acidified endosomal vesicles into the cytosol. To this end, the B-components form heptameric pores in endosomal membranes, which serve as translocation channels for the A-components.Here we demonstrate that a 7-fold symmetrical positively charged ß-cyclodextrin derivative, per-6-S-(3-aminomethyl)benzylthio-ß-cyclodextrin, protects cultured cells from intoxication with C2 and iota toxins in a concentration-dependent manner starting at low micromolar concentrations. We discovered that the compound inhibited the pH-dependent membrane translocation of the A-components of both toxins in intact cells. Consistently, the compound strongly blocked transmembrane channels formed by the B-components of C2 and iota toxin in planar lipid bilayers in vitro. With C2 toxin, we consecutively ruled out all other possible inhibitory mechanisms showing that the compound did not interfere with the binding of the toxin to the cells or with the enzyme activity of the A-component.Conclusions/SignificanceThe described ß-cyclodextrin derivative was previously identified as one of the most potent inhibitors of the binary lethal toxin of Bacillus anthracis both in vitro and in vivo, implying that it might represent a broad-spectrum inhibitor of binary pore-forming exotoxins from pathogenic bacteria

Skeletal evidence for variable patterns of handedness in chimpanzees, human hunter–gatherers, and recent British populations

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/98285/1/nyas12067.pd

Structured bimanual actions and hand transfers reveal population-level right-handedness in captive gorillas

There is a common prevailing perception that humans possess a species-unique population-level right-hand bias that has evolutionary links with language. New theories suggest that an early evolutionary division of cognitive function gave rise to a left-hemisphere bias for behaviours underpinned by structured sequences of actions. However, studies of great ape handedness have generated inconsistent results and considerable debate. Additionally, the literature places a heavy focus on chimpanzees, revealing a paucity of handedness findings from other great ape species, and thus limiting the empirical evidence with which we can evaluate evolutionary theory. We observed handedness during spontaneous naturalistic bimanual actions in a captive, biological group of 13 western lowland gorillas, Gorilla gorilla gorilla. Our results demonstrated a significant group-level right-handed bias for bimanual actions as well as for a novel measure of handedness: hand transfer. The two measures revealed similar patterns of handedness, such that a right-hand bias for the majority of individuals was found across both measures. Our findings suggest that human population-level right-handedness is a behavioural trait linked with left-hemisphere dominance for the processing of structured sequences of actions, and was inherited by a common ancestor of both humans and apes