Towards Development of a Non-Toxigenic Clostridioides difficile Oral Spore Vaccine against Toxigenic C. difficile.

Clostridioides difficile is an opportunistic gut pathogen which causes severe colitis, leading to significant morbidity and mortality due to its toxins, TcdA and TcdB. Two intra-muscular toxoid vaccines entered Phase III trials and strongly induced toxin-neutralising antibodies systemically but failed to provide local protection in the colon from primary C. difficile infection (CDI). Alternatively, by immunising orally, the ileum (main immune inductive site) can be directly targeted to confer protection in the large intestine. The gut commensal, non-toxigenic C. difficile (NTCD) was previously tested in animal models as an oral vaccine for natural delivery of an engineered toxin chimera to the small intestine and successfully induced toxin-neutralising antibodies. We investigated whether NTCD could be further exploited to induce antibodies that block the adherence of C. difficile to epithelial cells to target the first stage of pathogenesis. In NTCD strain T7, the colonisation factor, CD0873, and a domain of TcdB were overexpressed. Following oral immunisation of hamsters with spores of recombinant strain, T7-0873 or T7-TcdB, intestinal and systemic responses were investigated. Vaccination with T7-0873 successfully induced intestinal antibodies that significantly reduced adhesion of toxigenic C. difficile to Caco-2 cells, and these responses were mirrored in sera. Additional engineering of NTCD is now warranted to further develop this vaccine.

Mimicking Native Display of CD0873 on Liposomes Augments Its Potency as an Oral Vaccine against Clostridioides difficile.

Mucosal vaccination aims to prevent infection mainly by inducing secretory IgA (sIgA) antibody, which neutralises pathogens and enterotoxins by blocking their attachment to epithelial cells. We previously demonstrated that encapsulated protein antigen CD0873 given orally to hamsters induces neutralising antibodies locally as well as systemically, affording partial protection against Clostridioides difficile infection. The aim of this study was to determine whether displaying CD0873 on liposomes, mimicking native presentation, would drive a stronger antibody response. The recombinant form we previously tested resembles the naturally cleaved lipoprotein commencing with a cysteine but lacking lipid modification. A synthetic lipid (DHPPA-Mal) was designed for conjugation of this protein via its N-terminal cysteine to the maleimide headgroup. DHPPA-Mal was first formulated with liposomes to produce MalLipo; then, CD0873 was conjugated to headgroups protruding from the outer envelope to generate CD0873-MalLipo. The immunogenicity of CD0873-MalLipo was compared to CD0873 in hamsters. Intestinal sIgA and CD0873-specific serum IgG were induced in all vaccinated animals; however, neutralising activity was greatest for the CD0873-MalLipo group. Our data hold great promise for development of a novel oral vaccine platform driving intestinal and systemic immune responses.

A Multi-Factorial Observational Study on Sequential Fecal Microbiota Transplant in Patients with Medically Refractory Clostridioides difficile Infection.

Fecal microbiota transplantation (FMT) is highly effective in recurrent Clostridioides difficile infection (CDI); increasing evidence supports FMT in severe or fulminant Clostridioides difficile infection (SFCDI). However, the multifactorial mechanisms that underpin the efficacy of FMT are not fully understood. Systems biology approaches using high-throughput technologies may help with mechanistic dissection of host-microbial interactions. Here, we have undertaken a deep phenomics study on four adults receiving sequential FMT for SFCDI, in which we performed a longitudinal, integrative analysis of multiple host factors and intestinal microbiome changes. Stool samples were profiled for changes in gut microbiota and metabolites and blood samples for alterations in targeted epigenomic, metabonomic, glycomic, immune proteomic, immunophenotyping, immune functional assays, and T-cell receptor (TCR) repertoires, respectively. We characterised temporal trajectories in gut microbial and host immunometabolic data sets in three responders and one non-responder to sequential FMT. A total of 562 features were used for analysis, of which 78 features were identified, which differed between the responders and the non-responder. The observed dynamic phenotypic changes may potentially suggest immunosenescent signals in the non-responder and may help to underpin the mechanisms accompanying successful FMT, although our study is limited by a small sample size and significant heterogeneity in patient baseline characteristics. Our multi-omics integrative longitudinal analytical approach extends the knowledge regarding mechanisms of efficacy of FMT and highlights preliminary novel signatures, which should be validated in larger studies.

Colonisation Factor CD0873, an Attractive Oral Vaccine Candidate against Clostridioides difficile.

Clostridioides difficile is the main cause of health-care-associated infectious diarrhoea. Toxins, TcdA and TcdB, secreted by this bacterium damage colonic epithelial cells and in severe cases this culminates in pseudomembranous colitis, toxic megacolon and death. Vaccines in human trials have focused exclusively on the parenteral administration of toxin-based formulations. These vaccines promote toxin-neutralising serum antibodies but fail to confer protection from infection in the gut. An effective route to immunise against gut pathogens and stimulate a protective mucosal antibody response (secretory immunoglobulin A, IgA) at the infection site is the oral route. Additionally, oral immunisation generates systemic antibodies (IgG). Using this route, two different antigens were tested in the hamster model: The colonisation factor CD0873 and a TcdB fragment. Animals immunised with CD0873 generated a significantly higher titre of sIgA in intestinal fluid and IgG in serum compared to naive animals, which significantly inhibited the adherence of C. difficile to Caco-2 cells. Following challenge with a hypervirulent isolate, the CD0873-immunised group showed a mean increase of 80% in time to experimental endpoint compared to naïve animals. Survival and body condition correlated with bacterial clearance and reduced pathology in the cecum. Our findings advocate CD0873 as a promising oral vaccine candidate against C. difficile.

Corrigendum: Variant Signal Peptides of Vaccine Antigen, FHbp, Impair Processing Affecting Surface Localization and Antibody-Mediated Killing in Most Meningococcal Isolates.

[This corrects the article DOI: 10.3389/fmicb.2019.02847.].

Variant Signal Peptides of Vaccine Antigen, FHbp, Impair Processing Affecting Surface Localization and Antibody-Mediated Killing in Most Meningococcal Isolates.

Meningococcal lipoprotein, Factor H binding protein (FHbp), is the sole antigen of the Trumenba vaccine (Pfizer) and one of four antigens of the Bexsero vaccine (GSK) targeting Neisseria meningitidis serogroup B isolates. Lipidation of FHbp is assumed to occur for all isolates. We show in the majority of a collection of United Kingdom isolates (1742/1895) non-synonymous single nucleotide polymorphisms (SNPs) in the signal peptide (SP) of FHbp. A single SNP, common to all, alters a polar amino acid that abolishes processing: lipidation and SP cleavage. Whilst some of the FHbp precursor is retained in the cytoplasm due to reduced binding to SecA, remarkably some is translocated and further surface-localized by Slam. Thus we show Slam is not lipoprotein-specific. In a panel of isolates tested, the overall reduced surface localization of the precursor FHbp, compared to isolates with an intact SP, corresponded with decreased susceptibility to antibody-mediated killing. Our findings shed new light on the canonical pathway for lipoprotein processing and translocation of important relevance for lipoprotein-based vaccines in development and in particular for Trumenba.

First record of the root knot nematode, Meloidogyne minor in New Zealand with description, sequencing information and key to known species of Meloidogyne in New Zealand.

Meloidogyne minor Karssen et al. 2004 was collected from perennial ryegrass (Lolium perenne L.) growing in a sports ground in Christchurch, New Zealand. This is a new record for M. minor, the first report of this nematode occurring in New Zealand, and the second report from the southern hemisphere (after Chile). In general, the New Zealand isolate of M. minor corresponds well to the descriptions of M. minor given by Karssen et al. (2004). The New Zealand isolate is characterized by having a female with dorsally curved stylet, 13-14 µm long, with transversely ovoid knobs slightly sloping backwards from shaft; rounded perineal pattern; and male with stylet 16-19 µm long, large transversely ovoid knobs sloping slightly backwards from shaft; head region not set off, labial disc elevated, lateral lips prominent; and second stage juvenile 370-390 µm long, with hemizonid posterior but adjacent to excretory pore; tail 53-63 µm long; and a distinct hyaline tail terminus 14-18 µm long. In addition, molecular phylogeny using near full length small subunit (SSU), D2/D3 expansion segments of the large subunit (LSU), the internal transcribed spacer region (ITS1 and 2), and the intergenic spacer (IGS2) of the ribosomal rDNA supports the identification.

Insight into proteomic investigations of Neisseria meningitidis serogroup C strain L91543 from analysis of its genome sequence.

Here, we describe the draft sequence of a virulent isolate of Neisseria meningitidis strain L91543, belonging to serogroup C. The findings from previous proteomic and metabolomic studies of this strain can now be further interpreted with genomic analysis. Comparative analysis of the genome sequence revealed close similarity and localized synteny with the genome sequence of N. meningitidis serogroup C strain, FAM18. Polymorphisms were identified in the signal peptide sequence of factor H binding protein, a target for new meningococcal vaccines, which may result in its inefficient translocation across the cytoplasmic membrane affecting its processing (lipidation and cleavage of the signal peptide) and transportation to the outer membrane in strain L91543. This would explain the unusual proteomic data for factor H binding protein for this strain. NadA, another target for new vaccines, and the MtrR regulator, which controls expression of NadA, both contain SNPs between strains L91543 and FAM18. The genome sequence data were generated using Ion Torrent PGM sequencing, assembled into 50 contigs with 64× coverage and annotated with 2262 genes, 14 rRNAs and 56 tRNAs. The availability of the genome of N. meningitidis strain L91543 will aid our understanding of the proteome of this organism, importantly its vaccine antigens.

Genetic odyssey to generate marked clones in Drosophila mosaics.

Chimaeras, fanciful beasts that drew their force from being composed of parts of disparate animals, have stimulated our collective imagination for centuries. In modern terms, chimaeras are composite animals consisting of genetically distinct cell populations and are called "mosaics" if the different cell types have emerged from the same zygote. Phenotypic studies of chimeric animals formed from invertebrates, amphibians, birds, and mammals have provided many fundamental insights into biological processes, most notably in developmental biology. Many methods for generating both chimaeras and a range of markers for tracing their lineages have been developed over the years. Our laboratory has been intimately involved in the development of methods that facilitate the creation of genetic mosaics in Drosophila. Here, we review our contributions to the development of this field and discuss a number of approaches that will improve further the tool kit for generating mosaic animals.

Deliberate ignorance: a systematic review of blinding in nursing clinical trials.

BACKGROUND: Blinding is recommended widely as a strategy in randomized controlled trials (RCTs) to reduce biases that can result from awareness of who is receiving the intervention being tested. The absence of blinding, especially when the primary outcomes are subjective, has been found to be associated with inflated estimates of treatment effects, yet little is known about the use of blinding in nursing RCTs. OBJECTIVES: The purposes of this study were (a) to describe the extent to which nurse researchers state that they used blinding as a bias-reduction strategy or explain why it was not used, (b) to identify the groups that are blinded when blinding is used, (c) to assess whether the term blinding or masking is more prevalent, and (d) to explore factors that might affect the use or acknowledgement of blinding in nursing trials. METHODS: Data regarding blinding were extracted systematically and coded from a consecutive sample of 199 RCTs published in 16 nursing journals in 2007 to 2009. RESULTS: Blinding, the term used predominantly in nursing, was reported to have been used in 33% of the studies. Rates of blinding ranged from 2.5% for data analysts to 28% for data collectors. The absence of blinding was discussed as a limitation in only 13% of the studies in which blinding did not occur. The use or mention of blinding was related strongly to whether the journal in which the article was published had endorsed the Consolidated Standards for Reporting Trials guidelines (CONSORT). CONCLUSIONS: Nurse researchers conducting RCTs should be more diligent in following the CONSORT guidelines regarding the use and description of blinding. Although it is often impossible to blind study participants and intervention agents, the blinding of data collectors, data analysts, and nonresearch healthcare staff should be pursued.

The twin spot generator for differential Drosophila lineage analysis.

In Drosophila melanogaster, widely used mitotic recombination-based strategies generate mosaic flies with positive readout for only one daughter cell after division. To differentially label both daughter cells, we developed the twin spot generator (TSG) technique, which through mitotic recombination generates green and red twin spots that are detectable after the first cell division as single cells. We propose wide applications of TSG to lineage and genetic mosaic studies.

Nurse characteristics and inferences about children's pain.

The purpose of this study was to describe pediatric nurses' projected responses to children's pain as described in vignettes of hospitalized children and to explore nurse characteristics that might influence those responses. A survey was mailed to a national random sample of 700 RNs, and 334 nurses responded. The survey included case reports of three hospitalized school-aged children experiencing pain. Nurses were asked to rate their perceptions of the children's pain levels and to indicate how much analgesia they would recommend. Contrary to earlier studies, in response to the scenarios, nurses in this sample perceived high levels of pain, said they would administer doses of analgesia close to the maximum prescribed by physicians, and recommended an array of non-pharmacologic methods to treat pain. Variation in pain perceptions and decisions was not related to key personal and professional characteristics of the nurses, including their education level, race/ethnicity, age, years of clinical experience, and receipt of continuing education about pain. Findings from this large national study suggest that most nurses would make appropriate decisions relating to the treatment of children's pain, perhaps reflecting changes in the emphasis on pain management.

Stereotyping and nurses' recommendations for treating pain in hospitalized children.

The purpose of this study was to examine whether nurses' recommendations for managing children's pain were influenced by stereotypes based on children's personal attributes. Three vignettes, in which hospitalized children's sex, race, and attractiveness were experimentally manipulated, were mailed to a national random sample of 700 pediatric nurses; 334 nurses responded. Responses to vignette questions indicated little evidence of stereotyping. Nurses perceived similar levels of pain and recommended similar pain treatments, regardless of sex, race, and attractiveness. Nurses, on average, perceived children's pain at levels consistent with the children's self-reports and recommended assertive analgesic and non-pharmacologic pain management strategies. The results appear consistent with prevailing views on providing adequate pain treatment for children.

Signature-tagged transposon mutagenesis identifies novel Mycobacterium tuberculosis genes involved in the parasitism of human macrophages.

Using signature-tagged transposon mutagenesis, we isolated 23 Mycobacterium tuberculosis mutants, corresponding to 21 genes or genetic regions, attenuated in their ability to parasitize human macrophages. Mutants disrupted in the ABC transporter-encoding genes Rv0986 and Rv0987 were further characterized as being impaired in their ability to bind to host cells.

Comparative investigation of the pathogenicity of three Mycobacterium tuberculosis mutants defective in the synthesis of p-hydroxybenzoic acid derivatives.

p-Hydroxybenzoic acid derivatives (p-HBADs) are glycoconjugates secreted by all Mycobacterium tuberculosis isolates whose contribution to pathogenicity remains to be determined. The pathogenicity of three transposon mutants of M. tuberculosis deficient in the biosynthesis of some or all forms of p-HBADs was studied. Whilst the mutants grew similarly to the wild-type strain in macrophages and C57BL/6 mice, two of the mutants induced a more severe and diffuse inflammation in the lungs. The lack of production of some or all forms of p-HBADs in these two mutants also correlated with an increased secretion of the pro-inflammatory cytokines tumour-necrosis factor alpha, interleukin 6 and interleukin 12 in vivo. We propose that the loss of production of p-HBADs by tubercle bacilli results in their diminished ability to suppress the pro-inflammatory response to infection and that this ultimately provokes extensive pulmonary lesions in the C57BL/6 model of tuberculosis infection.

Development of a One-Step Real-Time Polymerase Chain Reaction Assay for Diagnosis of Phytophthora ramorum.

ABSTRACT Phytophthora ramorum is a recently described pathogen causing bleeding cankers, dieback, and leaf blight on trees and shrubs in parts of Europe and North America, where the disease is commonly known as sudden oak death. This article describes the development of a single-round real-time polymerase chain reaction (PCR) assay based on TaqMan chemistry, designed within the internal transcribed spacer 1 region of the nuclear ribosomal (nr)RNA gene for detection of P. ramorum in plant material. Unlike previously described methods for the molecular detection of P. ramorum, this assay involves no post amplification steps or multiple rounds of PCR. The assay was found to have a limit of detection of 10 pg of P. ramorum DNA, and could detect P. ramorum in plant material containing 1% infected material by weight within 36 cycles of PCR. The assay also was used to test DNA from 28 other Phytophthora spp. to establish its specificity for P. ramorum. A quick and simple method was used to extract DNA directly from host plant material, and detection of P. ramorum was carried out in multiplex with an assay for a gene from the host plant in order to demonstrate whether amplifiable DNA had been extracted. Amplifiable DNA was extracted from 84.4% of samples, as demonstrated by amplification of host plant DNA. The real-time protocol was used to test 320 plant samples (from 19 different plant species) from which DNA extraction had been successful, and was shown to give results comparable with a traditional isolation technique for diagnosis of P. ramorum in plant material from common U.K. hosts.

p-Hydroxybenzoic acid synthesis in Mycobacterium tuberculosis.

Glycosylated p-hydroxybenzoic acid methyl esters and structurally related phenolphthiocerol glycolipids are important virulence factors of Mycobacterium tuberculosis. Although both types of molecules are thought to be derived from p-hydroxybenzoic acid, the origin of this putative biosynthetic precursor in mycobacteria remained to be established. We describe the characterization of a transposon mutant of M. tuberculosis deficient in the production of all forms of p-hydroxybenzoic acid derivatives. The transposon was found to be inserted in Rv2949c, a gene located in the vicinity of the polyketide synthase gene pks15/1, involved in the elongation of p-hydroxybenzoate to phenolphthiocerol in phenolic glycolipid-producing strains. A recombinant form of the Rv2949c enzyme was produced in the fast-growing non-pathogenic Mycobacterium smegmatis and purified to near homogeneity. The recombinant enzyme catalyzed the removal of the pyruvyl moiety of chorismate to form p-hydroxybenzoate with an apparent K(m) value for chorismate of 19.7 microm and a k(cat) value of 0.102 s(-1). Strong inhibition of the reaction by p-hydroxybenzoate but not by pyruvate was observed. These results establish Rv2949c as a chorismate pyruvate-lyase responsible for the direct conversion of chorismate to p-hydroxybenzoate and identify Rv2949c as the sole enzymatic source of p-hydroxybenzoic acid in M. tuberculosis.

Digalactoside expression in the lipopolysaccharide of Haemophilus influenzae and its role in intravascular survival.

Digalactoside (galalpha-1-4 galbeta) structures of the lipopolysaccharide (LPS) of Haemophilus influenzae are implicated in virulence. A confounding factor is that tetranucleotide repeats within the lic2A, lgtC, and lex2 genes mediate phase-variable expression of the digalactosides. By deleting these repeats, we constructed recombinant strains of RM153 constitutively expressing either one or two LPS digalactosides. Expression of two digalactosides, rather than one, was associated with increased virulence of H. influenzae in vivo.

Elucidation of the monoclonal antibody 5G8-reactive, virulence-associated lipopolysaccharide epitope of Haemophilus influenzae and its role in bacterial resistance to complement-mediated killing.

The phase-variable locus lex2 is required for expression of a Haemophilus influenzae lipopolysaccharide (LPS) epitope of previously unknown structure. This epitope, which is reactive with monoclonal antibody (MAb) 5G8, has been associated with virulence of type b strains. When strain RM118 (from the same source as strain Rd), in which the lex2 locus and MAb 5G8 reactivity are absent, was transformed with lex2 DNA, transformants that were reactive with MAb 5G8 were obtained. Surprisingly, the 5G8 reactivity of these transformants was phase variable, although the lex2 locus lacked tetrameric repeats and was constitutively expressed. This phase variation was shown to be the result of phase-variable expression of phosphorylcholine (PCho) such that MAb 5G8 reacted only in the absence of PCho. Structural analysis showed that, compared to RM118, the lex2 transformant had acquired a tetrasaccharide, Gal-alpha1,4-Gal-beta1,4-Glc-beta1,4-Glc-beta1,4, linked to the proximal heptose (HepI). A terminal GalNAc was detected in a minority of glycoforms. LPS derived from a mutant of RM7004, a virulent type b strain which naturally expresses lex2 and has LPS containing the same tetrasaccharide linked to HepI as the sole oligosaccharide extension from the inner core, confirmed that GalNAc is not a part of the MAb 5G8-reactive epitope. Thus, MAb 5G8 specifically binds to the structure Gal-alpha1,4-Gal-beta1,4-Glc-beta1,4-Glc-beta attached via a 1,4 linkage to HepI of H. influenzae LPS, and we show that the ability to synthesize this novel tetrasaccharide was associated with enhanced bacterial resistance to complement-mediated killing.