The emergence of ‘hypervirulence’ in Clostridium difficile

The impact of Clostridium difficile-associated disease (CDAD) in healthcare settings throughout the developed world is considerable in terms of mortality, morbidity, and disease management. The incidence of CDAD has risen dramatically since the turn of this century, concomitant with the emergence of so-called hypervirulent strains which are thought to cause a more severe disease, higher relapse rates, and increased mortality. Pre-eminent amongst hypervirulent strains are those belonging to ribotype 027, which were first reported in Canada in 2003 and shortly thereafter in the UK. Since its arrival in Europe, it has spread rapidly and has now been reported in 16 member states and Switzerland. The physiological factors responsible for the rapid emergence of hypervirulent C. difficile strains remain unclear. It is known that they produce a binary toxin (CDT) in addition to toxins A and B, that they are resistant to fluoroquinolones due to mutations in gyrA, and that they are resistant to erythromycin. Representative strains have been suggested to produce more toxin A and B in the ‘laboratory flask’ (most likely due to a frameshift mutation in the repressor gene tcdC), to be more prolific in terms of spore formation, and also exhibit increased adherence to human intestinal epithelial cells due to altered surface proteins. However, the contribution of these and other as yet unidentified factors to the rapid spread of certain C. difficile variants (e.g., ribotypes 027 and 078) remains unclear at present. The advent of ClosTron technology means that it is now possible to construct genetically stable isogenic mutants of C. difficile and carry out reverse genetic studies to elucidate the role of specific gene loci in causing disease. The identification of virulence factors using this approach should help lead to the rational development of therapeutic countermeasures against CDAD.     

Molecular epidemiology and resistance profiles of Clostridium difficile in a tertiary care hospital in Spain

Epidemiological surveillance of Clostridium difficile infection has gained importance in recent years as a result of the rapid spread of epidemic strains, including hypervirulent strains and strains with reduced susceptibility to antimicrobials. The molecular epidemiology and antimicrobial susceptibility of C. difficile in the reference hospital of the Balearic Islands (Spain) is reported in this study. One hundred isolates of toxigenic C. difficile from different patients were selected using rapid dual EIA screening test. All isolates were characterized through toxin profile, PCR ribotyping and, in addition, multi-locus sequence typing (MLST) was performed on fifty selected strains. MICs to metronidazole, vancomycin, erythromycin and moxifloxacin were also determined. A total of 43 different ribotypes were distinguished, with higher prevalence of ribotype 014 (34%). Twenty one per cent of the isolates expressed binary toxin and it is noteworthy that 62% of these were identified as the hypervirulent ribotype 078, the second most prevalent ribotype found in our hospital (13%). A total of 20 different sequence types (STs) were found, including a new described allele and ST. MLST data showed a clear concordance between some ribotypes and STs, mainly represented by ribotype 014/ST-2, ribotype 078/ST-11 and ribotype 001/ST-3. Phylogenetic analysis also revealed that most of the isolates were genetically related, forming a large clonal complex. Finally, ribotypes 078 (ST-11) and 001 (ST-3) were associated with higher resistance to erythromycin and to erythromycin and moxifloxacin, respectively. All these data suggest that the combination of ribotyping and MLST is a good tool for the surveillance of the changing epidemiology of C. difficile. A wide dissemination of clones has been observed in our setting, ribotype 014 (ST-2) being the most prevalent followed by the hypervirulent ribotype 078 (ST-11) and ribotype 001 (ST-3), their spread in our setting probably influenced by their higher resistance.     

A multidomain galectin involved in innate immune response of pearl oyster Pinctada fucata

Galectins could specifically bind to β-galactoside residues and play crucial roles in innate immune responses of vertebrates and invertebrates. In this study, the cDNA of a galectin with multiple carbohydrate-recognition domains (CRDs) was cloned from pearl oyster Pinctada fucata (designated as PoGal). PoGal cDNA was 2138 bp long and consisted of a 5′-untranslated region (UTR) of 120 bp, a 3′-UTR of 350 bp with two cytokine RNA instability motifs (ATTTA), and an open reading frame (ORF) of 1668 bp encoding a polypeptide of 555 amino acids with an estimated molecular mass of 63.4 kDa and a theoretical isoelectric point of 4.8. PoGal contained four CRDs, each CRD of PoGal all had the conserved carbohydrate-binding motifs H-NPR and WG-ER. PoGal shared 43.7% and 62.9% identity to those of bay scallop and eastern oyster, respectively, which were only two galectins with four CRDs. The phylogenetic analysis revealed that all galectins with four CRDs formed a single clade. PoGal mRNA was constitutively expressed in all detected tissues, and the expression level of PoGal mRNA was significantly up-regulated in digestive gland, mantle, haemocyte, gonad and intestine after Vibrio alginolyticus stimulation. The expression profile analysis showed that the expression level of PoGal mRNA was significantly up-regulated at 4, 8 and 12 h after V. alginolyticus stimulation. These results suggested that PoGal was a constitutive and inducible acute-phase protein that perhaps involved in innate immune response of pearl oyster.     

天然免疫细胞对急性HBV感染作用机制研究进展

研究表明HBV感染能引起肝脏免疫损伤。过去认为适应性免疫应答在机体抗HBV感染及其引起的肝脏病变中发挥着主要作用。现在研究证实被誉为"机体天然免疫核心器官"的肝脏拥有大量天然免疫细胞,在HBV感染过程中扮演着重要角色,而且很有可能直接决定着急性HBV感染的临床结局和预后。急性HBV感染是感染HBV发病后的初期阶段,是乙肝治愈和防止转为慢性乙肝的关键。研究肝脏天然免疫细胞在急性HBV感染中的作用及机制对阐明HBV免疫致病机制、指导临床科学开展抗病毒治疗、防止急性感染的慢性化发展等方面有非常重要的意义。     

Risk factors for recurrence of Clostridium difficile infection: effect of vancomycin-resistant enterococci colonization

Recurrent Clostridium difficile infection (CDI) is one of the most difficult problems in healthcare infection control. We evaluated the risk factors associated with recurrence in patients with CDI. A retrospective cohort study of 84 patients with CDI from December 2008 through October 2010 was performed at Pusan National University Yangsan Hospital. Recurrence occurred in 13.1% (11/84) of the cases and in-hospital mortality rate was 7.1% (6/84). Stool colonization with vancomycin-resistant enterococci (VRE) (P = 0.006), exposure to more than 3 antibiotics (P = 0.009), low hemoglobin levels (P = 0.025) and continued use of previous antibiotics (P = 0.05) were found to be more frequent in the recurrent group. Multivariate analysis indicated that, stool VRE colonization was independently associated with CDI recurrence (odds ratio, 14.519; 95% confidence interval, 1.157-182.229; P = 0.038). This result suggests that stool VRE colonization is a significant risk factor for CDI recurrence.     

The innate immune response under the control of the LXR pathway

Macrophages play essential roles in infection and resolution of inflammation. This review summarizes recent findings that suggest a relevant role for the nuclear receptor liver X receptor (LXR) in the evolution of immune responses. By exerting both positive and negative regulation of specific macrophage gene expression networks, LXRs display anti-inflammatory activities and promote macrophage survival in bacterial infection settings. Agonists that activate the LXR pathway may be used to enhance innate immunity to highly virulent pathogens that otherwise induce macrophage apoptosis as a means to subvert host immune defense.     

Biology and host response to Cyprinid herpesvirus 3 infection in common carp

Viruses from the family Alloherpesviridae form an aquatic clade of herpesviruses infecting fish and amphibia. Diseases caused by these herpesviruses are of increasing importance because of the high morbidity and mortality associated with the infection, and the difficulties in diagnosing latently infected carriers. Cyprinid herpesvirus 3 (CyHV-3) induces a severe disease and mortality in common carp and thus greatly affects carp aquaculture and trade. This review summarises advancements in the understanding of the infection process and the current knowledge on immune responses of carp to CyHV-3. A focus is laid on host genetics and immunity responsible for resistance/survival from the disease and on the viral mechanisms accountable for evasion of carp immune responses. As current knowledge of immune responses to CyHV-3 is still limited, perspectives for future studies are outlined. Analysing CyHV-3 fish-host interactions will be useful and thought-provoking for a basic understanding of fish immune responses.     

Characterization of virulence factors and clonal diversity of Enterococcus faecalis isolates from treated dental root canals

The high prevalence of Enterococcus faecalis in root canal treated teeth with post-treatment disease, as evidenced by both molecular and traditional culturing methods, suggests that this species may be a key player in endodontic treatment failure. This study aimed to detect virulence factors by phenotypic and western blotting tests, and virulence genes by PCR from 20 clinical strains of E. faecalis isolated from treated root canals of teeth with (10) or without (10) apical periodontitis. Moreover, genomic diversity of these strains was assessed by pulsed-field gel electrophoresis (PFGE) and rep-PCR. All 20 strains presented the gelE gene (gelatinase), but 10 of them did not hydrolyze gelatin. Seven of the 10 gelatinase-producing isolates were recovered from root canals with lesions, which suggests a role for this virulence factor in the pathogenesis of post-treatment disease. The esp gene was expressed only in cases where gelatinase production was negative. The other virulence genes were found in 90% (efaA and ace genes), 45% (agg gene) and 95% (cpd gene) of the E. faecalis isolates. As for PFGE and rep-PCR, no specific clonal type of E. faecalis was found in association with teeth with or without disease, revealing the interindividual clonal diversity of endodontic infections.     

Candidate innate immune system gene expression in the ecological model Daphnia

The last ten years have witnessed increasing interest in host-pathogen interactions involving invertebrate hosts. The invertebrate innate immune system is now relatively well characterised, but in a limited range of genetic model organisms and under a limited number of conditions. Immune systems have been little studied under real-world scenarios of environmental variation and parasitism. Thus, we have investigated expression of candidate innate immune system genes in the water flea Daphnia, a model organism for ecological genetics, and whose capacity for clonal reproduction facilitates an exceptionally rigorous control of exposure dose or the study of responses at many time points. A unique characteristic of the particular Daphnia clones and pathogen strain combinations used presently is that they have been shown to be involved in specific host-pathogen coevolutionary interactions in the wild. We choose five genes, which are strong candidates to be involved in Daphnia-pathogen interactions, given that they have been shown to code for immune effectors in related organisms. Differential expression of these genes was quantified by qRT-PCR following exposure to the bacterial pathogen Pasteuria ramosa. Constitutive expression levels differed between host genotypes, and some genes appeared to show correlated expression. However, none of the genes appeared to show a major modification of expression level in response to Pasteuria exposure. By applying knowledge from related genetic model organisms (e.g. Drosophila) to models for the study of evolutionary ecology and coevolution (i.e. Daphnia), the candidate gene approach is temptingly efficient. However, our results show that detection of only weak patterns is likely if one chooses target genes for study based on previously identified genome sequences by comparison to homologues from other related organisms. Future work on the Daphnia-Pasteuria system will need to balance a candidate gene approach with more comprehensive approaches to de novo identify immune system genes specific to the Daphnia-Pasteuria interaction.     

Flagellin from Marinobacter algicola and Vibrio vulnificus activates the innate immune response of gilthead seabream

Adjuvants have emerged as the best tools to enhance the efficacy of vaccination. However, the traditional adjuvants used in aquaculture may cause adverse alterations in fish making necessary the development of new adjuvants able to stimulate the immune system and offer strong protection against infectious pathogens with minimal undesirable effects. In this respect, flagellin seems an attractive candidate due to its ability to strongly stimulate the immune response of fish. In the present study, we have evaluated the ability of recombinant flagellin from Marinobacter algicola (MA) and Vibrio vulnificus (Vvul), a non-pathogenic and a pathogenic bacteria, respectively, to stimulate the innate immune system of gilthead seabream (Sparus aurata L.) and compare the effect with that of the classical flagellin from Salmonella enterica serovar Typhimurium (Salmonella Typhimurium, STF). Intraperitoneal injection of MA and Vvul resulted in a strong inflammatory response characterized by increased reactive oxygen species production and the infiltration of acidophilic granulocytes at the injection site. Interestingly, however, only flagellin from MA consistently induced the expression of the gene encoding pro-inflammatory interleukin-1β. These effects were further confirmed in vitro, where a dose-dependent activation of macrophages and acidophilic granulocytes by MA and Vvul flagellins was observed. In contrast, STF flagellin was found to be less potent in both in vivo and in vitro experiments. Our results suggest the potential use of MA and Vvul flagellins as immunostimulants and adjuvants for fish vaccination.     

The structurally similar,penta-acylated lipopolysaccharides of Porphyromonas gingivalis and Bacteroides elicit strikingly different innate immune responses

Lipid A structural modifications can substantially impact the host's inflammatory response to bacterial LPS. Bacteroides fragilis, an opportunistic pathogen associated with life-threatening sepsis and intra-abdominal abscess formation, and Bacteroides thetaiotaomicron, a symbiont pivotal for proper host intestinal tissue development, both produce an immunostimulatory LPS comprised of penta-acylated lipid A. Under defined conditions, Porphyromonas gingivalis, an oral pathogen associated with periodontitis, also produces an LPS bearing a penta-acylated lipid A. However, this LPS preparation is 100–1000 times less potent than Bacteroides LPS in stimulating endothelial cells. We analyzed Bacteroides and P. gingivalis lipid A structures using MALDI-TOF MS and gas chromatography to determine the structural basis for this phenomenon. Even though both Bacteroides and P. gingivalis lipid A molecules are penta-acylated and mono-phosphorylated, subtle differences in mass and fatty acid content could account for the observed difference in LPS potency. This fatty acid heterogeneity is also responsible for the peak “clusters” observed in the mass spectra and obfuscates the correlation between LPS structure and immunostimulatory ability. Further, we show the difference in potency between Bacteroides and P. gingivalis LPS is TLR4-dependent. Altogether, the data suggest subtle changes in lipid A structure may profoundly impact the host's innate immune response.     

The role of the innate immune response in autoimmune disease

Autoimmune diseases are the clinical correlate of a dysregulation of the immune system, involving multiple steps and multiple components of both the innate and the adaptive immune system. Innate immune cells are sensitive to a very limited repertoire of foreign "patterns" that bind to selective "pattern recognition receptors". In contrast, adaptive auto-reactive T or B cells bear receptors specific for antigens including "self" antigens and are rendered non-reactive by several "quality control" mechanisms. Under special conditions, activation of cells of the innate immune system can break the state of inactivity of auto-reactive cells of the adaptive immune system, thereby provoking autoimmune disease. Here we review examples to illustrate how innate immune activation influences autoimmune disease and point to the implications for the treatment of human autoimmune disease.     

RNAi and antiviral defense in Drosophila: Setting up a systemic immune response

RNA interference (RNAi) controls gene expression in eukaryotic cells and thus, cellular homeostasis. In addition, in plants, nematodes and arthropods it is a central antiviral effector mechanism. Antiviral RNAi has been well described as a cell autonomous response, which is triggered by double-stranded RNA (dsRNA) molecules. This dsRNA is the precursor for the silencing of viral RNA in a sequence-specific manner. In plants, systemic antiviral immunity has been demonstrated, however much less is known in animals. Recently, some evidence for a systemic antiviral response in arthropods has come to light. Cell autonomous RNAi may not be sufficient to reach an efficient antiviral response, and the organism might rely on the spread and uptake of an RNAi signal of unknown origin. In this review, we offer a perspective on how RNAi-mediated antiviral immunity could confer systemic protection in insects and we propose directions for future research to understand the mechanism of RNAi-immune signal sorting, spreading and amplification.     

An immunohistochemical assessment of the cutaneous immune response to louse infestation in cattle

Skin samples were taken from 10 experimental cattle exposed naturally, during a period extending over two winters, to Bovicola bovis and Solenoptes capillatus, five becoming infested and five being protected from infestation by repeated treatment with ectoparasiticides. Skin sections were examined histopathologically and immunohistochemically for expression of the immune cell markers CD3, CD4, CD8 and class II antigens of the major histocompatibility complex (MHC). Louse-infested cattle had a mixed infiltration of the superficial dermis and perifollicular regions with eosinophils and mononuclear cells. The skin of infested cattle differed from that of non-infested cattle in showing significantly more cells expressing CD3, CD4 and MHC class II (P<0.05). Many of the MHC class II(+) cells had dendritic morphology, suggesting active antigen presentation within the lesions. Louse infestations have previously been thought to produce a type 1 hypersensitivity response, mediated by Th2 lymphocytes. However, the increased number of lymphocytes and antigen-presenting cells observed in the present study suggests that in chronic infestation there is activation of local cell-mediated (Th1) immunity.     

f57f4.4p::gfp as a fluorescent reporter for analysis of the C. elegans response to bacterial infection

Host defense mechanisms are multi-layered and involve constitutive as well as inducible components. The dissection of these complex processes can be greatly facilitated using a reporter gene strategy with a transparent animal. In this study, we use Caenorhabditis elegans as a model host and introduce a new pathogen-inducible fluorescent reporter involving the promoter of f57f4.4, a gene encoding a putative component of the glycocalyx. We show that this reporter construct does not respond to heavy metal or hypertonic environments, but is specifically and locally induced in the intestine upon Photorhabus luminescens and Pseudomonas aeruginosa infections. We further demonstrate that its upregulation requires live pathogens as well as elements of the nematode p38 MAP kinase and TGF-beta pathways. In addition to introducing a new tool for the study of the interactions between C. elegans and a pathogen, our results suggest a role for the glycocalyx in gut immunity.     

Contribution of Toll-like receptors to the control of hepatitis B virus infection by initiating antiviral innate responses and promoting specific adaptive immune responses

It is well accepted that adaptive immunity plays a key role in the control of hepatitis B virus (HBV) infection. In contrast, the contribution of innate immunity has only received attention in recent years. Toll-like receptors (TLRs) sense pathogen-associated molecule patterns and activate antiviral mechanisms, including intracellular antiviral pathways and the production of antiviral effector interferons (IFNs) and pro-inflammatory cytokines. Experimental results from in vitro and in vivo models have demonstrated that TLRs mediate the activation of cellular signaling pathways and the production of antiviral cytokines, resulting in a suppression of HBV replication. However, HBV infection is associated with downregulation of TLR expression on host cells and blockade of the activation of downstream signaling pathways. In primary HBV infection, TLRs may slow down HBV infection, but contribute only indirectly to viral clearance. Importantly, TLRs may modulate HBV-specific T- and B-cell responses in vivo, which are essential for the termination of HBV infection. Thus, TLR agonists are promising candidates to act as immunomodulators for the treatment of chronic HBV infection. Antiviral treatment may recover TLR expression and function in chronic HBV infection and may increase the efficacy of therapeutic approaches based on TLR activation. A combined therapeutic strategy with antiviral treatment and TLR activation could facilitate the restoration of HBV-specific immune responses and thereby, achieve viral clearance in chronically infected HBV patients.