Complete genome sequence of the Clostridium difficile laboratory strain 630Δerm reveals differences from strain 630, including translocation of the mobile element CTn5

Background

Clostridium difficile strain 630Δerm is a spontaneous erythromycin sensitive derivative of the reference strain 630 obtained by serial passaging in antibiotic-free media. It is widely used as a defined and tractable C. difficile strain. Though largely similar to the ancestral strain, it demonstrates phenotypic differences that might be the result of underlying genetic changes. Here, we performed a de novo assembly based on single-molecule real-time sequencing and an analysis of major methylation patterns.

Results

In addition to single nucleotide polymorphisms and various indels, we found that the mobile element CTn5 is present in the gene encoding the methyltransferase rumA rather than adhesin CD1844 where it is located in the reference strain.

Conclusions

Together, the genetic features identified in this study may help to explain at least part of the phenotypic differences. The annotated genome sequence of this lab strain, including the first analysis of major methylation patterns, will be a valuable resource for genetic research on C. difficile.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1252-7) contains supplementary material, which is available to authorized users.     

Are Rapid Immunoassays for in vivo Detection of Toxin A Sufficient for Diagnostic Purposes of Clostridium difficile -Associated Diseases?

One hundred and fifty-five stool specimens of patients suspected for Clostridium difficile -associated diarrhoea, colitis or pseudomembranous colitis (PMC) were investigated. All patients were pre-treated with antibiotics, suffered from watery diarrhoea and abdominal pain and were hospitalized in different hospital units. Units varied from departments of surgery, internal medicine, intensive care, paediatry, dermatology, orthopaedy to gastroenterology. Fifty C. difficile strains were isolated from the faecal samples. Clostridium difficile toxin detection was done directly in the stool samples, and also in cultured C. difficile strains (in vivo and in vitro, respectively). We observed clear differences between in vivo and in vitro toxin A detection by using commercial rapid immuno-enzymatic tests: from 25 in vivo toxin A-negative samples, 17 were positive in vitro. This observation suggests that culturing of C. difficile on selective medium is mandatory for adequate toxin detection and necessary for confirming the presence of toxin-producing C. difficile. This is especially important among patients with clinical symptoms and history of pretreatment with antibiotics and when in vivo toxin A detection is negative. It was established that toxin gene detection by PCR is optimal and PCR results were concordant with results of other in vitro assays. Genotyping by using AP-PCR and PCR ribotyping showed heterogeneity among the toxigenic C. difficile strains cultured from in vivo toxin A-negative stool samples.     

Methanogen Colonisation Does Not Significantly Alter Acetogen Diversity in Lambs Isolated 17?h After Birth and Raised Aseptically

Reductive acetogenesis is not competitive with methanogenesis in adult ruminants, whereas acetogenic bacteria are the dominant hydrogenotrophs in the early rumen microbiota. The ecology of hydrogenotrophs in the developing rumen was investigated using young lambs, raised in sterile isolators, and conventional adult sheep. Two lambs were born naturally, left with their dams for 17?h and then placed into a sterile isolator and reared aseptically. They were inoculated with cellulolytic bacteria and later with Methanobrevibacter sp. 87.7 to investigate the effect of methanogen establishment on the rumen acetogen population since they lacked cultivable representatives of methanogens. Putative acetogens were investigated by acetyl-CoA synthase and formyltetrahydrofolate synthetase gene analysis and methanogens by methyl coenzyme reductase A gene analysis. Unexpectedly, a low abundant but diverse population of methanogens (predominantly Methanobrevibacter spp.) was identified in isolated lambs pre-inoculation with Mbb. sp 87.7, which was similar to the community structure in conventional sheep. In contrast, potential acetogen diversity in isolated lambs and conventional sheep was different. Potential acetogens affiliated between the Lachnospiraceae and Clostridiaceae in conventional sheep and with the Blautia genus and the Lachnospiraceae in isolated lambs. The establishment of Mbb. sp. 87.7 (1,000-fold increase in methanogens) did not substantially affect acetogen diversity.     

Prevalence of Clostridium spp. and Clostridium difficile in children with acute diarrhea in São Paulo city,Brazil

Species of Clostridium are widely distributed in the environment, inhabiting both human and animal gastrointestinal tracts. Clostridium difficile is an important pathogen associated with outbreaks of pseudomembranous colitis and other intestinal disorders, such as diarrhea. In this study, the prevalence of Clostridium spp. and C. difficile, from hospitalized children with acute diarrhea, was examined. These children were admitted to 3 different hospitals for over 12 months. Eighteen (20%) and 19 (21%) stool specimens from children with (90) and without (91) diarrhea respectively, were positive to clostridia. Only 10 C. difficile strains were detected in 5.5% of the stool samples of children with diarrhea. None healthy children (without diarrhea) harbored C. difficile. From these 10 C. difficile, 9 were considered as toxigenic and genotyped as tcdA+/tcdB+ or tcdA-/tcdB+, and 1 strain as nontoxigenic (tcdA-/tdcB-). They were detected by the citotoxicity on VERO cells and by the multiplex-polymerase chain reaction. Thirty clinical fecal extracts produced minor alterations on VERO cells. The presence of C. difficile as a probable agent of acute diarrhea is suggested in several countries, but in this study, the presence of these organisms was not significant. More studies will be necessary to evaluate the role of clostridia or C. difficile in diarrhoeal processes in children.     

Why Does Clostridium acetireducens Not Use Interspecies Hydrogen Transfer for Growth on Leucine?

Clostridium acetireducens is the first reported anaerobic bacterium that is dependent on acetate as an electron acceptor for growth on branched-chain amino acids and alanine. The fermentation pathway of leucine and its deamination product α-ketoisocaproate were studied in this organism. Addition of Methanobacterium formicicum to pure cultures of C. acetireducens stimulated the degradation of α-ketoisocaproate but not the degradation of leucine, indicating that the electrons produced during the oxidative deamination of leucine were not transferred to hydrogen. This conclusion is supported by the observed low NAD(P)H ferredoxin reductase activity. Not only acetate but also crotonate proved to be an appropriate electron sink for the regeneration of NAD(P)⁺ in this bacterium. Interestingly, C. acetireducens was shown to form polyhydroxybutyrate during growth on leucine plus acetate. Received: 2 December 1996 / Accepted: 4 March 1997     

Thrombin generation is increased in patients with Clostridium difficile colitis – a pilot study

Background: There are only limited data in the literature on the thrombotic risk of patients with Clostridium difficile (CD) colitis, although this disease is widespread throughout the world.

Objective: The aim of this study was to explore thrombin generation in these patients – the best way to evaluate their coagulation.

Methods: A prospective observational study was conducted during 15 months on hospitalized patients with CD colitis. Thrombin generation was performed in platelet-poor plasma using a Ceveron^® alpha analyzer and was compared with a group of volunteer control subjects.

Results: Thirty-three patients and 51 control subjects were enrolled in the study. Two biomarkers – mean velocity index and peak thrombin – were significantly higher in patient group, compared to the control subjects (p?=?0.010, respectively, p?=?0.0395). This pattern of thrombin generation suggests that patients with CD colitis without septic shock have a potential thrombotic risk. The mean velocity index significantly correlated with the estimated related risk of death according to the Charlson age-comorbidity index.

Conclusions: The higher values of thrombin generation suggest that CD colitis increases the thromboembolic risk. The pattern of thrombin generation could identify patients with particularly higher thromboembolic risk. They are potential candidates for thromboprophylaxis strategies and monitorization.     

Auranofin disrupts selenium metabolism in Clostridium difficile by forming a stable Au–Se adduct

Clostridium difficile is a nosocomial pathogen whose incidence and importance are on the rise. Previous work in our laboratory characterized the central role of selenoenzyme-dependent Stickland reactions in C. difficile metabolism. In this work we have identified, using mass spectrometry, a stable complex formed upon reaction of auranofin (a gold-containing drug) with selenide in vitro. X-ray absorption spectroscopy supports the structure that we proposed on the basis of mass-spectrometric data. Auranofin potently inhibits the growth of C. difficile but does not similarly affect other clostridia that do not utilize selenoproteins to obtain energy. Moreover, auranofin inhibits the incorporation of radioisotope selenium (⁷⁵Se) in selenoproteins in both Escherichia coli, the prokaryotic model for selenoprotein synthesis, and C. difficile without impacting total protein synthesis. Auranofin blocks the uptake of selenium and results in the accumulation of the auranofin–selenide adduct in the culture medium. Addition of selenium in the form of selenite or l-selenocysteine to the growth medium significantly reduces the inhibitory action of auranofin on the growth of C. difficile. On the basis of these results, we propose that formation of this complex and the subsequent deficiency in available selenium for selenoprotein synthesis is the mechanism by which auranofin inhibits C. difficile growth. This study demonstrates that targeting selenium metabolism provides a new avenue for antimicrobial development against C. difficile and other selenium-dependent pathogens. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Conflict in Protected Areas: Who Says Co-Management Does Not Work?

Natural resource-related conflicts can be extremely destructive and undermine environmental protection. Since the 1990s co-management schemes, whereby the management of resources is shared by public and/or private sector stakeholders, have been a main strategy for reducing these conflicts worldwide. Despite initial high hopes, in recent years co-management has been perceived as falling short of expectations. However, systematic assessments of its role in conflict prevention or mitigation are non-existent. Interviews with 584 residents from ten protected areas in Colombia revealed that co-management can be successful in reducing conflict at grassroots level, as long as some critical enabling conditions, such as effective participation in the co-management process, are fulfilled not only on paper but also by praxis. We hope these findings will re-incentivize global efforts to make co-management work in protected areas and other common pool resource contexts, such as fisheries, agriculture, forestry and water management.     

High Cholesterol Diet Induces IL-1β Expression in Adult but Not Larval Zebrafish

Recently, it has been demonstrated that high cholesterol diet induced hypercholesterolemia and vascular lipid oxidation and accumulation in zebrafish larvae, suggesting that zebrafish is a new promising atherosclerosis model in addition to mouse models. However, up to date, there was no report regarding inflammatory cytokine expression during the lipid accumulation in zebrafish larva and adult fish. In this study, we first demonstrated the expression levels of IL-1β and TNF-α in high cholesterol diet (HCD)-fed zebrafish larvae, and found that although HCD induced vascular lipid accumulation, the cytokine expressions in the larvae were not changed by HCD. Furthermore, there was no significant difference in leukocyte accumulation in vessels between control and HCD fed group. But prolonged HCD induced IL-1β expression in spleen and liver compared to those of control zebrafish, and produced very early stage of fatty streak lesion in dorsal aorta of 19 week HCD-fed zebrafish. These results indicate that HCD induced hypercholesterolemia and atherosclerotic changes in zebrafish are very early stage, and suggest the necessity of the generation of mutant zebrafish having a disruption in a lipid metabolism-related gene leading to severe hypercholesterolemia and advanced atherosclerosis.     

Nitrogen Fertilization Does Significantly Increase Yields of Stands of Miscanthus × giganteus and Panicum virgatum in Multiyear Trials in Illinois

The C4 perennial grasses Miscanthus × giganteus and Panicum virgatum (switchgrass) are emerging bioenergy crops. They are attractive because they are productive and recycle nutrients to the overwintering belowground rhizomes, before the dry shoots are harvested. They are therefore expected to require minimal fertilizer inputs, a desirable trait for any crop. Until now, Europe has had the only long-term trials of M. × giganteus, and these have either shown no or a small effect of nitrogen fertilization, while trials of P. virgatum in the USA have shown a clear positive effect of N fertilization. This study exploited the first long-term trials of M. × giganteus in the USA, and first side-by-side comparison with P. virgatum, to test the hypothesis that N fertilization would only improve yields of the latter. A split-plot N fertility treatment (0, 67, 134, and 202 kg(N)?ha^?1) was added to >5-year-old replicated stands of the two crops at seven locations on contrasting soils in the US Midwest. Averaged across all locations, M. × giganteus yields increased significantly from 23.4 Mg ha^?1 with no N fertilization to 28.9 Mg ha^?1 (+25 %) at a N application rate of 202 kg ha^?1. P. virgatum also showed significant yield increases from 10.33 Mg ha^?1 at 0 kg(N)?ha^?1 to 13.6 Mg ha^?1 (+32 %) at 202 kg(N)?ha^?1. Both species therefore responded to N fertilization and to a similar extent. The increase per unit of added N was small compared to crops such as Zea mays and unlikely to be economically worthwhile. Nitrogen fertilization arrested most of the long-term yield decline that would otherwise have occurred in P. virgatum, but eliminated only about 40 % of the decline observed in M. × giganteus, suggesting additional causal factors for long-term yield decline in this crop. While the crops responded to nitrogen addition at some locations, they did not at others. Therefore a one-case-fits-all optimum fertilization rate cannot be prescribed.     

Endothelial Dysfunction in Tristetraprolin-deficient Mice Is Not Caused by Enhanced Tumor Necrosis Factor-α Expression

Cardiovascular events are important co-morbidities in patients with chronic inflammatory diseases like rheumatoid arthritis. Tristetraprolin (TTP) regulates pro-inflammatory processes through mRNA destabilization and therefore TTP-deficient mice (TTP^−/− mice) develop a chronic inflammation resembling human rheumatoid arthritis. We used this mouse model to evaluate molecular signaling pathways contributing to the enhanced atherosclerotic risk in chronic inflammatory diseases. In the aorta of TTP^−/− mice we observed elevated mRNA expression of known TTP targets like tumor necrosis factor-α (TNF-α) and macrophage inflammatory protein-1α, as well as of other pro-atherosclerotic mediators, like Calgranulin A, Cathepsin S, and Osteopontin. Independent of cholesterol levels TTP^−/− mice showed a significant reduction of acetylcholine-induced, nitric oxide-mediated vasorelaxation. The endothelial dysfunction in TTP^−/− mice was associated with increased levels of reactive oxygen and nitrogen species (RONS), indicating an enhanced nitric oxide inactivation by RONS in the TTP^−/− animals. The altered RONS generation correlates with increased expression of NADPH oxidase 2 (Nox2) resulting from enhanced Nox2 mRNA stability. Although TNF-α is believed to be a central mediator of inflammation-driven atherosclerosis, genetic inactivation of TNF-α neither improved endothelial function nor normalized Nox2 expression or RONS production in TTP^−/− animals. Systemic inflammation caused by TTP deficiency leads to endothelial dysfunction. This process is independent of cholesterol and not mediated by TNF-α solely. Thus, other mediators, which need to be identified, contribute to enhanced cardiovascular risk in chronic inflammatory diseases.     

A Recombination Directionality Factor Controls the Cell Type-Specific Activation of σK and the Fidelity of Spore Development in Clostridium difficile

The strict anaerobe Clostridium difficile is the most common cause of nosocomial diarrhea, and the oxygen-resistant spores that it forms have a central role in the infectious cycle. The late stages of sporulation require the mother cell regulatory protein σ^K. In Bacillus subtilis, the onset of σ^K activity requires both excision of a prophage-like element (skin^Bs) inserted in the sigK gene and proteolytical removal of an inhibitory pro-sequence. Importantly, the rearrangement is restricted to the mother cell because the skin^Bs recombinase is produced specifically in this cell. In C. difficile, σ^K lacks a pro-sequence but a skin^Cd element is present. The product of the skin^Cd gene CD1231 shares similarity with large serine recombinases. We show that CD1231 is necessary for sporulation and skin^Cd excision. However, contrary to B. subtilis, expression of CD1231 is observed in vegetative cells and in both sporangial compartments. Nevertheless, we show that skin^Cd excision is under the control of mother cell regulatory proteins σ^E and SpoIIID. We then demonstrate that σ^E and SpoIIID control the expression of the skin^Cd gene CD1234, and that this gene is required for sporulation and skin^Cd excision. CD1231 and CD1234 appear to interact and both proteins are required for skin^Cd excision while only CD1231 is necessary for skin^Cd integration. Thus, CD1234 is a recombination directionality factor that delays and restricts skin^Cd excision to the terminal mother cell. Finally, while the skin^Cd element is not essential for sporulation, deletion of skin^Cd results in premature activity of σ^K and in spores with altered surface layers. Thus, skin^Cd excision is a key element controlling the onset of σ^K activity and the fidelity of spore development.     

Does Virus-Induced Lysis Contribute Significantly to Bacterial Mortality in the Oxygenated Sediment Layer of Shallow Oxbow Lakes?

Despite the recognition that viruses are ubiquitous components of aquatic ecosystems, the number of studies on viral abundance and the ecological role of viruses in sediments is scarce. In this investigation, the interactions between viruses and bacteria were studied in the oxygenated silty sediment layer of a mesotrophic oxbow lake. A long-term study (13 months) and a diel study revealed that viruses are a numerically important and dynamic component of the microbial community. The abundance and decay rates ranged from 4.3 × 10⁹ to 7.2 × 10⁹ particles ml of wet sediment⁻¹ and from undetectable to 22.2 × 10⁷ particles ml⁻¹ h⁻¹, respectively, and on average the values were 2 orders of magnitude higher than the values for the overlying water. In contrast to our expectations, viruses did not contribute significantly to the bacterial mortality in the sediment, since on average only 6% (range, 0 to 25%) of the bacterial secondary production was controlled by viruses. The low impact of viruses on the bacterial community may be associated with the quantitatively low viral burden that benthic bacteria have to cope with compared to the viral burden with which bacterial assemblages in the water column are confronted. The virus-to-bacterium ratio of the sediment varied between 0.9 and 3.2, compared to a range of 5.0 to 12.4 obtained for the water column. We speculate that despite high numbers of potential hosts, the possibility of encountering a host cell is limited by the physical conditions in the sediment, which is therefore not a favorable environment for viral proliferation. Our data suggest that viruses do not play an important role in the processing and transfer of bacterial carbon in the oxygenated sediment layer of the environment investigated.