Importance of Toxin A, Toxin B, and CDT in virulence of an epidemic Clostridium difficile strain

Clostridium difficile infection is the main cause of healthcare-acquired diarrhea in the developed world. In addition to the main virulence factors toxin A and B, epidemic, PCR Ribotype 027 strains, such as R20291, produce a third toxin, CDT. To develop effective medical countermeasures, it is important to understand the importance of each toxin. Accordingly, we created all possible combinations of isogenic toxin mutants of R20291 and assessed their virulence. We demonstrated that either toxin A or toxin B alone can cause fulminant disease in the hamster infection model and present tantalizing data that C. difficile toxin may also contribute to virulence

What's a SNP between friends: the influence of single nucleotide polymorphisms on virulence and phenotypes of Clostridium difficile strain 630 and derivatives

Clostridium difficile is a major cause of antibiotic induced diarrhoea worldwide, responsible for significant annual mortalities and represents a considerable economic burden on healthcare systems. The two main C. difficile virulence factors are toxins A and B. Isogenic toxin B mutants of two independently isolated erythromycin-sensitive derivatives (630E and 630Δerm) of strain 630 were previously shown to exhibit substantively different phenotypes. Compared to 630, strain 630E and its progeny grow slower, achieve lower final cell densities, exhibit a reduced capacity for spore-formation, produce lower levels of toxin and are less virulent in the hamster infection model. By the same measures, strain 630Δerm and its derivatives more closely mirror the behaviour of 630. Genome sequencing revealed that 630Δerm had acquired seven unique Single Nucleotide Polymorphisms (SNPs) compared to 630 and 630E, while 630E had nine SNPs and a DNA inversion not found in the other two strains. The relatively large number of mutations meant that the identification of those responsible for the altered properties of 630E was not possible, despite the restoration of three mutations to wildtype by allelic exchange and comparative RNAseq analysis of all three strains. The latter analysis revealed large differences in gene expression between the three strains, explaining in part why no single SNP could restore the phenotypic differences. Our findings suggest that strain 630Δerm should be favoured over 630E as a surrogate for 630 in genetic-based studies. They also underline the importance of effective strain curation and the need to genome re-sequence master seed banks wherever possible

Characterization of the impact of rpoB mutations on the in vitro and in vivo competitive fitness of Clostridium difficile and susceptibility to fidaxomicin

ObjectivesTo establish the role of specific, non-synonymous SNPs in the RNA polymerase β subunit (rpoB) gene in reducing the susceptibility of Clostridium difficile to fidaxomicin and to explore the potential in vivo significance of rpoB mutant strains.MethodsAllelic exchange was used to introduce three different SNPs into the rpoB gene of an erythromycin-resistant derivative (CRG20291) of C. difficile R20291. The genome sequences of the created mutants were determined and each mutant analysed with respect to growth and sporulation rates, toxin A/B production and cytotoxicity against Vero cells, and in competition assays. Their comparative virulence and colonization ability was also assessed in a hamster infection model.ResultsThe MIC of fidaxomicin displayed by three mutants CRG20291-TA, CRG20291-TG and CRG20291-GT was substantially increased (>32, 8 and 2 mg/L, respectively) relative to that of the parent strain (0.25 mg/L). Genome sequencing established that the intended mutagenic substitutions in rpoB were the only changes present. Relative to CRG20291, all mutants had attenuated growth, were outcompeted by the parental strain, had lower sporulation and toxin A/B production capacities, and displayed diminished cytotoxicity. In a hamster model, virulence of all three mutants was significantly reduced compared with the progenitor strain, whereas the degree of caecum colonization was unaltered.ConclusionsOur study demonstrates that particular SNPs in rpoB lead to reduced fidaxomicin susceptibility. These mutations were associated with a fitness cost in vitro and reduced virulence in vivo

Altered stress responses in adults born by Caesarean section

peer-reviewedBirth by Caesarean-section (C-section), which increases the risk for metabolic and immune disorders, disrupts the normal initial microbial colonisation of the gut, in addition to preventing early priming of the stress and immune-systems.. Animal studies have shown there are enduring psychological processes in C-section born mice. However, the long-term impact of microbiota-gut-brain axis disruptions due to birth by C-section on psychological processes in humans is unknown. Forty age matched healthy young male university students born vaginally and 36 C-section delivered male students were recruited. Participants underwent an acute stressor, the Trier social stress test (TSST), during a term-time study visit. A subset of participants also completed a study visit during the university exam period, representing a naturalistic stressor. Participants completed a battery of cognitive tests and self-report measures assessing mood, anxiety, and perceived stress. Saliva, blood, and stool samples were collected for analysis of cortisol, peripheral immune profile, and the gut microbiota. Young adults born by C-section exhibit increased psychological vulnerability to acute stress and a prolonged period of exam-related stress. They did not exhibit an altered salivary cortisol awakening response to the TSST, but their measures of positive affect were significantly lower than controls throughout the procedure. Both C-section and vaginally-delivered participants performed equally well on cognitive assessments. Most of the initial effects of delivery mode on the gut microbiome did not persist into adulthood as the gut microbiota profile showed modest changes in composition in adult vaginally-delivered and C-sectioned delivered subjects. From an immune perspective, concentrations of IL-1β and 1L-10 were higher in C-section participants. These data confirm that there is a potential enduring effect of delivery mode on the psychological responses to acute stress during early adulthood. The mental health implications of these observations require further study regarding policies on C-section use

Multiple-Locus Variable Number Tandem Repeat Analysis of Staphylococcus Aureus: Comparison with Pulsed-Field Gel Electrophoresis and spa-Typing

(MRSA) is required to study the routes and rates of transmission of this pathogen. Currently available typing techniques are either resource-intensive or have limited discriminatory ability. Multiple-locus variable number tandem repeat analysis (MLVA) may provide an alternative high throughput molecular typing tool with high epidemiological resolution.-sequence typing and PFGE, at the MLVA complex level with group separation values of 95.1% and 89.2%. MLVA could not discriminate between pig-related MRSA strains isolated from humans and pigs, corroborating the high degree of relationship. MLVA was also superior in the grouping of MRSA isolates previously assigned to temporal-spatial clusters with indistinguishable SpaTypes, demonstrating its enhanced epidemiological usefulness. that yields discrete and unambiguous data that can be used to assign biological meaningful genotypes and complexes and can be used for interlaboratory comparisons in network accessible databases. Results suggest that MLVA offsets the disadvantages of other high discriminatory typing approaches and represents a promising tool for hospital, national and international molecular epidemiology

Rhesus Macaques (Macaca mulatta) Are Natural Hosts of Specific Staphylococcus aureus Lineages

Currently, there is no animal model known that mimics natural nasal colonization by Staphylococcus aureus in humans. We investigated whether rhesus macaques are natural nasal carriers of S. aureus. Nasal swabs were taken from 731 macaques. S. aureus isolates were typed by pulsed-field gel electrophoresis (PFGE), spa repeat sequencing and multi-locus sequence typing (MLST), and compared with human strains. Furthermore, the isolates were characterized by several PCRs. Thirty-nine percent of 731 macaques were positive for S. aureus. In general, the macaque S. aureus isolates differed from human strains as they formed separate PFGE clusters, 50% of the isolates were untypeable by agr genotyping, 17 new spa types were identified, which all belonged to new sequence types (STs). Furthermore, 66% of macaque isolates were negative for all superantigen genes. To determine S. aureus nasal colonization, three nasal swabs from 48 duo-housed macaques were taken during a 5 month period. In addition, sera were analyzed for immunoglobulin G and A levels directed against 40 staphylococcal proteins using a bead-based flow cytometry technique. Nineteen percent of the animals were negative for S. aureus, and 17% were three times positive. S. aureus strains were easily exchanged between macaques. The antibody response was less pronounced in macaques compared to humans, and nasal carrier status was not associated with differences in serum anti-staphylococcal antibody levels. In conclusion, rhesus macaques are natural hosts of S. aureus, carrying host-specific lineages. Our data indicate that rhesus macaques are useful as an autologous model for studying S. aureus nasal colonization and infection prevention

Mutations in Zebrafish lrp2 Result in Adult-Onset Ocular Pathogenesis That Models Myopia and Other Risk Factors for Glaucoma

The glaucomas comprise a genetically complex group of retinal neuropathies that typically occur late in life and are characterized by progressive pathology of the optic nerve head and degeneration of retinal ganglion cells. In addition to age and family history, other significant risk factors for glaucoma include elevated intraocular pressure (IOP) and myopia. The complexity of glaucoma has made it difficult to model in animals, but also challenging to identify responsible genes. We have used zebrafish to identify a genetically complex, recessive mutant that shows risk factors for glaucoma including adult onset severe myopia, elevated IOP, and progressive retinal ganglion cell pathology. Positional cloning and analysis of a non-complementing allele indicated that non-sense mutations in low density lipoprotein receptor-related protein 2 (lrp2) underlie the mutant phenotype. Lrp2, previously named Megalin, functions as an endocytic receptor for a wide-variety of bioactive molecules including Sonic hedgehog, Bone morphogenic protein 4, retinol-binding protein, vitamin D-binding protein, and apolipoprotein E, among others. Detailed phenotype analyses indicated that as lrp2 mutant fish age, many individuals—but not all—develop high IOP and severe myopia with obviously enlarged eye globes. This results in retinal stretch and prolonged stress to retinal ganglion cells, which ultimately show signs of pathogenesis. Our studies implicate altered Lrp2-mediated homeostasis as important for myopia and other risk factors for glaucoma in humans and establish a new genetic model for further study of phenotypes associated with this disease

Pleiotropic Effects of Sox2 during the Development of the Zebrafish Epithalamus

The zebrafish epithalamus is part of the diencephalon and encompasses three major components: the pineal, the parapineal and the habenular nuclei. Using sox2 knockdown, we show here that this key transcriptional regulator has pleiotropic effects during the development of these structures. Sox2 negatively regulates pineal neurogenesis. Also, Sox2 is identified as the unknown factor responsible for pineal photoreceptor prepatterning and performs this function independently of the BMP signaling. The correct levels of sox2 are critical for the functionally important asymmetrical positioning of the parapineal organ and for the migration of parapineal cells as a coherent structure. Deviations from this strict control result in defects associated with abnormal habenular laterality, which we have documented and quantified in sox2 morphants