Gill Douce

Emergence and global spread of epidemic healthcare-associated Clostridium difficile

Epidemic C. difficile (027/BI/NAP1) has rapidly emerged in the past decade as the leading cause of antibiotic-associated diarrhea worldwide. However, the key events in evolutionary history leading to its emergence and the subsequent patterns of global spread remain unknown. Here, we define the global population structure of C. difficile 027/BI/NAP1 using whole-genome sequencing and phylogenetic analysis. We show that two distinct epidemic lineages, FQR1 and FQR2, not one as previously thought, emerged in North America within a relatively short period after acquiring the same fluoroquinolone resistance–conferring mutation and a highly related conjugative transposon. The two epidemic lineages showed distinct patterns of global spread, and the FQR2 lineage spread more widely, leading to healthcare-associated outbreaks in the UK, continental Europe and Australia. Our analysis identifies key genetic changes linked to the rapid transcontinental dissemination of epidemic C. difficile 027/BI/NAP1 and highlights the routes by which it spreads through the global healthcare system.

Construction and Immunological Characterization of a Novel Nontoxic Protective Pneumolysin Mutant for Use in Future Pneumococcal Vaccines

ABSTRACT Pneumolysin, the pore-forming toxin produced by Streptococcus pneumoniae, may have an application as an immunogenic carrier protein in future pneumococcal conjugate vaccines. Most of the 90 S. pneumoniae serotypes identified produce pneumolysin; therefore, this protein may confer non-serotype-specific protection against pneumococcal infections such as pneumonia, meningitis, and otitis media. However, as pneumolysin is highly toxic, a nontoxic form of pneumolysin would be a more desirable starting point in terms of vaccine production. Previous pneumolysin mutants have reduced activity but retain residual toxicity. We have found a single amino acid deletion that blocks pore formation, resulting in a form of pneumolysin that is unable to form large oligomeric ring structures. This mutant is nontoxic at concentrations greater than 1,000 times that of the native toxin. We have demonstrated that this mutant is as immunogenic as native pneumolysin without the associated effects such as production of the inflammatory mediators interleukin-6 and cytokine-induced neutrophil chemoattractant KC, damage to lung integrity, and hypothermia in mice. Vaccination with this mutant protects mice from challenge with S. pneumoniae. Incorporation of this mutant pneumolysin into current pneumococcal vaccines may increase their efficacy.

Distinctive profiles of infection and pathology in hamsters infected with Clostridium difficile strains 630 and B1.

ABSTRACT Currently, the Golden Syrian hamster is widely considered an important model of Clostridium difficile disease, as oral infection of this animal pretreated with antibiotics reproduces many of the symptoms observed in humans. Two C. difficile strains, B1 and 630, showed significant differences in the progression and severity of disease in this model. B1-infected hamsters exhibited more severe pathology and a shorter time to death than hamsters infected with 630. Histological changes in the gut did not correlate with absolute numbers of C. difficile bacteria, but there were clear differences in the distribution of bacteria within gut tissues. Light, scanning, and transmission electron microscopy revealed high numbers of B1 bacteria at the mucosal surface of the tissue, whereas 630 bacteria were more frequently associated with the crypt regions. Both B1 and 630 bacteria were frequently observed within polymorphonuclear leukocytes, although, interestingly, a space frequently separated B1 bacteria from the phagosome wall, a phenomenon not observed with 630. However, pilus-like structures were detected on 630 located in the crypts of the gut tissue. Furthermore, B1 bacteria, but not 630 bacteria, were found within nonphagocytic cells, including enterocytes and muscle cells.

Transmission of IgA and IgG monoclonal antibodies to mucosal fluids following intranasal or parenteral delivery

Background: The efficacy by which passive antibodies can reach the lungs could be important for the outcome of immunotherapy of respiratory pulmonary infections. We examined how transmission to a number of mucosal sites is affected by the route of inoculation. Methods: Transmission of newly raised IgA class Mabs against mycobacterial surface antigens to saliva, lung or vaginal lavage, bile and serum of BALB/c mice was compared with existing IgG Mabs. ELISA was used for testing body fluids obtained 1–24 h after intranasal or intravenous inoculation and 1–7 days following back-pack tumour growth of hybridomas. Results: Intranasal inoculation resulted in a rapid rise and high levels of both IgA and IgG class Mabs in lung lavage. In contrast, following intravenous Mab injection or back-pack tumour growth of hybridoma cells, effective lung transmission was observed for the IgG1 and IgG2b MAbs, but not for the IgA Mabs. The secretory component was acquired by the transmitted IgA MAbs in the mucosal fluids, but not in the serum. Nevertheless, the time course of mucosal IgA antibody levels was similar to that of the tested IgG Mabs. Furthermore, the relative proportion of transmission to saliva and bile varied between individual Mabs indicating a role of tissue-specific, immunoglobulin class-unrelated mechanisms. Conclusions: Intranasal, rather than parenteral inoculation of mice is required for the efficient delivery of IgA antibodies against respiratory pulmonary pathogens. Interestingly, IgA-secretory component complexing of intranasally applied Mabs did not significantly influence their persistence in the lungs.

Genetic Detoxification of Bacterial Toxins: A New Approach to Vaccine Development

Chemically detoxified bacterial toxins (toxoids) have been successfully used as vaccines for the prevention of many bacterial infectious diseases. Today, nontoxic derivatives of bacterial toxins can be obtained by mutagenesis of the toxin genes. These genetically inactivated toxins are superior to the classical toxoids both in safety and in immunogenicity and therefore they should replace the old toxoids in the existing vaccines. In addition, they represent a novel class of immunogens with unique properties, some of which may be used for innovative approaches to vaccination.

New vaccines against bacterial toxins.

Chemically detoxified bacterial toxins are the main or the sole components of several vaccines such as diphtheria, tetanus, pertussis, and cholera. Other toxins are good candidates for novel vaccines, such as the vacuolating cytotoxin of Helicobacter pylori 1. Recently, genetic manipulation of the toxin’s genes has been used to obtain molecules that are already non toxic and do not require chemical treatment for detoxification. This approach has been successful for pertussis2, diphtheria3, cholera and LT toxins4. Preclinical and clinical studies have shown that these molecules have several advantages over the conventional chemically detoxified vaccines. Generally, they are much more immunogenic and induce an immune response, that recognizes better the native toxin molecules. Non toxic derivatives of cholera toxin and LT, in addition to being good candidates for antidiarrheal vaccines, are also mucosal adjuvants5. Therefore, genetically detoxified bacterial toxins not only are good candidates to improve existing vaccines, but they represent new tools for the development of innovative vaccines targeted to the mucosal system.