Status of vaccine research and development for Clostridium difficile.

Clostridium difficile associated disease is fundamentally associated with dysbiosis of the gut microbiome as a consequence of antibiotic use. This is because this sporulating, obligate anaerobe germinates and proliferates rapidly in the dysbiotic gut, which is an indirect consequence of their use. During its growth, C. difficile produces two toxins, toxin A (TcdA) and toxin B (TcdB), which are responsible for the majority of clinical symptoms associated with the disease. Three parenterally delivered vaccines, based on detoxified or recombinant forms of these toxins, have undergone or are undergoing clinical trials. Each offers the opportunity to generate high titres of toxin neutralising antibodies. Whilst these data suggest these vaccines may reduce primary symptomatic disease, they do not in their current form reduce the capacity of the organism to persist and shed from the vaccinated host. The current progress of vaccine development is considered with advantages and limitations of each highlighted. In addition, several alternative approaches are described that seek to limit C. difficile germination, colonisation and persistence. It may yet prove that the most effective treatments to limit infection, disease and spread of the organism will require a combination of therapeutic approaches. The potential use and efficacy of these vaccines in low and middle income countries will be depend on the development of a cost effective vaccine and greater understanding of the distribution and extent of disease in these countries.

Biological and genetic characterization of TnphoA mutants of Salmonella typhimurium TML in the context of gastroenteritis.

TnphoA transposon insertion mutants of phoN-negative derivatives of Salmonella typhimurium TML (of human gastroenteritic origin) were selected by growing mutagenized recipient bacteria under a variety of growth conditions. Ninety-seven individual mutants, which expressed alkaline phosphatase, were collected and tested for their ability to invade HEp-2 cells. Seven smooth mutants had a reduced ability to invade HEp-2 cells, and three smooth mutants were consistently more invasive than their corresponding parental strains. One rough mutant was of similar invasiveness and two were of reduced invasiveness when compared with that of parental strains. The seven smooth hypoinvasive mutants, the three smooth hyperinvasive mutants, and the three rough mutant strains were tested for their abilities to invade ileal enterocytes by the rabbit ileal invasion assay described previously (3). All smooth mutants exhibited parental levels of invasiveness. The rough mutants were hypoinvasive in the rabbit ileal invasion assay. The HEp-2 system is therefore not a good predictor of behavior in gut tissue in this model. DNA sequences flanking the transposon were determined for five mutants which were hypoinvasive in the HEp-2 cell assay. The mutations were found to be insertions in two previously identified invasion genes, invG and invH, and in a gene not normally associated with invasion, pagC. These observations lead one to be cautious in the interpretation of the biological significance of data obtained from invasion of tissue culture monolayers when extrapolated to gut tissue.

Quantitative studies of invasion of rabbit ileal mucosa by Salmonella typhimurium strains which differ in virulence in a model of gastroenteritis.

An asymmetric organ culture system in which ileal tissues, freshly removed from rabbits, can be maintained structurally and functionally for up to 4 h has been developed. The composition of the solutions used to maintain ileal tissue in vitro were as follows. The serosal surface was bathed in the World Health Organization (WHO) rehydration formulation: NaCl, 60 mM; NaHCO3, 30 mM; KCl, 20 mM; and glucose, 111 mM. The mucosal surface was bathed in the same solution with two important changes: all the sodium was replaced by choline, which is not absorbed, and tissue culture medium (consisting of commercial minimal essential medium to which was added fetal calf serum and glutamine to final concentrations of 10% [vol/vol] and 2.0 mM, respectively) was added to the choline-containing medium to a final concentration of 10% (vol/vol). The initial invasiveness (first 2 h) of seven strains of Salmonella typhimurium differing in virulence (defined in terms of clinical origin or the ability to induce fluid loss in monkeys or rabbit ileal loops) was assessed quantitatively in an in vitro invasion assay with the organ culture system. The virulent strains (TML, W118, and WAKE) were found to be about 25- to 100-fold more invasive than the avirulent strains (SL1027, M206, LT7, and Thax-1). Thus, a clear correlation between initial mucosal invasion and virulence of S. typhimurium in a model which is relevant to human gastroenteritis was established. This is the first time, to our knowledge, that quantitative studies of invasiveness have been carried out in vitro on freshly isolated functioning gut.

Invasion of HEp-2 cells by strains of Salmonella typhimurium of different virulence in relation to gastroenteritis.

Experiments to measure the invasiveness of seven strains of Salmonella typhimurium for HEp-2 cells showed that high inocula (100 bacteria/HEp-2 cell), as used by most workers to synchronise events and to increase the number of bacteria which invade, resulted in recovery of significantly less than 1% of the original inoculum after treatment with gentamicin to kill extracellular bacteria. Also, the cell culture medium became acidic, and microscopic examination of Giemsa-stained monolayers immediately following gentamicin treatment revealed high concentrations of bacteria associated with the cells. Moreover, with bacterium-cell interaction beyond 2 h, many HEp-2 cells became rounded, especially with virulent strains W118 and TML. Thus, the biological significance of the quantitative data was uncertain. The fall in pH and the rounding of HEp-2 cells were prevented by the use of a low (1:1) bacterium: cell ratio; but the recovery of bacteria after treatment with gentamicin was still lower than expected by microscopic examination. After treatment of cells with Triton X-100 to release bacteria, many remained bound to residual cell nuclei. Additional treatment with a rubber policeman, and vigorous pipetting to disperse aggregates of bacteria and cell debris, increased the recovery to c. 10% of the initial inoculum after interaction for 2 h, and 30-80% after 4 h, depending on the strain and experimental conditions. The pattern of invasiveness, but not the absolute count, was highly reproducible on different days and in different hands. However, after interaction exceeding 2 h, the distribution of bacteria was uneven, many cells having no associated organisms, others showing microcolonies.(ABSTRACT TRUNCATED AT 250 WORDS)