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Effects of antibiotic administration on the composition of the human intestinal microbiota, the prevalence of antibiotic resistant bacteria and the development of antibiotic-associated diarrhoea

Final Report Summary - AMIDIM (Effects of antibiotic administration on the composition of the human intestinal microbiota ? and the development of antibiotic-associated diarrhoea)

EU Marie Curie Individual Fellowship PIEF-GA-2008-220815 [AMIDIM]

The project AMIDIM was initiated to address the impact of long-term antibiotic treatment on human health by establishing effects on the composition of the human gut microbiota, on the spread of bacterial antibiotic resistance genes, and on the colonisation by virulent drug resistant opportunistic pathogenic bacteria. Acne patients, who are frequently prescribed long-term oral antibiotic treatment (usually derivatives of the broad-spectrum antibiotic tetracycline) for three to six months, were a suitable study group of 'health individuals undergoing extensive antibiotic therapy'. Since acne persists from puberty until the early twenties, and often longer, most sufferers undergo several courses of treatment which exerts considerable selection pressure on bacterial populations, both of the causative bacterium Propionibacterium acnes and the commensal microbiota. Furthermore, since tetracyclines are incompletely absorbed in the upper GIT tract, high concentrations reach the colon and perturbations in the normal intestinal microbiota can be apparent within two days.

The research was targeted through two research objectives:

Objective 1: Monitoring the diversity of the colonic microbial community in order to predict the effect of long-term antibiotic treatment, and identify any links to the onset of diarrhoea.

Overall changes in the numbers of bacteria resistant to specific tetracycline derivatives were assessed initially by culturing bacteria anaerobically on nutrient-rich media, plus or minus antibiotics. More bacteria were able to grow in the presence of antibiotics during and immediately after antibiotic therapy than prior to, or four weeks later. Variations in the dynamics of the colonic microbial community population were monitored qualitatively using density gradient gel electrophoresis (DGGE). Antibiotic treatment altered the composition of the microbiota, compared to the pre-treatment control samples, and the composition had not returned to baseline even one month after the cessation of antibiotic treatment. However, the inter-individual variation was a greater discriminatory factor in the microbial profile than the antibiotic, and there were no consistencies in the changes in the bacterial profiles. None of the patients reported any symptoms of diarrhoea during the study, and no C. difficile isolates were detected at any point, in any individual, using selective plating. This indicated that there were either no C. difficile bacteria present, or that numbers were below detection limits. Establishing the levels of residual doxycycline in faecal samples indicated that although levels were high immediately after therapy ceased, doxycycline was virtually undetectable one month later, even though the microbiota had not fully recovered by this time.

Objective 2: Global gene expression analysis of C. difficile in response to iron availability and antibiotics utilising DNA microarrays based on the genome sequence of C. difficile.

C. difficile was able to utilise various compounds as sources of iron for growth, even in the presence of iron chelators, including both inorganic and organic iron sources. Tetracyclines are known to have a strong iron-chelating activity and we found that the derivatives doxycycline and minocycline were more effective than tetracycline itself. A genomic microarray based on the genome sequence of C. difficile strain 630 was used to monitor differential gene expression in response to various concentrations of iron in the presence of the iron chelating agents DPP and minocycline. The microarray experiments were carried out using mRNA purified from bacterial cultures grown to mid-exponential phase, using cells exposed to mixtures of the inhibitor and the iron sources at various concentrations for various times.