Clostridium difficile is considered the leading cause of antibiotic‐associated disease worldwide. In the past decade, a large number of studies have focused on identifying the main sources of contamination in order to elucidate the complete life cycle of the infection. Hospitals, animals and retail foods have been considered as potential vectors. However, the prevalence of C. difficile in these types of samples was found to be rather low, suggesting that other contamination routes must exist. This study explores the presence of C. difficile in the natural environment and the seasonal dynamics of the bacterium. C. difficile was isolated from a total of 45 samples out of 112 collected (40.2%) on 56 sampling points. A total of 17 points were positive only during the winter sampling (30.4%), 10 were positive only during the summer sampling (17.9%) and 9 sampling points (16.1%) were positive in both summer sampling and winter sampling. Spore counts in soil samples ranged between 50 and 250 cfu/g for 24.4% of the positive samples, with the highest concentrations detected in samples collected in the forest during winter campaign (200–250 cfu/g). A total of 17 different PCR ribotypes were identified, and 15 of them had the genes coding for toxins A and B. Most of those ribotypes had not previously been found or had been isolated only sporadically (<1% of samples) from hospitals in Belgium. Regarding antimicrobial susceptibility, most of the resistant strains were found during the summer campaign. These findings bear out that C. difficile is present in the natural environment, where the bacterium undergoes seasonal variations.

248 21 Main text: 7082 22 23 24 25 26 2 ABSTRACT 27 HIV-2 is the second causative agent of AIDS and is commonly considered as an attenuated form 28 of retroviral infection. Most of HIV-2-infected individuals display a slow-progressing disease, 29 lower viral loads and a stronger immunological control of viral infection as compared with HIV-30

Background: Mycobacterium abscessus is notorious for being intrinsically resistant to most antibiotics. Antibiotic efflux is one of the mechanisms used by M. abscessus to pump out antibiotics from their cells. Inhibiting efflux pumps (EPs) can be an attractive strategy to enhance the activity of drugs. The objective of this study is to determine the activity of EP inhibitors (EPIs) to enhance the efficacy of the new drug bedaquiline against M. abscessus clinical isolates. Methods: A total of 31 phenotypically and genotypically identified M. abscessus subsp. abscessus, M. abscesss subsp. massiliense, and M. abscessus subsp. bolletii clinical isolates were studied. The contribution of EPs was determined by investigating the minimum inhibitory concentration (MIC) levels of bedaquiline reduction in the absence and presence of EPIs verapamil and reserpine using the resazurin microtiter assay. Results: The observed bedaquiline MIC reduction by verapamil was observed in 100% isolates and by reserpine in 54.8% isolates. Bedaquiline MIC was 4–32-fold using verapamil with M. abscessus subsp. bolletii showing the highest fold change and between 2- and 4-fold using reserpine. Conclusions: The results obtained in this study confirm that bedaquiline MIC decreased in the presence of EPIs verapamil and reserpine in clinical isolates of M. abscessus. Verapamil was the most effective EPI. As shown in previous studies, verapamil may have clinical potential as adjunctive therapy to enhance the effect of bedaquiline.

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