Characterizing Antimicrobial Resistance in Clinically Relevant Bacteria Isolated at the Human/Animal/Environment Interface Using Whole-Genome Sequencing in Austria.

Antimicrobial resistance (AMR) is a public health issue attributed to the misuse of antibiotics in human and veterinary medicine. Since AMR surveillance requires a One Health approach, we sampled nine interconnected compartments at a hydrological open-air lab (HOAL) in Austria to obtain six bacterial species included in the WHO priority list of antibiotic-resistant bacteria (ARB). Whole genome sequencing-based typing included core genome multilocus sequence typing (cgMLST). Genetic and phenotypic characterization of AMR was performed for all isolates. Eighty-nine clinically-relevant bacteria were obtained from eight compartments including 49 E. coli, 27 E. faecalis, 7 K. pneumoniae and 6 E. faecium. Clusters of isolates from the same species obtained in different sample collection dates were detected. Of the isolates, 29.2% were resistant to at least one antimicrobial. E. coli and E. faecalis isolates from different compartments had acquired antimicrobial resistance genes (ARGs) associated with veterinary drugs such as aminoglycosides and tetracyclines, some of which were carried in conjugative and mobilizable plasmids. Three multidrug resistant (MDR) E. coli isolates were found in samples from field drainage and wastewater. Early detection of ARGs and ARB in natural and farm-related environments can identify hotspots of AMR and help prevent its emergence and dissemination along the food/feed chain.

Removal of extracellular free DNA and antibiotic resistance genes from water and wastewater by membranes ranging from microfiltration to reverse osmosis.

Free DNA in the effluent from wastewater treatment plants has recently been observed to contain antibiotic resistance genes (ARGs), which may contribute to the spread of antibiotic resistance via horizontal gene transfer in the receiving environment. Technical membrane systems applied in wastewater and drinking water treatment are situated at central nodes between the environmental and human related aspects of the "One Health" approach and are considered as effective barriers for antibiotic resistant bacteria. However, they are not evaluated for their permeability for ARGs encoded in free DNA, which may result, for example, from the release of free DNA after bacterial die-off during particular treatment processes. This study examined the potential and principle mechanisms for the removal of free DNA containing ARGs by technical membrane filtration. Ten different membranes, varied by the charge (neutral and negative) and the molecular weight cut off (in a range from microfiltration to reverse osmosis), were tested for the removal of free DNA (pure supercoiled and linearized plasmids encoding for ARGs and free linear chromosomal DNA with a broader fragment size spectrum) in different water matrices (distilled water and wastewater treatment plant effluent). Our results showed that membranes with a molecular weight cut off smaller than 5000 Da (ultrafiltration, nanofiltration and reverse osmosis) could retain ≥99.80% of free DNA, both pure plasmid and linear fragments of different sizes, whereas microfiltration commonly applied in wastewater treatment showed no retention. Size exclusion was identified as the main retention mechanism. Additionally, surface charging of the membrane and adsorption of free DNA on the membrane surface played a key role in prevention of free DNA permeation. Currently, majority of the applied membranes is negatively charged to prevent adsorption of natural organic matter. In our study, negatively charged membranes showed lower retention of free DNA compared to neutral ones due to repulsion of free DNA molecules, reduced adsorption and decreased blockage of the membrane surface. Therefore, the applied membrane may not be as an effective barrier for ARGs encoded in free DNA, as it would be predicted based only on the molecular weight cut off. Thus, careful considerations of membrane's specifications (molecular weight cut-off and charge) are required during design of a filtration system for retention of free DNA.

Transformation of pleomorphic adenoma to carcinoma ex pleomorphic adenoma of the parotid gland is independent of p53 mutations.

BACKGROUND AND OBJECTIVES: This retrospective study was performed to evaluate the status of p53 in pleomorphic adenomas and carcinomas ex pleomorphic adenoma in the parotid gland. As loss or mutation of p53 can cause malignant transformation, the possible degeneration of pleomorphic adenomas to carcinomas ex pleomorhic adenoma was investigated by mutational analysis. METHODS: Twenty-five Patients including 14 patients with pleomorphic adenomas and 11 patients with carcinoma ex pleomorphic adenoma of the parotid gland were examined for p53 status. DNA was extracted out of paraffin-embedded tissue and PCR was performed for the coding exons 2-11. Denaturing gradient gel electrophoresis (DGGE) was carried out for mutational analysis and DNA sequencing was performed in case of a suspected mutation. RESULTS: Fourteen pleomorphic adenomas and 11 carcinomas ex pleomorphic adenoma were screened for p53 status and potent mutations. Subsequent sequencing of the distinct exons showed no mutation. CONCLUSION: We could not detect mutations of p53 neither in benign nor malignant parotid tumors and we therefore assume that p53 plays no role in the transformation from pleomorphic adenoma to carcinoma ex pleomorphic adenoma.

Oxidative stress: potential of distinct peroxide determination systems.

When reactive oxygen species attack biological structures, peroxides, which are short-lived oxidative intermediates, are generated. We evaluated the potential of two different, commercially available peroxide activity assays (Pox-Act and d-ROMS) to see whether the results were associated with the clinical condition of subjects who were participating in a routine health care program. Furthermore, we determined the total antioxidant status (TAS) and the titer of autoantibodies against oxidized low-density lipoprotein (oLAb) to verify the hydroperoxide measurements. Subjects with medical conditions (hereafter referred to as patients) had significantly increased serum peroxide levels compared to healthy subjects. The d-ROMS kit indicated that 86% of subjects had an increased level of total peroxides. Although the assays had a significant correlation (p<0.001), 34% of the subjects had an increased total peroxide concentration in the Pox-Act assay that was clearly associated with clinical symptoms. Furthermore, the sensitivity of the Pox-Act assay was 35 times higher than that of the d-ROMS kit. In subjects with medical conditions, there was a trend toward a decreased TAS and a slightly increased oLAb titer in comparison to healthy subjects, but this was not statistically significant. The Pox-Act assay seems to be a valuable tool for the determination of total peroxides, while the results from the d-ROMS kit should be considered with caution.