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      Low metabolic activity of biofilm formed by Enterococcus faecalis isolated from healthy humans and wild mallards (Anas platyrhynchos)

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          Abstract

          It is widely known that Enterococcus faecalis virulence is related to its biofilm formation. Although Enterococci are common commensal organisms of the gastrointestinal tract, the difference between commensal and pathogen strains remain unclear. In this study, we compare the biochemical profile of the biofilms formed by two groups of medical and two groups of commensal strains. The medical strains were isolated as pathogens from infections of urinary tract and other infections (wounds, pus and bedsores), and the commensal strains were taken from faeces of healthy volunteers and faeces of wild mallards ( Anas platyrhynchos) living in an urban environment. The properties of biofilms formed by medical and commensal strains differed significantly. Commensal strains showed lower metabolic activity and glucose uptake and higher biofilm biomass than the medical ones. Consistent with glucose uptake experiments, we found that the glucose dehydrogenase gene was more expressed in medical strains. These results indicate that higher metabolic activity and lower protein concentration of E. faecalis cells within biofilms are formed during infections.

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          Molecular screening of Enterococcus virulence determinants and potential for genetic exchange between food and medical isolates.

          Enterococci are used as starter and probiotic cultures in foods, and they occur as natural food contaminants. The genus Enterococcus is of increased significance as a cause of nosocomial infections, and this trend is exacerbated by the development of antibiotic resistance. In this study, we investigated the incidence of known virulence determinants in starter, food, and medical strains of Enterococcus faecalis, E. faecium, and E. durans. PCR and gene probe strategies were used to screen enterococcal isolates from both food and medical sources. Different and distinct patterns of incidence of virulence determinants were found for the E. faecalis and E. faecium strains. Medical E. faecalis strains had more virulence determinants than did food strains, which, in turn, had more than did starter strains. All of the E. faecalis strains tested possessed multiple determinants (between 6 and 11). E. faecium strains were generally free of virulence determinants, with notable exceptions. Significantly, esp and gelE determinants were identified in E. faecium medical strains. These virulence determinants have not previously been identified in E. faecium strains and may result from regional differences or the evolution of pathogenic E. faecium. Phenotypic testing revealed the existence of apparently silent gelE and cyl genes. In E. faecalis, the trend in these silent genes mirrors that of the expressed determinants. The potential for starter strains to acquire virulence determinants by natural conjugation mechanisms was investigated. Transconjugation in which starter strains acquired additional virulence determinants from medical strains was demonstrated. In addition, multiple pheromone-encoding genes were identified in both food and starter strains, indicating their potential to acquire other sex pheromone plasmids. These results suggest that the use of Enterococcus spp. in foods requires careful safety evaluation.
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              An improved colorimetric assay for cell proliferation and viability utilizing the tetrazolium salt XTT.

              A new tetrazolium salt XTT, sodium 3'-[1-[(phenylamino)-carbonyl]-3,4-tetrazolium]-bis(4-methoxy-6- nitro)benzene-sulfonic acid hydrate, was evaluated for use in a colorimetric assay for cell viability and proliferation by normal activated T cells and several cytokine dependent cell lines. Cleavage of XTT by dehydrogenase enzymes of metabolically active cells yields a highly colored formazan product which is water soluble. This feature obviates the need for formazan crystal solubilization prior to absorbance measurements, as required when using other tetrazolium salts such as MTT. Bioreduction of XTT by all the murine cells examined was not particularly efficient, but could be potentiated by addition of electron coupling agents such as phenazine methosulfate (PMS) or menadione (MEN). Optimal concentrations of PMS or MEN were determined for the metabolism of XTT by the T cell lines HT-2 and 11.6, NFS-60 a myeloid leukemia, MC/9 a mast cell line and mitogen activated splenic T cells. When used in combination with PMS, each of these cells generated higher formazan absorbance values with XTT than were observed with MTT. Thus the use of XTT in colorimetric proliferation assays offer significant advantages over MTT, resulting from reduced assay time and sample handling, while offering equivalent sensitivity.
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                Author and article information

                Contributors
                +48-58-3412016 , +48-58-3412016 , w.meissner@univ.gda.pl
                Journal
                Ann Microbiol
                Ann. Microbiol
                Annals of Microbiology
                Springer Berlin Heidelberg (Berlin/Heidelberg )
                1590-4261
                1869-2044
                14 February 2013
                14 February 2013
                2013
                : 63
                : 1477-1482
                Affiliations
                [ ]Avian Ecophysiology Unit, Department of Vertebrate Ecology and Zoology, University of Gdańsk, ul. Wita Stwosza 59, 80-308 Gdańsk, Poland
                [ ]Department of Medical Microbiology, Medical University of Gdańsk, ul. Do Studzienki 38, 80-227 Gdańsk, Poland
                [ ]IBB-Institute for Biotechnology and Bioengineering, Centre of Biological Engineering, University of Minho, Campus de Gualtar, 4710-057 Braga, Portugal
                [ ]Department of Laboratory and Microbiological Diagnostics, Kościerzyna Medical Center, ul. Piechowskiego 36, 83-400 Kościerzyna, Poland
                Article
                611
                10.1007/s13213-013-0611-3
                3832767
                24294189
                5b4a340a-9f78-45a0-a28f-5a15d45fe815
                © The Author(s) 2013

                Open Access This article is distributed under the terms of the Creative Commons Attribution License which permits any use, distribution, and reproduction in any medium, provided the original author(s) and the source are credited.

                History
                : 14 July 2012
                : 23 January 2013
                Categories
                Original Article
                Custom metadata
                © Springer-Verlag Berlin Heidelberg and the University of Milan 2013

                Microbiology & Virology
                enterococcus faecalis strains,metabolic activity,biofilm,protein concentration

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