Critical physiological factors influencing the outcome of antimicrobial testing according to ISO 22196 / JIS Z 2801.

Bactericidal materials gained interest in the health care sector as they are capable of preventing material surfaces from microbial colonization and subsequent spread of infections. However, commercialization of antimicrobial materials requires proof of their efficacy, which is usually done using in vitro methods. The ISO 22196 standard (Japanese test method JIS Z 2801) is a method for measuring the antibacterial activity of daily goods. As it was found reliable for testing the biocidal activity of antimicrobially active materials and surface coatings most of the laboratories participating in this study used this protocol. Therefore, a round robin test for evaluating antimicrobially active biomaterials had to be established. To our knowledge, this is the first report on inaugurating a round robin test for the ISO 22196 / JIS Z 2801. The first round of testing showed that analyses in the different laboratories yielded different results, especially for materials with intermediate antibacterial effects distinctly different efficacies were noted. Scrutinizing the protocols used by the different participants and identifying the factors influencing the test outcomes the approach was unified. Four critical factors influencing the outcome of antibacterial testing were identified in a series of experiments: (1) incubation time, (2) bacteria starting concentration, (3) physiological state of bacteria (stationary or exponential phase of growth), and (4) nutrient concentration. To our knowledge, this is the first time these parameters have been analyzed for their effect on the outcome of testing according to ISO 22196 / JIS Z 2801. In conclusion, to enable assessment of the results obtained it is necessary to evaluate these single parameters in the test protocol carefully. Furthermore, uniform and robust definitions of the terms antibacterial efficacy / activity, bacteriostatic effects, and bactericidal action need to be agreed upon to simplify communication of results and also regulate expectations regarding antimicrobial tests, outcomes, and materials.

MALDI-TOF mass spectrometry fingerprinting: A diagnostic tool to differentiate dematiaceous fungi Stachybotrys chartarum and Stachybotrys chlorohalonata.

Stachybotrys chartarum and Stachybotrys chlorohalonata are two closely related species. Unambiguous identification of these two species is a challenging task if relying solely on morphological criteria and therefore smarter and less labor-intensive approaches are needed. Here we show that even such closely related species of fungi as S. chartarum and S. chlorohalonata are unequivocally discriminated by their highly reproducible MALDI-TOF-MS fingerprints (matrix assisted laser desorption/ionization time-of-flight mass spectrometry fingerprints). We examined 19 Stachybotrys and one Aspergillus isolate by MALDI-TOF-MS. All but one isolate produced melanin containing conidia on malt extract agar. Mass spectra were obtained in good quality from the analysis of hyaline and darkly pigmented conidia by circumventing the property of melanin which causes signal suppression. MALDI-TOF fingerprint analysis clearly discriminated not only the two morphologically similar species S. chartarum and S. chlorohalonata from each other but separated them precisely from Stachybotrys bisbyi and Aspergillus versicolor isolates. Furthermore, even S. chartarum chemotypes A and S could be differentiated into two distinct groups by their MALDI-TOF fingerprints. The chemotypes of S. chartarum isolates were identified by trichodiene synthase 5 (tri5) sequences prior to mass spectra analysis. Additionally, species identities of all isolates were verified by their 18S rRNA and tri5 gene sequences.

Steroidobacter denitrificans gen. nov., sp. nov., a steroidal hormone-degrading gammaproteobacterium.

A denitrifying bacterium, designated strain FS(T), was isolated from anoxic digested sludge on oestradiol [17beta-oestra-1,3,5(10)-triene-3,17-diol] or testosterone (17beta-hydroxyandrost-4-en-3-one) as the sole source of carbon and energy with nitrate as the electron acceptor. Strain FS(T) represents the first known bacterium to grow anaerobically on both oestradiol (C-18) and testosterone (C-19). Steroidal hormones were degraded completely by nitrate reduction to dinitrogen monoxide, which was further reduced to dinitrogen in stationary-phase cultures. Gram-negative cells were slightly curved rods, 0.3-0.5 x 0.6-1.6 microm in size, motile, non-fermentative, non-spore-forming and catalase- and oxidase-positive, showing optimal growth at pH 7.0, 28 degrees C and 0.1% (w/v) NaCl. Beside steroidal hormones, the bacterium utilized only a narrow range of organic substrates with nitrate as the electron acceptor, including several fatty acids and glutamate. No aerobic or anaerobic growth occurred on liquid or solid complex media. Phylogenetic analysis of the 16S rRNA gene sequence showed that strain FS(T) has no known close relatives and represents a distinct lineage within the Gammaproteobacteria. Together with the genera Nevskia, Hydrocarboniphaga, Solimonas and Sinobacter (less than 88% 16S rRNA gene sequence similarity to strain FS(T)), it forms a phylogenetic cluster separated from the families Chromatiaceae, Ectothiorhodospiraceae and Xanthomonadaceae. The quinone system of strain FS(T) consisted exclusively of ubiquinone Q-8. The dominant polar lipids were diphosphatidylglycerol and phosphatidylethanolamine. Spermidine in combination with putrescine and traces of sym-homospermidine were the basic polyamines. The major fatty acids detected in testosterone- or heptanoate-grown cells were C(15:0) and C(17:1)omega8c, minor hydroxylated fatty acids were C(11:0) 3-OH and C(12:0) 3-OH. The G+C content of the DNA was 61.9 mol%. Based on the high 16S rRNA gene sequence divergence and different phenotypic properties from previously described gammaproteobacteria in combination with chemotaxonomic data, strain FS(T) is considered to represent a new genus and species, for which the name Steroidobacter denitrificans gen. nov., sp. nov. is proposed. The type strain of Steroidobacter denitrificans is FS(T) (=DSM 18526(T) =JCM 14622(T)).