Tigecycline resistance in clinical isolates of Enterococcus faecium is mediated by an upregulation of plasmid-encoded tetracycline determinants tet(L) and tet(M).

OBJECTIVES: Tigecycline represents one of the last-line therapeutics to combat multidrug-resistant bacterial pathogens, including VRE and MRSA. The German National Reference Centre for Staphylococci and Enterococci has received 73 tigecycline-resistant Enterococcus faecium and Enterococcus faecalis isolates in recent years. The precise mechanism of how enterococci become resistant to tigecycline remains undetermined. This study documents an analysis of the role of efflux pumps in tigecycline resistance in clinical isolates of Enterococcus spp. METHODS: Various tigecycline MICs were found for the different isolates analysed. Tigecycline-resistant strains were analysed with respect to genome and transcriptome differences by means of WGS and RT-qPCR. Genes of interest were cloned and expressed in Listeria monocytogenes for verification of their functionality. RESULTS: Detailed comparative whole-genome analyses of three isogenic strains, showing different levels of tigecycline resistance, revealed the major facilitator superfamily (MFS) efflux pump TetL and the ribosomal protection protein TetM as possible drug resistance proteins. Subsequent RT-qPCR confirmed up-regulation of the respective genes. A correlation of gene copy number and level of MIC was inferred from further qPCR analyses. Expression of both tet(L) and tet(M) in L. monocytogenes unequivocally demonstrated the potential to increase tigecycline MICs upon acquisition of either locus. CONCLUSIONS: Our results indicate that increased expression of two tetracycline resistance determinants, a tet(L)-encoded MFS pump and a tet(M)-encoded ribosomal protection protein, is capable of conferring tigecycline resistance in enterococcal clinical isolates.

Application of culture-independent methods to assess the bacteria removal efficiency of subsurface flow constructed wetlands.

The bacteriologic treatment efficiency of vertical and horizontal subsurface flow constructed wetlands (SFCWs) was analysed in two multistage wastewater treatment systems by culture dependent and independent methods. When assessed with standard cultivation procedures, bacteria removal efficiency of the vertical and horizontal SFCWs was similar. However, microscopic enumerations of the wastewater bacteria after DNA staining revealed a completely different removal pattern: bacteria removal efficiency of the horizontal SFCWs was in general low and erratic, whereas the vertical SFCWs displayed high bacteria removal rates. The discrepancies in the results obtained by bacteria enumeration and cultivation was due to a strong decrease in bacterial culturability after treatment by the horizontal SFCWs, leading to overestimation of the real bacterial concentrations in these effluents. Additionally, a PCR based approach for the detection of the enteropathogenic bacteria Campylobacter jejuni and Yersinia enterocolitica was tested in the wastewater samples. The methods were specific and reproducible in the analysed samples and could be carried out within 12 h, proving very adequate as an alternative to cultivation. This work recommends a review of the current standard methodology for wastewater quality surveillance, as well as of the design of SFCW.

Rapid screening and dereplication of bacterial isolates from marine sponges of the sula ridge by intact-cell-MALDI-TOF mass spectrometry (ICM-MS).

Rapid grouping of bacterial isolates is critical in comprehensive microbial studies of environmental samples or screening programmes e.g. in unknown marine environments where large numbers of strains have to be isolated on different growth media. Sets of bacteria have been cultured from the marine sponges Isops phlegraei, Haliclona sp. 1, Phakellia ventilabrum and Plakortis sp. growing at a depth of about 300 m on the Sula Ridge close to the Norwegian coast. We employed Intact-Cell MALDI-TOF (ICM) mass spectrometry to achieve a rapid proteometric clustering of a subset of the strain collection including 456 isolates. Cluster analysis of mass spectra resolved the strains into 11 groups corresponding to species of Alteromonas (15), Bacillus (3), Colwellia (31), Erythrobacter (19), Marinobacter (14), Marinococcus (6), Pseudoalteromonas (297), Pseudomonas (56), Roseobacter (3), Sphingomonas (2) and Vibrio (10) as verified by 16 S rDNA analysis. A further discrimination into subgroups was demonstrated for different isolates from the genus Pseudoalteromonas. The approach described here permits the rapid identification of isolates for dereplication, and the selection of strains representing rare species for subsequent characterization.

Toxicity and microcystin content of extracts from a Planktothrix bloom and two laboratory strains.

Toxic effects and microcystin content from various extracts of a Planktothrix agardhii bloom and two different strains of Planktothrix agardhii, HUB 076, and NIVA 34 were investigated. Extracts were obtained with solvents of different polarity such as hexane, dichloromethane, methanol, and water. Additionally, different pre-treatments were used to break the cells before extraction. Acute toxicity was determined with the fairy shrimp Thamnocephalus platyurus, subchronic effects were detected in embryos and larvae of the zebrafish Danio rerio. The extracts affected the test species to a different extent. Effects were severe in polar extracts (water and methanol) of all strains tested. Although the strain NIVA 34 did not contain any microcystins, aqueous extracts of this strain showed the highest acute toxicity to the crustacean species tested (LC50= 0.08 mg ml(-1)). In contrast aqueous extracts of the Planktothrix bloom containing high amounts of microcystins were less acutely toxic (LC50 = 0.46 mg ml(-1)). Our results indicate the existence of further toxic metabolites in different Plankorothrix agardhii strains.

Microbiological aspects of a bioreactor with submerged membranes for aerobic treatment of municipal wastewater.

An aerobic membrane bioreactor treating municipal wastewater at complete biomass retention was studied in respect of microbiological parameters over a period of 380 days. The results were compared to those obtained from a conventional activated sludge wastewater treatment plant (WWTP) treating the same wastewater. Microscopically, significant changes in the structure of the flocs and of the ratio between free suspended and aggregated cells could be observed. The presence of filamentous bacteria varied from almost not present to very high numbers. With the exception of short periods after changes in operating conditions, protozoa and metazoa were rarely present in the sludge community. The rate of oxygen consumption and the cell detectability by fluorescence in situ hybridizatio (FISH) with rRNA-targeted oligonucleotide probes were used to assess the physiological state of the bacterial cells Oxygen consumption rates of sludge samples obtained from both the conventional and membrane filtration plant wer determined without and after addition of different energy and carbon sources. In contrast to the conventional activate sludge, a pronounced increase in respiration activity upon the addition of organic substrates could be observed in th membrane filtration sludge. In situ probing with the Bacteria-specific probe EUB338 visualized 40-50% of all DAPI stainable bacteria in the membrane bioreactor, compared to 80% cells detectable by FISH in the conventional activate sludge. These results suggest that bacteria present in the highly concentrated biomass of the membrane reactor use the energy supplied for their maintenance metabolism and were not in a physiological state characteristic for growth This assumption could explain the zero net biomass production observed in the reactor.

Performance of a bioreactor with submerged membranes for aerobic treatment of municipal waste water.

Aerobic treatment of municipal waste water in a membrane bioreactor was studied for 535 d. Apart from sampling, sludge was retained completely by a submerged hollow fibre membrane with a pore-size of 0.2 microm. The pilot plant comprised an anoxic zone to enable denitrification. The maximum liquid hold-up of the plant was 3.9 m3. In this study the reactor performance and the stability of the process and the membrane capacity were investigated. A stable flux of 181 m(-2)h(-1) could be realised with a mean transmembrane pressure difference of 0.3bar with air-bubbling and backflushing the membrane and cleaning it in place every two months for one or two hours. For about 140d, a flux of 271 m(-2)h(-1) was achieved, but cleaning became necessary more often. The hydraulic retention time (HRT) varied between 10.4 and 15.6h. Accordingly the volumetric loading rate was between 1.1 and 1.7kg CODm(-3)d(-1). No inoculum was used. The mixed liquor suspended solids (MLSS) concentration gradually increased to 18-20g MLSSl(-1). The feed to microorganism (F/M) ratio varied according to the operation conditions but decreased against a value of 0.07 kg COD kg(-1) MLSSd(-1). Treatment performance was very stable and on a high level. The COD was reduced by 95%. Nitrification was complete and up to 82% of the total nitrogen could be denitrified.

Sulfophthalimide as major metabolite formed from sulfonated phthalocyanine dyes by the white-rot fungus Bjerkandera adusta.

The reaction products formed during the decolorization of the sulfophthalocyanine textile dyes Reactive blue 15 (RB15) and Reactive blue 38 (RB38) by the white-rot fungus Bjerkandera adusta were analyzed by high-performance liquid chromatography with diode array detection and with liquid chromatography-electrospray ionization-tandem mass spectrometry. Sulfophthalimides (SPI; 3 and 4) were identified as major metabolites by comparison with synthesized reference compounds. SPI was formed from both dyes in fungal cultures and by incubation with its purified manganese peroxidase and lignin peroxidase. Quantitative assessment of the SPI formed from RB15 accounted for approximately 60% of the theoretical amount.

Adequacy of in situ glass slides and direct sand extractions to assess the microbiota within sand columns used for drinking water treatment.

Historically, Cholodny-Rossi buried glass slide techniques have been used to study the microbiota of subsurface environments, yet the bias of such a technique has not been compared against direct sand extraction using modern in situ probing. Over a period of 34 wk, four separate 4-m-deep sand columns receiving raw lake water were examined to compare direct extraction of sand filter biofilm material against in situ glass slide biofilms. Significantly different DAPI direct counts and fluorescent in situ hybridization signals for major phylogenetic groups were observed. Not only were lower proportions (P < 0.001) of EUB338-probed DAPI cells observed on in situ glass slides, but also fewer gamma-Proteobacteria (12%-21%) and more alpha-Proteobacteria (16%-33%) when compared to direct sand extracts. Hence, investigators of the microbial ecology of even simple sand biofilms must consider the inherent biases from "accepted" methods and seek further independent methods to identify those which may be most accurate.

Bacterial dehalorespiration with chlorinated benzenes.

Chlorobenzenes are toxic, highly persistent and ubiquitously distributed environmental contaminants that accumulate in the food chain. The only known microbial transformation of 1,2,3,5-tetrachlorobenzene (TeCB) and higher chlorinated benzenes is the reductive dechlorination to lower chlorinated benzenes under anaerobic conditions observed with mixed bacterial cultures. The lower chlorinated benzenes can subsequently be mineralized by aerobic bacteria. Here we describe the isolation of the oxygen-sensitive strain CBDB1, a pure culture capable of reductive dechlorination of chlorobenzenes. Strain CBDB1 is a highly specialized bacterium that stoichiometrically dechlorinates 1,2,3-trichlorobenzene (TCB), 1,2,4-TCB, 1,2,3,4-TeCB, 1,2,3,5-TeCB and 1,2,4,5-TeCB to dichlorobenzenes or 1,3,5-TCB. The presence of chlorobenzene as an electron acceptor and hydrogen as an electron donor is essential for growth, and indicates that strain CBDB1 meets its energy needs by a dehalorespiratory process. According to their 16S rRNA gene sequences, strain CBDB1, Dehalococcoides ethenogenes and several uncultivated bacteria form a new bacterial cluster, of which strain CBDB1 is the first, so far, to thrive on a purely synthetic medium.

Heterogeneity in biofilms.

Biofilms, accumulations of microorganisms at interfaces, have been described for every aqueous system supporting life. The structure of these microbial communities ranges from monolayers of scattered single cells to thick, mucous structures of macroscopic dimensions (microbial mats; algal-microbial associations; trickling filter biofilms). During recent years the structure of biofilms from many different environments has been documented and evaluated by use of a broad variety of microscopic, physico-chemical and molecular biological techniques, revealing a generally complex 3D structure. Parallel to these investigations more and more complex mathematical models and simulations were developed to explain the development, structures, and interactions of biofilms. The forces determining the spatial structure of biofilms, including microcolonies, extracellular polymeric substances (EPS), and channels, are still the subject of controversy. To achieve conclusive explanations for the structures observed in biofilms the cooperation of both fields of investigation, modelling and experimental research, is necessary. The expanding field of molecular techniques not only allows more and more detailed documentation of the spatial distribution of species, but also of functional activities of single cells in their biofilm environment. These new methods will certainly reveal new insights in the mechanisms involved in the developmental processes involved in the formation and behavior of biofilms.

Microbiological safety of drinking water.

Emerging pathogens in drinking water have become increasingly important during the decade. These include newly-recognized pathogens from fecal sources such as Cryptosporidium parvum, Campylobacter spp., and rotavirus, as well as pathogens that are able to grow in water distribution systems, like Legionella spp., mycobacteria, and aeromonads. To perform a risk analysis for the pathogens in drinking water, it is necessary to understand the ecology of these organisms. The ecology of the drinking-water distribution system has to be evaluated in detail, especially the diversity and physiological properties of water bacteria. The interactions between water bacteria and (potential) pathogens in such diverse habitats as free water and biofilms are essential for the survival or growth of hygienically relevant organisms in drinking water. Results of epidemiological studies together with ecological data are the basis for effective resource protection, water treatment, and risk assessment.

Behavior of sulfate reducing bacteria under oligotrophic conditions and oxygen stress in particle-free systems related to drinking water.

The response of sulfate reducing bacteria (SRB) to oxygen stress under oligotrophic conditions in particle-free systems was studied in (i) sterile Berlin drinking water; (ii) mineral medium; and (iii) in coculture experiments with aerobic bacteria. Using a polyphasic approach including anaerobic cultivation, fluorescent in situ hybridization (FISH) and digital image analysis, the behavior of the strains zt3l and zt10e, isolated from Berlin groundwater and affiliated to the family Desulfovibrionaceae, was compared to the type strains Desulfomicrobium baculatum and Desulfovibrio desulfuricans. Anaerobic deep agar dilution series were performed for the determination of cell culturability. FISH and subsequent digital image analysis of probe-conferred fluorescence intensities were used for the assessment of metabolic activity. For the in situ identification of both isolates in coculture tests, two strain-specific oligonucleotides were developed and evaluated. The total cell counts of stressed SRB in drinking water decreased during the course of the assay dependent on the strain. Both environmental isolates could be cultured for a longer period than cells of D. baculatum and D. desulfuricans, respectively. The FISH intensities showed a strain-specific behavior. When exposed to simultaneous oxygen stress and carbon limitation in mineral medium, total cell counts of all four strains remained constant throughout a period of 72 days. The rate of culturability differed between the investigated strains. The decrease of metabolic activity as assessed by FISH was a strain-specific property. Exposure of SRB to oxygen stress and carbon starvation in coculture experiments with Aquabacterium commune resulted in strain dependent prolonged culturability and a delayed decrease of the metabolic activity compared to pure culture tests for all strains tested. Total cell counts of SRB were constant throughout the whole experiment.

Widefield deconvolution epifluorescence microscopy combined with fluorescence in situ hybridization reveals the spatial arrangement of bacteria in sponge tissue.

Widefield deconvolution epifluorescence microscopy (WDEM) combined with fluorescence in situ hybridization (FISH) was performed to identify and characterize single bacterial cells within sections of the mediterranean sponge Chondrosia reniformis. Sponges were embedded in paraffin wax or plastic prior to the preparation of thin sections, in situ hybridization and microscopy. Serial digital images generated by widefield epifluorescence microscopy were visualized using an exhaustive photon reassignment deconvolution algorithm and three-dimensional rendering software. Computer processing of series of images taken at different focal planes with the deconvolution technique provided deblurred three-dimensional images with high optical resolution on a submicron scale. Results from the deconvolution enhanced widefield microscopy were compared with conventional epifluorescent microscopical images. By the application of the deconvolution algorithm on digital image data obtained with widefield epifluorescence microscopy after FISH, the occurrence and spatial arrangement of Desulfovibrionaceae closely associated with micropores of Chondrosia reniformis could be visualized.

Bacterial growth based on reductive dechlorination of trichlorobenzenes.

An anaerobic mixed bacterial culture was enriched for bacteria dechlorinating 1,2,3- and 1,2,4-trichlorobenzene (TCB) to dichlorobenzenes by exclusive use of non-fermentable substrates and the application of vancomycin. Growth and dechlorination occurred in a purely synthetic medium with formate or hydrogen, acetate, and TCB. Neither acetogenesis nor methanogenesis was detected in the culture. Repeated subculturing maintaining high dechlorinating activities was also achieved when only hydrogen and TCB were supplied. This indicated that reductive dechlorination of TCB was the primary energy conservating process. The number of dechlorinating bacteria was strictly limited by the amount of TCB supplied in the medium. In addition, the dechlorinating activity could be maintained only in the presence of TCB. A most probable number analysis showed that the dechlorinating species amounted to at least 6 x 10(5) cells per ml at a total cell number of about 2 x 10(6) cells per ml. Vitamin B12 significantly stimulated the dechlorinating activity.

Phylogenetic Composition, Spatial Structure, and Dynamics of Lotic Bacterial Biofilms Investigated by Fluorescent in Situ Hybridization and Confocal Laser Scanning Microscopy.

> Abstract The phylogenetic composition, three-dimensional structure and dynamics of bacterial communities in river biofilms generated in a rotating annular reactor system were studied by fluorescent in situ hybridization (FISH) and confocal laser scanning microscopy (CLSM). Biofilms grew on independently removable polycarbonate slides exposed in the reactor system with natural river water as inoculum and sole nutrient and carbon source. The microbial biofilm community developed from attached single cells and distinct microcolonies via a more confluent structure characterized by various filamentous bacteria to a mature biofilm rich in polymeric material with fewer cells on a per-area basis after 56 days. During the different stages of biofilm development, characteristic microcolonies and cell morphotypes could be identified as typical features of the investigated lotic biofilms. In situ analysis using a comprehensive suite of rRNA-targeted probes visualized individual cells within the alpha-, beta-, and gamma-Proteobacteria as well as the Cytophaga-Flavobacterium group as major parts of the attached community. The relative abundance of these major groups was determined by using digital image analysis to measure specific cell numbers as well as specific cell area after in situ probing. Within the lotic biofilm community, 87% of the whole bacterial cell area and 79% of the total cell counts hybridized with a Bacteria specific probe. During initial biofilm development, beta-Proteobacteria dominated the bacterial population. This was followed by a rapid increase of alpha-Proteobacteria and bacteria affiliated to the Cytophaga-Flavobacterium group. In mature biofilms, alpha-Proteobacteria and Cytophaga-Flavobacteria continued to be the prevalent bacterial groups. Beta-Proteobacteria constituted the morphologically most diverse group within the biofilm communities, and more narrow phylogenetic staining revealed the importance of distinct phylotypes within the beta1-Proteobacteria for the composition of the microbial community. The presence of sulfate-reducing bacteria affiliated to the Desulfovibrionaceae and Desulfobacteriaceae confirmed the range of metabolic potential within the lotic biofilms.http://link.springer-ny.com/link/service/journals/00248/bibs/37n4p225.html

Aquabacterium gen. nov., with description of Aquabacterium citratiphilum sp. nov., Aquabacterium parvum sp. nov. and Aquabacterium commune sp. nov., three in situ dominant bacterial species from the Berlin drinking water system.

Three bacterial strains isolated from biofilms of the Berlin drinking water system were characterized with respect to their morphological and physiological properties and their taxonomic position. Phenotypically, the bacteria investigated were motile, Gram-negative rods, oxidase-positive and catalase-negative, and contained polyalkanoates and polyphosphate as storage polymers. They displayed a microaerophilic growth behaviour and used oxygen and nitrate as electron acceptors, but not nitrite, chlorate, sulfate or ferric iron. The substrates metabolized included a broad range of organic acids but no carbohydrates at all. The three species can be distinguished from each other by their substrate utilization, ability to hydrolyse urea and casein, cellular protein patterns and growth on nutrient-rich media as well as their temperature, pH and NaCl tolerances. Phylogenetic analysis, based on 16S rRNA gene sequence comparison, revealed that the isolates are affiliated to the beta 1-subclass of Proteobacteria. The isolates constitute three new species with internal levels of DNA relatedness ranging from 44.9 to 51.3%. It is proposed that a new genus, Aquabacterium gen. nov., should be created, including Aquabacterium citratiphilum sp. nov., Aquabacterium parvum sp. nov. and Aquabacterium commune sp. nov. The type species of the new genus is Aquabacterium commune. The type strain of A. citratiphilum is strain B4T (= DSM 11900T), the type strain of A. parvum is strain B6T (= DSM 11968T) and the type strain of A. commune is strain B8T (= DSM 11901T).

Transformation of industrial dyes by manganese peroxidases from Bjerkandera adusta and Pleurotus eryngii in a manganese-independent reaction.

We investigated the transformation of six industrial azo and phthalocyanine dyes by ligninolytic peroxidases from Bjerkandera adusta and other white rot fungi. The dyes were not oxidized or were oxidized very little by Phanerochaete chrysosporium manganese peroxidase (MnP) or by a chemically generated Mn3+-lactate complex. Lignin peroxidase (LiP) from B. adusta also showed low activity with most of the dyes, but the specific activities increased 8- to 100-fold when veratryl alcohol was included in the reaction mixture, reaching levels of 3.9 to 9.6 U/mg. The B. adusta and Pleurotus eryngii MnP isoenzymes are unusual because of their ability to oxidize aromatic compounds like 2,6-dimethoxyphenol and veratryl alcohol in the absence of Mn2+. These MnP isoenzymes also decolorized the azo dyes and the phthalocyanine complexes in an Mn2+-independent manner. The reactions with the dyes were characterized by apparent Km values ranging from 4 to 16 microM and specific activities ranging from 3.2 to 10.9 U/mg. Dye oxidation by these peroxidases was not increased by adding veratryl alcohol as it was in LiP reactions. Moreover, the reaction was inhibited by the presence of Mn2+, which in the case of Reactive Black 5, an azo dye which is not oxidized by the Mn3+-lactate complex, was found to act as a noncompetitive inhibitor of dye oxidation by B. adusta MnP1.

Purification and characterization of peroxidases from the dye-decolorizing fungus Bjerkandera adusta.

A peroxidase oxidizing Mn2+ (MnP) is described for the first time in Bjerkandera adusta, a fungus efficiently degrading xenobiotic compounds. The MnP appeared as two isoenzymes, which were purified to homogeneity together with two lignin peroxidases (LiP). Their N-terminal sequences were identical, but the MnP isoenzymes showed more basic isoelectric points and differences in amino acid composition and catalytic properties. The B. adusta LiP is similar to LiP from Phanerochaete chrysoporium. However, the interest of the MnP described here is related to its ability to catalyze Mn(2+)-mediated as well as Mn(2+)-independent reactions on aromatic compounds, which may be of use for applications in biotechnology and environmental technology.

A study on reducing substrates of manganese-oxidizing peroxidases from Pleurotus eryngii and Bjerkandera adusta.

A novel peroxidase, oxidizing Mn2+ and different aromatic compounds, was isolated. Hydroquinones, substituted phenols, dyes, other aromatic compounds and Mn2+ were compared as reducing substrates, and conclusions presented in the light of a molecular model built by homology modeling. The enzymes showed the fastest reaction rates with Mn2+, but the highest affinity corresponded to hydroquinones and dyes. Oxidation of Reactive Black 5 (an azo-dye not oxidized by Mn3+) was non-competitively inhibited by Mn2+. These findings, together with identification of putative Mn-binding site (involving Glu36, Glu40, Asp175 and inner heme propionate) and long-range electron transfer pathways, indicate that different sites are involved in substrate oxidation.

Physiological characterization of a bacterial consortium reductively dechlorinating 1,2,3- and 1,2,4-trichlorobenzene.

A bacterial mixed culture reductively dechlorinating trichlorobenzenes was established in a defined, synthetic mineral medium without any complex additions and with pyruvate as the carbon and energy source. The culture was maintained over 39 consecutive transfers of small inocula into fresh media, enriching the dechlorinating activity. In situ probing with fluorescence-labeled rRNA-targeted oligonucleotide probes revealed that two major subpopulations within the microbial consortium were phylogenetically affiliated with a sublineage within the Desulfovibrionaceae and the gamma subclass of Proteobacteria. The bacterial consortium grew by fermentation of pyruvate, forming acetate, propionate, CO2, formate, and hydrogen. Acetate and propionate supported neither the reduction of trichlorobenzenes nor the reduction of sulfate when sulfate was present. Hydrogen and formate were used for sulfate reduction to sulfide. Sulfate strongly inhibited the reductive dechlorination of trichlorobenzenes. However, when sulfate was depleted in the medium due to sulfate reduction, dechlorination of trichlorobenzenes started. Similar results were obtained when sulfite was present in the cultures. Molybdate at a concentration of 1 mM strongly inhibited the dechlorination of trichlorobenzenes. Cultures supplied with molybdate plus sulfate did not reduce sulfate, but dechlorination of trichlorobenzenes occurred. Supplementation of electron-depleted cultures with various electron sources demonstrated that formate was used as a direct electron donor for reductive dechlorination, whereas hydrogen was not.