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  • 1. Adamsson, I
    et al.
    Edlund, Charlotta
    Södertörn University, Avdelning Naturvetenskap.
    Seensalu, R
    Engstrand, L
    The use of AP-PCR and flaA-RFLP typing to investigate treatment failure in Helicobacter pylori infection2000In: Clinical Microbiology and Infection, ISSN 1198-743X, E-ISSN 1469-0691, Vol. 6, no 5, 265-267 p.Article in journal (Refereed)
  • 2. Agvald-Öhman, C
    et al.
    Wernerman, J
    Nord, C E
    Edlund, Charlotta
    Södertörn University, Avdelning Naturvetenskap.
    Anaerobic bacteria commonly colonize the lower airways of intubated ICU patients2003In: Clinical Microbiology and Infection, ISSN 1198-743X, E-ISSN 1469-0691, Vol. 9, no 5, 397-405 p.Article in journal (Refereed)
    Abstract [en]

    Objectives To investigate respiratory tract colonization by aerobic and anaerobic bacteria in mechanically ventilated patients. Methods Bacterial colonization of the stomach and the respiratory tract was qualitatively and quantitatively analyzed over time in 41 consecutive mechanically ventilated patients in a Swedish intensive care unit (ICU), with special emphasis on elucidation of the role of anaerobic bacteria in the lower respiratory tract. Samples were taken from the oropharynx, gastric juice, subglottic space and trachea within 24 h (median 14 h) of intubation, and then every third day until day 18 and every fifth day until day 33. Results The patients were often heavily colonized with microorganisms not considered to belong to a healthy normal oropharyngeal and gastric flora on admission to the ICU. A majority harbored enterococci, coagulase-negative staphylococci and Candida spp. in at least one site on day 1. Anaerobic bacteria, mainly peptostreptococci and Prevotella spp., were isolated from subglottic and/or tracheal secretions in 59% of the patients. Different routes of tracheal colonization for different groups of microorganisms were found. Primary or concomitant colonization of the oropharynx with staphylococci, enterococci, enterobacteria and Candida was often seen, while Pseudomonas spp., other non-fermenting Gram-negative rods and several anaerobic species often primarily colonized the trachea, indicating exogenous or direct gastrointestinal routes of colonization. Conclusions Mechanically ventilated patients were heavily colonized in their lower airways by potential pathogenic microorganisms, including a high load of anaerobic bacteria. Different routes of colonization were shown for different species.

  • 3. Backhed, F
    et al.
    Normark, S
    Schweda, Elke K H
    Södertörn University, Avdelning Naturvetenskap.
    Oscarson, S
    Richter-Dahlfors, A
    Structural requirements for TLR4-mediated LPS signalling: a biological role for LPS modifications2003In: Microbes and infection, ISSN 1286-4579, E-ISSN 1769-714X, Vol. 5, no 12, 1057-1063 p.Article in journal (Refereed)
    Abstract [en]

    Cells of the mucosal lining are the first to encounter invading bacteria during infection, and as such, they have developed numerous ways of detecting microbial intruders. Recently, we showed that epithelial cells recognize lipopolysaccharide (LPS) through the CD14-Toll-like receptor (TLR)-4 complex. Here, we identify the substructures of LPS that are recognized by the TLR4 receptor complex. In contrast to lipid A, the O-antigen does not mediate an inflammatory response; rather it interferes with the lipid A recognition. An Escherichia coli strain genetically modified to express penta-acylated lipid A not only showed reduced immunogenicity, but was also found to inhibit proinflammatory signalling induced by wild-type E. coli (hexa-acylated lipid A) as well as LPS from other bacteria of the Enterobacteriaceae family. Furthermore, penta-acylated LPS from Pseudomonas aeruginosa acted as an antagonist to hexa-acylated E. coli LPS, as did E. coli, as shown by its inhibitory effect on IL-8 production in stimulated cells. Hypo-acylated lipidA, such as that of P. aeruginosa, is found in several species within the gut microflora as well as in several bacteria causing chronic infections. Thus, our results suggest that the composition of the microflora may be important in modulating pro-inflammatory signalling in epithelial cells under normal as well as pathologic conditions.

  • 4.
    Backman, Agneta
    et al.
    Södertörn University, School of Chemistry, Biology, Geography and Environmental Science. Karolinska Institutet.
    Jansson, Janet K
    Södertörn University, School of Chemistry, Biology, Geography and Environmental Science. SLU.
    Degradation of 4-chlorophenol at low temperature and during extreme temperature fluctuations by Arthrobacter chlorophenolicus A62004In: Microbial Ecology, ISSN 0095-3628, E-ISSN 1432-184X, Vol. 48, no 2, 246-253 p.Article in journal (Refereed)
    Abstract [en]

    Low average temperatures and temperature fluctuations in temperate soils challenge the efficacy of microbial strains used for clean up of pollutants. In this study, we investigated the cold tolerance of Arthrobacter chlorophenolicus A6, a microorganism previously shown to degrade high concentrations of 4-chlorophenol at 28degreesC. Luciferase activity from a luc-tagged derivative of the strain (A6L) was used to monitor the metabolic status of the population during 4-chlorophenol degradation. The A6L strain could degrade 200-300 mug mL(-1) 4-chlorophenol in pure cultures incubated at 5degreesC, although rates of degradation, growth and the metabolic status of the cells were lower at 5degreesC compared to 28degreesC. When subjected to temperature fluctuations between 5 and 28degreesC, A6L continued to degrade 4-chlorophenol and remained active. In soil microcosm experiments, the degradation rates were significantly faster the first week at 28degreesC, compared to 5degreesC. However, this difference was no longer seen after 7 days, and equally low 4-chlorophenol concentrations were reached after 17 days at both temperatures. During 4-chlorophenol degradation in soil, CFU and luciferase activity values remained constant at both 5 and 28degreesC. However, once most of the 4-chlorophenol was degraded, both values decreased by 1-1.5 logarithmic values at 28degreesC, whereas they remained constant at 5degreesC, indicating a high survival of the cells at low temperatures. Because of the ability of A. chlorophenolicus A6 to degrade high concentrations of 4-chlorophenol at 5degreesC, together with its tolerance to temperature fluctuations and stress conditions found in soil, this strain is a promising candidate for bioaugmentation of chlorophenol-contaminated soil in temperate climates.

  • 5.
    Backman, Agneta
    et al.
    Södertörn University, School of Chemistry, Biology, Geography and Environmental Science. Karolinska Institutet.
    Maraha, Ninwe
    Södertörn University, School of Chemistry, Biology, Geography and Environmental Science. Karolinska Institutet.
    Jansson, Janet K
    Södertörn University, School of Chemistry, Biology, Geography and Environmental Science. SLU.
    Impact of temperature on the physiological status of a potential bioremediation inoculant, Arthrobacter chlorophenolicus A62004In: Applied and Environmental Microbiology, ISSN 0099-2240, E-ISSN 1098-5336, Vol. 70, no 5, 2952-2958 p.Article in journal (Refereed)
    Abstract [en]

    Arthrobacter chlorophenolicus A6 (A6) can degrade large amounts of 4-chlorophenol in soil at 5 and 28degreesC. In this study, we investigated the effects of temperature on the physiological status of this bacterium in pure culture and in soil. A derivative of A6 tagged with the gfp gene (encoding green fluorescent protein [GFP]) was used to specifically quantify A6 cells in soil. In addition, cyano-ditolyl-tetrazoliumchloride was used to stain GFP-fluorescent cells with an active electron transfer system ("viable cellis") whereas propidium iodide (PI) was used to stain cells with damaged membranes ("dead cells"). Another derivative of the strain (tagged with the firefly luciferase gene [luc]) was used to monitor the metabolic activity of the cell population, since the bioluminescence phenotype is dependent on cellular energy reserves. When the cells were incubated in soil at 28degreesC, the majority were stained with PI, indicating that they had lost their cell integrity. In addition, there was a corresponding decline in metabolic activity and in the ability to be grown in cultures on agar plates after incubation in soil at 28degreesC, indicating that the cells were dying under those conditions. When the cells were incubated in soil at 5degreesC, by contrast, the majority of the cells remained intact and a large fraction of the population remained metabolically active. A similar trend towards better cell survival at lower temperatures was found in pure-culture experiments. These results make A. chlorophenolicus A6 a good candidate for the treatment of chlorophenol-contaminated soil in cold climates.

  • 6. Ban, L.
    et al.
    Didon, Andrea
    Södertörn University, School of Life Sciences.
    Jonsson, L. M. V.
    Glinwood, R.
    Delp, Gabriele
    Södertörn University, School of Life Sciences.
    An improved detection method for the Rhopalosiphum padi virus (RhPV) allows monitoring of its presence in aphids and movement within plants2007In: Journal of Virological Methods, ISSN 0166-0934, E-ISSN 1879-0984, Vol. 142, no 1-2, 136-142 p.Article in journal (Refereed)
    Abstract [en]

    Rhopalosiphumpadi virus (RhPV) is an insect RNA virus that infects aphids, reducing their lifespan and fecundity. It can be transmitted vertically between aphids and horizontally via the plant. An improved detection method for the virus in aphids and plants using RT-PCR was developed; this allowed individual aphids to be tested for RhPV. Testing of R. padi aphids collected from different sites in Sweden revealed the presence of RhPV in wild aphid populations for the first time in Europe. Virus could be detected in several life stages of R. padi, including sexual individuals and eggs, establishing an over-wintering route for the virus. Using RT-PCR, systemic transport of the virus in plants was tracked. Virus spread from the aphid feeding site to all parts of the plant, including roots, within 7 days, and could be acquired by virus-free aphids feeding on the same plant.

  • 7. Beskow, Anne
    et al.
    Wright, Anthony P. H.
    Södertörn University, School of Life Sciences.
    Comparative analysis of regulatory transcription factors in Schizosaccharomyces pombe and budding yeasts2006In: Yeast, ISSN 0749-503X, E-ISSN 1097-0061, Vol. 23, no 13, 929-935 p.Article in journal (Refereed)
    Abstract [en]

    Regulatory transcription factors (rTFs), which bind specific DNA sequences in the regulatory regions of genes and subsequently activate or repress transcription, play a central role in programming genomic expression. The number of rTFs in a species might therefore reflect its functional complexity. For simple organisms like yeast, a relatively small number of rTFs might be expected that is fairly constant between yeast species. We show that the budding yeast, Saccharomyces cerevisiae, contains 201 rTfs, which is one of the largest rTF numbers found in yeast species for which genome sequences are available. This is a much higher number than the 129 rTFs found in the fission yeast, Schizosaccharomyces pombe, which is currently the yeast with the lowest number of rTFs. Comparative analysis of several different budding yeast species shows that most of the 'extra' rTFs found in S. cerevisiae were probably acquired as a result of a whole genome duplication (WGD) event that occurred in an ancestor of a subset of budding yeast species. However, we also show that budding yeast species that have not been affected by the WGD contain a greater number of rTFs than S. pombe (mean = 145). Thus, two or more mechanisms have led to the 60% increase in rTFs in S. cerevisiae compared to S. pombe. This difference may correlate with a more extensive functional divergence in budding yeasts compared to fission yeasts. The relatively small number of rTFs in S. pombe make this organism an attractive model for global studies of mechanisms that programme gene expression.

  • 8.
    Browall, Sarah
    Södertörn University College, School of Life Sciences.
    Comparative genomic analyse by microarray technology of pneumococci with different potential to cause disease.2007Independent thesis Basic level (professional degree), 20 points / 30 hpStudent thesis
    Abstract [en]

    Streptococcus pneumoniae is a gram-positive bacterium that can be found in both healthy carriers as well as in people that have developed disease. One of the major virulence factors of pneumococci is their polysaccharide capsule. Based on the capsule that surrounds the bacteria, pneumococci are divided into at least 90 different serotypes. Some serotypes seem to be more related to virulence than others.

    I have with comparative genome hybridization microarray technique, studied gene differences between 18 epidemiological well-characterised pneumococcal strains with different potential to cause disease. A microarray chip based on two sequenced pneumococcal genomes, R6 and TIGR4, has already been designed. According to Hierarchical clustering, both the serotype and the genetic type as assessed by MLST (sequence type or ST) seem to have impact on the relationship of clinical isolates. Most clinical isolates of the same serotype are clustered together except for serotype 14 isolates that seem to be more divergent. Further more the number of genes that are divergent between clinical isolates compared to R6 and TIGR4 differ from 65 to 289. Preliminary results indicate that although there is diversity among clinical isolates some are more closely related to each other then others. Absent genes seem to be evenly distributed among all 18 clinical isolates tested but hypothetical genes and genes for cell envelope are two groups of role categories that are absent to the largest extent in all isolates.

    According to results from microarray analysis, a gene region, spr0112-spr1015- is present in all type 9V isolates and absent in many isolates of serotype 14, 19F and 7F. These results have been confirmed by polymerase chain reaction (PCR).

    Conserved genes in a region around the capsule genes have been sequenced to identify marker genes for a capsulular switch between serotype 9V and 14. Preliminary results of the sequencing showed that as much as 750kb might have been transferred in the event of capsular switch.

  • 9.
    Bürglin, Thomas R.
    Södertörn University, School of Life Sciences.
    Evolution of hedgehog and hedgehog-related genes, their origin from Hog proteins in ancestral eukaryotes and discovery of a novel Hint motif2008In: BMC Genomics, ISSN 1471-2164, E-ISSN 1471-2164, Vol. 9, 127- p.Article in journal (Refereed)
    Abstract [en]

    Background: The Hedgehog (Hh) signaling pathway plays important roles in human and animal development as well as in carcinogenesis. Hh molecules have been found in both protostomes and deuterostomes, but curiously the nematode Caenorhabditis elegans lacks a bona-fide Hh. Instead a series of Hh-related proteins are found, which share the Hint/Hog domain with Hh, but have distinct N-termini. Results: We performed extensive genome searches such as the cnidarian Nematostella vectensis and several nematodes to gain further insights into Hh evolution. We found six genes in N. vectensis with a relationship to Hh: two Hh genes, one gene with a Hh N-terminal domain fused to a Willebrand factor type A domain (VWA), and three genes containing Hint/Hog domains with distinct novel N-termini. In the nematode Brugia malayi we find the same types of hh-related genes as in C. elegans. In the more distantly related Enoplea nematodes Xiphinema and Trichinella spiralis we find a bona-fide Hh. In addition, T. spiralis also has a quahog gene like C. elegans, and there are several additional hh-related genes, some of which have secreted N-terminal domains of only 15 to 25 residues. Examination of other Hh pathway components revealed that T. spiralis - like C. elegans - lacks some of these components. Extending our search to all eukaryotes, we recovered genes containing a Hog domain similar to Hh from many different groups of protists. In addition, we identified a novel Hint gene family present in many eukaryote groups that encodes a VWA domain fused to a distinct Hint domain we call Vint. Further members of a poorly characterized Hint family were also retrieved from bacteria. Conclusion: In Cnidaria and nematodes the evolution of hh genes occurred in parallel to the evolution of other genes that contain a Hog domain but have different N-termini. The fact that Hog genes comprising a secreted N-terminus and a Hog domain are found in many protists indicates that this gene family must have arisen in very early eukaryotic evolution, and gave rise eventually to hh and hh-related genes in animals. The results indicate a hitherto unsuspected ability of Hog domain encoding genes to evolve new N-termini. In one instance in Cnidaria, the Hh N-terminal signaling domain is associated with a VWA domain and lacks a Hog domain, suggesting a modular mode of evolution also for the N-terminal domain. The Hog domain proteins, the inteins and VWA-Vint proteins are three families of Hint domain proteins that evolved in parallel in eukaryotes.

  • 10. Chen, Nansheng
    et al.
    Mah, Allan
    Blacque, Oliver E.
    Chu, Jeffrey
    Phgora, Kiran
    Bakhoum, Mathieu W.
    Newbury, C. Rebecca Hunt
    Khattra, Jaswinder
    Chan, Susanna
    Go, Anne
    Efimenko, Evgeni
    Södertörn University, School of Life Sciences. Karolinska Institute.
    Johnsen, Robert
    Phirke, Prasad
    Södertörn University, School of Life Sciences. Karolinska Institute.
    Swoboda, Peter
    Södertörn University, School of Life Sciences. Karolinska Institute.
    Marra, Marco
    Moerman, Donald G.
    Leroux, Michel R.
    Baillie, David L.
    Stein, Lincoln D.
    Identification of ciliary and ciliopathy genes in Caenorhabditis elegans through comparative genomics2006In: Genome Biology, ISSN 1465-6906, E-ISSN 1474-760X, Vol. 7, no 12, R126- p.Article in journal (Refereed)
    Abstract [en]

    Background: The recent availability of genome sequences of multiple related Caenorhabditis species has made it possible to identify, using comparative genomics, similarly transcribed genes in Caenorhabditis elegans and its sister species. Taking this approach, we have identified numerous novel ciliary genes in C. elegans, some of which may be orthologs of unidentified human ciliopathy genes. Results: By screening for genes possessing canonical X-box sequences in promoters of three Caenorhabditis species, namely C. elegans, C. briggsae and C. remanei, we identified 93 genes ( including known X-box regulated genes) that encode putative components of ciliated neurons in C. elegans and are subject to the same regulatory control. For many of these genes, restricted anatomical expression in ciliated cells was confirmed, and control of transcription by the ciliogenic DAF-19 RFX transcription factor was demonstrated by comparative transcriptional profiling of different tissue types and of daf-19(+) and daf-19(-) animals. Finally, we demonstrate that the dye-filling defect of dyf-5( mn400) animals, which is indicative of compromised exposure of cilia to the environment, is caused by a nonsense mutation in the serine/threonine protein kinase gene M04C9.5. Conclusion: Our comparative genomics-based predictions may be useful for identifying genes involved in human ciliopathies, including Bardet-Biedl Syndrome ( BBS), since the C. elegans orthologs of known human BBS genes contain X-box motifs and are required for normal dye filling in C. elegans ciliated neurons.

  • 11.
    Dealtry, Simone
    et al.
    Julius Kühn-Institut – Federal Research Centre for Cultivated Plants (JKI), Institute for Epidemiology and Pathogen Diagnostics, Braunschweig, Germany .
    Ding, Guo-Chun
    Julius Kühn-Institut – Federal Research Centre for Cultivated Plants (JKI), Institute for Epidemiology and Pathogen Diagnostics, Braunschweig, Germany .
    Weichelt, Viola
    Julius Kühn-Institut – Federal Research Centre for Cultivated Plants (JKI), Institute for Epidemiology and Pathogen Diagnostics, Braunschweig, Germany.
    Dunon, Vincent
    ivision of Soil and Water Management, KU Leuven, Heverlee, Belgium .
    Schlüter, Andreas
    Center for Biotechnology (CeBiTec), Institute for Genome Research and Systems Biology, Bielefeld University, Bielefeld, Germany.
    Martini, María Carla
    BBM (Instituto de Biotecnología y Biología Molecular), CCT-CONICET-La Plata, Departamento de Ciencias Biológicas, Facultad de Ciencias Exactas, Universidad Nacional de La Plata, La Plata, Argentina .
    Papa, María Florencia Del
    IBBM (Instituto de Biotecnología y Biología Molecular), CCT-CONICET-La Plata, Departamento de Ciencias Biológicas, Facultad de Ciencias Exactas, Universidad Nacional de La Plata, La Plata, Argentina .
    Lagares, Antonio
    IBBM (Instituto de Biotecnología y Biología Molecular), CCT-CONICET-La Plata, Departamento de Ciencias Biológicas, Facultad de Ciencias Exactas, Universidad Nacional de La Plata, La Plata, Argentina.
    Amos, Gregory Charles Auton
    School of Life Sciences, University of Warwick, Warwick, United Kingdom .
    Wellington, Elizabeth Margaret Helen
    chool of Life Sciences, University of Warwick, Warwick, United Kingdom .
    Gaze, William Hugo
    School of Life Sciences, University of Warwick, Warwick, United Kingdom .
    Sipkema, Detmer
    Laboratory of Microbiology, Wageningen University, Wageningen, The Netherlands .
    Sjöling, Sara
    Södertörn University, School of Natural Sciences, Technology and Environmental Studies, Biology.
    Springael, Dirk
    Division of Soil and Water Management, KU Leuven, Heverlee, Belgium.
    Heuer, Holger
    Julius Kühn-Institut – Federal Research Centre for Cultivated Plants (JKI), Institute for Epidemiology and Pathogen Diagnostics, Braunschweig, Germany .
    van Elsas, Jan Dirk
    University of Groningen, Groningen, The Netherlands .
    Thomas, Christopher
    School of Biosciences, University of Birmingham, Edgbaston, Birmingham, Warwick, United Kingdom .
    Smalla, Kornelia
    Julius Kühn-Institut – Federal Research Centre for Cultivated Plants (JKI), Institute for Epidemiology and Pathogen Diagnostics, Braunschweig, Germany .
    Cultivation-Independent Screening Revealed Hot Spots of IncP-1, IncP-7 and IncP-9 Plasmid Occurrence in Different Environmental Habitats.2014In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 9, no 2, e89922Article in journal (Refereed)
    Abstract [en]

    IncP-1, IncP-7 and IncP-9 plasmids often carry genes encoding enzymes involved in the degradation of man-made and natural contaminants, thus contributing to bacterial survival in polluted environments. However, the lack of suitable molecular tools often limits the detection of these plasmids in the environment. In this study, PCR followed by Southern blot hybridization detected the presence of plasmid-specific sequences in total community (TC-) DNA or fosmid DNA from samples originating from different environments and geographic regions. A novel primer system targeting IncP-9 plasmids was developed and applied along with established primers for IncP-1 and IncP-7. Screening TC-DNA from biopurification systems (BPS) which are used on farms for the purification of pesticide-contaminated water revealed high abundances of IncP-1 plasmids belonging to different subgroups as well as IncP-7 and IncP-9. The novel IncP-9 primer-system targeting the rep gene of nine IncP-9 subgroups allowed the detection of a high diversity of IncP-9 plasmid specific sequences in environments with different sources of pollution. Thus polluted sites are "hot spots" of plasmids potentially carrying catabolic genes.

  • 12.
    Degerholm, Jenny
    et al.
    Stockholm University.
    Gundersen, Kjell
    Stockholm University.
    Bergman, Birgitta
    Stockholm University.
    Söderbäck, Erik
    Stockholm University.
    Phosphorus-limited growth dynamics in two Baltic Sea cyanobacteria, Nodularia sp and Aphanizomenon sp.2006In: FEMS Microbiology Ecology, ISSN 0168-6496, E-ISSN 1574-6941, Vol. 58, no 3, 323-332 p.Article in journal (Refereed)
    Abstract [en]

    Rates of carbon (C) specific growth and nitrogen (N(2)) fixation were monitored in cultures of Baltic Sea Nodularia and Aphanizomenon exposed to gradual limitation by inorganic phosphorus (P). Both cyanobacteria responded by decreased cellular P content followed by lowered rates of growth and N(2) fixation. C-specific growth and cellular N content changed faster in Aphanizomenon both when inorganic P was lowered as well as during reintroduction of P. Aphanizomenon also showed a more rapid increase in N-specific N(2) fixation associated with increased C-specific growth. When ambient concentrations of inorganic P declined, both cyanobacteria displayed higher rates of alkaline phosphatase (APase) activity. Lower substrate half-saturation constants (K(M)) and higher V(max) : K(M) ratio of the APase enzyme associated with Nodularia suggest a higher affinity for dissolved organic P (DOP) substrate than Aphanizomenon. Aphanizomenon, which appears more sensitive to changes in ambient dissolved inorganic P, may be adapted to environments with elevated concentrations of P or repeated intrusions of nutrient-rich water. Nodularia on the other hand, with its higher tolerance to increased P starvation may have an ecological advantage in stratified surface waters of the Baltic Sea during periods of low P availability.

  • 13. Dryselius, Rikard
    et al.
    Nikravesh, Abbas
    Kulyté, Agné
    Södertörn University, School of Life Sciences. Karolinska Institute.
    Goh, Shan
    Good, Liam
    Variable coordination of cotranscribed genes in Escherichia coli following antisense repression2006In: BMC Microbiology, ISSN 1471-2180, E-ISSN 1471-2180, Vol. 6, 97- p.Article in journal (Refereed)
    Abstract [en]

    Background: A majority of bacterial genes belong to tight clusters and operons, which complicates gene functional studies using conventional knock-out methods. Antisense agents can down-regulate the expression of genes without disrupting the genome because they bind mRNA and block its expression. However, it is unclear how antisense inhibition affects expression from genes that are cotranscribed with the target. Results: To examine the effects of antisense inhibition on cotranscribed genes, we constructed a plasmid expressing the two reporter genes gfp and DsRed as one transcriptional unit. Incubation with antisense peptide nucleic acid (PNA) targeted to the mRNA start codon region of either the upstream gfp or the downstream DsRed gene resulted in a complete expression discoordination from this artificial construct. The same approach was applied to the three cotranscribed genes in the endogenously expressed lac-operon (lacZ, Y and A) and partial downstream expression coordination was seen when the lacZ start codon was targeted with antisense PNA. Targeting the lacY mRNA start codon region showed no effect on the upstream lacZ gene expression whereas expression from the downstream lacA gene was affected as strongly as the lacY gene. Determination of lacZ and lacY mRNA levels revealed a pattern of reduction that was similar to the Lac-proteins, indicating a relation between translation inhibition and mRNA degradation as a response to antisense PNA treatment. Conclusion: The results show that antisense mediated repression of genes within operons affect cotranscribed genes to a variable degree. Target transcript stability appears to be closely related to inhibition of translation and presumably depends on translating ribosomes protecting the mRNA from intrinsic decay mechanisms. Therefore, for genes within operons and clusters it is likely that the nature of the target transcript will determine the inhibitory effects on cotranscribed genes. Consequently, no simple and specific methods for expression control of a single gene within polycistronic operons are available, and a thorough understanding of mRNA regulation and stability is required to understand the results from both knock-down and knock-out methods used in bacteria.

  • 14. Dzieciatkowska, Monika
    et al.
    Liu, Xin
    Heikema, Astrid P.
    Houliston, R. Scott
    van Belkum, Alex
    Schweda, Elke K. H.
    Södertörn University, School of Life Sciences.
    Gilbert, Michel
    Richards, James C.
    Li, Jianjun
    Rapid method for sensitive screening of oligosaccharide epitopes in the lipooligosaccharide from Campylobacter jejuni strains isolated from Guillain-Barre syndrome and Miller Fisher syndrome patients2008In: Journal of Clinical Microbiology, ISSN 0095-1137, E-ISSN 1098-660X, Vol. 46, no 10, 3429-3436 p.Article in journal (Refereed)
    Abstract [en]

    Campylobacter jejuni lipooligosaccharide (LOS) can trigger Guillain-Barre syndrome (GBS) due to its similarity to human gangliosides. Rapid and accurate structural elucidation of the LOS glycan of a strain isolated from a GBS patient could help physicians determine the spectrum of anti-ganglioside antibodies likely to be found and therefore provide valuable assistance in establishing an appropriate course of treatment. The ability of implemented mass spectrometry-based approaches in a clinical setting has been limited by the laborious and time-consuming nature of the protocols, typically 3 to 4 days, used to prepare LOS. In order to improve the analytical throughput, microwave-assisted enzymatic digestion was investigated. In this study, the bacterial cells were suspended in 50 mu l of 20 mM ammonium acetate buffer containing DNase and RNase and treated by direct microwave irradiation for 3 min. Then, proteinase K was added and the samples were again microwaved. The intact LOS samples were analyzed using electrophoresis-assisted open-tubular liquid chromatography-mass spectrometry. The reliability of the rapid, high-throughput technique was demonstrated through analysis of LOS glycans from 73 C. jejuni strains. The structure was elucidated using material from a single colony. The total time for sample preparation and MS analysis is less than 60 min.

  • 15.
    Edlund, Anna
    et al.
    Södertörn University, School of Life Sciences. SLU.
    Jansson, Janet K.
    SLU.
    Changes in active bacterial communities before and after dredging of highly polluted Baltic Sea sediments2006In: Applied and Environmental Microbiology, ISSN 0099-2240, E-ISSN 1098-5336, Vol. 72, no 10, 6800-6807 p.Article in journal (Refereed)
    Abstract [en]

    Bacteria residing in sediments have key functions in the marine food web. However, it has been difficult to correlate the identity and activity of bacteria in sediments due to lack of appropriate methods beyond cultivation-based techniques. Our aim was to use a combination of molecular approaches, bromodeoxyuridine incorporation and immunocapture, terminal restriction fragment length polymorphism, and cloning and sequencing of 16S rRNA genes to assess the composition of growing bacteria in Baltic Sea sediments. The study site was a highly polluted area off the Swedish coast. The sediments were sampled in two consecutive years, before and after remediation, by dredging of the top sediments. Levels of polyaromatic hydrocarbons (PAHs), mercury, and polychlorinated biphenyls were dramatically reduced as a result of the cleanup project. The compositions of growing members of the communities were significantly different at the two sampling periods. In particular, members from the class Deltaproteobacteria and genus Spirochaeta were more dominant before dredging, but members of the classes Gammaproteobacteria and the Flavobacteria represented the most dominant growing populations after dredging. We also cultivated isolates from the polluted sediments that could transform the model PAH compound, phenanthrene. Some of these isolates were confirmed as dominant growing populations by the molecular methods as well. This suite of methods enabled us to link the identity and activity of the members of the sediment communities.

  • 16.
    Edlund, Anna
    et al.
    Södertörn University, School of Life Sciences.
    Jansson, Janet K.
    Use of bromodeoxyuridine immunocapture to identify psychrotolerant phenanthrene-degrading bacteria in phenanthrene-enriched polluted Baltic Sea sediments2008In: FEMS Microbiology Ecology, ISSN 0168-6496, E-ISSN 1574-6941, Vol. 65, no 3, 513-525 p.Article in journal (Refereed)
    Abstract [en]

    The aim of this study was to enrich and identify psychrotolerant phenanthrene-degrading bacteria from polluted Baltic Sea sediments. Polyaromatic hydrocarbon (PAH)-contaminated sediments were spiked with phenanthrene and incubated for 2 months in the presence of bromodeoxyuridine that is incorporated into the DNA of replicating cells. The bromodeoxyuridine-incorporated DNA was extracted by immunocapture and analyzed by terminal-restriction fragment length polymorphism and 16S rRNA gene cloning and sequencing to identify bacterial populations that were growing. In addition, degradation genes were quantified in the bromodeoxyuridine-incorporated DNA by real-time PCR. Phenanthrene concentrations decreased after 2 months of incubation in the phenanthrene-enriched sediments and this reduction correlated to increases in copy numbers of xylE and phnAc dioxygenase genes. Representatives of Exiguobacterium, Schewanella, Methylomonas, Pseudomonas, Bacteroides and an uncultured Deltaproteobacterium and a Gammaproteobacterium dominated the growing community in the phenanthrene-spiked sediments. Isolates that were closely related to three of these bacteria (two pseudomonads and an Exiguobacterium sp.) could reduce phenanthrene concentrations in pure cultures and they all harbored phnAc dioxygenase genes. These results confirm that this combination of culture-based and molecular approaches was useful for identification of actively growing bacterial species with a high potential for phenanthrene degradation.

  • 17.
    Edlund, Charlotta
    et al.
    Södertörn University, Avdelning Naturvetenskap. Karolinska Institute.
    Alvan, G
    Barkholt, L
    Vacheron, F
    Nord, C E
    Pharmacokinetics and comparative effects of telithromycin (HMR 3647) and clarithromycin on the oropharyngeal and intestinal microflora2000In: Journal of Antimicrobial Chemotherapy, ISSN 0305-7453, E-ISSN 1460-2091, Vol. 46, no 5, 741-749 p.Article in journal (Refereed)
    Abstract [en]

    The pharmacokinetics in plasma and saliva of a new ketolide, telithromycin (HMR 3647), and the effect on the normal oropharyngeal and intestinal microflora were studied in healthy volunteers and compared with those of clarithromycin. Ten subjects received 800 mg telithromycin perorally once daily and 10 other subjects received 500 mg clarithromycin bid for 10 days. Blood, saliva and faecal specimens were collected at defined intervals before, during and after administration for pharmacokinetic and microbiological analyses. In subjects receiving telithromycin, the mean C(max), AUC and C(24) (24 h) in saliva exceeded the values obtained from plasma, while saliva and serum pharmacokinetic parameters were in the same range for the clarithromycin group. The quantitative ecological disturbances in the normal microflora during administration of telithromycin were moderate and comparable to those associated with clarithromycin administration. No overgrowth of yeasts or Clostridium difficile occurred. Emergence of resistant strains was seen in both treatment groups. Administration of both telithromycin and clarithromycin was associated with significant increases in MICs for intestinal Bacteroides isolates, which persisted 2 weeks after discontinuation of treatment. In addition, a significant emergence of highly clarithromycin-resistant a-haemolytic streptococci, intestinal enterococci and Enterobacteriaceae was detected at day 10 in the clarithromycin group. In conclusion, administration of telithromycin resulted in high drug levels in saliva, which indicates a good therapeutic profile for throat infections. Telithromycin seems to have a more favourable ecological profile compared with clarithromycin in terms of resistance development in the normal microflora.

  • 18.
    Edlund, Charlotta
    et al.
    Södertörn University, Avdelning Naturvetenskap. Karolinska Institute.
    Nord, C E
    Effect on the human normal microflora of oral antibiotics for treatment of urinary tract infections2000In: Journal of Antimicrobial Chemotherapy, ISSN 0305-7453, E-ISSN 1460-2091, Vol. 46, 41-48 p.Article in journal (Refereed)
    Abstract [en]

    Oral administration of antibiotics for treatment of urinary tract infections (UTIs) can cause ecological disturbances in the normal intestinal microflora. Poorly absorbed drugs can reach the colon in active form, suppress susceptible microorganisms and disturb the ecological balance. Suppression of the normal microflora may lead to reduced colonization resistance with subsequent overgrowth of pre-existing, naturally resistant microorganisms, such as yeasts and Clostridium difficile. New colonization by resistant potential pathogens may also occur and may spread within the body or to other patients and cause severe infections. It is therefore important to learn more about the ecological effects of antibacterial agents on the human microflora. The impact on intestinal microorganisms of oral antibiotics used for the treatment of UTIs is reviewed here. Ampicillin, amoxycillin and co-amoxiclav suppress both the aerobic and anaerobic intestinal microflora with overgrowth of ampicillin-resistant Enterobacteriaceae. Pivmecillinam also affects the intestinal microflora, suppressing Escherichia coli, but does not have a major effect on the anaerobic microflora. Several orally administered cephalosporins, such as cefixime, cefpodoxime, cefprozil and ceftibuten, reduce the number of Enterobacteriaceae and increase the number of enterococci. Colonization with C. difficile has also been observed. Fluoroquinolones eliminate or strongly suppress intestinal Enterobacteriaceae, but affect enterococci and anaerobic bacteria only slightly. When antimicrobial agents are prescribed for the treatment of UTIs, not only the antimicrobial spectrum of the agent but also the potential ecological disturbances, including the risk of emergence of resistant strains, should be considered.

  • 19.
    Edlund, Charlotta
    et al.
    Södertörn University, Avdelning Naturvetenskap. Karolinska Institute.
    Nord, C E
    The evaluation and prediction of the ecologic impact of antibiotics in human phase I and II trials2001In: Clinical Microbiology and Infection, ISSN 1198-743X, E-ISSN 1469-0691, Vol. 7, 37-41 p.Article in journal (Refereed)
  • 20.
    Elväng, Annelie M.
    et al.
    Stockholms universitet.
    Westerberg, Karolina
    Stockholms universitet.
    Jernberg, Cecilia
    Södertörn University, Avdelning Naturvetenskap. Karolinska Institutet.
    Jansson, Janet K
    Södertörn University, Avdelning Naturvetenskap.
    Use of green fluorescent protein and luciferase biomarkers to monitor survival and activity of Arthrobacter chlorophenolicus A6 cells during degradation of 4-chlorophenol in soil2001In: Environmental Microbiology, ISSN 1462-2912, E-ISSN 1462-2920, Vol. 3, no 1, 32-42 p.Article in journal (Refereed)
    Abstract [en]

    The recently isolated novel species Arthrobacter chlorophenolicus A6 is capable of growth on and degradation of high concentrations of 4-chlorophenol (up to 350 mug ml(-1)) as the sole carbon and energy source, This strain shows promise for bioremediation of environmental sites contaminated with high levels of chlorophenols. In this study, green fluorescent protein (gfp) or luciferase (luc) genes were used as biomarkers for monitoring cell number and activity, respectively, during degradation of 4-chlorophenol by A. chlorophenolicus cells. The individual marked strains, Arthrobacter chlorophenolicus A6L (luc-tagged) and Arthrobacter chlorophenolicus A6G (gfp-tagged), were monitored during degradation of 250 mug ml(-1) 4-chlorophenol in pure culture and 175 mug g(-1) 4-chlorophenol in soil microcosms. Both gene-tagged strains were capable of cleaning up the contaminated soil during 9 d incubation. During the bioremediation experiments, the luc-tagged cells were monitored using luminometry and the gfp tagged cells using flow cytometry, in addition to selective plate counting for both strains. The cells remained at high population levels in the soil (evidenced by GFP-fluorescent cell counts) and the A. chlorophenolicus A6L population was metabolically active (evidenced by luciferase activity measurements). These results demonstrate that the Arthrobacter chlorophenolicus A6 inoculum is effective for cleaning-up soil containing high concentrations of 4-chlorophenol.

  • 21. Engel, Katja
    et al.
    Ashby, Deborah
    Brady, Sean F.
    Cowan, Don A.
    Doemer, John
    Edwards, Elizabeth A.
    Fiebig, Klaus
    Martens, Eric C.
    McCormac, Dennis
    Mead, David A.
    Miyazaki, Kentaro
    Moreno-Hagelsieb, Gabriel
    O'Gara, Fergal
    Reid, Alexandra
    Rose, David R.
    Simonet, Pascal
    Sjöling, Sara
    Södertörn University, School of Natural Sciences, Technology and Environmental Studies, Biology.
    Smalla, Kornelia
    Streit, Wolfgang R.
    Tedman-Jones, Jennifer
    Valla, Svein
    Wellington, Elizabeth M. H.
    Wu, Cheng-Cang
    Liles, Mark R.
    Neufeld, Josh D.
    Sessitsch, Angela
    Charles, Trevor C.
    Meeting Report: 1st International Functional Metagenomics Workshop May 7-8, 2012, St. Jacobs, Ontario, Canada2013In: Standards in Genomic Sciences, ISSN 1944-3277, E-ISSN 1944-3277, Vol. 8, no 1, 106-111 p.Article in journal (Other academic)
    Abstract [en]

    This report summarizes the events of the 1st International Functional Metagenomics Workshop. The workshop was held on May 7 and 8, 2012, in St. Jacobs, Ontario, Canada and was focused on building an international functional metagenomics community, exploring strategic research areas, and identifying opportunities for future collaboration and funding. The workshop was initiated by researchers at the University of Waterloo with support from the Ontario Genomics Institute (OGI), Natural Sciences and Engineering Research Council of Canada (NSERC) and the University of Waterloo.

  • 22.
    Fang, Hong
    et al.
    Södertörn University, Avdelning Naturvetenskap. Karolinska Univesity Hospital, Huddinge.
    Edlund, Charlotta
    Södertörn University, Avdelning Naturvetenskap. Karolinska Univesity Hospital, Huddinge.
    Hultenby, K
    Karolinska Univesity Hospital, Huddinge.
    Hedberg, M
    Karolinska Univesity Hospital, Huddinge.
    Effects of cefoxitin on the growth and morphology of Bacteroides thetaiotaomicron strains with different cefoxitin susceptibility2002In: Anaerobe, ISSN 1075-9964, E-ISSN 1095-8274, Vol. 8, no 2, 55-61 p.Article in journal (Refereed)
    Abstract [en]

    To examine the effects of cefoxitin on bacterial growth and cell morphology, two pairs of Bacteroides thetaiotaomicron strains (238, 238 m and 1186, 1186 m) with different susceptibilities to this antibiotic were investigated in the present study. B. thetaiotaomicron 238m and 1186m were resistant laboratory mutants originating from the susceptible wild-type strains B. thetaiotaomicron 238 and 1186, respectively. It has been shown, in a previous study, that the mutant strains had alterations in their penicillin-binding proteins (PBPs) as compared to the parent strains. In the present study, strains 238 and 238m presented almost identical genomic fingerprints by PCR, so did strains 1186 and 1186m, which indicates that the parent and mutant strains have similar genomic background. In comparison with the parent strains, the growth rate of mutant strains was slower in cultures without antibiotic. The growth patterns challenged with cefoxitin were also different between the parent and the mutant strains. In case of the morphological responses to cefoxitin, the mutant strains were more resistant to the effect of cefoxitin than the parent strains. In conclusion, the growth patterns and the morphological changes induced by cefoxitin, of the investigated strains, were associated with the properties of PBPs. The resistant mutants with deficiency in PBPs grew slower than the susceptible parent strains, and cefoxitin caused filamentation at sub-MIC in B. thetaiotaomicron.

  • 23. Fang, Hong
    et al.
    Edlund, Charlotta
    Södertörn University, Avdelning Naturvetenskap.
    Nord, C E
    Hedberg, M
    Selection of cefoxitin-resistant Bacteroides thetaiotaomicron mutants and mechanisms involved in beta-lactam resistance2002In: Clinical Infectious Diseases, ISSN 1058-4838, E-ISSN 1537-6591, Vol. 35, S47-S53 p.Article in journal (Refereed)
    Abstract [en]

    The beta-lactam antibiotics are the most widely used of all the groups of antimicrobials, but beta-lactam resistance is increasingly common among members of the Bacteroides fragilis group. Three major mechanisms are involved in beta-lactam resistance, and they act together in certain instances. In the present study, 2 resistant mutants (238m and 1186m) of Bacteroides thetaiotaomicron, obtained from clinical isolates (238 and 1186) by selection with increasing concentrations of cefoxitin, showed decreased susceptibilities to cefoxitin and other beta-lactam antibiotics. Alterations in both penicillin-binding proteins (PBPs) and outer-membrane proteins (OMPs) were observed in the mutants in comparison with their parent strains. The similar alteration in OMPs was also observed in clinical isolates. In conclusion, the beta-lactam-resistant mutants of B. thetaiotaomicron with deficiency in both PBPs and OMPs can be selected for by exposure to cefoxitin, and several mechanisms are involved in the beta-lactam resistance in the strains investigated.

  • 24. Hao, Limin
    et al.
    Johnsen, Robert
    Lauter, Gilbert
    Södertörn University, School of Life Sciences. Karolinska Institute.
    Baillie, David
    Bürglin, Thomas R.
    Södertörn University, School of Life Sciences. Karolinska Institute.
    Comprehensive analysis of gene expression patterns of hedgehog-related genes2006In: BMC Genomics, ISSN 1471-2164, E-ISSN 1471-2164, Vol. 7, 280- p.Article in journal (Refereed)
    Abstract [en]

    Background: The Caenorhabditis elegans genome encodes ten proteins that share sequence similarity with the Hedgehog signaling molecule through their C-terminal autoprocessing Hint/Hog domain. These proteins contain novel N-terminal domains, and C. elegans encodes dozens of additional proteins containing only these N-terminal domains. These gene families are called warthog, groundhog, ground-like and quahog, collectively called hedgehog (hh)-related genes. Previously, the expression pattern of seventeen genes was examined, which showed that they are primarily expressed in the ectoderm. Results: With the completion of the C. elegans genome sequence in November 2002, we reexamined and identified 61 hh-related ORFs. Further, we identified 49 hh-related ORFs in C. briggsae. ORF analysis revealed that 30% of the genes still had errors in their predictions and we improved these predictions here. We performed a comprehensive expression analysis using GFP fusions of the putative intergenic regulatory sequence with one or two transgenic lines for most genes. The hh-related genes are expressed in one or a few of the following tissues: hypodermis, seam cells, excretory duct and pore cells, vulval epithelial cells, rectal epithelial cells, pharyngeal muscle or marginal cells, arcade cells, support cells of sensory organs, and neuronal cells. Using time-lapse recordings, we discovered that some hh-related genes are expressed in a cyclical fashion in phase with molting during larval development. We also generated several translational GFP fusions, but they did not show any subcellular localization. In addition, we also studied the expression patterns of two genes with similarity to Drosophila frizzled, T23D8.1 and F27E11.3A, and the ortholog of the Drosophila gene dally-like, gpn-1, which is a heparan sulfate proteoglycan. The two frizzled homologs are expressed in a few neurons in the head, and gpn-1 is expressed in the pharynx. Finally, we compare the efficacy of our GFP expression effort with EST, OST and SAGE data. Conclusion: No bona-fide Hh signaling pathway is present in C. elegans. Given that the hh-related gene products have a predicted signal peptide for secretion, it is possible that they constitute components of the extracellular matrix (ECM). They might be associated with the cuticle or be present in soluble form in the body cavity. They might interact with the Patched or the Patched-related proteins in a manner similar to the interaction of Hedgehog with its receptor Patched.

  • 25.
    Hjort, Karin
    et al.
    Södertörn University, School of Natural Sciences, Technology and Environmental Studies, Biology. Uppsala University.
    Presti, Ilaria
    University of Insubria.
    Elväng, Annelie
    Södertörn University, School of Natural Sciences, Technology and Environmental Studies, Biology.
    Marinelli, Flavia
    University of Insubria.
    Sjöling, Sara
    Södertörn University, School of Natural Sciences, Technology and Environmental Studies, Biology.
    Bacterial chitinase with phytopathogen control capacity from suppressive soil revealed by functional metagenomics2014In: Applied Microbiology and Biotechnology, ISSN 0175-7598, E-ISSN 1432-0614, Vol. 98, no 6, 2819-2828 p.Article in journal (Refereed)
    Abstract [en]

    Plant disease caused by fungal pathogens results in vast crop damage globally. Microbial communities of soil that is suppressive to fungal crop disease provide a source for the identification of novel enzymes functioning as bioshields against plant pathogens. In this study, we targeted chitin-degrading enzymes of the uncultured bacterial community through a functional metagenomics approach, using a fosmid library of a suppressive soil metagenome. We identified a novel bacterial chitinase, Chi18H8, with antifungal activity against several important crop pathogens. Sequence analyses show that the chi18H8 gene encodes a 425-amino acid protein of 46 kDa with an N-terminal signal peptide, a catalytic domain with the conserved active site F175DGIDIDWE183, and a chitinase insertion domain. Chi18H8 was expressed (pGEX-6P-3 vector) in Escherichia coli and purified. Enzyme characterization shows that Chi18H8 has a prevalent chitobiosidase activity with a maximum activity at 35 °C at pH lower than 6, suggesting a role as exochitinase on native chitin. To our knowledge, Chi18H8 is the first chitinase isolated from a metagenome library obtained in pure form and which has the potential to be used as a candidate agent for controlling fungal crop diseases. Furthermore, Chi18H8 may also answer to the demand for novel chitin-degrading enzymes for a broad range of other industrial processes and medical purposes.

  • 26. Hood, D W
    et al.
    Randle, G
    Cox, A D
    Makepeace, K
    Li, J J
    Schweda, Elke K H
    Södertörn University, School of Chemistry, Biology, Geography and Environmental Science.
    Richards, J C
    Moxon, E R
    Biosynthesis of cryptic lipopolysaccharide glycoforms in Haemophilus influenzae involves a mechanism similar to that required for O-antigen synthesis2004In: Journal of Bacteriology, ISSN 0021-9193, E-ISSN 1098-5530, Vol. 186, no 21, 7429-7439 p.Article in journal (Refereed)
    Abstract [en]

    It is generally thought that mucosal bacterial pathogens of the genera Haemophilus, Neisseria, and Moraxella elaborate lipopolysaccharide (LPS) that is fundamentally different from that of enteric organisms that express O-specific polysaccharide side chains. Haemophilus influenzae elaborates short-chain LPS that has a role in the pathogenesis of H. influenzae infections. We show that the synthesis of LPS in this organism can no longer be as clearly distinguished from that in other gram-negative bacteria that express an O antigen. We provide evidence that a region of the H. influenzae genome, the hmg locus, is involved in the synthesis of glycoforms in which tetrasaccharide units are added en bloc, not stepwise, to the normal core glycoforms, similar to the biosynthesis of an O-antigen.

  • 27.
    Hårdeman, Fredrik
    et al.
    Södertörn University, School of Life Sciences. Karolinska Institute.
    Sjöling, Sara
    Södertörn University, School of Life Sciences.
    Metagenomic approach for the isolation of a novel low-temperature-active lipase from uncultured bacteria of marine sediment2007In: FEMS Microbiology Ecology, ISSN 0168-6496, E-ISSN 1574-6941, Vol. 59, no 2, 524-534 p.Article in journal (Refereed)
    Abstract [en]

    A novel lipase was isolated from a metagenomic library of Baltic Sea sediment bacteria. Prokaryotic DNA was extracted and cloned into a copy control fosmid vector (pCC1FOS) generating a library of > 7000 clones with inserts of 24-39 kb. Screening for clones expressing lipolytic activity based on the hydrolysis of tributyrin and p-nitrophenyl esters, identified 1% of the fosmids as positive. An insert of 29 kb was fragmented and subcloned. Subclones with lipolytic activity were sequenced and an open reading frame of 978 bp encoding a 35.4-kDa putative lipase/esterase h1Lip1 (DQ118648) with 54% amino acid similarity to a Pseudomomas putida esterase (BAD07370) was identified. Conserved regions, including the putative active site, GDSAG, a catalytic triad (Ser148, Glu242 and His272) and a HGG motif, were identified. The h1Lip1 lipase was over expressed, (pGEX-6P-3 vector), purified and shown to hydrolyse p-nitrophenyl esters of fatty acids with chain lengths up to C-14. Hydrolysis of the triglyceride derivative 1,2-di-O-lauryl-rac-glycero-3-glutaric acid 6'-methylresorufin ester (DGGR) confirmed that h1Lip1 was a lipase. The apparent optimal temperature for h1Lip1, by hydrolysis of p-nitrophenyl butyrate, was 35 degrees C. Thermal stability analysis showed that h1Lip1 was unstable at 25 degrees C and inactivated at 40 degrees C with t(1/2) < 5 min.

  • 28.
    Jernberg, Cecilia
    et al.
    Södertörn University, Avdelning Naturvetenskap. Karolinska Institutet.
    Jansson, Janet K
    Södertörn University, Avdelning Naturvetenskap.
    Impact of 4-chlorophenol contamination and/or inoculation with the 4-chlorophenol-degrading strain, Arthrobacter chlorophenolicus A6L, on soil bacterial community structure2002In: FEMS Microbiology Ecology, ISSN 0168-6496, E-ISSN 1574-6941, Vol. 42, no 3, 387-97 p.Article in journal (Refereed)
    Abstract [en]

    The 4-chlorophenol-degrading strain, Arthrobacter chlorophenolicus A6L (chromosomally tagged with the firefly luciferase gene, luc) was inoculated into 4-chlorophenol-contaminated soil to assess the impact of bioaugmentation with a biodegrading strain on the indigenous microbiota. Simultaneously, the impact of 4-chlorophenol alone, or inoculation with A. chlorophenolicus into non-contaminated soil, was addressed. Using terminal restriction fragment length polymorphism (T-RFLP) several significant changes were detected in community fingerprint patterns obtained from soil microcosms treated under the different conditions. The relative abundances of some populations, as judged by the relative intensity of terminal restriction fragments, were significantly impacted by either 4-chlorophenol, A. chlorophenolicus inoculation, or by a combination of both inoculation and 4-chlorophenol contamination. Some populations were significantly stimulated and others were significantly repressed when compared to control soil with no additions. For several peaks, the positive or negative impact imposed by the treatments increased over the 13-day incubation period. Some members of the bacterial community were specifically sensitive to A. chlorophenolicus inoculation or to 4-chlorophenol contamination, whereas other populations remained relatively unaffected by any of the treatments. The A. chlorophenolicus inoculum was also monitored by T-RFLP and was found to have a significantly higher relative abundance in soil contaminated with 4-chlorophenol. These results were substantiated by a high correlation to luciferase activity measurements and the number of colony forming units of the inoculum. Therefore, the A. chlorophenolicus A6L population was positively stimulated by the presence of the 4-chlorophenol substrate (180 microg g(-1) soil) that it catabolized during the first 8 days of the incubation period as a carbon and energy source. Together, these results demonstrate that specific populations in the soil bacterial community rapidly fluctuated in response to specific disturbances and the resulting shifts in the community may therefore represent an adjustment in community structure favoring those populations best capable of responding to novel stress scenarios.

  • 29.
    Johnsson, Anna
    et al.
    Södertörn University, School of Life Sciences, Molecular biology. Karolinska Institutet.
    Xue-Franzen, Yongtao
    Södertörn University, School of Life Sciences, Molecular biology. Karolinska Intitutet.
    Lundin, Maria
    Södertörn University, School of Life Sciences, Molecular biology. Karolinska Institutet.
    Wright, Anthony P. H.
    Södertörn University, School of Life Sciences, Molecular biology. Karolinska Institutet.
    Stress-specific role of fission yeast Gcn5 histone acetyltransferase in programming a subset of stress response genes2006In: Eukaryotic Cell, ISSN 1535-9778, E-ISSN 1535-9786, Vol. 5, no 8, 1337-1346 p.Article in journal (Refereed)
    Abstract [en]

    Gcn5 is a coactivator protein that contributes to gene activation by acetylating specific lysine residues within the N termini of histone proteins. Gcn5 has been intensively studied in the budding yeast, Saccharomyces cerevisiae, but the features of genes that determine whether they require Gcn5 during activation have not been conclusively clarified. To allow comparison with S. cerevisiae, we have studied the genome-wide role of Gcn5 in the distantly related fission yeast, Schizosaccharomyces pombe. We show that Gcn5 is specifically required for adaptation to KCl- and CaCl2-mediated stress in S. pombe. We have characterized the genome-wide gene expression responses to KCl stress and show that Gcn5 is involved in the regulation of a subset of stress response genes. Gcn5 is most clearly associated with KCl-induced genes, but there is no correlation between Gcn5 dependence and the extent of their induction. Instead, Gen5-dependent KCl-induced genes are specifically enriched in four different DNA motifs. The Gcn5-dependent KCl-induced genes are also associated with biological process gene ontology terms such as carbohydrate metabolism, glycolysis, and nicotinamide metabolism that together constitute a subset of the ontology parameters associated with KCl-induced genes.

  • 30.
    Laurencon, Anne
    et al.
    Université de Lyon, France.
    Dubruille, Raphaelle
    Université de Lyon, France / University of Massachusetts Medical School, USA.
    Efimenko, Evgeni
    Södertörn University, School of Life Sciences. Karolinska Institute.
    Grenier, Guillaume
    Université de Lyon, France.
    Bissett, Ryan
    Université de Lyon, France / University of Glasgow, UK.
    Cortier, Elisabeth
    Université de Lyon, France.
    Rolland, Vivien
    Université de Lyon, France.
    Swoboda, Peter
    Södertörn University, School of Life Sciences. Karolinska Institute.
    Durand, Benedicte
    Université de Lyon, France.
    Identification of novel regulatory factor X (RFX) target genes by comparative genomics in Drosophila species2007In: Genome Biology, ISSN 1465-6906, E-ISSN 1474-760X, Vol. 8, no 9, R195- p.Article in journal (Refereed)
    Abstract [en]

    Background: Regulatory factor X (RFX) transcription factors play a key role in ciliary assembly in nematode, Drosophila and mouse. Using the tremendous advantages of comparative genomics in closely related species, we identified novel genes regulated by dRFX in Drosophila. Results: We first demonstrate that a subset of known ciliary genes in Caenorhabditis elegans and Drosophila are regulated by dRFX and have a conserved RFX binding site (X-box) in their promoters in two highly divergent Drosophila species. We then designed an X-box consensus sequence and carried out a genome wide computer screen to identify novel genes under RFX control. We found 412 genes that share a conserved X-box upstream of the ATG in both species, with 83 genes presenting a more restricted consensus. We analyzed 25 of these 83 genes, 16 of which are indeed RFX target genes. Two of them have never been described as involved in ciliogenesis. In addition, reporter construct expression analysis revealed that three of the identified genes encode proteins specifically localized in ciliated endings of Drosophila sensory neurons. Conclusion: Our X-box search strategy led to the identification of novel RFX target genes in Drosophila that are involved in sensory ciliogenesis. We also established a highly valuable Drosophila cilia and basal body dataset. These results demonstrate the accuracy of the X-box screen and will be useful for the identification of candidate genes for human ciliopathies, as several human homologs of RFX target genes are known to be involved in diseases, such as Bardet-BiedI syndrome.

  • 31. Lerner, S A
    et al.
    Dudek, E J
    Boisvert, W E
    Berndt, Kurt D
    Effect of highly potent antipseudomonal beta-lactam agents alone and in combination with aminoglycosides against Pseudomonas aeruginosa1984In: Reviews Of Infectious Diseases, ISSN 0162-0886, Vol. 6, no Supplement 3, S678-88 p.Article in journal (Refereed)
    Abstract [en]

    The activities of ticarcillin, cefsulodin, ceftazidime, aztreonam, and imipenem, formerly known as N-formimidoyl thienamycin, were evaluated alone and in combination with aminoglycosides against 56 clinical isolates of Pseudomonas aeruginosa, which were characterized by aminoglycoside susceptibility and content of aminoglycoside-modifying enzymatic activities. All beta-lactam agents were highly active against aminoglycoside-susceptible isolates, and with few exceptions the aminoglycoside-resistant isolates were susceptible to all of the beta-lactam agents except ticarcillin. Combinations of the beta-lactam agents with aminoglycosides frequently acted synergistically, but the effect of different beta-lactam agents in combination with an aminoglycoside against individual strains was unpredictable. The presence or absence of an aminoglycoside-modifying enzymatic activity had no observed effect on synergism with tobramycin. Killing-curve experiments with strains in the presence of concentrations of a beta-lactam and an aminoglycoside that were not bactericidal alone (one-fourth the minimal bactericidal concentration) showed synergistic bactericidal action against four strains that were tested. The results demonstrate the great activity of these newer antipseudomonal beta-lactam agents and their potential for synergism with aminoglycosides.

  • 32.
    Lowder, M
    et al.
    University of North Carolina at Charlotte, Charlotte, USA.
    Unge, A
    Stockholms universitet.
    Maraha, Ninwe
    Södertörn University, Avdelning Naturvetenskap.
    Jansson, Janet K
    Södertörn University, Avdelning Naturvetenskap. Stockholms universitet.
    Swiggett, J
    Carolinas Medical Center, Charlotte, USA.
    Oliver, J D
    University of North Carolina at Charlotte, Charlotte, USA.
    Effect of starvation and the viable-but-nonculturable state on green fluorescent protein (GFP) fluorescence in GFP-tagged Pseudomonas fluorescens A5062000In: Applied and Environmental Microbiology, ISSN 0099-2240, E-ISSN 1098-5336, Vol. 66, no 8, 3160-3165 p.Article in journal (Refereed)
    Abstract [en]

    The green fluorescent protein (GFP) gene, gfp, of the jellyfish Aequorea victoria is being used as a reporter system for gene expression and as a marker for tracking prokaryotes and eukaryotes. Cells that have been genetically altered with the gfp gene produce a protein that fluoresces when it is excited by UV light. This unique phenotype allows gth-tagged cells to be specifically monitored by nondestructive means, In this study we determined whether a gfp-tagged strain of Pseudomonas fluorescens continued to fluoresce under conditions under which the cells were starved, viable but nonculturable (VBNC), or dead. Epifluorescent microscopy, flow cytometry, and spectrofluorometry were used to measure fluorescence intensity in starved, VBNC, and dead or dying cells. Results obtained by using how cytometry indicated that microcosms containing VBNC cells, which were obtained by incubation under stress conditions (starvation at 37.5 degrees C), fluoresced at an intensity that mas at least 80% of the intensity of nonstressed cultures, Similarly, microcosms containing starved cells incubated at 5 and 30 degrees C had fluorescence intensities that were 90 to 110% of the intensity of nonstressed cells. VBNC cells remained fluorescent during the entire 6-month incubation period. in addition, cells starved at 5 or 30 degrees C remained fluorescent for at least 11 months. Treatment of the cells with UV light or incubation at 39 or 50 degrees C resulted in a loss of GFP from the cells. There was a strong correlation between cell death and leakage of GFP from the cells, although the extent of leakage varied depending on the treatment, Most dead cells were not GFP fluorescent, but a small proportion of the dead cells retained some GFP at a lower concentration than the concentration in live cells, Our results suggest that gfp-tagged cells remain fluorescent following starvation and entry into the VBNC state but that fluorescence is lost when the cells die, presumably because membrane integrity is lost.

  • 33.
    Lu, Zexun
    et al.
    Södertörn University, School of Chemistry, Biology, Geography and Environmental Science.
    Tombolini, R
    Woo, S
    Zeilinger, S
    Lorito, M
    Jansson, Janet K
    Södertörn University, School of Chemistry, Biology, Geography and Environmental Science. SLU.
    In vivo study of Trichoderma-pathogen-plant interactions, using constitutive and inducible green fluorescent protein reporter systems2004In: Applied and Environmental Microbiology, ISSN 0099-2240, E-ISSN 1098-5336, Vol. 70, no 5, 3073-3081 p.Article in journal (Refereed)
    Abstract [en]

    Plant tissue colonization by Trichoderma atroviride plays a critical role in the reduction of diseases caused by phytopathogenic fungi, but this process has not been thoroughly studied in situ. We monitored in Situ interactions between gfp-tagged biocontrol strains of T. atroviride and soilborne plant pathogens that were grown in cocultures and on cucumber seeds by confocal scanning laser microscopy and fluorescence stereomicroscopy. Spores of T. atroviride adhered to Pythium ultimum mycelia in coculture experiments. In mycoparasitic interactions of T. atroviride with P. ultimum or Rhizoctonia solani, the mycoparasitic hyphae grew alongside the pathogen mycelia, and this was followed by coiling and formation of specialized structures similar to hooks, appressoria, and papillae. The morphological changes observed depended on the pathogen tested. Branching of T. atroviride mycelium appeared to be an active response to the presence of the pathogenic host. Mycoparasitism of P. ultimum by T. atroviride occurred on cucumber seed surfaces while the seeds were germinating. The interaction of these fungi on the cucumber seeds was similar to the interaction observed in coculture experiments. Green fluorescent protein expression under the control of host-inducible promoters was also studied. The induction of specific Trichoderma genes was monitored visually in cocultures, on plant surfaces, and in soil in the presence of colloidal chitin or Rhizoctonia by confocal microscopy and fluorescence stereomicroscopy. These tools allowed initiation of the mycoparasitic gene expression cascade to be monitored in vivo.

  • 34.
    Lund, Bodil
    et al.
    Södertörn University, Avdelning Naturvetenskap. Huddinge University Hospital, Karolinska Institutet.
    Adamsson, I
    Huddinge University Hospital, Karolinska Institutet.
    Edlund, Charlotta
    Södertörn University, Avdelning Naturvetenskap. Huddinge University Hospital, Karolinska Institutet.
    Gastrointestinal transit survival of an Enterococcus faecium probiotic strain administered with or without vancomycin2002In: International Journal of Food Microbiology, ISSN 0168-1605, E-ISSN 1879-3460, Vol. 77, no 1-2, 109-115 p.Article in journal (Refereed)
    Abstract [en]

    The primary aim of this study was to evaluate if an ingested probiotic, containing viable Enterococcus faecium could survive gastrointestinal transit and if so, correlate the amount of the recovered probiotic strain with the host's own enterococci. The second aim was to investigate if simultaneous vancomycin intake influenced the survival and persistence of the probiotic strain and the stability of endogenous enterococci strains. Twenty healthy volunteers were given the probiotic product once daily for 10 days. Half of the subjects were simultaneously given vancomycin. Isolates of E. faecium strains were genotypically or phenotypically analysed with pulsed-field gel electrophoresis (PFGE) and the PhenePlate(TM) system, respectively. In eight of the ten volunteers given only the probiotic, the ingested E. faecium could be detected on day 10, while in none on day 31. From subjects given both probiotic and vancomycin no ingested E. faecium could be detected on day 10 or day 31. The estimated amount of ingested E. faecium recovered from faeces on day 10 ranged from 1.2 x 10(3) to 4.2 x 10(6) colony forming units per gram faeces, which in several cases were a substantial part of the total amount of E. faecium. The E. faecium isolated before probiotic plus vancomycin administration showed no close relationship to the ones isolated 3 weeks after ceased intake in any subjects. In conclusion, the ingested E. faecium strain can survive gastrointestinal transit. After intake, the E. faecium probiotic strain might become a large part of the total E, faecium population. The occurrence of the probiotic strain in the human gut seems to be transient after intake stop. Re-colonization of E. faecium after simultaneous probiotic plus vancomycin intake occurs mainly with strains without close genetic relationship to the strains harboured before treatment or to the ingested E. faecium strain.

  • 35.
    Lund, Bodil
    et al.
    Södertörn University, Avdelning Naturvetenskap. Huddinge University Hospital, Karolinska Institutet.
    Agvald-Öhman, C
    Huddinge University Hospital, Karolinska Institutet.
    Hultberg, Anna
    Södertörn University, Avdelning Naturvetenskap. Huddinge University Hospital, Karolinska Institutet.
    Edlund, Charlotta
    Södertörn University, Avdelning Naturvetenskap. Huddinge University Hospital, Karolinska Institutet.
    Frequent transmission of enterococcal strains between mechanically ventilated patients treated at an intensive care unit2002In: Journal of Clinical Microbiology, ISSN 0095-1137, E-ISSN 1098-660X, Vol. 40, no 6, 2084-2088 p.Article in journal (Refereed)
    Abstract [en]

    The objectives of this investigation were to study the respiratory tract colonization and transmission of enterococci between 20 patients treated with mechanical ventilation at an intensive care unit (ICU), to compare genotyping with phenotyping, and to determine the antibiotic susceptibilities of the isolated enterococci. Samples were collected from the oropharynx, stomach, subglottic space, and trachea within 24 It of intubation, every third day until day 18, and thereafter every fifth day until day 33. Enterococcal isolates (n = 170) were analyzed by pulsed-field gel electrophoresis and with the PhenePlate (PhP) system. The antimicrobial susceptibilities to five agents were determined. Seventeen of the 20 subjects were colonized with enterococci in the respiratory tract; 12 were colonized in the lower respiratory tract. Genotype analyses suggested that 13 patients were involved in a transmission event, including all patients intubated more than 12 days. In conclusion, colonization of resistant enterococci in the respiratory tract of intubated patients treated at an ICU was common. Transmission of enterococci between patients occurred frequently. Prolonged intubation period seems to be a risk factor for enterococcal cross-transmission.

  • 36.
    Lund, Bodil
    et al.
    Södertörn University, School of Life Sciences. Karolinska Univesity Hospital, Huddinge.
    Billström, H
    Karolinska Univesity Hospital, Huddinge.
    Edlund, Charlotta
    Södertörn University, School of Life Sciences. Karolinska Univesity Hospital, Huddinge.
    Increased conjugation frequencies in clinical Enterococcus faecium strains harbouring the enterococcal surface protein gene esp2006In: Clinical Microbiology and Infection, ISSN 1198-743X, E-ISSN 1469-0691, Vol. 12, no 6, 588-591 p.Article in journal (Refereed)
    Abstract [en]

    This study compared the in-vitro ability of Enterococcus faecium isolates of different origin to acquire vanA by conjugation in relation to the occurrence of the esp gene. In total, 29 clinical isolates (15/29 esp+), 30 normal intestinal microflora isolates (2/30 esp+) and one probiotic strain (esp-) were studied with a filter-mating assay. Conjugation events were confirmed by PCR and pulsed-field gel electrophoresis. Among the infection-derived isolates, the esp+ isolates had higher conjugation frequencies compared with esp- isolates (p < 0.001), with a median value of 6.4 x 10(-6) transconjugants/donor. The probiotic strain was shown to acquire vanA vancomycin resistance in in-vitro filter mating experiments.

  • 37.
    Lund, Bodil
    et al.
    Södertörn University, Avdelning Naturvetenskap. Huddinge University Hospital, Karolinska Institutet.
    Edlund, Charlotta
    Södertörn University, Avdelning Naturvetenskap. Huddinge University Hospital, Karolinska Institutet.
    Bloodstream isolates of Enterococcus faecium enriched with the enterococcal surface protein gene, esp, show increased adhesion to eukaryotic cells2003In: Journal of Clinical Microbiology, ISSN 0095-1137, E-ISSN 1098-660X, Vol. 41, no 11, 5183-5185 p.Article in journal (Refereed)
    Abstract [en]

    Infection-derived Enterococcus faecium strains enriched with esp had increased ability to adhere to Caco-2 cells (P < 0.05) and were less genetically diverse than esp-negative isolates. esp-negative E. faecium fecal isolates from healthy individuals adhered significantly better than esp-negative infection isolates (P < 0.05), indicating additional factors of importance to adhesion.

  • 38.
    Lund, Bodil
    et al.
    Södertörn University, Avdelning Naturvetenskap. Karolinska Institutet, Huddinge University Hospital.
    Edlund, Charlotta
    Södertörn University, Avdelning Naturvetenskap. Karolinska Institutet, Huddinge University Hospital.
    Probiotic Enterococcus faecium strain is a possible recipient of the vanA gene cluster2001In: Clinical Infectious Diseases, ISSN 1058-4838, E-ISSN 1537-6591, Vol. 32, no 9, 1384-1385 p.Article in journal (Refereed)
    Abstract [en]

    The characteristics of Enterococcus faecium have led to concern regarding the safety of probiotics that contain this bacterium. The results of an in vitro filter mating assay indicate that a probiotic E. faecium strain might be a potential recipient of vancomycin resistance genes.

  • 39.
    Lund, Bodil
    et al.
    Södertörn University, Avdelning Naturvetenskap.
    Edlund, Charlotta
    Södertörn University, Avdelning Naturvetenskap.
    Rynnel-Dagoo, B
    Lundgren, Y
    Sterner, J
    Nord, C E
    Ecological effects on the oro- and nasopharyngeal microflora in children after treatment of acute otitis media with cefuroxime axetil or amoxycillin-clavulanate as suspensions2001In: Clinical Microbiology and Infection, ISSN 1198-743X, E-ISSN 1469-0691, Vol. 7, no 5, 230-237 p.Article in journal (Refereed)
    Abstract [en]

    Objective To evaluate if the extent of normal microflora disturbances differed between treatment with amoxycillin-clavulanate administered in an active form and cefuroxime axetil administered as an inactive prodrug. Methods Twenty-eight children, 0.5-5 years old, diagnosed with acute otitis media (AOM), were treated with either amoxycillin-clavulanate (13.3 mg/kg 3 times daily) or cefuroxime axetil (15 mg/kg twice daily) for 7 days. Saliva samples and nasopharyngeal swabs were collected before, directly after and 2 weeks after treatment. The saliva samples were quantitatively and qualitatively analyzed and the nasopharyngeal swabs were qualitatively analyzed. All isolated strains were tested for beta -lactamase production. Results Both treatment regimens gave rise to similar alterations of the normal oropharyngeal microflora. In both groups, the amount of Streptococcus salivarius was significantly reduced (P < 0.05). The most common causative pathogens of acute otitis were S. pneumoniae, Haemophilus influenzae and Moraxella catarrhalis. On the day of enrollment, approximately half of the patients, in both groups, were infected with more than one pathogen. The rate of infection or colonization with more than one potential pathogen was low on day 7 but recurred 2 weeks after treatment to similar levels as on day 0. The total number of patients with reinfection, recolonization or recurrence of pathogens on day 21 was 11/12 in the amoxycillin-clavulanate group and 4/7 in the cefuroxime axetil group. The most common <beta>-lactamase producer was M. catarrhalis. Conclusion The local high concentration of antibiotics in the oropharynx immediately after intake of antibiotic suspensions seem to have little or no impact on the extent of disturbance of the microflora in this region. Children of this age group seem prone to either reinfection, recolonization or persistence of pathogens within 2 weeks after treatment. Furthermore, co-infection with more than one pathogen seems common in children with AOM and infection with beta -lactamase producing microorganisms occurs frequently.

  • 40.
    Löfmark, Sonja
    et al.
    Södertörn University, School of Chemistry, Biology, Geography and Environmental Science. Karolinska Institutet.
    Fang, Hong
    Karolinska Institutet.
    Hedberg, Maria
    Karolinska Institutet.
    Edlund, Charlotta
    Södertörn University, School of Chemistry, Biology, Geography and Environmental Science. Karolinska Institutet.
    Inducible metronidazole resistance and nim genes in clinical Bacteroides fragilis group isolates2005In: Antimicrobial Agents and Chemotherapy, ISSN 0066-4804, E-ISSN 1098-6596, Vol. 49, no 3, 1253-1256 p.Article in journal (Refereed)
    Abstract [en]

    Nitroimidazole resistance (nim) genes were detected in 2% of 1,502 clinical Bacteroides fragilis group strains isolated from 19 European countries, and a novel nim gene was identified. High metronidazole resistance could be induced in nim-positive strains, which emphasizes the importance of acknowledging metronidazole resistance in the clinical setting.

  • 41.
    Löfmark, Sonja
    et al.
    Karolinska Institute.
    Jernberg, Cecilia
    Södertörn University, School of Life Sciences. Karolinska Institute.
    Jansson, Janet K.
    Swedish University of Agricultural Sciences.
    Edlund, Charlotta
    Karolinska Institute / Medical Products Agency.
    Clindamycin-induced enrichment and long-term persistence of resistant Bacteroides spp. and resistance genes2006In: Journal of Antimicrobial Chemotherapy, ISSN 0305-7453, E-ISSN 1460-2091, Vol. 58, no 6, 1160-1167 p.Article in journal (Refereed)
    Abstract [en]

    Objectives: The aim was to study the long-term consequences of 1 week clindamycin administration regarding selection and persistence of resistance, resistance determinants and diversity of the Bacteroides spp. in the intestinal microflora. Methods: A total of 1306 Bacteroides isolates were collected from constitutively cultured faecal samples during a 2 year period from eight healthy volunteers. The strains were identified by biochemical and genotyping methods. MIC values were determined by the agar dilution method and presence of resistance genes was screened by real-time PCR. Results: Ecological changes in the intestinal microflora persisting up to 24 months were recorded after a 7 day clindamycin administration to four healthy volunteers. Compared to a control group, not exposed to clindamycin, an enrichment and stabilization of resistant Bacteroides strains and resistance determinants were discovered up to 2 years after clindamycin exposure. Conclusions: The results indicate that even a short-term antibiotic administration can cause long-term alterations in the commensal microbiota of individual subjects, detectable 2 years after dosing. The recorded selection and persistence of resistant strains and resistance genes, illustrates the importance of increasing our knowledge of the role of the abundant intestinal microbial community as a reservoir for spread of resistance.

  • 42.
    Maraha, Ninwe
    et al.
    Södertörn University, School of Chemistry, Biology, Geography and Environmental Science. Karolinska Institutet.
    Backman, Agneta
    Södertörn University, School of Chemistry, Biology, Geography and Environmental Science. Karolinska Institutet.
    Jansson, Janet K
    Södertörn University, School of Chemistry, Biology, Geography and Environmental Science. SLU.
    Monitoring physiological status of GFP-tagged Pseudomonas fluorescens SBW25 under different nutrient conditions and in soil by flow cytometry2004In: FEMS Microbiology Ecology, ISSN 0168-6496, E-ISSN 1574-6941, Vol. 51, no 1, 123-132 p.Article in journal (Refereed)
    Abstract [en]

    Pseudomonas fluorescens SBW25, a plant growth promoting bacterium. has been widely studied due to its potential as an inoculum for improving crop yields. Environmental inoculants are usually applied oil seeds or directly to soil and to effectively promote plant growth they need to be viable and active. However, it is difficult to study the physiological status of specific microorganisms in complex environments, such as soil. In this study, our aim was to use molecular tools to specifically monitor the physiological status of P. fluorescens SBW25 in soil and ill pure cultures incubated under different nutritional conditions. The cells were previously tagged with marker genes (encoding green fluorescent protein and bacterial luciferase) to specifically track the cells in environmental samples. The physiological status of the cells was determined using the viability stains 5-cyano-2,3-ditolyl-tetrazolium chloride (CTC) and propidium iodide (PI), which stain active and dead cells, respectively. Luciferase activity was used to monitor the metabolic activity of the population. Most of the cells died after incubation for nine days in nutrient rich medium. By contrast when incubated under starvation conditions, most of the population was not stained with CTC or PI (i.e. intact but inactive cells), indicating that most of the cells were presumably dormant. In soil, a large fraction of the SBW25 cell population became inactive and died, as determined by a decline in luciferase activity and CTC-stained cells, an increase in PI-stained cells, and an inability of the cells to be cultured oil agar medium. However, approximately 60% of the population was unstained, presumably indicating that the cells entered a state of dormancy in soil similar to that observed under starvation conditions in pure cultures. These results demonstrate the applicability of this approach for monitoring the physiological status of specific cells under stress conditions, such as those experienced by environmental inoculants in soil.

  • 43. Muleta, Diriba
    et al.
    Assefa, Fassil
    Hjort, Karin
    Södertörn University, School of Life Sciences, Molecular biology.
    Roos, Stefan
    Granhall, Ulf
    Characterization of Rhizobacteria isolated from Wild Coffea arabica L.2009In: Engineering in Life Sciences, ISSN 1618-0240, E-ISSN 1618-2863, Vol. 9, no 2, 100-108 p.Article in journal (Refereed)
    Abstract [en]

    Rhizobacteria from wild Arabica coffee Populations (Coffea arabica L.) in southwestern Ethiopia were isolated and characterized. The main purpose was to identify coffee-associated rhizobacteria and evaluate their potential in synthesizing the phytohormone indole acetic acid (IAA) and in degrading the ethylene precursor 1-aminocyclopropane-1-carboxylate (ACC). A total of 878 bacterial isolates were screened, of which 395 (45%) isolates were preliminarily characterized using metabolic identification kits (API). Both Gram-negative and Gram-positive bacteria were isolated, with the former group predominating (63% of cases). Based on pre-screening results of the biochemical tests, 51 of the isolates were subjected to PCR-RFLP (Restriction Fragment Length Polymorphism) analysis that yielded ten groups, of which 24 isolates were identified by 16S rRNA gene sequencing. The major genera identified were Pseudomonas (six species) and Bacillus (four species). Single species of Erwinia, Ochrobactrum and Serratia were also identified. The Erwinia sp., Serratia marcescens and many Pseudomonas spp. produced IAA, and some isolates (all Pseudomonas spp.) were also able to degrade ACC. Several of the microbes found in association with wild Arabica coffee bushes have potential agronomic importance, like e.g. Bacillus thuringiensis, which deserve further testing. According to these in vitro Studies, Isolates of Erwinia, Serratia and Pseudomonas are of particular interest in inoculant development due to their plant growth promoting traits.

  • 44.
    Nesme, Joseph
    et al.
    Université de Lyon, Ecully, France / Helmholtz Zentrum München Deutsches Forschungszentrum für Gesundheit und Umwelt (GmbH), Neuherberg, Germany.
    Achouak, Wafa
    Aix-Marseille Université, Saint-Paul-lez-Durance, France.
    Agathos, Spiros N
    Catholic University of Louvain, Louvain-la-Neuve, Belgium / Yachay Tech University, Urcuquí, Ecuador.
    Bailey, Mark
    Centre for Ecology and Hydrology, Oxford, UK.
    Baldrian, Petr
    Institute of Microbiology of the Czech Academy of Sciences, Praha, Czech Republic.
    Brunel, Dominique
    Centre National de Génotypage, Evry, France.
    Frostegård, Åsa
    Norwegian University of Life Sciences, Aas, Norway.
    Heulin, Thierry
    Aix-Marseille Université, Saint-Paul-lez-Durance, France.
    Jansson, Janet K
    Pacific Northwest National Laboratory, Richland, WA, USA.
    Jurkevitch, Edouard
    The Hebrew University of Jerusalem, Rehovot, Israel.
    Kruus, Kristiina L
    Technical Research Centre of Finland, Espoo, Finland.
    Kowalchuk, George A
    Utrecht University, Utrecht, Netherlands.
    Lagares, Antonio
    Universidad Nacional de La Plata, La Plata, Argentina.
    Lappin-Scott, Hilary M
    Swansea University, Swansea, UK.
    Lemanceau, Philippe
    Université de Bourgogne, Dijon, France.
    Le Paslier, Denis
    Université d'Evry Val d'Essonne, Evry, France.
    Mandic-Mulec, Ines
    University of Ljubljana, Ljubljana, Slovenia.
    Murrell, J Colin
    University of East Anglia, Norwich, UK.
    Myrold, David D
    Oregon State University, Corvallis, OR, USA.
    Nalin, Renaud
    NALINOV, Dremil Lafage, France.
    Nannipieri, Paolo
    University of Florence, Florence, Italy.
    Neufeld, Josh D
    University of Waterloo, Waterloo, ON, Canada.
    O'Gara, Fergal
    National University of Ireland, Cork, Ireland / Curtin University, Perth, WA, Australia.
    Parnell, John J
    National Ecological Observatory Network, Boulder, CO, USA.
    Pühler, Alfred
    Bielefeld University, Bielefeld, Germany.
    Pylro, Victor
    René Rachou Research Centre, Belo Horizonte, Brazil.
    Ramos, Juan L
    Consejo Superior de Investigaciones Científicas, Granada, Spain.
    Roesch, Luiz F W
    Federal University of Pampa, São Gabriel, Brazil.
    Schloter, Michael
    Helmholtz Zentrum München Deutsches Forschungszentrum für Gesundheit und Umwelt (GmbH), Neuherberg, Germany.
    Schleper, Christa
    University of Vienna, Vienna, Austria.
    Sczyrba, Alexander
    Bielefeld University, Bielefeld, Germany.
    Sessitsch, Angela
    AIT Austrian Institute of Technology GmbH, Tulln, Austria.
    Sjöling, Sara
    Södertörn University, School of Natural Sciences, Technology and Environmental Studies, Environmental Science.
    Sørensen, Jan
    niversity of Copenhagen, Frederiksberg, Denmark.
    Sørensen, Søren J
    University of Copenhagen, Copenhagen, Denmark.
    Tebbe, Christoph C
    Thünen-Institute of Biodiversity, Braunschweig, Germany.
    Topp, Edward
    University of Western Ontario, London, ON, Canada.
    Tsiamis, George
    University of Patras, Agrinio, Greece.
    van Elsas, Jan Dirk
    University of Groningen, Groningen, Netherlands.
    van Keulen, Geertje
    Swansea University, Swansea, UK.
    Widmer, Franco
    Institute for Sustainability Sciences, Agroscope, Zürich, Switzerland.
    Wagner, Michael
    University of Vienna, Vienna, Austria.
    Zhang, Tong
    The University of Hong Kong, Hong Kong, China.
    Zhang, Xiaojun
    Shanghai Jiao Tong University, Shanghai, China.
    Zhao, Liping
    Shanghai Jiao Tong University, Shanghai, China.
    Zhu, Yong-Guan
    Chinese Academy of Sciences, Xiamen, China.
    Vogel, Timothy M
    Université de Lyon, Ecully, France.
    Simonet, Pascal
    Swansea University, Swansea, UK.
    Back to the Future of Soil Metagenomics2016In: Frontiers in Microbiology, ISSN 1664-302X, E-ISSN 1664-302X, Vol. 7, 73Article in journal (Refereed)
  • 45.
    Nordin, Karolina
    et al.
    Södertörn University, School of Life Sciences. Stockholm University.
    Unell, M
    Jansson, Janet K
    Södertörn University, School of Life Sciences. SLU.
    Novel 4-chlorophenol degradation gene cluster and degradation route via hydroxyquinol in Arthrobacter chlorophenolicus A62005In: Applied and Environmental Microbiology, ISSN 0099-2240, E-ISSN 1098-5336, Vol. 71, no 11, 6538-6544 p.Article in journal (Refereed)
    Abstract [en]

    Arthrobacter chlorophenolicus A6, a previously described 4-chlorophenol-degrading strain, was found to degrade 4-chlorophenol via hydroxyquinol, which is a novel route for aerobic microbial degradation of this compound. In addition, 10 open reading frames exhibiting sequence similarity to genes encoding enzymes involved in chlorophenol degradation were cloned and designated part of a chlorophenol degradation gene cluster (cph genes). Several of the open reading frames appeared to encode enzymes with similar functions; these open reading frames included two genes, cphA-I and cphA-H, which were shown to encode functional hydroxyquinol 1,2-dioxygenases. Disruption of the cphA-I gene yielded a mutant that exhibited negligible growth on 4-chlorophenol, thereby linking the cph gene cluster to functional catabolism of 4-chlorophenol in A. chlorophenolicus A6. The presence of a resolvase pseudogene in the cph gene cluster together with analyses of the G+C content and codon bias of flanking genes suggested that horizontal gene transfer was involved in assembly of the gene cluster during evolution of the ability of the strain to grow on 4-chlorophenol.

  • 46. Oh, H
    et al.
    Edlund, Charlotta
    Södertörn University, Avdelning Naturvetenskap.
    Mechanism of quinolone resistance in anaerobic bacteria2003In: Clinical Microbiology and Infection, ISSN 1198-743X, E-ISSN 1469-0691, Vol. 9, no 6, 512-517 p.Article in journal (Refereed)
    Abstract [en]

    Several recently developed quinolones have excellent activity against a broad range of aerobic and anaerobic bacteria and are thus potential drugs for the treatment of serious anaerobic and mixed infections. Resistance to quinolones is increasing worldwide, but is still relatively infrequent among anaerobes. Two main mechanisms, alteration of target enzymes (gyrase and topoisomerase IV) caused by chromosomal mutations in encoding genes, or reduced intracellular accumulation due to increased efflux of the drug, are associated with quinolone resistance. These mechanisms have also been found in anaerobic species. High-level resistance to the newer broad-spectrum quinolones often requires stepwise mutations in target genes. The increasing emergence of resistance among anaerobes may be a consequence of previous widespread use of quinolones, which may have enriched first-step mutants in the intestinal tract. Quinolone resistance in the Bacteroides fragilis group strains is strongly correlated with amino acid substitutions at positions 82 and 86 in GyrA (equivalent to positions 83 and 87 of Escherichia coli ). Several studies have indicated that B. fragilis group strains possess efflux pump systems that actively expel quinolones, leading to resistance. DNA gyrase seems also to be the primary target for quinolones in Clostridium difficile , since amino acid substitutions in GyrA and GyrB have been detected in resistant strains. To what extent other mechanisms, such as mutational events in other target genes or alterations in outer-membrane proteins, contribute to resistance among anaerobes needs to be further investigated.

  • 47. Oh, H
    et al.
    El Amin, N
    Davies, T
    Appelbaum, P C
    Edlund, Charlotta
    Södertörn University, Avdelning Naturvetenskap.
    gyrA Mutations associated with quinolone resistance in Bacteroides fragilis group strains2001In: Antimicrobial Agents and Chemotherapy, ISSN 0066-4804, E-ISSN 1098-6596, Vol. 45, no 7, 1977-1981 p.Article in journal (Refereed)
    Abstract [en]

    Mutations in the gyrA gene contribute considerably to quinolone resistance in Escherichia coli. Mechanisms for quinolone resistance in anaerobic bacteria are less well studied. The Bacteroides fragilis group are the anaerobic organisms most frequently isolated from patients with bacteremia and intraabdominal infections. Forty-four clinafloxacin-resistant and-susceptible fecal and clinical isolates of the B. fragilis group (eight Bacteroides fragilis, three Bacteroides ovatus, five Bacteroides thetaiotaomicron, six Bacteroides uniformis, and 22 Bacteroides vulgatus) and six ATCC strains of the B. fragilis group were analyzed as follows: (i) determination of susceptibility to ciprofloxacin, levofloxacin, moxifloxacin, and clinafloxacin by the agar dilution method and (ii) sequencing of the gyrA quinolone resistance-determining region (QRDR) located between amino acid residues equivalent to Ala-67 through Gln-106 in E. coli. Amino acid substitutions were found at hotspots at positions 82 (n = 15) and 86 (n = 8). Strains with Ser82Leu substitutions (n = 13) were highly resistant to all quinolones tested. Mutations in other positions of gyrA were also frequently found in quinolone-resistant and -susceptible isolates. Eight clinical strains that lacked mutations in their QRDR were susceptible to at least two of the quinolones tested. Although newer quinolones have good antimicrobial activity against the B. fragilis group, quinolone resistance in B. fragilis strains can be readily selected in vivo. Mutational events in the QRDR of gyrA seem to contribute to quinolone resistance in Bacteroides species.

  • 48. Oh, H
    et al.
    Hedberg, M
    Wade, D
    Edlund, Charlotta
    Södertörn University, Avdelning Naturvetenskap.
    Activities of synthetic hybrid peptides against anaerobic bacteria: Aspects of methodology and stability2000In: Antimicrobial Agents and Chemotherapy, ISSN 0066-4804, E-ISSN 1098-6596, Vol. 44, no 1, 68-72 p.Article in journal (Refereed)
    Abstract [en]

    The increasing problem of antibiotic resistance among pathogenic bacteria requires development of new antimicrobial agents. One line of investigation is the synthesis of antimicrobial hybrid peptides, The aim of the present investigation was to determine the in vitro activities of 16 cecropin-melittin hybrid peptides (CAMEL analogues) against 60 anaerobic bacterial strains, to compare their activities with those of seven clinically used antimicrobial agents, and to compare different methods for anaerobic susceptibility testing of these peptides. The stability of one of the peptides, temporin B, with different stereoisomeric configurations was investigated in a fecal milieu. The CAMEL analogues showed antimicrobial activity against the anaerobic bacteria, with MICs ranging from 0.125 to 32 mu g/ml. The overall activities (the MICs at which 90% of isolates are inhibited) of the CAR IEL analogues against anaerobic bacteria were mainly inferior to those of imipenem, clindamycin, and piperacillin but were equal to or superior to those of metronidazole, cefoxitin, ciprofloxacin, and chloramphenicol. The agarose dilution method was found to be an accurate method for the testing of large numbers of bacterial strains. The D isomer of temporin B was inactivated more slowly in feces than the L isomer. This study shows that the CAMEL analogues are potential agents for the treatment of anaerobic infections.

  • 49.
    Oh, Herin
    et al.
    Södertörn University, Avdelning Naturvetenskap. Karolinska Institutet.
    Hedberg, M
    Karolinska Institutet.
    Edlund, Charlotta
    Södertörn University, Avdelning Naturvetenskap. Karolinska Institutet.
    Efflux-mediated fluoroquinolone resistance in the Bacteroides fragilis group2002In: Anaerobe, ISSN 1075-9964, E-ISSN 1095-8274, Vol. 8, no 5, 277-282 p.Article in journal (Refereed)
    Abstract [en]

    In order to investigate the role of efflux pumps in fluoroquinolone resistance, 35 Bacteroides fragilis group isolates with various resistance patterns against ciprofloxacin, levofloxacin, moxifloxacin and clinafloxacin were studied. The gyrA genotypes were known in all isolates studied. The accumulation of ciprofloxacin with and without carbonyl cyanide m-chlorophenylhydrazone (CCCP) was investigated using a silicon oil based fluorometric assay. Seventeen of 25 multiquinolone-resistant strains had significantly increased ciprofloxacin accumulation in the presence of CCCP compared to the corresponding susceptible type strains. Ten of the resistant isolates with increased efflux had no target mutations at positions 82 or 86 of their GyrA subunits. Strains with highly enhanced efflux were consequently shown to have a significant decrease of quinolone MIC values in the presence of efflux pump inhibitor. The results of the present study propose that high levels of resistance to older as well as newer fluoroquinolones, could be explained by increased activity of efflux pumps in B. fragilis group strains.

  • 50.
    Rahman, Mokhlasur
    et al.
    Södertörn University, School of Life Sciences.
    Abd, H.
    Romling, U.
    Sandström, G.
    Mollby, R.
    Aeromonas-Acanthamoeba interaction and early shift to a viable but nonculturable state of Aeromonas by Acanthamoeba2008In: Journal of Applied Microbiology, ISSN 1364-5072, E-ISSN 1365-2672, Vol. 104, no 5, 1449-1457 p.Article in journal (Refereed)
    Abstract [en]

    Aims: To investigate the hypothesis that amoeba may comprise a significant environmental reservoir for Aeromonas, Acanthamoeba-Aeromonas interaction experiments were performed. Methods and Results: Acanthamoeba were grown in monoculture and co-cultures with three different species of Aeromonas. Survival, invasion and viable but nonculturable state experiments were performed. We showed that at a low initial bacterial cell density, growth of Aeromonas spp. was inhibited by Acanthamoeba castellanii, while A. castellanii growth was unaffected. In contrast, a high initial bacterial cell density, Aeromonas hydrophila AEW44 and Aeromonas veronii biovar sobria AEW104 suppressed the growth of A. castellanii. Fluorescent and phase-contrast microscopic observations of GFP tagged Aer. hydrophila AEW44 demonstrated that the bacterial cells aggregated on A. castellanii cells after 15 min of incubation and internalized. Aeromonas hydrophila AEW44 cells were found to be actively moving. Interestingly, Aer. hydrophila AEW44 cells shifted more rapidly to a viable but nonculturable form when co-cultured with A. castellanii than in monoculture. Conclusions: We demonstrated that Aeromonas spp. are able to interact with and to infect the protozoan A. castellanii under laboratory conditions. Significance and Impact of the Study: Free-living amoeba might play a role as reservoir for Aeromonas, and thus may increase the transmission of Aeromonas by acting as a vehicle.

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