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  • 1. Ahlinder, Jon
    et al.
    Ohrman, Caroline
    Svensson, Kerstin
    Lindgren, Petter
    Johansson, Anders
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Infectious Diseases.
    Forsman, Mats
    Larsson, Pär
    Sjödin, Andreas
    Increased knowledge of Francisella genus diversity highlights the benefits of optimised DNA-based assays2012In: BMC Microbiology, ISSN 1471-2180, E-ISSN 1471-2180, Vol. 12, p. 220-Article in journal (Refereed)
    Abstract [en]

    Background: Recent advances in sequencing technologies offer promising tools for generating large numbers of genomes, larger typing databases and improved mapping of environmental bacterial diversity. However, DNA-based methods for the detection of Francisella were developed with limited knowledge about genetic diversity. This, together with the high sequence identity between several Francisella species, means there is a high risk of false identification and detection of the highly virulent pathogen Francisella tularensis. Moreover, phylogenetic reconstructions using single or limited numbers of marker sequences often result in incorrect tree topologies and inferred evolutionary distances. The recent growth in publicly accessible whole-genome sequences now allows evaluation of published genetic markers to determine optimal combinations of markers that minimise both time and laboratory costs. Results: In the present study, we evaluated 38 previously published DNA markers and the corresponding PCR primers against 42 genomes representing the currently known diversity of the genus Francisella. The results highlight that PCR assays for Francisella tularensis are often complicated by low specificity, resulting in a high probability of false positives. A method to select a set of one to seven markers for obtaining optimal phylogenetic resolution or diagnostic accuracy is presented. Conclusions: Current multiple-locus sequence-typing systems and detection assays of Francisella, could be improved by redesigning some of the primers and reselecting typing markers. The use of only a few optimally selected sequence-typing markers allows construction of phylogenetic topologies with almost the same accuracy as topologies based on whole-genome sequences.

  • 2.
    Ahmad, Irfan
    et al.
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Department of Microbiology, Tumor and Cell Biology, Karolinska Institutet; Department of Allied Health Sciences, University of Health Sciences.
    Cimdins, Annika
    Beske, Timo
    Römling, Ute
    Detailed analysis of c-di-GMP mediated regulation of csgD expression in Salmonella typhimurium2017In: BMC Microbiology, ISSN 1471-2180, E-ISSN 1471-2180, Vol. 17, article id 27Article in journal (Refereed)
    Abstract [en]

    Background: The secondary messenger cyclic di-GMP promotes biofilm formation by up regulating the expression of csgD, encoding the major regulator of rdar biofilm formation in Salmonella typhimurium. The GGDEF/EAL domain proteins regulate the c-di-GMP turnover. There are twenty-two GGDEF/EAL domain proteins in the genome of S. typhimurium. In this study, we dissect the role of individual GGDEF/EAL proteins for csgD expression and rdar biofilm development. Results: Among twelve GGDEF domains, two proteins upregulate and among fifteen EAL domains, four proteins down regulate csgD expression. We identified two additional GGDEF proteins required to promote optimal csgD expression. With the exception of the EAL domain of STM1703, solely, diguanylate cyclase and phosphodiesterase activities are required to regulate csgD mediated rdar biofilm formation. Identification of corresponding phosphodiesterases and diguanylate cyclases interacting in the csgD regulatory network indicates various levels of regulation by c-di-GMP. The phosphodiesterase STM1703 represses transcription of csgD via a distinct promoter upstream region. Conclusion: The enzymatic activity and the protein scaffold of GGDEF/EAL domain proteins regulate csgD expression. Thereby, c-di-GMP adjusts csgD expression at multiple levels presumably using a multitude of input signals.

  • 3.
    Bergh Drott, Johanna
    et al.
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Virology.
    Alexeyev, Oleg
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Virology.
    Bergström, Patrik
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Virology.
    Elgh, Fredrik
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Virology.
    Olsson, Jan
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Virology.
    Propionibacterium acnes infection induces upregulation of inflammatory genes and cytokine secretion in prostate epithelial cells2010In: BMC Microbiology, ISSN 1471-2180, E-ISSN 1471-2180, Vol. 10, p. 126-Article in journal (Refereed)
    Abstract [en]

    Background: The immune stimulating bacterium Propionibacterium acnes is a frequent colonizer of benign and malignant prostate tissue. To understand the pathogenesis of the earliest phase of this infection, we examined the P. acnes triggered immune response in cultivated prostate epithelial cells.

    Results: Prostate epithelial cells are triggered to secrete IL-6, IL-8 and GM-CSF when infected with P. acnes. The secretion of cytokines is accompanied by NFκB related upregulation of the secreted cytokines as well as several components of the TLR2-NFκB signaling pathway.

    Conclusions: P. acnes has potential to trigger a strong immune reaction in the prostate glandular epithelium. Upon infection of prostate via the retrograde urethral route, the induced inflammatory reaction might facilitate bacterial colonization deeper in the prostate tissue where persistent inflammation may impact the development of prostate diseases as hyperplasia and/or malignancy.

  • 4. Björndal, Asa Szekely
    et al.
    Szekely, Laszlo
    Elgh, Fredrik
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Virology.
    Ebola virus infection inversely correlates with the overall expression levels of promyelocytic leukaemia (PML) protein in cultured cells2003In: BMC Microbiology, ISSN 1471-2180, E-ISSN 1471-2180, Vol. 3, p. 6-Article in journal (Refereed)
    Abstract [en]

    We have established a simple fixation and immunofluorescence staining procedure that allows specific co-detection and precise sub-cellular localization of the PML nuclear bodies and the Ebola virus encoded proteins NP, VP35 and VP40 in formaldehyde treated cells. Interferon-alpha treatment delays virus production in vitro. Intact PML bodies may play an anti-viral role in Ebola infected cells.

  • 5.
    Bröms, Jeanette E.
    et al.
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Clinical Bacteriology. Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    Ishikawa, Takahiko
    Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Wai, Sun N.
    Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Sjöstedt, Anders
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Clinical Bacteriology. Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    A functional VipA-VipB interaction is required for the type VI secretion system activity of Vibrio cholerae O1 strain A15522013In: BMC Microbiology, ISSN 1471-2180, E-ISSN 1471-2180, Vol. 13, p. 96-Article in journal (Refereed)
    Abstract [en]

    Background: Many Gram-negative bacteria rely on a type VI secretion system (T6SS) to infect eukaryotic cells or to compete against other microbes. Common to these systems is the presence of two conserved proteins, in Vibrio cholerae denoted VipA and VipB, which have been shown to interact in many clinically relevant pathogens. In this study, mutagenesis of a defined region within the VipA protein was used to identify residues important for VipB binding in V. cholerae O1 strain A1552. Results: A dramatically diminished interaction was shown to correlate with a decrease in VipB stability and a loss of hemolysin co-regulated protein (Hcp) secretion and rendered the bacterium unable to compete with Escherichia coli in a competition assay. Conclusions: This confirms the biological relevance of the VipA-VipB interaction, which is essential for the T6SS activity of many important human pathogens.

  • 6.
    Drobni, Mirva
    et al.
    Umeå University, Faculty of Medicine, Odontology, Cariology.
    Hallberg, K
    Öhman, Ulla
    Umeå University, Faculty of Medicine, Odontology, Cariology.
    Birve, Anna
    Umeå University, Faculty of Medicine, Odontology, Cariology.
    Persson, Karina
    Umeå University, Faculty of Medicine, Odontology, Cariology.
    Johansson, Ingegerd
    Umeå University, Faculty of Medicine, Odontology, Cariology.
    Strömberg, Nicklas
    Umeå University, Faculty of Medicine, Odontology, Cariology.
    Sequence analyses of fimbriae subunit FimA proteins on Actinomyces naeslundii genospecies 1 and 2 and Actinomyces odontolyticus with variant carbohydrate binding specificities.2006In: BMC Microbiology, ISSN 1471-2180, E-ISSN 1471-2180, Vol. 43, no 6Article in journal (Refereed)
    Abstract [en]

    BACKGROUND: Actinomyces naeslundii genospecies 1 and 2 express type-2 fimbriae (FimA subunit polymers) with variant Galbeta binding specificities and Actinomyces odontolyticus a sialic acid specificity to colonize different oral surfaces. However, the fimbrial nature of the sialic acid binding property and sequence information about FimA proteins from multiple strains are lacking. RESULTS: Here we have sequenced fimA genes from strains of A.naeslundii genospecies 1 (n = 4) and genospecies 2 (n = 4), both of which harboured variant Galbeta-dependent hemagglutination (HA) types, and from A.odontolyticus PK984 with a sialic acid-dependent HA pattern. Three unique subtypes of FimA proteins with 63.8-66.4% sequence identity were present in strains of A. naeslundii genospecies 1 and 2 and A. odontolyticus. The generally high FimA sequence identity (> 97.2%) within a genospecies revealed species specific sequences or segments that coincided with binding specificity. All three FimA protein variants contained a signal peptide, pilin motif, E box, proline-rich segment and an LPXTG sorting motif among other conserved segments for secretion, assembly and sorting of fimbrial proteins. The highly conserved pilin, E box and LPXTG motifs are present in fimbriae proteins from other Gram-positive bacteria. Moreover, only strains of genospecies 1 were agglutinated with type-2 fimbriae antisera derived from A. naeslundii genospecies 1 strain 12104, emphasizing that the overall folding of FimA may generate different functionalities. Western blot analyses with FimA antisera revealed monomers and oligomers of FimA in whole cell protein extracts and a purified recombinant FimA preparation, indicating a sortase-independent oligomerization of FimA. CONCLUSION: The genus Actinomyces involves a diversity of unique FimA proteins with conserved pilin, E box and LPXTG motifs, depending on subspecies and associated binding specificity. In addition, a sortase independent oligomerization of FimA subunit proteins in solution was indicated.

  • 7.
    Enow Oben Ayuk, Constance
    et al.
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Oscarsson, Jan
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Zlatkov, Nikola
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Westermark, Marie
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Duperthuy, Marylise
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Wai, Sun Nyunt
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Uhlin, Bernt Eric
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Elevated recombinant clyA gene expression in the uropathogenic Escherichia coli strain 536, a clue to explain pathoadaptive mutations in a subset of extraintestinal E. coli strains2014In: BMC Microbiology, ISSN 1471-2180, E-ISSN 1471-2180, Vol. 14, p. 216-Article in journal (Refereed)
    Abstract [en]

    There are at least four different variants of ΔclyA, suggesting that such deletions in clyA have arisen at more than one occasion. On the basis of this occurrence of the truncated clyA genes, we considered that there may be a patho-adaptive selection for deletions in clyA in extraintestinal pathogenic E. coli. In E. coli K-12 the clyA gene has been viewed as “cryptic” since it is tightly silenced by the nucleoid structuring protein H-NS. We constructed a restored clyA+ locus in derivatives of the UPEC strain 536 for further investigation of this hypothesis and, in particular, how the gene would be expressed. Our results show that the level of clyA+ expression is highly increased in the UPEC derivatives in comparison with the non-pathogenic E. coli K-12. Transcription of the clyA+ gene was induced to even higher levels when the SfaX regulatory protein was overproduced. The derivative with a restored clyA+ locus displayed a somewhat slower growth than the parental UPEC strain 536 when a sub-inhibitory concentration of the antimicrobial peptide Polymyxin B was added to the growth medium.

  • 8.
    Forsell, Joakim
    et al.
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology.
    Bengtsson-Palme, Johan
    Department of Infectious Diseases, Institute of Biomedicine, The Sahlgrenska Academy, and Centre for Antibiotic Resistance Research (CARe), University of Gothenburg, Gothenburg, Sweden.
    Angelin, Martin
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Infectious Diseases.
    Johansson, Anders
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Clinical Bacteriology. Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    Evengård, Birgitta
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Infectious Diseases.
    Granlund, Margareta
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology.
    The relation between Blastocystis and the intestinal microbiota in Swedish travellers2017In: BMC Microbiology, ISSN 1471-2180, E-ISSN 1471-2180, Vol. 17, article id 231Article in journal (Refereed)
    Abstract [en]

    Background: Blastocystis sp. is a unicellular eukaryote that is commonly found in the human intestine. Its ability to cause disease is debated and a subject for ongoing research. In this study, faecal samples from 35 Swedish university students were examined through shotgun metagenomics before and after travel to the Indian peninsula or Central Africa. We aimed at assessing the impact of travel on Blastocystis carriage and seek associations between Blastocystis and the bacterial microbiota.

    Results: We found a prevalence of Blastocystis of 16/35 (46%) before travel and 15/35 (43%) after travel. The two most commonly Blastocystis subtypes (STs) found were ST3 and ST4, accounting for 20 of the 31 samples positive for Blastocystis. No mixed subtype carriage was detected. All ten individuals with a typable ST before and after travel maintained their initial ST. The composition of the gut bacterial community was not significantly different between Blastocystis-carriers and non-carriers. Interestingly, the presence of Blastocystis was accompanied with higher abundances of the bacterial genera Sporolactobacillus and Candidatus Carsonella. Blastocystis carriage was positively associated with high bacterial genus richness, and negatively correlated to the Bacteroides-driven enterotype. These associations were both largely dependent on ST4 – a subtype commonly described from Europe – while the globally prevalent ST3 did not show such significant relationships.

    Conclusions: The high rate of Blastocystis subtype persistence found during travel indicates that long-term carriage of Blastocystis is common. The associations between Blastocystis and the bacterial microbiota found in this study could imply a link between Blastocystis and a healthy microbiota as well as with diets high in vegetables. Whether the associations between Blastocystis and the microbiota are resulting from the presence of Blastocystis, or are a prerequisite for colonization with Blastocystis, are interesting questions for further studies.

  • 9.
    Forslund, Anna-Lena
    et al.
    Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Salomonsson, Emelie Näslund
    Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Golovliov, Igor
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Clinical Bacteriology. Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    Kuoppa, Kerstin
    Michell, Stephen
    Titball, Richard
    Oyston, Petra
    Noppa, Laila
    FOI.
    Sjöstedt, Anders
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Clinical Bacteriology. Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    Forsberg, Åke
    Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    The type IV pilin, PilA, is required for full virulence of Francisella tularensis subspecies tularensis2010In: BMC Microbiology, ISSN 1471-2180, E-ISSN 1471-2180, Vol. 10, p. 227-Article in journal (Refereed)
    Abstract [en]

    This suggests that expression and surface localisation of PilA contribute to virulence in the highly virulent type A strain, while PilT was dispensable for virulence in the mouse infection model.

  • 10. Garcia-Aljaro, Cristina
    et al.
    Melado-Rovira, Silvia
    Milton, Debra L.
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Blanch, Anicet R.
    Quorum-sensing regulates biofilm formation in Vibrio scophthalmi2012In: BMC Microbiology, ISSN 1471-2180, E-ISSN 1471-2180, Vol. 12, p. 287-Article in journal (Refereed)
    Abstract [en]

    Background: In a previous study, we demonstrated that Vibrio scophthalmi, the most abundant Vibrio species among the marine aerobic or facultatively anaerobic bacteria inhabiting the intestinal tract of healthy cultured turbot (Scophthalmus maximus), contains at least two quorum-sensing circuits involving two types of signal molecules (a 3-hydroxy-dodecanoyl-homoserine lactone and the universal autoinducer 2 encoded by luxS). The purpose of this study was to investigate the functions regulated by these quorum sensing circuits in this vibrio by constructing mutants for the genes involved in these circuits.

    Results: The presence of a homologue to the Vibrio harveyi luxR gene encoding a main transcriptional regulator, whose expression is modulated by quorum-sensing signal molecules in other vibrios, was detected and sequenced. The V. scophthalmi LuxR protein displayed a maximum amino acid identity of 82% with SmcR, the LuxR homologue found in Vibrio vulnificus. luxR and luxS null mutants were constructed and their phenotype analysed. Both mutants displayed reduced biofilm formation in vitro as well as differences in membrane protein expression by mass-spectrometry analysis. Additionally, a recombinant strain of V. scophthalmi carrying the lactonase AiiA from Bacillus cereus, which causes hydrolysis of acyl homoserine lactones, was included in the study.

    Conclusions: V. scophthalmi shares two quorum sensing circuits, including the main transcriptional regulator luxR, with some pathogenic vibrios such as V. harveyi and V. anguillarum. However, contrary to these pathogenic vibrios no virulence factors (such as protease production) were found to be quorum sensing regulated in this bacterium. Noteworthy, biofilm formation was altered in luxS and luxR mutants. In these mutants a different expression profile of membrane proteins were observed with respect to the wild type strain suggesting that quorum sensing could play a role in the regulation of the adhesion mechanisms of this bacterium.

  • 11.
    Honn, Marie
    et al.
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Clinical Bacteriology.
    Lindgren, Helena
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Clinical Bacteriology.
    Sjöstedt, Anders
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Clinical Bacteriology. Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    The role of MglA for adaptation to oxidative stress of Francisella tularensis LVS2012In: BMC Microbiology, ISSN 1471-2180, E-ISSN 1471-2180, Vol. 12, p. 14-Article in journal (Refereed)
    Abstract [en]

    Background: The Francisella tularensis protein MglA performs complex regulatory functions since it influences the expression of more than 100 genes and proteins in F. tularensis. Besides regulating the igl operon, it has been suggested that it also regulates several factors such as SspA, Hfq, CspC, and UspA, all important to stress adaptation. Therefore, it can be hypothesized that MglA plays an important role for Francisella stress responses in general and for the oxidative stress response specifically.

    Results: We investigated the oxidative stress response of the Delta mglA mutant of the live vaccine strain (LVS) of F. tularensis and found that it showed markedly diminished growth and contained more oxidized proteins than the parental LVS strain when grown in an aerobic milieu but not when grown microaerobically. Moreover, the Delta mglA mutant exhibited an increased catalase activity and reduced expression of the fsl operon and feoB in the aerobic milieu. The mutant was also found to be less susceptible to H2O2. The aberrant catalase activity and gene expression was partially normalized when the Delta mglA mutant was grown in a microaerobic milieu.

    Conclusions: Altogether the results show that the Delta mglA mutant exhibits all the hallmarks of a bacterium subjected to oxidative stress under aerobic conditions, indicating that MglA is required for normal adaptation of F. tularensis to oxidative stress and oxygen-rich environments.

  • 12.
    Johnning, Anna
    et al.
    Department of Infectious Diseases, Institute of Biomedicine, University of Gothenburg, Gothenburg, Sweden; Department of Mathematical Sciences, Chalmers University of Technology, Gothenburg, Sweden.
    Kristiansson, Erik
    Department of Mathematical Sciences, Chalmers University of Technology, Gothenburg, Sweden .
    Angelin, Martin
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Infectious Diseases.
    Marathe, Nachiket
    Department of Infectious Diseases, Institute of Biomedicine, University of Gothenburg, Gothenburg, Sweden; Microbial Culture Collection, National Centre for Cell Science, Pune, India .
    Shouche, Yogesh S.
    Microbial Culture Collection, National Centre for Cell Science, Pune, India .
    Johansson, Anders
    Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    Larsson, D. G. Joakim
    Department of Infectious Diseases, Institute of Biomedicine, University of Gothenburg, Gothenburg, Sweden .
    Quinolone resistance mutations in the faecal microbiota of Swedish travellers to India2015In: BMC Microbiology, ISSN 1471-2180, E-ISSN 1471-2180, Vol. 15, article id 235Article in journal (Refereed)
    Abstract [en]

    Background: International travel contributes to the spread of antibiotic resistant bacteria over the world. Most studies addressing travel-related changes in the faecal flora have focused on specific mobile resistance genes, or depended on culturing of individual bacterial isolates. Antibiotic resistance can, however, also spread via travellers colonized by bacteria carrying chromosomal antibiotic resistance mutations, but this has received little attention so far. Here we aimed at exploring the abundance of chromosomal quinolone resistance mutations in Escherichia communities residing in the gut of Swedish travellers, and to determine potential changes after visiting India. Sweden is a country with a comparably low degree of quinolone use and quinolone resistance, whereas the opposite is true for India. Methods: Massively parallel amplicon sequencing targeting the quinolone-resistance determining region of gyrA and parC was applied to total DNA extracted from faecal samples. Paired samples were collected from 12 Swedish medical students before and after a 4-15 week visit to India. Twelve Indian residents were included for additional comparisons. Methods known resistance mutations were common in Swedes before travel as well as in Indians, with a trend for all mutations to be more common in the Indian sub group. There was a significant increase in the abundance of the most common amino acid substitution in GyrA (S83L, from 44 to 72 %, p = 0.036) in the samples collected after return to Sweden. No other substitution, including others commonly associated with quinolone resistance (D87N in GyrA, S80I in ParC) changed significantly. The number of distinct genotypes encoded in each traveller was significantly reduced after their visit to India for both GyrA (p = 0.0020) and ParC (p = 0.0051), indicating a reduced genetic diversity, similar to that found in the Indians. Conclusions: International travel can alter the composition of the Escherichia communities in the faecal flora, favouring bacteria carrying certain resistance mutations, and, thereby, contributes to the global spread of antibiotic resistance. A high abundance of specific mutations in Swedish travellers before visiting India is consistent with the hypothesis that these mutation have no fitness cost even in the absence of an antibiotic selection pressure.

  • 13.
    Karched, Maribasappa
    et al.
    Umeå University, Faculty of Medicine, Department of Odontology.
    Ihalin, Rikka
    Eneslätt, Kjell
    Zhong, D
    Oscarsson, Jan
    Umeå University, Faculty of Medicine, Department of Odontology, Oral Microbiology.
    Wai, Sun Nyunt
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Chen, C
    Asikainen, Sirkka
    Umeå University, Faculty of Medicine, Department of Odontology.
    Vesicle-independent extracellular release of a proinflammatory outer membrane lipoprotein in free-soluble form2008In: BMC Microbiology, ISSN 1471-2180, E-ISSN 1471-2180, Vol. 28, no 8:18Article in journal (Refereed)
    Abstract [sv]

    Aggregatibacter actinomycetemcomitans is an oral bacterium associated with aggressively progressing periodontitis. Extracellular release of bacterial outer membrane proteins has been suggested to mainly occur via outer membrane vesicles. This study investigated the presence and conservation of peptidoglycan-associated lipoprotein (AaPAL) among A. actinomycetemcomitans strains, the immunostimulatory effect of AaPAL, and whether live cells release this structural outer membrane lipoprotein in free-soluble form independent of vesicles. RESULTS: The pal locus and its gene product were confirmed in clinical A. actinomycetemcomitans strains by PCR-restriction fragment length polymorphism and immunoblotting. Culturing under different growth conditions revealed no apparent requirement for the AaPAL expression. Inactivation of pal in a wild-type strain (D7S) and in its spontaneous laboratory variant (D7SS) resulted in pleiotropic cellular effects. In a cell culture insert model (filter pore size 0.02 um), AaPAL was detected from filtrates when strains D7S and D7SS were incubated in serum or broth in the inserts. Electron microscopy showed that A. actinomycetemcomitans vesicles (0.05-0.2 um) were larger than the filter pores and that there were no vesicles in the filtrates. The filtrates were immunoblot negative for a cytoplasmic marker, cyclic AMP (cAMP) receptor protein. An ex vivo model indicated cytokine production from human whole blood stimulated by AaPAL. CONCLUSIONS: Free-soluble AaPAL can be extracellularly released in a process independent of vesicles.

  • 14.
    Lindgren, Marie
    et al.
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Clinical Bacteriology. Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    Bröms, Jeanette E.
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Clinical Bacteriology. Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    Meyer, Lena
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Clinical Bacteriology. Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    Golovliov, Igor
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Clinical Bacteriology. Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    Sjöstedt, Anders
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Clinical Bacteriology. Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    The Francisella tularensis LVS ΔpdpC mutant exhibits a unique phenotype during intracellular infection2013In: BMC Microbiology, ISSN 1471-2180, E-ISSN 1471-2180, Vol. 13, article id 20Article in journal (Refereed)
    Abstract [en]

    Background: A prerequisite for the virulence of the facultative intracellular bacterium Francisella tularensis is effective intramacrophage proliferation, which is preceded by phagosomal escape into the cytosol, and ultimately leads to host cell death. Many components essential for the intracellular life cycle are encoded by a gene cluster, the Francisella pathogenicity island (FPI), constituting a type VI secretion system.

    Results: We characterized the FPI mutant ΔpdpC of the live vaccine strain (LVS) of F. tularensis and found that it exhibited lack of intracellular replication, incomplete phagosomal escape, and marked attenuation in the mouse model, however, unlike a phagosomally contained FPI mutant, it triggered secretion of IL-1β, albeit lower than LVS, and markedly induced LDH release.

    Conclusions: The phenotype of the ΔpdpC mutant appears to be unique compared to previously described F. tularensis FPI mutants.

  • 15.
    Lindmark, Barbro
    et al.
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Rompikuntal, Pramod Kumar
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Vaitkevicius, Karolis
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Song, Tianyan
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Mizunoe, Yoshimitsu
    Uhlin, Bernt Eric
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Guerry, Patricia
    Wai, Sun Nyunt
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Outer membrane vesicle-mediated release of cytolethal distending toxin (CDT) from Campylobacter jejuni2009In: BMC Microbiology, ISSN 1471-2180, E-ISSN 1471-2180, Vol. 16, no 9, p. 220-Article in journal (Refereed)
    Abstract [en]

    BACKGROUND: Cytolethal distending toxin (CDT) is one of the well-characterized virulence factors of Campylobacter jejuni, but it is unknown how CDT becomes surface-exposed or is released from the bacterium to the surrounding environment.

    RESULTS: Our data suggest that CDT is secreted to the bacterial culture supernatant via outer membrane vesicles (OMVs) released from the bacteria. All three subunits (the CdtA, CdtB, and CdtC proteins) were detected by immunogold labeling and electron microscopy of OMVs. Subcellular fractionation of the bacteria indicated that, apart from the majority of CDT detected in the cytoplasmic compartment, appreciable amounts (20-50%) of the cellular pool of CDT proteins were present in the periplasmic compartment. In the bacterial culture supernatant, we found that a majority of the extracellular CDT was tightly associated with the OMVs. Isolated OMVs could exert the cell distending effects typical of CDT on a human intestinal cell line, indicating that CDT is present there in a biologically active form.

    CONCLUSION: Our results strongly suggest that the release of outer membrane vesicles is functioning as a route of C. jejuni to deliver all the subunits of CDT toxin (CdtA, CdtB, and CdtC) to the surrounding environment, including infected host tissue.

  • 16.
    Oscarsson, Jan
    et al.
    Umeå University, Faculty of Medicine, Department of Odontology, Oral Microbiology.
    Karched, Maribasappa
    Umeå University, Faculty of Medicine, Department of Odontology, Oral Microbiology.
    Thay, Bernard
    Umeå University, Faculty of Medicine, Department of Odontology, Oral Microbiology.
    Chen, Casey
    Asikainen, Sirkka
    Umeå University, Faculty of Medicine, Department of Odontology, Oral Microbiology.
    Proinflammatory effect in whole blood by free soluble bacterial components released from planktonic and biofilm cells2008In: BMC Microbiology, ISSN 1471-2180, E-ISSN 1471-2180, Vol. 8, no 206, p. 13-Article in journal (Refereed)
    Abstract [en]

    BackgroundAggregatibacter actinomycetemcomitans is an oral bacterium associated with aggressive forms of periodontitis. Increasing evidence points to a link between periodontitis and cardiovascular diseases, however, the underlying mechanisms are poorly understood. This study investigated the pathogenic potential of free-soluble surface material, released from live planktonic and biofilm A. actinomycetemcomitans cells.

    Results: By employing an ex vivo insert model (filter pore size 20 nm) we demonstrated that the A. actinomycetemcomitans strain D7S and its derivatives, in both planktonic and in biofilm life-form, released free-soluble surface material independent of outer membrane vesicles. This material clearly enhanced the production of several proinflammatory cytokines (IL-1β, TNF-α, IL-6, IL-8, MIP-1β) in human whole blood, as evidenced by using a cytokine antibody array and dissociation-enhanced-lanthanide-fluorescent-immunoassay. In agreement with this, quantitative real-time PCR indicated a concomitant increase in transcription of each of these cytokine genes. Experiments in which the LPS activity was blocked with polymyxin B showed that the stimulatory effect was only partly LPS-dependent, suggesting the involvement of additional free-soluble factors. Consistent with this, MALDI-TOF-MS and immunoblotting revealed release of GroEL-like protein in free-soluble form. Conversely, the immunomodulatory toxins, cytolethal distending toxin and leukotoxin, and peptidoglycan-associated lipoprotein, appeared to be less important, as evidenced by studying strain D7S cdt/ltx double, and pal single mutants. In addition to A. actinomycetemcomitans a non-oral species, Escherichia coli strain IHE3034, tested in the same ex vivo model also released free-soluble surface material with proinflammatory activity.

    ConclusionA. actinomycetemcomitans, grown in biofilm and planktonic form, releases free-soluble surface material independent of outer membrane vesicles, which induces proinflammatory responses in human whole blood. Our findings therefore suggest that release of surface components from live bacterial cells could constitute a mechanism for systemic stimulation and be of particular importance in chronic localized infections, such as periodontitis.

  • 17. Siller, Maria
    et al.
    Janapatla, Rajendra P
    Pirzada, Zaid A
    Hassler, Christine
    Zinkl, Daniela
    Charpentier, Emmanuelle
    Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Functional analysis of the group A streptococcal luxS/AI-2 system in metabolism, adaptation to stress and interaction with host cells2008In: BMC Microbiology, ISSN 1471-2180, E-ISSN 1471-2180, Vol. 8, p. 188-Article in journal (Refereed)
    Abstract [en]

    Background

    The luxS/AI-2 signaling pathway has been reported to interfere with important physiological and pathogenic functions in a variety of bacteria. In the present study, we investigated the functional role of the streptococcal luxS/AI-2 system in metabolism and diverse aspects of pathogenicity including the adaptation of the organism to stress conditions using two serotypes of Streptococcus pyogenes, M1 and M19.

    Results

    Exposing wild-type and isogenic luxS-deficient strains to sulfur-limited media suggested a limited role for luxS in streptococcal activated methyl cycle metabolism. Interestingly, loss of luxS led to an increased acid tolerance in both serotypes. Accordingly, luxS expression and AI-2 production were reduced at lower pH, thus linking the luxS/AI-2 system to stress adaptation in S. pyogenes. luxS expression and AI-2 production also decreased when cells were grown in RPMI medium supplemented with 10% serum, considered to be a host environment-mimicking medium. Furthermore, interaction analysis with epithelial cells and macrophages showed a clear advantage of the luxS-deficient mutants to be internalized and survive intracellularly in the host cells compared to the wild-type parents. In addition, our data revealed that luxS influences the expression of two virulence-associated factors, the fasX regulatory RNA and the virulence gene sibA (psp).

    Conclusion

    Here, we suggest that the group A streptococcal luxS/AI-2 system is not only involved in the regulation of virulence factor expression but in addition low level of luxS expression seems to provide an advantage for bacterial survival in conditions that can be encountered during infections.

  • 18. Sullivan, Patrick F
    et al.
    Allander, Tobias
    Lysholm, Fredrik
    Goh, Shan
    Persson, Bengt
    Jacks, Andreas
    Evengård, Birgitta
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Infectious Diseases.
    Pedersen, Nancy L
    Andersson, Björn
    An unbiased metagenomic search for infectious agents using monozygotic twins discordant for chronic fatigue2011In: BMC Microbiology, ISSN 1471-2180, E-ISSN 1471-2180, Vol. 11, p. 2-Article in journal (Refereed)
    Abstract [en]

    Current, impairing chronic fatigue was not robustly associated with viremia detectable in serum.

  • 19.
    Vestman, Nelly Romani
    et al.
    Umeå University, Faculty of Medicine, Department of Odontology.
    Timby, Niklas
    Umeå University, Faculty of Medicine, Department of Clinical Sciences, Paediatrics.
    Holgerson, Pernilla Lif
    Umeå University, Faculty of Medicine, Department of Odontology.
    Kressirer, Christine A
    Claesson, Rolf
    Umeå University, Faculty of Medicine, Department of Odontology.
    Domellöf, Magnus
    Umeå University, Faculty of Medicine, Department of Clinical Sciences, Paediatrics.
    Öhman, Carina
    Umeå University, Faculty of Medicine, Department of Odontology, School of Dentistry.
    Tanner, Anne CR
    Hernell, Olle
    Umeå University, Faculty of Medicine, Department of Clinical Sciences, Paediatrics.
    Johansson, Ingegerd
    Umeå University, Faculty of Medicine, Department of Odontology, School of Dentistry.
    Characterization and in vitro properties of oral lactobacilli in breastfed infants2013In: BMC Microbiology, ISSN 1471-2180, E-ISSN 1471-2180, Vol. 13, p. 193-Article in journal (Refereed)
    Abstract [en]

    Background: Lactobacillus species can contribute positively to general and oral health and are frequently acquired by breastfeeding in infancy. The present study aimed to identify oral lactobacilli in breast and formula-fed 4 month-old infants and to evaluate potential probiotic properties of the dominant Lactobacillus species detected. Saliva and oral swab samples were collected from 133 infants who were enrolled in a longitudinal study (n=240) examining the effect of a new infant formula on child growth and development. Saliva was cultured and Lactobacillus isolates were identified from 16S rRNA gene sequences. Five L. gasseri isolates that differed in 16S rRNA sequence were tested for their ability to inhibit growth of selected oral bacteria and for adhesion to oral tissues. Oral swab samples were analyzed by qPCR for Lactobacillus gasseri.

    Results: 43 (32.3%) infants were breastfed and 90 (67.7%) were formula-fed with either a standard formula (43 out of 90) or formula supplemented with a milk fat globule membrane (MFGM) fraction (47 out of 90). Lactobacilli were cultured from saliva of 34.1% breastfed infants, but only in 4.7% of the standard and 9.3% of the MFGM supplemented formula-fed infants. L. gasseri was the most prevalent (88% of Lactobacillus positive infants) of six Lactobacillus species detected. L. gasseri isolates inhibited Streptococcus mutans binding to saliva-coated hydroxyapatite, and inhibited growth of S. mutans, Streptococcus sobrinus, Actinomyces naeslundii, Actinomyces oris, Candida albicans and Fusobacterium nucleatum in a concentration dependent fashion. L. gasseri isolates bound to parotid and submandibular saliva, salivary gp340 and MUC7, and purified MFGM, and adhered to epithelial cells. L. gasseri was detected by qPCR in 29.7% of the oral swabs. Breastfed infants had significantly higher mean DNA levels of L. gasseri (2.14 pg/uL) than infants fed the standard (0.363 pg/uL) or MFGM (0.697 pg/uL) formula.

    Conclusions: Lactobacilli colonized the oral cavity of breastfed infants significantly more frequently than formulafed infants. The dominant Lactobacillus was L. gasseri, which was detected at higher levels in breastfed than formula-fed infants and displayed probiotic traits in vitro.

  • 20.
    Zeng, Qing-Yin
    et al.
    Umeå University, Faculty of Science and Technology, Molecular Biology (Faculty of Science and Technology).
    Hansson, Per
    Wang, Xiao-Ru
    Specific and sensitive detection of the conifer pathogen Gremmeniella abietina by nested PCR2005In: BMC Microbiology, ISSN 1471-2180, E-ISSN 1471-2180, Vol. 5, p. 65-Article in journal (Refereed)
    Abstract [en]

    BACKGROUND: Gremmeniella abietina (Lagerb.) Morelet is an ascomycete fungus that causes stem canker and shoot dieback in many conifer species. The fungus is widespread and causes severe damage to forest plantations in Europe, North America and Asia. To facilitate early diagnosis and improve measures to control the spread of the disease, rapid, specific and sensitive detection methods for G. abietina in conifer hosts are needed. RESULTS: We designed two pairs of specific primers for G. abietina based on the 18S rDNA sequence variation pattern. These primers were validated against a wide range of fungi and 14 potential conifer hosts. Based on these specific primers, two nested PCR systems were developed. The first system employed universal fungal primers to enrich the fungal DNA targets in the first round, followed by a second round selective amplification of the pathogen. The other system employed G. abietina-specific primers in both PCR steps. Both approaches can detect the presence of G. abietina in composite samples with high sensitivity, as little as 7.5 fg G. abietina DNA in the host genomic background. CONCLUSION: The methods described here are rapid and can be applied directly to a wide range of conifer species, without the need for fungal isolation and cultivation. Therefore, it represents a promising alternative to disease inspection in forest nurseries, plantations and quarantine control facilities.

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