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  • 1.
    Claesson, Carina
    et al.
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Klinisk mikrobiologi. Linköpings universitet, Hälsouniversitetet.
    Monstein, Hans-Jürg
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Klinisk mikrobiologi. Linköpings universitet, Hälsouniversitetet. Östergötlands Läns Landsting, Laboratoriemedicinskt centrum.
    Nilsson, Lennart
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Klinisk mikrobiologi. Linköpings universitet, Hälsouniversitetet.
    Molecular typing and detection of the aap and atlE genes and ica operon in multi-drug resistant and susceptible coagulase negative staphylococciManuskript (preprint) (Annet vitenskapelig)
    Abstract [en]

    The aims of the present study were to identify clinical isolates of multidrug resistant (MDR) and susceptible coagulase negative staphylococci (CoNS), (n=76) to the species level by rpoB amplicon sequencing and to detect and compare the presence of the atlE and aap genes and the ica operon between MDR (n=26) and susceptible (n=27) Staphylococcus epidermidis isolates. Detection of the atlE and aap genes and ica operon was carried out using PCR amplification. Most of the isolates were S. epidermidis, both among the MDR and susceptible CoNS. Staphylococcus haemolyticus was the only other species found in the MDR group. All MDR and 96% of the susceptible S. epidermidis isolates carried the atlE gene. The ica operon was present in about 30% of both the MDR and susceptible S. epidermidis isolates. By comparison, aap gene carriage was more common among susceptible S. epidermidis isolates (44%) than the MDR S. epidermidis isolates (27%). The atlE gene was the only gene that was found alone in the S. epidermidis genome. About 25% of the S. epidermidis isolates carried the atlE and aap genes and the ica operon simultaneously. In conclusion, rpoB gene amplicon sequencing is an easy and reliable method to identify CoNS isolates at the species and subspecies level. Both MDR and susceptible S. epidermidis isolates were found to be well equipped with adhesion and aggregation genes which might help them to adhere to artificial surfaces, colonize hospitalised patients and cause biofilm-related infections.

  • 2. Evertsson, U
    et al.
    Monstein, Hans-Jurg
    Linköpings universitet, Hälsouniversitetet. Linköpings universitet, Institutionen för hälsa och miljö. Östergötlands Läns Landsting, Laboratoriemedicinskt centrum, Klinisk mikrobiologi.
    Johansson, AG
    Detection and identification of fungi in blood using broad-range 28S rDNA PCR amplification and species-specific hybridisation2000Inngår i: Acta Pathologica, Microbiologica et Immunologica Scandinavica (APMIS), ISSN 0903-4641, E-ISSN 1600-0463, Vol. 108, nr 5, s. 385-392Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    The aim of the present study was to develop a PCR-based method to detect and identify fungi directly from human venous blood. We used broad-range PCR primers that targeted a part of the large subunit 28S rRNA genes. To obtain species-specific hybridisation probes, type strains of Candida albicans, C. glabrata, C. krusei, C. parapsilosis, C. tropicalis and Cryptococcus neoformans were PCR amplified, and the amplicons were analysed by gene sequencing. Based on the sequence analysis, species-specific probes that targeted variable regions were designed and used in hybridisation analyses. Between 2 to 10 fungal cells/ml of spiked blood samples could be detected and correctly identified to species. We applied the technique to blood samples obtained from two patients with or two patients without verified candidaemia. The three samples of candidaemia patients were correctly identified to species level, and those of the negative patients remained negative. This method is a potential tool for diagnosis of systemic invasive candidiasis.

  • 3.
    Fransén, Karin
    et al.
    Linköpings universitet, Institutionen för biomedicin och kirurgi, Cellbiologi. Linköpings universitet, Hälsouniversitetet.
    Klintenäs, Maria
    Linköpings universitet, Institutionen för biomedicin och kirurgi, Cellbiologi. Linköpings universitet, Hälsouniversitetet.
    Österström, Anna
    Linköpings universitet, Institutionen för biomedicin och kirurgi, Cellbiologi. Linköpings universitet, Hälsouniversitetet.
    Dimberg, Jan
    Department of Natural Science and Biomedicine, University College of Health Sciences, Jönköping, Sweden.
    Monstein, Hans-Jürg
    Östergötlands Läns Landsting, Laboratoriemedicinskt centrum, Molekylärbiologiska tekniklaboratoriet.
    Söderkvist, Peter
    Linköpings universitet, Institutionen för biomedicin och kirurgi, Cellbiologi. Linköpings universitet, Hälsouniversitetet.
    Mutation analysis of the BRAF, ARAF and RAF-1 genes in human colorectal adenocarcinomas2004Inngår i: Carcinogenesis, ISSN 0143-3334, E-ISSN 1460-2180, Vol. 25, nr 4, s. 527-533Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Colorectal cancer is a multi-step process characterized by a sequence of genetic alterations in cell growth regulatory genes, such as the adenomatous polyposis coli, KRAS, p53 and DCC genes. In the present study mutation analysis was performed with SSCA/direct sequencing of the hot-spot regions in exons 11 and 15 for the BRAF gene and exons 1–2 for the KRAS gene in 130 primary colorectal cancer tumors and correlated with clinico-pathological and mutational data. We also performed mutation analysis of the corresponding conserved regions in the ARAF and RAF-1 genes. Mutations in the BRAF and KRAS genes were found in 11.5 and 40% of the tumors, respectively. One germline exonic and nine germline intronic genetic variants were found in the ARAF and RAF-1 genes. All of the BRAF mutations were located in the kinase domain of the conserved region 3 in exon 15 of the BRAF gene. One novel somatic mutation was also identified in the BRAF gene. The majority of the BRAF mutations were found in colon compared with rectal tumors (P = 0.014). In agreement with others, a statistically significant correlation between BRAF mutations and microsatellite instability could be found. A negative correlation was also evident between mutations in the BRAF and KRAS genes, which supports earlier studies where somatic mutations in these genes are mutually exclusive. Collectively, our results provide support for the idea that activation of the MAP kinase pathway, especially via BRAF and KRAS mutations, is of critical importance for the development of colorectal cancer.

  • 4. Grahn, Niclas
    et al.
    Hmani-Aifa, Mounira
    Fransén, Karin
    Linköpings universitet, Hälsouniversitetet. Linköpings universitet, Institutionen för biomedicin och kirurgi, Avdelningen för medicinsk cellbiologi.
    Söderkvist, Peter
    Linköpings universitet, Hälsouniversitetet. Linköpings universitet, Institutionen för biomedicin och kirurgi, Avdelningen för medicinsk cellbiologi.
    Monstein, Hans-Jurg
    Linköpings universitet, Hälsouniversitetet. Linköpings universitet, Institutionen för biomedicin och kirurgi, Avdelningen för medicinsk cellbiologi. Östergötlands Läns Landsting, Laboratoriemedicinskt centrum, Molekylärbiologiska tekniklaboratoriet.
    Molecular identification of Helicobacter DNA present in human colorectal adenocarcinomas by 16S rDNA PCR amplification and pyrosequencing analysis2005Inngår i: Journal of Medical Microbiology, ISSN 0022-2615, E-ISSN 1473-5644, Vol. 54, nr 11, s. 1031-1035Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Seroepidemiological studies have indicated that Helicobacter pylori infection might be a possible risk factor for colorectal adenocarcinoma (CRC) development. However, limited information is available as to whether or not Helicobacter species are present in CRC tissues. In this study the presence of Helicobacter DNA in 77 CRC biopsies was investigated by means of a Helicobacter species-specific 16S rDNA PCR assay and real-time DNA pyrosequencing of the 16S rDNA variable V3 region. Pyrosequencing revealed the presence of Helicobacter DNA sequences in 21 of 77 biopsy specimens (27%). 16S rDNA sequences corresponding to H. pylori 26695 and H. pylori J99 were most commonly found. Intriguingly, one sequence belonged to Helicobacter mustelae, previously identified in ferrets. No significant correlations were found in the prevalence of Helicobacter DNA between colon and rectum tumour biopsies (P = 0.815), nor between Dukes' classes A/B and C/D (P = 0.262). 16S rDNA PCR amplification combined with pyrosequencing analysis of 16S rDNA variable V3 regions provides a powerful molecular tool to identify Helicobacter species in human biopsy specimens. © 2005 SGM.

  • 5.
    Gustavsson, O.
    et al.
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Avdelningen för mikrobiologi och molekylär medicin. Linköpings universitet, Medicinska fakulteten. Region Östergötland, Diagnostikcentrum, Klinisk mikrobiologi. Innlandet Hospital Trust, Norway.
    Johansson, A. V.
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Avdelningen för mikrobiologi och molekylär medicin. Linköpings universitet, Medicinska fakulteten.
    Monstein, Hans-Jurg
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Avdelningen för mikrobiologi och molekylär medicin. Linköpings universitet, Medicinska fakulteten. Region Östergötland, Diagnostikcentrum, Klinisk mikrobiologi.
    Nilsson, Lennart E
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Avdelningen för mikrobiologi och molekylär medicin. Linköpings universitet, Medicinska fakulteten.
    Bredberg, A.
    Innlandet Hospital Trust, Norway; Lund University, Sweden.
    A wide spectrum of fastidious and ampicillin-susceptible bacteria dominate in animal-caused wounds2016Inngår i: European Journal of Clinical Microbiology and Infectious Diseases, ISSN 0934-9723, E-ISSN 1435-4373, Vol. 35, nr 8, s. 1315-1321Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    The main purpose of this study was to assess the actual occurrence of Gram-negative oxidase-positive bacteria (GNOP) in human wounds caused by animals, mostly cat and dog bites and scratches, and with signs of infection. We report a prospective series of 92 wound samples. Routine culturing was combined with a procedure optimised for fastidious GNOP. All GNOP isolates were identified by 16S rDNA sequencing to the species level. We observed a more prominent role of GNOP, including at least 30 species mostly in the families Flavobacteriaceae, Neisseriaceae and Pasteurellaceae, and less of Staphylococcus aureus and streptococci. The antibiotic susceptibility pattern was investigated, as GNOP are associated with sudden onset of serious infections, making an early decision on antibiotic treatment vital. All GNOP isolates judged to be clinically relevant displayed susceptibility to ampicillin and meropenem, but resistance to oxacillin, clindamycin and gentamicin was frequent. Our findings emphasise the need to cover GNOP as recommended in guidelines, and not only common wound pathogens, when treating an animal-caused wound.

  • 6.
    Hällgren, Anita
    et al.
    Department ofMolecularandClinicalMedicine Linköping University.
    Claesson, Carina
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Klinisk mikrobiologi. Linköpings universitet, Hälsouniversitetet.
    Saeedi, Baharak
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Klinisk mikrobiologi. Linköpings universitet, Hälsouniversitetet. Östergötlands Läns Landsting, Laboratoriemedicinskt centrum, Klinisk mikrobiologi.
    Monstein, Hans-Jurg
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Klinisk mikrobiologi. Linköpings universitet, Hälsouniversitetet. Östergötlands Läns Landsting, Laboratoriemedicinskt centrum.
    Hanberger, Håkan
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Infektionsmedicin. Linköpings universitet, Hälsouniversitetet. Östergötlands Läns Landsting, Medicincentrum, Infektionskliniken i Östergötland.
    Nilsson, Lennart
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Klinisk mikrobiologi. Linköpings universitet, Hälsouniversitetet.
    Molecular detection of aggregation substance, enterococcal surface protein, and cytolysin genes and in vitro adhesion to urinary catheters of Enterococcus faecalis and E. faecium of clinical origin2009Inngår i: International Journal of Medical Microbiology, ISSN 1438-4221, E-ISSN 1618-0607, Vol. 299, nr 5, s. 323-332Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    It has been hypothesized that nosocomial enterococci might have virulence factors that enhance their ability to colonise hospitalised patients. The objectives of this study were to investigate the prevalence of genes encoding 3 virulence factors: aggregation substance (asa1), enterococcal surface protein (esp), and 5 genes within the cytolysin operon (cylA, cylB, cylM, cylL(L), cylL(S)) and cytolysin production in 115 enterococcal clinical isolates (21 Enterococcus faecium and 94 E. faecalis). Adhesion to siliconized latex urinary catheters in relation to presence of esp was analysed in a subset of isolates. The isolates were previously characterised by pulsed-field gel electrophoresis (PFGE). esp was the only virulence gene found in E. faecium. It was found in 71% of the 21 E. faecium isolates. asa1, esp, and the cyl operon were found in 79%, 73% and 13% respectively, of the 94 E. faecalis isolates. There was a complete agreement between presence of the cyl operon and phenotypic cytolysin production. Isolates belonging to a cluster of genetically related isolates carried esp and asa1 more often when compared to unique isolates. No difference was found with respect to cyl genes. E. faecalis isolates adhered with higher bacterial densities than E. faecium. E. faecalis isolates within the same PFGE cluster adhered with similar bacterial densities, but there was no association between adhesion and the presence of esp when isolates within the same cluster were compared. In conclusion, E. faecalis isolates with high-level gentamicin resistance (HLGR) belonging to clusters of genetically related isolates widely distributed in Swedish hospitals, were likely to carry both esp and asa1. Adhesion was not affected by esp.   

  • 7.
    Hällgren, Anita
    et al.
    Linköpings universitet, Institutionen för molekylär och klinisk medicin, Klinisk mikrobiologi. Linköpings universitet, Institutionen för molekylär och klinisk medicin, Infektionsmedicin. Linköpings universitet, Hälsouniversitetet.
    Claesson, Carina
    Linköpings universitet, Institutionen för molekylär och klinisk medicin, Klinisk mikrobiologi. Linköpings universitet, Hälsouniversitetet.
    Saeedi, Baharak
    Linköpings universitet, Institutionen för molekylär och klinisk medicin, Klinisk mikrobiologi. Linköpings universitet, Hälsouniversitetet.
    Monstein, Hans-Jürg
    Linköpings universitet, Institutionen för biomedicin och kirurgi. Linköpings universitet, Hälsouniversitetet.
    Hanberger, Håkan
    Linköpings universitet, Institutionen för molekylär och klinisk medicin, Infektionsmedicin. Linköpings universitet, Hälsouniversitetet.
    Nilsson, Lennart E.
    Linköpings universitet, Institutionen för molekylär och klinisk medicin, Klinisk mikrobiologi. Linköpings universitet, Hälsouniversitetet.
    Frequency of aggregation substance, cytolysin and enterococcal surface protein in vitro adhesion to urinary catheters of E. faecalis and E. faecium of clinical originManuskript (preprint) (Annet vitenskapelig)
    Abstract [sv]

    Enterococcal isolates, 21 E. faecium and 94 E. faecalis, isolated from blood cultures, rectal specimens and various other clinical samples were examined for the presence of the virulence factors hemolysin/cytolysin, aggregation substance (asa1) and enterococcal surface protein (esp). The isolates were previously characterized by pulsed-field gel electrophoresis (PFGE). Adhesion to siliconized latex urinary catheters was analysed in 14 clinical isolates and 3 control strains. Densities of adhering bacteria were determined by a bioluminescence assay of bacterial ATP. The only virulence factor found in E. faecium, esp, was found in 71% of the 21 E. faceium isolates. Cytolysin production, asa1 and esp were found in 13%, 79% and 73%, respectively, of the 94 E. faecalis isolates. Isolates belonging to a cluster of genetically related isolates differed significantly with respect to carriage of esp and asa1 compared to unique isolates, with the virulence factors more commonly found among clustered isolates (p<0.01). No difference was found with respect to cytolysio production (p = 0.76). E. faecalis isolates adhered with higher bacterial densities than E. faecium. E. faecalis isolates within the same PFGE cluster adhered with similar bacterial densities, but there was no association between adhesion and the presence of esp when isolates within the same cluster were compared (p = 0.38 and 0.64).

  • 8.
    Hällgren, Anita
    et al.
    Linköpings universitet, Institutionen för molekylär och klinisk medicin, Klinisk mikrobiologi. Linköpings universitet, Hälsouniversitetet.
    Saeedi, Baharak
    Linköpings universitet, Institutionen för molekylär och klinisk medicin, Klinisk mikrobiologi. Linköpings universitet, Hälsouniversitetet.
    Nilsson, Maud
    Linköpings universitet, Institutionen för molekylär och klinisk medicin, Klinisk mikrobiologi. Linköpings universitet, Hälsouniversitetet.
    Monstein, Hans-Jürg
    Linköpings universitet, Institutionen för biomedicin och kirurgi. Linköpings universitet, Hälsouniversitetet.
    Isaksson, Barbro
    Linköpings universitet, Institutionen för molekylär och klinisk medicin, Klinisk mikrobiologi. Linköpings universitet, Hälsouniversitetet.
    Hanberger, Håkan
    Linköpings universitet, Institutionen för molekylär och klinisk medicin, Infektionsmedicin. Linköpings universitet, Hälsouniversitetet.
    Nilsson, Lennart
    Linköpings universitet, Institutionen för molekylär och klinisk medicin, Klinisk mikrobiologi. Linköpings universitet, Hälsouniversitetet.
    Genetic relatedness among Enterococcus faecalis with transposon-mediated high-level gentamicin resistance in Swedish intensive care units2003Inngår i: Journal of Antimicrobial Chemotherapy, ISSN 0305-7453, E-ISSN 1460-2091, Vol. 52, nr 2, s. 162-167Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    We studied 45 isolates of Enterococcus faecalis with high-level gentamicin resistance (HLGR), all but one concomitantly resistant to ciprofloxacin, and 25 ciprofloxacin-resistant isolates without HLGR for genetic relatedness using pulsed-field gel electrophoresis (PFGE). E. faecalis were isolated from patients admitted to intensive care units at eight hospitals in southern Sweden from December 1996 through December 1998. Genomic analysis by PFGE resulted in three clusters of genetically related isolates (designated clusters I, II and III) and 23 unique clones. Cluster I was found predominantly in the eastern and central parts of southern Sweden and clusters II and III in south-western Sweden. Among the 45 isolates with HLGR, 69% belonged to cluster I, 20% to cluster II, and 11% had unique PFGE patterns, which suggests that the majority of isolates with HLGR are closely related. Among the 25 ciprofloxacin-resistant isolates without HLGR, 68% had unique PFGE patterns, 12% belonged to cluster I and 20% to cluster III, which suggests the ciprofloxacin-resistant isolates are not related. All isolates with HLGR contained the aac(6)Ie-aph(2)Ia gene, which was carried on a Tn5281-like transposon in all isolates except one. We conclude that HLGR in E. faecalis was mainly due to dissemination of genetically related clones during the time studied, and that HLGR in these isolates was due to the presence of the aac(6)Ie-aph(2)Ia gene.

  • 9.
    Jonasson, Jon
    et al.
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Molekylär och immunologisk patologi. Linköpings universitet, Hälsouniversitetet. Östergötlands Läns Landsting, Laboratoriemedicinskt centrum, Klinisk patologi och klinisk genetik.
    Monstein, Hans-Jurg
    Linköpings universitet, Hälsouniversitetet. Linköpings universitet, Institutionen för klinisk och experimentell medicin, Klinisk mikrobiologi. Östergötlands Läns Landsting, Laboratoriemedicinskt centrum, Molekylärbiologiska tekniklaboratoriet.
    Commentary: Classification, identification and subtyping of bacteria based on pyrosequencing and signature matching of 16S rDNA fragments APMIS 110, 263-270: Commentary2007Inngår i: APMIS: Acta pathologica, microbiologica et immunologica Scandinavica. Supplementum, ISSN 0903-465X, E-ISSN 1600-5503, Vol. 115, nr 5, s. 678-679s. 678-679Artikkel i tidsskrift (Annet vitenskapelig)
    Abstract [en]

    [No abstract available]

  • 10.
    Jonsson, K.
    et al.
    Jönsson, K., Department of Laboratory Medicine, Division of Clinical Microbiology, Karolinska Hospital, S-171 76 Stockholm, Sweden.
    Guo, B.P.
    Dept. of Microbiol./Molec. Genet., Harvard Medical School, 200 Longwood Avenue, Boston, MA 02115, United States.
    Monstein, Hans-Jurg
    Linköpings universitet, Hälsouniversitetet. Linköpings universitet, Institutionen för klinisk och experimentell medicin, Klinisk mikrobiologi. Östergötlands Läns Landsting, Laboratoriemedicinskt centrum, Molekylärbiologiska tekniklaboratoriet.
    Mekalanos, J.J.
    Dept. of Microbiol./Molec. Genet., Harvard Medical School, 200 Longwood Avenue, Boston, MA 02115, United States.
    Kronvall, G.
    Department of Laboratory Medicine, Division of Clinical Microbiology, Karolinska Hospital, S-171 76 Stockholm, Sweden.
    Molecular cloning and characterization of two Helicobacter pylori genes coding for plasminogen-binding proteins2004Inngår i: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 101, nr 7, s. 1852-1857Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Helicobacter pylori binds a number of host cell proteins, including the plasma protein plasminogen, which is the proenzyme of the serine protease plasmin. Two H. pylori plasminogen-binding proteins have been described, however, no genes were identified. Here we report the use of a phage display library to clone two genes from the H. pylori CCUG 17874 genome that mediate binding to plasminogen. DNA sequence analysis of one of these genes revealed 96.6% homology with H. pylori 26695 HP0508. A subsequent database search revealed that the amino acid sequence of a lysine-rich C-terminal segment of HP0508 is identical to the C terminus of HP0863. Recombinant proteins expressed from HP0508 and HP0863 bound plasminogen specifically and in a lysine-dependent manner. We designate these genes pgbA and pgbB, respectively. These proteins are expressed by a variety of H. pylori strains, have surface-exposed domains, and do not inhibit plasminogen activation. These results indicate that pgbA and pgbB may allow H. pylori to coat its exterior with plasminogen, which subsequently can be activated to plasmin. The surface acquisition of protease activity may enhance the virulence of H. pylori.

  • 11.
    Karlsson, Anneli
    et al.
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Kirurgi. Linköpings universitet, Hälsouniversitetet.
    Ryberg, Anna
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Klinisk mikrobiologi. Linköpings universitet, Hälsouniversitetet. Östergötlands Läns Landsting, Diagnostikcentrum, Klinisk mikrobiologi.
    Dehnoei, Marjan Nosouhi
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Klinisk mikrobiologi. Linköpings universitet, Hälsouniversitetet. Östergötlands Läns Landsting, Diagnostikcentrum, Klinisk mikrobiologi.
    Borch, Kurt
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Kirurgi. Linköpings universitet, Hälsouniversitetet. Östergötlands Läns Landsting, Centrum för kirurgi, ortopedi och cancervård, Kirurgiska kliniken US.
    Monstein, Hans-Jürg
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Klinisk mikrobiologi. Linköpings universitet, Hälsouniversitetet. Östergötlands Läns Landsting, Diagnostikcentrum, Klinisk mikrobiologi.
    Association between cagA and vacA genotypes and pathogenesis in a Helicobacter pylori infected population from South-eastern Sweden2012Inngår i: BMC Microbiology, ISSN 1471-2180, E-ISSN 1471-2180, Vol. 12, nr 129Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    UNLABELLED: ABSTRACT:

    BACKGROUND: Chronic gastritis, peptic ulcer disease, and gastric cancer have been shown to be related to infection with Helicobacter pylori (H. pylori). Two major virulence factors of H. pylori, CagA and VacA, have been associated with these sequelae of the infection. In this study, total DNA was isolated from gastric biopsy specimens to assess the cagA and vacA genotypes.

    RESULTS: Variations in H. pylori cagA EPIYA motifs and the mosaic structure of vacA s/m/i/d regions were analysed in 155 H. pylori-positive gastric biopsies from 71 individuals using PCR and sequencing. Analysis of a possible association between cagA and vacA genotypes and gastroduodenal pathogenesis was made by logistic regression analysis. We found that H. pylori strains with variation in the number of cagA EPIYA motif variants present in the same biopsy correlated with peptic ulcer, while occurrence of two or more EPIYA-C motifs was associated with atrophy in the gastric mucosa. No statistically significant relation between vacA genotypes and gastroduodenal pathogenesis was observed.

    CONCLUSIONS: The results of this study indicate that cagA genotypes may be important determinants in the development of gastroduodenal sequelae of H. pylori infection. In contrast to other studies, vacA genotypes were not related to disease progression or outcome. In order to fully understand the relations between cagA, vacA and gastroduodenal pathogenesis, the mechanisms by which CagA and VacA act and interact need to be further investigated.

  • 12.
    Karlsson, Anneli
    et al.
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Cellbiologi. Linköpings universitet, Hälsouniversitetet.
    Ryberg, Anna
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Klinisk mikrobiologi. Linköpings universitet, Hälsouniversitetet.
    Nosouhi Dehnoei, Marjan
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Klinisk mikrobiologi. Linköpings universitet, Hälsouniversitetet.
    Borch, Kurt
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Kirurgi. Linköpings universitet, Hälsouniversitetet. Östergötlands Läns Landsting, Centrum för kirurgi, ortopedi och cancervård, Kirurgiska kliniken i Östergötland.
    Monstein, Hans-Jürg
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Klinisk mikrobiologi. Linköpings universitet, Hälsouniversitetet. Östergötlands Läns Landsting, Diagnostikcentrum, Klinisk mikrobiologi.
    Variation in number of cagA EPIYA-C phosphorylation motifs between cultured Helicobacter pylori and biopsy strain DNA2012Inngår i: Infection, Genetics and Evolution, ISSN 1567-1348, E-ISSN 1567-7257, Vol. 12, nr 1, s. 175-179Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    The Helicobacter pylori cagA gene encodes a cytotoxin which is activated by phosphorylation after entering the host epithelial cell. Phosphorylation occurs on specific tyrosine residues within EPIYA motifs in the variable 3'-region. Four different cagA EPIYA motifs have been defined according to the surrounding amino acid sequence; EPIYA-A, -B, -C and -D. Commonly, EPIYA-A and -B are followed by one or more EPIYA-C or -D motif. Due to observed discrepancies in cagA genotypes in cultured H. pylori and the corresponding DNA extracts it has been suggested that genotyping assays preferentially should be performed directly on DNA isolated from biopsy specimens. Gastric biopsies randomly selected from a Swedish cohort were homogenised and used for both direct DNA isolation and for H. pylori specific culturing and subsequent DNA isolation. In 123 of 153 biopsy specimens, the cagA EPIYA genotypes were in agreement with the corresponding cultured H. pylori strains. A higher proportion of mixed cagA EPIYA genotypes were found in the remaining 30 biopsy specimens. Cloning and sequencing of selected cagA EPIYA amplicons revealed variations in number of cagA EPIYA-C motifs in the mixed amplicons. The study demonstrates that culturing of H. pylori introduces a bias in the number of EPIYA-C motif. Consistent with other H. pylori virulence genotyping studies, we suggest that cagA EPIYA analysis should be performed using total DNA isolated from biopsy specimens.

  • 13. Karpati, F
    et al.
    Giedraitis, V
    Thore, M
    Lindman, R
    Monstein, Hans-Jurg
    Linköpings universitet, Hälsouniversitetet. Linköpings universitet, Institutionen för hälsa och miljö. Östergötlands Läns Landsting, Laboratoriemedicinskt centrum, Klinisk mikrobiologi.
    Hjelte, L
    Jonasson, Jon
    Linköpings universitet, Hälsouniversitetet. Linköpings universitet, Institutionen för hälsa och miljö. Östergötlands Läns Landsting, Laboratoriemedicinskt centrum, Klinisk mikrobiologi.
    Arbitrarily primed PCR and sequencing of 16S rDNA for epidemiological typing and species identification of Burkholderia cepacia isolates from Swedish patients with cystic fibrosis reveal genetic heterogeneity2001Inngår i: Acta Pathologica, Microbiologica et Immunologica Scandinavica (APMIS), ISSN 0903-4641, E-ISSN 1600-0463, Vol. 109, nr 5, s. 389-400Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    To investigate whether arbitrarily primed (AP)-PCR and/or 16S rDNA sequencing could be used as rapid methods for epidemiological typing and species identification of clinical Burkholderia isolates from patients with cystic fibrosis (CF), a total of 39 clinical B. cepacia isolates, including 33 isolates from 14 CF patients, were fingerprinted. ERIC-2 primer was used for AP-PCR. The AP-PCR clustering analysis resulted in 14 different clusters at a 70% similarity level. The AP-PRC patterns were individual despite considerable similarities. To sequence rDNA, a broad-range PCR was applied. The PCR product included four variable loops (V8, V3, V4 and V9) of the 16S ribosomal small subunit RNA gene. The multiple sequence alignment produced 12 different patterns, 5 of them including more than one isolate. Heterogeneity of the bases in the V3 region, indicating the simultaneous presence of at least two different types of 16S rRNA genes in the same cell, was revealed in 10 isolates. Most of the CF patients were adults who had advanced disease at follow-up. Both the sequencing and the AP-PCR patterns revealed genetic heterogeneity of isolates between patients. According to the results obtained, AP-PCR could advantageously be used for epidemiological typing of Burkholderia, whereas partial species identification could effectively be obtained by sequencing of the V3 region of the 16S RNA gene.

  • 14. Kolak, M.
    et al.
    Karpati, F.
    Stockholm Cystic Fibrosis Centre, Huddinge University Hospital, Stockholm, Sweden.
    Monstein, Hans-Jurg
    Linköpings universitet, Hälsouniversitetet. Linköpings universitet, Institutionen för klinisk och experimentell medicin, Klinisk mikrobiologi. Östergötlands Läns Landsting, Laboratoriemedicinskt centrum, Molekylärbiologiska tekniklaboratoriet.
    Jonasson, Jon
    Linköpings universitet, Hälsouniversitetet. Linköpings universitet, Institutionen för klinisk och experimentell medicin, Molekylär och immunologisk patologi. Östergötlands Läns Landsting, Laboratoriemedicinskt centrum, Klinisk patologi och klinisk genetik.
    Molecular typing of the bacterial flora in sputum of cystic fibrosis patients2003Inngår i: International Journal of Medical Microbiology, ISSN 1438-4221, E-ISSN 1618-0607, Vol. 293, nr 4, s. 309-317Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Despite recent advances in therapy, lower airway infections remain the major cause of morbidity and mortality in cystic fibrosis (CF) patients. Bacterial colonisation of the lower airways in CF is limited to a few bacterial species, commonly Staphylococcus aureus, Pseudomonas aeruginosa and Haemophilus influenzae. Burkholderia cepacia colonisation is much rarer, but it has been thought to be associated with more advanced lung disease and increased mortality. A rapid characterisation of the bacterial flora in sputum of CF patients is of great importance for proper treatment. The aim of this study was to establish bacterial profiles and to identify pathogenic bacteria in respiratory specimens by means of molecular methods including temporal temperature gradient gel electrophoresis (TTGE) and DNA sequencing of PCR amplicons derived from 16S rDNA variable V3 and V6 regions. Sputa of 13 CF patients (7 males/6 females, age 19-59 years) collected at the Stockholm CF centre were analysed. TTGE revealed the presence of complex bacterial profiles in all samples. The V3 and V6 PCR amplicons were cloned and sequenced by real-time DNA PyrosequencingTM. DNA from Staphylococcus aureus, Haemophilus influenzae, and Pseudomonas aeruginosa, respectively, was identified together with sequences from normal oral cavity flora. The results were in reasonable agreement with those obtained by conventional bacterial culture, considering that only known CF pathogens are included in routine reports. However, the methodology seems too elaborate to be introduced into daily routine.

  • 15.
    Monstein, Hans-Jurg
    et al.
    Linköpings universitet, Hälsouniversitetet. Linköpings universitet, Institutionen för hälsa och miljö.
    Ahrne, A
    Molin, G
    Nikpour-Badr, S
    Linköpings universitet, Hälsouniversitetet. Linköpings universitet, Institutionen för hälsa och miljö.
    Jonasson, Jon
    Linköpings universitet, Hälsouniversitetet. Linköpings universitet, Institutionen för hälsa och miljö. Östergötlands Läns Landsting, Laboratoriemedicinskt centrum, Klinisk mikrobiologi.
    Identification of enterococcal isolates by temperature gradient gel electrophoresis and partial sequence analysis of PCR-amplified 16S rDNA variable V6 regions2001Inngår i: Acta Pathologica, Microbiologica et Immunologica Scandinavica (APMIS), ISSN 0903-4641, E-ISSN 1600-0463, Vol. 109, nr 3, s. 209-216Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Based on partial sequence analysis of PCR-amplified 16S rDNA variable V6 regions of 14 enterococcal type strains, Enterococcus faecalis, Enterococcus mundtii, Enterococcus gallinarum, Enterococcus avium, Enterococcus raffinosus and Enterococcus saccharolyticus showed characteristic sequence motifs which made it possible to separate them into six individual species lines. Furthermore, two species cluster groups could be identified, including (i) Enterococcus faecium, Enterococcus durans, Enterococcus hirae, Enterococcus malodoratus, and (ii) Enterococcus casseliflavus/Enterococcus flavescens, Enterococcus pseudoavium, Enterococcus dispar and Enterococcus sulfureus. There were identical DNA sequences in the V6 region within each group. Temporal temperature gradient gel electrophoresis (TTGE) of the PCR products from 16 type strains, 12 enterococcal reference strains and 8 clinical isolates revealed that a single nucleotide divergence in DNA sequences was sufficient for separation, identification and division of the studied enterococcal strains into corresponding TTGE profiles. It was concluded that partial DNA sequence analysis and TTGE profiling of PCR-amplified 16S rDNA variable V6 regions provide useful tools for the identification of clinically important Enterococcus spp.

  • 16.
    Monstein, Hans-Jurg
    et al.
    Linköpings universitet, Hälsouniversitetet. Linköpings universitet, Institutionen för biomedicin och kirurgi, Avdelningen för medicinsk cellbiologi. Östergötlands Läns Landsting, Laboratoriemedicinskt centrum, Molekylärbiologiska tekniklaboratoriet.
    Balkhed Östholm, Åse
    Linköpings universitet, Hälsouniversitetet. Linköpings universitet, Institutionen för molekylär och klinisk medicin, Klinisk mikrobiologi. Östergötlands Läns Landsting, Hjärt- och Medicincentrum, Infektionskliniken i Östergötland.
    Nilsson, MV
    Nilsson, M
    Dornbusch, Kathrine
    Linköpings universitet, Hälsouniversitetet. Linköpings universitet, Institutionen för molekylär och klinisk medicin, Klinisk mikrobiologi.
    Nilsson, Lennart
    Linköpings universitet, Hälsouniversitetet. Linköpings universitet, Institutionen för molekylär och klinisk medicin, Klinisk mikrobiologi.
    Multiplex PCR amplification assay for the detection of blaSHV, blaTEM and blaCTX-M genes in Enterobacteriaceae2007Inngår i: Acta Pathologica, Microbiologica et Immunologica Scandinavica (APMIS), ISSN 0903-4641, E-ISSN 1600-0463, Vol. 115, nr 12, s. 1400-1408Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Extended-spectrum β-lactamases (ESBLs) are often mediated by bla-SHV, blaTEM and blaCTX-M genes in Enterobacteriaceae and other Gram-negative bacteria. Numerous molecular typing methods, including PCR-based assays, have been developed for their identification. To reduce the number of PCR amplifications needed we have developed a multiplex PCR assay which detects and discriminates between bla-SHV, blaTEM and blaCTX-M PCR amplicons of 747, 445 and 593 bp, respectively. This multiplex PCR assay allowed the identification of bla-SHV, blaTEM and blaCTX-M genes in a series of clinical isolates of Enterobacteriaceae with previously characterised ESBL phenotype. The presence of blaSHV, blaTEM and blaCTX-M genes was confirmed by partial DNA sequence analysis. Apparently, the universal well-established CTX-M primer pair used here to reveal plasmid-encoded blaCTX-M genes would also amplify the chromosomally located K-1 enzyme gene in all Klebsiella oxytoca strains included in the study.

  • 17.
    Monstein, Hans-Jurg
    et al.
    Linköpings universitet, Hälsouniversitetet. Linköpings universitet, Institutionen för hälsa och miljö.
    de la Cour, CD
    Jonasson, Jon
    Linköpings universitet, Hälsouniversitetet. Linköpings universitet, Institutionen för hälsa och miljö. Östergötlands Läns Landsting, Laboratoriemedicinskt centrum, Klinisk mikrobiologi.
    Probing 23S ribosomal RNA cleavage sites in coccoid Helicobater pylori.2001Inngår i: Helicobacter, ISSN 1083-4389, E-ISSN 1523-5378, Vol. 6, nr 2, s. 100-109Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Background. Previous studies have revealed that extensive nonrandom fragmentation of ribosomal RNA occurs during conversion of Helicobacter pylori to the coccoid form. The 16S rRNA fragmentation has been characterised in some detail. The aim of the present study was to define corresponding cleavage-sites in the 3'-half of the 23S rRNA molecule. Materials and Methods. Northern blot analysis using 23S rRNA specific antisense riboprobes and a 5'-end- labelled oligonucleotide probe was used to analyse the 23S rRNA fragmentation pattern in coccoid H. pylori type strain CCUG 17874T and H. pylori 26695, for which the genome has been sequenced. A double- stranded cDNA-dependent (ds-cDNA) primer- extension analysis technique using 23S rRNA ds-cDNA and a primer targeting the vicinity of the peptidyl-transferase centre was used to determine cleavage sites at the nucleotide level. Results. We report here the mapping of putative cleavage sites within domains IV and V, enclosing the peptidyl transferase centre, in the 3'-half of the 23S rRNA molecule. Three cleavage sites were located in domain IV. Two other cleavage sites were located in the peptidyl transferase centre, and one presumptive multiple-break site between helices 77 and 78 in domain V. The DNA motifs were different from the postulated A + U rich single-strand cleavage sites recognised by RNase E, which has been implicated in rRNA degradation in Escherichia coli. Conclusions. The present analysis suggests that a hitherto unknown mechanism is responsible for the nonrandom fragmentation of rRNA in coccoid H. pylori, which may have important consequences for the growth, and survival of the bacterium.

  • 18.
    Monstein, Hans-Jurg
    et al.
    Linköpings universitet, Hälsouniversitetet. Linköpings universitet, Institutionen för klinisk och experimentell medicin, Klinisk mikrobiologi. Östergötlands Läns Landsting, Laboratoriemedicinskt centrum, Molekylärbiologiska tekniklaboratoriet.
    Ellnebo-Svedlund, Katarina
    Linköpings universitet, Hälsouniversitetet. Linköpings universitet, Institutionen för biomedicin och kirurgi.
    Molecular typing of Helicobacter pylori by virulence-gene based multiplex PCR and RT-PCR analysis2002Inngår i: Helicobacter, ISSN 1083-4389, E-ISSN 1523-5378, Vol. 7, nr 5, s. 287-296Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Background. In recent years, numerous PCR amplification typing methods have been developed for the identification of H. pylori specific virulence genes. To reduce the number of PCR amplifications needed we have previously developed virulence gene based multiplex PCR and RT-PCR assays. Aim. The aim of the present study was to characterise Helicobacter pylori strains by means of virulence-gene based multiplex PCR and reverse-transcription PCR. Subjects. Helicobacter pylori clinical reference strains HP-HJM 1-25 originally obtained from routine cultures of clinical gastric biopsy specimens from dyspeptic patients were used in the present study. Methods. Helicobacter pylori multiplex PCR and RT-PCR assays were carried out using primer pairs targeting the cagA, vacA, ureA, ureI, hspA (hsp60), and sodB genes and their cDNA. Results. Unexpected multiplex PCR and RT-PCR patterns were observed by ethidium bromide staining of agarose gels and Southern blot hybridisation analysis. DNA sequence analysis revealed a complex pattern of point mutations, deletions and insertions in the sodB, cagA and vacA genes, respectively. Mutations occurring in the PCR and hybridisation primer binding sites might explain some of the discrepancies previously observed in the expression of these genes. Furthermore, the present data show that coexpression of cagA and vacA transcripts of different sizes may take place in the same strain. Conclusions. The multiplex PCR and RT-PCR assay described allows rapid characterisation of H. pylori virulence genes at the DNA and RNA (cDNA) levels. However, extensive DNA sequence analysis seems necessary if one wants to reveal details of mutations occurring in the cagA and vacA genes.

  • 19.
    Monstein, Hans-Jurg
    et al.
    Linköpings universitet, Hälsouniversitetet. Linköpings universitet, Institutionen för biomedicin och kirurgi, Avdelningen för medicinsk cellbiologi. Östergötlands Läns Landsting, Laboratoriemedicinskt centrum, Molekylärbiologiska tekniklaboratoriet.
    Grahn, Niclas
    Linköpings universitet, Hälsouniversitetet. Linköpings universitet, Institutionen för biomedicin och kirurgi, Avdelningen för medicinsk cellbiologi.
    Ohlsson, Bodil
    Proenkephalin-A mRNA is widely expressed in tissues of the human gastrointestinal tract2006Inngår i: European Surgical Research, ISSN 0014-312X, E-ISSN 1421-9921, Vol. 38, nr 5, s. 464-468Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Background: It has been shown that the non-opioid effects of Met-enkephalin, which is derived from proenkephalin-A, are mediated through a specific opioid growth factor (OGF) receptor which is assumed to be involved in the control of cell growth. The expression and tissue location of proenkephalin-A mRNA in the gastrointestinal tract remains largely unknown. Methods: In this study we have analyzed the expression of proenkephalin-A mRNA in the human esophagus, gastrointestinal tract and surrounding organs by means of reverse-transcriptase PCR (RT-PCR). Results: The present study demonstrates proenkephalin-A mRNA expression in the human esophagus, gastrointestinal tract, pancreas, and gallbladder. Conclusion: The present study demonstrates proenkephalin-A mRNA expression in regions of the human esophagus, gastrointestinal tract, pancreas, and gallbladder tissues which provides information for the future mapping of proenkephalin-A mRNA and protein expression/co-expression at the cellular level. Copyright © 2006 S. Karger AG.

  • 20.
    Monstein, Hans-Jurg
    et al.
    Linköpings universitet, Hälsouniversitetet. Linköpings universitet, Institutionen för klinisk och experimentell medicin, Klinisk mikrobiologi. Östergötlands Läns Landsting, Laboratoriemedicinskt centrum, Molekylärbiologiska tekniklaboratoriet.
    Grahn, Niclas
    Linköpings universitet, Hälsouniversitetet. Linköpings universitet, Institutionen för klinisk och experimentell medicin, Cellbiologi.
    Truedsson, M.
    Department of Internal Medicine, University Hospital Malmö, S-205 02 Malmö, Sweden.
    Ohlsson, B.
    Department of Internal Medicine, University Hospital Malmö, S-205 02 Malmö, Sweden.
    Oxytocin and oxytocin-receptor mRNA expression in the human gastrointestinal tract: A polymerase chain reaction study2004Inngår i: Regulatory Peptides, ISSN 0167-0115, E-ISSN 1873-1686, Vol. 119, nr 1-2, s. 39-44Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Background/aim: Oxytocin (OT) has a wide range of effects throughout the body. However, the role of OT on the gastrointestinal (GI) tract has to be settled. So far, the few studies performed reveal no conclusive results. The aim of this study was to examine the expression of OT and OT-receptor mRNA in the human GI tract. Material and methods: Full-thickness biopsies from all segments of the GI tract and the gallbladder were collected during operations at the Department of Surgery, Malmö University Hospital. Biopsies were taken and put immediately into fluid nitrogen and stored at -70°C until total RNA was extracted after mechanical tissue homogenization. Subsequently, poly A + mRNA was isolated from the total RNA extract using an automated nucleic acid extractor and converted into single-stranded cDNA. PCR amplifications were carried out using gene-specific OT and OT-receptor primers. The specificity of the PCR amplicons was further confirmed by Southern blot analyses using gene specific OT and OT-receptor hybridization probes. Results: Expression of OT and OT-receptor mRNA was detected in nearly all segments of the GI tract analyzed. In most of the biopsy specimens analyzed, co-expression of both OT and OT-receptor mRNA appeared to take place. Conclusion: The present study demonstrates that OT and OT-receptor mRNAs are expressed throughout the GI tract. A possible physiological and/or pathophysiological role of OT and OT-receptor expression in the human GI tract and the cellular location of its expression remain to be shown. © 2004 Elsevier B.V. All rights reserved.

  • 21.
    Monstein, Hans-Jurg
    et al.
    Linköpings universitet, Hälsouniversitetet. Linköpings universitet, Institutionen för biomedicin och kirurgi, Avdelningen för medicinsk cellbiologi. Östergötlands Läns Landsting, Laboratoriemedicinskt centrum, Molekylärbiologiska tekniklaboratoriet.
    Grahn, Niclas
    Linköpings universitet, Hälsouniversitetet. Linköpings universitet, Institutionen för biomedicin och kirurgi, Avdelningen för medicinsk cellbiologi.
    Truedsson, Mikael
    Ohlsson, Bodil
    Progastrin-releasing peptide and gastrin-releasing peptide receptor mRNA expression in non-tumor tissues of the human gastrointestinal tract2006Inngår i: World Journal of Gastroenterology, ISSN 1007-9327, E-ISSN 2219-2840, Vol. 12, nr 16, s. 2574-2578Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Aim: To investigate the expression of gastrin-releasing peptide (GRP) and GRP-receptor mRNA in non-tumor tissues of the human esophagus, gastrointestinal tract, pancreas and gallbladder using molecular biology techniques. Methods: Poly A+ mRNA was isolated from total RNA extracts using an automated nucleic acid extractor and, subsequently, converted into single-stranded cDNA (ss-cDNA). PCR amplifications were carried out using gene-specific GRP and GRP-receptor primers. The specificity of the PCR amplicons was further confirmed by Southern blot analyses using gene-specific GRP and GRP-receptor hybridization probes. Results: Expre ssion of GRP and GRP-receptor mRNA was detected at various levels in nearly all segments of the non-tumor specimens analysed, except the gallbladder. In most of the biopsy specimens, co-expression of both GRP and GRP-receptor mRNA appeared to take place. However, expression of GRP mRNA was more prominent than was GRP-receptor mRNA. Conclusion: GRP and GRP-receptor mRNAs are expressed throughout the gastrointestinal tract and provides information for the future mapping and determination of its physiological importance in normal and tumor cells. © 2006 The WJG Press. All rights reserved.

  • 22.
    Monstein, Hans-Jurg
    et al.
    Linköpings universitet, Hälsouniversitetet. Linköpings universitet, Institutionen för hälsa och miljö. Östergötlands Läns Landsting, Laboratoriemedicinskt centrum, Klinisk mikrobiologi.
    Johansson, Yvonne
    Linköpings universitet, Hälsouniversitetet. Linköpings universitet, Institutionen för hälsa och miljö. Östergötlands Läns Landsting, Laboratoriemedicinskt centrum, Klinisk mikrobiologi.
    Jonasson, Jon
    Linköpings universitet, Hälsouniversitetet. Linköpings universitet, Institutionen för hälsa och miljö. Östergötlands Läns Landsting, Laboratoriemedicinskt centrum, Klinisk mikrobiologi.
    Detection of vancomycin resistance genes combined with typing of Enterococci by means of multiplex PCR amplification and multiple primer DNA sequencing2000Inngår i: Acta Pathologica, Microbiologica et Immunologica Scandinavica (APMIS), ISSN 0903-4641, E-ISSN 1600-0463, Vol. 108, nr 1, s. 67-73Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    A multiplex PCR assay for the detection of vancomycin resistance (van) genes in enterococci was established. Primers targeting the 16S rRNA gene were included in the reaction mixture. Multiple-primer DNA sequencing of the PCR products provided species identification through partial nucleotide sequences of 16S rRNA genes, as well as confirmation of the correct identification of vanA, vanB, vanC-1, and vanC-2/3 genotypes. Thirty-nine enterococcal clinical isolates and type strains were examined for the presence of vancomycin resistance determinants. Twelve other isolates from a clinical reference collection (some of them having vanA, vanB, vanC-1, or vanC-2/3 genotypes) were used as controls. Hybridization and partial DNA sequence analysis of multiplex PCR products revealed that none of the clinical isolates had a vanA genotype and only one had a vanB genotype, vanC- 1 was found in three clinical isolates, and vanC-2/3 in one. Results obtained with the reference and type strains were in agreement with earlier results.

  • 23.
    Monstein, Hans-Jurg
    et al.
    Linköpings universitet, Hälsouniversitetet. Linköpings universitet, Institutionen för hälsa och miljö.
    Jonasson, Jon
    Linköpings universitet, Hälsouniversitetet. Linköpings universitet, Institutionen för hälsa och miljö. Östergötlands Läns Landsting, Laboratoriemedicinskt centrum, Klinisk mikrobiologi.
    Differential virulence-gene mRNA expression in coccoid forms of Helicobacter pylori2001Inngår i: Biochemical and Biophysical Research Communications - BBRC, ISSN 0006-291X, E-ISSN 1090-2104, Vol. 285, nr 2, s. 530-536Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Controversy exists whether coccoid forms of Helicobacter pylori maintain transcriptional and translational processes. The aim of the present study was to investigate mRNA levels in coccoid H. pylori and, if possible, to establish a correlation with the state of nonrandom fragmentation of rRNA in those cells. For that purpose, UreA, UreI, CagA, VacA, SodB, and Hsp60 mRNA levels in bacillary and coccoid forms of H. pylori CCUG 17874T, H. pylori 26695, and H. pylori J99, respectively, were studied by means of a multiplex reverse-transcription PCR assay and Southern blot analysis of the RT-PCR-amplified products. Nonrandom fragmentation of 23S rRNA was assessed by a recently described assay. Virulence-gene-derived mRNA transcripts were visualized in DNase I-treated RNA preparations. All three strains revealed the presence of different mRNA patterns in bacillary and coccoid forms. Putative promoter sequences similar to the consensus Escherichia coli -10 hexamer TATAAA box were present in all six virulence genes analyzed. Moreover, the decrease seen in mRNA levels during conversion into the coccoid form appeared to correlate with the 23S rRNA nonrandom fragmentation pattern. The present data indicate that modulation of virulence-gene expression is differently regulated in bacillary and coccoid H. pylori.

  • 24.
    Monstein, Hans-Jurg
    et al.
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Klinisk mikrobiologi. Linköpings universitet, Hälsouniversitetet. Östergötlands Läns Landsting, Laboratoriemedicinskt centrum, Klinisk mikrobiologi.
    Karlsson, Anneli
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Cellbiologi. Linköpings universitet, Hälsouniversitetet.
    Ryberg, Anna
    Linköpings universitet, Institutionen för klinisk och experimentell medicin. Linköpings universitet, Hälsouniversitetet. Östergötlands Läns Landsting, Laboratoriemedicinskt centrum, Klinisk mikrobiologi.
    Borch, Kurt
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Kirurgi. Linköpings universitet, Hälsouniversitetet. Östergötlands Läns Landsting, Kirurgi- och onkologicentrum, Kirurgiska kliniken i Östergötland med verksamhet i Linköping, Norrköping och Motala.
    Application of PCR amplicon sequencing using a single primer pair in PCR amplification to assess variations in Helicobacter pylori CagA EPIYA tyrosine phosphorylation motifs2010Inngår i: BMC Research Notes, ISSN 1756-0500, E-ISSN 1756-0500, Vol. 3, nr 35Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Background

    The presence of various EPIYA tyrosine phosphorylation motifs in the CagA protein of Helicobacter pylori has been suggested to contribute to pathogenesis in adults. In this study, a unique PCR assay and sequencing strategy was developed to establish the number and variation of cagA EPIYA motifs.

    Findings

    MDA-DNA derived from gastric biopsy specimens from eleven subjects with gastritis was used with M13- and T7- sequence-tagged primers for amplification of the cagA EPIYA motif region. Automated capillary electrophoresis using a high resolution kit and amplicon sequencing confirmed variations in the cagA EPIYA motif region. In nine cases, sequencing revealed the presence of AB, ABC, or ABCC (Western type) cagA EPIYA motif, respectively. In two cases, double cagA EPIYA motifs were detected (ABC/ABCC or ABC/AB), indicating the presence of two H. pylori strains in the same biopsy.

    Conclusion

    Automated capillary electrophoresis and amplicon sequencing using a single, M13- and T7-sequence-tagged primer pair in PCR amplification enabled a rapid molecular typing of cagA EPIYA motifs. Moreover, the techniques described allowed for a rapid detection of mixed H. pylori strains present in the same biopsy specimen.

  • 25.
    Monstein, Hans-Jurg
    et al.
    Linköpings universitet, Hälsouniversitetet. Linköpings universitet, Institutionen för hälsa och miljö.
    Nikpour-Badr, S
    Linköpings universitet, Hälsouniversitetet. Linköpings universitet, Institutionen för hälsa och miljö.
    Jonasson, Jon
    Linköpings universitet, Hälsouniversitetet. Linköpings universitet, Institutionen för hälsa och miljö. Östergötlands Läns Landsting, Laboratoriemedicinskt centrum, Klinisk mikrobiologi.
    Rapid molecular identification and subtyping of Helicobacter pylori by pyrosequencing of the 16S rDNA variable V1 and V3 regions2001Inngår i: FEMS Microbiology Letters, ISSN 0378-1097, E-ISSN 1574-6968, Vol. 199, nr 1, s. 103-107Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    We describe here the use of real-time DNA sequence analysis of Helicobacter pylori 16S rRNA gene fragments by pyrosequencingÖ for rapid molecular identification and subtyping of clinical isolates based on DNA sequence heterogeneity within the variable V1 and V3 regions. Six individual 16S rDNA V1 alleles (position 75-100) were identified in 23 clinical isolates obtained from gastric biopsy specimens. Eleven of these revealed sequence identities with H. pylori 26695 and one was identical with the rrn genes in strain J99. The other V1 alleles showing single or double nucleotide mutations or single nucleotide insertions could be divided into four groups with 5, 4, 1, and 1 isolates each. Two out of 25 isolates demonstrated single C to T transitions in the V3 region (position 990-1020). The present findings show that subtle DNA sequence variation occurs sufficiently often in the 16S rDNA variable V1 and V3 regions of H. pylori to provide a consistent system for subtyping.

  • 26.
    Monstein, Hans-Jurg
    et al.
    Linköpings universitet, Hälsouniversitetet. Linköpings universitet, Institutionen för klinisk och experimentell medicin, Klinisk mikrobiologi. Östergötlands Läns Landsting, Laboratoriemedicinskt centrum, Molekylärbiologiska tekniklaboratoriet.
    Ohlsson, B
    Axelson, J
    Malmo Univ Hosp, Dept Surg, MAS, SE-20502 Malmo, Sweden Linkoping Univ Hosp, LMO, Mol Biol Lab, S-58185 Linkoping, Sweden.
    Differential expression of gastrin, cholecystokinin-A and cholecystokinin-B receptor mRNA in human pancreatic cancer cell lines2001Inngår i: Scandinavian Journal of Gastroenterology, ISSN 0036-5521, E-ISSN 1502-7708, Vol. 36, nr 7, s. 738-743Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Background: It has been assumed that gastrin stimulates the growth of pancreatic cancer in an autocrine way through co-expression of gastrin and the cholecystokinin-B receptor (CCK-BR). However, pancreatic cancer cell lines established directly from patients have revealed a great heterogeneity in cell proliferation when exposed to CCK, gastrin and their receptor antagonists. The aim of this study was therefore to examine co-expression of CCK-A and CCK-B receptor (CCK-AR and CCK-BR), and gastrin mRNA as well as the secretion of CCK and gastrin peptides in these cell lines. Methods: Fourteen cell lines were established from primary pancreatic cancers or their metastases. Total RNA was isolated from the cell lines and reverse-transcribed into single-stranded cDNA. A PCR technique based on Tag polymerase-antibody interaction and CCK-AR, CCK-BR and gastrin-specific primers, followed by Southern blot analysis, were the methods used. The incubation mediums were analysed for the presence of secreted CCK/proCCK and gastrin/progastrin peptides by specific radioimmunoassays (RIA). Results: By means of nested Reverse-Transcribed Polymerase Chain Reaction (nested RT-PCR), combined with Southern blot analysis of the PCR amplified products, CCK-AR and gastrin mRNA co-expression was detected in cell Lines LPC-6p and LPC-10m, whereas CCK-BR and gastrin mRNA could be detected in cell lines LPC-8p and LPC-12m. A low level of secreted CCK peptides was detected in cell line LPC-6p. which also expressed CCK-AR mRNA. In no other cases were CCK or gastrin peptides detected in the cell culture mediums. Conclusion: The lack of CCK-BR and gastrin mRNA co-expression, and not detectable levels of secreted CCK and gastrin in culture media, does not lend support to the hypothesis that concomitant gene-expression of CCK receptors and gastrin or CCK are essential to maintaining pancreatic cancer cell proliferation.

  • 27.
    Monstein, Hans-Jurg
    et al.
    Linköpings universitet, Hälsouniversitetet. Linköpings universitet, Institutionen för biomedicin och kirurgi, Avdelningen för medicinsk cellbiologi. Östergötlands Läns Landsting, Laboratoriemedicinskt centrum, Molekylärbiologiska tekniklaboratoriet.
    Olsson, Crister
    Nilsson, Isabelle
    Linköpings universitet, Institutionen för medicin och vård. Linköpings universitet, Hälsouniversitetet.
    Grahn, Niclas
    Linköpings universitet, Hälsouniversitetet. Linköpings universitet, Institutionen för biomedicin och kirurgi, Avdelningen för medicinsk cellbiologi.
    Benoni, Cecilia
    Ahrné, Siv
    Multiple displacement amplification of DNA from human colon and rectum biopsies: Bacterial profiling and identification of Helicobacter pylori-DNA by means of 16S rDNA-based TTGE and pyrosequencing analysis2005Inngår i: Journal of Microbiological Methods, ISSN 0167-7012, E-ISSN 1872-8359, Vol. 63, nr 3, s. 239-247Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Amplifying bacterial DNA by PCR from human biopsy specimens has sometimes proved to be difficult, mainly due to the low amount of bacterial DNA present. Therefore, nested or semi-nested 16S rDNA PCR amplification has been the method of choice. In this study, we evaluate the potential use of whole genome amplification of total DNA isolated from human colon and rectum biopsy specimens, followed by 16S rDNA PCR amplification of multiple displacement amplified (MDA)-DNA. Subsequently, a H. pylori-specific 16S rDNA variable V3 region PCR assay was applied directly on MDA-DNA and, combined with pyrosequencing analysis, the presence of H. pylori in some biopsies from colon in patients with microscopic colitis was confirmed. Furthermore, temporal temperature gradient gel electrophoresis (TTGE) of 16S rDNA amplicons using primers flanking variable regions V3, V4, and V9, was used to establish bacterial profiles from individual biopsies. A variation of the bacterial profiles in the colonic mucosa in microscopic colitis and in normal rectal mucosa was observed. In conclusion we find the MDA technique to be a useful method to overcome the problem of insufficient bacterial DNA in human biopsy specimens. © 2005 Elsevier B.V. All rights reserved.

  • 28.
    Monstein, Hans-Jurg
    et al.
    Linköpings universitet, Institutionen för molekylär och klinisk medicin, Klinisk mikrobiologi. Linköpings universitet, Hälsouniversitetet.
    Quenadu, Michael
    Laboratory of Food hygiene, Department of Food Thecnology, University of Lund.
    Samuelsson, Annika
    Linköpings universitet, Institutionen för molekylär och klinisk medicin, Klinisk mikrobiologi. Linköpings universitet, Hälsouniversitetet.
    Ahrne, Siv
    Laboratory of Food hygiene, Department of Food Thecnology, University of Lund.
    Isaksson, Barbro
    Linköpings universitet, Institutionen för molekylär och klinisk medicin, Klinisk mikrobiologi. Linköpings universitet, Hälsouniversitetet.
    Jonasson, Jon
    Linköpings universitet, Institutionen för molekylär och klinisk medicin, Klinisk mikrobiologi. Linköpings universitet, Hälsouniversitetet.
    Division of the genus Enterococcus into species groups using PCR-based molecular typing methods1998Inngår i: Microbiology, ISSN 1350-0872, E-ISSN 1465-2080, Vol. 144, nr 5, s. 1171-1179Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Broad-range 16S rDNA PCR (BR-PCR) applied to DNA from 32 clinical enterococcal isolates and 12 other enterococci from a clinical reference collection followed by species-specific hybridization analysis identified 25 strains of Enterococcus faecalis and 19 Enterococcus species. Randomly amplified polymorphic DNA (RAPD) analysis using UPGMA clustering on the same material revealed four different clusters at a similarity level of 49%. Based on partial 16S rDNA sequence analysis of variable regions V4 and V9, it was possible to divide the 19 type strains specifying the genus Enterococcus into 12 different 16S rDNA species groups. The type strain distribution then served as a template for the analysis of the other 44 strains which were assigned to four different species groups (a-d) based on their 16S rDNA motifs. There was good agreement with the RAPD clusters. Species group a was an individual species line containing 25 strains that were identified as E. faecalis. Group b also represented an individual species line of 12 strains identified as E. faecium. The remaining seven strains that formed species groups c and d could not be fully identified to species by this analysis. It was concluded that BR-PCR of 16S rDNA followed by partial sequence analysis of the PCR products is a reliable technique for the identification and classification of enterococci. Further division of unresolved species groups should be achievable if regions other than V4 and V9 of 16S rDNA are also analysed

  • 29.
    Monstein, Hans-Jurg
    et al.
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Klinisk mikrobiologi. Linköpings universitet, Hälsouniversitetet. Östergötlands Läns Landsting, Diagnostikcentrum, Klinisk mikrobiologi.
    Ryberg, Anna
    Linköpings universitet, Institutionen för klinisk och experimentell medicin. Linköpings universitet, Hälsouniversitetet.
    Karlsson, Anneli
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Cellbiologi. Linköpings universitet, Hälsouniversitetet.
    Assessment of the mosaic structure in the Helicobacter pylori cagA gene 3 -region using an improved polymerase chain reaction-based assay2012Inngår i: Diagnostic microbiology and infectious disease, ISSN 0732-8893, E-ISSN 1879-0070, Vol. 73, nr 3, s. 281-283Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    The mosaic structure of the cagA gene has been suggested to affect Helicobacter pylori CagA-associated pathogenesis. An improved polymerase chain reaction assay allowed for a rapid and detailed molecular analysis of the cagA gene 3-region in a single amplification step, followed by amplicon sequencing using universal M13 and T7 sequencing primers.

  • 30.
    Monstein, Hans-Jurg
    et al.
    Östergötlands Läns Landsting, Laboratoriemedicinskt centrum, Klinisk mikrobiologi. Linköpings universitet, Hälsouniversitetet.
    Tiveljung, Annika
    Linköpings universitet, Institutionen för molekylär och klinisk medicin, Klinisk mikrobiologi. Linköpings universitet, Hälsouniversitetet.
    Kraft, C. H.
    Linköpings universitet, Institutionen för molekylär och klinisk medicin, Klinisk mikrobiologi. Linköpings universitet, Hälsouniversitetet.
    Borch, Kurt
    Linköpings universitet, Institutionen för biomedicin och kirurgi, Kirurgi. Linköpings universitet, Hälsouniversitetet.
    Jonasson, Jon
    Östergötlands Läns Landsting, Laboratoriemedicinskt centrum, Klinisk mikrobiologi. Linköpings universitet, Hälsouniversitetet.
    Profiling of bacterial flora in gastric biopsies from patients with Helicobacter pylori-associated gastritis and histologically normal control individuals by temperature gradient gel electrophoresis and 16S rDNA sequence analysis2000Inngår i: Journal of Medical Microbiology, ISSN 0022-2615, E-ISSN 1473-5644, Vol. 49, nr 9, s. 817-822Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    The aim of this study was to establish bacterial profiles in gastric biopsy specimens from patients with Helicobacter pylori-associated gastritis by means of temporal temperature gradient gel electrophoresis (TTGE) of PCR-amplified 16S rDNA fragments. Specimens from eight patients with asymptomatic gastritis and five histologically normal controls revealed a Helicobacter-specific band in the TTGE profile with increased amounts of Helicobacter-specific DNA in the biopsies from most of the gastritis patients. DNA from other genera including Enterococcus, Pseudomonas, Streptococcus, Staphylococcus and Stomatococcus was also found in the stomach. In the absence of gastric inflammation, Helicobacter spp. appeared to be part of a complex, presumably indigenous microbial flora found in the biopsy specimens from the stomach.

  • 31.
    Monstein, Hans-Jurg
    et al.
    Linköpings universitet, Hälsouniversitetet. Linköpings universitet, Institutionen för klinisk och experimentell medicin, Klinisk mikrobiologi. Östergötlands Läns Landsting, Laboratoriemedicinskt centrum, Molekylärbiologiska tekniklaboratoriet.
    Truedsson, M.
    Department of Clinical Sciences Malmö University.
    Ryberg, Anna
    Linköpings universitet, Hälsouniversitetet. Linköpings universitet, Institutionen för klinisk och experimentell medicin.
    Ohlsson, M.
    Department of Clinical Sciences Malmö University.
    Vasopressin receptor mRNA expression in the human gastrointestinal tract2008Inngår i: European Surgical Research, ISSN 0014-312X, E-ISSN 1421-9921, Vol. 40, nr 1, s. 34-40Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Background/Aim: Vasopressin and oxytocin are closely related peptides, and both exert effects on the gastrointestinal function. In the present study, we wanted to map the expression of vasopressin receptor mRNAs (V1a, V1b/V3, and V2) in nontumorous tissue biopsy specimens of human gastrointestinal tract and surrounding tissues. Methods: Total and polyA+ RNAs were isolated from human tissue biopsy specimens using an automated nucleic acid extractor and, subsequently, converted into single-stranded cDNA. Seminested PCR amplifications were carried out, using gene-specific V1a, V1b/V3, and V2 receptor primers. The PCR amplicons were partially sequenced to confirm their identity. Results: The present study demonstrated the expression of vasopressin receptor mRNAs in human gastrointestinal tract, pancreas, kidney, lung, brain, and ovary. The expression pattern varied between different parts of the gastrointestinal tract. In the colon ascendens, V1a receptor mRNA expression could not be detected in 3 out of 4 analyzed tissue biopsy specimens. On the other hand, all the vasopressin receptor mRNAs were expressed in all colon transversum biopsy samples. Conclusions: V1a, V1b/V3, and V2 receptor mRNAs are widely expressed throughout human gastrointestinal tract and surrounding tissues. The data obtained provide information for further mapping and determination of the physiological role of the vasopressin receptor mRNA expression in normal and tumorous tissues.

  • 32.
    Monstein, Hans-Jurg
    et al.
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Klinisk mikrobiologi. Linköpings universitet, Hälsouniversitetet.
    Tärnberg, Maria
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Klinisk mikrobiologi. Linköpings universitet, Hälsouniversitetet.
    Nilsson, Lennart
    Linköpings universitet, Hälsouniversitetet. Linköpings universitet, Institutionen för klinisk och experimentell medicin, Klinisk mikrobiologi.
    Molecular identification of blaCTX-M and blaOXY/K1 beta-lactamase genes in Enterobacteriaceae by sequencing of universal M13-sequence tagged PCR-amplicons.2009Inngår i: BMC Infectious Diseases, ISSN 1471-2334, E-ISSN 1471-2334, Vol. 9, nr 1, s. 7-Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    BACKGROUND: Plasmid encoded blaCTX-M enzymes represent an important sub-group of class A beta-lactamases causing the ESBL phenotype which is increasingly found in Enterobacteriaceae including Klebsiella spp. Molecular typing of clinical ESBL-isolates has become more and more important for prevention of the dissemination of ESBL-producers among nosocomial environment.

    METHODS: Multiple displacement amplified DNA derived from 20 K. pneumoniae and 34 K. oxytoca clinical isolates with an ESBL-phenotype was used in a universal CTX-M PCR amplification assay. Identification and differentiation of blaCTX-M and blaOXY/K1 sequences was obtained by DNA sequencing of M13-sequence-tagged CTX-M PCR-amplicons using a M13-specific sequencing primer.

    RESULTS: Nine out of 20 K. pneumoniae clinical isolates had a blaCTX-M genotype. Interestingly, we found that the universal degenerated primers also amplified the chromosomally located K1-gene in all 34 K. oxytoca clinical isolates. Molecular identification and differentiation between blaCTX-M and bla OXY/K1-genes could only been achieved by sequencing of the PCR-amplicons. In silico analysis revealed that the universal degenerated CTX-M primer-pair used here might also amplify the chromosomally located blaOXY and K1-genes in Klebsiella spp. and K1-like genes in other Enterobacteriaceae.

    CONCLUSION: The PCR-based molecular typing method described here enables a rapid and reliable molecular identification of blaCTX-M, and blaOXY/K1-genes. The principles used in this study could also be applied to any situation in which antimicrobial resistance genes would need to be sequenced.

  • 33.
    Monstein, Hans-Jürg
    et al.
    Linköpings universitet, Institutionen för molekylär och klinisk medicin, Klinisk mikrobiologi. Linköpings universitet, Hälsouniversitetet. Östergötlands Läns Landsting, Diagnostikcentrum, Klinisk mikrobiologi.
    Kihlström, Erik
    Linköpings universitet, Institutionen för molekylär och klinisk medicin, Klinisk mikrobiologi. Linköpings universitet, Hälsouniversitetet. Östergötlands Läns Landsting, Diagnostikcentrum, Klinisk mikrobiologi.
    Tiveljung, Annika
    Linköpings universitet, Institutionen för molekylär och klinisk medicin, Klinisk mikrobiologi. Linköpings universitet, Hälsouniversitetet.
    Detection and identification of bacteria using in-house broad range 16S rDNA PCR amplification and genus-specific DNA hybridization probes, located within variable regions of 16S rRNA genes1996Inngår i: Acta Pathologica, Microbiologica et Immunologica Scandinavica (APMIS), ISSN 0903-4641, E-ISSN 1600-0463, Vol. 104, nr 1-6, s. 451-458Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Broad range PCR amplification and genus-specific 16S ribosomal DNA hybridization was used to demonstrate that Chlamydia, Helicobacter and Mobiluncus hybridization probes, located within variable regions V3, V4, and V9 of the 16S rDNA, specifically bound to the corresponding PCR product obtained from pure cultures of the three genera. The sensitivity of the assay was determined by analysis of C. trachomatis serially diluted in urine. The detection limit was 1–10 elementary bodies using a hybridization probe derived from the variable region V3 of the 16S rRNA gene. A PCR product was furthermore formed in urine specimens not containing C. trachomatis, showing amplification of chlamydia also in the presence of DNA from the resident urethral flora that competes for annealing sites. Analysis of a restricted number of male urine specimens using the C. trachomatis-specific probe showed complete agreement with culture and a commercially available PCR kit. Our method not only has the capacity to detect C. trachomatis in microbiologically mixed urine samples but also the potential advantage of identifying other bacterial pathogens from the same PCR product by varying the hybridization probes.

  • 34.
    Monstein, Hans-Jürg
    et al.
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Avdelningen för mikrobiologi och molekylär medicin. Linköpings universitet, Hälsouniversitetet. Östergötlands Läns Landsting, Diagnostikcentrum, Klinisk mikrobiologi.
    Tärnberg, Maria
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Avdelningen för mikrobiologi och molekylär medicin. Linköpings universitet, Hälsouniversitetet.
    Persis, Shohreh
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Avdelningen för mikrobiologi och molekylär medicin. Linköpings universitet, Hälsouniversitetet.
    Johansson, Anders G
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Avdelningen för mikrobiologi och molekylär medicin. Linköpings universitet, Hälsouniversitetet. Östergötlands Läns Landsting, Diagnostikcentrum, Klinisk mikrobiologi.
    Comparison of a capillary gel electrophoresis-based multiplex PCR assay and ribosomal intergenic transcribed spacer-2 amplicon sequencing for identification of clinically important Candida species2014Inngår i: Journal of Microbiological Methods, ISSN 0167-7012, E-ISSN 1872-8359, Vol. 96, s. 81-83Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    The performance of a commercially available Seegene Seeplex STI Master Panel 3 multiplex PCR for Candida species identification was compared with an internal transcribed spacer 2 (ITS2) PCR assay. We found that the Seeplex assay was specific for identification of C. albicans, C. krusei, C. parapsilosis, C. glabrata, C. tropicalis and C. dubliniensis.

  • 35.
    Monstein, H.-J.
    et al.
    Östergötlands Läns Landsting, Laboratoriemedicinskt centrum, Klinisk mikrobiologi. Linköpings universitet, Hälsouniversitetet.
    Nilsson, Isabelle
    Linköpings universitet, Institutionen för molekylär och klinisk medicin, Klinisk mikrobiologi. Linköpings universitet, Hälsouniversitetet.
    Ellnebo-Svedlund, Katarina
    Linköpings universitet, Institutionen för molekylär och klinisk medicin, Klinisk mikrobiologi. Linköpings universitet, Hälsouniversitetet.
    Svensson, S.P.S.
    Linköpings universitet, Institutionen för medicin och vård, Farmakologi. Linköpings universitet, Hälsouniversitetet.
    Cloning and characterization of 5'-end alternatively spliced human cholecystokinin-B receptor mRNAs1998Inngår i: Receptors and Channels, ISSN 1060-6823, E-ISSN 1607-856X, Vol. 6, nr 3, s. 165-177Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    We report here the cloning and characterization of a 5'-end alternatively spliced human cholecystokinin-B (CCK-B) receptor mRNA. The 5'-end of this CCK-B receptor transcript (termed CCK-BRtx) consisted of exon Ia, present in the ordinary full-length CCK-B receptor mRNA (CCK-BRwt), and exon Ib, present in a previously described 5'-end alternatively spliced CCK-B receptor mRNA (CCK-BRt). A short open reading frame preceded the AUG translation initiation codon of the CCK-BRtx. Transfection of COS-7 cells with the CCK-BRtx or CCK-BRt cDNAs did not lead to the appearance of peptidergic and non-peptidergic binding sites. Cell free in vitro translation yielded proteins of approximately 44 kDa (CCK-B receptor) and 40 kDa (CCK-BRt receptor) whereas no 40 kDa product was detected from the cloned CCK-BRtx cDNA. Instead, a protein product of approximately 9 kDa was visualized, the size corresponding to the predicted protein encoded by the short open reading frame. The alternatively spliced CCK-B receptor transcripts were concomitantly expressed with the ordinary full-length CCK-B receptor mRNA in the brain, pancreas, and stomach. The possibility that such transcripts are translated in vivo into truncated CCK-B receptors is discussed.

  • 36.
    Nilsson, Isabelle
    et al.
    Linköpings universitet, Institutionen för medicin och vård, Farmakologi. Linköpings universitet, Hälsouniversitetet.
    Monstein, Hans-Jurg
    Östergötlands Läns Landsting, LMÖ - Laboratoriemedicin i Östergötland.
    Lindström, Erik
    Department of Pharmacology, Institute of Physiological Sciences, University of Lund, Lund, Sweden.
    Håkanson, Rolf
    Department of Pharmacology, Institute of Physiological Sciences, University of Lund, Lund, Sweden.
    Svensson, Samuel
    Linköpings universitet, Institutionen för medicin och vård, Farmakologi. Linköpings universitet, Hälsouniversitetet.
    Pharmacological analysis of CCK2 receptor ligands using COS-7 and SK-N-MC cells, expressing the human CCK2 receptor2002Inngår i: Regulatory Peptides, ISSN 0167-0115, E-ISSN 1873-1686, Vol. 103, nr 1, s. 29-37Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    A series of CCK2 receptor ligands were analysed with respect to their interaction with binding sites in the membranes of COS-7 cells and SK-N-MC cells transiently expressing the human CCK2 receptor (short isoform). The ligands were YF476, YM022, AG041R, L-740,093, JB93182, PD134308, and PD136450. Their binding was analysed by radioligand competition using [3H]L-365,260 as the labelled ligand. Saturation binding analysis indicated that [3H]L-365,260 interacted with a single class of binding sites. In competition binding experiments using COS-7-cell membranes, all seven ligands were incubated together with 2 nM [3H]L-365,260. The data for four of the compounds fitted a one-site model (pKi values: YM022: 9.2±0.02; YF476: 9.6±0.04; L-740,093: 9.2±0.01; and AG041R: 8.3±0.06), while the data for the three others fitted a two-site model (pKi values: JB93182: 8.8±0.04 and 6.0±0.15; PD134308: 9.0±0.04 and 6.1±0.15; and PD136450: 9.0±0.02 and 5.4±0.41). SK-N-MC cell membranes and 2 nM [3H]L-365,260 were incubated together with YM022, YF476, JB93182, and PD134308. The data for YM022 and YF476 fitted a one-site model (pKi values: YM022: 9.3±0.06; YF476: 9.4±0.02), while the data for JB93182 and PD134308 fitted a two-site model (pKi values: JB93182: 8.7±0.06 and 6.2±0.06; PD134308: 9.1±0.06 and 7.0±0.17). Competition binding experiments in the presence of the GTP-analogue guanylylimidodiphosphate, using either of the two cell types, produced similar binding data for PD134308 and JB93182 as in the absence of GTP-analogue. The human receptor seems to exist in a low and/or high affinity state. The shift from low to high affinity does not seem to reflect the degree of G protein coupling.

  • 37.
    Nilsson, Isabelle
    et al.
    Linköpings universitet, Institutionen för medicin och vård. Linköpings universitet, Hälsouniversitetet.
    Shabo, Ivan
    Linköpings universitet, Institutionen för biomedicin och kirurgi. Linköpings universitet, Hälsouniversitetet.
    Svanvik, Joar
    Linköpings universitet, Hälsouniversitetet. Linköpings universitet, Institutionen för biomedicin och kirurgi, Avdelningen för kirurgi. Östergötlands Läns Landsting, Kirurgi- och onkologicentrum, Kirurgiska kliniken i Östergötland med verksamhet i Linköping, Norrköping och Motala.
    Monstein, Hans-Jurg
    Linköpings universitet, Hälsouniversitetet. Linköpings universitet, Institutionen för biomedicin och kirurgi, Avdelningen för medicinsk cellbiologi. Östergötlands Läns Landsting, Laboratoriemedicinskt centrum, Molekylärbiologiska tekniklaboratoriet.
    Multiple displacement amplification of isolated DNA from human gallstones: Molecular identification of Helicobacter DNA by means of 16S rDNA-based pyrosequencing analysis2005Inngår i: Helicobacter, ISSN 1083-4389, E-ISSN 1523-5378, Vol. 10, nr 6, s. 592-600Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Background. Molecular typing of Helicobacter spp. in clinical biopsy specimens has become increasingly important. By means of nested polymerase chain reaction (PCR) amplification and Southern blot analysis of the PCR amplicons, we have shown that Helicobacter spp. DNA is present in human gallstones. In this study we have investigated the possibility of using multiple displacement amplification (MDA) of isolated gallstone DNA and pyrosequencing analysis for the molecular identification of Helicobacter spp. Materials and Methods. DNA isolated from the nucleus of 33 human gallstones and one control strain were used in a MDA assay. Subsequently, pyrosequencing analysis was performed either directly on MDA-DNA using primers flanking the Helicobacter spp. 16S rDNA variable V3 region or on PCR amplicons derived from broad-range primers flanking the 16S rDNA variable V3, V4, and V9 regions. Results. Pyrosequencing analysis of 16S rDNA derived from MDA-DNA revealed that Helicobacter spp.-like DNA was present in 25 of 33 (approximately 76%) gallstones. Using an H. pylori-specific Southern blot analysis, Helicobacter spp.-like DNA was present in 20 of 33 [approximately 61%] of the gallstones. Using MDA-DNA directly in pyrosequencing analysis, Helicobacter spp.-like DNA was present in 13 of 33 [approximately 39%] gallstones. Conclusions. We conclude that multiple displacement amplification combined with pyrosequencing enables a rapid and accurate molecular typing of Helicobacter spp. from small and precious biopsy specimens. © 2005 The Authors Journal compilation © 2005 Blackwell Publishing Ltd.

  • 38.
    Nilsson, Isabelle
    et al.
    Östergötlands Läns Landsting, LMÖ - Laboratoriemedicin i Östergötland. Linköpings universitet, Institutionen för medicin och vård, Farmakologi. Linköpings universitet, Hälsouniversitetet.
    Svensson, Samuel
    Linköpings universitet, Institutionen för medicin och vård, Farmakologi. Linköpings universitet, Hälsouniversitetet.
    Monstein, Hans-Jurg
    Östergötlands Läns Landsting, LMÖ - Laboratoriemedicin i Östergötland. Linköpings universitet, Institutionen för biomedicin och kirurgi. Linköpings universitet, Hälsouniversitetet.
    Molecular cloning of a putative Ciona intestinalis cionin receptor, a new member of the CCK/gastrin receptor family2003Inngår i: Gene, ISSN 0378-1119, E-ISSN 1879-0038, Vol. 323, nr 1-2, s. 79-88Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Cionin, a peptide showing similarities with cholecystokinin and gastrin has been shown to be expressed in the gut and neural ganglion of the protochordate Ciona intestinalis. The present report describes the cloning of a putative cionin receptor (CioR), a new member of the CCK/gastrin family from the gastrointestinal tract of C. intestinalis. mRNA from the stomach of C. intestinalis was isolated using a modified RNA extraction procedure and, subsequently, reverse-transcribed into single-stranded cDNA by means of rapid amplification of 5′- and 3′-cDNA ends (RACE-PCR), followed by full-length PCR amplification. The cloned full-length PCR amplicons contained a short upstream open-reading frame (uORF) coding for a putative 16 amino acid long peptide, followed by a long open reading frame encoding a 526 amino acid putative CioR protein. At the amino acid level, the putative CioR protein shared 35–40% homology with cloned mammalian, chicken, and Xenopus laevis CCK receptors. Phylogenetic analysis revealed that the chicken and X. laevis CCK receptors are orthologues of the mammalian CCK2 receptors whereas CioR protein forms a clade with vertebrate cholecystokinin receptors. Moreover, we found that the CioR cDNA and deduced amino acid sequences were found to correspond to the annotated CCK/gastrin-like receptor gene on Scaffold 117 (C. intestinalis draft genome project, Joint Genome Institute database; http://www.jgi.doe.gov).

  • 39.
    Nilsson, Isabelle
    et al.
    Östergötlands Läns Landsting, LMÖ - Laboratoriemedicin i Östergötland. Linköpings universitet, Institutionen för medicin och vård, Farmakologi. Linköpings universitet, Hälsouniversitetet.
    Svensson, Samuel
    Linköpings universitet, Institutionen för medicin och vård, Farmakologi. Linköpings universitet, Hälsouniversitetet.
    Monstein, Hans-Jürg
    Östergötlands Läns Landsting, LMÖ - Laboratoriemedicin i Östergötland. Linköpings universitet, Institutionen för biomedicin och kirurgi. Linköpings universitet, Hälsouniversitetet.
    Molecular cloning of an unusual bicistronic cholecystokinin receptor mRNA expressed in chicken brain: a structural and functional expression study2003Inngår i: Regulatory Peptides, ISSN 0167-0115, E-ISSN 1873-1686, Vol. 114, nr 1, s. 37-43Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    This report describes the molecular cloning and pharmacological characterization of a transiently expressed chicken brain cholecystokinin receptor (CCK-CHR) in COS-7 cells. A polymerase chain reaction (PCR)-based cloning strategy was applied using: (1) an initial PCR with deoxyinosine-containing primers designed to target conserved regions in CCK receptors, followed by (2) rapid amplification of cDNA ends (RACE), and (3) full-length PCR of the CCK-CHR cDNA. The full-length cloned bicistronic CCK-CHR cDNA contained a short upstream open reading frame (uORF) coding for a putative six-amino-acid-long peptide of unknown function, followed by a long open reading frame (lORF) encoding the 436-amino-acid-long CCK-CHR receptor protein. At the amino acid level, the CCK-CHR shared ∼50% homology with mammalian and Xenopus laevis CCK receptors. The pharmacological profile of CCK-CHR resembled that of CCK-B receptors using agonists (CCK-8, CCK-4, gastrin-17), whereas CCK-CHR showed higher affinity for the CCK-A receptor antagonist, devazepide, than for the CCK-B receptor antagonist, l-365,260. To the best of our knowledge, this is the first description and functional expression study of a cloned chicken CCK receptor cDNA.

  • 40.
    Nowrouzian, F.L.
    et al.
    Department of Clinical Bacteriology, Göteborg University, Göteborg, Sweden, Department of Clinical Bacteriology, Göteborg University, Guldhedsgatan 10, S-413 46 Göteborg, Sweden.
    Monstein, Hans-Jurg
    Linköpings universitet, Hälsouniversitetet. Linköpings universitet, Institutionen för klinisk och experimentell medicin, Klinisk mikrobiologi. Östergötlands Läns Landsting, Laboratoriemedicinskt centrum, Molekylärbiologiska tekniklaboratoriet.
    Wold, A.E.
    Department of Clinical Bacteriology, Göteborg University, Göteborg, Sweden.
    Adlerberth, I.
    Department of Clinical Bacteriology, Göteborg University, Göteborg, Sweden.
    Effect of human milk on type 1 and P-fimbrial mRNA expression in intestinal Escherichia coli strains2005Inngår i: Letters in Applied Microbiology, ISSN 0266-8254, E-ISSN 1472-765X, Vol. 40, nr 1, s. 74-80Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Aims: Escherichia coli from breastfed infants express more type 1 fimbriae and less P fimbriae than E. coli from bottle-fed infants. In this study we investigated the effect of human milk on production of mRNA for fimA (type 1 fimbriae) and papC (P fimbriae) in E. coli. Methods and Results: Production of adhesin gene mRNA was estimated using a reverse transcriptase polymerase chain reaction in E. coli strains under different culture conditions. More type 1 fimbrial mRNA was produced after culture in human milk (P = 0.001) or Luria broth (P = 0.014) than after culture on agar, whereas P-fimbrial mRNA production was similar under all tested growth conditions. When cultured on agar, E. coli strains carrying both the fim and pap operons produced less type 1 and P-fimbrial mRNA than strains that had only the fim or pap operons, respectively (P = 0.03 and 0.056). Significance and Impact of the Study: Environmental regulation of adhesin expression may be influenced by cross-talk between fimbrial operons.

  • 41.
    Ryberg, Anna
    et al.
    Linköpings universitet, Hälsouniversitetet. Linköpings universitet, Institutionen för klinisk och experimentell medicin, Medicinsk mikrobiologi.
    Borch, Kurt
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Kirurgi. Linköpings universitet, Hälsouniversitetet. Östergötlands Läns Landsting, Centrum för kirurgi, ortopedi och cancervård, Kirurgiska kliniken i Östergötland.
    Monstein, Hans-Jürg
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Klinisk mikrobiologi. Linköpings universitet, Hälsouniversitetet. Östergötlands Läns Landsting, Diagnostikcentrum, Klinisk mikrobiologi.
    Expression of multiple forms of 3'-end variant CCK2 receptor mRNAs in human pancreatic adenocarcinoma2011Inngår i: BMC research notes, ISSN 1756-0500, Vol. 4, s. 131-Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    BACKGROUND: Two main types of receptors for gastrin and cholecystokinin (CCK) have been cloned and identified. CCK1 (CCK-A) receptors are expressed in the pancreas, the gallbladder, and parts of the brain, while CCK2 (CCK-B/gastrin) receptors (CCK2R) are expressed in gastric glands and in most of the brain. A splice variant of the CCK2R designated CCKRi4sv (CCK-C), which is constitutively expressed in human pancreatic cancer cells, has also been described. The purpose of the present investigation was to study CCK2R, CCK2i4svR, and gastrin mRNA expression in human pancreatic adenocarcinoma on the assumption that co-expression of CCK2R and gastrin or constitutive CCK2i4svR mRNA expression plays a pivotal role in the progression of pancreatic cancer.

    FINDINGS: PCR amplification using CCK2R specific primer-pairs, followed by ethidium-bromide stained agarose gel electrophoresis revealed the expression of wild-type CCK2R mRNA in 12 of 17 biopsy specimens. A CCK2R intron 4 specific nested PCR assay revealed that CCK2i4svR mRNA was expressed in only one of the biopsy specimen. The authenticity of PCR amplicons was confirmed by cloning of selected amplicons and DNA sequence analysis. Moreover, we found that hitherto undescribed multiple forms of 3'-end variant CCK2R mRNAs with various deletions in the retained intron 4 and exon 5, tentatively generating truncated proteins, were expressed in the pancreatic adenocarcinomas.

    CONCLUSION: Cloning and DNA sequencing of selected amplicons revealed that CCK2R and multiple CCK2i4svR-like mRNAs are expressed in human pancreatic adenocarcinoma. The originally described CCK2i4svR mRNA was only expressed in one of 17 tumours and appears to be rarely expressed in pancreatic adenocarcinoma. We report that CCK2R- and gastrin mRNA co-expression may play a role in a portion, but not in all of these tumours, and that aberrant splicing takes places in these tissues generating multiple forms of 3'-end variant CCK2R mRNAs.

  • 42.
    Ryberg, Anna
    et al.
    Linköpings universitet, Institutionen för klinisk och experimentell medicin. Linköpings universitet, Hälsouniversitetet.
    Borch, Kurt
    Linköpings universitet, Hälsouniversitetet. Linköpings universitet, Institutionen för klinisk och experimentell medicin, Kirurgi. Östergötlands Läns Landsting, Kirurgi- och onkologicentrum, Kirurgiska kliniken i Östergötland med verksamhet i Linköping, Norrköping och Motala.
    Sun, Yi-Qian
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Kirurgi. Linköpings universitet, Hälsouniversitetet.
    Monstein, Hans-Jürg
    Linköpings universitet, Hälsouniversitetet. Linköpings universitet, Institutionen för klinisk och experimentell medicin, Klinisk mikrobiologi.
    Concurrent genotyping of Helicobacter pylori virulence genes and human cytokine SNP sites using whole genome amplified DNA derived from minute amounts of gastric biopsy specimen DNA2008Inngår i: BMC Microbiology, ISSN 1471-2180, E-ISSN 1471-2180, Vol. 8, s. 175-Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Background: Bacterial and cellular genotyping is becoming increasingly important in the diagnosis of infectious diseases. However, difficulties in obtaining sufficient amount of bacterial and cellular DNA extracted from the same human biopsy specimens is often a limiting factor. In this study, total DNA (host and bacterial DNA) was isolated from minute amounts of gastric biopsy specimens and amplified by means of whole genome amplification using the multiple displacement amplification (MDA) technique. Subsequently, MDA-DNA was used for concurrent Helicobacter pylori and human host cellular DNA genotyping analysis using PCR-based methods.

    Results: Total DNA was isolated from gastric biopsy specimens of 12 subjects with gastritis and 16 control subjects having a normal mucosa. The DNA was amplified using a multiple displacement amplification (MDA) kit. Next, concurrent genotyping was performed using H. pylori-specific virulence gene PCR amplification assays, pyrosequencing of bacterial 16S rDNA and PCR characterisation of various host genes. This includes Interleukin 1-beta (IL1B) and Interferon-gamma receptor (IFNGR1) SNP analysis, and Interleukin-1 receptor antagonist (IL1RN) variable tandem repeats (VNTR) in intron 2. Finally, regions of the vacA-gene were PCR amplified using M13-sequence tagged primers which allowed for direct DNA sequencing, omitting cloning of PCR amplicons. H. pylori specific multiplex PCR assays revealed the presence of H. pylori cagA and vacA genotypic variations in 11 of 12 gastritis biopsy specimens. Using pyrosequencing, 16S rDNA variable V3 region signatures of H. pylori were found in 11 of 12 individuals with gastritis, but in none of the control subjects. Similarly, IL1B and IFNGR1-SNP and IL1RN-VNTR patterns could be established in all individuals. Furthermore, sequencing of M13-sequence tagged vacA-PCR amplicons revealed the presence of highly diverse H. pylori vacA-s/i/m regions.

    Conclusion: The PCR-based molecular typing methods applied, using MDA-amplified DNA derived from small amounts of gastric biopsy specimens, enabled a rapid and concurrent molecular analysis of bacterial and host genes in the same biopsy specimen. The principles and technologies used in this study could also be applied to any situation in which human host and microbial genes of interest in microbial-host interactions would need to be sequenced.

  • 43.
    Ryberg, Anna
    et al.
    Linköpings universitet, Hälsouniversitetet. Linköpings universitet, Institutionen för klinisk och experimentell medicin, Medicinsk mikrobiologi.
    Olsson, Crister
    Malmö University Hospital.
    Ahrné, Siv
    Lund University.
    Monstein, Hans-Jürg
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Klinisk mikrobiologi. Linköpings universitet, Hälsouniversitetet. Östergötlands Läns Landsting, Laboratoriemedicinskt centrum, Klinisk mikrobiologi.
    Comparison of (GTG)5-oligonucleotide and ribosomal intergenic transcribed spacer (ITS)-PCR for molecular typing of Klebsiella isolates2011Inngår i: Journal of Microbiological Methods, ISSN 0167-7012, E-ISSN 1872-8359, Vol. 84, nr 2, s. 183-188Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Molecular typing of Klebsiella species has become important for monitoring dissemination of β-lactamase-producers in hospital environments. The present study was designed to evaluate poly-trinucleotide (GTG)5- and rDNA intergenic transcribed spacer (ITS)-PCR fingerprint analysis for typing of Klebsiella pneumoniae and Klebsiella oxytoca isolates. Multiple displacement amplified DNA derived from 19 K. pneumoniae (some with an ESBL-phenotype), 35 K. oxytoca isolates, five K. pneumoniae, two K. oxytoca, three Raoultella, and one Enterobacter aerogenes type and reference strains underwent (GTG)5 and ITS-PCR analysis. Dendrograms were constructed using cosine coefficient and the Neighbour joining method. (GTG)5 and ITS-PCR analysis revealed that K. pneumoniae and K. oxytoca isolates, reference and type strains formed distinct cluster groups, and tentative subclusters could be established. We conclude that (GTG)5 and ITS-PCR analysis combined with automated capillary electrophoresis provides promising tools for molecular typing of Klebsiella isolates.

  • 44.
    Sun, Yi-Qian
    et al.
    Linköpings universitet, Hälsouniversitetet. Linköpings universitet, Institutionen för biomedicin och kirurgi, Avdelningen för kirurgi.
    Monstein, Hans-Jurg
    Linköpings universitet, Hälsouniversitetet. Linköpings universitet, Institutionen för biomedicin och kirurgi, Avdelningen för medicinsk cellbiologi. Östergötlands Läns Landsting, Laboratoriemedicinskt centrum, Molekylärbiologiska tekniklaboratoriet.
    Ryberg, Anna
    Borch, Kurt
    Linköpings universitet, Hälsouniversitetet. Linköpings universitet, Institutionen för biomedicin och kirurgi, Avdelningen för kirurgi. Östergötlands Läns Landsting, Kirurgi- och onkologicentrum, Kirurgiska kliniken i Östergötland med verksamhet i Linköping, Norrköping och Motala.
    Multiple strand displacement amplification of DNA isolated from human archival plasma/serum: Identification of cytokine polymorphism by pyrosequencing analysis2007Inngår i: Clinica Chimica Acta, ISSN 0009-8981, E-ISSN 1873-3492, Vol. 377, nr 1-2, s. 108-113Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Background: DNA isolation from formalin-fixed paraffin-embedded tissue appears to be problematic due to degradation caused by fixative. Our aim was to investigate if the isolated genomic DNA from archival plasma/serum, combined with multiple strand displacement amplification (MDA) can be used for genotyping. Methods: Nine archival plasma/serum samples and freshly frozen gastric biopsies from the same nine H. pylori-infected subjects were used for DNA isolation. Subsequently, MDA-DNA derived from the plasma/serum samples and DNA isolated from the antrum biopsies were analyzed by PCR amplification and pyrosequencing for the presence of interleukin-1beta gene (IL-1B) single nucleotide polymorphism (SNP). In addition, Southern blot and pyrosequencing analysis of H. pylori-specific PCR amplicons were performed. Results: IL-1B SNP profiles obtained from the plasma/serum MDA-DNA and antrum biopsy DNA were identical. A C/C genotype was observed in 7 of 9 samples, and 2 of 9 revealed a C/T genotype for IL-1B - 511. Similarly, 7 of 9 had a T/T, and 2 of 9 had a C/T genotype for IL-1B - 31, 4 of 9 had a C/C, 4 of 9 had a C/T, and 1 of 9 had a T/T genotype, respectively, for IL-1B + 3954. Moreover, pyrosequencing analysis revealed the presence of H. pylori 26695 and J99-like 16S rDNA variable V3 region sequence motifs in the antrum biopsies but not in the plasma/serum samples. Conclusions: We conclude that MDA combined with pyrosequencing enables a rapid and accurate molecular typing of cytokine single nucleotide polymorphisms from archival plasma/serum samples. © 2006 Elsevier B.V. All rights reserved.

  • 45.
    Sun, Yi-Qian
    et al.
    Linköpings universitet, Institutionen för biomedicin och kirurgi, Kirurgi. Linköpings universitet, Hälsouniversitetet.
    Monstein, Hans-Jürg
    Östergötlands Läns Landsting, LMÖ - Laboratoriemedicin i Östergötland.
    Nilsson, Lennart
    Linköpings universitet, Institutionen för molekylär och klinisk medicin, Klinisk mikrobiologi. Linköpings universitet, Hälsouniversitetet.
    Petersson, Fredrik
    Pathology Research Department, Ryhov Hospital, Jönköping, Sweden.
    Borch, Kurt
    Linköpings universitet, Institutionen för biomedicin och kirurgi, Kirurgi. Linköpings universitet, Hälsouniversitetet.
    Profiling and identification of eubacteria in the stomach of Mongolian gerbils with and without Helicobacter pylori infection2003Inngår i: Helicobacter, ISSN 1083-4389, E-ISSN 1523-5378, Vol. 8, nr 2, s. 149-157Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Background. Mongolian gerbils are frequently used to study Helicobacter pylori-induced gastritis and its consequences. The presence of an indigenous bacterial flora with suppressive effect on H. pylori may cause difficulties with establishing this experimental model.

    Aim. The aim of the present study was to determine bacterial profiles in the stomach of Mongolian gerbils with and without (controls) H. pylori infection.

    Methods. Gastric tissue from H. pylori ATCC 43504 and CCUG 17874 infected and control animals were subjected to microbial culturing and histology. In addition, gastric mucosal samples from H. pylori ATCC 43504 infected and control animals were analyzed for bacterial profiling by temporal temperature gradient gel electrophoresis (TTGE), cloning and pyrosequencing of 16S rDNA variable V3 region derived PCR amplicons.

    Results. Oral administration of H. pylori ATCC 43504, but not CCUG 17874, induced colonization and gastric inflammation in the stomach of Mongolian gerbils. Temporal temperature gradient gel electrophoresis (TTGE) and partial 16S rDNA pyrosequencing revealed the presence of DNA representing a mixed bacterial flora in the stomach of both H. pylori ATCC 43504 infected and control animals. In both cases, lactobacilli appeared to be dominant.

    Conclusion. These findings suggest that indigenous bacteria, particularly lactobacilli, may have an impact on the colonization and growth of H. pylori strains in the stomach of Mongolian gerbils.

  • 46.
    Sun, Yi-Qian
    et al.
    Linköpings universitet, Institutionen för biomedicin och kirurgi, Kirurgi. Linköpings universitet, Hälsouniversitetet.
    Petersson, Fredrik
    Pathology Research Department, Ryhov Hospital, Jönköping.
    Monstein, Hans-Jürg
    Östergötlands Läns Landsting, LMÖ - Laboratoriemedicin i Östergötland.
    Söderholm, Johan D
    Linköpings universitet, Institutionen för biomedicin och kirurgi, Kirurgi. Linköpings universitet, Hälsouniversitetet.
    Rehfeld, Jens F
    Department of Clinical Biochemistry, Rigshospitalet, University of Copenhagen.
    Borch, Kurt
    Linköpings universitet, Institutionen för biomedicin och kirurgi, Kirurgi. Linköpings universitet, Hälsouniversitetet.
    Long-term morpho-functional development of Helicobacter pylori-induced gastritis in Mongolian gerbils2005Inngår i: Scandinavian Journal of Gastroenterology, ISSN 0036-5521, E-ISSN 1502-7708, Vol. 40, nr 10, s. 1157-1167Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    OBJECTIVE:

    Epidemiological studies have shown that Helicobacter pylori infection with associated chronic gastritis is the main risk factor for development of gastric cancer. The aim of this study was to investigate the long-term development of H. pylori-induced gastritis in Mongolian gerbils in terms of morphology, gastrin secretion, epithelial proliferation and gene expression of pro-inflammatory cytokines.

    MATERIAL AND METHODS:

    A total of 133 gerbils were inoculated with H. pylori and 62 served as controls. The gerbils were killed at different time-points between 6 and 94 weeks after inoculation. Serum concentrations of anti-H. pylori IgG and gastrin were determined by enzyme-linked immunoabsorbent assay (ELISA) and radioimmunoassay (RIA), respectively. Epithelial proliferation was evaluated immunohistochemically after labeling with 5-bromo-2'-deoxy-uridine. Gene expression of beta-actin, interleukin-1beta (IL-1beta) and tumor necrosis factor-alpha (TNF-alpha) were measured by real-time reverse transcriptase-polymerase chain reaction (RT-PCR). Histological parameters of gastritis were assessed semiquantitatively and expressed as a "gastritis score".

    RESULTS:

    Serum concentrations of anti-H. pylori IgG and gastrin increased over time. Epithelial proliferation in the antrum was increased 6 weeks after inoculation, followed by increased proliferation in the corpus 32 weeks after inoculation. Gene expression of IL-1beta and TNF-alpha were increased in H. pylori-infected gerbils. Beta-actin was not a reliable endogenous control for RT-PCR. With time, gastritis expanded from the antrum to the corpus and the gastritis score increased to reach a peak 32 weeks after inoculation. Pseudopyloric metaplasia (loss of specialized cells) was a characteristic feature in the corpus mucosa. Gastric ulcers, but neither dysplasia nor carcinoma, were observed during 94 weeks of infection.

    CONCLUSIONS:

    Long-term H. pylori infection in Mongolian gerbils led to progressive gastritis, glandular atrophy, hypergastrinemia, increased epithelial proliferation and elevated gene expression of pro-inflammatory cytokines.

  • 47.
    Tiveljung, Annika
    et al.
    Linköpings universitet, Institutionen för molekylär och klinisk medicin, Klinisk mikrobiologi. Linköpings universitet, Hälsouniversitetet.
    Backström, Jan
    Linköpings universitet, Institutionen för molekylär och klinisk medicin, Klinisk mikrobiologi. Linköpings universitet, Hälsouniversitetet.
    Forsum, Urban
    Linköpings universitet, Institutionen för molekylär och klinisk medicin, Klinisk mikrobiologi. Linköpings universitet, Hälsouniversitetet. Östergötlands Läns Landsting, Diagnostikcentrum, Klinisk mikrobiologi.
    Monstein, Hans-Jürg
    Linköpings universitet, Institutionen för molekylär och klinisk medicin, Klinisk mikrobiologi. Linköpings universitet, Hälsouniversitetet. Östergötlands Läns Landsting, Diagnostikcentrum, Klinisk mikrobiologi.
    Broad-range PCR amplification and DNA sequence analysis reveals variable motifs in 16S rRNA genes of Mobiluncus species1995Inngår i: Acta Pathologica, Microbiologica et Immunologica Scandinavica (APMIS), ISSN 0903-4641, E-ISSN 1600-0463, Vol. 103, nr 7-8, s. 755-763Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Using DNA primers based on highly conserved regions of bacterial 16S ribosomal RNA genes, a technique was established for detection of Mobiluncus species by polymerase chain reaction (PCR) and hybridization analysis. Part of the 16S rRNA genes of Mobiluncus mulieris, Mobiluncus curtisii and uncharacterized Mobiluncus strains were analyzed by broad-range PCR amplification and direct DNA sequencing analysis. Sequence comparison of the partial 16S rRNA genes of Mobiluncus curtisii, Mobiluncus mulieris and atypical Mobiluncus strains studied indicated genus and species-specific motifs within the variable regions V3, V4 and V9 of 16S ribosomal DNAs. A Mobiluncus curtisii-specific primer, located within the variable region V3 of the 16S rRNA gene, was designed for Southern blot hybridization analysis of broad-range PCR products. Broad-range amplification combined with a M. curtisii-specific hybridization probe, Mob V3, distinguished between Mobiluncus curtisii, Mobiluncus mulieris, and atypical Mobiluncus strains.

  • 48.
    Tiveljung, Annika
    et al.
    Linköpings universitet, Institutionen för molekylär och klinisk medicin, Klinisk mikrobiologi. Linköpings universitet, Hälsouniversitetet.
    Borch, Kurt
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Kirurgi. Linköpings universitet, Hälsouniversitetet. Östergötlands Läns Landsting, Centrum för kirurgi, ortopedi och cancervård, Kirurgiska kliniken i Östergötland.
    Jonasson, Jon
    Linköpings universitet, Institutionen för molekylär och klinisk medicin, Klinisk mikrobiologi. Linköpings universitet, Hälsouniversitetet. Östergötlands Läns Landsting, Diagnostikcentrum, Klinisk mikrobiologi.
    Mårdh, Sven
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Cellbiologi. Linköpings universitet, Hälsouniversitetet.
    Petersson, Fredrik
    Ryhov Hospital, Jönköping.
    Monstein, Hans-Jürg
    Linköpings universitet, Institutionen för molekylär och klinisk medicin, Klinisk mikrobiologi. Linköpings universitet, Hälsouniversitetet. Östergötlands Läns Landsting, Diagnostikcentrum, Klinisk mikrobiologi.
    Identification of Helicobacter in gastric biopsies by PCR based on 16S rDNA sequences: a matter of little significance for the prediction of H. pylori-associated gastritis?1998Inngår i: Journal of Medical Microbiology, ISSN 0022-2615, E-ISSN 1473-5644, Vol. 47, nr 8, s. 695-704Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    The aim of the present study was to correlate molecular evidence of the presence of Helicobacter pylori in gastric biopsy samples, based on analysis of 16S rDNA, vacuolating toxin (vacA), urease A (ureA) and cagA genes, with the clinical, histological and serological findings in patients with H. pylori-associated gastritis. Fresh biopsy samples were collected from the gastric antrum and corpus of 22 asymptomatic volunteers with or without H. pylori-associated gastritis. Total DNA was extracted from the biopsy material and subjected to 16S rDNA PCR amplification, Southern blotting and 16S rDNA sequence analysis of the PCR products. The vacA, ureA and cagA genes were characterised by PCR amplification and Southern blot analysis. Based on partial 16S rDNA sequence analysis, DNA belonging to the genus Helicobacter was detected in gastric biopsy samples from 20 of 22 subjects, including seven of nine histologically and serologically normal controls. Six of 20 partial 16S rDNA sequences revealed variations within variable regions V3 and V4 that deviated from those of the H. pylori type strain ATCC 4350T and, therefore, possibly represented other species of Helicobacter. VacA genes identical with those of the type strain were found predominantly in the subjects with H. pylori gastritis, and all the patients except one were found to be cagA-positive. There was no evidence of false positive PCR reactions. In conclusion, the PCR-based molecular typing methods used here were apparently too sensitive when applied to the detection of H. pylori in human gastric tissues. The lack of quantitative analysis makes them inappropriate as clinical tools for the diagnosis of H. pylori-associated gastritis, despite the fact that they provide a qualitative and sensitive tool for the detection and characterisation of H. pylori in the gastrointestinal tract.

  • 49.
    Tiveljung, Annika
    et al.
    Linköpings universitet, Institutionen för molekylär och klinisk medicin, Klinisk mikrobiologi. Linköpings universitet, Hälsouniversitetet.
    Forsum, Urban
    Linköpings universitet, Institutionen för molekylär och klinisk medicin, Klinisk mikrobiologi. Linköpings universitet, Hälsouniversitetet. Östergötlands Läns Landsting, Diagnostikcentrum, Klinisk mikrobiologi.
    Monstein, Hans-Jürg
    Linköpings universitet, Institutionen för molekylär och klinisk medicin, Klinisk mikrobiologi. Linköpings universitet, Hälsouniversitetet. Östergötlands Läns Landsting, Diagnostikcentrum, Klinisk mikrobiologi.
    Classification of the genus Mobiluncus based on comparative partial 16S rRNA gene analysis1996Inngår i: International Journal of Systematic Bacteriology, ISSN 0020-7713, Vol. 46, nr 1, s. 332-336Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    On the basis of partial 16S rRNA gene sequences and the results of Southern blot analyses, we confirmed the division of the genus Mobiluncus into the species Mobiluncus curtisii and Mobiluncus mulieris. Division of M. curtisii into M. curtisii subsp. curtisii and M. curtisii subsp. holmesii was not supported by our data.

  • 50.
    Tiveljung, Annika
    et al.
    Linköpings universitet, Institutionen för molekylär och klinisk medicin, Klinisk mikrobiologi. Linköpings universitet, Hälsouniversitetet.
    Söderholm, Johan D.
    Linköpings universitet, Institutionen för biomedicin och kirurgi, Kirurgi. Linköpings universitet, Hälsouniversitetet.
    Olaison, Gunnar
    Linköpings universitet, Institutionen för biomedicin och kirurgi, Kirurgi. Linköpings universitet, Hälsouniversitetet.
    Jonasson, Jon
    Östergötlands Läns Landsting, Laboratoriemedicinskt centrum, Klinisk mikrobiologi. Linköpings universitet, Hälsouniversitetet.
    Monstein, Hans-Jürg
    Östergötlands Läns Landsting, Laboratoriemedicinskt centrum, Klinisk mikrobiologi. Linköpings universitet, Hälsouniversitetet.
    Presence of eubacteria in biopsies from Crohn's disease inflammatory lesions as determined by 16S rRNA gene-based PCR1999Inngår i: Journal of Medical Microbiology, ISSN 0022-2615, E-ISSN 1473-5644, Vol. 48, nr 3, s. 263-268Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    The aim of this study was to search for putative microbial agents in Crohn's disease (CD) tissues by bacterial broad-range 16S rDNA PCR combined with genus- and species-specific DNA hybridisation analysis. Biopsies taken both surgically and endoscopically from the terminal ileum of 11 CD patients and 11 control patients were investigated. Significant amounts of eubacteria were demonstrated in biopsies taken endoscopically from both affected and unaffected individuals; the biopsies taken at surgery from control patients were negative. Three of five biopsies taken surgically from CD patients harboured Helicobacter spp.-, Mycobacterium paratuberculosis-, Listeria monocytogenes- and Escherichia coli-like 16S rDNA sequences. These findings show the importance of the sampling method chosen when combined with molecular typing of eubacteria in intestinal tissues. The mixed bacterial flora found in the surgical biopsies from CD patients supports the idea that the enteric microflora enters primary lesions where secondary bacterial colonisers may elicit a chronic inflammatory syndrome.

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