| 1. |
- Nilsson, Anna, et al.
(författare)
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Survival of Campylobacter jejuni and C. coli water isolates in lake and well water
- 2018
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Ingår i: Acta Pathologica, Microbiologica et Immunologica Scandinavica (APMIS). - : Wiley. - 0903-4641 .- 1600-0463. ; 126:9, s. 762-770
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Tidskriftsartikel (refereegranskat)abstract
- The role of water for transmission of Campylobacter jejuni and C. coli to humans might be underestimated, as factors important for bacterial viability in water are largely unknown. We have studied water survival of seven C. jejuni and eight C. coli isolates originally isolated from Swedish waters, together with selected reference strains, over eight days at 4 °C in the dark in untreated water collected from a local lake and a private well. To study seasonality, lake water samples were collected during spring and autumn. Samples for culturable bacterial counts were taken on days 2, 4, 6, and 8 and compared to the start inoculum. For C. jejuni, a significantly better survival was observed in autumn than in spring lake water. Furthermore, C. jejuni had a significantly better survival than C. coli in autumn lake and well water samples; the rate of culturability loss was almost double for C. coli in autumn lake water. These findings contribute to a better understanding on the seasonality of waterborne Campylobacter infections and the general predominance of C. jejuni.
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| 2. |
- Nilsson, Anna, 1986-
(författare)
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Characterization of Campylobacter jejuni and Campylobacter coli water isolates
- 2018
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Campylobacter jejuni and C. coli are together the most common cause of bacterial gastroenteritis in the European Union. Campylobacter can be transmitted to humans via contaminated water, but it is largely unknown how these bacteria survive in water.The aim of this thesis was to better understand the water survival strategies and pathogenic potential of Campylobacter water isolates. For this purpose, C. jejuni and C. coli, originally isolated from incoming water at surface water plants, were characterized using whole genome sequencing, phenotypical assays, water survival experiments and an in vitro infection model.C. jejuni water isolates included both common and uncommon sequence types for human pathogens, whereas C. coli isolates were assigned to clades 2 and 3, associated with environmental sources. For C. jejuni, comparative genomics revealed genes involved in oxidative and aerobic stress response. In C. coli, various carbon metabolism-related sequences were identified in clade 2 isolates and in clade 3 isolates, oxidative stress and putative virulence genes were detected. All water isolates were motile and the majority of C. jejuni isolates, but none of C. coli isolates, were able to form biofilm. C. jejuni survived better than C. coli in untreated well and lake water. Furthermore, in contrast to C. coli, a seasonal difference in survival was observed for C. jejuni with better survival in lake water collected during autumn than in spring. When tested in an in vitro infection model, all water isolates adhered to and induced IL-8 response in HT-29 cells indicating pathogenic potential. However, C. coli clade 3 isolates demonstrated a strong cytotoxic effect on human HT-29 cells leading to rapid cell death. This novel phenomenon was not observed for C. coli clade 2 or C. jejuni isolates.This is, to the best of our knowledge, the first study on Campylobacter water isolates characterized using genomic, phenotypical and in vitro infection analyses. These findings suggest that some Campylobacter isolates might survive better than others in water and water survival patterns shown here help us further understand the seasonality and predominance of water-related C. jejuni infections.
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| 3. |
- Malmberg, Christer, et al.
(författare)
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Faster results, higher precision: Evaluating the QuickMIC rapid AST assay in a clinical setting
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Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- The rapidly changing landscape of antimicrobial resistance poses a challenge for empirical therapy and highlights the need for fast antibiotic susceptibility diagnostics to guide treatment. Traditional methods for antibiotic susceptibility testing (AST) of bacteria such as broth microdilution (BMD) or the disc diffusion method (DDM) are comparatively slow and with high variability. Rapid AST methods under development often trade speed for resolution, sometimes only measuring responses at a single antibiotic concentration. QuickMIC is a recently developed lab-on-a-chip system for rapid AST. Here we evaluate the performance of the QuickMIC method with regard to speed, precision and accuracy in comparison to traditional diagnostic methods. 151 blood cultures of clinical Gram-negative isolates with a high frequency of resistant bacteria were tested with the QuickMIC system and compared with BMD for 12 antibiotics. To investigate sample turnaround time and functionality in a clinical setting, another 41 clinical blood culture samples were acquired from the Uppsala University Hospital and analyzed on site in the clinical laboratory with the QuickMIC system and compared with DDM for 8 antibiotics routinely used in the clinical laboratory. The overall essential agreement between MIC values obtained by QuickMIC and BMD was 83.2%, with times to result of 3 h 2 min (SD: 24.8 min) for the QuickMIC method. For the clinical dataset, the categorical agreement was 94.9%, and the total turnaround time as compared to routine diagnostics reduced by 40% (33 h vs. 55 h). Interexperiment variability was low (average SD: 44.6% from target MIC) compared to the acceptable standard of ±1 log2 unit (-50% to +100% deviation from target MIC) in BMD. We conclude that the QuickMIC method can be used to rapidly guide therapy, and may be especially valuable for antibiotics where wildtype and resistant MIC distributions are close or overlapping or in settings with high background resistance.
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| 4. |
- Malmberg, Christer, 1984-, et al.
(författare)
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The T2Bacteria panel and rapid AST with bacteria pre-sampling for combined ID and AST before blood culture positivity
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Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- Background: Rapid diagnostic methods are important for antibiotic stewardship and for improving quality of care in severe infections. New non-culture based methods for bacterial identification (ID) within hours directly from blood sampling are becoming available, such as the T2Bacteria panel. However, blood cultures remain necessary for bacteria isolation and follow-up analysis such as antibiotic susceptibility testing (AST). QuickMIC is a rapid diagnostic tool under development, capable of AST at very low bacterial concentrations. Here we evaluate a combined rapid ID/AST diagnostic workflow using the T2Bacteria panel and the QuickMIC AST system.Materials/methods: Two diagnostic workflows were simulated, a “standard” workflow using blood culture followed by MALDI-TOF MS (MS) and AST by broth microdilution (BMD); or a “rapid” workflow using T2Bacteria followed by AST using QuickMIC. Clinically derived strains of Escherichia coli (n = 5), Klebsiella pneumoniae (n = 9), Acinetobacter baumannii (n = 6), Pseudomonas aeruginosa (n = 5), Enterococcus faecalis (n=1) and Staphylococcus aureus (n =12) were inoculated in horse blood and used to simultaneously start blood cultures as well as analyses using the T2Bacteria panel. After identification of bacteria-containing blood cultures using the T2Bacteria panel, the cultures were sampled for analysis using rapid AST using QuickMIC. Further, after blood culture positivity, MS was performed. Specificity, sensitivity and turnaround times were compared between the two workflows, and QuickMIC results were compared to results obtained using the BMD method with regard to categorical and essential agreement.Results: The rapid diagnostic workflow was significantly faster than the standard workflow (9.5±2.5h vs. 52.9±0.4h, p<0.001), and significantly faster for Gram-negative compared to Gram-positive bacteria (7.4±0.6h vs 12.2±0.4h, p<0.001). For 68% of the samples, the rapid ID/AST result was available before blood culture positivity (86% for Gram-negatives, 45% for Gram-positives). For 100% of the samples, ID/AST results from the rapid workflow were available before ID by MS. Diagnostic sensitivity/specificity at the species level were 94.7%/99.5% and 97.4%/100% for analysis from the T2Bacteria panel or MS, respectively. QuickMIC results took on average 167±15 min, and categorical agreement to BMD was 70.9% (Gram-negative bacteria) and 72.8% (Gram-positive bacteria).Conclusions: We conclude that QuickMIC has the potential to be a suitable companion diagnostic to the T2Bacteria panel for delivering rapid ID/AST results to enable same-workshift results for improved antibiotic stewardship and quality of care.
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| 5. |
- Johansson, Cecilia, et al.
(författare)
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Liver dendritic cells present bacterial antigens and produce cytokines upon Salmonella encounter.
- 2004
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Ingår i: Journal of immunology (Baltimore, Md. : 1950). - 0022-1767. ; 172:4, s. 2496-2503
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Tidskriftsartikel (refereegranskat)abstract
- The capacity of murine liver dendritic cells (DC) to present bacterial Ags and produce cytokines after encounter with Salmonella was studied. Freshly isolated, nonparenchymal liver CD11c(+) cells had heterogeneous expression of MHC class II and CD11b and a low level of CD40 and CD86 expression. Characterization of liver DC subsets revealed that CD8alpha(-)CD4(-) double negative cells constituted the majority of liver CD11c(+) ( approximately 85%) with few cells expressing CD8alpha or CD4. Flow cytometry analysis of freshly isolated CD11c(+) cells enriched from the liver and cocultured with Salmonella expressing green fluorescent protein (GFP) showed that CD11c(+) MHC class II(high) cells had a greater capacity to internalize Salmonella relative to CD11c(+) MHC class II(low) cells. Moreover, both CD8alpha(-) and CD8alpha(+) liver DC internalized bacteria with similar efficiency after both in vitro and in vivo infection. CD11c(+) cells enriched from the liver could also process Salmonella for peptide presentation on MHC class I and class II to primary, Ag-specific T cells after internalization requiring actin cytoskeletal rearrangements. Flow cytometry analysis of liver CD11c(+) cells infected with Salmonella expressing GFP showed that both CD8alpha(-) and CD8alpha(+) DC produced IL-12p40 and TNF-alpha. The majority of cytokine-positive cells did not contain bacteria (GFP(-)) whereas only a minor fraction of cytokine-positive cells were GFP(+). Furthermore, only approximately 30-50% of liver DC containing bacteria (GFP(+)) produced cytokines. Thus, liver DC can internalize and process Salmonella for peptide presentation to CD4(+) and CD8(+) T cells and elicit proinflammatory cytokine production upon Salmonella encounter, suggesting that DC in the liver may contribute to immunity against hepatotropic bacteria.
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| 6. |
- Dhanjal, Soniya, et al.
(författare)
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Heterogeneous Nuclear Ribonucleoprotein C Proteins Interact with the Human Papillomavirus Type 16 (HPV16) Early 3'-Untranslated Region and Alleviate Suppression of HPV16 Late L1 mRNA Splicing.
- 2015
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Ingår i: Journal of Biological Chemistry. - 0021-9258 .- 1083-351X. ; 290:21, s. 13354-71
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Tidskriftsartikel (refereegranskat)abstract
- In order to identify cellular factors that regulate human papillomavirus type 16 (HPV16) gene expression, cervical cancer cells permissive for HPV16 late gene expression were identified and characterized. These cells either contained a novel spliced variant of the L1 mRNAs that bypassed the suppressed HPV16 late, 5'-splice site SD3632; produced elevated levels of RNA-binding proteins SRSF1 (ASF/SF2), SRSF9 (SRp30c), and HuR that are known to regulate HPV16 late gene expression; or were shown by a gene expression array analysis to overexpress the RALYL RNA-binding protein of the heterogeneous nuclear ribonucleoprotein C (hnRNP C) family. Overexpression of RALYL or hnRNP C1 induced HPV16 late gene expression from HPV16 subgenomic plasmids and from episomal forms of the full-length HPV16 genome. This induction was dependent on the HPV16 early untranslated region. Binding of hnRNP C1 to the HPV16 early, untranslated region activated HPV16 late 5'-splice site SD3632 and resulted in production of HPV16 L1 mRNAs. Our results suggested that hnRNP C1 controls HPV16 late gene expression.
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| 7. |
- Johansson, Cecilia, et al.
(författare)
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Elevated neutrophil, macrophage and dendritic cell numbers characterize immune cell populations in mice chronically infected with Salmonella.
- 2006
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Ingår i: Microbial pathogenesis. - : Elsevier BV. - 0882-4010 .- 1096-1208. ; 41:2-3, s. 49-58
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Tidskriftsartikel (refereegranskat)abstract
- The present study characterizes immune cell populations in mice chronically infected with Salmonella. Mice were characterized as chronically infected based on persistently high titers of Salmonella-reactive immunoglobulins in the serum >6 months after a single oral dose of S. enterica serovar Typhimurium. These mice had a visibly enlarged spleen but not liver, while both organs harbored bacteria and had increased total cellularity up to 11 months post-infection. Flow cytometry analysis revealed significantly elevated numbers of neutrophils, dendritic cells (DC) and macrophages in the spleen of chronically infected mice. In contrast, no significant increase in the absolute number of T and B cells was apparent in the spleen and DX5+ cells, which includes NK cells, some NK T cells and possibly some activated T cells, appears to correlate with chronic Salmonella infection in the liver but not the spleen. In situ analyses revealed that CD8alpha+ DC and Gr-1+ cells (neutrophils) increased in the splenic red pulp of chronically infected mice. In addition, Gr-1+ cells, CD68+ cells and CD11c+ cells (DC), the latter lacking detectable staining for CD8alpha and CD4, accumulated around hepatic blood vessels and in the hepatic network in the liver of mice chronically harboring bacteria. These data provide insight into changes that occur within immune cell populations, most notably within splenic and hepatic phagocytic cell populations, that accompany chronic infection with the intracellular bacterium Salmonella.
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| 8. |
- Johansson, Cecilia
(författare)
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Proteins involved in HPV-16 pre-mRNA processing
- 2010
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Ingår i: Current Topics in Virology. ; 8, s. 17-27
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Tidskriftsartikel (övrigt vetenskapligt/konstnärligt)abstract
- Self-inactivating retroviruses are an attractive vector choice for long term and stable expression of therapeutic genes because of their ability to integrate into the host genome. However, their utility as a human therapeutic platform is dependent on stringent criteria for safety and efficacy. Therefore, targeting the vector to a specific cell or tissue type is of paramount importance. Numerous virus-engineering strategies have been developed that either modify the viral surface glycoproteins or regulate transgene expression in a tissue-specific manner. The former, termed transductional targeting, involves modifying the viral glycoproteins that are responsible for binding to specific cellular receptors and facilitating entry into the host cell. The goal of this strategy is to develop a targeted, injectable retroviral based vector that will deliver the transgene to a specific cell or tissue type while leaving non-target tissue unharmed. Recently, the field has seen a technical advance with the demonstration that retroviruses can be pseudotyped with the envelope glycoproteins of the measles virus. The measles hemagglutinin (H) protein is responsible for receptor recognition, and the fusion (F) protein catalyzes lipid membrane mixing and viral entry. The natural tropism of the measles H protein for its receptors has been completely eliminated, allowing retargeting by the display of ligands such as growth factors or single-chain antibodies (scFvs) on the C-terminal end. We summarize the progress to date with retargeting retroviral cores via pseudotyping with measles virus H and F glycoproteins.
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| 9. |
- Johansson, Cecilia, et al.
(författare)
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Staphylococcus argenteus and Staphylococcus schweitzeri are cytotoxic to human cells in vitro due to high expression of alpha-hemolysin Hla
- 2019
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Ingår i: Virulence. - : Taylor & Francis. - 2150-5594 .- 2150-5608. ; 10:1, s. 502-510
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Tidskriftsartikel (refereegranskat)abstract
- Staphylococcus argenteus and Staphylococcus schweitzeri are newly identified species of the S. aureus-related complex. S. argenteus, as occurring globally and showing significant prevalence and comparable infection and morbidity rates compared to S. aureus, is becoming clinically important. Whole genome sequencing has revealed the presence of several virulence genes but the molecular mechanisms of S. argenteus infection and virulence are largely unknown. Here, we studied the effect of a previously characterized clinical S. argenteus isolate on human cells in vitro. The clinical isolate, together with the S. argenteus type strain MSHR1132T and the S. schweitzeri type strain FSA084T, had a cytotoxic effect on the cells, which showed necrotic cell death after a few hours of treatment. The protein causing the cytotoxic effect was purified and identified by mass spectrometry as alpha-hemolysin, Hla, which is awell-known pore-forming toxin in S.aureus. The cytotoxic effect could be blocked with an antibody against Hla. S.argenteus showed 12-15 fold higher expression levels of hla at the RNA level and 4-6 fold higher expression levels at the protein level compared to S.aureus. The higher expression levels of hla were supported by higher RNA levels of the regulatory factors sarA and saeR. Also, the RNAIII component of the accessory gene regulator (agr) quorum sensing system was 8,000-10,000 fold higher in the S.argenteus isolates compared to S.aureus. This is the first study on the effect of S.argenteus on ahuman cell line and strengthens the idea of significant virulence of S.argenteus.
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| 10. |
- Somberg, Monika, 1979-, et al.
(författare)
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Serine/arginine-rich protein 30c activates human papillomavirus type 16 L1 mRNA expression via a bimodal mechanism
- 2011
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Ingår i: Journal of General Virology. - : Microbiology Society. - 0022-1317 .- 1465-2099. ; 92:10, s. 2411-2421
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Tidskriftsartikel (refereegranskat)abstract
- Two splice sites on the human papillomavirus type 16 (HPV-16) genome are used exclusively by the late capsid protein L1 mRNAs: SD3632 and SA5639. These splice sites are suppressed in mitotic cells. This study showed that serine/arginine-rich protein 30c (SRp30c), also named SFRS9, activated both SD3632 and SA5639 and induced production of L1 mRNA. Activation of HPV-16 L1 mRNA splicing by SRp30c required an intact arginine/serine-repeat (RS) domain of SRp30c. In addition to this effect, SRp30c could enhance L1 mRNA production indirectly by inhibiting the early 3′-splice site SA3358, which competed with the late 3′-splice site SA5639. SRp30c bound directly to sequences downstream of SA3358, suggesting that SRp30c inhibited the enhancer at SA3358 and caused a redirection of splicing to the late 3′-splice site SA5639. This inhibitory effect of SRp30c was independent of its RS domain. These results suggest that SRp30c can activate HPV-16 L1 mRNA expression via a bimodal mechanism: directly by stimulating splicing to late splice sites and indirectly by inhibiting competing early splice sites.
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