TY - GEN A1 - Sydow, Katharina A1 - Eger, Elias A1 - Schwabe, Michael A1 - Heiden, Stefan E. A1 - Bohnert, Jürgen A. A1 - Franzenburg, Sören A1 - Jurischka, Christoph A1 - Schierack, Peter A1 - Schaufler, Katharina T1 - Geno- and Phenotypic Characteristics of a Klebsiella pneumoniae ST20 Isolate with Unusual Colony Morphology T2 - Microorganisms N2 - Klebsiella pneumoniae is a common member of the intestinal flora of vertebrates. In addition to opportunistic representatives, hypervirulent (hvKp) and antibiotic-resistant K. pneumoniae (ABR-Kp) occur. While ABR-Kp isolates often cause difficult-to-treat diseases due to limited therapeutic options, hvKp is a pathotype that can infect healthy individuals often leading to recurrent infection. Here, we investigated the clinical K. pneumoniae isolate PBIO3459 obtained from a blood sample, which showed an unusual colony morphology. By combining whole-genome and RNA sequencing with multiple in vitro and in vivo virulence-associated assays, we aimed to define the respective Klebsiella subtype and explore the unusual phenotypic appearance. We demonstrate that PBIO3459 belongs to sequence type (ST)20 and carries no acquired resistance genes, consistent with phenotypic susceptibility tests. In addition, the isolate showed low-level virulence, both at genetic and phenotypic levels. We thus suggest that PBIO3459 is an opportunistic (commensal) K. pneumoniae isolate. Genomic comparison of PBIO3459 with closely related ABR-Kp ST20 isolates revealed that they differed only in resistance genes. Finally, the unusual colony morphology was mainly associated with carbohydrate and amino acid transport and metabolism. In conclusion, our study reveals the characteristics of a Klebsiella sepsis isolate and suggests that opportunistic representatives likely acquire and accumulate antibiotic resistances that subsequently enable their emergence as ABR-Kp pathogens. KW - virulence KW - next-generation sequencing KW - biofilm KW - exopolysaccharides Y1 - 2022 U6 - https://doi.org/10.3390/microorganisms10102063 SN - 2076-2607 VL - 10 IS - 10 ER - TY - GEN A1 - Lopens, Steffi A1 - Wunsch, Ewa A1 - Milkiewicz, Malgorzata A1 - Röber, Nadja A1 - Zarske, Grit A1 - Nasser, Abdullah A1 - Conrad, Karsten A1 - Laass, Martin W. A1 - Rödiger, Stefan A1 - Krawczyk, Marcin A1 - Roggenbuck, Dirk A1 - Milkiewicz, Piotr T1 - PR3-ANCAs Detected by Third-Generation ELISA Predicts Severe Disease and Poor Survival in Primary Sclerosing Cholangitis T2 - Diagnostics N2 - A highly sensitive detection of anti-neutrophil cytoplasmic antibodies to serine proteinase-3 (PR3-ANCAs) aids in the serological diagnosis of autoimmune liver disorders and the prediction of severity in primary sclerosing cholangitis (PSC). Here, we evaluate a novel third-generation ELISA for the detection of PR3-ANCAs. In total, 309 patients with PSC, 51 with primary biliary cholangitis (PBC), and 120 healthy blood donors (BD) were analyzed. For the survival analysis in PSC, the outcome was defined as liver-transplantation-free survival during the follow-up. Positive PR3-ANCA levels were found in 74/309 (24.0%) of patients with PSC. No BDs and one patient with PBC demonstrated PR3-ANCA positivity. PR3-ANCAs were revealed as independent predictors for a poor PSC outcome (study endpoint: liver transplantation/death, log-rank test, p = 0.02). PR3-ANCA positivity, lower albumin levels, and higher bilirubin concentrations were independent risks of a poor survival (Cox proportional-hazards regression analysis, p < 0.05). The Mayo risk score for PSC was associated with PR3-ANCA positivity (p = 0.01) and the disease severity assessed with a model of end-stage liver disease (MELD) and extended MELD-Na (p < 0.05). PR3-ANCAs detected by a third-generation ELISA are diagnostic and prognostic markers for PSC. Their wider use could help to identify patients who are at-risk of a more severe disease. KW - primary sclerosing cholangitis KW - antibodies against serine protease proteinase-3 KW - survival KW - health-related quality of life KW - liver biochemistry KW - disease severity Y1 - 2022 U6 - https://doi.org/10.3390/diagnostics12112682 SN - 2075-4418 VL - 12 IS - 11 ER - TY - GEN A1 - Khan, Muhammad Moman A1 - Sidorczuk, Katarzyna A1 - Becker, Juliane A1 - Aleksandrowicz, Adrianna A1 - Baraniewicz, Karolina A1 - Ludwig, Christina A1 - Ali, Aamir A1 - Kingsley, Robert A. A1 - Schierack, Peter A1 - Kolenda, Rafał T1 - Characterization of clumpy adhesion of Escherichia coli to human cells and associated factors influencing antibiotic sensitivity T2 - Microbiology Spectrum N2 - Escherichia coli intestinal infection pathotypes are characterized by distinct adhesion patterns, including the recently described clumpy adhesion phenotype. Here, we identify and characterize the genetic factors contributing to the clumpy adhesion of E. coli strain 4972. In this strain, the transcriptome and proteome of adhered bacteria were found to be distinct from planktonic bacteria in the supernatant. A total of 622 genes in the transcriptome were differentially expressed in bacteria present in clumps relative to the planktonic bacteria. Seven genes targeted for disruption had variable distribution in different pathotypes and nonpathogenic E. coli, with the pilV and spnT genes being the least frequent or absent from most groups. Deletion (Δ) of five differentially expressed genes, flgH, ffp, pilV, spnT, and yggT, affected motility, adhesion, or antibiotic stress. ΔflgH exhibited 80% decrease and ΔyggT depicted 184% increase in adhesion, and upon complementation, adhesion was significantly reduced to 13%. ΔflgH lost motility and was regenerated when complemented, whereas Δffp had significantly increased motility, and reintroduction of the same gene reduced it to the wild-type level. The clumps produced by Δffp and ΔspnT were more resistant and protected the bacteria, with ΔspnT showing the best clump formation in terms of ampicillin stress protection. ΔyggT had the lowest tolerance to gentamicin, where the antibiotic stress completely eliminated the bacteria. Overall, we were able to investigate the influence of clump formation on cell surface adhesion and antimicrobial tolerance, with the contribution of several factors crucial to clump formation on susceptibility to the selected antibiotics. KW - Infectious Diseases KW - Cell Biology KW - Microbiology (medical) KW - Genetics KW - General Immunology and Microbiology KW - Ecology KW - Physiology Y1 - 2024 U6 - https://doi.org/10.1128/spectrum.02606-23 SN - 2165-0497 ER - TY - GEN A1 - Khan, Muhammad Moman A1 - Mushtaq, Muhammad Ahmed A1 - Abbas, Nayyar A1 - Fatima, Fariha A1 - Gibbon, Marjorie J. A1 - Schierack, Peter A1 - Mohsin, Mashkoor T1 - Occurrence, antimicrobial resistance and genomic features of Klebsiella pneumoniae from broiler chicken in Faisalabad, Pakistan T2 - Frontiers in Veterinary Science N2 - IntroductionThe dissemination of antimicrobial resistance (AMR) in critical priority pathogens is a significant threat. Non-clinical reservoirs of AMR, such as agriculture and food production facilities, may contribute to the transmission of clinically relevant pathogens such as multidrug-resistant (MDR) Klebsiella pneumoniae. There is currently very limited knowledge regarding the population structure and genomic diversity of K. pneumoniae in poultry production in Pakistan.MethodsWe explored healthy broilers in a commercial farm from Faisalabad, Pakistan, and identified six K. pneumoniae strains from 100 broiler birds. We characterized the strains, determining clonality, virulence and antimicrobial resistance genes using next generation sequencing.ResultsThe evaluation of antimicrobial susceptibility revealed that all the strains were MDR. Genomic analysis showed that 3/6 strains belonged to ST152, harbouring acquired resistance aminoglycosides [aadA2, aph(4′)-Ia], β-lactams (blaSHV-187, blaLAP2), fosfomycin (fosA6), tetracycline (tetA), trimethoprim (dfrA12), quinolone (qnrS1), sulphonamides (sul2) and phenicol (floR). All the strains harboured the efflux pump genes oqxA, oqxB, emrR, kpnG, kpnH, kpnF, baeR, mtdB and mtdC. All six strains encoded identical virulence profiles possessing six genes, i.e., ureA, iutA, entB, allS, fimH and mrkD. Phylogenomic analysis of the dominant sequence type (ST152) present in our dataset with publicly available genomes showed that the isolates clustered to strains mainly from human sources and could pose a potential threat to food safety and public health.DiscussionThe combination of these findings with antimicrobial use data would allow a better understanding of the selective pressures that may be driving the spread of AMR. This is the first report of MDR K. pneumoniae isolated from broiler hens in Pakistan, and the finding suggests that routine surveillance of WHO critical priority pathogens in such settings would be beneficial to the development of effective control strategies to reduce AMR. KW - antimicrobial resistance KW - poultry KW - genomics KW - sequence type Y1 - 2024 U6 - https://doi.org/10.3389/fvets.2024.1433124 SN - 2297-1769 VL - 11 PB - Frontiers Media S.A. ER - TY - GEN A1 - Brauckhoff, Tim A1 - Kieffer, Coline A1 - Rödiger, Stefan T1 - Extracting Insights from biological images T2 - Journal of Open Source Software Y1 - 2024 U6 - https://doi.org/10.21105/joss.07074 SN - 2475-9066 VL - 9 IS - 102 ER - TY - GEN A1 - Liedtke, Victoria A1 - Rose, Laura A1 - Hiemann, Rico A1 - Nasser, Abdullah A1 - Rödiger, Stefan A1 - Bonaventura, Alena A1 - Winkler, Laura A1 - Sowa, Mandy A1 - Stöckle, Michael A1 - Schierack, Peter A1 - Junker, Kerstin A1 - Roggenbuck, Dirk T1 - Over-Expression of LEDGF/p75 in HEp-2 Cells Enhances Autoimmune IgG Response in Patients with Benign Prostatic Hyperplasia—A Novel Diagnostic Approach with Therapeutic Consequence? T2 - International Journal of Molecular Sciences N2 - Lens epithelium-derived growth factor splice variant of 75 kDa (LEDGF/p75) is an autoantigen over-expressed in solid tumors and acts as a stress-related transcriptional co-activator. Participation of autoimmune responses in the pathophysiology of benign prostatic hyperplasia (PBH) and a corresponding immunosuppressive therapy by TNFalpha antagonists has been recently suggested. Thus, autoAb testing could aid in the diagnosis of BPH patients profiting from such therapy. We generated CRISPR/Cas9 modified HEp-2 LEDGF knock-out (KO) and HEp-2 LEDGF/p75 over-expressing (OE) cells and examined IgG autoantibody reactivity to LEDGF/p75 in patients with prostate cancer (PCa, n = 89), bladder cancer (BCa, n = 116), benign prostatic hyperplasia (BPH, n = 103), and blood donors (BD, n = 60) by indirect immunofluorescence assay (IFA). Surprisingly, we could not detect elevated binding of autoAbs against LEDGF/p75 in cancer patients, but autoAb reactivity to LEDGF/p75 OE cells in about 50% of patients with BPH was unexpectedly significantly increased. Furthermore, a line immunoassay enabling the detection of 18 different autoAbs revealed a significantly increased occurrence of anti-dsDNA autoAbs in 34% of BPH patients in contrast to tumor patients and BD. This finding was confirmed by anti-mitochondrial (mDNA) autoAb detection with the Crithidia luciliae immunofluorescence test, which also showed a significantly higher prevalence (34%) of anti-mDNA autoAbs in BPH. In summary, our study provided further evidence for the occurrence of autoimmune responses in BPH. Furthermore, LEDGF/p75 over-expression renders HEp-2 cells more autoantigenic and an ideal target for autoAb analysis in BPH with a potential therapy consequence. KW - LEDGF/p75 KW - autoimmunity KW - CRISPR/Cas9 KW - dsDNA KW - mDNA Y1 - 2023 U6 - https://doi.org/10.3390/ijms24076166 SN - 1422-0067 VL - 24 IS - 7 ER - TY - GEN A1 - Roei, Tulchinsky A1 - Boris, Gilburd A1 - Yehuda, Shovman A1 - Milena, Tocut A1 - Eleanor, Zeruya A1 - Ariel, Binyaminov A1 - Tima, Davidson A1 - Büttner, Thomas A1 - Nasser, Abdullah A1 - Michel, Juliane A1 - Rödiger, Stefan A1 - Schierack, Peter A1 - Howard, Amital A1 - Roggenbuck, Dirk A1 - Yehuda, Shoenfeld A1 - Ora, Shovman T1 - CytoBead ANA 2 assay : a novel method for the detection of antinuclear antibodies T2 - Scientific reports N2 - Detection of anti-nuclear autoantibodies (ANA) is based on a two-step algorithm including indirect immunofluorescence (IIF) on HEp2 cells and subsequent reflex/confirmatory testing for specific autoantibodies. Simultaneous cell- and microbead-based autoantibody detection by IIF may be utilized for the evaluation of systemic autoimmune rheumatic diseases (SARDs). In the present study, we assessed the performance of CytoBead ANA 2 in the detection of ANA and ANA-specific autoantibodies, compared to ANA IIF and BioPlex™ 2200. We also tested the ability of CytoBead ANA DFS-70 to identify dense-fine speckled (DFS) pattern associated with anti-DFS70 antibodies in non-SARDs patients. Hundred-twelve routine sera samples were assessed by manual CytoBead ANA 2 for the presence of ANA and specific autoantibodies. In parallel, these samples were analyzed by HEp2 ANA IIF test and a subsequent multiplexed assay BioPlex™ 2200 ANA. Twenty-nine ANA-positive samples obtained from non-SARDs patients and exhibiting DFS pattern by ANA IIF were further tested by CytoBead ANA DFS-70. A substantial agreement was observed between classical ANA IIF and manual CytoBead ANA 2 for the detection of ANA (k = 0.74). Discordant results were mainly associated with the presence of anti-SSA/Ro antibodies detected by CytoBead ANA 2 in ANA IIF negative patients. A good to almost perfect agreement was found between CytoBead ANA 2 and BioPlex™ 2200 for detection of specific antibodies with kappa values ranging from 0.70 to 0.90. Twenty samples (68.9%) obtained from 29 ANA IIF positive without SARDs patients exhibited DFS pattern in CytoBead ANA DFS-70, which confirmed the presence of anti-DFS70 antibodies. The diagnostic performance of manual CytoBead ANA 2 for ANA screening and detection of ANA specific antibodies is comparable to the diagnostic performance of ANA IIF followed by BioPlex™ 2200. This novel one-step assay enables simultaneous ANA screening and confirmation and represents a promising alternative approach to the time-consuming and costly two-tier ANA analysis. KW - Anti-nuclear antibodies KW - Anti-DFS70 antibodies KW - Indirect immunofluorescence KW - Multiplexed assay KW - CytoBead technology Y1 - 2025 U6 - https://doi.org/10.1038/s41598-025-04583-3 SN - 2045-2322 VL - 15 IS - 1 SP - 1 EP - 11 PB - Nature Publishing Group UK CY - London ER -