• Complete Genome Sequencing of Isolates from Malaysia Reveals Massive Genome Rearrangement but High Conservation of Virulence-Associated Genes.

      Ramli, Siti Roszilawati; Bunk, Boyke; Spröer, Cathrin; Geffers, Robert; Jarek, Michael; Bhuju, Sabin; Goris, Marga; Mustakim, Sahlawati; Pessler, Frank; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany. (Frontiers, 2021-09-15)
      The ability of Leptospirae to persist in environments and animal hosts but to cause clinically highly variable disease in humans has made leptospirosis the most common zoonotic disease. Considering the paucity of data on variation in complete genomes of human pathogenic Leptospirae, we have used a combination of Single Molecule Real-Time (SMRT) and Illumina sequencing to obtain complete genome sequences of six human clinical L. interrogans isolates from Malaysia. All six contained the larger (4.28-4.56 Mb) and smaller (0.34-0.395 Mb) chromosome typical of human pathogenic Leptospirae and 0-7 plasmids. Only 24% of the plasmid sequences could be matched to databases. We identified a chromosomal core genome of 3318 coding sequences and strain-specific accessory genomes of 49-179 coding sequences. These sequences enabled detailed genomic strain typing (Genome BLAST Distance Phylogeny, DNA-DNA hybridization, and multi locus sequence typing) and phylogenetic classification (whole-genome SNP genotyping). Even though there was some shared synteny and collinearity across the six genomes, there was evidence of major genome rearrangement, likely driven by horizontal gene transfer and homologous recombination. Mobile genetic elements were identified in all strains in highly varying numbers, including in the rfb locus, which defines serogroups and contributes to immune escape and pathogenesis. On the other hand, there was high conservation of virulence-associated genes including those relating to sialic acid, alginate, and lipid A biosynthesis. These findings suggest (i) that the antigenic variation, adaption to various host environments, and broad spectrum of virulence of L. interrogans are in part due to a high degree of genomic plasticity and (ii) that human pathogenic strains maintain a core set of genes required for virulence.
    • Oral Phenotype and Salivary Microbiome of Individuals With Papillon-Lefèvre Syndrome.

      Lettieri, Giulia Melo; Santiago, Luander Medrado; Lettieri, Giancarlo Crosara; Borges, Luiz Gustavo Dos Anjos; Marconatto, Letícia; de Oliveira, Laudimar Alves; Damé-Teixeira, Nailê; Salles, Loise Pedrosa; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany. (Frontiers, 2021-08-26)
      Papillon-Lefèvre syndrome (PLS) is an autosomal recessive rare disease, main characteristics of which include palmoplantar hyperkeratosis and premature edentulism due to advanced periodontitis (formerly aggressive periodontitis). This study aimed to characterize the oral phenotype, including salivary parameters, and the salivary microbiome of three PLS sisters, comparatively. Two sisters were toothless (PLSTL1 and PLSTL2), and one sister had most of the teeth in the oral cavity (PLST). Total DNA was extracted from the unstimulated saliva, and the amplicon sequencing of the 16S rRNA gene fragment was performed in an Ion PGM platform. The amplicon sequence variants (ASVs) were obtained using the DADA2 pipeline, and the taxonomy was assigned using the SILVA v.138. The main phenotypic characteristics of PLS were bone loss and premature loss of primary and permanent dentition. The PLST sister presented advanced periodontitis with gingival bleeding and suppuration, corresponding to the advanced periodontitis as a manifestation of systemic disease, stage IV, grade C. All three PLS sisters presented hyposalivation as a possible secondary outcome of the syndrome. Interestingly, PLST salivary microbiota was dominated by the uncultured bacteria Bacterioidales (F0058), Fusobacterium, Treponema, and Sulfophobococcus (Archaea domain). Streptococcus, Haemophilus, and Caldivirga (Archaea) dominated the microbiome of the PLSTL1 sister, while the PLSTL2 had higher abundances of Lactobacillus and Porphyromonas. This study was the first to show a high abundance of organisms belonging to the Archaea domain comprising a core microbiome in human saliva. In conclusion, a PLST individual does have a microbiota different from that of the periodontitis' aggressiveness previously recognized. Due to an ineffective cathepsin C, the impairment of neutrophils probably provided a favorable environment for the PLS microbiome. The interactions of Bacteroidales F0058, Caldivirga, and Sulfophobococcus with the microbial consortium of PLS deserves future investigation. Traditional periodontal therapy is not efficient in PLS patients. Unraveling the PLS microbiome is essential in searching for appropriate treatment and avoiding early tooth loss.
    • The Small Protein YmoA Controls the Csr System and Adjusts Expression of Virulence-Relevant Traits of .

      Böhme, Katja; Heroven, Ann Kathrin; Lobedann, Stephanie; Guo, Yuzhu; Stolle, Anne-Sophie; Dersch, Petra; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany. (Frontiers, 2021-08-03)
      Virulence gene expression of Yersinia pseudotuberculosis changes during the different stages of infection and this is tightly controlled by environmental cues. In this study, we show that the small protein YmoA, a member of the Hha family, is part of this process. It controls temperature- and nutrient-dependent early and later stage virulence genes in an opposing manner and co-regulates bacterial stress responses and metabolic functions. Our analysis further revealed that YmoA exerts this function by modulating the global post-transcriptional regulatory Csr system. YmoA pre-dominantly enhances the stability of the regulatory RNA CsrC. This involves a stabilizing stem-loop structure within the 5'-region of CsrC. YmoA-mediated CsrC stabilization depends on H-NS, but not on the RNA chaperone Hfq. YmoA-promoted reprogramming of the Csr system has severe consequences for the cell: we found that a mutant deficient of ymoA is strongly reduced in its ability to enter host cells and to disseminate to the Peyer's patches, mesenteric lymph nodes, liver and spleen in mice. We propose a model in which YmoA controls transition from the initial colonization phase in the intestine toward the host defense phase important for the long-term establishment of the infection in underlying tissues.
    • A simplified LC-MS/MS method for the quantification of the cardiovascular disease biomarker trimethylamine-N-oxide and its precursors

      Rox, Katharina; Rath, Silke; Pieper, Dietmar H.; Vital, Marius; Brönstrup, Mark; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany. (Elsevier BV, 2021-03)
      Trimethylamine-N-oxide (TMAO) has emerged as a potential biomarker for atherosclerosis and the development of cardiovascular diseases (CVDs). Although several clinical studies have shown striking associations of TMAO levels with atherosclerosis and CVDs, TMAO determinations are not clinical routine yet. The current methodology relies on isotope-labeled internal standards, which adds to pre-analytical complexity and costs for the quantification of TMAO and its precursors carnitine, betaine or choline. Here, we report a liquid chromatography-tandem mass spectrometry based method that is fast (throughput up to 240 samples/day), consumes low sample volumes (e.g., from a finger prick), and does not require isotope-labeled standards. We circumvented the analytical problem posed by the presence of endogenous TMAO and its precursors in human plasma by using an artificial plasma matrix for calibration. We cross-validated the results obtained using an artificial matrix with those using mouse plasma matrix and demonstrated that TMAO, carnitine, betaine and choline were accurately quantified in ‘real-life’ human plasma samples from healthy volunteers, obtained either from a finger prick or from venous puncture. Additionally, we assessed the stability of samples stored at −20 °C and room temperature. Whereas all metabolites were stable at −20 °C, increasing concentrations of choline were determined when stored at room temperature. Our method will facilitate the establishment of TMAO as a routine clinical biomarker in hematology in order to assess the risk for CVDs development, or to monitor disease progression and intervention effects.
    • Free human DNA attenuates the activity of antimicrobial peptides in atopic dermatitis.

      Kopfnagel, Verena; Dreyer, Sylvia; Zeitvogel, Jana; Pieper, Dietmar H; Buch, Anna; Sodeik, Beate; Rademacher, Franziska; Harder, Jürgen; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany. (John Wiley & Sons LTD., 2021-06-27)
      Background: The high susceptibility of AD patients to microbial skin infections has been attributed to a deficient antimicrobial peptide (AMP) expression, which is contradicted by a growing amount of recent studies clearly demonstrating that AMP expression is not impaired in lesional skin of AD patients. The reasons for the high susceptibility of AD patients to microbial infections are still unknown. Methods: The influence of self-DNA on the antimicrobial activity of RNase 7, LL-37, and hBD2 has been investigated using antibacterial and antiviral assays. The amount of self-DNA on skin has been analyzed by skin rinsings and subsequent quantification using dsDNA assays. DNA source was identified by qPCR. Results: Complex formation of the AMPs with self-DNA significantly impaired their antibacterial activity against Staphylococcus aureus and their antiviral activity against HSV-1. The inhibition of the antibacterial activity was dependent on the DNA concentration but not on the length of the DNA molecules. Of note, we detected significant higher amounts of cell-free self-DNA in skin rinses taken from lesional AD skin compared to skin rinses from non-lesional skin and from normal skin of healthy donors. Consequently, rinse solution from AD lesional skin prevented antibacterial activity of LL-37. Conclusion: Our study indicates that extracellular self-DNA is released in considerable amounts in AD skin lesions and AMP-self-DNA-complex formation leads to a significant loss of antibacterial and antiviral activity in atopic dermatitis. Studies on strategies to reduce the amount of extracellular DNA in AD are needed to identify possible methods relevant in clinical settings.
    • Broad Spectrum Antibiotic Xanthocillin X Effectively Kills Acinetobacter baumannii via Dysregulation of Heme Biosynthesis

      Hübner, Ines; Shapiro, Justin A.; Hoßmann, Jörn; Drechsel, Jonas; Hacker, Stephan M.; Rather, Philip N.; Pieper, Dietmar H.; Wuest, William M.; Sieber, Stephan A.; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany. (American Chemical Society (ACS), 2021-01-20)
      Isonitrile natural products exhibit promising antibacterial activities. However, their mechanism of action (MoA) remains largely unknown. Based on the nanomolar potency of xanthocillin X (Xan) against diverse difficult-to-treat Gram-negative bacteria, including the critical priority pathogen Acinetobacter baumannii, we performed in-depth studies to decipher its MoA. While neither metal binding nor cellular protein targets were detected as relevant for Xan’s antibiotic effects, sequencing of resistant strains revealed a conserved mutation in the heme biosynthesis enzyme porphobilinogen synthase (PbgS). This mutation caused impaired enzymatic efficiency indicative of reduced heme production. This discovery led to the validation of an untapped mechanism, by which direct heme sequestration of Xan prevents its binding into cognate enzyme pockets resulting in uncontrolled cofactor biosynthesis, accumulation of porphyrins, and corresponding stress with deleterious effects for bacterial viability. Thus, Xan represents a promising antibiotic displaying activity even against multidrug resistant strains, while exhibiting low toxicity to human cells.
    • Eradication of chronic HCV infection: improvement of dysbiosis only in patients without liver cirrhosis.

      Wellhöner, Freya; Döscher, Nico; Woelfl, Franziska; Vital, Marius; Plumeier, Iris; Kahl, Silke; Potthoff, Andrej; Manns, Michael Peter; Pieper, Dietmar Helmut; Cornberg, Markus; et al. (Wiley, 2021-01-07)
      It is well accepted that liver diseases and their outcomes are associated with intestinal microbiota but causality is difficult to establish. The intestinal microbiota is altered in patients with hepatitis C. As chronic HCV infection can now be cured in almost all patients, it is an ideal model to study the influence of liver disease on the microbiota. We aimed to analyze prospectively the changes in the gut microbiome in patients who received direct acting antivirals (DAA) and achieved sustained virological response (SVR). Amplicon sequencing of the V1-V2 region in the 16S rRNA gene was performed in stool samples of patients with chronic hepatitis C. Patients in the treatment group received direct acting antivirals (n=65) whereas in the control group no DAA were given (n=33). Only patients achieving SVR were included. The alpha diversity increased numerical but not significantly from baseline to SVR24/48 (2.784±0.248 vs. 2.846±0.224; p= 0.057). When stratifying for the presence of liver cirrhosis, a significant increase in diversity was only seen in patients without cirrhosis. Differences in the microbial community structure induced by the achievement of SVR were only observed in patients without liver cirrhosis. In patients with liver cirrhosis and in the control group, no significant differences were observed. In conclusion, the achievement of SVR24/48 in patients with chronic HCV was associated with changes in the intestinal microbiota. However, these changes were only seen in patients without liver cirrhosis. A major role of liver remodeling on the intestinal microbiota is indicated by the dynamics of the intestinal microbial community structure depending on the stage of fibrosis in patients resolving chronic hepatitis C.
    • Lethal Neonatal Respiratory Failure by Perinatal Transmission of Ureaplasma Parvum after Maternal PPROM.

      Zöllkau, Janine; Pieper, Dietmar H; Pastuschek, Jana; Makarewicz, Oliwia; Mentzel, Hans-Joachim; Dawczynski, Kristin; Schleußner, Ekkehard; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany. (Thieme Verlag, 2020-12-18)
      A primiparous pregnant woman was admitted due to preterm premature rupture of membranes (PPROM) at 27+0 week of gestational age (WGA). Conventional vaginal microbiological analysis had no pathological finding. Management decisions based on national guidelines included antenatal corticoids, tocolytics and antibiotics. Unstoppable efforts of preterm labor in 28+0 WGA and supposed amniotic infection syndrome necessitated emergency cesarean section. The preterm infant underwent NICU therapy, developed an early-onset neonatal sepsis and therapy-refractory pulmonary insufficiency with consecutive right heart failure, resulting in death on the 36th day of life. Microbiota analyses by 16Sr DNA sequencing was performed from maternal vaginal swabs and from neonatal pharyngeal swabs. Maternal antibiotic treatment resulted in depletion of physiological vaginal colonization with Lactobacillus crispatus. Ureaplasma parvum became the dominant vaginal microorganism at delivery and was detected in high relative abundance in the neonatal specimen. Progressive radiological air-space changes and interstitial pathologies associated with Ureaplasma infection (bronchopulmonary dysplasia type III) were seen early at the 3rd and distinctly from 14th day of life. This clearly demonstrates the need of vaginal colonization diagnostics in PPROM patients and awareness of the consecutive risks in the preterm. Vaginal microbiome analysis may allow individualized and targeted maternal and fetal diagnostic, prophylactic and therapeutic strategies to identify, protect and treat the high-risk neonates after PPROM.
    • Cohort Profile: The LoewenKIDS Study - life-course perspective on infections, the microbiome and the development of the immune system in early childhood.

      Gottschick, Cornelia; Raupach-Rosin, Heike; Langer, Susan; Hassan, Lamiaa; Horn, Johannes; Dorendorf, Evelyn; Caputo, Mahrrouz; Bittner, Martina; Beier, Lea; Rübsamen, Nicole; et al. (Oxford Academic, 2019-02-27)
      [Noabstract available]
    • Impact of dental cement on the peri-implant biofilm-microbial comparison of two different cements in an in vivo observational study.

      Korsch, Michael; Marten, Silke-Mareike; Walther, Winfried; Vital, Marius; Pieper, Dietmar H; Dötsch, Andreas; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany. (Wiley, 2018-08-20)
      Background: The type of cement used in cemented fixed implant-supported restorations influences formation of undetected excess cement and composition of the peri-implant biofilm. Excess cement and dysbiosis of the biofilm involve the risk of peri-implant inflammation. Purpose: The aim of the study was to investigate the impact of two different cements on the peri-implant biofilm and inflammation. Materials and methods: In an observational study, the suprastructures of 34 patients with cemented fixed implant-supported restorations were revised. In 20 patients, a methacrylate cement (Premier Implant cement [PIC]) and in 14 patients, a zinc oxide eugenol cement (Temp Bond [TB]) were used. After revision, TB was used for recementation. During revision and follow-up after 1 year, microbial samples were obtained. Results: Excess cement was found in 12 (60%) of the 20 patients with PIC. Suppuration was observed in two (25%) implants with PIC without excess cement (PIC-) and in all 12 (100%) implants with PIC and excess cement (PIC+). Implants cemented with TB had neither excess cement nor suppuration. The taxonomic analysis of the microbial samples revealed an accumulation of periodontal pathogens in the PIC patients independent of the presence of excess cement. Significantly, fewer oral pathogens occurred in patients with TB compared to patients with PIC. TB was used in all cases (PIC and TB) for recementation. In the follow-up check, suppuration was not found around any of the implants with PIC-, only around one implant with PIC+ and around one implant with TB. Bacterial species associated with severe periodontal infections that were abundant in PIC- and PIC+ samples before the revision were reduced after 1 year to levels found in the TB samples. Conclusions: The revision and recementation with TB had a positive effect on the peri-implant biofilm in cases with PIC. The cementation of suprastructures on implants with TB is an alternative method to be considered.
    • Toward Biorecycling: Isolation of a Soil Bacterium That Grows on a Polyurethane Oligomer and Monomer.

      Espinosa, María José Cárdenas; Blanco, Andrea Colina; Schmidgall, Tabea; Atanasoff-Kardjalieff, Anna Katharina; Kappelmeyer, Uwe; Tischler, Dirk; Pieper, Dietmar H; Heipieper, Hermann J; Eberlein, Christian; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany. (Frontiers, 2020-03-27)
      The fate of plastic waste and a sustainable use of synthetic polymers is one of the major challenges of the twenty first century. Waste valorization strategies can contribute to the solution of this problem. Besides chemical recycling, biological degradation could be a promising tool. Among the high diversity of synthetic polymers, polyurethanes are widely used as foams and insulation materials. In order to examine bacterial biodegradability of polyurethanes, a soil bacterium was isolated from a site rich in brittle plastic waste. The strain, identified as Pseudomonas sp. by 16S rRNA gene sequencing and membrane fatty acid profile, was able to grow on a PU-diol solution, a polyurethane oligomer, as the sole source of carbon and energy. In addition, the strain was able to use 2,4-diaminotoluene, a common precursor and putative degradation intermediate of polyurethanes, respectively, as sole source of energy, carbon, and nitrogen. Whole genome sequencing of the strain revealed the presence of numerus catabolic genes for aromatic compounds. Growth on potential intermediates of 2,4-diaminotoluene degradation, other aromatic growth substrates and a comparison with a protein data base of oxygenases present in the genome, led to the proposal of a degradation pathway.
    • Microbial Community Structure Along a Horizontal Oxygen Gradient in a Costa Rican Volcanic Influenced Acid Rock Drainage System.

      Arce-Rodríguez, Alejandro; Puente-Sánchez, Fernando; Avendaño, Roberto; Libby, Eduardo; Mora-Amador, Raúl; Rojas-Jimenez, Keilor; Martínez, María; Pieper, Dietmar H; Chavarría, Max; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany. (Springer Nature, 2020-06-22)
      We describe the geochemistry and microbial diversity of a pristine environment that resembles an acid rock drainage (ARD) but it is actually the result of hydrothermal and volcanic influences. We designate this environment, and other comparable sites, as volcanic influenced acid rock drainage (VARD) systems. The metal content and sulfuric acid in this ecosystem stem from the volcanic milieu and not from the product of pyrite oxidation. Based on the analysis of 16S rRNA gene amplicons, we report the microbial community structure in the pristine San Cayetano Costa Rican VARD environment (pH = 2.94-3.06, sulfate ~ 0.87-1.19 g L-1, iron ~ 35-61 mg L-1 (waters), and ~ 8-293 g kg-1 (sediments)). San Cayetano was found to be dominated by microorganisms involved in the geochemical cycling of iron, sulfur, and nitrogen; however, the identity and abundance of the species changed with the oxygen content (0.40-6.06 mg L-1) along the river course. The hypoxic source of San Cayetano is dominated by a putative anaerobic sulfate-reducing Deltaproteobacterium. Sulfur-oxidizing bacteria such as Acidithiobacillus or Sulfobacillus are found in smaller proportions with respect to typical ARD. In the oxic downstream, we identified aerobic iron-oxidizers (Leptospirillum, Acidithrix, Ferrovum) and heterotrophic bacteria (Burkholderiaceae bacterium, Trichococcus, Acidocella). Thermoplasmatales archaea closely related to environmental phylotypes found in other ARD niches were also observed throughout the entire ecosystem. Overall, our study shows the differences and similarities in the diversity and distribution of the microbial communities between an ARD and a VARD system at the source and along the oxygen gradient that establishes on the course of the river.
    • Potential TMA-Producing Bacteria Are Ubiquitously Found in Mammalia.

      Rath, Silke; Rud, Tatjana; Pieper, Dietmar H; Vital, Marius; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany. (Frontiers, 2019-01-01)
      Human gut bacteria metabolize dietary components such as choline and carnitine to trimethylamine (TMA) that is subsequently oxidized to trimethylamine-N-oxide (TMAO) by hepatic enzymes. Increased plasma levels of TMAO are associated with the development of cardiovascular and renal disease. In this study, we applied gene-targeted assays in order to quantify (qPCR) and characterize (MiSeq) bacterial genes encoding enzymes responsible for TMA production, namely choline-TMA lyase (CutC), carnitine oxygenase (CntA) and betaine reductase (GrdH) in 89 fecal samples derived from various mammals spanning three dietary groups (carnivores, omnivores and herbivores) and four host orders (Carnivora, Primates, Artiodactyla and Perissodactyla). All samples contained potential TMA-producing bacteria, however, at low abundances (<1.2% of total community). The cutC gene was more abundant in omnivores and carnivores compared with herbivores. CntA was almost absent from herbivores and grdH showed lowest average abundance of all three genes. Bacteria harboring cutC and grdH displayed high diversities where sequence types affiliated with various taxa within Firmicutes dominated, whereas cntA comprised sequences primarily linked to Escherichia. Composition of TMA-forming communities was strongly influenced by diet and host taxonomy and despite their high correlation, both factors contributed uniquely to community structure. Furthermore, Random Forest (RF) models could differentiate between groups at high accuracies. This study gives a comprehensive overview of potential TMA-producing bacteria in the mammalian gut demonstrating that both diet and host taxonomy govern their abundance and composition. It highlights the role of functional redundancy sustaining potential TMA formation in distinct gut environments.
    • Identification of benzene-degrading Proteobacteria in a constructed wetland by employing in situ microcosms and RNA-stable isotope probing.

      Nitz, Henrike; Duarte, Márcia; Jauregui, Ruy; Pieper, Dietmar H; Müller, Jochen A; Kästner, Matthias; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany. (Springer, 2019-12-23)
      Constructed wetlands (CWs) are effective ecological remediation technologies for various contaminated water bodies. Here, we queried for benzene-degrading microbes in a horizontal subsurface flow CW with reducing conditions in the pore water and fed with benzene-contaminated groundwater. For identification of relevant microbes, we employed in situ microcosms (BACTRAPs, which are made from granulated activated carbon) coupled with 13C-stable isotope probing and Illumina sequencing of 16S rRNA amplicons. A significant incorporation of 13C was detected in RNA isolated from BACTRAPs loaded with 13C-benzene and exposed in the CW for 28 days. A shorter incubation time did not result in detectable 13C incorporation. After 28 days, members from four genera, namely Dechloromonas, Hydrogenophaga, and Zoogloea from the Betaproteobacteria and Arcobacter from the Epsilonproteobacteria were significantly labeled with 13C and were abundant in the bacterial community on the BACTRAPs. Sequences affiliated to Geobacter were also numerous on the BACTRAPs but apparently those microbes did not metabolize benzene as no 13C label incorporation was detected. Instead, they may have metabolized plant-derived organic compounds while using the BACTRAPs as electron sink. In representative wetland samples, sequences affiliated with Dechloromonas, Zoogloea, and Hydrogenophaga were present at relative proportions of up to a few percent. Sequences affiliated with Arcobacter were present at < 0.01% in wetland samples. In conclusion, we identified microbes of likely significance for benzene degradation in a CW used for remediation of contaminated water.
    • Risk factors and Predictors of Mortality in Streptococcal Necrotizing Soft-Tissue Infections: A Multicenter Prospective Study.

      Bruun, Trond; Rath, Eivind; Bruun Madsen, Martin; Oppegaard, Oddvar; Nekludov, Michael; Arnell, Per; Karlsson, Ylva; Babbar, Anshu; Bergey, Francois; Itzek, Andreas; et al. (Oxford University Press (OUP), 2020-01-10)
      Background Necrotizing soft-tissue infections (NSTI) are life-threatening conditions often caused by β-hemolytic streptococci, group A streptococcus (GAS) in particular. Optimal treatment is contentious. The INFECT cohort includes the largest set of prospectively enrolled streptococcal NSTI cases to date. Methods From the INFECT cohort of 409 adults admitted with NSTI to five clinical centers in Scandinavia, patients culture-positive for GAS or Streptococcus dysgalactiae (SD) were selected. Risk factors were identified by comparison with a cohort of non-necrotizing streptococcal cellulitis. The impact of baseline factors and treatment on 90-day mortality was explored using Lasso regression. Whole-genome sequencing of bacterial isolates was used for emm typing and virulence gene profiling. Results The 126 GAS NSTI cases and 27 cases caused by SD constituted 31% and 7% of the whole NSTI cohort, respectively. When comparing to non-necrotizing streptococcal cellulitis, streptococcal NSTI was associated to blunt trauma, absence of pre-existing skin lesions, and a lower BMI. Septic shock was significantly more frequent in GAS (65%) compared to SD (41%) and polymicrobial, non-streptococcal NSTI (46%). Age, male sex, septic shock, and no administration of intravenous immunoglobulin (IVIG) were among factors associated with 90-day mortality. Predominant emm types were emm1, emm3 and emm28 in GAS and stG62647 in SD.
    • Correlation between immunoglobulin dose administered and plasma neutralization of streptococcal superantigens in patients with necrotizing soft tissue infections.

      Bergsten, Helena; Madsen, Martin Bruun; Bergey, Francois; Hyldegaard, Ole; Skrede, Steinar; Arnell, Per; Oppegaard, Oddvar; Itzek, Andreas; Perner, Anders; Svensson, Mattias; et al. (Oxford University Press, 2020-01-09)
      Analyses of plasma collected pre- and post-administration of intravenous immunoglobulin G (IVIG) from patients with Group A Streptococcus necrotizing soft tissue infections demonstrated a negative correlation between IVIG dose and toxin-triggered T-cell proliferation (r=-0.67, p<0.0001). One 25g-dose IVIG was sufficient to yield patient plasma neutralizing activity against streptococcal superantigens.
    • Importance of superoxide dismutase A and M for protection of Staphylococcus aureus in the oxidative stressful environment of cystic fibrosis airways.

      Treffon, Janina; Chaves-Moreno, Diego; Niemann, Silke; Pieper, Dietmar Helmut; Vogl, Thomas; Roth, Johannes; Kahl, Barbara C; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany. (Wiley, 2020-01-02)
      Staphylococcus aureus is one of the earliest pathogens that persists the airways of cystic fibrosis (CF) patients and contributes to increased inflammation and decreased lung function. In contrast to other staphylococci, S. aureus possesses two superoxide dismutases (SODs), SodA and SodM, with SodM being unique to S. aureus. Both SODs arm S. aureus for its fight against oxidative stress, a byproduct of inflammatory reactions. Despite complex investigations it is still unclear, if both enzymes are crucial for the special pathogenicity of S. aureus. To investigate the role of both SODs during staphylococcal persistence in CF airways, we analyzed survival and gene expression of S. aureus CF isolates and laboratory strains in different CF-related in vitro and ex vivo settings. Bacteria located in inflammatory and oxidized CF sputum transcribed high levels of sodA and sodM. Especially expression values of sodM were remarkably higher in CF sputum than in bacterial in vitro cultures. Interestingly, also S. aureus located in airway epithelial cells expressed elevated transcript numbers of both SODs, indicating that S. aureus is exposed to oxidative stress at various sites within CF airways. Both enzymes promoted survival of S. aureus during PMN killing and seem to act compensatory, thereby giving evidence that the interwoven interaction of SodA and SodM contributes to S. aureus virulence and facilitates S. aureus persistence within CF airways.
    • High Nuclease Activity of Long Persisting Isolates Within the Airways of Cystic Fibrosis Patients Protects Against NET-Mediated Killing.

      Herzog, Susann; Dach, Felix; de Buhr, Nicole; Niemann, Silke; Schlagowski, Jannik; Chaves-Moreno, Diego; Neumann, Claudia; Goretzko, Jonas; Schwierzeck, Vera; Mellmann, Alexander; et al. (Frontiers, 2019-01-01)
      Staphylococcus aureus is one of the first and most prevalent pathogens cultured from the airways of cystic fibrosis (CF) patients, which can persist there for extended periods. Airway infections in CF patients are characterized by a strong inflammatory response of highly recruited neutrophils. One killing mechanism of neutrophils is the formation of neutrophil extracellular traps (NETs), which capture and eradicate bacteria by extracellular fibers of neutrophil chromatin decorated with antimicrobial granule proteins. S. aureus secretes nuclease, which can degrade NETs. We hypothesized, that S. aureus adapts to the airways of CF patients during persistent infection by escaping from NET-mediated killing via an increase of nuclease activity. Sputum samples of CF patients with chronic S. aureus infection were visualized by confocal microscopy after immuno-fluorescence staining for NET-specific markers, S. aureus bacteria and overall DNA structures. Nuclease activity was analyzed in sequential isogenic long persisting S. aureus isolates, as confirmed by whole genome sequencing, from an individual CF patient using a FRET-based nuclease activity assay. Additionally, some of these isolates were selected and analyzed by qRT-PCR to determine the expression of nuc1 and regulators of interest. NET-killing assays were performed with clinical S. aureus isolates to evaluate killing and bacterial survival depending on nuclease activity. To confirm the role of nuclease during NET-mediated killing, a clinical isolate with low nuclease activity was transformed with a nuclease expression vector (pCM28nuc). Furthermore, two sputa from an individual CF patient were subjected to RNA-sequence analysis to evaluate the activity of nuclease in vivo. In sputa, S. aureus was associated to extracellular DNA structures. Nuclease activity in clinical S. aureus isolates increased in a time-and phenotype-dependent manner. In the clinical isolates, the expression of nuc1 was inversely correlated to the activity of agr and was independent of saeS. NET-mediated killing was significantly higher in S. aureus isolates with low compared to isolates with high nuclease activity. Importantly, transformation of the clinical isolate with low nuclease activity with pCM28nuc conferred protection against NET-mediated killing confirming the beneficial role of nuclease for protection against NETs. Also, nuclease expression in in vivo sputa was high, which underlines the important role of nuclease within the highly inflamed CF airways. In conclusion, our data show that S. aureus adapts to the neutrophil-rich environment of CF airways with increasing nuclease expression most likely to avoid NET-killing during long-term persistence.
    • MAIT Cells Are Major Contributors to the Cytokine Response in Group A Streptococcal Toxic Shock Syndrome.

      Emgård, Johanna; Bergsten, Helena; McCormick, John K; Barrantes, Israel; Skrede, Steinar; Sandberg, Johan K; Norrby-Teglund, Anna; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany. (National Academy of Sciences, 2019-11-26)
      Streptococcal toxic shock syndrome (STSS) is a rapidly progressing, life-threatening, systemic reaction to invasive infection caused by group A streptococci (GAS). GAS superantigens are key mediators of STSS through their potent activation of T cells leading to a cytokine storm and consequently vascular leakage, shock, and multiorgan failure. Mucosal-associated invariant T (MAIT) cells recognize MR1-presented antigens derived from microbial riboflavin biosynthesis and mount protective innate-like immune responses against the microbes producing such metabolites. GAS lack de novo riboflavin synthesis, and the role of MAIT cells in STSS has therefore so far been overlooked. Here we have conducted a comprehensive analysis of human MAIT cell responses to GAS, aiming to understand the contribution of MAIT cells to the pathogenesis of STSS. We show that MAIT cells are strongly activated and represent the major T cell source of IFNγ and TNF in the early stages of response to GAS. MAIT cell activation is biphasic with a rapid TCR Vβ2-specific, TNF-dominated response to superantigens and a later IL-12- and IL-18-dependent, IFNγ-dominated response to both bacterial cells and secreted factors. Depletion of MAIT cells from PBMC resulted in decreased total production of IFNγ, IL-1β, IL-2, and TNFβ. Peripheral blood MAIT cells in patients with STSS expressed elevated levels of the activation markers CD69, CD25, CD38, and HLA-DR during the acute compared with the convalescent phase. Our data demonstrate that MAIT cells are major contributors to the early cytokine response to GAS, and are therefore likely to contribute to the pathological cytokine storm underlying STSS.
    • Diversity and community composition of particle-associated and free-living bacteria in mesopelagic and bathypelagic Southern Ocean water masses: Evidence of dispersal limitation in the Bransfield Strait

      Milici, Mathias; Vital, Marius; Tomasch, Jürgen; Badewien, Thomas H.; Giebel, Helge A.; Plumeier, Iris; Wang, Hui; Pieper, Dietmar H.; Wagner-Döbler, Irene; Simon, Meinhard; et al. (Wiley-Blackwell, 2017-05-01)
      The Southern Ocean constitutes about 10% of the global oceans' volume and is characterized by high primary production. Particulate organic matter (POM) is exported from the photic zone to the deep ocean and sustains life of particle associated (PA) and free-living (FL) bacterial communities in the dark realm. Little is known about the composition and diversity of PA and FL bacterial communities below the photic zone and how they differ among various regions of the Southern Ocean. Therefore, we investigated the composition of small (3–8 μm) and large (> 8 μm) PA and FL (0.2–3 μm) bacterial communities between 500 m and 3600 m in the Bransfield Strait, Drake Passage, and the south Atlantic Ocean featuring also Southern Ocean water masses. PA bacterial communities had a higher OTU richness and evenness than FL ones. Taxonomic analysis revealed a different community composition between FL and PA bacteria. A large number of OTUs belonging to diverse phyla (Bacteroidetes, Planctomycetes, Betaproteobacteria, Deltaproteobacteria, and Verrucomicrobia) were significantly enriched on particles; in contrast very few bacterial lineages were FL specialists. Life-style (FL vs. PA) and region (Bransfield basin vs. other regions) strongly influenced bacterial communities. Depth explained only marginal fraction of the total variation (∼ 12%), suggesting that selective processes driven by depth have a smaller effect in the Southern Ocean when compared to life-style (25%) and region (31%). Overall these data indicate a strong influence of isolated water masses such as the basin of the Bransfield Strait on the composition of bacterial communities in the dark ocean. © 2017 The Authors Limnology and Oceanography published by Wiley Periodicals, Inc. on behalf of Association for the Sciences of Limnology and Oceanography