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- ItemArgon Humidification Exacerbates Antimicrobial and Anti-MRSA kINPen Plasma Activity(Basel : MDPI, 2023) Clemen, Ramona; Singer, Debora; Skowski, Henry; Bekeschus, SanderGas plasma is a medical technology with antimicrobial properties. Its main mode of action is oxidative damage via reactive species production. The clinical efficacy of gas plasma-reduced bacterial burden has been shown to be hampered in some cases. Since the reactive species profile produced by gas plasma jets, such as the kINPen used in this study, are thought to determine antimicrobial efficacy, we screened an array of feed gas settings in different types of bacteria. Antimicrobial analysis was performed by single-cell analysis using flow cytometry. We identified humidified feed gas to mediate significantly greater toxicity compared to dry argon and many other gas plasma conditions. The results were confirmed by inhibition zone analysis on gas-plasma-treated microbial lawns grown on agar plates. Our results may have vital implications for clinical wound management and potentially enhance antimicrobial efficacy of medical gas plasma therapy in patient treatment.
- ItemCaspase-1 inflammasome activity in patients with Staphylococcus aureus bacteremia(Oxford : Wiley-Blackwell, 2019) Rasmussen, Gunlög; Idosa, Berhane Asfaw; Bäckman, Anders; Monecke, Stefan; Strålin, Kristoffer; Särndahl, Eva; Söderquist, BoThe inflammasome is a multiprotein complex that mediates caspase-1 activation with subsequent maturation of the proinflammatory cytokines IL-1ß and IL-18. The NLRP3 inflammasome is known to be activated by Staphylococcus aureus, one of the leading causes of bacteremia worldwide. Inflammasome activation and regulation in response to bacterial infection have been found to be of importance for a balanced host immune response. However, inflammasome signaling in vivo in humans initiated by S. aureus is currently sparsely studied. This study therefore aimed to investigate NLRP3 inflammasome activity in 20 patients with S. aureus bacteremia (SAB), by repeated measurement during the first week of bacteremia, compared with controls. Caspase-1 activity was measured in monocytes and neutrophils by flow cytometry detecting FLICA (fluorescent-labeled inhibitor of caspase-1), while IL-1ß and IL-18 was measured by Luminex and ELISA, respectively. As a measure of inflammasome priming, messenger RNA (mRNA) expression of NLRP3, CASP1 (procaspase-1), and IL1B (pro-IL-1ß) was analyzed by quantitative PCR. We found induced caspase-1 activity in innate immune cells with subsequent release of IL-18 in patients during the acute phase of bacteremia, indicating activation of the inflammasome. There was substantial interindividual variation in caspase-1 activity between patients with SAB. We also found an altered inflammasome priming with low mRNA levels of NLRP3 accompanied by elevated mRNA levels of IL1B. This increased knowledge of the individual host immune response in SAB could provide support in the effort to optimize management and treatment of each individual patient. © 2019 The Authors. Microbiology and Immunology published by The Societies and John Wiley & Sons Australia, Ltd
- ItemPhenotypic and Molecular Detection of Biofilm Formation in Staphylococcus aureus Isolated from Different Sources in Algeria(Basel : MDPI, 2020) Achek, Rachid; Hotzel, Helmut; Nabi, Ibrahim; Kechida, Souad; Mami, Djamila; Didouh, Nassima; Tomaso, Herbert; Neubauer, Heinrich; Ehricht, Ralf; Monecke, Stefan; El-Adawy, HosnyStaphylococcus aureus is an opportunistic bacterium causing a wide variety of diseases. Biofilm formation of Staphylococcus aureus is of primary public and animal health concern. The purposes of the present study were to investigate the ability of Staphylococcus aureus isolated from animals, humans, and food samples to form biofilms and to screen for the presence of biofilmassociated and regulatory genes. In total, 55 Staphylococcus aureus isolated from sheep mastitis cases (n = 28), humans (n = 19), and from food matrices (n = 8) were identified using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). The ability of Staphylococcus aureus for slime production and biofilm formation was determined quantitatively. A DNA microarray examination was performed to detect adhesion genes (icaACD and biofilmassociated protein gene (bap)), genes encoding microbial surface components recognizing adhesive matrix molecules (MSCRAMMs), regulatory genes (accessory gene regulator (agr) and staphylococcal accessory regulator (sarA)), and the staphylococcal cassette chromosome mec elements (SCCmec). Out of 55 Staphylococcus aureus isolates, 39 (71.0%) and 23 (41.8%) were producing slime and biofilm, respectively. All Staphylococcus aureus strains isolated from food showed biofilm formation ability. 52.6% of the Staphylococcus aureus strains isolated from sheep with mastitis, and 17.9% of isolates from humans, were able to form a biofilm. Microarray analysis typed the Staphylococcus aureus into 15 clonal complexes. Among all Staphylococcus aureus isolates, four of the human isolates (21.1%) harbored the mecA gene (SCCmec type IV) typed into 2 clonal complexes (CC22-MRSA-IV and CC80-MRSA-IV) and were considered as methicillin-resistant, while two of them were slime-producing. None of the isolates from sheep with mastitis harbored the cna gene which is associated with biofilm production. The fnbB gene was found in 100%, 60% and 40% of biofilm-producing Staphylococcus aureus isolated from food, humans, and sheep with mastitis, respectively. Three agr groups were present and agr group III was predominant with 43.6%, followed by agr group I (38.2%), and agr group II (18.2%). This study revealed the capacity of Staphylococcus aureus isolates to form biofilms and highlighted the genetic background displayed by Staphylococcus aureus isolates from different sources in Algeria. © 2020 by the authors. Licensee MDPI, Basel, Switzerland.
- ItemSequence Analysis of Novel Staphylococcus aureus Lineages from Wild and Captive Macaques(Basel : Molecular Diversity Preservation International (MDPI), 2022) Monecke, Stefan; Roberts, Marilyn C.; Braun, Sascha D.; Diezel, Celia; Müller, Elke; Reinicke, Martin; Linde, Jörg; Joshi, Prabhu Raj; Paudel, Saroj; Acharya, Mahesh; Chalise, Mukesh K.; Feßler, Andrea T.; Hotzel, Helmut; Khanal, Laxman; Koju, Narayan P.; Schwarz, Stefan; Kyes, Randall C.; Ehricht, RalfStaphylococcus aureus is a widespread and common opportunistic bacterium that can colonise or infect humans as well as a wide range of animals. There are a few studies of both methicillin-susceptible S. aureus (MSSA) and methicillin-resistant S. aureus (MRSA) isolated from monkeys, apes, and lemurs, indicating a presence of a number of poorly or unknown lineages of the pathogen. In order to obtain insight into staphylococcal diversity, we sequenced strains from wild and captive individuals of three macaque species (Macaca mulatta, M. assamensis, and M. sylvanus) using Nanopore and Illumina technologies. These strains were previously identified by microarray as poorly or unknown strains. Isolates of novel lineages ST4168, ST7687, ST7688, ST7689, ST7690, ST7691, ST7692, ST7693, ST7694, ST7695, ST7745, ST7746, ST7747, ST7748, ST7749, ST7750, ST7751, ST7752, ST7753, and ST7754 were sequenced and characterised for the first time. In addition, isolates belonging to ST2990, a lineage also observed in humans, and ST3268, a MRSA strain already known from macaques, were also included into the study. Mobile genetic elements, genomic islands, and carriage of prophages were analysed. There was no evidence for novel host-specific virulence factors. However, a conspicuously high rate of carriage of a pathogenicity island harbouring edinB and etD2/etE as well as a higher number of repeat units within the gene sasG (encoding an adhesion factor) than in human isolates were observed. None of the strains harboured the genes encoding Panton–Valentine leukocidin. In conclusion, wildlife including macaques may harbour an unappreciated diversity of S. aureus lineages that may be of clinical relevance for humans, livestock, or for wildlife conservation, given the declining state of many wildlife populations.
- ItemThe Staphylococcus aureus extracellular matrix protein (Emp) has a fibrous structure and binds to different extracellular matrices(Berlin : Nature Pulishing, 2017) Geraci, Jennifer; Neubauer, Svetlana; Pöllath, Christine; Hansen, Uwe; Rizzo, Fabio; Krafft, Christoph; Westermann, Martin; Hussain, Muzaffar; Peters, Georg; Pletz, Mathias W.; Löffler, Bettina; Makarewicz, Oliwia; Tuchscherr, LorenaThe extracellular matrix protein Emp of Staphylococcus aureus is a secreted adhesin that mediates interactions between the bacterial surface and extracellular host structures. However, its structure and role in staphylococcal pathogenesis remain unknown. Using multidisciplinary approaches, including circular dichroism (CD) and Fourier transform infrared (FTIR) spectroscopy, transmission electron (TEM) and immunogold transmission electron microscopy, functional ELISA assays and in silico techniques, we characterized the Emp protein. We demonstrated that Emp and its truncated forms bind to suprastructures in human skin, cartilage or bone, among which binding activity seems to be higher for skin compounds. The binding domain is located in the C-terminal part of the protein. CD spectroscopy revealed high contents of β-sheets (39.58%) and natively disordered structures (41.2%), and TEM suggested a fibrous structure consisting of Emp polymers. The N-terminus seems to be essential for polymerization. Due to the uncommonly high histidine content, we suggest that Emp represents a novel type of histidine-rich protein sharing structural similarities to leucine-rich repeats proteins as predicted by the I-TASSER algorithm. These new findings suggest a role of Emp in infections of deeper tissue and open new possibilities for the development of novel therapeutic strategies.
- ItemLong-Term Sinonasal Carriage of Staphylococcus aureus and Anti-Staphylococcal Humoral Immune Response in Patients with Chronic Rhinosinusitis(Basel : MDPI, 2021) Thunberg, Ulrica; Hugosson, Svante; Ehricht, Ralf; Monecke, Stefan; Müller, Elke; Cao, Yang; Stegger, Marc; Söderquist, BoWe investigated Staphylococcus aureus diversity, genetic factors, and humoral immune responses against antigens via genome analysis of S. aureus isolates from chronic rhinosinusitis (CRS) patients in a long-term follow-up. Of the 42 patients who provided S. aureus isolates and serum for a previous study, 34 could be included for follow-up after a decade. Clinical examinations were performed and bacterial samples were collected from the maxillary sinus and nares. S. aureus isolates were characterized by whole-genome sequencing, and specific anti-staphylococcal IgG in serum was determined using protein arrays. S. aureus was detected in the nares and/or maxillary sinus at both initial inclusion and follow-up in 15 of the 34 respondents (44%). Three of these (20%) had S. aureus isolates from the same genetic lineage as at inclusion. A low number of single-nucleotide polymorphisms (SNPs) were identified when comparing isolates from nares and maxillary sinus collected at the same time point. The overall change of antibody responses to staphylococcal antigens over time showed great variability, and no correlation was found between the presence of genes encoding antigens and the corresponding anti-staphylococcal IgG in serum; thus our findings did not support a role, in CRS, of the specific S. aureus antigens investigated.
- ItemEvolution and Global Transmission of a Multidrug-Resistant, Community-Associated Methicillin-Resistant Staphylococcus aureus Lineage from the Indian Subcontinent(Washington D.C. : American Society for Microbiology, 2019) Steinig, Eike J.; Duchene, Sebastian; Robinson, D. Ashley; Monecke, Stefan; Yokoyama, Maho; Laabei, Maisem; Slickers, Peter; Andersson, Patiyan; Williamson, Deborah; Kearns, Angela; Goering, Richard V.; Dickson, Elizabeth; Ehricht, Ralf; Ip, Margaret; O'Sullivan, Matthew V.N.; Coombs, Geoffrey; Petersen, Andreas; Brennan, Gráinne I.; Shore, Anna C.; Coleman, David C.; Pantosti, Annalisa; de Lencastre, Herminia; Westh, Henrik; Kobayashi, Nobumichi; Heffernan, Helen; Strommenger, Birgit; Layer, Franziska; Weber, Stefan; Aamot, Hege Vangstein; Skakni, Leila; Peacock, Sharon J.; Sarovich, Derek; Harris, Simon; Parkhill, Julian; Massey, Ruth C.; Holden, Mathew T.G.; Bentley, Stephen; Tong, Stephen Y.C.The evolution and global transmission of antimicrobial resistance have been well documented for Gram-negative bacteria and health care-associated epidemic pathogens, often emerging from regions with heavy antimicrobial use. However, the degree to which similar processes occur with Gram-positive bacteria in the community setting is less well understood. In this study, we traced the recent origins and global spread of a multidrug-resistant, community-associated Staphylococcus aureus lineage from the Indian subcontinent, the Bengal Bay clone (ST772). We generated whole-genome sequence data of 340 isolates from 14 countries, including the first isolates from Bangladesh and India, to reconstruct the evolutionary history and genomic epidemiology of the lineage. Our data show that the clone emerged on the Indian subcontinent in the early 1960s and disseminated rapidly in the 1990s. Short-term outbreaks in community and health care settings occurred following intercontinental transmission, typically associated with travel and family contacts on the subcontinent, but ongoing endemic transmission was uncommon. Acquisition of a multidrug resistance integrated plasmid was instrumental in the emergence of a single dominant and globally disseminated clade in the early 1990s. Phenotypic data on biofilm, growth, and toxicity point to antimicrobial resistance as the driving force in the evolution of ST772. The Bengal Bay clone therefore combines the multidrug resistance of traditional health care-associated clones with the epidemiological transmission of community-associated methicillin-resistant S. aureus (MRSA). Our study demonstrates the importance of whole-genome sequencing for tracking the evolution of emerging and resistant pathogens. It provides a critical framework for ongoing surveillance of the clone on the Indian subcontinent and elsewhere.
- ItemPhylodynamic signatures in the emergence of community-associated MRSA(Washington, DC : National Acad. of Sciences, 2022) Steinig, Eike; Aglua, Izzard; Duchene, Sebastian; Meehan, Michael T.; Yoannes, Mition; Firth, Cadhla; Jaworski, Jan; Drekore, Jimmy; Urakoko, Bohu; Poka, Harry; Wurr, Clive; Ebos, Eri; Nangen, David; Müller, Elke; Mulvey, Peter; Jackson, Charlene; Blomfeldt, Anita; Aamot, Hege Vangstein; Laman, Moses; Manning, Laurens; Earls, Megan; Coleman, David C.; Greenhill, Andrew; Ford, Rebecca; Stegger, Marc; Syed, Muhammad Ali; Jamil, Bushra; Monecke, Stefan; Ehricht, Ralf; Smith, Simon; Pomat, William; Horwood, Paul; Tong, Steven Y. C.; McBryde, EmmaCommunity-associated, methicillin-resistant Staphylococcus aureus (MRSA) lineages have emerged in many geographically distinct regions around the world during the past 30 y. Here, we apply consistent phylodynamic methods across multiple community-associated MRSA lineages to describe and contrast their patterns of emergence and dissemination. We generated whole-genome sequencing data for the Australian sequence type (ST) ST93-MRSA-IV from remote communities in Far North Queensland and Papua New Guinea, and the Bengal Bay ST772-MRSA-V clone from metropolitan communities in Pakistan. Increases in the effective reproduction number (Re) and sustained transmission (Re > 1) coincided with spread of progenitor methicillin-susceptible S. aureus (MSSA) in remote northern Australian populations, dissemination of the ST93-MRSA-IV genotype into population centers on the Australian East Coast, and subsequent importation into the highlands of Papua New Guinea and Far North Queensland. Applying the same phylodynamic methods to existing lineage datasets, we identified common signatures of epidemic growth in the emergence and epidemiological trajectory of community-associated S. aureus lineages from America, Asia, Australasia, and Europe. Surges in Re were observed at the divergence of antibiotic-resistant strains, coinciding with their establishment in regional population centers. Epidemic growth was also observed among drug-resistant MSSA clades in Africa and northern Australia. Our data suggest that the emergence of community-associated MRSA in the late 20th century was driven by a combination of antibiotic-resistant genotypes and host epidemiology, leading to abrupt changes in lineage-wide transmission dynamics and sustained transmission in regional population centers.
- ItemMyxobacteria-Derived Outer Membrane Vesicles: Potential Applicability Against Intracellular Infections(Basel : MDPI, 2020) Goes, Adriely; Lapuhs, Philipp; Kuhn, Thomas; Schulz, Eilien; Richter, Robert; Panter, Fabian; Dahlem, Charlotte; Koch, Marcus; Garcia, Ronald; Kiemer, Alexandra K.; Müller, Rolf; Fuhrmann, GregorIn 2019, it was estimated that 2.5 million people die from lower tract respiratory infections annually. One of the main causes of these infections is Staphylococcus aureus, a bacterium that can invade and survive within mammalian cells. S. aureus intracellular infections are difficult to treat because several classes of antibiotics are unable to permeate through the cell wall and reach the pathogen. This condition increases the need for new therapeutic avenues, able to deliver antibiotics efficiently. In this work, we obtained outer membrane vesicles (OMVs) derived from the myxobacteria Cystobacter velatus strain Cbv34 and Cystobacter ferrugineus strain Cbfe23, that are naturally antimicrobial, to target intracellular infections, and investigated how they can affect the viability of epithelial and macrophage cell lines. We evaluated by cytometric bead array whether they induce the expression of proinflammatory cytokines in blood immune cells. Using confocal laser scanning microscopy and flow cytometry, we also investigated their interaction and uptake into mammalian cells. Finally, we studied the effect of OMVs on planktonic and intracellular S. aureus. We found that while Cbv34 OMVs were not cytotoxic to cells at any concentration tested, Cbfe23 OMVs affected the viability of macrophages, leading to a 50% decrease at a concentration of 125,000 OMVs/cell. We observed only little to moderate stimulation of release of TNF-alpha, IL-8, IL-6 and IL-1beta by both OMVs. Cbfe23 OMVs have better interaction with the cells than Cbv34 OMVs, being taken up faster by them, but both seem to remain mostly on the cell surface after 24 h of incubation. This, however, did not impair their bacteriostatic activity against intracellular S. aureus. In this study, we provide an important basis for implementing OMVs in the treatment of intracellular infections.
- ItemBiochemical Analysis of Leukocytes after In Vitro and In Vivo Activation with Bacterial and Fungal Pathogens Using Raman Spectroscopy(Basel : MDPI, 2021) Pistiki, Aikaterini; Ramoji, Anuradha; Ryabchykov, Oleg; Thomas-Rueddel, Daniel; Press, Adrian T.; Makarewicz, Oliwia; Giamarellos-Bourboulis, Evangelos J.; Bauer, Michael; Bocklitz, Thomas; Popp, Juergen; Neugebauer, UteBiochemical information from activated leukocytes provide valuable diagnostic information. In this study, Raman spectroscopy was applied as a label-free analytical technique to characterize the activation pattern of leukocyte subpopulations in an in vitro infection model. Neutrophils, monocytes, and lymphocytes were isolated from healthy volunteers and stimulated with heat-inactivated clinical isolates of Candida albicans, Staphylococcus aureus, and Klebsiella pneumoniae. Binary classification models could identify the presence of infection for monocytes and lymphocytes, classify the type of infection as bacterial or fungal for neutrophils, monocytes, and lymphocytes and distinguish the cause of infection as Gram-negative or Gram-positive bacteria in the monocyte subpopulation. Changes in single-cell Raman spectra, upon leukocyte stimulation, can be explained with biochemical changes due to the leukocyte’s specific reaction to each type of pathogen. Raman spectra of leukocytes from the in vitro infection model were compared with spectra from leukocytes of patients with infection (DRKS-ID: DRKS00006265) with the same pathogen groups, and a good agreement was revealed. Our study elucidates the potential of Raman spectroscopy-based single-cell analysis for the differentiation of circulating leukocyte subtypes and identification of the infection by probing the molecular phenotype of those cells.