TY - JOUR A1 - Peters, Simon A1 - Kaiser, Lena A1 - Fink, Julian A1 - Schumacher, Fabian A1 - Perschin, Veronika A1 - Schlegel, Jan A1 - Sauer, Markus A1 - Stigloher, Christian A1 - Kleuser, Burkhard A1 - Seibel, Juergen A1 - Schubert-Unkmeir, Alexandra T1 - Click-correlative light and electron microscopy (click-AT-CLEM) for imaging and tracking azido-functionalized sphingolipids in bacteria JF - Scientific Reports N2 - Sphingolipids, including ceramides, are a diverse group of structurally related lipids composed of a sphingoid base backbone coupled to a fatty acid side chain and modified terminal hydroxyl group. Recently, it has been shown that sphingolipids show antimicrobial activity against a broad range of pathogenic microorganisms. The antimicrobial mechanism, however, remains so far elusive. Here, we introduce 'click-AT-CLEM', a labeling technique for correlated light and electron microscopy (CLEM) based on the super-resolution array tomography (srAT) approach and bio-orthogonal click chemistry for imaging of azido-tagged sphingolipids to directly visualize their interaction with the model Gram-negative bacterium Neisseria meningitidis at subcellular level. We observed ultrastructural damage of bacteria and disruption of the bacterial outer membrane induced by two azido-modified sphingolipids by scanning electron microscopy and transmission electron microscopy. Click-AT-CLEM imaging and mass spectrometry clearly revealed efficient incorporation of azido-tagged sphingolipids into the outer membrane of Gram-negative bacteria as underlying cause of their antimicrobial activity. KW - antimicrobials KW - biological techniques KW - imaging KW - microbiology KW - microbiology techniques KW - microscopy Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-259147 VL - 11 IS - 1 ER - TY - JOUR A1 - Duske, Helene A1 - Claus, Heike A1 - Krone, Manuel A1 - Lâm, Thiên-Trí T1 - Prevalence of piperacillin/tazobactam resistance in invasive \(Haemophilus\) \(influenzae\) in Germany JF - JAC-Antimicrobial Resistance N2 - Background Haemophilus influenzae (Hi) is a Gram-negative bacterium that may cause sepsis or meningitis, treatment of which mainly includes β-lactam antibiotics. Since 2019 EUCAST breakpoints for piperacillin/tazobactam have been available. Little is known about the prevalence and mechanisms of piperacillin/tazobactam resistance in Hi. Objectives To provide reliable prevalence data for piperacillin/tazobactam resistance in Hi in Germany, to evaluate different antibiotic susceptibility testing methods and to examine possible resistance mechanisms. Methods According to EUCAST breakpoints, the MIC for piperacillin/tazobactam resistance is >0.25 mg/L. All invasive Hi in Germany from 2019 were examined by gradient agar diffusion (GAD) for piperacillin/tazobactam susceptibility. Piperacillin/tazobactam broth microdilution (BMD), piperacillin GAD on tazobactam-containing agar [piperacillin GAD on Mueller–Hinton agar with horse blood (MH-F)/tazobactam) and piperacillin/tazobactam agar dilution (AD) were used for confirmation. Phenotypic testing was complemented by ftsI sequencing. Results Piperacillin/tazobactam GAD resulted in 2.9% (21/726) resistant Hi. BMD did not confirm piperacillin/tazobactam resistance. Two strains were found resistant by AD, of which one was also resistant using piperacillin GAD on MH-F/tazobactam. Overall, we found two strains with a piperacillin/tazobactam MIC >0.25 mg/L in at least two different tests (0.3%). Both were β-lactamase-producing amoxicillin/clavulanate-resistant with PBP3 mutations characterized as group III-like+. Relevant PBP3 mutations occurred in six strains without phenotypic piperacillin/tazobactam resistance. These mutations suggest a reduced efficacy of β-lactam antibiotics in these isolates. Conclusions Piperacillin/tazobactam resistance prevalence in invasive Hi is low in Germany. Reduced susceptibility was correlated with PBP3 mutations, in particular with group III mutations. KW - microbiology KW - immunology KW - generalized anxiety disorder KW - haemophilus influenzae KW - agar KW - Germany KW - piperacillin KW - piperacillin/tazobactam KW - tazobactam KW - Haemophilus influenzae Y1 - 2024 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-350424 SN - 2632-1823 VL - 6 IS - 1 ER -