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- ItemDetection of Protein Glycosylation Using Tip-Enhanced Raman Scattering(Columbus, Ohio : American Chemical Society, 2016) Cowcher, David P.; Deckert-Gaudig, Tanja; Brewster, Victoria L.; Ashton, Lorna; Deckert, Volker; Goodacre, RoystonThe correct glycosylation of biopharmaceutical glycoproteins and their formulations is essential for them to have the desired therapeutic effect on the patient. It has recently been shown that Raman spectroscopy can be used to quantify the proportion of glycosylated protein from mixtures of native and glycosylated forms of bovine pancreatic ribonuclease (RNase). Here we show the first steps toward not only the detection of glycosylation status but the characterization of glycans themselves from just a few protein molecules at a time using tip-enhanced Raman scattering (TERS). While this technique generates complex data that are very dependent on the protein orientation, with the careful development of combined data preprocessing, univariate and multivariate analysis techniques, we have shown that we can distinguish between the native and glycosylated forms of RNase. Many glycoproteins contain populations of subtly different glycoforms; therefore, with stricter orientation control, we believe this has the potential to lead to further glycan characterization using TERS, which would have use in biopharmaceutical synthesis and formulation research.
- ItemMolecular characterisation of extended-spectrum ß-lactamase producing Escherichia coli in wild birds and cattle, Ibadan, Nigeria(London : BioMed Central, 2021) Fashae, Kayode; Engelmann, Ines; Monecke, Stefan; Braun, Sascha D.; Ehricht, RalfBackground: Antimicrobial resistance (AMR) is an increasing global health concern reducing options for therapy of infections and also for perioperative prophylaxis. Many Enterobacteriaceae cannot be treated anymore with third generation cephalosporins (3GC) due to the production of certain 3GC hydrolysing enzymes (extended spectrum beta-lactamases, ESBLs). The role of animals as carriers and vectors of multi-resistant bacteria in different geographical regions is poorly understood. Therefore, we investigated the occurrence and molecular characteristics of ESBL-producing Escherichia coli (E. coli) in wild birds and slaughtered cattle in Ibadan, Nigeria. Cattle faecal samples (n = 250) and wild bird pooled faecal samples (cattle egrets, Bubulcus ibis, n = 28; white-faced whistling duck, Dendrocygna viduata, n = 24) were collected and cultured on cefotaxime-eosin methylene blue agar. Antimicrobial susceptibility was determined by agar diffusion assays and all 3GC resistant isolates were genotypically characterised for AMR genes, virulence associated genes (VAGs) and serotypes using DNA microarray-based assays. Results: All 3GC resistant isolates were E. coli: cattle (n = 53), egrets (n = 87) and whistling duck (n = 4); cultured from 32/250 (12.8%), 26/28 (92.9%), 2/24(8.3%), cattle, egrets and whistling duck faecal samples, respectively. blaCTX-M gene family was prevalent; blaCTX-M15 (83.3%) predominated over blaCTX-M9 (11.8%). All were susceptible to carbapenems. The majority of isolates were resistant to at least one of the other tested antimicrobials; multidrug resistance was highest in the isolates recovered from egrets. The isolates harboured diverse repositories of other AMR genes (including strB and sul2), integrons (predominantly class 1) and VAGs. The isolates recovered from egrets harboured more AMR genes; eight were unique to these isolates including tetG, gepA, and floR. The prevalent VAGs included hemL and iss; while 14 (including sepA) were unique to certain animal isolates. E. coli serotypes O9:H9, O9:H30 and O9:H4 predominated. An identical phenotypic microarray profile was detected in three isolates from egrets and cattle, indicative of a clonal relationship amongst these isolates. Conclusion: Wild birds and cattle harbour diverse ESBL-producing E. coli populations with potential of inter-species dissemination and virulence. Recommended guidelines to balance public health and habitat conservation should be implemented with continuous surveillance. © 2021, The Author(s).
- ItemCharacterisation of S. aureus/MRSA CC1153 and review of mobile genetic elements carrying the fusidic acid resistance gene fusC([London] : Macmillan Publishers Limited, part of Springer Nature, 2021) Monecke, Stefan; Müller, Elke; Braun, Sascha D.; Armengol-Porta, Marc; Bes, Michèle; Boswihi, Samar; El-Ashker, Maged; Engelmann, Ines; Gawlik, Darius; Gwida, Mayada; Hotzel, Helmut; Nassar, Rania; Reissig, Annett; Ruppelt-Lorz, Antje; Senok, Abiola; Somily, Ali M.; Udo, Edet E.; Ehricht, RalfWhile many data on molecular epidemiology of MRSA are available for North America, Western Europe and Australia, much less is known on the distribution of MRSA clones elsewhere. Here, we describe a poorly known lineage from the Middle East, CC1153, to which several strains from humans and livestock belong. Isolates were characterised using DNA microarrays and one isolate from the United Arab Emirates was sequenced using Nanopore technology. CC1153 carries agr II and capsule type 5 genes. Enterotoxin genes are rarely present, but PVL is common. Associated spa types include t504, t903 and t13507. PVL-positive CC1153-MSSA were found in Egyptian cattle suffering from mastitis. It was also identified among humans with skin and soft tissue infections in Saudi Arabia, France and Germany. CC1153-MRSA were mainly observed in Arabian Gulf countries. Some isolates presented with a previously unknown SCCmec/SCCfus chimeric element in which a mec B complex was found together with the fusidic acid resistance gene fusC and accompanying genes including ccrA/B-1 recombinase genes. Other isolates carried SCCmec V elements that usually also included fusC. Distribution and emergence of CC1153-MRSA show the necessity of molecular characterization of MRSA that are resistant to fusidic acid. These strains pose a public health threat as they combine resistance to beta-lactams used in hospitals as well as to fusidic acid used in the community. Because of the high prevalence of fusC-positive MRSA in the Middle East, sequences and descriptions of SCC elements harbouring fusC and/or mecA are reviewed. When comparing fusC and its surrounding regions from the CC1153 strain to available published sequences, it became obvious that there are four fusC alleles and five distinct types of fusC gene complexes reminiscent to the mec complexes in SCCmec elements. Likewise, they are associated with different sets of ccrA/B recombinase genes and additional payload that might include entire mec complexes or SCCmec elements.
- ItemHigh resolution spectroscopy reveals fibrillation inhibition pathways of insulin([London] : Macmillan Publishers Limited, part of Springer Nature, 2016) Deckert-Gaudig, Tanja; Deckert, VolkerFibril formation implies the conversion of a protein’s native secondary structure and is associated with several neurodegenerative diseases. A better understanding of fibrillation inhibition and fibril dissection requires nanoscale molecular characterization of amyloid structures involved. Tip-enhanced Raman scattering (TERS) has already been used to chemically analyze amyloid fibrils on a sub-protein unit basis. Here, TERS in combination with atomic force microscopy (AFM), and conventional Raman spectroscopy characterizes insulin assemblies generated during inhibition and dissection experiments in the presence of benzonitrile, dimethylsulfoxide, quercetin, and β-carotene. The AFM topography indicates formation of filamentous or bead-like insulin self-assemblies. Information on the secondary structure of bulk samples and of single aggregates is obtained from standard Raman and TERS measurements. In particular the high spatial resolution of TERS reveals the surface conformations associated with the specific agents. The insulin aggregates formed under different inhibition and dissection conditions can show a similar morphology but differ in their β-sheet structure content. This suggests different aggregation pathways where the prevention of the β-sheet stacking of the peptide chains plays a major role. The presented approach is not limited to amyloid-related reasearch but can be readily applied to systems requiring extremely surface-sensitive characterization without the need of labels.