Accurate Detection of the Four Most Prevalent Carbapenemases in E. coli and K. pneumoniae by High-Resolution Mass Spectrometry

Background: At present, phenotypic growth inhibition techniques are used in routine diagnostic microbiology to determine antimicrobial resistance of bacteria. Molecular techniques such as PCR are often used for confirmation but are indirect as they detect particular resistance genes. A direct technique would be able to detect the proteins of the resistance mechanism itself. In the present study targeted high resolution mass spectrometry assay was developed for the simultaneous detection of KPC, OXA-48-like, NDM, and VIM carbapenemases. Methods: Carbapenemase specific target peptides were defined by comparing available sequences in GenBank. Selected peptide sequences were validated using 62 Klebsiella pneumoniae and Escherichia coli isolates containing: 16 KPC, 21 OXA-48-like, 16 NDM, 13 VIM genes, and 21 carbapenemase negative isolates. Results: For each carbapenemase, two candidate peptides were validated. Method validation was performed in a blinded manner for all 83 isolates. All carbapenemases were detected. The majority was detected by both target peptides. All target peptides were 100% specific in the tested isolates and no peptide carry-over was detected. Conclusion: The applied targeted bottom-up mass spectrometry technique is able to accurately detect the four most prevalent carbapenemases in a single analysis

Lipids Are the Preferred Substrate of the Protist Naegleria gruberi, Relative of a Human Brain Pathogen

Naegleria gruberi is a free-living non-pathogenic amoeboflagellate and relative of Naegleria fowleri, a deadly pathogen causing primary amoebic meningoencephalitis (PAM). A genomic analysis of N. gruberi exists, but physiological evidence for its core energy metabolism or in vivo growth substrates is lacking. Here, we show that N. gruberi trophozoites need oxygen for normal functioning and growth and that they shun both glucose and amino acids as growth substrates. Trophozoite growth depends mainly upon lipid oxidation via a mitochondrial branched respiratory chain, both ends of which require oxygen as final electron acceptor. Growing N. gruberi trophozoites thus have a strictly aerobic energy metabolism with a marked substrate preference for the oxidation of fatty acids. Analyses of N. fowleri genome data and comparison with those of N. gruberi indicate that N. fowleri has the same type of metabolism. Specialization to oxygen-dependent lipid breakdown represents an additional metabolic strategy in protists. Bexkens et al. show that N. gruberi amoebae live preferably on lipids, for which they need oxygen, a lifestyle largely unknown among protists. This challenges existing views about its energy metabolism, with implications for treatment of its pathogenic relative, N. fowleri, the brain-eating agent of primary amoebic me

Biochemical Adaptations in Host-Parasite Interactions

In this thesis, multiple novel insights into the molecular mechanisms involved in hostparasite interactions are presented. The main topic is the energy metabolism of S. mansoni and its interaction with the immune system of the host

Persistent contamination of a duodenoscope working channel in a non-clinical simulated ERCP setting

Background To mitigate duodenoscope contamination, recent design enhancements have primarily focused on the distal tip. However, the working channels remain unchanged, which may be linked to biofilm formation. We assessed the persistence of microorganisms, indicative of biofilm formation, in reprocessed duodenoscopes in a nonclinical endoscopic retrograde cholangiopancreatography (ERCP) simulation setting. Methods Three new duodenoscopes were over-soiled in non-clinical ERCP simulations followed by reprocessing. After 40 tests, the Pseudomonas aeruginosa strain in the soil (Pa-type 1) was switched to a different P. aeruginosa strain (Pa-type 2) for 20 subsequent tests. Cultures of the tip and working channel were acquired after high level disinfection and overnight storage. Results One duodenoscope showed persistent growth of P. aeruginosa from the fifth test until the end of the study. Pa-type 1 remained present until the end of the study in the cultures of this duodenoscope, even after discontinuation of exposure to that specific strain. The other two duodenoscopes only showed incidental contamination. Conclusion Persistent contamination by Pa-type 1 was seen in one out of three duodenoscopes after exposure to supraphysiological levels of gut microorganisms. No clear explanation was found for this persistent contamination as exposure and handling were identical and no abnormalities of this particular duodenoscope were identified by borescope inspection

Characterization of staphylococcus roterodami sp. Nov., a new species within the staphylococcus aureus complex isolated from a human foot infection

This article introduces a new Staphylococcus species cultivated from a human foot wound infection in a Dutch traveller returning from the island of Bali, Indonesia: Staphylococcus roterodami sp. nov. Based on the genomic sequence, there is strong molecular evidence for assigning the strain to a novel species within the S. aureus complex. Differences in cellular fatty acid spectrum and biochemical tests underline these findings. Its ecological niche and pathogenicity require further study. The type strain is DSM111914T (JCM34415T)

Schistosoma mansoni does not and cannot oxidise fatty acids, but these are used for biosynthetic purposes instead

Adult schistosomes, parasitic flatworms that cause the tropical disease schistosomiasis, have always been considered to be homolactic fermenters and, in their energy metabolism, strictly dependent on carbohydrates. However, more recent studies suggested that fatty acid β-oxidation is essential for egg production by adult female Schistosoma mansoni. To address this conundrum, we performed a comprehensive study on the lipid metabolism of S. mansoni. Incubations with [14C]-labelled fatty acids demonstrated that adults, eggs and miracidia of S. mansoni did not oxidise fatty acids, as no 14CO2 production could be detected. We then re-examined the S. mansoni genome using the genes known to be involved in fatty acid oxidation in six eukaryotic model reference species. This showed that the earlier automatically annotated genes for fatty acid oxidation were in fact incorrectly annotated. In a further analysis we could not detect any genes encoding β-oxidation enzymes, which demonstrates that S. mansoni cannot use this pathway in any of its lifecycle stages. The same was true for Schistosoma japonicum and all other schistosome species that have been sequenced. Absence of β-oxidation, however, does not imply that fatty acids from the host are not metabolised by schistosomes. Adult schistosomes can use and modify fatty acids from their host for biosynthetic purposes and incorporate those in phospholipids and neutral lipids. Female worms deposit large amounts of these lipids in the eggs they produce, which explains why interference with the lipid metabolism in females will disturb egg formation, even though fatty acid β-oxidation does not occur in schistosomes. Our analyses of S. mansoni further revealed that during the development and maturation of the miracidium inside the egg, changes in lipid composition occur which indicate that fatty acids deposited in the egg by the female worm are used for phospholipid biosynthesis required for membrane formation in the developing miracidium