Long-Term Persistence of blaCTX-M-15 in Soil and Lettuce after Introducing Extended-Spectrum ß-Lactamase (ESBL)-Producing Escherichia coli via Manure or Water.

The number of environmental antibiotic-resistant bacteria (ARB) has increased dramatically since the start of antibiotic mass production for broad bacterial infection treatment in 1944. Nowadays, ARB and their resistance-determining genes (ARGs) are readily detected in all environments, including the human food chain. A highly relevant food group in this context is fresh produce, frequent raw consumption of which facilitates direct transfer of ARB and ARGs to the consumer. Here, we investigate the persistence of an extended-spectrum ß-lactamase (ESBL)-producing Escherichia coli (E. coli) pEK499 and its clinically most important ARG (blaCTX-M-15), after introduction via irrigation water or manure into a lettuce-growing system. Culturable ESBL-producing E. coli persisted longest in soil and when introduced via manure (until 9 weeks after introduction), while being undetectable on lettuce beyond day 7. In contrast, qPCR detection of blaCTX-M-15 was much more frequent: introduction via water significantly increased blaCTX-M-15 on lettuce until week 4, as opposed to manure, which affected the soil in the long-term (9 weeks) while leading to blaCTX-M-15 detection on lettuce until day 7 only. Our findings demonstrate long-term persistence of undesired ARB and ARG after their introduction via both irrigation and amendment. Such an understanding of the persistence kinetics of an ESBL-producing E. coli and plasmid-encoded blaCTX-M-15 aids the determination of critical actions in order to mitigate their transfer to the consumer.

Distinct changes in all major components of the neurovascular unit across different neuropathological stages of Alzheimer's disease.

In the brain capillaries, endothelial cells, pericytes, astrocytes and microglia form a structural and functional complex called neurovascular unit (NVU) which is critically involved in maintaining neuronal homeostasis. In the present study, we applied a comprehensive immunohistochemical approach to investigate the structural alterations in the NVU across different Alzheimer's disease (AD) neuropathological stages. Post-mortem human cortical and hippocampal samples derived from AD patients and non-demented elderly control individuals were immunostained using a panel of markers representing specific components of the NVU including Collagen IV (basement membrane), PDGFR-ß (pericytes), GFAP (astrocytes), Iba1 (microglia), MRC1 (perivascular macrophages) and lectin as an endothelial cell label. Astrocytes (GFAP) and microglia (Iba1) were quantified both in the whole visual-field and specifically within the NVU, and the sample set was additionally analyzed using anti-tau (AT8) and three different anti-Aß (clones G2-10, G2-11, 4G8) antibodies. Analyses of lectin labeled sections showed an altered vascular distribution in AD patients as revealed by a reduced nearest distance between capillaries. Within the NVU, a Braak-stage dependent reduction in pericyte coverage was identified as the earliest structural alteration during AD progression. In comparison to non-demented elderly controls, AD patients showed a significantly higher astrocyte coverage within the NVU, which was paralleled by a reduced microglial coverage around capillaries. Assessment of perivascular macrophages moreover demonstrated a relocation of these cells from leptomeningeal arteries to penetrating parenchymal vessels in AD patients. Collectively, the results of our study represent a comprehensive first in-depth analysis of AD-related structural changes in the NVU and suggest distinct alterations in all components of the NVU during AD progression.

The amyloid-ß degradation intermediate Aß34 is pericyte-associated and reduced in brain capillaries of patients with Alzheimer's disease.

An impairment of amyloid ß-peptide (Aß) clearance is suggested to play a key role in the pathogenesis of sporadic Alzheimer's disease (AD). Amyloid degradation is mediated by various mechanisms including fragmentation by enzymes like neprilysin, matrix metalloproteinases (MMPs) and a recently identified amyloidolytic activity of ß-site amyloid precursor protein cleaving enzyme 1 (BACE1). BACE1 cleavage of Aß40 and Aß42 results in the formation of a common Aß34 intermediate which was found elevated in cerebrospinal fluid levels of patients at the earliest disease stages. To further investigate the role of Aß34 as a marker for amyloid clearance in AD, we performed a systematic and comprehensive analysis of Aß34 immunoreactivity in hippocampal and cortical post-mortem brain tissue from AD patients and non-demented elderly individuals. In early Braak stages, Aß34 was predominantly detectable in a subset of brain capillaries associated with pericytes, while in later disease stages, in clinically diagnosed AD, this pericyte-associated Aß34 immunoreactivity was largely lost. Aß34 was also detected in isolated human cortical microvessels associated with brain pericytes and its levels correlated with Aß40, but not with Aß42 levels. Moreover, a significantly decreased Aß34/Aß40 ratio was observed in microvessels from AD patients in comparison to non-demented controls suggesting a reduced proteolytic degradation of Aß40 to Aß34 in AD. In line with the hypothesis that pericytes at the neurovascular unit are major producers of Aß34, biochemical studies in cultured human primary pericytes revealed a time and dose dependent increase of Aß34 levels upon treatment with recombinant Aß40 peptides while Aß34 production was impaired when Aß40 uptake was reduced or BACE1 activity was inhibited. Collectively, our findings indicate that Aß34 is generated by a novel BACE1-mediated Aß clearance pathway in pericytes of brain capillaries. As amyloid clearance is significantly reduced in AD, impairment of this pathway might be a major driver of the pathogenesis in sporadic AD.