Counts of mesophilic aerobic, mesophilic anaerobic, thermophilic aerobic sporeforming bacteria and persistence of Bacillus cereus spores throughout cocoa powder processing chain.

Sporeforming bacteria are a concern in some food raw materials, such as cocoa powder. Samples (n = 618) were collected on two farms and at several stages during cocoa powder manufacture in three commercial processing lines to determine the impact of each stage on bacterial spore populations. Mesophilic aerobic, mesophilic anaerobic, thermophilic aerobic, and Bacillus cereus spore populations were enumerated in all the samples. Genetic diversity in B. cereus strains (n = 110) isolated from the samples was examined by M13 sequence-based PCR typing, partial sequencing of the panC gene, and the presence/absence of ces and cspA genes. The counts of different groups of sporeforming bacteria varied amongst farms and processing lines. For example, the counts of mesophilic aerobic spore-forming (MAS) populations of cocoa bean fermentation were lower than 1 log spore/g in Farm 1 but higher than 4 log spore/g in Farm 2. B. cereus isolated from cocoa powder was also recovered from cocoa beans, nibs, and samples after roasting, refining, and pressing, which indicated that B. cereus spores persist throughout cocoa processing. Phylogenetic group IV was the most frequent (73%), along with processing. Strains from phylogenetic group III (14 %) did not show the ces gene's presence.

Culturable microbiological profile of a non-sterile drugs pharmaceutical production environment

Abstract For places where non-sterile drug production occurs, regulatory bodies recommend monitoring of the environmental bioburden. This procedure provides information regarding possible microbiological risks to which the products may be exposed, so that subsequent action measures may be implemented. The aim of the present work was to quantify and characterize the microorganisms present in Grade D (ISO 8) cleanrooms of a Brazilian pharmaceutical industry, identifying any possible seasonal climatic influences on these environments. Sampling was performed by surface and air monitoring, over 12 months during the year 2019, in rooms that were in operation. For both sampling methods, no statistically significant differences in bacteria and fungi counts were found between months or seasonal periods. Microorganisms that presented higher incidence included Staphylococcus epidermidis (15%) and Micrococcus spp. (13%), common to the human microbiota, and the fungi Cladosporium sp. (23%) and Penicillium sp. (21%), typical of the external environment. The results showed that microbial contamination in the Grade D cleanrooms was within the permissible maximum levels and remained similar throughout the year. Microbiological quality control in the clean areas of the pharmaceutical industry investigated was considered effective, with regular maintenance being necessary to keep bioburden levels controlled.

Protocol for rapid obtention and fractionation of anaerobic bacterial conditioned media to study calcium signaling in enteroendocrine cells.

Gut microbiota influences neurodevelopment, behavior and contributes to neurodegenerative disorders. One possible mechanism is the direct modulation of calcium (Ca2+) signaling and protein homeostasis in enteroendocrine cells (EECs), a component of the gut epithelium. Here, we present a protocol to isolate fractions of conditioned media (CM) from the anaerobic bacteria Akkermansia muciniphila and the utilization of this CM to monitor Ca2+ fluctuation in EECs by imaging. This protocol can be adapted and applied to various bacterial cultures and cell types. For complete details on the use and execution of this protocol, please refer to Amorim Neto et al. (2022).

Transcellular propagation of fibrillar α-synuclein from enteroendocrine to neuronal cells requires cell-to-cell contact and is Rab35-dependent.

Parkinson's disease (PD) is a neurodegenerative condition featured by motor dysfunction, death of midbrain dopaminergic neurons and accumulation of α-synuclein (αSyn) aggregates. Growing evidence suggests that PD diagnosis happens late in the disease progression and that the pathology may originate much earlier in the enteric nervous system (ENS) before advancing to the brain, via autonomic fibers. It was recently described that a specific cell type from the gut epithelium named enteroendocrine cells (EECs) possess many neuron-like properties including αSyn expression. By facing the gut lumen and being directly connected with αSyn-containing enteric neurons in a synaptic manner, EECs form a neural circuit between the gastrointestinal tract and the ENS, thereby being a possible key player in the outcome of PD in the gut. We have characterized the progression and the cellular mechanisms involved in αSyn pre-formed fibrils (PFFs) transfer from EECs to neuronal cells. We show that brain organoids efficiently internalize αSyn PFF seeds which triggers the formation of larger intracellular inclusions. In addition, in the enteroendocrine cell line STC-1 and in the neuronal cell line SH-SY5Y, αSyn PFFs induced intracellular calcium (Ca2+) oscillations on an extracellular Ca2+ source-dependent manner and triggered αSyn fibrils internalization by endocytosis. We characterized the spread of αSyn PFFs from enteroendocrine to neuronal cells and showed that this process is dependent on physical cell-to-cell contact and on Rab35 GTPase. Lastly, inhibition of Rab35 increases the clearance of αSyn fibrils by redirecting them to the lysosomal compartment. Therefore, our results reveal mechanisms that contribute to the understanding of how seeded αSyn fibrils promote the progression of αSyn pathology from EECs to neuronal cells shifting the focus of PD etiology to the ENS.

Akkermansia muciniphila induces mitochondrial calcium overload and α -synuclein aggregation in an enteroendocrine cell line.

The gut microbiota influence neurodevelopment, modulate behavior, and contribute to neurodegenerative disorders. Several studies have consistently reported a greater abundance of Akkermansia muciniphila in Parkinson disease (PD) fecal samples. Therefore, we investigated whether A.muciniphila-conditioned medium (CM) could initiate α-synuclein (αSyn) misfolding in enteroendocrine cells (EEC) - a component of the gut epithelium featuring neuron-like properties. We found that A. muciniphila CM composition is influenced by the ability of the strain to degrade mucin. Our in vitro experiments showed that the protein-enriched fraction of mucin-free CM induces RyR-mediated Ca2+ release and increased mitochondrial Ca2+ uptake leading to ROS generation and αSyn aggregation. Oral administration of A. muciniphila cultivated in the absence of mucin to mice led to αSyn aggregation in cholecystokinin (CCK)-positive EECs but no motor deficits were observed. Noteworthy, buffering mitochondrial Ca2+ reverted the damaging effects observed. These molecular insights offer evidence that bacterial proteins can induce αSyn aggregation in EECs.

Molecular and cellular basis of hyperassembly and protein aggregation driven by a rare pathogenic mutation in DDX3X.

Current studies estimate that 1-3% of females with unexplained intellectual disability (ID) present de novo splice site, nonsense, frameshift, or missense mutations in the DDX3X protein (DEAD-Box Helicase 3 X-Linked). However, the cellular and molecular mechanisms by which DDX3X mutations impair brain development are not fully comprehended. Here, we show that the ID-linked missense mutation L556S renders DDX3X prone to aggregation. By using a combination of biophysical assays and imaging approaches, we demonstrate that this mutant assembles solid-like condensates and amyloid-like fibrils. Although we observed greatly reduced expression of the mutant allele in a patient who exhibits skewed X inactivation, this appears to be enough to sequestrate healthy proteins into solid-like ectopic granules, compromising cell function. Therefore, our data suggest ID-linked DDX3X L556S mutation as a disorder arising from protein misfolding and aggregation.

Illuminating the Brain With X-Rays: Contributions and Future Perspectives of High-Resolution Microtomography to Neuroscience.

The assessment of three-dimensional (3D) brain cytoarchitecture at a cellular resolution remains a great challenge in the field of neuroscience and constant development of imaging techniques has become crucial, particularly when it comes to offering direct and clear obtention of data from macro to nano scales. Magnetic resonance imaging (MRI) and electron or optical microscopy, although valuable, still face some issues such as the lack of contrast and extensive sample preparation protocols. In this context, x-ray microtomography (µCT) has become a promising non-destructive tool for imaging a broad range of samples, from dense materials to soft biological specimens. It is a new supplemental method to be explored for deciphering the cytoarchitecture and connectivity of the brain. This review aims to bring together published works using x-ray µCT in neurobiology in order to discuss the achievements made so far and the future of this technique for neuroscience.

Interaction of Aspergillus flavus and A. parasiticus with Salmonella spp. isolated from peanuts.

Although Aspergillus flavus and Aspergillus parasiticus are the main microorganisms of concern in peanuts, due to aflatoxin contamination, several Salmonella outbreaks from this product have been reported over the last ten decades. Thus, it is important to understand the relationship between microorganisms to predict, manage and estimate the diversity in the peanut supply chain. The purpose of this study was to evaluate aflatoxin production during the co-cultivation of Aspergillus section Flavi and Salmonella both isolated from peanuts. Three strains of A. section Flavi: A. flavus producing aflatoxin B, A. flavus non-producing aflatoxin and A. parasiticus producing aflatoxin B and G were co-cultivated with seven serotypes of Salmonella of which six were isolated from the peanut supply chain (S. Muenster, S. Miami, S. Glostrup, S. Javiana, S. Oranienburg and S. Yoruba) and one was S. Typhimurium ATCC 14028. First of all, each Salmonella strain was inoculated by pour plate (ca. 5 log cfu/mL) in PDA (potato dextrose agar). Then, each pre-cultured fungus was inoculated in the center of the petri dish. The plates were incubated at 30 °C and the fungal colony diameter was measured once a day for 7 days. As a control each Aspergillus strain was cultivated in the absence of Salmonella culture. All three strains of Aspergillus with absence of Salmonella (control) reached the maximum colony diameter and their growth rate was influenced when co-cultivated (p < 0.05) with all Salmonella serotypes tested. The maximum inhibition in the colony diameter was 20% for A. flavus aflatoxin B producer and A. parasiticus, and 18% for A. flavus non- aflatoxin producer when cultivated with Salmonella. However, no significant difference (p < 0.05) in reduction of colony diameter was observed among the Salmonella serotypes. Aflatoxin production was determined previously, by using the agar plug technique on thin layer chromatography (TLC). The production of aflatoxin G by A. parasiticus in co-cultivation with Salmonella was not observed. On the other hand, A. flavus preserved their characteristics of aflatoxin B production. The quantification of aflatoxin reduction by Salmonella interaction was evaluated using HPLC method. There was a maximum reduction of aflatoxin production of 88.7% and 72.9% in A. flavus and A. parasiticus, respectively, when cultivated with Salmonella. These results indicate that some serotypes of Salmonella may interfere with aflatoxin production and fungal growth of A. flavus and A. parasiticus in the peanut supply chain.

Genetic diversity, antimicrobial resistance and virulence profile of Salmonella isolated from the peanut supply chain.

Thirty-Eight Salmonella isolates recovered from different stages of the peanut supply chain in three Brazilian States (São Paulo, Minas Gerais and Bahia) were subtyped by pulsed-field gel electrophoresis (PFGE) and characterized by phenotypic and genotypic tests for antimicrobial resistance and virulence genes. The isolates were distributed into seven PFGE pulsotypes. All the isolates were resistant to sulfonamide. However, only one isolate from a production site in Minas Gerais had resistance to two types of antimicrobials (sulfonamide and ampicillin). Furthermore, the isolates had intermediary resistance to kanamycin (16/38), streptomycin (14/38) and ceftazidime (12/38). Four isolates had the antimicrobial resistance gene related to phenicols (floR) and 37 related to aminoglycosides (strA). The blashv gene related to ß-lactams was detected in isolates recovered from all the production regions. Six virulence genes (invA, sefA, sivH, mgtC, ssaQ and agfA) were observed in all isolates. The sopE gene was detected in 24 isolates, avrA in 12. The gtgB, ipfA and rck genes were not detected. The results showed that the pulsotype 1 was restricted to Minas Gerais whereas the pulsotype 7 was present in São Paulo and Bahia. In addition, most of the isolates were not multidrug resistant.