Rehydration of Freeze Substituted Brain Tissue for Pre-embedding Immunoelectron Microscopy.

Electron microscopy (EM) volume reconstruction is a powerful tool for investigating the fundamental structure of brain circuits, but the full potential of this technique is limited by the difficulty of integrating molecular information. High quality ultrastructural preservation is necessary for EM reconstruction, and intact, highly contrasted cell membranes are essential for following small neuronal processes through serial sections. Unfortunately, the antibody labeling methods used to identify most endogenous molecules result in compromised morphology, especially of membranes. Cryofixation can produce superior morphological preservation and has the additional advantage of allowing indefinite storage of valuable samples. We have developed a method based on cryofixation that allows sensitive immunolabeling of endogenous molecules, preserves excellent ultrastructure, and is compatible with high-contrast staining for serial EM reconstruction.

The Cantaloupe Farm Environment Has a Diverse Genetic Pool of Antibiotic-Resistance and Virulence Genes.

Cantaloupes contaminated with pathogens have led to many high-profile outbreaks and illnesses. Since bacterial virulence genes (VGs) can act in tandem with antibiotic-resistance and mobile genetic elements, there is a need to evaluate these gene reservoirs in fresh produce, such as cantaloupes. The goal of this study was to assess the distribution of antibiotic-resistance, virulence, and mobile genetic elements genes (MGEGs) in cantaloupe farm environments. A total of 200 samples from cantaloupe melons (n = 99), farm workers' hands (n = 66), and production water (n = 35) were collected in México. Each sample was assayed for the presence of 14 antibiotic-resistance genes, 15 VGs, and 5 MGEGs by polymerase chain reaction. Our results indicated that tetracycline (tetA and tetB) (18% of cantaloupe, 45% of hand samples) and sulfonamide (sul1) (30% of cantaloupe, 71% of hand samples) resistance genes were frequently detected. The colistin resistance gene (mcr1) was detected in 10% of cantaloupe and 23% of farm workers' hands. Among VGs, Salmonella genes invA and spiA were the most abundant. There was a significantly higher likelihood of detecting antibiotic-resistance, virulence, and MGEGs on hands compared with water samples. These results demonstrate a diverse pool of antibiotic-resistance and VGs in cantaloupe production.

Analysis of Bacterial Communities by 16S rRNA Gene Sequencing in a Melon-Producing Agro-environment.

Cantaloupe melons, which have been responsible of an increasing number of foodborne disease outbreaks, may become contaminated with microbial pathogens during production. However, little information is available on the microbial populations in the cantaloupe farm environment. The purpose of this work was to characterize the bacterial communities present on cantaloupe farms. Fruit, soil, and harvester hand rinsates were collected from two Mexican cantaloupe farms, each visited three times. Microbiome analysis was performed by sequencing 16sRNA and analyzed using qiime2 software. Correlations were determined between sample type and microbial populations. The α and ß diversity analysis identified 2777 sequences across all samples. The soil samples had the highest number and diversity of unique species (from 130 to 1329 OTUs); cantaloupe (from 112 to 205 OTUs), and hands (from 67 to 151 OTUs) had similar diversity. Collectively, Proteobacteria was the most abundant phyla (from 42 to 95%), followed by Firmicutes (1-47%), Actinobacteria (< 1 to 23%), and Bacteroidetes (< 1 to 4.8%). The most abundant genera were Acinetobacter (20-58%), Pseudomonas (14.5%), Erwinia (13%), and Exiguobacterium (6.3%). Genera with potential to be pathogenic included Bacillus (4%), Salmonella (0.85%), Escherichia-Shigella (0.38%), Staphylococcus (0.32%), Listeria (0.29%), Clostridium (0.28%), and Cronobacter (0.27%), which were found at lower frequencies. This study provides information on the cantaloupe production microbiome, which can inform future research into critical food safety issues such as antimicrobial resistance, virulence, and genomic epidemiology.