Standing up FungiNet: Genomic epidemiology and surveillance of fungal diseases

Objectives: With the advent of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) sequencing, the public health landscape for genomic epidemiology and surveillance has transformed for a variety of pathogens. For fungal diseases, the U. S. Centers for Disease Control and Prevention (CDC) is working with global partners to stand up FungiNet, a network that aims to equip scientists with laboratory, bioinformatics, and informatics resources to harness genomic data. FungiNet partners will use genomic and epidemiologic data to detect outbreaks, identify introductions, and characterize transmission of fungal infections. In 2022, FungiNet aims to onboard nine state and local health departments in the United States and two global partners, the Instituto Nacional de Salud in Colombia and the National Institute for Communicable Diseases in South Africa, with a focus on Candida auris. Method(s): To streamline the onboarding process, CDC generated standardized operating procedures (SOPs) specific to C. auris. For DNA extraction, SOPs were created for workflows using the Zymo Research Quick-DNA TM (ZR) Fungal/Bacterial Miniprep, Qiagen Dneasy Blood and Tissue, and Epicentre (Illumina) MasterPure Yeast DNA Purification kits. For library preparation and Illumina sequencing, PulseNet methods used for foodborne pathogens were validated for C. auris. For NCBI data submissions, required data elements were defined. For SNP and phylogenetic analyses, the bioinformatics workflow My-coSNP was adapted to use Nextflow software and the Terra platform. For visualization with epidemiologic data, guidance documents and tutorials for Microreact were created. Finally, for data reporting, processes are being designed in REDCap and in laboratory information management systems to rapidly share genomic-related data. Result(s): To date, 11 partners have committed to building capacity for C. auris genomic sequencing and analysis as a FungiNet partner. Of these, seven have validated methods for DNA extraction, and nine have generated high-quality sequencing data. Only one partner has installed and locally run MycoSNP, and none have submitted raw sequence data to NCBI. Conclusion(s): Currently, 11 FungiNet partners are working to onboard C. auris genomic sequencing and bioinformatics analysis in 2022. This process is complex, requiring several laboratories, bioinformatics, and informatics workflows. For many partners, bioinformatics analysisand NCBIsubmission are themost challenging activities with the installationof MycoSNPand the ability to batch upload data to NCBI as the main barriers. Next steps will focus on the validation of informatics methods to link genomic and epidemiologic data.

Inhibitory effects of fucoidan from Laminaria japonica against some pathogenic bacteria and SARS-CoV-2 depend on its large molecular weight.

Fucoidan is a highly sulfated polysaccharide with a wide range of bioactivities, including anti-pathogenic activity. However, the relationship between structure and activity of fucoidan in inhibiting pathogen infections remains unclear. Here, different-molecular-weight fucoidans were prepared by photocatalytic degradation followed by membrane ultrafiltration, and their chemical structures and anti-pathogenic microbiota activity were compared. Results showed that photocatalytic degradation could effectively degrade fucoidan while its structure block and sulfate groups were not destroyed obviously. Fucoidan (90.8 kDa) of 5 mg/mL could inhibit the growth of S. aureus, S. typhimurium and E. coli, but its degradation products, Dfuc1 (19.2 kDa) and Dfuc2 (5.5 kDa), demonstrated lower inhibitory effect. In addition, compared to Dfuc1 and Dfuc2, fucoidan showed stronger capability to prevent the adhesion of S. aureus, L. monocytogenes, V. parahaemolyticus and S. typhimurium to HT-29 cells. Moreover, the inhibitory effect against SARS-CoV-2 and the binding activity to S protein were also positively correlated to molecular weight. These results indicate that natural fucoidan with higher molecular weight are more effective to inhibit these pathogenic bacteria and SARS-CoV-2, providing a better understanding of the relationship between structure and activity of fucoidan against pathogenic microbiota.

Qualitative and Quantitative Detection of Potentially Virulent Vibrio parahaemolyticus in Drinking Water and Commonly Consumed Aquatic Products by Loop-Mediated Isothermal Amplification.

Vibrio parahaemolyticus can cause acute gastroenteritis, wound infection, and septicemia in humans. In this study, a simple, specific, and user-friendly diagnostic tool was developed for the first time for the qualitative and quantitative detection of toxins and infection process-associated genes opaR, vpadF, tlh, and ureC in V. parahaemolyticus using the loop-mediated isothermal amplification (LAMP) technique. Three pairs of specific inner, outer, and loop primers were designed for targeting each of these genes, and the results showed no cross-reaction with the other common Vibrios and non-Vibrios pathogenic bacteria. Positive results in the one-step LAMP reaction (at 65 °C for 45 min) were identified by a change to light green and the emission of bright green fluorescence under visible light and UV light (302 nm), respectively. The lowest limit of detection (LOD) for the target genes ranged from 1.46 × 10-5 to 1.85 × 10-3 ng/reaction (25 µL) for the genomic DNA, and from 1.03 × 10-2 to 1.73 × 100 CFU/reaction (25 µL) for the cell culture of V. parahaemolyticus. The usefulness of the developed method was demonstrated by the fact that the bacterium could be detected in water from various sources and commonly consumed aquatic product samples. The presence of opaR and tlh genes in the Parabramis pekinensis intestine indicated a risk of potentially virulent V. parahaemolyticus in the fish.