A novel CRISPRi platform to study the role of essential genes in antifungal drug-resistant Candida albicans isolates

With the emergence of antifungal resistant Candida albicans strains, the need for new antifungal drugs is critical in combating this fungal pathogen. Investigating essential genes in C. albicans is a vital step in characterizing putative antifungal drug targets. As some of these essential genes are conserved between fungal organisms, developed therapies targeting these genes have the potential to be broad range antifungals. In order to study these essential genes, classical genetic knockout or CRISPR-based approaches cannot be used as disrupting essential genes leads to lethality in the organism. Fortunately, a variation of the CRISPR system (CRISPR interference or CRISPRi) exists that enables precise transcriptional repression of the gene of interest without introducing genetic mutations. CRISPRi utilizes an endonuclease dead Cas9 protein that can be targeted to a precise location but lacks the ability to create a double-stranded break. The binding of the dCas9 protein to DNA prevents the binding of RNA polymerase to the promoter through steric hindrance thereby reducing expression. We recently published the novel use of this technology in C. albicans and are currently working on expanding this technology to large scale repression of essential genes. Through the construction of an essential gene CRISPRi-sgRNA library, we can begin to study the function of essential genes under different conditions and identify genes that are involved in critical processes such as drug tolerance in antifungal resistant background strains. These genes can ultimately be characterized as putative targets for novel antifungal drug development, or targeted as a means to sensitize drug-resistant strains to antifungal treatment.

Microbial Quality and Aflatoxin Levels of Bread and Flour Products Vended in Akure Metropolis, Ondo State, Nigeria

Aim: This study evaluated the microbial quality characteristics of bread and flour-made products vended for human consummation in Akure metropolis. Methods: The sample products including bread, buns, puff puff, meat pie and cake collected from different locations were analysed using standard microbiological methods to enumerate the bacterial and fungal consortia. Macro and micro-morphological identification of the implicated fungi in the food samples were done via standard techniques. The presence and quantity of some aflatoxin types were also investigated using standard techniques. Results: The fungal organisms enumerated include species of Fusarium, Aspergillus, Cladosporium, Mucor, Sacharomyces cerevisiae, Rhizopus and Penicillium. Bacteria consortium implicated in sample products include; Staphylococcus aureus, Bacillus sp., Escherichia coli, Clostridium sp., Pseudomonas aeruginosa and the likes. The levels of aflatoxin B1 and B2 produced were predominantly associated with Aspergillus flavus enumerated from bread products which serve as a rider to the aflatoxin contamination in vended flour products. Conclusion: The toxicity and potency of aflatoxins make them a primary health hazard and as well accountable for losses associated with contamination of processed foods and ready-to-eat foods. It is recommended that bakers should implement the use of heat-treated flour in the production process of ready-to-eat products for human safety.

Molecular Characterization of Post Harvest Fungus Associated with Spoilt Broccoli

Broccoli (Brassica oleracea var. italica) is a nutritional vegetable that looks like a small tree. Despite the fact that it is extensively loaded with arrays of vitamins, minerals, fiber and antioxidants, it has been observed that it has a short lifespan of not more than 2-5 days due to post-harvest deterioration. A study was conducted to isolate and identify the common fungal pathogens causing post-harvest deterioration of broccoli crown. Diseased broccoli crowns were collected from Ogunabali Fruit Garden Market in D-Line, Port Harcourt Local Government Area of Rivers State. Fungal isolates were collected and morphologically identified. The DNA of the most common fungal isolate, BC-3B was molecularly characterized using Internal Transcribed Spacer 4 and 5 (ITS-4 and 5) molecular markers. The morphological studies revealed that the BC-3B isolate was an Aspergillus niger. The BC-3B isolate DNA sequence was aligned using Basic Local Alignment Search Tool for Nucleotide (BLASTN) 2.8.0 version of National Center for Biotechnology Information (NCBI) database. The molecular weight of the DNA of the isolates was over 600base pairs. Based on sequence similarity, it was observed that the broccoli isolate BC-3B was 93% identical to Aspergillus niger. From the above results, these findings showed that Aspergillus niger is the causal fungal pathogen of post-harvest rot of broccoli. Phylogenetic tree was constructed to access the relationship between the isolates obtained from this study. This study has provided information on some of the fungal organisms found in broccoli. It is anticipated that this result will provide information for disease control approach for alleviating the post-harvest losses of broccoli caused by Aspergillus niger and provide a foundation for further study of possible harm of consuming diseased broccoli.

257. Outcomes in Fungal Prosthetic Joint Infection

Background There is a paucity of data in the literature regarding the most effective treatment and related outcomes of fungal prosthetic joint infection. The majority of reported cases are treated using a two stage revision method in which the initial prosthesis is removed and the patient is treated with systemic antifungal medications and potentially irrigation of the joint cavity with antifungal and antibacterial agents. Alternately, others have suggested a one-stage revision in order to prevent a period of significant functional impairment and potentially improved functional outcomes and lower overall costs. Cases of radical removal of the prosthesis without replacement have been reported. To date, the largest studies of total hip arthroplasty and total knee arthroplasty have included 37 and 45 patients, respectively. Methods A retrospective record review of patients admitted within two health systems between January 1, 2007 and December 31, 2018 with prosthetic joints and a deep culture of the joint positive for fungal organisms was performed. Results Eighteen patients fit criteria. Nine patients had knee replacements and nine patients had hip replacements. The average age at time of infection was 61. Ten patients were female. Average BMI was 32.1. Twelve presented with a painful joint, eight presented with drainage, and one with dehiscence. Average WBC count was 9.3, average ESR was 47, and average CRP was 11.8. All patients were noted with Candida species. Eight patients were treated with two stage revisions, three received one stage revisions as destination therapy. One required amputation. All but one patient was associated with concurrent bacterial infection. Of the twelve patients that had known outcomes, six were noted with cure and six were noted with relapse. All patients that were cured received two stage exchange or girdlestone procedure. Conclusion In our study, fungal prosthetic joint infection was associated with poor outcomes. All of the patients in our study were noted with Candida species, which is in concordance with the known literature. Two stage exchange was associated with better outcomes. Disclosures All Authors: No reported disclosures

Antimicrobial Resistance (AMR) in COVID-19 Patients: A Systematic Review and Meta-Analysis (November 2019 - June 2021)

BackgroundPneumonia from SARS-CoV-2 is difficult to distinguish from other viral and bacterial etiologies. Broad-spectrum antimicrobials are frequently prescribed to patients hospitalized with COVID-19 which potentially acts as a catalyst for the development of antimicrobial resistance (AMR). ObjectivesWe conducted a systematic review and meta-analysis during the first 18 months of the pandemic to quantify the prevalence and types of resistant co-infecting organisms in patients with COVID-19 and explore differences across hospital and geographic settings.MethodsWe searched MEDLINE, Embase, Web of Science (BioSIS), and Scopus from November 1, 2019 to May 28, 2021 to identify relevant articles pertaining to resistant co-infections in patients with laboratory confirmed SARS-CoV-2. Patient- and study-level analyses were conducted. We calculated pooled prevalence estimates of co-infection with resistant bacterial or fungal organisms using random effects models. Stratified meta-analysis by hospital and geographic setting was also performed to elucidate any differences. ResultsOf 1331 articles identified, 38 met inclusion criteria. A total of 1959 unique isolates were identified with 29% (569) resistant organisms identified. Co-infection with resistant bacterial or fungal organisms ranged from 0.2 to 100% among included studies. Pooled prevalence of co-infection with resistant bacterial and fungal organisms was 24% (95% CI: 8-40%; n=25 studies: I2=99%) and 0.3% (95% CI: 0.1-0.6%; n=8 studies: I2=78%), respectively. Among multi-drug resistant organisms, methicillin-resistant Staphylococcus aureus, carbapenem-resistant Acinetobacter baumannii, Klebsiella pneumoniae, Pseudomonas aeruginosa and Candida auris were most commonly reported. Stratified analyses found higher proportions of AMR outside of Europe and in ICU settings, though these results were not statistically significant. Patient-level analysis demonstrated >50% (n=58) mortality, whereby all but 6 patients were infected with a resistant organism. ConclusionsDuring the first 18 months of the pandemic, AMR was moderately prevalent in COVID-19 patients and varied by hospital and geography although there was substantial heterogeneity. Given the variation in patient populations within these studies, clinical settings, practice patterns, and definitions of AMR, further research is warranted to quantify AMR in COVID-19 patients to improve surveillance programs, infection prevention and control practices and antimicrobial stewardship programs globally.

Invasive Fungal Sinusitis: Clinical Pathologic Characteristics of Acute vs. Chronic Infections

Introduction/Objective Invasive fungal sinusitis is a serious condition requiring early diagnosis and treatment. It has been classified into acute, chronic and granulomatous forms. This study aims to investigate clinical pathologic aspects such as a) Frequency of mass forming lesions, b) Frequency of granulomatous reaction, c) Frequency of acute inflammatory reaction and d) Frequency of angioinvasion, perineural invasion and necrosis, to determine if these factors allow a more meaningful clinical-pathologic classification. Methods/Case Report Cases of invasive fungal sinusitis with surgical pathology specimens available in our laboratory since January 1, 2006 to date were gathered. Electronic medical records, histopathologic diagnostic material and laboratory fungal identification results were reviewed. Results (if a Case Study enter NA) Thirty-one cases of invasive fungal sinusitis were found: Twenty-two were acute (< 4wk duration) and 9 chronic. Patient comorbidities in acute cases were malignancies: 45%, diabetes mellitus: 26% and solid organ transplant: 10%. Among patients with malignancies, 5 cases had relapsed/refractory acute myeloid leukemia with neutropenia < 1000/uL. Patients with diabetes mellitus exhibited an average HbA1c of 10.0%. Two out of 3 transplant patients had graft versus host disease. The most common causative fungi were species of Aspergillus, Candida and mucormycetes. By contrast, a third of the chronic cases had a history of recreational drug use and six presented with space occupying lesions seen on imaging studies. Upon histologic review, four of these showed granulomas and the majority of cases exhibited extensive necrosis. Among necrotic cases, perineural and vascular invasion by fungal organisms was identified. Conclusion We report the contrasting clinical pathologic characteristics of acute and chronic invasive fungal sinusitis in a series of cases treated at University affiliated tertiary/quaternary-care Hospitals. Acute invasive sinusitis is usually a complication of severe systemic diseases. Chronic cases are caused by various medical conditions including the use of recreational drug and may mimic neoplasms on imaging.

Bioprospecting Trichoderma: A Systematic Roadmap to Screen Genomes and Natural Products for Biocontrol Applications

Natural products derived from microbes are crucial innovations that would help in reaching sustainability development goals worldwide while achieving bioeconomic growth. Trichoderma species are well-studied model fungal organisms used for their biocontrol properties with great potential to alleviate the use of agrochemicals in agriculture. However, identifying and characterizing effective natural products in novel species or strains as biological control products remains a meticulous process with many known challenges to be navigated. Integration of recent advancements in various “omics” technologies, next generation biodesign, machine learning, and artificial intelligence approaches could greatly advance bioprospecting goals. Herein, we propose a roadmap for assessing the potential impact of already known or newly discovered Trichoderma species for biocontrol applications. By screening publicly available Trichoderma genome sequences, we first highlight the prevalence of putative biosynthetic gene clusters and antimicrobial peptides among genomes as an initial step toward predicting which organisms could increase the diversity of natural products. Next, we discuss high-throughput methods for screening organisms to discover and characterize natural products and how these findings impact both fundamental and applied research fields.

Nutritional changes induced by fungi on cowpea (Vigna unguiculata L. Walp) seeds

Seeds are usually infected by microorganisms and pests during storage, causing deterioration and reduction in the nutritive and market value of these seeds. In this study, the proximate composition of Vigna unguiculata seeds inoculated with different fungal organisms was determined to ascertain the level of deterioration caused by fungi on the seeds. The fungi used in the study were Botryodiplodia theobromae, Fusarium oxysporum, Rhizopus stolonifer and Aspergillus niger. There was a significant increase (p<0.05, 0.008) in the protein content of seeds inoculated with fungi. Fusarium oxysporum (29.45%) caused the highest increase in protein followed by Aspergillus niger (28.14%), Botryodiplodia theobromae (27.85%) and Rhizopus stolonifer (27.50%). The increase could be attributed to the proteineous content of the fungal mycelia. There was a significant increase (p<0.05, 0.005/0.014) in moisture and ash content of inoculated seeds respectively. Fusarium oxysporum caused the highest increase in ash (7.93) while Rhizopus stolonifer (5.4) caused the lowest increase. The increase in ash content is due to the presence of minerals like potassium and phosphorus in the mycelia of the fungi. There was a significant decrease (p<0.05, 0.019) in the carbohydrate, lipid, fibre and dry matter content of fungi-inoculated seeds when compared with the control. Fusarium oxysporum (36.6) caused the highest decrease while Rhizopus stolonifer (43.2) caused the lowest decrease in dry matter of inoculated seeds. Decrease in dry matter may be as a result of production of enzymes by these fungi.

Screening of Phylloplane Fungal Flora of Some Medicinal Plants in Durg- Bhilai Region of Chhattisgarh State

The leaf surface fungal flora is subjected not only to the influence of the host, but also to its own factors. An important aspect is the production of self incubatory products as well as self stimulating products by the fungal organisms present on the leaf surface. Amongst microorganisms fungi can be a sole cause of spoilage and substantially decrease the quality, grade and price of plant materials. In the present studies leaves were found mainly infected with Aspergillus niger, A. favus , Curvularia lunata, Chaetomium globosom, Penicillum citrinum and Fusarium oxysporum as these six were found on the leaves throughout the study and their incidence degree were quite high.