Dung beetle-associated yeasts display multiple stress tolerance: a desirable trait of potential industrial strains.

BACKGROUND: Stress-tolerant yeasts are highly desirable for cost-effective bioprocessing. Several strategies have been documented to develop robust yeasts, such as genetic and metabolic engineering, artificial selection, and natural selection strategies, among others. However, the significant drawbacks of such techniques have motivated the exploration of naturally occurring stress-tolerant yeasts. We previously explored the biodiversity of non-conventional dung beetle-associated yeasts from extremophilic and pristine environments in Botswana (Nwaefuna AE et.al., Yeast, 2023). Here, we assessed their tolerance to industrially relevant stressors individually, such as elevated concentrations of osmolytes, organic acids, ethanol, and oxidizing agents, as well as elevated temperatures. RESULTS: Our findings suggest that these dung beetle-associated yeasts tolerate various stresses comparable to those of the robust bioethanol yeast strain, Saccharomyces cerevisiae (Ethanol Red™). Fifty-six percent of the yeast isolates were tolerant of temperatures up to 42 °C, 12.4% of them could tolerate ethanol concentrations up to 9% (v/v), 43.2% of them were tolerant to formic acid concentrations up to 20 mM, 22.7% were tolerant to acetic acid concentrations up to 45 mM, 34.0% of them could tolerate hydrogen peroxide up to 7 mM, and 44.3% of the yeasts could tolerate osmotic stress up to 1.5 M. CONCLUSION: The ability to tolerate multiple stresses is a desirable trait in the selection of novel production strains for diverse biotechnological applications, such as bioethanol production. Our study shows that the exploration of natural diversity in the search for stress-tolerant yeasts is an appealing approach for the development of robust yeasts.

Prevalence, molecular and phenotypic profiles of arboreal associated Cryptococcus neoformans in Botswana.

Mopane tree (Colophospermum mopane) is one of the main ecological niches of Cryptococcus neoformans, an opportunistic fungal pathogen that causes cryptococcosis primarily on immunocompromised hosts after inhalation of basidiospores from the environment. Hence, we investigated the prevalence, and phenotypically (antifungal resistance and biofilm formation capacity) and genotypically (mating type and genetic structure) characterized C. neoformans isolated from C. mopane, Acacia tortilis, Adansonia digitata and Ziziphus mucronata in Botswana. We report 7.1% and 2.9% prevalence of C. neoformans in C. mopane and other trees, respectively. All tested C. neoformans isolates were determined to be non-WT to fluconazole. Most isolates (65%) of C. neoformans isolates were biofilm producers. Mating type determination revealed a higher proportion of the globally rare MATa allele (53%) and a single MATα/MATa hybrid. The observed genotypeswere VNI (71%), VNB (23%) and VNB/VNB hybrids (6%). Native trees other than C. mopane are alternative ecological niches of antifungal resistant C. neoformans, and this represents a serious public health concern,and this represents a serious public health concern, especially for high-risk populations. Prevalence of C. neoformans on native trees and the observed emergence of hybrids (evidence of sexual recombination) highlight the need for increased surveillance and risk assessment within a One Health paradigm.

Determining Potential Link between Environmental and Clinical Isolates of Cryptococcus neoformans/Cryptococcus gattii Species Complexes Using Phenotypic and Genotypic Characterisation.

Opportunistic infections due to Cryptococcus neoformans and C. gattii species complexes continue to rise unabated among HIV/AIDS patients, despite improved antifungal therapies. Here, we collected a total of 20 environmental and 25 presumptive clinical cryptococcal isolates from cerebrospinal fluid (CSF) samples of 175 patients enrolled in an ongoing clinical trial Ambition 1 Project (Botswana-Harvard Partnership). Identity confirmation of the isolates was done using MALDI-TOF MS and PCR. We describe the diversity of the isolates by PCR fingerprinting and sequencing (Oxford Nanopore Technology) of the intergenic spacer region. Mating types of the isolates were determined by amplification of the MAT locus. We report an unusual prevalence of 42.1% of C. neoformans x C. deneoformans hybrids Serotype AD (n = 16), followed by 39.5% of C. neoformans Serotype A (n = 15), 5.3% of C. deneoformans, Serotype D (n = 2), 7.9% of C. gattii (n = 3), and 5.3% of C. tetragattii (n = 2) in 38 representative isolates that have been characterized. Mating type-specific PCR performed on 38 representative environmental and clinical isolates revealed that 16 (42.1%) were MATa/MATα hybrids, 17 (44.7%) were MATα, and five (13.2%) possessed MATa mating type. We used conventional and NGS platforms to demonstrate a potential link between environmental and clinical isolates and lay a foundation to further describe mating patterns/history in Botswana.

Phenotypic and genotypic profiling reveals a high prevalence of methicillin-resistant Staphylococcus aureus isolated from hospitals, houseflies and adjacent informal food retailers in Botswana.

The increasing occurrence of methicillin-resistant Staphylococcus aureus (MRSA) in the environment, food and healthcare systems is a global public health concern. MRSA is reported to cause food poisoning, osteomyelitis and pyogenic infections of the skin, and consequently has been categorized as a high-priority pathogen by the World Health Organization. Here, we determined the presence of MRSA in clinical (n=56), food (n=150) and housefly samples (n=970) collected from two hospitals in Botswana. Characterization based on phenotypic (antimicrobial resistance, biofilm production) and genotypic (antimicrobial resistance genes and integrons) profiles were performed on all isolates. Of the total samples tested, 64 were positive for MRSA following conventional culture methods and PCR amplification of the mecA and mecC genes for confirmation of presumptive MRSA isolates. The confirmed isolates included 71 % (95 % CI 83.2-59.6) from clinical, 9 % (95 % CI 14--4.8) from food, and 1 % (95 % CI 1.6-0.4) collected from housefly samples. In total 89 % (n=57) isolates in the current study showed a multidrug resistance phenotype, among these, resistance to ß-lactams and glycoside antibiotic classes were predominant. Genotypic characterization showed the domination of the blatem gene (95 %) followed by fox (63 %) and tetO (19 %) whilst vanA was only reported in 13 % of the isolates. Integrons were detected in 50 % (32/64) of the total MRSA isolates, and we report a high prevalence of etd gene, detected in 67 % (43/64) of the isolates followed by eta 38 % (24/64) whilst tsst-1 (3%) was the least detected genetic determinant. The genes etb and PVL were not detected in a ll the tested MRSA isolates. We provide the first report on the prevalence of MRSA isolated from the clinical-food-vector nexus harbouring biofilm and blatem genes, and antibiotic resistance profiles in Botswana. These results are significant for risk-assessment analysis and the development of improved MRSA infection prevention and control strategies.

Biosurfactant production by halophilic yeasts isolated from extreme environments in Botswana.

Nine morphologically distinct halophilic yeasts were isolated from Makgadikgadi and Sua pans, as pristine and extreme environments in Botswana. Screening for biosurfactant production showed that Rhodotorula mucilaginosa SP6 and Debaryomyces hansenii MK9 exhibited the highest biosurfactant activity using Xanthocercis zambesiaca seed powder as a novel and alternative inexpensive carbon substrate. Chemical characterization of the purified biosurfactants by Fourier Transform Infra-Red spectroscopy suggested that the biosurfactant from R. mucilaginosa SP6 was a rhamnolipid-type whereas the biosurfactant from D. hansenii MK9 was a sophorolipid-type. The two biosurfactants exhibited antimicrobial activities against eight pathogenic bacteria and fungal strains (Proteus vulgaris, Escherichia coli, Klebsiella pneumoniae, Staphylococcus aureus, Micrococcus luteus, Cryptococcus neoformans, Candida albicans and Aspergilus niger). The sophorolopid-type biosurfactant was found to be the most potent among the antimicrobial drug resistant strains tested. The findings open up prospects for the development of environmentally friendly antimicrobial drugs that use an inexpensive source of carbon to reduce the costs associated with the production of biosurfactants.

Antioxidant and antimicrobial studies on fused-ring pyrazolones and isoxazolones.

A series of 3-nitrochalcones have been synthesized enroute towards fused ring pyrazolones and isoxazolones. Base catalyzed condensation of the chalcones with ethylacetoacetate yielded cyclohexenones in good yields (74-76%). The treatment of cyclohexenones with hydrazine hydrate or hydroxylamine chloride in the presence of a base afforded the corresponding fused-ring pyrazolinones (70-78% yield) and isoxazolinones (58-66% yield). The newly synthesized compounds were characterized by IR, 1D and 2D NMR and HRMS spectral analysis. The compounds were screened for their antioxidant and antimicrobial activities. Pyrazolinones showed good DPPH radical scavenging and iron metal chelating properties. The para-hydroxy group was important for a compound to have enhanced antioxidant activity. Pyrazolinones and isoxazolinone exhibited a wider range of antimicrobial activities compared to cyclohexenones. Pyrazolinones and isoxazolinone bearing a thiophene ring were the most potent type of compounds against Bacillus subtilis and Candida albicans with MIC values of 0.313-1.25 µg/mL. Some of the synthesized compounds were found to have promising antioxidant, metal chelation and antimicrobial activities.

Enterotoxigenic and antibiotic resistance determination of Staphylococcus aureus strains isolated from food handlers in Gaborone, Botswana.

The prevalence, antibiotic resistance, and enterotoxigenic potential of Staphylococcus aureus strains from different anatomical sites on food handlers in Gaborone, Botswana, were determined. Of a total of 200 food handlers tested, 115 (57.5%) were positive for S. aureus. Of the 204 S. aureus isolates, 63 (30.9%), 91 (44.6%), and 50 (24.5%) were isolated from the hand, nasal cavity, and face, respectively, and 43 (21%) of the isolates were enterotoxigenic. The most prevalent enterotoxin was type A, which accounted for 34.9% of all the enterotoxigenic strains, and enterotoxin D was produced by the fewest number of strains (9.3%). Resistance to methicillin was encountered in 33 (22.4%) of the penicillin G-resistant isolates, and 9 (27.3%) of these methicillin-resistant isolates also were resistant to vancomycin. Nineteen antibiotic resistance profiles were determined, and the nasal cavity had the highest diversity of resistance profiles. The nasal cavity also had the highest number of resistant strains, 77 (53%), whereas the hand and face had 49 (32%) and 24 (16.0%) resistant strains, respectively. To reduce the Staphylococcus carriage rate among food handlers, training coupled with a commitment to high standards of personal and environmental hygiene is recommended.