Antifungal Effect of Triclosan On Aspergillus Fumigatus: Quorum Quenching Role As A Single Agent And Synergy With Liposomal Amphotericin-B

The purpose of this research was to determine Aspergillus fumigatus conidial viability and its biofilm formation upon treatment with triclosan and amphotericin-B loaded liposomes. A. fumigatus was treated with the antimicrobials, triclosan and liposomal amphotericin-B (L-AMB), in single and combined supplementation. To quantify the cells’ viability upon treatments, resazurin-based viability assay was performed. Confocal laser scanning microscopy was done by applying FUN-1 stain to screen the role of the agents on extracellular polymeric substances. Total A. fumigatus biomass upon treatments was estimated by using crystal violet-based assay. To study the agents’ effect on the conidial viability, flow cytometry analysis was performed. Expression levels of A. fumigatus genes encoding cell wall proteins, α-(1,3)-glucans and galactosaminogalactan were analysed by real-time polymerase chain reaction assay. A synergistic interaction occurred between triclosan and L-AMB when they were added sequentially (triclosan+L-AMB) at their sub-minimum inhibitory concentrations, the triclosan and L-AMB MICs were dropped to 0.6 and 0.2 mg/L, respectively, from 2 and 1 mg/L. Besides, L-AMB and triclosan contributed to the down-regulation of α-(1,3)-glucan and galactosaminogalactan in A. fumigatus conidia and resulted in less conidia aggregation and mycelia adhesion to the biotic/abiotic surfaces; A. fumigatus conidia -became hydrophilic upon treatment, as a result of rodlet layer being masked by a hydrophilic layer or modified by the ionic strength of the rodlet layer. In A. fumigatus, the potential mechanisms of action for L-AMB might be through killing the cells and for triclosan through interrupting the cells’ development as a consequence of quorum quenching.

Evaluation of indigenous fungal entomopathogens and aqueous leaf extract of Annona muricata against Polyphagotarsonemus latus infesting Jatropha curcas in Indonesia

Puspitarini RD, Afandhi A, Fernando I. 2021. Evaluation of indigenous fungal entomopathogens and aqueous leaf extract of Annona muricata against Polyphagotarsonemus latus infesting Jatropha curcas in Indonesia. Biodiversitas 22: 2648-2655. The broad mite Polyphagotarsonemus latus (Banks) (Acari: Tarsonemidae) has been known to cause a great deal of injury on Jatropha curcas (L.) plantation in Indonesia. Isolation of indigenous fungal entomopathogens from rhizosphere soils, followed by pathogenicity assay was conducted to find an effective isolate for controlling P. latus. Additionally, the potential combination of the selected isolate and soursop (Annona muricata L.) aqueous leaf extract (SLE) was investigated. A total of 24 isolates were obtained by using the insect bait method. Four fungal isolates, namely Paecilomyces sp. 1., Lecanicillium sp., Beauveria sp., and Fusarium sp. 1, showed high conidial viability and were chosen as representatives to assess their pathogenicity against P. latus. Among the tested isolates, Beauveria sp. which had the highest conidial viability among the tested isolates, needed a shorter period to completely kill the tested mites. However, the compatibility test revealed the deleterious effect of SLE on Beauveria sp. SLE at all concentrations showed a very toxic effect on the fungus, therefore the two must be applied separately. Our results provide useful information on the effectiveness of indigenous entomopathogenic fungi Beauveria sp. and aqueous leaf extract of A. muricata as an alternative tool to control the broad mite P. latus on the J. curcas plantation.

Comparative eco-physiology revealed extensive enzymatic curtailment, lipases production and strong conidial resilience of the bat pathogenic fungus Pseudogymnoascus destructans

The genus Pseudogymnoascus encompasses soil psychrophilic fungi living also in caves. Some are opportunistic pathogens; nevertheless, they do not cause outbreaks. Pseudogymnoascus destructans is the causative agent of the white-nose syndrome, which is decimating cave-hibernating bats. We used comparative eco-physiology to contrast the enzymatic potential and conidial resilience of P. destructans with that of phylogenetically diverse cave fungi, including Pseudogymnoascus spp., dermatophytes and outdoor saprotrophs. Enzymatic potential was assessed by Biolog MicroArray and by growth on labelled substrates and conidial viability was detected by flow cytometry. Pseudogymnoascusdestructans was specific by extensive losses of metabolic variability and by ability of lipid degradation. We suppose that lipases are important enzymes allowing fungal hyphae to digest and invade the skin. Pseudogymnoascus destructans prefers nitrogenous substrates occurring in bat skin and lipids. Additionally, P. destructans alkalizes growth medium, which points to another possible virulence mechanism. Temperature above 30 °C substantially decreases conidial viability of cave fungi including P. destructans. Nevertheless, survival of P. destructans conidia prolongs by the temperature regime simulating beginning of the flight season, what suggests that conidia could persist on the body surface of bats and contribute to disease spreading during bats active season.

KERAPATAN DAN VIABILITAS KONIDIA BEAUVERIA BASSIANA DAN METARHIZIUM ANISOPLIAE PADA MEDIA IN VITRO PH RENDAH

Conidial density and viability of Beauveria bassiana and Metarhizium anisopliae grown on the low-pH in vitro medium. Liquid bioinsecticide with active ingredient from conidial entomopathogenic fungus has major constraints, namely short shelf life due to declining conidial viability and density is caused by low pH in the bioinsecticide carrier. This experiment aimed to measure the loss of conidial viability and density of Beauveria bassiana and Metarhizium anisopliae isolates grown on in vitro medium with low pH. Entomopathogenic fungus isolates were used as much as 28 isolates grown on in vitro medium at low pH, namely pH 5, 4, 3, and 2. The results showed that the fungus isolate that had the highest conidial density on in vitro medium at pH 5 was found on isolates of B. bassiana with code BPcMs (2.583 x 109 conidia mL-1), while the lowest one was found on isolates of B. bassiana with code of BWS Pantura (0.825x109 conidia mL-1). All isolate conidial density from in vitro medium with pH 2 decreased regularly. Conidial density of BPcMs isolate decreased to 2.483 x 109conidia mL-1, as well as BWS Pantura isolate also decreased to 0.425x109 conidia mL-1. The highest conidial viability at pH 5 was found on isolates of B. bassiana with code of BPcMs (51.572%), while the lowest conidial viability was found on isolate of B. bassiana with BTmPc code (15.040%). At pH 2, almost isolates tested had low conidial viability. The conidial viability of isolates BPcMs decreased to 47.037%%, while the isolates BTmPc also decreased to 12.778%. Therefore, the lower of the pH of the in vitro medium was, the lower of conidial viability and density of B. bassiana and M. anisopliae was

Flow Cytometry Is a Powerful Tool for Assessment of the Viability of Fungal Conidia in Metalworking Fluids

ABSTRACT Fungal contamination of metalworking fluids (MWF) is a dual problem in automated processing plants because resulting fungal biofilms obstruct cutting, drilling, and polishing machines. Moreover, some fungal species of MWF comprise pathogens such as Fusarium solani. Therefore, the development of an accurate analytical tool to evaluate conidial viability in MWF is important. We developed a flow cytometric method to measure fungal viability in MWF using F. solani as the model organism. To validate this method, viable and dead conidia were mixed in several proportions and flow was cytometrically analyzed. Subsequently, we assessed the fungicidal activity of two commercial MWF using flow cytometry (FCM) and compared it with microscopic analyses and plating experiments. We evaluated the fungal growth in both MWF after 7 days using quantitative PCR (qPCR) to assess the predictive value of FCM. Our results showed that FCM distinguishes live from dead conidia as early as 5 h after exposure to MWF, whereas the microscopic germination approach detected conidial viability much later and less accurately. At 24 h, microscopic analyses of germinating conidia and live/dead analyses by FCM correlated well, although the former consistently underestimated the proportion of viable conidia. In addition, the reproducibility and sensitivity of the flow cytometric method were high and allowed assessment of the fungicidal properties of two commercial MWF. Importantly, the obtained flow cytometric results on viability of F. solani conidia at both early time points (5 h and 24 h) correlated well with fungal biomass measurements assessed via a qPCR methodology 7 days after the start of the experiment. IMPORTANCE This result shows the predictive power of flow cytometry (FCM) in assessing the fungicidal capacity of MWF formulations. It also implies that FCM can be implemented as a rapid detection tool to estimate the viable fungal load in an industrial processing matrix (MWF).

Distinct Roles of Myosins in Aspergillus fumigatus Hyphal Growth and Pathogenesis

Myosins are a family of actin-based motor proteins found in many organisms and are categorized into classes based on their structures. Class II and V myosins are known to be important for critical cellular processes, including cytokinesis, endocytosis, exocytosis, and organelle trafficking, in the model fungiSaccharomyces cerevisiaeandAspergillus nidulans. However, the roles of myosins in the growth and virulence of the pathogenAspergillus fumigatusare unknown. We constructed single- and double-deletion strains of the class II and class V myosins inA. fumigatusand found that while the class II myosin (myoB) is dispensable for growth, the class V myosin (myoE) is required for proper hyphal extension; deletion ofmyoEresulted in hyperbranching and loss of hyphal polarity. BothmyoBandmyoEare necessary for proper septation, conidiation, and conidial germination, but onlymyoBis required for conidial viability. Infection with the ΔmyoEstrain in the invertebrateGalleria mellonellamodel and also in a persistently immunosuppressed murine model of invasive aspergillosis resulted in hypovirulence, while analysis of bronchoalveolar lavage fluid revealed that tumor necrosis factor alpha (TNF-α) release and cellular infiltration were similar compared to those of the wild-type strain. The ΔmyoEstrain showed fungal growth in the murine lung, while the ΔmyoBstrain exhibited little fungal burden, most likely due to the reduced conidial viability. These results show, for the first time, the important role these cytoskeletal components play in the growth of and disease caused by a known pathogen, prompting future studies to understand their regulation and potential targeting for novel antifungal therapies.

A putative methyltransferase, mtrA, contributes to development, spore viability, protein secretion and virulence in the entomopathogenic fungus Beauveria bassiana

The filamentous fungus, Beauveria bassiana, is a ubiquitously distributed insect pathogen, currently used as an alternative to chemical pesticides for pest control. Conidiospores are the means by which the fungus disseminates in the environment, and these cells also represent the infectious agent most commonly used in field applications. Little, however, is known concerning the molecular basis for maintenance of spore viability, a critical feature for survival and persistence. Here, we report on the role of a putative methyltransferase, BbmtrA, in conidial viability, normal fungal growth and development, and virulence, via characterization of a targeted gene knockout strain. Loss of BbmtrA resulted in pleiotropic effects including reduced germination, growth and conidiation, with growing mycelia displaying greater branching than the WT parent. Conidial viability dramatically decreased over time, with <5 % of the cells remaining viable after 30 days as compared with >80 % of the WT. Reduced production of extracellular proteins was also observed for the ΔBbmtrA mutant, including protease/peptidases, glycoside hydrolases and the hyd1 hydrophobin. The latter was further confirmed by hyd1 gene expression analysis. Insect bioassays using the greater wax moth, Galleria mellonella, further revealed that the ΔBbmtrA strain was attenuated in virulence and failed to sporulate on host cadavers. These data support a global role for mtrA in fungal physiological processes.