In Vitro Investigation of the Impact of Bacterial-Fungal Interaction on Carbapenem-Resistant Klebsiella pneumoniae.

Fungal-bacterial co-culturing is a potential technique for the production of secondary metabolites with antibacterial activity. Twenty-nine fungal species were screened in a co-culture with carbapenem-resistant Klebsiella pneumoniae at different temperatures. A temperature of 37 ° showed inhibition of bacterial growth. Antimicrobial susceptibility testing for K. pneumoniae was conducted to compare antibiotic resistance patterns before and after the co-culture. Genotypic comparison of the K. pneumonia was performed using next generation sequencing (NGS). It was shown that two out of five K. pneumoniae, with sequence type ST 101 isolates, lost bla-OXA48, bla-CTX-M-14, tir, strA and strB genes after the co-culture with Scopulariopsis brevicaulis fungus. The other three isolates (ST 383 and 147) were inhibited in the co-culture but did not show any changes in resistance. The total ethyl acetate extract of the fungal-bacterial co-culture was tested against K. pneumoniae using a disc diffusion method. The concentration of the crude extract was 0.97 mg/µL which resulted in total inhibition of the bacteria. Using chromatographic techniques, the purified compounds were identified as 11-octadecenoic acid, 2,4-Di-tert-butylphenol, 2,3-Butanediol and 9-octadecenamide. These were tested against K. pneumoniae using the well diffusion method at a concentration of 85 µg/µL which resulted in total inhibition of bacteria. The co-culture results indicated that bacteria under chemical stress showed variable responses and induced fungal secondary metabolites with antibacterial activities.

Petroleum Hydrocarbon Bioremediation Using Native Fungal Isolates and Consortia.

Crude oil spills as a result of natural disasters or extraction and transportation operations are common nowadays. Oil spills have adverse effects on both aquatic and terrestrial ecosystems and pose a threat to human health. This study have been concerned with studying the capability of six fungal species (Curvularia brachyspora, Penicillium chrysogenum, Scopulariopsis brevicaulis, Cladosporium sphaerospermum, Alternaria alternata, and Stemphylium botryosum) and three fungal consortia (FC), FC1 (P. chrysogenum and C. brachyspora), FC2 (S. brevicaulis and S. botryosum), and FC3 (S. brevicaulis, S. botryosum, and C. sphaerospermum), to remediate petroleum hydrocarbons (PHs). Qualitative and quantitative changes in polyaromatic hydrocarbons (PAHs) and saturated hydrocarbons (SH) mixtures and the patterns of PHs degradation have been examined using HPLC and GC. Studying the GC chromatogram of C. sphaerospermum revealed severe degradation of SHs exhibited by this species, and the normal-paraffin and isoparaffin degradation percentage have been valued 97.19% and 98.88%, respectively. A. alternata has shown the highest significant (at P ˂ 0.05) PAH degradation percent reaching 72.07%; followed by P. chrysogenum, 59.51%. HPLC data have revealed that high-molecular-weight PAH percent/total PAHs decreased significantly from 98.94% in control samples to 68.78% in samples treated with A. alternata. FC1 and FC2 consortia have exhibited the highest significant PH deterioration abilities than did the individual isolates, indicating that these fungal consortia exhibited positive synergistic effects. The study supports the critical idea of the potential PAH and SH biodegradation as a more ecologically acceptable alternative to their chemical degradation.

Insights into Asparaginase from Endophytic Fungus Lasiodiplodia theobromae: Purification, Characterization and Antileukemic Activity.

Endobiotic fungi are considered as a reservoir of numerous active metabolites. Asparaginase is used as an antileukemic drug specially to treat acute lymphoblastic leukaemia. The presented study aims to optimize the media conditions, purify, characterize, and test the antileukemic activity of the asparaginase induced from Lasiodiplodia theobromae. The culture medium was optimized using an experiment designed by The Taguchi model with an activity ranging from 10 to 175 IU/mL. Asparaginase was induced with an activity of 315 IU/mL. Asparaginase was purified with a specific activity of 468.03 U/mg and total activity of 84.4 IU/mL. The purified asparaginase showed an approximate size of 70 kDa. The purified asparaginase showed an optimum temperature of 37 °C and an optimum pH of 6. SDS reduced the activity of asparaginase to 0.65 U/mL while the used ionic surfactants enhanced the enzyme activity up to 151.92 IU/mL. The purified asparaginase showed a Km of 9.37 µM and Vmax of 127.00 µM/mL/min. The purified asparaginase showed an IC50 of 35.2 ± 0.7 IU/mL with leukemic M-NFS-60 cell lines and CC50 of 79.4 ± 1.9 IU/mL with the normal WI-38 cell line. The presented study suggests the use of endophytic fungi as a sustainable source for metabolites such as asparaginase, provides an opportunity to develop a facile, eco-friendly, cost-effective, and rapid synthesis of antileukemic drugs, which have the potential to be used as alternative and reliable sources for potent anticancer agents.

Antimicrobial and Immunomodulatory Effect of Gum Arabic on Human and Bovine Granulocytes Against Staphylococcus aureus and Escherichia coli.

Gum arabic (GA) is a traditional herbal medicine from Acacia Senegal (L.) Willdenow trees, which consist of a complex mixture of polysaccharides and glycoproteins. It is used in daily applications for several diseases and is considered to protect against bacterial infections. The detailed mechanisms behind these observations are still unclear. In this study, we investigated the direct antibacterial activity of GA water and ethanol extracts against Staphylococcus (S.) aureus or Escherichia (E.) coli and the immunomodulating properties of those extracts on granulocytes as a first line of defense against bacteria. Firstly, the direct antimicrobial effect of GA was tested on three different S. aureus strains and two E. coli strains. The growth of bacteria was analyzed in the presence of different GA concentrations over time. GA water as well as ethanol extracts showed a significant growth inhibition in a concentration-dependent manner in the case of S. aureus Newman, S. aureus Rd5, and E. coli 25922, but not in the case of S. aureus USA300 and E. coli K1. Transmission electron microscopic analysis confirmed an antibacterial effect of GA on the bacteria. Secondly, the immunomodulatory effect of GA on the antimicrobial activity of bovine or human blood-derived granulocytes was evaluated. Interestingly, water and ethanol extracts enhanced antimicrobial activity of granulocytes by the induction of intracellular ROS production. In line with these data, GA increased the phagocytosis rate of E. coli. No effect was seen on neutrophil extracellular trap (NET) formation that mediates killing of extracellular bacteria such as S. aureus. In conclusion, we show that GA exhibits a direct antibacterial effect against some S. aureus and E. coli strains. Furthermore, GA boosts the antimicrobial activities of granulocytes and increases intracellular ROS production, which may lead to more phagocytosis and intracellular killing. These data might explain the described putative antimicrobial activity of GA used in traditional medicine.

In Vitro Testing of Crude Natural Plant Extracts from Costa Rica for Their Ability to Boost Innate Immune Cells against Staphylococcus aureus.

The increasing occurrence of antibiotic-resistant Staphylococcus (S.) aureus tremendously limits the antibiotic-based treatment options; therefore, an open discussion of alternative treatment strategies is urgently needed. The use of naturally derived materials might become a more promising concept, not only as directly acting antimicrobials, but also for stimulation of the immune system. Costa Rican plant extracts were screened for their ability to enhance the antimicrobial activity of human blood-derived cells against S. aureus infections. We identified three plant extracts which significantly reduced the growth of S. aureus in the presence of human blood without directly acting as antibacterials: Byrsonima crassifolia acetone bark extract, Mandevilla veraguasensis acetone vine extract and Verbesina oerstediana acetone bark extract (VEOEBA). The effect of VEOEBA was studied in more detail, and revealed that VEOEBA increases the antimicrobial activity of neutrophils by enhancing the formation of neutrophil extracellular traps.