Effect of the variation in the extracellular concentration of l-arginine in the physiology of Leishmania (Viannia) braziliensis and its susceptibility to some antileishmanial drugs.

The knowledge about amino acid metabolism in trypanosomatids is a valuable source of new therapeutic targets. l-arginine is an essential amino acid for Leishmania parasites, and it participates in the synthesis of polyamines, a group of essential nutrients used for nucleic acids, proteins biosynthesis, and redox modulation necessary for proliferation. In the present study, we evaluated the effect of changes in the availability of this amino acid on promastigotes and intracellular amastigotes on U937 macrophages and showed that the absence of l-arginine in culture medium negatively influences the growth and infectivity of Leishmania (Viannia) braziliensis, causing a decrease in the percentage of the infected cells and parasite load tested through light microscopy. In addition, the absence of l-arginine resulted in the parasite's inability to regulate its reactive oxygen species (ROS) production, which persisted for up to 24 h by flow cytometry following the probe H2DCF-DA dye. Moreover, the differentiation of promastigote to amastigote in axenic culture was more significant at low concentrations of l-arginine suggesting that this depletion induces a stress environment to increase this transformation under axenic conditions. No association was established between the availability of l-arginine and the effectiveness of antileishmanial drugs. All these results confirm the importance of l-arginine in L. braziliensis life cycle vital processes, such as its replication and infectivity, as documented in other Leishmania species. Based on these results, we proposed that the l-arginine uptake/metabolism route is possible in exploring new antileishmanial drugs.

Non-destructive quantification of anaerobic gut fungi and methanogens in co-culture reveals increased fungal growth rate and changes in metabolic flux relative to mono-culture.

BACKGROUND: Quantification of individual species in microbial co-cultures and consortia is critical to understanding and designing communities with prescribed functions. However, it is difficult to physically separate species or measure species-specific attributes in most multi-species systems. Anaerobic gut fungi (AGF) (Neocallimastigomycetes) are native to the rumen of large herbivores, where they exist as minority members among a wealth of prokaryotes. AGF have significant biotechnological potential owing to their diverse repertoire of potent lignocellulose-degrading carbohydrate-active enzymes (CAZymes), which indirectly bolsters activity of other rumen microbes through metabolic exchange. While decades of literature suggest that polysaccharide degradation and AGF growth are accelerated in co-culture with prokaryotes, particularly methanogens, methods have not been available to measure concentrations of individual species in co-culture. New methods to disentangle the contributions of AGF and rumen prokaryotes are sorely needed to calculate AGF growth rates and metabolic fluxes to prove this hypothesis and understand its causality for predictable co-culture design. RESULTS: We present a simple, microplate-based method to measure AGF and methanogen concentrations in co-culture based on fluorescence and absorbance spectroscopies. Using samples of < 2% of the co-culture volume, we demonstrate significant increases in AGF growth rate and xylan and glucose degradation rates in co-culture with methanogens relative to mono-culture. Further, we calculate significant differences in AGF metabolic fluxes in co-culture relative to mono-culture, namely increased flux through the energy-generating hydrogenosome organelle. While calculated fluxes highlight uncertainties in AGF primary metabolism that preclude definitive explanations for this shift, our method will enable steady-state fluxomic experiments to probe AGF metabolism in greater detail. CONCLUSIONS: The method we present to measure AGF and methanogen concentrations enables direct growth measurements and calculation of metabolic fluxes in co-culture. These metrics are critical to develop a quantitative understanding of interwoven rumen metabolism, as well as the impact of co-culture on polysaccharide degradation and metabolite production. The framework presented here can inspire new methods to probe systems beyond AGF and methanogens. Simple modifications to the method will likely extend its utility to co-cultures with more than two organisms or those grown on solid substrates to facilitate the design and deployment of microbial communities for bioproduction and beyond.

Synthesis and Biological Evaluation of a Novel C8-Pyrrolobenzodiazepine (PBD) Adenosine Conjugate. A Study on the Role of the PBD Ring in the Biological Activity of PBD-Conjugates.

Here we sought to evaluate the contribution of the PBD unit to the biological activity of PBD-conjugates and, to this end, an adenosine nucleoside was attached to the PBD A-ring C8 position. A convergent approach was successfully adopted for the synthesis of a novel C8-linked pyrrolo(2,1-c)(1,4)benzodiazepine(PBD)-adenosine(ADN) hybrid. The PBD and adenosine (ADN) moieties were synthesized separately and then linked through a pentynyl linker. To our knowledge, this is the first report of a PBD connected to a nucleoside. Surprisingly, the compound showed no cytotoxicity against murine cells and was inactive against Mycobacterium aurum and M. bovis strains and did not bind to guanine-containing DNA sequences, as shown by DNase I footprinting experiments. Molecular dynamics simulations revealed that the PBD-ADN conjugate was poorly accommodated in the DNA minor groove of two DNA sequences containing the AGA-PBD binding motif, with the adenosine moiety of the ligand preventing the covalent binding of the PBD unit to the guanine amino group of the DNA duplex. These interesting findings shed further light on the ability of the substituents attached at the C8 position of PBDs to affect and modulate the biological and biophysical properties of PBD hybrids.

Culturing Periprosthetic Joint Infection: Number of Samples, Growth Duration, and Organisms.

BACKGROUND: Owing to the difficulty isolating microorganisms in periprosthetic joint infection (PJI), current guidelines recommend that 3-5 intraoperative samples be cultured and maintained for 3-14 days. We investigated (1) the optimal number of culture samples and growth duration to diagnose PJI and (2) the microbiology profile at our institution. METHODS: A retrospective review of 711 patients (329 hips, 382 knees) with PJI that met Musculoskeletal Infection Society criteria from 2000 to 2014 was performed. Two thousand two hundred ninety aerobic and anaerobic cultures were analyzed. A manual chart review collected demographic, surgical, and microbiological data. Microbiology profiles were trended. Logistic regression analysis was performed to determine statistical significance. RESULTS: Obtaining 5 samples provided the greatest yield positive cultures for diagnosing PJI. The percentage of positive cultures overall was 62.6% and stratified by organism type: antibiotic resistant (80.0%), Staphylococcus aureus (76.0%), gram negative (58.9%), Pseudomonas (52.0%), variant PJI organisms (28.2%), Propionibacterium acnes (20.0%), and Escherichia coli (8.0%). Although most organisms were cultured in 5 days or less, 10.8 days were needed for Propionibacterium acnes, 6.6 for variant PJI organisms, and 5.2 for coagulase-negative Staphylococcus. At 3 days, only 42.2% of cultures turned positive compared with 95.0% at 8 days. There was a significant decrease in time in gram-positive PJIs and an increase in culture-negative PJIs. CONCLUSION: The optimal number of cultures and growth duration depended on the type of organism. This study provides evidence that 5 samples should be obtained and held for at least 8 days given that the type of organisms is likely to be unknown at the time of surgery.

Recent Advances in Diagnosis and Treatment Approaches in Fungal Keratitis: A Narrative Review.

Fungal keratitis represents a potentially sight-threatening infection associated with poor prognosis, as well as financial burden. Novel diagnostic methods include polymerase-chain-reaction (PCR)-based approaches, metagenomic deep sequences, in vivo confocal microscopy, and antifungal susceptibility testing. The ideal therapeutic approaches and outcomes have been widely discussed in recent times, with early therapy being of the utmost importance for the preservation of visual acuity, minimizing corneal damage and reducing the scar size. However, combination therapy can be more efficacious compared to monotherapy. Understanding the pathogenesis, early diagnosis, and prevention strategies can be of great importance. In this narrative, we discuss the recent progress that may aid our understanding of the diagnosis, treatment, and prevention of mycotic keratitis.

The Effect of Colored and White Light on Growth and Phycobiliproteins, Chlorophyll and Carotenoids Content of the Marine Cyanobacteria Phormidium sp. and Cyanothece sp. in Batch Cultures.

Two local marine cyanobacteria, Phormidium sp. and Cyanothece sp., were batch-cultured under 18-19.5 °C, at 40 ppt salinity, using white LED light of low (40 µmol photons/m2/s) and high (160 µmol/m2/s) intensity and, additionally, blue, green and red LED light. Yield was highest in high white light in both species (2.15 g dw/L in Phormidium, 1.47 g/L in Cyanothece), followed by green light (1.25 g/L) in Cyanothece and low white and green (1.26-1.33 g/L) in Phormidium. Green light maximized phycocyanin in Phormidium (0.45 mg/mL), while phycoerythrin was enhanced (0.17 mg/mL) by blue light and allophycocyanin by all colors (~0.80 mg/mL). All colors maximized phycocyanin in Cyanothece (~0.32 mg/mL), while phycoerythrin and allophycocyanin peaked under green light (~0.138 and 0.38 mg/mL, respectively). In Phormidium, maximization of chlorophyll-a (9.3 µg/mL) was induced by green light, while total carotenoids and b-carotene (3.05 and 0.89 µg/mL, respectively) by high white light. In Cyanothece, both white light intensities along with green maximized chlorophyll-a (~9 µg/mL) while high white light and green maximized total carotenoids (2.6-3.0 µg/mL). This study strongly indicates that these cyanobacteria can be cultured at the first stage under white light to accumulate sufficient biomass and, subsequently, under colored light for enhancing phycobiliproteins.

A new technique to evaluate Acidithiobacillus thiooxidans growth during a bioleaching process based on DNA quantification.

The potential of Acidithiobacillus (Thiobacillus) genus members, namely Acidithiobacillus ferrooxidans and Acidithiobacillus thiooxidans, for bioleaching purposes is known. Specifically, previous studies have shown the potential of A. thiooxidans strain DSM 26636 used in bioleaching processes to remove metals in high-metal-content matrices. All Acidithiobacillus growth-monitoring techniques available to date, including sulfate production, commonly used, present disadvantages. Thus, the current work shows a technique based on DNA quantification to evaluate the growth of A. thiooxidans DSM 26636, which is useful even in the presence of a high-metal-content residue. This proposed methodology may represent a functional complementary tool to evaluate Acidithiobacillus growth to develop biometallurgical applications.

Urine Flow Cytometry Parameter Cannot Safely Predict Contamination of Urine-A Cohort Study of a Swiss Emergency Department Using Machine Learning Techniques.

BACKGROUND: Urine flow cytometry (UFC) analyses urine samples and determines parameter counts. We aimed to predict different types of urine culture growth, including mixed growth indicating urine culture contamination. METHODS: A retrospective cohort study (07/2017-09/2020) was performed on pairs of urine samples and urine cultures obtained from adult emergency department patients. The dataset was split into a training (75%) and validation set (25%). Statistical analysis was performed using a machine learning approach with extreme gradient boosting to predict urine culture growth types (i.e., negative, positive, and mixed) using UFC parameters obtained by UF-4000, sex, and age. RESULTS: In total, 3835 urine samples were included. Detection of squamous epithelial cells, bacteria, and leukocytes by UFC were associated with the different types of culture growth. We achieved a prediction accuracy of 80% in the three-class approach. Of the n = 126 mixed cultures in the validation set, 11.1% were correctly predicted; positive and negative cultures were correctly predicted in 74.0% and 96.3%. CONCLUSIONS: Significant bacterial growth can be safely ruled out using UFC parameters. However, positive urine culture growth (rule in) or even mixed culture growth (suggesting contamination) cannot be adequately predicted using UFC parameters alone. Squamous epithelial cells are associated with mixed culture growth.

Culture growth of testate amoebae under different silicon concentrations.

Testate amoebae with self-secreted siliceous shell platelets ("idiosomes") play an important role in terrestrial silicon (Si) cycles. In this context, Si-dependent culture growth dynamics of idiosomic testate amoebae are of interest. Clonal cultures of idiosomic testate amoebae were analyzed under three different Si concentrations: low (50µmolL-1), moderate/site-specific (150µmolL-1) and high Si supply (500µmolL-1). Food (Saccharomyces cerevisiae) was provided in surplus. (i) Shell size of four different clones of idiosomic testate amoebae either decreased (Trinema galeata, Euglypha filifera cf.), increased (E. rotunda cf.), or did not change (E. rotunda) under the lowest Si concentration (50µmolSiL-1). (ii) Culture growth of idiosomic Euglypha rotunda was dependent on Si concentration. The more Si available in the culture medium, the earlier the entry into exponential growth phase. (iii) Culture growth of idiosomic Euglypha rotunda was dependent on origin of inoculum. Amoebae previously cultured under a moderate Si concentration revealed highest sustainability in consecutive cultures. Amoebae derived from cultures with high Si concentrations showed rapid culture growth which finished early in consecutive cultures. (iv) Si (diluted in the culture medium) was absorbed by amoebae and fixed in the amoeba shells resulting in decreased Si concentrations.

Viability of stressed Mycobacterium tuberculosis and association with multidrug resistance.

This study investigated biological characteristics of recovered stressed M. tuberculosis isolates that failed to grow in differential culture media for phenotypic identification and in culture media containing anti-tuberculosis drugs for drug-susceptibility testing, despite of having grown in primary culture. It represents an improvement in the diagnosis of MDR tuberculosis and tuberculosis control.

Evolution and role of corded cell aggregation in Mycobacterium tuberculosis cultures.

The aim of this study was to evaluate the evolution and role of corded cell aggregation in Mycobacterium tuberculosis cultures according to growth time and conditions. Thus, in standard culture using aerated 7H9 Middlebrook broth supplemented with 0.05% Tween 80, a dramatic CFU decrease was observed at the end of the exponential phase. This phase was followed by a stable stationary phase that led to dissociation between the optical density (O.D.) and CFU values, together with the formation of opaque colonies in solid culture. Further analysis revealed that this was due to cording. Scanning electron microscopy showed that cording led to the formation of very stable coiled structures and corded cell aggregations which proved impossible to disrupt by any of the physical means tested. Modulation of cording with a high but non-toxic concentration of Tween 80 led to a slower growth rate, avoidance of a sudden drop-off to the stationary phase, the formation of weaker cording structures and the absence of opaque colonies, together with a lower survival at later time-points. An innovative automated image analysis technique has been devised to characterize the cording process. This analysis has led to important practical consequences for the elaboration of M. tuberculosis inocula and suggests the importance of biofilm formation in survival of the bacilli in the extracellular milieu.

Viability of stressed Mycobacterium tuberculosis and association with multidrug resistance

This study investigated biological characteristics of recovered stressed M. tuberculosis isolates that failed to grow in differential culture media for phenotypic identification and in culture media containing anti-tuberculosis drugs for drug-susceptibility testing, despite of having grown in primary culture. It represents an improvement in the diagnosis of MDR tuberculosis and tuberculosis control.